Abstract:
The present invention relates to novel peptides and mixtures thereof which have been shown anti-tumor activity. Further, the invention relates to methods for identifying such compounds as well as to methods for their production. DNA encoding said peptides, vectors, host organisms, pharmaceutical preparations and antibodies that specifically bind with said peptide are also a part of the present invention.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to novel peptides and mixtures thereof which have been shown anti-tumor activity. Furthermore, the invention relates to methods for identifying such compounds as well as to methods for their production. DNA encoding said peptides, vectors, host organisms, pharmaceutical preparations and antibodies that specifically bind with said peptide are also a part of the present invention. 
       BACKGROUND OF THE INVENTION 
       [0002]    The major therapeutic approaches, with respect of the treatment of breast cancer, have evolved around endocrine therapies. Tamoxifen has been widely applied, but at a cost of several gynaecological and vasomotor symptoms 2 . Other therapeutic approaches, such as non-specific aromatase inhibitors, have also been associated with adverse side effects. As a result of this, emerging third-generation aromatase inhibitors such as letrozole, anastrozole and exemestane have increasingly been applied 2 . However, given their synthetic and foreign nature to the body&#39;s numerous mechanisms, their side-effects still proves to be “heavy-duty” for the patient&#39;s recovery and personal and psychosocial well-being. Thus there remains a need for improved therapeutic approaches of treating cancer, such as breast cancer, with less adverse side effects. 
         [0003]    During the recent years, acupuncture has been increasingly used for treating long- and short-termed pathologies in human patients 17 . Acupuncture has been combined with chemotherapy in cancer treatment trials 25-30 , and has also been used for treating nausia caused by chemotherapy 32-34 . Furthermore, in 2003 it was reported that acupuncture may have a positive effect when treating breast cancer 38 . However, the reason why acupuncture has a positive effect agains breast cancer cells has not previously been disclosed. 
         [0004]    Based on a theory that acupuncture treatment may stimulate the formation/secretion of certain factors that inhibit proliferation of said breast cancer cells, the inventors of the present invention initiated a study that resulted in the identification of 12 peptides. Each of said 12 peptides were demonstrated to affect the proliferation of breast cancer cells, and a mixture of said 12 peptides was shown to inhibit the proliferation of the cancer cells. Said 12 peptides, hereinafter referred to as SEQIDNO1-12, have not previously been disclosed. However, sequences that are similar to SEQIDNO1, SEQIDNO2, SEQIDNO10 and SEQIDNO11 have been disclosed in the prior art. 
         [0005]    EMBO J, 1987, 6(9):2767-2771 disclose a DNA sequence encoding a peptide that consists of 36 amino acids. Even though this peptide may be considered similar to SEQIDNO1 of the present invention, the prior art does not suggest a function of said peptide. 
         [0006]    WO2003046556 relates to the identification of 3 peptides that may be used as disease markers. One of these peptides consists of 17 amino acids. This peptide differs from SEQIDNO2 in that it has a His residue in position 17. Although the two sequences are quite similar, the prior art does not disclose a function of the peptide, except that it may be used as a disease marker. 
         [0007]    WO2005116607 relates to a method for the identification of Hb J-Toronto signature peptides. One of these signature peptides consists of 23 amino acids, wherein amino acid 1-11 in this peptide is identical to amino acid 3-13 in SEQIDNO10. In addition to the fact that SEQIDNO10 of the present invention is significantly shorter than the disclosed peptide, the prior art does not mention anything about the peptide&#39;s function. 
         [0008]    WO2005114221 disclose hundreds of peptides that have been isolated from prostate cancer tissue. One of these peptides consists of 19 amino acids. This peptide differs from SEQIDNO11 in that it has two additional amino acids. Although the two sequences may be considered similar, the prior art does not suggest or mention a function of the peptide. 
         [0009]    As mentioned above, there remains a need for improved therapeutic approaches of treating cancer, such as breast cancer. One such alternative approach is acupuncture treatment. In fact, acupuncture has been demonstrated to have a positive effect against breast cancer cells. However, the factors that are responsible for the positive effect of acupuncture treatment have not previously been disclosed. 
         [0010]    One object of the present invention is to provide a method for the identification of the factors that are formed and/or secreted as a result of acupuncture treatment. It is also an object of the present invention to isolate or synthesise said factors. Said factors may then be used in the treatment of various disorders. 
       SUMMARY OF THE INVENTION 
       [0011]    A first aspect of the present invention relates to a peptide comprising an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQIDNO5, SEQIDNO7, SEQIDNO8, SEQIDNO1 to SEQIDNO4, SEQIDNO6 and SEQIDNO9 to SEQIDNO12; or conservative modifications thereof. 
         [0012]    A second aspect of the present invention relates to a nucleic acid encoding said peptide and a third and fourth aspect of the present invention relates to a vector comprising said nucleic acid and a suitable host organism comprising said nucleic acid and/or said vector respectively. 
         [0013]    A fifth aspect of the present invention relates to a method of producing said peptide, comprising cultivating said host organism and isolating the peptide. 
         [0014]    A sixth aspect of the present invention relates to a composition comprising a peptide according to the first aspect of the present invention. 
         [0015]    Another aspect of the present invention relates to said peptide, said nucleic acid, said vector or said composition for medical use. 
         [0016]    Further, the present invention relates to the use of said peptide, said nucleic acid, said vector or said composition for manufacturing a medicament for the treatment of cancer. 
         [0017]    The present invention also relates to an antibody or antibody fragment that specifically binds with said peptide. 
         [0018]    Furthermore, the present invention relates to a pharmaceutical formulation comprising said peptide, said nucleic acid, said vector and/or said composition; and a pharmaceutical acceptable vehicle. 
         [0019]    The present invention also relates to a method of identifying potential drugs, comprising the following steps:
   a) sampling of blood from a patient that suffers from a disease   b) stimulating a specific acupuncture point for a predetermined period of time   c) sampling of blood from the patient that has been subjected to acupuncture treatment   d) isolating a fraction of the blood sample obtained in step c) that has a protein/peptide content that is significantly different from the protein/peptide content in the corresponding fraction of the blood sample obtained in step a).   e) sequencing of the protein(s)/peptide(s) that is/are present in the fraction obtained in to step d).   
 
         [0025]    Another aspect of the present invention relates to a method, preferably an in vitro method, of identifying potential drugs, comprising the following steps:
   a) isolating the fraction of a blood sample A that has a protein/peptide content that is significantly different from the protein/peptide content in the corresponding fraction of blood sample B, wherein blood sample A has been sampled from a patient, prior to acupuncture treatment, that suffers from a disease and blood sample B has been sampled from the same patient subsequent to acupuncture treatment, wherein said acupuncture treatment involves stimulation of a specific acupuncture point for a predetermined period of time;   b) sequencing of the protein(s)/peptide(s) that is/are present in the fraction obtained in step a).   
 
         [0028]    Preferred embodiments of the present invention are set forth in the dependent claims. 
     
    
     
       DESCRIPTION OF THE FIGURES 
         [0029]    
         FIG. 1 
       
           [0030]    Shows the controlling sequences of the body processes according to Chinese acupuncture. In this system heart controls lung, lung controls liver, liver controls spleen/stomach, spleen/stomach controls kidney and kidney controls heart. If a disorder is related to the stomach, e.g. breast cancer 38,39 , then the liver does not control the stomach properly 38 . Consequently the liver has to be treated by stimulating an acupuncture point belonging to the liver meridian, e.g. LV03-T/LV03 (see  FIG. 2 ). 
           [0031]    
         FIG. 2 
       
           [0032]    Shows the location of the LV03-T/LV03 (distal to point Liver 3 between Os metatarsale 1 and 2) acupuncture point on the human foot. 
           [0033]    
         FIG. 3 
       
           [0034]    First dimension analysis in the 2D-HPLC run (HPCF). The S 0  line shows the protein content in the sample before acupuncture treatment, and the S 1  line shows the protein content in the sample after acupuncture treatment. 
           [0035]    
         FIG. 4 
       
           [0036]    RP-HPLC plots for the second dimension, illustrating the signals (UV-absorbance) as intensity bands. The bands to the right illustrates the signals that were obtained from the S 1  sample (after acupuncture treatment), and the bands to the left illustrates the signals that were obtained from the S 0  sample (before acupuncture treatment). The central portion shows the signals as peaks of the RP-HPLC analysis. 
           [0037]    
         FIG. 5 
       
           [0038]    SEQ ID NO:1 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0039]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0040]    
         FIG. 6 
       
           [0041]    SEQ ID NO:2 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0042]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0043]    
         FIG. 7 
       
           [0044]    SEQ ID NO:3 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0045]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0046]    
         FIG. 8 
       
           [0047]    SEQ ID NO:4 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0048]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0049]    
         FIG. 9 
       
           [0050]    SEQ ID NO:5 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0051]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0052]    
         FIG. 10 
       
           [0053]    SEQ ID NO:6 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0054]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0055]    
         FIG. 11 
       
           [0056]    SEQ ID NO:7 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0057]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0058]    
         FIG. 12 
       
           [0059]    SEQ ID NO:8 on a T47D breast cancer cell line. Decreased fluorescent readings is relatively to the DC5 sample indicates reduced cell growth. 
           [0060]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0061]    
         FIG. 13 
       
           [0062]    SEQ ID NO:9 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0063]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0064]    
         FIG. 14 
       
           [0065]    SEQ ID NO:10 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0066]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0067]    
         FIG. 15 
       
           [0068]    SEQ ID NO:11 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0069]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0070]    
         FIG. 16 
       
           [0071]    SEQ ID NO:12 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. 
           [0072]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0073]    
         FIG. 17 
       
           [0074]    Mixture of SEQ ID NO:1 through SEQ ID NO:12 on Tamoxifen resistant TMX2-28 cells. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. The concentration of each of the 12 peptides in a specific sample is 1/12 of the concentration that is defined in the figure. 
           [0075]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
           [0076]    
         FIG. 18 
       
           [0077]    Mixture of SEQ ID NO:1 through SEQ ID NO:12 on a MCF-7 breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. The concentration of each of the 12 peptides in a specific sample is 1/12 of the concentration that is defined in the figure. 
           [0078]    DC5=Medium, DMSO=Dimethylsulfoxide. 
           [0079]    
         FIG. 19 
       
           [0080]    Mixture of SEQ ID NO:1 through SEQ ID NO:12 on a T47D breast cancer cell line. Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. The concentration of each of the 12 peptides in a specific sample is 1/12 of the concentration that is defined in the figure. 
           [0081]    DC5=Medium, Bap=Benzo[α]pyrene, DMSO=Dimethylsulfoxide. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0082]    According to Thoresen, 2003 38 , all the organs and organ processes may be involved in a sophisticated feed-back control system for cell-growth. This feed-back control system is described in Chinese acupuncture as the control-cycle. In this system, heart controls lung, lung controls liver, liver controls spleen/stomach, spleen/stomach controls kidney and kidney controls heart ( FIG. 1 ). Accordingly, if a disease is related to the stomach, then the liver does not control the stomach properly 38 . In order to treat said disease, an acupuncture point belonging to the liver median should be stimulated. Based on this theory, it might be possible that said acupuncture treatment stimulates the secretion of certain factors that may have a positive effect against said disease. 
         [0083]    The present invention relates to a method of identifying potential drugs. Said method involves sampling of blood (S 0 ) from a patient, e.g. from a patient suffering from breast cancer. Subsequently, a needle is applied to stimulate a specific acupuncture point for a predetermined period of time. In case the patient suffers from breast cancer, an acupuncture point belonging to the liver median (e.g. LV03-T/LV03) should be stimulated. Said predetermined period of time is preferably 1-60 minutes, even more preferably 1-30 minutes and most preferably 1-20 minutes. Subsequent to the acupuncture treatment, a blood sample is collected (S 1 ). 
         [0084]    The blood samples S 0  and S 1  are then fractionated in order to obtain a fraction of compounds that are filtered through a cut off filter, such as a 50 kDa, 40 kDa, 30 kDa, 20 kDa, 10 kDa or a 5 kDa cut off filter. Preferably, said cut off filter is a 10 kDa cut off filter. Said fractionation may e.g. be performed as explained in example 1. 
         [0085]    Initial protein content estimates with the absorbance method (A 280 -A 320 ) indicated a protein content of 0.48 mg/ml in the 10 kDa S 0  fraction (before stimulus) and a protein content of 1.02 mg/ml in the 10 kDa S 1  fraction (after stimulus). The increase in protein content indicates that a significant effect was mediated by the acupuncture treatment. 
         [0086]    To further study the effect of acupuncture treatment, the two fractions (10 kDa S 0  and 10 kDa S 1 ) were independently subjected to a method that is suitable to identify the part of the fraction that is responsible for the observed change in protein content. The use of 2D-HPLC, as explained in example 1, is one example of such a suitable method. Said change in protein content may be an increase or a decrease, such as an increase. 
         [0087]    The parts of the 10 kDa S 1  fraction that was demonstrated a higher or lower protein content compared with the corresponding part of the 10 kDa S 0  fraction were independently subjected to a method that is suitable for peptide identification. The use of electrospray mass spectral analysis, as explained in example 1, is one example of such a suitable method. 
         [0088]    Preferably, the parts of the 10 kDa S 1  fraction that was demonstrated a 10% increase/decrease in protein content relatively to the corresponding part of the 10 kDa S 0  fraction were independently subjected to a method that is suitable for peptide identification. Even more preferably, the parts of the 10 kDa S 1  fraction that was demonstrated a 20% increase/decrease in protein content relatively to the corresponding part of the 10 kDa S 0  fraction were independently subjected to a method that is suitable for peptide identification. 
         [0089]    A 10% or 20% increase/decrease in protein content should be understood as a 10% or 20% increase/decrease in the signal intensity obtained by the method described in example 1 (sample analysis). 
         [0090]    The sequence of each identified peptide was then estimated, e.g. by using BioWorks SeQuest Analysis Software package (Shevchenko and Chernushevich, 1997), as explained in example 1. Based on the estimated peptide sequences, twelve different peptides were synthesized. 
         [0091]    A first aspect of the present invention relates to a peptide comprising an amino acid sequence having at least 60% sequence identity with an amino acid sequence selected from the group consisting of SEQIDNO1 to SEQIDNO12, preferably SEQIDNO5, SEQIDNO7 or SEQIDNO8; or conservative modifications thereof. Preferably said sequence identity is at least 70%, more preferably at least 80% and even more preferably at least 90%, such as 100%. Preferably, all of said derivatives and variants of SEQIDNO1 have a biological activity that is similar to the peptide represented by SEQIDNO1. The same applies to SEQIDNO2-12. 
         [0092]    One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservative modification” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. Typically conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Histidine (H), Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). 
         [0093]    In one embodiment according to the first aspect of the present invention, said peptide consists of an amino acid sequence having at least 60% sequence identity with an amino acid sequence selected from the group consisting of SEQIDNO1 to SEQIDNO12, preferably SEQIDNO5, SEQIDNO7 or SEQIDNO8; or conservative modifications thereof. Preferably said sequence identity is at least 70%, more preferably at least 80% and even more preferably at least 90%, such as 100%. Preferably, all of said derivatives and variants of SEQIDNO1 have a biological activity that is similar to the peptide represented by SEQIDNO1. The same applies to SEQIDNO2-12. 
         [0094]    In one embodiment according to the second aspect of the present invention, said amino acid sequence is selected from the group consisting of: 
       SEQIDNO2 to SEQIDNO12; 
     SEQIDNO1 and SEQIDNO3-12; 
     SEQIDNO1 to SEQIDNO9 and SEQIDNO11-12; 
     SEQIDNO1 to SEQIDNO10 and SEQIDNO12; 
       [0095]    or conservative modifications thereof. 
         [0096]    In another embodiment according to the first aspect of the present invention, said peptides do not comprise an amino acid sequence selected from the group consisting of: 
         [0000]    
       
         
               
               
             
           
               
                   
                 MTPFASPVAPLDPLLKYGRGQGPVSSASGTTTDLG; 
               
               
                   
                   
               
               
                   
                 KVGAHAGEYG AEALERH; 
               
               
                   
                   
               
               
                   
                 VLSPADKTNVKAAWGKVGAHAGE; 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 RTLAGENQTAFEIEELNRK. 
               
             
          
         
       
     
         [0097]    While some of the peptides according to the first aspect of the present invention have been shown to inhibit proliferation of TMX-2-28, MCF-7 and T47D cells, others induced growth and yet others apparently had no significant effect ( FIG. 5-16  and table 1-12). However, a linear dose-response curve was observed after 24 hours of incubation with a mixture of said peptides, which indicates a strong growth inhibitory effect ( FIG. 16-19 ). Accordingly, the peptides according to the first aspect of the present invention may be useful individually or in mixtures. 
         [0098]    A sixth aspect of the present invention relates to a composition comprising a peptide according to the first aspect of the present invention. Preferably, said composition comprises at least two of said peptides, and even more preferably one of the at least two peptides is the peptide represented by SEQIDNO5, SEQIDNO7 or SEQIDNO8. Most preferably said composition comprises all of the three last-mentioned peptides. One example of such a composition is a composition comprising each and all of the twelve peptides according to the first aspect of the present invention. 
         [0099]    In one embodiment according to the sixth aspect of the present invention, said composition comprises the peptides represented by SEQIDNO2 to SEQIDNO7, SEQIDNO10 and SEQIDNO11. In another embodiment, said composition comprises the peptides represented by SEQIDNO1 to SEQIDNO7, SEQIDNO10 and SEQIDNO11. In yet another embodiment, said composition comprises the peptides represented by SEQIDNO2 to SEQIDNO8, SEQIDNO10 and SEQIDNO11. 
         [0100]    A second aspect of the present invention relates to a nucleic acid molecule encoding the peptide according to the first aspect of the present invention; or conservative modifications thereof. Said nucleic acid may be DNA or RNA. The nucleic acid sequence can be deduced by the skilled artisan on the basis of the disclosed amino acid sequences. 
         [0101]    With respect to the nucleic acid sequences, conservative modifications refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical is or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, often silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences. 
         [0102]    A third aspect of the present invention relates to a vector comprising the nucleic acid according to the second aspect of the present invention. The vector can be of any type suitable e.g. for expression of said peptides or propagation of genes encoding said peptides in a particular organism. The specific choice of vector depends on the host organism and is known to a person skilled in the art. 
         [0103]    A fourth aspect of the present invention relates to a suitable host organism comprising the nucleic acid according to the second aspect of the present invention, and/or the vector according to the third aspect of the present invention. The host organism may be of eukaryotic or prokaryotic origin. 
         [0104]    A fifth aspect of the present invention relates to a method of producing the peptide according to the first aspect of the present invention comprising cultivating the host organism according to the fourth aspect of the present invention and isolating the peptide. 
         [0105]    An seventh aspect of the present invention relates to the peptide according to the first aspect of the present invention, the composition according to the sixth aspect of the present invention, the nucleic acid according to the second aspect of the present invention or the vector according to the third aspect of the present invention for medical use. 
         [0106]    A eighth aspect of the present invention relates to the use of the peptide according to the first aspect of the present invention, the composition according to the sixth aspect of the present invention, the nucleic acid according to the second aspect of the present invention or the vector according to the third aspect of the present invention for manufacturing a medicament for the treatment of cancer. Preferably said cancer is breast cancer or intestinal cancer (e.g. colon cancer). 
         [0107]    A ninth aspect of the present invention relates to an antibody or an antibody fragment that specifically binds with the peptide according to the first aspect of the present invention. 
         [0108]    A tenth aspect of the present invention relates to a pharmaceutical formulation comprising the peptide according to the first aspect of the present invention, the composition according to the sixth aspect of the present invention, the nucleic acid according to the second aspect of the present invention or the vector according to the third aspect of the present invention. 
         [0109]    Having now fully described the present invention in some detail by way of illustration and example for purpose of clarity of understanding, it will be obvious to one of ordinary skill in the art that same can be performed by modifying or changing the invention by with a wide and equivalent range of conditions, formulations and other parameters thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. 
       EXAMPLES 
       [0110]    The following examples are meant to illustrate how to make and use the invention. They are not intended to limit the scope of the invention in any manner or to any degree. 
       Example 1 
     Identification of 12 Peptides, Hereinafter Refereed to as SEQIDNO1-12 
     Acupuncture and Sample Collection 
       [0111]    A sample (S 0 ) of 7 ml blood was collected from a female patient aged 43 with breast cancer with spreading to the bones. The sample was collected in a solution of Guanidinium Chloride to yield a final concentration of 6M Guanidinium Chloride. The acupuncture needle, which was sterilized and ideally of gold or stainless steel, was inserted in the designated point LV03-T/LV03 ( FIG. 2 ). The second sample, S 1 , was taken 1 minute after insertion of the needle and mixed with Guanidinium Chloride. 
       Sample Fractionation 
       [0112]    Samples were then fractionated in 5 steps at 3000 rpm in a Eppendorff Centrifuge, first with a spin-column filter of 0.45 μm to remove larger intracellular components, second with a 0.20 μm spin-column filter to remove cellular components, third at a 300 kDa cut-off to remove large proteins and eventual cellular bodies, then 100 kDa to remove larger and small proteins and at last 10 kDa cut off to obtain an oligopeptide fraction. 
       Sample Analysis 
       [0113]    100 μL of the 10 kDa fractions of the S 0  and S 1  samples were analysed with respect to the total amount of protein, using the absorbance method (A 280 -A 320 ). The result of this analysis indicates that the protein content in the 10 kDa fraction of the S 0 -sample (before stimulus) was around 0.48 mg/ml and that the protein content in the 10 kDa fraction of S 1 -sample (after stimulus) was around 1.02 mg/ml. The increase in protein content indicates that a significant effect was mediated by the acupuncture treatment. 
       2D-HPLC Analysis 
       [0114]    The 10 kDa fractions of the S 0  and S 1  samples were then subjected to 2D liquid chromatography with a Beckman&#39;s Proteome Lab™ PF 2D system. The samples were cleared at 100,000 g centrifugation and exchanged with start buffer (6M urea, 25 mM bis-tris, 0.2% n-octyl-β- D -glucopyranoside, pH 8.5 with ammonium hydroxide) on a PD10 column. The samples were then subjected to separation on a HPCF (High Performance ChromatoFocusing) column pre-equilibrated with the “start buffer”. Samples were monitored for UV absorbance (280 nm) and pH in a flow cell during the entire run. 1 mg of sample was injected with a flow rate of 0.2 ml/minute. After the initial 35 min start buffer load, an isocratic gradient was generated by switching to the eluent buffer (6M urea, 10% v/v Polybuffer™ 74 (GE Healthcare), 0.2% n-octyl-β- D -glucopyranoside, pH4 with iminodiacetic acid) for 95 min. Fraction collection occurred every 5 min or if a pH change of 0.3 was recorded. Upon completion of the pH gradient, a 20 minute 1M NaCl was run during which fractions were collected for 5 min intervals. 
         [0115]    The run in the first dimension (IEC) revealed significant differences between the S 0  and S 1  samples, which were largely observed in the run-period ranging between 0-35 min (see  FIG. 3 ). The fractions identified in the 0-35 min time-interval from the first dimension was therefore subjected to to separation by HPRP (High Performance Reverse Phase). 
         [0116]    A HPRP column was equilibrated with 0.1% TFA, and 200 ul of the selected HPCF fractions were injected at a flow rate of 0.750 ml/mn. Fractions were monitored for UV absorbance at 214 nm. Immediately after injection, an 0-100% acetonitrile/0.8% TFA gradient was run for 30 min. HPRP fractions were then collected on a Gilson fraction collector set at 0.25-0.5 minute per fraction. UV absorbance data from samples were imported into the ProteoVue™ software package for a visual display of the UV absorbance. Comparison between two samples will be done by importing separate ProteoVue™ analysis representing two different sample into the DeltaVue™ software package. This allows direct comparison of the UV absorbance profile of two samples analyzed under identical run parameters. The run in the second dimension revealed significant differences between the S 0  and S 1  samples, as shown in  FIG. 4 , lane 2, 3 and 4. 
         [0117]    Proteins were subjected to in-solution tryptic digestion (Schevchenko and Schevchenko, 2003), on the Genomic Solution™ (Ann Arbor, Mich.) ProPrep™ robot in the Department of Biochemistry, Molecular Biology and Biophysics&#39; Proteomic Analysis Core facility, University of Minnesota. Samples were reduced in the presence of 10 mM DTT/25 mM NH 4 HCO 3  at 60° for 30 minutes followed with the addition of iodacetamide (55 mM final concentration)/25 mM NH 4 HCO 3  and incubated for 30 minutes at 25°. Tryptic digestion in the presence of 12 ng/μl trypsin (Promega, Madison, Wis.) in 25 mM NH 4 HCO 3 , 5 mM CaCl 2  at 37° was preformed for 10 hours. Formic acid was added to a final concentration of 0.1% w/v to stop the digestion. The sample was then frozen and concentrated in a speed vacuum. 
       Electrospray Mass Spectral Analysis 
       [0118]    Samples were rehydrated in loading buffer (30 μl of 98:2 water:ACN, 0.1% trifluoroacetic acid) and loaded onto a Michrom C-18 nanotrap by sample aspiration of 27.5 μl into a 100 μl sample loop using load buffer as the transfer reagent on a Michrom BioResources Paradigm AS1. The column was switched in-line with a capillary column allowing peptides elution at 350 nl/min with the Michrom BioResourceMS4. The capillary column (75 μm internal diameter) was packed in-house to 12 cm length with 5 m, 200 Å pore size C 18  particles (Michrom BioResources, Auburn, Calif.) as described in (Mosely et al., 1997). Peptides were eluted with a linear gradient with 100% solvent A (95:5 water:ACN, 0.1% formic acid), to a final solvent B (5:95 water:ACN, 0.1% formic acid). The LC system was online with ThermoFinnigan. (ABI, Inc., Foster City, Calif.) LTQ ion trap mass spectrometer (MS). An electrospray spray voltage of 2250 V was applied distal to the analytical column. The instrument&#39;s calibration is monitored using the [M+2H] 2+  average peak at 811 m/z (Sigma-Aldrich, Inc., St. Louis, Mo.). As peptides eluted from the column they were focused into the mass spectrometer where product ion spectra were collected in a data dependent acquisition (DDA) mode. 
       Sequence Analysis 
       [0119]    Mass spectra were analyzed using BioWorks SeQuest Analysis Software package. (Shevchenko and Chernushevich, 1997) Product ion mass spectra were searched using BioWorks (ABI, Inc., Foster City, Calif.) against a human database (Apr. 22, 2004) for protein identification. The following search parameters were used: 2 trypsin missed cleave site; peptide and product ion tolerance=1.0 Dalton; variable amino acids incorporated into the search were carbamidomethyl cysteine, singly oxidized methionine; and deamidation of glutamine and asparagine. 12 peptides were identified, hereinafter referred to as SEQIDNO1 to SEQIDNO 12. 
       Peptide Synthesis 
       [0120]    Peptides SEQ ID NO:1 to SEQ ID NO:12 were synthesized using conventional peptide synthesis equipment. 
       Example 2 
     Anti Tumor Activity 
       [0121]    Stock solutions for each of the twelve synthesised peptides were prepared in basic culture medium (DMEM) at a concentration of 0.01 M. Several of the compounds did not go 100% into solution at this concentration and continued effort was directed towards appropriately diluting the peptides using 0.1% dimethylsulfoxide (DMSO) for the highest peptide concentrations. 
         [0122]    Re-feed media were prepared to yield the following final six concentrations of each of the 12 peptides: 5×10 −3 , 5×10 −4 , 5×10 −5 , 5×10 −6 , 5×10 −7 , and 5×10 −8 M. The mixtures were then prepared containing equal volumes of peptides SEQ ID NO:1 through SEQ ID NO:12 (equal volumes of said re-feed media) to yield the following final six concentrations: 5×10 −3 , 5×10 −4 , 5×10 −5 , 5×10 −6 , 5×10 −7 , and 5×10 −8 M. With that, the 5×10 −3 M mixture contains about 0.42×10 −3 M of each peptide. Furthermore, mixtures containing equal volumes of peptides SEQ ID NO:2-SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:11 were also prepared (equal volumes of said re-feed media) to yield the following final six concentrations: 5×10 −3 , 5×10 −4 , 5×10 −5 , 5×10 −6 , 5×10 −7 , and 5×10 −8  M. With that, the 5×10 −3 M mixture contains about 0.42×10 −3 M of each of said 8 peptides. 
         [0123]    MCF-7 and T47D cells were seeded into 96-well plates at a density of 10,000 cells per well in 90 μL of appropriate culture medium. TMX2-28 cells were seeded into 96-well plates at a density of 5,000 cells per well in 90 μL of appropriate culture medium. Plates were placed in a 37° C., 5% CO 2  incubator and cells were allowed to attach to the bottom of the well for 24 hours after which 10 μL of the appropriate refeed medium was added. Each concentration was tested in duplicate in each cell line. In addition, DC5, DMSO and either tamoxifen or BaP were present on each plate. Cells were then incubated for 24 hours. Subsequently 10 μL of AlmarBlue (Biosource) was added to each well. After three hours, the fluorescence was detected using a Packard Instrument Plate Reader with 535/20 excitation and 590/20 emission filters. 
         [0124]    As shown in  FIG. 5-16  and table 1-12, some of the peptides of the present invention inhibited proliferation of TMX-2-28, MCF-7 and T47D cells, others induced growth and yet others apparently had no significant effect. Further, as shown in  FIG. 17-19  and table 13, a mixture of the 12 peptides was shown to inhibit the proliferation of the cells in a dose-dependent manner. Furthermore, a mixture of SEQIDNO2-7+SEQIDNO10-11 was also found to inhibit cell growth (table 14). These results are particularly interesting in light of the fact that the incubation with the peptide mixture was for 24 hours only, a time-period that was insufficient to decrease the proliferation by either Tamoxifen or Benzo[α]pyrene. 
       Tables 
       [0125]      
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 SEQIDNO1 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 74129 
                 73649 
                 72975 
                 74503 
                 75445 
                 71409 
                 52745 
                 70158 
                 72202 
               
               
                 72743 
                 71774 
                 71640 
                 75697 
                 77164 
                 71588 
                 59998 
                 73977 
                 69445 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 69131 
                 65474 
                 65860 
                 62613 
                 63693 
                 62279 
                 62147 
                 62484 
                 60560 
               
               
                 67176 
                 66009 
                 64140 
                 62686 
                 62463 
                 60719 
                 61921 
                 65064 
                 54197 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 5) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 65814 
                 64794 
                 63241 
                 63036 
                 62633 
                 55767 
                 68709 
                 67910 
                 66572 
               
               
                 66151 
                 65757 
                 63091 
                 62709 
                 62689 
                 59593 
                 69129 
                 63991 
                 64694 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 SEQIDNO2 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 70121 
                 70162 
                 69083 
                 72557 
                 72061 
                 68910 
                 52745 
                 70158 
                 72202 
               
               
                 70743 
                 68517 
                 71861 
                 73134 
                 73603 
                 72372 
                 59998 
                 73977 
                 69445 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 68630 
                 66492 
                 65617 
                 63699 
                 63539 
                 63298 
                 62147 
                 62484 
                 60560 
               
               
                 65876 
                 66554 
                 65531 
                 63627 
                 63477 
                 62132 
                 61921 
                 65064 
                 54197 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 6) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 66780 
                 67257 
                 63525 
                 64064 
                 62463 
                 57744 
                 68709 
                 67910 
                 66572 
               
               
                 68474 
                 66607 
                 67453 
                 66021 
                 63876 
                 60399 
                 69129 
                 63991 
                 64694 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 SEQIDNO3 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 68963 
                 69237 
                 71007 
                 72558 
                 72686 
                 69066 
                 52745 
                 70158 
                 72202 
               
               
                 67344 
                 66454 
                 67496 
                 69648 
                 69197 
                 70821 
                 59998 
                 73977 
                 69445 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 63831 
                 64053 
                 64217 
                 62452 
                 61563 
                 62603 
                 62147 
                 62484 
                 60560 
               
               
                 65178 
                 64928 
                 64899 
                 66233 
                 63132 
                 61621 
                 61921 
                 65064 
                 54197 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 7) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 61359 
                 66736 
                 65079 
                 64329 
                 61766 
                 59482 
                 68709 
                 67910 
                 66572 
               
               
                 61718 
                 61724 
                 58554 
                 59141 
                 61031 
                 59451 
                 69129 
                 63991 
                 64694 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 SEQIDNO4 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 68299 
                 67939 
                 69917 
                 70744 
                 71761 
                 70255 
                 52745 
                 70158 
                 72202 
               
               
                 67310 
                 68552 
                 70618 
                 72469 
                 72585 
                 70823 
                 59998 
                 73977 
                 69445 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 64475 
                 63637 
                 65699 
                 64278 
                 64878 
                 61356 
                 62147 
                 62484 
                 60560 
               
               
                 66716 
                 66597 
                 66767 
                 64885 
                 64702 
                 61062 
                 61921 
                 65064 
                 54197 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 8) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 65000 
                 66449 
                 66808 
                 65859 
                 66040 
                 57800 
                 68709 
                 67910 
                 66572 
               
               
                 63427 
                 66303 
                 65677 
                 65314 
                 64619 
                 59394 
                 69129 
                 63991 
                 64694 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 SEQIDNO5 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 27056 
                 62564 
                 67405 
                 69438 
                 70141 
                 69376 
                 60075 
                 71189 
                 72712 
               
               
                 29836 
                 67140 
                 69619 
                 69605 
                 70591 
                 67302 
                 61350 
                 71192 
                 67410 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 56679 
                 61089 
                 61618 
                 61677 
                 62738 
                 57537 
                 62999 
                 71558 
                 65433 
               
               
                 54497 
                 62458 
                 65571 
                 66372 
                 64281 
                 60120 
                 67975 
                 68854 
                 65607 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 9) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 54287 
                 61500 
                 63089 
                 64043 
                 62339 
                 61350 
                 67072 
                 68276 
                 64397 
               
               
                 54068 
                 65566 
                 64735 
                 64408 
                 63402 
                 61820 
                 68671 
                 68910 
                 60369 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 SEQIDNO6 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 68949 
                 70355 
                 70389 
                 71096 
                 70707 
                 62308 
                 60075 
                 71189 
                 72712 
               
               
                 68566 
                 69358 
                 69887 
                 70512 
                 71208 
                 64367 
                 61350 
                 71192 
                 67410 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 69282 
                 69071 
                 67961 
                 66967 
                 65221 
                 60855 
                 62999 
                 71558 
                 65433 
               
               
                 66723 
                 69114 
                 70454 
                 68329 
                 65987 
                 61454 
                 67975 
                 68854 
                 65607 
               
               
                   
               
             
          
           
               
                 T47D cells T47D cells (illustrated in FIG. 10) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 60992 
                 57868 
                 66902 
                 65508 
                 65244 
                 61392 
                 67072 
                 68276 
                 64397 
               
               
                 68394 
                 68983 
                 68862 
                 66620 
                 64730 
                 63577 
                 68671 
                 68910 
                 60369 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are 5 positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 SEQIDNO7 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 58887 
                 64931 
                 68010 
                 68325 
                 67109 
                 62010 
                 60075 
                 71189 
                 72712 
               
               
                 60090 
                 66366 
                 67591 
                 70586 
                 70471 
                 66328 
                 61350 
                 71192 
                 67410 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 64171 
                 67257 
                 66311 
                 63995 
                 65755 
                 60589 
                 62999 
                 71558 
                 65433 
               
               
                 62704 
                 64433 
                 66105 
                 67019 
                 66292 
                 62763 
                 67975 
                 68854 
                 65607 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 11) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 62688 
                 68041 
                 66479 
                 65388 
                 64353 
                 64124 
                 67072 
                 68276 
                 64397 
               
               
                 60767 
                 64777 
                 60908 
                 63864 
                 63685 
                 63134 
                 68671 
                 68910 
                 60369 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 SEQIDNO8 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 63114 
                 67475 
                 68833 
                 70559 
                 70603 
                 68676 
                 60075 
                 71189 
                 72712 
               
               
                 65457 
                 69324 
                 70414 
                 72074 
                 73593 
                 68864 
                 61350 
                 71192 
                 67410 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 64002 
                 68720 
                 69897 
                 68985 
                 68423 
                 61653 
                 62999 
                 71558 
                 65433 
               
               
                 64485 
                 70447 
                 70376 
                 70404 
                 68263 
                 66015 
                 67975 
                 68854 
                 65607 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 12) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 62688 
                 68041 
                 66479 
                 65388 
                 64353 
                 64124 
                 67072 
                 68276 
                 64397 
               
               
                 60767 
                 64777 
                 60908 
                 63864 
                 63685 
                 63134 
                 68671 
                 68910 
                 60369 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 SEQIDNO9 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 71608 
                 69255 
                 69234 
                 67663 
                 67229 
                 68009 
                 56076 
                 67625 
                 65584 
               
               
                 70176 
                 69328 
                 68280 
                 69155 
                 67807 
                 65131 
                 57832 
                 68347 
                 62965 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 67879 
                 65563 
                 65046 
                 63071 
                 57728 
                 62556 
                 63504 
                 64678 
                 63670 
               
               
                 68176 
                 69218 
                 65931 
                 65962 
                 62231 
                 61065 
                 65399 
                 63807 
                 61809 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 13) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 67492 
                 63181 
                 62848 
                 63130 
                 60146 
                 65094 
                 50994 
                 64632 
                 63285 
               
               
                 77427 
                 64535 
                 62359 
                 68689 
                 64467 
                 67070 
                 60368 
                 63641 
                 63372 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                 SEQIDNO10 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 74950 
                 70143 
                 68926 
                 68674 
                 67904 
                 64358 
                 56076 
                 67625 
                 65584 
               
               
                 67668 
                 70096 
                 71006 
                 69541 
                 70174 
                 65678 
                 57832 
                 68347 
                 62965 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 66888 
                 67041 
                 68442 
                 67343 
                 62238 
                 62410 
                 63504 
                 64678 
                 63670 
               
               
                 71581 
                 68134 
                 69458 
                 65315 
                 64184 
                 63264 
                 65399 
                 63807 
                 61809 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 14) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 58905 
                 62235 
                 61232 
                 61576 
                 62058 
                 63213 
                 50994 
                 64632 
                 63285 
               
               
                 62040 
                 61799 
                 61422 
                 62488 
                 61865 
                 64672 
                 60368 
                 63641 
                 63372 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                 SEQIDNO11 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 66600 
                 67045 
                 66568 
                 64371 
                 65587 
                 64433 
                 56076 
                 67625 
                 65584 
               
               
                 67446 
                 66528 
                 64759 
                 65373 
                 66707 
                 65703 
                 57832 
                 68347 
                 62965 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 65114 
                 65947 
                 64058 
                 63837 
                 60430 
                 60886 
                 63504 
                 64678 
                 63670 
               
               
                 72373 
                 68983 
                 64030 
                 64861 
                 65118 
                 63795 
                 65399 
                 63807 
                 61809 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 15) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 61976 
                 61546 
                 62448 
                 62557 
                 62018 
                 63935 
                 50994 
                 64632 
                 63285 
               
               
                 62453 
                 64267 
                 60200 
                 63900 
                 64649 
                 65696 
                 60368 
                 63641 
                 63372 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                 SEQIDNO12 activity studies. 
               
               
                   
               
             
             
               
                 TMX2-28 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 64801 
                 67335 
                 69868 
                 67062 
                 68382 
                 66458 
                 56076 
                 67625 
                 65584 
               
               
                 66313 
                 67800 
                 69586 
                 68845 
                 67167 
                 66191 
                 57832 
                 68347 
                 62965 
               
               
                   
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 Tamoxifen 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 64634 
                 67452 
                 67604 
                 65967 
                 64530 
                 59810 
                 63504 
                 64678 
                 63670 
               
               
                 66223 
                 68292 
                 67923 
                 68650 
                 64993 
                 61717 
                 65399 
                 63807 
                 61809 
               
               
                   
               
             
          
           
               
                 T47D cells (illustrated in FIG. 16) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 BaP 
                 DMSO 
                 DC5 
               
               
                   
               
               
                 57184 
                 62230 
                 64065 
                 64023 
                 65019 
                 62179 
                 50994 
                 64632 
                 63285 
               
               
                 62215 
                 62786 
                 63826 
                 65295 
                 65289 
                 65227 
                 60368 
                 63641 
                 63372 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
               
                   
               
               
                 Activity studies on a mixture comprising SEQIDNO1-12. 
               
               
                   
               
             
             
               
                 TMX2-28 (illustrated in FIG. 17) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 DC5 
                 DMSO 
                 BaP 
               
               
                   
               
               
                 19618 
                 19938 
                 19149 
                 24420 
                 26842 
                 27997 
                 26593 
                 29683 
                 28167 
               
               
                 17966 
                 16195 
                 21134 
                 25207 
                 25027 
                 25967 
                 27751 
                 29462 
                 27829 
               
               
                   
               
             
          
           
               
                 MCF-7 (illustrated in FIG. 18) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 DC5 
                 DMSO 
                 Tamoxifen 
               
               
                   
               
               
                 15917 
                 19169 
                 20614 
                 23282 
                 23677 
                 25949 
                 26653 
                 27209 
                 28761 
               
               
                 14811 
                 19624 
                 21839 
                 24219 
                 25099 
                 24607 
                 25764 
                 26113 
                 26110 
               
               
                   
               
             
          
           
               
                 T47D (illustrated in FIG. 19) 
               
             
          
           
               
                 Concentration(Molarity) 
                 Controls 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
                 DC5 
                 DMSO 
                 BaP 
               
               
                   
               
               
                 19911 
                 19942 
                 24175 
                 24407 
                 23984 
                 23633 
                 25127 
                 24113 
                 20732 
               
               
                 22269 
                 20661 
                 23915 
                 24233 
                 23571 
                 22438 
                 25805 
                 24706 
                 21197 
               
               
                   
               
               
                 Decreased fluorescent readings relatively to the DC5 sample indicates reduced cell growth. Bap (1 × 10 −6  M) and tamoxifen (5 × 10 −6  M) are positive controls. 
               
               
                 DC5 = Medium, Bap = Benzo[a]pyrene, DMSO = Dimethylsulfoxide. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 Activity studies on a mixture 
               
               
                 comprising SEQIDNO2-7 + SEQIDNO10-11. 
               
               
                 Concentration(Molarity) 
               
             
          
           
               
                 5 × 10 −4   
                 5 × 10 −5   
                 5 × 10 −6   
                 5 × 10 −7   
                 5 × 10 −8   
                 5 × 10 −9   
               
               
                   
               
             
          
           
               
                 TMX2-28 cells 
               
             
          
           
               
                 18046 
                 18061 
                 24875 
                 25179 
                 27474 
                 28777 
               
               
                 16175 
                 24487 
                 27451 
                 25866 
                 26799 
                 28223 
               
             
          
           
               
                 MCF-7 cells 
               
             
          
           
               
                 16309 
                 19277 
                 22516 
                 23270 
                 24537 
                 24714 
               
               
                 20269 
                 23225 
                 23895 
                 23205 
                 24024 
                 25069 
               
             
          
           
               
                 T47D cells 
               
             
          
           
               
                 19565 
                 22096 
                 22449 
                 23776 
                 23486 
                 24580 
               
               
                 21973 
                 22865 
                 23128 
                 23092 
                 23427 
                 24804 
               
               
                   
               
               
                 Decreased fluorescent readings indicates reduced cell growth. 
               
             
          
         
       
     
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         33. Dundee J W, Ghaly R G, Bill K M, Chestnutt W N, Fitzpatrick K T, Lynas A G. (1989). Effect of stimulation of the P6 antiemetic point on postoperative nausea and vomiting. Brit. J. Anaesth. 63:612-8. 
         34. Aglietti L, Roila F, Tonato M, Basurto C, Bracarda S, Picciafuoco M, Ballatori E, Del Favero A. (1990). A pilot study of metoclopramide, dexamethasone, diphenhydramine and acupuncture in women treated with cisplatin. Cancer Chemother. Pharmacol. 26:239-40 
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         39. Ross J (1995). Acupuncture Point Combinations: the Key to Clinical Success. 1st Edition, Churchill Livingstone ISBN-10: 0443050066