Amin M. Kamel

Improvingthe decision-making process in structural modification of drug candidates: reducing toxicity

The rule of three, relating to activity-exposure-toxicity, presents the single most difficult challenge in the design and advancement of drug candidates to the development stage. Absorption, distribution, metabolism and excretion (ADME) studies are widely used in drug discovery to optimize this balance of properties necessary to convert lead compounds into drugs that are both safe and effective for human patients. Idiosyncratic drug reactions (IDRs; referred to as type B reactions, which are mainly caused by reactive metabolites) are one type of adverse drug reaction that is important to human health and safety. This review highlights the strategies for the decision-making process involving substructures that, when found in drugs, can form reactive metabolites and are involved in toxicities in humans; the tools used to reduce IDRs are also discussed. Several examples are included to show how toxicity studies have influenced and guided drug design. Investigations of reactive intermediate formation in subcellular fractions with the use of radiolabeled reagents are also discussed.

Mass spectral characterization of tetracyclines by electrospray ionization, H/D exchange, and multiple stage mass spectrometry

Electrospray ionization (ESI) and collisionally induced dissociation (CID) mass spectra were obtained for five tetracyclines and the corresponding compounds in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The number of labile hydrogens, x, could easily be determined from a comparison of ESI spectra obtained with N2 and with ND3 as the nebulizer gas. CID mass spectra were obtained for [M + H]+ and [M – H]− ions and the exchanged analogs, [M(Dx) + D]+ and [M(Dx) − D]−, and produced by ESI using a Sciex API-IIIplus and a Finnigan LCQ ion trap mass spectrometer. Compositions of product ions and mechanisms of decomposition were determined by comparison of the MSN spectra of the un-deuterated and deuterated species. Protonated tetracyclines dissociate initially by loss of H2O (D2O) and NH3 (ND3) if there is a tertiary OH at C-6. The loss of H2O (D2O) is the lower energy process. Tetracyclines without the tertiary OH at C-6 lose only NH3 (ND3) initially. MSN experiments showed easily understandable losses of HDO, HN(CH3)2, CH3 - N=CH2, and CO from fragment ions. The major fragment ions do not come from cleavage reactions of the species protonated at the most basic site. Deprotonated tetracyclines had similar CID spectra, with less fragmentation than those observed for the protonated tetracyclines. The lowest energy decomposition paths for the deprotonated tetracyclines are the competitive loss of NH3 (ND3) or HNCO (DNCO). Product ions appear to be formed by charge remote decompositions of species de-protonated at the C-10 phenol.

Electrospray ionization mass spectrometry of tetracycline, oxytetracycline, chlorotetracycline, minocycline, and methacycline.

The effects of mobile-phase additives and analyte concentration on electrospray ionization mass spectra of a series of tetracyclines were investigated in both positive and negative ion modes. Only [M + H](+) and [M - H](-) ions were observed. The greatest sensitivity as [M + H](+) ions was obtained with 1% acetic acid and the greatest sensitivity as [M - H](-) ions was obtained using 50 mM ammonium hydroxide. Sensitivities in the positive ion mode were greater than those in the negative ion mode. The sensitivity as [M + H](+) showed no systematic variation with pH; however, the sensitivity as [M - H](-) did increase with increasing pH. A larger linear range was observed for [M - H](-) than for [M + H](+) ions. Both [M + Na](+) and [M + H](+) ions were observed with 0.5 mM sodium acetate and sodium iodide, but no adduct ions were observed with ammonium acetate. Some M(2)H(+) ions were observed at higher concentrations. Cluster ions, Na(NaOAc)(n)(+) or Na(NaI)(n)(+), but no sample ions were observed using 5 mM salts. The data suggest that mechanisms in addition to solution ionization are involved in the formation of the ESI sample ions. The utility of mobile phases containing 1% HOAc or 50 mM NH(4)OH was demonstrated for chromatographic separations.

Collisionally-induced dissociation of purine antiviral agents: mechanisms of ion formation using gas phase hydrogen/deuterium exchange and electrospray ionization tandem mass spectrometry.

Electrospray ionization (ESI) and collision-induced dissociation (CID) mass spectra were obtained for two purine nucleoside antiviral agents (acycloguanosine and vidarabine) and one purine nucleotide (vidarabine monophosphate) and for the corresponding compounds in which the labile hydrogens were replaced by deuterium gas-phase exchange. The number of labile hydrogens, x, was determined from a comparison of ESI spectra obtained with N2 and with ND3 as the nebulizer gas. CID mass spectra were obtained for [M + H]+ and [M – H]− ions and the exchanged analogs, [M(Dx) + D]+ and [M(Dx) – D]−, produced by ESI using a Sciex API-IIIplus mass spectrometer. Compositions of product ions were determined and mechanisms of decomposition elucidated by comparison of the CID mass spectra of the undeuterated and deuterated species. Protonated purine antiviral agents dissociate through rearrangement decompositions of base-protonated [M + H]+ ions by cleavage of the glycosidic bonds to give the protonated bases with a sugar moiety as the neutral fragment. Cleavage of the same bonds with charge retention on the sugar moiety gives low abundance ions, due to the low proton affinity of the sugar moiety compared with that of the purine base. CID of protonated purine bases [B + H]+ occurs through two major pathways: (1) elimination of NH3 (ND3) and (2) loss of NH2CN (ND2CN). Minor pathways include elimination of HNCO (DNCO), loss of CO and loss of HCN (DCN). Deprotonated acycloguanosine and vidarabine exhibit the deprotonated base [B – H]− as a major fragment from glycosidic bond cleavage and charge delocalization on the base. Deprotonated vidarabine monophosphate, however, shows predominantly phosphate-related product ions. CID of deprotonated guanine shows two principal pathways: (1) elimination of NH3 (ND3) and (2) loss of NH2CN (ND2CN). Minor pathways include elimination of HNCO (DNCO), loss of CO and loss of HCN (DCN). The dissociation reactions of deprotonated adenine, however, proceed by elimination of HCN and elimination of NCHNH (NCHND). The mass spectra of the antiviral agents studied in this paper may be useful in predicting reaction pathways in other heteroaromatic ring decompositions of nucleosides and nucleotides.

Identification of the degradation product of ezlopitant, a non-peptidic substance P antagonist receptor, by hydrogen deuterium exchange, electrospray ionization tandem mass spectrometry (ESI/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy.

The degradation product of ezlopitant was isolated from low specific activity material and identified by solution phase hydrogen/deuterium (H/D) exchange and electrospray ionization tandem mass spectrometry (ESI/MS/MS) to be an isopropyl peroxide analog of ezlopitant. The structure of the degradant was further confirmed by nuclear magnetic resonance (NMR) spectroscopy utilizing complete 1H and 13C assignments. Studies were also performed to identify the factors responsible for the oxidative degradation of ezlopitant, which included salt form, storage conditions and salt formation solvent. Of all the variable studies over a 3 weeks period, only a change in the salt form prevented this oxidative degradation.

Metabolism, pharmacokinetics and excretion of a potent tachykinin NK1 receptor antagonist (CP-122,721) in rat: Characterization of a novel oxidative pathway

The metabolism, pharmacokinetics and excretion of a potent and selective substance P receptor antagonist, (+)-(2S,3S)-3-(2-methoxy-5-trifluoromethoxybenzlamino)-2-phenylpiperidine, CP-122,721, have been studied in rat following oral administration of a single dose of [14C]CP-122,721. Total recovery of the administered dose was 84.1 ± 1.1% for male rat and 80.9 ± 2.7% for female rat. Approximately 81% of the administered radioactivity recovered in urine and faeces were excreted in the first 72 h. Absorption of CP-122,721 was rapid in both male and female rat, as indicated by the rapid appearance of radioactivity in plasma. The plasma concentrations of total radioactivity were always much greater than unchanged drug, indicating early formation of metabolites. CP-122,721 t1/2 was 3.1 and 2.2 h for male and female rat, respectively. The plasma concentrations of CP-122,721 reached a peak of 941 and 476 ng ml−1 for male and female rat, respectively, at 0.5 h post-dose. Based on AUC0–tlast, only 1.5% of the circulating radioactivity was attributable to unchanged drug (average of male and female rats) and the balance, approximately 98.5% of the plasma radioactivity was due to metabolites. The major metabolic pathways of CP-122,721 were due to O-demethylation, aromatic hydroxylation and indirect glucuronidation. The minor metabolic pathways included aliphatic oxidation at the piperidine moiety and aliphatic oxidation at the benzylic position of the trifluoromethoxy anisole moiety. In addition, a novel oxidative metabolite resulting from ipso substitution by the oxygen atom and trifluoromethoxy elimination followed by glucuronide conjugation was also identified.

Reduction and methylation of ziprasidone by glutathione, aldehyde oxidase, and thiol S-methyltransferase in humans: an in vitro study.

In humans, approximately two-thirds of the metabolic clearance of ziprasidone proceeds through reduction of the N-S bond of the benzisothiazole moiety, which is followed by methylation of the resulting thiophenol to the major metabolite S-methyldihydroziprasidone. The objective of this study was to gain an understanding of the underlying mechanism of this clearance route. Incubation of ziprasidone in human liver cytosol yielded the reduction product dihydroziprasidone. Heating the cytosol prior to incubation prevented the reaction, and the reaction was saturable (KM = 3.9 µM; Vmax = 0.24 nmol/min/mg protein), supporting that it was enzyme catalyzed. It was partially stimulated by 2-hydroxypyrimidine and inhibited by menadione, supporting a role for aldehyde oxidase in this reaction. However, incubation of ziprasidone with reduced glutathione, even in the absence of cytosol, readily yielded dihydroziprasidone indicating that there is also a chemical reduction component to this clearance pathway. The pathway was reversible because incubation of dihydroziprasidone in cytosol or with oxidized glutathione in buffer yielded ziprasidone. The methylation of dihydroziprasidone was observed in human liver microsomes in the presence of S-adenosylmethionine (KM = 14 µM; Vmax = 0.032 nmol/min/mg protein). This reaction was inhibited by 2,3-dichloro-α-methylbenzylamine supporting that thiol methyltransferase is the enzyme responsible for this reaction. Thus, the main metabolic pathway for ziprasidone in humans occurs via chemical reduction and aldehyde oxidase catalyzed reduction, followed by thiol methyltransferase catalyzed methylation.

In vitro-in vivo correlation for intrinsic clearance for CP-409,092 and sumatriptan: a case study to predict the in vivo clearance for compounds metabolized by monoamine oxidase.

Oxidative deamination of the GABAA partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the clearance of these two compounds. Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A. The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro intrinsic clearance (CLint) values were determined and compared to the corresponding in vivo oral clearance (CLPO) values. The overall objective was to determine whether an in vitro-in vivo correlation (IVIVC) could be described for compounds cleared by MAO-A. The intrinsic clearance, CLint, of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CLint, values were calculated as 0.008 and 0.002 ml/mg/min for CP-409,092 and sumatriptan, respectively). A similar correlation was observed from the in vivo metabolic data where the unbound oral clearance, CL(u)PO, values in humans were calculated as 724 and 178 ml/min/kg for CP-409,092 and sumatriptan, respectively. The present work demonstrates that it is possible to predict in vivo metabolic clearance from in vitro metabolic data for drugs metabolized by the enzyme monoamine oxidase.

Metabolism, pharmacokinetics and excretion of the GABAA receptor partial agonist [14C]CP-409,092 in rats

The metabolism and excretion of a GABAA partial agonist developed for the treatment of anxiety, CP-409,092; 4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide, were studied in rats following intravenous and oral administration of a single doses of [14C]CP-409,092. The pharmacokinetics of CP-409,092 following single intravenous and oral doses of 4 and 15 mg kg−1, respectively, were characterized by high clearance of 169 ± 18 ml min−1 kg−1, a volume of distribution of 8.99 ± 1.46 l kg−1, and an oral bioavailability of 2.9% ± 3%. Following oral administration of 100 mg kg−1 [14C]CP-409,092, the total recovery was 89.1% ± 3.2% for male rats and 89.3% ± 0.58% for female rats. Approximately 87% of the radioactivity recovered in urine and faeces were excreted in the first 48 h. A substantial portion of the radioactivity was measured in the faeces as unchanged drug, suggesting poor absorption and/or biliary excretion. There were no significant gender-related quantitative/qualitative differences in the excretion of metabolites in urine or faeces. The major metabolic pathways of CP-409,092 were hydroxylation(s) at the oxo-tetrahydro-indole moiety and oxidative deamination to form an aldehyde intermediate and subsequent oxidation to form the benzoic acid. The minor metabolic pathways included N-demethylation and subsequent N-acetylation and oxidation. The present work demonstrates that oxidative deamination at the benzylic amine of CP-409,092 and subsequent oxidation to form the acid metabolite seem to play an important role in the metabolism of the drug, and they contribute to its oral clearance and low exposure.

Determination of the substance P receptor antagonist CP-122, 721 in plasma by narrow-bore high-performance liquid chromatography-ionspray tandem mass spectrometry

A simple, highly sensitive and specific LC-MS-MS assay was developed for the determination of CP-122,721 (I) in rat and human plasma. I and a structural analog, CP-129,943 (II, internal standard), were extracted from plasma with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were reconstituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC column and a mobile phase of acetonitrile-20 mM ammonium acetate, pH 5 (50:50, v/v). Column effluents were monitored by ionspray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to product ion combinations of m/z 381-->205 and 395-->219 was used to quantitate I and II, respectively. The assay exhibited a linear dynamic range of 0.2-100 ng/ml. Absolute recoveries from plasma were above 80% for both I and II. The precision and accuracy values for the method were within +/-3 and +/-9%, respectively. Sample analysis times were less than 5 min from one injection to the next. The assay has proved to be applicable to the pharmacokinetic study of I in rats.