Development and validation of an ultra-high performance liquid chromatography coupled to tandem mass spectrometry method for the quantification of the antileishmanial drug paromomycin in human skin tissue.

Bioanalytical assay development and validation procedures were performed to quantify antiprotozoal drug paromomycin in human skin tissue by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Paromomycin, an aminoglycoside drug, is administered intra-muscularly and used in the treatment of multiple clinical presentations of the neglected tropical disease leishmaniasis. It is currently studied in the treatment of post-kala-azar dermal leishmaniasis, a disease where the Leishmania parasites divide and reside in the skin. We present a target-site bioanalytical method to accurately quantify paromomycin in human skin tissue, with the clinical purpose of quantifying paromomycin in skin biopsies from post-kala-azar dermal leishmaniasis patients originating from Sudan. Enzymatic digestion using collagenase A incubated at 37 °C overnight was employed as homogenization method to produce skin tissue homogenates. Further sample preparation was performed by protein precipitation using trichloroacetic acid and a dilution step. Final extracts were injected onto a C18 analytical column and isocratic heptafluorobutyric acid ion-pair separation and elution were employed. The chromatography system was coupled to a triple quadrupole mass spectrometer for detection. The method was validated in digestion solution over a linear range from 5 to 1000 ng/mL (r2 ≥ 0.9967) with the assay performance of accuracy and precision within acceptable criteria values as stated by the EMA guidelines. Furthermore, matrix effects were observed in human skin tissue and were corrected by the multiple deuterated paromomycin internal standard. No substantial IS-normalized matrix effect was detected along with relatively high sample preparation recovery. Consequently, digestion solution matrix serving as the preparation of calibration standards can be used as surrogate matrix for human skin tissue, which is convenient given the limited availability of control matrix. Finally, paromomycin was accurately quantified in skin of post-kala-azar dermal leishmaniasis patients originating from clinical trials in Sudan.

Geographical Variability in Paromomycin Pharmacokinetics Does Not Explain Efficacy Differences between Eastern African and Indian Visceral Leishmaniasis Patients.

INTRODUCTION: Intramuscular paromomycin monotherapy to treat visceral leishmaniasis (VL) has been shown to be effective for Indian patients, while a similar regimen resulted in lower efficacy in Eastern Africa, which could be related to differences in paromomycin pharmacokinetics. METHODS: Pharmacokinetic data were available from two randomized controlled trials in VL patients from Eastern Africa and India. African patients received intramuscular paromomycin monotherapy (20 mg/kg for 21 days) or combination therapy (15 mg/kg for 17 days) with sodium stibogluconate. Indian patients received paromomycin monotherapy (15 mg/kg for 21 days). A population pharmacokinetic model was developed for paromomycin in Eastern African and Indian VL patients. RESULTS: Seventy-four African patients (388 observations) and 528 Indian patients (1321 observations) were included in this pharmacokinetic analysis. A one-compartment model with first-order kinetics of absorption and elimination best described paromomycin in plasma. Bioavailability (relative standard error) was 1.17 (5.18%) times higher in Kenyan and Sudanese patients, and 2.46 (24.5%) times higher in Ethiopian patients, compared with Indian patients. Ethiopian patients had an approximately fourfold slower absorption rate constant of 0.446 h-1 (18.2%). Area under the plasma concentration-time curve for 24 h at steady-state (AUCτ,SS) for 15 mg/kg/day (median [interquartile range]) was higher in Kenya and Sudan (172.7 µg·h/mL [145.9-214.3]) and Ethiopia (230.1 µg·h/mL [146.3-591.2]) compared with India (97.26 µg·h/mL [80.83-123.4]). CONCLUSION: The developed model provides detailed insight into the pharmacokinetic differences among Eastern African countries and India, however the resulting differences in paromomycin exposure do not seem to explain the geographical differences in paromomycin efficacy in the treatment of VL patients.

Safety and immunogenicity of ChAd63-KH vaccine in post-kala-azar dermal leishmaniasis patients in Sudan.

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic, stigmatizing skin condition occurring frequently after apparent clinical cure from visceral leishmaniasis. Given an urgent need for new treatments, we conducted a phase IIa safety and immunogenicity trial of ChAd63-KH vaccine in Sudanese patients with persistent PKDL. LEISH2a (ClinicalTrials.gov: NCT02894008) was an open-label three-phase clinical trial involving sixteen adult and eight adolescent patients with persistent PKDL (median duration, 30 months; range, 6-180 months). Patients received a single intramuscular vaccination of 1 × 1010 viral particles (v.p.; adults only) or 7.5 × 1010 v.p. (adults and adolescents), with primary (safety) and secondary (clinical response and immunogenicity) endpoints evaluated over 42-120 days follow-up. AmBisome was provided to patients with significant remaining disease at their last visit. ChAd63-KH vaccine showed minimal adverse reactions in PKDL patients and induced potent innate and cell-mediated immune responses measured by whole-blood transcriptomics and ELISpot. 7/23 patients (30.4%) monitored to study completion showed >90% clinical improvement, and 5/23 (21.7%) showed partial improvement. A logistic regression model applied to blood transcriptomic data identified immune modules predictive of patients with >90% clinical improvement. A randomized controlled trial to determine whether these clinical responses were vaccine-related and whether ChAd63-KH vaccine has clinical utility is underway.

Blood Parasite Load as an Early Marker to Predict Treatment Response in Visceral Leishmaniasis in Eastern Africa.

BACKGROUND: To expedite the development of new oral treatment regimens for visceral leishmaniasis (VL), there is a need for early markers to evaluate treatment response and predict long-term outcomes. METHODS: Data from 3 clinical trials were combined in this study, in which Eastern African VL patients received various antileishmanial therapies. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative polymerase chain reaction (qPCR) before, during, and up to 6 months after treatment. The predictive performance of pharmacodynamic parameters for clinical relapse was evaluated using receiver-operating characteristic curves. Clinical trial simulations were performed to determine the power associated with the use of blood parasite load as a surrogate endpoint to predict clinical outcome at 6 months. RESULTS: The absolute parasite density on day 56 after start of treatment was found to be a highly sensitive predictor of relapse within 6 months of follow-up at a cutoff of 20 parasites/mL (area under the curve 0.92, specificity 0.91, sensitivity 0.89). Blood parasite loads correlated well with tissue parasite loads (ρ = 0.80) and with microscopy gradings of bone marrow and spleen aspirate smears. Clinical trial simulations indicated a > 80% power to detect a difference in cure rate between treatment regimens if this difference was high (> 50%) and when minimally 30 patients were included per regimen. CONCLUSIONS: Blood Leishmania parasite load determined by qPCR is a promising early biomarker to predict relapse in VL patients. Once optimized, it might be useful in dose finding studies of new chemical entities.

Visceral leishmaniasis relapse hazard is linked to reduced miltefosine exposure in patients from Eastern Africa: a population pharmacokinetic/pharmacodynamic study.

BACKGROUND: Low efficacy of miltefosine in the treatment of visceral leishmaniasis was recently observed in Eastern Africa. OBJECTIVES: To describe the pharmacokinetics and establish a pharmacokinetic/pharmacodynamic relationship for miltefosine in Eastern African patients with visceral leishmaniasis, using a time-to-event approach to model relapse of disease. METHODS: Miltefosine plasma concentrations from 95 patients (48 monotherapy versus 47 combination therapy) were included in the population pharmacokinetic model using non-linear mixed effects modelling. Subsequently a time-to-event model was developed to model the time of clinical relapse. Various summary pharmacokinetic parameters (various AUCs, Time > EC50, Time > EC90), normalized within each treatment arm to allow simultaneous analysis, were evaluated as relapse hazard-changing covariates. RESULTS: A two-compartment population model with first-order absorption fitted the miltefosine pharmacokinetic data adequately. Relative bioavailability was reduced (-74%, relative standard error 4.7%) during the first week of treatment of the monotherapy arm but only the first day of the shorter combination regimen. Time to the relapse of infection could be described using a constant baseline hazard (baseline 1.8 relapses/year, relative standard error 72.7%). Miltefosine Time > EC90 improved the model significantly when added in a maximum effect function on the baseline hazard (half maximal effect with Time > EC90 6.97 days for monotherapy). CONCLUSIONS: Miltefosine drug exposure was found to be decreased in Eastern African patients with visceral leishmaniasis, due to a (transient) initial lower bioavailability. Relapse hazard was inversely linked to miltefosine exposure. Significantly lower miltefosine exposure was observed in children compared with adults, further urging the need for implementation of dose adaptations for children.

Insights into the possible role of IFNG and IFNGR1 in Kala-azar and Post Kala-azar Dermal Leishmaniasis in Sudanese patients.

BACKGROUND: Little is known about the parasite/host factors that lead to Post Kala-azar Dermal Leishmaniasis (PKDL) in some visceral leishmaniasis (VL) patients after drug-cure. Studies in Sudan provide evidence for association between polymorphisms in the gene (IFNGR1) encoding the alpha chain of interferon-γ receptor type I and risk of PKDL. This study aimed to identify putative functional polymorphisms in the IFNGR1 gene, and to determine whether differences in expression of interferon-γ (IFNG) and IFNGR1 at the RNA level are associated with pathogenesis of VL and/or PKDL in Sudan. METHODS: Sanger sequencing was used to re-sequence 841 bp of upstream, exon1 and intron1 of the IFNGR1 gene in DNA from 30 PKDL patients. LAGAN and SYNPLOT bioinformatics tools were used to compare human, chimpanzee and dog sequences to identify conserved noncoding sequences carrying putative regulatory elements. The relative expression of IFNG and IFNGR1 in paired pre- and post-treatment RNA samples from the lymph nodes of 24 VL patients, and in RNA samples from skin biopsies of 19 PKDL patients, was measured using real time PCR. Pre- versus post-treatment expression was evaluated statistically using the nonparametric Wilcoxon matched pairs signed-rank test. RESULTS: Ten variants were identified in the 841 bp of sequence, four of which are novel polymorphisms at -77A/G, +10 C/T, +18C/T and +91G/T relative to the IFNGR1 initiation site. A cluster of conserved non-coding sequences with putative regulatory variants was identified in the distal promoter of IFNGR1. Variable expression of IFNG was detected in lymph node aspirates of VL patients before treatment, with a marked reduction (P = 0.006) in expression following treatment. IFNGR1 expression was also variable in lymph node aspirates from VL patients, with no significant reduction in expression with treatment. IFNG expression was undetectable in the skin biopsies of PKDL cases, while IFNGR1 expression was also uniformly low. CONCLUSIONS: Uniformly low expression of IFN and IFNGR1 in PKDL skin biopsies could explain parasite persistence and is consistent with prior demonstration of genetic association with IFNGR1 polymorphisms. Identification of novel potentially functional rare variants at IFNGR1 makes an important general contribution to knowledge of rare variants of potential relevance in this Sudanese population.

Visceral leishmaniasis-hepatitis B/C coinfections: a rising necessity to triage patients for treatment.

BACKGROUND AND OBJECTIVES: Visceral leishmaniasis (VL) is a life-threatening infection caused by Leishmania species. In Sudan, VL is caused by L donovani. Most drugs used to treat VL, especially pentavalent antimony compounds (sodium stibogluconate, SSG), are potentially hepatotoxic. A number of fatal catastrophes happened because patients with VL-hepatitis B/C coinfection were indiscriminately treated with SSG in settings where VL and viral hepatitis coexist. This study aimed to study biochemical and hematological parameters of patients with VL-hepatitis B/C coinfections with the aim to modify treatment protocols to reduce coinfection.added morbidity and mortality. DESIGN AND SETTINGS: This was a prospective analytical, hospital-based, and case-controlled study. The study was done at Kassab Hospital and Professor Elhassan Centre for tropical medicine during the period of February 2008 to April 2013. MATERIALS AND METHODS: Following informed consent by the participants, 78 parasitologically confirmed VL patients with either hepatitis B or C or both and 528 sex- and age-unmatched VL patients without hepatitis B/C coinfection (control group) were enrolled sequentially. Diagnosis of hepatitis B or C was made using immunochromatographic test kits and confirmed by an enzyme-linked immunosorbent assay. RESULTS: VL patients with hepatitis B/C coinfections had significantly increased levels of AST, ALT, and total bilirubin compared to the control group (P=.0001 for all), with significantly decreased levels of albumin and platelets counts (P=.0029 for both). CONCLUSION: VL-hepatitis B/C coinfections are an emerging entity that needs anti-leishmanial treatment modification. Alternative treatments like paromomycin and amphotericin B (AmBisome) could be reserved for these patients.

Safety and efficacy of single dose versus multiple doses of AmBisome for treatment of visceral leishmaniasis in eastern Africa: a randomised trial.

BACKGROUND: Anti-leishmanial drug regimens that include a single dose AmBisome could be suitable for eastern African patients with symptomatic visceral leishmaniasis (VL) but the appropriate single dose is unknown. METHODOLOGY: A multi-centre, open-label, non-inferiority, randomized controlled trial with an adaptive design, was conducted to compare the efficacy and safety of a single dose and multiple doses of AmBisome for the treatment of VL in eastern Africa. The primary efficacy endpoint was definitive cure (DC) at 6 months. Symptomatic patients with parasitologically-confirmed, non-severe VL, received a single dose of AmBisome 7.5 mg/kg body weight or multiple doses, 7 times 3 mg/kg on days 1-5, 14, and 21. If interim analyses, evaluated 30 days after the start of treatment following 40 or 80 patients, showed the single dose gave significantly poorer parasite clearance than multiple doses at the 5% significance level, the single dose was increased by 2·5 mg/kg. In a sub-set of patients, parasite clearance was measured by quantitative reverse transcriptase (qRT) PCR. PRINCIPAL FINDINGS: The trial was terminated after the third interim analysis because of low efficacy of both regimens. Based on the intention-to-treat population, DC was 85% (95%CI 73-93%), 40% (95%CI 19-64%), and 58% (95%CI 41-73%) in patients treated with multiple doses (n = 63), and single doses of 7·5 (n = 21) or 10 mg/kg (n = 40), respectively. qRT-PCR suggested superior parasite clearance with multiple doses as early as day 3. Safety data accorded with the drug label. CONCLUSIONS: The tested AmBisome regimens would not be suitable for VL treatment across eastern Africa. An optimal single dose regimen was not identified. TRIALS REGISTRATION: www.clinicaltrials.govNCT00832208.

The economic burden of visceral leishmaniasis in Sudan: an assessment of provider and household costs.

Visceral leishmaniasis (VL) is a neglected parasitic disease that is fatal if left untreated and is endemic in eastern Sudan. We estimated the direct and indirect costs of treatment of VL from the perspective of the provider and the household at three public hospitals in Gedaref State. The median total cost for one VL episode was estimated to be US$450. Despite the free provision of VL drugs at public hospitals, households bore 53% of the total cost of VL with one episode of VL representing 40% of the annual household income. More than 75% of households incurred catastrophic out-of-pocket expenditures. The length of treatment of 30 days led to important costs for both health providers and households. Alternative treatment regimens that reduce the duration of treatment are urgently needed.

Immunogenicity and immune modulatory effects of in silico predicted L. donovani candidate peptide vaccines.

Visceral leishmaniasis (VL) is a serious parasitic disease for which control measures are limited and drug resistance is increasing. First and second generation vaccine candidates have not been successful. The goal of the present study was to select possibly immunogenic L. donovani donovani GP63 peptides using immunoinformatics tools and to test their immunogenicity in vitro. The amino acid sequence of L. donovani donovani GP63 [GenBank accession: ACT31401] was screened using the EpiMatrix algorithm for putative T cell epitopes that would bind to the most common HLA class II alleles (DRB1*1101 and DRB1*0804) among at-risk populations. Four T cell epitopes were selected from nine potential candidates. Stimulation of whole blood from healthy volunteers using the peptides separately produced mean IFN-γ and IL-4 levels that were not significantly different from negative controls, while the pooled peptides produced a moderate IFN-γ increase in some volunteers. However, mean IL-10 levels were significantly reduced for all individuals compared with controls. The immunogenicity of these epitopes may be harnessed most effectively in a vaccine delivered in combination with immune-modulating adjuvants.

Safety and efficacy of miltefosine alone and in combination with sodium stibogluconate and liposomal amphotericin B for the treatment of primary visceral leishmaniasis in East Africa: study protocol for a randomized controlled trial.

BACKGROUND: Treatment options for visceral leishmaniasis (VL) in East Africa are far from satisfactory due to cost, toxicity, prolonged treatment duration or emergence of parasite resistance. Hence there is a need to explore alternative treatment protocols such as miltefosine alone or in combinations including miltefosine, sodium stibogluconate (SSG) or liposomal amphotericin B. The aim of this trial is to identify regimen(s) which are sufficiently promising for future trials in East Africa. METHODS/DESIGN: A phase II randomized, parallel arm, open-labelled trial is being conducted to assess the efficacy of each of the three regimens: liposomal amphotericin B with SSG, Liposomal amphotericin B with miltefosine and miltefosine alone. The primary endpoint is cure at day 28 with secondary endpoint at day 210 (6 months). Initial cure is a single composite measure based on parasitologic evaluation (bone marrow, spleen or lymph node aspirate) and clinical assessment. Repeated interim analyses have been planned after recruitment of 15 patients in each arm with a maximum sample size of 63 for each. These will follow group-sequential methods (the triangular test) to identify when a regimen is inadequate (<75% efficacy) or adequate (>90% efficacy). We describe a method to ensure consistency of the sequential analysis of day 28 cure with the non-sequential analysis of day 210 cure. DISCUSSION: A regimen with adequate efficacy would be a candidate for treatment of VL with reasonable costs. The design allows repeated testing throughout the trial recruitment period while maintaining good statistical properties (Type I & II error rates) and reducing the expected sample sizes. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01067443.