A novel retinoic acid receptor-selective retinoid, ALRT1550, has potent antitumor activity against human oral squamous carcinoma xenografts in nude mice.

We have identified a novel retinoid, ALRT1550, that potently and selectively activates retinoic acid receptors (RARs). ALRT1550 binds RARs with Kd values of approximately equal to 1-4 nM, and retinoid X receptors with low affinities (Kd approximately equal to 270-556 nM). We studied the effects of ALRT1550 on cellular proliferation in squamous carcinoma cells. ALRT1550 inhibited in vitro proliferation of UMSCC-22B cells in a concentration-dependent manner with an IC50 value of 0.22 +/- 0.1 (SE) nM. 9-cis-Retinoic acid (ALRT1057), a pan agonist retinoid that activates RARs and retinoid X receptors, inhibited proliferation with an IC50 value of 81 +/- 29 nM. In vivo, as tumor xenografts in nude mice, UMSCC-22B formed well-differentiated squamous carcinomas, and oral administration (daily, 5 days/week) of ALRT1550, begun 3 days after implanting tumor cells, inhibited tumor growth by up to 89% in a dose-dependent manner over the range of 3-75 micrograms/kg. ALRT1550 (30 micrograms/kg) also inhibited growth of established tumors by 72 +/- 3% when tumors were allowed to grow to approximately equal to 100 mm3 before dosing began. In comparison, 9-cis retinoic acid at 30 mg/kg inhibited growth of established tumors by 73 +/- 5%. Interestingly, retinoids did not appear to alter tumor morphologies in UMSCC-22B tumors. Notably, ALRT1550 produced a therapeutic index of approximately equal to 17 in this model, indicating a separation between doses that inhibited tumor growth and that induced symptoms of hypervitaminosis A. In summary, ALRT1550 potently inhibits cellular proliferation in vitro and in vivo in this squamous cell carcinoma tumor model. These data support additional study of ALRT1550 for its potential for improving anticancer therapy in human clinical trials.

Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3.

BACKGROUND: The secosteroid 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) acts through the vitamin D receptor (VDR) to elicit many activities that make it a promising drug candidate for the treatment of a number of diseases, including cancer and psoriasis. Clinical use of 1,25(OH)2D3 has been limited by hypercalcemia elicited by pharmacologically effective doses. We hypothesized that structurally distinct, nonsecosteroidal mimics of 1,25(OH)2D3 might have different activity profiles from vitamin D analogs, and set out to discover such compounds by screening small-molecule libraries. RESULTS: A bis-phenyl derivative was found to activate VDR in a transactivation screening assay. Additional related compounds were synthesized that mimicked various activities of 1,25(OH)2D3, including growth inhibition of cancer cells and keratinocytes, as well as induction of leukemic cell differentiation. In contrast to 1, 25(OH)2D3, these synthetic compounds did not demonstrate appreciable binding to serum vitamin D binding protein, a property that is correlated with fewer calcium effects in vivo. Two mimics tested in mice showed greater induction of a VDR target gene with less elevation of serum calcium than 1,25(OH)2D3. CONCLUSIONS: These novel VDR modulators may have potential as therapeutics for cancer, leukemia and psoriasis with less calcium mobilization side effects than are associated with secosteroidal 1,25(OH)2D3 analogs.

Boc-CCK-4 derivatives containing side-chain ureas as potent and selective CCK-a receptor agonists.

Novel Boc-CCK-4 derivatives were communicated recently as having high potency and selectivity for the CCK-A receptor (Shiosaki et al. J. Med. Chem. 1990, 33, 2950-2952). While Boc-CCK-4 binds selectively to the CCK-B receptor, replacement of the methionine with an N epsilon-substituted lysine dramatically reversed receptor selectivity, leading to the development of this novel series of tetrapeptides. A detailed structure-activity analysis of a series of urea-substituted tetrapeptides, represented by the general structure Boc-Trp-Lys(N epsilon-CO-NHR)-Asp-Phe-NH2, revealed that a number of substituted phenyl, naphthyl, and aliphatic urea residues in the lysine side chain yielded potent and selective CCK-A ligands. These tetrapeptides elicit full agonist responses in stimulating pancreatic amylase release that are effectively blocked by a selective CCK-A receptor antagonist. Conversion of the urea to a thiourea significantly reduced CCK-A binding potency as did replacement of the lysine with the homologous ornithine or homolysine. Tetrapeptides that were partial agonists (less than 80% efficacy) in phosphoinositide (PI) hydrolysis relative to CCK-8 did not exhibit high-dose inhibition of amylase secretion in guinea pig acini.

Synthesis of retinoid X receptor-specific ligands that are potent inducers of adipogenesis in 3T3-L1 cells.

A novel series of oxime ligands has been synthesized that displays potent, specific activation of the retinoid X receptors (RXRs). The oximes of 3-substituted (tetramethyltetrahydronaphthyl)carbonylbenzoic acids are readily available by condensation with hydroxyl- or methoxylamine; alkylation of the hydroxyl oxime provides a variety of analogues. Oximes and variously substituted oxime derivatives demonstrate high binding affinity for the RXRs and specific RXR activation and, hence, are called rexinoids. These oxime rexinoids are activators of the RXR:PPARgamma heterodimer and are potent inducers of differentiation of 3T3-L1 preadipocytes to adipocytes. We have recently reported that ligands which activate the RXR:PPARgamma heterodimer in this manner are effective in the treatment of type II diabetes (non-insulin-dependent diabetes mellitus, NIDDM). Thus, these new oxime rexinoids are potential therapeutic agents for the treatment of metabolic disorders, such as obesity and diabetes.

Cholecystokinin antagonists: (R)-tryptophan-based hybrid antagonists of high affinity and selectivity for CCK-A receptors.

The intriguing structural similarities of glutamic acid based cholecystokinin (CCK) antagonists (A-64718 and A-65186) and the benzodiazepine CCK antagonist MK-329 (L-364,718) have been reported. Efforts to include the weak CCK antagonist benzotript into this construct utilizing a similar approach have resulted in a novel series of benzotript-based hybrid antagonists N alpha-(3'-quinolylcarbonyl)-(R)-tryptophan di-n-pentylamide (9, A-67396), N alpha-(4',8'-dihydroxy-2'-quinolylcarbonyl)-(R)-tryptophan di-n-pentylamide (23, A-70276), and N alpha-(3'-quinolylcarbonyl)-(R)-5'-hydroxytryptophan di-n-pentylamide (36, A-71134) which possess respectively binding affinities of 23, 21, and 11 nM for the pancreatic CCK-A receptor and which inhibit CCK8-induced amylase secretion. Compound 9 possesses a selectivity of greater than 500-fold for the pancreatic CCK-A receptor over the CCK-B receptor.

Development of potent and selective CCK-A receptor agonists from Boc-CCK-4: tetrapeptides containing Lys(N epsilon)-amide residues.

A series of Boc-CCK-4 derivatives represented by the general structure Boc-Trp-Lys(N epsilon-COR)-Asp-Phe-NH2, where R is an aromatic, heterocyclic, or aliphatic group, are potent and selective CCK-A receptor agonists. These amide-bearing compounds complement the previously described urea-based tetrapeptides (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842); structure-activity studies revealed parallel as well as divergent trends between these two series. A significant correlation was observed between pancreatic binding affinity and the resonance constant R of the phenyl substituent in one particular series of derivatives. Sulfation of phenolic amides appended onto the epsilon-amino group of the lysine did not affect affinity for the CCK-A receptor in contrast to the 500-fold increase in binding potency observed upon sulfation of CCK-8, suggesting that the lysine appendage and the sulfated tyrosine in CCK-8, both key structural elements that impart high affinity for the CCK-A receptor, are interacting differently with the receptor. The amide-bearing tetrapeptides are full agonists relative to CCK-8 in stimulating pancreatic amylase release while being partial agonists in eliciting phosphoinositide (PI) hydrolysis. Both effects were blocked by selective CCK-A receptor antagonists.

Discovery of novel retinoic acid receptor agonists having potent antiproliferative activity in cervical cancer cells.

Retinoic acid receptor (RAR) active retinoids have proven therapeutically useful for treating certain cancers and dermatological diseases. Herein, we describe the discovery of two new RAR active trienoic acid retinoids, (2E,4E,6E)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10a, ALRT1550) and (2E,4E,6Z)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10b, LG100567). ALRT1550 is a RAR selective retinoid which exhibits exceptional potency in both competitive binding and cotransfection assays. Moreover, it is the most potent antiproliferative retinoid described to date and thus has implications for the treatment of certain cancers. LG100567 is a potent panagonist which activates both RARs and retinoid X receptors.

Characterization of the novel CCK analogs JMV-180, JMV-320, and JMV-332 in H345 cells.

The interaction of the novel CCK analogs JMV-180, JMV-320, and JMV-332 with CCK-B/gastrin receptors on small cell lung cancer (SCLC) cells was investigated. JMV-180, JMV-320, and JMV-332 potently inhibited specific binding of 125I-CCK-8 to CCK-B/gastrin receptors expressed on the SCLC cell line NCI-H345 (H345) with IC50 values of 4.9, 1.8, and 7.0 nM, respectively. JMV-320 and JMV-332 stimulated intracellular calcium ([Ca2+]i) release in a dose-dependent manner in cells preloaded with indo-1. JMV-180 did not stimulate [Ca2+]i but inhibited the [Ca2+]i release elicited by 10 nM CCK-8 in a dose-dependent manner. These data indicate that JMV-320 and JMV-332 function as CCK-B/gastrin receptor agonists while JMV-180 functions as a CCK-B/gastrin receptor antagonist in H345 cells.

Effects of selective CCK receptor agonists on food intake after central or peripheral administration in rats.

In this paper report the effects of peripheral (intraperitoneal, i.p.) and central (intracerebroventricular, i.c.v.) injection of selective cholecystokinin (CCK) receptor agonists on food intake in the rat. Stimulation of peripheral and central CCK-A receptors by the selective CCK-A receptor agonist A-71623 suppressed intakes of a liquid diet in both deprived and sated rats. In contrast, i.c.v., but not i.p., injections of the selective CCK-B receptor agonist A-63387, reduced food intakes, although on a molar basis the effect was much less than that seen with A-71623. Although these results stress the relative importance of the CCK-A receptor in the effects of exogenous CCK-8 administration on feeding, stimulation of the CCK-B receptor may still be involved in the control of feeding following the endogenous release of CCK.

Centrally administered CCK-8 suppresses activity in mice by a "peripheral-type" CCK receptor.

Cholecystokinin octapeptide (CCK-8) administered either systemically (IP) or centrally (ICV) suppresses several types of behavior in mice including exploratory locomotion, rearing and grooming. At doses equimolar to those active for CCK-8, neither desulfated CCK-8 (CCK-8-DS), nor the protected C-terminus tetrapeptide fragment, BOC-CCK-4, is behaviorally active when administered either centrally or systemically. A potent and selective antagonist to the peripheral type (Type A) CCK receptor, A-65186, when given systemically, blocked the effects of systemically administered CCK-8, but failed to block the effects of ICV administered CCK-8. Central administration of A-65186 blocked the effects of ICV administered CCK-8. These results demonstrate that administration of exogenous CCK-8 to mice can suppress exploratory locomotion by acting either centrally or peripherally and that in either case the demonstrated behavioral effects are mediated via a "peripheral" type (Type A) CCK receptor.

A-71623, a selective CCK-A receptor agonist, suppresses food intake in the mouse, dog, and monkey.

The anorectic actions of cholecystokinin (CCK)-8 and of a selective CCK-A agonist, A-71623, were examined in CD1 mice, beagle dogs, and cynomolgus monkeys. A-71623 suppressed intakes in all species tested, and the effects were blocked by a selective CCK-A antagonist, A-70104. In the dog only, CCK-8 was more potent on a molar basis compared to A-71623, although the effects of both CCK agonists were more short-lived in the dog compared to the other species tested. Our results support other evidence suggesting that the anorectic actions of exogenous application of CCK-8 in these species are mediated via stimulation of the CCK-A receptor subtype.

Thyrotropin releasing hormone augments growth of spinal cord transplants in oculo.

The effects of thyrotropin releasing hormone (TRH) on spinal cord growth were evaluated using the in oculo transplant model. The growth of fetal spinal cord allografts, placed into the anterior eye chamber of Sprague-Dawley rats, was markedly augmented by acute exposure of the graft and host animal to TRH at the time of transplantation. No significant growth augmentation was seen after equimolar administration of a mixture of the amino acids that comprise the TRH molecule. It is concluded that acutely administered TRH, at the time of grafting, elicits a significant stimulation of the growth of spinal cord tissue. Our data strengthen the rationale for continued clinical trials of this peptide in spinal cord injury.

Analysis of combinatorial chemistry samples by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method has been developed that can evaluate the purity of samples generated in combinatorial chemistry libraries. This method uses an open tube capillary (27 cm x 50 microm) along with a run buffer composed of sodium dodecyl sulfate (SDS), hydroxypropyl-beta-cyclodextrin, and sodium tetraborate coupled with UV detection. Neutral compounds and compounds that were insoluble in aqueous buffers could be analyzed under these conditions in approximately 3 min. The concentration of SDS and the concentration of hydroxypropyl-beta-cyclodextrin effected the separation. The affect on selectivity resulting from the addition of an organic modifier to the run buffer was examined. The low background absorbency of the run buffer made for easy detection of compounds that absorbed at low UV wavelengths. The quick analysis time made this suitable for analysis of combinatorial chemistry samples.