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Metabolism by N-Acetyltransferase 1 In Vitro and in Healthy Volunteers: A Prototype for Targeted Inhibition

From the Division of Clinical Pharmacology and Medical Toxicology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, and the Laboratory of Clinical Pharmacology, Food and Drug Administration, Rockville, Maryland.

Address for reprints: Louis R. Cantilena, Jr, MD, PhD, Division of Clinical Pharmacology and Medical Toxicology, Building 53, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Inhibition of drug metabolism is generally avoided but can be useful in limited circumstances, such as reducing the formation of toxic metabolites. Acetylation is a major pathway for drug elimination that can also convert substrates into toxic species, including carcinogens. Sulfamethoxazole, a widely used antibiotic, is metabolized via arylamine N-acetyltransferase 1. p-Aminosalicylate, used for antitubercular treatment, is also metabolized by N-acetyltransferase 1 and could potentially inhibit sulfamethoxazole metabolism. Human hepatocytes from 4 donors were incubated in vitro with sulfamethoxazole and paminosalicylate at clinically achievable concentrations. p-Aminosalicylate competitively reduced the acetylation of sulfamethoxazole in vitro by 61% to 83% at 200 µM. Four healthy volunteers were studied following doses of 500 mg sulfamethoxazole either alone or during administration of paminosalicylate (4 g ter in die). Plasma concentrations of paminosalicylate exceeded 100 µM. With each subject as his or her own control, p-aminosalicylate reduced by 5-fold the ratio of plasma concentrations of acetylsulfamethoxazole relative to parent drug (P < .001). Metabolic drug-drug interaction studies in vitro successfully predicted inhibition of acetylation via N-acetyltransferase 1 in vivo. Although no specific toxic species was investigated in this work, the potential was demonstrated for improving the therapeutic index of drugs that have toxic metabolites.

Key Words: NAT1 • p-aminosalicylate • drug metabolism • drug interactions • sulfamethoxazole

Unlike the wide variety of isoforms for cytochromes P450 and UDP-glucuronyl transferases, there are only 2 forms of human NAT—namely, NAT1 and NAT2. Although NAT1 and NAT2 have overlapping specificities, they also have unique and distinct activities.¹ Expression varies substantially among individuals for both NAT1 and NAT2. NAT2 is strongly polymorphic; indeed, it was the first enzyme for which polymorphism in human drug metabolism was appreciated, with several tests for identifying "fast" and "slow" acetylators.² More recently, the polymorphism of NAT1 has also been established.³

Acetylation of substrates by NAT is a double-edged sword. On one side, conjugation via NAT plays an important role in drug detoxification and elimination from the body. On the other side, it can convert substrates into more toxic species, including carcinogens.^4,5 Clearance of parent compounds is reduced and their respective concentrations are higher in slow metabolizers compared to rapid metabolizers. As a result, slow metabolizers are generally more prone to those adverse drug reactions linked to parent drug concentrations. Conversely, fast acetylators have higher risk of adverse consequences when these clinical events are linked to the relative exposure to acetylated metabolites. Although the usual therapeutic goal is to avoid drug-drug interactions, in the case of formation of toxic metabolites, optimized therapeutics might involve deliberate inhibition of acetylation, which could lead to an improved therapeutic index of the agent.

Although drugs that are NAT2 substrates tend to be more familiar (eg, isoniazid, procainamide, hydralazine, dapsone, aminoglutethimide, sulfamethazine), NAT1 substrates are also in clinical use. Sulfamethoxazole, one of the most widely prescribed drugs in the world, and p-aminosalicylate, used clinically as an antitubercular agent, are both eliminated from the body primarily by NAT1 metabolism.¹

Metabolic drug-drug interactions are now widely studied in human liver microsomes or hepatocytes in vitro for CYP enzymes, with the intent to predict inhibitory behavior in vivo. In the present study, we used intact human hepatocytes to determine the potential for p-aminosalicylate to competitively inhibit the acetylation of sulfamethoxazole at concentrations achievable with standard clinical doses. We then investigated whether the same phenomenon would be observed in vivo in healthy volunteers. Although we were generally motivated by the potential advantage of reducing exposure to acetylated metabolites, sulfamethoxazole and p-aminosalicylate were chosen as model drugs because of their widespread clinical utilization rather than because of a specific issue related to their metabolites.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Chemicals
Sulfamethoxazole, p-aminosalicylate, acetyl-L-carnitine, and carnitine-acetyltransferase were obtained from Sigma Chemical Co (St. Louis, Mo). p-Acetamidosalicylate and acetylsulfamethoxazole were chemically synthesized by acetylation of the parent compound through reaction with acetic anhydride. The acetylated products were confirmed by liquid chromatography/mass spectrometry (LC/MS). All other reagents used were of analytical grades.

Metabolism In Vitro
Cryopreserved human hepatocytes from 4 individual donors were thawed and prepared for the experiment exactly as described by the protocol provided by the supplier, In Vitro Technologies (Baltimore, Md). Before the addition of drug or inhibitor, the hepatocytes were equilibrated in Williams E medium with alanyl-L-glutamine at 37°C for 30 minutes. After addition of drug and/or inhibitor in the final volume of 500 µL, the hepatocytes were further incubated for 4 hours. The reaction was terminated by the addition of 15% perchloric acid to a final concentration of 1.6%. After centrifugation in a microfuge at 14,000 rpm, up to 50 µL was injected on the high-performance liquid chromatography (HPLC) column. Viability of the hepatocytes was determined at the beginning and end of the incubation. Viability was at least 50%, consistent with the vendor's specifications.

We also attempted to investigate acetylation in human liver cytosolic preparations but found unacceptable within-day and between-day variability.

Inhibition of Acetylation
Parallel incubations were conducted in hepatocytes for drug alone versus drug plus potential inhibitors. Percent inhibition was calculated from the ratio of [metabolite]/[parent] with and without the potential inhibitor.

HPLC Conditions
The parent drugs and acetylated metabolites were separated by reversed-phase HPLC on a Waters Symmetry C18 5-µm, 4.6 x 150-mm column at the elution rate of 1 mL/min. The mobile phase consisted of 1% acetic acid (v/v), with a gradient for acetonitrile from 7% to 19% over 16 minutes followed by 19% to 37% over the next 10 minutes. The column was equilibrated for 10 minutes under the initial condition of 7% acetonitrile after each injection. Drugs and their N-acetylated metabolites were quantitated by ultraviolet (UV) absorption at 268 nm using a diode-array detector.

Determination of Drugs and Metabolites in Plasma
Plasma was diluted 1:3 v/v in deionized water, and perchloric acid (15%) was added to yield a final concentration of perchloric acid of 1.6%. After centrifugation in a microfuge at 14,000 rpm, 20 µL was injected onto the HPLC column. The standard curve was constructed from blank plasma spiked with a range of known amounts of sulfamethoxazole, acetylsulfamethoxazole, p-aminosalicylate, and p-acetamidosalicylate.

Clinical Administration of Sulfamethoxazole and p-Aminosalicylate
The clinical study was a single-center, nonrandomized, open-label outpatient and inpatient design. The clinical protocol and informed consent document were approved by the institutional review board of the Uniformed Services University of the Health Sciences and the Research in Humans Subjects Committee of the Food and Drug Administration prior to enrollment.

Subjects. Subjects were recruited from the general community by responding to IRB-approved advertisements. To be eligible, male or female volunteers had to be between the ages of 18 and 45 years; be within 30% of ideal body weight; be in good health based on history as well as physical and clinical laboratory examinations; be able to provide written informed consent; not have used prescription or nonprescription drugs for 14 days prior to enrollment or oral contraceptives for 30 days prior to enrollment; not take more than 1 multivitamin per day (use of nutritional supplements or herbal remedies was not permitted); not be HIV positive; for women, not be pregnant or breastfeeding; not have any positive findings on a urine toxicology screen; not be positive for hepatitis B or C; and not have used any tobacco-containing products for 1 month. Six consenting healthy volunteers were screened. The first volunteer completed a preliminary version of the protocol. Subject 2 was not enrolled. Table I shows the demographic data for the 4 subjects who were enrolled and completed the protocol as described below.

Clinical protocol. Subjects were confined in an inpatient clinical pharmacology unit on study days 3 and 5 to facilitate intensive pharmacokinetic blood sampling on these specific study days. Subjects ingested a single dose of 500 mg sulfamethoxazole on each of days 1 to 5. Sulfamethoxazole was administered with 200 mL of water in the fasting state (a minimum of a 2-hour fast was required).

On study days 4 and 5, p-aminosalicylate (4 g tid) was administered mixed with applesauce. Plasma samples for pharmacokinetic analyses were obtained on study days 3 and 5. The schedule for pharmacokinetic samples was based on sulfamethoxazole dosing (predose and 0.5, 1, 2, 4, 8, 12, 18, and 24 hours). The ratios of AUC values for metabolite/parent were compared for day 3 and day 5 using the paired t test.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Human Hepatocytes
When 200 µM of sulfamethoxazole was incubated with hepatocytes, acetylsulfamethoxazole was the sole product formed (Figure 1A). When 200 µM of paminosalicylate was coincubated with 200 µM of sulfamethoxazole, p-acetaminosalicylate was also produced (Figure 1B), but acetylsulfamethoxazole formation was reduced by 75% ± 10% in hepatocytes from 4 separate donors (Figure 2). Over a concentration range of 50 to 800 µM of p-aminosalicylate, formation of acetylsulfamethoxazole was reduced by 70% to 90% (Figure 2).

View larger version (25K): [in this window] [in a new window]   Figure 1. High-performance liquid chromatography (HPLC) separation of sulfamethoxazole (SMX) and p-aminosalicylate (PAS), along with their acetylated metabolites (Ac) following incubation with human hepatocytes: (A) sulfamethoxazole alone, and (B) sulfamethoxazole plus p-aminosalicylate.

View larger version (12K): [in this window] [in a new window]   Figure 2. Inhibition of acetylation of sulfamethoxazole by paminosalicylate. For 1 human donor, mean inhibition ± SD was determined in triplicate for hepatocytes in vitro at a range of paminosalicylate concentrations from 50 to 800 µM (). At 200 µMof p-aminosalicylate, the mean inhibition ± SD was determined for hepatocytes in vitro from 4 separate human donors (). For comparison, the mean inhibition ± SD is shown at a representative concentration of 100 µM for 3 healthy volunteers studied in vivo ().

Clinical Studies
As shown in Figure 3, there was a substantial reduction for all subjects in the plasma concentrations of acetylsulfamethoxazole that were observed on day 5 compared with day 3, indicating a high degree of inhibition of metabolism of sulfamethoxazole to acetylsulfamethoxazole by p-aminosalicylate. On average, the ratio of plasma concentrations of acetylsulfamethoxazole, relative to parent drug, decreased 5-fold (80%), which was statistically significant (P < .001; Table II). Although there was substantial intersubject variation in exposure to sulfamethoxazole, the use of each subject as his or her own control provided a very striking result in only 4 subjects.

View larger version (18K): [in this window] [in a new window]   Figure 3. Concentrations of sulfamethoxazole (SMX) and acetylsulfamethoxazole in plasma (µM) versus time, in days. When paminosalicylate (PAS) was started on day 4, the AUC ratio of acetylsulfamethoxazole/sulfamethoxazole decreased an average of 5-fold.

At the usual clinically employed doses (4 g, 3 times daily), p-aminosalicylate concentrations in plasma were measured (Figure 4) and verified to exceed the levels shown to inhibit NAT1 in our laboratory experiments with human hepatocytes in vitro. The wide fluctuations in p-aminosalicylate concentrations between doses support the use of tid, or an every 8-hour dosing schedule, to maintain p-aminosalicylate concentrations sufficient for inhibition of the NAT1 enzyme. The drug label for p-aminosalicylate lists hypersensitivity and gastrointestinal intolerance as the most common adverse events associated with use of the product. No adverse events were seen in this small clinical study that were temporally related to the short-term use of this agent.

View larger version (19K): [in this window] [in a new window]   Figure 4. Concentrations of p-aminosalicylate (PAS) in plasma obtained from the 4 subjects at various times on day 5. The dosage regimen for p-aminosalicylate was 4 g every 6 hours on both days 4 and 5.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The majority of metabolism-based drug-drug interactions can lead to unintended excess of the parent drug, frequently causing adverse drug events and nearly always requiring an adjustment of doses or a discontinuation of one of the agents. Less commonly, there are therapeutic situations for which there are some advantages to blocking metabolism. Several examples of intentional therapeutic inhibition of drug metabolism are listed in Table III.

Among the various motivations for the intentional inhibition of metabolism described in Table III, blocking the formation of a toxic metabolite is highly relevant for NAT. In some cases, the acetylated metabolite generated by NAT has been shown to be definitively more toxic than the parent drug. In other cases, there is not a direct link to a specific metabolite, but there is a strong association between high rates of acetylation and adverse reactions.¹³

As an extension of this category, even in the absence of drug therapy, there have been several linkages reported between acetylation rates and predisposition to diseases as diverse as cancers and lupus.^3,4 In certain situations, individuals with a rapid acetylation phenotype have a 10-fold or greater risk of disease than individuals with a slow acetylation phenotype. For these situations, it is a reasonable hypothesis that inhibition of NAT could be beneficial to patients. If safe and effective inhibitors of NAT were identified, then it would be possible to test the hypothesis with clinical intervention studies.

Sulfamethoxazole and p-aminosalicylate are excellent choices for the clinical investigation of NAT1. As a widely used antibiotic, the human safety and kinetics of sulfamethoxazole are well established. In the absence of p-aminosalicylate, the relative exposure to sulfamethoxazole and acetylsulfamethoxazole in this study was similar to that reported by other authors.¹⁴

We recognized that p-aminosalicylate was the leading candidate for an inhibitor of NAT1 because its plasma concentrations during chronic therapy¹⁵ are approximately 10-fold higher than its Ki for NAT1 inhibition. Thus, it was plausible that p-aminosalicylate could be used safely as an inhibitor of NAT1, even for chronic use. Indeed, one of the arguments for the safety of an intentional blockade of NAT1 would be that there are already patients who have unintentionally inhibited NAT1 with p-aminosalicylate on a chronic basis.

Human hepatocytes have readily acetylated drugs known to be substrates for NAT.¹⁶ Thus, these preparations can be used as a screening tool for selective inhibitors. The precise relationship is uncertain for the effective in vitro concentrations of a NAT inhibitor and the actual plasma concentrations required in vivo to produce meaningful NAT inhibition.

Nonetheless, in this work, metabolic drug-drug interaction studies in vitro successfully predicted inhibition of acetylation via NAT1 in vivo. The inhibition was substantial in each subject, with an average decrease in relative exposure of 5-fold. The use of each subject as his or her own control provided an efficient proof-of-concept for the predictivity of data in vitro. Of course, a much larger number of subjects would be required to forecast population-wide effects.

Although others have used competitive inhibition of NAT in the lab as a tool for the ex vivo phenotyping of NAT1 versus NAT2,¹⁷ to our knowledge, this is the first study to show intentional inhibition of NAT in humans based on in vitro testing. Further study of other inhibitors and the potential clinical benefit from intentional inhibition is required.

	ACKNOWLEDGEMENTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We thank Lawrence Anderson for mass spectral identification of the chemically acetylated sulfamethoxazole and p-aminosalicylate.

	FOOTNOTES

 
Supported by a grant from the Office of the Assistant Secretary for Health, Department of Health and Human Services.

DOI: 10.1177/0091270004270224

Submitted for publication July 30, 2004; Revised version accepted August 17, 2004.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Request Reprints Citing Articles Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Cantilena, L. R. Articles by Collins, J. M. Search for Related Content PubMed PubMed Citation Articles by Cantilena, L. R. Articles by Collins, J. M. Social Bookmarking                   What's this?