New approaches to treatment of various cancers based on cytotoxic analogs of LHRH,somatostatin and bombesin

The development of targeted cytotoxic analogs of hypothalamic peptides for the therapy of various cancers is reviewed and various oncological studies on experimental tumors are summarized. Novel therapeutic modalities for breast, prostate and ovarian cancer consist of the use of targeted cytotoxic analogs of LH-RH containing doxorubicin (DOX) or 2-pyrrolino-DOX. The same radicals have been incorporated into cytotoxic analogs of somatostatin which can be also targeted to receptors for this peptide in prostatic, mammary, ovarian, renal and lung cancers, brain tumors and their metastases. A targeted cytotoxic analog of bombesin containing 2-pyrrolino-DOX has also been synthesized and successfully tried in experimental models of prostate cancer, small cell lung carcinoma and brain tumors. The development of these new classes of peptide analogs should lead to a more effective treatment for various cancers.     

Biological activity and receptor binding characteristics to various human tumors of acetylated somatostatin analogs.

Several somatostatin analogs with recently synthesized acetylated N terminus were assayed in vivo for their effects on sodium pentobarbital-stimulated growth hormone (GH) levels in fed male rats and gastrin-releasing peptide (14-27)-stimulated gastrin levels in fasted male rats. The binding characteristics of these analogs to somatostatin receptors were also examined in various human tumors and normal tissues. The analog RC-101-I, injected at a dose of 0.1 micrograms/100 g body wt, significantly suppressed GH release (P less than 0.01) for at least 2 hr. Analog RC-160-II caused the longest inhibition of GH release, greater than that induced by nonacetylated parent analog RC-160, with GH levels showing significant suppression (P less than 0.01) for more than 3 hr. Analogs RC-160-II and RC-101-I and RC-160, injected at a dose of 1.0 micrograms/100 g body wt, significantly (P less than 0.01) suppressed gastrin-releasing peptide (14-27)-stimulated serum gastrin. Analog RC-101-I was active in this test at a dose of 0.1 micrograms/100 g body wt. RC-160-II showed significant binding to somatostatin-14 receptors in all investigated tissues (human colon, human colon cancer, breast cancer, human pancreas and pancreatic cancer, human prostate and prostate cancer, and rat cerebral cortex), but there were marked variations in binding affinities among various normal and cancerous tissues. The highest affinity was found in membranes of colon cancer (Ka = 18.4 nM-1) and breast cancer (Ka = 12.46 nM-1). The binding affinity of RC-160-II to somatostatin receptors in membranes of the breast cancer was similar to that of RC-160. RC-101-I showed higher binding affinity to somatostatin-14 receptors than RC-160 in human breast, pancreatic, and prostate cancer. With the exception of breast cancer tissue, the binding affinity of RC-101-I was significantly lower than that of RC-160-II in membranes of all investigated tissues. It can be concluded that acetylated somatostatin analogs RC-101-I and RC-160-II possess prolonged and enhanced biological activities in suppressing serum GH and gastrin in rats. Significant variations in binding affinities for these analogs in different tissues and various tumors suggest that differences may exist between somatostatin receptors in normal versus malignant tissues. This raises the possibility that some of these analogs could be used more selectively in the treatment of various neoplasms.     

Position 4 substituted somatostatin analogs: increased binding to somatostatin receptors in pituitary and brain

Somatostatin (S-14) analogs with Phe4 substitutions bound to pituitary and cerebrocortical S-14 receptors more avidly than did S-14. The 2-4 fold greater affinities of the Phe4 S-14 as well as analogs with structural modification of the Phe4 residue for binding to pituitary S-14 receptors showed good correlation with their reported potencies for in vivo Gh inhibition. In the cerebral cortex, [Phe4] S-14, [Phe4, D-Trp8] S-14 and [F5-Phe4] S14 were 2-3 times more potent while [p-NH2-Phe4] S-14 was 6 times more potent compared to S-14 in binding to S-14 receptors. The increased binding affinities of the Phe4 analogs in these two tissues does not appear to be due to differential stability of the analogs compared to S-14 under the experimental conditions used. [Thr4] S-14 exhibited very low binding in both these tissues. Thus structural modification of the position 4 moiety of the S-14 molecule does not result in dissociated affinities for binding to S-14 receptors in the brain and the pituitary. The increased receptor binding affinities of the Phe4 analogs in the cerebral cortex suggest that they may be more potent than S-14 in the CNS.     

Mu-opiate binding and morphine antagonism by octapeptide analogs of somatostatin

A series of cyclic conformationally restrained octapeptide analogs of somatostatin were examined for their ability to inhibit the binding of tritiated mu, kappa, and delta opiate receptor ligands. Several of these substances were found to have high affinity for mu opiate receptors while having very low affinity for both kappa and delta receptors. Previous suggestions that somatostatin analogs exhibit opiate antagonist activity led to a study of the ability of the two most potent compounds to inhibit morphine analgesia in rats after intracerebroventricular injection. One of the compounds significantly antagonized morphine analgesia although the other displayed severe toxicity. These two compounds differed in that the very toxic compound had previously been found to possess significant somatostatin activity. It thus appears that the structural requirements for toxicity and somatostatin activity can be differentiated from those for opiate activity.     

Purification of a pituitary receptor for somatostatin. The utility of biotinylated somatostatin analogs.

A somatostatin (SRIF) receptor and its associated Gi regulatory proteins was purified from GH4C1 rat pituitary cells by: 1) saturation of the membrane-bound receptor with biotinyl-NH-[Leu8,D-Trp22,Tyr25] SRIF28 (bio-S28); 2) solubilization of receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine (D.L); 3) adsorption of solubilized receptor-ligand complex to immobilized streptavidin; and 4) elution of receptor and G-protein by GTP. The receptor, a glycoprotein with an average M(r) of 85,000, was then purified to substantial homogeneity on immobilized wheat germ agglutinin. The 85-kDa glycoprotein was identified as a SRIF receptor by several criteria. (a) It had the same size as the chemically cross-linked R.[125I]L complex. (b) Yield of the purified protein increased and plateaued in the same range of bio-S28 concentrations where specific high affinity binding reached saturation. (c) It was copurified with appropriate G-protein subunits. The 85-kDa receptor and two other proteins with M(r) values of 35,000 and 40,000, the sizes of G beta and G alpha, did not appear in eluates from control streptavidin columns done with SRIF receptors loaded with nonbiotinylated S14. The 40-kDa protein was identified as a Gi alpha by ADP-ribosylation from [32P]NAD catalyzed by pertussis toxin. (d) Both the chemically cross-linked R.[125I]L complex and SRIF receptor purified from [35S]methionine-labeled GH4C1 cells were reduced in size to about 38 kDa by endoglycosidase F. (e) Amino acid sequence from the purified receptor was nearly identical with that of a recently cloned SRIF receptor subtype.     

Synthesis and binding potencies of cyclic hexapeptide analogs of somatostatin incorporating acidic and basic peptoid residues.

The synthesis, binding affinity, and structure-activity relationships of compounds related to the cyclic hexapeptide, c[Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11], L-363,301 (the numbering in the sequence refers to the position of the residue in native somatostatin) is reported. The Pro residue in this compound is replaced with the peptoid residues Nasp [N-(2-carboxyethyl) glycine], Ndab [N-(2-aminoethyl) glycine] and Nlys [N-(4-aminobutyl) glycine]. This series of compounds enables us to draw conclusions about the influence of positively or negatively charged residues in the bridging region on the binding affinity towards the isolated human somatostatin receptors. A loss of binding to the recombinant human somatostatin (hsst) receptors in the Nasp analog compared with L-363,301 and compared with the Ndab and Nlys analogs clearly demonstrates that the presence of an acidic residue in the bridging region is unfavorable for binding to the hsst receptors. Comparison between the Ndab analog and the Nlys analog suggests that the presence of a basic residue in the bridging region might be advantageous for binding to the hsst5 receptor provided that the residue bearing the basic group extends far enough to allow for interaction with the receptor, while the length of the basic peptoid residue does not influence binding to the hsst2 receptor. These results are useful for the design of hsst5 selective somatostatin analogs.     

The binding of substrate analogs to phosphotriesterase

Phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the detoxification of organophosphates such as the widely utilized insecticide paraoxon and the chemical warfare agent sarin. The three-dimensional structure of the enzyme is known from high resolution x-ray crystallographic analyses. Each subunit of the homodimer folds into a so-called TIM barrel, with eight strands of parallel beta-sheet. The two zinc ions required for activity are positioned at the C-terminal portion of the beta-barrel. Here, we describe the three-dimensional structure of PTE complexed with the inhibitor diisopropyl methyl phosphonate, which serves as a mimic for sarin. Additionally, the structure of the enzyme complexed with triethyl phosphate is also presented. In the case of the PTE-diisopropyl methyl phosphonate complex, the phosphoryl oxygen of the inhibitor coordinates to the more solvent-exposed zinc ion (2.5 A), thereby lending support to the presumed catalytic mechanism involving metal coordination of the substrate. In the PTE-triethyl phosphate complex, the phosphoryl oxygen of the inhibitor is positioned at 3.4 A from the more solvent-exposed zinc ion. The two structures described in this report provide additional molecular understanding for the ability of this remarkable enzyme to hydrolyze such a wide range of organophosphorus substrates.     

Absence of binding of targeted analogs of somatostatin carrying cytotoxic radicals or radionuclides to growth hormone secretagogue receptors on human myocardium

Various peripheral human tissues express receptors for growth hormone secretagogue (GHS), the highest density being in the myocardium. It was also reported that some octapeptide analogs of somatostatin (SRIH) can displace radiolabeled Tyr-Ala-hexarelin from GHS receptors on the human pituitary and heart. Thus, it is possible that radionuclide analogs of SRIH such as OctreoScan and recently developed cytotoxic SRIH analogs containing doxorubicin (DOX) intended for targeted tumor therapy, could bind to these GHS receptors, compromising the safety of compounds of this type. Therefore, we determined the binding of OctreoScan and two cytotoxic SRIH analogs consisting of octapeptide carrier RC-121 and DOX (AN-162) or 2-pyrrolino-DOX (AN-238) to human myocardium specimens. None of these compounds displayed specific binding to the human heart indicating that the clinical use of SRIH analogs linked to anthracyclines or radionuclides should not be associated with increased cardiac side effects.     

The mode of binding of potential transition-state analogs to acetylcholinesterase.

Phenylacetone, 4-phenyl-2-butanone, and 4-oxopentyltrimethylammonium chloride were tested as potential transition state analogs for eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). Phenylacetone is a competitive inhibitor of the enzyme but not a transition state analog, since its binding constant is similar to that for the substrate phenyl acetate. 4-Phenyl-2-butanone binds 6-18 times more tightly than the inhibitors 4-phenyl-2-butanol and N-benzylacetamide and the substrate benzyl acetate and also blocks inactivation of the enzyme with methanesulfonyl fluoride. However, its binding is independent of pH in the range 5-7.5, whereas both V and V/Km for benzyl acetate hydrolysis decrease with decreasing pH in this range. These data indicate a specific but weak interaction between the ketone carbonyl and the enzyme, but probably do not justify considering this compound a transition state analog. 4-oxopentyltrimethylammonium iodide has previously been shown to bind about 125 times more strongly than the substrate acetylcholamine. It also binds about 375 times more strongly than the alcohol 4-hydroxypentyltrimethylammonium iodide. Furthermore, the ketone protects the enzyme from inactivation by methansulfony fluoride, while the corresponding quaternary ammonium alcohol accelerates this inactivation reaction. This additional information confirms that the ketone is a transition state analog.     

Synthesis and binding potencies of cyclohexapeptide somatostatin analogs containing naphthylalanine and arylalkyl peptoid residues

We report the synthesis, binding affinities to the recombinant human somatostatin receptors, and structure‐activity relationship studies of compounds related to the cyclic hexapeptide, c‐[Pro⁶‐Phe⁷‐D‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹], L‐363,301 (the numbering in the sequence refers to the position of the residues in native somatostatin). The Pro residue in this compound is replaced with the arylalkyl peptoid residues Nphe (N‐benzylglycine), (S)βMeNphe [(S)‐N‐[(α‐methyl)benzyl]glycine] or (R)βMeNphe [(R)‐N‐[(α‐methyl)benzyl]glycine] and l ‐1‐naphthylalanine is incorporated into either position 7 or 11 of the parent compound. The synthesis and binding data of the Nnal⁶ ([N‐naphthylmethyl]glycine) analog of L‐363,301 is also reported. The incorporation of the Nnal residue into position 6 of L‐363,301 resulted in an analog with weaker binding affinities to all hsst receptors but enhanced selectivity towards the hsst2 receptor compared with the parent compound. The other compounds bind effectively to the hsst2 receptor but show some variations in the binding to the hsst3 and hsst5 receptors resulting in different ratios of binding affinities to the hsst5 and hsst2 or hsst3 and hsst2, respectively. The incorporation of the Nphe residue into position 6 and the Nal residue into position 7 of L‐363,301 led to a compound which binds potently to the hsst2 and has increased selectivity towards this receptor (weaker binding to hsst3 and hsst5 receptors) compared with the parent compound. The analogs with β‐methyl chiral substitutions in the aromatic peptoid side chain and Nal in position 7 or 11 bind effectively to the hsst2 and hsst5 receptors. They exhibit similar ratios of binding affinities to the hsst5 and hsst2 receptors as observed for L‐363,301. There are however minor differences in binding to the hsst3 receptor among these analogs. These studies allow us to investigate the influence of additional hydrophobic groups on the binding activity to the isolated human somatostatin receptors and the results are important for the design of other somatostatin analogs. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Measles virus modulates human T-cell somatostatin receptors and their coupling to adenylyl cyclase.

The possible role of immunomodulatory peptide somatostatin (SRIF) in measles virus (MV)-induced immunopathology was addressed by analysis of SRIF receptors and their coupling to adenylyl cyclase in mitogen-stimulated Jurkat T cells and human peripheral blood mononuclear cells (PBMC). SRIF-specific receptors were assayed in semipurified membrane preparations by using SRIF14 containing iodinated tyrosine at the first position in the amino acid chain ([125I]Tyr1) as a radioligand. A determination of receptor number by saturation of radioligand binding at equilibrium showed that in Jurkat cells, MV infection led to a dramatic decrease in the total receptor number. The virus-associated disappearance of one (Ki2 = 12 +/- 4 nM [mean +/- standard error of the mean [SEM]]; n = 4) of two somatostatin binding sites identified in control Jurkat cells (Ki1 = 78 +/- 3 pM and Ki2 = 12 +/- 4 nM [mean +/- SEM]; n = 4) was also observed. Almost identical results were obtained for phytohemagglutinin-activated human PBMC. In the absence of MV infection, two somatostatin binding sites were present (Ki1 = 111 +/- 31 pM and Ki2 = 17 +/- 2 nM [mean +/- SEM]; n = 2), whereas in MV-infected cells, only the high-affinity (Ki1 = 48 +/- 15 pM [mean +/- SEM]; n = 2) binding site remained. In addition, MV infection reinforced the inhibitory effects of SRIF on adenylyl cyclase activity, since maximal inhibition at 1 microM peptide was 11% +/- 4% in control cells versus 25% +/- 3% (P < 0.05) in infected Jurkat cells. Moreover, MV infection severely impaired the capacity of adenylyl cyclase to be activated directly (by forskolin) or indirectly (via Gs protein-coupled vasoactive intestinal peptide receptor). An assessment of [methyl-3H]thymidine incorporation showed that SRIF increased proliferative responses to mitogens only in control cells, not in MV-infected cells. Altogether, our data emphasize that MV-associated alteration of SRIF transduction appears to be related to the loss of SRIF-dependent increase of mitogen-induced proliferation.     

An in vivo OctreoScan-negative adrenal pheochromocytoma expresses somatostatin receptors and responds to somatostatin analogs treatment in vitro.

A 52-yr-old woman presented with hypertension, elevated urinary vanillylmandelic acid, metanephrines, normetanephrines, and plasma chromogranin A (CgA), but normal urinary catecholamine levels. Abdominal ultrasonography and subsequent MRI imaging showed a 3 cm nodular lesion of the right adrenal gland also visualized by 123I-meta-iodobenzylguanidine scintigraphy consistent with a pheochromocytoma (PC). Her OctreoScan was negative. The patient underwent right adrenalectomy and histological examination showed a PC. The adrenal medulla tissue was examined for somatostatin (SRIH) receptor subtypes 1 to 5 (SSTR1 to 5) expression by RT-PCR. Cultured tumor cells were treated with either SRIH, Lanreotide (Lan), or an SSTR2 (BIM-23 120) or SSTR5 (BIM-23 206) selective agonist. CgA secretion was measured in the medium by ELISA and catecholamine levels by HPLC after 6h. Cell viability was assessed after 48h. RT-PCR analysis showed that SSTR1, 2, 3 and 4 were expressed. CgA secretion was significantly reduced by SRIH (- 80 %), Lan (- 35 %), and the SSTR2 selective agonist (- 65 %). Norepinephrine secretion was reduced by SRIH (- 66 %), Lan (- 40 %), and BIM-23 120 (- 70 %). Epinephrine and dopamine secretion was also inhibited by treatment with SRIH (- 90 % and - 93 %, respectively) and BIM-23 120 (- 33 % and - 75 %, respectively) but not by Lan. Cell viability was also significantly reduced by SRIH (- 30 %), Lan (- 10 %), and the SSTR2 selective agonist (- 20 %). The SSTR5 selective agonist did not modify either CgA and catecholamine secretion or cell viability. Our data show that SSTRs may be present in a PC although OctreoScan is negative in vivo, and that SRIH and its analogs may reduce both differentiated and proliferative functions in chromaffin cells in vitro. These findings suggest that SRIH analogs with enhanced SSTR2 affinity might be useful in the medical therapy of PC, even when an OctreoScan is negative.     

Structure-function relationships of somatostatin analogs

Objective of peptide chemistry has always been the production of analogues for clinical application. Advantages sought over natural peptides are (a) reduced molecular size; (b) prolonged biological half-life, and (c) enhanced specificity. After elucidation of the active core of somatostatin a number of analogues have been synthetized. Among them SMS 201-995, an octapeptide, was selected for further development because of its high potency and prolonged plasma clearance. Procedures extending the duration of action of somatostatin derivatives such as enhancement of lipophilicity and amino acid substitution are described, and factors influencing the specificity of such substances are succinctly analyzed.     

Extended retro-inverso analogs of somatostatin

An extended retro-inverso modification was introduced at the central six residues of the somatostatin molecule, the region of internal enzymatic degradations. The synthesis of the analog [Ala⁴,g-Phe⁶-r-D -Phe⁷-r-D -Trp⁸-r-D -Lys⁹-r-D -Thr¹⁰-m-R,S-Phe¹¹]-somatostatin required a unique strategy accommodating the unusual structure. Side-chain protection based on the t-butyl group in combination with Fmoc and Nps α-amino protection was employed. The key component containing the gem-diaminoalkyl residue was generated by an iodobenzene bistrifluoroacetate-mediated reaction. The separation of diastereomers of the cyclic tetradecapeptide in highly pure form was accomplished by high-performance liguid chromatography on a semipreparative scale. The analogs exhibited very low potency in the growth hormone inhibition test in vitro. This is interpreted as the consequence of the complex structural changes created by the extended retro-inverso modification.     

Synthesis and characterization of multiply-tyrosinated, multiply-iodinated somatostatin analogs.

Radio-labeled somatostatin analogs have recently gained popularity as agents useful in intraoperative tumor localization, external scintigraphy and in situ radiotherapy. We have synthesized and characterized a series of novel N-terminally extended multiply-tyrosinated somatostatin analogs that possess high binding affinity for somatostatin receptors, exhibit biological activity comparable to the native peptide and retain these characteristics after iodination. These analogs can be radio-iodinated to high specific activities. Following radioiodination, these analogs exhibit minimal radiolysis and may be clinically useful for tumor localization, scanning and therapy.     

Calcium-dependence of somatostatin binding to receptors

Binding of 125I-[Tyr11] somatostatin (S14) and 125I-[Tyr1]S14 has been studied in pancreatic acini and cerebral cortex. Ca2+-dependence of somatostatin binding to receptors was observed only with the highly non degradable iodinated analog 125I-[Tyr11] somatostatin but not with 125I-[Tyr1] somatostatin. The inhibitory activity of S14 on secretin-stimulated cAMP cellular content was decreased when Ca2+ concentration in the medium was reduced to 30 nM, indicating that the Ca2+ dependence of somatostatin binding to receptors is also present with the native peptide.     

Natural peptides and their analogs. XXXV. Synthesis and properties of [Ala5, Orn9]somatostatin

A total chemical synthesis of [Ala5, Orn9]somatostatin has been performed. This structural analogue of natural somatostatin inhibits the release of somatotropin, insulin and glucagon, but shows no inhibitory effect on secretion of prolactin.     

Development of specific and non-specific somatostatin analogs

Biological activity of six somatostatin analogs has been investigated. In these analogs, disulfide bond is replaced by ethylene bond cyclized with alpha-amino suberic acid. In addition, they contain unique D-configuration in both Trp8 and Cys14 moiety with dicarba substitution. An analog of the short chain length, C omega 7-cyclo (Phe6-Phe7-D-Trp8-Lys9-Thr10-Phe11-D-Asu14) (analog 4) has suppressive effect for GH, but not for other hormones. Analog 6, C omega 9-cyclo(Asn5-Phe6-Phe7-D-Trp8-Lys9-Thr10-Ph e11-Thr12-D-Asu14), has suppressed GH and insulin secretion, but not for gastrin and glucagon. Analog 1, C omega 11-cyclo (Lys4-Asn5-Phe6-Phe7-D-Trp8-Lys9-Thr10-Phe11- Thr12-Ser13-D-Asu14] and 5, C omega 9-cyclo (Lys4-Asn5-Phe6-Phe7-D-Trp8-Lys9-Thr10-Phe11-D-+ ++Asu14) have broad suppressive effect for GH, gastrin, insulin and glucagon release after arginine infusion. The shortest analog, analog 2, C omega 5-cyclo (Phe7-D-Trp8-Lys9-Thr10-D-Asu14) has weak suppressive effect of GH, insulin and glucagon secretion, and it is suggested that Phe6 and Phe11 are necessary for the appearance of suppressive effect of GH. Specific analog, analog 4, may be useful for the future treatment for acromegaly and diabetic retinopathy. Nonspecific analogs, 1 and 5 are candidates for the clinical application of wide variety.