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Effects of Indinavir in a Preliminary Phase I Study on Dogs with Stage III Slenic Hemangiosarcoma

ENRICO P. SPUGNINI1, VINCENZO ESPOSITO2,3, ANGELA M. GROEGER3,4, ROBERTO CASSANDRO2, NICOLETTA ONORI5, ANTONIO CHIRIANNI2 and ALFONSO BALDI1,6

1SAFU Department, Regina Elena Cancer Institute, Rome; 2Third Division D. Cotugno Hospital, Naples, Italy; 3International Society for the Study of Comparative Oncology (ISSCO), Silver Spring, MD, U.S.A.; 4Department of Cardio-Thoracic Surgery, University of Vienna, Vienna, Austria; 5Service of Pharmacy, Campus BioMedico University, Rome; 6Department of Biochemistry, Pathology Section, Second University of Naples, Naples, Italy

Abstract. HIV protease inhibitors are antiretroviral drugs able To critically evaluate the results obtained from cell to prevent production of infectious particles. It has been shown cultures and rodents and to better assess the degree of that these protease inhibitors are able to inhibit cancer- angiogenesis inhibition, a modified preclinical phase I/II promoted angiogenesis in patients affected by Kaposi’s trial in dogs with spontaneously occurring splenic sarcoma. A preliminary phase I study on dogs with stage III hemangiosarcoma (HA5) was undertaken. HA5 of the splenic hemangiosarcoma was designed in order to evaluate spleen is an aggressive soft tissue neoplasm that originates the efficacy and toxicity of the protease inhibitor Indinavir to from cancer endothelial cells and leads to the death of most delay the progression of this advanced neoplasm. The results affected dogs within 6 months from diagnosis (10, 11). The suggest that Indinavir is potentially beneficial in dogs affected adoption of Indinavir for the treatment of canine splenic by microscopic residual disease. HA5 is based on the fact that the tumor strongly produces angiogenic factors such as VEGF and bFGF (12). HIV protease inhibitors are antiretroviral drugs able to block the active site of HIV aspartyl protease, preventing Materials and Methods the production of infectious viral particles. Their use in combination with nucleoside inhibitors of HIV reverse Phase I study on dogs with stage III splenic hemangiosarcoma. transcriptase (highly active antiretroviral therapies; Cohorts of 3 dogs were enrolled in an escalation study of oral HAART) has led to impressive clinical outcome in AIDS Indinavir. Eligible dogs were required to have histologically patients (1). Recent publications suggest that protease confirmed stage III splenic HA5 according to the World inhibitors (PIs) used in the therapy of HIV-1 patients could Health Organization (WHO) staging system (Table I) and an have anti-neoplastic properties through a mechanism of owner-signed informed consent form detailing the risks, inhibition of the growth of tumor-induced blood vessels, as benefits and responsibilities associated with participation in suggested by the regression of Kaposi’s sarcomas (KS) in this trial. The staging process involved complete blood cell HIV-1 HHV8 (human herpesvirus 8) co-infected patients count, serum biochemistry, coagulation profile, urinalysis, (2-5). Numerous studies have shown that cytokines and thoracic radiographs (3 projections), abdominal growth factors play a major role in tumor growth, ultrasonography and cardiac ecography. Enrollment had to angiogenesis, inhibition of host defenses and the metastatic be performed within 3 weeks after surgical splectomy, at cascade (4, 5). Among others, basic fibroblast growth factor which time the dogs, average weight 30 kg, were scheduled to (bFGF) and vascular endothelial growth factor (VEGF) are receive 400 mg of Indinavir per os on an every-other-day basis highly expressed in many human and canine tumors (6-9). for a month (15 doses), as extrapolated from human and murine studies. If the protocol was well tolerated and the recheck imaging studies evidenced tumor responses or stable disease, the dogs would receive the treatment with the same Correspondence to: Enrico P. Spugnini, SAFU Department, Regina Elena Cancer Institute, Via delle Messi d’ oro, 156, 00158 Rome, schedule for another month. The following cohort was Italy. Fax: + 390652662505, e-mail: [email protected] planned to be treated on an every-day basis and, if the treatment was not associated with toxicities, the daily Key Words: Hemangiosarcoma, Indinavir, dog. schedule was to be increased by a 10% factor. Tumor

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Table I. Modified WHO staging system for canine splenic HA5. PI therapy due to reduced angiogenic activity or to excessive tumor mass. The role of Indinavir in the bleeding Stage I Tumor confined to the spleen observed in the dogs cannot be completely ruled out, Stage II Splenic tumor with ruptured capsule however, this phenomenon is less frequently encountered or spread to regional lymph node since the adoption of new and safer PIs (1). Further studies are warranted to identify new antiangiogenic molecules to Stage III Splenic tumor with measurable metastases control this aggressive canine neoplasm; a promising or multicentric disease strategy could be the association of such compounds with conventional anthracycline therapy to increase the action of the two drugs.

Acknowledgements progression or the occurrence of unacceptable hematological or gasto-intestinal toxicities were considered end-points. We thank Merck Sharpe & Dohme Italia for the gift of Indinavir and I.S.S.C.O. for its continuous support. A.B. was supported by grants from FUTURA-onlus; E.P.S. was partially supported by Results Grant 2005 of the Italian Ministry of Health.

The purpose of this pilot study was to evaluate the efficacy References and toxicity of Indinavir in a canine HA5 model. All the 3 subjects of the first cohort (2 German Shepherd and a 1 Carpenter CC, Cooper DA and Fischl MA: Antiretroviral mixed breed dog) died due to massive hemorrhage 14, 20 therapy in adults: updated recommendations of the International AIDS society-USA Panel. JAMA 283: 381-390, and 3 days after the enrollment. This could be ascribed to 2000. diffuse omental bleeding due to tumor spread, as reported 2 Aversa SM, Cattelan AM, Salvagno L, Crivellari G, Banna G, in the literature, to paraneoplastic disseminated Trevenzoli M, Chiarion-Sileni V and Monfardini S: Treatments intravascular coagulation (DIC), or to PI-induced bleeding. of AIDS-related Kaposi's sarcoma. Crit Rev Oncol Hematol 53: Coagulation profiles performed at the time of the 253-265, 2005. hemorrhages were within normal limits, thus excluding 3 Lebbe C, Blum L, Pellet C, Blanchard G, Verola O, Morel P, DIC. The profuse bleeding observed in the 3 dogs is more Danne O and Calvo F: Clinical and biological impact of antiretroviral therapy with protease inhibitors on HIV-related consistent with tumor progression in consideration of the Kaposi's sarcoma. AIDS 2: F45-F49, 1998. tumor type and advanced stage. Moreover, the PI Indinavir 4 Cattelan AM, Calabro ML, Aversa SM, Zanchetta M, has been reported to be associated with bleeding in Meneghetti F, De Rossi A and Chieco-Bianchi L: Regression humans, usually localized at the site of muscles, joints, soft of AIDS-related Kaposi's sarcoma following antiretroviral tissue, oral mucosa and urinary tract, while intracranial, therapy with protease inhibitors: biological correlates of clinical intra-thoracic and abdominal bleeding have rarely been outcome. Eur J Cancer 35: 1809-1815, 1999. described (13, 14). 5 Sgadari C, Barillari G, Toschi E, Carlei D, Bacigalupo I, Baccarini S, Palladino C, Leone P, Bagarini R, Malavasi L, CAfaro A, Falchi M, Valdembri D, Rezza G, Bussolino F, Discussion Monini P and Ensoli B: HIV protease inhibitors are potent anti-angiogenic molecules and promote regression of Kaposi On the basis of the cell culture investigations and the sarcoma. Nature Med 8: 225-232, 2002. preclinical studies conducted in vitro on cell lines and in 6 Hanahan D and Folkman J: Patterns and emerging mechanisms vivo on ectopic murine tumor xenografts (2-5), a pilot study of the angiogenic switch during tumorigenesis. Cell 86: 353-364, for the evaluation of efficacy and toxicity of Indinavir in a 1996. 7 Singh RK, Bucana CD, Gutman M, Fan D, Wilson MR and canine HA5 model was set up. Stage III HA5 is extremely Fidler IJ: Organ site-dependent expression of basic fibroblast metastatic and its usual outcome after splenectomy is the growth factor in human renal cell carcinoma. Am J Pathol 145: death of the animal. The addition of a doxorubicin-based 365-374, 1994. protocol, albeit efficacious, failed to significantly extend the 8 Fosmire SP, Dickerson EB, Scott AM, Bianco SR, Pettengill survival; the average survival of stage III dogs receiving MJ, Meylemans H, Padilla M, Frazer-Abel AA, Akhtar N, antiblastic drugs was 87 to 107 days (15, 16). The massive Getzy DM, Wojcieszyn J, Breen M, Helfand SC and Modiano and fatal bleeding of the first cohort of 3 dogs has led to JF: Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium. Lab Invest 84: 562-572, 2004. the termination of this study in consideration of the limited 9 Clifford CA, Hughes D, Beal MW, Mackin AJ, Henry CJ, efficacy of Indinavir in dogs with stage III splenic HA5. Shofer FS and Sorenmo KU: Plasma vascular endothelial This could be ascribed to the advanced stage of the growth factor concentrations in healthy dogs and dogs with neoplasm in our subjects, making them less responsive to hemangiosarcoma. J Vet Intern Med 15: 131-135, 2001.

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