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Background In Thailand, HIV transmission is well characterized in large urban centers such as Bangkok and Chiang Mai but less so outside of these areas. We assessed HIV incidence and associated risk factors in two other locations. Methods Participants assigned male sex at birth were enrolled in Nakhon Ratchasima and Ratchaburi. HIV and syphilis testing and sociobehavioral questionnaires occurred over 18 months. HIV incidence rates and 95% confidence intervals (CIs) were estimated using a Poisson distribution. Cox proportional hazards models were used to estimate unadjusted and adjusted hazard ratios (aHRs) and 95% CIs for associations between potential risk factors and HIV seroconversion. Results A total of 1003 participants were enrolled. Overall HIV incidence was 1.56 per 100 person-years (95% CI:1.02–2.44) and similar at both sites. In the fully adjusted model, receptive anal sex in the past 6 months was associated with seroconversion (aHR:3.05, 95% CI:1.00-9.32), while sex with a sex worker in the past six months was associated with reduced risk of seroconversion (aHR:0.11, 95% CI:0.01–0.80). In the parsimoniously adjusted model, receptive anal sex (aHR:3.35, 95% CI:1.30–8.63) and STI diagnosis in the past six months (aHR:3.39, 95% CI:1.11–10.29) were associated with seroconversion, while sex with a sex worker in the past six months was associated with reduced risk of seroconversion (aHR:0.12, 95% CI:0.02–0.70). Conclusions Recent receptive anal sex practices were associated with HIV acquisition in these populations, highlighting the continued need for interventions encouraging safer anal sex practices to reduce HIV incidence.

Background: In order to assess feasibility for future HIV prevention trials, we measured HIV incidence, retention, and assessed risk factors for seroconversion among men who have sex with men (MSM) and transgender women (TGW) in Bangkok, Thailand between 2017-2019. Methods: We conducted an 18-month prospective cohort study of HIV-uninfected Thai cisgender men and transgender women who reported sex with men in the past six months. HIV and syphilis testing and computer-based behavioral questionnaires were administered at each visit. We utilized Poisson regression to calculate HIV incidence rates. A survival random forest model identified the most predictive risk factors for HIV seroconversion and then used in a survival regression tree model to elucidate hazard ratios for individuals with groups of selected risk factors. Cox proportional hazards (PH) regression evaluated the strength of association between individual covariates and risk of seroconversion. Findings: From April 2017-October 2019, 1,184 participants were screened, 167 were found ineligible, and 1,017 enrolled. Over the 18-month study, visit retention was 93·4% (95% CI 91·6%-94·8%) and HIV incidence was 3·73 per 100 person-years (95% CI 2·79-5·87). Utilizing survival regression tree modeling, those who were 18-20 years of age, reported sexual attraction to mostly or only men, and had five or more lifetime sexual partners were 4·9 times more likely to seroconvert compared to other cohort participants. Factors associated with HIV incidence utilizing Cox PH regression included sexual attraction to mostly or only men (adjusted hazard ratio (aHR) 14·9 (95% CI 20·1-107·9), younger age (18-19 years, aHR 10·88 (95% CI 4·12-28·7), five or greater lifetime sexual partners (aHR 2·0, 95%CI 1·1-3·6), inconsistent condom use with casual partners (aHR 2·43, 95% CI 1·3-4·5), and prior HIV testing (adjusted HR 2·0, 95% CI 1·1-3·5). Interpretation: HIV incidence remains high among Bangkok-based MSM and TGW. These key populations have high retention and are ideal candidates for efficacy evaluation of future prevention strategies. Funding Statement: This work was supported by the US Army Medical Research and Materiel Command (Military Infectious Diseases Research Program) (cooperative agreements W81XWH-11-2-0174 and W81XWH-07-2-0067 with the Henry M. Jackson Foundation for the Advancement of Military Medicine), the US Army Medical Materiel Development Activity, and the National Institute of Allergy and Infectious Disease (interagency agreement Y1-AI-2642-12 with the US Army Medical Research and Materiel Command) and the Division of AIDS Interagency Agreements (DAIDS IAA Y1-AI-2642-16). Declaration of Interests: T.C. reports grants from U.S. Army, grants from NIH, M.R. and S.V. reports grants from US Army Medical Research and Materiel Command with Henry M. Jackson Foundation for the Advancement of Military Medicine, during the conduct of the study. All other authors report no potential conflicts. Ethics Approval Statement: The study was reviewed and approved by the institutional review boards at Walter Reed Army Institute of Research (WRAIR), Faculty of Tropical Medicine at Mahidol University, and the Royal Thai Army. All participants provided written informed consent prior to enrollment.

Background We measured Human Immunodeficiency (HIV) incidence, retention, and assessed risk factors for seroconversion among two previously unreported cohorts of men who have sex with men (MSM) and Transgender Women (TGW) in Bangkok, Thailand between 2017 and 2019. Methods We conducted an 18-month prospective cohort study of HIV-uninfected Thai cisgender men and TGW aged between 18 and 35 years who reported sex with men in the past six months and at least one additional risk factor for HIV infection. HIV and syphilis testing and computer-based behavioral questionnaires were administered at each visit. We utilized Poisson regression to calculate HIV incidence rates. A survival random forest model identified the most predictive risk factors for HIV sero-conversion and then used in a survival regression tree model to elucidate hazard ratios for individuals with groups of selected risk factors. Cox proportional hazards (pH) regression evaluated the strength of association between individual covariates and risk of sero-conversion. Findings From April 2017-October 2019, 1,184 participants were screened, 167 were found ineligible, and 1,017 enrolled. Over the 18-month study, visit retention was 93·4% (95% CI 91·6%–94·8%) and HIV incidence was 3·73 per 100 person-years (95% CI 2·79–5·87). Utilizing survival regression tree modeling, those who were 18–20 years of age, reported sexual attraction to mostly or only men, and had five or more lifetime sexual partners were 4·9 times more likely to seroconvert compared to other cohort participants. Factors associated with HIV incidence utilizing Cox pH regression included sexual attraction to mostly or only men (adjusted hazard ratio (aHR) 14·9 (95% CI 20·1–107·9), younger age (18–19 years, aHR 10·88 (95% CI 4·12–28·7), five or greater lifetime sexual partners (aHR 2·0, 95%CI 1·1–3·6), inconsistent condom use with casual partners (aHR 2·43, 95% CI 1·3–4·5), and prior HIV testing (adjusted HR 2·0, 95% CI 1·1–3·5). Interpretation Interpretation HIV incidence remains high among Bangkok-based MSM and TGW. These key populations expressed high interest in participating in efficacy evaluation of future prevention strategies and had high retention in this 18 month study. Funding Funding US National Institute of Allergy and Infectious Diseases (NIAID), Division of AIDS Interagency Agreements (DAIDS) and U.S. Department of the Army.

The worst Ebola virus disease (EVD) outbreak in history has resulted in more than 28,000 cases and 11,000 deaths. We present the final results of two phase 1 trials of an attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV)-based vaccine candidate designed to prevent EVD.We conducted two phase 1, placebo-controlled, double-blind, dose-escalation trials of an rVSV-based vaccine candidate expressing the glycoprotein of a Zaire strain of Ebola virus (ZEBOV). A total of 39 adults at each site (78 participants in all) were consecutively enrolled into groups of 13. At each site, volunteers received one of three doses of the rVSV-ZEBOV vaccine (3 million plaque-forming units [PFU], 20 million PFU, or 100 million PFU) or placebo. Volunteers at one of the sites received a second dose at day 28. Safety and immunogenicity were assessed.The most common adverse events were injection-site pain, fatigue, myalgia, and headache. Transient rVSV viremia was noted in all the vaccine recipients after dose 1. The rates of adverse events and viremia were lower after the second dose than after the first dose. By day 28, all the vaccine recipients had seroconversion as assessed by an enzyme-linked immunosorbent assay (ELISA) against the glycoprotein of the ZEBOV-Kikwit strain. At day 28, geometric mean titers of antibodies against ZEBOV glycoprotein were higher in the groups that received 20 million PFU or 100 million PFU than in the group that received 3 million PFU, as assessed by ELISA and by pseudovirion neutralization assay. A second dose at 28 days after dose 1 significantly increased antibody titers at day 56, but the effect was diminished at 6 months.This Ebola vaccine candidate elicited anti-Ebola antibody responses. After vaccination, rVSV viremia occurred frequently but was transient. These results support further evaluation of the vaccine dose of 20 million PFU for preexposure prophylaxis and suggest that a second dose may boost antibody responses. (Funded by the National Institutes of Health and others; rVSV∆G-ZEBOV-GP ClinicalTrials.gov numbers, NCT02269423 and NCT02280408 .).

Background: The RV144 phase 3 vaccine trial in Thailand demonstrated that ALVAC-HIV (vCP1521) and AIDSVAX® B/E administration over six months resulted in a 31% efficacy in preventing HIV acquisition. Subsequent boosting of RV144 vaccine recipients 6 to 8 years later improved the magnitude and quality of immune responses, expanding subdominant memory B cells. Methods: RV306 is a double-blind, placebo-controlled, randomized clinical trial of the primary RV144 vaccine series with additional AIDSVAX® B/E with or without ALVAC-HIV boosting at months 12, 15, or 18 in healthy volunteers. Findings: Late boosting was safe, and improved antigen-specific plasma IgG responses to HIV-1 envelope and the first and second variable loops (V1V2), an inverse correlate of HIV risk in RV144, without increasing HIV-specific plasma IgA, a direct correlate of risk. Late boosting improved Tier 1 neutralization and HIV-1 envelope-specific CD4+ T cell polyfunctionality, an independent correlate contributing to RV144 vaccine efficacy. Late boosting was also required to maintain antigen-specific CD4+ T cell proliferation. Interestingly, increasing the interval between priming and boosting improved several humoral and cellular immune responses. Interpretation: Taken together, these results suggest that additional boosting of the RV144 regimen with longer intervals between the primary vaccination series and late boost improved immune responses and may improve the efficacy of preventing HIV acquisition. Funding Statement: Cooperative Agreement # W81XWH-18-2-0040 in collaboration with the US Army and DAIDS/NIAID/NIH. Declaration of Interests: J. T., and C. D. are employees of Sanofi Pasteur. S.P. was an employee of Sanofi Pasteur during the conduct of this study and analysis. F. S. is an employee of Global Solutions for Infectious Diseases. All other authors report no potential conflicts. The opinions expressed herein are those of the authors and do not represent the official position of the US Army or the Department of Defense. Trade names are used for identification purposes only and do not imply endorsement. Ethics Approval Statement: The study was approved by ethical review boards at the Walter Reed Army Institute of Research, Thai Ministry of Public Health, Royal Thai Army Medical Department, Faculty of Tropical Medicine, Mahidol University, Chiang Mai University, and Chulalongkorn University Faculty of Medicine. This study was conducted in accordance with Good Participatory Practice principles.

The triterpene quassinoid ailanthinone is a structurally intricate natural product possessing highly potent antimalarial activity against multidrug resistance plasmodium parasites. Although the mechanism of action of ailanthinone is not completely understood, it is presumed to disrupt regular ribosomal functions by inhibiting parasite protein synthesis. Natural scarcity and low solubility of many quassinoids have impeded their development as potential clinical candidates, but exquisite potency of ailanthinone against Plasmodium remains compelling in the global fight against malaria. Herein, we report the highly selective synthesis of 1-hydroxyl derivatives of ailanthinone, including ester, carbonate, carbamate and sulfonate derivatives. The key feature of the synthesis is a one-step regioselective modification of the 1-hydroxyl group in favor of two other hydroxyl groups at C12 and C13. Derivatives were obtained via direct reaction with acyl/sulfonyl chlorides in the presence of a tertiary amine base without any protection-deprotection. In vitro antimalarial evaluations of these derivatives were compared with chloroquine and mefloquine against the Plasmodium falciparum clones, D6, W2, and TM91C235. The results demonstrate that modification of the 1-hydroxyl group is achievable, and the antimalarial activity of these derivatives relative to the parent product is significantly retained, thus paving the way for synthesis of derivatives with improved biological availability and/or increased potency.

Azithromycin, a first member of the azalide family of macrolides, while having substantial antimalarial activity, failed as a single agent for malaria prophylaxis. In this paper we present the first analogue campaign to identify more potent compounds from this class. Ureas and thioureas of 15-membered azalides, N′′-substituted 9a-(N′-carbamoyl-β-aminoethyl), 9a-(N′- thiocarbamoyl-β-aminoethyl), 9a-[N′-(β- cyanoethyl)-N′-(carbamoyl-β-aminoethyl)], 9a-[N′- (β-cyanoethyl)-N′-(thiocarbamoyl-β-aminoethyl)], 9a-{; ; ; ; N′-[β-(ethoxycarbonyl)ethyl]-N′(carbamoyl-β- aminoethyl)}; ; ; ; , and 9a-[N′-(β-amidoethyl)-N′- (carbamoyl-β-aminoethyl)] of 9-deoxo-9-dihydro-9a- aza-9a-homoerythromycin A, were synthesized and their biological properties evaluated. The results obtained indicate a substantial improvement of the in vitro activity against P. falciparum (up to 88 times over azithromycin), particularly for compounds containing both sugars on the macrocyclic ring and aromatic moiety on 9a- position. The improved in vitro activity was not confirmed in the mouse model, likely due to an increase in lipophilicity of these analogues leading to a higher volume of distribution. Overall, with increased in vitro activity, promising PK properties, and modest in vivo efficacy, this series of molecules represents a good starting platform for the design of novel antimalarial azalides.

A series of 15-membered azalide urea and thiourea derivatives has been synthesized and evaluated for their in vitro antimalarial activity against chloroquine-sensitive (D6), chloroquine/pyremethamine resistant (W2) and multidrug resistant (TM91C235) strains of Plasmodium falciparum. We have developed an effective automated synthetic strategy for the rapid synthesis of urea/thiourea libraries of a macrolide scaffold. Compounds have been synthesized using a solution phase strategy with overall yields of 50-80%. Most of the synthesized compounds had inhibitory effects. The top 10 compounds were 30-65 times more potent than azithromycin, an azalide with antimalarial activity, against all three strains.

The synthesis of the chimeric molecules consisting of two pharmacophores, tetraoxane and 7-chloro-4-aminoquinoline, is reported. The tetraoxanes 2, 4, and 8 show relatively potent in vitro antimalarial activities, with IC90 values for the Plasmodium falciparum strain W2 of 2.26, 12.44, and 10.74 nM, respectively. In addition, two compounds, 2 and 4, cured mice in a modified Thompson test for antimalarial blood stage activity, with a minimum curative dose of 80 mg/kg, a minimum active dose of 20 mg/kg/day, and a maximum tolerated dose of gt 960 mg/kg.

The development of widespread drug resistance to chloroquine (CQ(a)) has resulted in severe health issues for countries in malaria endemic regions. The antimalarial properties of artemisinin 2 and of other peroxides, such as 1,2,4,5-tetraoxacy-cloalkanes (tetraoxanes), have recently begun to be exploited in the development of new approaches to fighting CQ-resistant strains of malaria. New tetraoxanes employing a steroidal backbone have now been prepared that are highly active, are inexpensive, and demonstrate low toXieity.(5,6) A part of our research in this field is focused on the development of a new type of tetraoxane with nonidentical substituents(6) that utilize a steroid and small cyclohexylidene carriers possessing secondary amide bonds. Also, during our work in this field we discovered that tetraoxanes are unusually stable, even. at pH 1.6,(6c) a characteristic that subsequently allowed the synthesis of many interesting derivatives. This communication encompasses the synthesis of various amino-functionalized antimalarials based on the appreciable stability of the tetraoxane moiety to reaction conditions such as reductive amination and LiAlH4 reduction. Their respective antimalarial activities and the pronounced antiproliferative activity of certain products are reported along with in vitro metabolism studies.

Several dicyclohexylidene tetraoxanes were prepared in order to gain a further insight into structure-activity relationship of this kind of antimalarials. The tetraoxanes 2-5, obtained as a cis/trans mixture, showed pronounced antimalarial activity against Plasmodium falciparum chloroquine susceptible D6, chloroquine resistant W2 and multidrug-resistant TM91C235 (Thailand) strains. They have better than or similar activity to the corresponding desmethyl dicyclohexylidene derivatives. Two chimeric endoperoxides with superior antimalarial activity to the natural product ascaridole were also synthesized.

Tetra-acetamide pyrroloquinazolinediamine (PQD-A4) and bis-ethylcarbamyl pyrroloquinazolinediamine (PQD-BE) are new derivatives of pyrroloquinazolinediamine (PQD) and are being investigated as potential chemotherapeutic agents for the treatment of malaria. Comparative studies to assess the therapeutic indices of PQD-A4, PQD-BE, and PQD were conducted in Plasmodium berghei -infected rats following daily intragastric dosing for three consecutive days. Artesunate (AS), a standard drug for treatment of severe malaria, was used as a comparator. The minimum doses required to clear malaria parasitemia were 156 μmol/kg of body weight for AS and 2.4 μmol/kg for PQD, PQD-4A, and PQD-BE. The maximum tolerated dose (MTD) of AS was 625 μmol/kg, and its therapeutic index was calculated to be 4. The MTDs of PQD-A4, PQD-BE, and PQD were found to be 190, 77, and 24 μmol/kg, respectively, yielding therapeutic indices of 80, 32, and 10, respectively. Although PQD-A4 and PQD-BE are only half as potent as PQD based on their curative effects, the two new derivatives, PQD-4A and PQD-BE, are 8.0-fold and 3.2-fold safer, respectively, than their parent compound when they are dosed for three consecutive days. Oral PQD-A4 and PQD-BE are 44 to 70 times more potent on an mg basis than intravenous AS. As assessed from the therapeutic index over 3 days, PQD-A4, PQD-BE, and PQD administered orally are 20.0, 8.0, and 2.5 times safer than AS given intravenously. The results indicate that PQD-4A is a promising candidate for antimalarial treatment.

Tryprostatin A 1 and B 2 are indole alkaloid-based fungal products that act in the G2/M phase of the cell cycle. Tryprostatin A and B as well as their two enantiomers and four diastereomers have been synthesized via a common strategy. As a measure of cytotoxicity, these eight stereoisomers were assayed for their growth inhibitory properties in human breast, prostate, and lung cancer cell lines. The ability of the tryprostatins and the tryprostatin stereoisomers to induce topoisomerase II-mediated DNA relaxation or to inhibit tubulin polymerization was also examined. Although none of the stereoisomers were significantly active in topoisomerase II- or tubulin-based assays, ds2-try B 11 was found to exhibit a cytotoxicity profile more potent than etoposide 3 in the human cancer cell lines examined. In addition, ds2-try B 11 is comprised of an L-tryptophan derivative coupled to a D-proline moiety, the latter stereochemistry of which may enhance the activity of 11 and potential analogues in vivo.

This study evaluated the pharmacokinetics of topical creams containing 15% paromomycin (“paromomycin alone”) and 15% paromomycin plus 0.5% gentamicin (WR 279,396) in patients with cutaneous leishmaniasis. The investigational creams were applied topically to all lesions once daily for 20 days. Plasma samples were analyzed for simultaneous quantitation of paromomycin and gentamicin isomers and total gentamicin. Pharmacokinetic parameters for gentamicin could not be calculated because detectable levels were rarely evident. After one application, the paromomycin area under the concentration-time curve from 0 to 24 h (AUC 0–24 ) was 2,180 ± 2,621 ng · h/ml (mean ± standard deviation [SD]) for the paromomycin-alone group and 975.6 ± 1,078 ng · h/ml for the WR 279,396 group. After 20 days of application, the paromomycin AUC 0–24 and maximum concentration of drug ( C max ) were 5 to 6 times greater than those on day 1 for both treatment groups. For the paromomycin-alone group, the AUC 0–24 was 8,575 ± 7,268 ng · h/ml and the C max was 1,000 ± 750 ng/ml, compared with 6,037 ± 3,956 ng · h/ml and 660 ± 486 ng/ml for the WR 279,396 group, respectively. Possibly due to large intersubject variability, no differences ( P ≥ 0.05) in the AUC 0–24 or C max were noted between treatment or between sites on day 1 or 20. The percentage of dose absorbed on day 20 was 12.0% ± 6.26% and 9.68% ± 6.05% for paromomycin alone and WR 279,396, respectively. Paromomycin concentrations in plasma after 20 days of application were 5 to 9% of those after intramuscular administration of 15 mg/kg of body weight/day to adults for the systemic treatment of visceral leishmaniasis. Effective topical treatment of cutaneous leishmaniasis appears to be possible with limited paromomycin and gentamicin systemic absorption, thus avoiding drug accumulation and toxicity. (The work described here has been registered at ClinicalTrials.gov under registration no. NCT01032382 and NCT01083576.)

International audience; BACKGROUND: There is a need for a simple and efficacious treatment for cutaneous leishmaniasis with an acceptable side-effect profile. METHODS: We conducted a randomized, vehicle-controlled phase 3 trial of topical treatments containing 15% paromomycin, with and without 0.5% gentamicin, for cutaneous leishmaniasis caused by Leishmania major in Tunisia. We randomly assigned 375 patients with one to five ulcerative lesions from cutaneous leishmaniasis to receive a cream containing 15% paromomycin-0.5% gentamicin (called WR 279,396), 15% paromomycin alone, or vehicle control (with the same base as the other two creams but containing neither paromomycin nor gentamicin). Each lesion was treated once daily for 20 days. The primary end point was the cure of the index lesion. Cure was defined as at least 50% reduction in the size of the index lesion by 42 days, complete reepithelialization by 98 days, and absence of relapse by the end of the trial (168 days). Any withdrawal from the trial was considered a treatment failure. RESULTS: The rate of cure of the index lesion was 81% (95% confidence interval [CI], 73 to 87) for paromomycin-gentamicin, 82% (95% CI, 74 to 87) for paromomycin alone, and 58% (95% CI, 50 to 67) for vehicle control (P

Novel classes of antimalarial drugs are needed due to emerging drug resistance. Azithromycin, the first macrolide investigated for malaria treatment and prophylaxis, failed as a single agent and thus novel analogues were envisaged as the next generation with improved activity. We synthesized 42 new 9a-N substituted 15-membered azalides with amide and amine functionalities via simple and inexpensive chemical procedures using easily available building blocks. These compounds exhibited marked advances over azithromycin in vitro in terms of potency against Plasmodium falciparum (over 100-fold) and high selectivity for the parasite and were characterized by moderate oral bioavailability in vivo. Two amines and one amide derivative showed improved in vivo potency in comparison to azithromycin when tested in a mouse efficacy model. Results obtained for compound 6u, including improved in vitro potency, good pharmacokinetic parameters, and in vivo efficacy higher than azithromycin and comparable to chloroquine, warrant its further development for malaria treatment and prophylaxis.

BACKGROUND: Injectable artesunate (AS) can cause fetal death and teratogenic effects in animals at levels below the human therapeutic dose. Similar toxicity has also been found for oral artemisinins in various animal species, but has not been found in humans. METHODS: Studies on tissue distribution (5 mg/kg) and toxicokinetics (TK, 30 mg/kg × 3) were conducted in pregnant (GD11–13) and non-pregnant rats. RESULTS: TK profiles of AS showed that the two groups of rats were similar after a single AS dose but were significantly different after multiple doses. Following a daily dose for 3 days, no change in AS concentration was found in the pregnant animals, but a significant concentration decline was seen in the non-pregnant rats on day 3. In addition, a higher conversion rate of AS to dihydroartemisinin (DHA) was detected in the pregnant rats after either single or multiple doses compared to non-pregnant controls. The ratios of AUCDHA/AUCAS were 0.99–1.02 for the pregnant rats and 0.42–0.48 for non-pregnant animals, resulting in a total AUCDHA D1–3 that was about 3.7-fold higher in pregnant rats (15,049 ng·h/ml) than in non-pregnant rats (4,015 ng·h/ml). Furthermore, the tissue/blood partition coefficients demonstrated that the level of radiolabeled AS that was distributed into uterus, placenta, and ovary was 2–4-fold higher than in blood. CONCLUSIONS: TK data showed that unchanged AS and DHA in the blood of pregnant rats were 1.53- and 3.74-fold, respectively, higher than those of non-pregnant animals, which all likely relate to the severe embryotoxicity of AS, even with a low-dosage regimen in pregnant animals. Birth Defects Res (Part B), 2008. Published 2008 Wiley-Liss, Inc.

Tryprostatin A is an inhibitor of breast cancer resistance protein, consequently a series of structure-activity studies on the cell cycle inhibitory effects of tryprostatin A analogues as potential antitumor antimitotic agents have been carried out. These analogues were assayed for their growth inhibition properties and their ability to perturb the cell cycle in tsFT210 cells. SAR studies resulted in the identification of the essential structural features required for cytotoxic activity. The absolute configuration L-Tyr-L-pro in the diketopiperazine ring along with the presence of the 6-methoxy substituent on the indole moiety of 1 was shown to be essential for dual inhibition of topoisomerase II and tubulin polymerization. Biological evaluation also indicated the presence of the 2-isoprenyl moiety on the indole scaffold of 1 was essential for potent inhibition of cell proliferation. Substitution of the indole N(a)-H in 1 with various alkyl or aryl groups, incorporation of various L-amino acids into the diketopiperazine ring in place of L-proline, and substitution of the 6-methoxy group in 1 with other functionality provided active analogues. The nature of the substituents present on the indole N(a)-H or the indole C-2 position influenced the mechanism of action of these analogues. Analogues 68 (IC(50)=10 microM) and 67 (IC(50)=19 microM) were 7-fold and 3.5-fold more potent, respectively, than 1 (IC(50)=68 microM) in the inhibition of the growth of tsFT210 cells. Diastereomer-2 of tryprostatin B 8 was a potent inhibitor of the growth of three human carcinoma cell lines: H520 (IC(50)=11.9 microM), MCF-7 (IC(50)=17.0 microM) and PC-3 (IC(50)=11.1 microM) and was equipotent with etoposide, a clinically used anticancer agent. Isothiocyanate analogue 71 and 6-azido analogue 72 were as potent as 1 in the tsFT210 cell proliferation and may be useful tools in labeling BCRP.

We report on the initial result of the coupling of 4-amino-7-chloroquinoline with steroidal and adamantane constituents to provide small molecules with excellent in vitro antimalarial activities (IC90 (W2) down to 6.74 nM). The same entities also inhibit the botulinum neurotoxin serotype A light chain metalloprotease at low micromolar levels (7-31 microM). Interestingly, structural features imparting increased antimalarial activity also provide increased metalloprotease inhibition, thus allowing for simultaneous compound optimizations against distinct targets.

Eleven new tetraoxanes possessing cholic acid-derived carrier and isopropylidene moiety were synthesized and were tested in vitro and in vivo. In vitro screening revealed that nine of them were more potent against CQ-resistant W2 than CQ-susceptible D6 strain and that two of them were equally or more potent than artemisinin and mefloquine against multi- drug resistant TM91C235 strain. Amine 8 cured all mice at the dose of 160 mg/kg/day, while the anilide 9 exhibited MCD lt = 20 mg/kg/day. The diol 13 was most potent antiproliferative with GI(50), TGI, LC50 MG_MID 0.98 mu M, 3.80 mu M, 11.22 mu M, respectively. All tested compounds showed no toxic effects. (c) 2008 Elsevier Ltd. All rights reserved.

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.

A series of acid-stable carboxamide derivatives of 2-guanidinoimidazolidinedione (5a-c and 6a-c) were prepared as potential malaria prophylactic and radical cure agents. The new compounds showed moderate to good causal prophylactic activity in mice infected with Plasmodium yoelii sporozoites. Three compounds were further tested for causal prophylactic activity in Rhesus monkeys infected with Plasmodium cynomolgi sporozoites, and all showed a delay in patency from 13 to 40 days at 30 mg/kg/day x 3 days by IM dosing. Two out of four compounds tested for radical curative activity in Rhesus showed cure at 30 mg/kg/day x 3 days. The other two compounds showed delay in relapse from 16 to 68 days. Conversion of new carboxamides (5 and 6) to s-triazine derivatives (7) was demonstrated in mouse and human microsomal preparations and in rat plasma. The results suggest the metabolites, s-triazine derivatives 7, may be the active species of the new carboxamides 5a-c and 6a-c prepared in this study.

The synthesis of deoxycholic acid (DCA)- and cholic acid (CA)-derived mixed tetraoxanes revealed that N-(2-dimethylamino)ethyl derivatives are potent antimalarials in vitro and in vivo. The tetraoxanes presented in this paper are dual inhibitors: besides curing mice in vivo without observed toxic effects, they kill cancer cell lines at very low concentrations. For example, DCA and CA derivatives 16 and 25 cured 3/5 (160 mg/kg/day) and 2/5 (40 mg/kg/day, MTD960 mg/kg), respectively, and they were extremely active against melanoma LOX IMVI cancer, LC50 = 22 nM and 69 nM, respectively.

Objectives To assess the antimalarial pharmacodynamics and pharmacokinetics of the novel dihydrofolate reductase (DHFR) inhibitor, JPC2056 and its principal active metabolite JPC2067 in cynomolgus monkeys using an in vivo-in vitro model. Methods In a two-phase crossover design, five cynomolgus monkeys were administered a single dose (20 mg/kg) and multiple doses (20 mg/kg daily for 3 days) of JPC2056. Plasma samples collected from treated monkeys were assessed for in vitro antimalarial activity against Plasmodium falciparum lines having wild-type (D6), double-mutant (K1) and quadruple-mutant (TM90-C2A) DHFR-thymidylate synthase (TS) and a P. falciparum line transformed with a Plasmodium vivax dhfr-ts quadruple-mutant allele (D6-PvDHFR). Plasma JPC2056 and JPC2067 concentrations were measured by LC-mass spectrometry. Results The mean inhibitory dilution (ID(90)) of monkey plasma at 3 h after drug administration against D6, K1 and TM90-C2A was, respectively, 1253, 585 and 869 after the single-dose regimen and 1613, 1120 and 1396 following the multiple-dose regimen. Less activity was observed with the same monkey plasma samples against the D6-PvDHFR line, with a mean ID(90) of 53 after multiple dosing. Geometric mean plasma concentrations of JPC2056 and JPC2067 at 3 h after drug administration were, respectively, 113 and 12 ng/mL after the single dose and 150 and 17 ng/mL after multiple dosing. The mean elimination half-life of JPC2056 was shorter than its metabolite after both regimens (single dose, 7.3 versus 11.8 h; multiple doses, 6.6 versus 11.1 h). Conclusions The high potency of JPC2056 against P. falciparum DHFR-TS quadruple-mutant lines provides optimism for the future development of JPC2056 for the treatment of malaria infections.

Pyrroloquinazolinediamine (PQD) derivatives such as tetra-acetamide PQD (PQD-A4) and bis-ethylcarbamyl PQD (PQD-BE) were much safer (with therapeutic indices of 80 and 32, respectively) than their parent compound, PQD (therapeutic index, 10). Further evaluation of PQD-A4 and PQD-BE in single and multiple pharmacokinetic (PK) studies as well as corresponding toxicity studies was conducted with rats. PQD-A4 could be converted to two intermediate metabolites (monoacetamide PQD and bisacetamide PQD) first and then to the final metabolite, PQD, while PQD-BE was directly hydrolyzed to PQD without precursor and intermediate metabolites. Maximum tolerant doses showed that PQD-A4 and PQD-BE have only 1/12 and 1/6, respectively, of the toxicity of PQD after a single oral dose. Compared to the area under the concentration-time curve for PQD alone (2,965 ng·h/ml), values measured in animals treated with PQD-A4 and PQD-BE were one-third (1,047 ng·h/ml) and one-half (1,381 ng·h/ml) as high, respectively, after an equimolar dosage, suggesting that PQD was the only agent to induce the toxicity. Similar results were also shown in multiple treatments; PQD-A4 and PQD-BE generated two-fifths and three-fifths, respectively, of PQD concentrations, with 8.8-fold and 3.8-fold safety margins, respectively, over the parent drug. PK data indicated that the bioavailability of oral PQD-A4 was greatly limited at high dose levels, that PQD-A4 was slowly converted to PQD via a sequential three-step process of conversion, and that PQD-A4 was significantly less toxic than the one-step hydrolysis drug, PQD-BE. It was concluded that the slow and smaller release of PQD was the main reason for the reduction in toxicity and that the active intermediate metabolites can still maintain antimalarial potency. Therefore, the candidate with multiple-step hydrolysis of PQD could be developed as a safer potential agent for malaria treatment.

The synthesis of deoxycholic acid (DCA)- and cholic acid (CA)-derived mixed tetraoxanes revealed that N-(2-dimethylamino)ethyl derivatives are potent antimalarials in vitro and in vivo. The tetraoxanes presented in this paper are dual inhibitors: besides curing mice in vivo without observed toxic effects, they kill cancer cell lines at very low concentrations. For example, DCA and CA derivatives 16 and 25 cured 3/5 (160 mg/kg/day) and 2/5 (40 mg/kg/day, MTD >960 mg/kg), respectively, and they were extremely active against melanoma LOX IMVI cancer, LC50 = 22 nM and 69 nM, respectively.

Investigation of a series of 1-phenyl-3-aryl-2-propen-1-ones resulted in the identification of nine inhibitors with submicromolar efficacy against at least one Plasmodium falciparum strain in vitro. These inhibitors were inactive when given orally in a Plasmodium berghei infected mouse model. Significant compound degradation occurred upon their exposure to a liver microsome preparation, suggesting metabolic instability may be responsible for the lack of activity in vivo.

Mixed tetraoxanes 5a and 13 synthesized from cholic acid and 4-oxocyclohexanecarboxylic acid were as active as artemisinin against chloroquine-susceptible, chloroquine-resistant, and multidrug-resistant Plasmodium falciparum strains (IC50, IC90). Most active 13 is metabolically stable in in vitro metabolism studies. In vivo studies on tetraoxanes with a C(4'') methyl group afforded compound 15, which cured 4/5 mice at 600 and 200 mg, kg(-1), day(-1), and 2/5 mice at 50 mg, kg(-1), day(-1), showing no toxic effects. Tetraoxane 19 was an extremely active antiproliferative with LC50 of 17 nM and maximum tolerated dose of 400 mg/kg. In Fe(II)-induced scission of tetraoxane antimalarials only RO center dot radicals were detected by EPR experiments. This finding and the indication of Fe(IV)=O species led us to propose that RO center dot radicals are probably capable of inducing the parasite's death. Our results suggest that C radicals are possibly not the only lethal species derived from peroxide prodrug antimalarials, as currently believed.

Mefloquine is one of the drugs approved by the FDA for malaria chemoprophylaxis. Mefloquine is also approved for the treatment of malaria and is widely used for this purpose in combination with artesunate. However, the clinical utility of the compound has been compromised by reports of adverse neurological effects in some patients. In the present study, the potential neurological effects of mefloquine were investigated with six 7-week-old female rats given a single oral dose of the compound. Potential mefloquine-induced neurological effects were monitored using a standard functional observational battery, automated open field tests, automated spontaneous activity monitoring, a beam traverse task, and histopathology. Plasma mefloquine concentrations were determined 72 h after dosing by using liquid chromatography-mass spectrometry. Mefloquine induced dose-related changes in endpoints associated with spontaneous activity and impairment of motor function and caused degeneration of specific brain stem nuclei (nucleus gracilis). Increased spontaneous motor activity was observed only during the rats' normal sleeping phase, suggesting a correlate to mefloquine-induced sleep disorders. The threshold dose for many of these effects was 187 mg/kg of body weight. This dose yielded plasma mefloquine concentrations after 72 h that are similar to those observed in humans after the treatment dose. Collectively, these data suggest that there may be a biological basis for some of the clinical neurological effects associated with mefloquine.

The high level of attrition of drugs in clinical development has led pharmaceutical companies to increase the efficiency of their lead identification and development through techniques such as combinatorial chemistry and high-throughput (HTP) screening. Since the major reasons for clinical drug candidate failure other than efficacy are pharmacokinetics and toxicity, attention has been focused on assessing properties such as metabolic stability, drug-drug interactions (DDI), and absorption earlier in the drug discovery process. Animal studies are simply too labor-intensive and expensive to use for evaluating every hit, so it has been necessary to develop and implement higher throughput in vitro ADME screens to manage the large number of compounds of interest. The antimalarial drug development program at the Walter Reed Army Institute of Research, Division of Experimental Therapeutics (WRAIR / ET) has adopted this paradigm in its search for a long-term prophylactic for the prevention of malaria. The overarching goal of this program is to develop new, long half-life, orally bioavailable compounds with potent intrinsic activity against liver- and blood-stage parasites. From the WRAIR HTP antimalarial screen, numerous compounds are regularly identified with potent activity. These hits are now immediately evaluated using a panel of in vitro ADME screens to identify and predict compounds that will meet our specific treatment criteria. In this review, the WRAIR ADME screening program for antimalarial drugs is described as well as how we have implemented it to predict the ADME properties of small molecule for the identification of promising drug candidates.

Tolcapone is a catechol-O-methyltransferase (COMT) inhibitor used for control of motor fluctuations in Parkinson's disease (PD). Since its entry onto the market in 1998, tolcapone has been associated with numerous cases of hepatotoxicity, including three cases of fatal fulminant hepatic failure. The cause of this toxicity is not known; however, it does not occur with the use of the structurally similar drug entacapone. It is known that tolcapone is metabolized to amine (M1) and acetylamine (M2) metabolites in humans, but that the analogous metabolites were not detected in a limited human study of entacapone metabolism. We hypothesized that one or both of these tolcapone metabolites could be oxidized to reactive species and that these reactive metabolites might play a role in tolcapone-induced hepatocellular injury. To investigate this possibility, we examined the ability of M1 and M2 to undergo in vitro bioactivation by electrochemical and enzymatic methods. Electrochemical experiments revealed that M1 and M2 are more easily oxidized than the parent compound, in the order M1 > M2 > tolcapone. There was a general correlation between oxidation potential and the half-lives of the compounds in the presence of two oxidizing systems, horseradish peroxidase and myeloperoxidase. These enzymes catalyzed the oxidation of M1 and M2 to reactive species that could be trapped with glutathione (GSH) to form metabolite adducts (C1 and C2). Each metabolite was found to only form one GSH conjugate, and the structures were tentatively identified using LC-MS/MS. Following incubation of M1 and M2 with human liver microsomes in the presence of GSH, the same adducts were observed, and their structures were confirmed using LC-MS/MS and (1)H NMR. Experiments with chemical P450 inhibitors and cDNA-expressed P450 enzymes revealed that this oxidation is catalyzed by several P450s, and that P450 2E1 and 1A2 play the primary role in the formation of C1 while P450 1A2 is most important for the production of C2. Taken together, these data provide evidence that tolcapone-induced hepatotoxicity may be mediated through the oxidation of the known urinary metabolites M1 and M2 to reactive intermediates. These reactive species may form covalent adducts to hepatic proteins, resulting in damage to liver tissues, although this supposition was not investigated in this study.

The synthesis and biological activity of amino acid functionalized beta-carboline derivatives, which are structurally related to azatoxin and the tryprostatins, are reported. These compounds were assayed for their growth inhibition properties in H520 and PC3 cell lines and were examined for their abilities to inhibit topoisomerase II-mediated DNA relaxation.