Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins.

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.

Effects of N-hydroxy-N'-aminoguanidine isoquinoline in combination with other inhibitors of ribonucleotide reductase on L1210 cells.

The 1-isoquinolylmethylene derivative of N-hydroxy-N'-aminoguanidine (HAG) is the most potent agent of the recently synthesized series of HAG-derived ribonucleotide reductase inhibitors. To potentiate the effects of the HAG-isoquinoline drug [HAG-1-isoquinolylmethylene tosylate (HAG-IQ)], we combined it with other inhibitors of ribonucleotide reductase. Using mouse leukemia L1210 cell cultures, we tested drug combinations for their cytostatic and cytotoxic properties and for their effects on intracellular ribonucleotide reductase activity and nucleic acid synthesis. Deoxyguanosine or deoxyadenosine combined with HAG-IQ inhibited cell growth in an additive manner; three-drug combinations, HAG-IQ plus either deoxyguanosine/8-aminoguanosine or deoxyadenosine/deoxycoformycin, were strongly synergistic. When Desferal, an iron chelator, was added to these combinations, the four-drug combinations increased inhibition of cell growth and increased cytotoxicity. The intracellular target of these drug combinations in L1210 cells was the ribonucleotide reductase site. The formation of deoxycytidine from [14C]cytidine and incorporation into DNA were markedly inhibited by these drug combinations, while RNA synthesis was unaffected. These data show that the antiproliferative and cytotoxic effects of HAG-IQ, a potent inhibitor by itself, can be further potentiated in combinations with other ribonucleotide reductase inhibitors.

2,6-Diaminopurinedeoxyriboside as a prodrug of deoxyguanosine in L1210 cells.

The mode of action of the antiproliferative nucleoside analogue 2,6-diaminopurinedeoxyriboside (DAPdR) has been characterized in cultured L1210 cells. A marked concentration-dependent decrease in DNA synthesis and ribonucleotide reductase activity occurred in L1210 cells exposed to 0.05 to 1.0 mM DAPdR. Concomitantly, dGTP levels increased as much as 1100-fold as compared to untreated controls. Adenosine deaminase efficiently catalyzed DAPdR conversion to deoxyguanosine in vitro. In a comparative study, DAPdR and deoxyguanosine gave similar results. A 50% inhibition of cell growth during a 72-h incubation was achieved with 0.14 mM DAPdR or 0.26 mM deoxyguanosine. Deoxycytidine rescued the L1210 cells from DAPdR and deoxyguanosine toxicity to the same extent. DAPdR and deoxyguanosine counteracted the toxic effects of mycophenolic acid with the same efficiency. While DAPdR was not metabolized to its 5'-triphosphate, 2,6-diaminopurine was converted to 2,6-diaminopurineriboside 5'-triphosphate in L1210 cells; accordingly 50% inhibition of cell growth occurred at 0.015 mM 2,6-diaminopurine. Combinations of DAPdR with erythro-9-(2-hydroxy-3-nonyl)adenine or deoxycoformycin resulted in antagonism instead of an expected synergism. These data suggest that DAPdR exerts its toxicity on L1210 cells as a prodrug of deoxyguanosine.

Antiproliferative effects of the somatostatin analogue octreotide (SMS 201-995) on ZR-75-1 human breast cancer cells in vivo and in vitro.

The somatostatin analogue octreotide (SMS 201-995) inhibits secretion and growth of certain tumor cells, and current efforts are directed toward the elucidation of its mode of antiproliferative action. In this study, the effect of octreotide on the growth of ZR-75-1 human breast cancer cells has been characterized in immunodeficient nude mice and in cell culture. These results have been related to the expression of somatostatin receptors in vivo and in vitro. Continuous infusion of 10 micrograms/kg/h of octreotide yielded plasma levels of 5.7 ng/ml and elicited highly significant growth inhibitory effects on solid ZR-75-1 breast tumors in nude mice. After 2 and 4 weeks of treatment, tumor volumes in the octreotide group were 39.1 and 36.7% of those of control animals treated with vehicle, respectively. Autoradiographic studies demonstrated that 8 of 12 ZR-75-1 tumors studied were somatostatin receptor positive. When ZR-75-1 tumor cells were exposed in vitro to nanomolar concentrations of octreotide, a dose-dependent inhibition of cell growth was observed in the presence of 5% fetal calf serum or under serum-free conditions using epidermal growth factor, insulin-like growth factor type I, or insulin as growth stimulus. In parallel receptor-binding experiments, ZR-75-1 cells were shown to express specific high-affinity somatostatin receptors (Kd value = 0.9 nM, Bmax = 6000 sites/cell). From these experiments, we conclude that octreotide is a powerful inhibitor of ZR-75-1 tumor cell growth in nude mice and in culture. This inhibitory action of octreotide and the presence of somatostatin receptors on ZR-75-1 tumor cells in vitro and in vivo suggest a direct, somatostatin receptor-mediated effect of octreotide.

Somatostatin analogue octreotide enhances the antineoplastic effects of tamoxifen and ovariectomy on 7,12-dimethylbenz(alpha)anthracene-induced rat mammary carcinomas.

The efficacy of tamoxifen and ovariectomy in the management of breast cancer is limited by the resistance of many neoplasms to these endocrine therapies and by the fact that initially responding tumors often escape from control during long-term treatment. We evaluated the effect of coadministration of the somatostatin analogue octreotide, which has single agent activity in several in vivo and in vitro breast cancer models, on the antineoplastic actions of tamoxifen and ovariectomy on 7,12-dimethylbenz(alpha)anthracene-induced mammary tumors. Rats received tamoxifen (0.5 mg/kg twice weekly s.c.), octreotide (10 micrograms/kg/h for 6 weeks by osmotic minipump), or the combination 7 weeks following 7,12-dimethylbenz(alpha)anthracene administration. The number of tumors per animal and the sum of the volumes of palpable tumors per animal were significantly less in the combination treatment than in the others. In ovariectomized rats the marked regression of established tumors in the initial 4 weeks after ovariectomy was frequently followed by tumor regrowth. However, continuous infusion of octreotide (50 micrograms/kg/h for 6 weeks postovariectomy) significantly (P < 0.01) suppressed this regrowth. Our data suggest that octreotide enhances the antitumor effects of tamoxifen or ovariectomy in the 7,12-dimethylbenz(alpha)anthracene mammary cancer model.

Effects of N-hydroxy-N'-aminoguanidine derivatives on ribonucleotide reductase activity, nucleic acid synthesis, clonogenicity, and cell cycle of L1210 cells.

Derivatives of N-hydroxy-N'-aminoguanidine were recently shown to be efficient inhibitors of mammalian ribonucleotide reductase and cancer cell growth. We investigated the effects of the 1-isoquinolylmethylene and the 2-quinolylmethylene derivatives of N-hydroxy-N'-aminoguanidine on intracellular targets, cell viability, and cell cycle of L1210 mouse leukemia cells. A 2-h exposure of L1210 cells to either drug in the low micromolar concentration range led to inhibition of intracellular ribonucleotide reductase activity and DNA synthesis. After a 24-h incubation in the presence of these drugs, RNA synthesis was also markedly diminished. The clonogenicity of L1210 cells was inhibited after treatment with the drugs for 24 and 48 h, the I50 values being comparable to the drug concentrations required for 50% inhibition of DNA synthesis and cell proliferation. The isoquinoline compound was always more inhibitory to reductase activity, nucleic acid synthesis, and clonogenicity than the quinoline compound. As shown by flow cytometry, the N-hydroxy-N'-aminoguanidine isoquinoline derivative at 0.5-10 microM led to an elevation of G0/G1 cells and a decrease of G2/M and S cells. At 10 microM of the drug this shift remained unchanged over 48 h. L1210 cells treated with 0.5, 1, and 2 microM of the drug overcame the block after 4 to 12 h of exposure and progressed through S- and G2/M-phase in a synchronized manner.

sst2 somatostatin receptor expression reverses tumorigenicity of human pancreatic cancer cells.

Among the five cloned somatostatin receptor subtypes (sst1 to sst5), sst2 mediates the antiproliferative effect of somatostatin analogues in vitro. Somatostatin analogues have been shown to inhibit cell growth in vitro and in vivo in pancreatic cancer models that expressed sst2. We recently demonstrated the loss of sst2 gene expression in human pancreatic adenocarcinomas and most of the derived pancreatic cancer cell lines. In the present study, we corrected the sst2 defect in human pancreatic cancer BxPC-3 and Capan-1 cells by stable transfection with human sst2 cDNA. In the absence of exogenous ligand, both BxPC-3 and Capan-1 cells expressing sst2 showed a significant reduction in cell growth. This inhibitory effect was blocked by treatment with antiserum to somatostatin. sst2-expressing cells produced somatostatin-like immunoreactivity that mainly corresponded to somatostatin 14, indicating the induction of a negative autocrine loop. In other respects, sst2 expression in Capan-1 cells induced a significant reduction of clonogenicity in soft agar. Moreover, a significantly reduced (Capan-1 cells) or suppressed (BxPC-3 cells) tumor growth in athymic nude mice was observed. The reversal of tumorigenicity induced by the restoration of sst2 expression suggests that the loss of sst2 contributes to the malignancy of human pancreatic cancers.

Biochemical pharmacology and analysis of fluoropyrimidines alone and in combination with modulators.

After more than three decades since their introduction, fluoropyrimidines, especially FUra, are still a mainstay in the treatment of various solid malignancies. The antitumor effects of fluoropyrimidines are dependent upon metabolic activation. FdUMP, FUTP and FdUTP were identified as the key cytotoxic metabolites that interfere with the proper function of thymidylate synthase and nucleic acids. The relevance of these metabolites is cell-type specific. Recently, fluorouridine diphospho sugars have been detected, but the precise function of this class of metabolites is currently unknown. In mammalian systems fluoropyrimidines and their natural counterparts share the same metabolic pathways since the substrate properties in enzyme-catalyzed reactions are frequently comparable. Ongoing studies indicate that the metabolism and action of fluoropyrimidines exhibit circadian rhythms, which appear to be due to variations in the activity of metabolizing enzymes. Essential for the expanding knowledge of the pathways and effects of fluoropyrimidines has been the constant improvement of analytical methods. These include ligand binding techniques, numerous dedicated HPLC systems and 19F-NMR. Because the overall response rates achieved with fluoropyrimidines are modest, strategies based on biochemical modulation have been devised to enhance their therapeutic index. Biochemical modulators include a wide range of various compounds with different modes of action. In recently completed clinical trials, combinations of FUra with leucovorin, a precursor for 5,10-methylene tetrahydrofolate, or with levamisole, an anthelminthic with immunomodulatory activity, appeared to be superior to FUra alone. At the preclinical level combinations of fluoropyrimidines with, e.g. interferons or L-histidinol were demonstrated to be interesting candidates for further testing. The future therapeutic utility of fluoropyrimidines will depend on both the improvement of combination regimens currently used in the treatment of cancer patients and the judicious clinical implementation of promising experimental modulation strategies. Moreover, novel fluoropyrimidines with superior pharmacological properties may become important as part of or instead of modulation approaches.

Somatostatin analogs for diagnosis and treatment of cancer.

Somatostatin (SRIF) is a cyclic tetradecapeptide hormone initially isolated from ovine hypothalami. It inhibits endocrine and exocrine secretion, as well as tumor cell growth, by binding to specific cell surface receptors. Its potent inhibitory activity, however, is limited by its rapid enzymatic degradation and the consequent short plasma half-life. Octreotide is a short SRIF analog with increased duration of action compared to SRIF. Octreotide is approved for the treatment of acromegaly, amine precursor uptake and decarboxylation-omas, complications of pancreatic surgery and severe forms of diarrhea. Preclinical studies have focussed on the anticancer effects of octreotide and the related SRIF analogs BIM 23014 and RC-160. In vitro at nanomolar concentrations, these analogs inhibit the growth of tumor cells that express high affinity SRIF receptors. Accordingly, SRIF analogs, such as octreotide, potently inhibit the growth of SRIF receptor-positive tumors in various rodent models, and, in particular, xenotransplanted human tumors in nude mice. The range of cancers susceptible to octreotide and related SRIF analogs includes mammary, pancreatic, colorectal and lung malignancies. Moreover, an indirect antiproliferative effect of SRIF analogs is achievable in SRIF receptor-negative tumors, whose growth is driven by factors (gastrin, insulin-like growth factor-1, etc.) that are downregulated by SRIF. The use of radiolabeled somatostatin analogs represents a new diagnostic approach. [111In-DTPA]octreotide was developed for gamma camera imaging of SRIF receptor-positive malignancies, such as gasteroenteropancreatic tumors. Visualization of SRIF receptor-positive tumors in humans is emerging as an important methodology, both in tumor staging and predicting therapeutic response to octreotide. Recently, five SRIF receptor subtypes (SSTR1-5) have been cloned, all of which bind SRIF with high affinity. In contrast, SRIF receptor subtypes 1-5 have different binding profiles for short SRIF analogs. Octreotide, SSTR5, show moderate affinity for SSTR3 and fail to bind with high affinity to the other subtypes (SSTR1 and 4). Accordingly, the oncological profile of these three analogs is apparently similar. In conclusion, somatostatin analogs are a promising class of compounds for diagnosis and treatment of cancer. Current work is focussed on the identification of further SRIF receptor subtype-selective analogs with potential in oncology.

Regulation of CRH-induced secretion of ACTH and corticosterone by SOM230 in rats.

OBJECTIVE: Adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome is biochemically characterized by increased plasma concentrations of ACTH inducing hypersecretion of cortisol. Somatostatin is known to inhibit ACTH secretion, and in vitro data have shown the inhibition of ACTH secretion by agonists activating sst2 and sst5 receptors. The present study aimed to determine the inhibitory effect of the multireceptor ligand SOM230, compared with the sst2-preferring agonist octreotide, on corticotropin-releasing hormone (CRH)-stimulated secretion of ACTH and corticosterone in rats. METHODS: Secretion of ACTH and corticosterone was induced by i.v. application of CRH (0.5 microg/kg) in rats pretreated 1 h before by i.v. application of SOM230 (1, 3, or 10 microg/kg), octreotide (10 microg/kg) or NaCl 0.9%. RESULTS: SOM230 (3 and 10 microg/kg) inhibited CRH-induced ACTH release by 45+/-3% and 51+/-2%, respectively, and corticosterone release by 43+/-5% and 27+/-16%, respectively. 10 microg/kg of octreotide tended to be less potent at inhibiting ACTH release (34+/-6% inhibition) and did not alter the secretion of corticosterone. CONCLUSION: SOM230 has a stronger inhibitory effect on ACTH and corticosterone secretion than octreotide in rats. This difference can be explained by its higher affinity to sst1, sst3 and especially sst5 receptors compared with octreotide.

Coexpression of CD4 and CD8alpha on rat T-cells in whole blood: a sensitive marker for monitoring T-cell immunosuppressive drugs.

The aim of this study was to develop a new quantitative method for measuring in vitro the effects of T-cell immunosuppressive drugs by flow cytometry. Rat whole blood samples were stimulated with the T-cell mitogen succinylated concanavalin A in the presence or absence of different drugs. After 3 days, the expression of CD25 and CD8alpha in mitogen-stimulated CD4(+) cells increased 10- to 20-fold as measured by flow cytometry. Drug efficacy and potency was calculated based on dose-response curves of the drug-mediated decrease in CD4(+)/CD8alpha(+)/CD25(+) cells. The expression of CD8alpha in mitogen-stimulated CD4(+) cells was blocked completely by calcineurin inhibitors (cyclosporine A and FK-506), and partially by rapamycin and SDZ-RAD. The IC(50) (50% inhibitory concentration) values obtained were (mean+/-S.E.): 99.5+/-16.6 nM for cyclosporine A, 10.4+/-1.3 nM for FK-506, 1.8+/-0.7 nM for rapamycin, and 6.4+/-1.1 nM for SDZ-RAD. Our results show, for the first time, that CD8alpha, used as an activation antigen, is a sensitive marker for monitoring T-cell immunosuppression.

Novel antiproliferative agents derived from lavendustin A.

The active partial structure of the potent tyrosine kinase inhibitor lavendustin A was derivatized in the search for novel agents against cellular proliferation. The antiproliferative potential of the new derivatives was determined using the human keratinocyte cell line HaCaT as the primary test system. Whereas the lavendustin A partial structure is ineffective in inhibiting cell proliferation, esterification of its carboxylic acid function leads to measurable antiproliferative activity. Additional O-methylation of the 2,5-dihydroxyphenyl moiety yields activity in the micromolar range. Further substantial increases in activity are achieved by replacing the nitrogen with oxygen and carbon within the 2,5-dimethoxyphenyl series (but not within the 2,5-dihydroxyphenyl analogs) leading to 5-[2-(2,5-dimethoxyphenyl) ethyl]-2-hydroxybenzoic acid methyl ester (13) as the most potent analog identified to date. These increases in antiproliferative activity are paralleled, however, by the disappearance of activity against the epidermal growth factor receptor-associated tyrosine kinase, suggesting another mechanism of action.

Aromatic quinolinecarboxamides as selective, orally active antibody production inhibitors for prevention of acute xenograft rejection.

The prevention of xenograft rejection is substantially dependent on inhibiting antibodies (Ab) produced by B-cells independently of T-cell signals (TI-1). Due to their ubiquitous biochemical mechanisms of action, the immunosuppressants currently employed not only fail to discriminate between B- and T-cells but also have a narrow therapeutic window and, thus, their prolonged use in complex immunosuppressive regimens is problematic. By capitalizing on the target enzyme-bound (DHODH) structure 1b of one of these compounds, leflunomide, and modulating part of its multiple mechanisms of action to gain selectivity, the quinoline-8-carboxamide 3 was designed as a potentially weak enzyme inhibitor but effective immunosuppressant. Compound 3 fulfilled the mechanistic criteria set and had 10-fold B-cell over T-cell selectivity. Its pyridyl analogue 4 was found to be a highly potent and selective B-cell immunosuppressant with a 75-fold selectivity for B- over T-cells (as judged by the MLR data) and no general cytotoxicity at concentrations up to 160-fold higher than those required to inhibit B-cells. In the mouse, 4 effectively blocked TI-1 Ab production and suppressed Ab-mediated xenograft rejection in a xenotransplantation model under a once-daily dosing regimen, with efficacy down to 0.3 mg/kg/day po. These are the first data demonstrating the feasibility of the development of drugs specific for impeding Ab production.

Ribonucleotide reductase activity and growth of glutathione-depleted mouse leukemia L1210 cells in vitro.

L1210 cells treated with L-buthionine-(S/R)-sulfoximine (BSO) had glutathione (GSH) and non-protein thiol levels only 15% that of control. These GSH-depleted cells grew as well as the control L1210 cells and there was no decrease in ribonucleotide reductase activity in situ as measured by the conversion of [14C]cytidine to deoxytidine nucleotides and incorporation into DNA. Further, when these BSO-stressed cells were treated with hydroxyurea or IMPY, there was no potentiation of the inhibition caused by hydroxyurea or IMPY alone. These data indicate that the glutathione/glutaredoxin system of ribonucleotide reductase is not the sole carrier of reducing equivalents from NADPH for the reduction of the 2'-position of the corresponding ribonucleoside 5'-diphosphate; and that glutathione is not critical in regenerating the tyrosyl free-radical on the M2 subunit which is destroyed by the hydroxyurea or 2,3-dihydro-1H-pyrazolo-[2,3-alpha]imidazole (IMPY) treatment.

Substrate properties of 5-fluorouridine diphospho sugars detected in hepatoma cells.

Nucleotide sugars derived from 5-fluorouridine were studied in cultured AS-30D hepatoma cells as well as in kinetic enzyme assays in vitro in comparison with the physiologic uridine diphospho sugars. Hepatoma cells converted 5-fluoro [14C]uridine to 5-fluorouridine diphospho (FUDP) glucose, FUDP-galactose, FUDP-N-acetylglucosamine, FUDP-N-acetylgalactosamine, and trace amounts of FUDP-glucuronate, as analyzed by different systems of high-performance liquid chromatography. 5-Fluoro[14C]uridine and [14C]uridine, at concentrations of 5 microM in the culture medium, were phosphorylated by the cells during 60 min to similar amounts of FUTP and UTP, respectively, while the synthesis of [14]FUDP-sugars was reduced to 14% as compared to that of [14C]UDP-sugars. FUDP-sugars, synthesized by chemical and enzymatic procedures, were assayed in vitro as substrates for enzymes of UDP-sugar metabolism. Km and V values in a range comparable to that of the respective UDP-sugars were determined for FUDP-sugars in the reactions catalyzed by UDP-glucose pyrophosphorylase, galactose-1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, UDP-N-acetylglucosamine 2-epimerase, glycogen synthase, and UDP-glucose dehydrogenase. Our experiments in hepatoma cells and with enzymes in vitro have revealed additional reactions of FUDP-sugar metabolism demonstrating a metabolite pattern analogous to that of UDP-sugars. The amounts of FUDP-sugars formed relative to UDP-sugars in intact cells were smaller than suggested on the basis of their kinetic comparison in vitro.

Properties of N-hydroxy-N'-aminoguanidine derivatives as inhibitors of mammalian ribonucleotide reductase.

In previous studies, N-hydroxy-N'-aminoguanidine (HAG) derivatives were demonstrated to suppress growth and clonogenicity of tumor cells which correlated with the inhibition of ribonucleotide reductase and DNA synthesis. The present work has focused on the properties of five HAG derivatives as inhibitors of the ribonucleotide reductase from Ehrlich ascites tumor cells. HAG derivatives acted as non-competitive inhibitors of ribonucleotide reductase with respect to the substrates CDP and ADP. The apparent Ki values for the various HAG derivatives as inhibitors of CDP reductase ranged from 3.4 to 543 microM. However, the apparent Ki values for these inhibitors with respect to ADP reductase were 2- to 10-fold lower than the respective values for CDP reductase. After a preincubation of HAG derivatives and ribonucleotide reductase in the absence of substrates, an increased inhibition was observed. The activity of the inhibited enzyme could be restored by passage over a Sephadex G-25 column and subsequent incubation with dithioerythritol. The addition of either the non-heme iron subunit or the effector-binding subunit to the intact enzyme in the assay mixture resulted in a diminished inhibition of ADP reduction. Inhibition by HAG derivatives of ribonucleotide reductase activity in the test tube was not enhanced by iron chelators. However, a combination of HAG compounds and iron chelators synergistically inhibited the growth of L1210 cells.

Somatostatin receptors and the potential use of Sandostatin to interfere with vascular remodelling.

Graft vessel disease (GVD) is a major cause of graft loss after the first year following transplantation. GVD is a complex, multifunctional process that involves immunological as well as non-immunological events such as ischaemia/reperfusion injury. An important target cell to interfere with the development of GVD is the smooth muscle cell (SMC). Somatostatin (SRIF) analogues have been shown previously to inhibit the proliferation of SMC in vitro and in vivo. We provide evidence that Sandostatin, an octapeptide SRIF analogue that is known to have anti-proliferative properties on SMC proliferation, inhibits vascular remodelling in a rat angioplasty model. Furthermore, in two allotransplantation models, Sandostatin effectively interferes with the development of signs of chronic rejection/GVD. The role of the different SRIF receptor subtypes in chronic graft rejection is currently under investigation.

SOM230: a novel somatostatin peptidomimetic with broad somatotropin release inhibiting factor (SRIF) receptor binding and a unique antisecretory profile.

OBJECTIVE: The aim of the present study was to identify a small, metabolically stable somatotropin release inhibiting factor (SRIF) analog with a more universal binding profile similar to that of natural somatostatin, resulting in improved pharmacological properties and hence new therapeutic uses. DESIGN: A rational drug design approach was followed by synthesizing alanine-substituted SRIF-14 analogs to determine the importance of single amino acids in SRIF-14 for SRIF receptor subtype binding. The incorporation of structural elements of SRIF-14 in a stable cyclohexapeptide template in the form of modified unnatural amino acids resulted in the identification of the novel cyclohexapeptide SOM230. RESULTS: SOM230 binds with high affinity to SRIF receptor subtypes sst1, sst2, sst3 and sst5 and displays a 30- to 40-fold higher affinity for sst1 and sst5 than Sandostatin (octreotide; SMS 201-995) or Somatuline (BIM 23014). In vitro, SOM230 effectively inhibited the growth hormone releasing hormone (GHRH)-induced growth hormone (GH) release in primary cultures of rat pituitary cells with an IC(50) of 0.4+/-0.1 nmol/l (n=5). In vivo, SOM230 also potently suppressed GH secretion in rats. The ED(50) values determined at 1 h and 6 h post injection of SOM230 indicated its very long duration of action in vivo. This property was also reflected in pharmacokinetic studies comparing plasma levels of SMS 201-995 and SOM230 after subcutaneous application. Whereas SMS 201-995 had a terminal elimination half life of 2 h, this was markedly prolonged in SOM230-treated animals (t(1/2)=23 h). Furthermore, in rats SOM230 demonstrated a much higher efficacy in lowering plasma insulin-like growth factor-I (IGF-I) levels compared with SMS 201-995. The infusion of 10 microg/kg/h of SOM230 using subcutaneously implanted minipumps decreased plasma IGF-I levels far more effectively than SMS 201-995. After 126 days of continuous infusion of SOM230 plasma IGF-I levels were decreased by 75% of placebo-treated control animals. For comparison SMS 201-995, when used under the same experimental conditions, resulted in only a 28% reduction of plasma IGF-I levels, indicating a much higher efficacy for SOM230 in this animal model. It is important to note that the inhibitory effect of SOM230 was relatively selective for GH and IGF-I in that insulin and glucagon secretion was inhibited only at higher doses of SOM230. This lack of potent inhibition of insulin and glucagon release was also reflected in the lack of effect on plasma glucose levels. Even after high dose treatment over 126 days no obvious adverse side effects were noticed, including changes in plasma glucose levels. CONCLUSION: We have identified a novel short synthetic SRIF peptidomimetic, which exhibits high affinity binding to four of the five human SRIF receptor subtypes and has potent, long lasting inhibitory effects on GH and IGF-I release. Therefore SOM230 is a promising development candidate for effective GH and IGF-I inhibition and is currently under evaluation in phase 1 clinical trials.

Preclinical studies on the anticancer activity of the somatostatin analogue octreotide (SMS 201-995).

The antiproliferative effect of somatostatin-14 and its analogue, octreotide, on in vitro pancreatic and breast tumor cells has led to the suggestion that octreotide may have further oncological indications in addition to its use in the treatment of gastroentero-pancreatic (GEP) tumors. To extend these in vitro observations, we evaluated the effect of octreotide in rodent models of pancreatic and breast tumors. Octreotide at a dose of 5 micrograms or 50 micrograms twice a day in nude mice bearing solid MiaPaCa pancreatic tumors (subline 21) or ZR-75-1 breast tumors induced a significant inhibition of tumor growth from week 2 until the end of treatment at week 5. After 5 weeks, the mean volume of ZR-75-1 tumors in animals treated with the 50-micrograms regimen was 48% of that in controls. Autoradiographic studies showed that a high percentage (71%) of ZR-75-1 tumors were somatostatin receptor-positive. In addition, the growth of ZR-75-1 cells in vitro was significantly inhibited by octreotide. The drug was also tested in a second breast cancer model, 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumors in rats, and continuous administration of 10 micrograms/kg/h over 6 weeks led to an approximate 50% reduction in the number of tumors arising in the rat mammary gland. These data suggest that pancreatic and breast cancer may be among the malignant diseases clinically susceptible to octreotide.

Binding properties of somatostatin receptor subtypes.

In the past few years, five different somatostatin (SRIF) receptor subtypes (sst1.5) have been identified, which form a distinct group in the superfamily of G-protein-coupled receptors. The naturally occurring somatostatins SRIF-28, SRIF-25, and SRIF-14 all reveal high-affinity binding for sst1.5. In contrast, short synthetic analogs that are in clinical use, such as SMS 201-995, RC-160, or BIM 23014, primarily interact with the sst2 subtype. Some SRIF analogs were previously reported to be selective for one SRIF receptor subtype, eg, the sst2 (MK 678), the sst3 (BIM 23056), or the sst5 (BIM 23052, L362-855) subtype. However, when we studied the binding affinities of these SRIF analogs for human (h) sst1.5 expressed in either CHO or COS-1 cells, we were unable to confirm these previously reported selectivities. The absence of sst antagonists is a major drawback for investigating the functional role of each sst subtype. We used site-directed mutagenesis to identify amino acids that determine ligand specificity for sst2. A single Ser305 to Phe mutation in TM VII increased the affinity of hsst1, for SMS 201-995 nearly 100-fold, and when Gln291 was also exchanged to Asn in TM VII of hsst1, almost full sst2-like binding of SMS 201-995 was obtained. These data may aid in the design and synthesis of new selective type sst ligands. We have identified the expression of sst subtypes in nonclassical SRIF target tissue such as the lung. The pKi values for SRIF and various SRIF analogs in rat lung tissue preparations were in close correlation with those obtained for CHO cells expressing the sst4 subtype. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the predominant expression of mRNA specific for sst4 in mouse, rat, and human lung tissue, confirmed by autoradiographies of rat lung. No specific binding for [125I]Tyr3-SMS 201-995 was detected, since SMS 201-995 has low affinity for sst4. In contrast, specific binding of [125I]SRIF-28 to rat lung sections was demonstrated, which could be displaced by unlabelled SRIF-14 and SRIF-28, indicating specific, high affinity binding of this radioligand to sst4 receptors.