Analogue AN-238 Cancer and Its Metastases by Cytotoxic Somatostatin Inhibition of PC-3 Human Androgen-independent Prostate

We evaluated whether AN-238, the cytotoxic analogue of somatostatin (SST) consisting of the radical 2-pyrrolinodoxorubicin (AN-201) linked covalently to the SST octapeptide carrier RC-121 (D-Phe-Cys-Tyr-D-TrpLys-Val-Cys-Thr-NH2), could be used for targeting human primary and metastatic prostate carcinomas that express SST receptors (SSTRs). The antitumor activity and toxicity of AN-238 and its components were first characterized in nude mice bearing s.c. xenografts of PC-3 human androgen-independent prostate cancer. In experiment 1, AN-238 was injected once i.v. at 200 nmol/kg when the mean volume of s.c. tumors was about 30 mm. Administration of AN-238 inhibited tumor growth, as shown by a 74% decrease in tumor volume and by a 71% reduction in tumor weight after 7 weeks as compared with the control group. AN-201 at an equimolar dose did not show any antitumor activity. The mortality was 14.3% (one of seven mice) in the AN-238-treated group and 47% (three of seven mice) in mice that received AN-201. In experiment 2, two i.v. injections of AN-238 at 150 nmol/kg were given 10 days apart when the tumors measured 65–70 mm. A significant inhibition of tumor volume (62.3%; P < 0.001) and tumor weight (61.1%; P < 0.01) was observed after 4 weeks of treatment. AN-201, given alone at the same dose or coadministered with RC-121, had no significant effect on PC-3 tumors. The suppression of tumor growth induced by AN-238 was accompanied by a significant enhancement of apoptosis (P < 0.01). There were similar side effects in all treated groups, which included a transient loss of body weight and leukopenia. The effectiveness of AN-238 in a metastatic model was then investigated in animals implanted orthotopically with 2 3 10 PC-3 cells. Two i.v. injections of AN-238 or AN-201 at 150 nmol/kg were administered 10 days apart at 10 weeks after intraprostatic inoculation of PC-3 cells. After 4 weeks of treatment, the mean weight of primary tumors in animals receiving AN-238 was 77% lower (P < 0.01) than that in controls. This reduction was also significantly greater (P < 0.05) than that in animals given AN-201, which showed only a 34% inhibition (nonsignificant versus controls). All control animals and four of six (67%) mice treated with AN-201 developed metastases in the lymph nodes; however, no lymphatic spread of cancer was found in the AN-238-treated group. Using reverse transcription-PCR analysis, we demonstrated the expression of SSTR2 and SSTR5 in intraprostatic tumors and their metastases in lymph nodes as well as in s.c. tumors. The present study demonstrates the high efficacy of SSTR-targeted chemotherapy in a model of advanced human androgen-independent prostatic carcinoma, as shown by the inhibition of primary tumors and their metastases by the cytotoxic SST analogue AN-238. INTRODUCTION Prostate cancer is the most frequently diagnosed malignant neoplasm among American men and remains the second leading cause of cancer-related deaths (1, 2). In spite of major refinements in diagnosis, a significant number of prostate cancers are only detected at an advanced stage of the disease, with no possibility of cure by radical prostatectomy (2, 3). The principal approach to management of disseminated carcinoma of the prostate has continued to be essentially unchanged since 1941, when the effectiveness of androgen ablation was first reported by Huggins and Hodges (4). Although the rate of response to androgen deprivation is high (80%), the mean duration of the remission is only 18–36 months, and patients eventually die of hormone-refractory disease (5, 6). Conventional chemotherapy for prostate cancer that is no longer responsive to androgen deprivation was initially found to produce only marginal responses (7) but is now being reevaluated with interest as new agents and better supportive care become available (reviewed in Ref. 8). Nevertheless, palliation still remains the primary goal of current chemotherapy, and overall response rates are low and are associated with general toxicity. To overcome the problem of toxicity, attempts have been made to achieve site-specific drug delivery and improve the therapeutic index. Targeted chemotherapy using potent cytotoxic radicals conjugated to carrier molecules, such as antibodies (9, 10) or analogues of peptide hormones (reviewed in 11), that can be specifically recognized by tumor cells may improve therapy of androgen-independent tumors. Various tumors, including carcinoma of the prostate, express receptors for peptides such as LH-RH and SST (6, 11–14). Preclinical studies have shown that SST and its octapeptide analogues can inhibit the growth of prostate cancer and other malignancies (6, 11, 15–18). SSTRs were found on surgical specimens of primary and metastatic lesions of prostate cancer and visualized in vivo by scintigraphy with In-labeled octreotide (19, 20). Some clinical improvement was observed in patients with relapsed prostate cancer who were treated with SST analogues (21–24). The successful use of radiolabeled SST analogues for in vivo imaging of SSTR-positive tumors (20, 25, 26), as well as our own previous experience with cytotoxic analogues of LH-RH (6, 11) and a prototype peptide conjugate consisting of methotrexate linked to SST analogue RC-121 (27), encouraged us to design and synthesize a series of modern targeted cytotoxic analogues of SST (28). These conjugates consist of doxorubicin or its potent derivative 2-pyrrolinodoxorubicin (AN-201) (29) linked covalently to carrier SST octapeptide analogues such as RC-121 (30). The hybrids thus formed retain both the receptor affinity and the cytotoxic activity of their respective components (28, 31). Recently, Koppán et al. (31) demonstrated that one of these cytotoxic analogues, AN-238 containing AN-201 conjugated to RC-121, inhibited the growth of SSTR2positive Dunning R-3327-AT-1 anaplastic rat prostate cancer at nontoxic doses. Received 11/23/98; accepted 2/18/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by The Medical Research Service of the Veterans Affairs Department (A. V. S.), a CaPCURE Foundation Research Award (to A. V. S.), and a grant from ASTA Medica AG (Frankfurt am Main, Germany) to Tulane University School of Medicine (to A. V. S.). 2 On leave from the Department of Neuroendocrinology, Medical Center of Postgraduate Education, Warsaw, Poland. 3 To whom requests for reprints should be addressed, at Veterans Administration Medical Center, 1601 Perdido Street, New Orleans, LA 70112-1262. Phone: (504) 589-5230; Fax: (504) 566-1625. 4 The abbreviations used are: LH-RH, luteinizing hormone-releasing hormone; SST, somatostatin; SSTR, SST receptor; hSSTR, human SSTR; GAPDH, glyceraldehyde-3phosphate dehydrogenase; RT-PCR, reverse transcription-PCR; NOR, nuclear organizer region; BW, body weight.