Pharmacokinetics and Absorption, Distribution, Metabolism and Excretion of RGLS4326 in Mouse and Monkey, an Anti-miR-17 Oligonucleotide for the Treatment of Polycystic Kidney Disease.

RGLS4326 is a short oligonucleotide inhibitor of microRNA-17 (miR-17) that preferentially distributes to the kidney and displaces miR-17 from translationally active polysomes. Here, we present pharmacokinetics and absorption, distribution, metabolism, and excretion properties of RGLS4326 from mice and monkeys. RGLS4326 was absorbed rapidly after subcutaneous administration, distributed extensively to the kidney and liver, with preferential distribution to the kidney, and cleared rapidly from plasma by tissue uptake and renal excretion. Plasma exposure increased in a dose-proportional manner with no notable accumulation after repeat doses. Plasma protein binding of RGLS4326 across all species tested was between 79% and 96%. RGLS4326 predominantly distributed to the kidney with a long half-life (t1/2; t1/2 ranged from 8-11 days) and no marked (≤twofold) accumulation in kidney and liver after repeat doses. RGLS4326 was minimally metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species and underwent sequential hydrolysis from both 3' and 5' ends to produce chain-shortened metabolites. There were no human unique metabolites observed. Renal excretion was the major route of elimination of RGLS4326, and a significant fraction (50%-79%) of the dose was recovered intact in the urine of mice and monkeys across all dose levels. RGLS4326 is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, RGLS4326 exhibits low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT: Pharmacokinetics (PK) and absorption, distribution, metabolism, and excretion (ADME) properties of RGLS4326 were characterized in vivo and in vitro. RGLS4326 shows similar PK and ADME properties across mice and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in preferential exposure of RGLS4326 to the intended target organ (kidney) to drive maximum target engagement. These studies support the interpretation of toxicology and efficacy studies and help characterize the disposition of RGLS4326 in humans.

Considerations and recommendations for assessment of plasma protein binding and drug-drug interactions for siRNA therapeutics.

At the time of writing, although siRNA therapeutics are approved for human use, no official regulatory guidance specific to this modality is available. In the absence of guidance, preclinical development for siRNA followed a hybrid of the small molecule and biologics guidance documents. However, siRNA differs significantly from small molecules and protein-based biologics in its physicochemical, absorption, distribution, metabolism and excretion properties, and its mechanism of action. Consequently, certain reports typically included in filing packages for small molecule or biologics may benefit from adaption, or even omission, from an siRNA filing. In this white paper, members of the 'siRNA working group' in the IQ Consortium compile a list of reports included in approved siRNA filing packages and discuss the relevance of two in vitro reports-the plasma protein binding evaluation and the drug-drug interaction risk assessment-to support siRNA regulatory filings. Publicly available siRNA approval packages and the literature were systematically reviewed to examine the role of siRNA plasma protein binding and drug-drug interactions in understanding pharmacokinetic/pharmacodynamic relationships, safety and translation. The findings are summarized into two decision trees to help guide industry decide when in vitro siRNA plasma protein binding and drug-drug interaction studies are warranted.

In Vitro Metabolism of Slowly Cleared G Protein-Coupled Receptor 139 Agonist TAK-041 Using Rat, Dog, Monkey, and Human Hepatocyte Models (HepatoPac): Correlation with In Vivo Metabolism.

Hepatic metabolism of low-clearance compound TAK-041 was studied in two different in vitro model systems using rat, dog, monkey, and human suspended cryopreserved hepatocytes and HepatoPac micropatterned coculture model primary hepatocytes. The aim of this work was to investigate the most appropriate system to assess the biotransformation of TAK-041, determine any notable species difference in the rate and in the extent of its metabolic pathways, and establish correlation with in vivo metabolism. TAK-041 exhibited very low turnover in suspended cryopreserved hepatocyte suspensions for all species, with no metabolites observed in human hepatocytes. However, incubations conducted for up to 14 days in the HepatoPac model resulted in more robust metabolic turnover. The major biotransformation pathways of TAK-041 proceed via hydroxylation on the benzene ring fused to the oxotriazine moiety and subsequent sulfate, glucuronide, and glutathione conjugation reactions. The glutathione conjugate of TAK-041 undergoes further downstream metabolism to produce the cysteine S-conjugate, which then undergoes N-acetylation to mercapturic acid and/or conversion to ß-lyase-derived thiol metabolites. The minor biotransformation pathways include novel ring closure and hydrolysis, hydroxylation, oxidative N-dealkylation, and subsequent reduction. The HepatoPac model shows a notable species difference in the rate and in the extent of metabolic pathways of TAK-041, with dogs having the fastest metabolic clearance and humans the slowest. Furthermore, the model shows its suitability for establishing correlation with in vivo metabolism of low-turnover and extensively metabolized compounds such as TAK-041, displaying an extensive and unusual downstream sequential ß-lyase-derived thiol metabolism in preclinical species and human. SIGNIFICANCE STATEMENT: This study investigated the most appropriate in vitro system to assess the biotransformation of the low-turnover and extensively metabolized compound TAK-041, determine any notable species difference in the rate and in the extent of its metabolic pathways, and establish correlation with in vivo metabolism. The HepatoPac model was identified and showed its suitability for species comparison and establishing correlation, with in vivo metabolism displaying an extensive and unusual downstream sequential ß-lyase-derived thiol metabolism in preclinical species and human.

In Vitro Biotransformation of the Nrf2 Activator Bardoxolone: Formation of an Epoxide Metabolite That Undergoes Two Novel Glutathione-Mediated Metabolic Pathways: Epoxide Reduction and Oxidative Elimination of Nitrile Moiety.

Metabolism of bardoxolone methyl (BARD-Me), an oleanolic acid derivative, and its epoxide metabolite was studied in different in vitro systems. BARD-Me also undergoes glutathione (GSH)-adduct formation via direct nucleophilic attack at the ß-carbon of the α,ß-unsaturated ketone substituent on the A-ring. The presence of an electron-withdrawing nitrile residue on the α-carbon increases the α,ß-unsaturated ketone's susceptibility to nucleophilic attack by thiols. This allows BARD-Me to generate reversible adducts with the thiol groups of cysteine residues in target proteins without the potential toxic liabilities of irreversible covalent adduct formation. However, BARD-Me epoxide can also react with thiols irreversibly. Therefore, the epoxide was synthesized and its metabolic fate studied in vitro. BARD-Me epoxide was found to undergo two novel metabolic biotransformations: epoxide reduction and oxidative elimination of nitrile moiety. Both metabolic pathways proceed via nucleophilic attack of the thiol group of GSH at each of the two carbon atoms of the epoxide as evidenced by the formation of two ß-hydroxy sulfide regioisomers. Oxidative elimination of nitrile moiety proceeds via nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is ß to the cyano group to give a cyanohydrin metabolite, which spontaneously decomposes to release cyanide and the corresponding ketone. Nucleophilic attack of the thiol group of GSH at the epoxide carbon atom that is α to the cyano group results in the formation of the GSH monoadduct that undergoes intermolecular reduction with another GSH molecule, followed by elimination of oxidized GSH (GS-SG) and the formation of an enolate intermediate. Upon protonation, the enolate intermediate gives rise to hydroxylated BARD-Me, which is readily converted back to BARD-Me through the elimination of water. The chemical reactivity of the epoxide metabolite and the liberation of cyanide are of significant toxicological interest for the potential utility of BARD-Me as a therapeutic agent.

Epidemiology of urinary tract infections in the children in zagazig university children's hospital

Background: The most common bacterial infection among children is Urinary Tract Infection (UTI). Early diagnosis and good treatment of UTI is very important as the risk of renal damage is increased in children below the age of five years which result of morbidity. The aim of this study was to estimate the prevalence of urinary tract infection (UTI) in children attending Pediatric outpatient clinic in Zagazig University Children's Hospital. Also to determine related risk factors, isolate the organisms that cause UTI in children and antibiotics susceptibility patterns. Methods: This Cross sectional descriptive study, was conducted on 600 children, (377 males and 223 females) from two to seven years old attending to pediatric outpatient clinic ZUH , All patient groups were exposed to full medical history, physical examination, Dipstick analysis by using both nitrite and leukocyte esterase detector, Microscopic examinations and urine culture for positive cases.Results: The prevalence of UTI between children included in the current study was (7%). LE positive were 56 (9.3%), Nitrite positive were 47 (7.8%) and both LE and Nitrite positive were 17 (2.8%). Conclusion: The prevalence of UTI was 7 % in our study, E - Coli was detected to be the most common organism, Cefotaxime and Amikacin were detected to be the most common antibiotic sensitive to the isolates

Prediction of Mass Spectral Response Factors from Predicted Chemometric Data for Druglike Molecules.

A method is developed for the prediction of mass spectral ion counts of drug-like molecules using in silico calculated chemometric data. Various chemometric data, including polar and molecular surface areas, aqueous solvation free energies, and gas-phase and aqueous proton affinities were computed, and a statistically significant relationship between measured mass spectral ion counts and the combination of aqueous proton affinity and total molecular surface area was identified. In particular, through multilinear regression of ion counts on predicted chemometric data, we find that log10(MS ion counts) = -4.824 + c 1•PA + c 2•SA, where PA is the aqueous proton affinity of the molecule computed at the SMD(aq)/M06-L/MIDI!//M06-L/MIDI! level of electronic structure theory, SA is the total surface area of the molecule in its conjugate base form, and c 1 and c 2 have values of -3.912 × 10-2 mol kcal-1 and 3.682 × 10-3 Å-2. On a 66-molecule training set, this regression exhibits a multiple R value of 0.791 with p values for the intercept, c 1, and c 2 of 1.4 × 10-3, 4.3 × 10-10, and 2.5 × 10-6, respectively. Application of this regression to an 11-molecule test set provides a good correlation of prediction with experiment (R = 0.905) albeit with a systematic underestimation of about 0.2 log units. This method may prove useful for semiquantitative analysis of drug metabolites for which MS response factors or authentic standards are not readily available. Graphical Abstract ᅟ.

Metabolic Enzyme Sulfotransferase 1A1 Is the Trigger for N-Benzyl Indole Carbinol Tumor Growth Suppression.

In an attempt to identify novel therapeutics and mechanisms to differentially kill tumor cells using phenotypic screening, we identified N-benzyl indole carbinols (N-BICs), synthetic analogs of the natural product indole-3-carbinol (I3C). To understand the mode of action for the molecules we employed Cancer Cell Line Encyclopedia viability profiling and correlative informatics analysis to identify and ultimately confirm the phase II metabolic enzyme sulfotransferase 1A1 (SULT1A1) as the essential factor for compound selectivity. Further studies demonstrate that SULT1A1 activates the N-BICs by rendering the compounds strong electrophiles which can alkylate cellular proteins and thereby induce cell death. This study demonstrates that the selectivity profile for N-BICs is through conversion by SULT1A1 from an inactive prodrug to an active species that induces cell death and tumor suppression.

Inhibition of cytochrome P450 enzymes and biochemical aspects of mechanism-based inactivation (MBI).

Mechanism-based inactivation (MBI) often involves metabolic bioactivation of the xenobiotic by cytochrome P450s (CYPs) to an electrophilic reactive intermediate and results in quasi-irreversible or irreversible inactivation. Such reactive intermediate can cause quasi-irreversible inhibition through coordination to the ferrous state, Fe(II), of the P450 enzyme forming a tight noncovalent bond leading to the formation of metabolic-intermediate complex (MIC). By contrast, irreversible inactivation is one of the most common causes for the observed drug­drug interaction (DDI) and usually implies the formation of a covalent bond between the metabolite and the enzyme via alkylation of either the heme or the P450 apoprotein. Here we illustrate the important points of the current literature understanding of the mechanisms of inhibition of CYP enzymes with emphasis on general mechanistic aspects of MBI for some drugs/moieties associated with the phenomenon. Additionally, the utility of computational and in silico approaches to address bioactivation issues will be briefly discussed.

In vitro metabolism of the 5-hydroxytryptamine1B receptor antagonist elzasonan.

The metabolism of elzasonan has been examined in vitro using hepatic microsomes from human and recombinant heterologously expressed P450 enzymes (rCYP). Metabolism occurs primarily via oxidative N-demethylation to form M4 and oxidation reactions to form elzasonan N-oxide (M5) and 5-hydroxyelzasonan metabolite (M3). Additionally, elzasonan was shown to be metabolized to the novel cyclized indole metabolite (M6) which undergoes subsequent oxidation to form the iminium ion metabolite (M3a). The rCYP data was normalized relative to the levels of each CYP form in native human liver microsomes to better assess the contribution of each rCYP in the metabolism of elzasonan. Results demonstrated the involvement of CYP3A4 in the pathways leading to M3a, M3, M5 and M6 and CYP2C8 in the formation of M4. Kinetic constants for the formation of M3 were determined and correlation and inhibition studies suggested that CYP3A4 is primarily responsible for the formation of M3 and CYP2C19 plays a very minor role in its formation. Cytochrome b5 has shown to be an essential component in P450 3A4 catalyzed 5-hydroxyelzasonan formation and provides insights on the disconnect between human liver microsomes data and that of rCYP. Furthermore, rCYP3A4 containing b5 are useful models for predicting the rates for liver microsomes P450-dependent drug oxidations and should be utilized routinely.

Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines: identification of a rare stable quinone methide and subsequent GSH conjugate.

In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P450 (P450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution.

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 (K(M) = 3.9 µM; V(max) = 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 (K(M) = 14 µM; V(max) = 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 GABA(A) 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 (CL(int)) values were determined and compared to the corresponding in vivo oral clearance (CL(PO)) 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, CL(int), of CP-409,092 was approximately 4-fold greater than that of sumatriptan (CL(int), 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 5-hydroxytryptamine1b receptor antagonist elzasonan in humans.

The metabolism, pharmacokinetics, and excretion of a potent and selective 5-hydroxytryptamine(1B) receptor antagonist elzasonan have been studied in six healthy male human subjects after oral administration of a single 10-mg dose of [(14)C]elzasonan. Total recovery of the administered dose was 79% with approximately 58 and 21% of the administered radioactive dose excreted in feces and urine, respectively. The average t(1/2) for elzasonan was 31.5 h. Elzasonan was extensively metabolized, and excreta and plasma were analyzed using mass spectrometry and NMR spectroscopy to elucidate the structures of metabolites. The major component of drug-related material in the excreta was in the feces and was identified as 5-hydroxyelzasonan (M3), which accounted for approximately 34% of the administered dose. The major human circulating metabolite was identified as the novel cyclized indole metabolite (M6) and accounted for ∼65% of the total radioactivity. A mechanism for the formation of M6 is proposed. Furthermore, metabolism-dependent covalent binding of drug-related material was observed upon incubation of [(14)C]elzasonan with liver microsomes, and data suggest that an indole iminium ion is involved. Overall, the major metabolic pathways of elzasonan were due to aromatic hydroxylation(s) of the benzylidene moiety, N-oxidation at the piperazine ring, N-demethylation, indirect glucuronidation, and oxidation, ring closure, and subsequent rearrangement to form M6.

Collision induced dissociation studies of alkali metal adducts of tetracyclines and antiviral agents by electrospray ionization, hydrogen/deuterium exchange and multiple stage mass spectrometry.

The collision induced dissociation (CID) mass spectra were obtained for the X(+)-adducts (X=Na(+) or Li(+)) of five tetracyclines, four pyrimidine and three purine derivatives and their fully D-exchanged species in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The CID spectra were obtained for [M + Na](+) and [M + Li](+) and the exchanged analogs, [M(D) + Na](+) and [M(D) + Li](+), and compositions of product ions and mechanisms of decomposition were determined by comparison of the MS(n) spectra of the undeuterated and deuterated species. Metal ions are bound to the base of purine and pyrimidine antiviral agents and dissociate primarily to give the metal complexes of the base [B + X](+). For vidarabine monophosphate, however, the metal ions are bound to the phosphate group, resulting in unique and characteristic cleavage reactions not observed in the uncomplexed system, and dissociate through the loss of phosphate and/or phosphate metal ion complex. The [B + X](+) of these antiviral agents are relatively stable and show no or little fragmentation compared to [B + H](+). The CID of [B + X](+) of guanine derivative occurs mainly through elimination of NH(3) and that of trifluoromethyl uracil dissociates primarily through the loss of HF. For tetracyclines, metal ions are bound to ring A at the tricarbonylmethyl group and dissociate initially by the loss of NH(3)/ND(3) from [M(H) + X](+) and [M(D) + X](+). The CID spectra of [M + X](+) of tetracyclines are somewhat similar to those of [M + H](+). The dominant fragments from the metal complexes of these compounds are charge remote decompositions involving molecular rearrangements and the loss of small stable molecules. Additionally, tetracyclines and the antiviral agents show more selectivity towards Li+ ion than the corresponding complexes with Na(+) or K(+).

Mechanism of [m+h]+ formation in atmospheric pressure photoionization mass spectrometry: identification of propionitrile in acetonitrile with high mass accuracy measurement and tandem mass spectrometry and evidence for its involvement in the protonation phenomenon.

The role of propionitrile in the production of [M+H]+ under atmospheric pressure photoionization (APPI) was investigated. In dopant-assisted APPI using acetone and anisole, protonated acetone and anisole radical cations were the most prominent ions observed. In dopant-free or direct APPI in acetonitrile, however, a major ion in acetonitrile was detected and identified as propionitrile, using high accuracy mass measurement and collision induced dissociation studies. Vaporizing ca. 10(-5) M althiazide and bendroflumethazide under direct APPI in acetonitrile produced their corresponding protonated species [M+H]+. In addition to protonated acetonitrile, its dimers, and acetonitrile/water clusters, protonated propionitrile, propionitrile dimer, and propionitrile/water clusters were also observed. The role of propionitrile, an impurity in acetonitrile and/or a possible product of ion-molecule reaction, in the production of [M+H]+ of althiazide and bendroflumethazide was further investigated in the absence of dopant using propionitrile-d5. The formation of [M+D]+ species was observed, suggesting a possible role of propionitrile in the protonation process. Additionally, an increase in the [M+H]+ signal of althiazide and bendroflumethazide was observed as a function of propionitrile concentration in acetonitrile. Theoretical data from the literature supported the assumption that one possible mechanism, among others, for the formation of [M+H]+ could be attributed to photo-initiated isomerization of propionitrile. The most stable isomers of propionitrile, based on their calculated ionization energy (IE) and relative energy (DeltaE), were assumed to undergo proton transfer to the analytes, and mechanisms were proposed.

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

ESI and CID mass spectra were obtained for four pyrimidine nucleoside antiviral agents and the corresponding compounds in which the labile hydrogens were replaced by deuterium using gas-phase exchange. The number of labile hydrogens, x, was determined from a comparison of ESI spectra obtained with N(2) and with ND(3) as the nebulizer gas. CID mass spectra were obtained for [M + H](+) and [M - H](-) ions and the exchanged analogs, [M(D(x)) + D](+) and [M(D(x)) - D](-), produced by ESI using a SCIEX API-III(plus) mass spectrometer. Protonated pyrimidine 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 glycosidic bonds with charge retention on the sugar moiety eliminates the base moiety as a neutral molecule and produces characteristic sugar ions. CID of protonated pyrimidine bases, [B + H](+), occurs through three major pathways: (1) elimination of NH(3) (ND(3)), (2) loss of H(2)O (D(2)O), and (3) elimination of HNCO (DNCO). Protonated trifluoromethyl uracil, however, dissociates primarily through elimination of HF followed by the loss of HNCO. CID mass spectra of [M - H](-) ions of all four antiviral agents show NCO(-) as the principal decomposition product. A small amount of deprotonated base is also observed, but no sugar ions. Elimination of HNCO, HN(3), HF, CO, and formation of iodide ion are minor dissociation pathways from [M - H](-) ions.

Metabolism, pharmacokinetics, and excretion of the substance P receptor antagonist CP-122,721 in humans: structural characterization of the novel major circulating metabolite 5-trifluoromethoxy salicylic acid by high-performance liquid chromatography-tandem mass spectrometry and NMR spectroscopy.

The metabolism, pharmacokinetics, and excretion of a potent and selective substance P receptor antagonist, CP-122,721 [(+)-(2S,3S)-3-(2-methoxy-5-trifluoromethoxybenzylamino)-2-phenylpiperidine], have been studied in six healthy male human subjects [four extensive metabolizers (EMs) and two poor metabolizers (PMs) of CYP2D6) following oral administration of a single 30-mg dose of [14C]CP-122,721. Approximately 84% of the administered radioactivity was recovered from the urine and feces of the subjects over a period of 312 h. Approximately 80% of the dose for EM subjects was recovered within 48 h. PM subjects, however, excreted only about 45% of the dose in 48 h and required the full 312 h to achieve nearly 80% recovery. Absorption of CP-122,721 was rapid in both extensive and poor metabolizers, as indicated by the rapid appearance of radioactivity in serum. The serum concentrations of total radioactivity were always much greater than those of unchanged drug indicating early formation of metabolites. The average CP-122,721 t1/2 was 6.7 h and 45.0 h for EM and PM subjects, respectively. The serum concentrations of CP-122,721 reached a peak of 7.4 and 69.8 ng/ml for extensive and poor metabolizers, respectively. 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, O-dealkylation of the trifluoromethoxy group, N-dealkylation, and oxidative deamination. In addition to the major human circulating metabolite 5-trifluoromethoxy salicylic acid (TFMSA), all other circulating metabolites of CP-122,721 were glucuronide conjugates of oxidized metabolites. TFMSA was identified using high pressure liquid chromatography/tandem mass spectrometry and NMR and mechanisms were proposed for its formation. There are no known circulating active metabolites of CP-122,721.

High performance liquid chromatography/atmospheric pressure ionization/tandem mass spectrometry (HPLC/API/MS/MS) in drug metabolism and toxicology.

Studies of the metabolic fate of drugs and other xenobiotics in living systems may be divided into three broad areas: (1) elucidation of biotransformation pathways through identification of circulatory and excretory metabolites (qualitative studies); (2) determination of pharmacokinetics of the parent drug and/or its primary metabolites (quantitative studies); and (3) identification of chemically-reactive metabolites, which play a key role as mediators of drug-induced toxicities (mechanistic studies). Mass spectrometry has been regarded as one of the most important analytical tools in studies of drug metabolism, pharmacokinetics and biochemical toxicology. With the commercial introduction of new ionization methods such as those based on atmospheric pressure ionization (API) techniques and the combination of liquid chromatography-mass spectrometry (LC-MS), it has now become a truly indispensable technique in pharmaceutical research. Triple stage quadrupole and ion trap mass spectrometers are presently used for this purpose, because of their sensitivity and selectivity. API-TOF mass spectrometry has also been very attractive due to its enhanced full-scan sensitivity, scan speed, improved resolution and ability to measure the accurate masses for protonated molecules and fragment ions. This review aims to survey the utility of mass spectrometry in drug metabolism and toxicology and to highlight novel applications and future trends in this field.

The impact of recent innovations in the use of liquid chromatography-mass spectrometry in support of drug metabolism studies: are we all the way there yet?

Absorption, distribution, metabolism, excretion and toxicology (ADMET) studies are widely used in drug discovery and development to help obtain the optimal balance of properties necessary to convert lead compounds into drugs that are safe and effective for human use. Drug discovery efforts have been aimed at identifying and addressing metabolism issues at the earliest possible stage, by developing and applying innovative liquid chromatography-mass spectrometry (LC-MS)-based techniques and instrumentation, which are both faster and more accurate than prior techniques. Such new approaches are demonstrating considerable potential to improve the overall safety profile of drug candidates throughout the drug discovery and development process. These emerging techniques streamline and accelerate the process by eliminating potentially harmful candidates earlier and improving the safety of new drugs. In the area of drug metabolism, for example, revolutionary changes have been achieved by the combination of LC-MS with innovative instrumentation such as triple quadrupoles, ion traps and time-of-flight mass spectrometry. In turn, most ADMET studies have come to rely on LC-MS for the analysis of an ever-increasing workload of potential candidates. This article provides a discussion on the importance of LC-MS in supporting drug metabolism studies, and highlights the relative merits of current applications for LC-MS in drug metabolism testing and analysis. These applications include in vitro and in vivo testing, pharmacokinetic profiling, chiral separations, stable isotope labeling, metabolic activation testing, metabolite characterization and radiolabeled-drug testing.

Characterization of a novel metabolite intermediate of ziprasidone in hepatic cytosolic fractions of rat, dog, and human by ESI-MS/MS, hydrogen/deuterium exchange, and chemical derivatization.

Ziprasidone (Geodone), a novel atypical antipsychotic agent, is recently approved for the treatment of schizophrenia. It undergoes extensive metabolism in preclinical species and humans after oral administration, and only a very small amount of administered dose is excreted as unchanged drug. In vitro studies using human liver microsomes have shown that the oxidative metabolism of ziprasidone is mediated primarily by CYP3A4. However, coadministration of ziprasidone with ketoconazole, a CYP3A4 inhibitor, showed only a modest increase in its exposure. Therefore, in vitro metabolism of ziprasidone was investigated in hepatic cytosolic fractions to further understand its clearance mechanisms in preclinical species and humans. The major metabolite from incubation of ziprasidone in cytosolic fractions of rat, dog, and human was characterized by liquid chromatography-tandem mass spectrometry and found to be the product of reductive cleavage. Derivatization and hydrogen/deuterium exchange were used to deduce that the addition of two hydrogen atoms had occurred at the benzisothiazole moiety. Further studies to determine the enzyme involved in the formation of this metabolite are currently in progress. The identification of this novel metabolite in cytosol has clarified the clearance mechanism of ziprasidone in humans and preclinical species.