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Journal of Clinical Pharmacology, 2005; 45:346-351
© 2005 the American College of Clinical Pharmacology

DRUG TOXICITY

Differential Toxicity of Reactive Metabolites of Clindamycin and Sulfonamides in HIV-Infected Cells: Influence of HIV Infection on Clindamycin Toxicity In Vitro

John A. Wijsman, MSc, Gregory A. Dekaban, PhD and Michael J. Rieder, MD, PhD

From the Biotherapeutic Group, John P. Robarts Research Institute, London, Ontario, Canada.

Address for reprints: Dr Michael J. Rieder, Division of Clinical Pharmacology, Department of Paediatrics, Children's Hospital of Western Ontario, 800 Commissioner's Road East, London, Ontario, Canada.


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Hypersensitivity adverse drug reactions are much more common among patients with acquired immunodeficiency syndrome (AIDS) than in the general population. High rates of hypersensitivity reactions to clindamycin have been noted. To investigate the role of reactive metabolites in these reactions, the authors studied toxicity of clindamycin and sulphamethoxazole (SMX) and their metabolites in uninfected and human immunodeficiency virus (HIV)–infected MOLT3 cells. Infected and uninfected cells were incubated with clindamycin or sulphamethoxazole hydroxylamine in increasing concentrations; reactive metabolites were generated by coincubation of cells and drug with murine microsomes and a microsomal activating system. Over a concentration range of 0 to 400 µM SMX-HA, there was a significant concentration-dependent increase in cell death in HIV-infected compared to uninfected cells (28%±3% vs 8%±5% at 400 µM, P < .05). In contrast, coincubation of cells with clindamycin, microsomes, and a microsomal activating system, as well as combinations of primaquine or pyrimethamine, was not associated with an increase in cell death among infected compared to uninfected cells. No concentration-toxicity was demonstrated. These data support the role of reactive metabolites in adverse drug reactions to sulfonamides during HIV infection, whereas alternate mechanism(s) may be responsible for increased rates of adverse drug reactions to clindamycin among patients with AIDS.

Key Words: Clindamycin • sulphamethoxazole • adverse drug reactions • toxicity • HIV infection • hypersensitivity

Human immunodeficiency virus (HIV) infection is a worldwide pandemic, with more than 40 million people infected worldwide. Current therapy includes the use of drugs to control viral load and antimicrobials, such as the sulfonamides, to control important and common opportunistic infections. However, rates of adverse drug reactions (ADRs) to therapy—notably, sulfonamide therapy for opportunistic infections—are reported to be higher in HIV-infected patients than in the general population.1-4 In HIV-infected patients treated with sulphamethoxazole-trimethoprem, rates of ADR have been reported to reach 80% and are commonly in the range of 30 to 40%.2 In contrast, rates of adverse drug reactions in the general population are typically in the range of 5%. In the case of HIV infection, hypersensitivity adverse drug reactions are specifically more common than in the general population. In the general population, the commonest types of ADRs to sulfonamides are urticarial rashes or gastrointestinal complaints such as diarrhea. In contrast, among people living with acquired immunodeficiency syndrome (AIDS), the commonest ADRs have been serious rashes, including erythema multiforme; the incidence of Stevens-Johnson syndrome and toxic epidermal necrolyis is also markedly higher among people with AIDS rather than in the general population.

The development of an adverse drug reaction may require that therapy be stopped and therapeutic alternates used. Alternate therapy with clindamycin-primaquine has been found to be efficacious as both a treatment and prophylaxis but is also associated with a higher incidence of ADRs in HIV-infected patients with >50% ADR events reported.3 In contrast,4,5 rates of ADR to both of these drugs are less than 3% in the general population.

The pathogenesis of this increased sensitivity to drugs that accompanies HIV infection is unknown but may be related to decreased intracellular glutathi-one,1,2,6 concomitant drug therapy, presence of specific HIV proteins, altered immune function,6,7 or other as yet undetermined factors.3

There are high rates of ADRs to other anti-infective drugs used in the care of patients with AIDS. In the general population, therapy with clindamycin is associated with risk for gastrointestinal ADRs. In contrast, in the context of therapy for people living with AIDS, a study has demonstrated a high rate of rash in association with therapy with clindamycin/primaquine.5

We have previously shown that, in vitro, HIV infection produces a specific increase in the sensitivity of MOLT3 cells to the reactive hydroxylamine metabolite of sulphamethoxazole (SMX-HA).4 Similarly, peripheral blood mononuclear cells (PBMCs) from HIV-infected individuals are also more sensitive to the toxic effects of SMX-HA when tested in vitro.7 The mechanism of this increase in sensitivity to SMX-HA is unknown but clearly is not related to concomitant therapy in the HIV-infected cell line. Although the process of increased sensitivity to SMX-HA is not understood, considering the high rate of adverse drug reactions to clindamycin-containing preparations in HIV-infected patients, we conducted this study to ascertain whether HIV infection is sufficient to induce increased cellular sensitivity to clindamycin or its metabolites. As clindamycin is frequently coadministered with the anti-malarial agents primaquine or pyrimethamine to enhance the antimicrobial spectrum,5,6,8,9 sensitivity to these drugs as well as their combinations with clindamycin was assessed.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Cell Culture
MOLT3 and MOLT3IIIB cells were maintained in log growth in RPMI1640 containing 10% fetal calf serum (FCS), 50 µM 2-mercaptoethanol, penicillin (100 IU/mL), and streptomycin (100 µg/mL). MOLT-3 T lymphoblasts were infected with HIVIIIB by exposure to HIVIIIB-containing tissue culture supernatant. Chronic infection was demonstrated by formation of syncytia and presence of virus in supernatants using reverse transcriptase–polymerase chain reaction (RT-PCR).4

Drug Incubations
Cells were washed 2x in Hank's balanced salt solution (HBSS), resuspended in HBSS, and incubated in 96-well tissue culture plates at 50,000 cells/well. Cells were incubated at a final concentration of 400 µM SMX or 0, 12, 25, 50, 25, 100, or 400µM SMX-HA for 2 hours. The supernatant was removed and the cells incubated in medium for 18 hours. To expose cells to clindamycin, primaquine, pyrimethamine, or the combinations of clindamycin + primaquine or clindamicin + pyrimethamine, cells were incubated over a series of four 10x dilutions beginning at 1000 µM clindamycin, 15µM primaquine, 400 µM pyrimethamine, or combinations at the same concentrations. To expose the cells to metabolites of these drugs, parallel incubations in the presence of microsomes plus an activating system10,11 were performed.

Viability Assessment
Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye conversion. Eighteen hours following drug exposure, MTT was added to the wells to a final concentration of 1 mg/mL and incubated at 37°C for 2 hours. The formazan reaction product was solubolized overnight using 50% dimethylformamide/20% sodium dodecyl sulfate (SDS), and 96-well plates were read on a plate reader (Molecular Probes, Sunnyvale, Calif) at 590 nm. Viability was determined by comparison to a standard curve generated by plating cells at 0%, 25%, 50%, and 100% of the initial plating density. To determine the viability of cells exposed to microsomes, a parallel standard curve was generated in which the cells were exposed to microsomes.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
To determine the viability of the cell lines following exposure to the sulfamethoxazole metabolite, MOLT3 and MOLT3IIIB cells were incubated with SMX-HA at varying concentrations, washed, cultured overnight, and then incubated with MTT. The MOLT3 and MOLT3IIIB cells exposed to 400 µM SMX exhibited no differences in viability (97% ±3.4% vs 99% ± 1.2). In contrast, when these cells were exposed to a range of concentrations of SMX-HA, the MOLT3IIIB cells exhibited significantly higher rates of cell death at all concentrations (P < .05) (Figure 1). This toxicity is consistent with our previous work.4



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  		Figure 1. MOLT3 and the HIV-infected MOLT3IIIB cell lines exhibit concentration-dependent toxicity (P < .05) following exposure to sulphamethoxazole-hydroxylamine, one of the reactive metabolites of sulfamethoxazole. The MOLT3IIIB cell line exhibits significantly increased levels of toxicity at every concentration tested. This increase in sensitivity to sulphamethoxazole-hydroxylamine (SMX-HA) appears to be a direct consequence of HIV infection.

 

In separate experiments, the cells were exposed to clindamycin or its metabolites, washed, and incubated overnight, and the viability was determined using MTT dye conversion. MOLT3 and MOLT3IIIB cells exposed to clindamycin (1-1000 µM) did not exhibit a dose-dependent decrease in cell viability (107% ± 16% and 103% ± 3.5%, respectively, at 1000 µM; Figure 2). There was no increased toxicity in response to clindamycin + microsomes in either the MOLT3 or MOLT3IIIB cell lines. At 100 and 1000 µM clindamycin plus metabolites, MOLT3IIIB cells consistently exhibit slightly increased viability (110% ± 5% and 110% ± 4.7%, respectively).



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  		Figure 2. The MOLT3 and MOLT3IIIB cell lines were incubated with clindamycin over a range of concentrations for 2 hours alone or coincubated with microsomes to generate drug metabolites and assessed for viability after 18 hours. No toxicity was evident following exposure to clindamycin or metabolites in either cell line.

 

MOLT3 and MOLT3IIIB cells exposed to primaquine or its metabolites did exhibit a trend toward dose-dependent toxicity over the range tested with viability at 15µM in the range of 75% for both cell lines (Figure 3A). Coincubation with a microsomal activating system did not induce increased toxicity in either cell line. The combination of clindamycin and primaquine also induced a dose-dependent increase in toxicity with a cell viability at the dose of 1000 µM clindamycin, 15 µM primaquine of 47% and 35% in the MOLT3 cells, and 52% and 58% in the MOLT3IIIB cells when exposed to the drug combination or the combination plus the microsomal activating system (Figure 3B). Neither primaquine nor the combination of primaquine plus clindamycin established an increased toxicity in the MOLT3IIIB cell line compared to the uninfected MOLT3 cell line.






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  		Figure 3. The MOLT3 and MOLT3IIIB cell lines were incubated with primaquine, pyrimethamine, or combinations of primaquine or pyrimethamine with clindamycin for 2 hours alone or coincubated with microsomes to generate drug metabolites and assessed for viability after 18 hours. (A) Primaquine caused an equal decrease in viability in both cell lines at the highest concentration tested, 15 µM. (B) Pyrimethamine caused a slight but statistically insignificant toxicity in both cell lines. (C) The combination of clindamycin and primaquine induced a concentration-dependent increase in toxicity in both cell lines. No increased sensitivity was observed in the MOLT3IIIB cell line. (D) The viability of the MOLT3 and the MOLT3IIIB cell lines was not affected by exposure to the combination of clindamycin and pyrimethamine. The absence of increased toxicity to these drugs or their metabolites in the MOLT3IIIB cell line suggests that primary HIV infection does not induce an increased sensitivity to clindamycin.

 

Examination of cells exposed to pyrimethamine by the toxicity assay revealed a nonstatistically significant trend toward dose-dependent toxicity in MOLT3 cells, with viability decreasing to 83% ± 7% when exposed to 400 µM pyrimethamine (Figure 3C). The viability of the MOLT3IIIB cell line under similar conditions was 95% ± 0.2%. In the presence of the microsomal activating system, cellular viability increased in comparison to cells treated with drug alone in almost all cases. The drug combination of clindamycin plus pyrimethamine produced a toxicity pattern very similar to pyrimethamine alone (Figure 3D). Activation of the parent drug was not associated with increased toxicity at any concentration.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Despite the fact that adverse drug reactions are major complications of medical therapy—and indeed, in the developed world, are among the top five causes of death—there has until recently been relatively little appreciation for the mechanism(s) underlying drug hypersensitivity. This is especially true in the case of HIV infection, in which the mechanism(s) responsible for the marked increase in drug hypersensitivity remain largely unknown.

It is now appreciated that drug metabolism may be a fundamental pathophysiologic mechanism for many adverse events previously believed to be idiosyncratic; indeed, over the past decade, there has been considerable insight into the possible role(s) of reactive drug metabolites in the initiation and propagation of hypersensitivity adverse drug reactions.12-14 It is now appreciated that hypersensitivity adverse drug reactions to many common and important classes of drugs, such as the sulfonamides, aromatic anticonvulsants, novel antipsychotics, and the antimicrobial agent cefaclor, appear to be mediated, at least in part, by the oxidative metabolism of the parent drug to a reactive intermediate.11-13,15 The mechanisms by which these effects occur remain unclear but may involve cellular injury and dysfunction as well as initiation of undesired immunoogical events.

We have previously described increased sensitivity to sulfonamide reactive metabolites in the presence of HIV infection, a specific increase in sensitivity that appears to be an important determinant of adverse drug reactions in the setting of AIDS.4,7 This toxicity occurs at concentrations of 100 µM, concentrations that can be predicted to occur in vivo during high-dose therapy. The differential toxicity to sulfamethoxazole metabolites, observed between HIV-infected and uninfected cell lines, has not yet been explained. The results presented here confirm our earlier report of the differential toxicity to sulfamethoxazole hydroxylamine in HIV-infected MOLT3 cells. In this set of experiments, cellular viability was determined using MTT dye conversion, an assay that is both objective and more sensitive to cellular dysfunction than the trypan blue dye exclusion previously used. This sensitivity may be related to HIV-specific alterations in cellular homeostasis, which places the cells at increased risk for cellular injury from reactive chemical species.3,4

Bioactivation of sulfonamides appears to occur in the common N-terminal amine. Clindamycin consists of a derivative of the amino acid trans-L-4-n-propyl-hygrinic acid joined to a sulfur-containing octose derivative. Thus, there are several sites for drug activation. Primaquine is known to be extensively metabolized, including via oxidative metabolism to 8-(3-carboxyl-1-methylpropylamino)-6-methoxyquinolone, 5-hydroxy primaquine, and 5-hydroxy-6-desmethylprimaquine.16 Pyrimethamine is a 2.4-diaminopyrimidine, again a compound whose structure suggests the possibility of bioactivation to a reactive metabolite.

The clinical characteristics of the adverse reactions associated with clindamycin-containing agents in the setting of HIV infection are similar to those seen for hypersensitivity reactions to drugs that are believed to be mediated by reactive metabolites. Consequently, given the nature of the components in clindamycin preparations, we investigated whether activation of clindamycin was associated with cellular toxicity. To study this, we used a microsomal activating system that we have used in previous studies to establish the toxicity of the reactive metabolites of other compounds.

Although the chemistry of clindamycin, primaquine, and pyrimethamine and the clinical characteristics of the adverse reactions suggest that drug metabolism may be an important determinant of adverse reactions to clindamycin-containing preparations in the case of AIDS, our data do not support the role of reactive metabolites in adverse drug reactions in the setting of HIV infection. There was a trend toward concentration-dependent toxicity when clindamycin was combined with either primaquine or pyrimethamine, but this toxicity was not increased in the presence of HIV infection or by activation to reactive intermediates.

This work is further support for the role of reactive drug metabolites in the development of adverse drug reactions to sulfonamides in the setting of HIV infection. The mechanisms responsible for the increased rate of adverse drug reactions to clindamycin-containing preparations in the setting of HIV infection remain unclear. It is possible that perturbations in immunity secondary to HIV infection may contribute to the increased rate of adverse drug reactions to clindamycin-containing preparations, and this approach is currently the subject of research in our laboratory.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
This research was supported by a grant from the Medical Research Council of Canada. Dr Dekaban is a Career Scientist of the Ontario Ministry of Health.


   FOOTNOTES
 
Study performed at the John P. Robarts Research Institute. This research was supported by a grant from the Medical Research Council of Canada.

DOI: 10.1177/0091270004272670

Submitted for publication January 8, 2004; Revised version accepted October 28, 2004.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

1. Lehmann DF, Holohan PD, Blair DC. Comparisons of oxidative metabolism and reductive capacity in sulfonamide-tolerant and - intolerant patients with human immunodeficiency virus. J Clin Pharmacol. 1996;36: 1149-1153.[Abstract]

2. van der Ven AJ, Koopmans PP, Vree TB, van der Meer JW. Drug intolerance in HIV disease. [Review] [39 refs]. J Antimicrob Chemother. 1994;34: 1-5.[Free Full Text]

3. Koopmans PP, van der Ven AL, Vree TB, van der Meer JW. [Drug reactions occur frequently in patients with HIV infection]. [Review] [51 refs] [Dutch]. Nederlands Tijdschrift voor Geneeskunde 1995;139: 985-988.[Medline] [Order article via Infotrieve]

4. Rieder MJ, Krause R, Bird IA, Dekaban GA. Toxicity of sulfonamide-reactive metabolites in HIV-infected, HTLV-infected, and noninfected cells. JAIDS. 1995;8: 134-140.

5. Toma E, Thorne A, Singer J, Raboud J, Lemieux C, Trottier S, et al. Clindamycin with primaquine vs. trimethoprim-sulfamethoxazole therapy for mild and moderately severe Pneumocystis carinii pneumonia in patients with AIDS: a multicenter, double-blind, randomized trial (CTN 004). CTN-PCP Study Group. Clin Infect Dis. 1998;27: 524-530.[Web of Science][Medline] [Order article via Infotrieve]

6. Caumes E, Bocquet H, Guermonprez G, Rogeaux O, Bricaire F, Katlama C, et al. Adverse cutaneous reactions to pyrimethamine/sulfadiazine and pyrimethamine/clindamycin in patients with AIDS and toxoplasmic encephalitis. Clin Infect Dis. 1995;21: 656-658.[Web of Science][Medline] [Order article via Infotrieve]

7. Carr A, Tindall B, Penny R, Cooper DA. In vitro cytotoxicity as a marker of hypersensitivity to sulphamethoxazole in patients with HIV. Clin Exp Immunol. 1993;94: 21-25.[Web of Science][Medline] [Order article via Infotrieve]

8. Barber BA, Pegram PS, High KP. Clindamycin/primaquine as prophylaxis for Pneumocystis carinii pneumonia. Clin Infect Dis. 1996; 23: 718-722.[Web of Science][Medline] [Order article via Infotrieve]

9. Safrin S, Finkelstein DM, Feinberg J, Frame P, Simpson G, Wu A, et al. Comparison of three regimens for treatment of mild to moderate Pneumocystis carinii pneumonia in patients with AIDS: a double-blind, randomized, trial of oral trimethoprim-sulfamethoxazole, dapsone-trimethoprim, and clindamycin-primaquine. ACTG 108 Study Group [see comments]. Ann Intern Med. 1996;124: 792-802.[Abstract/Free Full Text]

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11. Tschen AC, Rieder MJ, Oyewumi LK, Freeman DJ. The cytotoxicity of clozapine metabolites: implications for predicting clozapine-induced agranulocytosis. Clin Pharmacol Ther. 1999;65: 526-532.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

12. Kearns GL, Wheeler JG, Rieder MJ, Reid J. Serum sickness-like reaction to cefaclor: lack of in vitro cross-reactivity with loracarbef. Clin Pharmacol Ther. 1998;63: 686-693.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

13. Hess DA, Sisson ME, Suria H, Wijsman J, Puvanesasingham R, Madrenas J, et al. Cytotoxicity of sulfonamide reactive metabolites: apopotosis and selective toxicity of CD(8+) cells by the hydryoxylamine of sulfamethoxazole. Fed Am Soc Exp Biol J. 1999;13: 1688-1698.

14. Schnyder B, Burkhart C, Schnyder-Frutig K, von Greyerz S, Naisbitt DJ, Pirmohamed M, et al. Recognition of sulfamethoxazole and its reactive metabolites by drug-specific CD4+ T cells from allergic individuals. J Immunol. 2000;164: 6647-6654.[Abstract/Free Full Text]

15. Knowles SR, Shapiro LE, Shear NH. Anticonvulsant hypersensitivity syndrome: incidence, prevention and management. Drug Saf. 1999;21: 489-501.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

16. Mihaly GW, Ward SA, Edwards G, Orme ML, Breckinridge AM. Pharmacokinetics of primaquine in man: identification of the carboxylic acid derivative as a major plasma metabolite. Br J Clin Pharmacol. 1984;17: 441-446.[Web of Science][Medline] [Order article via Infotrieve]
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