Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug.

BACKGROUND: Human African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10-100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations. METHODOLOGY: To do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study. PRINCIPAL FINDINGS: Screening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline2 receptor. The reduction in insulin secretion in MIN6 ß-cells by pentamidine may be secondary to pentamidine-mediated activation of ß-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine. SIGNIFICANCE: These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.

Oral pentamidine-loaded poly(d,l-lactic-co-glycolic) acid nanoparticles: an alternative approach for leishmaniasis treatment.

Leishmaniasis is a group of diseases caused by a protozoa parasite from one of over 20 Leishmania species. Depending on the tissues infected, these diseases are classified as cutaneous, mucocutaneous and visceral leishmaniasis. For the treatment of leishmaniasis refractory to antimony-based drugs, pentamidine (PTM) is a molecule of great interest. However, PTM displays poor bioavailability through oral routes due to its two strongly basic amidine moieties, which restricts its administration by a parenteral route and limits its clinical use. Among various approaches, nanotechnology-based drug delivery systems (nano-DDS) have potential to overcome the challenges associated with PTM oral administration. Here, we present the development of PTM-loaded PLGA nanoparticles (NPs) with a focus on the characterization of their physicochemical properties and potential application as an oral treatment of leishmaniasis. NPs were prepared by a double emulsion methodology. The physicochemical properties were characterized through the mean particle size, polydispersity index (PdI), zeta potential, entrapment efficiency, yield process, drug loading, morphology, in vitro drug release and in vivo pharmacological activity. The PTM-loaded PLGA NPs presented with a size of 263 ± 5 nm (PdI = 0.17 ± 0.02), an almost neutral charge (-3.2 ± 0.8 mV) and an efficiency for PTM entrapment of 91.5%. The release profile, based on PTM dissolution, could be best described by a zero-order model, followed by a drug diffusion profile that fit to the Higuchi model. In addition, in vivo assay showed the efficacy of orally given PTM-loaded PLGA NPs (0.4 mg kg-1) in infected BALB/c mice, with significant reduction of organ weight and parasite load in spleen (p-value < 0.05). This work successfully reported the oral use of PTM-loaded NPs, with a high potential for the treatment of visceral leishmaniasis, opening a new perspective to utilization of this drug in clinical practice.

inPentasomes: An innovative nose-to-brain pentamidine delivery blunts MPTP parkinsonism in mice.

Preclinical and clinical evidences have demonstrated that astroglial-derived S100B protein is a key element in neuroinflammation underlying the pathogenesis of Parkinson's disease (PD), so much as that S100B inhibitors have been proposed as promising candidates for PD targeted therapy. Pentamidine, an old-developed antiprotozoal drug, currently used for pneumocystis carinii is one of the most potent inhibitors of S100B activity, but despite this effect, is limited by its low capability to cross blood brain barrier (BBB). To overcome this problem, we developed a non-invasive intranasal delivery system, chitosan coated niosomes with entrapped pentamidine (inPentasomes), in the attempt to provide a novel pharmacological approach to ameliorate parkinsonism induced by subchronic MPTP administration in C57BL-6 J mice. inPentasomes, prepared by evaporation method was administered daily by intranasal route in subchronic MPTP-intoxicated rodents and resulted in a dose-dependent manner (0.001-0.004 mg/kg) capable for a significant Tyrosine Hydroxylase (TH) positive neuronal density rescue in both striatum and substantia nigra of parkinsonian mice. In parallel, inPentasomes significantly decreased the extent of glial-related neuroinflammation through the reduction of specific gliotic markers (Iba-1, GFAP, COX-2, iNOS) with consequent PGE2 and NO2- release reduction, in nigrostriatal system. inPentasomes-mediated S100B inhibition resulted in a RAGE/NF-κB pathway downstream inhibition in the nigrostriatal circuit, causing a marked amelioration of motor performances in intoxicated mice. On the basis of our results, chitosan coated niosomes loaded with pentamidine, the inPentasome system, self-candidates as a promising new intranasal approach to mitigate parkinsonism in humans and possibly paves the way for a possible clinical repositioning of pentamidine as anti-PD drug.

Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis.

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.

Development of a liposome formulation for improved biodistribution and tumor accumulation of pentamidine for oncology applications.

Pentamidine isethionate, widely used for the treatment of parasitic infections, has shown strong anticancer activity in cancer cells and models of melanoma and lung cancer. Systemic administration of pentamidine is associated with serious toxicities, particularly renal, affecting as many as 95% of patients (O'Brien et al., 1997). This work presents the development of a liposome pentamidine formulation for greater tumor accumulation and lower drug exposure to vulnerable tissues. Liposomes formulated with saturated/unsaturated phospholipids of different chain lengths, varying cholesterol content, and surface PEG were explored to understand the effects of such variations on drug release, encapsulation efficiency, stability and in vivo performance. Saturated phospholipids with longer chain lengths, higher cholesterol content and PEG resulted in greater stability. The optimal formulation obtained showed significantly lower clearance rate (3.6 ± 1.2 mL/h/Kg) and higher AUC0-inf (348 ± 31 µmol/L × h) in vivo when compared to free drug (414 ± 138 mL/h/Kg and 2.58 ± 0.74 µmol/L × h, respectively). In tumor-bearing mice, liposomal delivery decreased kidney drug levels by up to 5-fold at 6 and 24h post-administration. Tumor drug exposure was up to 12.7-fold greater with liposomal administration compared to free drug. Overall, the liposomal pentamidine formulation developed has significant potential for the treatment of solid tumors.

In vivo SPECT imaging of [¹²³I]-labeled pentamidine pro-drugs for the treatment of human African trypanosomiasis, pharmacokinetics, and bioavailability studies in rats.

UNLABELLED: Pentamidine is an effective antiparasitic agent and approved drug for the treatment of African trypanosomiasis (sleeping sickness). However, pentamidine suffers from poor orally bioavailability and lacks central nervous system (CNS) delivery. Therefore its applicability is limited to intravenous or intramuscular treatment of the first stage of the African trypanosomiasis. For this reason, several new pentamidine pro-drugs have been developed with the aim of providing improved orally availability and CNS penetration. AIM: this work aims to measure and to compare the distribution, bioavailability, and ability to cross the blood-brain barrier of [(123)I]-labeled pentamidine and its pro-drugs, N,N'-dihydroxypentamidine and N,N'­bis(succinyloxy) pentamidine, using SPECT (single photon emission computed tomography) after intravenously and per orally administration in rats. METHODS: a total of 60 male Sprague Dawley rats were examined. Each [(123)I]-labeled substance (n=3) was applied to 12 rats (n=6 i.v. and n=6 orally). In two additional test series both [(123)I]iodopentamidine (n=6) and N,N'-bis(succinyloxy)-[(123)I]iodopentamidine (n=6) were administered orally together with the non-radioactive homologues. To evaluate the in vivo stability of the labeled compounds, [(123)I]NaI solution was administered intravenously (n=6) and orally (n=6). In vivo SPECT images were acquired after 30 min, 4h, and 24h and blood samples were taken over 24h. The SPECT images were fusioned with previously acquired magnetic resonance images. After the last SPECT the rats were perfused, sacrificed and the organ γ-radiation levels were determined with a γ-counter. Analysis and quantification of the reconstructed SPECT images was performed using the region of interest technique. RESULTS AND CONCLUSION: the data showed a highly improved oral bioavailability of the [(123)I]-labeled pro-drugs compared to [(123)I]-labeled pentamidine. While [(123)I]iodopentamidine was mainly renally eliminated the pro-drugs were primarily metabolized in the liver and underwent biliary elimination. Considering pentamidine's nephrotoxicity this feature has to be seen as an advantage of the pro-drug principle. Moreover, a significantly higher concentration in the brain was detected after intravenous injection of N,N'-dihydroxy[(123)I]iodopentamidine compared to [(123)I]iodopentamidine. The feasibility of an effective treatment of second stage African trypanosomiasis, in which the parasites already infected the brain, with the herein investigated pro-drugs remains to be clarified with infected animals in additional in vivo studies.

In vitro and in vivo evaluation of 28DAP010, a novel diamidine for treatment of second-stage African sleeping sickness.

African sleeping sickness is a neglected tropical disease transmitted by tsetse flies. New and better drugs are still needed especially for its second stage, which is fatal if untreated. 28DAP010, a dipyridylbenzene analogue of DB829, is the second simple diamidine found to cure mice with central nervous system infections by a parenteral route of administration. 28DAP010 showed efficacy similar to that of DB829 in dose-response studies in mouse models of first- and second-stage African sleeping sickness. The in vitro time to kill, determined by microcalorimetry, and the parasite clearance time in mice were shorter for 28DAP010 than for DB829. No cross-resistance was observed between 28DAP010 and pentamidine on the tested Trypanosoma brucei gambiense isolates from melarsoprol-refractory patients. 28DAP010 is the second promising preclinical candidate among the diamidines for the treatment of second-stage African sleeping sickness.

Pharmacokinetic comparison to determine the mechanisms underlying the differential efficacies of cationic diamidines against first- and second-stage human African trypanosomiasis.

Human African trypanosomiasis (HAT), a neglected tropical disease, is fatal without treatment. Pentamidine, a cationic diamidine, has been used to treat first-stage (hemolymphatic) HAT since the 1940s, but it is ineffective against second-stage (meningoencephalitic, or central nervous system [CNS]) infection. Novel diamidines (DB75, DB820, and DB829) have shown promising efficacy in both mouse and monkey models of first-stage HAT. However, only DB829 cured animals with second-stage infection. In this study, we aimed to determine the mechanisms underlying the differential efficacies of these diamidines against HAT by conducting a comprehensive pharmacokinetic characterization. This included the determination of metabolic stability in liver microsomes, permeability across MDCK and MDR1-MDCK cell monolayers, interaction with the efflux transporter MDR1 (P-glycoprotein 1 or P-gp), drug binding in plasma and brain, and plasma and brain concentration-time profiles after a single dose in mice. The results showed that DB829, an azadiamidine, had the highest systemic exposure and brain-to-plasma ratio, whereas pentamidine and DB75 had the lowest. None of these diamidines was a P-gp substrate, and the binding of each to plasma proteins and brain differed greatly. The brain-to-plasma ratio best predicted the relative efficacies of these diamidines in mice with second-stage infection. In conclusion, pharmacokinetics and CNS penetration influenced the in vivo efficacies of cationic diamidines against first- and second-stage HAT and should be considered when developing CNS-active antitrypanosomal diamidines.

Evaluation of (131)I-pentamidine for scintigraphy of experimentally Leishmania tropica-infected hamsters.

We aimed to assess the ability of (131)I-Pentamidine scintigraphy to detect the lesions of Leishmania tropica infection. An experimental model of cutaneous leishmaniasis was developed. The presence of cutaneous leishmaniasis was confirmed. Pentamidine was radioiodinated with (131)I. The radiolabeled pentamidine was validated by the requisite quality control tests to check its radiolabeling efficiency, in vitro stability. (131)I-Pentamidine (activity: 18.5 MBq/100 µl) was injected intracardiacally into infected hamsters. Static whole body images of the hamsters were acquired under the gamma camera at 5 and 30 min, 2, 6 and 24 h following the administration. On the scintigrams, anatomically adjusted regions of interest (ROIs) were drawn over the right feet (target) and left feet (not-target) and various organs. Accumulation of (131)I-Pentamidine at sites of infection is expressed as the target to non-target (T/NT) ratio. The results T/NT ratio decreased with time. In concluding the (131)I-Pentamidine has poor sensitivity in detection of L. tropica infection.

Effect of cimetidine on pentamidine induced hyperglycemia in rats.

The antiprotozoal agent pentamidine, used for the treatment of Pneumocystis jirovecii pneumonia (PCP), is known to cause abnormalities in blood glucose homeostasis, such as hypoglycemia and hyperglycemia. Pentamidine has been reported to be a substrate of organic cation transporter 1 (OCT1). We investigated the combination effects of cimetidine, an OCT1 inhibitor, on the pharmacokinetics of pentamidine and on pentamidine-induced hyperglycemia. Pentamidine was infused intravenously to rats for 20 min at a dose of 7.5 or 15 mg/kg and serum samples were obtained periodically. The serum concentration of glucose did not change significantly after pentamidine infusion at 7.5mg/kg, while it increased with pentamidine at 15 mg/kg, and the maximal concentration of glucose was 167 ± 36 mg/dl, 30 min after the start of pentamidine infusion. Cimetidine (50mg/kg) enhanced the pentamidine-induced elevation of glucose concentration and the maximal concentration of glucose was 208 ± 33 mg/dl in the pentamidine 15 mg/kg treated group. Cimetidine combination significantly reduced total body clearance of pentamidine and increased pentamidine concentrations in the liver, kidneys, and lungs. A significant correlation was found between changes in serum glucose concentrations and serum concentrations of pentamidine 30 min after the start of pentamidine infusion. These results suggest that the hyperglycemic effect of pentamidine is dependent on the concentration of pentamidine and can be enhanced by cimetidine combination.

Systematic exploration of synergistic drug pairs.

Drug synergy allows a therapeutic effect to be achieved with lower doses of component drugs. Drug synergy can result when drugs target the products of genes that act in parallel pathways ('specific synergy'). Such cases of drug synergy should tend to correspond to synergistic genetic interaction between the corresponding target genes. Alternatively, 'promiscuous synergy' can arise when one drug non-specifically increases the effects of many other drugs, for example, by increased bioavailability. To assess the relative abundance of these drug synergy types, we examined 200 pairs of antifungal drugs in S. cerevisiae. We found 38 antifungal synergies, 37 of which were novel. While 14 cases of drug synergy corresponded to genetic interaction, 92% of the synergies we discovered involved only six frequently synergistic drugs. Although promiscuity of four drugs can be explained under the bioavailability model, the promiscuity of Tacrolimus and Pentamidine was completely unexpected. While many drug synergies correspond to genetic interactions, the majority of drug synergies appear to result from non-specific promiscuous synergy.

New prodrugs of the antiprotozoal drug pentamidine.

Pentamidine is an effective antimicrobial agent that is approved for the treatment of African trypanosomiasis but suffers from poor oral bioavailability and central nervous system (CNS) penetration. This work deals with the development and systematic characterisation of new prodrugs of pentamidine. For this reason, numerous prodrugs that use different prodrug principles were synthesised and examined in vitro and in vivo. Another objective of the study was the determination of permeability of the different pentamidine prodrugs. While some of the prodrug principles applied in this study are known, such as the conversion of the amidine functions into amidoximes or the O-alkylation of amidoximes with a carboxymethyl residue, others were developed more recently and are described here for the first time. These newly developed methods aim to increase the affinity of the prodrug for the transporters and mediate an active uptake via carrier systems by conjugation of amidoximes with compounds that improve the overall solubility of the prodrug. The different principles chosen resulted in several pentamidine prodrugs with various advantages. The objective of this investigation was the systematic characterisation and evaluation of eight pentamidine prodrugs in order to identify the most appropriate strategy to improve the properties of the parent drug. For this reason, all prodrugs were examined with respect to their solubility, stability, enzymatic activation, distribution, CNS delivery, and oral bioavailability. The results of this work have allowed reliable conclusions to be drawn regarding the best prodrug principle for the antiprotozoal drug pentamidine.

Synthesis and biological evaluation of L-valine-amidoximeesters as double prodrugs of amidines.

In general, drugs containing amidines suffer from poor oral bioavailability and are often converted into amidoxime prodrugs to overcome low uptake from the gastrointestinal tract. The esterification of amidoximes with amino acids represents a newly developed double prodrug principle creating derivatives of amidines with both improved oral availability and water solubility. N-valoxybenzamidine (1) is a model compound for this principle, which has been transferred to the antiprotozoic drug pentamidine (8). Prodrug activation depends on esterases and mARC and is thus independent from activation by P450 enzymes. Therefore, drug-drug interactions or side effects will be minimized. The synthesis of these two compounds was established, and their biotransformation was studied in vitro and in vivo. Bioactivation of N-valoxybenzamidine (1) and N,N'-bis(valoxy)pentamidine (7) via hydrolysis and reduction has been demonstrated in vitro with porcine and human subcellular enzyme preparations and the mitochondrial Amidoxime Reducing Component (mARC). Moreover, activation of N-valoxybenzamidine (1) by porcine hepatocytes was studied. In vivo, the bioavailability in rats after oral application of N-valoxybenzamidine (1) was about 88%. Similarly, N,N'-bis(valoxy)pentamidine (7) showed oral bioavailability. Analysis of tissue samples revealed high concentrations of pentamidine (8) in liver and kidney.

Three optimized and validated (using accuracy profiles) LC methods for the determination of pentamidine and new analogs in rat plasma.

Three novel LC-UV methods for the determination of pentamidine (PTMD) and two of its new analogs in rat plasma are described. The chromatographic conditions (wavelength, acetonitrile percentage in the mobile phase, internal standard) were optimized to have an efficient selectivity. A pre-step of extraction was simultaneously developed for each compound. For PTMD, a solid phase extraction (SPE) with Oasis(®) HLB cartridges was selected, while for the analogs we used protein precipitation with acetonitrile. SPE for PTMD gave excellent results in terms of extraction yield (99.7 ± 2.8) whereas the recoveries for the analogs were not so high but were reproducible as well (64.6 ± 2.6 and 36.8 ± 1.6 for analog 1 and 2, respectively). By means of a recent strategy based on accuracy profiles (ß-expectation tolerance interval), the methods were successfully validated. ß was fixed at 95% and the acceptability limits at ± 15% as recommended by the FDA. The method was successfully validated for PTMD (29.6-586.54 ng/mL), analog 1 (74.23-742.3 ng/mL) and analog 2 (178.12-890.6 ng/mL). The first concentration level tested was considered as the LLOQ (lower limit of quantification) for PTMD and analog 1 whereas for analog 2, the LLOQ was not the first level tested and was raised to 178.12 ng/mL.

Diamidines for human African trypanosomiasis.

Aromatic diamidines are potent trypanocides. Pentamidine, a diamidine, has been used for more than 60 years to treat human African trypanosomiasis (HAT); however, the drug must be administered parenterally and is active against first-stage HAT only, prior to the parasites causing neurological deterioration through invasion of the CNS. A major research effort to design novel diamidines has led to the development of orally active prodrugs and, remarkably, a new generation of compounds that can penetrate the CNS. In this review, progress in the development of diamidines for the treatment of HAT is discussed.

Inhibitory effects on cytochrome p450 enzymes of pentamidine and its amidoxime pro-drug.

Pentamidine is an antimicrobial drug, intravenously used in the treatment of trypanosomiasis, leishmaniasis or pneumocystis pneumonia. To elucidate potential drug interactions with pentamidine and N,N'-dihydroxypentamidine, respectively, the cytochrome P450 (CYP450) inhibitory properties of these compounds were determined. The study was performed in vitro by using human liver microsomes and marker substrates of several CYP450 isoenzymes. Marker activities were investigated by high-performance liquid chromatography in presence of known selective inhibitors or at different concentrations of pentamidine and N,N'-dihydroxypentamidine, respectively. No or only minor influence on CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4 marker activities could be observed, suggesting that neither of the tested substances would exert a significant effect on hepatic CYP450 isoenzymes responsible for the metabolism of co-administrated drugs in vivo. However, in vivo studies are needed before final conclusions can be drawn.

Loss of the high-affinity pentamidine transporter is responsible for high levels of cross-resistance between arsenical and diamidine drugs in African trypanosomes.

Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable cross-resistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [(3)H]pentamidine. In particular, the high-affinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1(-/-) line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4',6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 muM pentamidine. No overexpression of the Trypanosoma brucei brucei ATP-binding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance.

Diacetyldiamidoximeester of pentamidine, a prodrug for treatment of protozoal diseases: synthesis, in vitro and in vivo biotransformation.

Pentamidine is an effective antimicrobial agent. To increase its poor oral bioavailability due to the strong basic amidine functionality, the less basic O-acetylamidoxime prodrug, the diacetyldiamidoximeester, was used, which has greatly improved lipophilicity. The objectives of this investigation were the synthesis of all potential metabolites of the double prodrug, the conformational analysis of its structure, and to study the in vitro and in vivo biotransformation by ester cleavage and N-reduction to pentamidine via four intermediate metabolites. The biotransformation of diacetyldiamidoximeester to pentamidine involving the reduction of the amidoxime function and the ester cleavage could be demonstrated. The kinetic parameters were determined. Amidoximes were efficiently metabolized by several enzyme systems located in microsomes and mitochondria of different organs including the final formation of the active metabolite pentamidine. The formation of pentamidine after oral administration of the diacetyldiamidoximeester to rats could be demonstrated as well.

Accumulation and intracellular distribution of antitrypanosomal diamidine compounds DB75 and DB820 in African trypanosomes.

The aromatic diamidine pentamidine has long been used to treat early-stage human African trypanosomiasis (HAT). Two analogs of pentamidine, DB75 and DB820, have been shown to be more potent and less toxic than pentamidine in murine models of trypanosomiasis. The diphenyl furan diamidine, DB75, is the active metabolite of the prodrug DB289, which is currently in phase III clinical trials as a new orally active candidate drug to treat first-stage HAT. The new aza analog, DB820, is the active diamidine of the prodrug DB844, currently undergoing preclinical evaluation as a new candidate to treat HAT of the central nervous system. The exact mechanisms of antitrypanosomal activity of aromatic dications remain poorly understood, with multiple mechanisms hypothesized. Pentamidine is known to be actively transported into trypanosomes and binds to DNA within the nucleus and kinetoplast. A long-hypothesized mechanism of action has been that DNA binding ultimately leads to interference with DNA-associated enzymes. Both DB75 and DB820 are intensely fluorescent, which provides an important tool for determining the kinetics of accumulation and intracellular distribution in trypanosomes. We show in the current study that DB75 and DB820 rapidly accumulate and strongly concentrate within trypanosomes, with intracellular concentrations over 15,000-fold higher than mouse plasma concentrations. Both compounds initially accumulate in the DNA-containing nucleus and kinetoplast, but at later time points, they concentrate in non-DNA-containing cytoplasmic organelles. Analyses of the kinetics of uptake and intracellular distribution are necessary to begin to define antitrypanosomal mechanisms of action of DB75, DB820, and other aromatic diamidines.

Characterization of the choline carrier of Plasmodium falciparum: a route for the selective delivery of novel antimalarial drugs.

New drugs are urgently needed to combat the growing problem of drug resistance in Plasmodium falciparum malaria. The infected erythrocyte is a multicompartmental system, and its transporters are of interest as drug targets in their own right and as potential routes for the delivery of antimalarial drugs. Choline is an important nutrient that penetrates infected erythrocyte membranes through the endogenous carrier and through parasite-induced permeability pathways, but nothing is known about its transport into the intracellular parasite. Here we present the first characterization of choline transport across the parasite membrane. Transport exhibits Michaelis-Menten kinetics with an apparent K(m) of 25.0 +/- 3.5 muM for choline. The carrier is inhibitor-sensitive, temperature-dependent, and Na(+)-independent, and it is driven by the proton-motive force. Highly active bis-amidine and bis-quaternary ammonium compounds are also known to penetrate the host erythrocyte membrane through parasite-induced permeability pathways. Here, we demonstrate that the parasite choline transporter mediates the delivery of these compounds to the intracellular parasite. Thus, the induced permeability pathways in the host erythrocyte membrane and the parasite choline transporter described here form a cooperative transport system that shows great promise for the selective targeting of new agents for the chemotherapy of malaria.