Pharmacokinetics and pharmacodynamics of stereoisomeric drugs with particular reference to bioequivalence determination.

Drugs containing one or more chiral centres exist in stereoisomeric molecular forms. Most commonly, drugs containing a single asymmetric carbon atom exist in two enantiomeric forms, designated as eutomer (the more potent) and distomer (the less potent). As well as differences in potency and other pharmacodynamic properties, most members of enantiomeric pairs commonly differ also in their pharmacokinetic profiles. This article reviews factors underlying differences in pharmacological properties of enantiomers. The relevance of such differences for studies designed to evaluate the bioequivalence of products containing chiral drugs is also reviewed.

Residues of veterinary drugs at injection sites.

Residues of veterinary drugs have potential implications for human food safety and international trade in animal-derived food commodities. A particular concern is the slow depletion of residues of some injectable formulations from the site of administration. Licensing authorities have adopted different approaches to the human food safety assessment of injection site residues. European agencies apply the maximum residue limit (MRL) for muscle to muscle at the injection site and specify a withdrawal period sufficient to ensure the ingestion of a 300 g portion of muscle, if comprised entirely of injection site tissue, does not exceed the acceptable daily intake. The agencies in Australia, Canada and the USA also exclude injection site residues from the MRL-setting process. These agencies evaluate the risk to consumers posed by potential acute manifestations resulting from the infrequent ingestion of injection site residues based on acute dietary exposure considerations. While all of these approaches protect the safety of consumers, the adoption of different approaches has potential implications for residue surveillance programs in the international trade in meat. In particular, when an exporting country establishes standards for residues at injection sites based on acute dietary exposure considerations and the importing country assesses these residues against the MRL for muscle, the unnecessary condemnation of meat and disruption to market access may result. The latter may represent a potential economical impost to the exporting country. An internationally harmonized approach to the risk analysis of residues of veterinary drugs at injection sites, which protects the safety of consumers and facilitates the international trade in meat, is needed.

N-and O-acetylation of aromatic and heterocyclic amine carcinogens by human monomorphic and polymorphic acetyltransferases expressed in COS-1 cells.

Human monomorphic and polymorphic arylamine acetyltransferases (EC 2.3.1.5) were expressed in monkey kidney COS-1 cells and used to study the N- and O-acetylation of a number of carcinogenic amines and their N-hydroxy metabolites. The monomorphic enzyme N-acetylated the aromatic amines, 2-aminofluorene and 4-aminobiphenyl, and also O-acetylated their N-hydroxy derivatives. None of the food-derived heterocyclic amines (Glu-P-1, PhIP, IQ, MeIQx) were substrates and their N-hydroxy metabolites were poorly O-acetylated by this isozyme. By contrast, the polymorphic acetyltransferase catalyzed the N-acetylation of both aromatic amines, and to a lesser extent, Glu-P-1 and PhIP. However, all six N-hydroxy amine substrates were readily O-acetylated to form DNA-bound adducts by the polymorphic isozyme. These data suggest that, for the heterocyclic amine carcinogens, rapid acetylator individuals will be predisposed to their genotoxicity.

Direct O-acetylation of N-hydroxy arylamines by acetylsalicylic acid to form carcinogen-DNA adducts.

Acetylsalicylic acid (aspirin) has been shown to acetylate a number of drugs and biological macromolecules. Since enzymatic O-acetylation of N-hydroxy arylamines is regarded as an important activation step for DNA adduct formation, we initially examined the ability of aspirin to serve as an acetyl donor for this reaction, using rabbit liver cytosol. Instead, a direct non-enzymatic reaction was observed. Arylamine-DNA binding was enhanced from 3- to 25-fold at pH 7 by addition of aspirin to reactions containing the N-hydroxy derivatives of 2-aminofluorene (AF), 4-aminobiphenyl, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, but not for 2-amino-3-methylimidazo[4,5-f]quinoline or 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. Further studies with N-hydroxy-AF showed that reaction rates were first order with respect to both aspirin (0.1-10 mM) and N-hydroxy-AF (0.01-0.1 mM) concentrations. In contrast, aspirin had no effect on reactions conducted at pH 5 where N-hydroxy-AF is known to undergo protonation and react with DNA to form high levels of N-(deoxyguanosin-8-yl)-AF. N-Acetylation of AF by aspirin under these conditions was also negligible. However, the formation of the adduct from N-hydroxy-AF occurred at high yield (64-82%) at pH 7 with either DNA or 2'-deoxyguanosine. HPLC analyses showed only an aspirin-dependent loss of N-hydroxy-AF and concomitant adduct formation, with no detectable formation of solvolysis products. This indicated that the reaction proceeds to a significant extent only upon addition of the nucleophile, and suggests the formation of an O-tetrahedral intermediate that is in equilibrium with both the N-hydroxy derivative and the reactive N-acetoxy arylamine. Thus, the apparent O-acetylation of certain N-hydroxy arylamines selectively by aspirin offers a convenient route for the synthesis of arylamine-DNA adducts. The potential biological significance of this reaction in vivo is also discussed.

Distribution of acetyltransferase activities in the intestines of rapid and slow acetylator rabbits.

Epidemiological studies have shown that there is a significantly greater proportion of the rapid acetylator phenotype in patients with colorectal tumors than in controls; phenotype-related differences in bioactivation of dietary or environmental amines in the intestinal epithelium have been suggested as a mechanism for this effect. In the present study, we have used hepatic and intestinal cytosols to compare N-acetyltransferase (NAT1 and NAT2), O-acetyltransferase (OAT) and arylhydroxamic acid N,O-acyltransferase (AHAT) distribution in rapid and slow acetylator rabbits. The ratio (rapid/slow) for p-aminobenzoic acid acetylation (a selective substrate for NAT1) was 6 in liver, 1.7-2 in small intestine and 1.3-1.5 in large intestine while the ratio of sulfamethazine acetylation (a selective substrate for NAT2) was 150 in liver, 16-22 in small intestine and 1.8-2.5 in large intestine. The ratios (rapid/slow) for DNA binding of N-hydroxy-3,2'-dimethyl-4-aminobiphenyl and N-hydroxy-4-aminobiphenyl (primarily substrates for OAT) were 82-84 in liver, 13-20 in small intestine and 3.8-5.3 in large intestine and for DNA binding of N-hydroxy-2-acetylamidofluorene (a substrate for AHAT), the ratio was 432 in liver, 32-161 in small intestine and 8.8-13.5 in large intestine. The data show also that NAT1 activity is uniformly distributed along the intestinal tract whereas NAT2 activity is highest in the small intestine. In addition, hepatic and intestinal OAT and AHAT but not NAT1 activities in the rabbit intestine are similarly distributed to activities for NAT2, suggesting that NAT2, OAT and AHAT activities are properties of a single protein in the rapid acetylator phenotype. Moreover, OAT and AHAT activities were much higher in tissues from the rapid than the slow phenotype. The data support the hypothesis that phenotype-dependent metabolic activation of N-OH heterocyclic or aromatic amines to reactive acetoxy metabolites may be involved in the etiology of colorectal cancer.

Immunological evidence for N-acetyltransferase isozymes in the rabbit.

An immunological evaluation of N-acetyltransferase (NAT) (EC 2.3.1.5) in liver, duodenum, lung, and kidney of the rabbit is described. Polyclonal antibodies to hepatic NAT isolated from rapid acetylator rabbits were raised in a goat and utilized for immunoblot analyses and enzyme inhibition studies. Immunoblot analyses demonstrated that hepatic and duodenal cytosols from rapid but not slow acetylator rabbits contained an immunoreactive 33-kDa protein. No immunoreactivity was observed for lung or kidney cytosols from either rapid or slow acetylators. The inhibition of sulfamethazine and p-aminobenzoic acid acetylation by polyclonal antibodies was investigated using cytosols from rapid and slow acetylator rabbits. With rapid acetylator cytosols, maximal inhibition of hepatic, duodenal, and lung NAT activities was 94.4 +/- 9.0%, 92.5 +/- 8.5%, and 28.3 +/- 2.4%, respectively, for sulfamethazine (500 mM) acetylation and 90.1 +/- 8.0%, 80.2 +/- 6.4%, and 26.7 +/- 3.1%, respectively, for p-aminobenzoic acid (500 microM) acetylation. Using 25 microM p-aminobenzoic acid as substrate, maximal inhibition of NAT activity was 32.0 +/- 2.1% with liver cytosol and 5.8 +/- 0.16% with duodenal cytosol, whereas no inhibition of lung NAT activity was observed. Kidney NAT activity was not inhibited by the polyclonal antibodies. With slow acetylator cytosols, no inhibition of NAT activities was observed. It is concluded that at least two NATs are present in liver, duodenum, and lung of rapid acetylator rabbits. Furthermore, the principal NAT in liver and duodenum is immunologically related to the minor form of lung NAT and is antigenically distinct from kidney NAT of rapid acetylators. Hepatic, duodenal, lung, and kidney NAT(s) of slow acetylator rabbits is (are) immunologically distinct from the major hepatic NAT in rapid acetylators. The data support the model in which the hepatic polymorphism in rabbits is caused by the total lack of the major rapid acetylator hepatic NAT in the phenotypic slow acetylator animal. These observations may have significant implications in the organ-specific toxicities of carcinogens that undergo metabolic activation via N-acetylation.

Metabolism of drugs and other xenobiotics in the gut lumen and wall.

Metabolism in the gut lumen and wall can decrease the bioavailability and the pharmacological effects of a wide variety of drugs. Bacterial flora in the gut, the environmental pH and oxidative or conjugative enzymes present in the intestinal epithelial cells can all contribute to the process. Bacterial biotransformation is greatest in the colon, while gut wall metabolism is generally highest in the jejunum and decreases distally. Gut wall metabolism may be induced or inhibited by dietary or environmental xenobiotics or by co-administered drugs. Recent evidence suggests that some drugs, food-derived mutagens and other xenobiotics can be metabolized by gut flora and/or gut wall enzymes to reactive species which may cause tumors.

In vivo mechanisms for the enhanced acetylation of sulfamethazine in the rabbit after hydrocortisone treatment.

The administration of hydrocortisone (HC; 75 mg/kg i.v. daily for 10 days) to rabbits significantly increased the metabolic clearance (Clm) of sulfamethazine (SMZ) from 1.92 +/- 0.05 to 2.85 +/- 0.06 liters/hr/kg (P less than 0.01). However, p.o. availability of SMZ was similar before (0.42 +/- 0.06) and after (0.39 +/- 0.03) steroid treatment. Furthermore, total liver blood flow as measured by the radioactive microsphere technique was increased (P less than 0.01) from 3.64 +/- 0.15 to 5.12 +/- 0.54 liters/hr/kg by HC treatment. Estimates of presystemic intestinal extraction and hepatic extraction were derived. These data predicted that intestinal extraction ranged from 0.11 to 0.15 in untreated rabbits and from 0.11 to 0.16 in HC-treated rabbits. Moreover, intestinal clearance was shown to contribute less than 4% to Clm of SMZ on both study days. By comparison, estimates of hepatic extraction ranged from 0.51 to 0.53, and from 0.54 to 0.56 in untreated and HC-treated rabbits, respectively. Based on the concept of a well-stirred, perfusion-limited model of hepatic elimination, the HC-induced increases in hepatic enzyme activity and blood flow were shown to make comparable contributions to the increase in hepatic clearance. It is concluded that the liver is the major site of SMZ acetylation in untreated and HC-treated, rapid acetylator rabbits. Furthermore, the increase in Clm of SMZ observed in vivo after HC treatment is attributable primarily to increases in hepatic N-acetyltransferase activity and liver blood flow.

Measurement of organ blood flow in the rabbit.

A surgical procedure to facilitate the radioactive microsphere technique for simultaneously measuring cardiac output and its regional distribution has been developed. The procedure allows the use of either anesthetized or conscious rabbits. Organ blood flows in untreated and saline-treated conscious rabbits and saline-treated anesthetized rabbits were quantified and compared. First, comparison of data from the untreated and saline-treated conscious rabbits demonstrated a significantly lower hepatic blood flow in the untreated animals. Moreover, liver blood flow in the untreated animals was markedly less than literature values. Since the untreated rabbits were not handled on a regular basis prior to the blood flow study, it is suggested that anxiety may have caused the redistribution of splanchnic circulation. Secondly, comparison of the data from the conscious and anesthetized saline-treated rabbits demonstrated that total liver blood flow was similar and in agreement with literature values. Hence, either the conscious or anesthetized animal can be used to estimate liver blood flow. By contrast, blood flow to the kidney and caecum was significantly altered by anesthesia (propanidid: nitrous oxide: halothane), indicating that blood flow to these organs is best studied in the placid conscious rabbit.

Effect of intra-articular glucocorticoids on the disposition of sulphadimidine in chronic osteoarthritis patients.

1. The disposition of sulphadimidine (15 mg kg-1 orally) was investigated in six chronic osteoarthritis patients (four slow and two fast acetylators) prior to and 4 days following intra-articular administration of glucocorticoids. 2. The mean (+/- s.e. mean) renal clearance of sulphadimidine was increased from 0.03 +/- 0.01 to 0.07 +/- 0.02 ml min-1 kg-1 (P = 0.01) following the administration of intra-articular steroid. 3. Mean metabolic clearance and volume of distribution data were similar on the two study days. However, two of the slow acetylators showed marked increases (63% and 193%) in metabolic clearance following steroid treatment.

Induction of sulfamethazine acetylation by hydrocortisone in the rabbit.

Daily intravenous administration of hydrocortisone (HC) to rabbits resulted in a marked time-dependent increase in the metabolic clearance of sulfamethazine (SMZ). Kinetic analysis of the plasma concentration-time profiles for SMZ indicated that HC treatment significantly increased the rate of acetylation of SMZ without altering the renal clearances or volumes of distribution for either parent drug or the N-acetyl metabolite N-acetylsulfamethazine (NASMZ). Total body clearance of NASMZ was also unaltered. Doses of HC ranging from 25 to 150 mg/kg were equally effective in enhancing the in vivo acetylation rate of SMZ. Moreover, the induction of SMZ acetylation was reversible when treatment with the steroid was terminated. The in vitro rate of SMZ acetylation was measured in the cytosol of various organs from controls and rabbits treated with HC for 10 days. HC did not alter the Michaelis-Menten parameters for N-acetyltransferase (NAT) activity in kidney, lung, or gut. Similarly, the Km for SMZ acetylation in liver cytosol was not affected by the steroid. However, the Vmax estimates of hepatic NAT activity were significantly increased after HC treatment. At 0.5 mM SMZ and variable [acetyl-CoA], Vmax increased 3-fold from 82 +/- 20 to 244 +/- 43 mumol/min/organ, while at 0.5 mM acetyl-CoA and variable [SMZ], Vmax increased 2.8-fold from 75 +/- 19 to 208 +/- 41 mumol/min/organ. This increase was primarily due to a 69% increase in liver weight since Vmax expressed per mg of cytosolic protein was similar in both groups. The endogenous acetyl CoA concentrations were significantly increased by HC in liver and lung, but not in gut and kidney.(ABSTRACT TRUNCATED AT 250 WORDS)