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All reported GnRH receptor mutants (causing human hypogonadotropic hypogonadism) are misfolded proteins that cannot traffic to the plasma membrane. Pharmacoperones correct misfolding and rescue mutants, routing them to the plasma membrane where they regain function. Because pharmacoperones are often peptidomimetic antagonists, these must be removed for receptor function after rescue; in vivo this necessitates pulsatile pharmacoperone administration. As an antecedent to in vivo studies, we determined whether pharmacoperones need to be present at the time of synthesis or whether previously misfolded proteins could be refolded and rescued. Accordingly, we blocked either protein synthesis or intra-cellular transport. Biochemical and morphological studies using 12 mutants and 10 pharmacoperones representing three different chemical classes show that previously synthesized mutant proteins, retained by the quality control system (QCS), are rescued by pharmacoperones, showing that pharmacoperone administration in vivo likely need not consider whether the target protein is being synthesized at the time of drug administration.

Purpose: The angiogenic phenotype of malignant cancers has been established as a target for cancer therapy. ABT-526 and ABT-510, two peptide mimetics of thrombospondin-1 (TSP-1), block angiogenesis in vitro and in vivo and slow tumor growth in mice. To guide the clinical development of these drugs, translational studies in dogs with naturally occurring cancers were initiated., Experimental Design: A prospective open-label trial using ABT-510 or ABT-526 in pet dogs with measurable malignant spontaneously arising tumors. Endpoints included safety, pharmacokinetics, antitumor activity, and preliminary assessment of changes in circulating endothelial cell populations., Results: Two-hundred and forty-two dogs were sequentially entered to this open-label trial. The elimination half-life for ABT-510 and ABT-526 was 0.7 and 0.8 h, respectively (range, 0.5-1 h). No dose-limiting toxicities were seen in any dogs (N = 242). Forty-two dogs receiving peptide had objective responses (>50% reduction in tumor size; n = 6) or significant disease stabilization. Most objective responses were seen after 60 days of exposure to the TSP-1 peptide. Antitumor activity was similar for both peptides and was seen in several histologies, including mammary carcinoma, head and neck carcinoma, soft tissue sarcoma, cutaneous T-cell lymphoma, and non-Hodgkin's lymphoma. Assessment of circulating endothelial cell populations in a small subset of dogs suggested that effective exposure to TSP-1 peptides may be associated with reductions in circulating endothelial cells., Conclusions: These results support the safety and activity of ABT-526 and ABT-510 in dogs with naturally occurring malignant cancers. Data from this preclinical trial support the development of TSP-1 mimetic peptides as anticancer agents.

Antibacterial SAR for a series of macrolides derived from erythromycin A that are potent LHRH antagonists was developed in an attempt to eliminate the antibiotic activities of these compounds. Increasing the size of the alkyl substituents on the desosamine 3'-amine resulted in potent LHRH antagonists that were inactive against staphylococcal bacteria strains, and were significantly (>10-fold) less active against streptococcal bacteria strains. Complete elimination of antibacterial activities could be achieved by replacement of one or both methyl groups on the 3'-amine with a large alkyl substituent.

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies., (Copyright 2002 Wiley-Liss, Inc.)

After almost two decades, the research on LHRH antagonists has produced a number of decapeptides that are currently in clinical studies. The structures of these antagonists, unlike the agonists, differ substantially from that of LHRH. Five of the ten amino acids are unnatural and of D configuration. The structural combination of a hydrophobic N-terminus (residues 1, 2, and 3) and a basic/hydrophilic C-terminus (residues 6 and 8) was thought to be responsible for some HR reactions encountered with the second generation of LHRH antagonists. This side effect was greatly reduced by substituting the appropriate combination of amino acids at positions 5, 6, and 8. The next hurdle in the drug development of LHRH antagonists was solubility and aggregation. In the case of A-75998, water solubility was improved by 12- to 25-fold via substitution of NMeTyr at position 5. However, based on DLS analysis, the aqueous solutions still contained some large aggregates that were not visible to the naked eye. This formation of aggregates was eliminated on formulating A-75998 in Encapsin. In men, a single s.c. dose of 2 mg of A-75998 suppressed T to the castrate levels for over 30 hr. Other LHRH antagonists including ganirelix and cetrorelix are also in phase I/II clinical studies. Clinical studies with cetrorelix in prostate cancer; in vitro fertilization, and benign prostate hypotrophy have been reported.

A novel series of octapeptide LHRH antagonists was designed on the basis of the structure of the (2-9) fragment of a LHRH agonist. By adopting a systematic SAR study, we were able to improve first the in vitro activity and then the in vivo LH suppression, raising them up to the range of the decapeptide antagonists NalGlu (51) and A-75998 (50), resulting in A-76154 (49). The octapeptide antagonist A-76154 is the most potent reduced-size LHRH antagonist reported. It suppresses LH in the castrated rat by over 80% for a period of 4 h following sc bolus administration of 30 micrograms/kg.

Each peptide bond in leuprolide (1), deslorelin (13), and nafarelin (24) was separately substituted with N-methyl. The synthesized compounds were tested for in vitro receptor binding, LH release, and stability against chymotrypsin and intestinal degradation. The NMe-Ser4 (30), NMe-Leu7 (33), and Sar10 (35) analogues of nafarelin had pD2 values 2-, 20-, 9-fold higher than their respective parent. All the other N-methyl agonists were less active. For the first time, conversion of LHRH agonists to antagonists was observed as a result of N-methyl substitution in the peptide backbone. [NMe-Phe2,DLeu6,Pro9NHEt]LHRH (4), [NMe-1Nal3,DLeu6,Pro9NHEt]LHRH (6), [NMe-His2,DTrp6,Pro9NHEt]LHRH (14), [NMe-Phe2,DNal6]LHRH (27), and [D2Nal6,NMe-Arg8]LHRH (34) exhibited antagonist responses. Substitutions of NMe-1Nal3, NMe-Ser4, or NMe-Tyr5 in leuprolide rendered the 3-4 peptide bond in these compounds completely stable to chymotrypsin. Examination of the three-dimensional structure of leuprolide when bound to the active site of chymotrypsin, reveals the NH's of residues 3 and 5 are involved in hydrogen bond interactions with the enzyme. N-Methylation at these positions is not only disrupting the hydrogen bond interactions, but is also sterically preventing the substrate from fitting in the enzyme's active site. All the compounds in the leuprolide series were also tested against intestinal degradation using an in vitro rat jejunum sac assay. In this model the pattern of stabilization was similar, but not identical, to that against chymotrypsin. The pharmacokinetics of all the analogues in the leuprolide series and of several others in the deslorelin and nafarelin series were determined. The clearance values of all the three NMe-Tyr5 analogues, 8, 20, and 31 were lower than their respective parents. These slower clearances suggest lower rates of metabolism.

A number of 5-substituted 3-[1-(2-benzoxazolyl)hydrazino]propanenitrile analogues have been studied as inhibitors of the rat pleural reverse passive Arthus reaction, and quantitative structure-activity relationships (QSAR) of these analogues have been examined. The QSAR equations indicate that hydrophilic substituents at the 5-position produce more potent compounds, while electron-releasing groups decrease activity. The results supplement QSAR data we previously obtained from the dermal reverse passive Arthus reaction.

Quantitative structure-activity relationships (QSAR) of the 1-phenyl-3-aminopyrazoline analogues as inhibitors of immune complex-induced inflammation have been studied. The correlation suggests that the overall size of the phenyl substituents are of importance, and bulky groups have negative effects on potency. The negative steric effects are gradually increased from ortho to meta to para positions. The negative steric effects were sometimes altered by the electronic effects of the substituents. Electron-releasing groups on the phenyl ring increased potency, while electron-withdrawing groups decreased it. Ortho substituents, however, have unaccounted for additional deleterious effects described here with an indicator variable. The octanol-water partition coefficient (log P) and dissociation constants (pKa) of the 1-(m-trifluoromethylphenyl)-3-aminopyrazoline analogue have been experimentally determined.

3-[1-(2-Benzoxazolyl)hydrazino]propanenitrile derivatives were evaluated in the dermal and pleural reverse passive Arthus reactions in the rat. In the pleural test these compounds were effective in reducing exudate volume and accumulation of white blood cells. This pattern of activity was similar to that of hydrocortisone and different from that of indomethacin. The structural requirements for inhibiting the Arthus reactions were studied by systematic chemical modification of 1. These structure-activity relationship studies revealed that nitrogen 1' of the hydrazino group is essential for activity and must be electron rich, whereas chemical modifications of other sites of 1 had only a modest effect on activity.

The structural requirements for inhibition of RBL-1 (rat basophilic leukemia) 5-lipoxygenase by 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15-HETE, 1) were studied by systematic chemical modifications of the molecule at the hydroxyl and carboxyl groups, the double bonds, and the carboxylate and omega side chains. The most potent inhibitors were analogues that contained a 5,8-cis,cis-diene system and acted as alternate substrates for the enzyme. However, several analogues in which the 5,8-diene had been reduced were also found to inhibit the enzyme. Inhibition of 5-lipoxygenase by 15-hydroxyeicosa-11,13-dienoic acid (15-HEDE) analogues was optimal in compounds that generally contained a free carboxyl group, a carboxylate side chain of nine carbons, an omega side chain of five or six carbons, a cis,trans- or trans,cis-11,13-diene or 11,13-diyne system, and a 15-hydroxyl group. Conversion of 15-HEDE to its 16-membered lactone reduced but did not eliminate 5-lipoxygenase inhibitory activity. In contrast, a 3- to 10-fold enhancement of activity occurred when 5,15-diHETE (58) or 5-HETE (56) were cyclized to their respective delta-lactones. Molecular modeling of 15-HEDE analogues, modified in the C11-C15 region, showed that inactive analogues protrude into regions in space not occupied by active analogues. These structural studies indicate that multiple regions are important for 5-lipoxygenase inhibition by both 15-HETE and 15-HEDE analogues and that no single region plays a predominant role in inhibition.

The octanol-water partition coefficients (log P) and the dissociation constants (pKa) of 3-[1-(2-benzoxazolyl)hydrazino]propanenitrile analogues have been determined, and quantitative structure--activity relationships (QSAR) of the analogues as inhibitors of immune complex-induced inflammation have been studied. A significant correlation is observed between log P and pi substituent constants, and between pKa and inductive-field (F) and resonance (R) constants. The QSAR equations indicate that smaller substituents both at the 5-position and/or at the side chain tend to make the compound more potent, while an electron-withdrawing group at the side chain tends to make the compound less potent. The predicted potencies of 14 of 18 additional monosubstituted and all six disubstituted analogues agree reasonably well with the observed activities.

2-[(Phenylthio)methyl]pyridine derivatives inhibited the dermal reverse passive Arthus reaction (RPAR) in the rat. In the same model, indomethacin was inactive, and hydrocortisone was active. Compounds Ia-d also significantly reduced exudate volume and white blood cell accumulation in the pleural RPAR. This pattern of activity was similar to that of hydrocortisone and different from that of indomethacin.

A series of reduced-size hexapeptide analogues of LH-RH were synthesized that contain the residues corresponding to amino acid positions 4-9 and are linked to various carboxylic acids in place of residue 3. These compounds were tested in vitro in the rat pituitary receptor binding and LH release assays. A wide range of binding affinities was obtained up to and exceeding that of LH-RH. Both agonists and antagonists were obtained. From the SAR studies, it appears that a very precise size, length, and shape of the substituent at position 3 is required to achieve agonist activity, whereas the structural requirements for antagonist activity appear to be much less stringent. Depending on the nature of the substituent at positions 6 and 4, the biological response switches from antagonist to agonist or vice versa. The results suggest that conformational changes at position 6 or 4 feed back to the substituent at position 3, which induces the change from agonist to antagonist. The most potent compounds in the series were tested in vivo and found to be active.

A group of novel 4-substituted phenylacetic acids were prepared and coupled with several 7-amino-delta-3-cephems to afford a family of parenterally active cephalosporins. A compound designated 13I had the broadest spectrum of activity and the highest potency of the group against both Gram-positive and Gram-negative bacteria. The activity of 13I included high potency against penicillinase-producing staphylococci and activity against anaerobes, including Bacteroides fragilis.