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Transgender women (TGW) have been underrepresented in trials and use gender-affirming hormonal therapies (GAHT) that may alter renal function by significantly increasing creatinine clearance. Population pharmacokinetic (popPK) models and simulations would aid in understanding potential differences in emtricitabine/tenofovir disproxil fumarate (F/TDF) parent-metabolite concentrations in TGW on GAHT when compared to cisgender men (CGM) not exposed to GAHT.Pharmacokinetic (PK) data from a Phase 1, open-label clinical trial with directly observed therapy of daily F/TDF consisting of 8 TGW and 8 CGM was utilized for model building. PopPK analysis was performed using nonlinear mixed effects modelling (NONMEM 7.5.0). Covariates of body weight, creatinine clearance, and gender were evaluated. Final models were subjected to Monte Carlo simulations to compare drug exposure following once daily and on-demand (IPERGAY 2 + 1 + 1) dosing of F/TDF.Tenofovir (TFV) and emtricitabine PK were best described by a 2-compartment model, first-order absorption/elimination with absorption lag time. Parent models were linked to their metabolites by first order formation and elimination. Creatinine clearance was a significant covariate influencing clearance in both models. Simulations demonstrated that at least 2, weekly 2 + 1 + 1 cycles of on-demand dosing in TGW on GAHT is necessary for TFV-diphosphate to reach similar exposure after the initial week of on-demand dosing in CGM not on GAHT.PopPK models of TFV, emtricitabine and intracellular metabolites in TGW were established. Dose simulations revealed that TGW should be treated for at least 2 weeks to have comparable exposures to CGM.

Chemoprophylaxis is currently the best available prevention from malaria, but its efficacy is compromised by non-adherence to medication. Here we develop a long-acting injectable formulation of atovaquone solid drug nanoparticles that confers long-lived prophylaxis against Plasmodium berghei ANKA malaria in C57BL/6 mice. Protection is obtained at plasma concentrations above 200 ng ml-1 and is causal, attributable to drug activity against liver stage parasites. Parasites that appear after subtherapeutic doses remain atovaquone-sensitive. Pharmacokinetic–pharmacodynamic analysis indicates protection can translate to humans at clinically achievable and safe drug concentrations, potentially offering protection for at least 1 month after a single administration. These findings support the use of long-acting injectable formulations as a new approach for malaria prophylaxis in travellers and for malaria control in the field., Long-acting antimalarials could provide improved prophylaxis and treatment options in the field. Here, Bakshi et al. develop a long-acting injectable atovaquone nanomedicine that prevents malaria infection prophylactically for up to 4 weeks in mice with no evidence for generation of resistant parasites.

Antibacterial drugs are an important component of malaria therapy. We studied the interactions of clindamycin, tetracycline, chloramphenicol, and ciprofloxacin against Plasmodium falciparum under static and dynamic conditions. In microtiter plate assays (static conditions), and as expected, parasites displayed the delayed death response characteristic for apicoplast-targeting drugs. However, rescue by isopentenyl pyrophosphate was variable, ranging from 2,700-fold for clindamycin to just 1.7-fold for ciprofloxacin, suggesting that ciprofloxacin has targets other than the apicoplast. We also examined the pharmacokinetic-pharmacodynamic relationships of these antibacterials in an in vitro glass hollow-fiber system that exposes parasites to dynamically changing drug concentrations. The same total dose and area under the concentration-time curve (AUC) of the drug was deployed either as a single short-lived high peak (bolus) or as a constant low concentration (infusion). All four antibacterials were unambiguously time-driven against malaria parasites: infusions had twice the efficacy of bolus regimens, for the same AUC. The time-dependent efficacy of ciprofloxacin against malaria is in contrast to its concentration-driven action against bacteria. In silico simulations of clinical dosing regimens and resulting pharmacokinetics revealed that current regimens do not maximize time above the MICs of these drugs. Our findings suggest that simple and rational changes to dosing may improve the efficacy of antibacterials against falciparum malaria.

Antibacterial drugs are an important component of malaria therapy. We studied the interactions of clindamycin, tetracycline, chloramphenicol, and ciprofloxacin against Plasmodium falciparum under static and dynamic conditions. In microtiter plate assays (static conditions), and as expected, parasites displayed the delayed death response characteristic for apicoplast-targeting drugs. However, rescue by isopentenyl pyrophosphate was variable, ranging from 2,700-fold for clindamycin to just 1.7-fold for ciprofloxacin, suggesting that ciprofloxacin has targets other than the apicoplast. We also examined the pharmacokinetic-pharmacodynamic relationships of these antibacterials in an in vitro glass hollow-fiber system that exposes parasites to dynamically changing drug concentrations. The same total dose and area under the concentration-time curve (AUC) of the drug was deployed either as a single short-lived high peak (bolus) or as a constant low concentration (infusion). All four antibacterials were unambiguously time-driven against malaria parasites: infusions had twice the efficacy of bolus regimens, for the same AUC. The time-dependent efficacy of ciprofloxacin against malaria is in contrast to its concentration-driven action against bacteria. In silico simulations of clinical dosing regimens and resulting pharmacokinetics revealed that current regimens do not maximize time above the MICs of these drugs. Our findings suggest that simple and rational changes to dosing may improve the efficacy of antibacterials against falciparum malaria.

Oral preexposure prophylaxis (PrEP) trials report disparate efficacy attributed to variable adherence. HPTN 066 was conducted to establish objective, quantitative benchmarks for discrete, regular levels of adherence using directly observed dosing of tenofovir (TFV) disoproxil fumarate (TDF)/emtricitabine (FTC). Healthy, HIV-uninfected men and women were randomized to one of four oral regimens of fixed-dose TDF 300 mg/FTC 200 mg tablet for 5 weeks with all doses observed: one tablet weekly (one/week), one tablet twice weekly (two/week), two tablets twice weekly (four/week), or one tablet daily (seven/week). Trough serum TFV and FTC, peripheral blood mononuclear cell (PBMC), and CD4(+) TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) concentrations were determined throughout dosing and 2 weeks after the last dose. Rectosigmoidal, semen, and cervicovaginal samples were collected for drug assessment at end of dosing and 2 weeks later in a subset of participants. The 49 enrolled participants tolerated the regimens well. All regimens achieved steady-state concentrations by the second dose for serum TFV/FTC and by 7 days for PBMC TFV-DP/FTC-TP. Steady-state median TFV-DP predose concentrations demonstrated dose proportionality: one/week 1.6 fmol/10(6) PBMCs, two/week 9.1, four/week 18.8, seven/week, 36.3. Further, TFV-DP was consistently quantifiable 2 weeks after the last dose for the ≥4/week regimens. Adherence benchmarks were identified using receiver operating characteristic curves, which had areas under the curve ≥0.93 for all analytes in serum and PBMCs. Intersubject and intrasubject coefficients of variation (%CV) ranged from 33% to 63% and 14% to 34%, respectively, for all analytes in serum and PBMCs. Steady-state PBMC TFV-DP was established earlier and at lower concentrations than predicted and was the only analyte demonstrating predose concentration dose proportionality. Steady-state daily dosing serum TFV and PBMC TFV-DP was consistent with highly effective PrEP clinical trials. HPTN 066 provides adherence benchmarks for oral TFV/FTC regimens to assist interpreting study outcomes.

Antiretroviral drug concentrations at sites of HIV exposure are important drivers that influence the development of HIV pre-exposure chemoprophylaxis strategies and regimens. We assessed the effect of collection method—in the presence or absence of tissue culture medium—on tenofovir (TFV) and tenofovir diphosphate (TFV-DP) concentrations in colonic biopsies. We find significant baseline interbiopsy variation in TFV (38% CV) and TFV-DP (33% CV) concentrations. Incubation in medium leads to a fluid absorption-driven twofold increase in tissue weight with a concomitant 75% decrease in weight-adjusted tissue TFV concentrations 120 min post-incubation. In contrast, adjusted TFV-DP concentrations decrease by only 25% during the same period, with this difference not achieving statistical significance. Although colonic biopsies should be collected in the absence of medium for accurate TFV concentrations, the presence of medium does not significantly impact TFV-DP-dependent pharmacokinetic or pharmacodynamic assays. Appropriate assessment of tissue drug concentrations should account for biopsy collection method and drug mechanism of action.

Although preexposure prophylaxis with oral tenofovir (TFV) disoproxil fumarate/emtricitabine reduces HIV acquisition rates, poor adherence to and acceptability of daily vaginal gels have led to development of vaginal film formulations to improve adherence and, potentially, to enable episodic use.In this 2-arm, cross-over study of a fast-dissolving tenofovir film (40 mg) compared with a previously studied semisolid tenofovir 1% gel (40 mg), 10 healthy women received a single vaginal dose of each study product. Clinical, pharmacokinetic, and antiviral assessments were performed over 1 week after dose.Nine of 10 participants experienced mild to moderate adverse effects, similar between products, with no severe adverse events or events attributed to study products. TFV concentrations after film dosing exceeded concentrations after gel dosing in plasma between 8 and 24 hours (P ≤ 0.02). TFV concentrations in cervicovaginal fluid and both TFV and TFV diphosphate concentrations in cervical tissue homogenates were higher after film dosing (all P values0.04). The differences ranged from median (interquartile range) 2.9-fold (1.1, 9.0; midvaginal cervicovaginal fluid) to 4.4-fold (2.9, 7.7; plasma). Neither film nor gel demonstrated reduced cervical tissue biopsy infectivity after ex vivo HIV challenge.Single-dose tenofovir film demonstrated consistently higher concentrations in plasma and cervicovaginal samples when compared with gel during the first day after dosing. Single-dose cervical tissue TFV-diphosphate concentrations at 5 hours exceeded steady-state concentrations previously reported with daily oral Truvada dosing. Tenofovir film may provide an alternative to tenofovir oral and gel formulations. Clinical efficacy remains to be tested.

HIV seroconversion outcomes in preexposure prophylaxis (PrEP) trials of oral tenofovir (TFV)-containing regimens are highly sensitive to drug concentration, yet less-than-daily dosing regimens are under study. Description of TFV and its active moiety, TFV diphosphate (TFV-DP), in blood, vaginal tissue, and colon tissue may guide the design and interpretation of PrEP clinical trials. Six healthy women were administered a single oral dose of 300 mg tenofovir disoproxil fumarate (TDF) and 4.3 mg (12.31 MBq, 333 μCi) (14)C-TDF slurry. Blood was collected every 4 h for the first 24 h, then at 4, 8, 11, and 15 days postdosing. Colonic and vaginal samples (tissue, total and CD4(+) cells, luminal fluid and cells) were collected 1, 8 and 15 days postdose. Samples were analyzed for TFV and TFV-DP. Plasma TFV demonstrated triphasic decay with terminal elimination half-life median [interquartile range (IQR)] 69 h (58-77). Peripheral blood mononuclear cell (PBMC) TFV-DP demonstrated biphasic peaks (median 12 h and 96 h) followed by a terminal 48 h (38-76) half-life; Cmax was 20 fmol/million cells (2-63). One day postdose, the TFV-DP paired colon:vaginal tissue concentration ratio was 1 or greater in all subjects' tissue homogenates, median 124 (range 1-281), but was not sustained. The ratio was lower and more variable in cells extracted from tissue. Among all sample types, TFV and TFV-DP half-life ranged from 23 to 139 h. PBMC TFV-DP rose slowly in the hours after dosing indicating that success with exposure-driven dosing regimens may be sensitive to timing of the dose prior to exposure. Colonic tissue homogenate TFV-DP concentrations were greater than in vaginal homogenate at 24 h, but not in cells extracted from tissue. These and the other pharmacokinetic findings will guide the interpretation and design of future PrEP trials.

While preexposure prophylaxis with oral tenofovir/emtricitabine reduces HIV acquisition rates, poor adherence to and acceptability of vaginal gels and the potential for evolving drug resistance have led to development of vaginal film formulations and other antiretroviral drugs, respectively, including the non-nucleoside reverse transcriptase inhibitor dapivirine. In this two-arm crossover study of a novel fast-dissolving dapivirine film and a previously studied semisolid dapivirine gel, 10 healthy women received a single 1.25 mg vaginal dose of each study product; one withdrew after the first dose. Clinical, pharmacokinetic, and antiviral pharmacodynamic assessments (ex vivo HIV-BaL challenge of tissue explants) were performed over 168 h postdose. Six of ten participants experienced mild to moderate adverse effects, similar between products, with no severe adverse events or adverse events attributed to study products. There were no statistically significant differences in plasma, cervicovaginal fluid (CVF), or cervical tissue dapivirine concentrations between the gel and film (all p > .05). CVF dapivirine concentrations were 1.5 and 6 log10 greater than tissue and plasma concentrations, respectively (p

Knowledge of pharmacokinetic/pharmacodynamic (PK/PD) relationships can enhance the speed and economy of drug development by enabling informed and rational decisions at every step, from lead selection to clinical dosing. For anti-infective agents in particular, dynamic in vitro hollow-fiber cartridge experiments permit exquisite control of kinetic parameters and the study of their consequent impact on pharmacodynamic efficacy. Such information is of great interest for the cost-restricted but much-needed development of new antimalarial drugs, especially since the major human pathogen Plasmodium falciparum can be cultivated in vitro but is not readily available in animal models. This protocol describes the materials and procedures for determining the PK/PD relationships of antimalarial compounds.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Rectally applied drugs have been imaged using radioisotopes and magnetic resonance contrast agents. However, prior studies have not described the distribution and clearance of rectally-applied drugs in quantitative terms with respect to complex three dimensional paths through the gastrointestinal tract. Such tools would allow statistical comparisons of candidate products in development or comparison of drug product with the distribution of a drug target, for example, HIV infected seminal fluid. WHAT THIS STUDY ADDS • New quantitative spatial parameters, derived from three dimensional curve fitting, have been successfully applied in this study to quantify the distribution of rectally-applied gels. Indirect assessment using nuclear medicine techniques avoided the distortion and redistribution associated with sigmoidoscopic sampling. Thus, these measurements can be repeated over time to create concentration–distance–time surfaces to describe rectal product distribution and clearance within the gastrointestinal lumen to inform microbicide and other rectal product development. AIMS We sought to describe quantitatively the distribution of rectally administered gels and seminal fluid surrogates using novel concentration–distance parameters that could be repeated over time. These methods are needed to develop rationally rectal microbicides to target and prevent HIV infection. METHODS Eight subjects were dosed rectally with radiolabelled and gadolinium-labelled gels to simulate microbicide gel and seminal fluid. Rectal doses were given with and without simulated receptive anal intercourse. Twenty-four hour distribution was assessed with indirect single photon emission computed tomography (SPECT)/computed tomography (CT) and magnetic resonance imaging (MRI), and direct assessment via sigmoidoscopic brushes. Concentration–distance curves were generated using an algorithm for fitting SPECT data in three dimensions. Three novel concentration–distance parameters were defined to describe quantitatively the distribution of radiolabels: maximal distance (Dmax), distance at maximal concentration (DCmax) and mean residence distance (Dave). RESULTS The SPECT/CT distribution of microbicide and semen surrogates was similar. Between 1 h and 24 h post dose, the surrogates migrated retrograde in all three parameters (relative to coccygeal level; geometric mean [95% confidence interval]): maximal distance (Dmax), 10 cm (8.6–12) to 18 cm (13–26), distance at maximal concentration (DCmax), 3.8 cm (2.7–5.3) to 4.2 cm (2.8–6.3) and mean residence distance (Dave), 4.3 cm (3.5–5.1) to 7.6 cm (5.3–11). Sigmoidoscopy and MRI correlated only roughly with SPECT/CT. CONCLUSIONS Rectal microbicide surrogates migrated retrograde during the 24 h following dosing. Spatial kinetic parameters estimated using three dimensional curve fitting of distribution data should prove useful for evaluating rectal formulations of drugs for HIV prevention and other indications.

Many antiretroviral (ARV) drugs have large blood plasma-to-seminal plasma (BP/SP) concentration ratios. Concern exists that these drugs do not adequately penetrate the male genital tract (MGT), resulting in the MGT becoming a “pharmacological sanctuary” from these agents, with ineffective MGT concentrations despite effective blood concentrations. Efavirenz (EFV) is the most highly protein-bound ARV drug, with >99% binding in blood plasma and the largest BP/SP total EFV concentration ratio, reportedly ranging from 11 to 33. To evaluate protein binding as an explanation for the differences between the drug concentrations in blood and semen, we developed a novel ultrafiltration method, corrected for the duration of centrifugation, to measure protein binding in the two matrices. In six subjects, protein-free EFV concentrations were the same in blood and semen; the median (interquartile range (IQR)) protein-free EFV SP/BP ratio was 1.21 (0.99–1.35); EFV protein binding was 99.82% (99.79–99.86) in BP and 95.26% (93.24–96.67) in SP. This shows that the MGT is not a sanctuary from EFV. Clinical Pharmacology & Therapeutics (2011) 90 1, 151–156. doi:10.1038/clpt.2011.99

African trypanosomiasis (sleeping sickness), caused by protozoan Trypanosoma brucei species, is a debilitating disease that is lethal if untreated. Available drugs are antiquated, toxic, and compromised by emerging resistance. The indenoisoquinolines are a class of noncamptothecin topoisomerase IB poisons that are under development as anticancer agents. We tested a variety of indenoisoquinolines for their ability to kill T. brucei . Indenoisoquinolines proved trypanocidal at submicromolar concentrations in vitro. Structure-activity analysis yielded motifs that enhanced potency, including alkylamino substitutions on N-6, methoxy groups on C-2 and C-3, and a methylenedioxy bridge between C-8 and C-9. Detailed analysis of eight water-soluble indenoisoquinolines demonstrated that in trypanosomes the compounds inhibited DNA synthesis and acted as topoisomerase poisons. Testing these compounds on L1210 mouse leukemia cells revealed that all eight were more effective against trypanosomes than against mammalian cells. In preliminary in vivo experiments one compound delayed parasitemia and extended survival in mice subjected to a lethal trypanosome challenge. The indenoisoquinolines provide a promising lead for the development of drugs against sleeping sickness.

Hydroxyurea (HU) significantly enhances the antiretroviral effects of the adenosine analog reverse transcriptase inhibitor dideoxyinosine (ddI). This is believed to be due to a reduction in intracellular de-oxyadenosine triphosphate (dATP) concentrations resulting from HU-mediated inhibition of ribonucleotide reductase (RnR). The effect of combined HU-ddI treatment on intracellular dATP pools in vivo has not been examined. We measured intracellular dATP concentrations in peripheral blood mononuclear cells (PBMCs) from 69 HIV-infected patients receiving 1000 or 1500 mg HU daily for 14 days, 200 mg ddI twice daily for 14 days, or a combination of the two drugs. Median intracellular dATP concentrations decreased from base-line to day 14 by 46% in the ddI + 1000 mg HU arm and by 62% in the ddI + 1500 mg HU arm. When compared to the HU monotherapy arms, these changes proved statistically significant (p = 0.018; stratified Wilcoxon rank-sum test). These findings support reduced intracellular dATP as the mechanism of ddI-HU synergistic activity, and indicate that changes in intracellular nucleotides contribute to HU activity and toxicity in patients. Since a significant reduction in dATP was measurable only when ddI was combined with HU, the antiretroviral activity of ddI may be more complex than previously assumed.

Understanding the distribution of microbicide and human immunodeficiency virus (HIV) within the gastrointestinal tract is critical to development of rectal HIV microbicides. A hydroxyethylcellulose-based microbicide surrogate or viscosity-matched semen surrogate, labeled with gadolinium-DTPA (diethylene triamine pentaacetic acid) and 99mTechnetium-sulfur colloid, was administered to three subjects under varying experimental conditions to evaluate effects of enema, coital simulation, and microbicide or semen simulant over 5 h duration. Quantitative assessment used single photon emission computed tomography (SPECT)/computed tomography (CT) and magnetic resonance imaging (MRI) imaging, and sigmoidoscopic sampling. Over 4 h, radiolabel migrated cephalad in all studies by a median (interquartile range) of 50% (29–102%; P

Objective To determine the feasibility of using quantitative changes in vaginal permeability to small molecules as a measure of candidate microbicide toxicity. Study design Controlled, open-labeled, prospective study. Seven healthy women received a single vaginal dose of hydroxyethylcellulose gel (HEC), nonoxynol-9 (N-9) or K-Y Jelly. Each gel was radiolabeled with a small molecule ( 99m Tc-DTPA) followed by 12-h blood and urine collection. Pharmacokinetic (PK) parameters of 99m Tc-DTPA were calculated to compare the impact of each gel on vaginal permeability. Each woman served as her own control. The Friedman test with post hoc Wilcoxon test was used to detect differences among the gels. Results Vaginal permeability of 99m Tc-DTPA was highest for the N-9 radiolabel. N-9 plasma area under the concentration curve was 2.7-fold higher (p=.04), and peak concentration was threefold higher (p=.04) compared to HEC. There were no significant PK parameter differences between HEC and K-Y Jelly or between N-9 and K-Y Jelly. Cumulative dose-adjusted median (interquartile range) 12-h timed urine gamma activity was 66.70×10 −4 μCi (27.90–152.00) following HEC dosing, 103.00×10 −4 μCi (98.20–684.00) following N-9 gel dosing and 20.30×10 −4 μCi (11.10–55.90) following K-Y gel dosing. The differences between urine HEC and K-Y Jelly (p=.047) and between N-9 and K-Y Jelly (p=.016) were statistically significant. Conclusions It is feasible to measure differences in vaginal permeability among vaginal gels using a radiolabeled small molecule, though there are permeability differences that require a nuanced understanding of gel composition to interpret the results. Implications Establishing the safety of both vehicle and active pharmaceutical ingredient is an essential task in microbicide development, to be determined as soon as possible. This study suggests that a combination of microbicide toxicity assessments, that is, cervicovaginal permeability, inspection and histopathology, may need to be studied simultaneously.

Diseases caused by parasitic protozoa present a health problem of immense magnitude, and there is an urgent need for safe and effective new therapies. DNA topoisomerases are clinically relevant targets for anti-cancer and anti-bacterial agents. Inhibitor studies on parasite topoisomerases have revealed that these enzymes have great promise as molecular targets for anti-parasitics, and have helped to dissect the basic biology of DNA topoisomerases in these organisms. This review provides a brief introduction to DNA topoisomerases and anti-topoisomerase drugs, and an overview of studies on protozoal DNA topoisomerases and their inhibitors.

Malaria presents a tremendous public health burden, and new therapies are needed. Massive compound libraries screened against Plasmodium falciparum have yielded thousands of lead compounds, resulting in an acute need for rational criteria to select the best candidates for development. We reasoned that, akin to antibacterials, antimalarials might have an essential pharmacokinetic requirement for efficacy: action governed either by total exposure or peak concentration (AUC/CMAX), or by duration above a defined minimum concentration [time above minimum inhibitory concentration (TMIC)]. We devised an in vitro system for P. falciparum, capable of mimicking the dynamic fluctuations of a drug in vivo. Using this apparatus, we find that chloroquine is TMIC-dependent, whereas the efficacy of artemisinin is driven by CMAX. The latter was confirmed in a mouse model of malaria. These characteristics can explain the clinical success of two antimalarial drugs with widely different kinetics in humans. Chloroquine, which persists for weeks, is ideally suited for its TMIC mechanism, whereas great efficacy despite short exposure (t1/2 in blood 3 hours or less) is attained by CMAX-driven artemisinins. This validated preclinical model system can be used to select those antimalarial lead compounds whose CMAX or TMIC requirement for efficacy matches pharmacokinetics obtained in vivo. The apparatus can also be used to explore the kinetic dependence of other pharmacodynamic endpoints in parasites.

The objective of this study was to develop an in vitro pharmacodynamic (PD) system to test the impact of protein binding on antiretroviral (ARV) drug effect and intracellular ARV distribution. CD4(+) T cells were isolated from peripheral blood mononuclear cells (PBMCs) and exposed to varying and physiologically relevant concentrations of human serum albumin (HSA) and the ARV drugs efavirenz (EFV), raltegravir (RAL), etravirine (ETR), and enfuvirtide (ENF). The effect of varying extracellular protein concentration on the intracellular distribution of EFV, RAL, and ETR was assessed using ultraperformance liquid chromatography tandem mass spectrometry. HIV infectivity was assessed using an HIV-1 reporter virus expressing an Env-green fluorescent protein (GFP) and quantified using flow cytometry. Increasing extracellular HSA concentration was associated with increased relative infectivity for all drugs tested as well as decreased intracellular concentrations for EFV, RAL, and ETR. Median-effect plots indicate linearity between log10 antiviral effect (fraction of virus affected divided by fraction unaffected) and log10 intracellular drug concentration. The median [interquartile range (IQR)] slope (m) of the median-effect plots was 2.97 (2.26-5.85) for EFV, 3.52 (3.11-3.74) for ETR, and 2.39 (2.15-3.74) for RAL. The intracellular ARV concentrations associated with half-maximal antiviral effect (IC50) of EFV, ETR, and RAL were 1.2 (0.51-5.39), 39.06 (30.10-51.76), and 4.67 (3.91-5.02) ng/ml, respectively. This study demonstrates a significant reduction in cell penetration and antiviral effect of highly bound ARVs due to increasing extracellular concentration of HSA. This study is therefore the first to demonstrate experimentally how protein binding impacts intracellular distribution and the efficacy of ARVs.

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing., Author Summary Trypanosoma brucei is a protozoan parasite that causes cattle disease and human sleeping sickness in Africa. Trypanosomes are primitive eukaryotes with atypical biological features. One well-studied example is their mitochondrial genome, known as kinetoplast DNA or kDNA. kDNA, resembling medieval chain mail, is a giant network of interlocked DNA rings known as minicircles and maxicircles. Here we study ethidium bromide, a drug used for over 50 years for treating cattle infected with trypanosomes. EB's killing mechanism has been elusive, and many thought it could not involve kDNA. We now report that EB kills these parasites by blocking the initiation of minicircle replication, and, at higher drug concentration, it also blocks nuclear replication.

We evaluated the causal prophylactic antimalarial activity of a single oral dose of pafuramidine (DB289), an experimental prodrug of active metabolite DB75, in a randomized, double-blind, placebo-controlled, outpatient study. Sixteen healthy volunteers were dosed and challenged in a single cohort. Subjects were randomly assigned to one of three treatment arms: 100 mg pafuramidine eight days before challenge, 100 mg pafuramidine the day before challenge, or placebo. Challenge was by the bites of Plasmodium falciparum-infected Anopheles gambiae. Malaria developed in 15 persons but did not develop in one person in the day -8 pafuramidine treatment arm. Plasma levels of DB75 were lower than expected, and as intended were too low to provide suppressive prophylaxis at the earliest appearance of erythrocytic parasites. We conclude that a single dose of 100 mg pafuramidine does not adequately protect non-immune individuals against P. falciparum and shows no clinically or statistically significant evidence of causal prophylactic activity.