Determining tissue distribution of the oral antileishmanial agent miltefosine: a physiologically-based pharmacokinetic modeling approach.

Miltefosine (MTS) is the only approved oral drug for treating leishmaniasis caused by intracellular Leishmania parasites that localize in macrophages of the liver, spleen, skin, bone marrow, and lymph nodes. MTS is extensively distributed in tissues and has prolonged elimination half-lives due to its high plasma protein binding, slow metabolic clearance, and minimal urinary excretion. Thus, understanding and predicting the tissue distribution of MTS help assess therapeutic and toxicologic outcomes of MTS, especially in special populations, e.g., pediatrics. In this study, a whole-body physiologically-based pharmacokinetic (PBPK) model of MTS was built on mice and extrapolated to rats and humans. MTS plasma and tissue concentration data obtained by intravenous and oral administration to mice were fitted simultaneously to estimate model parameters. The resulting high tissue-to-plasma partition coefficient values corroborate extensive distribution in all major organs except the bone marrow. Sensitivity analysis suggests that plasma exposure is most susceptible to changes in fraction unbound in plasma. The murine oral-PBPK model was further validated by assessing overlay of simulations with plasma and tissue profiles obtained from an independent study. Subsequently, the murine PBPK model was extrapolated to rats and humans based on species-specific physiological and drug-related parameters, as well as allometrically scaled parameters. Fold errors for pharmacokinetic parameters were within acceptable range in both extrapolated models, except for a slight underprediction in the human plasma exposure. These animal and human PBPK models are expected to provide reliable estimates of MTS tissue distribution and assist dose regimen optimization in special populations.

Skin pharmacokinetics of miltefosine in the treatment of post-kala-azar dermal leishmaniasis in South Asia.

INTRODUCTION: Post-kala-azar dermal leishmaniasis (PKDL) arises as a dermal complication following a visceral leishmaniasis (VL) infection. Current treatment options for PKDL are unsatisfactory, and there is a knowledge gap regarding the distribution of antileishmanial compounds within human skin. The present study investigated the skin distribution of miltefosine in PKDL patients, with the aim to improve the understanding of the pharmacokinetics at the skin target site in PKDL. METHODS: Fifty-two PKDL patients underwent treatment with liposomal amphotericin B (20 mg/kg) plus miltefosine (allometric dosing) for 21 days. Plasma concentrations of miltefosine were measured on study days 8, 15, 22 and 30, while a punch skin biopsy was taken on day 22. A physiologically based pharmacokinetic (PBPK) model was developed to evaluate the distribution of miltefosine into the skin. RESULTS: Following the allometric weight-based dosing regimen, median miltefosine concentrations on day 22 were 43.73 µg/g (IQR: 21.94-60.65 µg/g) in skin and 33.29 µg/mL (IQR: 25.9-42.58 µg/mL) in plasma. The median individual concentration ratio of skin to plasma was 1.19 (IQR: 0.79-1.9). In 87% (45/52) of patients, skin exposure was above the suggested EC90 PK target of 10.6 mg/L associated with in vitro susceptibility. Simulations indicated that the residence time of miltefosine in the skin would be more than 2-fold longer than in plasma, estimated by a mean residence time of 604 versus 266 hours, respectively. CONCLUSION: This study provides the first accurate measurements of miltefosine penetration into the skin, demonstrating substantial exposure and prolonged retention of miltefosine within the skin. These findings support the use of miltefosine in cutaneous manifestations of leishmaniasis. In combination with parasitological and clinical data, these results are critical for the future optimization of combination therapies with miltefosine in the treatment of PKDL.

Population pharmacokinetics of a combination of miltefosine and paromomycin in Eastern African children and adults with visceral leishmaniasis.

OBJECTIVES: To improve visceral leishmaniasis (VL) treatment in Eastern Africa, 14- and 28-day combination regimens of paromomycin plus allometrically dosed miltefosine were evaluated. As the majority of patients affected by VL are children, adequate paediatric exposure to miltefosine and paromomycin is key to ensuring good treatment response. METHODS: Pharmacokinetic data were collected in a multicentre randomized controlled trial in VL patients from Kenya, Sudan, Ethiopia and Uganda. Patients received paromomycin (20 mg/kg/day for 14 days) plus miltefosine (allometric dose for 14 or 28 days). Population pharmacokinetic models were developed. Adequacy of exposure and target attainment of paromomycin and miltefosine were evaluated in children and adults. RESULTS: Data from 265 patients (59% ≤12 years) were available for this pharmacokinetic analysis. Paromomycin exposure was lower in paediatric patients compared with adults [median (IQR) end-of-treatment AUC0-24h 187 (162-203) and 242 (217-328) µg·h/mL, respectively], but were both within the IQR of end-of-treatment exposure in Kenyan and Sudanese adult patients from a previous study. Cumulative miltefosine end-of-treatment exposure in paediatric patients and adults [AUCD0-28 517 (464-552) and 524 (456-567) µg·day/mL, respectively] and target attainment [time above the in vitro susceptibility value EC90 27 (25-28) and 30 (28-32) days, respectively] were comparable to previously observed values in adults. CONCLUSIONS: Paromomycin and miltefosine exposure in this new combination regimen corresponded to the desirable levels of exposure, supporting the implementation of the shortened 14 day combination regimen. Moreover, the lack of a clear exposure-response and exposure-toxicity relationship indicated adequate exposure within the therapeutic range in the studied population, including paediatric patients.

Pharmacokinetic study of the novel phosphocholine derivative 3-dibutylaminopropylphosphonic acid by LC-MS coupling.

CRP is an important mediator of the inflammatory response. Pro-inflammatory CRP effects are mediated by pCRP* and mCRP, dissociation products of the native pCRP. The concentration of pCRP during inflammation may rise up to concentrations 1000-fold from baseline. By prevention of the conformational change from pCRP to pCRP*, pro-inflammatory immune responses can be inhibited and local tissue damage reduced. 3-(Dibutylamino)propylphosphonic acid (C10m) is a new substance that can suppress ischemic-reperfusion injury by targeting CRP in the complement cascade. It hampers dissociation of pCRP into its monomers, thus preventing exacerbation of tissue inflammation subsequent to reperfusion injury. In this study, the pharmacokinetics and metabolism of the new drug candidate C10m was investigated. A sensitive and selective method for detection of C10m and its metabolites from plasma and urine was developed with LC-MS and LC-MS/MS coupling. The LLOQ is at 0.1 µg mL-1 and recovery at 87.4% ± 2.8%. Accuracy and precision were within 15% coefficient of variation and nominal concentrations, respectively. Concentration time profile after i.v. bolus injection of C10m was analyzed by LC-MS/MS. Bioavailability has shown to be below 30%. Most likely due to the compounds' very polar chemical properties, no phase-I or phase-II metabolism could be observed. Absence of phase-I metabolism was cross-checked by performing microsomal incubations. Our study revealed that C10m is rapidly eliminated via urine excretion and that half-times appear to be increased with coadministration of the target pCRP.

Evaluation of release and pharmacokinetics of hexadecylphosphocholine (miltefosine) in phosphatidyldiglycerol-based thermosensitive liposomes.

Hexadecylphosphocholine (HePC, Miltefosine) is a drug from the class of alkylphosphocholines with an antineoplastic and antiprotozoal activity. We previously reported that HePC uptake from thermosensitive liposomes (TSL) containing 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) into cancer cells is accelerated at mild hyperthermia (HT) resulting in increased cytotoxicity. In this study, we compared HePC release of different TSL formulations in serum. HePC showed rapid but incomplete release below the transition temperature (Tm) of investigated TSL formulations in serum. Short heating (5 min) to 42 °C increased HePC release from DPPG2-TSL (Tm = 41 °C) by a factor of two in comparison to body temperature (37 °C). Bovine serum albumin (BSA) induced HePC release from DPPG2-TSL comparable to serum. Furthermore, multilamellar vesicles (MLV) were capable to extract HePC from DPPG2-TSL in a concentration- and temperature-dependent manner. Repetitive exposure of DPPG2-TSL to MLV at 37 °C led to a fast initial release of HePC which slowed down after subsequent extraction cycles finally reaching approx. 50% HePC release. A pharmacokinetic study in rats revealed a biphasic pattern with an immediate clearance of approx. 50% HePC whereas the remaining 50% HePC showed a prolonged circulation time. We speculate that HePC located in the external leaflet of DPPG2-TSL is rapidly released upon contact with suitable biological acceptors. As demonstrated by MLV transfer experiments, asymmetric incorporation of HePC into the internal leaflet of DPPG2-TSL might improve HePC retention in presence of complex biological media and still give rise to HT-induced HePC release.

Enhancing the in vitro and in vivo activity of itraconazole against breast cancer using miltefosine-modified lipid nanocapsules.

Itraconazole (ITC), a well-tolerated antifungal drug, exerts multiple anticancer effects which justified its preclinical and clinical investigation as potential anti-cancer agent with reduced side effects. Enhancement of ITC anti-cancer efficacy would bring valuable benefits to patients. We propose herein lipid nanocapsules (LNCs) modified with a subtherapeutic dose of miltefosine (MFS) as a membrane bioactive amphiphilic additive (M-ITC-LNC) for the development of an ITC nanoformulation with enhanced anticancer activity compared with ITC solution (ITC-sol) and unmodified ITC-LNC. Both LNC formulations showed a relatively small size (43-46 nm) and high entrapment efficiency (>97%), though ITC release was more sustained by M-ITC-LNC. Cytotoxicity studies revealed significantly greater anticancer activity and selectivity of M-ITC-LNC for MCF-7 breast cancer cells compared with ITC-sol and ITC-LNC. This trend was substantiated by in vivo findings following a 14 day-treatment of murine mammary pad Ehrlich tumors. M-ITC-LNC showed the greatest enhancement of the ITC-induced tumor growth inhibition, proliferation, and necrosis. At the molecular level, the tumor content of Gli 1, caspase-3, and vascular endothelial growth factor verified superiority of M-ITC-LNC in enhancing the ITC antiangiogenic, apoptotic, and Hedgehog pathway inhibitory effects. Finally, histopathological and biochemical analysis indicated greater reduction of ITC systemic toxicity by M-ITC-LNC. Superior performance of M-ITC-LNC was attributed to the effect of MFS on the structural and release properties of LNC coupled with its distinct bioactivities. In conclusion, MFS-modified LNC provides a simple nanoplatform integrating the potentials of LNC and MFS for enhancing the chemotherapeutic efficacy of ITC and possibly other oncology drugs.

Preparation, Pharmacokinetics, and Antitumor Potential of Miltefosine-Loaded Nanostructured Lipid Carriers.

BACKGROUND: The purpose of this study was to investigate the suitability of nanostructured lipid carriers (NLCs) loaded with miltefosine (HePC) as an anticancer drug for the treatment of breast cancer. METHODS: HePC-NLCs were prepared using a microemulsion technique and then evaluated for particle size, polydispersity index (PDI), incorporation efficiency, in vitro release of entrapped drug, and hemolytic potential. Furthermore, pharmacokinetic, biodistribution, and liver toxicity analyses were performed in Sprague-Dawley rats, and antitumor efficacy was evaluated in Michigan Cancer Foundation-7 (MCF-7) and squamous cell carcinoma-7 (SCC-7) cells in vitro and in tumour-bearing BALB/c mice in vivo. Advanced analyses including survival rate, immunohistopathology, and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays were performed to evaluate apoptosis in vivo. RESULTS: The average particle size of the HePC-NLCs was 143 ± 16 nm, with a narrow PDI (0.104 ± 0.002), and the incorporation efficiency was found to be 91 ± 7%. The NLCs released HePC in a sustained manner, and this release was significantly lower than that of free drug. The in vitro hemolytic assay demonstrated a significantly reduced hemolytic potential (~9%) of the NLCs compared to that of the test formulations. The HePC-NLCs demonstrated enhanced pharmacokinetic behaviour over free drug, including extended blood circulation and an abridged clearance rate in rats. Furthermore, the HePC-NLCs exhibited higher cytotoxicity than the free drug in MCF-7 and SCC-7 cells. Moreover, the HePC-NLCs showed significantly enhanced (P < 0.005) antitumor activity compared to that of the control and free drug-treated mouse groups. Tumour cell apoptosis was also confirmed, indicating the antitumor potential of the HePC-NLCs. CONCLUSION: These findings demonstrate the ability of NLCs as a drug delivery system for enhanced pharmacokinetic, antitumor, and apoptotic effects, most importantly when loaded with HePC.

Pharmacokinetic / pharmacodynamic relationships of liposomal amphotericin B and miltefosine in experimental visceral leishmaniasis.

BACKGROUND: There is a continued need to develop effective and safe treatments for visceral leishmaniasis (VL). Preclinical studies on pharmacokinetics and pharmacodynamics of anti-infective agents, such as anti-bacterials and anti-fungals, have provided valuable information in the development and dosing of these agents. The aim of this study was to characterise the pharmacokinetic and pharmacodynamic properties of the anti-leishmanial drugs AmBisome and miltefosine in a preclinical disease model of VL. METHODOLOGY / PRINCIPAL FINDINGS: BALB/c mice were infected with L. donovani (MHOM/ET/67/HU3) amastigotes. Groups of mice were treated with miltefosine (orally, multi-dose regimen) or AmBisome (intravenously, single dose regimen) or left untreated as control groups. At set time points groups of mice were killed and plasma, livers and spleens harvested. For pharmacodynamics the hepatic parasite burden was determined microscopically from tissue impression smears. For pharmacokinetics drug concentrations were measured in plasma and whole tissue homogenates by LC-MS. Unbound drug concentrations were determined by rapid equilibrium dialysis. Doses exerting maximum anti-leishmanial effects were 40 mg/kg for AmBisome and 150 mg/kg (cumulatively) for miltefosine. AmBisome displayed a wider therapeutic range than miltefosine. Dose fractionation at a total dose of 2.5 mg/kg pointed towards concentration-dependent anti-leishmanial activity of AmBisome, favouring the administration of large doses infrequently. Protein binding was >99% for miltefosine and amphotericin B in plasma and tissue homogenates. CONCLUSION / SIGNIFICANCE: Using a PK/PD approach we propose optimal dosing strategies for AmBisome. Additionally, we describe pharmacokinetic and pharmacodynamic properties of miltefosine and compare our findings in a preclinical disease model to available knowledge from studies in humans. This approach also presents a strategy for improved use of animal models in the drug development process for VL.

Topical treatment of cutaneous leishmaniasis with novel amphotericin B-miltefosine co-incorporated second generation ultra-deformable liposomes.

The present study aims to optimize and evaluate amphotericin B (AmB) and miltefosine (MTF) co-loaded second generation ultra-deformable liposomes (SGUDLs) for the topical treatment of cutaneous leishmaniasis (CL). The development of an effective topical drug formulation against CL is desirable because of its non-invasive nature, which may potentially enhance the patient adherence and treatment accessibility. AmB-MTF co-loaded SGUDLs were prepared and characterized for size, entrapment efficiency (EE) and elasticity. The optimized formulation was then subjected to ex-vivo permeation studies in addition to cytotoxicity and anti-leishmanial assays. The co-loaded SGUDLs had an average size of 139.7 ± 1.7 nm and high EE of 77.8 ± 3.9% with respect to AmB. The ex-vivo permeation of co-loaded SGUDLs exhibited 6.15-fold higher permeation of AmB. A synergistic interaction was observed between AmB and MTF, and anti-leishmanial activity of co-loaded SGUDLs against amastigotes of Lesihmania mexicana indicated 8.62 and 6.12-fold lower IC50 values of AmB and MTF as compared to plain drug solutions, respectively. The results of the in-vivo study displayed a significant reduction in the parasitic burden in an infected BALB/c experimental model of CL. In conclusion, AmB-MTF co-loaded SGUDLs could be an effective topical treatment option against CL.

Accelerated Blood Clearance of Lipid Nanoparticles Entails a Biphasic Humoral Response of B-1 Followed by B-2 Lymphocytes to Distinct Antigenic Moieties.

Accelerated blood clearance (ABC) is a phenomenon in which certain pharmaceutical agents are rapidly cleared from the blood upon second and subsequent administrations. ABC has been observed for many lipid-delivery vehicles, including liposomes and lipid nanoparticles (LNP). Previous studies have demonstrated a role for humoral responses against the polyethylene glycol motifs in clearance, but significant gaps remain in our understanding of the mechanism of ABC, and strategies for limiting the impact of ABC in a clinical setting have been elusive. mRNA therapeutics have great promise, but require chronic administration in encapsulating delivery systems, of which LNP are the most clinically advanced. In this study, we investigate the mechanisms of ABC for mRNA-formulated LNP in vivo and in vitro. We present evidence that ABC of mRNA-formulated LNP is dramatic and proceeds rapidly, based on a previously unrecognized ability of LNP to directly activate B-1 lymphocytes, resulting in the production of antiphosphorylcholine IgM Abs in response to initial injection. Upon repeated injections, B-2 lymphocytes also become activated and generate a classic anti-polyethylene glycol adaptive humoral response. The ABC response to phosphorylcholine/LNP-encapsulated mRNA is therefore a combination of early B-1 lymphocyte and later B-2 lymphocyte responses.

In Silico Docking of Alkylphosphocholine Analogs to Human Serum Albumin Predicts Partitioning and Pharmacokinetics.

Alkylphosphocholine (APC) analogs are a novel class of broad-spectrum tumor-targeting agents that can be used for both diagnosis and treatment of cancer. The potential for clinical translation for APC analogs will strongly depend on their pharmacokinetic (PK) profiles. The aim of this work was to understand how the chemical structures of various APC analogs impact binding and PK. To achieve this aim, we performed in silico docking analysis, in vitro and in vivo partitioning experiments, and in vivo PK studies. Our results have identified 7 potential high-affinity binding sites of these compounds on human serum albumin (HSA) and suggest that the size of the functional group directly influences the albumin binding, partitioning, and PK. Namely, the bulkier the functional groups, the weaker the agent binds to albumin, the more the agent partitions onto lipoproteins, and the less time the agent spends in circulation. The results of these experiments provide novel molecular insights into the binding, partitioning, and PK of this class of compounds and similar molecules as well as suggest pharmacological strategies to alter their PK profiles. Importantly, our methodology may provide a way to design better drugs by better characterizing the PK profile for lead compound optimization.

Preclinical Characterization of 86/90Y-NM600 in a Variety of Murine and Human Cancer Tumor Models.

We characterize the in vivo biodistribution and tumor selectivity of 86Y-NM600, a theranostic alkylphosphocholine radiometal chelate with broad tumor selectivity, in a variety of preclinical cancer models. Methods: Mice bearing flank tumors (representative of lung, pancreatic, prostate, liver, skin, and lymphoid cancers) were injected intravenously with 9.25 MBq of 86Y-NM600 and imaged longitudinally over 4-5 d using small-animal PET/CT. Percentage injected activity per gram (%IA/g) for each volume of interest was measured at each time point for the organs of interest. Mice were euthanized after the final time point, and the tumor and organs of interest were counted with an automatic γ-counter. Absorbed doses delivered by 90Y-NM600 per injected activity (Gy/MBq) were estimated. Mice bearing B78 flank tumors were injected with a prescription of 90Y-NM600 that delivered 2.5 Gy of absorbed tumor dose and was compared with an equivalent absorbed dose delivered via external-beam radiotherapy using tumor volume as a measure of response. Histology and complete blood counts were analyzed in naïve C57BL/6 mice that were injected with 9.25 MBq of 90Y-NM600 at 5, 10, and 28 d after injection. Results: PET imaging showed consistent tumor accumulation and retention across all tumor models investigated, with little off-target retention of NM600 except in the liver, as is characteristic of hepatobiliary metabolism. The tumor uptake was highest in the pancreatic and lymphoid cancer models, reaching peak concentrations of 9.34 ± 2.66 %IA/g (n = 3) and 9.10 ± 0.13 %IA/g (n = 3), respectively, at approximately 40-48 h after injection. These corresponded to tumor dose estimates of 2.72 ± 0.33 Gy/MBq and 2.67 ± 0.32 Gy/MBq, respectively. In the toxicity study, there were no visible signs of acute toxicity by histology, and perturbation of hematologic parameters was transient when observed, returning to pretherapy levels after 28 d. Conclusion: NM600 is a theranostic agent with a unique ability to selectively target a variety of cancer types, presenting a unique opportunity for PET image-guided targeted radionuclide therapy and combination with immunotherapies.

Ultralong circulating choline phosphate liposomal nanomedicines for cascaded chemo-radiotherapy.

Cancer radiation therapy (RT) is limited by endogenous DNA repair of tumor cells and microenvironmental hypoxia in tumor tissues. Herein, we demonstrated an effective cancer chemo-radiotherapy strategy based on choline phosphate liposomal nanomedicines, which inhibit the intrinsic radioresistance of RT and concomitantly harness the RT-induced hypoxia to produce additional toxicity to overcome post-RT radioresistance. To achieve this strategy, a radiotherapy sensitizer, vorinostat, and a hypoxia-activated banoxantrone dihydrochloride (AQ4N) were simultaneously delivered to a tumor using liposomes composed of an inverted polarity lipid 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe). The DOCPe liposomes exhibited a longer blood circulation time and enhanced tumor accumulation, compared to their zwitterionic phosphocholine counterpart. The RT was sensitized by vorinostat to kill non-tolerant normoxic tumor cells efficiently. The irradiation aggravated hypoxia-activated AQ4N to further potentiate RT treatment. This chemo-radiotherapy combination showed excellent tumor treatment efficacy and is promising for future clinical translation.

Pharmacokinetics, Safety, and Efficacy of an Allometric Miltefosine Regimen for the Treatment of Visceral Leishmaniasis in Eastern African Children: An Open-label, Phase II Clinical Trial.

BACKGROUND: Convenient, safe, and effective treatments for visceral leishmaniasis in Eastern African children are lacking. Miltefosine, the only oral treatment, failed to achieve adequate efficacy, particularly in children, in whom linear dosing (2.5 mg/kg/day for 28 days) resulted in a 59% cure rate, with lower systemic exposure than in adults. METHODS: We conducted a Phase II trial in 30 children with visceral leishmaniasis, aged 4-12 years, to test whether 28 days of allometric miltefosine dosing safely achieves a higher systemic exposure than linear dosing. RESULTS: Miltefosine accumulated during treatment. Median areas under the concentration time curve from days 0-210 and plasma maximum concentration values were slightly higher than those reported previously for children on linear dosing, but not dose-proportionally. Miltefosine exposure at the start of treatment was increased, with higher median plasma concentrations on day 7 (5.88 versus 2.67 µg/mL). Concentration-time curves were less variable, avoiding the low levels of exposure observed with linear dosing. The 210-day cure rate was 90% (95% confidence interval, 73-98%), similar to that previously described in adults. There were 19 treatment-related adverse events (AEs), but none caused treatment discontinuation. There were 2 serious AEs: both were unrelated to treatment and both patients were fully recovered. CONCLUSIONS: Allometric miltefosine dosing achieved increased and less-variable exposure than linear dosing, though not reaching the expected exposure levels. The new dosing regimen safely increased the efficacy of miltefosine for Eastern African children with visceral leishmaniasis. Further development of miltefosine should adopt allometric dosing in pediatric patients. CLINICAL TRIALS REGISTRATION: NCT02431143.

Pharmacokinetics and disposition of miltefosine in healthy mice and hamsters experimentally infected with Leishmania infantum.

Miltefosine is the only currently available oral drug for treatment of leishmaniasis. However, information on the pharmacokinetics (PK) of miltefosine is relatively scarce in animals. PK parameters and disposition of the molecule was determined in healthy NMRI mice and Syrian hamsters infected and treated with different miltefosine doses and regimens. Long half-life of the molecule was confirmed and differential pattern of accumulation of the drug was observed in analyzed organs in mice and hamster. Long treatment schedules produced miltefosine levels over IC50 value against L. infantum intracellular amastigotes for at least 24 days in spleen and liver of infected hamsters. The observed differential pattern of organ accumulation of the drug in mice and hamster supports the relevance of both species for translational research on chemotherapy of leishmaniasis.

Simultaneous population pharmacokinetic modelling of plasma and intracellular PBMC miltefosine concentrations in New World cutaneous leishmaniasis and exploration of exposure-response relationships.

Objectives: Leishmania parasites reside within macrophages and the direct target of antileishmanial drugs is therefore intracellular. We aimed to characterize the intracellular PBMC miltefosine kinetics by developing a population pharmacokinetic (PK) model simultaneously describing plasma and intracellular PBMC pharmacokinetics. Furthermore, we explored exposure-response relationships and simulated alternative dosing regimens. Patients and methods: A population PK model was developed with NONMEM, based on 339 plasma and 194 PBMC miltefosine concentrations from Colombian cutaneous leishmaniasis patients [29 children (2-12 years old) and 22 adults] receiving 1.8-2.5 mg/kg/day miltefosine for 28 days. Results: A three-compartment model with miltefosine distribution into an intracellular PBMC effect compartment best fitted the data. Intracellular PBMC distribution was described with an intracellular-to-plasma concentration ratio of 2.17 [relative standard error (RSE) 4.9%] and intracellular distribution rate constant of 1.23 day-1 (RSE 14%). In exploring exposure-response relationships, both plasma and intracellular model-based exposure estimates significantly influenced probability of cure. A proposed PK target for the area under the plasma concentration-time curve (day 0-28) of >535 mg·day/L corresponded to >95% probability of cure. In linear dosing simulations, 18.3% of children compared with 2.8% of adults failed to reach 535 mg·day/L. In children, this decreased to 1.8% after allometric dosing simulation. Conclusions: The developed population PK model described the rate and extent of miltefosine distribution from plasma into PBMCs. Miltefosine exposure was significantly related to probability of cure in this cutaneous leishmaniasis patient population. We propose an exploratory PK target, which should be validated in a larger cohort study.

Pharmacodynamics and cellular accumulation of amphotericin B and miltefosine in Leishmania donovani-infected primary macrophages.

Objectives: We examined the in vitro pharmacodynamics and cellular accumulation of the standard anti-leishmanial drugs amphotericin B and miltefosine in intracellular Leishmania donovani amastigote-macrophage drug assays. Methods: Primary mouse macrophages were infected with L. donovani amastigotes. In time-kill assays infected macrophages were exposed to at least six different concentrations of serially diluted drugs and the percentage of infected macrophages was determined after 6, 12, 24, 48, 72 and 120 h of exposure. Cellular drug accumulation was measured following exposure to highly effective drug concentrations for 1, 6, 24, 48 and 72 h. Data were analysed through a mathematical model, relating drug concentration to the percentage of infected cells over time. Host cell membrane damage was evaluated through measurement of lactate dehydrogenase release. The effect of varying the serum and albumin concentrations in medium on the cellular accumulation levels of miltefosine was measured. Results: Amphotericin B was more potent than miltefosine (EC50 values of 0.65 and 1.26 µM, respectively) and displayed a wider therapeutic window in vitro. The kinetics of the cellular accumulation of amphotericin B was concentration- and formulation-dependent. At an extracellular concentration of 10 µM miltefosine maximum cellular drug levels preceded maximum anti-leishmanial kill. Miltefosine induced membrane damage in a concentration-, time- and serum-dependent manner. Its cellular accumulation levels increased with decreasing amounts of protein in assay medium. Conclusions: We have developed a novel approach to investigate the cellular pharmacology of anti-leishmanial drugs that serves as a model for the characterization of new drug candidates.

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.

A phase I study of single-agent perifosine for recurrent or refractory pediatric CNS and solid tumors.

The PI3K/Akt/mTOR signaling pathway is aberrantly activated in various pediatric tumors. We conducted a phase I study of the Akt inhibitor perifosine in patients with recurrent/refractory pediatric CNS and solid tumors. This was a standard 3+3 open-label dose-escalation study to assess pharmacokinetics, describe toxicities, and identify the MTD for single-agent perifosine. Five dose levels were investigated, ranging from 25 to 125 mg/m2/day for 28 days per cycle. Twenty-three patients (median age 10 years, range 4-18 years) with CNS tumors (DIPG [n = 3], high-grade glioma [n = 5], medulloblastoma [n = 2], ependymoma [n = 3]), neuroblastoma (n = 8), Wilms tumor (n = 1), and Ewing sarcoma (n = 1) were treated. Only one DLT occurred (grade 4 hyperuricemia at dose level 4). The most common grade 3 or 4 toxicity at least possibly related to perifosine was neutropenia (8.7%), with the remaining grade 3 or 4 toxicities (fatigue, hyperglycemia, fever, hyperuricemia, and catheter-related infection) occurring in one patient each. Pharmacokinetics was dose-saturable at doses above 50 mg/m2/day with significant inter-patient variability, consistent with findings reported in adult studies. One patient with DIPG (dose level 5) and 4 of 5 patients with high-grade glioma (dose levels 2 and 3) experienced stable disease for two months. Five subjects with neuroblastoma (dose levels 1 through 4) achieved stable disease which was prolonged (≥11 months) in three. No objective responses were noted. In conclusion, the use of perifosine was safe and feasible in patients with recurrent/refractory pediatric CNS and solid tumors. An MTD was not defined by the 5 dose levels investigated. Our RP2D is 50 mg/m2/day.

Plasma and cerebrospinal fluid pharmacokinetics of select chemotherapeutic agents following intranasal delivery in a non-human primate model.

The blood-brain barrier (BBB) limits entry of most chemotherapeutic agents into the CNS, resulting in inadequate exposure within CNS tumor tissue. Intranasal administration is a proposed means of delivery that can bypass the BBB, potentially resulting in more effective chemotherapeutic exposure at the tumor site. The objective of this study was to evaluate the feasibility and pharmacokinetics (plasma and CSF) of intranasal delivery using select chemotherapeutic agents in a non-human primate (NHP) model. Three chemotherapeutic agents with known differences in CNS penetration were selected for intranasal administration in a NHP model to determine proof of principle of CNS delivery, assess tolerability and feasibility, and to evaluate whether certain drug characteristics were associated with increased CNS exposure. Intravenous (IV) temozolomide (TMZ), oral (PO) valproic acid, and PO perifosine were administered to adult male rhesus macaques. The animals received a single dose of each agent systemically and intranasally in separate experiments, with each animal acting as his own control. The dose of the agents administered systemically was the human equivalent of a clinically appropriate dose, while the intranasal dose was the maximum achievable dose based on the volume limitation of 1 mL. Multiple serial paired plasma and CSF samples were collected and quantified using a validated uHPLC/tandem mass spectrometry assay after each drug administration. Pharmacokinetic parameters were estimated using non-compartmental analysis. CSF penetration was calculated from the ratio of areas under the concentration-time curves for CSF and plasma (AUCCSF:plasma). Intranasal administration was feasible and tolerable for all agents with no significant toxicities observed. For TMZ, the degrees of CSF drug penetration after intranasal and IV administration were 36 (32-57) and 22 (20-41)%, respectively. Although maximum TMZ drug concentration in the CSF (Cmax) was lower after intranasal delivery compared to IV administration due to the lower dose administered, clinically significant exposure was achieved in the CSF after intranasal administration with the lower doses. This was associated with lower systemic exposure, suggesting increased efficiency and potentially lower toxicities of TMZ after intranasal delivery. For valproic acid and perifosine, CSF penetration after intranasal delivery was similar to systemic administration. Although this study demonstrates feasibility and safety of intranasal drug administration, further agent-specific studies are necessary to optimize agent selection and dosing to achieve clinically-relevant CSF exposures.