Publications

2015
The Company presented a poster at the American Association for Cancer Research in Philadelphia, Pennsylvania on April 18, 2015.

Assessment of the antitumor activity of leech (huridinaria manillensis) saliva extract in prostate cancer

Amr E. Ammar, Mohamed H. Hassona, Gray R. Meckling, Leslie G. Chan, Mei Y. Chin, Abdulrahman Abdualkader, Mohamed Alaama, Ahmed Merzouk, Abulbashar Helaluddin, Abbas Ghawi, Omer Kucuk, Emma S. Guns. UBC, Vancouver, BC, Canada; International Islamic University of Malaysia, Kuantan, Malaysia;Winship Cancer Institute of Emory University, Atlanta, GA

Abstract

Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. Leech saliva contains a large number of peptides and proteins, which have anti-thrombotic, antimicrobial, antitumor and anti-metastatic activities. Currently, leech therapy has an established role as an important tool in microsurgery, reconstructive surgery and salvage of grafted tissues.

Leech saliva extract (BPS-001) was prepared as an aqueous solution from frozen lyophilized powder. LSE (BPS-001) IC50 was determined in-vitro in five prostate cancer cell lines using MTS cell viability assay. In-vivo efficacy of LSE was determined in LNCaP and 22RV-1 in nude mice xenograft models. Mice were injected with 2×106 LNCaP or 22RV-1 cells subcutaneously; the mice were castrated in both studies to resemble castration resistant prostate cancer. After castration, mice were divided into four groups of 6-8 each. Mice were subcutaneously injected with either LSE (BPS-001) (5 mg/kg) once a week, LSE (BPS-001) (5 mg/kg) twice a week, docetaxel (10 mg/kg) or vehicle twice a week. PSA, tumor volume, and weight were measured weekly in the LNCaP model; and in the 22RV-1 tumor volume and weights were measured twice weekly. After four weeks of treatment, mice were euthanized, tumors and organs were collected for transcriptome and histopathological analysis.

LSE (BPS-001) induced cell death in a panel of prostate cancer cell lines including LNCaP, PC3 and 22RV-1. IC50 values of were determined as 22 μg/ml in both LNCaP and PC3 cells and 53 μg/ml in 22RV-1cells. Furthermore, in vivostudies show that LSE (BPS-001) once and twice weekly regimens both caused significant decrease in PSA and tumor volume compared to control. There was no significant difference between the antitumor activity of LSE and docetaxel. Interestingly, once weekly treatment with LSE (BPS-001) was associated with significant weight gain (due to good dietary intake) at several time points. Immunohistochemical staining (IHC) showed significant increase in caspase-3 and significant decrease in P21, Ki-67, and PCNA expression in the LSE (BPS-001) treated mice compared to the control group. Transcriptome analysis of tumor samples showed that LSE (BPS-001) had significant immunomodulatory, anti-inflammatory, along with significant effects on cell-cell adhesion, induction of glutathione transferase and inhibition of certain growth factors. Consequently, these effects led to significant cell cycle arrest, increase in apoptosis and decrease in proliferation.

LSE (BPS-001) has significant in vitro and in vivo anti-tumor activity with no apparent side effects. This can be attributed, at least partly, to its ability to inhibit cellular proliferation and induce apoptosis through its modulation of immunity, cell-cell adhesion, and inflammation.

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The Company presented a poster at the American Society of Clinical Oncology in Chicago, Illinois on May 30, 2015.

Assessment of the antitumor activity of leech (huridinaria manillensis) saliva extract in prostate cancer.

Mohamed Hessein, Amr Ammar, Mei Chin, Abdulrahman Abdualkader, Mohamed Alaama, Ahmed Merzouk, Abul Bashar Helaluddin, Abbas Ghawi, Omer Kucuk, Emma Guns; Vancouver Prostate Centre, UBC, Vancouver, BC, Canada; International Islamic University Malaysia, Kuantan, Pahang, Malaysia; The Winship Cancer Institute of Emory University, Atlanta, GA

Abstract

Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. Leech saliva contains a large number of peptides and proteins, which have anti-thrombotic, antimicrobial, antitumor and anti-metastatic activities. Currently, leech therapy has an established role as an important tool in microsurgery, reconstructive surgery and salvage of grafted tissues. Methods: LSE (BPS-001) IC50 was determined in-vitro in five prostate cancer cell lines using MTS cell viability assay. In-vivoefficacy of LSE (BPS-001) was determined in LNCaP and 22RV-1 in nude mice xenograft models. Castrated tumor bearing mice were divided into four groups of 6-8 each. Mice were subcutaneously injected with either LSE (BPS-001) (5 mg/kg) once a week, LSE (BPS-001) (5 mg/kg) twice a week, docetaxel (10 mg/kg) or vehicle twice a week for four weeks. PSA, tumor volume, and weight were measured periodically. Results: In-vitro, LSE (BPS-001) induced cell death in a panel of five prostate cancer cell lines. In vivo studies showed that LSE (BPS-001) once and twice weekly regimens both caused significant decrease in PSA and tumor volume compared to control with no significant difference from docetaxel treated group. Interestingly, once weekly treatment was associated with significant weight gain (due to good dietary intake) at several time points. Immunohistochemical staining (IHC) showed significant decrease in P21and Ki-67 expression in the LSE (BPS-001) treated mice compared to the control group in the LNCaP model. Active caspase-3 was increased at once and twice weekly doses in both models confirming the pro-apoptotic properties of LSE (BPS-001). Transcriptome analysis of tumor samples showed that LSE (BPS-001) had significant immunomodulatory, anti-inflammatory, along with significant effects on cell-cell adhesion, induction of glutathione transferase, inhibition of certain growth factors, and modulating several transcription factors. Conclusions: LSE (BPS-001) has significant in vitro and in vivo anti-tumor activity with no apparent side effects. This can be attributed, at least partly, to its ability to inhibit cellular proliferation, induce apoptosis and arrest cell cycle through its modulation of immunity, cell-cell adhesion, and inflammation.

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2014
The Company presented a poster at the SIO International Oncology Conference in Houston, Texas on October 28, 2014.

In Vivo Assessment of the Therapeutic Effects of Lyophilized Leech Saliva Extract From (Huridinaria manillensis) on Different Tumor Xenograft Models in Nude Mice

M.D.H Hassona; A.E. Ammar; T.C. Gao; M.Y. Chin; A.M. Abdualkader; M. Alaama; A. Merzouk; O. Kucuk, E.S. Guns*
Vancouver Prostate Centre, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada ; Pharmaceutical Chemistry Department, Faculty of Pharmacy, International Islamic University Malaysia, Kuantan, Pahang, Malaysia; Biopep Solutions Inc., Vancouver, BC, Canada; Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, Georgia, USA

Abstract

Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. A large number of peptides and proteins have been identified and characterized in leech saliva extract (BPS-001) including anti-thrombotic agents, cancer metastasis inhibitors and anti-microbials. Currently, leech therapy is established as an important tool in microsurgery and reconstructive operations having demonstrated superior clinical outcomes for the optimal salvage of grafted tissues.

In the current study, we have determined the toxicity of LSE (BPS-001) from (Huridinaria manillensis) in CD1 mice. We used 5 groups of mice, 3 mice per group, which were subcutaneously injected with vehicle or 1,5,10 and 20 mg/kg of LSE (BPS-001) twice weekly for 4 weeks. After 4 weeks, we collected blood samples for CBC as well as liver and kidney function tests. In addition, liver, spleen, kidney, lung and brain tissues were collected for further toxicological analysis (ongoing). To evaluate the in vivo efficacy of LSE (BPS-001) in the PC3 tumor model, we used 4 groups of male nude mice, six mice per group, which were subcutaneously injected with 0.5 mg/kg or 1 mg/kg LSE (BPS-001), docetaxel (15 mg/kg) as a positive control or vehicle, respectively. Currently, we are determining the effect of LSE (BPS-001) on the LNCaP xenograft mouse model (ongoing study).

The data demonstrate that LSE (BPS-001) is safe when administered up to 20 mg/kg with no toxicity. There is a significant decrease in the growth of PC3 xenografts with either docetaxel or LSE (BPS-001) (1 mg/kg) treatment compared to the vehicle-treated control mice. There was no significant difference between the anti-tumor activity of docetaxel and LSE (BPS-001) (1 mg/kg). There was a significant decrease in the body weight of docetaxel-treated mice while there was no change in the body weight of BPS-001-treated mice
To our knowledge this is the first report of LSE (BPS-001) as a safe biological agent with significant anti-tumor activity in the PC3 prostate cancer xenograft model with no apparent side effects.

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2013

In – vivo Anti – hyperglycemic Activity of Saliva Extract From the Tropical Leech Hirudinaria Manillensis

Abdualkader, Abdualrahman Mohammed, Ghawi, Abbas Mohammad, Alaama, Mohamed, Awang, Mohamed, Merzouk, Ahmed

Abstract

The anticoagulant effect of leech saliva was traditionally employed in the treatment of diabetes mellitus complications such as peripheral vascular complications. This study was carried out to examine the effect of leech saliva extract (BPS-001) on blood glucose levels in alloxan-induced diabetic rats. First, LSE (BPS-001) was collected from leeches which were fed on a phagostimulatory solution. Second, total protein concentration was estimated using the Bradford assay. Third, diabetic rats were injected subcutaneously (sc) with LSE (BPS-001) at doses of 500 and 1000 µg·kg 1body weight (bw). Other diabetic rats were injected sc with insulin at doses of 10 and 20 U·kg 1bw. Another group was injected simultaneously with LSE (BPS-001) (250µg·kg 1bw) and insulin (10 U·kg 1bw). Fasting blood glucose (FBG) concentrations were monitored during a study period of eight hours at regular intervals. Findings showed that both doses of LSE (BPS-001) resulted in a significant and gradual decrease in FBG starting from 10%−18% downfall after two hours of injection reaching the maximal reduction activity of 58% after eight hours. Remarkably, LSE (BPS-001) was sufficient to bring the rats to a near norm-glycemic state. The high dose of insulin induced a severe hypoglycemic condition after 2−4 h of injection. The lower dose was able to decline FBG for 2−6 h in rats which became diabetic again after 8 h. On the other hand, the concurrent injection of low doses of LSE (BPS-001) and insulin produced a hypoglycemic effect with all rats showing normal FBG levels. Taken together, these findings indicated that the subcutaneous injection of LSE (BPS-001) of the medicinal Malaysian leech was able to provide better glycemic control compared with insulin. Moreover, the synergism between LSE (BPS-001) and insulin suggests that LSE (BPS-001) could be utilized as an adjuvant medication in order to reduce insulin dosage or to achieve better control of blood glucose.

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2012

Anticancer Effects of Medical Leech Saliva Extract BPS-001

Ahmed Merzouk, Abbas Mohammad Ghawi, Abdualrahman M. Abdualkader, Abubakar Danjuma Abdullahi and Mohamed Alaama

Abstract

Leech saliva contains biologically active compounds that are mainly proteins & peptides. Small cell lung cancer (SCLC) is a form of cell lung carcinoma. In this study a modified and smooth extraction method of saliva was used without leech scarification. Trying to find out the biological activity of medical Malaysian Leech Saliva Extract as cytotoxic in vitro, the SW 1271 cell line was grown and maintained in Leibovitz’s L-15 medium supplemented with 10% foetal bovine serum at 37°C in normal atmospheric air. Serial dilutions of LSE (BPS-001) were added to the cell line SW 1271 media for testing the cytotoxic activities. Result revealed that the LSE(BPS-001) has a cytotoxic activity against small cell lung cancer(SW 1271 cell line) with IC50 of 119.844 μg/ml compared with IC50 values of two reference standard drugs Irinotecan (5.81 μg/ml) and Carboplatin(18.754 μg/ml). In a combination regimen, LSE (BPS-001) reduced the IC50 of Carboplatin & Irinotecan by 65% & 11.5% respectively. Carboplatin reduced the IC50 of LSE (BPS-001) by 4.6%, while Irinotecan reduced it by 57%. This results provides a promising novel agent for treatment of small cell lung cancer (SCLC) at least in vitro, more researches are needed.

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Free Radical Scavenging Activity of the Medicinal Malaysian Leech Saliva Extract (BPS-001), Hirudinaria Manillensis

Abbas Mohammad Ghawi, Abdualrahman M. Abdualkader, Ahmed Merzouk and Mohamed Alaama

Abstract

Antioxidants from natural sources have been arisen as prophylactic and therapeutical agents in many life-threatening disorders such as cancer and cardiovascular diseases. Leech therapy has been used since the extreme old ages in a variety of abnormalities. The current study was executed to examine the antioxidant activity of the salivary gland secretion of the medicinal Malaysian leech using DPPH free radical scavenging activity method. Leech saliva extract (BPS-001) was collected from starved leeches after feeding them on the phagostimulatory solution of 0.001M arginine in 0.15 M sodium chloride. Total protein concentration was 78.753 ± 2.406 μg/ml. A series of different dilutions of LSE (BPS-001) were mixed with DPPH in a methanolic medium and the changes in absorbance were measured at 516nm. Results showed that LSE (BPS-001) expressed a free radical scavenging activity with IC50 of 7.282 μg/ml compared with 5.803 μg/ml of L-ascorbic acid as a positive control. Therefore, this study revealed that the proteomic contents of LSE (BPS-001) are promising natural antioxidants.

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Season Variation and Stavaction Period Influence on the Antithrombotic Activity of Leech Saliva Extract from Medicinal Malaysian Leech Hirudinaria Manillensis

2012 Abbas Mohammad Ghawi, Abdualrahman M Abdualkader, Ahmed Merzouk and Mohamed

Abstract

Leech therapy has been practiced for a wide range of therapeutical purposes since the extreme old ages. Nowadays, leech application in plastic and microsurgery has been considered as a promising tool. In Malaysia, traditional physicians have used the medicinal leeches as an effective remedy for bloodletting and many body disorders. Leech saliva extract (BPS-001) was collected after feeding leeches on the phagostimulatory solution through parafilm membrane. The total protein concentration was estimated using Bradford assay. The antithrombin activity was evaluated using the amidolytic assay of the synthetic substrate S-2238 and thrombin time assay in vitro. It was found that LSE (BPS-001) could inhibit thrombin-medicated hydrolysis of the substrate. The extract effectively prolonged thrombin time of the citrated plasma in a linear dose-dependent manner. It was found that the extract collected during the dry season was more biologically active than those collected during the rainy season. Likewise, results revealed that the longer the starvation period, the lower the antithrombin activity. For effective utilization of leech therapy or leech products, we recommend to be used during the dry season and after a starvation period not more than 16 weeks.

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