[CANCER RESEARCH 58, 4132-4137, September 15. 1998)

Targeted Cytotoxic Analogue of Somatostatin AN-238 Inhibits Growth ofAndrogen-independent Dunning R-3327-AT-1 Prostate Cancer in Ratsat Nontoxic Doses1

Miklos Koppan,2 Attila Nagy, Andrew V. Schally,3 Jose M. Arencibia, Artur Plonowski, and Gabor Halmos

Endocrine, PoH'peptide and Cancer Institute, Veterans Affairs Medical Center, New Orleans, Louisiana 70146; and Department of Medicine, Tulane University School of

Medicine, New Orleans, Louisiana 70112

ABSTRACT

Receptors for somatostatin (SST) that are found on prostate cancersmight be used for targeting of chemotherapeutic agents. Thus, doxorubi-cin derivative 2-pyrrolinodoxorubicin (AN-201) can be linked to SST

analogue RC-121 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2) to formtargeted cytotoxic SST analogue AN-238. In this study, we evaluated theeffects of AN-238 on the growth of SST receptor (SSTR)-positive andro-gen-independent Dunning R-3327-AT-1 prostate cancers in Copenhagenrats. The dose range and tumor growth-inhibitory effects of AN-238 andAN-201 were investigated in preliminary experiments. Administration ofcytotoxic radical AN-201 at single i.v. doses of 110, 125, and 150 nmol/kgresulted in 0, 77.7, and 100% mortality, respectively, within 6-10 days.Four weeks after the injection of 110 nmol/kg AN-201, mean tumor

volume was reduced by 35.1% (P < 0.05), as compared with controls. Incontrast, a single i.v. injection of analogue AN-238 at a dose of 300

nmol/kg was nontoxic and remarkably potent in inhibiting the growth ofDunning AT-1 tumors, resulting in a 85.9% (/' < 0.01) reduction in tumor

volume after 4 weeks. Treatment with AN-238 extended the survival timeof tumor-bearing rats from 52.0 ±3.75 to 91.8 ±3.70 days, corresponding

to a 76.5% (P < 0.01) increase. In a comprehensive experiment, wecompared the effects of radical AN-201 at 115 nmol/kg, analogue AN-238at 115 and 300 nmol/kg, carrier SST analogue RC-121 at 300 nmol/kg, anda mixture of AN-201 and RC-121 at doses of 300 nmol/kg administered i.v.Administration of AN-201 at 115 nmol/kg led to 90.0% mortality in 12days, but animals treated with 115 nmol/kg of AN-238 showed no signs oftoxicity, their tumor volume was reduced by 40.0% (/' < 0.05), and their

tumor weight was reduced by 42.8% (P < 0.01) after 4 weeks, as compared with controls. The dose of 300 nmol/kg of AN-238 was also nontoxicand diminished tumor volume by 80.9% (/' < 0.01) and tumor weight by

82.0% (P < 0.01). No reduction in tumor growth or toxic effects wasobserved with carrier RC-121, but after the injection of unconjugatedmixture of AN-201 and RC-121 at doses of 300 nmol/kg, all rats diedwithin 4 days. Specific high-affinity receptors for SST were found onDunning R-3327-AT-1 tumor membranes by radioligand binding assayand were identified by reverse transcription-PCR as SSTR2. Our studyindicates that cytotoxic SST analogue AN-238 can be targeted to SSTRs ontumors and produces a powerful inhibition of the growth of Dunning-AT-1 rat prostate cancer at doses that are nontoxic, whereas its cytotoxiccomponent, 2-pyrrolinodoxorubicin, is toxic and ineffective.

INTRODUCTION

Carcinoma of the prostate is the most common malignant tumor inmen, and in the United States, -185,000 new cases and -39,000

deaths from this disease are estimated for 1998 (1). About 40% of new

Received 4/17/98; accepted 7/17/98.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 with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by a grant from ASTA Medica AG (Frankfurt am Main.

Germany) and CaPCURE to Tulane University School of Medicine and by the MedicalResearch Service of the Veterans Affairs Department (to A. V. S.).

2 Present address: Department of Human Anatomy. University Medical School of

Pecs, Pecs, H-7643 Hungary.1To whom requests for reprints should be addressed, at Veterans Affairs Medical

Center, 1601 Perdido Street, New Orleans, LA 70146. Phone: (504)589-5230: Fax:(504)566-1625.

diagnoses are made at an advanced stage of the disease, when cure byradical prostatectomy is not possible (2). The current therapeuticmethods for the management of advanced prostate cancer are palliative and based upon androgen deprivation (3-5). However, castration,antiandrogens, or analogues of luteinizing hormone-releasing hor

mone can only provide a remission (4), and most patients will relapsefrom androgen control in 18-36 months (3-5). Therapeutic optionsare limited for patients who relapse. The treatment of androgen-

independent prostate cancer is a major oncological challenge, and newtherapeutic approaches must be developed. Low response rates withhigh toxicity are found with conventional chemotherapy (6). Weeklyadministration of doxorubicin, which is one of the agents used in thetherapy of advanced androgen-independent prostate cancer, produces

a weak response with significant toxic effects (7). A local delivery ofchemotherapeutic agents to malignant cells would greatly reduce theirtoxicity while enhancing the tumoricidal effect (3). Thus, targetedcytotoxic agents could be formed by linking various cytotoxic radicalsto peptide hormone carriers that have specific binding sites on tumortissues (3).

Various primary human tumors, such as endocrine, mammary,gastric, renal, and prostatic tumors, and tumor cell lines were shownto possess SSTRs4 (3, 8-14). Experimental oncological studies dem

onstrated that SST and its octapeptide analogues, such as octreotide orRC-160, can inhibit the growth of some of these malignancies (3, 15,

16). Most of the octapeptide SST analogues bind selectively to SSTR2and SSTRS subtypes (17). Although SSTR2 subtype was not found inprimary prostate cancer specimens using in situ hybridization, theexpression of octreotide-preferring SSTRs on metastatic hormone-

refractory prostatic adenocarcinoma has been verified (18, 19). Inaddition, the expression of SSTRS subtype in epithelial cells fromprimary cultures of surgically removed prostate cancer specimens wasdemonstrated by the RT-PCR method (9). High-affinity binding ofoctapeptides RC-160 and RC-121 was also described on human

prostate cancer specimens removed by radical prostatectomy (14).Tumor growth inhibition and direct antiproliferative effect of SST orits analogues have also been demonstrated in rat prostate cancermodels, in human prostatic cancer cell lines xenografted into nudemice, and in vitro (11, 20-24). Clinical studies have demonstrated

improvement in performance status and bone pain, fall in PSA, andprolongation of survival in patients with metastatic hormone-refrac

tory prostate cancer after treatment with some SST analogues (25, 26).The demonstration of SSTRs on various cancers stimulated the

development of radiolabeled octapeptide analogues for the localization of tumors (3, 16, 27, 28). Thus, ['"in-DTPA-D-Phe'joctreotide

was successfully used for scintigraphic imaging of different cancers in> 1000 patients (29). These findings and our previous experience withthe antitumor effects of an early targeted cytotoxic SST analogue,AN-51, consisting of RC-121 and methotrexate (30), prompted us to

develop modern, targeted cytotoxic analogues of SST. Recently, wedescribed the synthesis and evaluation of such SST analogues con-

4 The abbreviations used are: SSTR, somatostatin receptor; SST. somatostatin; RT-

PCR. reverse transcription-PCR.

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CYTOTOXIC SOMATOSTATIN ANALOGUE IN PROSTATE CANCI.R

taining doxorubicin or its intensely potent derivative 2-pyrrolinodoxo-

rubicin (31 ). These compounds were developed for therapy of cancersthat possess receptors for SST (31).

Here, we evaluated the antiproliferative effects of targeted cyto-toxic SST analogue AN-238 in rats bearing the rapidly growing andhormone-insensitive Dunning R-3327-AT-1 prostate cancer, whichwas previously shown to bind SST octapeptides such as RC-160 (24).The effects of analogue AN-238 were compared to those exerted byits components, carrier SST analogue RC-121 and cytotoxic radicalAN-201.

MATERIALS AND METHODS

Peptides and Cytotoxic Agent. The SST analogue RC-121 (o-Phe-

Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2) and 2-pyrrolinodoxorubicin (AN-201)

were synthesized in our laboratory as described (32, 33). The cytotoxicconjugate AN-238 was made by coupling one molecule of 2-pyrrolinodoxorubicin- 14-O-hemiglutarate to the NH2 terminus of [Lys(yV-(9-fluorenyD-methoxycarbonyl)5]RC-121, followed by deprotection and purification (31).

The SST analogue RC-160 was synthesi/.ed by Debiopharm (Lausanne. Swit

zerland). Before i.v. injection, all compounds were dissolved in 20 fil of 0.1 Nacetic acid and diluted with 5% (w/v) aqueous D-mannitol solution (Sigma

Chemical Co.. St. Louis, MO).Animals. Six-week-old male Copenhagen rats were purchased from the

National Cancer Institute (Frederick Cancer Research and Development Center, Frederick. MD). The rats were housed four per cage in a temperature-controlled room with a 12-h light/12-h dark schedule and were given water and

standard rat chow ad libitum. All studies were performed in accordance withinstitutional guidelines of animal care and the welfare of animals in experimental neoplasia.

Experimental Protocols. The Dunning R-3327-AT-1 tumor cells were

originally kindly provided by Dr. John T. Isaacs (Department of Urology.James Buchanan Brady Urological Institute, The Johns Hopkins Hospital,Baltimore. MD) and maintained in tissue culture as described (24). DunningR-3327-AT-1 tumor cells (3 X IO6) were injected s.c. into three rats. After 2

weeks, resulting tumors were aseptically dissected and mechanically minced;3-mm' pieces of tumor tissue were transplanted s.c. by trocar needle into rats

under methoxyflurane anesthesia (Metofane; Pittman-Moore. Mundelein. IL).The experiments started when tumors had grown to ~70 mm' in volume. In

the first experiment, single i.v. injections of AN-201 at 110. 125. and 150

nmol/kg doses were given to rats (six to nine per group) through the jugularvein under anesthesia. Tumor growth was measured weekly using microcali-

pers. Body weights and deaths related to toxicity were recorded. In a secondexperiment, groups of five rats were injected i.v. with either 300 nmol/kg ofAN-238 or vehicle (controls). Tumor volumes and body weights were meas

ured weekly, and survival time in both groups was determined. In a third,comprehensive experiment, six groups of rats were used. The first group,composed of 10 rats, received an i.v. injection of AN-201 at 115 nmol/kg. Thesecond and third groups received AN-238 at 115 nmol/kg (seven rats) and 300nmol/kg (eight rats). Other groups were injected with the mixture of AN-201and carrier SST analogue RC-121 at the doses of 300 nmol/kg (four rats) orcarrier SST analogue RC-121 at 300 nmol/kg (five rats). The control group,

consisting of eight rats, was given the injection vehicle. This experiment lastedfor 4 weeks, and tumor volumes were recorded weekly. Blood samples werecollected once a week from the tail vein with Unopette microcollection kit(Becton Dickinson, Franklin Lakes, NJ). WBC and platelet counts weredetermined manually using a hemacytometer. The changes in WBC andplatelet count were evaluated on the basis of results previously reponed in rats(34), and values out of that normal range were expressed as a percentage of thecorresponding value found in controls. The toxicity was evaluated on the basisof changes in WBC and platelet counts and body weights of animals. Tumorweight was measured at autopsy, and tumor burden was calculated (mg oftumor weight/g of body weight). Samples of each tumor were snap-frozen andstored at -70°C for preparation of membranes for receptor assay and for

extraction of RNA for RT-PCR.Receptor Assay. Binding of octapeptide RC-160 to SSTR on tumor mem

brane preparations was determined by ligand competition assays using I25I-

labeled RC-160, as reported previously (31). The LIGAND PC computerizedcurve-fitting program of Munson and Rodbard (35) was used to determine the

type of receptor binding, the dissociation constant (Aid). and the maximalbinding capacity (Bmm) of receptors (35). Receptor binding affinity of cytotoxic SST analogue AN-238 to tumor membranes was measured in displacement experiments based on competitive inhibition of I25I-RC-I6() bindingusing various concentrations of AN-238 (10~h-10~12 M). IC50, defined as the

dose causing 50% inhibition of I25I-RC-16() binding, was calculated by a

computerized curve fitting program (36).RT-PCR. The expression of SSTR2 and SSTR5 subtypes and ß-actin

(internal control) in tumor tissue was assessed. Total RNA was extracted usingRNAzol B (Tel-Test, Inc., Friendswood, TX) and quantified by spectropho-tometer at 260 nm. All reagents used for RT-PCR were purchased fromPerkin-Elmer (Norwalk, CT) unless otherwise specified. To avoid genomic

DNA contamination, all samples were subjected to DNase treatment beforeRT-PCR. RNA (500 ng) was treated with 0.3 unit of RQ1 RNase-free DNase

(Promega, Madison, WI) in a total reaction volume of 19 /ul. containing 5 mMMgCU, 50 mM KC1. 10 mM Tris-HCl (pH 8.3). 1 min each dNTP, 1 unit/jilRNase inhibitor, and 2.5 /XMrandom hexamers at 37°Cfor 30 min. Afterdenaturation at 99°Cfor 5 min. l PL\of 50 units/jxl murine leukemia virus

reverse transcriptase was added, and reverse transcription was carried out forl h at 42°Cand terminated by denaturation of the enzyme at 99°Cfor 5 min.

Negative controls were run. w ith the addition of 1 jil of diethyl pyrocarbonate-

treated water instead of murine leukemia virus reverse transcriptase.Five fj.1 of the reverse transcribed products were amplified in a 25-¿tl

reaction volume containing 1.5 mM MgCU 50 mM KG, 10 mM Tris-HCl (pH

8.3), 0.2 mM each dNTP, 0.12 UM each sense and antisense primer, and 0.3 PL\of 5 units//il AmpliTaq DNA polymerase. In accordance with the publishedsequences (37. 38) and a previous study (39). the following primers were used:SSTR2 sense. 5f-AGAGCAGCCATGGAGTTGACCTCTG: SSTR2 anti-sense, 5'-CATCTGCGATGGCCAGGTT: SSTR5 sense, 5'-AAGCTGACAG-CATGGAGCCCCTC: SSTR5 antisense, 5'-CGTCAGCCACGGCCAGGTT:ß-actinsense, 5'-GTCACCCACACTGTGCCCATCT; and /3-actin antisense,5'-ACAGAGTACTTGCGCTCAGGAG. The samples were denatured for 2

min at 94°Cand then amplified in Robocycler (Stratagene, La Jolla, CA) for

30 cycles (SSTR2), 40 cycles (SSTR5), or 20 cycles (ß-actin) with a stepprogram of 94°Cfor 1 min. 60°Cfor 1 min. and 72°Cfor 1 min. After a finalstep of 72°C for 10 min. 5 fil of the PCR samples were subjected to

electrophoresis in 8% polyacrylamide gel and stained by silver. As a reference,brain tissue from a normal rat was used.

Statistical Analysis. Statistical analyses of the data were performed usingDuncan's multiple range test (40). Data were expressed as mean ±SE.

RESULTS

Toxicity and Tumor Growth Inhibition. In the first experiment,different i.v. doses of cytotoxic radical AN-201 were given to ratsbearing Dunning R-3327-AT-1 tumors. AN-201 at 110 nmol/kg re

duced the body weight by 11.8% (P < 0.05), 1 week after injection,as compared with controls, but no deaths occurred in this group.However, 77.7% and 100% mortality occurred within 6-10 days, after

administration of 125 and 150 nmol/kg of the cytotoxic radical,respectively (Fig. 1). All deaths were preceded by significant loss inbody weight. Four weeks after injection of 110 nmol/kg of AN-201,

the tumor volume was reduced by 35.1% (P < 0.05), but there was nosignificant decrease in tumor weight, as compared with controls.

In the second experiment, a single injection of cytotoxic SSTanalogue AN-238 at a dose of 300 nmol/kg had no significant effect

on the body weight and no deaths occurred due to toxicity (Fig. 1), but4 weeks later, an 85.9% (P < 0.01) decrease in tumor volume wasfound, as compared with controls. AN-238 also extended the meansurvival time of tumor-bearing rats from 52.0 ±3.75 to 91.8 ±3.70

days (P < 0.01), corresponding to a 76.5% increase.The third comprehensive experiment was designed to compare the

antiproliferative effects and toxicity of single i.v. injections of radicalAN-201, hybrid analogue AN-238, carrier SST analogue RC-121, and

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CYTOTOXIC SOMATOSTATIN ANALOGUE IN PROSTATE CANCER

100

80 -

3- 60 -

40 -

20 -

110 125 150 115 115 300"AN-201 §AN-201 §AN-238

Fig. I. Toxicity-related mortality in Copenhagen rats bearing Dunning R-3327-AT-1prostate cancers after treatment with cytoloxic radical AN-201 or cytotoxic analogue ofSST AN-238. #, rats injected i.v. with AN-201 at doses of 110, 125. and 150 nmol/kg inthe first experiment. §.rats injected i.v. with AN-201 at 115 nmol/kg or AN-238 at 115and 300 nmol/kg in the third experiment. Toxicity-related deaths occurred within 6-12days after injection.

the unconjugated mixture of AN-201 and RC-121. AN-201, injected

at 115 nmol/kg, resulted in a 32.2% (P < 0.01) weight loss, accompanied by a 90.6% (P < 0.01) decrease in WBCs during the firstweek. In some of the rats, diarrhea occurred, and four of eight rats had

thrombocytopenia. Because of toxicity of AN-201, 9 of 10 animalsdied within 12 days (Fig. 1). Analogue AN-238, given at 115 nmol/kg,

did not affect the body weight or WBC and platelet count, and noneof the rats died (Fig. 1). After 4 weeks, when the experiment ended,in the group treated with 115 nmol/kg of AN-238, the tumor size was

reduced by 40.0% (P < 0.05), tumor weight was reduced by 42.8%(P < 0.01), and tumor burden was reduced by 37.0% (P < 0.05), as

compared with controls (Fig. 2 and Table 1). Rats that received 300nmol/kg of analogue AN-238 gained somewhat less weight than the

controls during the first week of the experiment, and 7 days after theinjection, the average body weight was 10.5% (P < 0.01) lower thanin the control group. At that time, the average WBC decreasedtransiently by 56.2% (P < 0.01) but returned to normal values duringthe second week. Platelet counts were normal, no diarrhea was observed, and none of the rats died (Fig. 1). Administration of analogueAN-238 at the dose of 300 nmol/kg decreased tumor volume by

80.9% (P < 0.01), tumor weight by 82.0% (P < 0.01), and tumorburden by 81.4% (P < 0.01) 4 weeks after the injection (Fig. 2 andTable 1). Single injection of carrier SST analogue RC-121 at 300

nmol/kg did not influence the body weight or tumor growth, and WBCand platelet counts in that group did not differ from the controls.Administration of unconjugated mixture of RC-121 and AN-201 at

doses of 300 nmol/kg killed all rats in 4 days.Receptor Assay and RT-PCR. Results of the ligand competition

assays on SSTRs are shown in Table 2. In the control group, a singleclass of specific, high-affinity, and low-capacity binding sites forradiolabeled RC-160 was found, with a mean dissociation constant

(Kj) of 9.0 nM and a mean maximal binding capacity (ßmax)of 299.9fmol/mg membrane protein. In the groups treated with 115 or 300nmol/kg AN-238, the affinity and capacity of binding sites for RC-160

were not affected, and K¿sand ßmaxswere not significantly differentfrom those of controls (Table 2). The IC50 of analogue AN-238 was

Fig. 2. Effect of single i.v. injection of cytotoxic analogue of SSTAN-238 or its components on tumor growth in rats bearing DunningR-3327-AT-1 prostate cancers. Bars. SE. t. rats treated with mixture ofAN-201 and RC-121 at doses of 300 nmol/kg died within 4 days. %,ratsinjected with AN-201 at the dose of 115 nmol/kg died within 12 daysafter injection. Last tumor measurement for that group was made on day8. *, P < 0.05 versus control; **. P < 0.01 versus control; +.P< 0.05versus AN-238 at 115 nmol/kg.

E

o

15000 -

12000 -

9000 -

6000 -

3000 -

0 -

-O- contro)AN-201 at 115 nmol/kg, i.v.AN-238 at 115 nmol/kg, i.v.AN-238 at 300 nmol/kg, i.v.

-O- RC-121 * AN-201 at 300 nmol/kg, i.v-O- RC-121 at 300 nmol/kg. i.v.

**

tÃŽ

15 22

Experimental days

29

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CYTOTOXIC SOMATOSTATIN ANALOGUE IN PROSTATE CANCER

Table 1 Effect of single i.v. injection of vehicle (control), carrier SST analogue RC-I2I. and cyloloxic SST analogue AN-238 on lunuir volume, tumor weight, tumor burden, andtumor doubling time in rats bearing androgen-independent Dunning AT-1 rumors"

Tumor volume(mm3)GroupsControl

RC-121AN-238(115 nmol/kg)

AN-238 (300 nmol/kg)Initial73.3

±12.557.0 ±13.273.4 ±17.858.9 ±16.1Final1"11463

±251311852 ±31576874 ±864'2181 ±4301'-'Tumor

weight*1(g)11.4

±2.9310.4 ±2.996.51 ±1.31''2.05 ±0.48^Tumor

burden(mg/g bodymass)54.0

±12.948.5 ±15.134.3 ±5.9e

10.8 ±2.fTumor

doublinglime3.99

±0.143.86 ±0.494.39 ±0.235.18 ±0.39'

" Values are expressed as mean ±SE.* Recorded after 4 weeks.c P < 0.05 vs. control.dP < 0.01 vs. control.' P < 0.05 vi. AN-238 at 115 nmol/kg.

fP < 0.01 vi. AN-238 at 115 nmol/kg.

Table 2 Binding characteristics ofSSTRs rm membranes of Dunning AT-1 rat prostatecancer after in vivo treatment with cytotoxic analogue of SST AN-238 and its

carrier, RC-121"

GroupControl

RC-121AN-238(115 nmol/kg)

AN-238 (300 nmol/kg)K¿

(nM)9.09

±2.717.88 ±2.526.48 ±0.247.07 ±0.71SSSTRs¿Jmax

(fmol/mgprotein)299.9

±5.1292.5 ±30.93 13.0 ±62.9297.4 ±36.6

"Binding characteristics were obtained from 12-point displacement experiments.[I25I]RC-160 was used as a radioligand. All values represent mean ±SE of two to three

independent experiments, each done in duplicate or triplicate.

1.63 ±0.33 nM. This IC50 represents a remarkably high bindingaffinity of AN-238 to SSTR.

Using RT-PCR analysis, we detected mRNA for SSTR2 in Dunning R-3327-AT-1 prostate cancer (Fig. 3). Products of the expected

size of 277 bp for SSTR2 were found in the control group, as well asin groups treated with 115 nmol and 300 nmol/kg cytotoxic analogueAN-238 or with carrier RC-121 (Fig. 3). Densitometric analyses did

not show a significant difference between the expression of SSTR2from control and treated tumor specimens. mRNA for SSTR5 was notdetected in tumor samples. No PCR products were amplified from thenegative controls, ruling out the possibility of genomic DNA contamination (data not shown). Amplification with ß-actin-specific primers

produced a single product of 542 bp from all samples (Fig. 3),confirming that there was no degradation in the RNA preparation.PCR products of the expected sizes of 277 bp for SSTR2 and 261 bpfor SSTR5 were found in rat brain using the same primers andexperimental conditions (data not shown).

DISCUSSION

During the past several decades, many chemotherapeutic agentshave been evaluated in patients with advanced prostatic carcinomawho had become unresponsive to hormonal therapy. However, theoverall results have been unsatisfactory because prostate cancer appears to be resistant to most chemotherapeutic agents and because useof these drugs is strongly limited by their systemic toxicity (6, 7).Targeted chemotherapy might improve the responses. The findingsthat tumor cells can express various membrane receptors makes itpossible to design targeted anticancer agents consisting of a carriermolecule that can bind to the receptors and a highly active cytotoxicradical. Such compounds could be delivered more selectively tocancer cells, causing stronger tumor growth inhibition while sparinghealthy tissues.

Receptors for SST and mRNAs for SSTR subtypes have beenextensively investigated on diverse tumors. These studies revealedthat a significant proportion of human tumors can express the oc-

tapeptide SST analogue-preferring subtypes SSTR2 and SSTR5 (17,

31). It has also been demonstrated that SST and its octapeptideanalogues, such as octreotide or RC-160, can inhibit the growth of

some of these tumors (3, 15, 16, 23). Prostatic carcinoma is alsoknown to express SSTRs (9-14), but conflicting data exist about the

expression of SSTR2 and SSTRS subtypes. Although SSTR2 was notfound in primary prostate cancer specimens using in situ hybridization, the octreotide-preferring SSTRs were revealed on métastasesofhormone-refractory prostatic adenocarcinoma (18, 19). In addition,

cultured epithelial cells from prostate cancer specimens were demonstrated to express SSTRS (9). High-affinity binding of the octapep-tides RC-160 and RC-121 was also described on surgically removed

human prostate cancer specimens (14).Recently, we synthesized a powerful cytotoxic SST analogue, AN-

238 (31 ), by linking 2-pyrrolinodoxorubicin, a derivative of doxoru-bicin that is 500-1000 times more potent in vitro (33), to octapeptideSST analogue RC-121 through an ester bond. In this study, weinvestigated the antiproliferative effects and toxicity of AN-238, as

compared with its components, in Copenhagen rats bearing DunningR-3327-AT-1 rat prostate cancer. This rapidly growing, androgen-

insensitive anaplastic tumor is considered to be representative of theadvanced human prostatic carcinoma (41). Inhibitory effects as wellas high-affinity binding of RC-160 have been previously shown in this

model (24).Our results demonstrate that AN-238 can significantly inhibit

growth of Dunning R-3327-AT-1 tumors in vivo after a single i.v.injection. Analogue AN-238 is much less toxic than its cytotoxicradical AN-201 and more potent in inhibiting tumor growth thanAN-201 or the carrier peptide. In the first experiment, the highlypotent derivative of doxorubicin, AN-201, had only a weak inhibitory

effect on Dunning tumors at the dose of 110 nmol/kg, 4 weeks aftera single i.v. injection, and produced no deaths among animals. However, in the comprehensive third experiment, 90% of rats injected with115 nmol/kg of AN-201 died in 12 days. This suggests that the highest

234SSTR2

234ß-actin

542 bp

277 bp

Fig. 3. Expression of SSTR2 subtype and ß-actin(internal control) in Dunning AT-1tumors in rats after a single i.v. injection of vehicle (control. Lane lì,RC-121 at 300nmol/kg (Lane 2), AN-238 at 115 nmol/kg (Lane 3). or AN-238 at 300 nmol/kg (Lane 4),as revealed by RT-PCR. The PCR products shown are from one representative tumor fromeach group. PCR products were subjected to electrophoresis on 8% polyacrylamide gelsand stained with silver. The PCR products were of the expected sizes of 277 bp for SSTR2and 542 bp for ß-actin.

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CYTOTOXIC SOMATOSTATIN ANALOGUE IN PROSTATE CANCl-.K

nontoxic dose for AN-201 in Copenhagen rats bearing Dunningtumors may be ~ 110 nmol/kg. Similar variations in toxicity were also

found with the methoxymorpholinyl derivative of doxorubicin (FCE23762), which highly resembles AN-201, in preclinical trials in nude

mice (42). Our finding that a single injection of a highly potentcytotoxic agent like AN-201 at sublethal dose had little, if any, effecton the growth of the Dunning AT-1 tumor, is in general agreement

with the weak responses seen in patients with advanced stage prostatecancer after systemic administration of many different chemothera-

peutic agents, including doxorubicin (6, 7). However, the targeting ofcytotoxic radical AN-201 greatly improved the responses of DunningAT-1 tumors. Thus, cytotoxic SST analogue AN-238 at 115 nmol/kg

significantly reduced the tumor weight by 42.8% (P < 0.01), indicating that a localized delivery of the cytotoxic radical AN-201 to thetumor tissue can be achieved when it is linked to RC-121. This more

selective delivery could be attributed to a targeting action of the SSTcarrier. This view is in accordance with biodistribution studies demonstrating a fast and significant accumulation of an 9"Y-labeled SST

analogue designed for targeted radiotherapy in pancreatic tumor expressing SSTR (43). AN-238 at the dose of 300 nmol/kg caused a

significantly greater reduction in tumor weight than when given at 115nmol/kg (82.0% versus 42.8%, P < 0.01). Such an effect could beexplained by an increased accumulation of the cytotoxic radical in thetumor when a higher dose is given. It is noteworthy that a singleinjection of AN-238 at a dose of 300 nmol/kg, which is about threetimes higher than a lethal dose of AN-201, caused no mortality or

toxicity. Although it is well known that SSTRs are widely present invarious normal tissues, such as the gastrointestinal tract or the pituitary gland (17), it could be speculated that the treatment with cytotoxic SST analogue AN-238 does not affect the ability of the undif-

ferentiated or the resting cells in the gastrointestinal tract to replacethe damaged cells by mitotic divisions. Preliminary clinical studieshave shown no or only low-grade toxicity to the pituitary, the kidney,

and the bone marrow after administration of radiolabeled SST analogues to cancer patients, whereas a significant reduction in tumorprogression was achieved (44, 45).

A single injection of SST analogue RC-121 at the dose of 300

nmol/kg had no effect on the tumor growth. This finding is inaccordance with the fact that SST analogues have to be administereddaily in high doses for many weeks or by minipumps to showantitumor effect. Recently, depot formulations of several SST analogues have been introduced in clinical trials that can produce highcirculating levels of the SST analogue for prolonged periods (16).

The radiolabeled ligand competition assay confirmed the presenceof specific octapeptide-binding receptors on Dunning AT-1 tumor

membranes. Receptor assay also demonstrated a high binding affinityof cytotoxic SST analogue AN-238 to these tumors, proving that thebinding property of carrier SST analogue RC-121 is fully preserved inconjugate AN-238. Because there were no reductions in the concen

tration and the binding affinity of SSTRs in tumors after administration of AN-238, it is likely that a repetitive treatment with the

conjugate could be used to better inhibit tumor growth. Studies withmultiple dose regimens are planned for the future. Using RT-PCRanalyses, we identified the SSTR2 subtype in the Dunning AT-1

tumors. This subtype is known to bind octapeptide analogues withhigh affinity (17). On the basis of binding of AN-238 to DunningAT-1 tumor membranes and the expression of SSTR2, we couldconclude that the superior effect of AN-238, as compared to AN-201,

and the accompanying lack of toxicity might result from more specificdelivery of the cytotoxic conjugate AN-238 to SSTR2 on tumor cells.

To confirm this proposed mechanism of action and before any clinicaltrials, biodistribution studies must be performed with AN-238 andAN-201. Further studies on androgen-independent DU-145 and PC-3

human prostate cancers, as well as on human prostate cancer specimens, are in progress to investigate whether SSTR expression onhuman prostate cancers is comparable to that found on the DunningAT-1 model.

Our findings suggest that targeted cytotoxic SST analogues couldbe more efficacious and less toxic than presently used systemicchemotherapeutic agents for the therapy of patients with advancedmetastatic prostate cancer who no longer respond to androgen deprivation.

ACKNOWLEDGMENTS

The authors thank Dr. Ren-Zhi Cai for the synthesis of SST analogueRC-121 and Damon Bowe, Dr. Ilona Hoffmann, and Patricia Armatis for their

experimental assistance.

REFERENCES

22.

tandis, S. H.. Murray. T.. Dolden. S., and Wingo. P. A. Cancer slatisiics, 1998. CACancer J. Clin., 48: 6-29. 1998.Schroder. F. H.. and Boyle, P. Screening of prostate cancer: necessity or nonsense?Eur. J. Cancer, 29A: 656-661. 1993.Schally, A. V.. and Comaru-Schally. A. M. Hypothalamic and other peptidc hormones. In: Holland. J. F., Frei, E., III. Bast. R. C., Jr., Kufe, D. E.. Morton. D. L., andWeichselbaum, R. R. (eds.). Cancer Medicine. Ed. 4. pp. 1067-1086. Baltimore:Williams & Wilkins. 1997.Crawford, E. D. Hormonal therapy of prostatic carcinoma. Defining the challenge.Cancer, (if, (Suppl. 5): 1035-1038, 1990.

Sogani, P. C.. and Fair, W. R. Treatment of advanced prostatic cancer. Uro!. Clin.North Am.. 14: 353-371, 1987.Raghavan. D. Non-hormone chemotherapy for prostate cancer: principles of treatmentand application to the testing of new drugs. Semin. Oncol.. 15: 371-389. 1988.Rangel. C.. Matzkin. H.. and Solovay. M. S. Experience with weekly doxorubicinIAdriamycin) in hormone-refractory stage D2 prostate cancer. Urology. 39: 577-582.1992.Reuhi. J. C.. Krenning, E. P.. Lamberts S. W. J.. and Kvols. L. In vitro detection ofsomalostatin receptors in human tumors. Metabolism, 41 (Suppl. 2): 104-110. 1992.Sinisi, A. A., Bellastella, A.. Prezioso. D., Nicchio, M. R., Lotti, T.. Salvatore, M.,and Pasquali. D. Different expression patterns of somatostatin receptor subtypes incultured epithelial cells from human normal prostate and prostate cancer. J. Clin.Endocrinol. Melab.. «2:2566-2569. 1997.

Munkelewitz. R. A.. Whyard, T. C., Waltzer. W. C.. Meek. A. G., and Hod. Y.Expression of somatostatin receptors in human prostatic cell lines. J. Urol.. 757(Suppl. 4): 9. 1997.Brevini. T. A.. Bianchi. R., and Motta. M. Direct inhibitory effect of somatostatin onthe growth of the human prostatic cancer cell line LNCaP: possible mechanism ofaction. J. Clin. Endocrinol. Metab., 77: 626-631, 1993.Taylor, J. E.. Theveniau. M. A., Bashirzadeh. R.. Reisine, T.. and Eden. P. A.Detection of somatostatin receptor subtype 2 (SSTR2) in established tumors andtumor cell lines: evidence for SSTR2 heterogeneity. Peptides. 15: 1229-1236. 1994.Taloud. R., Degeorges. A.. Prévost.G., Hoepffner. J. L., Gauville, C.. Millot. G..Thomas. F.. and Calvo. F. Somatostatin receptors in prostate tissues and derived cellcultures, and the in vitro inhibitory effect of BIM-23014 analog. Mol. Cell. Endocrinol., 113: 195-204. 1995.Srkalovic. G.. Cai, R. Z.. and Schally. A. V. Evaluation of receptors for somatostatinin various tumors using different analogs. J. Clin. Endocrinol. Metab., 70: 661-669.

1990.Weckbecker. G.. Raulf, F., Stolz, B., and Bruns. C. Somatostatin analogs for diagnosis and Ireatment of cancer. Pharmacol. Ther.. 60: 245-264. 1993.Pollak. M. N.. and Schally. A. V. Mechanism of antineoplastic action of somatostatinanalogs. Proc. Soc. Exp. Biol. Med., 217: 143-152. 1998.Patel. Y. C.. Greenwood. M. T.. Panetta. R.. Demchyshyn. L.. Niznik, H., and Srikant.S. B. The somatostatin receptor family. Life Sci., 57: 1249-1265, 1995.Reubi. J. C.. Waser. B.. Schaer. J-C.. and Markwalder. R. Somatostatin receptors inhuman prostate and prostate cancer. J. Clin. Endocrinol. Metab., 80: 2806-2814,1995.Nilsson. S.. Reubi. J. C.. Kalkner, K-M.. Laissue. J. A.. Horisberger. U., Olerud. C..and Westlin. J-E. Metastatic hormone-refractory prostatic adenocarcinoma expressessomatostatin receptors and is visualized in vivo by ['"ln]-labeled DTPA-D-[Phe')-

octreotide scintigraphy. Cancer Res.. 55 (Suppl. 23): 5805s-5810s. 1995.Pinski. J.. Halmos. G.. and Schally. A. V. Somatostatin analog RC-160 and bombesin/gastrin-releasing peptide antagonist RC-3095 inhibit the growth of androgen-independent DU-145 human prostate cancer line in nude mice. Cancer Lett., 71: 189-196.1993.Pinski, J.. Schally. A. V., Halmos. G., and Szepeshàzi. K. Effect of somatostatinanalog RC-160 and bombesin/gastrin-releasing peptide antagonist RC-3095 ongrowth of PC-3 human prostate-cancer xenografts in nude mice. Int. J. Cancer. 55:963-967. 1993.

Yano. T.. Pinski. J.. Szepeshàzi, K., Milovanovic, S. R., Groot. K., and Schally, A. V.Effect of microcapsules of luteinizing hormone-releasing hormone antagonist SB-75

4136

on June 10, 2020. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

CYTOTOXIC SOMATOSTATIN ANALOGUE IN PROSTATE CANCI-R

and somatostatin analog RC-160 on endocrine status and tumor growth in theDunning R-3327H rat prostate cancer model. Prostate, 20: 297-310, 1992.

23. Serially, A. V. Oncological applications of somatostatin analogues. Cancer Res.. 48:6977-6985. 1988.

24. Pinski, J., Reile, H., Halmos, G., Groot, K.. and Schally, A. V. Inhibitory effects ofsomatostatin analogue RC-160 and bomhesin/gastrin releasing peptide antagonistRC-3095 on the growth of the androgen-independent Dunning R-3327-AT-I ratprostate cancer. Cancer Res.. 54: 169-174, 1994.

25. Gonzales-Barcena. D., Molina-Ayala, M. A.. Cortez-Moralez. A., Vadillo-Buenfil. I..Cardenas-Comejo, I., Comaru-Schally. A. M.. and Schally. A. V. Response toadministration of somatostatin analog RC-160 (Vapreotide) in patients with advanced

prostatic cancer at relapse time. p. 421. 80th Annual Meeting of the EndocrineSociety. New Orleans. LA. June 24-27. 1998.

26. Maulard. C., Richaud. P.. Droz. J. P.. Jessueld. D., Dufour-Esquerre, F., and Housset.M. Phase l-ll study of the somatostatin analogue lanreotide in hormone-refractoryprostate cancer. Cancer Chem. Pharmacol., 36: 259-262, 1995.

27. Breeman. W. A. P.. Hofland. L. J.. van der Plujim. M.. van Kotsveld. P. M., de Jong.M.. Setyono-Han. B.. Bakker. W. H.. Kwekkeboom. D. J., Visser. T. J.. Lamberts. S.W. J.. and Krenning. E. P. A new radiolabelled somatostatin analog |'"ln-DTPA-D-Phe'l RC-160: preparation, biological activity, receptor scintigraphy in rats andcomparison with [lnIn-DTPA-D-Phe']octreotide. Eur. J. Nucí.Med.. 21: 328-335,

1994.28. Bakker. W. H, Krenning, E. P., Reubi. J. C.. Breeman, W. A. P.. Setyono-Han. B., de

Jong. M., Kooij. P. P. M., Bruns, C., van Hagen. M., Marbach. P.. Visser, T. J.. Pless,J., and Lamberts, S, W. J. In vivo application of ['"In-DTPA-D-Phe']octreotide for

detection of somatostatin receptor-positive tumors in rats. Life Set., 49: 1593-1601.

1991.29. Krenning. E. P.. Kwekkeboom. D. J.. Bakker. W. H.. Breeman. W. A., Kooij, P. P..

Oei. H. Y., van Hagen, M., Postema. P. T., de Jong, M., Reubi, J. C.. Visser, T. J.,Reijs. A. E. M.. Hofland. L. J.. Köper.J. W., and Lamberts. S. W. J. Somaloslalinreceptor scintigraphy with ["'in-DTPA-n-Phe']- and [I:'l-Tyr']oclreotide: the Rot

terdam experience with more than 1000 patients. Eur. J. Nucí.Med.. 20: 716-731.

1993.30. Radulovic, S., Nagy, A.. Szoke. B.. and Schally, A. V. Cytotoxic analog of soma

tostatin containing methotrexate inhibits growth of MIA PaCa-2 human pancreaticcancer xenografts in nude mice. Cancer Lett.. 62: 263-271, 1992.

31. Nagy, A.. Schally. A. V.. Halmos. G.. Armatis, P., Cai, R. Z., Csernus. V.. Kovács.M.. Koppán. M.. Szepesházi. K., and Kahán,Z. Synthesis and biological evaluationof cytotoxic analogs of somatostatin containing doxorubicin or its intensely potentderivative. 2-pyrrolinodoxorubicin. Proc. Nati. Acad. Sci. USA. 95: 1794-1799.1998.

32. Cai. R. Z.. Szoke. B.. Lu. R.. Fu. D.. Redding. T. W., and Schally, A. V. Synthesisand biological activity of highly potent octapeplide analogs of somatostatin. Proc.Nati. Acad. Sci. USA, 83: 1896-1900. 1986.

33. Nagy. A.. Armatis. P.. and Schally A. V. High-yield conversion of doxorubicin to2-pyrrolinodoxorubicin. an analog 500-1000 times more potent: structure-activityrelationship of daunosamine-modified derivatives of doxorubicin. Proc. Nati. Acad.Sci. USA, 93: 2464-2469. 1996.

34. Creskoff. A. J.. Fitz-Hugh. T.. Jr.. and Farris, E. J. Hcmatology of the rat: methods

and standards. In: E. J. Farris and J. Q. Griffiths (eds.). The Rat in LaboratoryInvestigation, pp. 406-420. Philadelphia: J. B. Lippincott Co.. 1949.

35. Munson. P. J.. and Rodbard. D. Ligand: a versatile computerized approach forcharacterization of ligand binding systems. Anal. Biochem., 107: 220-239, 1980.

36. McPherson. G. A. Analysis of radioligand binding experiments. A collection ofcomputer programs for the IBM PC. J. Pharmacol. Methods, 14: 213-228. 1985.

37. Kluxen, F. K.. Bruns. C.. and Lubert, H. Expression cloning of a rat brain somatostatin receptor cDNA. Proc. Nati. Acad. Sci. USA. 89: 4618-4622. 1992.

38. O'Carrol. A. M.. Lolait. S. J.. Konig. M., and Mahan. L. C. Molecular cloning and

expression of a pituitary somatostatin receptor with preferential affinity for soma-tostatin-28. Mol. Pharmacol.. 42: 939-946, 1992.

39. Murata. T.. Takizawa. T.. Fuñaba. M., Fujimura. H.. Murata. E., and Torii. K.Quantitation of mouse and rat ß-actinmRNA by competitive polymerase chainreaction using capillary electrophoresis. Anal. Biochem., 244: 172-174. 1997.

40. Steel. R. G.. and Torrie, J. Principles and Procedures of Statistics, p. 114. New York:McGraw-Hill, Inc., 1976.

41. Isaacs. J. T. Hormonally responsive versus unresponsive progression of proslaliccancer to antiandrogen therapy as studied with the Dunning R-3327-AT and -G ratadenocarcinomas. Cancer Res., 42: 5010-5014. 1982.

42. Ripamonti. M.. Pezzoni, G.. Pesenti. E., Pastori. A.. Farao. M.. Bargiolti, A., Suarato.A.. Spreafico. F.. and Grandi. M. In wYo anti-tumor activity of FCE 23762, amethoxymorpholinyl derivative of doxorubicin active on doxorubicin-resistant tumour cells. Br. J. Cancer. 65: 703-707. 1992.

43. Stolz, B.. Smith-Jones. P.. Albert. R.. Tolcsvai. L., Briner, U., Ruser. G., Macke. H.,Weckbecker, G., and Bruns. C. Somatostatin analogues for somalosialin-receplor-mediated radiotherapy of cancer. Digestion. 57 (Suppl. 1): 17-21. 1996.

44. Wiseman. G. A., and Kvols. L. K. Therapy of neuroendocrine tumors with radiola-beled MIBG and somatostatin analogues. Semin. Nucí.Med., 25: 272-278. 1995.

45. Otte. A.. Mueller-Brand. J.. Delias. S.. Nitsche. E. U.. Herrmann, R.. and Maecke.H. R. Yttrium-90-labeled somatostatin-analogue for cancer treatment. Lancet. 351:417-418. 1998.

4137

on June 10, 2020. © 1998 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1998;58:4132-4137. Cancer Res   Miklos Koppan, Attila Nagy, Andrew V. Schally, et al.   Prostate Cancer in Rats at Nontoxic DosesGrowth of Androgen-independent Dunning R-3327-AT-1 Targeted Cytotoxic Analogue of Somatostatin AN-238 Inhibits

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