METHOD OF TREATING CARCINOMAS
FIELD OF THE INVENTION
The present invention relates to the use of peptide, nucleotide-peptide mixtures for treating carcinomas and preventing their spread.
BACKGROUND OF THE INVENTION
The use of peptones, peptides, proteins and nucleic acid mixtures to produce antiviral agents was originated in 1934. After some years of experimentation, such an antiviral agent was produced by using bovine serum albumin in combination with peptone, and ribonucleic acid. This procedure formed an antiviral biotic agent which is nontoxic, free from anaphylactogenic properties and is miscible with tissue fluids and blood sera. This agent was described as a "lipopeptide-nucleic acid compound" and registered under trademark RETICULOSE® by Chemico Laboratories, Inc. Physician Desk Reference, p 651, i960. RETICULOSE® was reported as an antiviral agent for treating a variety of human viral infections, such as influenza, herpes, hepatitis A and B. RETICULOSE® was last sold in the United States in 1964.
RETICULOSE® was described in U.S. Patent No.5,849,196, December 15, 1999 and U.S. Patent No. 6,312,602, November 6, 2001 both issued to Kochel. This peptide nucleotide mixture was described in both Kochel U.S. Patents as a mixture of peptides, nucleotides and nucleotide peptides having molecule weights in a range from about 1 to 25 kDa and which absorbs light in at least two bands having a maximum value of 205 nm and 260 nm, which mixture results from casein, blood albumin, beef peptone, nucleic acid, and a base wherein components of said mixture having molecular weights outside of the range from about 1-25 kDa are removed.
As disclosed in U.S. Patent 5,849,196, the starting materials for making RETICULOSE® consist of, by weight, 40-50% of casein, 1-10% of blood albumin, 15-4096 of beef peptone, 10-25% of sodium hydroxide. These starting materials are suspended in water which yields a ratio of proteins (casein, peptone and blood albumin) to water equaling to about 4.3 to about 100 by weight. After an autoclaving treatment of the mixture of the
starting materials, the resulting solution is filtered and pH is adjusted to approximately 8.5 and then to 7.8, after which the neutralized solution is filtered again. The pH is further adjusted to approximately 7.5 after the solution is diluted. Such process yields a aqueous mixture of peptides, nucleic acids, having molecular weights in a range of approximately 1 to 25 KDa.
As taught by U.S. Patent 5,849,196, the components over 15 KDa of the conventional composition REΗCULOSE® are more effective in treating viral diseases such as HIV, influenza virus, herpes simplex virus, etc. while the components in a range of approximately 1 to 15 KDa function as phagocytosis inhibitors. In U.S. Patent 6,303,153, which issued October 16, 2001 to Friedland and Hirschman, a new liquid mixture of nucleorides, peptides and nucleotide peptides called Product R was produced for use as an antiviral agent. As disclosed in U.S. Patent No. 6,303,153, this liquid mixture was produced from a mixture of a protein combination of casein, beef peptone and bovine serum albumin, with a plant RNA and a base in water with the ratio of said protein combination to said water being in a range from about 1.5/100 to about 2.5/100 by weight. The mixture was heated to an elevated temperature to form an aqueous solution. Upon formation of this solution, the insoluble elements are removed. Since this mixture is administered to patients, one adjusts the pH of the solution to a physiologically acceptable pH for administration.
In U.S. Patent 6,528,098 issued March 4, 2003 to Freidland et al., it was found that the active ingredient of Product R was formed from a mixture of
NHa - Lys Val Leu Pro Val Pro Gin Lys Ala Val Pro Tyr Pro Gin Arg Asp I Met Pro He Gin Ala Phe Leu Leu Tyr Gin Glu Pro Val Leu Gly - COOH
and a nucleotide diphosphodiester-peptide of the formula:
wherein R is a peptide having the sequence :
NH2-Gly Glu He Pro Asp Ala Gly Gly Arg He Val Asp Tyr Tyr Val Gly Phe Ser Asp Ser Val-COOH, and which is connected to the diphosphodiester moiety to form the nucleotide peptide by the hydroxy group of the serine molecule.
Product R is an immunomodulating agent useful for treating a wide range of viral infections, such as infections of human immunodeficiency virus (HIV), herpes simplex virus and adenovirus. It has become known that Product R is effective in stimulating the production of chemokines, including interferon gamma, interleukin-6, interleukin-i
and tumor necrosis factor-alpha in monocytes and lymphocytes (S.Z. Hirschman and C.W. Chen, J. Invest. Med.44, 347-351,1996). It has also been found that Product R upregulates the expression of cytokines such as interleukin-8 and macrophage chemoattractant protein-i (D-A. Lazzarino, M. de Diego, S.Z. Hirschman, K.Y. Zhang, S. Shaikh, E. Musi, L. Liaw and R.J. Alexander, Cytokine, 14, 234-239, 2000). Product R also downregulates the expression of the chemokine receptors CXCR4 and CCR5 in H-9 T-lymphocytes; these receptors serve as coreceptors for HIV-i on target T-cells (DA. Lazzarino, M.de Diego, E. Musi, S.Z. Hirschman and R.J. Alexander {Immunology Letters, 74, 189-195, 2000). A clinical trial using Product R in HIV-infected patients has been conducted and has demonstrated the efficacy of the drug in decreasing viral loads and increase CD4+ and CD8+ cell counts (P.N. Levett, S.Z. Hirschman, T.C. Roach, H. Broome, R.J. Alexander and H.S. Fraser (HTVClin. Trials, 3, 272-278, 2002).
The malignant progression of cancers involves two separate, but interactive, cellular processes: loss of differentiated properties and a loss of proper tissue compartmentalization (metastases). In the case of dedifferentiation, the loss of cell-cell attachment, altered cell substratum attachment and altered cytoskeletal organization all play a key role. In metastasis, the same is true, but also cell locomotion, proteolysis and the ability to survive and proliferate at distant sites are essential (AM. Valles, G. C. Tucker, J.P. Thiery, Cell ReguL, 1, 975 ,1990 and D. Rusciano. M.M. Burger, Bioessays, 14, 185, 1992).
Chemokines that normally guide infection-fighting cells to their targets have been shown to play an important role in breast cancer metastasis (A Muller, B. Homey, H. Soto, N. Ge, D. Catron, M.E.. Buchanan, T. McClanahan, E. Murphy, W. Yuan, S.N. Wagner, S.N. Barrera, A Mohar, E. Verastegul and A Zlotnik, Nature, 410, 50-56, 2001). Cytokines induce cytoskeletal rearrangement, firm adhesiom to endothelial cells and directional migration, through their interaction with G-protein coupled receptors. The chemokine receptors CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumors and metastases while normal tissue cells do not express these receptors. Signaling through these chemokine receptors mediates actin
polymerization and induces chemotactic and invasive responses. As seen above, Product R downregulates the CXR4 receptor.
Cell adhesion is a pivotal step that occurs in early tumor metastasis. The process is mediated by integrins, which are typically upregulated by cytokines. Studies have shown that the cytokines IL-ibeta and TNF-alpha decrease the expression of aibi integrins on human osteosarcoma cells, thereby increasing their cellular adhesion characteristics. An agent that stimulates the production of these cytokines may therefore inhibit the cellular adhesion properties of metastasis cancer cells. Product Rhas been shown to enhance the expression of these cytokines in peripheral blood monocytes. Many tumor types are now known to metastasize and invade by an over-expression of cytokine receptors, such as CXCR4, CCR5 and IL-8 receptors.
Expression of CXCR4 cytokine receptors have been implicated in the invasive ability and/or metastasis of many types of cancers. Among the cancers are included:
Breast Cancer (A. Muller, Nature, 410: 50-56, 2001);
Thyroid Cancer (J.H. Hwang, et al, J. Clin. Endocrin. Metab., 88: 408-416, 2003);
Brain Cancer (S. Barbero, A Bajetto, et al, Ann. NYAcad. Set, 973-60-69 ,2002);
Ovarian Cancer (C. Scotton, D. Millikem, et al, Br. J. Cancer, 85, 891-897,2001);
Colon Cancer (LS. Zelenberg, L. Ruuls-Van Stalle, E. Roos, Cancer Res., 63, 3833-3839, 2003);
Prostate Cancer (Y. X. Sun, J. Wang, et al , J. Cell. Biochem., 89, 462-473, 2003);
Squamous Cell Carcinoma (D. Uchida, N.M. Begum, et al, Exp. Cell Res. 288-302, 2003);
Chronic Lymphocytic Leukemia (J. Barrentina, J. Junca, et al, Ann. Hematol, 82, 500- 505, 2002); and
Acute Lymphocytic Leukemia (J. Juraez, KF bradstock, et al, Leukemia, 17, 1294-1300, 2003).
Expression of CCR5 has been implicated in the invasiveness and metastasis for example:
Nasopharyngeal Cancer: (K.F. Tang, S.Y. Tan, et al, Human Pathol., 32, 42-49, 2001). Breast Cancer (S. Manes, E. Mira et al, J. Exp. Med., 198, 1381-1389).
Multiple Myeloma (M. Abe, K. Hiuara, et al , Blood, 100, 2195-2202, 2002).
SUMMARY OF THE INVENTION
In accordance with this invention, it has been found that compositions containing an active ingredient formed from a mixture of peptides, nucleotides and nucleotide peptides can be used to treat patients suffering from carcinomas of the breast or cervix. This composition can be used not only to treat patients with breast or cervical cancer but to treat patients who have already had these cancers removed or treated to prevent the metastasis spread of these cancer cells. In accordance with this invention, it has been found that this active ingredient is capable of effectively inhibiting the invasion and migration of cancer cells of the breast and cervix.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 demonstrates the inhibition of Cell Invasion in MDA-MB-231, MCF-7 and MCF-12A Breast cancer cells upon treatment with the active ingredient.
Figure 2 shows the dose response of therapeutic Product R in MDA-MB-231 Breast Tumor Cell Lines.
Figure 3 shows the dose response of therapeutic Product R in SiHa Cervical carcinoma cells.
Figure 4 shows photomicrographs demonstrating the inhibition of cell invasion in MDA- MB-231 Breast Cancer cell lines.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a method of treating patients with carcinomas of the breast or cervix by administering to these patients a composition containing an active ingredient which is composed of a liquid peptide, nucleotide and nucleotide peptide mixture. Among the carcinomas treated are adenocarcinomas and squamous cell carcinomas. Breast carcinomas are predominantly adenocarcinomas, whereas cervical carcinomas are mainly squamous cell carcinomas.
In this respect the treatment of carcinomas of the breast or cervix with a composition containing these active ingredients, in accordance with this invention, can retard and/or reverse the progression of these carcinomas. The active ingredient is generally administered parenterally to patients in anaqueous medium. In this manner these carcinomas after treatment, in accordance with this invention, can be reduced in size and in many cases, completely disappear. On the other hand, not only does the composition of this invention retard the development of these carcinomas but it also inhibits the invasion and metastic potential of these cancer cells. In this case, the administration of compositions containing the active ingredients, in accordance with this invention, to patients who have been diagnosed as having either carcinomas of the breast and/or skin or who have had these carcinomas surgically or chemotherapeutically treated can be treated with the active ingredient of this invention to prevent the spread or metastasis of these carcinomas.
In accordance with this invention, the active ingredient for use in this invention is formed from a mixture of the peptide having the sequence given in Formula I and the nucleotide diphosphodiester-peptide of Formula II as described hereinbefore. In accordance with this embodiment, a mixture of the peptide having the sequence of Formula I with a nucleotide diphosphodiester-peptide of Formula II can be used to form the active ingredient of the composition which is administered to the patient. On the other hand, this mixture of the peptide of Formula I and the nucelotide diphosphodiester-peptide of Formula II can be applied in a peptide, nucleotide and nucleotide peptide mixture such as in the Product R mixture in which it is formed.
In accordance with the method of this invention, the active ingredient which is used to treat carcinomas, in accordance with this invention, can be either a simple mixture of the peptide of Formula I with the nucleotide diphosphodiester-peptide of Formula II or can be isolated from Product R or can be prepared by any other conventional means. However, if desired, the active ingredient can be the entire Product R composition which is a liquid mixture which contains the active ingredient formed from the peptide having the sequence of Formula I and the nucleotide diphosphdiester-peptide of Formula II.
On the other hand, the active ingredient can be the antiviral mixture of a peptide, nucleotide, and nucleotide peptide such as RETICULOSE® disclosed in Kochel, U.S. Patents 5,849,196 and 6,312,602. This includes the original RETICULOSE® product as well as the improved product disclosed in these Kochel patents.
In accordance with a preferred embodiment of this invention, the active ingredient in the composition used in accordance with this invention is formed from the peptide of Formula I and the nucleotide diphosphdiester-peptide of Formula II. These peptides are prepared from Product R in accordance with the disclosure of U.S. Patent 6,528,098 issued March 4, 2003 to Freidland and Hirschman. The disclosure of the '098 patent, with regard to preparation of Product R, its characterization and the preparation and isolation of the components I and II, are incorporated herein. In the '098 patent, the peptide of Formula I is referred as Peptide A, whereas the nucleotide diphosphodiester- peptide of Formula II is referred to as Peptide B. As see from Figure 20 in the '098 patent, both Peptide A and Peptide B, when isolated and tested alone are less active than Product R. On the other hand, when Peptide A and Peptide B are combined, the combined activity of the mixture of these two components was comparable with Product R. This demonstrates that these two peptide- nucleotide components are the key ingredients which combine when mixed so as to be responsible for the major part of the biological activity of Product R. In accordance with this invention, it has been found that this combination of these two components administered to patients for treatment retard the progression of carcinomas of the cervix or breast.
In accordance with this invention, the components of Formula I and Formula II can be present together in admixture in any effective amount as the active ingredient in the composition to retard the progression of carcinomas of the cervix or breast when applied in accordance with this invention. It has been found that best results are achieved when the active ingredient is formed from about 0.5 to about 2.0 moles of the protein per mole of the nucleotide peptide. Molar amounts of from about 0.8 moles to about 1.2 moles of the protein per mole of said nucleotide peptide are preferred, with molar amounts of the nucleotide peptide to the protein in a mole ratio of about 1 to 1 being especially preferred. On the other hand, the active ingredient can be administered as Product R. which contains the mixture of the components I and II. Product R is produced in accordance with the disclosures in U.S. Patent 6,303,153 and 6,528,098. As see from these patents, Product R is a mixture of peptides, nucleotides and nucleotide peptides and absorbs light at wavelengths of 230 nm to 260 nm and 280 nm resulting in a 260 nm/280 nm absorption ratio of about 1.998 and a 260 nm/230 nm absorption ratio of about 1.359. The nucleotides in said active ingredient mixture result from a plant RNA and the peptides in said active ingredient mixture result from a mixture of casein, beef peptone and bovine serum albumin. The molecules of nucleotides and peptides in Product R have non-uniformly distributed molecular weights of no more than 14 KDa. The Product R is produced in accordance with the disclosure of the aforementioned U.S. patents from a mixture of a protein combination consisting of casein, beef peptone and bovine serum albumin with a plant RNA and a base. This mixture is placed in an aqueous medium to form the Product R. In forming the Product R, the weight ratio of the protein combination which consists of the casein, beef peptone and bovine serum albumin to the water is a range from about 1.5/100 to about 2.5/100 parts by weight. The mixture is processed at elevated temperatures and pressures to form a solution. After forming a solution the insoluble elements are removed and the solution may be further diluted with water. This liquid form of Product R may be used as the active ingredient in the therapeutic compositions. In forming the therapeutic composition to be administered, it is preferred to adjust the pH of the solution to a physiologically acceptable pH for administering to humans. In carrying out this method, molecules in
Product R generally have non-unifoπnly distributed molecular weights in the range of no more than 14 KDa, with molecular weights in the range of no more than 8 KDa being preferred with molecules of molecular weights of substantially 5.2 KDa and 4.3 KDa being especially preferred. It has been discovered that the active ingredient formed from the mixture of the compound of formula I and the compound of formula II or from peptide, nucleotide mixtures such as RETICULOSE®, as described in the Kochel patents, when administered to patients in accordance with this invention is effective in retarding the progression of carcinomas by shrinking the tumor size of carcinomas and in many cases causing them to disappear and preventing their recurrence. Also, the administration of these active ingredients prevents the metastasis or spread of the carcinomas and can be used in follow up therapy after these carcinomas have been treated either surgically or by chemotherapy to prevent metastasis and/or recurrences of these carcinomas. The active ingredient when in the form of the mixture of compounds I and II or as Product R or as RETICULOSE® is in the form of a liquid and can be administered in an aqueous solution.
The treatment of this invention can be effected with the active ingredients alone or in combination with other measures such as surgery, radiation therapy, hormone therapy or treatment with standard chemotherapy (cytostatic and cytotoxic agents) or biological response modifiers (interferons, interleukins, agents or other cytokines).
These pharmaceutical compositions of the invention contain said active ingredient in association with a compatible pharmaceutically acceptable carrier material. Any conventional carrier material can be utilized. The carrier material can be an organic or inorganic inert carrier material suitable for administration to patients. Suitable carriers include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene-glycols, petroleum jelly and the like. Furthermore, the pharmaceutical preparations may contain other pharmaceutically active agents. Additional additives such as flavoring agents, preservatives, stabilizers, emulsifying
agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.
In treating carcinomas of the cervix and breast to retard the progression of these carcinomas, the aforementioned active ingredient is administered to patients effected by the carcinomas in an amount effective for retarding the progression of these carcinomas. Of course, the amount will be dependent upon the amount and size of these carcinomas and the requirement of the patients. In retarding the progression of these carcinomas, one can see a reduction both in the size, amount and recurrence of these carcinomas and in some cases a disappearance of all of these carcinomas. In accordance with another embodiment of this invention, the active ingredients are administered for treating patients who have had a treated primary malignancy wherein the primary malignancy is a carcinoma of the breast or cervix. It has been found that the administration of these active ingredients to such patients inhibits both the recurrence of the primary malignancy and the development of a secondary malignancy. This primary malignancy may be treated by removal through surgery or other means such as chemotherapy. In accordance with this embodiment of the invention the administration of the active ingredient can provide adjuvant therapy to prevent spread of the first malignancy to a different site. Furthermore, the treatment in accordance with this invention also retards or inhibits development of a secondary malignancy. The method of this invention may be utilized to prevent a second malignancy where the first malignancy is a carcinoma of the breast or cervix. In using the method of this invention for adjuvant therapy to prevent the development of a secondary malignancy, the composition of this invention is administered to provide the active ingredient in an amount sufficient to inhibit the reoccurrence of the primary malignancy or the development of a secondary malignancy.
The aforementioned active ingredients are prepared as a liquid, preferably as a injectable aqueous solution containing from 7 mg to 10 mg of the mixture of peptides, nucleotides and nucleotide peptides per ml of solution. These active ingredients are preferably administered to the patient parenterally, while other administration routes
such as nasal spray or ingestion maybe employed. For the injectable aqueous solution containing from 7 mg to 10 mg of the mixture of peptides, nucleotides and nucleotide peptides per ml of solution, a suitable effective dose of these active ingredients is in the range of from about 2.5 microliters to about 30 microliters of this solution per kilogram of body weight per day, preferably in the range of about 10 microliters to about 20 microliters per kilogram of body weight per day. Generally the active ingredient which is a mixture of peptides, nucleotides and nucleotide peptides is administered at a dosage amount of from about 15 micrograms to about 210 micrograms per kilogram of body weight per day in a sterile injectable IV formulation. The desired dose may be administered as two, three or more sub-doses at appropriate intervals, generally equally spread in time, throughout the day. Preferably, the full daily dose is administered in one administration. Alternatively, these active ingredients may be administered to the patients according to the conventional dosages or any dosages that are apparent to a person of ordinary skill in the art. In accordance with this invention, the amounts of the ingredient to be applied and the frequency of application per day will depend upon the observation and judgment of the physician who is administering the treatment with the active ingredients in accordance with the method of this invention
Compositions containing the aforementioned active ingredient may be administered by an suitable injection route including, but not limited to intravenously, intraperitoneally, subcutaneously, intramuscularly, and intradermally, etc. The presently preferred route of administration is intramuscularly. It will be appreciated that the preferred route may vary with, for example, the condition and age of the recipient. Any conventional injectable method and formulation administering peptides such as EPO and interferon α can be used to administer the aforementioned active ingredient
The composition containing the active ingredient can be used in therapy in conjunction with other medicaments including corticosteroid, gamma globulin, or vitamins, antiviral agents such as interferon or interleukin, etc. While it is possible for compositions containing the aforementioned active ingredients to be administered as part of a pharmaceutical formulation, it is preferable to present it alone, although it may be administered at about the same time as one or more other pharmaceuticals are
independently administered. If compositions containing the aforementioned active ingredient are administered as part of a pharmaceutical formulation, the formulations of the present invention comprise at least one administered ingredient, i.e., compositions containing the aforementioned active ingredient, as above defined, together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The formulations may conveniently be presented in unit-dose or multi-dose containers, e.g. sealed ampules and vials. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction of the administered ingredient.
The invention will be better understood from the following examples, which are illustrative of the invention and no limiting. In the Examples peptide of designates Peptide A as the peptide of the sequence of formula I and Peptide B designates the nucleotide diphosphodiester-peptide of the formula II.
In the Examples all of the cell lines were obtained from the ATCC. The cell line numbers as deposited at the ATCC are as follows:
MCF-7 ATCC HTB-26 MCF-12A ATCC CRL-10782 MDA-MB-231 ATCC HTB-26 SiHa HTB-35 EXAMPLES Example I Preparation of Product R
First, the starting materials casein, beef peptone, RNA, BSA, and sodium hydroxide are suspended in proportions of, by weight, 35-50% (casein), 15-40% (beef peptone), 10- 25% (yeast RNA), 1-10% (BSA) and 5-25% (sodium hydroxide) in an appropriate volume
of distilled water. While any RNA is suitable for the intended purpose of the present invention, plant RNA is preferred and yeast RNA is the most preferred. The ratio of total proteins versus the volume of distilled water is generally about 1.5-2.5 to about 100 by weight, preferably about 2.2 to about 100 by weight. This means that every 1.5-2.5 grams of the total proteins are suspended in about 100 milliliters of distilled water.
The suspension, as prepared, above is then autoclaved at a pressure of approximately 5- 15 lbs., preferably 8-10 lbs. under an elevated temperature in a range, for example about 150 - 3000 F, preferably about 200-230°F, over a period of approximately 2-10 hours, preferably more than 3 hours. Under such conditions RNA is completely hydrolyzed into nucleotides. After autoclaving, the solution is cooled to room temperature, and then allowing to stay at a temperature of 30 to 8°C for at least 12 hours to precipitate insoluble elements. Alternatively, the cooled solution maybe centrifuged at a temperature below 8°C to remove the precipitates.
The resulting solution is then filtered through a 2 micron and a 0.45 micron filter under an inert gas such as nitrogen or argon at a pressure of about 1-6 psi. In a similar manner the solution is filtered again through a pyrogen retention filter, preferably 0.2 micron. After the above filtration, the solution is cooled at 30 to 8°C again for about 12 hours and filtered again in the same way as described above.
The resulting filtrate is then assayed for total nitrogen content using methods known to a person of ordinary skill in the art such as Kjeldahl method, J.G.C.D. Kjeldahl, Z. Anal. Chem., Vol. 22, P366 (1883). Based on the assay, the filtrate is then diluted with chilled distilled water to an appropriate volume having a preferred total nitrogen content ranging from 165 to 210 mg/ml.
The pH of the diluted solution is then adjusted with HCI to a physiologically acceptable pH, preferably to about 7.3 to 7.6, after which the dilution solution is filtered again through a 0.2 micron filter under an inert gas as described above. The resulted solution containing the peptide of the sequence of formula I and the nucleotide diphosphodiester-peptide of formula II is used as the active ingredient in Examples 3 and 4.
Example 2 Purification of Product R Peptides Prepared in F-κ?τnp|ι» T
The product solution produced in Example 1 (6 ml) was lyophilized and the lyophilization cake was dissolved in a total volume of 1.5 mL of Nanopure water to make up a 4X concentration. A BioGel P-b column (16 x 51 cm) was equilibrated with 0.05X Dulbecco's phosphate buffered saline, Ca++ and Mg++ free (PBS). The resulting product was applied to the column. The column was eluted with 0.05X PBS, 1 ml fractions were collected. Fractions were tested for the presence of Peptide-A and Peptide-B by the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) technique, followed by silver staining of the gel.
Fractions containing Peptide-A were pooled, and then lyophilized. Fractions containing Peptide B were not completely free of Peptide A Fractions containing >8o% of Peptide- B were pooled and lyophilized. The above process was repeated several times until sufficient amounts of individual peptides were obtained for structural studies.
Combined pools of lyophilized Peptide-A were dissolved in Nanopure water, concentrated and further purified by centrifugation through a Centriplus 3 (MWCO-3 kDa) filter assembly (Millipore Corporation, USA). The protein content of the concentrate was assayed using the BioRed DC Protein assay Kit. The purity of the Peptide-A was re-examined by SDS-PAGE, and the sample was lyophilize and the lyophilization cake was stored refrigerated at 4°C until use for structural studies.
Combined pools of lyophilize Peptide-B were dissolved in a small volume of Nanopure water, dialyzed against 10 mM Tris-HCl, pH 7.4 in a Spectra/Por Dispo Dialyzer (MWCO-ikDa), obtained from Spectrum Microgren, USA The protein content was assayed by use of the BioRad DC Protein assay kit and by SDS-PAGE, as mentioned for Peptide-A The dialysate was lyophilized and the lyophilization cake of Peptide-B was stored refrigerated at 4°C until use for structural studies.
Peptide-A was analyzed by NMR spectroscopy, N-terminal sequencing by the Edman method and by mass spectroscopy.
The purified and separated Peptide-A sample was available as a lyophilized cake following dialysis as described above for Peptide-B. Example 3
To determine the effect of Product R on the growth of breast cancer cells, MCF-7 and MDA-MB-231 cells (obtained from American Type Culture Collection, Rockville, MD) were treated with Product R (10% and 20%) over a course of 5 days. Product R inhibited cell replication at all concentrations used and the cell replication was inhibited as early as 24 hours.
A 6 well plate of seed cells was washed 2 times with fresh Dulbecco's modified Eagle medium. 5 mL of trypsin solution was added and the plate was incubated at 37°C for 4 min. Ten milliliters of medium was added and the cells were transferred to a 50 mL centrifuge tube. They were then rinsed with 2x10 mL of culture medium. Cells in log phase were trypsinized, washed 3 times with PBS buffer, re-suspended in complete medium, and counted. The cell suspension was diluted to 2.5x104 cells/mL. 4 mL of cell suspension was placed in each well of a 6 well plate (1x10 s cells/well). One plate was used for each of the 1 day, 3 day and 5 day study and samples were run in triplicate. Concentrations of 5%, 10% and 20% Product R were used in each plate. A 20% solution of phosphate-buffered saline (PBS) served as the control. Cell counts were carried out after 24 hours, at 3 days and at 5 days. Cells were counted under a microscope using a hemocytometer after trypsinization, washing 2x with culture medium and resuspending in media without fetal bovine serum.
Table 1 shows the growth inhibition results for the experiment using MDA-MB-231 breast cancer cell lines.
Table 1. Cell Survival of Product R (PR)-treated MDA-MB-231 Breast Cancer Cells
Example 4 Inhibition Of Migration And Invasion Of Cancer Cells By Product R For these experiments, MDA-MB-231, MCF-7, MCF-12A breast cancer cells and SiHa cells were treated with 10% or 20% Product R for 48 hours. MATRIGEL Basement Membrane Matrix extracted from the Engelebreth-Holm-Swaπn (EHS) mouse tumor was used as a reconstituted basement membrane in vitro (H.K. Kleinman, M.L. McGarvey, et al, Biochemistry, 25, 312, 1986). This reconstituted basement membrane has been shown to ccontain collagen IV, laminin, entactin, heparin sulfate and proteoglycan and provides a biologically active extracellular matrix for in vitro invasion assays. The enzymatic degradation of type IV collagen has been strongly implicated in the cancer cell metastatic invasion process (LA Iiotta, et al, Cancer Metastasis Rev., 1, 277, 1982). The invasion chambers consisted of cell culture inserts (9mm in diameter) containing 125 μg/cm
2 protein concentration placed in a 24-well plate. This protein concentration minimized the preferential migration through the center of the membrane due to meniscus formation by the matrix. The insert contained a 8μm pore size microporous polyethylene terephthalate (PET) membrane with a uniform layer of MATRIGEL ™ matrix that occluded the membrane pores.
Invasion assays were performed using the invasive breast cancer cell lines MDA-MB- 231, MDA-MB-361 and MCF-7 and SiHa cervical carcinoma cell lines. Cells were pretreated by seeding in complete medium containing 5%, 10% or 20% Product R. Cells were also pretreated in the presence of an equal amount of phosphate buffered saline
(PBS) as a control. MATRIGEL™ matrix chambers were rehydrated for 2 hours in a humidified tissue culture incubator (37°C» 5% COa atm.) by the addition of warm (37°C) bicarbonate based culture medium to the interior of the inserts and the bottom of the wells. An equal number of control inserts were used in each experiment. The control inserts did not contain the MATRIGEL matrix and measured cell migration only. Cell suspensions in culture medium containing 5x104 cells/ml were prepared. Cells were seeded (2.5 x ιo« cells) onto the MATRIGEL™ matrix in the upper compartment and a chemoattractant (fibronectin or conditioned medium) was present in the lower compartment. The chambers were incubated for 22 hours in a humidified tissue culture incubator, at 37°C, 5%CO2 atmosphere. Invasive cells attached to and invaded the matrix, passing through the membrane pores, and emerged on the underside of the membrane, while non-invasive cells were blocked from migrating through the occluded pores. The MATRIGEL™ matrix and the non-invading cells were removed from the upper surface of the membranes by scrubbing with a cotton swab. The cells on the lower surface of the membrane were fixed and stained with Diff-Quik™ stain. Slides were prepared of each membrane by removing the membrane from the insert housing using a sharp scalpel blade and placing it bottom side down on a microscope slide on which a small drop of immersion oil had been placed. A second small drop of oil and a cover slip were placed on top of the membrane.
Visual counting of random fields under a light microscope quantitated the cellular invasion. This was facilitated by photographing the membrane through the microscope. Cells were counted in fields in the center of the membrane as well as cells in the membrane periphery for true representation of the cell number throughout the membrane. Results
Figure 1 demonstrates the ability of Product R to effectively inhibit the invasive capability of MDA-MB-231, MCF-7 and MCF-12A breast cancer tumor cells. There is a marked and uniform propensity of all the three cancer cell types to lose their invasive or metastatic properties upon treatment with a 10% dilution of Product R.
Figure 2 demonstrates the dose-response of Product R treatment in MDA-MB-231 breast cancer cell lines. A clear dose-response relationship is noted in going from a 5% to a 20% diluted Product R solution upon both the migration and invasive capabilities of the breast cancer cells relative to a PBS control. This shows that Product R limits greatly the migration and invasive characteristics of breast cancer cells that aid such cells in the metastasis process.
Figure 3 demonstrates the dose-response of Product R treatment in SiHa cervical carcinoma cell lines. A clear dose-response relationship is noted in going from a 5% to a 20% diluted solution of Product R upon both the migration and invasive capabilities of the uterine cervical cancer cells relative to a PBs control. This shows that Product R limits greatly the migration and invasive characteristics of cervical cancer cells that aid such cells in the metastatic process.
Figure 4 shows photomicrographs of the effects of Product R on the inhibition of cell invasion in MDA-MB-231 breast cancer cell lines. Results show that relative to a PBS control, cells treated with 10% Product R show a greatly diminished propensity to invade the MATRIGEL™ matrix. At a 20% Product R concentration, the inhibition of cell invasion is even more pronounced as evidenced by the few cells observed in the microscopic field that were able to invade through the biological matrix. This demonstrates the clear utility of Product R in inhibiting the invasive action of metastatic cancer cells.
Table 1 shows the effect of Product R on the growth inhibition of MDA-MB-231 breast cancer cell lines. Product R shows a clear dose-dependent effect on the survival of these cells relative to a PBS control.