Methyl-substitution of an iminohydantoin spiropiperidine ß-secretase (BACE-1) inhibitor has a profound effect on its potency.

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 µM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.

Local delivery of gene vectors from bare-metal stents by use of a biodegradable synthetic complex inhibits in-stent restenosis in rat carotid arteries.

BACKGROUND: Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. METHODS AND RESULTS: We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37 degrees C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial Ad(GFP) expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. CONCLUSIONS: Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature.

Effects of growth hormone replacement therapy on 1,25-dihydroxyvitamin D and calcium metabolism.

To elucidate the changes in mineral metabolism and blood concentrations of calciotropic hormones which accompany GH therapy, we studied 12 GH-deficient children for 5 days before and for 1 week after high dose (5 IU/day) GH therapy, and again at 1 month, 3 months, and 1 yr of replacement therapy (0.1 IU/kg to a maximum dose of 2 IU three times weekly). All responded with acceleration of height velocity, and bone ages advanced appropriately. Fasting serum ionized calcium levels did not change: 4.11 +/- 0.06 (SEM) mg/dl before, 4.19 +/- 0.05 for the week of high dose therapy, and 4.20 +/- 0.14 during replacement therapy. Likewise, fasting serum parthormone did not vary: 38.9 +/- 2.6 muleq/ml before to 44.1 +/- 9.2 at 1 yr. Twenty four-hour nephrogenous cyclic AMP (NcAMP) did not vary over the first week (1.2 +/- 0.7 nmol/dl glomerular filtrate before, 1.3 +/- 0.4 after 1 week), but increased to 5.3 +/- 1.9 after 1 yr (alpha less than 0.001). The response of ionized calcium and parathormone to a standardized disodium EDTA infusion of 50 mg/kg also did not change. The mean fasting serum calcitonin level was not different before therapy (29.4 +/- 2.8 pg/ml), after 1 week (21.5 +/- 1.8), or after 1 yr (42.4 +/- 11.0). However, the mean serum 1,25-dihydroxyvitamin D concentration rose from 33.1 +/- 3.3 pg/ml before therapy to 68.3 +/- 12.3 on the seventh day of high dose therapy (alpha less than 0.01), returning to pretherapy values by 1 month. We conclude that high dose GH therapy in GH-deficient children raises 1,25-dihydroxyvitamin D concentration acutely, but that long term, physiological replacement therapy does not cause such an effect. Because NcAMP excretion rose in the absence of an increase in serum parathormone concentration, we conclude that GH sensitizes the kidney to a cAMP-mediated effect of parathormone.

The effect of supplemental ascorbic acid on serum vitamin B12 levels in myelomeningocele patients.

Serum levels of ascorbic acid and vitamin B12 were analyzed in 40 myelomeningocele children to study the effect of supplemental ascorbic acid on serum vitamin B12 levels. The experimental group was composed of 20 children receiving ascorbic acid for urinary acidification: 10 received an average of 1.8 g daily, 10 received an average of 1.5 g daily (the amount depending on the requirement needs for urinary acidification) half of each group received ascorbic acid for less than 3 yr (an average of 2.1 yr) and half received ascorbic acid for more than 3 yr (an average of 4.3 yr). The control group consisted of 20 myelomeningocele children not receiving supplemental ascorbic acid. Both groups were matched for age, sex, race, and physical activity. Dietary levels of ascorbic acid and B12 were calculated to rule out their influence on serum levels. Results showed that the experimental group with supplemental ascorbic acid produced significantly higher ascorbic acid values than the control group. The serum B12 levels of the experimental group were not significantly different than those of the control groups and these children showed neither a deficient serum levels of B12, anemia, nor elevated mean corpuscular volume. Hemoglobin levels were slightly higher for the experimental group. Dietary calculations of B12 and ascorbic acid were not significantly greater than the Recommended Daily Allowance ruling out any influence of diet on serum levels. No evidence of vitamin B12 deficiency developed in 20 myelomeningocele children receiving daily mean doses of 1.65 g of supplemental ascorbic acid. In view of our findings, it is highly improbable that megadoses of supplemental ascorbic acid would induce vitamin B12 deficiency in man.

Potent noncovalent thrombin inhibitors that utilize the unique amino acid D-dicyclohexylalanine in the P3 position. Implications on oral bioavailability and antithrombotic efficacy.

In an effort to prepare orally bioavailable analogs of our previously reported thrombin inhibitor 1, we have synthesized a series of compounds that utilize the unique amino acid D-dicyclohexylalanine as a P3 ligand. The resulting compounds are extremely potent and selective thrombin inhibitors, and the N-terminal Boc derivative 8 exhibited excellent oral bioavailability and pharmacokinetics in both rats and dogs. The des-Boc analog 6 was not orally bioavailable in rats. The high level of oral bioavailability observed with 8 appears to be a direct function of its increased lipophilicity versus other close analogs. Although increased lipophilicity may serve to increase the oral absorption of tripeptide thrombin inhibitors, it also appears to have detrimental effects on the antithrombotic properties observed with the compounds. Compound 6 performed extremely well in our in vivo antithrombotic assay, while the much more lipophilic but essentially equipotent analog 8 performed poorly. We have found that in general with this series of thrombin inhibitors as well as with other unreported series, increased lipophilicity and the associated increases in plasma protein binding have detrimental effects on 2X APTT values and subsequent performance in in vivo antithrombotic models.

Identification of MK-944a: a second clinical candidate from the hydroxylaminepentanamide isostere series of HIV protease inhibitors.

Recent results from human clinical trials have established the critical role of HIV protease inhibitors in the treatment of acquired immune-deficiency syndrome (AIDS). However, the emergence of viral resistance, demanding treatment protocols, and adverse side effects have exposed the urgent need for a second generation of HIV protease inhibitors. The continued exploration of our hydroxylaminepentanamide (HAPA) transition-state isostere series of HIV protease inhibitors, which initially resulted in the identification of Crixivan (indinavir sulfate, MK-639, L-735,524), has now yielded MK-944a (L-756,423). This compound is potent, is selective, and competitively inhibits HIV-1 PR with a K(i) value of 0.049 nM. It stops the spread of the HIV(IIIb)-infected MT4 lymphoid cells at 25.0-50.0 nM, even in the presence of alpha(1) acid glycoprotein, human serum albumin, normal human serum, or fetal bovine serum. MK-944a has a longer half-life in several animal models (rats, dogs, and monkeys) than indinavir sulfate and is currently in advanced human clinical trials.

Discovery and development of the novel potent orally active thrombin inhibitor N-(9-hydroxy-9-fluorenecarboxy)prolyl trans-4-aminocyclohexylmethyl amide (L-372,460): coapplication of structure-based design and rapid multiple analogue synthesis on solid support.

Early studies in these laboratories of peptidomimetic structures containing a basic P1 moiety led to the highly potent and selective thrombin inhibitors 2 (Ki = 5.0 nM) and 3 (Ki = 0.1 nM). However, neither attains significant blood levels upon oral administration to rats and dogs. With the aim of improving pharmacokinetic properties via a more diverse database, we devised a resin-based route for the synthesis of analogues of these structures in which the P3 residue is replaced with a range of lipophilic carboxylic amides. Assembly proceeds from the common P2-P1 template 7 linked via an acid-labile carbamate to a polystyrene support. Application of the methodology in a repetitive fashion afforded several interesting analogues out of a collection of some 200 compounds. Among the most potent of the group, N-(9-hydroxy-9-fluorenecarboxy)-prolyl trans-4-aminocyclohexylmethyl amide (L-372,460 8, Ki = 1.5 nM), in addition to being fully efficacious in a rat model of arterial thrombosis at an infusion rate of 10 micrograms/kg/min, exhibits oral bioavailability of 74% in dogs, and oral bioavailability of 39% in monkeys with a serum half-life of just under 4 h. On the basis of its favorable biological properties, inhibitor 8 has been subject to further evaluation as a possible treatment for thrombogenic disorders.

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position.

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.

Synthesis of a series of potent and orally bioavailable thrombin inhibitors that utilize 3,3-disubstituted propionic acid derivatives in the P3 position.

As part of an effort to prepare efficacious and orally bioavailable analogs of the previously reported thrombin inhibitors 1a, b, we have synthesized a series of compounds that utilize 3,3-disubstituted propionic acid derivatives as P3 ligands. By removing the N-terminal amino group, the general oral bioavailability of this class of compounds was enhanced without excessively increasing the lipophilicity of the compounds. The overall properties of the molecules could be drastically altered depending on the nature of the groups substituted onto the 3-position of the P3 propionic acid moiety. A number of the compounds exhibited good oral bioavailability in rats and dogs, and numerous compounds were efficacious in a rat FeCl3-induced model of arterial thrombosis. Compound 7, the 3,3-diphenylpropionic acid derivative, showed the best overall profile of in vivo and in vitro activity. Molecular modeling studies suggest that these compounds bind in the thrombin active site in a manner essentially identical to that previously reported for compound 1a.

Discovery of a novel, selective, and orally bioavailable class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position.

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

Evaluation and comparison of two fully automated radioassay systems with distinctly different modes of analysis.

Two fully automated radioimmunoassay systems with batch and sequential modes of analysis were used to assay serum thyroxine, triiodothyronine, and digoxin. The results obtained were compared with those obtained by manual methods. The batch system uses antibody coated tubes while the sequential system uses immobilized antibody chambers for the separation of bound from free ligands. In accuracy, both systems compared favorable with the established manual methods, but the sequential system showed better precision than the batch system. There was a statistically significant carryover of thyroxine in the sequential system when there were at least six-fold differences in the concentrations of thyroxine in adjacent samples, but the carryover was not significant in the batch system. Compared with the batch system, the sequential system has a shorter throughtime for individual samples (time from aspiration of the sample to the printout of results) but a longer interval for final overall printout of assay results (lower throughput).

Clinical significance of serum vitamin B12 measured by radioassay using pure intrinsic factor.

Serum vitamin B12 (B12) levels of 53 patients (15 with pernicious anemia) and 42 healthy volunteers were determined using crude intrinsic factor (IF), pure IF, and a mixture of crude IF + R-protein blocking agent (block IF). The radioassay using pure IF showed less sample-to-sample variation in nonspecific binding than the radioassay using block IF. The mean B12 levels in 42 healthy subjects were significantly higher with crude IF (499 +/- 23 pg/ml, 1 s.e.m.) than with pure IF (408 +/- 29 pg/ml) or with block IF (407 +/- 22 pg/ml). B12 levels were abnormally low in all 15 patients with pernicious anemia by pure IF (less than 100 pg/ml), in 14 patients by block IF (less than 150 pg/ml), and in only seven patients by crude IF (less than 200 pg/ml). Our data confirm previous reports that B12 deficiency can be diagnosed more reliably by measuring serum B12 levels with etiher pure IF or block IF.

Serum osteocalcin measurements in prostate carcinoma patients with skeletal deposits shown by bone scintigram: comparison with serum PSA/PAP measurements.

The correlation of technetium-99m-HMDP bone scintigraphic findings, serum osteocalcin as a measure of bone turnover and prostate-specific antigen (PSA) and/or prostate acid phosphatase (PAP) was determined in 19 men with bone metastasis due to prostatic carcinoma. Six of the 19 patients with metastases on bone scan showed elevation of osteocalcin. These patients had extensive metastatic disease. All 19 men with positive bone scans had high serum PSA and/or PAP levels. Serum osteocalcin measurement is less sensitive to detection of bone deposits than PSA/PAP measurements (p less than 0.0008).

Metabolite-P450 complex formation by methylenedioxyphenyl HIV protease inhibitors in rat and human liver microsomes.

P450 complex formation and the unusual pharmacokinetics of methylenedioxyphenyl HIV protease inhibitors were examined by in vitro studies using human and rat liver microsomes and by in vivo oral dosing studies. In vitro spectral studies indicated that the formation of a P450 complex having absorbance maxima at 425 and 456 nm was time and concentration dependent; 27-60% of the total P450 was complexed in dexamethasone-induced rat liver microsomes after a 30-min incubation with 100 microM HIV protease inhibitors. Methoxy substitution on the phenyl ring of the methylenedioxyphenyl moiety increased formation of the P450 complex, whereas chlorine substitution markedly decreased the P450 complexation. Kinetic studies on the P450 complex formation indicated that both methoxy and chlorine substitution affected the maximum complex formation rate (Vmax), while it had little effect on Km values (approximately 10 microM). This complexation in human liver microsomes was inhibited markedly by an anti-CYP3A1 antibody. Furthermore, the P450 complex formation resulted in a time-dependent loss of CYP3A-catalyzed marker activities (testosterone 2beta/6beta-hydroxylase) in both rat and human liver microsomes. Collectively, these results point to the involvement of CYP3A isoforms in P450 complexation by methylenedioxyphenyl HIV protease inhibitors. Additionally, after oral administration to rats, one of these HIV protease inhibitors (Compound I), which complexed P450 to the greatest extent, showed no elimination over a period of 500 min after administration of the highest dose. It is suggested that formation of a quasi-irreversible metabolite-CYP3A complex with methylenedioxyphenyl HIV protease inhibitors was responsible for the CYP3A-selective time-dependent loss of catalytic function and the unusual dose-dependent pharmacokinetics after oral administration.