3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors: oxime ether analogs of pravastatin.

Pravastatin, a potent anti-hypercholesteremic drug, has been developed by Bristol-Myers Squibb for treatment of hypercholesterolemia and other related diseases. Several structurally related compounds (SQ 31,554, SQ 31,879, SQ 31,947, SQ 32,391, SQ 32,770, SQ 32,390 and SQ 32,469) modified at the 3-position of the hexahydronaphthalene ring system of pravastatin were prepared in the course of developing the basic reagents for a radioimmunoassay of the parent drug. The biological activity of these analogs was comparable to pravastatin itself. Indeed, one member of this series was found to be several times more potent than pravastatin.

An affinity column method for determination of the immunoreactivity of 131I-chimeric L6 monoclonal antibody and comparison to in vivo tumor localization.

An affinity column method was developed to determine the immunoreactivity of 131I-ChiL6 (chimeric L6 monoclonal antibody), a candidate for radioimmunotherapy. This method involved assessing the binding of the radiolabeled antibody to antigen containing membranes bound to a Reacti-gel agarose matrix. The immunoreactivity determined by the affinity column method correlated to other in vitro binding assays including the Lindmo infinite antigen excess method. In tumor-bearing mice which had been injected with 131I-ChiL6, which possessed high immunoreactivities (90-82%), a high tumor uptake (13.5-10.5% ID/g) was observed. A decrease in tumor uptake (5.2-4.8% ID/g) was observed with 131I-ChiL6 samples of low immunoreactivity (42% and 31%, respectively). While a moderate loss of immunoreactivity (4-18%) of the 131I-ChiL6 samples could be detected by the affinity column method, the loss of tumor uptake in vivo observed was not as significant. This method was found to be an efficient and sensitive method for detecting damage to the antibody during radiolabeling and applicable as a quality control method for clinical trials. This rapid method, compared to the other in vitro binding assays (including the Lindmo infinite antigen excess method) has distinct advantages as a quality control method since it requires less manipulation and can be semi-automated.

Production and characterization of monoclonal antibodies to the carboxyl-terminal heptapeptide of endothelin-1.

Two cell lines, RR5.ET-1 and RR1.ET-1, that produce monoclonal antibodies specific for the carboxyl-terminal heptapeptide of endothelin-1 (ET-1) have been cloned and stabilized. An RIA was developed to facilitate the evaluation and characterization of these monoclonal antibodies. The affinity constant of each MAb for ET-1, as determined by Scatchard analysis, was 5.74 x 10(8) M-1 for RR5.ET-1 and 4.15 x 10(7) M-1 for RR1.ET-1. The antibodies reacted specifically with the carboxyl-terminus (ET15-21) and did not cross-react with the amino-terminal amino acids (ET1-16). As expected, the antibodies cross-reacted with endothelin-2 (ET-2) and endothelin-3 (ET-3), and to a lesser extent, with the closely related sarafotoxins. Both MAbs retained about 55% reactivity with the ET-1 terminal sequence of Asp-Ile-Ile-Trp (ET18-21) but had no reactivity with the ET sequence His-Leu-Asp-Ile-Ile-Trp-Val-Asn (ET16-23) nor with Big ET-1 (ET1-39). These data strongly suggest that the terminal four amino acids of ET-1 are included in the MAb binding site. More importantly, the terminal Trp21 must be free, not linked to Val22 to retain reactivity with either of the MAbs.

Radioimmunoassay for hydroxyphosphinyl-3-hydroxybutanoic acid (SQ 33,600), a hypocholesterolemia agent.

A specific and sensitive radioimmunoassay (RIA) for the measurement of SQ 33,600 in biological fluids has been developed. The assay utilizes a SQ 33,600 polyclonal antibody, [125I]iodohistamine-SQ 33,600 radiolabel, and standards in serum. Satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free radiolabel was achieved by employing polyethylene glycol-goat anti-rabbit gamma-globulin (PEG-GARG) separant. A quantitative recovery in serum and urine of the exogenous analyte was obtained at all concentrations of SQ 33,600 tested. Intra-assay coefficients of variation (CVs) were 6.19 and 5.57% for the low and high controls, respectively. Interassay CVs were 6.64 and 6.06% for the low and medium controls, respectively. Results from the parallelism studies were acceptable for both serum and urine samples. Comparison of results from samples which were assayed by RIA and high-performance liquid chromatography (HPLC) demonstrated a significant correlation (r = 0.994; HPLC = 1.09 RIA + 57.98; n = 45). The present RIA has been successfully used to assay clinical specimens from pharmacokinetic studies.

A specific radioimmunoassay for the measurement of gadoteridol, a contrast agent for magnetic resonance imaging in biological fluids.

Gadoteridol, a nonionic gadolinium chelate, is currently being evaluated for contrast-enhanced magnetic resonance imaging. A radioimmunoassay (RIA) has been developed for the measurement of gadoteridol in biological fluids. The RIA has a range of 0 to 25 micrograms/mL and has the sensitivity to detect 0.05 microgram/mL of gadoteridol. Satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free radiolabel was achieved with 12.5% polyethylene glycol. A quantitative recovery of the exogenous analyte was obtained at all concentrations of gadoteridol tested. Linearity in both serum and urine was satisfactory. Intraassay coefficients of variation were 6.4 and 2.8% for the low and medium controls, respectively. Interassay coefficients of variation were 5.4, 3.8, and 12.2% for the low, medium, and high controls, respectively. Cross reactivities of the ligand 5 and the calcium salt 6 were 37 and 29%, respectively. Clinical samples from the ascending dosage studies were analyzed by the gadoteridol RIA. The results obtained from the serum specimens demonstrated an excellent linear proportionality between drug concentration in blood and administered dosage of gadoteridol. Cumulative urinary excretion data showed that 94% of the drug was excreted in the urine within 24 h.

A radioimmunoassay for the new antiviral agent 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil.

A specific and sensitive radioimmunoassay (RIA) for the measurement of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) in biological fluids has been developed. The assay has a range of 2.5-1,000 ng/ml and 10-1,000 ng/ml for serum and urine, respectively, and has the sensitivity to detect 2.5 and 25 ng/ml of BV-araU in serum and urine, respectively. A satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free ligand was achieved by employing polyethylene glycol-goat anti-rabbit gamma globulin separant. A quantitative recovery of the exogenous analyte was obtained at all concentrations of BV-araU tested. The assay is specific for the parent drug and is not affected by the presence of its metabolite, BV-U (bromovinyl uracil) or serum components (nucleotides, nucleosides, or sugars). Intraassay coefficients of variation were 3.1-4.4% and 2.5-4.2% for serum and urine controls, respectively. Interassay variability was < 8.6% for all serum and urine controls. Linear regression analysis showed that the correlation between RIA and high-pressure liquid chromatography was excellent (r = 0.997). The ascending dosage studies have been analyzed by the BV-araU RIA, and results indicate that the values of area under the serum concentration-time curve increased proportionally with the administered dose of BV-araU up to 80 mg. Cumulative urinary excretion data showed that approximately 50% of unchanged BV-araU was excreted in the urine within 24 h.

Radioimmunoassay for ceronapril, a new angiotensin-converting enzyme inhibitor, and its application to a pharmacokinetic study in healthy male volunteers.

Ceronapril is a member of a new chemical class of angiotensin-converting enzyme inhibitors being developed by The Bristol-Myers Squibb Pharmaceutical Research Institute. A radioimmunoassay (RIA) has been developed for the measurement of ceronapril in biological fluids. The RIA has a range of 0 to 500 ng/ml and has the sensitivity to detect 1.0 ng/ml of ceronapril. Satisfactory zero binding and sensitivity were obtained after a 2-h incubation at room temperature or overnight at 4 degrees C. Separation of the antibody-bound and free radiolabel was achieved by employing polyethylene glycol-goat anti-rabbit gamma-globulin separant. A quantitative recovery of the exogenous analyte was obtained at all concentrations of ceronapril tested. Intraassay coefficients of variance (CV's) were 3.9% and 4.6% for the low and medium controls, respectively. A highly significant statistical correlation between RIA and [14C]TLRC was observed for both plasma and urine samples. Clinical samples from the ascending dosage studies have been analyzed by the ceronapril RIA. The maximum concentration and the area under the plasma concentration-time curve did not increase in a dose-proportional manner for doses above 100 mg.

Oral bioavailability and anti-simian varicella virus efficacy of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) in monkeys.

BV-araU (1-beta-D-arabinofuranosyl-E-5-[2-bromovinyl]uracil) has potent antiviral activity against varicella zoster virus in cell culture and is undergoing clinical evaluation. In the present study, pharmacokinetic parameters and the efficacy of BV-araU against infection with simian varicella virus (SVV) were evaluated in African green monkeys. Pharmacokinetic parameters were determined by analysis of the BV-araU content of sera obtained after oral and intravenous administration to normal monkeys. Peak serum concentrations showed dose proportionality, with the 0.1 mg/kg dose resulting in a peak serum concentration of 0.05 micrograms/ml, the approximately ED50 value for the SVV inoculum in cell culture. BV-araU administered orally twice daily at 0.1 mg/kg for 10 days starting 48 h after intratracheal SVV infection prevented vesicular rash development and suppressed viremia. Effective therapy could be initiated 96 h after infection. Taken together, these results indicate that BV-araU is effective oral therapy at doses that achieve peak serum levels equivalent to the ED50 for SVV in cell culture.

Pharmacokinetics, safety, and pharmacologic effects of fosinopril sodium, an angiotensin-converting enzyme inhibitor in healthy subjects.

The pharmacokinetics, pharmacodynamics, and safety of fosinopril sodium (SQ 28,555), a new orally active angiotensin-converting enzyme (ACE) inhibitor, was evaluated in 73 healthy men in two separate studies. In study I, doses ranging from 10 to 640 mg were administered once daily for 3 days to seven groups of five subjects each. Serum aldosterone levels, ACE activity, and sitting blood pressure were determined, as were pharmacokinetic parameters of fosinoprilat, the active diacid of fosinopril. In a dose-tolerance study (study II), 80 and 160 mg of the drug were administered in doses of 40 mg bid and 80 mg bid for 2 weeks. Pharmacokinetics were determined on days 1 and 14, and blood pressure and ACE activity were measured daily. One hour after all doses of fosinopril, serum ACE activity was undetectable. Peak blood levels of fosinoprilat occurred at about 3 hours after dosing, and linear kinetics of the diacid were observed. ACE activity remained undetectable for more than 24 hours after the treatment was stopped in study II. Serum aldosterone levels were decreased by 50% of baseline values in both studies. In study I, maximal reductions in mean blood pressure occurred approximately 6 hours postdose; once-daily doses of 20 mg or greater achieved reductions of 11.3 to 21.6% (P less than or equal to .05, compared with placebo reductions). Fosinopril was well tolerated. Subjects reported only mild gastrointestinal complications at doses of 80 mg/day or higher. These data show that fosinopril is a safe and effective inhibitor of ACE with a long duration of action on serum ACE activity.

A radioimmunoassay for SQ 27,519, the active phosphinic acid-carboxylic diacid of the prodrug fosinopril in human serum.

Fosinopril (SQ 28,555) is a member of a new chemical class of angiotensin converting enzyme inhibitors being developed by The Squibb Institute for Medical Research. During or following absorption, fosinopril, a prodrug, is hydrolyzed pharmacologically to the active diacid, SQ 27,519. A specific radioimmunoassay (RIA) for the measurement of SQ 27,519 in human serum has been developed. The assay utilizes a specific SQ 27,519 antibody, 125I-iodohistamine-SQ 27,519 radiolabel, and human serum standards. Satisfactory zero binding and assay sensitivity are achieved after a 2-h incubation at room temperature. Separation of the antibody-bound and free radiolabeled antigens is achieved by using polyethylene glycol-goat anti-rabbit gamma globulin separant. Recovery efficiencies ranged from 97.2 to 109.4%. The assay exhibited little or no cross-reactivity with captopril. Cross-reactivities for prodrug (SQ 28,555) and phenolic SQ 27,519 were 5 and 9%, respectively. Intra-assay variability (3.3-5.6%) and interassay variability (7.1-6.6%) were observed. Linear regression analysis indicates that RIA and [14C] thin-layer radiochromatography (TLRC) methods gave a highly significant correlation (RIA = 1.0 [14C]TLRC + 0.17, r = 0.991). Pharmacokinetic profiles of patient sera containing SQ 27,519 obtained by RIA and [14C]TLRC are identical. The RIA has been used routinely in support of the bioavailability and pharmacokinetic studies of fosinopril in humans.