The Pharmacokinetic Profile of Palovarotene: An Open-Label Phase I Trial Investigating the Effect of Food and Potential for Drug-Drug Interaction in Healthy Participants.

BACKGROUND AND OBJECTIVES: Palovarotene is under development for the treatment of fibrodysplasia ossificans progressiva (FOP). The objectives of this study were to evaluate palovarotene pharmacokinetics under fed versus fasted conditions and its induction potential towards cytochrome P450 3A4 (CYP3A4) substrate, midazolam. METHODS: In this phase I, open-label trial (NCT04829773), palovarotene pharmacokinetics were characterized after repeated once-daily dosing. In one cohort, healthy participants received three doses of palovarotene 20 mg on Days 1, 6, and 11, as whole capsules under fasted or fed conditions, or sprinkled on food under fed conditions. In another cohort, individuals received midazolam 2 mg on Days 1 and 15 and a daily dose of palovarotene 20 mg on Days 2-15. Palovarotene and midazolam pharmacokinetics, including area under the concentration-time curve from time zero to infinity (AUC(0-∞)) and maximum observed plasma drug concentration (Cmax), were assessed. Adverse events (AEs) were recorded. RESULTS: Overall, 23 participants completed each part. Palovarotene Cmax and AUC(0-∞) increased by 16.5% and 39.7% under fed versus fasted conditions. Pharmacokinetics were comparable between the whole capsule and sprinkled on food, under fed conditions. Midazolam AUC(0-∞) and Cmax decreased by 13.3% and 9.7% upon palovarotene co-administration over 14 days, less than that required to be considered a weak CYP3A4 inducer. Plasma palovarotene exposures were comparable after single and multiple doses. No serious AEs were reported. CONCLUSIONS: These data support palovarotene administration after a meal, as a whole capsule or sprinkled on food. Palovarotene at 20 mg/day is a not a clinical inducer of CYP3A4. These results provide insights into palovarotene pharmacokinetics, aiding optimization of administration for patients with FOP. CLINICAL TRIALS REGISTRATION NUMBER: NCT04829773.

Fibrodysplasia ossificans progressiva, also known as FOP, is a very rare genetic condition where bone forms in places it is not usually found, such as in the muscles, tendons, and ligaments. Retinoids are molecules that the body produces from vitamin A to aid normal bone development. Palovarotene is a therapeutic retinoid that has been developed for the treatment of FOP. This article describes a clinical trial where people without FOP received oral palovarotene to determine how it is absorbed and broken down (metabolized) by the body when taken after a meal or after fasting (a period of not eating) as a whole capsule or when sprinkled on food. The trial also examined how palovarotene might interact with other treatments that are broken down by the body in the same way as palovarotene.The trial found that the amount of palovarotene that circulates in the blood increased more when taken after a meal compared with after fasting. Palovarotene was metabolized by the body in a similar way when taken as a whole capsule or when sprinkled on food. This finding is important as some people with FOP have difficulty swallowing. At a 20 mg dose, palovarotene was unlikely to interact with other treatments that are metabolized in the same way. No serious side effects were reported.These results show that palovarotene should be taken after a meal, either as a whole capsule or sprinkled on food.

A Pharmacokinetic, Safety, and Tolerability Trial of Palovarotene in Healthy Japanese and Non-Japanese Participants.

BACKGROUND AND OBJECTIVE: Palovarotene is an oral, selective retinoic acid receptor gamma agonist under investigation for fibrodysplasia ossificans progressiva (FOP). Palovarotene is primarily metabolized by cytochrome P450 (CYP) 3A4. Differences in CYP-mediated metabolism of CYP substrates have been observed between Japanese and non-Japanese individuals. This phase I trial (NCT04829786) compared the pharmacokinetic profile of palovarotene in healthy Japanese and non-Japanese participants and evaluated the safety of single doses. METHODS: Healthy Japanese and non-Japanese participants were matched individually (1:1) and randomized to receive a single oral dose of palovarotene 5 or 10 mg, followed by the alternate dose after a 5-day washout period. Maximum plasma drug concentration (Cmax) and area under the plasma concentration-time curve (AUC) were assessed. Estimates of the geometric mean difference between dose and Japanese and non-Japanese groups were calculated for natural log-transformed Cmax and AUC parameters. Adverse events (AEs), serious AEs, and treatment-emergent AEs were recorded. RESULTS: Eight pairs of matched non-Japanese and Japanese individuals and two unmatched Japanese individuals participated. Mean plasma concentration-time profiles were similar between the two cohorts at both dose levels, demonstrating that palovarotene absorption and elimination are similar irrespective of dose level. The pharmacokinetic parameters of palovarotene were similar between groups at both dose levels. Cmax and AUC values were dose-proportional between doses in each group. Palovarotene was well tolerated; there were no deaths or AEs leading to treatment discontinuation. CONCLUSIONS: Japanese and non-Japanese groups had similar pharmacokinetic profiles, indicating that palovarotene dose adjustments are not necessary for Japanese patients with FOP.

Translational approach from preclinical to clinical: comparison of dose finding methods of a new Bcl2 inhibitor using PK-PD modeling and interspecies extrapolation.

The attrition rate of anticancer drugs during the clinical development remains very high. Interspecies extrapolation of anticancer drug pharmacodynamics (PD) could help to bridge the gap between preclinical and clinical settings and to improve drug development. Indeed, when combined with a physiologically-based-pharmacokinetics (PBPK) approach, PD interspecies extrapolation could be a powerful tool for predicting drug behavior in clinical trials. The present study aimed to explore this field for anticipating the clinical efficacy of a new Bcl-2 inhibitor, S 55746, for which dose ranging studies in xenografted mice and clinical data from a phase 1 trial involving cancer patients were available. Different strategies based on empirical or more mechanistic assumptions (based on PBPK-PD modelling) were developped and compared: the Rocchetti approach (ROC); the Orthogonal Rocchetti approach (oROC), a variant of ROC based on an orthogonal regression; the Consistent across species approach, bringing out an efficacy parameter assumed to be consistent across species; and the Scaling species-specific parameters approach, assuming the concentration-efficacy link is the same in mice as in humans, after allometric scaling. Empirical approaches (ROC and oROC) gave similar predictive performances and seemed to overestimate the active S 55746 dose compared to mechanistic approaches, while strategies elaborated from semi-mechanistic concepts and PBPK-PD modelling did not seem to be invalidated by clinical efficacy data. Also, empirical methods only predict a single dose level for the subsequent clinical studies, whereas mechanism-based strategies are more informative about the dose response relationship, highlighting the potential interest of such approaches in drug development.

Prediction of Human Nonlinear Pharmacokinetics of a New Bcl-2 Inhibitor Using PBPK Modeling and Interspecies Extrapolation Strategy.

S 55746 ((S)-N-(4-hydroxyphenyl)-3-(6-(3-(morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzo[d][1,3]dioxol-5-yl)-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide) is a new selective Bcl-2 (B-cell lymphoma 2) inhibitor developed by Servier Laboratories and used to restore apoptosis functions in cancer patients. The aim of this work was to develop a translational approach using physiologically based (PB) pharmacokinetic (PK) modeling for interspecies extrapolation to anticipate the nonlinear PK behavior of this new compound in patients. A PBPK mouse model was first built using a hybrid approach, defining scaling factors (determined from in vitro data) to correct in vitro clearance parameters and predicted Kp (partition coefficient) values. The qualification of the hybrid model using these empirically determined scaling factors was satisfactorily completed with rat and dog data, allowing extrapolation of the PBPK model to humans. Human PBPK simulations were then compared with clinical trial data from a phase 1 trial in which the drug was given orally and daily to cancer patients. Human PBPK predictions were within the 95% prediction interval for the eight dose levels, taking into account both the nonlinear dose and time dependencies occurring in S 55746 kinetics. Thus, the proposed PK interspecies extrapolation strategy, based on preclinical and in vitro information and physiologic assumptions, could be a useful tool for predicting human plasma concentrations at the early stage of drug development.

Tumor Growth Inhibition Modelling Based on Receptor Occupancy and Biomarker Activity of a New Bcl-2 Inhibitor in Mice.

The Bcl-2 inhibitor S 55746 ((S)-N-(4-hydroxyphenyl)-3-(6-(3-(morpholinomethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzo[d][1,3]dioxol-5-yl)-N-phenyl-5,6,7,8-tetrahydroindolizine-1-carboxamide) is able to restore apoptosis functions impaired by tumorigenesis in mice. Data from pharmacokinetic (PK), biomarker, and tumor growth studies in a xenograft mouse model were considered for population modeling. The aim of the modeling exercise was to link the kinetics of the drug to the biomarker and tumor-size time profiles to better understand its dose-effect relationship. The PK, caspase kinetics, and tumor dynamics were successfully characterized by the proposed pharmacokinetic-pharmacodynamic model. The nonlinear plasma PK was best described by a two-compartment disposition model with both saturable absorption and elimination. Caspase was activated above the effective drug-concentration threshold (CTHRE ), at which near-maximal activity was reached. Increasing the dose did not increase the activation but better sustained it. Tumor growth followed a biphasic pattern, with caspase having an all-or-none inhibiting effect, consistent with the bistability property of the caspase pathway. For tumor eradication, the CTHRE in plasma was 2876 ng ml-1, and the relative caspase activity threshold (CaspTHRE) was 46.5. There was a strong relationship between the time spent above these thresholds and tumor growth inhibition. Tumor growth was inhibited by 50% when CaspTHRE was exceeded 13.8% of the time and when CTHRE was exceeded 8.1% of the time per dosing. This semimechanistic approach, based on experimental mice data and in vitro parameters, provides an interesting tool to quantify or simulate antitumor effects and, eventually, to plan phase 1 studies.

PEGylated cyclodextrins as novel siRNA nanosystems: correlations between polyethylene glycol length and nanoparticle stability.

Silencing disease-related genes in the central nervous system (CNS) using short interfering RNA (siRNA) holds great promise for treating neurological disorders. Yet, delivery of RNAi therapeutics to the brain poses major challenges to non-viral systems, especially when considering systemic administration. Cationic nanoparticles have been widely investigated for siRNA delivery, but the tendency of these to aggregate in physiological environments limits their intravenous application. Thus, strategies to increase the stability of nanoparticles have been developed. Here, we investigated the ability of modified cationic amphiphilic or PEGylated amphiphilic cyclodextrins (CD) to formulate stable CD.siRNA nanoparticles. To this end, we describe a simple method for post-modification of pre-formed cationic CD.siRNA nanoparticles at their surface using PEGylated CDs of different PEG lengths. PEGylated CD.siRNA nanoparticles presented reduced surface charges and increased stability in physiological salt conditions. Stability of PEGylated CD.siRNA nanoparticles in vitro increased with both PEG length and PEG density at the surface. Furthermore, in a comparative pharmacokinetic study, increased systemic exposure and reduced clearance were achieved with CD-formulations when compared to naked siRNAs. However, no significant differences were observed among non-PEGylated and PEGylated CD.siRNAs suggesting that longer PEG lengths might be required for improving stability in vivo.

Differential nanotoxicological and neuroinflammatory liabilities of non-viral vectors for RNA interference in the central nervous system.

Progression of RNA interference-based gene silencing technologies for the treatment of disorders of the central nervous system (CNS) depends on the availability of efficient non-toxic nanocarriers. Despite advances in the field of nanotechnology undesired and non-specific interactions with different brain-cell types occur and are poorly investigated. To this end, we studied the cytotoxic and neuroinflammatory effects of widely-used transfection reagents and modified amphiphilic ß-cyclodextrins (CDs). All non-viral vectors formed positively charged nanoparticles with distinctive physicochemical properties. Differential and significant cytotoxic effects were observed among commercially available cationic vectors, whereas CDs induced limited disruptions of cellular membrane integrity and mitochondrial dehydrogenase activity. Interestingly, murine derived BV2 microglia cells and a rat striatal in vitro model of Huntington's disease (ST14A-HTT120Q) were more susceptible to toxicity than human U87 astroglioma cells. BV2 microglia presented significant increases in cytokine, toll-like receptor 2 and cyclooxygenase-2 gene expression after transfection with selected commercial vectors but not with CD.siRNA nanoparticles. Non-viral siRNA nanoparticles formulated with G6 polyamidoamine (PAMAM) also significantly increased cytokine gene expression in the brain following injections into the mouse striatum. Together our data identify modified CDs as nanosystems that enable siRNA delivery to the brain with low levels of cytotoxicity and immunological activation.

Cationic and PEGylated Amphiphilic Cyclodextrins: Co-Formulation Opportunities for Neuronal Sirna Delivery.

Optimising non-viral vectors for neuronal siRNA delivery presents a significant challenge. Here, we investigate a co-formulation, consisting of two amphiphilic cyclodextrins (CDs), one cationic and the other PEGylated, which were blended together for siRNA delivery to a neuronal cell culture model. Co-formulated CD-siRNA complexes were characterised in terms of size, charge and morphology. Stability in salt and serum was also examined. Uptake was determined by flow cytometry and toxicity was measured by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Incorporation of a PEGylated CD in the formulation had significant effects on the physical and biological properties of CD.siRNA complexes. Co-formulated complexes exhibited a lower surface charge and greater stability in a high salt environment. However, the inclusion of the PEGylated CD also dramatically reduced gene silencing efficiency due to its effects on neuronal uptake. The co-formulation strategy for cationic and PEGylated CDs improved the stability of the CD.siRNA delivery systems, although knockdown efficiency was impaired. Future work will focus on the addition of targeting ligands to the co-formulated complexes to restore transfection capabilities.

Self-assembling modified ß-cyclodextrin nanoparticles as neuronal siRNA delivery vectors: focus on Huntington's disease.

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a toxic Huntingtin (HTT) protein. The use of short interfering RNAs (siRNAs) to silence the mutant protein is one of the most promising therapeutic strategies under investigation. The biggest caveat to siRNA-based approaches is the lack of efficient and nontoxic delivery vectors for siRNA delivery to the central nervous system. In this study, we investigated the potential of modified amphiphilic ß-cyclodextrins (CDs), oligosaccharide-based molecules, as novel siRNA neuronal carriers. We show that CDs formed nanosize particles which were stable in artificial cerebrospinal fluid. Moreover, these complexes were able to reduce the expression of the HTT gene in rat striatal cells (ST14A-HTT120Q) and in human HD primary fibroblasts. Only limited toxicity was observed with CD·siRNA nanoparticles in any of the in vitro models used. Sustained knockdown effects were observed in the striatum of the R6/2 mouse model of HD after single direct injections of CD·siRNA nanoparticles. Repeated brain injections of CD·siRNA complexes resulted in selective alleviation of motor deficits in this mouse model. Together these data support the utility of modified ß-CDs as efficient and safe siRNA delivery vectors for RNAi-based therapies for neuropsychiatric and neurodegenerative disorders.

In vitro investigations of the efficacy of cyclodextrin-siRNA complexes modified with lipid-PEG-Octaarginine: towards a formulation strategy for non-viral neuronal siRNA delivery.

PURPOSE: Development of RNA interference based therapeutics for neurological and neurodegenerative diseases is hindered by a lack of non-viral vectors with suitable properties for systemic administration. Amphiphilic and cationic cyclodextrins (CD) offer potential for neuronal siRNA delivery. We aimed to improve our CD-based siRNA formulation through incorporation of a polyethyleneglycol (PEG) shielding layer and a cell penetrating peptide, octaarginine (R8). METHODS: CD.siRNA complexes were modified by addition of an R8-PEG-lipid conjugate. Physical properties including size, charge and stability were assessed. Flow cytometry was used to determine uptake levels in a neuronal cell model. Knockdown of an exogenous gene and an endogenous housekeeping gene were used to assess gene silencing abilities. RESULTS: CD.siRNA complexes modified with R8-PEG-lipid exhibited a lower surface charge and greater stability to a salt-containing environment. Neuronal uptake was increased and significant reductions in the levels of two target genes were achieved with the new formulation. However, the PEG layer was not sufficient to protect against serum-induced aggregation. CONCLUSIONS: The R8-PEG-lipid-CD.siRNA formulation displayed enhanced salt-stability due to the PEG component, while the R8 component facilitated transfection of neuronal cells and efficient gene silencing. Further improvements will be investigated in the future in order to optimise stability in serum and enhance neuronal specificity.

Anisamide-targeted cyclodextrin nanoparticles for siRNA delivery to prostate tumours in mice.

A hepta-guanidino-ß-cyclodextrin (G-CD), its hepta-PEG conjugate (G-CD-PEG), and the corresponding anisamide-terminated PEG conjugate (G-CD-PEG-AA) have been synthesised and compared as delivery vectors for siRNA to prostate cancer cells and tumours in vivo. The G-CD-PEG-AA.siRNA formulations (in which anisamide targets the sigma receptor), but not the non-targeted formulations, induced prostate cell-specific internalisation of siRNA resulting in approximately 80% knockdown in vitro of the reporter gene, luciferase. Following intravenous administration of the anisamide-targeted formulation in a mouse prostate tumour model significant tumour inactivation with corresponding reductions in the level of vascular endothelial growth factor (VEGF) mRNA were achieved, without demonstrating enhanced toxicity. This data imply significant potential for anisamide-conjugated cyclodextrin vectors for targeted delivery of therapeutic siRNAs in the treatment of prostate cancer.

A click chemistry route to 2-functionalised PEGylated and cationic ß-cyclodextrins: co-formulation opportunities for siRNA delivery.

A new approach to the synthesis of amphiphilic ß-cyclodextrins has used 'click' chemistry to selectively modify the secondary 2-hydroxyl group. The resulting extended polar groups can be either polycationic or neutral PEGylated groups and these two amphiphile classes are compatible in dual cyclodextrin formulations for delivery of siRNA. When used alone with an siRNA, a cationic cyclodextrin was shown to have good transfection properties in cell culture. Co-formulation with a PEGylated cyclodextrin altered the physicochemical properties of nanoparticles formed with siRNA. Improved particle properties included lower surface charges and reduced tendency to aggregate. However, as expected, the transfection efficiency of the cationic vector was lowered by co-formulation with the PEGylated cyclodextrin, requiring future surface modification of particles with targeting ligands for effective siRNA delivery.

Drug delivery and imaging with polydiacetylene micelles.

This concept article summarizes our recent findings regarding photopolymerized micelles obtained from the self-assembly of diacetylene-containing amphiphiles. Their synthesis and characterization are presented as well as some biomedical applications, such as tumor imaging and drug delivery. Finally, ongoing studies and future challenges are briefly discussed.

Tumor-targeted polydiacetylene micelles for in vivo imaging and drug delivery.

In vivo tumor targeting and drug delivery properties of small polymerized polydiacetylene (PDA) micelles (∼10 nm) is investigated in a murine MDA-MB-231 xenograft model of breast cancer. Three micelles with different surface coatings are synthesized and tested for their ability to passively target tumor through the enhanced permeability and retention effect. After injection (24 h), fluorescence diffuse optical tomographic imaging indicates a tumor uptake of nearly 3% of the injected dose for the micelles with a 2 kDa poly(ethylene glycol) (PEG)-coating (PDA-PEG2000). The uptake of PDA micelles in tumors is confirmed by co-localization with [(18) F]-fluorodeoxyglucose (FDG) positron emission tomography. Although FDG has a higher diffusion rate in tumors, 40 ± 19% of the retained micelles is co-registered with the tumor volume visualized by FDG. Finally, PDA-PEG2000 micelles are loaded with the hydrophobic anticancer drug paclitaxel and used in vivo to inhibit tumor growth. These findings demonstrate the potential of PDA-PEG2000 micelles for both in vivo tumor imaging and drug delivery applications.

Enhanced drug loading in polymerized micellar cargo.

A new drug carrier system based on self-assembly and polymerization of polydiacetylenic amphiphiles is described. Although classical amphiphiles can help in solubilizing hydrophobic molecules upon self-arrangement into a variety of nanometric structures, a greater effect on drug loading was observed for our polymerized micelles as compared to the non-polymerized analogues. This permitted higher aqueous solubilization of lipophilic drugs with low micelle concentration. (14)C labeling of a model drug on one side and of the amphiphile on the other side permitted assessment, after intravenous injection, of biodistribution and excretion profiles of the drug cargo.

Poly-6-cationic amphiphilic cyclodextrins designed for gene delivery.

A new series of amphiphilic cyclodextrins containing cationic groups at the 6-positions and alkyl or biolabile ester groups at the 2-positions has been synthesised. Selective 2-O-allylation followed by photochemical addition of lipophilic thiols made it possible to control lipophilicity in these mesomolecules and allow solubility and self-assembly in water. The cationic groups are cysteamine-derived, while the alkyl and ester groups are C(1)-C(16) and benzyl ester groups. This is a new general synthetic route to a potentially wide range of polycationic cyclodextrins capable of acting as gene delivery vectors by condensing DNA and forming liquid crystalline complexes with oligonucleotides.

Recent advances in the field of nanometric drug carriers.

Over the past few years, health and medicine have been domains where nanotechnologies have shown great promise, in particular in the area of drug carriers and drug targeting. Many active substances suffer from poor solubility, instability in biological medium and low bioavailability. Inaccurate distribution and accumulation of the drug in the body could lead to some side effects possibly detrimental to drug development. With the advent of nanosciences applied to medicine, new tools are becoming available, giving rise to a whole range of drug carriers with different properties and functionalities. Nanocarriers should play a crucial role in the controlled and sustained delivery of drugs. Various types of functional nanosystems are currently being explored and the aim of this review is to give an overview of the most recent advances in the field of nanometric drug carriers, including future strategies and perspectives.