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Role of Specific Cytochrome P450 Isoforms in the Conversion of Phenoxypropoxybiguanide Analogs in Human Liver Microsomes to Potent Antimalarial Dihydrotriazines

Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland (D.S.D., M.P.K., K.S.S., C.O.A., J.C.S., W.K.M., D.R.S., T.W.S.); and Jacobus Pharmaceutical Company, Princeton, New Jersey (G.A.S., D.P.J.)

(Received December 14, 2006; accepted November 13, 2007)

	Abstract

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Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

We have investigated several potential antimalarial drug candidates based on the previously described phenoxypropoxybiguanide analogs (Canfield et al., 1993; Jensen et al., 2001). All of these prodrug analogs have displayed a high level of antimalarial activity in the in vivo mouse model (data not shown) and the respective active metabolites have displayed a high degree of potency in in vitro efficacy testing. Formation of the active triazine metabolites of these PS-15-like analogs has previously been demonstrated in human and animal liver microsomes (Shearer et al., 2005). Metabolic formation of CG from PG has been extensively investigated in vitro and in clinical patients (Watkins et al., 1984; Yeo et al., 1994). Taken together, data in the literature suggest that PG is metabolized primarily to CG by CYP2C19 with a minor contribution by CYP3A4 and possibly CYP2D6 (Helsby et al., 1990; Birkett et al., 1994; Wright et al., 1995; Funck-Brentano et al., 1997; Coller et al., 1999; Lu et al., 2000). The role of CYP2C19 in the conversion of PG to the active metabolite CG has raised several concerns in that approximately 15 to 25% of Asian populations are classified as poor metabolizers of CYP2C19 substrates (Jurima et al., 1985; Wilkinson et al., 1989; Desta et al., 2002; Hoskins et al., 2003). In addition, several protease pump inhibitors such as omeprazole have been shown to competitively inhibit metabolism by CYP2C19 and have been shown to reduce CG plasma concentrations in human patients (Funck-Brentano et al., 1997). The chemical structures of PG, PS-15, and JPC-2056 and the respective active dihydrotriazine metabolites are displayed in Fig. 1. The production of the active dihydrotriazine metabolites for PS-15 and JPC-2056 as well as other antifolate analogs in this series in pooled human liver microsomes has previously been examined [pooled human liver microsomes (pHLM)] (Shearer et al., 2005). In the studies described here we used human recombinant P450 isoforms to identify the most likely isoforms involved in the conversion of PG, PS-15, and JPC-2056 to the respective active metabolites. These studies are followed by P450 inhibition studies using chemical and specific P450-inhibiting antibodies to determine the relative contribution and importance of each isoform in the production of the active metabolites by pHLM. As previously described, it is well established in the literature by both in vitro and in vivo studies that PG is primarily converted to CG by CYP2C19, following in importance by CYP3A4 and possibly CYP2D6, respectively. Given the structural similarities of the triazine motifs between PS-15 and JPC-2056, PG was included in each study for comparison.

View larger version (9K): [in this window] [in a new window]   FIG. 1. Structures of proguanil, PS-15, JPC-2056, and there respective active metabolites (cycloguanil, WR99210, and JPC-2067).

	Materials and Methods

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Compounds. PG, CG, PS-15, and WR99210 were obtained from the chemical inventory system at Walter Reed Army Institute of Research. The novel PS-15 analog, JPC2056, and the corresponding active metabolite were obtained from Jacobus Pharmaceutical Company (Princeton, NJ). The internal standard JPC-2062 was also obtained from Jacobus Pharmaceutical Company. Ketoconazole, ticlopidine, β-NADP, magnesium chloride, glucose 6-phosphate (G6P), and glucose 6-phosphate dehydrogenase (G6PD) were purchased from Sigma-Aldrich (St. Louis, MO).

Microsomal Assay. Each compound was prepared in a stock solution of acetonitrile and water and then added to a mixture containing NADPH regeneration buffer (β-NADP⁺, G6P, and MgCl₂) and pHLM (BD Biosciences, San Jose, CA) to a final volume of 225 µl. The mixture was incubated for 5 min at 37°C and then initiated by the addition of 25 µl of G6PD. Final component concentrations were 1.25 mM β-NADP⁺, 3.3 mM G6P, 3.3 mM MgCl₂, 1 unit/ml G6PD, and 1 mg/ml microsomal protein. Acetonitrile concentrations in the final reaction were 0.1% or less. Each reaction was maintained at 37°C until termination at the specified time point (90 min) by the addition of an equal volume of ice-cold acetonitrile containing analog JPC-2062 as an internal standard. A minimum of six reactions were conducted for each compound. Production of metabolites was initially evaluated in pHLM at concentrations ranging from 1 to 50 µM. Production of active metabolites for each prodrug was determined to be linear within this concentration range (Fig. 2).

View larger version (14K): [in this window] [in a new window]   FIG. 2. Production of active metabolites (cycloguanil, WR99210, and JPC-2067) from proguanil, PS-15, and JPC-2056 in pooled human liver microsomes. Each parent compound was incubated for 90 min over initial substrate concentrations ranging from 0.1 to 50 µM.

Isoform-Specific Metabolism for Antifolate Analogs. Human CYP1A2, CYP2C9*1 (Arg₁₄₄), CYP2C19, CYP2D6, and CYP3A4 recombinant enzymes systems containing reductase and cytochrome b₅ obtained from BD Biosciences were used to identify the specific isoforms capable of metabolizing the antifolate analogs. In this assay, isoforms were diluted in NADPH regeneration buffer (final assay concentrations of 1.25 mM β-NADP, 3.3 mM G6P, and 3.3 mM MgCl₂) to a CYP450 concentration of 20 pmol for CYP1A2, CYP2C9, CYP2D6, and CYP3A4 isoforms and 40 pmol for CYP2C19. The test compounds were added to the mixture to obtain a final concentration of 10 µM. The final concentration of acetonitrile in the assays was 0.1% or less. The mixture was incubated for 5 min at 37°C and then initiated by the addition of 25 µl of G6PD (1 unit/ml final assay concentration). The reaction was stopped after a 60-min reaction time by addition of 1 volume of ice-cold acetonitrile containing a 125 nM concentration of Jacobus Pharmaceutical analog JPC-2062 as internal standard. The samples were centrifuged for 5 min at 13,000 rpm, and the supernatant was transferred to clean vials for LC-MS/MS analysis.

Chemical Inhibition Studies. Selective chemical inhibition studies were performed to investigate the roles of CYP2C19 and CYP3A4 in the metabolism of the antifolate analogs. The well characterized inhibitors ticlopidine (TCP) (K_i = 1.2 µM), ketoconazole (KTZ) (K_i = 0.2 µM), and quinidine (QDN) (K_i = 0.3 µM) were used to evaluate the roles of CYP2C19-, CYP3A4-, and CYP2D6-specific isoforms in the metabolism of the antifolate analogs, respectively. The metabolism of each analog was tested in the presence of each chemical inhibitor at two concentrations (the lowest concentration was close to the K_i reported for the specific enzyme and the highest concentration was approximately 10- to 20-fold higher than the reported K_i. For the chemical inhibitors KTZ and QDN, concentrations of 0.5 and 5 µM were used. For Experiments with TCP concentrations of 1.25 and 10 µM were used. Each antifolate analog was tested at a single concentration of 50 µM, which is approximately equal to the K_m for PS-15 or K_m/2 for PG and JPC-2056 (Shearer et al., 2005). The substrate concentration and concentration of the pHLM used in these reactions were determined previously to be within the linear range for production of the active metabolites. Each test compound was added into a mixture of pHLM and NADPH regeneration buffer containing various concentrations of the appropriate chemical inhibitor. Final assay conditions contained organic solvent concentrations of 0.1% or less. This complete mixture was initiated with G6PD and incubated for 90 min following standard metabolism procedures described above.

Antibody Inhibition Study for Antifolates Analogs. Monoclonal antibodies against specific P450 isoforms (CYP1A2, CYP2C8, CYP2C19, CYP2D6, and CYP3A4) were preincubated for 15 min with pHLM at room temperature. NADPH regeneration buffer containing test compound (final assay concentrations were 1.25 mM β-NADP, 3.3 mM G6P, and 3.3 mM MgCl₂) was added to the microsomes (1 mg/ml final concentration)/antibodies mixture and incubated for 5 min at 37°C in a water bath. The reaction was initiated by the addition of G6PD (1 unit/ml final assay concentration) and incubated for 90 min following standard metabolism procedures described above. All drugs were tested at 50 µM, and the amounts of antibodies were selected per the manufacturer's specifications to produce significant inhibition of each isoform. Control samples contained preimmune IgG from rabbit. The inhibitory monoclonal antibodies (IgG₁) against human CYP1A2, CYP2C19, CYP2D6, and CYP2C8 and the inhibitory monoclonal antibodies (IgM) against human CYP3A4 were purchased from XenoTech, LLC (Kansas City, KS). Control preimmune IgG from rabbit was also purchased from XenoTech, LLC.

	Results

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Metabolism of Antifolate Analogs by Pooled Human Liver Microsomes. The production of active metabolites was observed in pHLM over substrate concentrations ranging from 1 to 50 µM. Formation of cycloguanil, WR99210, and JPC-2067 was observed to be linear over this concentration range. Substrate concentration-dependent production of the active metabolites in pHLM is exhibited in Fig. 2. At the substrate concentrations used in these experiments, both WR99210 and JPC-2067 were generated at a higher rate than was cycloguanil. At the lowest, but perhaps most physiologically relevant concentration of 1 µM, JPC-2067 and WR99120 were generated at rates of approximately 3- and 4-fold higher than cycloguanil, respectively.

Metabolism of Antifolate Analogs by Selected P450 Isoforms. To identify possible P450 isoforms involved in the production of each active metabolite and focus the chemical and antibody inhibition studies, human recombinant P450 isoforms were used in initial experiments. Each parent compound was individually incubated with one of the five primary drug-metabolizing P450s: recombinant human CYP1A2, CYP2C9*1 (Arg₁₄₄), CYP2C19, CYP2D6, or CYP3A4 (Fig. 3). Production of CG from PG was observed after incubation with expressed CYP2C19 and CYP3A4 isoforms as previously reported. (Helsby et al., 1990; Birkett et al., 1994; Wright et al., 1995; Funck-Brentano et al., 1997; Coller et al., 1999; Lu et al., 2000). Interestingly, incubation of PG in CYP2D6 isoforms produced a similar amount of CG as was observed in the CYP3A4 isoforms (as expressed as picomoles per minute per picomole of enzyme). No CG could be detected after incubation of PG in CYP1A2 or CYP2C9*1(Arg₁₄₄) isoforms. The highest production of WR99210 (the active dihydrotriazine of PS-15) was observed after incubation in expressed CYP3A4 isoforms. CYP2D6 and CYP2C19 isoforms also produced a significant amount of WR99210, whereas a relatively small amount of WR99210 could be detected after incubation in CYP1A2. Conversion of JPC-2056 to JPC-2067 was observed after incubation in the CYP3A4, CYP2C19, and CYP2D6 isoforms. In comparison with PS-15, conversion of JPC-2056 to JPC-2067 after incubation with CYP2C19 and CYP2D6 was relatively minor compared with the production of JPC-2067 after incubation with the CYP3A4 isoform. A similar amount of JPC-2067 was converted in the presence of CYP1A2 as was observed for WR99210. The active metabolite of JPC-2056 was not observed after incubation with the CYP2C9*1(Arg₁₄₄) isoform. Based on these results, the chemical inhibition studies focused on the role of P450 isoforms CYP2C19, CYP3A4, and CYP2D6.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Production of active metabolites (cycloguanil, WR99210, and JPC-2067) from proguanil, PS-15, and JPC-2056 in the presence of selected recombinant P450 isoforms. Each parent compound was incubated for 60 min with an initial concentration of 10 µM.

Inhibition by Selective Chemical Inhibitors. Selective and potent chemical inhibitors for CYP2C19, CYP3A4, and CYP2D6 were used to further examine the role of these isoforms in the conversion of PG, PS-15, and JPC-2056 to their respective active metabolites in pHLM. First, the potent CYP2C19 inhibitor TCP (K_i = 1.2 µM) was used to investigate the role of CYP2C19 (Ko et al., 2000; Ha-Duong et al., 2001). TCP was a potent inhibitor of the conversion of PG to CG with more than 50% inhibition at 10 µM. TCP was slightly less potent in inhibiting the production of WR99210 and JPC-2067 from their respective parent compounds (approximately 40%). The selective CYP3A4 inhibitor KTZ (K_i = 0.2 µM) (Zhang et al., 2002) was used to investigate the role of CYP3A4. KTZ (5 µM), inhibited the production of CG and WR99210 from their respective parent compounds by approximately 45%, whereas the production of JPC-2067 from JPC-2056 was inhibited by more than 60%. The selective CYP2D6 inhibitor QDN (K_i = 0.3 µM) was used to investigate the role of CYP2D6 (Otton et al., 1984). Activation of PG to CG was not significantly inhibited in the presence of 5 µM QDN. In contrast, 5 µM QDN inhibited the production of WR99210 by more than 60%. Formation of JPC-2067 from JPC-2056 was moderately inhibited in the presence of 5 µM QDN. The results of the chemical inhibition studies are presented in Fig. 4 and summarized in Table 1.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Inhibition of the metabolic activation of each analog to their respective active metabolites by the following specific P450 inhibitors: the 2C19 inhibitor TCP, the 3A4 inhibitor KTZ, and the 2D6 inhibitor QDN. Each parent compound was incubated for 90 min in pHLM with an initial concentration of 50 µM.

Inhibition by Selective Monoclonal Antibodies. Monoclonal antibodies against CYP1A2, CYP2C8, CYP2C19, CYP2D6, and CYP3A4 were used to evaluate the contribution of the respective P450 enzymes to the in vitro metabolism of antifolate analogs in pHLM. Activation of PG to CG was most significantly inhibited by the presence of anti-CYP2C19 antibodies. The production of CG was moderately inhibited by the CYP3A4-specific antibodies (approximately 30%) but not the anti-CYP2D6, anti-CYP1A2, or anti-CYP2C8 antibodies. In contrast, production of WR99210 was not significantly inhibited by anti-CYP2C19 antibodies, whereas the production of JPC-2067 was only slightly inhibited by anti-CYP2C19. However, the activation of both PS-15 and JPC-2056 to their respective active metabolites was markedly reduced in the presence of the CYP3A4 antibodies. Anti-CYP3A4 antibodies inhibited WR99210 and JPC-2056 production by approximately 70% and 80%, respectively. Anti-CYP2D6 antibodies also significantly inhibited the production of WR99210 and JPC-2067 by 35 and 25%, respectively. No significant inhibition of WR99210 production was observed in the presence of the CYP2C8- or CYP1A2-specific antibodies. A small but significant level of inhibition (approximately 10–15%) in the production of JPC-2056 was observed in the presence of the CYP1A2 and CYP2C8 antibodies. Inhibition of CG, WR99210, and JPC-2067 formation by selective P450 monoclonal antibodies is summarized in Fig. 5 and Table 1.

View larger version (21K): [in this window] [in a new window]   FIG. 5. Inhibition of the metabolic activation of each analog to their respective active metabolites by selected, specific P450s inhibiting monoclonal antibodies. Each parent compound was incubated for 90 min in pHLM with an initial concentration of 50 µM.

	Discussion

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Inhibitors of Plasmodium dihydrofolate reductase are an important class of antimalarial drugs in clinical use today. Unfortunately, drug resistance to these agents has emerged owing to a series of point mutations in the pfDHFR-TS enzyme. Efforts have been made to develop novel, third-generation antifolate analogs that retain efficacy against mutant pfDHFR-TS enzymes and with antifolate-resistant and antifolate-sensitive strains of the malaria parasite. Analog JPC-2056 is a biguanide selected as a development candidate because of the high in vitro efficacy of its active metabolite, JPC-2067, against P. falciparum strains D6 and W2. Because JPC-2056 is a prodrug similar to PG in that it requires metabolic activation for antimalarial activity, a detailed understanding of its metabolism including the role of different metabolizing enzymes is critical to the clinical development of this compound. Thus, this study was initiated to investigate the role of specific P450 isoforms in the conversion of JPC-2056 to its active and primary metabolite. A series of experiments using pHLM, recombinant P450 isoforms, selective P450 chemical inhibitors, and specific anti-P450 inhibitory antibodies were conducted to elucidate which P450 isoforms are primarily involved in the production of the active metabolite of JPC-2056. Because JPC-2056 and its metabolite are structurally similar to PG and CG, respectively, PG was included in these studies as a reference compound. The previous clinical candidate for this series of compounds, PS-15, was also included for comparison.

Whereas the structures of PG, PS-15, and JPC-2067 are remarkably similar at the triazine site of metabolic conversion, initial experiments indicated that there was a significant difference in the rate of formation of the respective metabolites. The metabolic conversion of PG to CG has been extensively characterized in several studies both in vitro and in vivo (Watkins et al., 1984; Birkett et al., 1994; Funck-Brentano et al., 1997; Coller et al., 1999; Lu et al., 2000). The bulk of data in the literature indicate that the primary isoform responsible for the conversion of PG to CG is CYP2C19; however, several studies have indicated possible contributions from both CYP3A4 and CYP2D6 (Birkett et al., 1994; Funck-Brentano et al., 1997; Coller et al., 1999; Lu et al., 2000). The results for PG were similar to previous observations in that CYP2C19, CYP2D6, and CYP3A4 are important isoforms in the conversion of PG to CG. These results were confirmed by the chemical and antibody inhibition studies in pHLM. CG production was potently inhibited by the CYP2C19 inhibitor TCP and the anti-CYP2C19 antibody. The production of CG was also reduced by approximately 50% by the CYP3A4 inhibitor KTZ and approximately 30% by the anti-CYP3A4 antibody. Although a moderate amount of CG was produced in the presence of the recombinant CYP2D6 isoform, chemical inhibition studies in pHLM did not reveal a significant role of CYP2D6.

Initial experiments with the recombinant enzyme systems revealed that isoforms CYP2C19, CYP3A4, CYP2D6, and possibly CYP1A2 were important in the conversion of both PS-15 and JPC-2056 to their respective active metabolites. However, relative to the production of CG from PG, studies with the recombinant isoforms indicated that CYP3A4 may be more predominant than CYP2C19 or CYP2D6. Chemical and antibody inhibition studies confirmed these early indications provided by the recombinant enzyme system in that production of the active metabolites from PS-15 and JPC-2056 was also potently inhibited by the CYP3A4 inhibitor KTZ and the anti-CYP3A4 antibodies (45–60 and 70–80%, respectively). The recombinant enzyme studies also indicated that CYP2D6 may be more important in the conversion of PS-15 to WR99210 relative to the production of either CG or JPC-2067 from the respective parent compounds. Chemical and antibody inhibition studies also confirmed these results in that both the CYP2D6 inhibitor QDN and the anti-CYP2D6 antibodies significantly inhibited the production of WR99210, whereas production of CG was not inhibited and the production JPC-2067 was only moderately inhibited in these experiments. In contrast with CG, WR99210 and JPC-2067 formation was not significantly inhibited by TCP at a concentration of 1.25 µM. However, both WR99210 and JPC-2067 production was inhibited by 45 and 43%, respectively, with 10 µM TCP (Fig. 4; Table 1). TCP is also a relatively potent inhibitor of CYP2D6 with a reported K_i of 3.4 µM (Ko et al., 2000; Ha-Duong et al., 2001). WR99210 and JPC-2067 production in pHLM is clearly somewhat dependent on CYP2D6 on the basis of the data presented herein. For this reason, the high percent inhibition of WR99210 and JPC-2067 formation by 10 µM TCP may be due predominantly to CYP2D6 inhibition and/or a combination of CYP2D6 and CYP2C19 inhibition. Given the low inhibition of WR99210 and JPC-2067 formation in the presence of the anti-CYP2C19 antibody, the former is more likely the case. Interestingly, moderate amounts of WR99210 and JPC-2067 were produced in the presence of the recombinant CYP1A2 isoform; however, production of these metabolites was not significantly inhibited by anti-CYP1A2 antibodies in pHLM. Chemical inhibition studies were not performed to further investigate the role of CYP1A2 in the production of WR99210 and JPC-2067.

The series of experiments described here indicate that (in vitro) CYP3A4 is the primary enzyme responsible for the activation of JPC-2056 to JPC-2067. On the basis of CYP3A4 inhibition studies with KTZ and anti-CYP3A4 antibodies, CYP3A4 appears to be responsible for approximately 70% of the production of JCP-2067 in pHLM. CYP2D6 may be responsible for as much as 20 to 30% of the remaining JPC-2067 formation, whereas CYP2C19 and perhaps CYP1A2 may play a minor role as well. Interestingly, although CYP3A4 also appeared to play the major role in the metabolism of the previous clinical candidate and structurally similar compound PS-15, some of the evidence suggests that CYP2C19 and particularly CYP2D6 were also important enzymes.

The primary purpose of this work was to identify the major P450 isoforms(s) involved in the in vitro metabolism of JPC-2056. The results presented here indicate clearly that CYP3A4 is the major enzyme involved in these reactions. Unlike its precursor, PG, the metabolic activation of JPC-2056 to JPC-2067 appears to be much less dependent on the activity of CYP2C19. If confirmed in vivo, these results could play an important role in the consideration of clinical studies and treatment using JPC-2056 in the future. When we consider the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, it is encouraging that the current clinical prodrug candidate from this series appears to be less dependent on these enzymes for metabolic activation. In addition, CYP3A4 is generally expressed in human liver and gut at much higher concentrations than CYP2C19. The in vitro experiments presented here indicate that JPC-2056 may be converted at a 3- to 4-fold higher rate than PG perhaps as a result of higher levels of CYP3A4 in the pHLM. It will be interesting to observe how this difference will affect the pharmacokinetics of JPC-2056 and its active metabolite JPC-2067 with respect to PG in the clinical setting. However, in light of the apparent importance of CYP3A4, possible drug-drug interactions with potent CYP3A4 inhibitors will have to be considered in treatment with JPC-2056.

	Footnotes

 
This material has been reviewed by the Walter Reed Army Institute of Research, and there is no objection to its publication. The opinions or assertions contained herein are the personal views of the authors and are not to be construed as official or reflecting the views of the Department of the U.S. Army or the U.S. Department of Defense.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.013920.

ABBREVIATIONS: PG, proguanil; CG, cycloguanil; pfDHFR-TS, Plasmodium falciparum dihydrofolate reductase-thymidylate synthase; pHLM, pooled human liver microsomes; G6P, glucose-6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PAR, peak area ratio; QC, quality control; TCP, ticlopidine; KTZ, ketoconazole; QDN, quinidine.

¹ Current affiliation: GlaxoSmithKline, Inc., Research Triangle Park, NC 27709.

Address correspondence to: Dr. Todd W. Shearer, GlaxoSmithKline, Inc., Metabolic CEDD DMPK, 5 Moore Drive, MAIN A3409, Research Triangle Park, NC 27709. E-mail: todd.shearer{at}yahoo.com

	References

Top Abstract Materials and Methods Results Discussion References

 


Baldwin SJ, Bloomer JC, Smith GJ, Ayrton AD, Clarke SE, and Chenery RJ (1995) Ketoconazole and sulphaphenazole as the respective selective inhibitors of P4503A and 2C9. Xenobiotica 25: 261–270.[Medline]

Birkett DJ, Rees D, Andersson T, Gonzalez FJ, Miners JO, and Veronese ME (1994) In vitro PG activation to cycloguanil by human liver microsomes is mediated by CYP3A isoforms as well as by S-mephenytoin hydroxylase. Br J Clin Pharmacol 37: 413–420.[Medline]

Biswas S (2001) Plasmodium falciparum dihydrofolate reductase Val-16 and Thr-108 mutation associated with in vivo resistance to antifolate drug: a case study. Indian J Malariol 38: 76–83.[Medline]

Canfield CJ, Milhous WK, Ager AL, Rossan RN, Sweeney TR, Lewis NJ, and Jacobus DP (1993) PS-15: a potent, orally active antimalarial from a new class of folic acid antagonists. Am J Trop Med Hyg 49: 121–126.[Abstract/Free Full Text]

Coller JK, Somogyi AA, and Bochner F (1999) Comparison of (S)-mephenytoin and proguanil oxidation in vitro: contribution of several CYP isoforms. Br J Clin Pharmacol 48: 158–167.[CrossRef][Medline]

Curtis J, Maxwell CA, Msuya FH, Mkongewa S, Alloueche A, and Warhurst DC (2002) Mutations in dhfr in Plasmodium falciparum infections selected by chlorproguanil-dapsone treatment. J Infect Dis 186: 1861–1864.[CrossRef][Medline]

Delfino RT, Santos-Filho OA, and Figueroa-Villar JD (2002) Molecular modeling of wild-type and antifolate resistant mutant Plasmodium falciparum DHFR. Biophys Chem 98: 287–300.[CrossRef][Medline]

Desta Z, Zhao X, Shin JG, and Flockhart DA (2002) Clinical significance of the cytochrome P450 2C19 genetic polymorphism. Clin Pharmacokinet 41: 913–958.[CrossRef][Medline]

Eskandarian AA, Keshavarz H., Basco LK, and Mahboudi F (2002) Do mutations in Plasmodium falciparum dihydropteroate synthase and dihydrofolate reductase confer resistance to sulfadoxine-pyrimethamine in Iran? Trans R Soc Trop Med Hyg 96: 96–98.[CrossRef][Medline]

Funck-Brentano C, Becquemont L, Lenevu A, Roux A, Jaillon P, and Beaune P (1997) Inhibition by omeprazole of proguanil metabolism: mechanism of the interaction in vitro and prediction of in vivo results from the in vitro experiments. J Pharmacol Exp Ther 280: 730–738.[Abstract/Free Full Text]

Ha-Duong NT, Dijols S, Macherey AC, Goldstein JA, Dansette PM, and Mansuy D (2001) Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19. Biochemistry 40: 12112–12122.[CrossRef][Medline]

Hastings MD and Sibley CH (2002) Pyrimethamine and WR99210 exert opposing selection on dihydrofolate reductase from Plasmodium vivax. Proc Natl Acad Sci U S A 99: 13137–13141.[Abstract/Free Full Text]

Helsby NA, Ward SA, Howells RE, and Breckenridge AM (1990) In vitro metabolism of the biguanide antimalarials in human liver microsomes: evidence for a role of the mephenytoin hydroxylase (P450 MP) enzyme. Br J Clin Pharmacol 30: 287–291.[Medline]

Jensen NP, Ager AL, Bliss RA, Canfield CJ, Kotecka BM, Rieckmann KH, Terpinski J, and Jacobus DP (2001) Phenoxypropoxybiguanides, prodrugs of DHFR-inhibiting diaminotriazine antimalarials. J Med Chem 44: 3925–3931.[CrossRef][Medline]

Jurima M, Inaba T, Kadar D, and Kalow W (1985) Genetic polymorphism of mephenytoin p(4')-hydroxylation: difference between Orientals and Caucasians. Br J Clin Pharmacol 19: 483–487.[Medline]

Kinyanjui SM, Mberu EK, Winstanley PA., Jacobus DP, and Watkins WM (1999) The antimalarial dihydrotriazine WR99210 and the prodrug PS-15: folate reversal of in vitro activity against Plasmodium falciparum and a non-antifolate mode of action of the prodrug. Am J Trop Med Hyg 60: 943–947.[Abstract]

Knight DJ, Mamalis P, and Peters W (1982) The antimalarial activity of N-benzyloxydihydrotriazines. III. The activity of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichloropropyloxy)-1,3,5-triazine hydrobromide (BRL 51084) and hydrochloride (BRL 6231). Ann Trop Med Parasitol 76: 1–7.[Medline]

Ko JW, Desta Z, Soukhova NV, Tracy T, and Flockhart DA (2000) In vitro inhibition of the cytochrome P450 (CYP450) system by the antiplatelet drug ticlopidine: potent effect on CYP2C19 and CYP2D6. Br J Clin Pharmacol 49: 343–351.[CrossRef][Medline]

Lu AH, Shu Y, Huang SL, Wang W, Ou-Yang DS, and Zhou HH (2000) In vitro proguanil activation to cycloguanil is mediated by CYP2C19 and CYP3A4 in adult Chinese liver microsomes. Acta Pharmacol Sin 21: 747–752.[Medline]

Milhous WK, Weatherly NF, Bowdre JH, and Desjardins RE (1985) In vitro activities of and mechanisms of resistance to antifol antimalarial drugs. Antimicrob Agents Chemother 27: 525–530.[Abstract/Free Full Text]

Otton SV, Inaba T, and Kalow W (1984) Competitive inhibition of sparteine oxidation in human liver by β-adrenoceptor antagonists and other cardiovascular drugs. Life Sci 34: 73–80.[CrossRef][Medline]

Peterson DS, Milhous WK, and Wellems TE (1990) Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria. Proc Natl Acad Sci U S A 87: 3018–3022.[Abstract/Free Full Text]

Phillips-Howard PA and Wood D (1996) The safety of antimalarial drugs in pregnancy. Drug Saf 14: 131–145.[Medline]

Rastelli G, Sirawaraporn W, Sompornpisut P, Vilaivan T, Kamchonwongpaisan S, Quarrell R, Lowe G, Thebtaranonth Y, and Yuthavong Y (2000) Interaction of pyrimethamine, cycloguanil, WR99210 and their analogues with Plasmodium falciparum dihydrofolate reductase: structural basis of antifolate resistance. Bioorg Med Chem 8: 1117–1128.[CrossRef][Medline]

Rieckmann KH (1973) The in vitro activity of experimental antimalarial compounds against strains of Plasmodium falciparum with varying degrees of sensitivity to pyrimethamine and chloroquine, in Chemotherapy of Malaria and Resistance to Antimalarials, World Health Organization Technical Report Series. World Health Organization, Geneva, Switzerland.

Rieckmann KH, Yeo AE, and Edstein MD (1996) Activity of PS-15 and its metabolite, WR99210, against Plasmodium falciparum in an in vivo-in vitro model. Trans R Soc Trop Med Hyg 90: 568–571.[CrossRef][Medline]

Salsali M, Holt A, and Baker GB (2004) Inhibitory effects of the monoamine oxidase inhibitor tranylcypromine on the cytochrome P450 enzymes CYP2C19, CYP2C9, and CYP2D6. Cell Mol Neurobiol 24: 63–76.[CrossRef][Medline]

Shearer TW, Kozar MP, O'Neil MT, Smith PL, Schiehser GA, Jacobus DP, Diaz DS, Yang YS, Milhous WK, and Skillman DR (2005) In vitro metabolism of phenoxypropoxybiguanide analogues in human liver microsomes to potent antimalarial dihydrotriazines. J Med Chem 48: 2805–2813.[CrossRef][Medline]

Taavitsainen P, Juvonen R, and Pelkonen O (2001) In vitro inhibition of cytochrome P450 enzymes in human liver microsomes by a potent CYP2A6 inhibitor, trans-2-phenylcyclopropylamine (tranylcypromine), and its nonamine analog, cyclopropylbenzene. Drug Metab Dispos 29: 217–222.[Abstract/Free Full Text]

Wang JS, Wen X, Backman JT, Taavitsainen P, Neuvonen PJ, and Kivisto KT (1999) Midazolam -hydroxylation by human liver microsomes in vitro: inhibition by calcium channel blockers, itraconazole and ketoconazole. Pharmacol Toxicol 85: 157–161.[Medline]

Warhurst DC (1999) Drug resistance in Plasmodium falciparum malaria. Infection 27: S55–58.[CrossRef][Medline]

Warhurst DC (2002) Resistance to antifolates in Plasmodium falciparum, the causative agent of tropical malaria. Sci Prog 85: 89–111.[Medline]

Watkins WM, Sixsmith DG, and Chulay JD (1984) The activity of proguanil and its metabolites, cycloguanil and p-chlorophenylbiguanide, against Plasmodium falciparum in vitro. Ann Trop Med Parasitol 78: 273–278.[Medline]

Wilkinson GR, Guengerich FP, and Branch RA (1989) Genetic polymorphism of S-mephenytoin hydroxylation. Pharmacol Ther 43: 53–76.[CrossRef][Medline]

Wright JD, Helsby NA, and Ward SA (1995) The role of S-mephenytoin hydroxylase (CYP2C19) in the metabolism of the antimalarial biguanides. Br J Clin Pharmacol 39: 441–444.[Medline]

Yeo AE, Edstein MD, Shanks GD, and Rieckmann KH (1994) A statistical analysis of the antimalarial activity of proguanil and cycloguanil in human volunteers. Ann Trop Med Parasitol 88: 587–594.[Medline]

Yuthavong Y (2002) Basis for antifolate action and resistance in malaria. Microbes Infect 4: 175–182.[CrossRef][Medline]

Yuvaniyama J, Chitnumsub P, Kamchonwongpaisan S, Vanichtanankul J, Sirawaraporn W, Taylor P, Walkinshaw MD, and Yuthavong Y (2003) Insights into antifolate resistance from malarial DHFR-TS structures. Nat Struct Biol 10: 357–365.[CrossRef][Medline]

Zhang W, Kilicarslan T, Tyndale RF, and Sellers EM (2001) Evaluation of methoxsalen, tranylcypromine, and tryptamine as specific and selective CYP2A6 inhibitors in vitro. Drug Metab Dispos 29: 897–902.[Abstract/Free Full Text]

Zhang W, Ramamoorthy Y, Kilicarslan T, Nolte H, Tyndale RF, and Sellers EM (2002) Inhibition of cytochromes P450 by antifungal imidazole derivatives. Drug Metab Dispos 30: 314–318.[Abstract/Free Full Text]

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