0021-972X/90/7003-0661$02.00/0
`Journal of Clinical Endocrinology and Metabolism
`Copyright© 1990 by The Endocrine Society
`
`Vol. 70,No. 3
`Printed in U.S.A.
`
`Evaluation of Receptors for Somatostatin in Various
`Tumors Using Different Analogs*
`
`GORDAN SRKALOVIC, REN-ZHI CAI, and ANDREW V. SCHALLY
`Endocrine, Polypeptide, and Cancer Institute, Veterans Administration Medical Center, and the
`Department of Medicine, Tulane University Medical School, New Orleans, Louisiana 70146
`
`ABSTRACT. The binding characteristics of several somato(cid:173)
`statin (88-14) analogs developed in our laboratory were exam(cid:173)
`ined in various human and animal tumors and normal tissues.
`In rat cerebral cortex and human breast cancer membranes the
`interaction of 88-14 with its binding sites was rapid, specific,
`saturable, linear with protein concentrations, and dependent on
`time and temperature. Analysis of kinetic and equilibrium ex(cid:173)
`perimental data showed that the interaction of [125I-Tyr11]SS-14
`with the binding sites in all normal and tumoral tissue specimens
`was consistent with the presence of a single class of noncooper(cid:173)
`ative binding sites. Superactive octapeptide analogs of somato-
`
`statin-containing hexapeptide sequences Cys-Phe-o-Trp-Lys(cid:173)
`
`Thr-C~s or Cys-Tyr-o-Trp-Lys-Val-Cys showed significant
`binding affinities to SS-14 receptors. Among these analogs, D-
`
`Trp-Cys-Phe-o-Trp-Lys-Thr-Cys-Thr-NH2 (RC-98-I) showed
`the highest binding affinity to normal human pancreatic tissue
`and human pancreatic adenocarcinoma. In contrast, 8andostatin
`(8M8 201-995) bound only to normal pancreas, not to human
`
`pancreatic cancers. Analog RC-98-I also showed a high binding
`to human and rat prostate cancers. In human epithelial ovarian
`cancers and an arrhenoblastoma, analogs o-Phe-Cys-Phe-D(cid:173)
`Trp-Lys-Thr-Cys-Trp-NH2 (RC-95-I), o-Phe-Cys-Tyr-o-Trp(cid:173)
`Lys-Val-Cys-Thr-NH2 (RC-121) and o-Phe-Cys-Tyr-o-Trp(cid:173)
`Lys-Val-Cys-Trp-NH2 (RC-160) appeared to be the most potent
`in displacing labeled 88-14. Analogs Ac-Phe-Cys-Phe-o-Trp(cid:173)
`Lys-Thr-Cys-Thr-NH2 (RC-101-I) as well as RC-121, RC-160,
`and RC-95-I, but not 8M8-201-995, showed high binding affinity
`in human breast cancers. In specimens of human meningioma
`the highest binding was found with analogs RC-121, RC-95-I,
`and RC-101-I. Since marked variations in binding affinities were
`noted for several analogs in the tissues of origin and the tumors,
`this suggests that differences may exist between somatostatin
`receptors not only in normal vs. cancerous tissues, but also
`among various tumors. Our findings also imply that some ana(cid:173)
`logs could be therapeutically superior to others in the treatment
`of certain tumors. (J Clin Endocrinol Metab 70: 0000, 1990)
`
`SOMATOSTATIN has many biological actions, in(cid:173)
`
`hibits a large variety of cells, and appears to be an
`endogenous antiproliferative agent (1-4). Various studies
`demonstrated inhibitory effects of somatostatin in pa(cid:173)
`tients with acromegaly, endocrine pancreatic tumors
`such as insulinomas and glucagonomas, and ectopic tu(cid:173)
`mors such as gastrinomas and vasoactive intestinal pep(cid:173)
`tide-producing tumors (1, 2, 5-8). However, the half-life
`of somatostatin is very short, so that its therapeutic use
`is impractical (1, 5, 6). Several groups, including ours,
`designed and synthesized somatostatin analogs with
`more selective and prolonged activities (1, 5, 6, 9-12).
`Because of a wide spectrum of activities, including
`suppression of the secretions of the pituitary, pancreas,
`stomach, and gut, interference with growth factors, and
`
`Received August 7, 1989.
`Address all correspondence and requests for reprints to: Dr. Andrew
`V. Schally, Veterans Administration Medical Center, 1601 Perdido
`Street, New Orleans, Louisiana 70146.
`* This work was supported by NIH Grants CA-40003, CA-40004,
`and CA-40077; the Medical Service of the V.A.; the G. Harold and
`Leila Y. Mathers Foundation; and the U.S. Cancer Research Council
`(to A.V.S.).
`
`possible direct antiproliferative effects on some tissues,
`somatostatin analogs might inhibit various tumors
`through multiple mechanisms (1-5, 12-14). Direct anti(cid:173)
`proliferative actions of somatostatin analogs are most
`likely mediated by specific receptors located on tumor
`cells. High affinity binding sites for somatostatin and its
`analogs have been found in normal tissues and in tumors
`(2, 6, 12).
`Previously, we demonstrated inhibitory effects of our
`analogs RC-121 and RC-160 on the growth of prostate,
`breast, and pancreatic cancers (4, 15, 16). Our other
`octapeptide analogs were also designed for antitumor
`activity (10, 11). In the course of detailed studies on these
`new peptides, we analyzed their receptor-binding char(cid:173)
`acteristics in an attempt to select the most suitable
`analog for the treatment of tumors originating from
`different tissues. Since it is possible that distinct soma(cid:173)
`tostatin receptors are expressed in the normal vs. can -
`cerous cells (17), we performed binding studies on various
`normal tissue and tumor specimens obtained from hu(cid:173)
`mans and experimental animals. The present report gives
`an account of our work.
`
`661
`
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`662
`
`Peptides
`
`· Materials and Methods
`
`SRKALOVIC, CAI, AND SCHALLY
`
`JCE& M,1990
`Vol 70•Noa
`
`] SS-14 were provided
`Somatostatin-14 (SS-14) and [Tyr11
`by Debio:gharm. S.A. (Lausanne, Switzerland). SMS-201-995
`(o-Phe-C~s-Phe-D-Trp-Lys-Thr-Cys-Thr-OL) (9) was kindly
`provided by Sandoz Ltd. (Basel, Switzerland). Octapeptide
`analogs of SS-14 shown in Table 1 were synthesized using
`standard solid phase procedures and purified by high pressure
`liquid chromatography (HPLC), as previously reported (10, 11).
`
`Buffers
`
`The homogenization buffer consisted of 0.3 M sucrose, 25
`mM Tris base, 0.25 mM phenylmethylsulfonylfluoride, 1 mM
`EGTA, 10 mM monothioglycerol and Trasylol (aprotinin) at
`10,000 kallikrein inactivator units/liter (pH 7.5). Receptor
`binding of SS-14 was carried out in 50 mM Tris base, 5 mM
`MgCl2, 0.2% BSA, Trasylol (100,000 U/liter), bacitracin (20
`mg/L), and 0.25 mM phenylmethylsulfonylfluoride (pH 7.5).
`The wash buffer was the same as assay buffers, but did not
`contain BSA. The chemicals used for buffers were purchased
`from Sigma Chemical Co. (St. Louis, MO).
`
`Preparation of membranes
`
`Normal tissues from experimental animals were obtained
`from adult female and male Sprague-Dawley rats (Charles
`River Breeding Laboratories, Wilmington, MA) and Syrian
`golden hamsters (NCI, Frederick Cancer Research Facility,
`Frederick 0 MD). R-3327H Dunning rat prostate adenocarci(cid:173)
`noma were provided by Dr. Norman Altman (Papanicolaou
`Cancer Research Institute, Miami, FL) (4, 18). Pancreatic
`cancers in hamsters were induced with N-nitrosobis-(2-oxopro(cid:173)
`pyl)-amine (BOP) as previously described (15). Normal human
`specimens were obtained from Laboratory Services of the V.A.
`Medical Center (New Orleans, LA). Autopsies were performed
`6-18 h after death. Before completing the receptor assays, all
`autopsy specimens were carefully examined by our pathologist
`(Dr. Bela Szende). Only those specimens that did not show
`signs of postmortem autolysis were selected for receptor studies.
`The age of patients from whom the normal prostate specimens
`
`TABLE 1. The structures of somatostatin analogs synthesized in our
`laboratory and tested in receptor binding assays
`
`Code
`
`Structure
`
`RC-121
`
`o-Phe-Cys-Tyr-o-Trp-Lys-Val-Cys-Thr-NH2
`
`RC-160
`
`o-Phe-Cys-Tyr-o-Trp-Lys-Val-Cys-Trp-NH 2
`
`RC-95-1
`
`o-Phe-Cys-Phe-o-Trp-Lys-Thr-Cys-Trp-NH2
`
`RC-98-1
`
`o-Trp-Cys-Phe-o-Trp-Lys-Thr-Cys-Thr-NH2
`
`RC-101-1
`
`AcPhe-Cys-Phe-o-Trp-Lys-Thr-Cys-Thr-NH2
`
`were obtained ranged from 58-62 yr, and in the case of normal
`pancreas 64-75 yr. Active normal ovary (premenopausal) was
`obtained from a 36-yr-old woman, while inactive (postmeno(cid:173)
`pausal) ovary was obtained from a 64-yr-old woman at autopsy,
`Specimens of human pancreatic adenocarcinoma were ob(cid:173)
`tained from Dr. V. -Hollander, Mount Sinai Medical Center
`(New York, NY). The age of pancreatic cancer patients ranged
`from 65-69 yr. Human prostate cancers were obtained after
`radicalprostatectomy from Dr. J.E. Pontes, Section ofUrologic
`Oncology, the Cleveland Clinic Foundation (Cleveland, OH).
`The average prostate cancers Gleason grade was 7 (range, 5_
`9). The mean age of prostate cancer patients was 64.6 (range,
`56-72 yr). Breast cancer specimens were obtained from Dr. J,
`L. Wittliff after biopsies performed in women, 28-82 yr of age,
`suspected of having breast cancer. Breast biopsies were per(cid:173)
`formed at James Graham Brown Cancer Center, University of
`Louisville, School of Medicine (Louisville, KY). Ovarian can(cid:173)
`cers (poorly differentiated ovarian adenocarcinomas and ar(cid:173)
`rhenoblastoma) were obtained from Dr. Eric L. Jenison (OB/
`GYN and Gynecologic Oncology and Reconstructive Pelvic
`Surgery, Akron, OH) and Dr. J. Bellina, the Omega Interna(cid:173)
`tional Institute (New Orleans, LA). The age of ovarian cancer
`patients was 48-60 yr. Human cancer specimens were frozen
`and shipped on dry ice to the Endocrine, Polypeptide, and
`Cancer Institute (New Orleans, LA) for membrane receptor
`assays. The specimens were stored at -70 C until processed.
`Cerebral cortex tissue of male Sprague-Dawley rats was used
`as positive controls for SS-14 binding.
`Samples were cleaned of fat and connective tissue, cut intb
`small slices, and homogenized in 5 vol sucrose buffer using an
`Ultra-Turrax homogenizer (Tissumizer, Tekmar, Cincinnati,
`OH) at maximal speed 5 for 5-s strokes at O C. The homogenate
`was centrifuged at 500 X g for 10 min at 4 C. The supernatant
`containing the crude membrane fractions was then ultracentri(cid:173)
`fuged at 70,000 X g for 45 min at 4 C (Beckman Preparative
`Ultracentrifuge, Beckman Instruments, Inc., Palo Alto, CA).
`The resulting pellet was resuspended in wash buffer and used
`for the receptor binding studies. Protein concentration was
`determined by the Bio-Rad protein assay kit (Richmond, CA)
`according to Bradford (19).
`For the radioiodination of [Tyr11]SS-14, a modification of
`the chloramine-T of Greenwood et al. (20) method was used.
`An aliquot of the peptide (5 µg in 5 µl 0.01 N acetic acid) was
`mixed with 40 µL 0.5 M phosphate buffer and 1 mCi 1251 Na in
`a volume of 2 µL. Ten microliters of chloramine-T were added.
`The reaction lasted 30 s at room temperature arid was stopped
`by adding 10 µL cysteine (5.1 mg/mL in 0.5 M phosphate buffer)
`and diluting with 500 µL 0.5 M phosphate buffer. The purifi(cid:173)
`cation of the labeled peptide was carried out using the HPLC
`with a C-18 column (W-Porex 5C18, 250 X 4.6 mm id; Pheno(cid:173)
`menex, Rancho Palos Verdes, CA). The specific activity was
`78-141.8 µCi/µg.
`The binding assay of SS-14 was conducted as described
`previously (18, 21). Binding reactions were performed in 12 X
`75-mm polypropylene round-bottom culture tubes at 21 C,
`using a competitive inhibition method. Displacement curves
`were obtained with 100-140 pM iodinated [125I]SS-14 in the
`presence of increasing amounts of unlabeled SS-14 (150 pM to
`
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`SOMATOSTATIN RECEPTORS IN TUMORS
`
`663
`
`1.5 µM). The procedure was as follows. Fifty microliters of
`unlabeled SS-14 in incubation buffer and 50 µL labeled ligand
`were placed into the tube and vortexed, and 50 µL of the
`suspension containing the membrane receptor fraction were
`added. In each binding experiment, one set of tubes was used
`with 50 µL wash buffer instead of membrane to determine the
`radioactivity bound to nonmembrane particles. Each assay
`point was performed in triplicate. The tubes were incubated for
`120 min at 21 C. The reaction was terminated by rapid filtration
`through glass fiber filters (Filtermats-Receptor Binding, Ska(cid:173)
`tron, Inc., Lier, Norway), prewetted in assay buffer and pre(cid:173)
`soaked for at least 3 h in 0.5% polyethylenimine solution to
`minimize filter adsorption. A semiautomatic cell-harvesting
`system (Skatron) with 10-s washing time was used. The radio(cid:173)
`activity of filters was counted in an automatic ,y-counter (Mi(cid:173)
`cromedic System, Inc., Huntsville, AL). The specificity of SS-
`14 binding to its receptors was investigated using competitive
`inhibition studies with labeled SS-14 and unlabeled o-Trp 6
`-
`LHRH (160 pM to 1.6 µM) and epidermal growth factor (EGF;
`0.1-100 ng).
`
`Mathematical analysis of the binding data
`
`The counts of radioactivity obtained from filters or tubes
`containing membrane particles were corrected by deducting the
`background count from assays in which no membranes were
`added. The Ligand-PC computerized curve-fitting program of
`Munson and Rodbard (22) was used to determine the types of
`receptor binding, dissociation constant (Kd), and the maximal
`binding capacity of receptors (BmaJ, Analysis of kinetic studies
`was made using a collection of radioligand binding analysis
`programs by G. A. McPherson (Elsevier-BIOSOFT, Cambridge,
`United Kingdom).
`
`Results
`
`Binding kinetics
`
`Rat cerebral cortex membranes. As shown in Fig. 1, bind(cid:173)
`ing of [125I-Tyr11]SS-14 in rat cerebral cortex membranes
`was time and temperature dependent. At 21 C, specific
`binding increased with time to reach maximal values
`after 120 min of incubation (Fig. 1, A). At 4 C maximal
`specific binding was also reached after 120 min, but
`remained below that observed of 21 C. Consequently, all
`subsequent experiments were performed at 21 C. After
`reaching equilibrium, binding remained constant for at
`least an additional 120 min. The rate of ligand binding
`during association experiments followed pseudofirst or(cid:173)
`der kinetics. Dissociation of [125I-Tyr11]SS-14 from rat
`cerebral cortex membranes was also dependent on time
`(Fig. 1, B). The rate of decline of [121I-Tyr11]SS-14 bind(cid:173)
`ing followed simple first order kinetics. Calculations of
`association (K 1) and dissociation (K_ 1) rate constants
`gave values of 7.06 X 108 M-1 min-1 and 0.0195 min-1,
`respectively. The Kd value from kinetic behavior was
`calculated as K-i/K1 = 2.75 X 10-11 M. Data from these
`kinetic studies suggested that only one class of receptors
`
`10.0
`
`A
`
`K_ 1 :"' 0.0196 m1n-l
`K1 = 1.oe z 108 u- 1 m.ln- 1
`Kd = 2.75 z 10-11 II.
`
`8.0
`
`6.0
`
`4.0
`
`2.0
`
`g
`....
`
`,-<
`I
`(fl
`(fl
`
`lO
`C\l
`,-<
`~
`'-'
`
`~ z p
`
`0
`a::i
`
`{~~
`
`•
`
`"
`
`~ ~
`!Ila( ... )
`
`I ...
`
`0
`
`•
`
`6
`
`240
`
`60
`
`120
`
`180
`TIME (min)
`FIG. 1. Association and dissociation kinetics of [125I-Tyr11]SS-14 bind(cid:173)
`ing to membranes from rat cerebral cortex. The membranes were
`incubated in 0.15 mL with 115 pmol [125I-Tyr11]SS-14 for the time
`intervals indicated in the absence (total binding) and presence (non(cid:173)
`specific binding) of 1.5 µmol unlabeled 88-14. Specific binding repre(cid:173)
`sents the difference between total and nonspecific binding. In A the
`time course of specific binding after incubation at 21 C (0-0) and 4
`C (L'.-L) is represented. In the experiment shown in B, dissociation
`was initiated by the addition of 3.0 µmol unlabeled 88-14 after 120-
`min incubation at 21 C. Specific binding (0-0) represents the differ(cid:173)
`ence between total and nonspecific binding. All values given represent
`the extent of binding, expressed as a percentage of the total radioactiv(cid:173)
`ity added to each tube. Each point is the average value ± SE of triplicate
`determinations.
`
`for SS-14 is present on rat cerebral cortex membranes.
`
`Rat anterior pituitary and human breast cancer mem(cid:173)
`branes. Specific binding of [125I-Tyr11]SS-14 to mem(cid:173)
`branes of rat anterior pituitary and human breast cancer
`was also time dependent (Figs. 2 and 3). In both tissues
`the rate of ligand binding during association experiments
`followed pseudofirst order of kinetics (Figs. 2A and 3A).
`Association rate constants (K1) were 1.43 X 107 M-1 min- 1
`for binding to rat anterior pituitary membranes and 3.2
`X 107 M-1 min-1 for binding to human breast cancer
`membranes. Dissociation rate constants were 0.0145 and
`, respectively. From kinetic studies Kd val(cid:173)
`0.0218 min- 1
`ues were estimated to be 1.02 X 10-9 M for [125I-Tyr11
`]
`SS-14 binding to rat anterior pituitary membranes and
`6.81 x 10-10 M for binding to human breast cancer
`membranes. Monoexponential model was selected as the
`most appropriate for binding to both rat anterior pitui(cid:173)
`tary membranes and human breast cancer tissue.
`
`Dependence of f 25I-Tyr11]SS-14 binding to rat cerebral
`cortex on plasma membrane concentrations
`
`125I-Tyr11]SS-14 binding to rat cerebral cortex mem(cid:173)
`[
`branes increased in a linear fashion as a function of
`plasma membrane protein concentration over a range of
`0-200 µg membrane protein in a 0.15-mL incubation
`volume. Specific binding decreased slightly at higher
`protein concentrations (200-400 µg), probably due to
`
`NOVARTIS EXHIBIT 2100
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`Page 3 of 9
`
`

`

`g u
`
`.. n
`l u
`I ..
`.. .
`. i
`
`K_ 1 - 0.01'5 m.1n-l
`,.. 1.43 X 107 m.ln-l y-l
`
`K1
`Kd "'1.02s: 10-9 w:
`
`0
`
`6
`
`664
`
`6.0 A
`
`g
`,..,
`'<!<
`I
`Ul
`
`C\l
`
`Ul '°
`s
`z :::,
`
`A
`
`0
`i:Il
`
`5,0
`
`4.0
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`0
`
`SRKALOVIC, CAI, AND SCHALL Y
`
`JCE & M• 1990
`Vol 70, No 3
`Comparison of affinities of SS-14 and its analogs to mem(cid:173)
`brane receptors in various normal and tumoral tissues
`
`mac...,,
`0
`
`1
`
`The analysis of displacement curves of [1251 -Tyr11
`] SS-
`14 by unlabeled SS-14 in all tissue specimens investi(cid:173)
`gated suggested that the labeled SS-14 was bound to one
`class of noncooperative binding sites. Binding of [125J_
`Tyr11]8S-14 to different tissue specimens was specific
`since unlabeled D-Trp6-LHRH (160 pmol to 1.6 µmol)
`and EGF (0.1-100 ng) were unable to displace labeled
`SS-14 from its binding sites.
`Homologous displacement assay of [125l-Tyr11]8S-14
`by unlabeled SS-14 in rat cerebral cortex revealed that
`the labeled peptide was bound to one class of binding
`sites with high affinity (Kd = 2.7 X 10-11 M) and low
`capacity (Bmax = 1.95 X 10-14 mol/mg membrane pro(cid:173)
`tein). When various somatostatin octapeptide analogs
`were examined in heterologous displacement assay, RC-
`121 and RC-95-I showed the highest affinity to receptors
`for SS-14 in rat cerebral cortex membranes (Table 2). In
`this tissue, SMS-201-995 showed 2 times less affinity
`than SS-14 and about 16 times less affinity than RC-121
`and RC-95-I.
`In membranes from human meningioma specimens,
`the highest affinities for SS-14 receptors were shown by
`RC-101-I and RC-95-I, as based by displacement of [125I(cid:173)
`Tyr11]SS-14 in heterologous assays (Table 2). In these
`experiments the affinities of RC-101-I and RC-95-I were
`92 and 44 times higher, respectively, than that of SS-14.
`In normal human pancreas and human pancreatic
`cancer specimens, analysis of homologous displacement
`of [125l-Tyr11]8S-14 by unlabeled SS-14 suggested that
`the labeled peptide was bound to one class of low capacity
`binding sites with Bmax values of 4.5 X 10-14 and 2.7 X
`
`TABLE 2. Displacement of [125I-Tyr11 ]SS-14 with 88-14 and its analogs
`from rat cerebral cortex and human meningioma membrane receptors
`
`Tissue
`
`Rat cerebral cortex
`
`Peptides
`(unlabeled)
`
`88-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`SMS-201-995
`
`K.
`(nM- 1)
`
`36.41
`296.38
`4.78
`292.04
`ND
`23.21
`17.6
`
`Human meningioma
`
`6.55
`88-14
`24.88
`RC-121
`3.98
`RC-160
`287.34
`RC-95-I
`3.36
`RC-98-I
`598.78
`RC-101-I
`K., Affinity constant of ligand for membrane receptors; ND, no
`displacement. The results of each experiment were calculated from
`data of triplicate tubes of a IO-point assay. The coefficients of variations
`of the determinations ranged from 10-30%.
`
`60
`
`120
`
`180
`TIME (min)
`
`240
`
`FIG. 2. Association and dissociation kinetics of [126I-Tyr11]SS-14 bind(cid:173)
`ing to membranes from rat anterior pituitary. The membranes were
`incubated in 0.15 mL with 115 pmol [126I-Tyr11]SS-14 at 21 C for the
`time intervals indicated in the absence (total binding) and presence
`(nonspecific binding) of 1.5 µmol unlabeled 88-14. Specific binding
`(0-0) represents the difference between total and nonspecific binding,
`as indicated in A. In the experiment illustrated in B, dissociation was
`initiated by the addition of 3.0 µmol unlabeled 88-14 after 120-min
`incubation at 21 C. Specific binding (0-0) represents the difference
`between total and nonspecific binding. All values given represent the
`extent of binding expressed as a percentage of the total radioactivity
`added to each tube. Each point is the average value ± SE of triplicate
`determinations.
`
`15.o A
`
`u B
`
`g
`,..,
`'<!<
`
`12.0
`
`I
`Ul
`
`Ul '° C\l
`,..,
`c..
`A z :::,
`
`0
`i:Il
`
`9.0
`
`6.0
`
`3.0
`
`0.0
`0
`
`K_ 1 ,.. 0.0210 m1n-l
`K1
`• 3.2 I 10? }Cl mln-l
`
`Kd
`
`• 8.01 z 10-lO U:
`
`0
`
`•
`
`• Q
`
`0
`
`60
`
`120
`
`180
`TIME (min)
`
`240
`
`FIG. 3. Association and dissociation kinetics of [126I-Tyr11]SS-14 bind(cid:173)
`ing to membranes from human breast cancer specimens. The mem(cid:173)
`branes were incubated in 0.15 mL with 115 pmol [126I-Tyr11]SS-14 at
`21 C for the time intervals indicated in the absence (total binding) and
`presence (nonspecific binding) of 1.5 µmol unlabeled 88-14. Specific
`binding (0-0) represents the difference between total and nonspecific
`binding as indicated in A. In the study shown 'in B, dissociation was
`initiated by the addition of 3.0 µmol unlabeled 88-14 after 120-min
`incubation at 21 C. Specific binding (0-0) represents the difference
`between total and nonspecific binding. All values given represent the
`extent of binding expressed as a percentage of the total radioactivity
`added to each tube. Each point is the average value ± SE of triplicate
`determinations.
`
`[1 25l-Tyr11]8S-14.
`increasing dissociation of labeled
`Therefore, plasma membrane protein concentrations of
`30-100 µgin a 0.15-mL incubation volume were used in
`subsequent experiments.
`
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`SOMATOSTATIN RECEPTORS IN TUMORS
`
`665
`
`10-13 mol/mg membrane protein, respectively. The affin(cid:173)
`ity in pancreatic cancers (Kct = 3.5 X 10-9 M; Fig. 4) was
`lower than that in normal pancreas (Kct = 2.9 X 10-9 M).
`Analog RC-98-1 was highly potent in displacing [125I(cid:173)
`Tyr11]SS-14 from membranes of both normal and carci(cid:173)
`nomatous pancreatic tissue (Table 3). SMS-201-995
`showed high affinity to SS-14 receptors in normal human
`pancreatic tissue, but its affinity to receptors in human
`pancreatic cancers was very poor (288 times less than
`SS-14). In the MIA PaCa-2 human pancreatic cancer
`cell line heterologous displacement assays showed that
`RC-98-1 and RC-160 had the highest affinities for SS-14
`receptors. As can be seen in Table 3, labeled SS-14 was
`also bound to normal pancreas and BOP-induced pan(cid:173)
`creatic cancer in Syrian hamsters. Among the analogs
`investigated, RC-121 showed the highest potency for
`displacing [125I-Tyr11]SS_-14 from its binding sites on
`BOP-induced pancreatic cancer membranes. However,
`the affinities of RC-121 and other analogs for SS-14
`receptors on BOP-induced cancer membranes were much
`lower than that of SS-14 (Table 3).
`The displacement of [125I-Tyr11]SS-14 from binding
`sites in membranes of normal human prostate and hu(cid:173)
`man prostate cancer specimens by increasing concentra(cid:173)
`tions of unlabeled SS-14 and Scatchard analysis of these
`data suggested that in both cases the labeled peptide was
`bound to one class of binding sites. However, in normal
`prostate we found receptors with low affinity (Kct = 1.4
`) and high capacity (Bmax = 1.5 X 10-12
`X 10-8
`) and in
`prostate cancer, high affinity (Kct = 4.0 X 10-10
`) and low
`capacity (Bmax = 1.4 X 10-14 mol/mg membrane protein;
`Fig. 5). Among the analogs tested, RC--121 showed the
`highest affinity for SS-14-binding sites on membranes of
`normal human prostate (Table 4). Affinity of SS-14 for
`its receptors on normal human prostate was 95 times
`
`2.4 A
`
`LOO B
`
`i uo
`
`I.IQ
`
`LOO i
`
`1.00
`
`....
`
`0.00
`0
`
`..
`
`..
`
`C\I
`I
`0
`rl
`M
`
`~ 0
`~
`~ 0
`
`i:i:i
`
`2.3
`
`2.2
`
`2.1
`
`LOO . ..
`
`a-U.,IOOIID,plll
`
`...
`
`2.0 +----t----+---+----+---+-----11----~
`-9.5
`-9.0
`-8.5
`-8.0
`-7.5
`-7.0
`-6.5
`-6.0
`SS-14, LOG TOTAL (M)
`FIG. 4. A, Displacement of [125I-Tyr11]SS-14 by increasing amounts of
`unlabeled SS-14 using membranes of human pancreatic cancer. The
`individual data points represent the average value ± SE of triplicate
`determinations. B, Scatchard plot analysis of data from displacement
`curves illustrated in A. The Scatchard nonspecific bound to free ratio
`was 0.02114.
`
`TABLE 3. Displacement of [125I-Tyr11
`] SS-14 with SS-14 and its analogs
`from membrane receptors on human and experimental pancreatic
`cancers and normal tissues
`
`Tissue
`
`Normal human pancreas
`
`Human pancreatic cancer
`
`MIA PaCa-2 human pancreatic
`cancer cell line
`
`Normal hamsters pancreas
`
`BOP-induced pancreatic cancer in
`hamsters
`
`Peptides
`(unlabeled)
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`SMS-201-995
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`SMS-201-995
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`
`K.
`(nM- 1)
`
`0.34
`0.64
`0.44
`0.22
`1.33
`0.04
`83.11
`
`0.29
`0.52
`0.48
`0.03
`2.68
`0.02
`0.001
`
`0.03
`0.01
`0.13
`ND
`0.09
`0.02
`
`0.44
`0.14
`1.70
`0.13
`0.80
`0.43
`
`1.01
`0.38
`0.17
`0.16
`O.D7
`0.22
`
`K 0 , Affinity constant of ligand for membrane receptors; ND, no
`displacement. The results of each experiment were calculated from
`data of triplicate tubes of a 10-point assay. The coefficients of variations
`of the determination ranged from 8-24%.
`
`lower than that of RC-121. In human prostate cancer,
`SS-14, RC-98-1, and RC-95-1 showed the highest affinity
`to SS-14-binding sites. In normal human prostate, hu(cid:173)
`man prostate cancer, and Dunning R3327H rat prostate
`tumor, analog SMS-201-995 showed markedly lower af(cid:173)
`finity to SS-14 receptors than the other octapeptide
`analogs.
`We were unable to detect receptors for SS-14 in spec(cid:173)
`imens of normal human postmenopausal ovary and nor(cid:173)
`mal rat ovary (not shown). In normal premenopausal
`ovary, SS-14 was bound to receptors with low affinity
`(Table 5). The analysis of displacement curves of [125I(cid:173)
`Tyr11]SS-14 by unlabeled SS-14 and its analogs from
`ovarian adenocarcinoma and arrhenoblastoma indicated
`
`NOVARTIS EXHIBIT 2100
`Par v. Novartis, IPR 2016-01479
`Page 5 of 9
`
`

`

`2.0 A
`
`1.0
`
`B
`
`TABLE 5. Displacement of [125I-Tyr11]SS-14 with SS-14 and analogs
`from membrane receptors on normal human ovary and ovarian cancers
`
`SRKALOVIC, CAI, AND SCHALL Y
`
`JCE & M• 1990
`Vol 70, No 3
`
`666
`
`N
`I
`0
`.--<
`
`1.8
`
`1.6
`
`1.4
`
`0·\.o
`
`u
`
`a.o
`
`a.o
`
`1.0
`U
`m-tt.,aoom,,u
`
`I.O
`
`Normal human ovary (premenopausal)
`
`Tissue
`
`Adenocarcinoma of ovary
`
`Peptides
`(unlabeled)
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`SMS-201-995
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`SMS-201-995
`
`0.06
`1.21
`0.03
`0.02
`0.21
`0.15
`0.01
`
`0.45
`1.10
`0.55
`1.80
`0.11
`0.004
`0.003
`
`1.2+----+-------,1------+----+------l
`-10.0
`-6.0
`-5.0
`-7.0
`-9.0
`-8.0
`SS-14, LOG TOTAL (M)
`
`Fm. 5. A, Displacement of [125I-Tyr11]SS-14 of increasing amounts of
`unlabeled SS-14 using membranes of human prostate cancer. The
`individual data points represent the average value ± SE of triplicate
`determinations. B, Scatchard plot analysis of data from displacement
`curves illustrated in A. The Scatchard nonspecific bound to free ratio
`was 0.01362.
`
`TABLE 4; Displacement of [125I-Tyr11]SS-14with SS-14 and its analogs
`from membrane receptors on normal human prostate, human prostate
`cancer, and Dunning tumor R3327H
`
`Tissue
`
`Normal human prostate
`
`Human prostate cancer
`
`Peptides
`(unlabeled)
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`SMS-201-995
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`SMS-201-995
`
`K.
`(nM-1 )
`
`0.07
`6.76
`ND
`ND
`0.10
`0.27
`0.08
`
`2.48
`0.21
`0.12
`2.76
`2.27
`0.36
`0.04
`
`Dunning tumor
`R3327H
`
`SS-14
`2.29
`RC-121
`5.37
`RC-160
`0.2
`ND
`RC-95-I
`RC-98-I
`1.17
`14.23
`RC-101-I
`0.43
`SMS-201-995
`K., Affinity constant of ligand for membrane receptors; ND, no
`displacement. The results of each experiment were calculated from
`data of triplicate tubes of a 10-point assay. The coefficients of variations
`of the determination ranged from 8-24%.
`
`that analog RC~95-I had the highest affinity (Table 5).
`Again, SMS-201-995 showed the lowest affinity to SS-
`14-binding sites on ovarian cancer membranes compared
`to SS-14 and other analogs.
`In membranes of human breast cancer specimens [125I(cid:173)
`Tyr11] SS-14 showed binding to one class of noncooper(cid:173)
`ative, high affinity, and low capacity binding sites (Kd =
`
`Arrhenoblastoma
`
`0.81
`8.90
`8.96
`30.47
`1.84
`1.28
`K. Affinity constant of ligand for membrane receptors; ND, no
`displacement. The results of each experiment were calculated from
`data of triplicate tubes of a 10-point assay. The coefficients of variations
`of the determination ranged from 15-24%.
`
`SS-14
`RC-121
`RC-160
`RC-95-I
`RC-98-I
`RC-101-I
`
`TABLE 6. Displacement of [125I-Tyr11]SS-14 with SS-14 and analogs
`from human breast cancer membrane receptors
`
`Tissue
`
`Peptides
`(unlabeled)
`
`K.
`(nM-1 )
`
`Human breast cancer
`
`1.40
`SS-14
`RC-121
`14.10
`12.00
`RC-160
`RC-95-I
`74.25
`1.44
`RC-98-I
`RC-101-I
`121.16
`0.10
`SMS-201-995
`K., Affinity constant of ligand for membrane receptors; ND, no
`displacement. The results of each experiment were calculated from
`data of triplicate tubes of a 10-point assay. The coefficients of variations
`of the determination ranged from 10-15%.
`7.0 X 10-10 M; Bmax = 9.5 X 10-14 moljmg membrane
`protein). The analysis of heterologous displacement
`curves demonstrated that analog RC-101-I had the high(cid:173)
`est affinity to SS-14-binding sites on breast cancer mem(cid:173)
`branes. Analogs RC-95-I, RC-121, and RC-160 also
`showed high affinity to SS-14 receptors in breast cancers.
`The affinity to SS-14-binding sit(;)s of SMS-201-995 was
`14 times lower than that of SS-14 (Table 6).
`
`Discussion
`Background of this study
`Analog SMS-201-995 and other somatostatin analogs
`have been used for the treatment of acromegaly and
`
`NOVARTIS EXHIBIT 2100
`Par v. Novartis, IPR 2016-01479
`Page 6 of 9
`
`

`

`SOMATOSTATIN RECEPTORS IN TUMORS
`
`667
`
`endocrine tumors of the gastroenteropancreatic system,
`including carcinoid tumors, insulinomas, glucagonomas,
`gastrinomas, and vasoactive intestinal peptide-secreting
`tumors (5, 12, 23-25). Attempts are being made to use
`modern somatostatin analogs for the therapy of human
`breast cancer, prostate cancer, and carcinoma of exocrine
`pancreas (5, 12). However, the potential of modern so(cid:173)
`matostatin analogs in treatment of various malignancies
`still remains to be established. Experimental evidence
`suggests that modern somatostatin analogs could provide
`a useful palliative form of treatment for some tumors.
`The fall in G H levels induced by somatostatin analogs
`could, through mechanisms involving endogenous growth
`factors, especially insulin-like growth factor-I (IGF -I),
`be of major importance for the inhibition of growth of
`various tumors (4, 14). IGF-I and -II and other growth
`factors, including EGF, and transforming growth factor,
`appear to be involved in the proliferation of both normal
`and neoplastic cells (5, 12, 26-28). We have demonstrated
`that our somatostatin analog RC-160, alone or in com(cid:173)
`bination with the LHRH agonist D-Trp6-LHRH, can
`inhibit the growth of BOP-induced ductal pancreatic
`cancers in hamsters (5, 12, 15), Dunning prostate tumors
`in rats (4), and florid MXT mammary tumors in mice
`(16). Somatostatin analogs have been also tried in ex(cid:173)
`perimental models of chondrosarcomas and osteosarco(cid:173)
`mas (5, 12, 23). In the MIA PaCa-2 human pancreatic
`cancer cell line, somatostatin reverses the stimulatory
`effect of EGF on the phosphorylation of the tyrosine
`kinase portion of the EGF receptor and on cell growth
`(29). Superactive octapeptide analogs RC-160 and RC-
`121 exhibit high activity on dephosphorylation of EGF
`receptor ( 17) and inhibit the EG F -induced growth of
`cultured MIA PaCa-2 c