Carmen Fernandez-Metzler

Mechanistic investigation of ionization suppression in electrospray ionization

We show results from experiments designed to determine the relative importance of gas phase processes and solution phase processes into ionization suppression observed in biological sample extracts. The data indicate that gas phase reactions leading to the loss of net charge on the analyte is not likely to be the most important process involved in ionization suppression. The results point to changes in the droplet solution properties caused by the presence of nonvolatile solutes as the main cause of ionization suppression in electrospray ionization of biological extracts.

Kinesin spindle protein (KSP) inhibitors. 9. Discovery of (2S)-4-(2,5-difluorophenyl)-n-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the treatment of taxane-refractory cancer.

Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.

Non-Peptide αvβ3 Antagonists. Part 4: Potent and Orally Bioavailable Chain-Shortened RGD Mimetics

Abstract Potent non-peptidic α v β 3 antagonists have been prepared where deletion of an amide bond from an earlier series of linear RGD-mimetics provides a novel series of chain-shortened α v β 3 antagonists with significantly improved oral pharmacokinetics. These chain-shortened α v β 3 antagonists represent structurally novel integrin inhibitors.

The development and cross-validation of methods based on radioimmunoassay and LC/MS-MS for the quantification of the Class III antiarrhythmic agent, MK-0499, in human plasma and urine

An analytical method based on radioimmunoassay (RIA) has been developed for the determination of the antiarrhythmic agent, MK-0499, in plasma and urine. Owing to the potency of the drug, the specificity of this assay in human plasma could not be adequately determined using conventional RIA procedures. A highly specific procedure, based on LC/MS-MS, was developed to cross-validate the RIA. The lower quantifiable limits of the RIA and LC/MS-MS-based methods were 0.05 and 0.013 ng ml-1, respectively. Cross-validation data, compared using paired students t-test regression analysis, showed excellent correlation between methods. The mass spectrometric assay was also used to simultaneously measure plasma concentrations of unlabeled and 14C-labeled MK-0499 following administration of the drug at high specific activity to volunteers.

Non-Peptide αvβ3 antagonists. Part 6: Design and synthesis of αvβ3 antagonists containing a pyridone or pyrazinone central scaffold

Abstract Two novel series of small-molecule RGD mimetics containing either a substituted pyridone or pyrazinone central constraint were prepared. Modification of the β-alanine 3-substituent produced compounds that are potent and selective α v β 3 antagonists and exhibit a range of physicochemical properties.

Determination of chiral sulfoxides in plasma by normal-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A sensitive and specific assay has been developed and validated for the separation of a chiral sulfoxide drug candidate with simultaneous determination of the corresponding sulfide and sulfone in plasma by normal-phase LC-MS-MS. Separation was achieved on a Chiralpak AD (100 x 2.1 mm) column with a 2-propanol-hexane (80:20) mobile phase within 7 min. Aqueous mobile phase (2-propanol-10 mM ammonium acetate, 75:25) was added post-column prior to introduction into the heated nebulizer interface of a Sciex API 3 plus mass spectrometer, to avoid the explosion hazard of hexane-containing mobile phases in the presence of a corona discharge. The linear range of this assay was 5-2500 ng/ml. The accuracy and precision of the chiral sulfoxides, the sulfide and the sulfone were within +/- 15% across the linear range. The limit of quantitation for each component was 5 ng/ml based on the extraction of 0.25 ml plasma. The recovery for each component was between 82 and 120%. This assay was sufficiently sensitive and specific to support pre-clinical development studies in rats, dogs and monkeys.

Disposition of a novel and potent αvβ3 antagonist in animals, and extrapolation to man

1. The disposition of 3-{2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl) propyl]-imidazolidin-1-yl}-3(S)-(6-methoxy-pyridin-3-yl)propionic acid (compound A), a potent and selective αvβ3 antagonist, was characterized in several animal species in support of its selection for preclinical safety studies and potential clinical development. 2. Compound A exhibited marked species differences in pharmacokinetics; the plasma clearances and bioavailabilities ranged from 33–47 ml min−1 kg−1 in rats and mice to 4–9 ml min−1 kg−1 in dogs and monkeys, and about 20% in rats to 70–80% in dogs and monkeys, respectively. Both the intravenous (i.v.) and oral kinetics of compound A were linear over the dose range studied in dogs (0.1–5 mg kg−1 i.v. and 0.25–20 mg kg−1 orally [p.o.]) and rats (1–30 mg kg−1 i.v. and 4–160 mg kg−1 p.o.). 3. Compound A was eliminated substantially by urinary excretion; the urinary recovery of the unchanged drug was 67% in rhesus, 48% in dogs and about 30% in rats. In these animal species, biotransformation was modest. 4. Following i.v. administration of [14C]-compound A to rats, the radioactivity rapidly distributed to all tissues investigated, with high levels of the radioactivity detected in liver, kidney and intestine soon after the drug administration. The radioactivity declined rapidly, with less than 1% of the i.v. dose remaining at 30-h post-dose. 5. Compound A was moderately bound to plasma proteins, with unbound fractions of 26, 20, 14 and 5% for rats, dogs, monkeys and humans, respectively. It was bound primarily to human α1-acid glycoprotein (about 85% binding at 0.1% concentration), as compared with human albumin (< 50% binding at 4% concentration). 6. Using simple allometry, compound A was predicted to exhibit relatively low clearance (1–3 ml min−1 kg−1) and low volume of distribution (0.1–0.3 l kg−1) in humans. Based on the predicted values, compound A was projected to exhibit a favourable oral pharmacokinetic profile in humans, with good bioavailability (50–80%). These predicted values provided a basis for compound selection for further development.

Determination of MK-852, a new fibrinogen receptor antagonist, in plasma and urine by radioimmunoassay

MK-852 is a novel fibrinogen receptor antagonist. A sensitive and specific radioimmunoassay has been developed for the determination of this drug candidate in plasma and urine. The immunogen was prepared by coupling to albumin via a dinitrophenylene bridge and the radioligand by reaction of the drug with the 125I-labelled Bolton-Hunter reagent. The method was specific and no immunoreactive material other than parent drug was detectable in plasma from dosed volunteers. The direct assay using 0.05 ml of plasma is sensitive to 0.2 ng ml-1 without matrix interference and has sufficient sensitivity, precision, accuracy, and selectivity for the analysis of clinical samples. The lower quantifiable limit in (diluted) urine is 50 ng ml-1.

Renal elimination of a novel and potent αvβ3 integrin antagonist in animals

Compound A (3-{2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]napthyridin-2-yl)propyl]-imidazolidin-1-yl}-3(S)-(6-methoxy-pyridin-3-yl)propionic acid), a hydrophilic zwitterion, is a potent and selective αvβ3 integrin antagonist currently under clinical development for the treatment of osteoporosis. The mechanism of renal excretion of compound A was investigated using a combination of in vivo and in vitro approaches. In rats, renal excretion of compound A involved tubular secretion; ratios between renal clearance, corrected for unbound fraction in plasma (CLr,u) and glomerular filtration rate (GFR) were greater than unity (2–5). The tubular secretion of compound A was saturable at high plasma levels (> 26 μM), and was inhibited significantly, although modestly (about twofold) by relatively high plasma concentrations of the organic anion PAH (160 µM) and the cation cimetidine (about 400 μM), but not by the P-gp inhibitor quinidine (about 50 μM). However, compound A (about 100 μM) had a minimal effect on CLr/GFRs for cimetidine and PAH. In rhesus monkeys, renal elimination of compound A also involved tubular secretion, with a CLr,u/GFR ratio of about 30. The renal secretion of compound A was not affected by either cimetidine (about 120 μM) or PAH (about 80 μM). Similarly, compound A (about 40 μM) had a minimal effect on the renal tubular secretion of both cimetidine and PAH. At the doses studied, neither rat nor monkey plasma protein binding of compound A, cimetidine or PAH was affected in the presence of each other. In vitro transport studies showed that compound A was not a substrate for P-gp in the Caco-2, human MDR1 and mouse mdr1a transfected LLC-PK1 cell lines. In an uptake study using rOAT1 and rOAT3 transfected HEK cell lines, compound A was shown to be a substrate for rat OAT3 (Km = 15 μM), but not rat OAT1. The results suggest that the tubular secretion of compound A is not mediated by P-gp, but rather is mediated, at least in part, via the organic anion transporter OAT3, the renal transporter shown to be capable of transporting both the organic anion PAH and the organic cation cimetidine. Although there is a possibility for pharmacokinetic interactions between compound A and substrates or inhibitors of OAT3, at the renal excretion level, the magnitude of interaction would likely be modest in humans at clinically relevant doses.