Combined recirculation of the rat liver and kidney: Studies with enalapril and enalaprilat

Combined recirculation of the rat liver (L) and kidney (IPK) at 10 ml min^–1 per organ (LK) was developed to examine the hepatorenal handling of the precursor-metabolite pair: [¹⁴C]-enalapril and [³H]enalaprilat. Loading doses followed by constant infusion of [¹⁴C]enalapril and preformed [³H]enalaprilat to the reservoirs of the IPK or the LK preparation was used to achieve steady stale conditions. In both organs, enalapril was mostly metabolized to its dicarboxylic acid metabolite, enalaprilat, which was excreted unchanged. At steady state, the fractional excretion for [¹⁴C]enalapril (FE=0.45 to 0.48) and preformed [³H]enalaprilat (FE{pmi}=1.1) were constant and similar for both the IPK and LK. The additivity of clearance was demonstrated in the LK preparation, namely, the total clearance of enalapril was the sum of its hepatic and renal clearances. However, the apparent fractional excretion for fanned [¹⁴C]enalaprilat, FE{mi} and the apparent urinary clearance were time-dependent and higher than the corresponding values for preformed [³H]enalaprilat in both the IPK and LK. The FE{mi} and urinary clearance values further differed between the IPK and LK. Biliary clearance of formed vs. preformed enalaprilat displayed the same discrepant trends as observed for FE{mi} vs. FE{pmi} for the LK. These observations on the time-dependent and variable excretory clearance (urinary or biliary) of the formed metabolite vs. the constant, and much reduced, excretory clearance of the preformed metabolite are due to dual contributions to formed metabolite excretion: the nascently formed, intracellular metabolite which immediately underwent excretion and the formed metabolite which reentered the circulation, behaved as a preformed species. When data for the IPK and LK preparations were modeled with a physiological model with parameters previously reported for the L and IPK, all data, including metabolite excretory clearances, were well predicted. Model simulations revealed that the apparent FE{mi} differed between the LK and IPK preparations when the liver was present as an additional metabolite formation organ; the apparent excretory (urinary orGlossary k⁰ infusion rate into the reservoir - C_R reservoir concentration - C_Out,k and C_Out,L venous concentrations for the kidney and liver - C_p,k and c_P,L concentrations in renal and hepatic plasma, respectively - C_k and C_L concentrations in kidney and liver tissue, respectively - C_U and C_Bile concentrations in urine and bile, respectively - CL _b ⁱⁿ andCL _b ^ef influx and efflux clearances, respectively, at the basolateral membrane of the renal tubular cell - C _l ⁱⁿ and CL _l ^ef influx and efflux clearances, respectively, at the luminal membrane of the renal tubular cell - CL _int,K ^m renal metabolic intrinsic clearance of the drug - CL _d ⁱⁿ and CL _d ^ef influx and efflux clearances, respectively, at the sinusoidal membrane - CL _int ^m,L hepatic metabolic intrinsic clearance of the drug - CL _int,L ^b biliary intrinsic clearance - V_R plasma reservoir volume - V_P,K and V_P,L plasma volumes of the kidney and liver, respectively - V_K and V_L tissue volumes of the kidney and liver, respectively - V_U and V_Bile volumes of urine and bile, respectively - Q_K and Q_L total renal and hepatic plasma flow rates, respectively - GFR glomerular filtration rate - Q_U and Q_Bile urine and bile flow rates, respectively - f_P, f_K, and f_L unbound fractions in plasma and kidney and liver tissue, respectively This work was supported by the Medical Research Council of Canada. I. A. M. de Lannoy was a recipient of the Ontario Graduate Scholarship from the Ontario Ministry of Health; K. S. Pang was a recipient of the Faculty Development Award, Medical Research Council.     

Presence of a diffusional barrier on metabolite kinetics: enalaprilat as a generated versus preformed metabolite

Studies in the once-through perfused rat liver with the simultaneous delivery of 14 C-enalapril and its polar diacid metabolite, 3H-enalaprilat, revealed different extents of elimination (exclusively by biliary excretion) for the generated (14C-enalaprilat) and preformed (3H-enalaprilat) metabolite (18 and 5% dose) [Pang, Cherry, Terrell, and Ulm: Drug Metab. Dispos. 12, 309-313 (1984)]. The present re-examination of data provided an explanation for these discrepant observations: enalaprilat, being a polar dicarboxylic acid, encounters more of a diffusional barrier than its precursor, enalapril, an ethyl ester of enalaprilat. Programs written in Fortran 77 on mass balance relationships were employed to simulate data upon varying the diffusional clearances for drug (CLd) and metabolite [CLd(mi)] from 1 to 5000 ml/min. The metabolic and biliary intrinsic clearances for drug and metabolite were found by trial and error such that the combinations of all clearance parameters yielded data similar to enalaprilat, and 3H-enalaprilat. Our finding indicated that the diffusional clearance for enalaprilat was low (2 ml/min) compared to that of enalapril (75 ml/min). The presence of a diffusional barrier for enalaprilat retards entry of the preformed metabolite into hepatocytes but prevents efflux of the intracellularly formed generated metabolite into sinusoidal blood, thereby enhancing generated metabolite elimination.     

Sequential first-pass elimination of a metabolite derived from a precursor

We examined data from our previous studies in which we not only delivered perfusate containing tracer concentrations of [¹⁴C]phenacetin and its metabolite [³H]acetaminophen under constant perfusate flow (10 ml/ min/ liver) into the rat liver preparation just once, but also recirculated fresh reservoir perfusate containing a tracer dose of [¹⁴C]phenacetin through the same rat liver preparation. From the single-pass studies, estimates of f_m, the fractional rate of conversion for [¹⁴C]phenacetin to form [¹⁴C]acetaminophen, and F_(M.P), the apparent availability of [¹⁴C]acetaminophen, were obtained by determining the concentrations of [¹⁴C]acetaminophen in the perfusate before and after incubation with Glusulase. These estimates were f_m=0.871±0.16 and F_(M.P)=0.43±0.10. These and the steady-state clearance values of phenacetin (9.1±0.8ml/min) and acetaminophen (6.7±0.7ml/min) from the single-pass studies were used to predict the concentrations of [¹⁴C]acetaminophen in the reservoir perfusate on recirculation of [¹⁴C]phenacetin. We found that the sequential first-pass elimination of the metabolite must be considered when the metabolite is highly extracted by the liver. If we had neglected to take this into account, the fractional rate of conversion of a precursor to form a metabolite and the rate of formation of the metabolite would have been underestimated by the factor F_(M.P).     

Drug pharmacokinetics and the carbon dioxide breath test

The interrelationship of the pharmacokinetics of a drug and the expiration of carbon dioxide formed as a metabolite have been studied. The pharmacokinetic characteristics of the drug that affect the usefulness of the carbon dioxide excretion as a measure of liver function were examined by means of computer simulations. The parent drug extraction ratio, fraction demethylated, volume of distribution, and absorption rate of an oral dosage form all contribute to the carbon dioxide breath test result. A drug that would be a useful substrate when the carbon dioxide breath test is used as a probe for changes in liver function should be at least 50% metabolized by demethylation, have a hepatic extraction ratio of 0.2–0.5, and be administered in a form that is rapidly absorbed.Appendix b. symbols CL _c net clearance of formaldehyde to carbon dioxide - CL _int,f intrinsic hepatic clearance of formation of formaldehyde from parent drug (bound and unbound to plasma proteins) - CL _int,p intrinsic hepatic clearance of total parent drug (bound and unbound to plasma proteins) - CL _sys,f systemic hepatic clearance of formation of formaldehyde from parent drug,Q _H CL _int,f /(Q _H +CL _int,p ) - CL _sys,p systemic hepatic clearance of parent drug,Q _H CL _int,p /(Q _H +CL _int,p ) - E extraction ratio,CL _int,p /(Q _H +CL _int,p - F I-E fraction escaping first-pass metabolism,Q _H/(Q _H +CL _int,p - fm fraction of parent drug metabolized by demethylation to formaldehyde,CL _int,f /CL _int,p - HCHO amount of formaldehyde - [HCHO] concentration of formaldehyde - _a absorption rate constant - M _i metabolite of P formed by routes other than demethylation - M ₁ metabolite of P formed by demethylation - P amount of parent drug in the body - [P] concentration of parent drug measured in arterial blood - P _A amount of parent drug at absorption site - P _L amount of parent drug in the liver - Q _H hepatic blood flow - V _F volume of distribution of formaldehyde - V _p volume of distribution of parent drug     

A physiological model for renal drug metabolism: Enalapril esterolysis to enalaprilat in the isolated perfused rat kidney

A physiologically based kidney model was developed to describe the metabolism of enalapril and explain the observed discrepancies between generated and preformed enalaprilat (metabolite) elimination in the constant flow single-pass and recirculating isolated perfused rat kidney preparations (IPKs) as a result of the differing points of origin of the metabolite within the kidney, subsequent to the simultaneous delivery of¹⁴Cenalapril and³H-enalaprilat. The model incorporated clearances for diffusion/transport of drug and metabolite across the basolateral and luminal membranes of the renal cells, an intrinsic clearance for renal drug metabolism, in addition to physiological variables such as perfusate flow rate, glomerular filtration rate, and urine flow rate. Nonlinear curve fitting of single-pass and recirculating data was performed to estimate the rate-limiting step in the renal elimination of enalaprilat. Through fitting and simulation procedures, we were able to predict metabolic and excretory events for enalapril (renal extraction ratio 0.25–0.3; fractional excretion, FE, was less than unity) and the relatively constant pattern of urinary excretion of preformed enalaprilat (extraction ratio 0.07; FE1). The extraction ratio of the intrarenally formed enalaprilat in single-pass IPK was about twofold that for the preformed metabolite, whereas the FEs of generated enalaprilat in recirculating IPKs were >1, and tended to increase, then decrease with perfusion time. These observations were explained by the optimized parameters which indicated that efflux from cell to lumen was rate-controlling in the excretion of enalaprilat, and another small transport barrier also existed at the basolateral membrane; the lower extraction ratio of preformed enalaprilat was due to its poor transmembrane clearance at the basolateral membrane. The variable FEs for generated enalaprilat vs. the relatively constant FE for preformed metabolite in the recirculating IPK was explained by the changing contributions of both circulating and intrarenal metabolite to metabolite excretion.This work was supported by the Medical Research Council of Canada. I. A. M. de Lannoy was a recipient of the Ontario Graduate Scholarship, and K. S. Pang is a recipient of the Faculty Development Award from MRC, Canada M5S 2S2.     

Identification of the major biliary metabolite of (+)-catechin in the rat

1. Following oral administration of [U-¹⁴C]-(+)-catechin to the rat, the major biliary metabolite was shown to be the glucuronide of 3′-O-methyl-(+) catechin by chromatography and mass spectrometry.

2. [methyl-¹⁴C]-O-Methyl-(+)-catechin was formed by incubation of (+)-catechin with S-adenosyl-L-[methyl-¹⁴C]methionine in vitro in both liver homogenates and in the presence of purified catechol-O-methyl transferase.

3. Alkaline micro-fusion techniques have been used to determine the position of the O-methyl substituent in the B-ring of the isolated O-methyl-(+)-catechin glucuronide.     

Disposition of enalapril and its diacid metabolite, enalaprilat, in a perfused rat liver preparation. Presence of a diffusional barrier for enalaprilat into hepatocytes

Enalaprilat (MK-422), a new and potent angiotensin- converting enzyme inhibitor, and its monoethyl ester precursor, enalapril, were studied in a single pass perfused rat liver preparation under constant perfusate flow (10 ml/min) at concentrations of 0.29-0.41 microM for 14C-enalapril and 0.01-0.015 microM for 3H- enalaprilat . During their simultaneous delivery to the same rat liver preparation, the steady state hepatic extraction ratio of 14C-enalapril was high (0.861 +/- 0.02) and 14C- enalaprilat appeared rapidly in effluent perfusate plasma. Of the enalapril dose, 22.7 +/- 6.9% appeared in bile. 14C- Enalaprilat accounted for 79% of the total radioactivity in bile (18% of dose) whereas 14C-enalapril was present only as 10% of the total (2.3% of dose). By contrast, the steady state hepatic extraction of 3H- enalaprilat was very low (0.053) and the disappearance was virtually identical to the appearance of 3H- enalaprilat in bile. These findings suggest that diffusional barrier exists for enalaprilat as the preformed metabolite, which hinders penetration into hepatocytes, and therefore, elimination. The precursor, enalapril, effectively brings enalaprilat into hepatocytes were more extensive biliary excretion of the generated metabolite takes place. This account adds to our further understanding of metabolite kinetics; in addition to the uneven distribution of enzyme system and the intrinsic clearance for metabolite formation and elimination, the presence of a diffusional barrier is another important determinant which may cause deviations between the kinetics of a generated and performed metabolite.     

Synthesis of 14C‐labeled and 13C‐,15N‐labeled dasatinib and its piperazine N‐dealkyl metabolite

Dasatinib (SPRYCEL^®) is a multiple kinase inhibitor approved for the treatment of chronic myelogenous leukemia and Philadelphia chromosome‐positive acute lymphoblastic leukemia in patients with resistance to prior therapy, including imatinib mesylate (Gleevec^®). Radiolabeled dasatinib and its piperazine N‐dealkyl metabolite were synthesized to investigate absorption, distribution, metabolism, and elimination of the compounds in humans and animals. These compounds were prepared following a three‐step sequence, which included thiazole carboxamide formation via cyclization of labeled thiourea with a brominated oxyacrylamide precursor. In the final step a common intermediate was converted to either [¹⁴C]dasatinib or the radiolabeled piperazine N‐dealkyl metabolite with labeling in the aminothiazole ring. Syntheses of both compounds were achieved with radiochemical purities in excess of 98%. Stable‐labeled dasatinib and the piperazine N‐dealkyl metabolite were also needed for use as mass spectral internal standards in support of bioanalytical assays. By following the same route used for the carbon‐14 synthesis, [¹³C₄, ¹⁵N₂]dasatinib and the [¹³C₄, ¹⁵N₂]metabolite were prepared with labeling in both the dichloropyrimidine and thiazole ring systems. This convergent process introduced stable isotope labeling through (1, 2, 3‐¹³C₃) diethyl malonate and [¹³C,¹⁵N₂]thiourea. Copyright © 2008 John Wiley & Sons, Ltd.     

Metabolism and disposition of gemfibrozil in Wistar and multidrug resistance-associated protein 2-deficient TR− rats

1. The roles of multidrug resistance-associated protein (Mrp) 2 deficiency and Mrp3 up-regulation were evaluated on the metabolism and disposition of gemfibrozil.

2. Results from in vitro studies in microsomes showed that the hepatic intrinsic clearance (CL_int) for the oxidative metabolism of gemfibrozil was slightly higher (1.5-fold) in male TR^? rats, which are deficient in Mrp2, than in wild-type Wistar rats, whereas CL_int for glucuronidation was similar in both strains.

3. The biliary excretion of intravenously administered [¹⁴C]gemfibrozil was significantly impaired in TR^? rats compared with Wistar rats (22 versus 93% of the dose excreted as the acyl glucuronides over 72?h). Additionally, the extent of urinary excretion of radioactivity was much higher in TR^? than in Wistar rats (78 versus 2.6% of the dose).

4. There were complex time-dependent changes in the total radioactivity levels and metabolite profiles in plasma, liver and kidney, some of which appeared to be related to the up-regulation of Mrp3.

5. Overall, it was demonstrated that alterations in the expression of the transporters Mrp2 and Mrp3 significantly affected the excretion as well as the secondary metabolism and distribution of [¹⁴C]gemfibrozil.     

Pharmacokinetic–Pharmacodynamic Modeling of the Hydroxy Lerisetron Metabolite L6-OH in Rats: An Integrated Parent–Metabolite Model

Purpose The twofold aim of this study was to characterize in vivo in rats the pharmacokinetics (PK) and pharmacodynamics (PD) of L6-OH, a metabolite of lerisetron with in vitro pharmacological activity, and evaluate the extent to which L6-OH contributes to the overall effect. Methods The PK of L6-OH was determined directly postmetabolite i.v. dose (PK-1), and also simultaneously for L (lerisetron concentration) and for generated L6-OH after lerisetron dose (200 μg kg⁻¹, i.v.), using Nonlinear Mixed Effects Modeling with an integrated parent–metabolite PK model (PK-2). Surrogate effect was measured by inhibition of serotonin-induced bradycardia. Protein binding was assayed via ultrafiltration and all quantification was performed via liquid chromatography-electrospray ionization-mass spectrometry. Results L6-OH showed elevated plasma and renal clearances, and volume of distribution (PK-1). The in vivo potency (PD) of L6-OH was high (EC₅₀ = 0.098 ng mL⁻¹ and EC_50unbound = 0.040 ng mL⁻¹). Total clearance for L (PK-2) in the presence of generated L6-OH (CL_L = CL_→L6-OH + CL_n) was 0.0139 L min⁻¹. Most of this clearance was L6-OH formation (F_c = 99.6%), but only an 8.6% fraction of L6-OH was released into the bloodstream. The remainder undergoes biliar and fecal elimination. The parameters estimated from PK-2 were used to predict concentrations of L6-OH (Cp_L6) generated after a lerisetron therapeutic dose (10 μg kg⁻¹) in the rat. These concentrations are needed for the PD model and are below the quantification limit. Cp_L6max was less than the EC₅₀ of L6-OH. Conclusions We conclude that after lerisetron administration, L6-OH is extensively formed in the rat but it is quickly eliminated; therefore, besides being equipotent with the parent drug, the L6-OH metabolite does not influence the effect of lerisetron.     

Facile synthesis of stable isotope‐labeled antibacterial agent RWJ‐416457 and its metabolite

Synthesis of multiple stable isotope‐labeled antibacterial agent RWJ‐416457, (N‐{3‐[3‐fluoro‐4‐(2‐methyl‐2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide), and its major metabolite, N‐{3‐[4‐(2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐3‐fluoro‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide, is described. The stable isotope‐labeled [¹³CD₃]RWJ‐416457 was prepared readily by acetylation of the precursor amine, 5‐aminomethyl‐3‐[3‐fluoro‐4‐(2‐methyl‐2,6‐dihydro‐4H‐pyrrolo[3,4‐c]pyrazol‐5‐yl)‐phenyl]‐oxazolidin‐2‐one with CD₃¹³COCl in pyridine. Synthesis of the stable isotope‐labeled metabolite involved a construction of multiple isotope‐labeled pyrazole ring. N,N‐dimethyl(formyl‐¹³C,D)amide dimethyl acetal was first prepared by treating N,N‐dimethyl(formyl‐¹³C,D)amide with dimethyl sulfate, followed by sodium methoxide. Then, N‐{3‐[3‐fluoro‐4‐(3‐oxo‐pyrrolidin‐1‐yl)‐phenyl]‐2‐oxo‐oxazolidin‐5‐ylmethyl}‐acetamide was condensed with N,N‐dimethyl(formyl‐¹³C,D)amide dimethyl acetal, and the resultant β‐ketoenamine intermediate underwent pyrazole ring formation with hydrazine‐¹⁵N₂, to give the [¹³C¹⁵N₂D]‐labeled metabolite. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of [2H, 13C] and [14C] labeled vasopressin V1a/V2 receptor antagonist RWJ‐676070 and its stable labeled N‐des‐benzoyl metabolite

Stable isotope‐labeled ([¹³C₂, D₃]) and carbon 14‐labeled vasopressin receptor antagonist RWJ‐676070, a spirobenzazepine amide, were prepared in separate syntheses for use in drug metabolism studies. The stable isotopically labeled sample was prepared starting from [¹³C₂, D₆]dimethyl sulfate and [¹³C]copper (I) cyanide in nine steps with a 14% overall isotopic yield. The ¹⁴C label was introduced in five steps starting from [¹⁴C]potassium cyanide to provide material having a specific activity of 58 mCi/mmol (2.15 GBq/mmol). Selective hydrolysis of the metabolically labile amide bond in [¹³C₂, D₃]RWJ‐676070 gave the corresponding labeled metabolite in one step. Copyright © 2008 John Wiley & Sons, Ltd.     

Metabolite to parent drug concentration ratio as a function of parent drug extraction ratio: Cases of nonportal route of administration

The ratio of metabolite to parent drug concentration (Cm/Cp)of a medium or high extraction ratio (E>0.1)drug administered intravenously has been shown to depend on intrinsic clearance of drug by other metabolic routes (CL_r,int)as well as on organ blood flow (Q).In contrast, for a low extraction ratio drug given intravenously or for any drug given by a portal route, this ratio is equal to the ratio of formation clearance (CL_f,int)and metabolite clearance (CL_m,int).The sensitivity of Cm/Cpto changes in CL_r,int and CL_f,int has been analyzed quantitatively. It was shown to be dependent on the fraction metabolized to that particular metabolite (fm).Supported in part by National Institutes of Health National Research Service Awards GM07348 and GM-07750 from the National Institute of General Medical Sciences and NINCDS Research Grant NS-04053 and NINCDS Research Contract NO l-NS-1-2282.     

A review of metabolite kinetics

The importance of metabolites as active and toxic entities in drug therapy evokes the need for an examination of metabolite kinetics after drug administration. In the present review, emphasis is placed on single-compartmental characteristics for a drug and its primary metabolites under linear kinetic conditions. The determination of the first-order elimination rate constants for drug and metabolite are also detailed. For any ithprimary metabolite miformed solely in liver, kinetic parameters with respect to primary metabolite formation under first-order conditions require a comparison of the areas under the metabolite concentration-time curve after drug and preformed metabolite administrations. These area ratios hold regardless of the number of noneliminating compartments for the drug and metabolite. These parameters include f_mi and g_mi,the fractions of total body clearance that respectively furnishes mito the general circulation and forms mi,and h_mi,the fraction of hepatic clearance responsible for the formation of mi.Moreover, the fraction of dose d_mi converted to form miis defined with respect to the route of drug administration. The inherent assumption of these estimates, however, requires that the extent of sequential elimination of the generated mibe identical to the extent of metabolism of preformed mi.Discrepancies have been found, and may be attributed mostly to the uneven distribution of drug-metabolizing activities as well as to the presence of diffusional barriers. Other linear systems that involve miformation from multiple organs are briefly described.     

Nonlinear elimination and hepatic concentration of conjugation- metabolite of valproate in guinea-pigs

The plasma clearance and metabolic rate characteristics of valproic acid (VPA) were studied using guinea-pigs placed on various (0.08-9 μmol ml^?1 = 11–1303 μg ml^?1) steady-state plasma concentrations (C_ss) by constant intravenous (i.v.) infusion. The total clearance (CL) was significantly decreased at plasma concentration of 0.61 μmol ml^?1 (88 μg ml^?1). The metabolic clearance of VPA was apparently biphasic. The maximum metabolic rate (V_max) and the Michaelis-Menten constant (K_m) for the primary (V_maxl, K_ml) and the secondary (V_max2, K_m2) pathways were V_maxl = 1.52 μmol min ^?1kg^?1, K_ml = 0.15 μmol ml^?1, V_max2 = 24.98 μmol min ^?1 kg^?1 and K_m2 = 11.70 μmol ml^?1, respectively. The K_ml value was within clinical therapeutic concentration range. The formation of conjugated VPA (cjVPA) metabolite in liver was shown to be saturable. Plasma protein binding of VPA was also nonlinear. The dose-dependent decrease in metabolic clearance was counterbalanced by the increased unbound fraction (f_u), resulting in a relatively constant apparent clearance of VPA over a wide concentration range. The hepatic concentration of VPA was not significantly different from the plasma unbound concentration, again over a wide concentration range. The biliary and hepatic concentrations of VPA were not significantly different; but the concentration ratio of cjVPA in bile compared with that of VPA in liver decreased against hepatic concentration of VPA, which suggests a saturable conjugation rate. The K_m value estimated from hepatic cjVPA production as a function of plasma VPA concentration was comparable with the K_ml value. These results implied that the primary metabolic parameters may describe the conjugation pathway which is nonlinear within the clinical therapeutic concentration range.     

Human mass balance,metabolite profile and identification of metabolic enzymes of [14C]ASP015K,a novel oral janus kinase inhibitor

Abstract

1.?The human mass balance of ¹⁴C-labelled ASP015K ([¹⁴C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [¹⁴C]ASP015K (100?mg, 3.7?MBq) in solution. [¹⁴C]ASP015K was rapidly absorbed with t_max of 1.6 and 1.8?h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5′-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components.

2.?In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT).

3.?The in vitro intrinsic clearance (CL_{int_in?vitro}) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CL_{int_in vitro} of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms.     

The relation between type of renal disease and renal drug clearance in children

Summary It is generally assumed that the renal clearance of drugs in patients with renal impairment are affected to a similar extent regardless of the type of renal disease (intact nephron hypothesis). We have studied the effect of underlying renal disease on the pharmacokinetics of cefotaxime and desacetylcefotaxime in two groups of children (ages 7 to 16 y) with varying degrees of renal dysfunction.Patients in group 1 (n=5) had intrinsic renal disease and those in group 2 (n=5) had extrinsic renal disease, as identified by the primary renal lesion. After a single intravenous dose of cefotaxime timed blood and urine samples were collected for 24 h; cefotaxime and desacetylcefotaxime were measured by HPLC.There were no significant differences between the groups in age, body surface area, urine output, creatinine clearance, total body clearance, nonrenal clearance, renal clearance, and volume of distribution at steady state of cefotaxime, and renal clearance of desacetylcefotaxime. However, the renal clearance: creatinine clearance (CL_R:CL_CR) ratios for both cefotaxime [1.34 in group 1 vs. 0.51 in group 2] and desacetylcefotaxime [1.58 in group 1 vs. 0.75 in group 2] were statistically significant between the two groups. Group 1 patients had an average CL_R:CL_CR ratio greater than 1 for both the parent compound and the metabolite, suggesting that net tubular secretion was still intact, despite a diminished glomerular filtration rate (CL_CR=24 ml·min^–1·1.73 m^–2). In contrast, patients in group 2 (CL_CR=49) ml·min^–1·1.73 m^–2) had an average CL_R:CL_CR ratio less than 1 for both cefotaxime and desacetylcefotaxime, suggesting that renal tubular transport mechanisms did not remain functional in these patients.Our findings suggest that the effect of renal insufficiency on the renal elimination of cefotaxime and its metabolite desacetylcefotaxime may depend on the cause of renal insufficiency.     

Pharmacokinetics of temocapril and temocaprilat after 14 once daily oral doses of temocapril in hypertensive patients with varying degrees of renal impairment

Aims The aim of this study was to determine the potential influence of renal impairment on the pharmacokinetics of temocapril and its pharmacologically active diacid metabolite, temocaprilat. Methods Non-compartmental pharmacokinetics were assessed in four groups of hypertensive patients (n=8 per group, four investigational centres) with normal (creatinine clearance determined via 24 h urine sampling, CL_CR, ≥60 ml min⁻¹ ) and impaired renal function (CL_CR 40–59, 20–39, <20 ml min⁻¹ ) after 14 once daily oral doses of 10 mg temocapril hydrochloride. Results For temocapril, there were no statistically significant differences in median t_max or mean C_max, AUC_SS, t_½,Z, CL/F between the four groups. Renal clearance, CL_R, for temocapril showed a linear decreasing trend with decreasing CL_CR [ mean (s.d.): 32.2 (10.7) to 3.7 (3.0) ml min⁻¹]. Steady-state for temocaprilat was reached on day 5. For temocaprilat, no statistically significant differences in mean C_max or median t_max were detected. With decreasing mean CL_CR, mean AUC_SS for temocaprilat increased statistically significantly although only 2.4-fold [mean (s.d.): 2115 (565) to 4989 (2338) ng ml⁻¹ h] and t_½,Z was prolonged [mean (s.d.): 15.2 (1.2) to 20.0 (7.5) h]. CL_R for temocaprilat showed a linear decreasing trend with decreasing CL_CR [mean (s.d.): 20.2 (4.3) to 3.0 (1.8) ml min⁻¹]. Conclusions These results indicate that impaired renal function has only a limited effect on the pharmacokinetics of temocapril and its active metabolite, temocaprilat. This may be attributed to the dual, i.e. renal and biliary, elimination pathway of the drug.     

Synthesis of a highly potent leukocyte function‐associated antigen‐1 antagonist and its metabolite labeled with stable isotopes and carbon‐14, part 2

(S)‐2‐[(R)‐7‐(3,5‐Dichlorophenyl)‐5‐methyl‐6‐oxo‐5‐(4‐trifluoromethoxybenzyl)‐6,7‐dihydro‐5H‐imidazo[1,2‐a]imidazole‐3‐sulfonylamino]‐proprionamide (1), a potent lymphocyte function‐associated antigen‐1 antagonist and its sulfonamide metabolite (2) labeled with stable isotopes and carbon‐14 were prepared for Drug Metabolism and PharmacoKinetics and other studies. A long linear route was used to prepare [¹³C₂, ²H₃]‐(1) using [3,3,3‐²H]‐D‐alanine and [¹³C₂]‐glycine in 15 steps and 2.5% overall yield. With the availability of [¹³C₆]‐3,5‐dichloroaniline, the sulfonamide [¹³C₆]‐(2) was prepared in 12 steps and in 5.6% overall yield. For the carbon‐14 synthesis, a six‐step synthesis gave both compounds [¹⁴C]‐(1) and [¹⁴C]‐(2) from the common sulfonyl chloride intermediate [¹⁴C]‐(15) in 18% and 4% radiochemical yields and specific activities of 44 and 40.5 mCi/mmol, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Syntheses of (R,S)‐naproxen and its 6‐O‐desmethylated metabolite labelled with 2H

Naproxen, a well‐known non‐steroidal anti‐inflammatory drug, and its 6‐O‐desmethylated metabolite have been labelled with ²H. (R,S)‐Naproxen 7 labelled with ²H was obtained in five steps using the commercially available [²H₃]iodomethane 5 as the stable labelled reagent. The demethylation of 7 using 48% HBr in 1‐butyl‐3‐methylimidazolium tetrafluoborate gave the corresponding ²H‐labelled 6‐O‐desmethylated metabolite 8. Copyright © 2008 John Wiley & Sons, Ltd.