Craig K. Svensson

Therapeutic drug monitoring in saliva. An update.

SummaryThis article re-examines the issue of salivary therapeutic drug monitoring (STDM). The anatomy and physiology of saliva and the salivary glands, as well as the effects of disease and drugs on salivary secretion and composition, are discussed briefly. Drugs for which therapeutic drug monitoring (TDM) has been shown useful are individually considered to determine if salivary drug concentrations (Csal) are reflective of plasma free drug concentrations (Cup). That is, is the Csal/Cup ratio time- and concentration-independent, as supported by a review of literature data? The primary determinant which appears to govern the potential utility of STDM for many of the drugs is the pKa of the drug. Drugs which are not ionisable or are un-ionised within the salivary pH range (phenytoin, carbamazepine, theophylline) are candidates for STDM based on current literature data. Digoxin and cyclosporin are potential candidates for STDM; however, further studies are necessary to confirm these preliminary findings. On the basis of current literature data, STDM does not appear to be useful for other drugs therapeutically monitored in serum/plasma.

Clinical Pharmacokinetics of Nicotine

SummaryNicotine intake is considered to be a major factor in sustaining tobacco addiction. For this reason, nicotine gum has recently been introduced as an adjuvant to smoking cessation. The introduction of nicotine as a ‘therapeutic’ entity necessitates a careful examination of its clinical pharmacokinetics.Insufficient data exist to quantitatively assess the absorption of nicotine after oral administration. Based upon physicochemical and pharmacokinetic principles, the oral bioavailability of nicotine would be expected to be less than 20%. The limited data available in the literature appear to support this conclusion. Absorption from the oral mucosa is the principal site of nicotine absorption in subjects who chew tobacco or nicotine gum. Absorption by this route is highly pH dependent. Nicotine is also readily absorbed from the nasal mucosa, and after topical administration.Nicotine distributes extensively into body tissues with a volume of distribution ranging from 1.0 to 3.0 L/kg. Nicotine has been shown to transfer across the placenta and into breast milk in humans. Plasma protein binding is negligible, ranging from 4.9 to 20%.The predominant route of nicotine elimination is hepatic metabolism. Although a number of metabolites of nicotine have been identified, it is unclear whether any of these compounds contribute to the pharmacological effect of nicotine. Nicotine is also excreted unchanged in urine in a pH-dependent fashion. With urinary pH less than 5, an average 23% of the nicotine dose is excreted unchanged. When urinary pH is maintained above 7.0, unchanged nicotine urinary excretion drops to 2%.After intravenous administration, nicotine exhibits biexponential decline in plasma. Total plasma clearance ranges from 0.92 to 2.43 L/min. During urinary acidification, renal clearance averages 0.20 L/min. Non-renal blood clearance averages 1.2 L/min, indicating that nicotine elimination is dependent on hepatic blood flow.The literature is devoid of information regarding the effect of disease on the pharmacokinetics of nicotine. Based upon the drug’s pharmacokinetics in healthy smokers, it would be anticipated that disease states which alter hepatic blood flow may have the greatest impact on nicotine pharmacokinetics. In addition, drugs which alter hepatic blood flow may cause significant alterations in the systemic clearance of nicotine.Dependence on smoking appears to be related, at least in part, to the achievement of a rapid rise in plasma nicotine concentrations. If this assessment is correct, the most desirable adjuvant for smoking cessation would be one that closely mimics this pattern of plasma nicotine concentrations. Thus, the slow rise in plasma concentrations after chewing nicotine gum may suggest a pharmacokinetic explanation for the relatively high failure rate with this method of smoking cessation. It appears that because the rate of nicotine absorption is even slower than with the gum formulation, transdermal preparations are unlikely to be a satisfactory alternative to smoking. Further investigations are, therefore,equired to determine a formulation which gives the desired plasma nicotine concentration profile.One of the major effects of smoking on drug therapy is the induction of drug-metabolising enzymes. However, the effects on drug metabolising capacity when a subject changes from smoking to nicotine gum has not yet been studied. The effect nicotine itself has on drug metabolism in humans is also unknown.Considerable work remains to define adequately the clinical pharmacokinetics of nicotine. Determination of factors which influence the efficacy of nicotine as an adjuvant in smoking cessation may prove beneficial in reducing the number of tobacco consumers worldwide.

Biotransformation of Drugs in Human Skin

Although it is the largest organ of the human body, skin is often not considered in discussions of drug metabolism. However, there is growing evidence that most common drug-metabolizing enzymes are expressed in the skin. Evidence for expression of cytochromes P450, flavin monooxygenases, glutathione-S-transferases, N-acetyltransferases, and sulfotransferases in human skin and skin cells are presented. Additional discussion is focused on the evidence of actual metabolism of drugs. Finally, the potential clinical implications of metabolism within the skin are discussed briefly.

Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: II. Expression and Role of Flavin-Containing Monooxygenases and Peroxidases

Arylamine compounds, such as sulfamethoxazole (SMX) and dapsone (DDS), are metabolized in epidermal keratinocytes to arylhydroxylamine metabolites that auto-oxidize to arylnitroso derivatives, which in turn bind to cellular proteins and can act as antigens/immunogens. Previous studies have demonstrated that neither cytochromes P450 nor cyclooxygenases mediate this bioactivation in normal human epidermal keratinocytes (NHEKs). In this investigation, we demonstrated that methimazole (MMZ), a prototypical substrate of the flavin-containing monooxygenases (FMOs), attenuated the protein haptenation observed in NHEKs exposed to SMX or DDS. In addition, recombinant FMO1 and FMO3 were able to bioactivate both SMX and DDS, resulting in covalent adduct formation. Western blot analysis confirmed the presence of FMO3 in NHEKs, whereas FMO1 was not detectable. In addition to MMZ, 4-aminobenzoic acid hydrazide (ABH) also attenuated SMX- and DDS-dependent protein haptenation in NHEKs. ABH did not alter the bioactivation of these drugs by recombinant FMO3, suggesting its inhibitory effect in NHEKs was due to its known ability to inhibit peroxidases. Studies confirmed the presence of peroxidase activity in NHEKs; however, immunoblot analysis and reverse transcription-polymerase chain reaction indicated that myeloperoxidase, lactoperoxidase, and thyroid peroxidase were absent. Thus, our results suggest an important role for FMO3 and yet-to-be identified peroxidases in the bioactivation of sulfonamides in NHEKs.

Acetylator phenotype and genotype in patients infected with HIV : discordance between methods for phenotype determination and genotype

The acetylator phenotype and genotype of AIDS patients, with and without an acute illness, was compared with that of healthy control subjects (30 per group). Two probe drugs, caffeine and dapsone, were used to determine the phenotype in the acutely ill cohort. Polymerase chain reaction amplification and restriction fragment length polymorphism analysis served to distinguish between the 26 known NAT2 alleles and the 21 most common NAT1 alleles. The distribution (%) of slow:rapid acetylator phenotype seen among acutely ill AIDS patients differed with the probe substrate used: 70:30 with caffeine versus 53:47 with dapsone. Phenotype assignment differed considerably between the two methods and there were numerous discrepancies between phenotype and genotype. The NAT2 genotype distribution was 45:55 slow:rapid. Control subjects, phenotyped only with caffeine, were 67:33 slow:rapid versus 60:40 genotypically. Stable AIDS patients, phenotyped only with dapsone, were 55:45 slow:rapid versus 46:54 genotypically. Following resolution of their acute infections, 12 of the acutely ill subjects were rephenotyped with dapsone. Phenotype assignment remained unchanged in all cases. The distribution of NAT1 alleles was similar in all three groups. It is evident from the amount of discordance between caffeine phenotype and dapsone phenotype or genotype that caution should be exercised in the use of caffeine as a probe for NAT2 in acutely ill patients. It is also clear that meaningful study of the acetylation polymorphism requires both phenotypic and genotypic data.

Acetylator Phenotype and Genotype in HIV‐Infected Patients with and without Sulfonamide Hypersensitivity

Adverse reactions to sulfonamides occur at a higher frequency in patients infected with the human immunodeficiency virus (HIV) than noninfected patients. Some studies have suggested that patients with the slow acetylator phenotype are predisposed to these reactions, whereas other studies suggest that the slow acetylator genotype is not a predisposing factor. To rationalize these seemingly contradictory observations, the authors determined the N‐acetyltransferase 2(NAT2) genotype and phenotype in patients with and without a history of hypersensitivity reactions to sulfonamides. HIV‐infected patients with a history of a delayed‐type hypersensitivity reaction to trimethoprim‐sulfamethoxazole were enrolled, along with a group of AIDS patients with no history of hypersensitivity (delayed or immediate). NAT2 phenotype was determined in both groups using dapsone, while the genotype was determined using a polymerase chain reaction‐restriction fragment length polymorphism assay. Ten of 14 patients (71%) with a history of hypersensitivity exhibited the slow acetylator phenotype, while 8 of 14 patients (57%) without such a history exhibited this same phenotype (odds ratio [OR] = 1.9, 95% confidence interval [CI] = 0.4–9.0; p = 0.69, Fishers Exact Test). While 9 of 14 patients (64%) with a history of hypersensitivity exhibited a slow acetylator genotype, only 4 of 14 patients (29%) without such a history exhibited this genotype (ns). There were more instances of discordance between deduced and actual phenotype in the nonhypersensitive patients (n = 4) than in the hypersensitive patients (n = 1). The reported higher frequency of the slow acetylator phenotype among patients with a history of hypersensitivity to sulfonamides does not appear to be explained by metabolic changes that would cause discordance between acetylator genotype and phenotype.

Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: I. Expression and Role of Cytochromes P450

Cutaneous drug reactions (CDRs) are among the most common adverse drug reactions and are responsible for numerous minor to life-threatening complications. Several arylamine drugs, such as sulfamethoxazole (SMX) and dapsone (DDS), undergo bioactivation, resulting in adduction to cellular proteins. These adducted proteins may initiate the immune response that ultimately results in a CDR. Recent studies have demonstrated that normal human epidermal keratinocytes (NHEKs) can bioactivate these drugs, resulting in protein haptenation. We sought to identify the enzyme(s) responsible for this bioactivation in NHEKs. Using immunofluorescence confocal microscopy and an adduct-specific enzyme-linked immunosorbent assay (ELISA), we found that N-acetylation of the primary amine of SMX and DDS markedly reduced the level of protein haptenation in NHEKs. Detection of mRNA and/or protein confirmed the presence of CYP3A4, CYP3A5, and CYP2E1 in NHEKs. In contrast, although a faint band suggestive of CYP2C9 protein was detected in one NHEK sample, a CYP2C9 message was not detectable. We also examined the ability of chemical inhibitors of cytochromes P450 (aminobenzotriazole and 1-dichloroethylene) and cyclooxygenase (indomethacin) to reduce protein haptenation when NHEKs were incubated with SMX or DDS by either confocal microscopy or ELISA. These inhibitors did not significantly attenuate protein adduction with either SMX or DDS, indicating that cytochromes P450 and cyclooxygenase do not play important roles in the bioactivation of these xenobiotics in NHEKs and thus suggesting the importance of other enzymes in these cells.

Longitudinal distribution of arylamine N-acetyltransferases in the intestine of the hamster, mouse, and rat. Evidence for multiplicity of N-acetyltransferases in the intestine.

Experimental and clinical evidence indicates that AcCoA:arylamine N-acetyltransferases (NATs; EC 2.3.1.5) are involved in the bioactivation and inactivation of a wide variety of arylamine, hydrazine, and carcinogenic arylamine xenobiotics. Longitudinal distribution of NATs in the intestine of the hamster, mouse, and two strains of rat was examined utilizing the model arylamine substrates procainamide(PA) and p-aminobenzoic acid (PABA) for the monomorphic (NAT1) and polymorphic (NAT2) enzymes in the rodent. NAT1 and NAT2 were distributed quite differently in each species examined. In particular, rat intestinal NATs were distributed equally throughout the intestinal tract. In contrast, hamster intestinal NATs decreased in activity from the proximal small intestine to the distal large intestine. Mouse NAT2 activity was highest in the cecum, whereas NAT1 was highest in the proximal small intestine. Although these model substrates have been shown to be selective for NATs, they are not specific. Therefore, a series of biochemical studies were undertaken to evaluate NAT multiplicity in the intestine of the F-344 rat. To assess multiplicity of NAT expression, selective inhibition, differential sensitivity to heat inactivation, and kinetic analysis were performed on intestinal cytosol. Eadie-Hofstee transformation of PA N-acetylation yielded a curvilinear plot indicative that a low affinity-high capacity enzyme aside from NAT1 (presumably NAT2) was contributing to PA N-acetylation activity. PA activity was found to exhibit approximately 4- to 5-fold greater thermostability than PABA activity. Furthermore, PA acetylation could be inhibited selectively with vinyl fluorenyl ketone (2.5 to 5 microM) but not with methotrexate (up to 2 mM). Taken together, these studies suggest the expression of both NAT1 and NAT2 in the intestine of the F-344 rat.

Enhanced Xenobiotic-Induced Hepatotoxicity and Kupffer Cell Activation by Restraint-Induced Stress

We tested the hypothesis that environmental stress is a predisposing factor for liver injury by examining the effect of acute restraint on liver injury provoked by carbon tetrachloride (CCl4) and allyl alcohol. Mice were immobilized using Plexiglas restraint cages, producing a form of psychogenic stress, whereas other animals were allowed to roam free. Serum alanine aminotransferase levels were elevated significantly in restrained animals after administration of varying doses of CCl4 or allyl alcohol that did not produce liver injury in unrestrained animals. This enhanced liver injury after CCl4 was seen in both male and female mice. The duration of acute restraint was found to be important because a period of 2.5 h of restraint enhanced hepatotoxicity, whereas shorter periods of restraint did not significantly increase liver injury. Serum corticosterone concentrations increased, whereas hepatic glutathione content decreased during and after acute restraint. In addition, delay in administration of CCl4 until 5 h after completion of restraint also produced an elevated level of liver injury compared with that seen in free roaming animals. Immunohistochemical examination of the livers showed significantly enhanced Kupffer cell activation in restrained mice compared with that of free roaming mice. These observations suggest that induction of psychogenic stress may increase the susceptibility to liver injury observed with classic hepatotoxicants and may represent an important predisposing factor to liver injury after xenobiotic exposure. The underlying mechanism seems to be increased macrophage activation in the liver, which may subsequently sensitize hepatocytes to xenobiotics and thus enhance hepatotoxicity.

Arylacetamide deacetylase activity towards monoacetyldapsone: Species comparison, factors that influence activity, and comparison with 2-acetylaminofluorene and p-nitrophenyl acetate hydrolysis

The deacetylation of monoacetyldapsone (MADDS) was examined in liver microsomes and cytosol from male Sprague-Dawley rats, Golden Syrian hamsters, and Swiss Albino mice. All three rodent species demonstrated greater MADDS deacetylation activity in liver microsomes than in liver cytosol. Further investigations were conducted in hamsters. The velocity of MADDS deacetylation in major organs in the hamster was greatest in the intestine, followed by the liver and kidney. The effect of pretreatment with common inducers on liver microsomal deacetylation activity was also examined in the hamster. Phenobarbital, 100 mg/kg/day x 3 days, did not alter activity, while dexamethasone at the same dose reduced 2-acetylaminofluorene (2-AFF), MADDS, and p-nitrophenyl acetate (NPA) hydrolysis by at least 50%. Due to a previous report that KI activated the deacetylation of an arylacetamide in vitro (Khanna et al., J Pharmacol Exp Ther 262: 1225-1231, 1992), the effects of the halides KF, KCl, KBr and KI on MADDS hydrolysis in vitro were tested. Of the halides studied, only KF altered MADDS hydrolysis, resulting in an almost complete inhibition of deacetylase activity at 50 mM (with the initial concentration of MADDS at 0.6 mM) with an IC50 = 0.16 mM. Cornish-Bowden and Dixon plots indicated that the inhibition exerted by KF was non-competitive. The rank order of inhibitor potencies was constructed using phenylmethylsulfonyl fluoride (PMSF), bis(p-nitrophenyl)phosphate (BNPP), physostigmine, and KF with 2-AFF, MADDS, and NPA as substrates. Different rank order potencies were obtained for each of the substrates tested. The substrates 2-AFF, MADDS, and NPA did not act as competitive inhibitors on the hydrolysis rates of each other. Liver microsomal arylacetamide deacetylase activity was greater in male hamsters than in females with either MADDS or 2-AAF as substrates; however, hydrolysis of NPA was similar in both male and female hamsters. These data support the hypothesis that the enzyme which catalyzes the hydrolysis of MADDS differs from that catalyzing either 2-AAF or NPA hydrolysis.