Pafuramidine is a novel orally active antimalarial. To identify a combination partner, we measured the in vitro antimalarial activities of the active metabolite, DB75, with amodiaquine, artemisinin, atovaquone, azithromycin, chloroquine, clindamycin, mefloquine, piperaquine, pyronaridine, tafenoquine, and tetracycline. None of the drugs tested demonstrated antagonistic or synergistic activity in combination with pafuramidine

Meshnick, Steven R.

Purfield, Anne E.

Tidwell, Richard R.

PubMed

Purfield, A. E.

Tidwell, R. R.

Meshnick, S. R.

Carolina Digital Repository

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2008, p. 2253–2255 Vol. 52, No. 60066-4804/08/$08.000 doi:10.1128/AAC.01536-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.Interactions of DB75, a Novel Antimalarial Agent, with OtherAntimalarial Drugs In VitroAnne E. Purfield, Richard R. Tidwell, and Steven R. Meshnick*Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina 27599-7435Received 28 November 2007/Returned for modification 20 January 2008/Accepted 11 March 2008Pafuramidine is a novel orally active antimalarial. To identify a combination partner, we measured the invitro antimalarial activities of the active metabolite, DB75, with amodiaquine, artemisinin, atovaquone,azithromycin, chloroquine, clindamycin, mefloquine, piperaquine, pyronaridine, tafenoquine, and tetracycline.None of the drugs tested demonstrated antagonistic or synergistic activity in combination with pafuramidine.Pafuramidine (DB289), 2,5-bis-(4-amidinophenyl)furan-bis-O-methylamidoxime, is a promising new orally active antipar-asitic compound (9). A recent clinical study found that pafura-midine (200 mg/kg of body weight/day for 5 days) was effectivefor 96% (22 of 23) of Plasmodium falciparum-infected patients(9). In vivo, pafuramidine is metabolized into the active me-tabolite 2,5-bis-(4-amidinophenyl)furan (DB75) (10, 11). Pre-vious studies have shown that DB75 is effective against P.falciparum, Plasmodium vivax, Pneumocystis jirovecii, Trypano-soma brucei, and Leishmania spp. in vitro and in animal models(2, 7).Currently, the WHO recommends that all new antimalarialdrugs be used in combination with a second antimalarial drugto prevent the development of resistance to monotherapy. Thetwo partnered drugs cannot be antagonistic and should ideallybe synergistic (8). The purpose of this study was to characterizethe interactions of the active metabolite of pafuramidine,DB75, with potential antimalarial partner drugs.To measure in vitro drug sensitivity, we used 42-h microdi-lution checkerboard growth assays and cultured P. falciparum3D7 parasites as previously described (4). Serial dilutions ofDB75 were tested in combination with serial dilutions of eachpartner drug: amodiaquine, artemisinin, atovaquone, azithro-mycin, chloroquine, clindamycin, mefloquine, piperaquine,pyronaridine, tafenoquine, and tetracycline (3). Drug concen-trations ranged from 5- to 10-fold above and below the prede-termined 50% inhibitory concentration (IC50). For each part-ner drug, the assay was repeated two or three times. Theatovaquone-proguanil combination was used to validate themethod and to determine the threshold of the sum of frac-tional inhibitory concentrations (FIC) for synergism.Additionally, 66-h and 96-h exposure periods were used forselect drugs (tetracycline, clindamycin, azithromycin, and taf-enoquine) with long onsets in vitro. To measure growth in 66 h,the methods described above were followed except that a 0.4%parasite population was used and [3H]hypoxanthine was addedfor the final 18 h of incubation. Ring cultures synchronizedwith 0.3% sorbitol were used for the 96-h assay, and [3H]hy-poxanthine was added for the final 48 h.Results were expressed as FIC and as the mean sums of theFIC. DB75–partner drug interactions were defined as eitherindifferent (linear points on isobolograms; sum of FIC  1),synergistic (concave curves on isobolograms; sum of FIC 0.63) (atovaquone and proguanil), or antagonistic (convexcurves on isobolograms; sum of FIC  2.0) (1, 3).To find a potential partner for DB75, the compound wastested in combination with 11 current and investigational an-timalarials. DB75 manifested indifferent relationships with 10of the drugs tested in the 42-h assay by both the FIC andsum-of-FIC methods (Fig. 1; Table 1). As a positive control,atovaquone and proguanil showed clear synergy (sum of FIC 0.63).Three of these drugs (tetracycline, tafenoquine, andazithromcycin) also showed indifference even with extendedincubations (Fig. 1F, D, and B, respectively; Table 1).The clindamycin-DB75 combination was indifferent at 96 h(Fig. 1L; Table 1). As previously reported, clindamycin aloneexhibited a biphasic dose response by the 42- and 66-h assays(data not shown) (5). Complete inhibition was not achieved* Corresponding author. Mailing address: Department of Epidemi-ology, CB 7435, University of North Carolina, Chapel Hill, NC 27599-7435. Phone: (919) 966-7414. Fax: (919) 966-2089. E-mail: meshnick@unc.edu. Published ahead of print on 24 March 2008.TABLE 1. Results for antimalarial agents in combinationwith DB75Partner drugFIC (sum of FIC)a from the following assay:42-h 66-h 96-hClindamycin N/A (N/A) N/A (N/A) I (1.09  0.16)bTetracycline I (0.88  0.08) I (1.04  0.19)Atovaquone I (0.94  0.14)Tafenoquine I (1.00  0.06) I (1.05  0.06)Mefloquine I (1.08  0.14)Amodiaquine I (1.09  0.03)Artemisinin I (1.19  0.15)Piperaquine I (1.23  0.04)Azithromycin I (1.20  0.18) I (1.29  0.13)Pyronaridine I (1.20  0.02)Chloroquine I (1.31  0.12)Atovaquone andproguanilS (0.63)a FIC sums are reported as means  standard deviations from two to threeassays. S, synergistic; I, indifferent; N/A, not applicable. Mean DB75 IC50s standard deviations are 128  51 nM at 42 h, 139  43 nM at 66 h, and 3.7 0.76 nM at 96 h.b Synchronized rings were used for the 96 h assay.2253with clindamycin at these times, even with hemolytic concen-trations greater than 100 M; therefore, no IC50 could beextrapolated for the 42- or 66-h assay. However, at 96 h, asigmoidal dose response was evident for clindamycin alone;therefore, an FIC could be determined.In summary, the lack of interaction of DB75 with eitherchloroquine or atovaquone suggests that DB75 does not inter-act with hemozoin or mitochondria in the parasite, as has beenpreviously suggested (6). These data also suggest that pafura-midine could be successfully partnered with any of the 11antimalarials tested here. Follow-up studies in animal modelsare needed.Funding for this work was provided by Medicines for Malaria Ven-ture and the Bill and Melinda Gates Foundation.We acknowledge Carla Hand, Jesse Kwiek, Jeff Frelinger, CarrieBarnes, David Klapper, Stephanie Wallace, and Jaina Patel for tech-nical assistance.REFERENCES1. Berenbaum, M. C. 1978. A method for testing for synergy with any numberof agents. J. Infect. Dis. 137:122–130.2. Brendle, J. J., A. Outlaw, A. Kumar, D. W. Boykin, D. A. Patrick, R. R.Tidwell, and K. A. Werbovetz. 2002. Antileishmanial activities of severalclasses of aromatic dications. Antimicrob. Agents Chemother. 46:797–807.3. Canfield, C. J., M. Pudney, and W. E. Gutteridge. 1995. Interactions ofatovaquone with other antimalarial drugs against Plasmodium falciparum invitro. Exp. Parasitol. 80:373–381.4. Desjardins, R. E., C. J. Canfield, J. D. Haynes, and J. D. Chulay. 1979.Quantitative assessment of antimalarial activity in vitro by a semiautomatedmicrodilution technique. Antimicrob. Agents Chemother. 16:710–718.5. Seaberg, L. S., A. R. Parquette, I. Y. Gluzman, G. W. Phillips, Jr., T. F.Brodasky, and D. J. Krogstad. 1984. Clindamycin activity against chloro-quine-resistant Plasmodium falciparum. J. Infect. Dis. 150:904–911.6. Stead, A. M., P. G. Bray, I. G. Edwards, H. P. DeKoning, B. C. Elford, P. A.Stocks, and S. A. Ward. 2001. Diamidine compounds: selective uptake andtargeting in Plasmodium falciparum. Mol. Pharmacol. 59:1298–1306.7. Tidwell, R. R., S. K. Jones, J. D. Geratz, K. A. Ohemeng, C. A. Bell, B. J.Berger, and J. E. Hall. 1990. Development of pentamidine analogues as newagents for the treatment of Pneumocystis carinii pneumonia. Ann. N. Y.Acad. Sci. 616:421–441.FIG. 1. Isobolograms of DB75–partner drug interactions. (A, C, E, and G to K) Growth was measured by using 42-h [3H]hypoxanthine growthassays for all drugs tested in combination with DB75. (A) Atovaquone and proguanil were used in combination to show true synergy at 42 h. (B,D, and F) Azithromycin (B), tafenoquine (D), and tetracycline (F) were also tested in combination with DB75 for 66 h. (L) Clindamycin was testedat 96 h. A concave isobologram (A) indicates synergy; a convex one (none shown) indicates antagonism; and points along a straight line (B to L)indicate indifference.2254 NOTES ANTIMICROB. AGENTS CHEMOTHER.8. WHO. 2001. Antimalarial drug combination therapy. World Health Organi-zation, Geneva, Switzerland.9. Yeramian, P., S. R. Meshnick, S. Krudsood, K. Chalermrut, U. Silacham-roon, N. Tangpukdee, J. Allen, R. Brun, J. J. Kwiek, R. Tidwell, and S.Looareesuwan. 2005. Efficacy of DB289 in Thai patients with Plasmodiumvivax or acute, uncomplicated Plasmodium falciparum infections. J. Infect.Dis. 192:319–322.10. Zhou, L., K. Lee, D. R. Thakker, D. W. Boykin, R. R. Tidwell, and J. E. Hall.2002. Enhanced permeability of the antimicrobial agent 2,5-bis(4-amidino-phenyl)furan across Caco-2 cell monolayers via its methylamidoxime pro-drug. Pharm. Res. 19:1689–1695.11. Zhou, L., D. R. Thakker, R. D. Voyksner, M. Anbazhagan, D. W. Boykin,J. E. Hall, and R. R. Tidwell. 2004. Metabolites of an orally active antimi-crobial prodrug, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime,identified by liquid chromatography/tandem mass spectrometry. J. MassSpectrom. 39:351–360.VOL. 52, 2008 NOTES 2255

Interactions of DB75, a Novel Antimalarial Agent, with Other Antimalarial Drugs In Vitro

Pafuramidine is a novel orally active antimalarial. To identify a combination partner, we measured the in vitro antimalarial activities of the active metabolite, DB75, with amodiaquine, artemisinin, atovaquone, azithromycin, chloroquine, clindamycin, mefloquine, piperaquine, pyronaridine, tafenoquine, and tetracycline. None of the drugs tested demonstrated antagonistic or synergistic activity in combination with pafuramidine. Pafuramidine (DB289), 2,5-bis-(4-amidinophenyl)furan-bis-O-methylamidoxime, is a promising new orally active antipar-asitic compound (9). A recent clinical study found that pafura-midine (200 mg/kg of body weight/day for 5 days) was effective for 96 % (22 of 23) of Plasmodium falciparum-infected patients (9). In vivo, pafuramidine is metabolized into the active me-tabolite 2,5-bis-(4-amidinophenyl)furan (DB75) (10, 11). Pre-vious studies have shown that DB75 is effective against P

Anne E. Purfield

Richard R. Tidwell

Steven R. Meshnick

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Interactions of DB75, a Novel Antimalarial Agent, with Other Antimalarial Drugs In Vitro▿

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