TARGETED ONCOLYTIC PEPTIDE FOR TREATMENT OF OVARIAN CANCERS

First generation targeted oncolytic peptides consisted of synthetic lytic peptides conjugated to human chorionic gonadotropin or LHRH [5-7]. They selectively kill . needed to improve the survival of patient. EP-100 is a novel oncolytic peptide that is being developed to selectively target surface luteinizing hormone releasing hormone (LHRH) receptors. It has been reported that approximately 80 % human ovarian cancer cells and their recurrences over-express LHRH receptors. EP-100 consists of an LHRH receptor-targeting ligand conjugated to a novel lytic peptide (CLIP71). EP-100 was tested in vitro and in vivo for activity in LHRH receptor overexpressing OVCAR-3 cells. LHRH receptor-negative SKOV-3 ovarian cancer cells were used as negative controls. Cells were cultured in the presence of various concentrations (0.001-100 μM) of EP-100 or unconjugated CLIP 71 for 1 to 24h. Viability was measured by formazan conversion assays. In addition, LHRH receptor expression in cancer cells was determined by immunohistochemistry and quantified by a computerized image scoring system: 0 for no receptor expression and 3 for maximum receptor expression. In vivo efficacy of EP-100 was conducted in nude mice implanted with OVCAR-3 cells Nu/Nu , , Cell viability was determined using formazan conversion assays


Background continued
First generation targeted oncolytic peptides consisted of synthetic lytic peptides conjugated to human chorionic gonadotropin or LHRH [5][6][7].They selectively kill Ovarian cancer is the fifth leading cause of death from cancer in women.New therapies are needed to improve the survival of patient.EP-100 is a novel oncolytic peptide that is being developed to selectively target surface luteinizing hormone releasing hormone (LHRH) receptors.It has been reported that approximately 80 % human ovarian cancer cells and their recurrences over-express LHRH receptors.EP-100 consists of an LHRH receptor-targeting ligand conjugated to a novel lytic peptide (CLIP71).EP-100 was tested in vitro and in vivo for activity in LHRH receptor overexpressing OVCAR-3 cells.LHRH receptor-negative SKOV-3 ovarian cancer cells were used as negative controls.Cells were cultured in the presence of various concentrations (0.001-100 μM) of EP-100 or unconjugated CLIP 71 for 1 to 24h.Viability was measured by formazan conversion assays.In addition, LHRH receptor expression in cancer cells was determined by immunohistochemistry and quantified by a computerized image scoring system: 0 for no receptor expression and 3 for maximum receptor expression.In vivo efficacy of EP-100 was conducted in nude mice implanted with OVCAR-3 cells Nu/Nu LHRH receptors) were incubated for 1, 2, 6 and 24 hours with EP-100 or CLIP-71 Cell viability was determined using formazan conversion assays OVCAR-3 xenograft model: Nu/Nu female nude mice bearing OVCAR-3 xenografts.Treatment was initiated on day 33 after tumor cell injection and continued on days 41 and 47 with a final necropsy on day 52.The doses for the 3 weekly injections were 0.02, 0.2 and 2 mg/kg body weight, given as a bolus single intravenous injection via lateral tail vein.Treatment groups included controls (10 mice each) injected with saline or CLIP71 (2 mg/kg); and EP-100 (0.02 (N=10), 0.2 (N=10), and 2 mg/kg (N=9),), and Cisplatinum (10 mg/kg, 3qd (N=10),).Tumors were obtained on the day of the first treatment to determine baseline measurements (N=9).Serum CA125 was used as a measure of drug activity.
PET imaging was conducted in treated and untreated mice bearing OVCAR-3 xenografts.
LHRH receptor expression was determined through quantitative immunoperoxidase analysis on cell cultures and tumor sections.EP-100 is a novel oncolytic peptide that selectively targets surface LHRH receptors.EP-100, has been tested extensively in vitro and in vivo and shows no adverse effects in various species when administered in repeated injections.EP-100 is a fast acting compound, that lacks hemolytic activity and is not immunogenic.

Summary
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[μM] (p<0.005) with increasing incubation time.EP-100 is a fast acting agent (4.9 μM) compared to the unconjugated drug.
-express hCG or LHRH receptors within hours of contact with the cells.The direct contact and interaction with the cancer cell membrane causes their disruption and cell death by necrosis [5-7].Esperance Pharmaceuticals™ has developed a new generation of oncolytic peptides conjugated to LHRH, that regress xenografts of breast, ovarian and prostate cancers in nude mice.

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To test in vitro efficacy of EP-100 in ovarian cancer cell lines • To test in vivo efficacy of EP-100 in an OVCAR-of EP-100 correlated with LHRH receptor expression.2. EP-100 destroyed LHRH receptor expressing OVCAR-3 cells within an hour (4.9 μM) 3. Unconjugated drug (CLIP 71) was slow acting 4. LHRH receptor negative cells (SKOV-3) showed similar sensitivity to EP-100 and CLIP71 regardless of conjugation to LHRH (p<0.005).The IC 50 in LHRH receptor negative SKOV 3 cells for EP 100 was also higher 11.5 μM after 24 h of incubation compared with CLIP71 incubations resulted in IC 50 values of 50 μM (p<0.005).EP-100 regressed tumors significantly (p < 0.001) at doses as low as 0.2mg/kg with several mice remaining tumor free.CLIP71, cisplatinum or saline had no effect on tumor growth.Tumor regression was greatest in mice treated with EP-100 at 0.2 mg/kg (p<0.03vs baseline).Tumor free mice were found in groups 0.2 and 2 mg/kg of EP-100.Cisplatinum and CLIP71 were not effective in reducing tumor weights.Serum CA125 secretion was reduced in EP-100 treated mice at 0.2 and 2 mg/kg (p<0.0002)compared to saline controls.These results indicate that EP-100 selectively targets and kills only cancer cells that over-express LHRH receptors.They demonstrate that EP-100 is a potential therapy for multi-drug resistant ovarian cancer in humans.regardless of conjugation to LHRH 5. EP-100 is specific for cells that express LHRH receptors 6.In vivo EP-100 reduced OVCAR-3 xenografts in weekly injections at doses as low as 0.2 mg/kg bodyweight 7. Controls (saline) and cisplatinum or CLIP71 treated mice showed tumor growth 8. Tumor weights were reduced in EP-100 treated groups 9.As measure of tumor viability serum CA125 was reduced in treatment groups 10.LHRH receptor levels were reduced in EP-100 treated tumors Background Traditional non-targeted treatments are often associated with serious side effects, are systemically active and do not discriminate between cancer and normal cells in vital organs.Further, patient's tumors often develop multi-drug resistance and the recurrence of their cancers often presents a more aggressive form of the disease resulting in death.An alternative approach under development by Esperance Pharmaceuticals for killi -100 treated tumors were necrotic 12.Treated tumors lacked [ 18 F]-FDG uptake 13.All treatment groups tolerated the injections well 14.No changes were observed in serum chemistry or complete blood count parameters or et al, Anticancer Drug Design Figure 1: EP-100 activity correlates with LHRH receptor levels.EP-100 reached its maximal activity in OVCAR-3 (LHRH receptor positive) cells within one hour (4.9 μM), whereas unconjugated CLIP71 resulted in IC 50 values of 33, 36, 12.5 and 11.9 [μM] (p<0 005) with increasing incubation time EP-100 is a fast acting agent (4 9 μM) Figure 2: A, B) Effect of EP-100 and cisplatinum on growth (volume) of OVCAR-3 xenografts.Treatments were administered through lateral tail vein injection (D 33, 41 and 47) as single bolus injection.Necropsies were conducted on day 52.Data are presented as mean   SEM.Arrows show dosing.C) shows the median tumor weights, D) median changes in tumor weights compared to baseline values.Treatment response measured as tumor regression was greatest in mice treated with EP-100 at 0.2 mg/kg (p<0.03vs baseline).Reduced tumor weights compared to saline controls and CLIP71 (p<0.05) were obtained in the groups for 0.2 and 2mg/kg dosages of EP-100.Cisplatinum and CLIP 71 were not effective in reducing tumor weights.E) Tumor marker ovarian cancer antigen CA125 relative to tumor weights at necropsy from OVCAR-3-xenograft study, Treatment response as tumor viability from median CA125 secretion compared to saline controls was greatest in mice treated with EP- The SKOV-3 cell line (LHRH receptor negative) showed similar sensitivity regardless of conjugation to LHRH.EP-100 exhibited its maximal efficacy (11.5 μM) after 24 h of incubation whereas CLIP71 incubations resulted in IC 50 values of 86, 96, 53 and 50 [μM] (p<0.005) with increasing incubation time.Data are presented as mean ± SEM, N = 8.EP-100 is not an appropriate target for SKOV-3 cells that do not express LHRH receptors p g 100 at 0.2 and 2 mg/kg, (p<0.0002).F) Treatment response as median CA125 secretion/Tumor weight.G) Tumors and LHRH receptor levels in control and treated tumors, Histological section of treated and control tumors (H&E, 40 x).H) PET image of control and treated mice show lack of viable tumor cells after treatment.