John Veals

HPLC-API/MS/MS: a powerful tool for integrating drug metabolism into the drug discovery process

Abstract HPLC combined with atmospheric pressure ionization (API) mass spectrometry (MS) has become a very useful tool in the pharmaceutical industry. The technique of HPLC-API/MS/MS is becoming very important for both drug discovery and drug development programs. In the drug discovery area, it has three major uses: (1) rapid, quantitative method development, (2) metabolite identification, and (3) multi-drug analysis. The sensitivity of the API source and the selectivity provided by tandem mass spectrometry (MS/MS) enable rapid, quantitative method development for drugs in plasma. Early information on the metabolism of candidate drugs can guide structural modifications, thereby improving the activity and/or bioavailability.

Demonstration of the capabilities of a parallel high performance liquid chromatography tandem mass spectrometry system for use in the analysis of drug discovery plasma samples

There is a continuing need for increased throughput in the evaluation of new drug entities in terms of their pharmacokinetic (PK) parameters. This report describes an alternative procedure for increasing the throughput of plasma samples assayed in one overnight analysis: the use of parallel high performance liquid chromatography (HPLC) combined with tandem mass spectrometry (parallel LC/MS/MS). For this work, two HPLC systems were linked so that their combined effluent flowed into one tandem MS system. The parallel HPLC/APCI-MS/MS system consisted of two Waters 2690 Alliance systems (each one included an HPLC pump and an autosampler) and one Finnigan TSQ 7000 triple quadrupole mass spectrometer. Therefore, the simultaneous chromatographic separation of the plasma samples was carried out in parallel on two HPLC systems. The MS data system was able to deconvolute the data to calculate the results for the samples. Using this system, 20 compounds were tested in one overnight assay using the rapid rat PK screening model which includes a total of 10 standards plus samples and two solvent blanks per compound tested. This application provides an additional means of increasing throughput in the drug discovery PK assay arena; using this approach a two-fold increase in throughput can be achieved in the assay part of the drug discovery rat PK screening step.

High-performance liquid chromatographic determination and stability of 5-(3-methyltriazen-1-yl)-imidazo-4-carboximide, the biologically active product of the antitumor agent temozolomide, in human plasma

5-(3-Methyltriazen-1-yl)-imidazo-4-carboximide (MTIC) is a highly unstable compound which is believed to be the biologically active degradation product of the antitumor agent temozolomide. An HPLC method has been developed and validated for the analysis of MTIC in human plasma. Because of the instability of MTIC, sample processing was kept to minimal. The method involved precipitation of plasma protein with methanol followed by analysis of the supernatant using reversed-phase column and UV detection at 316 nm. The linearity (r>0.99), precision (C.V.<9%) and accuracy (bias<5%) were satisfactory. The lower limit of quantitation (LOQ) was 10 ng/ml. The recovery of MTIC and internal standard was > or = 86.7%. MTIC was stable in plasma though three freeze-thaw cycles, and was stable at 4 degrees C for 1 h and at -80 degrees C for at least 70 days. MTIC may be unstable at 10 degrees C in processed samples; therefore, samples were placed in the autosampler (10 degrees C) immediately prior to injection. By using this analytical method, MTIC was quantified in plasma of cancer patients (n=12) within 0.25-12 h after oral administration of temozolomide at 150 mg/m2. The mean maximum plasma concentration (Cmax) was 211 ng/ml which was observed at a mean Tmax of 1.88 h post dose. MTIC disappeared rapidly from plasma with an apparent in vivo half-life (t1/2) of 1.9 h similar to that of temozolomide. Following in vitro incubation of MTIC in human plasma at 25 degrees C, MTIC disappearance was bioexponential with estimated t1/2 values of 25 and 60 min for the first and second phases, respectively. Therefore, the elimination t1/2 of MTIC in human in vivo (1.9 h) was controlled by the rate of its formation from temozolomide.

Effect of dexamethasone on monoamine oxidase inhibition by iproniazid in rat brain

Chronic (6 days) dexamethasone administration caused a slight decrease of rat brain MAO enzyme activity which was reflected by lower levels of 14C-homovanillic acid (HVA) and increased levels of 14C-3-methoxytyramine (3MT) following intracisternal injections of 14C-dopamine (DA). Opposite results with dexamethasone were obtained in iproniazid (MAO-inhibited)-treated rats. In these animals, brain MAO enzyme activity was significantly increased by dexamethasone. This effect increased with the duration of dexamethasone treatment and appeared to be dose dependent. In the brain areas tested (hypothalamus, midbrain, cerebellum, pons and medulla, olfactory, rest of brain) increases of MAO enzyme activity were also indicated by lower levels of 14C-3MT and increased levels of 14C-HVA formed from intracisternally injected radiolabeled DA. Treatment with other glucocorticoids (16alpha-methyldichlorisone, 16beta-methylprednisone and prednisolone) had a similar effect on 14C-DA metabolism. On the other hand, desoxycorticosterone, progestone, estradiol and testosterone, did not exhibit this property. The data indicate that chronic glucocorticoid treatment may have a slight inhibitory effect on brain MAO and also has the ability to partially reverse or antagonize the inhibition of MAO caused by iproniazid.

Inhibition of dopamine uptake into synaptosomes of rat corpus striatum by chlorpheniramine and its structural analogs.

Abstract Pheniramine and its structural analogs were evaluated for their ability to inhibit dopamine uptake into synaptosomes of rat corpus striatum. A structural activity relationship has been observed. A chlorine or bromine substitution greatly facilitates the inhibitory activity of pheniramine which is further enhanced by demethylation or addition of another halogen. There was no appreciable difference in activity between d- and l- optical isomers of chlorpheniramine or brompheniramine. There was also no correlation between the antihistaminic potency of the compounds and their inhibitory activity on dopamine uptake.

Pharmacological profile of the NOP agonist and cough suppressing agent SCH 486757 (8-[Bis(2-Chlorophenyl)Methyl]-3-(2-Pyrimidinyl)-8-Azabicyclo[3.2.1]Octan-3-Ol) in preclinical models

We describe the pharmacological and pharmacokinetic profiles of SCH 486757, a nociceptin/orphanin FQ peptide (NOP) receptor agonist that has recently entered human clinical trials for cough. SCH 486757 selectively binds human NOP receptor (K(i)=4.6+/-0.61nM) over classical opioid receptors. In a guinea pig capsaicin cough model, SCH 486757 (0.01-1mg/kg) suppressed cough at 2, 4, and 6h post oral administration with a maximum efficacy occurring at 4h equivalent to codeine, hydrocodone, dextromethorphan and baclofen. The antitussive effects of SCH 486757 (3.0mg/kg, p.o.) was blocked by the NOP receptor antagonist J113397 (12mg/kg, i.p.) but not by naltrexone (10mg/kg, p.o.). SCH 486757 does not produce tolerance to its antitussive activity after a 5-day BID dosing regimen. After acute and chronic dosing paradigms, SCH 486757 (1mg/kg) inhibited capsaicin-evoked coughing by 46+/-9% and 40+/-11%, respectively. In a feline mechanically-evoked cough model, SCH 486757 produces a maximum inhibition of cough and expiratory abdominal electromyogram amplitude of 59 and 61%, respectively. SCH 486757 did not significantly affect inspiratory electromyogram amplitude. We examined the abuse potential of SCH 486757 (10mg/kg, p.o.) in a rat conditioned place preference procedure which is sensitive to classical drugs of abuse, such as amphetamine and morphine. SCH 486757 was without effect in this model. Finally, SCH 486757 displays a good oral pharmacokinetic profile in the guinea pig, rat and dog. We conclude that SCH 486757 has a favorable antitussive profile in preclinical animal models.

The effect of amantadine on radiolabeled biogenic amines in the rat brain

Abstract Intracisternally (i.ci.) administered radiolabeled dopamine, l-dopa or norepinephrine were used to study the mode of oction of amantadine as an anti-Parkinson agent. The data show thatpretreatment with amantadine resulted in higher radioactivity concentration in all brain areas investigated when radiolabeled dopamine was administered i.ci. This increase in the brain radioactivity was reflected in higher levels of dopamine and its metabolites. Amantadine had no effect on the brain radioactivity concentration in experiments in which radiolabeled l-dopa or norepinephrine were administered i.ci. Amantadine also did not affect MAO activity in rat brain and liver, and was shown to be a very weak inhibitor of dopamine or serotonin uptake into synaptosomes of rat corpus striatum. Possible modes of action of amantadine are discussed.

The excretion of rosaramicin in breast milk.

The excretion of rosaramicin, a macrolide antibiotic, was studied in the breast milk of ten lactating women. Breast milk and serum samples were collected for 48 hours after a single 250‐mg oral dose of rosaramicin. Mean serum half‐life, apparent volume of distribution, and oral clearance were 4.4 hours, 3.41 L/kg, and 6.34 mL/min/kg, respectively. Mean milk/serum ratio was 0.12 and the total amount of drug recovered over the first ten hours was 6.25 μg, approximately 0.0025% of the dose. A positive correlation between breast milk volume and breast milk clearance was found, suggesting that the amount of drug received by a nursing infant will depend on the volume of milk produced by the mother. Drug‐induced toxicity from the parent drug is unlikely to occur in nursing infants since the amount of rosaramicin that a nursing infant could ingest is small.

The effect of pheniramine and its structural analogues on 5-hydroxytryptamine in rat and mouse brain

Abstract A series of pheniramine analogues was tested for the ability to inhibit 5-HT uptake by synaptosomes of rat corpus striatum. A chlorine or bromine substitution on the aromatic ring greatly facilitated the inhibitory activity of pheniramine. 3,4-dichlorpheniramine was found to be the most active in this series and was also more effective than desipramine or amphetamine. In mice treated with 3,4-dichlorpheniramine, the rate of 5-HT synthesis in brain tissue was considerably reduced when compared to controls.

Analysis of isepamicin in human plasma by radioimmunoassay, microbiologic assay, and high-performance liquid chromatography.

Plasma concentrations of isepamicin, a new aminoglycoside antibiotic, were determined by radioimmunoassay (RIA), microbiological assay (MA), and high-performance liquid chromatography (HPLC) in healthy volunteers after administration of 7.5 mg/kg intramuscular dosages once daily for 10 days. Plasma samples were collected on days 1, 7, and 10. The limit of quantitation (LOQ) was 0.1 microg/ml for HPLC and RIA and 0.5 microg/ml for MA. The HPLC and RIA yielded superimposable plasma concentration-time curves, whereas the plasma concentrations obtained with MA appeared to be 20% to 30% lower. Regression analysis indicated good correlations among the three assays, with coefficients of correlation measuring 0.935 to 0.960 for RIA compared with HPLC, 0.925 to 0.945 for MA compared with HPLC, and 0.920 to 0.945 for RIA compared with MA.