CA2321256A1

CA2321256A1 - Pharmaceutical preparations and methods for inhibiting tumours

Info

Publication number: CA2321256A1
Application number: CA002321256A
Authority: CA
Inventors: Unknown
Original assignee: Procyon Biopharma Inc
Current assignee: Individual
Priority date: 2000-10-16
Filing date: 2000-10-16
Publication date: 2002-04-16
Also published as: ES2351783T3

Abstract

The invention provides pharmaceutical preparations for inhibiting growth of prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian or other cancers of epithelial secretion, or benign prostate hyperplasia (BPH). In one embodiment the pharmaceutical preparation includes human rHu PSP94 which may be administered in an appropriate dosage form, dosage quantity and dosage regimen to a patient suffering from prostate cancer. In another embodiment the pharmaceutical preparation includes a mixture of human rHu PSP94 and an anticancer drug which may be administered in an appropriate dosage form, dosage quantity and dosage regimen to a patient suffering from, for example gastrointestinal cancer. The anticancer drug of the latter mixture may be one selected from the group of drugs including mitomycin, idarubicin, cisplatin, 5-fluoro-uracil, methotrexate, adriamycin and daunomycin.

Description

PHARMACEUTICAL PREPARATIONS AND METHODS
FOR INHIBITING TUMOURS
FIELD OF THE INVENTION
The present invention relates to pharmaceutical preparations for use as tumour suppressive agents for tumours arising from cancers such as prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial and ovarian cancers, and benign prostate hyperplasia (BPH).
BACKGROUND OF THE INVENTION
The prostate gland, which is found exclusively in male mammals, produces several components of semen and blood and several regulatory peptides. The prostate gland comprises stroma and epithelium cells, the latter group consisting of columnar secretory cells and basal nonsecretory cells. A
proliferation of these basal cells as well as stroma cells gives rise to benign prostatic hyperplasia (BPH), which is one common prostate disease. Another common prostate disease is prostatic adenocarcinoma (CaP), which is the most common of the fatal pathophysiological prostate cancers, and involves a malignant transformation of epithelial cells in the peripheral region of the prostate gland. Prostatic adenocarcinoma and benign prostatic hyperplasia are two common prostate diseases which have a high rate of incidence in the aging human male population. Approximately one out of every four males above the age of 55 suffers from a prostate disease of some form or another. Prostate cancer is the second most common cause of cancer related death in elderly men, with approximately 96,000 cases diagnosed and about 26,000 deaths reported annually in the United States.
Studies of the various substances synthesized and secreted by normal, benign and cancerous prostates carried out in order to gain an understanding of the pathogenesis of the various prostate diseases reveal that certain of these substances may be used as immunohistochemical tumour markers in the diagnosis of prostate disease. The three predominant proteins or peptides secreted by a normal prostate gland are:
(1) Prostatic Acid Phosphatase (PAP); (2) Prostate Specific Antigen (PSA); and, (3) Prostate Secretary Protein of 94 amino acids (PSP94), which is also known as Prostatic Inhibin Peptide (PIP), Human Seminal Plasma Inhibin (HSPI), or (3-microseminoprotein (~3-MSP), and which is hereinafter referred to as pSp94.
PSP94 is a simple non-glycosylated cysteine-rich protein, and constitutes one of three predominant proteins found in human seminal fluid along with Prostate Specific Antigen (PSA) and Prostate Acid Phosphatase (PAP). PSP94 has a molecular weight of 10.7 kDa, and the complete amino acid sequence of this protein has already been determined (SEQ ID N0:1). The cDNA and gene for PSP94 have been cloned and characterized (Ulvsback, et al., Biochem. Biophys. Res. Comm., 164:1310, 1989; Green, et al., Biochem. Biophys. Res. Comm., 167:1184, 1990).
Immunochemical and in situ hybridization techniques have shown that PSP94 is located predominantly in prostate epithelial cells.
It is also present, however, in a variety of other secretory epithelial cells (Weiber, et al., Am. J. Pathol., 137:593, 1990). PSP94 has been shown to be expressed in prostate adenocarcinoma cell line, LNCap (Yang, et al., J. Urol., 160:2240, 1998). As well, an inhibitory effect of exogenous PSP94 on tumour cell growth has been observed both in vivo and in vitro (Garde, et al., Prostate, 22:225, 1993; Lokeshwar, et al., Cancer Res., 53:4855, 1993), suggesting that PSP94 could be a negative regulator for prostate carcinoma growth via interaction with cognate receptors on tumor cells.

Native PSP94 has been shown to have a therapeutic modality in treating hormone refractory PCa (and potentially other prostate indications).
Metabolic and immunohistochemical studies have shown that the prostate is a major source of PSP94. PSP94 is involved in the feedback control of, and acts to suppress secretion of, circulating follicle stimulating hormone (FSH) both in-vitro and in-vivo in adult male rats. PSP94 acts both at the pituitary as well as at the prostate site since both are provided with receptor sites for PSP94.
Both PSA and PAP have been studied as tumour markers in the detection of prostate disease, but since both exhibit elevated levels in prostates having benign prostatic hyperplasia (BPH), neither marker is specific and therefore they are of limited utility.
Recently, it has been shown that PSP94 concentrations in serum of patients with BPH or CaP are significantly higher than normal. The highest serum concentration of PSP99 observed in normal men is approximately 40 ng/ml, while in men with either BPH or CaP, serum concentrations of PSP94 have been observed in the range from 300-400 ng/ml. Because there exists some overlap in the concentrations of PSP94 in subjects having normal prostates and patients exhibiting either BPH or CaP, serum levels in and of themselves are of little value.
A major therapy in the treatment of prostate cancer is androgen-ablation. While most patients respond initially to this treatment, its effectiveness decreases over time, possibly because of the presence of a heterogenous population of androgen-dependant and androgen-independent cells to the androgen treatment, while any androgen insensitive cells present would continue to proliferate unabated.

Other forms of cancer which are currently exacting a heavy toll are breast cancer in women and cancer of the gastrointestinal tract. Currently, the use of various cancer drugs such as mitomycin, idarubicin, cisplatin, 5-fluoro-uracil, methotrexate, adriamycin and daunomycin form part of the therapy for treating such cancers. One drawback to such a therapeutic treatment is the presence of adverse side effects due to the drugs in the concentration ranges required for effective treatment.
Accordingly, it would be advantageous to find a more effective means of arresting the growth of prostate, breast and gastrointestinal cancer cells and tumours which can be used effectively against both androgen sensitive and androgen insensitive cells.
In our previous work, described in United States Patent No. 5,428,011, we provided pharmaceutical preparations of native human seminal plasma PSP94 for inhibiting in-vitro and in vivo cancerous prostate, gastrointestinal and breast tumours.
The pharmaceutical preparations included native human seminal plasma PSP94 which could be administered in an appropriate dosage form, dosage quantity and dosage regimen to a patient suffering from prostate cancer. In another embodiment, the pharmaceutical preparation included a mixture of human seminal plasma PSP94 and an anticancer drug which may be administered in an appropriate dosage form, dosage quantity and dosage regimen to a patient suffering from, for example gastrointestinal cancer.
PSP94 sourced from human seminal fluid carries with it significant risk of contamination with infectious agents (e. g., HIV, hepatitis (a, b, or c), and other viruses and/or prions).
Even with the use of harsh chemical treatment, total eradication of such agents cannot be guaranteed. Additionally, human seminal fluid is found in limited supply, thus making bulk production of PSP94 very difficult. Therefore, the acceptability of human or even xenogeneic sourced PSP94 may be very difficult for both the regulatory authorities and the marketplace.
Therefore, the use of recombinant technology for producing PSP94 would represent a significant advancement, as recombinant PSP94 could be produced both free of pathogens and in an unlimited supply. Furthermore, the material would be homogeneous from a single lot source, avoiding batch to batch variation.
SU1~ARY OF THE INVENTION
The present invention relates to the use of recombinant human rHu PSP94 (SEQ ID N0: 2) for inhibiting the growth of prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian and other cancers of epithelial secretion, or benign prostate hyperplasia (BPH). Preferably, the rHu PSP94 is used in a dosage range from about 500 picograms/kg/day to about 1 milligram/kg/day.
The recombinant human rHu PSP94 has been expressed in Pichia pastoris (host strain GS115 (his4)), is non-glycosylated and has 10 cystein residues. The molecular weight of rHu PSP94 was determined as 11.5 kDa, compared to 10.7 kDa for its native counterpart. An MTS cell proliferation assays showed that rHu PSP94 could produce a dose dependent decrease in cell numbers of PC-3 human prostate adenocarcinoma cell line.
The invention also relates to the use of at least one peptide selected from the group consisting of the decapeptide depicted in SEQ ID N0: 3, the peptide analogue depicted in SEQ
ID N0:4, the peptide analogue depicted in SEQ ID N0:5, and the peptide analogue depicted in SEQ ID N0:6, or a mixture thereof, for inhibiting growth of prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian and other cancers of epithelial secretion, or benign prostate hyperplasia (BPH).
Preferably, the peptide is used in a dosage range from about 250 nanograms/kg/day to about 1 milligram/kg/day.
In another aspect of the invention there is provided a method for treating a patient with prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian and other cancers of epithelial secretion, or benign prostate hyperplasia (BPH), comprising administering to the patient a pharmaceutical preparation comprising a peptide selected from the group consisting of rHu PSP94 (SEQ ID N0:2), the decapeptide depicted in SEQ ID N0: 3, the peptide analogue depicted in SEQ ID N0: 4, the peptide analogue depicted in SEQ ID N0: 5, and the peptide analogue depicted in SEQ ID N0:6.
The present invention also provides a composition for the use of inhibiting the growth tumours in patients suffering from prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian and other cancers of epithelial secretion as well as benign prostate hyperplasia (BPH). The composition comprises recombinant human rHu PSP94 (SEQ ID N0: 2), preferably present in a dosage range of about 5 nanograms/kg/day to about 10 micrograms/kg/day. The composition preferably also includes an anticancer drug. A variety of suitable anticancer drugs are known in the art. Examples include, without limitation, mitomycin, idarubicin, cisplatin, 5-fluoro-uracil, methotrexate, adriamycin and daunomycin.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts mass spectrometry analysis of peptide (7-21) (SEQ ID N0: 4).
Figure 2 depicts mass spectrometry analysis of peptide (31-45) (SEQ ID N0:5).
Figure 3 depicts mass spectrometry analysis of peptide (76-94) (SEQ ID N0:6) .
Figures 4 - 6 are graphs depicting the inhibitory activity of the decapeptide of SEQ ID N0: 3 on in-vitro PC-3 cells.
Figure 7 depicts a gel showing DNA fragmentation following treatment of PC-3 cells with the peptide depicted in SEQ ID N0:5.
Figure 8 is a graph depicting the results of an apoptosis assay with an ELISA plus kit following peptide treatment of ATCC-PC3 cells for 72 hours.
Figure 9 is a graph depicting growth of fibroblast cells when exposed to PSP94 and its analogues in-vitro for 72 hours.
Figure 10 is a graph depicting the effect of the peptides depicted in SEQ ID NOs:4 and 6 on the growth of ATCC-PC3 cells in-vitro at 72 hours.
Figure 11 is a graph depicting the effect of peptides of the invention on growth of ATCC-PC3(OPTI) cells at 72 hours of exposure in-vitro.

_ Figures 12 and 13 are graphs depicting the results of studies of the anti-tumor efficacy validation of rPSP94 against Mat Ly Lu (MLL) tumor implanted in nude mice.
Figure 14 is a graph depicting tumor growth reduction in rHu PSP94-treated mice.
Figures 15 - 17 are graphs depicting tumor growth reduction in decapeptide (SEQ ID N0:3)-treated mice.
DETAILED DESCRIPTION OF THE INVENTION
Various in-vivo and in-vitro experimental studies have been carried out and are summarized herein below to determine the efficacy of concentrations of rHu PSP94 (SEQ ID N0: 2) higher than native PSP99 concentrations secreted by the diseased prostate as tumour suppressive agent for arresting or inhibiting the growth of prostatic adenocarcinoma. Studies have also been carried out to determine the efficacy of decapeptide (SEQ ID
N0:3) and synthetic peptide analogues of rHu PSP94 (SEQ ID N0: 4, No. 5 and No.6) as tumour suppressive agents.
Studies were carried out using PC-3 human prostate adenocarcinoma line which can be maintained both in vivo as a xenograft in nude mice and in vitro as a cell line. In addition, a rat Dunning Mat LyLu prostate tumour, which is a pre-eminent animal model for the study of CaP, was also used. The Dunning tumour is a fast growing, poorly differentiated, transplantable tumour which can be maintained both in-vivo in the Copenhagen rat and in-vitro as a cell line.
The following examples are offered by way of illustration and not by way of limitation.

_ EXAMPLE 1 PREPARATION OF rHu PSP94 (SEQ ID NO: 2) rHu PSP9q was cloned and expressed in Pichia pastoris, and then purified and characterized as follows.
M~tvrinlc DEAF-cellulose (DE52) was from Whatman, Fairfield, NJ
and the dialysis tubing from BioLynx (Pierce Inc.). Broad-range molecular weight markers and Econo-pack columns fitted with flow adapters were from Bio-Rad Labs Ltd CA. Pellicon device was from Millipore, MA. Tris-HC1 was obtained from ICN, and MES from Sigma. Other reagents were obtained from standard commercial sources and were of reagent grade. ECL detection kit was purchased from Biolynx Canada and swine anti-rabbit IgG
alkaline-phosphatase conjugates from DAKO, Denmark. Rabbit polyclonal antiserum against PSP94 was a gift from the late Dr.
A. Sheth. Pichia expression Kit version G was from Invitrogen, Carlsbad, CA and the Non-Radioactive High Prime DIG labeling kit~ was from Boehringer Mannheim Indianapolis, IN. The MTS
assays were performed using Cell Titer Aqueous Non radioactive cell proliferation assay kit from Promega Madison, WI. MRX
microtiter plate reader was from Dynex technologies, Chantillly, VA. All primers were synthesized by Procyon Biopharma Inc.
London, ON.
Cell lines and Cell cultures P. pastoris host strain GS115 (his4) and all the Pichia related products were obtained from Invitrogen. PC3 (ATCC-# CRL 1435) cell line was from the American Type Cell Culture (ATCC) and maintained in OPTI MEM media withl0 o FBS.
All cell culture products were obtained from GIBCO BRL.

Cloning and Expression TA cloning vector (pCR TM 2.1) containing human PSP94 cDNA with 20 a.a. leader sequence used previously (9)(Baijal et.
al. 1999) was modified without its leader sequence to amplify human PSP94. The primers for the PCR reaction were designed to contain EcoRI restriction sites at either end. The 5' primer used was 5'-GGG AAG ~AAT TCT CAT GCT ATT TCA TA-3' and the 3' primer, 5'-TGG ATA TCT GCA GAA TT~C GGC-3'. The EcoRI cleavage signal has been designated with ~ and the +1 start site for PSP94 (Ser), has been underlined. The subcloning of the PSP94 insert was done in pPIC9 vector (Invitrogen), which contained EcoRI
restriction site at the 5' end, about 5-8 amino acids away from the cleavage site that removes the signal sequence of the PSP94 insert. The amplification of the product was done by PCR, using BM ExpandTM High Fidelity PCR System.
The PCR included 1 cycle for 12' @ 94 C, 25 cycles for 1' @ 94 C followed by 1' @ 55 C, 1' @ 72 C and final 10' @ 72 C.
The product was run on 1.5 o agarore gel and appropriate bands isolated using Pharmacia Sehphaglass Kit (Bandprep). The EcoRI
enzyme was used for the restriction digestions of both the plasmid and the PCR products followed by ligation and transformations, using DHSa cells. The isolated clones were selected for by ampicillin resistance and inserts were identified by restriction mappings. The constructs were sequenced (Robart's sequencing service, London ON) to identify PSP94 insert with correct orientation and reading frame as well scrutinized for point mutations.
To follow the time course of expression and to determine the optimum time for harvesting secreted PSP94, daily aliquots of the expression medium were removed beginning with the first day of induction, until the time of harvesting (96 h).
Expression of the protein was tracked by SDS PAGE with 15 % gel, followed by Western blot analysis using polyclonal antibody raised against native PSP94.
Screening for High Expressing Clones About 100 clones were picked and grown into 2 ml culture media to OD6oo of ~6. Total DNA was isolated for rapid dot blot technique to detect multiple integration by Southern blot analysis to screen for high PSP94 expressing clones. 200u1 of each culture specimens were denatured and blotted to a positively charged nylon membrane, placed in the dot blot apparatus (in duplicate) and air-dried. The membrane was soaked between two sheets of Whatman 3MM paper for 15 min in a solution with 50 mM EDTA, 2.5 o BME, pH 9, followed by 24 hours at 37 C
with 1 mg/ml Zymolyase 100T, 5min in 0.1 N NaOH, 1.5 M NaCl, 0.015M sodium citrate pH 7 and 2 x 5 min in 2xSSC. Finally the membrane was baked at 80 C for 45 min and UV exposed for l5min.
Human PSP94 cDNA probe was labeled with the non radioactive High Prime DIG labeling kit~ (Boehringer Mannheim) and was used for hybridization (9). All DIG labeling procedures were performed according to the manufacturer's protocol and the hybridization signals were detected on the Hyper film-ECL (Amersham Life Science Inc. Arlington Hts, IL).
Hybridization with DIG labeled cDNA probe (25ng/ul) was done for 2 days at 42 C in high SDS buffer (SDS 7 %;
formamide, 500; 5 X SSC; 50 mM sodium phosphate, pH 7.0; N-lauroyl-sarcosine, 0.1 0 (w/v)) and blocking reagent, 2 %. CSPD~
(Boehringer Mannheim) was used as the chemiluminescence substrate. The clone with highest signal intensity was used for all flasks shaken cultures.

Optimization of the Expression of the Protein in the Flask Shaken Cultures A single clone containing the PSP94 construct was selected for the optimum expression of the protein. 25m1 of BMG
medium was grown until the OD 60o measured between 2 and 6. The flask shaken cultures in I liter volumes of BMG media were inoculated with t25m1 culture and incubated in Baffled Erlenmeyer flasks for growth phase, until OD soo reached approximately 2.0 to 6Ø The culture was centrifuged for l5min at 2500 X g and the pellet collected. The growth in the induction phase was then carried out in BMM media by inoculating it with the collected cell pellet in Baffled flasks and grown for 6 days, as described by Invitrogen. The volume of BMM
varied based on the amount of pellet. Five ml of 1000 methanol was added per litre of culture, each day, around the same time, to a final concentration to 0.1 %. The plasmid without the insert served as a negative control.
To determine the optimum time for harvesting secreted PSP94, aliquots were monitored every 24 hrs for 6 days, beginning with the first day of induction. The levels of PSP94 protein were determined by measuring the cell densities at OD 600 and byl5 % Comassie stained SDS PAGE and Western blot analysis, using polyclonal antibody against native PSP94.
Sample Preparation The specimen from the flask shaken culture with the highest rPSP94 expression, post induction growth (96 hr), was centrifuged at 2500 X g for 20min. The supernatant was filtered through 0.8um filter and concentrated approximately 10-fold using the Pellicon unit (Millipore). The specimen was dialyzed against 0.05 mM Tris HC1 buffer, pH 8.0, using a Mr 3500 cut-off tubing. The final supernatant was tested by SDS PAGE and Western blot analysis and further used for purification.

Culture Conditions for Fermentation The fermentation was carried out at the Institute for Biological Sciences, National Research Council Ottawa ON Canada, following the manufacturer's protocol. The fermentation procedure was initiated by inoculating 7.5-liter medium with 625 ml of starter flask shaken culture material. The growth phase culture was carried out for 2 days in BMG medium until the OD 600 reached approx. 0.5. The initial growth phase was terminated and induction initiated by the addition of 100 o methanol, as described by inVitrogen. The culture was finally harvested at 95 hr after being on methanol for 67 hrs. The final volume of the culture was approximately 13.5 lit.
Sample Preparation using Fermentation Culture The large cell mass was removed by centrifugation and was performed at NRC. The cell free medium collected (9 lit) was further clarified by 0.2 a filtration Pellicon unit. The final 8.5 lit containing secreted rPSP94 was tested for rPSP94 expression and saved at -20 C for isolation and purification of the protein.
Protein Estimation The protein in the culture supernatants of the flask shaken and the fermentation was based on band intensities of various amounts of the supernatant samples on the SDS PAGE gels.
Standard curve were constructed by electrophoresis of increasing predetermined quantities of pure lyophilized PSP94 on the same gel. The initial estimate for rPSP94 at each step of purification was determined by OD z$o, Quantification of protein at the final steps of purification was done by BCA method, using bovine serum albumin as standard.

Lyophilization Samples of purified rPSP94 were dialyzed against deionized water using Mr 3000 cut-off membrane and lyophilized.
SDS gel electrophoresis SDS gel electrophoresis was performed using 150 acrylamide separating and 5o stacking gels, containing O.lo SDS
according to Laemmli under reducing conditions. Broad-range molecular weight markers were used for the estimation of molecular weights of the protein. Protein bands were stained with Coomassie Brilliant Blue R-250.
Western Blotting For immunoblotting, Mini Trans-Blot Electrophoretic Transfer Cell (Bio Rad) was used with Hi bond-C supper membrane (Amersham) and 85mm blotting papers. Protein samples, 0.4 ug each, run on SDS PAGE gel, as described earlier, were transferred to the membrane using transfer buffer (25 mM Tris, 192 mM Glycine, pH 8.3 and 20 o methanol) at 200 mA° for 2hrs at 4° C. Membranes were blocked 0/N by incubation in 20 (w/v) non-fat dry milk (skim milk) dissolved in 500 mM NaCl, 20 mM Tris-HC1, pH 7.5 (TBS) at room temp (RT). Immunoblots were washed three-times with TBS containing 0.02% (v/v) Tween 20 (TTBS).
The blot was subsequently incubated with anti PSP94 (1:2000 dilution) in TTBS with 2% skim milk for 2 hrs at RT. Membranes were washed thrice with TTBS, 5min each, incubated with secondary antibody, swine anti rabbit antibody HRP conjugated ((1:5000) in TTBS at RT and washed thrice with 0.02% TTBS 5 minutes each. Blots were developed using the ECL detection system, as per manufacturer's instructions, using the Super Signal Substrate, and exposed to a Hyperfim ECL from Amersham LS
for 5 to 20 sec. Pre-stained molecular weight markers were used for the estimation of molecular weights of the protein.

DE52 Column Chromatography After removal of P. pastoris cells from the fermentation culture, PSP94 containing supernatant was concentrated ~ ten fold, dialyzed and subjected to anion exchange chromatography. A column of DE52 with a bed volume ~ 40m1 (2.5 (id) X 8 cm (h) cm) was equilibrated with 0.05 M Tris-HC1, pH
8Ø The sample (25 ml) containing 15-20 mg PSP94 protein was applied to the DE52 column at a flow rate of 1 ml/min.
Impurities from the column were removed by washing it with 40-50 ml of the equilibrating buffer, and monitoring the A 280. This step was followed by 100-150 ml of 0.05 M Tris-HC1, pH 6.5 and most importantly till the pH of the wash was ~6.5. The column was further washed with 100- 150 ml of 0.05 M MES-acetate buffer, pH 6.5, until the A 28o value approached zero. Finally rPSP94 was eluted from the column with 0.05 M MES-acetate buffer, pH 5Ø Peak fractions were identified by A 280 determination, followed by SDS PAGE and Western blot analysis as described above. Fractions with high A 28o values (0.5 to 1.8) were pooled and dialyzed against water/PBS for storage at -20 C
and/or lyophilization.
Amino acid Composition The amino acid analysis of the DE52 purified flask shaken and the fermentation specimens was carried out at the Louisiana State University Medical Core Laboratory. The Perkin Elmer Biosystems Derivatizer-Analysis system was used with Spheri-5 PTC C-18 5u column and UV detection at OD 2s4~
Mass Spectral analysis Purified samples from the flask shaken and the fermentation cultures were analyzed using the PerSeptive Biosystems (Framingham, MA), with Voyager-DE MALDI-TOF mass spectrometer using 337 nm light from a nitrogen laser at the Louisiana State University Medical Core Laboratory. About 50 _ scans were averaged for each analysis. A sample from the native PSP94 was also analyzed under similar conditions for comparison.

EFFECT OF rHu PSP94 ON IN-VITRO PC-3 CELLS
(MTS ASSAY) The biological activity of the rHu PSP94 was determined by its growth inhibitory effects on the human prostate cancer cells PC-3. The cell proliferation was monitored on PC-3 cells using the MTS/PMS kit (Promega), which primarily measures the mitochondrial activity of live cells. In addition, a visual observation of the cells was also done to check the cell morphology, which could also result in inhibition of cell growth. These results are summarized in Table 1. The following conditions for MTS assay were used:
PC-3 (ATCC, Lot AT06), Passage Number n >_ 70, Cell line adapted to grow in serum-free OPTI-MEM. Continuous exposure for 72 hrs without changing medium (adding PSP94 at 2X concentration directly to wells and diluting it 1:2 to 1X to minimize cell manipulation and avoid detachment). Evaluation of rHu PSP94 demonstrated a substantial reduction in cell numbers ranging from 37-57o at 80 and 120 ~g/ml concentrations, respectively in 3 out of 4 experiments (Table 1). Results of Trypan Blue Exclusion Test demonstrated 62o inhibition of cell viability at 8 0 ~.g /ml .

TABLE 1: Effect of rHu PSP99 on in vitro PC-3 cells (MTS assay) Reference:
(Monthly Report June Activity 2000) xperiment# rHu PSP9 ~~Viability (control at pg/ml =
100_) 40 60___ 80 120 079-105 " 63 63 63 68 079-120 " 95 85 78 D

079-153 " 100 52 62 60 079-140 99.09.08 100 98 90 52 rHu PSP9 ~ Viability(control = 100 at ug/ml " 92 95 80 71 59 " 89 69 79 68 65 EFFECT OF rHu PSP94 ON IN-VITRO PC-3 CELLS
([3H]-THYMIDINE UPTAKE ASSAY) The vitro rHu PSP94 efficacy validation was assessed in the [3H]-Thymidine Uptake assay where PC-3 cells are exposed to various concentrations of rHu PSP94 for 72 hours and during the final 16 hrs of incubation the cells are pulsed with 1 ~Cu of [3H]-Thymidine. These results are summarized in Table 2.
Evaluation of rHu PSP94 demonstrated a 65o reduction in the o-age of cells incorporating [3H]-thymidine following treatment at 80 ~,g/ml for 72hrs. For comparison of sensitivities between [3H]-Thymidine Uptake and the MTS assays an additional plate was set aside for MTS assay evaluation and treated in parallel with the same lot of rHu PSP94. The results demonstrated a 35% reduction in cell viability (65o cells remaining viable) following treatment at 80 ~g/ml, indicating that the [3H]-Thymidine Uptake assay might be more sensitive than the MTS assay.

TABLE 2: Effect of rHu PSP94 on in vitro PC-3 cells ([3~H]-Thymidine uptake assay) [3~H]-Thymidine Uptake (~
of control) at xperiment PSP9 g/ml #

rHu PSP94 1 (00-133F) 94 101 98 79 35 n PSP9' 1 994135 (B2) 97 98 100 98 77 EFFECT OF DECAPEPTIDE AND OTHER PEPTIDE ANALOGUES ON

The synthetic decapeptide (SEQ ID NO: 3) has been shown herein to mimic the biological action of native nPSP94 (SEQ ID N0: 1) and therefore its effect on the PC-3 cells was studied in clonogenicity assay. Referring to Figures 4-6, the decapeptide (SEQ ID N0: 3) has a similar inhibitory action as nPSP94 (SEQ ID N0: 1) on in-vitro PC-3 cells was studied.
Specifically, a 40o colony count inhibition was observed with 1 ug/ml of the decapeptide (SEQ ID N0: 2) leading to a maximum of 60% inhibition at 10 ~g/ml.

DNA FRAGMENTATION ASSAY
DNA fragmentation assay measures the extent of apoptosis by the presence of DNA ladder on 1.2o Agarose gel following exposure of PC-3 to various concentrations of the peptide analogues. Peptide analogues used: peptide 7-21 (SEQ ID
N0: 4), peptide 31-45 (SEQ ID N0:5) and peptide 76-94 (SEQ ID
N0:6). Duration of treatment 72 hrs. DNA was isolated and run on 1.2o Agarose gel. Results demonstrated that all the peptides tested produced a DNA laddering effect characteristic of apoptosis. This effect was especially evident following treatment with peptide 31-45 (SEQ ID N0: 5) (Figure 7).

APOPTOSIS ASSAY BY ELISA PLUS
The three peptides analogues (SEQ ID N0: 4, No.5 and No. 6) and native PSP99 as a positive control were tested in ELISA plus assay to measure cell death through apoptosis. The PC-3 cells were treated with various concentrations of peptides for 72 hrs. It was observed that a dose dependent increase in the apoptotic cell death was noticed in all the 3 peptides analogues (SEQ ID N0: 4, No.5 and No. 6). The middle portion peptide (SEQ ID N0: 5) was more potent at 90 ~tm concentration (Figure 8). Apoptosis assay was done as per instructions in the ApopTag kit (Boerenger Mannheim).

The biological activity of the peptide analogues (SEQ
ID NO: 4, 5 and 6) was determined by their growth inhibitory effects on the human prostate cancer cells PC-3. The cell proliferation was monitored on PC-3 cells and control normal fibroblast using the MTS/PMS kit (Promega), which primarily measures the mitochondrial activity of live cells. In addition, a visual observation of the cells was also done to check the cell morphology, which could also result in inhibition of cell growth. These results are show in Figures 9 and 10. No cell inhibitory effect was observed against fibroblast cells following incubation with various peptide concentrations for 72 hrs (Figure 9 and Figure 11) whereas significant growth inhibition was observed especially with middle portion peptide fragment 31-45 (SEQ ID N0: 5) as well as the amino and carboxy terminus peptide fragments (SEQ ID NOs: 4 and 6, respectively) (Figure 10).
EXAMPLES 8 & 9 IN-VIVO EXPERIMENTS (Figures 12 & 13) (Study MLL-1) Anti-tumor Efficacy Validation of rPSP94 against Mat Ly Lu (MLL) Tumor Implanted in Nude Mice.
(Protocol based on S. Garde et al.; The Prostate, 22: 225-233, 1993 "Effect of Prostatic Inhibin Peptide (PIP) on Prostate Cancer Cell Growth In Vitro and In Vivo") Groups (n = 8 mice / group):
Group 1: Negative control PBS s.c.
- Group 2: Positive control Doxorubicin @ 5mg/kg i.v.
single bolus on Day 3 - Group 3: rPSP94 Batch # 00-133F @ l~g/kg s.c.
- Group 4: rPSP94 Batch # 00-133F @ 10~g/kg s.c.
- Group 5: rPSP99 Batch # 00-133F @ 100~g/kg s.c.
Schematic:
M M M M

T T T T
T . C . I . Tx................................ E-daily.......
............................Tx Terminate (Study MLL-2) Anti-tumor Efficacy Validation of rPSP94 against Mat Ly Lu (MLL) Tumor Implanted in SCID
Mice.
(Protocol based on S. Garde et al.; The Prostate, 22: 225-233, 1993 "Effect of Prostatic Inhibin Peptide (PIP) on Prostate Cancer Cell Growth In Vitro and In Vivo") Groups (n = 8 mice / group):
- Group l: Negative control PBS s.c.

- Group 2: Positive control Doxorubicin @ 5mg/kg i.v.
single bolus on Day 3 - Group 3: rPSP9q Batch # 00-133F @ l~g/kg s.c.
- Group 4: rPSP94 Batch # 00-133F @ 10~g/kg s.c.
- Group 5: rPSP94 Batch # 00-133F @ 100~g/kg s.c.
Schematic:
M M M M

T T T T
T . C . I . Tx................................ daily->.......
............................Tx Terminate Conclusions For Examples 8 & 9:
Difference in tumor size and growth in Nude vs SCID.
The tumors were smaller in SLID mice. Maybe due to some specific factors controlling tumor growth.
- Good tumor reduction with Doxorubicin (good positive control):
- Tumor Weight Reduction in Nude mice (MLL-1): 48$ p=0.006 - Tumor Weight Reduction in SCID mice (MLL
2): 82~ p=0.002 - Significant tumor reduction with rPSP94 @ l~,g/kg:
- Tumor Weight Reduction in Nude mice (MLL
1): 26$ p=0.042 - Tumor Weight Reduction in SLID mice (MLL-2): 65~ p=0.010 EXAMPLE 10 (Figure 14) PC3 human prostate tumor (ATCC 1435) obtained from ATCC was implanted into nude mice and tumor growth monitored for approximately 18 days. The PC-3 cells were injected once subcutaneously into the shoulder. Once tumor growth has been established (50 mm3 volume) the treatment with rHu PSP94 (SEQ ID

N0: 2) begun at qldxl4 s.c. based on the following assigned treatment groups (n= 8 per group):
reatmentTest and Dose Level*ose Conc. No of Animal roups control (~g/kg) (~g/mg) ticles (Neg PBS 0 0 8 ontrol) (Pos Doxorubicin 5000** 2500 8 ontrol) 3 rHu PSP9' 1 0.5 8 4 rHu PSP99 10 5 8 rHu PSP99 100 50 8 6 rHu PSP94 1000 500 8 5 Results of this experiment (Figure 14) demonstrated tumor growth reduction in the rHu PSP94 treated group of mice at a dosage level of 1 ~g/kg body weight per day similar to that of Doxorubicin (given at 5 mg/kg) a chemotherapeutic agent used as reference gold standard.
EXAMPLE 11 (Figures 15-17) PC3 human prostate tumor (ATCC 1435) obtained from ATCC was implanted bilateraly into nude mice and tumor growth monitored for approximately 18 days. The PC-3 cells were injected once subcutaneously into each flank. Once tumor growth has been established (0.25-0.50 cc volume) the treatment with decapeptide PB102 (SEQ ID N0:3), native PSP99 (PB100) (Sequence ID. 1) and control irrelevant decapepride PB111 - begun at qldxl4 s.c. based on the following randomly assigned treatment groups (n=4 per group):
reatmentTest and Dose I~evel*ose Conc. No of Animal roups control (~g/kg) (~g/mg) ticles (Neg PBS 0 0 4 ontrol) 3 PB102 1 0.5 4 (decapeptide) (decapeptide) (decapeptide) (decapeptide) 7 PB100 1 0.5 4 (nPSP94) (nPSP9) (nPSP99 ) (nPSP99) 12 (control) 1 0.5 4 13 (control) 10 5 4 14 (control) 100 50 4 (control) 1000 500 4 Results of this study (Figures 15-17) demonstrated 5 significant (p<0.05) tumor growth reduction in the decapeptide PB102- treated group of mice at a dosage level of 10 ~g/kg body weight per day.
All publications and patent applications cited in this 10 specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

SEQUENCE FISTING
SEQ ID No: 1 (nPSP94 sequence):
NH
SCYFIPNEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYETEISCCTLVSTPVGYDKDNC
QRIFKKEDCKYIVVEKKDPKKTCSVSEWII COOH
SEQ ID NO: 2 (rPSP sequence):

NH
EAEAYVEFSCYFIPNEGVPGDSTRKCMDLKGNKHPINSEWQTDNCETCTCYETEISCCTLVSTP
VGYDKDNCQRIFKKEDCKYIVVEKKDPKKTCSVSEWII COOH
SEQ ID NO: 3 (decapeptide) Tyr-Thr-Cys-Ser-Val-Ser-Glu-Pro-Gly-Ile SEQ ID N0: 4 (N-terminus peptide 7-21) NH
Asn-Glu-Gly-Val-Pro-Gly-Asp-Ser-Thr-Arg-Lys-Cys-Met-Asp-Leu SEQ ID N0: 5 (middle portion peptide 31-45) Glu-Trp-Gln-Thr-Asp-Asn-Cys-Glu-Thr-Cys-Thr-Cys-Tyr-Glu-Thr SEQ ID N0: 6 (carboxy-terminus 76-94) Ile-Val-Val-Glu-Lys-Lys-Asp-Pro-Lys-Lys-Thr-Cys-Ser-Val-Ser-Glu-Trp-Ile-Ile COOH

Claims

1. Use of human rHu PSP94 (SEQ ID NO: 2) for inhibiting growth of prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian or other cancers of epithelial secretion, or benign prostate hyperplasia (BPH), wherein the human rHu PSP94 is used in a dosage range from about 500 picograms/kg/day to about 1 milligrams/kg/day.

2. The use of human rHu PSP94 (SEQ ID NO: 2) according to claim 1 wherein the human rHu PSP94 is used in a dosage range from about 5 nanograms/kg/day to about 10 micrograms/kg/day.

3. The use of human rHu PSP94 (SEQ ID NO: 2) according to claim 1 wherein the human rHu PSP94 is used in a mixture comprising the human rHu PSP94 (SEQ ID NO: 2) and a pharmaceutically acceptable carrier.

4. The use of human rHu PSP94 (SEQ ID NO: 2) according to claim 3 wherein the human rHu PSP94 is used in a dosage range from about 5 nanograms/kg/day to about 500 nanograms/kg/day.

5. The use of human rHu PSP94 according to claim 3 wherein the pharmaceutically acceptable carrier includes time release encapsulation means for effecting continual dosing of the human rHu PSP99.

6. The use of human rHu PSP94 according to claim 5 wherein the time release means comprises a liposome.

7. The use of human rHu PSP94 according to claim 5 wherein the time release means comprises a polysaccharide.

8. The use of human rHu PSP94 according to claim 1 wherein human rHu PSP94 is used in a mixture including an anticancer drug.

9. The use of humans rHu PSP94 according to claim 8 wherein the anticancer drug is selected from the group consisting of mitomycin, idarubicin, cisplatin, 5-fluoro-uracil, methotrexate, adriamycin and daunomycin.

10. Use of at least one peptide selected from the group consisting of the synthetic decapeptide depicted in SEQ ID NO:3, the peptide analogue depicted in SEQ ID NO:4, the peptide analogue depicted in SEQ ID NO:5, and the peptide analogue depicted in SEQ ID NO:6, for inhibiting growth of prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian or other cancers of epithelial secretion, or benign prostate hyperplasia (BPH), wherein the peptide is used in a dosage range from about 250 nanograms/kg/day to about 1 milligrams/kg/day.

11. A method for treating a patient with prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian or other cancers of epithelial secretion, or benign prostate hyperplasia (BPH), the method comprising administering to the patient a pharmaceutical preparation comprising at least one peptide selected from the group consisting of human rHu PSP94 (SEQ ID NO:2), the decapeptide depicted in SEQ ID NO:3, the peptide analogue depicted in SEQ ID NO:4, the peptide analogue depicted in SEQ ID NO:5, and the peptide analogue depicted in SEQ ID NO:6).

12. The method of claim 11 wherein the pharmaceutical preparation includes an anticancer drug.

13. The method of claim 12 wherein the anticancer drug is selected from the group consisting of mitomycin, idarubicin, cisplatin, 5-fluoro-uracil, methotrexate, adriamycin and daunomycin.

14. The method according to claim 13 wherein the peptide is human rHu PSP94 (SEQ ID NO: 2) and wherein it is administered in a dosage range from about 25 picograms/kg/day to about 1 milligrams/kg/day.

15. The method according to claim 13 wherein the human rHu PSP94 is administered in a mixture including an anticancer drug, wherein the anticancer drug is selected from the group consisting of mitomycin, idarubicin, cisplatin, 5-fluoro-uracil, methotrexate, adriamycin and daunomycin.

16. The method according to claim 14 wherein the human rHu PSP94 is administered in a dosage range from about 5 nanograms/kg/day to about 10 micrograms/kg/day.

17. The method according to claim 16 wherein the human rHu PSP94 is administered in a mixture including an anticancer drug selected from the group consisting of mitomycin, idarubicin, cisplatin, 5-fluoro-uracil, methotrexate, adriamycin and daunomycin.

18. The method according to claim 16 wherein the human rHu PSP94 (SEQ ID NO: 2) is used in a mixture including a pharmaceutically acceptable carrier.

19. The method according to claim 18 wherein the pharmaceutically acceptable carrier includes time release encapsulation means for effecting continual dosing of the human rHu PSP94.

20. The method according to claim 19 wherein the time release encapsulation means comprises a liposome.

21. The method according to claim 19 wherein the time release encapsulation means comprises a polysaccharide.

22. The method according to claim 11 wherein the peptide is administered in a dosage range of about 25 nanograms/kg/day to about 50 micrograms/kg/day.

23. The method according to claim 22 wherein the pharmaceutically acceptable carrier includes time release encapsulation means for effecting continual dosing of the peptide.

24. A composition for inhibiting the growth tumours in patients suffering from prostatic adenocarcinoma, stomach cancer, breast cancer, endometrial, ovarian or other cancers of epithelial secretion, or benign prostate hyperplasia (BPH), comprising:
a) Human rHu PSP94 (SEQ ID NO: 2) present in a dosage range of about 5 nanograms/kg/day to about 10 micrograms/kg/day; and b) an anticancer drug.

25. A composition according to claim 24 wherein said anticancer drug is selected from the group consisting of mitomycin, idarubicin, cisplatin, 5-fluoro-uracil, methotrexate, adriamycin and daunomycin.

26. A composition according to claim 24 including a pharmaceutically acceptable carrier.

27. A composition according to claim 24 including a pharmaceutically acceptable carrier includes time release encapsulation means for effecting continual dosing of the composition.

28. A method for treating patients with diseases characterized by elevated levels of FSH comprising administering a pharmaceutical preparation in an appropriate dosage form, the pharmaceutical preparation comprising at least one peptide selected from the group consisting of human recombinant PSP94 (SEQ ID NO: 2), the decapeptide depicted in SEQ ID NO: 3, the peptide depicted in SEQ ID NO:4, the peptide depicted in SEQ ID
NO:5, and the peptide depicted in SEQ ID NO:6.