PATENT DOCUMENT

Abstract:
This invention provides a method for treating tumors in a subject, comprising administering to the subject an effective amount of a recombinant interferon, wherein the interferon has the amino acid sequence of SEQ ID NO:2 and is encoded by the nucleotide sequence SEQ ID NO:1, wherein the tumors are selected from the group consisting of skin cancer, basal cell carcinoma, liver cancer, thyroid cancer, rhinopharyngeal cancer, solid carcinoma, prostate cancer, esophageal cancer, pancreatic cancer, superficial bladder cancer, hemangioma, epidermoid carcinoma, cervical cancer, glioma, leucocythemia, acute leucocythemia, chronic leucocythemia, lymphadenoma, and polycythemia vera.

Full Description:
[0001]    The application is a continuation application of U.S. Ser. No. 12/246,153, filed Oct. 6, 2008 which is a continuation application of U.S. Ser. No. 11/077,813, filed Mar. 10, 2005, which is a continuation-in-part of U.S. Ser. No. 10/927,975, filed Aug. 26, 2004, which is a continuation-in-part of U.S. Ser. No. 10/650,365, filed Aug. 28, 2003, which is a continuation-in-part of Int&#39;l App&#39;l No. PCT/CN02/00128, filed Feb. 28, 2002, which claims priority of Chinese App&#39;l No. 01104367.9, filed Feb. 28, 2001. The contents of the above applications are hereby incorporated in their entireties by reference into this application. 
     
    
       [0002]    Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. 
       FIELD OF THE INVENTION 
       [0003]    This invention is related to a field of bioengineering. Specifically this invention relates to a recombinant super-compound interferon (rSIFN-co) or its equivalent with changed spatial configuration, high efficacy and low side effects. Therefore, high dose of rSIFN-co may be used. This invention also relates to a process to produce said super-compound interferon (rSIFN-co) or a pharmaceutical composition comprising said super-compound interferon (rSIFN-co) or its equivalent, and uses of said interferon or composition for anti-viral and anti-tumor therapy. 
       BACKGROUND OF THE INVENTION 
       [0004]    IFN-con is a new interferon molecule constructed with the most popular conservative amino acid found in natural human IFN-α subtypes using genetic engineering methods. U.S. Pat. Nos. 4,695,623 and 4,897,471 have described it. IFN-con had been proven to have broad-spectrum IFN activity and virus- and tumor-inhibition and natural killer cell activity. U.S. Pat. No. 5,372,808 by Amgen, Inc. addresses treatment Infergen® (interferon alfacon-1). Chinese Patent No. 97193506.8 by Amgen, Inc. addresses re-treatment of Infergen® (interferon alfacon-1) on hepatitis C. Chinese Patent No. 98114663.5 by Shenzhen Jiusheng Bio-engineering Ltd. addresses recombinant human consensus interferon-α treatment for hepatitis B and hepatitis C. 
         [0005]    The United States Food and Drug Administration (FDA) authorized Amgen to produce Infergen® (interferon alfacon-1) with  E. Coli . for clinical hepatitis C treatment at the end of 1997. 
         [0006]    Hepatitis B patients can be identified when detecting HBsAg and the HBeAg. IFN-α is commonly used in clinics to treat hepatitis B. IFN-α binds superficial cell membrane receptors, thus inhibiting DNA and RNA (ribonucleic acid) duplication and inducing some enzymes to prevent duplication of the virus in hepatitis-infected cells. All IFNs can inhibit DNA duplication of viruses, but they cannot inhibit the e and s antigen expression. 
         [0007]    An outbreak of atypical pneumonia, referred to as severe acute respiratory syndrome (SARS) and first identified in Guangdong Province, China, has spread to several countries. Similar cases were detected in patients in Hong Kong, Vietnam, and Canada from February and March 2003. The World Health Organization (WHO) issued a global alert for the illness. In mid-March 2003, SARS was documented in health care workers and household members who had cared for patients with severe respiratory illness in the Far East. Many of these cases could be traced through multiple chains of transmission to one health care worker from Guangdong Province who visited Hong Kong, where he was hospitalized with pneumonia and died. By late April 2003, thousands of SARS cases and hundreds of SARS-related deaths from over 25 countries around the world were reported to WHO. Most of these cases occurred through exposure to SARS patients in household or health care settings. This invention provides a method to prevent and/or treat SARS. This disclosure describes recombinant super-compound interferon (rSIFN-co), method to produce the same and uses thereof. Particularly, the super-compound interferon disclosed herein is capable of inhibiting, preventing and/or treating the hepatitis viruses, SARS virus, or virus-induced upper respiratory diseases, the Influenza virus, for example Avian Influenza virus and Ebola virus. 
         [0008]    In addition, rSIFN-co is effective in preventing and/or treating viral diseases and tumors with less side effects as compared to other available interferons. 
       SUMMARY OF THE INVENTION 
       [0009]    This invention provides a recombinant super-compound interferon (rSIFN-co) and its equivalent with changed spatial configuration, high efficacy and low side effects. Therefore, high dose of rSIFN-co may be used. 
         [0010]    This invention also provides artificial gene encoding for the super-compound interferon or its equivalent. 
         [0011]    This invention provides a vector comprising the gene which codes for the super-compound interferon or its equivalent. 
         [0012]    This invention provides an expression system comprising the vector comprising the gene which codes for the super-compound interferon or its equivalent. This invention also provides a host cell comprising the vector comprising the gene which codes for the recombinant super-compound interferon (rSIFN-co) or its equivalent. Said host cell may be eukaryotic or prokaryotic, such as  E. Coli.    
         [0013]    This invention provides a method for producing a recombinant super-compound interferon (rSIFN-co) with changed spatial configuration and enhanced antiviral activity comprising steps of:
   (a) Introducing nucleic acid molecule which codes for said interferon with preferred codons for expression to an appropriate host; and   (b) Placing the introduced host in conditions allowing expression of said interferon.   
 
         [0016]    This invention provides the method for producing recombinant super-compound interferon (rSIFN-co), further comprising recovery of the expressed interferon. 
         [0017]    This invention provides a method for inhibiting, preventing or treating viral diseases, or for inhibiting or treating tumors in a subject comprising administering to the subject an effective amount of the super-compound interferon or its equivalent. 
         [0018]    This invention provides the above-described method wherein super-compound interferon is administered orally, via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via a respirator. 
         [0019]    This invention provides the method to prevent or treat viral diseases wherein the viral diseases is hepatitis A, hepatitis B, hepatitis C, other types of hepatitis, infections of viruses caused by Epstein-Barr virus, Human Immunodeficiency Virus (HIV), Ebola virus, Severe Acute Respiratory Syndrome Virus (SARS), Influenza virus, Cytomegalovirus, herpes simplex viruses, or other types of herpes viruses, papovaviruses, poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T-cell leukemia viruses I, or human T-cell leukemia viruses II, or human T-cell leukemia virus III. 
         [0020]    This invention provides the method to prevent or treat viral diseases wherein the viral diseases are Human Immunodeficiency Virus (HIV) and Ebola virus. 
         [0021]    This invention provides a method for anti-hepatitis activities. It can inhibit HBV-DNA replication, HBsAg and HBeAg production. 
         [0022]    This invention provides a method to prevent or treat upper respiratory infection diseases. 
         [0023]    This invention provides a method to prevent or treat tumors or cancers wherein the tumor is skin cancer, basal cell carcinoma and malignant melanoma, renal cell carcinoma, liver cancer, thyroid cancer, rhinopharyngeal cancer, solid carcinoma, prostate cancer, stomach/abdominal cancer, esophageal cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, and superficial bladder cancer, hemangioma, epidermoid carcinoma, cervical cancer, non-small-cell lung cancer, small-cell lung cancer, glioma, leucocythemia, acute leucocythemia and chronic leucocythemia, chronica myelocytic leukemia, hairy cell leukemia, lymphadenoma, multiple myeloma, polycythemia vera, or Kaposi&#39;s sarcoma. 
         [0024]    This invention provides a method for preventing or treating virus-induced diseases in a subject comprising administering to the subject an effective amount of recombinant super-compound interferon or a functional equivalent thereof. 
         [0025]    The super-compound interferon (rSIFN-co) may be administered orally, via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via a respirator. 
         [0026]    This invention provides a method for inhibiting the causative agent of virus-induced diseases, comprising contacting the causative agent with an effective amount of super-compound interferon or its equivalent. 
         [0027]    This invention also provides a method for inhibiting virus-induced diseases, comprising contacting an effective amount of the super-compound interferon with said virus or cells. This contact could be direct or indirect. 
         [0028]    This invention provides a composition comprising an effective amount of the super-compound interferon capable of inhibiting, preventing or treating virus-induced diseases, and a suitable carrier. 
         [0029]    This invention provides a pharmaceutical composition comprising an effective amount of the recombinant super-compound interferon capable of inhibiting, preventing or treating virus-induced diseases in a subject, and a pharmaceutically acceptable carrier. 
         [0030]    This invention provides a method for preventing or treating tumors in a subject comprising administering to the subject an effective amount of recombinant super-compound interferon or a functional equivalent thereof. 
         [0031]    This invention provides a method for inhibiting tumors, comprising contacting the causative agent with an effective amount of super-compound interferon or its equivalent. 
         [0032]    This invention also provides a method for inhibiting tumors, comprising contacting an effective amount of the super-compound interferon with said virus or cells. This contact could be direct or indirect. 
         [0033]    This invention provides a composition comprising an effective amount of the super-compound interferon capable of inhibiting, preventing or treating tumors, and a suitable carrier. 
         [0034]    This invention provides a pharmaceutical composition comprising an effective amount of the recombinant super-compound interferon capable of inhibiting, preventing or treating tumors in a subject, and a pharmaceutically acceptable carrier. 
     
    
     
       DETAILED DESCRIPTION OF THE FIGURES 
         [0035]      FIG. 1 . rSIFN-co cDNA sequence designed according to  E. Coli . codon usage and deduced rSIFN-co amino acid sequence 
           [0036]      FIGS. 2A-B . Sequence of another super-compound interferon 
           [0037]      FIG. 3 . Diagram of pLac T7 cloning vector plasmid 
           [0038]      FIG. 4 . Diagram of pHY-4 expression vector plasmid 
           [0039]      FIG. 5 . Construction process of expression plasmid pHY-5 
           [0040]      FIG. 6-A . Circular Dichroism spectrum of Infergen® 
         (Tested by Analysis and Measurement Center of Sichuan University) 
         [0041]    Spectrum range: 250 nm-190 nm
 
Sensitivity: 2 m°/cm
 
Light path: 0.20 cm
 
         Equipment: Circular Dichroism J-500C 
         [0042]    Samples: contains 30 μg/ml IFN-con1, 5.9 mg/ml of NaCl and 3.8 mg/ml of Na 2 PO 4 , pH7.0. 
           [0043]    Infergen® (interferon alfacon-1), made by Amgen Inc., also known as consensus interferon, is marketed for the treatment of adults with chronic hepatitis C virus (HCV) infections. It is currently the only FDA-approved, bio-optimized interferon developed through rational drug design and the only interferon with data on the label specifically for non-responding or refractory patients. InterMune&#39;s sales force re-launched Infergen® in January 2002 with an active campaign to educate U.S. hepatologists about the safe and appropriate use of Infergen®, which represents new hope for the more than 50 percent of HCV patients who fail other currently available therapies. 
           [0044]      FIG. 6-B . Circular Dichroism spectrum of Infergen® From Reference [Journal of Interferon and Cytokine Research. 16:489-499 (1996)] 
           [0045]    Circular dichroism spectra of concensus interferon subforms. Concensus interferon was fractionated using an anion exchange column. Samples were dialyzed into 10 mM sodium phosphate, pH 7.4. Measurements were made on Jasco J-170 spectopolarimeter, in a cell thermostat at 15° C. (—), acylated form; (--) cis terminal form; ( . . . ), met terminal form. A. Far UV Spectrum. B. Near UV Spectrum. 
           [0046]      FIG. 6-C . Circular Dichroism spectrum of rSIFN-co 
           [0000]    Spectrum range: 320 nm-250 nm
 
Sensitivity: 2 m°/cm
 
Light path: 2 cm
 
         Equipment: Circular Dichroism J-500C 
         [0047]    Samples: contains 0.5 mg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml of Na 2 PO 4 , pH7.0. 
           [0048]      FIG. 6-D . Circular Dichroism spectrum of rSIFN-co 
           [0000]    Spectrum range: 250 nm-190 nm
 
Sensitivity: 2 m°/cm
 
Light path: 0.20 cm
 
         Equipment: Circular Dichroism J-500C 
         [0049]    Samples: contains 30 μg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml of Na 2 PO 4 , pH7.0. 
           [0050]    Clearly, as evidenced by the above spectra, the secondary or even tertiary structure of rSIFN-co is different from Infergen®. 
           [0051]      FIG. 7 . rSIFN-co Crystal I 
           [0052]      FIG. 8 . rSIFN-co Crystal II 
           [0053]      FIG. 9 . The X-ray Diffraction of rSIFN-co Crystal 
           [0054]      FIG. 10 . Comparison of Inhibition Effects of Different Interferons on HBV Gene Expression 
           [0055]      FIG. 11A-C . Recombinant Super-Compound Interferon Spray 
         Height: 90 mm 
         [0056]    Width: 25 mm (bottom), 6 mm (top) 
         Weight: 9 g 
         [0057]    Volume delivery: 0.1 ml 
           [0058]      FIG. 11D . Recombinant Super-Compound Interferon Spray 
           [0059]    When using the spray for the first time, take off the cap and discharge in the air several times until some liquid squirts out. Do not need to test spray for subsequent uses. To use, follow the illustrations shown in the figure, i.e.: (1) Pre-spray and (2) Press down on the nozzle to release the medication. 
           [0060]      FIG. 12 . Comparison of Anti-SARS Activity of Interferons: Left top panel is negative control i.e. no virus added. Right top panel is positive control i.e. virus is added, but no rSIFN-co added. Left bottom panel is rSIFN-co with SARS Virus. Right bottom panel is αIFN. 
           [0061]      FIGS. 13A-1 . Curves of Changes of Body Temperature in Group A (5 patients) 
           [0062]    This figure is the record of body temperature changes of 5 patients in Group A. 
           [0063]      FIGS. 13A-2 . Curves of Changes of Body Temperature in Group A (5 patients) 
           [0064]    This figure is the record of body temperature changes of the other 5 patients in Group A. 
           [0065]      FIGS. 13B-1 . Curves of Changes of Body Temperature in Group B (5 patients) 
           [0066]    This figure is the record of body temperature changes of 5 patients in Group B. 
           [0067]      FIGS. 13B-2 . Curves of Changes of Body Temperature in Group B (6 patients) 
           [0068]    This figure is the record of body temperature changes of the other 6 patients in Group B. 
           [0069]      FIG. 14 . Graph of Inhibition of Wild-Type HIV by rSIFN-co using EXCEL and Luciferase as Y axis and concentration of rSIFN-co as X axis. A clear inverse dose-dependent response has been shown. 
           [0070]      FIG. 15 . Graph of Inhibition of Drug Resistant HIV by rSIFN-co using EXCEL and Luciferase as Y axis and concentration of rSIFN-co as X axis. A clear inverse dose-dependent response has been shown. 
           [0071]      FIG. 16 . rSIFN-co Inhibition of Influenza Virus: on the left, the control well is shown with Influenza virus added and without interferon, the cells had obvious CPE, such as rounding of cells, cell necroses, decrease in reflective light and sloughing off. 
           [0072]    On the right, the experimental wells is shown containing Influenza virus and rSIFN-co at concentration 10 nanogram per milliliter (ng/ml) had morphology comparable to normal cells. 
           [0073]      FIGS. 17A-H . Clinical Report of a patient with mammary and ovarian cancers. These figures show an obvious anti-cancer effect of rSIFN-co on this patient. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0074]    Recombinant Super-Compound Interferon (rSIFN-co) 
         [0075]    This invention provides a recombinant super-compound interferon (rSIFN-co) or an equivalent thereof with changed spatial configuration. This invention reveals that proteins with the same primary sequence might have different biological activities. As illustrated in this application, proteins with identical amino acid sequences may have different activities. The efficacy of these proteins may sometimes be improved and, sometimes, proteins with changed spatial configuration would reveal new function. 
         [0076]    As defined herein, equivalents are molecules which are similar in function to the compound interferon. An equivalent could be a deletion, substitution, or replacement mutant of the original sequence. Alternatively, it is also the intention of this invention to cover mimics of the recombinant super-compound interferon (rSIFN-co). Mimics could be a peptide, polypeptide or a small chemical entity. 
         [0077]    The recombinant super-compound interferon (rSIFN-co) described herein includes but is not limited to interferon α, β, γ or ω. In an embodiment, it is IFN-1α, IFN-2β or other mutants. 
         [0078]    In another embodiment, the recombinant super-compound interferon (rSIFN-co) disclosed has higher efficacy than α, β, γ, ω or a combination thereof and as compared to the interferons disclosed in U.S. Pat. Nos. 4,695,623 and 4,897,471. This recombinant super-compound interferon (rSIFN-co) is believed to have unique secondary or tertiary structure, wherein the 3-dimensional change is the result of changes in its production process. (See e.g.  FIG. 6 .) 
         [0079]    The recombinant super-compound interferon (rSIFN-co) described herein has spatial structure change(s) resulting from the changes of its production process. 
       Lower Side Effects 
       [0080]    The recombinant super-compound interferon (rSIFN-co) possesses lower side effects when compared with other interferons. These lower side effects allow for higher dosages to be used on patients in need of interferon treatments. These lower side effects open the possibility of using rSIFN-co for prevention and/or treatment of other diseases. Accordingly, this invention provides the recombinant super-compound interferon (rSIFN-co) with less side effects when administered to a subject. 
         [0081]    This invention provides recombinant super-compound interferon (rSIFN-co) with less side effects as compared to all currently available interferons. 
         [0082]    This invention further provides a method for treating or preventing viral diseases or tumors in a subject comprising administering to the subject an effective amount of the rSIFN-co with less side effects as compared to all currently available interferons. Therefore, high dose of rSIFN-co may be used. In an embodiment, the effective amount of recombinant super-compound interferon is in nanogram level. 
         [0000]    Process to Produce rSIFN-co 
       Artificial Gene 
       [0083]    This invention also provides artificial gene encoding for the super-compound interferon or its equivalent. It is within the ordinary skill to design an artificial gene. Many methods for generating nucleotide sequence and other molecular biology techniques have been described previously. See for example, Joseph Sambrook and David W. Russell, Molecular Cloning: A laboratory Manual, December 2000, published by Cold Spring Harbor Laboratory Press. 
         [0084]    The recombinant super-compound interferon (rSIFN-co) may also be produced with its gene as artificially synthesized cDNA with adjustment of its sequence from the wild-type according to codon preference of  E. Coli . Extensive discussion of said codon usage (preference) may be found in U.S. Pat. No. 4,695,623. See e.g. column 6, line 41—column 7, line 35. 
       Vector 
       [0085]    This invention provides a vector comprising the gene which codes for the super-compound interferon or its equivalent. 
         [0086]    This invention provides an expression system comprising the vector comprising the gene which codes for the super-compound interferon or its equivalent. The cells include, but are not limited to, prokaryotic or eukaryotic cells. 
         [0087]    This invention also provides a host cell comprising the vector comprising the gene which codes for the recombinant super-compound interferon (rSIFN-co) or its equivalent. 
         [0088]    This invention provides a method for producing a recombinant super-compound interferon (rSIFN-co) with changed spatial configuration and enhanced antiviral activity comprising steps of:
   (a) Introducing nucleic acid molecule which codes for said interferon with preferred codons for expression to an appropriate host; and   (c) Placing the introduced host in conditions allowing expression of said interferon.   
 
         [0091]    This invention provides the method for producing recombinant super-compound interferon (rSIFN-co), further comprising recovery of the expressed interferon. 
       Expression System 
       [0092]    The above-described recombinant super-compound interferon (rSIFN-co) may be produced by a high-efficiency expression system which uses a special promoter, enhancer or other regulatory element. In an embodiment the promoter is inducible. Said inducible promoter includes but is not limited to P BAD , heat shock promoters or heavy metal inducible promoters. Heat shock promoters are activated by physical means, while other promoters are activated by chemical means, for example IPTG or Tetracyclin. IPTG is added to the cells to activate the downstream gene or removed to inactivate the gene. Tetracyclin is used to induce promoters or to regulate the strength of promoters. 
         [0093]    In an embodiment the promoter is P BAD . Since early nineties, the properties of the mechanism of expression and repression of P BAD  by AraC have been studied extensively, and their interactions have been dissected at the molecular level. See Schleif, R. S. 1992 DNA looping.  Annu. Rev. Biochem.  61:199-223. The AraC protein is both a positive and negative regulator, when present, it turns on the transcription from the P BAD  promoter, when absent, the transcription occurs at a very low rate. See Guzman, L. M. et al. (1995)  J. Bact.  177: 4121-4130. The efficacy and mechanism of P BAD  promoter is well known by other ordinary skilled artisans and is commercially-available. 
         [0094]    The commercially-available Invitrogen expression kit includes pBAD vectors&#39; designed to provide precise control of expression levels. The araBAD promoter initiates gene expression. It&#39;s both positively and negatively regulated by the product of the araC gene, a transcriptional regulator that forms a complex with L-arabinose. In the absence of arabinose, the AraC dimer contacts the O2 and I1 half sites of the araBAD operon, forming a 210 bp DNA loop. For maximum transcriptional activation, two events are required: first, Arabinose binds to AraC. The protein releases the O2 site and binds the I2 site, which is adjacent to the I1 site. This releases the DNA loop and allows transcription to begin. Second, the cAMP activator protein (CAP)-cAMP complex binds to the DNA and stimulates binding of AraC to I1 and I2. Basal expression levels can be repressed by introducing glucose to the growth medium. Glucose acts by lowering cAMP levels, which in turn decreases the binding of CAP. As cAMP levels are lowered, transcriptional activation is decreased. Invitrogen&#39;s pBAD vectors are specifically designed for maximum expression and ease of use. 
         [0095]    Nine pBAD vectors are currently available: pBAD102/D-TOPO®, pBAD202/D-TOPO®, pBAD-TOPO®, pBAD/Thio-TOPO®, pBAD/His, pBAD/Myc-His, pBAD-DEST49, pBAD/gIII and pBAD/Thio-E. with the following features in all pBAD vectors:
       1. araBAD promoter for dose-dependent regulation   2. araC gene for tight control of the araBAD promoter   3. Optimized ribosome binding site for increased translation efficiency   4. rrnB transcription termination region for efficient transcript       
 
         [0100]    The inducible promoters include but are not limited to heat shock promoters or heavy metal inducible promoters. 
         [0101]    This invention provides a process for production of recombinant super-compound interferon (rSIFN-co) comprising introducing an artificial gene with selected codon preference into an appropriate host, culturing said introduced host in an appropriate condition for the expression of said compound interferon and harvesting the expressed compound interferon. 
         [0102]    The process may comprise extraction of super recombinant super-compound interferon (rSIFN-co) from fermentation broth, collection of inclusion bodies, denaturation and renaturation of the harvested protein. 
         [0103]    The process may maintain the high efficacy even when the recombinant super-compound interferon (rSIFN-co) is used with an agent and in a particular concentration. The process also comprises separation and purification of the recombinant super-compound interferon (rSIFN-co). The process further comprises lyophilization of the purified recombinant super-compound interferon (rSIFN-co). The process comprises production of liquid injection of recombinant super-compound interferon (rSIFN-co). 
         [0104]    In one embodiment, recombinant super-compound interferon (rSIFN-co) was produced with recombinant techniques. On the condition of fixed amino acid sequence, the IFN DNA was redesigned according to the  E. Coli . codon usage and then the rSIFN-co gene was artificially synthesized. rSIFN-co cDNA was cloned into the high-expression vector of  E. Coli . by DNA recombinant techniques, and a high expression of rSIFN-co was gained by using of induce/activate-mechanism of L-arabinose to activate the transcription of P BAD  promoter. 
         [0105]    Compared with usual thermo-induction, pH induction and IPTG induction systems of genetic engineering, arabinose induction/activation system has some advantages: (1) Common systems relieve promoter function by creating a “derepression” pattern. Promoters then induce downstream gene expression. Temperature and pH change and the addition of IPTG cannot activate promoters directly. In the system disclosed herein, L-arabinose not only deactivates and represses but also activates the transcription of P BAD  promoter which induces a high expression of rSIFN-co. Therefore, the arabinose induction/activation system is a more effective expression system. (2) The relationship between Exogenous and L-arabinose dosage is linear. This means the concentration of arabinose can be changed to adjust the expression level of the exogenous gene. Therefore, it is easier to control the exogenous gene expression level in  E. Coli . by arabinose than by changing temperature and pH value. This characteristic is significant for the formation of inclusion bodies. (3) L-arabinose is resourceful, cheap and safe, which, on the contrary, are the disadvantages of other inducers such as IPTG. 
         [0106]    This embodiment creates an effective and resistant rSIFN-co-expressing  E. Coli . engineering strain with an L-arabinose induction/activation system. The strain is cultivated and fermented under suitable conditions to harvest the bacterial bodies. Inclusion bodies are then purified after destroying bacteria and washing repeatedly. The end result, mass of high-purity, spatial-configuration-changed rSIFN-co protein for this invention and for clinical treatment, was gained from denaturation and renaturation of inclusion bodies and a series of purification steps. Said purification would not effect the biological activity of the purified protein. 
         [0107]    The above-described recombinant super-compound interferon (rSIFN-co) possesses anti-viral or anti-tumor activity, and; therefore, is useful in inhibiting, preventing and treating viral diseases, inhibiting or treating tumors, or cancers. 
       Viral Diseases 
       [0108]    This invention provides a method for treating or preventing viral diseases or tumors in a subject comprising administering to the subject an effective amount of the recombinant super-compound interferon (rSIFN-co) or its equivalent. 
         [0109]    As used herein, viral diseases include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, other types of hepatitis, infections caused by Epstein-Barr virus, Human Immunodeficiency Virus (HIV), Ebola virus, Severe Acute Respiratory Syndrome Virus (SARS), Influenza virus, Cytomegalovirus, herpes simplex viruses, other herpes viruses, papovaviruses, poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T-cell leukemia virus I, human T-cell leukemia virus II, or human T-cell leukemia virus III. 
         [0110]    In an embodiment, the effective amount is at nanogram level. In another embodiment, the virus is Human Immunodeficiency Virus and the effective amount is as low as 4 nanograms per milliliter. In another embodiment, the virus is Influenza and the effective amount is as low as 10 nanogram per milliliter. 
       Inhibition of DNA Replication and Secretion of HBsAg and HBeAg of Hepatitis B Virus. 
       [0111]    The recombinant super-compound interferon (rSIFN-co) inhibits the DNA duplication and secretion of HBsAg and HBeAg of Hepatitis B Virus. 
       Severe Acute Respiratory Syndrome Virus (SARS) 
       [0112]    This invention provides a method for preventing or treating Severe Acute Respiratory Syndrome, or virus-induced upper respiratory diseases, of a subject comprising administering to the subject an effective amount of recombinant super-compound interferon (rSIFN-co) or a functional equivalent thereof. In an embodiment of the above method, the interferon is α, β, γ, ω or a combination thereof. 
         [0113]    The recombinant super-compound interferon (rSIFN-co) may be administered orally, via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via a spray or a respirator. In an embodiment rSIFN-co is administered subcutaneously or intramuscularly at a dose of higher than or equal to 10 Million International Unit per square meter of surface area. In another embodiment rSIFN-co is administered subcutaneously or intramuscularly at a dose of higher than or equal to 20 Million International Unit per square meter of surface area. In an embodiment, the interferon is delivered by a spray device. In a specific embodiment, the device is described in  FIG. 11 . In one of the embodiments, the interferon is lyophilized. 
         [0114]    This invention provides a method for inhibiting the causative agent of Severe Acute Respiratory Syndrome, or virus-induced upper respiratory diseases, comprising contacting the agent with an effective amount of recombinant super-compound interferon (rSIFN-co) or its equivalent. 
         [0115]    It is determined that the causative agent of SARS is a virus. See eg. Rota et al (2003), Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome. Science 1085952 and Marra, et al. (2003), The Genome Sequence of the SARS-Associated Coronavirus. Science 1085853. 
         [0116]    This invention also provides a method for inhibiting Severe Acute Respiratory Syndrome virus or Severe Acute Respiratory Syndrome virus-infected cells, or virus-induced upper respiratory diseases, or cells infected with viruses capable of inducing upper respiratory diseases, comprising contacting an effective amount of the recombinant super-compound interferon (rSIFN-co) with said virus or cell. This contact could be direct or indirect. 
         [0117]    This invention provides a composition comprising an effective amount of the recombinant super-compound interferon (rSIFN-co) capable of inhibiting Severe Acute Respiratory Syndrome virus or Severe Acute Respiratory Syndrome virus-infected cells, or virus-induced upper respiratory diseases, or cells infected with viruses capable of inducing upper respiratory diseases, and a suitable carrier. 
         [0118]    This invention provides a composition comprising an effective amount of the super-compound interferon capable of preventing or treating Severe Acute Respiratory Syndrome, or virus-induced upper respiratory diseases, of a subject and a suitable carrier. 
         [0119]    This invention provides a pharmaceutical composition comprising an effective amount of the recombinant super-compound interferon (rSIFN-co) capable of inhibiting Severe Acute Respiratory Syndrome virus or Severe Acute Respiratory Syndrome virus-infected cells, or virus-induced upper respiratory diseases, and a pharmaceutically acceptable carrier. 
         [0120]    This invention provides a pharmaceutical composition comprising an effective amount of the recombinant super-compound interferon (rSIFN-co) capable of preventing or treating Severe Acute Respiratory Syndrome, or virus-induced upper respiratory diseases, in a subject and a pharmaceutically acceptable carrier. 
         [0121]    This invention provides a device to deliver the above-described pharmaceutical composition. 
         [0122]    In a preferred embodiment, the subject is a human. As it can easily be appreciated, the super-compound interferon can be used in other animals or mammals. 
         [0123]    This invention provides a method for preventing Severe Acute Respiratory Syndrome or virus-induced upper respiratory diseases, in humans comprising application of the super-compound interferon three times a day via a spray which contains twenty micrograms of interferon, equal to ten million units of activity in three milliliter. 
       Viral Upper Respiratory Infection (VURI) 
       [0124]    Viral upper respiratory infection, alternative names common cold, colds. This is a contagious viral infection of the upper respiratory tract characterized by inflammation of the mucous membranes, sneezing, and a sore throat. It is usually caused by over 200 different viruses, known as rhinoviruses. Colds are not caused by the same viruses responsible for Influenza. Colds are spread through droplets from the coughing or sneezing of others with a cold or by hand contact with objects contaminated by someone with a cold. The incidence of colds is highest among children, and the incidence decreases with age because immunity to the virus causing the cold occurs after the illness. Gradually, immunity to a wide variety of viruses that cause colds is developed in adults. Children may have 10 colds a year, and adults may have 3 colds a year. 
         [0125]    The U.S. Centers for Disease Control and Prevention have estimated that the average annual incidence of upper respiratory tract infections (URIs) in the United States is 429 million episodes, resulting in more than $2.5 billion in direct and indirect healthcare costs. The common cold is most often caused by one of several hundred rhinoviruses (52%), but coronaviruses (8%) or the respiratory syncytial virus (7%) may also lead to infection. Other viruses, such as influenza (6%), parainfluenza, and adenoviruses, may produce respiratory symptoms, but these are often associated with pneumonia, fever, or chills. 
         [0126]    Colds occur in a seasonal pattern that usually begins in mid-September and concludes in late April to early May. The common cold is quite contagious and can be transmitted by either person-to-person contact or airborne droplets. Upper respiratory symptoms usually begin 1 to 2 days after exposure and generally last 1 to 2 weeks, even though viral shedding and contagion can continue for 2 to 3 more weeks. Symptoms may persist with the occurrence of complications such as sinusitis or lower respiratory involvement such as bronchitis or pneumonia. 
         [0127]    The common cold has a variety of overt symptoms, including malaise, nasal stuffiness, rhinorrhea, nonproductive cough, mild sore throat, and, in some cases, a low-grade fever. Because of the similarity of symptoms, a cold may be mistaken for perennial allergic rhinitis, but allergies can usually be ruled out because of the differences in chronicity. 
         [0128]    If a patient presents with a viral URI, the spectrum of remedies is extensive. Since most of these infections are self-limiting, clinicians usually recommend rest and fluids, but other treatments include environmental and nutritional therapies, over-the-counter and prescription decongestant and antihistamine products, new antihistamine and anticholinergic nasal formulations, and antibiotics. Table 1 lists commonly used cough and cold medications and their side effects. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 A Profile of Common Cough and Cold Medications and Their Side 
               
               
                 Effects 
               
             
          
           
               
                   
                   
                 Side Effects and Special 
               
               
                 Medication 
                 Purpose 
                 Considerations 
               
               
                   
               
               
                 Aerosolized beta2 
                 Reverse 
                 Raises heart rate and may 
               
               
                 agonists (eg, 
                 postinflammatory 
                 cause tremor 
               
               
                 albuterol) 
                 bronchospasm 
               
               
                 Alcohol-based 
                 Treat multiple 
                 Potential drowsiness and 
               
               
                 liquid combination 
                 symptoms 
                 coordination problems 
               
               
                 products 
               
               
                 Alpha1 agonists 
                 Decongestion 
                 May cause tachycardia, 
               
               
                 (oral) (eg, 
                   
                 nervousness, transient 
               
               
                 pseudoephedrine, 
                   
                 stimulation, dizziness, 
               
               
                 phenyl- 
                   
                 drowsiness, elevation of 
               
               
                 propanolamine) 
                   
                 blood pressure 
               
               
                 Anticholinergic 
                 Drying 
                 May cause nasal dryness and 
               
               
                 compounds: 
                   
                 occasional epistaxis 
               
               
                 Ipratropium 
               
               
                 bromide 
               
               
                 (topical) 
               
               
                 Other 
                 Drying 
                 May cause orthostasis, 
               
               
                 anticholinergics 
                   
                 dysfunction of heat 
               
               
                 (eg, 
                   
                 regulation, dry mouth, 
               
               
                 methscopolamine, 
                   
                 constipation 
               
               
                 atropine, 
               
               
                 hyoscyamine) 
               
               
                 Antihistamines 
                 Drying 
                 Drowsiness, dry mouth, 
               
               
                 (oral) (eg, 
                   
                 orthostatic hypertension 
               
               
                 chlorpheniramine, 
               
               
                 diphenhydramine) 
               
               
                 Benzonatate 
                 Cough suppression, 
                 Chewing can numb the 
               
               
                 capsules 
                 local anesthesia 
                 mouth; can cause sedation, 
               
               
                   
                   
                 dizziness 
               
               
                 Codeine, 
                 Cough suppression 
                 Drowsiness, constipation, 
               
               
                 hydrocodone 
                   
                 nausea 
               
               
                 Dextromethorphan 
                 Cough suppression 
                 Drowsiness possible, but 
               
               
                   
                   
                 side effects uncommon 
               
               
                 Guaifenesin 
                 Promote 
                 No side effects; must be 
               
               
                   
                 expectoration 
                 taken with lots of water to 
               
               
                   
                 (mucolysis) 
                 improve efficacy 
               
               
                 Topical 
                 Decongestion 
                 Local burning; prolonged use 
               
               
                 decongestants (eg, 
                   
                 may cause dependence 
               
               
                 oxymetazoline, 
               
               
                 phenylephrine) 
               
               
                 Zinc and vitamin C 
                 Possible reduction 
                 Possible taste disturbance, 
               
               
                 lozenges 
                 in symptom severity 
                 increase of oxalate stones 
               
               
                   
                 and duration 
                 if susceptible 
               
               
                   
               
             
          
         
       
     
       Prevention and Treatment of Upper Respiratory Tract Infections (URI) 
       [0129]    Nearly 70˜80% URI are caused by viruses such as respiratory Syncytical virus, adenovirus, rhinovirous, cox-sackie virus, corona virus and its variant, influenza A virus and its variant, influenza B virus and its variant, parainfluenza virus and its variant, or enterovirus and its variant. A main cause of URI in adults is from rhinovirous. For children, respiratory syncytical virus and parainfluenza virus are two leading causes of URI. 
         [0130]    Recombinant super-compound interferon (rSIFN-co) plays an important role in the fight against virus that causes URI. Super-compound interferon gains its anti-virus affects mainly via two mechanisms:
       1. Attach to surface of sensitive cells and induce them to produce anti-virus protein, then block the duplication and reproduction of viruses in vivo.   2. recombinant super-compound interferon (rSIFN-co) can adjust immune response, including T-cell immune response, activity of NK cell, the phagocytosis function of monokaryon, and even formation of some antibodies in vivo.       
 
         [0133]    In treatment for URI, recombinant super-compound interferon (rSIFN-co) can be directly applied to the affected area via a spray or a respiration. This method of treatment allows the interferon to reach the target cells first hand. Consequently, marketing the supply as a spray, rather than via oral or injection, would be safer and more effective for administrating the interferon. 
       Prevention and Treatment of SARS 
       [0134]    With the consent of the Sichuan (a province in China) working group on SARS prevention and control, the distribution of recombinant super-compound interferon (rSIFN-co) began in May of 2003. Super-compound interferon spray was allocated to doctors and nurses in hospitals, populated areas with a high risk for SARS, and to the National research group on prevention and control of SARS. Among the 3,000 users as of Dec. 19, 2003, there were no reports of any side effects connected to the use of the spray. Furthermore, none of the doctors and nurses, the people of Sichuan Province, or other organizations that have used the Super-compound interferon spray has been infected by SARS. 
         [0135]    Therefore, this invention provides a method for inhibiting, preventing or treating virus replication or virus-infected cells by contacting said virus or infected cells with an effective amount of the recombinant super-compound interferon (rSIFN-co) or its equivalent. 
       Prevention and Treatment of Tumors 
       [0136]    This recombinant super-compound interferon (rSIFN-co) is useful in inhibiting, preventing or treating the following cancers or tumors: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
             
             
               
                 Cancer 
                 Skin Cancer 
                 Basal Cell Carcinoma 
               
               
                   
                   
                 Malignant Melanoma 
               
               
                   
                 Renal cell carcinoma 
               
               
                   
                 Liver Cancer 
               
               
                   
                 Thyroid Cancer 
               
               
                   
                 Rhinopharyngeal Cancer 
               
               
                   
                 Solid Carcinoma 
                 Prostate Cancer 
               
               
                   
                   
                 Stomach/Abdominal Cancer 
               
               
                   
                   
                 Esophageal Cancer 
               
               
                   
                   
                 Rectal Cancer 
               
               
                   
                   
                 Pancreatic Cancer 
               
               
                   
                   
                 Breast Cancer 
               
               
                   
                 Ovarian Cancer &amp; 
               
               
                   
                 Superficial Bladder Cancer 
               
               
                   
                 Hemangioma 
               
               
                   
                 Epidermoid Carcinoma 
                 Cervical Cancer 
               
               
                   
                   
                 Non-small Cell Lung Cancer 
               
               
                   
                   
                 Small Cell Lung Cancer 
               
               
                   
                   
                 Glioma 
               
               
                 Malignant 
                 Leucocythemia 
                 Acute Leucocythemia 
               
               
                 Hemal 
                   
                 Chronic Leucocythemia 
               
               
                 Disease 
                 Chronic Myelocytic Leukemia 
               
               
                   
                 Hairy Cell Leukemia 
               
               
                   
                 Lymphadenoma 
               
               
                   
                 Multiple Myeloma 
               
               
                   
                 Polycythemia Vera 
               
               
                 Others 
                 Kaposi&#39;s Sarcoma 
               
               
                   
               
             
          
         
       
     
         [0137]    Accordingly, this invention provides a method for inhibiting tumor or cancer cell growth by contacting the recombinant super-compound interferon (rSIFN-co) or its equivalent with said tumor or cancer cells. 
       Formulation and Route of Administration 
       [0138]    This invention also provides the produced super-compound interferon by the above processes. 
         [0139]    This invention provides a composition comprising recombinant super-compound interferon (rSIFN-co) or its equivalent and a suitable carrier. 
         [0140]    This invention provides a pharmaceutical composition comprising the recombinant super-compound interferon (rSIFN-co) or its equivalent and a pharmaceutically acceptable carrier. 
         [0141]    This invention provides the above-described method wherein recombinant super-compound interferon (rSIFN-co) was administered via orally via vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal or mucosal administration, or by inhalation via a spray or a respirator. 
         [0142]    This invention provides the above-described method wherein recombinant super-compound interferon (rSIFN-co) was administered following the protocol of injections of 9 μg, 15 μg or 24 μg every two days, 3 times a week, for 24 weeks. 
         [0143]    It was surprising to find that recombinant super-compound interferon (rSIFN-co), the spatial structure of which has been changed, is not only a preparation to inhibit the DNA duplication of hepatitis B, but to inhibit the secretion of HBsAg and HBeAg on 2.2.15 cells. 
         [0144]    One objective of this invention is to offer a preparation of recombinant super-compound interferon (rSIFN-co) to directly inhibit the DNA duplication of hepatitis B viruses and the secretion of HBeAg and HBsAg of hepatitis B and decrease them to normal levels. 
       Formulation 
       [0145]    The following are some rSIFN-co preparations: tablets, capsules, liquids for oral consumption, pastes, injections, sprays, suppositories, and solutions. Injections are recommended. It is common to subcutaneously inject or vein-inject the medicine. The medicine carrier could be any acceptable medicine carrier, including carbohydrates, cellulosum, adhesive, collapse, emollient, filling, add-dissolving agent, amortization, preservative, thickening agent, matching, etc. 
         [0146]    This invention also provides a pharmaceutical composition comprising the above composition and a pharmaceutically acceptable carrier. 
         [0147]    For the purposes of this invention, “pharmaceutically acceptable carriers” means any of the standard pharmaceutical carriers. Examples of suitable carriers are well known in the art and may include, but are not limited to, any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution and various wetting agents. Other carriers may include additives used in tablets, granules, capsules, etc. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gum, glycols or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well-known conventional methods. 
         [0000]    Increase of the Half-Life of rSIFN-co 
       Pegylation 
       [0148]    Pegylation is the process by which polyethylene glycol chains are attached to protein and peptide drugs to increase pharmacokinetics by shielding these proteins and peptide drugs from proteolytic enzymes. See Harris and Chess,  Effect of pegylation on pharmaceuticals . Nat Rev Drug Discov. 2003 March; 2(3):214-21. 
         [0149]    Pegylations is a well-established method for increasing the circulating half-life of protein and liposomal pharmaceuticals based on large hydrodynamic volume of polyethylene glycols. These polyethylene glycols shield the proteins and peptide drugs from renal clearance, enzymatic degradation and immune system recognition, thus their half-life and making them more acceptable to patients. See Molineux,  Pegylation: engineering improved pharmaceuticals for enhanced therapy . Cancer Treat Rev. 2002 April; 28 Suppl A: 13-6. The author concludes that pegylation has beneficial effect on the quality of life of cancer patients. 
         [0150]    Pegylation of the interferon increases the amount of time the interferon remains in the body by increasing the size of the interferon molecule by decreasing the rate of absorption, prolonging the half-life and the rate of interferon clearance. Thus, the duration of biological activity is increased with pegylated interferon over nonpegylated interferon, thus providing an advantage over nonpegylated interferons with less frequent administration and comparable tolerability. The author states that monotherapy with pegylated interferon produces a better response in some patients than monotherapy with the nonpegylated formulation. See Baker,  Pegylated Interferons. Rev Gastroenterol Disord.  2001; 1(2):87-99. 
       Sustained Release or Controlled Release 
       [0151]    Sustained release delivery matrices and liposomes maybe used with rSIFN-co to create sustained release and controlled release formulation. See Robinson and Talmadge,  Sustained Release of Growth Factors . In Vivo 2002 November-December; 16(6): 535-40. The authors state that both pegylation and sustained release delivery matrices and liposomes improve the pharmacokinetic and pharmacodynamic properties of recombinant molecules, and thus by improving clinical efficacy these approaches increase patient compliance. 
         [0152]    This invention provides recombinant super-compound interferon (rSIFN-co) comprising an agent or encapsulated by an agent, capable of affecting the half-life or delivery of said interferon. In an embodiment this agent is polyethylene glycol (PEG). 
         [0153]    This invention further provides a method for treating or preventing viral diseases or tumors in a subject comprising administering to the subject an effective amount of the recombinant super-compound interferon (rSIFN-co) or its equivalent comprising an agent or encapsulated by an agent, capable of affecting the half-life or delivery of said interferon. In an embodiment this agent is polyethylene glycol (PEG). 
         [0154]    This invention will be better understood from the examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter. 
       EXPERIMENTAL DETAILS 
       [0155]    IFN-con is a new interferon molecule constructed according to conservative amino acids in human IFN-α subtype using genetic engineering methods. It has been proven that IFN-con has broad-spectrum IFN activity, such as high antivirus and tumor inhibition activity, especially for effectively treating hepatitis C. 
         [0156]      E. Coli . codon was used to redesign rSIFN-co cDNA and then artificially synthesize cDNA of rSIFN-co from published Infergen® (interferon alfacon-1) DNA sequences and deduced amino acid sequences ( FIG. 1 ). 
         [0157]    In order to get pure rSIFN-co protein, rSIFN-co cDNA was cloned into  E. Coli . high-expression vector, and L-arabinose, which can activate strong P BAD  promoter in vectors, was used to induce high expression of rSlFN-co gene. 
       Example 1 
     Synthesis of  E. Coli . cDNA Sequence 
       [0158]    Redesign of rSIFN-co cDNA Sequence 
         [0159]    rSIFN-co cDNA was redesigned according to the codon usage of  E. Coli . to achieve high expression in  E. Coli . Deduced amino acid sequence from the redesigned cDNA sequence of rSIFN-co is completely coincidental with primitive amino acid sequence of published Infergen® (interferon alfacon-1) ( FIG. 1 ). 
         [0000]    rSIFN-co cDNA Sequence Synthesis
 
rSIFN-co cDNA 5′-Terminus and 3′-Terminus Semi-Molecular Synthesis
 
         [0160]    Two semi-moleculars can be directly synthesized: rSIFN-co cDNA 5′-terminus 280 bp (fragment I) and 3′-terminus 268 bp (fragment II) by PCR. There are 41 bp overlapping among fragment II and fragment I. 
       (1) Chemical Synthesis Oligodeoxynucleotide Fragment: 
       [0161]      
         [0000]    
       
         
               
               
             
           
               
                 Oligomer A: 
                   
               
               
                 (SEQ ID NO: 10) 
                   
               
               
                 5′ATGTGCGACCTGCCGCAGACCCACTCCCTGGGTAACCGTCGTGCTCTGATCCTGCTGGCTCA 
                   
               
               
                   
               
               
                 GATGCGTCGTATCTCCCCGTTCTCCTGCCTGAAAGACCGTCACGAC3′ 
               
               
                   
               
               
                 Oligomer B: 
               
               
                 (SEQ ID NO: 11) 
                   
               
               
                 5′CTGAAAGACCGTCACGACTTCGGTTTCCCGCAGGAAGAATTCGACGGTAACCAGTTCCAGAAAGCT 
                   
               
               
                   
               
               
                 CAGGCTATCTCCGTTCTGCACGAAATGATCCAGCAGACCTTC3′ 
               
               
                   
               
               
                 Oligomer C: 
               
               
                 (SEQ ID NO: 12) 
                   
               
               
                 5′GCTGCTGGTACAGTTCGGTGTAGAATTTTTCCAGCAGGGATTCGTCCCAAGCAGCGGAGGAG 
                   
               
               
                   
               
               
                 TCTTTGGTGGAGAACAGGTTGAAGGTCTGCTGGATCATTTC3′ 
               
               
                   
               
               
                 Oligomer D: 
               
               
                 (SEQ ID NO: 13) 
                   
               
               
                 5′ATCCCTGCTGGAAAAATTCTACACCGAACTGTACCAGCAGCTGAACGACCTGGAAGCTTGCG 
                   
               
               
                   
               
               
                 TTATCCAGGAAGTTGGTGTTGAAGAAACCCCGCTGATGAAC3′ 
               
               
                   
               
               
                 Oligomer E: 
               
               
                 (SEQ ID NO: 14) 
                   
               
               
                 5′GAAGAAACCCCGCTGATGAACGTTGACTCCATCCTGGCTGTTAAAAAATACTTCCAGCGTAT 
                   
               
               
                   
               
               
                 CACCCTGTACCTGACCGAAAAAAAATACTCCCCGTGCGCTTGGG3′ 
               
               
                   
               
               
                 Oligomer F: 
               
               
                 (SEQ ID NO: 15) 
                   
               
               
                 5′TTATTCTTTACGACGCAGACGTTCCTGCAGGTTGGTGGACAGGGAGAAGGAACGCATGATTT 
                   
               
               
                   
               
               
                 CAGCACGAACAACTTCCCAAGCGCACGGGGAGTATTTTTTTTCGGTCAGG3′ 
               
             
          
         
       
     
         [0162]    PCR I for Fragment I: oligodeoxynucleotide B as template, oligodeoxynucleotide A and C as primers, synthesized 280 bp Fragment I. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 PCR I mixture (units: μl) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 sterilized distilled water 
                 39 
                   
               
               
                 10xPfu buffer (Stratagen American Ltd.) 
                 5 
               
               
                 dNTP mixture (dNTP concentration 2.5 mmol/L) 
                 2 
               
               
                 Oligomer A primer (25 μmol/L) 
                 1 
               
               
                 Oligomer C primer (25 μmol/L) 
                 1 
               
               
                 Oligomer B template (1 μmol/L) 
                 1 
               
               
                 Pfu DNA polymerase (Stratagen American Ltd.) (25 U/μl) 
                 1 
               
               
                 Total volume 
                 50 
                 μl 
               
               
                   
               
             
          
         
       
       
         
           
             PCR Cycle: 
             95° C.2m→(95° C.45s→65° C.1m→72° C.1m)×25 cycle→72° C.10m→4° C. 
             PCR II for Fragment II: oligodeoxynucleotide E as template, oligodeoxynucleotide D and F as primers, synthesized 268 bp Fragment II. 
           
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 PCR II mixture (units: μl) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 sterilized distilled water 
                 39 
                   
               
               
                 10xPfu buffer (Stratagen American Ltd.) 
                 5 
               
               
                 dNTP mixture (dNTP concentration 2.5 mmol/L) 
                 2 
               
               
                 Oligomer D primer (25 μmol/L) 
                 1 
               
               
                 Oligomer F primer (25 μmol/L) 
                 1 
               
               
                 Oligomer E template (1 μmol/L) 
                 1 
               
               
                 Pfu DNA polymerase (Stratagen American Ltd.) (25 U/μl) 
                 1 
               
               
                 Total volume 
                 50 
                 μl 
               
               
                   
               
             
          
         
       
       
         
           
             PCR cycle: the same as PCR I
 
Assembling of rSIFN-co cDNA
 
           
         
       
     
         [0167]    Fragment I and II were assembled together to get the complete cDNA molecular sequence of rSIFN-co using the overlapping and extending PCR method. Restriction enzyme Nde I and Pst I were introduced to clone rSIFN-co cDNA sequence into plasmid. 
         [0168]    (1) Chemical Synthesis Primers 
         [0000]    
       
         
               
               
             
           
               
                   
                 Oligomer G: 
               
               
                   
                 (SEQ ID NO: 16) 
               
               
                   
                 5′ATCGGCCATATGTGCGACCTGCCGCAGACCC3′ 
               
               
                   
                   
               
               
                   
                 Oligomer H: 
               
               
                   
                 (SEQ ID NO: 17) 
               
               
                   
                 5′ACTGCCAGGCTGCAGTTATTCTTTACGACGCAGACGTTCC3′ 
               
             
          
         
       
     
         [0169]    (2) Overlapping and Extending PCR 
         [0000]    
       
         
               
             
               
               
             
           
               
                   
               
               
                 PCR mixture (units: μl) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 sterilized distilled water 
                 38 
               
               
                 10xPfu buffer (Stratagen American Ltd.) 
                  5 
               
               
                 dNTP mixture (dNTP concentration 2.5 mmol/L) 
                  2 
               
               
                 primer G (25 μmol/L) 
                  1 
               
               
                 primer H (25 μmol/L) 
                  1 
               
               
                 *fragment I preduction (1 μmol/L) 
                  1 
               
               
                 *fragment II preduction (1 μmol/L) 
                  1 
               
               
                 Pfu DNA polymerase (Stratagen American Ltd.) (2.5 U/μl) 
                  1 
               
               
                 Total volume 
                 50μ 
               
               
                   
               
               
                 *Separate and purify PCR production with StrataPrep PCR purification kit produced by Stratagen American Ltd. And dissolve into sterilized distilled water. 
               
             
          
         
       
       
         
           
             PCR cycle: the same as PCR I
 
rSIFN-co Gene Clone and Sequence Analysis
 
           
         
       
     
         [0171]    pLac T7 plasmid as cloning vector. pLac T7 plasmid is reconstructed with pBluescript II KS(+) plasmid produced by Stratagen ( FIG. 3 ). 
         [0172]    Purified PCR production of rSIFN-co cDNA with StrataPrep PCR purification kit. Digest cDNA and pLac T7 plasmid with NdeI and PstI. Run 1% agarose gel electrophoresis and separate these double-digested DNA fragments. Recover 507 bp long rSIFN-co DNA fragment and 2.9 kb plasmid DNA fragment. Ligate these fragments by T4 DNA ligase to form a recombinant plasmid. Transform DH 5α competent cells (Gibco) with the recombinant plasmid, culture at 37° C. overnight. Identify the positive recombinant colony, named pHY-1. 
         [0173]    Run DNA sequencing with SequiTherm™ Cycle Sequencing Kit produced by American Epicentre Technologies Ltd using L1-COR Model 4000L. Primers are T7 and T3 common sequence primer, the DNA sequencing result matches theoretic design. 
         [0174]    Purify the rSIFN-co, sequence the N-terminus amino acids, the N-terminus amino acid sequence matches experimental design which is as follows: N-Cys-Asp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly-Asn-Arg-Arg-Ala-Leu- 
       Construction, Transformation, Identification, and Hereditary Stability of Expression Vector 
     Construction and Transformation of Expression Vector 
       [0175]    Digested  E. Coli . expression vector pHY-4 (see  FIG. 3 ) with Nde I to linearize and subsequently digest with Xba I. Run 1% agarose gel electrophoresis, and purify the 4.8 kb pHY-4 Nde I-Xba I digest fragment with QIAEX II kit produced by QIAGEN Germany Ltd. 
         [0176]    At the same time, the pHY-4 plasmid is double digested with Nde I-Xba I. Run 1% agarose gel electrophoresis and purify the 715 bp fragment. Ligate the rSIFN-co and pHY-4 fragments with T4 DNA ligase to construct the recombinant plasmid (See  FIG. 4 ). Transform DH 5α competent cells with the recombinant plasmid. Spread the transformed cells on LB plate with Amp, 37° C. culture overnight. 
       Positive Cloning Strain Screening 
       [0177]    Randomly choose  E. Coli . colonies from above LB-plate, screening the positive strains containing recombinant vector by endonuclease digesting and PCR analysis. Name one of the positive recombinant plasmid pHY-5, and name the strain containing pHY-5 plasmid PVIII. Amplify and store the positive strain with glycerol in −80° C. 
         [0000]    High Expression of rSIFN-co gene in  E. Coli . In pHY-5 plasmid, rSIFN-co gene is under the control of strong promoter P BAD . This promoter is positively and negatively regulated by the product of the gene araC. AraC is a transcriptional regulator that forms a complex with arabinose. In the absence of arabinose, the AraC dimer binds O 2  and I 1 , forming a 210 bp loop. This conformation leads to a complete inhibition of transcription. In the presence of arabinose, the dimer is released from O 2  and binds I 1  and I 2  leading to transcription. Arabinose binding deactivates, represses, and even activates the transcription of P BAD  promoter, which stimulates P BAD , inducing high expression of rSIFN-co. rSIFN-co expression level in PVIII is more than 50% of the total  E. Coli . protein. 
       Summary 
       [0178]    rSIFN-CO is a new interferon molecule artificially built according to the conservative amino acid of human a interferons. It has been proven as an effective anti-hepatitis drug. In order to get enough pure rSIFN-co protein, a stable recombinant  E. Coli . strain which highly expresses rSIFN-co protein was constructed. 
         [0179]    First, according to published Infergen® (interferon alfacon-1) amino acid sequence,  E. Coli . codon was used to synthesize the whole cDNA of rSIFN-co. This DNA fragment was sequenced, proving that the 501 bp codon sequence and TAA termination codon sequence are valid and identical to theocratic design. Subsequent analysis revealed that the N-terminus amino acid sequence and amino acid composed of rSIFN-co produced by the recombinant strain were both identical to the prediction. 
         [0180]    The rSIFN-co cDNA was cloned into  E. Coli . high-expression vector pHY-4 plasmid to construct the recombinant plasmid pHY-5 . E. Coli . LMG194 strain was further transformed with pHY-4 plasmid to get stable rSIFN-co high-expression transformant. This transformant was cultured for 30 generations. The heredity of pHY-5 recombinant plasmid in  E. Coli . LMG194 was normal and stable, and the expression of rSIFN-co was high and steady. 
         [0181]      E. Coli . LMG194, which contains recombinant pHY-5 plasmid, is actually an ideal high-expression engineering strain. 
       REFERENCES 
       [0000]    
       
         1. Blatt L M, Davis J M, Klein S B. et al. The biologic activity and molecular characterization of a novel synthetic interferon-alpha species, consensus interferon. Journal of Interferon and Cytokine Research, 1996; 16(7):489-499. 
         2. Alton, K. et al: Production characterization and biological effects of recombinant DNA derived human IFN-α and IFN-γ analogs. In: De Maeger E, Schellekens H. eds. The Biology of Interferon System. 2nd ed. Amsterdam: Elsevier Science Publishers, 1983: 119-128 
         3. Pfeffer L M. Biologic activity of natural and synthetic type 1 interferons. Seminars in Oncology, 1997; 24 (3 suppl 9):S9-63—S9-69. 
         4. Ozes O N, Reiter Z, Klein S, et al. A comparison of interferon-con1 with natural recombinant interferons-α antiviral, antiproliferative, and natural killer-inducing activities. J. Interferon Res., 1992; 12:55-59. 
         5. Heathcote E J L, Keeffe E B, Lee S S, et al. Re-treatment of chronic hepatitis C with consensus interferon. Hepatology, 1998; 27(4):1136-1143. 
         6. Klein M L, Bartley T D, Lai P H, et al. Structural characterization of recombinant consensus interferon-alpha. Journal of Chromatography, 1988; 454:205-215. 
         7. The Wisconsin Package, by Genetics Computer Group, Inc. Copyright 1992, Medison, Wis., USA 
         8. Nishimura, A et al: A rapid and highly efficient method for preparation of competent  E. coli  cells. Nuclei. Acids Res. 1990, 18:6169 
         9. All molecular cloning techniques used are from: Sambrook, J., E. F. Fritsch and T. Maniatis. Molecular Cloning: A laboratory manual, 2nd ed. CSH Laboratory Press, Cold Spring Harbour, N.Y. 1989. 
         10. Guzman, L. M et al: Tight regulation, modulation, and high-level express-ion by vectors containing the arabinose P BAD  promoter. J. Bacteriol. 1995, 177: 4121-4130.
 
rSIFN-co cDNA Sequence Designed According to  E. coli . Codon Usage and deduced rSIFN-co amino acid sequence
 
       
     
         [0000]    
       
         
               
               
               
             
           
               
                   
                 5′   11    21    31   41    51 
                   
               
               
                  +1 
                 M C D L P Q T H S L G N R R A L I L L A 
               
               
                   1 
                 ATGTGCGACC TGCCGCAGAC CCACTCCCTG GGTAACCGTC GTGCTCTGAT CCTGCTGGCT 
               
               
                   
                 TACACGCTGG ACGGCGTCTG GGTGAGGGAC CCATTGGCAG CACGAGACTA GGACGACCGA 
               
               
                   
               
               
                   
                 5′   71    81    91   101    111 
               
               
                  +1 
                 Q M R R I S P F S C L K D R H D F G F P 
               
               
                  61 
                 CAGATGCGTC GTATCTCCCC GTTCTCCTGC CTGAAAGACC GTCACGACTT CGGTTTCCCG 
               
               
                   
                 GTCTACGCAG CATAGAGGGG CAAGAGGACG GACTTTCTGG CAGTGCTGAA GCCAAAGGGC 
               
               
                   
               
               
                   
                 5′   131  141   151   161   171 
               
               
                  +1 
                 Q E E F D G N Q F Q K A Q A I S V L H E 
               
               
                 121 
                 CAGGAAGAAT TCGACGGTAA CCAGTTCCAG AAAGCTCAGG CTATCTCCGT TCTGCACGAA 
               
               
                   
                 GTCCTTCTTA AGCTGCCATT GGTCAAGGTC TTTCGAGTCC GATAGAGGCA AGACGTGCTT 
               
               
                   
               
               
                   
                 5′   191   201   211   221   231 
               
               
                  +1 
                 M I Q Q T F N L F S T K D S S A A W D E 
               
               
                 181 
                 ATGATCCAGC AGACCTTCAA CCTGTTCTCC ACCAAAGACT CCTCCGCTGC TTGGGACGAA 
               
               
                   
                 TACTAGGTCG TCTGGAAGTT GGACAAGAGG TGGTTTCTGA GGAGGCGACG AACCCTGCTT 
               
               
                   
               
               
                   
                 5′   251   261   271   281   291 
               
               
                  +1 
                 S L L E K F Y T E L Y Q Q L N D L E A C 
               
               
                 241 
                 TCCCTGCTGG AAAAATTCTA CACCGAACTG TACCAGCAGC TGAACGACCT GGAAGCTTGC 
               
               
                   
                 AGGGACGACC TTTTTAAGAT GTGGCTTGAC ATGGTCGTCG ACTTGCTGGA CCTTCGAACG 
               
               
                   
               
               
                   
                 5′      311    321     331   341   351 
               
               
                  +1 
                 V I Q E V G V E E T P L M N V D S I L A 
               
               
                 301 
                 GTTATCCAGG AAGTTGGTGT TGAAGAAACC CCGCTGATGA ACGTTGACTC CATCCTGGCT 
               
               
                   
                 CAATAGGTCC TTCAACCACA ACTTCTTTGG GGCGACTACT TGCAACTGAG GTAGGACCGA 
               
               
                   
               
               
                   
                 5′      371   381    391   401   411 
               
               
                  +1 
                 V K K Y F Q R I T L Y L T E K K Y S P C 
               
               
                 361 
                 GTTAAAAAAT ACTTCCAGCG TATCACCCTG TACCTGACCG AAAAAAAATA CTCCCCGTGC 
               
               
                   
                 CAATTTTTTA TGAAGGTCGC ATAGTGGGAC ATGGACTGGC TTTTTTTTAT GAGGGGCACG 
               
               
                   
               
               
                   
                 5′      431   441    451   461   471 
               
               
                  +1 
                 A W E V V R A E I M R S F S L S I N L Q 
               
               
                 421 
                 GCTTGGGAAG TTGTTCGTGC TGAAATCATG CGTTCCTTCT CCCTGTCCAC CAACCTGCAG 
               
               
                   
                 CGAACCCTTC AACAAGCACG ACTTTAGTAC GCAAGGAAGA GGGACAGGTG GTTGGACGTC 
               
               
                   
               
               
                   
                 5′    491   501 
               
               
                  +1 
                 E R L R R K E #            (SEQ ID NO: 1) 
               
               
                 481 
                 GAACGTCTGC GTCGTAAAGA ATAA (SEQ ID NO: 2) 
               
               
                   
                 CTTGCAGACG CAGCATTTCT TATT (SEQ ID NO: 3) 
               
             
          
         
       
     
       Example 2 
     Separation and Purification of rSIFN-co 
     1. Fermentation 
       [0192]    Inoculate the recombinant strain in LB media, shaking (200 rpm) under 37° C. overnight (approximate 18 h), then add 30% glycerol to the fermentation broth to get final concentration of 15%, allotted to 1 ml tube and kept in −20° C. as seed for production. 
         [0193]    Add 1% of the seed to LB media, shaking (200 rpm) under 37° C. overnight to enlarge the scale of the seed, then add to RM media with a ratio of 10%, culturing under 37° C. Add arabinose (20% solution) to 0.02% as an inductor when the OD 600  reaches about 2.0. 4 hours after that, stop the culture process, collect the bacteria by centrifuge, resuspend the pellet with buffer A, and keep in −20° C. overnight. Thaw and break the bacteria by homogenizer, then centrifuge. Wash the pellet with buffer B, buffer C, and distilled water to get a relatively pure inclusion bodies. 
       2. Denaturation and Renaturation 
       [0194]    Dissolve the inclusion body in Guanidine-HCl (or urea) of 6 mol/L. The solution will be a little cloudy. Centrifuge it at a speed of 10000 rpm. Determine the protein concentration of the supernatant. This supernatant is called “denaturation solution.” Add the denaturation solution to renaturation buffer, and keep the final protein concentration under 0.3 mg/ml. It is better to add the totally denatured solution in three steps instead of one step. Keep the solution overnight under 4° C. Afterwards, dialyze 10 mol/L, 5 mol/L PB buffer and distilled water, then adjust its pH by 2 mol/L HAc-NaAc. Let it stand, then filtrate. 
       3. Purification 
       [0000]    
       
         
           
             POROS HS/M anion exchange chromatography: 
           
         
       
     
         [0000]    
       
                 
         
             
             
         
       
     
         [0196]    Condense the eluted solution by POROS HS/M. Sometimes a purification by sephacryl S-100 step can be added to meet stricter purity requirements. 
       Note: 
       [0000]    
       
         Buffer A: 100 mmol/L Tris-HCl, pH 7.5-10 mmol/L EDTA-100 mmol/L NaCl 
         Buffer B: 50 mmol/L Tris-HCl, pH 7.5-1 mol/L Urea-10 mmol/L EDTA-0.5% Triton X-100 
         Buffer C: 50 mmol/L Tris-HCl, pH 7.5-2 mol/L Urea-10 mmol/L EDTA-0.5% Triton X-100 
         Buffer D: 1 mol/L NaCl- - -50 mmol/L Na 2 HPO 4  (pH 5.5) 
         Buffer E: 1 mol/L NaCl- - -50 mmol/L Na 2 HPO 4  (pH 5.0) 
         Buffer F: 1 mol/L NaCl- - -50 mmol/L Na 2 HPO 4  (pH 4.0) 
         Buffer G: 1 mol/L NaCl- - -50 mmol/L Na 2 HPO 4  (pH 3.6) 
         Renaturation buffer: 0.5 mol/L Arginine—150 mmol/L Tris-HCl, pH 7.5-0.2 mmol/L EDTA 
       
     
       LB Media: 1 L 
       [0205]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Tryptone 
                 10 g 
               
               
                   
                 Yeast extracts 
                  5 g 
               
               
                   
                 NaCl 
                 10 g 
               
               
                   
                   
               
             
          
         
       
     
       RM Media: 1 L 
       [0206]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Casein 
                  20 g 
               
               
                   
                 MgCl 
                   1 mmol/L (0.203 g) 
               
               
                   
                 Na 2 HPO 4   
                   4 g; 
               
               
                   
                 KH 2 PO 4   
                   3 g, 
               
               
                   
                 NaCl 
                 0.5 g 
               
               
                   
                 NH 4 Cl 
                   1 g 
               
               
                   
                   
               
             
          
         
       
     
         [0207]    After purification, the buffer was changed to PBS (pH 7.0) along with the step of condensing by POROS HS/M. This is called the “Protein Stock Solution.” It can be directly used in the preparation of injections or sprays, or stored at 2-8° C. 
       Formula for Injection: 
       [0208]      
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Solution 
                 Lyophilized powder 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Solution of rSIFN-co 
                 34.5 μg/ml 
                 34.5 μg/ml 
               
               
                   
                 PB (pH7.0) 
                   25 mmol/L 
                   10 mmol/L 
               
               
                   
                 Glycine 
                 — 
                  0.4 mol/L 
               
               
                   
                 NaCl 
                  0.1 mol/L 
                 — 
               
               
                   
                   
               
             
          
         
       
     
       For Spray: 
       [0209]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 EDTA 
                 0.01% 
               
               
                   
                 Tween 80 
                 0.05% 
               
               
                   
                 Trisodium citrate 
                 10 mmol/L 
               
               
                   
                 Glycerol 
                 1.26% 
               
               
                   
                 Sodium Chloride 
                 0.03% 
               
               
                   
                 Phenylmethanol 
                  0.5% 
               
               
                   
                 HSA 
                  0.1% 
               
               
                   
                 rSIFN-co 
                 10 μg/ml 
               
               
                   
                   
               
             
          
         
       
     
       Quality Control Process 
       [0210]    During purification, tests for protein content, protein purity, specific activity and pyrogen are conducted after each step. When the stock solution is obtained, all the tests listed in the table are done one after the other. 
         [0211]    The quality of the product is controlled according to “Chinese Requirements for Biologics.” 
       1. Original Protein Solution 
       [0212]      
         [0000]    
       
         
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Item of Test 
                 Method 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Protein Stock Solution: 
               
             
          
           
               
                   
                 Test for Protein Content 
                 Lowry 
               
               
                   
                 Test for Protein Purity 
                 Non-reductive SDS-PAGE 
               
               
                   
                   
                 (sodium dodecyl sulfate 
               
               
                   
                   
                 polyacrylamide gel 
               
               
                   
                   
                 electrophoresis) 
               
               
                   
                   
                 HPLC Analysis 
               
               
                   
                 Test for Molecular Weights 
                 Reductive SDS-PAGE 
               
               
                   
                 Test for Specific Activity 
                 According to Method in 
               
               
                   
                   
                 “Specific Activity Test of 
               
               
                   
                   
                 Interferon 
               
               
                   
                 Test for Leftover Exogenetic 
                 Using DNA Labeling and 
               
               
                   
                 DNA 
                 Detection Kit 
               
               
                   
                 Test for Activity of 
                 According to Method in 
               
               
                   
                 Leftover Antibiotics 
                 “Chemical and Other Test 
               
               
                   
                   
                 Methods for Biologics” 
               
               
                   
                 Test for Bacterial Endotoxin 
                 According to Method in 
               
               
                   
                   
                 “Requirements for Bacterial 
               
               
                   
                   
                 Endotoxin Test of Biologics” 
               
               
                   
                 Test for Isoelectronic Point 
                 Isoelectric Focusing 
               
               
                   
                   
                 Electrophoresis 
               
               
                   
                 Test for Identify 
                 UV spectrum (range of 
               
               
                   
                 Characteristics of the 
                 wavelength: 190-380 nm) 
               
               
                   
                 Protein 
               
               
                   
                   
                 Peptide Mapping (hydrolyzed 
               
               
                   
                   
                 by pancreatic enzyme, 
               
               
                   
                   
                 analyzed by C-18 column) 
               
               
                   
                   
                 N-terminal Sequence Test 
               
               
                   
                   
                 C-terminal Sequence Test 
               
               
                   
                   
                 Circular Dichroism 
               
               
                   
                   
                 Amino Acid Analysis 
               
             
          
           
               
                 Semi-finished Product 
               
             
          
           
               
                   
                 Test for Bacterial Endotoxin 
                 According to Method in 
               
               
                   
                   
                 “Requirements for Bacterial 
               
               
                   
                   
                 Endotoxin Test of Biologics” 
               
             
          
           
               
                 Product 
               
             
          
           
               
                   
                 Appearance Check 
                   
               
               
                   
                 Chemical 
                 According to Method in 
               
               
                   
                   
                 “Chemical and Other Test 
               
               
                   
                   
                 Methods for Biologics” 
               
               
                   
                 Test for Specific Activity 
                 According to Method in 
               
               
                   
                   
                 “Specific Activity Test of 
               
               
                   
                   
                 Interferon 
               
               
                   
                 Sterility Test 
                 According to Method in “c” 
               
               
                   
                 Abnormal Toxicity Test 
                 Test on Mouse 
               
               
                   
                 Pyrogen Test 
                 According to Method in 
               
               
                   
                   
                 “Requirements for Pyrogen 
               
               
                   
                   
                 Test of Biologics” 
               
               
                   
                 Test for Stability of 
               
               
                   
                 Product 
               
               
                   
                   
               
               
                   
                 Note: 
               
               
                   
                 “Chemical and Other Test Methods for Biologics”, “Requirements for Pyrogen Test of Biologics” and “Requirements for Bacterial Endotoxin Test of Biologics” all can be found in the “Chinese Requirements for Biologics.” “Chinese Requirements for Biologics,” PAN Zhengan, ZHANG Xinhui, DUAN Zhibing, et al. Chinese Biologics Standardization committee. Published by Chemical Industry Publishing Company, 2000. 
               
             
          
         
       
     
       Example 3 
     Stability of Lyophilized Powder of Recombinant Super-Compound Interferon Injection 
       [0213]    The stability experiments were carried out with samples of lyophilized powder of recombinant super-compound interferon (rSIFN-co) injection in two specifications and three batches. The experiments started in April 2000. 
       1. Sample Source 
       [0214]    Samples were supplied by Sichuan Huiyang Life-engineering Ltd., Sichuan Province. Lot: 990101-03, 990101-05, 990102-03, 990102-05, 990103-03, 990103-05 
       2. Sample Specifications 
       [0215]    Every sample in this experiment should conform with the requirements in the table below. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Standard of Samples in Experiment 
               
             
          
           
               
                 Items 
                 Standards 
               
               
                   
               
               
                 1. Appearance 
                 white loose powder 
               
               
                 2. Dissolving time 
                 dissolve rapidly in injection water (within 
               
               
                   
                 2 min) at room temperature 
               
               
                 3. Clarity 
                 colorless liquid or with little milk-like 
               
               
                   
                 glisten; should not be cloudy, impurity or 
               
               
                   
                 with indiscernible deposit 
               
               
                 4. pH value 
                 6.5~7.5 
               
               
                 5. Potency (IU/dose) 
                 80%~150% of indicated quantity (9 μg:  
               
               
                   
                 4.5 × 10 6  IU, 15 μg: 7.5 × 10 6  IU) 
               
               
                 6. Moisture 
                 no more than 3.0% ( W/W) 
               
               
                   
               
             
          
         
       
     
       3. Experimental Content 
       [0216]    Test samples at 2˜8° C.: The test samples were put into a 2˜8° C. refrigerator, then the above items of these samples were respectively tested in the 1 st , 3 rd , 6 th , 9 th , 12 th , 18 th , 24 th , 30 th , 36 th  month. The results were recorded. 
         [0217]    Test samples at 25° C.: The test samples were put into a thermostat at 25° C., then the above items of these samples were respectively tested in the 1 st , 3 rd , 6 th , 9 th , 12 th , 18 th , 24 th , 30 th  month. The results were recorded. 
         [0218]    Test samples at 37° C.: The test samples were put into a thermostat at 37° C., then the above items of these samples were respectively tested in the 1 st , 3 rd , 6 th , 9 th , 12 th , 18 th , 24 th  month. The results were recorded. 
       4. Results and Conclusion 
       [0000]    
       
         
           
             1) At 37° C., according to data collected at designated points during testing and compared with data before testing, the potency began descending from the 6 th  month and the changes in the three batches were similar. The appearance of other items had no changes. 
             2) At 25° C., according to data collected at designated points during testing and compared with data before the testing, the potency only had a little change, and the changes in the three batches were similar. The appearance of other items had no changes. 
             3) At 2-8° C., according to data collected at designated points during testing and compared with data before testing, the potency of the three batches all were stable. The appearance of other items also had no changes. 
             In conclusion, it is suggested that the lyophilized powder of recombinant super-compound interferon for injection should be better stored and transported at low temperatures. Without such conditions, the product can also be stored for short periods (i.e., 3 months) at room temperature. 
           
         
       
     
       Example 3.5 
     Production Flow Chart of rSIFN-co 
     1. Production 
       [0223]    1.1 Fermentation
       Use mixture of LB+M9 as culturing medium. The amount of innoculum will be 1.5%. Agitate to OD 600 =0.4 (about 3.5 hours) under 32° C., then raise temperature to 42° C. Continue the agitation for another 6 hours, the expression of rSIFN-co will reach the maximum level. The examination under scanning of the gel resulting from SDS-PAGE shows that the level of expression is up to 57%, which is the highest standard in China.       
 
         [0225]    1.2 Purification 
         [0000]    
       
                 
         
             
             
         
       
     
         [0226]    The purity of the product (rSIFN-co) from this production procedure is shown to 95% under the test of SDS-PAGE where molecular weight is 14.5 Kda. The reverse phase HPLC shows a single peak and the purity is up to 97%. Its specific activity is up to 1×10 9  IU/mg protein. 
         [0227]    1.3 Packaging and Inspection
       After HPLC purification, 2% human serum albumin, 1% sucrose and 1% glucose are added to the rSIFN-co. It is then separated and lyophilized into injection sample. When tested under the Wish-VVS inspection system, the result was 4.5×10 8  IU. When tested with aseptic inspection and pyrogen inspection under the standard requirement of China, the results were negative. This result complies with the requirements for IV injection.       
 
       2. Quality Control 
       [0229]    2.1 Biological Characteristics
       (1) When using LB+M9 to cultivate bacteria, the characteristics should match with the typical characteristics of E-coli bacteria. No other bacteria were detected.   (2) When smeared for Gram staining and inspected under a microscope, it is bacteria-negative.   (3) Reaction to antibiotics is the same as those original bacteria.   (4) Electron microscope inspection shows typical characteristics of E-coli bacteria. No mycoplasma, virus spore or other micro pollutes was detected.   (5) Biochemical reaction test shows characteristics of E-coli bacteria.       
 
         [0235]    2.2 Quality Control of Interferon Expression
       (1) Interferon expression (cultivated in an agitating platform) matches the amount of expression in original input bacteria.   (2) When tested with anti-interferon serum, a reaction is shown.   (3) Plasmid inspection: Restriction digest matched with the original plasmid.       
 
         [0239]    2.3 Bacteria Strain Product
       Bacteria strain product denotes the specimen from the original bacteria strain that was produced from the procedures shown in 1.2.   The bacteria strain product should be inspected as follows to make sure there is no derivation: Use LB to plate 2-3 pieces and cultivate. Separate and take 5-10 bacteria groups for the test of interferon expression. Repeat the test at least two (2) times. Only use the one which shows the highest % to be the bacteria strain product.       
 
         [0242]    2.4 Inoculum
       The inoculum denotes the chosen bacteria strain product after fermentation. The amount, cultivation time and most appropriate OD value of inoculum can be decided according to bacteria strain. An anti-polluted bacteria procedure should apply for whatever inoculum would be produced.       
 
         [0244]    2.5 Growing of Bacteria Strain
       Growing of bacteria strain would be done in a Bacteria Free room environment where no more than one bacterium is growing in the same room. Same culturing medium will be used for both bacteria strain and inoculum. The one used in rSIFN-co is LB.       
 
         [0246]    2.6 Fermentation
       (1) Fermentation only takes place in a clean fermentation room with a single bacteria fermentation environment.   (2) Cleaning of fermentation container and tube is done twice, before and after the insertion of culturing medium. Then, the container should be frozen to reach the appropriate temperature for inoculum.   (3) Avoid using antibiotic which might affect cell growth in the culturing medium.   (4) Fermentation parameters like temperature, pH value, dissolved oxygen and time required could be varied according to different types of bacterial strains.       
 
         [0251]    2.7 Bacteria Collection
       (1) Centrifuge the bacteria solution to collect bacteria or use another method. All apparatus should be cleaned before and after the operation. The waste solution should be drained after the cleaning procedure.   (2) The bacteria should be kept under 4-8° C. if they are going to be split within 24 hours. Otherwise, they should be kept under −30° C. Those are kept under such conditions can be used within 6 months.       
 
         [0254]    2.8 Bacteria Cell Lysis
       (1) Use appropriate buffer solution to balance the bacteria strain. Cell lysis can be done by physical, chemical or biological methods. Use centrifuge to precipitate the bacteria and apply cleaning solutions.   (2) If the chemical method is used to split cells, no solutions harmful to human beings should be used.       
 
         [0257]    2.9 Purification
       (1) Purification will get rid of most of the non-interferon contents. In the process of purification, no toxic materials should be found if extra elements are added.   (2) If using antibody affinity chromatography for purification, there should be an indication of the source and degree of purity. Also, inspection of small quality IgG should be performed.   (3) During the process of purification, clearance of pyrogen is critical. All apparatus should be checked to eliminate this interference.   (4) The highly concentrated interferon is known as “intermediate product”. After inspection and tests, add albumin to raise the concentration to 2% which is now known as “albumin intermediate product”. After examination and tests, it should be kept at −30° C. and never thawed before use. This product should be used within 6 months.   (5) The albumin that is used in this process should also fulfill tests and requirements such as: negativity under RBSAG inspection and an indication of the ratio among monomer, dimer and polymer.       
 
         [0263]    2.10 Production into Tube Product
       (1) Filtration: Use 0.22μ membrane to filter the bacteria. The product should be handled with aseptic techniques. Samples should be taken to test the value of the interferon.   (2) Dilution: Dilute the albumin intermediate product with 2% diluent. No preservative should be added. The product can be lyophilized after the aseptic inspection and pyrogen inspection.       
 
         [0266]    2.11 Lyophilization
       The lyophilization should not affect the activity of interferon, and the water content of said lyophilite will be maintained.       
 
         [0268]    2.12 Inspection
       There are two types of rSIFN-co made. One is for injection and the other for topical use. The specifications for the two are different. There are intermediate products and final products for each type. In the injection type, intermediate products include purified interferon, albumin intermediate product, and bacteria free albumin intermediate product. Final product from the injection type will denote only lyophilized product. The intermediate product in the topical type denotes only purified interferon. The final product from the topical type denotes only separated packed liquid formed lyophilized products.       
 
         [0270]    2.13 Packaging
       There is different packaging for the injection type and the topical type.       
 
         [0272]    2.14 Storage
       The product should be kept at 4° C. The purification solution should not be stored in a frozen state.       
 
         [0274]    2.15 Expiration
       The expiration period is two (2) years after the lyophilization procedure for lyophilized products. The expiration period is 6 months after individual packing for liquidated products.       
 
       Example 4 
     Preparation of rSIFN-co 
       [0276]      
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Preparation of lyophilized injection 
               
             
          
           
               
                   
                   
                 Lyophilized powder 
               
               
                   
                   
               
             
          
           
               
                   
                 Stock Solution of 
                 34.5 
                 μg/ml 
               
               
                   
                 rSIFN-co 
                   
                   
               
               
                   
                 PB (pH7.0) 
                 10  
                 mmol/L 
               
               
                   
                 Glycine 
                 0.4 
                 mol/L 
               
               
                   
                   
               
             
          
         
       
     
         [0277]    Preparation technique: Weigh materials according to recipe. Dissolve with sterile and pyrogen-free water. Filter through 0.22 μm membrane to de-bacterialize, preserve at 6-10° C. Fill in vials after affirming they are sterile and pyrogen-free, 0.3 ml/vial or 0.5 ml/vial, and lyophilize in freeze dryer. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                 Preparation of liquid injection 
               
             
          
           
               
                   
                   
                 Solution 
               
               
                   
                   
               
             
          
           
               
                   
                 Stock Solution of 
                 34.5  
                 μg/ml 
               
               
                   
                 rSIFN-co 
                   
                   
               
               
                   
                 PB (pH7.0) 
                 25  
                 mmol/L 
               
               
                   
                 NaCl 
                 0.1  
                 mol/L 
               
               
                   
                   
               
             
          
         
       
     
         [0278]    Preparation: Weigh materials according to recipe. Add to desired level with sterile and pyrogen-free water. Filter through 0.22 μm membrane to de-bacterialize, preserve at 6-10° C. Fill in airtight vial after affirming it is sterile and non-pyrogen at 0.3 ml/vial or 0.5 ml/vial. Store at 2-10° C., and protect from light. 
       Example 4.5 
     Acute Toxicity of rSIFN-co 
       [0279]    Treat mice with large dose (150 μg/kg, equal to 1000 times of the normal dose per kilo used in treatment of adult patients) of rSIFN-co at one time by intramuscular injection. Then observe and record their deaths and toxic reactions. Results show that: 24 hours after injection, no abnormal reaction had been recorded. The organs of the animals which had been selected to be killed also had no signs of abnormal changes. Those remaining mice were all kept alive and were normal after two weeks. The weights of mice in the experimental group and control group all increased, and the ratio of increase showed no obvious difference between the two groups (P&gt;0.05) according to their weights on the fourteenth day. No abnormal changes were seen from the main organs of those mice after two weeks. 
       1. Experimental Material 
     1.1 Animals 
       [0280]    40 healthy adult mice, weighing 18-22 g, half male and half female, qualified by Sichuan experiment animal control center. 
       1.2 Medicines 
       [0281]    rSIFN-co (Provided by Sichuan Huiyang Life-engineering Ltd.) sterilized solution, 0.15 mg/ml, Lot: 981201 
         [0282]    rSIFN-co was administered i.m. in saline. 
       2. Method 
       [0283]    Separate the 40 mice into two groups randomly, one for experimental medicine, another for control. Inject medicines or saline at the same ratio (0.1 ml/10 g) through muscle to each mouse according to which group they belong. (150 μg/kg of rSIFN-co for experimental group; and saline for control group). After injection, observe and record acute toxicity shown in mice. Kill half of the mice (male and female each half) to check whether there were any abnormal pathologic changes in their main organs, such as heart, spleen, liver, lung, kidney, adrenal gland, stomach, duodenum, etc. after 24 hours. Those that remain are kept and observed until the fourteenth day. Weigh all mice, kill them, and then observe the appearance of the organs listed above to see if there are any abnormalities. Take pathological tissue and examine it, using the examination to assess the difference in weight increases in the two groups. 
       3. Results 
       [0284]    Results show that there was no acute toxicity seen after all mice were treated with i.m. rSIFN-co with 150 μg/kg at a time, equal to 1000 times the normal dose per kilo used in treatment of adult patients. In the 14 days after injection, all mice lived well. They ate, drank, exercised, and excreted normally and showed normal hair conditions. None of them died. The observation of the main organs of the randomly selected mice shows no abnormal changes 24 hours after injection. 14 days after injection, all remaining mice were killed. Autopsies also showed no changes. The weights of mice in the two groups all increased, but no obvious difference was shown when accessed with statistic method (p&gt;0.05). See Table 6.1: 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6.1 
               
             
             
               
                   
               
               
                 Influence to weights of mice after injection of rSIFN-co 
               
             
          
           
               
                   
                   
                   
                 Weights 
                 Weights 
                 Increased 
               
               
                   
                   
                   
                 before 
                 after 
                 value of 
               
               
                   
                   
                   
                 injection 
                 injection 
                 weights 
               
               
                 Group 
                 Dose 
                 Animal 
                 (g) 
                 (g) 
                 (g) 
               
               
                   
               
             
          
           
               
                 Control 
                 0 
                 20 
                 19.8 ± 1.7 
                 30.8 ± 2.8 
                 11.0 ± 2.9 
               
               
                 rSIFN-co 
                 150 
                 20 
                 19.4 ± 1.7 
                 32.1 ± 3.3 
                 12.7 ± 4.3 
               
               
                   
               
             
          
         
       
     
       4. Conclusion 
       [0285]    Under conditions of this experiment, there were no toxic reactions in all mice after injection of rSIFN-co with 150 μg/kg. The conclusion can be reached that the maximum tolerable dose of i.m. in mice is 150 μg/kg, which is equal to 1000 times the normal dose per kilo used in treatment of adult patients. 
         [0000]    2002 rSIFN-co Drug Inspection Report: 
         [0286]    Nov. 14, 2002 rSIFN-co Drug Inspection Report by China Drugs &amp; Biological Products Inspection Laboratory. 
         [0287]    On Nov. 14, 2000, 80 vials of rSIFN-co each containing 9 μg (micrograms) provided by Sichuan Biotechnology Research Center were tested. rSIFN-co Drug was white in color with produced no precipitation when water was added. The pH value was 6.9 while the standard was between 6.5 to 7.5. The water content of rSIFN-co was 2.3% while the standard was smaller than 3.0%. Test for bacteria showed no bacterial grown. rSIFN-co passed pyrogen test. The toxicity test on mice showed no harm. Mice were alive and gained weight. The specific activity test was 6.0×10 6  IU/vial while the standard was between 3.6×10 6  IU/vial to 6.8×10 6  IU/vial. The identification test was positive. 
       Example 5 
       [0288]    Crystal Growth of rSIFN-co and Test of Crystallography Parameter 
         [0289]    Crystal of rSIFN-co. Two types of crystal were found after systematically trial and experiment. (See  FIGS. 7-9 ) 
       1. Crystal Growth 
       [0000]    
       
         
           
             Dissolve rSIFN-co protein with pure water (H 2 O) to 3 mg/ml in density. Search of crystallization by using Hampton Research Crystal Screen I and II which was made by Hampton Company. By using Drop Suspension Diffusion Method, liquid 500 μl, drop 1 μl protein+1 μl liquid, in 293K temperature. First 2 different types of small crystals were found as listed in Table 12.1. 
           
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 12.1 
               
             
             
               
                   
               
               
                 Screen of rSIFN-co Crystallin 
               
             
          
           
               
                 Condition 
                 I 
                 II 
               
               
                   
               
               
                 Diluent 
                 0.1M Tris-HCl 
                 0.1M HEPES 
               
               
                   
                 PH = 8.75 
                 PH = 7.13 
               
               
                 Precipitant 
                 17.5% (w/v) PEG550 MME  
                 10% (w/v) PEG6K 
               
               
                 Additives 
                 0.1M NaCl 
                 3% (w/v) MPD 
               
               
                 Temperature 
                 293K 
                 293K 
               
               
                 Crystal Size (mm) 
                 0.2 × 0.2 × 0.1 
                 0.6 × 0.02 × 0.02 
               
               
                 Crystallogram 
                 FIG. 7 
                 FIG. 8 
               
               
                   
               
             
          
         
       
     
       2. Data Collection and Processing 
       [0000]    
       
         
           
             Crystal I was used to collect X-Ray diffraction data and preliminary analysis of crystallography. Parameters were also tested. The diffraction data was collected under room temperature. Crystal I (Condition I) was inserted into a thin siliconized wall tube. Using BrukerAXS Smart CCD detector, the light source is CuKα (λ=1.5418 Å) generated by Nonius FR591 X-ray generator. Light power 2000 KW (40 kv×50 mA), wave length 1.00 Å, under explosion 60 second, Δφ=2°, the distance between crystal and detector was 50 mm. Data was processed for using Proteum Procedure Package by Bruker Company. See  FIG. 9  for crystal diffraction pattern (partially). See Table 12.2 for the result of the process. 
           
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 12.2 
               
             
             
               
                   
               
               
                 Results of Crystallography Parameters 
               
             
          
           
               
                   
                 Parameters 
                   
               
               
                   
                   
               
               
                   
                 a (Å) 
                  82.67 
               
               
                   
                 b (Å) 
                 108.04 
               
               
                   
                 c (Å) 
                 135.01 
               
               
                   
                 α (Å) 
                  90.00 
               
               
                   
                 β (Å) 
                  90.00 
               
               
                   
                 γ (Å) 
                  98.35 
               
               
                   
                 Space Group 
                 P2 or P2 1   
               
               
                   
                 Sharpness of separation 
                 5 Å 
               
               
                   
                 Asymmetric molecule # 
                  10 
               
               
                   
                 Dissolution 
                  57.6% 
               
               
                   
                   
               
             
          
         
       
     
         [0292]    Besides, there was no crystal growth of rSIFN-co based on previous publications. The closest result to the rSIFN-co was huIFN-α2b but the screen was very complicated. After seeding 3 times, crystal grew to 0.5×0.5×0.3 mm, sharpness of separation was 2.9 Å, space group was P2 1 . The crystals were also big, asymmetric molecule number was 6, and dissolution was about 60%. 
       Clinical Report 1: 
       [0293]    Evidence of effectiveness of rSIFN-co in healing cancer. See  FIGS. 17A-H . 
         [0294]    The ultra sound inspection showed an enlarged right ovary and abdominal fluid. The patient was suspected of having ovarian cancer. 
         [0295]    Western China No. 2 Hospital reported a patient with ovarian cancer and breast gland cancer diagnosed on Jul. 14, 2004. Her serum contained CA-125&gt;600 U/ml and CA-153&gt;250 U/ml. Also 2000 ml abdominal water was found. On Jul. 16, 2004, malignant cancer cells and low differential gland cancer cells (likely a low graded differential Adenocarcinoma) were found from the abdominal water and cancer cells and death materials were found from the mammary gland check up. On Aug. 4, 2004, it was concluded diagnosis as ovary cancer. 
         [0296]    The patient was treated with rSFIN-co starting Jul. 14, 2004. She was injected with 15 μg of rSFIN-co on Jul. 14, 2004, Jul. 16, 2004, Jul. 18, 2004, Jul. 20, 2004 and Jul. 22, 2004 respectively. She began chemotherapy on Jul. 22, 2004. On Aug. 3, 2004 abdominal surgery was performed. It was expected that her abdominal water would be more than 2000 ml. However, only 200 ml were recorded. On Aug. 4, 2004 the examination results showed she had mammary gland cancer, ovarian cancer of right and left ovary and lymphoma. She was treated with rSIFN-co and chemotherapy at the same time. She did not have operation on mammary glands. 
         [0297]    On Dec. 27, 2004 the examination report showed her CA-125 dropped to 5 U/ml and CA-153 dropped to 13 U/ml. On Feb. 25, 2005, her PET examination report from Daping Hospital, Third Military Medical University of PRC showed there was no obvious abnormal difference on metabolic reactions on her body and brain. The symptoms of her mammary gland cancer disappeared. No traces of cancer were found. 
       PET Imaging: 
       [0298]    On Feb. 25, 2005 PET imaging report on Feb. 25, 2005 of this 43 years old patient diagnosis with left side ovary cancer and was treated with rSIFN-co since Jul. 14, 2004; PET imaging was done at PET Center of the Daping Hospital, Third Military Medical University of PRC. 
         [0299]    Fasting patient was intravenously injected with  18 F-FDG14.8mCi. Brain images were taken 50 minutes after injection. The images were clear, no obvious abnormal increase or decrease of radiation were observed on cerebral epidermis, both sides of cerebellum, both sides of hypothalamus and basal.
       Whole body imaging was done 60 minutes after injection. The images were clear. No obvious abnormal increase or decrease of radiation on neck, lungs, mediastinum, liver, both sides of adrenals, abdominal lymph gland, pelvic cavity, bones.       
 
         [0301]    The image of heart was clear. 
         [0302]    Result: The FDG-PET images of the whole body and brain did not show abnormal FDG metabolic increase or decrease after five-and-half (5.5) months of rSIFN-co treatment of ovarian ovary cancer. 
         [0000]    Conclusion: Comparison of CA-153 and CA-125 levels before and after rSFIN-co treatment evidenced that rSFIN-co is effective against breast and ovarian cancer. 
       Clinical Report 2: 
       [0303]    A kidney cancer patient was treated in the following manner. In a half-month period, the patient was given 3 injections of 9 μg of rSIFN-co and 3 injections of 15 μg of rSIFN-co. In the one and a half months following these injections, he received 24 μg injections of rSIFN-co every day. A kidney biopsy showed no metastasis after this course of treatment. The patient showed a full recovery. Every half year after recovery, the patient received 15 μg injections of rSIFN-co 15 times over a one-month period. 
       Example 6 
     rSIFN-co Inhibits HBV-DNA Duplication and Secretion of HBsAg and HBeAg 
       [0304]    Materials 
         [0305]    Solvent and Dispensing Method: Add 1 ml saline into each vial, dissolve, and mix with MEM culture medium at different concentrations. Mix on the spot. 
         [0306]    Control drugs: IFN-α2b (Intron A) as lyophilized powder, purchased from Schering Plough. 3×10 6  IU each, mix to 3×10 6  IU/ml with culture medium; Infergen® (liquid solution), purchased from Amgen, 9 μg, 0.3 ml each, equal to 9×10 6  IU, and mix with 9×10 6  IU/ml culture medium preserve at 4° C.; 2.2.15 cell: 2.2.15 cell line of hepatoma (Hep G2) cloned and transfected by HBV DNA, constructed by Mount Sinai Medical Center. 
         [0307]    Reagent: MEM powder, Gibco American Ltd. cattle fetal blood serum, HycloneLab American Ltd. G-418 (Geneticin); MEM dispensing, Gibco American Ltd.; L-Glutamyl, imported and packaged by JING KE Chemical Ltd.; HBsAg and HBeAg solid-phase radioimmunoassay box, Northward Reagent Institute of Chinese Isotope Ltd.; Biograncetina, Northern China Medicine; And Lipofectin, Gibco American Ltd. 
         [0308]    Experimental goods and equipment: culture bottle, Denmark Tunclon™; 24-well and 96-well culture board, Corning American Ltd.; Carbon Dioxide hatching box, Shel-Lab American Ltd.; MEM culture medium 100 ml: 10% cattle fetal blood serum, 3% Glutamyl 1%, G418 380 μg/ml, biograncetina 50 U/ml. 
       Method: 
       [0309]    2.2.15 cell culture: Added 0.25% pancreatic enzyme into culture box with full of 2.2.15 cell, digest at 37° C. for 3 minutes, and add culture medium to stop digest and disturb it to disperse the cells, reproduce with ratio of 1:3. They will reach full growth in 10 days. 
         [0310]    Toxicity test: Set groups of different concentrations and a control group in which cells are not acted on with medicine. Digest cells, and dispense to a 100,000 cell/ml solution. Inoculate to 96-well culture board, 200 μl each well, culture at 37° C. for 24 h with 5% CO 2 . Test when simple cell layer grows. 
         [0311]    Dispense rSIFN-co to 1.8×10 7  IU/ml solution, then prepare a series of solutions diluted at two-fold gradients. Add into 96-well culture board, 3 wells per concentration. Change the solution every 4 days. Test cytopathic effect by microscope after 8 days. Fully destroy as 4, 75% as 3, 50% as 2, 25% as 1, zero as 0. Calculate average cell lesion and inhibition rate of different concentrations. Calculate TC 50  and TC 0  according to the Reed Muench method. 
         [0000]    
       
         
           
             
               TC 
               50 
             
             = 
             
               Antilog 
                
               
                 ( 
                 
                   B 
                   + 
                   
                     
                       
                         50 
                         - 
                         B 
                       
                       
                         A 
                         - 
                         B 
                       
                     
                     × 
                     C 
                   
                 
                 ) 
               
             
           
         
       
     
         [0312]    A=log&gt;50% medicine concentration, B=log&lt;50% medicine concentration, C=log dilution power 
         [0313]    Inhibition test for HBeAg and HBsAg: Separate into positive and negative HBeAg and HBsAg contrast groups, cell contrast group and medicine concentration groups. Inoculate 700,000 cells/ml of 2.2.15 cell into 6-well culture board, 3 ml each well, culture at 37° C. for 24 h with 5% CO 2 , then prepare 5 gradiently diluted solutions with 3-fold as the grade (Prepare 5 solutions, each with a different protein concentration. The concentration of Solution 2 is 3 times lower than that of Solution 1, the concentration of Solution 3 is 3 times lower than that of Solution 2, etc.) 4.5×10 6  IU/ml, 1.5×10 6  IU/ml, 0.5×10 6  IU/ml, 0.17×10 6 1 U/ml, and 0.056×10 6 1 U/ml, 1 well per concentration, culture at 37° C. for 24 h with 5% CO 2 . Change solutions every 4 days using the same solution. Collect all culture medium on the 8 th  day. Preserve at −20° C. Repeat test 3 times to estimate HBsAg and HBeAg with solid-phase radioimmunoassay box (Northward Reagent Institute of Chinese Isotope Ltd.). Estimate cpm value of each well with a γ-accounting machine. 
         [0314]    Effects calculation: Calculate cpm mean value of contrast groups and different-concentration groups and their standard deviation, P/N value such as inhibition rate, IC50 and SI. 
         [0000]    
       
         
           
             
               
                 1 
                 ) 
               
                
               Antigen 
                
               
                   
               
                
               inhibition 
                
               
                   
               
                
               
                 rate 
                  
                 
                   ( 
                   % 
                   ) 
                 
               
             
             = 
             
               
                 
                   A 
                   - 
                   B 
                 
                 A 
               
               × 
               100 
             
           
         
       
       
         
           
             A=cpm of control group; B=cpm of test group; 
             2) Counting the half-efficiency concentration of the medicine 
           
         
       
     
         [0000]    
       
         
           
             
               Antigen 
                
               
                   
               
                
               inhibition 
                
               
                   
               
                
               
                 IC 
                 50 
               
             
             = 
             
               Antilog 
                
               
                 ( 
                 
                   B 
                   + 
                   
                     
                       
                         50 
                         - 
                         B 
                       
                       
                         A 
                         - 
                         B 
                       
                     
                     × 
                     C 
                   
                 
                 ) 
               
             
           
         
       
     
         [0000]    A=log&gt;50% medicine concentration, B=log&lt;50% medicine concentration, C=log dilution power
       3) SI of interspace-conformation changed rSIFN-co effect on HBsAg and HBeAg in 2.2.15 cell culture       
 
         [0000]    
       
         
           
             SI 
             = 
             
               
                 TC 
                 50 
               
               
                 TC 
                 50 
               
             
           
         
       
       
         
           
             4) Estimate the differences in cpm of each dilution degree from the control group using student t test 
           
         
       
     
         [0319]    Southern blot: (1) HBV-DNA extract in 2.2.15 cell: Culture cell 8 days. Exsuction culture medium (Separate cells from culture medium by means of draining the culture medium). Add lysis buffer to break cells, then extract 2 times with a mixture of phenol, chloroform and isoamyl alcohol (1:1:1), 10,000 g centrifuge. Collect the supernatant adding anhydrous alcohol to deposit nucleic acid. Vacuum draw, re-dissolve into 20 μlTE buffer. (2) Electrophoresis: Add 6×DNA loading buffer, electrophoresis on 1.5% agarose gel, IV/cm, at fixed pressure for 14-18 h. (3) Denaturation and hybridization: respectively dip gel into HCl, denaturaion buffer and neutralization buffer. (4) Transmembrane: Make an orderly transfer of DNA to Hybond-N membrane. Bake, hybridize and expose with dot blot hybridization. Scan and analyze relative density with gel-pro software. Calculate inhibition rate and IC 50 . 
       Results 
       [0320]    Results from Tables 4.1, 4.2 and 4.3 show: After maximum innocuous concentration exponent culturing for 8 days with 2.2.15 cell, the maxima is 9.0±0×10 6  IU/ml average inhibition rate of maximum innocuous concentration rSIFN-co to HBeAg is 46.0±5.25% (P&lt;O. 001), IC 50  is 4.54±1.32×10 6  IU/ml, SI is 3.96; rate to HBsAg is 44.8±6.6%, IC 50  is 6.49±0.42×10 6  IU/ml, SI is 2.77. This shows that rSIFN-co can significantly inhibit the activity of HBeAg and HBsAg, but that the IFN of the contrast group and Infergen® cannot. It has also been proven in clinic that rSIFN-co can decrease HBeAg and HBsAg or return them to normal levels. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4.1 
               
               
                   
               
               
                 Results of inhibition rate of rSIFN-co to HBsAg and HBeAg 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Inhibition rate 
                 Average 
                   
                 1- 
                 Accumulated 
               
             
          
           
               
                 Concentration 
                 First 
                 Second 
                 Third 
                 First 
                 Second 
                 Third 
                 inhibition 
                   
                 Accu- 
                 inhibition 
               
               
                 (×10 4  IU/ml) 
                 well 
                 well 
                 well 
                 well 
                 well 
                 well 
                 rate 
                 Accumulation 
                 mulation 
                 rate 
               
               
                   
               
             
          
           
               
                 First batch: (rSIFN-co) 
               
               
                   
               
               
                 Inhibition effect to HBeAg 
               
             
          
           
               
                 900 
                 9026 
                 8976 
                 10476 
                 0.436227 
                 0.43935 
                 0.345659 
                 0.407079 
                 0.945909 
                 0.592921 
                 0.614693546 
               
               
                 300 
                 9616 
                 12082 
                 10098 
                 0.3993754 
                 0.245347 
                 0.369269 
                 0.337997 
                 0.5388299 
                 1.254924 
                 0.300392321 
               
               
                 100 
                 9822 
                 16002 
                 12800 
                 0.386508 
                 0.0005 
                 0.2005 
                 0.195836 
                 0.200833 
                 2.059088 
                 0.08867188 
               
               
                 33.33333 
                 15770 
                 19306 
                 16824 
                 0.014991 
                 0 
                 0 
                 0.004997 
                 0.0049969 
                 3.054091 
                 0.001633453 
               
               
                 11.11111 
                 19172 
                 22270 
                 18934 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4.054091 
                 0 
               
               
                 Control 
                 Cell 
                 16010 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 602.74446016 
               
             
          
           
               
                 Inhibition effect to HBsAg 
               
             
          
           
               
                 900 
                 7706 
                 7240 
                 7114 
                 0.342155 
                 0.381936 
                 0.392693 
                 0.372261 
                 0.922258 
                 0.627739 
                 0.595006426 
               
               
                 300 
                 8856 
                 7778 
                 9476 
                 0.2439816 
                 0.336008 
                 0.191053 
                 0.257014 
                 0.5499972 
                 1.370724 
                 0.286349225 
               
               
                 100 
                 10818 
                 10720 
                 10330 
                 0.07649 
                 0.084856 
                 0.118149 
                 0.093165 
                 0.292983 
                 2.27756 
                 0.113977019 
               
               
                 33.33333 
                 10744 
                 11114 
                 10570 
                 0.082807 
                 0.051221 
                 0.097661 
                 0.07723 
                 0.1998179 
                 3.20033 
                 0.058767408 
               
               
                 11.11111 
                 10672 
                 9352 
                 10810 
                 0.088953 
                 0.201639 
                 0.077173 
                 0.122588 
                 0.122588 
                 4.077742 
                 0.02918541 
               
               
                 Control 
                 Cell 
                 11714 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 641.7736749 
               
               
                   
               
             
          
           
               
                 Second batch: (rSIFN-co) 
               
               
                   
               
               
                 Inhibition effect to HBeAg 
               
             
          
           
               
                 900 
                 7818 
                 8516 
                 9350 
                 0.554378 
                 0.514592 
                 0.467054 
                 0.512008 
                 1.371181 
                 0.487992 
                 0.737521972 
               
               
                 300 
                 10344 
                 10628 
                 9160 
                 0.4103967 
                 0.394209 
                 0.477884 
                 0.427497 
                 0.8591731 
                 1.060496 
                 0.447563245 
               
               
                 100 
                 12296 
                 14228 
                 13262 
                 0.299134 
                 0.18901 
                 0.244072 
                 0.244072 
                 0.4316522 
                 1.816423 
                 0.19201839 
               
               
                 33.33333 
                 15364 
                 17414 
                 16188 
                 0.124259 
                 0.00741 
                 0.77291 
                 0.069653 
                 0.1876045 
                 2.74677 
                 0.063933386 
               
               
                 11.11111 
                 17386 
                 13632 
                 15406 
                 0.009006 
                 0.222982 
                 0.121865 
                 0.117951 
                 0.117951 
                 3.628819 
                 0.03148073 
               
               
                 Control 
                 Cell 
                 16962 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 365.9357846 
               
             
          
           
               
                 Inhibition effect to HBsAg 
               
             
          
           
               
                 900 
                 5784 
                 6198 
                 5792 
                 0.498265 
                 0.462353 
                 0.497571 
                 0.486063 
                 0.893477 
                 0.513937 
                 0.634835847 
               
               
                 300 
                 7150 
                 8534 
                 8318 
                 0.379771 
                 0.259715 
                 0.278452 
                 0.30598 
                 0.4074138 
                 1.207957 
                 0.252210647 
               
               
                 100 
                 9830 
                 11212 
                 10210 
                 0.147294 
                 0.027412 
                 0.11433 
                 0.096345 
                 0.101434 
                 2.111612 
                 0.04583464 
               
               
                 33.33333 
                 13942 
                 12368 
                 13478 
                 0 
                 0 
                 0 
                 0 
                 0.0050891 
                 3.111612 
                 0.001632835 
               
               
                 11.11111 
                 12418 
                 11634 
                 11352 
                 0 
                 0 
                 0.015267 
                 0.005089 
                 0.005089 
                 4.106523 
                 0.001237728 
               
               
                 Control 
                 Cell 
                   
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 611.0919568 
               
               
                   
               
             
          
           
               
                 Third batch: (rSIFN-co 
               
               
                   
               
               
                 Inhibition effect to HBeAg 
               
             
          
           
               
                 900 
                 9702 
                 9614 
                 8110 
                 0.428016 
                 0.433204 
                 0.521872 
                 0.461031 
                 1.316983 
                 0.538969 
                 0.709599543 
               
               
                 300 
                 8914 
                 10032 
                 8870 
                 0.4744723 
                 0.40856 
                 0.477066 
                 0.453366 
                 0.8559525 
                 1.085603 
                 0.440859127 
               
               
                 100 
                 16312 
                 12688 
                 13934 
                 0.038321 
                 0.251975 
                 0.178517 
                 0.156271 
                 0.402586 
                 1.929332 
                 0.172641621 
               
               
                 33.33333 
                 15080 
                 12814 
                 13288 
                 0.110954 
                 0.244547 
                 0.216602 
                 0.190701 
                 0.2463153 
                 2.738631 
                 0.082519158 
               
               
                 11.11111 
                 21928 
                 15366 
                 15728 
                 0 
                 0.094093 
                 0.072751 
                 0.0055615 
                 0.055615 
                 3.683017 
                 0.014875633 
               
               
                 Control 
                 Cell 
                 17544 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 382.0496935 
               
             
          
           
               
                 Inhibition effect to HBsAg 
               
             
          
           
               
                 900 
                 5616 
                 6228 
                 5346 
                 0.496864 
                 0.442035 
                 0.521054 
                 0.486651 
                 0.763125 
                 0.513349 
                 0.597838293 
               
               
                 300 
                 8542 
                 8590 
                 7096 
                 0.234725 
                 0.230425 
                 0.364272 
                 0.276474 
                 0.2764738 
                 1.236875 
                 0.182690031 
               
               
                 100 
                 11420 
                 11360 
                 11394 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2.236875 
                 0 
               
               
                 33.33333 
                 12656 
                 11582 
                 13110 
                 0 
                 0 
                 0 
                 0 
                 0 
                   
                 0 
               
               
                 11.11111 
                 13142 
                 12336 
                 13342 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4.236875 
                 0 
               
               
                 Control 
                 Cell 
                 11528 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 694.7027149 
               
               
                   
               
               
                 HBeAg: Average 
               
               
                 IC50: 450.2434 
               
               
                 SD: 132.315479 
               
               
                 HBsAg: Average 
               
               
                 IC50: 649.1894 
               
               
                 SD: 42.29580 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4.2 
               
             
             
               
                   
               
               
                 Results of inhibition rate of Intron A(IFN-α2b) to HBsAg and HBeAg 
               
             
          
           
               
                   
                 Inhibition rate 
                 Average 
                   
                   
                 Accumulated 
               
             
          
           
               
                 Concentration 
                 First 
                 Second 
                 Third 
                 First 
                 Second 
                 Third 
                 inhibition 
                 Accumula- 
                 1- 
                 inhibition 
               
               
                 (×10 4  IU/ml) 
                 well 
                 well 
                 well 
                 well 
                 well 
                 well 
                 rate 
                 tion 
                 Accumulation 
                 rate 
               
               
                   
               
             
          
           
               
                 Inhibition effect to HBeAg 
               
             
          
           
               
                 300 
                 14918 
                 11724 
                 9950 
                 0 
                 0.029711 
                 0.176529 
                 0.068747 
                 0.068747 
                 0.931253 
                 0.068746724 
               
               
                 100 
                 14868 
                 16890 
                 15182 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1.931253 
                 0 
               
               
                  33.33333 
                 16760 
                 21716 
                 16400 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2.931253 
                 0 
               
               
                  11.11111 
                 20854 
                 15042 
                 16168 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3.931253 
                 0 
               
               
                  3.703704 
                 12083 
                 12083 
                 12083 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4.931253 
                 0 
               
               
                 Control 
                 Cell 
                 17544 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 FALSE 
               
             
          
           
               
                 Inhibition effect to HBsAg 
               
             
          
           
               
                 300 
                 9226 
                 8196 
                 9658 
                 0.152489 
                 0.247106 
                 0.521054 
                 0.1708 
                 0.189295 
                 0.8292 
                 0.185857736 
               
               
                 100 
                 10946 
                 10340 
                 10828 
                 0 
                 0.050156 
                 0.364272 
                 0.018495 
                 0.0184947 
                 1.810705 
                 0.010110817 
               
               
                  33.33333 
                 12250 
                 12980 
                 13934 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2.810705 
                 0 
               
               
                  11.11111 
                 12634 
                 12342 
                 12000 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3.810705 
                 0 
               
               
                  3.703704 
                 10886 
                 10886 
                 10886 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4.810705 
                 0 
               
               
                 Control 
                 Cell 
                 10886 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 FALSE 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4.3 
               
               
                   
               
               
                 Results of inhibition rate of Infergen ® to HBsAg and HBeAg 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Inhibition rate 
                   
               
             
          
           
               
                 Concentration 
                 First 
                 Second 
                 Third 
                 First 
                 Second 
                 Third 
                 Average 
                   
                   
                 Accumulated 
               
               
                 (×10 4  IU/ml) 
                 well 
                 well 
                 well 
                 well 
                 well 
                 well 
                 inhibition rate 
                 Accumulation 
                 1-Accumulation 
                 inhibition rate 
               
               
                   
               
             
          
           
               
                 First batch: (Infergen ®) 
               
               
                   
               
               
                 Inhibition effect to HBeAg 
               
             
          
           
               
                 900 
                 14172 
                 12156 
                 17306 
                 0.091655 
                 0.220869 
                 0 
                 0.104175 
                 0.306157 
                 0.895825 
                 0.254710274 
               
               
                 300 
                 13390 
                 12288 
                 16252 
                 0.1417767 
                 0.212409 
                 0 
                 0.118062 
                 0.2019827 
                 1.777764 
                 0.102024519 
               
               
                 100 
                 14364 
                 18834 
                 14194 
                 0.079349 
                 0 
                 0.090245 
                 0.056531 
                 0.083921 
                 2.721232 
                 0.029916678 
               
               
                 33.33333 
                 15722 
                 16034 
                 16340 
                 0 
                 0 
                 0 
                 0 
                 0.0273897 
                 3.721232 
                 0.007306592 
               
               
                 11.11111 
                 17504 
                 17652 
                 14320 
                 0 
                 0 
                 0.082169 
                 0.02739 
                 0.02739 
                 4.693843 
                 0.005801377 
               
               
                 Control 
                 Cell 
                 15602 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 FALSE 
               
             
          
           
               
                 Inhibition effect to HBsAg 
               
             
          
           
               
                 900 
                 12080 
                 11692 
                 12234 
                 0 
                 0.01275 
                 0 
                 0.00425 
                 0.025163 
                 0.99575 
                 0.024647111 
               
               
                 300 
                 12840 
                 11484 
                 12350 
                 0 
                 0.030313 
                 0 
                 0.010104 
                 0.0209125 
                 1.985646 
                 0.010422073 
               
               
                 100 
                 12894 
                 14696 
                 15086 
                 0 
                 0 
                 0 
                 0 
                 0.010808 
                 2.985646 
                 0.003606955 
               
               
                 33.33333 
                 15032 
                 12928 
                 13020 
                 0 
                 0 
                 0 
                 0 
                 0.0108081 
                 3.985646 
                 0.002704416 
               
               
                 11.11111 
                 11794 
                 11984 
                 11508 
                 0.004137 
                 0 
                 0.028287 
                 0.010808 
                 0.010808 
                 4.974837 
                 0.002167838 
               
               
                 Control 
                 Cell 
                 11843 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 FALSE 
               
               
                   
               
             
          
           
               
                 Second batch: (Infergen ®) 
               
               
                   
               
               
                 Inhibition effect to HBeAg 
               
             
          
           
               
                 900 
                 6278 
                 6376 
                 6408 
                 0.200051 
                 0.187564 
                 0.183486 
                 0.190367 
                 0.274635 
                 0.809633 
                 0.253290505 
               
               
                 300 
                 7692 
                 9092 
                 6394 
                 0.0198777 
                 0 
                 0.18527 
                 0.068383 
                 0.0842678 
                 1.74125 
                 0.046161005 
               
               
                 100 
                 8960 
                 7474 
                 8190 
                 0 
                 0.047655 
                 0 
                 0.015885 
                 0.015885 
                 2.725365 
                 0.005794856 
               
               
                 33.33333 
                 8530 
                 8144 
                 9682 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3.725365 
                 0 
               
               
                 11.11111 
                 7848 
                 7848 
                 7848 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4.725365 
                 0 
               
               
                 Control 
                 Cell 
                 7848 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 FALSE 
               
             
          
           
               
                 Inhibition effect to HBsAg 
               
             
          
           
               
                 900 
                 12364 
                 12268 
                 12274 
                 0.036171 
                 0.043655 
                 0.043187 
                 0.041004 
                 0.140162 
                 0.958996 
                 0.12751773 
               
               
                 300 
                 11590 
                 12708 
                 13716 
                 0.0965076 
                 0.009355 
                 0 
                 0.035287 
                 0.0991581 
                 1.923709 
                 0.0490186 
               
               
                 100 
                 12448 
                 13468 
                 13982 
                 0.029623 
                 0 
                 0 
                 0.009874 
                 0.063871 
                 2.913834 
                 0.02144964 
               
               
                 33.33333 
                 12616 
                 11346 
                 12444 
                 0.016526 
                 0.115529 
                 0.029935 
                 0.053996 
                 0.0539965 
                 3.859838 
                 0.013796309 
               
               
                 11.11111 
                 12828 
                 12828 
                 12828 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4.859838 
                 0 
               
               
                 Control 
                 Cell 
                 12828 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 FALSE 
               
               
                   
               
             
          
           
               
                 Third batch: (Infergen ®) 
               
               
                   
               
               
                 Inhibition effect to HBeAg 
               
             
          
           
               
                 900 
                 7240 
                 6642 
                 6158 
                 0.064599 
                 0.14186 
                 0.204393 
                 0.136951 
                 0.217399 
                 0.863049 
                 0.201211735 
               
               
                 300 
                 11072 
                 8786 
                 6902 
                 0 
                 0 
                 0.108269 
                 0.03609 
                 0.0804479 
                 1.82696 
                 0.042176564 
               
               
                 100 
                 7016 
                 9726 
                 7552 
                 0.09354 
                 0 
                 0.024289 
                 0.039276 
                 0.044358 
                 2.787683 
                 0.015663017 
               
               
                 33.33333 
                 7622 
                 8866 
                 8676 
                 0.015245 
                 0 
                 0 
                 0.005082 
                 0.0050818 
                 3.782601 
                 0.001341671 
               
               
                 11.11111 
                 7740 
                 7740 
                 7740 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4.782601 
                 0 
               
               
                 Control 
                 Cell 
                 7740 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 FALSE 
               
             
          
           
               
                 Inhibition effect to HBsAg 
               
             
          
           
               
                 900 
                 11048 
                 11856 
                 11902 
                 0.04775 
                 0 
                 0 
                 0.015917 
                 0.015917 
                 0.984083 
                 0.015916796 
               
               
                 300 
                 13454 
                 12896 
                 11798 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1.984083 
                 0 
               
               
                 100 
                 12846 
                 13160 
                 12546 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2.984083 
                 0 
               
               
                 33.33333 
                 12680 
                 12458 
                 12360 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3.984083 
                 0 
               
               
                 11.11111 
                 11602 
                 11602 
                 11602 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4.984083 
                 0 
               
               
                 Control 
                 Cell 
                 11602 
                   
                 Blank 
                 0 
                   
                 Dilution 
                 3 
                 IC50 
                 FALSE 
               
               
                   
               
               
                 HBeAg: Average 
               
               
                 IC50: 0 
               
               
                 SD: 0 
               
               
                 HBsAg: Average 
               
               
                 IC50: 0 
               
               
                 SD: 0 
               
             
          
         
       
     
       Example 7 
     The Clinic Effects of Recombinant Super-Compound Interferon (rSIFN-co) 
       [0321]    The recombinant super-compound interferon (rSIFN-co) is an invention for viral disease therapy, especially for hepatitis. Meanwhile, it can inhibit the activity of EB viruses, VSV, Herpes simplex viruses, cornaviruses, measles viruses, et al. Using Wish cells/VSV system as the assay for anti-virus activity, the results showed that: the other rIFN, was 0.9×10 8  IU/mg, Intron A was 2.0×10 8  IU/mg and rSIFN-co was 9×10 8  IU/mg. The anti-viral activity of rSIFN-co is much higher than those of the former two. 
         [0322]    Under the permission of the State Food and Drug Administration (SFDA), People&#39;s Republic of China, the clinical trials have taken place in West China Hospital, Sichuan University, the Second Hospital of Chongqing Medical University, the First Hospital of School of Medical, Zhejiang University since the February 2003. The clinical treatment which focuses on hepatitis B is conducted under the guidance of the mutilcenter, double-blind random test. IFN-α1b was used as control, and the primary results showed the following: 
         [0000]    The Effect of rSIFN-co Compared with IFN-α1b in the Treatment of Chronic Active Hepatitis B 
       1. Standard of Patients Selection: 
       [0323]    Standards 1-4 are effective for both treatment with rSIFN-co (9 μg) and IFN-α1b (5 MU, 50 μg), and Standard 1-5 are for rSIFN-co (15 μg) treatment. 
       1). Age: 18-65 
       [0324]    2). HBsAg-test positive over last six months, HBeAg-test positive, PCR assay, HBV-DNA copies ≧10 5 /ml
 
3). ALT ≧two times the normal value
 
4). Never received IFN treatment; or received the Lamividine treatment but failed or relapsed
 
5) Once received other IFNs (3 MU or 5 MU) treatment six months ago following the standard of SFDA, but failed or relapsed
 
       2. Evaluation of the Effects: 
       [0325]    In reference to the recommendations from the Tenth China National Committee of Virus Hepatitis and Hepatopathy, the effects were divided into three degrees according to the ALT level, HBV-DNA and HBeAg tests.
   Response: ALT normal level, HBV-DNA negative, HBeAg negative   Partial response: ALT normal level, HBV-DNA or HBeAg negative   Non response: ALT, HBV-DNA and HBeAg unchanged   The response and partial response groups were considered effective cases.   
 
       3. Results of Clinic Trial: 
       [0330]    Group A: treatment with rSIFN-co (9 μg) 
         [0331]    Group B: treatment with IFN-α1b (5 MU, 50 μg) 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                   
                 HBsAg 
                 HBeAg 
                 HBV-DNA 
                   
               
               
                   
                   
                   
                   
                   
                 Transfer 
                 Transfer 
                 Transfer 
                 Heptal 
               
               
                   
                   
                   
                   
                   
                 to 
                 to 
                 to 
                 function 
               
               
                   
                   
                   
                   
                 Effective 
                 negative 
                 negative 
                 negative 
                 Recovery 
               
               
                 Period 
                 group 
                 Medicine 
                 cases 
                 Rate 
                 rate 
                 rate 
                 rate 
                 rate 
               
               
                   
               
             
             
               
                 8-12 
                 A 
                 rSIFN- 
                 32 
                 46.88 
                 9.38 
                 28.13 
                 37.50 
                 84.38 
               
               
                 week 
                   
                 co(9 μg) 
                   
                 (15)   
                 (3)   
                 (9)   
                 (12)   
                 (27)   
               
               
                   
                 B 
                 IFN-α1b 
                 32 
                 21.88 
                 0.00 
                  9.38 
                 15.63 
                 56.25 
               
               
                   
                   
                 (5MU, 50 μg) 
                   
                 (7)   
                 (0)   
                 (3)   
                 (5)   
                 (18)   
               
               
                 16-24 
                 A 
                 rSIFN- 
                 64 
                 54.69 
                 7.81 
                 25.00 
                 34.38 
                 90.63 
               
               
                 week 
                   
                 co(9 μg) 
                   
                 (35)   
                 (5)   
                 (16)   
                 (22)   
                 (58)   
               
               
                   
                 B 
                 IFN-α1b 
                 64 
                 25.00 
                 0.00 
                  9.38 
                 18.75 
                 78.13 
               
               
                   
                   
                 (5MU 50 μg) 
                   
                 (16)   
                 (0)   
                 (6)   
                 (12)   
                 (50)   
               
               
                   
               
             
          
         
       
     
         [0332]    In Group C, the cases were prior treatment of chronic active hepatitis B with other IFNs (3 MU or 5 MU) that failed or relapsed and then were treated with rSIFN-co (15 μg), subcutaneous injection, every one day, for 24 weeks. The total cases were 13. After 12 weeks treatment, 7 of 13 (53.85%) were effective. 3 of 13 (23.08%) HBeAg transferred to negative; 7 of 13 (53.85%) HBV-DNA transferred to negative; 11 of 13 (84.62%) heptal functions recovered to normal. 
         [0000]    4. The Side Effects of rSIFN-co Compared with IFN-α1b in the Treatment 
         [0333]    The side effects of IFN include fever, nausea, myalgia, anorexia, hair loss, leucopenia and thrombocytopenia, etc. The maximum dose of IFN-α1b is 5 MIU per time; the routine dose is 3 MIU. When taken the routine dose, 90% patients have I-II degree (WHO standard) side effects. They had fever lower than 38° C., nausea, myalgia, anorexia, etc. When taken at maximum dose, the rate of side effects did not rise obviously, but were more serious. The maximum dose of rSIFN-co is 24 μg, subcutaneous injection, every one day for 3 months. The routine dose is 9 μg. When routine doses were used, less than 50% of patients had I-II degree (WHO standard) side effects, including fever below 38° C., nausea, myalgia, anorexia, leucopenia and slight thrombocytopenia. With maximum dosage, about 50% patients suffered from leucopenia and thrombocytopenia after using rSIFN-co one month, but those side effects disappeared after stopping treatment for one week. It is safe for continued use. 
         [0000]    The Observations of rSIFN-co Treat Hepatitis C 
       1. Standard of Patients Selection 
       [0334]    1) age: 18-65
 
2) HCV antibody positive
 
3) ALT≧1.5 times of the normal value, last more than 6 months
 
       2. Evaluation of the Effects: 
       [0335]    Referring to the standard of Infergen® for treatment of hepatitis C and according to the ALT level and HCV-RNA test, divided the effects into three degree: 
         [0336]    Response: ALT normal level, HCV-RNA negative 
         [0337]    Partial response: ALT normal level, HCV-RNA unchanged 
         [0338]    Non response: ALT and HCV-RNA unchanged 
       3. Effects in Clinic 
       [0339]    The clinical trial was done at the same time with hepatitis B treatment. 46 cases received the treatment, 9 μg each time, subcutaneous injection, every day for 24 weeks. After treatment, 26 of 46 (56.52%) have obvious effects, 12 of 46 (26.09%) HCV-RNA transferred to negative, 26 of 46 (56.52%) heptal functions recovered to normal. 
       Example 8 
     Comparison of Inhibitory Effects of Different Interferons on HBV Gene Expression 
       [0340]    Hepatitis B virus (HBV) DNA contains consensus elements for transactivating proteins whose binding activity is regulated by interferons. Treatment of HBV-infected hepatocytes with interferons leads to inhibition of HBV gene expression. The aim of the present study was to characterize the effects of different interferons on HBV regulated transcription. Using transient transfection of human hepatoma cells with reporter plasmids containing the firefly luciferase gene under the control of HBV-Enhancer EnH I, Enh II and core promoter, Applicant studied the biological activities of three different interferons on transcription. 
       Materials and Methods 
       [0341]    1. Interferons: IFN-con1 (Infergen®), IFN-Hui-Yang (rSIFN-co) and IFNα-2b (Intron A).
 
2. Reporter plasmid: The DNA fragments containing HBV-Enhancer EnH I, Enh II and core promoter were prepared using PCR and blunt-end cloned into the Smal I site of the promoter- and enhancer-less firefly luciferase reporter plasmid pGL3-Basic (Promega, WI, USA). The resulting reporter plasmid was named as pGL3-HBV-Luc.
 
3. Cell Culture and DNA transfection: HepG2 cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin and 100 ug/ml streptomycin. The cells were kept in 30° C., 5% CO2 incubator. The cells were transfected with pGL3-HBV-Luc reporter plasmid using Boehringer&#39;s Lipofectin transfection kit. After 18 hours, the medium containing transfection reagents was removed and fresh medium was added with or without interferons. The cells were kept in culture for another 48 hours.
 
4. Luciferase Assay: Forty-eight hours after addition of interferon, the cells were harvested and cell lysis were prepared. The protein concentration of cell lysates were measured using Bio-Rad Protein Assay kit. The luciferase activity was measured using Promega&#39;s Luciferase Reporter Assay Systems according to the instructions of manufacturer.
 
       Results 
     Expression of Luciferase Activity in Different Interferon-Treated Cell Lysates 
       [0342]      
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 No treatment 
                 IFN-con1 
                 rSIFN-co 
                 IFNα-2b 
               
               
                   
                   
               
             
             
               
                   
                 100 
                 65 
                 32 
                 73 
               
               
                   
                   
               
             
          
         
       
     
         [0343]    This result shows that rSIFN-co inhibits most effectively on the expression of HBV gene expression of HB core Antigen. This data shows inhibitory effect of rSIFN-co is twice better than Infergen® and Intron A. See  FIG. 10 . 
       Example 9 
     Recombinant Super-Compound Interferon Spray 
       [0344]    Major component: Recombinant Super Compound Interferon 
         [0345]    Characteristic: Liquid, no insoluble material 
         [0346]    Pharmacology: Recombinant Super-Compound Interferon has a wide spectrum of anti-virus activity. Its effects are 5-20 times higher than those interferons (IFNs) which are available on the market. It can inhibit coronavirus growth in cell culture. In vitro test shows that rSIFN-co has an obvious anti-SARS virus activity. rSIFN-co effect to 10,000 and 1000 TCID 50 . The Inhibitory Indexes are 0.92 μg/ml and 0.18 μg/ml respectively. The Treatment Indexes (TI) are 151.28, 773.22 respectively. The mechanism is interruption of the combination reaction between the IFN and the correspondent receptor, and inducement of the expression of 2′5′-A synthesizenzyme, protein kinase R in the target cell, therefore inhibiting expression of the viral protein. IFN can induce expression of various anti-virus proteins to inhibit the reproduce of viral proteins, enhance the function of Natural Killer (NK) cell and other Immune regulative functions, and inhibit the invasion of viruses. 
         [0347]    Acute toxicity: All mice are alive after the maximum dose (1000 times to human dose) subcutaneous injection, did not observe LD50. 
         [0348]    Indication: Prevention of Severe Acute Respiratory Syndrome 
         [0349]    Dosage and Administration: Spray to both nasal cavity and throat, three times a day. 
         [0350]    Adverse reactions: There was no report of adverse reactions from the rSIFN-co spray. It did not induce allergy. If the stimulation is occasional, adverse gastrointestinal reaction is small, and no other obvious adverse reaction was noted during treatment, it is safe to continue use. All reactions will resolve themselves. 
         [0351]    Warning: Patients allergic to IFN and productions of  E. Coli . cannot use this product. 
         [0352]    Precautions: Before first use, spray twice to expel the air. If there is any cloudy precipitation material, if the product is expired, or there is material on the vial, do not use it. 
         [0353]    Pediatric Use: It is unclear. 
         [0354]    Geriatric Use: It is unclear. 
         [0355]    Nursing mothers and pregnant women: Use with care or under physician&#39;s supervision. 
         [0356]    Drug Interactions: It is unclear. 
         [0357]    Overdose: One-time dose of over 27 million of International Units have not produced any adverse effects. 
         [0358]    Supplied: 1 spray/pack, 20 ug (1×10 7  IU)/3 ml. See  FIGS. 11A-11D . 
         [0359]    Storage: Store at 4-8° C. Do not freeze, protect from light. 
         [0360]    Effective period: Approximately one year 
         [0361]    Manufacture: Manufactured by Sichuan Huiyang life-engineering Ltd. 
         [0362]    Address: 8 Yusa Road, Room 902, Building A
       Chengdu, 610017   Sichuan, P.R. China       
 
       Example 9-A 
     In Vitro Effect of a New-Style Recombinant Compound Interferon on SARS-Associated Coronavirus 
       [0365]    Sample supplied by: Huiyang Life Engineering Lt Company, SiChuan Province 
         [0366]    Experimenter: Molecular Biology Department, microorganism and epidemiology Institute, Academy of Military Medical Science 
         [0367]    Original data: Preserved in archive of Molecular Biology Department, microorganism and epidemiology Institute, Academy of Military Medical Science 
       1. Materials 
       [0368]    Medicine: New-type recombinant compound interferon, 9 μg each, supplied by Huiyang Life Engineering Lt Company, SiChuan Province, Lot number: 20020501. 
         [0369]    Cells: Vero E 6 , supplied by Molecular Biology Department of Microorganism and Epidemiology Institute, Academy of Military Medical Science. 
         [0370]    Virus: SARS-associated coronavirus, BJ-01, supplied by Molecular Biology Department of Microorganism and Epidemiology Institute, Academy of Military Medical Science. 
         [0371]    Cell medium: DMEM supplemented with 10% FBS. 
       2. Condition 
       [0372]    Virus was measured in grade 3 rd  laboratory of biosafety 
       3. Method 
       [0373]    CPE (cytopathic effect) assay of TCID 50 : 100 μl of Vero E 6  cells were plated in 96-well plates at 2×10 4  cells per well. After 24 hr incubation at 37° C., Vero E6 monolayer cells were treated with 9 levels of SARS-associated coronavirus dilution by 10-fold dilution, 4 wells per dilution. The cells were incubated at 37° C. and 5% CO 2 . CPE (cytopathic effect) was examined daily by microscopy. CPE less than 25% was determined as +, 26-50% as ++, 51-75% as +++, 76-100% as ++++. CPE was recorded. Then TCID 50  was calculated by Reed-Muench method. 
         [0374]    Cytotoxicity of medicine: Vero E 6  cells were inoculated into 96-well plates at 2×10 4  cells (100 ul) per well. After 24-hr incubation at 37° C., cells grew up to monolayer. The medicine was diluted into 36, 18, 9, 4.5, 2.259 μg/ml (final concentration) and added into wells each for 4 wells. The normal cells as control group were set. CPE of medicine group was daily observed during 5-day period, and then the concentration of medicine exhibiting no toxicity was determined. 
         [0375]    CPE assay of the activity of the medicine against SARS-associated coronavirus: 100 μl of Vero E 6  cells were plated in 96-well plates at 2×10 4  cells per well. After 24 hr incubation at 37° C., cells grew up to monolayer. The medicine at the maximal concentration exhibiting no cytotoxicity was diluted into 5 levels by 2-fold dilution and added into wells (100 μl per well). By incubation with 5% CO 2  at 37° C. for 24-hour, different concentration of virus (10 −3 , 10 −4 , 10 −5 ) were added. After treatment with virus for 48-72 hours, CPE was examined (CPE less than 25% was determined as +, 26-50% as ++, 51-75% as +++, 76-100% as ++++, normal cell as −). The cells were divided into the normal group, the medicine control group, and the different dilution of virus control group, 4 wells per group. CPE was examined daily. Till cytopathic effect was obviously exhibited in the virus control group, the anti-virus activity of interferon was evaluated. The experiment was repeated. IC 50  of the medicine was calculated by Reed-Muench method. 
       4. Results 
       [0376]    Toxicity of virus: TCID 50  of virus was 10 −8 . 
         [0377]    Cytotoxicity of medicine: the concentration of Recombinant compound interferon exhibiting no cytotoxicity was 18 μg/ml, the cells shape was similar with the control group, and no cytopathic effect was exhibited. 
         [0378]    The anti-virus effect of the medicine: Shown in Table 9-A.1 and Table 9-A.2 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 A.1, the anti-virus effect of new-type recombinant 
               
               
                 compound interferon (first experiment) 
               
             
          
           
               
                   
                 Concentration of 
                 CPE at different concentration  
               
               
                   
                 IFN 
                 of virus 
               
             
          
           
               
                   
                 (μg/ml) 
                 10 −3   
                 10 −4   
                 10 −5   
               
               
                   
                   
               
             
          
           
               
                   
                 18 
                 − 
                 − 
                 − 
               
               
                   
                 9 
                 − 
                 − 
                 − 
               
               
                   
                 4.5 
                 + + 
                 − 
                 − 
               
               
                   
                 2.25 
                 + + + 
                 + + 
                 − 
               
               
                   
                 1.125 
                 + + + + 
                 + + + + 
                 + + 
               
               
                   
                 Virus control 
                 + + + + 
                 + + + + 
                 + + + 
               
               
                   
                 group 
                   
                   
                   
               
               
                   
                 Normal group 
                 − 
                 − 
                 − 
               
               
                   
                 Medicine control 
                 − 
                 − 
                 − 
               
               
                   
                 group 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 A.2, the anti-virus effect of new-type recombinant 
               
               
                 compound interferon (second experiment) 
               
             
          
           
               
                   
                 Concentration of 
                 CPE at different concentration  
               
               
                   
                 IFN 
                 of virus 
               
             
          
           
               
                   
                 (μg/ml) 
                 10 −3   
                 10 −4   
                 10 −5   
               
               
                   
                   
               
             
          
           
               
                   
                 18 
                 − 
                 − 
                 − 
               
               
                   
                 9 
                 − 
                 − 
                 − 
               
               
                   
                 4.5 
                 + 
                 − 
                 − 
               
               
                   
                 2.25 
                 + + + 
                 + +  
                 − 
               
               
                   
                 1.125 
                 + + + + 
                 + + + + 
                 + + 
               
               
                   
                 Virus control 
                 + + + + 
                 + + + + 
                 + + + + 
               
               
                   
                 group 
                   
                   
                   
               
               
                   
                 Normal group 
                 − 
                 − 
                 − 
               
               
                   
                 Medicine control 
                 − 
                 − 
                 − 
               
               
                   
                 group 
               
               
                   
                   
               
             
          
         
       
     
       5. Conclusion 
       [0379]    The concentration of the new-type recombinant compound interferon exhibiting no cytotoxicity at 18 μg/ml. Its IC 50  were 1.27, 2.25, and 4.04 μg/ml respectively according to the concentration of 10 −5  (1000TCID 50 ), 10 −4  (1000TCID 50 ), 10 −3  (100000TCID 50 ) of SARS-associated coronavirus (Table 9-A.3). 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 A.3, IC 50  of IFN at different concentrations  
               
               
                 of virus 
               
             
          
           
               
                   
                 Dilution of virus 
                 IC50 of IFN (ug/ml) 
               
               
                   
                   
               
               
                   
                 10 −3   
                 4.04 
               
               
                   
                 10 −4   
                 2.25 
               
               
                   
                 10 −5   
                 1.27 
               
               
                   
                   
               
             
          
         
       
     
         [0380]    Principal: Jin-yan Wang 
         [0381]    Laboratory assistant: Yan-hong Zhao, Xiao-guang Ji, Xiao-yu Li. 
         [0382]    Original data: Preserved in archives of Molecular Biology Department, microorganism and epidemiology Institute, Academy of Military Medical Science 
         [0383]    Date: From May 12th to 30th, 2003 
       Example 9-B 
     In Vitro Effect of a New-Type Recombinant Compound Interferon and Recombinant Interferon α-2b Injection on SARS-Associated Coronavirus 
       [0384]    Sample (rSIFN-co) supplied by: Huiyang Life Engineering Ltd., Sichuan province 
         [0385]    Experimenter: Molecular Biology Department, Institute of microbiology and epidemiology, Academy of Military Medical Science 
         [0386]    Original data: Preserved in monument room of Molecular Biology Department, Institute of microbiology and epidemiology, Academy of Military Medical Science 
       1. Materials 
       [0387]    Medicine: New-type recombinant compound interferon (rSIFN-co), 618 μg/ml, supplied by Huiyang Life Engineering Ltd., SiChuan Province; Alfaron (recombinant interferon α-2b injection), supplied by Tianjin Hualida Biotechnology Co., Ltd. 30 ug/vial (300,0000 IU/vial), Lot Number: 20030105. 
         [0388]    Cells: Vero E 6 , supplied by Molecular Biology Department of Institute of microbiology and epidemiology, Academy of Military Medical Science. 
         [0389]    Virus: SARS-associated coronavirus, BJ-01, supplied by Molecular Biology Department of Institute of microbiology and epidemiology, Academy of Military Medical Science. 
         [0390]    Condition: Viruses were measured in grade 3 rd  laboratory of biosafety 
       2. Method 
       [0391]    TCID 50  was measured with CPE assay: Vero E 6  cells were inoculated in 96-well plates at 2×10 4  cells (100 μl) per well. After a 24-hr incubation at 37° C., Vero E6 monolayers were treated with 9 levels of SARS-associated coronavirus dilution by 10 times decreasing, each dilution per 4 wells. The cells were incubated at 37° C. and 5% carbon dioxide. CPE was examined daily by phase-contrast microscopy. CPE less than 25% was determined as +, 26-50% as ++, 51-75% as +++, 76-100% as ++++. CPE was recorded. Then TCID 50  was calculated by Reed-Muench method. 
         [0392]    TC 50  of IFNs were measured by MTT assay: Vero E 6  cells were inoculated in 96-well plates at 2×10 4  cells per well (100 μl). After 24-hr incubation at 37° C., the supernatant liquid was removed when cells grew up to monolayer, then Vero E 6  was treated with different concentration of IFNs, each dilution per 4 wells. Normal group was set. After 5-day observation, the cells were mixed with MTT for 4 hours. After that, remove the liquid, and then thereafter DMSO were added into cells for 0.5 hour. The OD 570nm  was measured by microplate reader. Finally, TC 50  was calculated by Reed-Muench method. 
         [0393]    The activity of the INFs against SARS-associated coronavirus was measured with MTT assay: 100 μl of Vero E 6  cells were inoculated in 96-well plates at 2×10 4  cells per well. After 24-hr incubation 37° C., cells became monolayer. The medicine dilution at the concentration of exhibiting no cytotoxicity was 5 times decreasing and there were 5 levels of dilution. Then each dilution was added to 4 wells, 100 ul per well. After 24-hour incubation at 37° C. and 5% CO 2 , IFN solution was removed, then different concentrations of virus dilution (10000, 1000, 100 TCID 50 ) were added into dishes, 4 wells per dilution. The cells were divided into the normal group, the medicine control group, and the different dilution of virus control group (10000, 1000, 100 TCID 50 ). The cells were incubated at 37° C. and 5% CO 2  for 48-72 hr, until cytopathic effect was exhibited in the virus control group, CPE was recorded (CPE less than 25% was determined as +, 26-50% as ++, 51-75% as +++, 76-100% as ++++, normal cell as −). The growth ability of cells was measured with MTT assay, and then the antivirus effect of the INFs was evaluated. The experiment was repeated 3 times. IC 50  of the medicine was calculated by Reed-Muench method. 
       3. Results 
       [0394]    TCID 50  of virus: TCID 50  of virus was 10 −7 . 
         [0395]    TC 50  of IFNs: The concentration of new-type recombinant compound interferon (rSIFN-co) exhibiting no cytotoxicity was 100 μg/ml, and that of recombinant IFNα-2b was 12.5 μg/ml, the cells shape was identical with the normal group at that concentration. TC50 of new-type recombinant compound interferon (rSIFN-co) was 139.18 μg/ml, that of recombinant IFNα-2b was 17.18 μg/ml. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 B.1 TC 50  of IFNs 
               
             
          
           
               
                   
                 TC 50  (μg/ml) 
                   
               
             
          
           
               
                   
                 1 st   
                 2 nd   
                 3 rd   
                 Mean value- 
               
               
                 IFN 
                 experiment 
                 experiment 
                 experiment 
                 (X ± SD, n = 3) 
               
               
                   
               
             
          
           
               
                 new-type 
                 141.42 
                 125.96 
                 150.08 
                 139.18 ± 12.22 
               
               
                 recombinant 
                   
                   
                   
                   
               
               
                 compound 
                   
                   
                   
                   
               
               
                 interferon 
                   
                   
                   
                   
               
               
                 IFNα-2b 
                 17.68 
                 15.75 
                 18.10 
                  17.18 ± 1.25 
               
               
                   
               
             
          
         
       
     
         [0396]    The anti-virus effect of the medicine: The anti-virus effects of two IFNs were observed in vitro. The results of the experiments are shown on the Table 9-B.2, and the results of TI are shown on the Table 9-B.3. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 B.2, The anti-virus activity of IFNs 
               
             
          
           
               
                   
                 Concentration 
                 IC 50  (μg/ml) 
               
             
          
           
               
                   
                 of 
                 1 st   
                 2 nd   
                 3 rd   
                 Mean value- 
               
               
                 IFNs 
                 virus (TCID 50 ) 
                 experiment 
                 experiment 
                 experiment 
                 (X ± SD, n = 3 
               
               
                   
               
             
          
           
               
                 new-type 
                 10000 
                 0.79 
                 1.04 
                 0.93 
                 0.92 ± 0.12 
               
               
                 recombinant 
                   
                   
                   
                   
                   
               
               
                 compound interferon 
                   
                   
                   
                   
                   
               
               
                 IFNα-2b 
                   
                 5.04 
                 4.56 
                 4.65 
                 4.75 ± 0.25 
               
               
                 new-type 
                 1000 
                 0.19 
                 0.18 
                 0.18 
                 0.18 ± 0.01 
               
               
                 recombinant 
                   
                   
                   
                   
                   
               
               
                 compound interferon 
                   
                   
                   
                   
                   
               
               
                 IFNα-2b 
                   
                 1.18 
                 1.19 
                 1.12 
                 1.16 ± 0.04 
               
               
                 new-type 
                 100 
                 0.08 
                 0.10 
                 0.11 
                 0.10 ± 0.02 
               
               
                 recombinant 
                   
                   
                   
                   
                   
               
               
                 compound interferon 
                   
                   
                   
                   
                   
               
               
                 IFNα-2b 
                   
                 0.33 
                 0.21 
                 0.30 
                 0.28 ± 0.06 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 B.3, The anti-virus activity of IFNs 
               
             
          
           
               
                   
                 Concentration 
                   
                   
                   
               
               
                   
                 of 
                   
                   
                 TI 
               
               
                 IFNs 
                 virus (TCID 50 ) 
                 TC 50  (μg/ml) 
                 IC 50  (μg/ml) 
                 (TC 50 /IC 50 ) 
               
               
                   
               
             
          
           
               
                 new-type 
                 10000 
                 139.18 
                 0.92 
                 151.28 
               
               
                 recombinant 
                   
                   
                   
                   
               
               
                 compound  
                   
                   
                   
                   
               
               
                 interferon 
                   
                   
                   
                   
               
               
                 IFNα-2b 
                   
                 17.18 
                 4.75 
                 3.62 
               
               
                 new-type 
                 1000 
                 139.18 
                 0.18 
                 773.22 
               
               
                 recombinant 
                   
                   
                   
                   
               
               
                 compound 
                   
                   
                   
                   
               
               
                 interferon 
                   
                   
                   
                   
               
               
                 IFNα-2b 
                   
                 17.18 
                 1.16 
                 14.78 
               
               
                 new-type 
                 100 
                 139.18 
                 0.10 
                 1391.80 
               
               
                 recombinant 
                   
                   
                   
                   
               
               
                 compound  
                   
                   
                   
                   
               
               
                 interferon 
                   
                   
                   
                   
               
               
                 IFNα-2b 
                   
                 17.18 
                 0.28 
                 61.36 
               
               
                   
               
             
          
         
       
     
       4. Conclusion 
       [0397]    The protection effect of new-type recombinant compound interferon (rSIFN-co) and IFNα-2b on Vero E 6  was observed in vitro, and the anti-virus ability of IFNs was manifested. IC 50  of new-type recombinant compound interferon on SARS-associated coronavirus at the concentration of 10000, 1000, and 100 was 0.92, 0.18, and 0.10 μg/ml in three experiments, TI of that was 151.28, 773.22, and 1391.80 respectively. IC 50  of IFNα-2b was 4.75, 1.16, and 0.28 μg/ml, TI (treatment index) of that was 3.62, 14.78, 61.36 respectively. 
         [0398]    Most importantly, the two tests (See the above Examples 9A &amp; 9B) of in vitro anti-SARS virus effect of rSIFN-co all testified that even the effective dose of rSIFN-co to inhibit SARS virus is 1/5 of that of Interferon α-2b which was used clinically in China at present, the Treatment Index (TI) of rSIFN-co is nearly 50 times of that of Interferon a-2b. (SEE: In vitro effect of a new-type recombinant compound interferon and recombinant interferon-α-2b injection on SARS-associated coronavirus. By The Institute of Microbiology &amp; Epidemiology, Academy of Military Medical Science) Also, see  FIG. 12 . 
         [0399]    Thirty thousand sprays of rSIFN-co had been used among front-line nurses and doctors, and people at high risk in Sichuan province. The result shows that none of the nurses and doctors infected SARS in Sichuan Province. 
         [0400]    Principal: Jin-yan Wang 
         [0401]    Laboratory assistant: Yan-hong Zhao, Xiao-guang Ji, Min Zhang, Jing-hua, Zhao. 
         [0402]    Date: From July 1st to 30th, 2003 
       Example 10 
     Side Effects and Changes in Body Temperature when Using rSIFN-co 
       [0403]    There are usually more side effects to using interferon. The side effects includes: nausea, muscle soreness, loss of appetite, hair loss, hypoleucocytosis (hypoleukmia; hypoleukocytosis; hypoleukia), and decrease in blood platelet, etc. 
       Method 
       [0404]    Sample patients are divided into two groups. 10 patients in Group A were injected with 9 μg rSIFN-co. 11 patients in Group B were injected with 9 μg Infergen®. Both groups were monitored for 48 hours after injections. First monitoring was recorded 1 hour after injection. After that, records were taken every 2 hours. 
         [0405]    Table 11.1 is the comparison of side effects between patients being injected with 9 μg of rSIFN-co and 9 μg of Infergen®. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 11.1 
               
             
             
               
                   
               
               
                 Side Effects 
               
             
          
           
               
                   
                   
                 rSIFN-co 
                 Infergen ® 
               
               
                   
                   
                 9 μg (Group A) 
                 9 μg (Group B) 
               
               
                   
                   
                 Person: n = 10 
                 Person: n = 11 
               
               
                 Body Systems 
                 Reactions 
                 Headcount 
                 Headcount 
               
               
                   
               
             
          
           
               
                 In General 
                 Feeble 
                 3 
                 3 
               
               
                   
                 Fever 
                 3 
                 6 
               
               
                   
                 Sole heat 
                 1 
                   
               
               
                   
                 frigolabile 
                 3 
                 4 
               
               
                   
                 Leg 
                   
                 3 
               
               
                   
                 strengthless 
                   
                   
               
               
                   
                 Mild lumbago 
                 2 
                 1 
               
               
                   
                 Body soreness 
                 4 
                 5 
               
               
                 Central Nervous 
                 Headache 
                 3 
                 6 
               
               
                 System/Peripheral 
                 Dizziness 
                 2 
                 11 
               
               
                 Nervous System 
                 Drowsiness 
                   
                 3 
               
               
                 Gastroenterostomy 
                 Apoclesis 
                 1 
                   
               
               
                   
                 Celiodynia 
                 1 
                   
               
               
                   
                 Diarrhea 
                 1 
                   
               
               
                 Musculoskeletal 
                 Myalgia 
                 1 
                 2 
               
               
                 system 
                   
                   
                   
               
               
                   
                 Arthralgia 
                 2 
                   
               
               
                 Respiratory 
                 Stuffy nose 
                 1 
                   
               
               
                 system 
                   
                   
                   
               
               
                 Paropsia 
                 Swollen Eyes 
                   
                 1 
               
               
                   
               
             
          
         
       
     
       Results 
       [0406]    For those patients who were injected with rSIFN-co, the side effects were minor. They had some common symptoms similar to flu, such as: headache, feebleness, frigolability, muscle soreness, hidrosis, arthralgia (arthrodynia; arthronalgia). The side effects of those patients whom were injected with Infergen® were worse than those injected with rSIFN-co. 
         [0407]    From  FIGS. 13A-1 ,  13 A- 2 ,  13 B- 1 , and  13 B- 2 , it was obvious that the body temperatures of sample patients in Group B were higher than the patients in Group A. It also reflected that the endurance of rSIFN-co was much better than Infergen®. 
       Example 11 
     Effects of Recombinant Super-Compound Interferon (rSIFN-co) on Ebola Virus 
       [0408]    Background: Ebola virus is a notoriously deadly virus that causes fearsome symptoms, the most prominent being high fever and massive internal bleeding. Ebola virus kills as many as 90% of the people it infects. It is one of the viruses capable of causing hemorrhagic (bloody) fever. There is no specific treatment for the disease. Currently, patients receive supportive therapy. This consists of balancing the patient&#39;s fluids and electrolytes, maintaining their oxygen level and blood pressure, and treating them for any complicating infections. Death can occur within 10 days of the onset of symptoms. 
       1. Materials 
       [0000]    
       
         
           
             1.1 Drugs: rSIFN-co, provided by Sichuan Biotechnology Research Center. 
             1.2 Virus: Ebola, supplied by The Academy of Military Medical Science, Institute of Microbiology Epidemiology. 
             1.3 Safety level of experiment: Viral experiments were carried under Biological Laboratory Safety System level 3. 
             1.4 Animals: 60 BALB/c mice 
           
         
       
     
       2 Method 
       [0000]    
       
         
           
             2.1 60 mice were randomly separated into 6 groups, each group consisting of 10 mice. Group 1 was treated with 1 μg/ of rSIFN-co on the day of inoculation with Ebola virus. Group 2 was treated with 1 μg/ of rSIFN-co day one (1) after inoculation with Ebola virus. Group 3 was treated with 1 μg/ of rSIFN-co on the day two (2) after inoculation with Ebola virus. Group 4 was treated with 1 μg/ of rSIFN-co on day three (3) after inoculation with Ebola virus. Group 5 was treated with 1 μg/ of rSIFN-co on day four (4) after inoculation with Ebola virus. Group 6 was not treated with rSIFN-co and this is designated as the control group. 
             2.2 Administration of the medication: 1 μg/ of rSIFN-co was administered once a day for six (6) consecutive days. 
           
         
       
     
       3 Results 
       [0000]    
       
         
           
             All ten (10) mice in group 6 (control group) died. All mice in groups one (1), two (2) and three (3) survived with no observable toxic effect. In groups four (4) and five (5), showed some effects. 
           
         
       
     
       4 Conclusion 
       [0000]    
       
         
           
             Clearly these result show effectiveness of rSIFN-co against Ebola virus. 
           
         
       
     
       Example 12 
     Anti-HIV Effects of Recombinant Super-Compound Interferon (rSIFN-co) 
     1. Materials 
       [0000]    
       
         
           
             1.1 Wild-Type HIV 
             1.2 Drug Resistant HIV 
             1.3 293-CD4-CCR5 cells 
             1.4 DMEM, Gibco 
             1.5 Fetal Bovine Seru, Gibco 
             1.6 rSIFN-co provided by Sichuan Biotechnology Research Center 
           
         
       
     
         [0423]    1.7 96-well plate, NUNC
       1.8 CO 2  incubator   1.9 Laminar Flow Hood   1.10 Fluorometer   1.1 IU V Absorbance Meter   1.12 Others       
 
       2. Method 
       [0000]    
       
         
           
             2.1 293-CD4-CCR5 cells in exponential (log) phase were obtained, digested with 0.25% pancreatin, stained with Trypan blue stain to determine cell number and diluted with DMEM to concentration of 2.0×10 5  cells per milliliter (cell/ml). 
             2.2 Each well of 96-well plate was filled with 100 μl (microliters) of 293-CD4-CCR5-DMEM suspension solution. The plate was placed into 5% carbon dioxide incubator at 37 degrees Celsius and observed the next day seventy percent (70%) of basal area of the well were recovered. 
             2.3 After supernatant was removed, 100 μl (microliters) of different concentrations of rSIFN-co were added to each well. Two controls were used: Phosphate Buffered Saline (PBS) and Growth Media. 
             2.4 The plate was placed into carbon dioxide incubator at 37 degrees Celsius for approximately 18 to 20 hours. 
             2.5 Experimental wells: Different concentrations of the Wild-Type HIV and Drug Resistant HIV viruses were placed into each well at 100 μl (microliters) per well. 
             Control wells: No virus was added, only 100 μl (microliters) of DMEM per well. 
             2.6 The plate was placed into carbon dioxide incubator at 37 degrees Celsius for approximately 24 hours. 
             2.7 Routine Luciferase assay was performed and protein concentrations of the supernatants were measured. Luciferase was measured in RLU/mg units. 
           
         
       
     
       3 Results 
       [0000]    
       
         
           
             rSIFN-co can inhibit HIV at level of ≧4 nanograms per milliliter (ng/ml). See Table 4 and  FIGS. 14-15 . When using Luciferase as Y axis and concentration of rSIFN-co as X axis, using EXCEL, it is clear that at level of rSIFN-co ≧4 nanograms per milliliter (ng/ml), the level of Luciferase activities are obviously lower than in PBS and Medium controls. A clear inverse dose-dependent response has been shown. 
           
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Comparison of Inhibition of Wild-Type HIV and Drug 
               
               
                 Resistant HIV by rSIFN-co 
               
             
          
           
               
                   
                 Concentration 
                 Luciferase Assay 
               
             
          
           
               
                   
                 of rSIFN-co 
                 Wild-Type HIV 
                 Drug Resistant HIV 
               
               
                   
                   
               
               
                   
                 Medium 
                 13500 + 2000 
                 18000 + 2000 
               
               
                   
                   1 μg/ml 
                  3000 + 200 
                  2800 + 800 
               
               
                   
                  500 ng/ml 
                  3000 + 600 
                  2800 + 900 
               
               
                   
                  250 ng/ml 
                  3400 + 400 
                  4000 + 600 
               
               
                   
                  125 ng/ml 
                  4300 + 200 
                  4100 + 600 
               
               
                   
                 62.5 ng/ml 
                  4300 + 400 
                  4100 + 1000 
               
               
                   
                   31 ng/ml 
                  5000 + 800 
                  5100 + 800 
               
               
                   
                   15 ng/ml 
                  7200 + 400 
                  6000 + 1500 
               
               
                   
                  7.5 ng/ml 
                  7000 + 800 
                  7700 + 1300 
               
               
                   
                   4 ng/ml 
                  9000 + 2000 
                  8900 + 2000 
               
               
                   
                 PBS 
                 13000 + 3000 
                 15100 + 2300 
               
               
                   
                 Medium 
                 16000 + 3600 
                 19000 + 2500 
               
               
                   
                   
               
               
                   
                 4 Conclusion: rSIFN-co is effective against both: Wild-Type HIV and Drug Resistant HIV. 
               
             
          
         
       
     
       Example 13 
     Anti-Influenza Effects of Recombinant Super-Compound Interferon (rSIFN-co) 
     1. Materials 
       [0000]    
       
         
           
             1.1. 10-day old chick embryonic membrane cells 
             1.2. SIFN-co provided by Sichuan Biotechnology Research Center 
             1.3. Influenza virus provided by Molecular Biology Department of Institute of microbiology and epidemiology, Academy of Military Medical Science. 
             1.4. DMEM, Gibco 
             1.5. Newborn Calf Serum 
             1.6. 96-well plate, NUNC 
             1.7. CO 2  incubator 
             1.8. Laminar Flow Hood 
             1.9. Inverted Microscope 
             1.10. Others 
           
         
       
     
       2. Method 
       [0000]    
       
         
           
             2.1 10-day old chick embryonic membrane cell in exponential (log) phase were obtained, digested with 0.25% pancreatin, stained with Trypan blue stain to determine cell number and diluted with DMEM to concentration of 2.0×10 5  cells per milliliter (cell/ml). 
             2.2 Each well of 96-well plate was filled with 100 μl (microliters) of 293-CD4-CCR5-DMEM suspension solution. The plate was placed into carbon dioxide incubator at 37 degrees Celsius. The next day cells grew to a monolayer. 
             2.3 After supernatant was removed, 100 μl (microliters) of different concentrations of rSIFN-co were added to each well. Two control wells: No rSIFN-co was added 
             2.4 The plate was placed into carbon dioxide incubator at 37 degrees Celsius for approximately 18 to 20 hours. 
             2.5 Experimental wells: Different concentrations of the Influenza virus were placed into each well at 100 microliters (μl) per well. 
             Control wells: No Influenza virus was added, only 100 μl (microliters) of DMEM per well. 
             2.6 The plate was placed into carbon dioxide incubator at 37 degrees Celsius for approximately 24 hours. 
             2.7 Cells were observed under inverted microscope. 
           
         
       
     
       3. Results 
       [0000]    
       
         
           
             3.1 Under inverted microscope, the cells in the control well with Influenza virus added and without interferon had obvious CPE, such as rounding of cells, cell necroses, decrease in reflective light and sloughing off. 
             3.2 Cells from the experimental wells containing rSIFN-co at concentration ≧10 nanogram per milliliter (ng/ml) had no CPE and morphology comparable to normal cells. See  FIG. 16 . 
             3.3 Control Wells without Influenza virus added and without interferon did not have any CPE. 
           
         
       
     
       4 Conclusion 
       [0000]    
       
         
           
             At concentration ≧10 nanogram per milliliter (ng/ml) rSIFN-co is effective against Influenza virus.