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Matched Legal Cases: ['Application No. 01104367', 'Application No. 2003248419', 'Application No. 2003248419', 'Application No. 2004279350', 'Application No. 2003248419', 'Application No. 2006257286', 'Application No. 200706014', 'Application No. 2', 'Application No. 2', 'Application No. 2', 'Application No. 2', 'Application No. 01104367', 'Application No. 01104367', 'Application No. 200680007733', 'Application No. 200480031909', 'Application No. 200480031909', 'Application No. 200480031910', 'Application No. 200680007733', 'Application No. 01104367', 'Application No. 200480031909', 'Application No. 200480031910', 'Application No. 200680007733', 'Application No. 01104367', 'Application No. 01104367', 'Application No. 01104367', 'Application No. 01104367', 'Application No. 02702211', 'Application No. 02702211', 'Application No. 02702211', 'Application No. 04809634', 'Application No. 04782529', 'Application No. 279', 'Application No. 310', 'Application No. 00310', 'Application No. 1214', 'Application No. 00311', 'Application No. 00311', 'Application No. 280', 'Application No. 2002', 'Application No. 2006', 'Application No. 2006', 'Application No. 2006', 'Application No. 2002', 'Application No. 200601204', 'Application No. 200601204', 'Application No. 200706014', 'Application No. 95107930', 'Application No. 095107930', 'Application No. 92', 'Application No. 92', 'Application No. 92']

Patent US8114395 - Treatment of viral diseases with recombinant interferon α - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsThis invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. The super-compound interferon possesses anti-viral or anti-tumor activity and therefore is useful to prevent and treat viral diseases and cancers. This invention also provides...http://www.google.com/patents/US8114395?utm_source=gb-gplus-sharePatent US8114395 - Treatment of viral diseases with recombinant interferon αAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS8114395 B2Publication typeGrantApplication numberUS 12/105,455Publication dateFeb 14, 2012Filing dateApr 18, 2008Priority dateFeb 28, 2001Fee statusPaidAlso published asCA2439503A1, CN1245215C, CN1311035A, DE60234085D1, EP1371373A1, EP1371373A4, EP1371373B1, US7364724, US8425896, US20040202641, US20080305080, US20110189128, WO2002080958A1Publication number105455, 12105455, US 8114395 B2, US 8114395B2, US-B2-8114395, US8114395 B2, US8114395B2InventorsGuangwen Wei, Rongbing Guo, Renhuai ZhangOriginal AssigneeSichuan Biotechnology Research CenterExport CitationBiBTeX, EndNote, RefManPatent Citations (52), Non-Patent Citations (165), Classifications (22), Legal Events (4) External Links: USPTO, USPTO Assignment, EspacenetTreatment of viral diseases with recombinant interferon α
US 8114395 B2Abstract
This invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. The super-compound interferon possesses anti-viral or anti-tumor activity and therefore is useful to prevent and treat viral diseases and cancers. This invention also provides an artificial gene which codes for the super-compound interferon or its equivalent. Finally, this invention provides methods to produce recombinant super-compound interferon or its equivalent and various uses of said interferon.
1. A method for treating a viral disease in a subject comprising administering to the subject an effective amount of a recombinant interferon which has the amino acid sequence of SEQ ID NO: 2 and is encoded by the nucleotide sequence of SEQ ID NO: 1.
2. The method of claim 1, wherein the interferon is administered orally, by vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal administration, mucosal administration, or by inhalation via an aspirator.
3. The method of claim 1, wherein the interferon is administered following the protocol of injecting 9 μg or 15 μg per day, 3 times a week, for a total of 24 weeks.
4. The method of claim 1, wherein the viral disease is hepatitis A, hepatitis B, hepatitis C, or other types of hepatitis.
5. The method of claim 1, wherein the viral disease is an infection caused by the Epstein-Barr virus, cytomegalovirus, herpes simplex virus, other types of herpes virus, papovavirus, poxvirus, picornavirus, adenovirus, rhinovirus, human T-cell leukemia virus I, human T-cell leukemia virus II, or human T-cell leukemia virus III.
7. The method of claim 1, wherein the recombinant interferon is formulated in a form selected from the group consisting of tablets, capsules, oral liquids, pastes, injections, sprays, suppositories, and solutions.
8. The method of claim 1, wherein the recombinant interferon inhibits the secretion of Hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg).
9. The method of claim 1, wherein the recombinant interferon is produced by a process comprising:
(1) introducing into an isolated host cell a polynucleotide comprising SEQ ID NO: 1 that encodes the recombinant interferon;
(2) culturing the host cell in an appropriate condition for the expression of the recombinant interferon; and
(3) harvesting the recombinant interferon, wherein the recombinant interferon has the amino acid sequence of SEQ ID NO: 2.
10. The method of claim 9, wherein the polynucleotide comprising SEQ ID NO: 1 is under the control of promoter pBAD in said process.
11. The method of claim 9, wherein the harvesting step comprises extracting the interferon from a fermentation broth and collection of inclusion bodies, followed by denaturation and renaturation of the interferon.
12. The method of claim 9, wherein the harvesting step comprises separation and purification of the recombinant interferon.
13. The method of claim 9, wherein the harvesting step comprises lyophilization of the recombinant interferon.
This application is a continuation of U.S. Ser. No. 10/650,365, filed 28 Aug. 2003 now U.S. Pat. No. 7,364,724 which claims priority of International Patent Application No. PCT/CN02/00128, filed on 28 Feb. 2002, which claims priority of Chinese Application No. 01104367.9, filed on 28 Feb. 2001, the contents of which are incorporated by reference here into this application.
Throughout this application, various references are referred to. 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.
rSIFN-co is a new interferon molecule constructed with the most popular conservative amino acid found in natural human α-IFN subtypes using genetic engineering methods. U.S. Pat. Nos. 4,695,623 and 4,897,471 have described it. rSIFN-co had been proved 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 rSIFN-co. Chinese Patent No. 97193506.8 by Amgen, Inc. addresses re-treatment of rSIFN-co on hepatitis C. Chinese Patent No. 98114663.5 by Shenzhen Jiusheng Bio-engineering Ltd. addresses treatment of rSIFN-co on hepatitis B and hepatitis C.
This invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. An equivalent is a molecule which is similar in function to the super-compound interferon. The super-compound interferon possesses anti-viral or anti-tumor activity. This invention also provides an artificial gene codes for the super-compound interferon or its equivalent.
This invention provides a process for production of recombinant super-compound interferon comprising introducing an artificial gene with selected codon preference into an appropriate host, culturing said introduced host in an appropriate condition permitting expression of said super-compound interferon and harvesting the expressed super-compound interferon.
This invention provides a composition comprising the recombinant super-compound interferon or its equivalent and a suitable carrier. This invention further provides a pharmaceutical composition comprising the recombinant super-compound interferon or its equivalent and a pharmaceutically acceptable carrier.
This invention provides a method for treating viral diseases or tumor in a subject comprising administering to the subject an effective amount of the super-compound interferon or its equivalent.
This invention provides the above-described method wherein super-compound interferon was administered via oral, vein injection, muscle injection, peritoneal injection, subcutaneous injection, nasal, mucosal administration, by inhalation via an inspirator.
FIG. 1. rSIFN-co cDNA sequence (SEQ ID NO: 1) designed according to E. coli codon usage and deduced rSIFN-co amino acid sequence (SEQ ID NO: 2)
FIG. 2. Sequence (SEQ ID NO: 4) of another super-compound interferon
FIG. 6-A. Circular Dichroism spectrum of Infergen®
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 in the label specifically for non-responding or refractory patients. InterMune'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. See www.intermune.com/wt/itmn/infergen, Aug. 27, 2003
FIG. 6-B. Human Alpha Species consensus IFN
Circular Diochroism spectra of consensus Interferon subforms. Consensus interferon was fractionated using an anion exchange column. Samples were dialyzed into 10 mM sodium phosphate, pH 7.4. Measurements were made on a Jasco J-170 spectopolarimeter, in a cell thermostat at 15° C. (-), acylated form; (- - -), cys terminal form; (---), met terminal form. i. Far UV spectrum. ii, Near UV spectrum.
Light Path: 1, 180-250 nm; ii, 250-320 nm--.
Circular Dichroism spectrum of Infergen® From Reference [Journal of Interferon and Cytokine Research. 16:489-499 (1996)]
This invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. This invention reveals that protein with same primary sequence might have different biological activities. As illustrated in the following example, this invention disclosed two proteins with identical amino acid sequence but with different activities. This activity may sometimes become improved efficacy and sometimes, the protein with changed spatial configuration would reveal new function.
In an embodiment, the super-compound interferon disclosed has higher efficacy than the interferon described in U.S. Pat. No. 4,695,623 or 4,897,471. This super-compound interferon is believed to have unique secondary or tertiary structure. (See e.g. FIG. 6)
The above-described super-compound interferon may be produced by a high efficiency expression system which uses a special promoter. In an embodiment, the promoter is PBAD. As it could be easily appreciated by other ordinary skilled artisan, other inducible promoter, such as heat shock promoter, may be used in this invention.
The super-compound interferon 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
The above described super-compound interferon possesses anti-viral or anti-tumor activity and therefore useful in preventing and treating viral diseases, tumors or cancers.
The virus diseases include but are not limited to hepatitis A, hepatitis B, hepatitis C, other types of hepatitis, infections caused by Epstein-Barr virus, Cytomegalovirus, herpes simplex viruses, other herpes viruses, papovaviruses, poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T cell leukaemia viruses I, human T cell leukaemia viruses II, or human T cell leukemia viruses III.
Therefore, this invention provides a method for inhibiting virus replication or virus infected cells by contacting said virus or infected cells with an effective amount of the super-compound interferon or its equivalent. This super-compound interferon is useful in preventing or treating the following cancers or tumors;
Tummy Cancer
Recta Cancer
Acute Leucocythemia
Chronic Leucocythemia
Accordingly, this invention provides a method for inhibiting tumor or cancer cell growth by contacting the super-compound interferon or its equivalent with said tumor or cancer cells. In a further embodiment, the super-compound interferon inhibits the DNA duplication and secretion of HBsAg and HBeAg of Hepatitis B Virus.
This invention also provides an artificial gene codes 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.
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.
This invention provides the above-described method wherein the viral diseases is hepatitis A, hepatitis B, hepatitis C, other types of hepatitis, infections of viruses caused by Epstein-Barr virus, Cytomegalovirus, herpes simplex viruses, or other type of herpes viruses, papovaviruses, poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T cell leukaemia viruses I, or human T cell leukaemia viruses II, or human T cell leukemia virus III.
This invention provides the above-described method wherein super-compound interferon was administered following the protocol of injection 9 μg or 15 μg per day, 3 times a week, total 24 weeks.
In one of the results of this invention, 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 PBAD promoter.
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. So 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 PBAD promoter which induce a high expression of rSIFN-co. Therefore, the arabinose induction/activation system is a more effective expression system. (2) The relation between Exogenous and L-arabinose dosage is linearity. 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.
The following are some rSIFN-co preparations: tablets, capsules, oral liquids, 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 acceptance medicine carrier, including carbohydrate, cellulosum, adhesive, collapse, emollient, filling, add-dissolve agent, amortization, preservative, add-thick agent, matching, etc.
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 and 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.
rSIFN-co is a new interferon molecule constructed according to conservative amino acid in human IFN-α subtype with genetic engineering method. It has been proven that rSIFN-co has broad-spectrum IFN activity, such as high antivirus and tumor inhibition activity, especially for effectively treating hepatitis C.
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 PBAD promoter in vectors, was used to induce high expression of rS1FN-co gene.
“Oligomer A:”
“Oligomer B:”
“Oligomer C:”
“Oligomer D:”
TGAAC3′
“Oligomer E:”
“Oligomer F:”
PCR I mixture
(units: μl)
sterilized distilled water
10xPfu buffer (Stratagen American Ltd.)
dNTP mixture (dNTP concentration 2.5 mmol/L)
Oligomer A primer (25 μmol/L)
Oligomer C primer (25 μmol/L)
Oligomer B template (1 μmol/L)
Pfu DNA polymerase (Stratagen American Ltd.) (25 U/μl)
2 m→(95° C.45 s→65° C.1 m→72° C.1 m)×25 cycle→72° C.10 m→4° C.
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
“Oligomer G:
5′ATCGGCCATATGTGCGACCTGCCGCAGACCC3′”
Oligomer H:
5′ACTGCCAGGCTGCAGTTATTCTTTACGACGCAGACGTTCC3′”
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
Purify the rSIFN-co, sequence the N-terminus amino acids, and N-terminus amino acid sequence (amino acids 2-16 of SEQ ID NO: 2) 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
High Expression of rSIFN-co Gene in E. Coli. In pHY-5 plasmid, rSIFN-co gene is under control of strong promoter PBAD. 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 O2 and I1 forming a 210 bp loop. This conformation leads to a complete inhibition of transcription. In the presence of arabinose, the dimer is released from O2 and binds I1 and I2 leading to transcription. Arabinose binding deactivates, represses and even activates the transcription of PBAD promoter, which stimulates PBAD inducing high expression of rSIFN-co rSIFN-co expression level in PVIII is more than 50% of the total E. Coli. protein.
RSIFN-CO is a new interferon molecule artificially built according to the conservative amino acid of human a interferons. It has been proven as a effective anti-hepatitis drug. In order to get enough pure rSIFN-co protein, a stable recombinant E. Coli. strain which high expresses rSIFN-co protein was constructed.
First, according to published rSIFN-co amino acid sequence, E. Coli. codon was used to synthesize whole cDNA of rSIFN-co. This DNA fragment was sequenced and proved that the 501 bp codon sequence and TAA termination codon sequence are valid and identical with 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.
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 3.0 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.
E. Coli. LMG194, which contains recombinant pHY-5 plasmid, is actually an ideal high-expression engineering strain.
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
4. Ozes O N, Reiter Z, Klein S, et al. A comparison of interferon-con1 with natural recombinant interferons-(:antiviral, antiproliferative, and natural killer-inducing activities. J. Interferon Res., 1992; 12:55-59.
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 PBAD promoter. J. Bacteriol. 1995, 177: 4121-4130.
rSIFN-co cDNA Sequence (SEQ ID NO: 1) Designed According to E. coli Codon Usage and Deduced rSIFN-co Amino Acid Sequence (SEQ ID NO: 2)
5′ 11 21 31 41 51
M C D L P Q T H S L G N R R A L I L L A
ATGTGCGACC TGCCGCAGAC CCACTCCCTG GGTAACCGTC GTGCTCTGAT CCTGCTGGCT
TACACGCTGG ACGGCGTCTG GGTGAGGGAC CCATTGGCAG CACGAGACTA GGACGACCGA
5′ 71 81 91 101 111
Q M R R I S P F S C L K D R H D F G F P
CAGATGCGTC GTATCTCCCC GTTCTCCTGC CTGAAAGACC GTCACGACTT CGGTTTCCCG
GTCTACGCAG CATAGAGGGG CAAGAGGACG GACTTTCTGG CAGTGCTGAA GCCAAAGGGC
5′ 131 141 151 161 171
Q E E F D G N Q F Q K A Q A I S V L H E
CAGGAAGAAT TCGACGGTAA CCAGTTCCAG AAAGCTCAGG CTATCTCCGT TCTGCACGAA
GTCCTTCTTA AGCTGCCATT GGTCAAGGTC TTTCGAGTCG GATAGAGGCA AGACGTGCTT
5′ 191 201 211 221 231
M I Q Q T F N L F S T K D S S A A W D E
ATGATCCAGC AGACCTTCAA CCTGTTCTCC ACCAAAGACT CCTCCGCTGC TTGGGACGAA
TACTAGGTCG TCTGGAAGTT GGACAAGAGG TGGTTTCTGA GGAGGCGACG AACCCTGCTT
5′ 251 261 271 281 291
S L L E K F Y T E L Y Q Q L N D L E A C
TCCCTGCTGG AAAAATTCTA CACCGAACTG TACCAGCAGC TGAACGACCT GGAAGCTTGC
AGGGACGACC TTTTTAAGAT GTGGCTTGAC ATGGTCGTCG ACTTGCTGGA CCTTCGAACG
5′ 311 321 331 341 351
V I Q E V G V E E T P L M N V D S I L A
GTTATCCAGG AAGTTGGTGT TGAAGAAACC CCGCTGATGA ACGTTGACTC CATCCTGGCT
CAATAGGTCC TTCAACCACA ACTTCTTTGG GGCGACTACT TGCAACTGAG GTAGGACCGA
5′ 371 381 391 401 411
V K K Y F Q R I T L Y L T E K K Y S P C
GTTAAAAAAT ACTTCCAGCG TATCACCCTG TACCTGACCG AAAAAAAATA CTCCCCGTGC
CAATTTTTTA TGAAGGTCGC ATACTGGGAC ATGGACTGGC TTTTTTTTAT GAGGGGCACG
5′ 431 441 451 461 471
A W E V V R A E I M R S F S L S T N L Q
GCTTGGGAAG TTGTTCGTGC TGAAATCATG CGTTCCTTCT CCCTGTCCAC CAACCTGCAG
CGAACCCTTC AACAAGCACG ACTTTAGTAC GCAAGGAAGA GGGACAGGTG GTTGGACGTC
5′ 491 501
E R L R R K E # (SEQ ID NO: 2)
GAACGTCTGC GTCGTAAAGA ATAA (SEQ ID NO: 1)
CTTGCAGACG CAGCATTTCT TATT (SEQ ID NO: 15)
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 denaturation 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.
Chelating sepharose fast flow: Add PB buffer of 0.2 mol/L (pH 6.6) and NaCl of 4 mol/L in the solution from HS to adjust solution pH to pH 6.0 and NaCl concentration to 1 mol/L.
Buffer B: 50 mmol/L Tris-HCl, pH 7.5-1 mol/L Urea-10 mmol/L EDTA-0.5. Triton X-100
Buffer D: 1 mol/L NaCl—50 mmol/L Na2HPO4 (pH 5.5)
Buffer E: 1 mol/L NaCl—50 mmol/L Na2HPO4 (pH 5.0)
Buffer F: 1 mol/L NaCl—50 mmol/L Na2HPO4 (pH 4.0)
Buffer G: 1 mol/L NaCl—50 mmol/L Na2HPO4 (pH 3.6)
LB Media:
RM Media:
1 mmol/L (0.203 g)
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 directly used in the preparation of injections or sprays, or stored at 2-8° C.
Solution Lyophilized powder Solution of rSIFN- 34.5 μg/ml 34.5 μg/ml co PB (pH 7.0) 25 mmol/L 10 mmol/L Glycine — 0.4 mol/L NaCl 0.1 mol/L — For spray: 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
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.
The quality of the product is controlled according to “Chinese Requirements for Biologics”
Test for Protein Content
Test for Protein Purity
Non-reductive SDS-PAGE
Test for Molecular Weights
Reductive SDS-PAGE
Test for Specific Activity
According to Method in
Test for Leftover Exogenetic
Using DNA Labeling and
Test for Activity of
Leftover Antibiotics
Test for Bacterial Endotoxin
Test for Isoelectronic Point
Test for Identify
UV spectrum (range of
wavelength: 190-380 nm)
According to Method in “c”
Abnormal Toxicity Test
Test on Mouse
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.
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 on April, 2000.
TABLE 1 Standard of Samples in Experiment Items Standards 1. Appearance white loose powder 2. Dissolving dissolve rapidly in injection water(within time 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 80%~150% of indicated quantity (9 μg: 4.5 × (IU/dose) 106 IU, 15 μg: 7.5 × 106 IU) 6. Moisture no more than 3.0% (W/W) 3. Experiment Content
15.3.1 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 1st, 3rd, 6th, 9th, 12th, 18th, 24th, 30th, 36th month. The results were recorded.
15.3.2 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 1st, 3rd, 6th, 9th, 12th, 18th, 24th, 30th month. The results were recorded.
15.3.3 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 1st, 3rd, 6th, 9th, 12th, 18th, 24th month. The results were recorded.
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.
Control drugs: IFN-α2b (Intron A) as lyophilized powder, purchased from Schering Plough. 3×106 U each, mix to 3×106 IU/ml with culture medium; Infergen® (liquid solution) purchased from Amgen, 9 μg, 0.3 ml each, equal to 9×106 IU, and mix with 9×106 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.
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.
Toxicity test: Set groups of different concentrations and a control group in which cell is not acted on with medicine. Digest cell, 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% CO2. Test when simple cell layer grows.
Dispense rSIFN-co to 1.8×107 IU/ml solution than 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 TC50 and TC0 according to the Reed Muench method.
TC 50 = Antilog ( B + 50 - B A - B × C ) A=log>50% medicine concentration, B=log<50% medicine concentration, C=log dilution power
A cpm of control group; B cpm of test group;
Antigen inhibition IC 50 = Antilog ( B + 50 - B A - B × C ) A log>50% medicine concentration, B=log<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: SI
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 μl TE 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, denaturation 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 IC50.
Results from Tables 1, 2 and 3 show: After maximum innocuous concentration exponent culturing for 8 days with 2.2.15 cell, the maxima is 9.0±0×106 IU/ml average inhibition rate of maximum innocuous concentration rSIFN-co to HBeAg is 46.0±5.25% (P<O 0.001), IC50 is 4.54±1.32×106 IU/ml, SI is 3.96; rate to HBsAg is 44.8±6.6%, IC50 is 6.49±0.42×106 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 proved in clinic that rSIFN-co can decrease HBeAg and HBsAg or return them to normal levels.
(×104 IU/ml)
0.436227
0.43935
0.345659
0.407079
0.945909
0.592921
0.614693546
0.3993754
0.245347
0.369269
0.337997
0.5388299
1.254924
0.300392321
0.386508
0.195836
0.200833
2.059088
0.08867188
0.004997
0.0049969
3.054091
0.001633453
4.054091
602.74446016
0.342155
0.381936
0.392693
0.372261
0.922258
0.627739
0.595006426
0.2439816
0.336008
0.191053
0.257014
0.5499972
1.370724
0.286349225
0.084856
0.118149
0.093165
0.292983
2.27756
0.113977019
0.082807
0.051221
0.097661
0.1998179
3.20033
0.058767408
0.201639
4.077742
0.02918541
641.7736749
0.554378
0.514592
0.467054
0.512008
1.371181
0.487992
0.737521972
0.4103967
0.394209
0.477884
0.8591731
1.060496
0.447563245
0.299134
0.244072
0.4316522
1.816423
0.19201839
0.124259
0.069653
0.1876045
2.74677
0.063933386
0.222982
0.121865
0.117951
3.628819
0.03148073
365.9357846
0.498265
0.462353
0.497571
0.486063
0.893477
0.513937
0.634835847
0.379771
0.259715
0.30598
0.4074138
1.207957
0.252210647
0.147294
0.027412
0.096345
2.111612
0.04583464
0.0050891
3.111612
0.001632835
0.015267
4.106523
0.001237728
611.0919568
0.428016
0.433204
0.521872
0.461031
1.316983
0.538969
0.709599543
0.4744723
0.40856
0.477066
0.453366
0.8559525
1.085603
0.440859127
0.038321
0.178517
0.156271
0.402586
1.929332
0.172641621
0.110954
0.216602
0.190701
0.2463153
2.738631
0.082519158
0.094093
0.072751
0.0055615
3.683017
0.014875633
382.0496935
0.496864
0.442035
0.521054
0.486651
0.763125
0.513349
0.597838293
0.234725
0.230425
0.364272
0.276474
0.2764738
1.236875
0.182690031
2.236875
4.236875
694.7027149
0.029711
0.176529
0.068747
0.931253
0.068746724
1.931253
2.931253
3.931253
4.931253
0.152489
0.247106
0.189295
0.185857736
0.050156
0.018495
0.0184947
1.810705
0.010110817
2.810705
3.810705
4.810705
0.091655
0.220869
0.104175
0.306157
0.895825
0.254710274
0.1417767
0.212409
0.118062
0.2019827
1.777764
0.102024519
0.090245
0.056531
2.721232
0.029916678
0.0273897
3.721232
0.007306592
0.082169
4.693843
0.005801377
0.025163
0.024647111
0.030313
0.010104
0.0209125
1.985646
0.010422073
2.985646
0.003606955
0.0108081
3.985646
0.002704416
4.974837
0.002167838
0.200051
0.187564
0.183486
0.190367
0.809633
0.253290505
0.0198777
0.068383
0.0842678
1.74125
0.046161005
0.047655
2.725365
0.005794856
3.725365
4.725365
0.043655
0.043187
0.041004
0.958996
0.12751773
0.0965076
0.035287
0.0991581
1.923709
0.0490186
0.029623
0.063871
2.913834
0.02144964
0.115529
0.029935
0.053996
0.0539965
3.859838
0.013796309
4.859838
0.064599
0.204393
0.136951
0.217399
0.863049
0.201211735
0.108269
0.0804479
1.82696
0.042176564
0.024289
0.044358
2.787683
0.015663017
0.0050818
3.782601
0.001341671
4.782601
0.984083
0.015916796
1.984083
2.984083
3.984083
4.984083
Stock Solution of
PB (pH 7.0)
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 it is sterile and pyrogen-free, 0.3 ml/vial or 0.5 ml/vial, and lyophilize in freeze dryer.
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. Storage at 2-10° C., and protect from light.
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 had no obvious difference between the two groups (P>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.
2.2 Medicines
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 remains were 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.
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>0.05). See Table 1:
TABLE 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 3. Conclusion
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 0.9×108 IU/mg, Intron A was 2.0×108 IU/mg and rSIFN-co was 9×108 IU/mg. The anti-viral activity of rSIFN-co is much higher than those of the former two.
Under the permission of the State Food and Drug Administration (SFDA), People'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 the 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:
The Effect of rSIFN-co Compared with IFN-α1b in the Treatment of Chronic Active Hepatitis B
1. Standard of patients selection: The standard 1-4 are effective to both treatment with rSIFN-co (9 μg) and IFN-α1b (5 MU, 50 μg), and the standard 1-5 are for rSIFN-co (15 μg) treatment.
2). HBsAg test positive last over six months, HBeAg test positive, PCR assay, HBV-DNA copies ≧105/ml
3). ALT≧two times of the normal value
4). Never received IFN treatment; or those 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
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 NBeAg unchanged The response and partial response groups consider as effective cases.
rSIFN-
co (9 μg)
(15) (3) (9) (12) (27) B
IFN-α1b
(5MU, 50 μg)
(7) (0) (3) (5) (18) 16-24
(35) (5) (16) (22) (58) B
(16) (0) (6) (12) (50) Group A: treatment with rSIFN-co (9 μg)
In Group C, the cases were chronic active hepatitis B treatment with other IFNs (3 MU or 5 MU) before but failed or relapsed and treated with rSIFN-co (15 μg), subcutaneous injection, every one day, last 24 weeks. The total cases are 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%) hepal functions recovered to normal.
The side effects of IFN include fever, nausea, myalgia, anorexia, hair lose, 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 are fever lower than 38° C., nausea, myalgia, anorexia, etc. When taken at maximum dose, the rate of side effects do not rise obviously, but are 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% patients have I-II degree (WHO standard) side effects, including fever below 38° C., nausea, myalgia, anorexia, leucopenia and thrombocytopenia slightly. With maximum dosage, about 50% patients suffered from leucopenia and thrombocytopenia after using rSIFN-co one month, but those side effects would disappear after stopping treatment for one week. It is safe for continue use.
1. Standard of Patient's Selection
3) ALT≧1.5 times of the normal value, last more than 6 months
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 (26.08%) HCV-RNA transferred to negative, 26 of 46 (56.52%) hepal functions recovered to normal.
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[Abstract only].Classifications U.S. Classification424/85.7, 530/351, 435/69.51, 514/4.3International ClassificationA61P35/02, C12N15/09, A61P31/20, C12N15/20, C07K14/555, A61P1/16, A61P31/18, A61K38/21, A61P31/14, A61P31/22, A61P31/12, A61P31/16, C12N15/21, C07K14/56, A61K38/00Cooperative ClassificationC07K14/555, A61K38/00European ClassificationC07K14/555Legal EventsDateCodeEventDescriptionSep 3, 2008ASAssignmentOwner name: SICHUAN BIOTECHNOLOGY RESEARCH CENTER, CHINAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WEI, GUANGWEN;GUO, RONGBING;ZHANG, RENHUAI;REEL/FRAME:021475/0115Effective date: 20030929Apr 22, 2014CCCertificate of correctionAug 5, 2015FPAYFee paymentYear of fee payment: 4Nov 20, 2015ASAssignmentOwner name: SUPERLAB FAR EAST LIMITED, VIRGIN ISLANDS, BRITISHFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SICHUAN BIOTECHNOLOGY RESEARCH CENTER;REEL/FRAME:037107/0576Effective date: 20151008RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services