[CANCER RESEARCH 60, 2996 –3001, June 1, 2000] Inhibition of Metastatic Renal Cell Carcinomas Expressing Somatostatin Receptors by a Targeted Cytotoxic Analogue of Somatostatin AN-238 1 Artur Plonowski, 2 Andrew V. Schally, 3 Attila Nagy, Hippokratis Kiaris, Francine Hebert, and Gabor Halmos Endocrine, Polypeptide and Cancer Institute, Veterans Administration Medical Center, New Orleans, Louisiana 70112-1262 [A. P., A. V. S., A. N., H. K., F. H., G. H.], and Section of Experimental Medicine, Department of Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112-2699 [A. P., A. V. S., A. N., H. K., G. H.] ABSTRACT The effectiveness of chemotherapy targeted to somatostatin (SST) re- ceptors based on cytotoxic SST analogue AN-238, consisting of 2-pyrro- linodoxorubicin (AN-201) linked to SST carrier octapeptide, was investi- gated in human renal cell carcinomas (RCCs). SST receptors, which showed high-affinity binding for AN-238, were found in the SW-839 RCC line with sst 2A subtype and in the 786-0 RCC line, which expressed the sst 5 subtype. CAKI-1 RCC, which does not express sst 2A or sst 5 , was used as a negative control for testing the specificity of SST receptor targeting. Using microsatellite analysis, AN-238 was shown to selectively inhibit the proliferation of 786-0 line, but not the CAKI-1 RCC line in vitro. The effects of three i.v. injections of 150 nmol/kg of AN-238 or AN-201, given on days 1, 8, and 21, were evaluated in groups of nude mice bearing s.c. xenografts of SW-839 and 786-0 RCC. After 5 weeks, the volumes of SW-839 and 786-0 RCC tumors were decreased by 67.2 (P < 0.05) and 78.3% (P < 0.01), respectively, whereas AN-201 had no significant effect on tumor growth. The inhibition of SST receptor-negative CAKI-1 tumors by AN-238 was only marginal. To investigate the efficacy of SST receptor- targeted chemotherapy in metastatic RCC, three i.v. injections of AN-238 or AN-201 at 150 nmol/kg were given at biweekly intervals to nude mice implanted with 786-0 tumors under the renal capsule. After 6 weeks, the weight of orthotopic tumors treated with AN-238 (55.3  44.3 mg) was significantly lower (87% reduction; P < 0.001) than that in the control group (414.2  41.0 mg) or in animals given AN-201 (270.2  60.3 mg; P < 0.05). Five of six animals (83%), both in the control and the AN-201 group, developed metastases to lymph nodes, but only one of seven mice (14%) given AN-238 showed lymphatic spread. Lung metastases were found in 83% of controls and 50% of AN-201 treated animals, but none occurred in mice treated with AN-238. This study demonstrates that targeted cytotoxic SST analogue AN-238 provides an effective therapy for chemoresistant neoplasms such as RCC. Because most clinical RCCs express SST receptors, this treatment modality might be beneficial to patients with metastatic disease. INTRODUCTION In the past two decades, the incidence rate of RCC 4 has increased by 45% (1). In the United States alone, 30,000 new cases of RCC and 12,000 deaths from this malignancy were estimated for 1999 (2). Approximately one-third of the RCC cases have documented meta- static spread at the time of the initial presentation, and 50% of patients with presumably organ-confined disease who are treated with radical nephrectomy are expected to develop metastases within 10 years (3). The prognosis for metastatic RCC remains dismal because of its resistance to both chemotherapy and radiotherapy (4). Immu- notherapy of advanced RCC has only a marginal effectiveness, whereas its side effects substantially decrease the quality of life (5–7). To overcome the problem of chemoresistance, efforts have been made to use cytotoxic drugs in combination with agents that can reverse the MDR mechanism (8). This approach, however, has led only to an increased toxicity to normal organs that are protected by MDR, such as bone marrow or brain (9). Alternatively, the MDR in cancer cells can be abolished by enhanced local accumulation of a cytotoxic agent. This can be achieved by targeted delivery of cyto- toxic radicals coupled to carriers such as monoclonal antibodies or analogues of peptide hormones that preferentially bind to malignant cells (10, 11). Thus, cytotoxic hybrids of peptide hormones such as LHRH, bombesin, or SST that can be targeted to their respective receptors on various tumors were shown to be more potent and less toxic than their unconjugated cytotoxic radicals (11). One of these cytotoxic analogues, AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier SST analogue RC-121, was recently dem- onstrated to strongly inhibit experimental SST receptor-positive tu- mors that are resistant to conventional chemotherapeutics (12, 13). Several previous reports demonstrated that most kidney neoplasms express receptors for SST (14 –16). Thus, 72% of specimens of human RCC were shown to bind the SST octapeptide analogue 125 I-[Tyr 3 ]- octreotide with high affinity (14). PCR analysis of another series of 13 samples of human RCC revealed that subtype 2 of the SST receptor (sst 2 ) was expressed in all of the specimens, whereas sst 1 and sst 4 were expressed in 85 and 46% of the specimens, respectively (15). In a clinical setting, the scintigraphy with radiolabeled octreotide was useful for the visualization of RCC in patients, especially in cases with multiple lesions (16). These findings suggest that targeted chemother- apy with cytotoxic SST analogues may have potential application in SST receptor-positive RCC. In the present study, we investigated the effectiveness of SST receptor-targeted chemotherapy in experimental models of human RCC. We first showed that cytotoxic SST analogue AN-238 binds selectively to SST receptor-positive RCC lines and inhibits their proliferation in vitro. We then demonstrated that analogue AN-238 inhibits the growth and metastatic dissemination of RCC in vivo. These findings, which suggest that targeted chemotherapy based on AN-238 could be considered for patients with advanced, SST recep- tor-positive RCC, are described herein. MATERIALS AND METHODS Chemicals The SST analogue RC-121 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH 2 ) was synthesized in our laboratory as described previously (17). The cytotoxic conjugate AN-238 was made by coupling one molecule of 2-pyrrolinodoxo- rubicin-14-O-hemiglutarate to the NH 2 terminus of [Lys(Fmoc) 5 ]RC-121 fol- lowed by deprotection and purification (18). The cytotoxic radical AN-201 was prepared as described previously (19). GHRH [hGHRH(1-29)NH 2 ] was syn- thesized in our laboratory. RC-160 (Vapreotide) was obtained from Debio- pharm (Lausanne, Switzerland). Received 12/6/99; accepted 4/3/00. 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 This work was supported by The Medical Research Service of the Veterans Affairs Department and a grant from ASTA Medica AG, Frankfurt am Main, Germany to Tulane University School of Medicine (both to A. V. S.). 2 On leave from Department of Neuroendocrinology, Medical Center of Postgraduate Education, Warsaw, Poland 04-158. 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: RCC, renal cell carcinoma; MDR, multidrug resistance; LHRH, luteinizing hormone-releasing hormone; SST, somatostatin; GHRH, growth hor- mone-releasing hormone; RT-PCR, reverse transcription-PCR; BW, body weight; GH, growth hormone; CE, carboxylesterase. 2996 Research. on February 8, 2016. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from