Integration of Biorelevant Pediatric Dissolution Methodology into PBPK Modeling to Predict In Vivo Performance and Bioequivalence of Generic Drugs in Pediatric Populations: a Carbamazepine Case Study.

This study investigated the impact of gastro-intestinal fluid volume and bile salt (BS) concentration on the dissolution of carbamazepine (CBZ) immediate release (IR) 100 mg tablets and to integrate these in vitro biorelevant dissolution profiles into physiologically based pharmacokinetic modelling (PBPK) in pediatric and adult populations to determine the biopredictive dissolution profile. Dissolution profiles of CBZ IR tablets (100 mg) were generated in 50-900 mL biorelevant adult fasted state simulated gastric and intestinal fluid (Ad-FaSSGF and Ad-FaSSIF), also in three alternative compositions of biorelevant pediatric FaSSGF and FaSSIF medias at 200 mL. This study found that CBZ dissolution was poorly sensitive to changes in the composition of the biorelevant media, where dissimilar dissolution (F2 = 46.2) was only observed when the BS concentration was changed from 3000 to 89 µM (Ad-FaSSIF vs Ped-FaSSIF 50% 14 BS). PBPK modeling demonstrated the most predictive dissolution volume and media composition to forecast the PK was 500 mL of Ad-FaSSGF/Ad-FaSSIF media for adults and 200 mL Ped-FaSSGF/FaSSIF media for pediatrics. A virtual bioequivalence simulation was conducted by using Ad-FaSSGF and/or Ad-FaSSIF 500 mL or Ped-FaSSGF and/or Ped-FaSSIF 200 mL dissolution data for CBZ 100 mg (reference and generic test) IR product. The CBZ PBPK models showed bioequivalence of the product. This study demonstrates that the integration of biorelevant dissolution data can predict the PK profile of a poorly soluble drug in both populations. Further work using more pediatric drug products is needed to verify biorelevant dissolution data to predict the in vivo performance in pediatrics.

Quantification of Fluid Volume and Distribution in the Paediatric Colon via Magnetic Resonance Imaging.

Previous studies have used magnetic resonance imaging (MRI) to quantify the fluid in the stomach and small intestine of children, and the stomach, small intestine and colon of adults. This is the first study to quantify fluid volumes and distribution using MRI in the paediatric colon. MRI datasets from 28 fasted (aged 0-15 years) and 18 fluid-fed (aged 10-16 years) paediatric participants were acquired during routine clinical care. A series of 2D- and 3D-based software protocols were used to measure colonic fluid volume and localisation. The paediatric colon contained a mean volume of 22.5 mL ± 41.3 mL fluid, (range 0-167.5 mL, median volume 0.80 mL) in 15.5 ± 17.5 discreet fluid pockets (median 12). The proportion of the fluid pockets larger than 1 mL was 9.6%, which contributed to 94.5% of the total fluid volume observed. No correlation was detected between all-ages and colonic fluid volume, nor was a difference in colonic fluid volumes observed based on sex, fed state or age group based on ICH-classifications. This study quantified fluid volumes within the paediatric colon, and these data will aid and accelerate the development of biorelevant tools to progress paediatric drug development for colon-targeting formulations.

Development of a Pediatric Relative Bioavailability/Bioequivalence Database and Identification of Putative Risk Factors Associated With Evaluation of Pediatric Oral Products.

Generally, bioequivalence (BE) studies of drug products for pediatric patients are conducted in adults due to ethical reasons. Given the lack of direct BE assessment in pediatric populations, the aim of this work is to develop a database of BE and relative bioavailability (relative BA) studies conducted in pediatric populations and to enable the identification of risk factors associated with certain drug substances or products that may lead to failed BE or different pharmacokinetic (PK) parameters in relative BA studies in pediatrics. A literature search from 1965 to 2020 was conducted in PubMed, Cochrane Library, and Google Scholar to identify BE studies conducted in pediatric populations and relative BA studies conducted in pediatric populations. Overall, 79 studies covering 37 active pharmaceutical ingredients (APIs) were included in the database: 4 bioequivalence studies with data that passed BE evaluations; 2 studies showed bioinequivalence results; 34 relative BA studies showing comparable PK parameters, and 39 relative BA studies showing differences in PK parameters between test and reference products. Based on the above studies, common putative risk factors associated with differences in relative bioavailability (DRBA) in pediatric populations include age-related absorption effects, high inter-individual variability, and poor study design. A database containing 79 clinical studies on BE or relative BA in pediatrics has been developed. Putative risk factors associated with DRBA in pediatric populations are summarized.

Characterisation of fasted state gastric and intestinal fluids collected from children.

Fundamental knowledge about the composition of intestinal fluids in paediatric populations is currently unavailable. This study aimed to characterise gastric and intestinal fluid from paediatric populations. Gastric and intestinal fluid samples were obtained during routine clinical endoscopy from paediatric patients at a large teaching hospital. These fluids were characterised to measure the pH; buffer capacity; osmolality; bile acid concentration and composition. A total of 55 children were recruited to the study aged from 11 months to 15 years of age where 53 gastric fluid samples and 40 intestinal fluid samples were obtained. pH values recorded ranged from pH 0.57 to 11.05 (median: 2.50) in gastric fluids and from 0.89 to 8.97 (median: 3.27) in intestinal fluids. The buffer capacity did not change significantly between gastric and intestinal fluids with median values of 12 mM/L/ΔpH for both fluids. Gastric fluid osmolality values ranged from 1 to 615 mOsm/kg, while intestinal fluid values ranged from 35 to 631 mOsm/kg. Gastric fluid bile acid concentrations ranged from 0.002 to 2.3 mM with a median value of 0.017 mM whilst intestinal fluid bile acid concentrations ranged from 0.0008 to 3.3 mM with a median value of 0.178 mM. Glycocholate; taurocholic acid; glycochenodeoxycholate and taurochenodeoxycholate were the most commonly identified bile acids within paediatric intestinal fluids. All compositional components were associated with large inter-individual variability. Further work is required to develop simulated paediatric media and to explore the impact of these media on drug solubility and dissolution.

In Silico Toxicology Data Resources to Support Read-Across and (Q)SAR.

A plethora of databases exist online that can assist in in silico chemical or drug safety assessment. However, a systematic review and grouping of databases, based on purpose and information content, consolidated in a single source, has been lacking. To resolve this issue, this review provides a comprehensive listing of the key in silico data resources relevant to: chemical identity and properties, drug action, toxicology (including nano-material toxicity), exposure, omics, pathways, Absorption, Distribution, Metabolism and Elimination (ADME) properties, clinical trials, pharmacovigilance, patents-related databases, biological (genes, enzymes, proteins, other macromolecules etc.) databases, protein-protein interactions (PPIs), environmental exposure related, and finally databases relating to animal alternatives in support of 3Rs policies. More than nine hundred databases were identified and reviewed against criteria relating to accessibility, data coverage, interoperability or application programming interface (API), appropriate identifiers, types of in vitro, in vivo,-clinical or other data recorded and suitability for modelling, read-across, or similarity searching. This review also specifically addresses the need for solutions for mapping and integration of databases into a common platform for better translatability of preclinical data to clinical data.

A critical review of adverse effects to the kidney: mechanisms, data sources, and in silico tools to assist prediction.

INTRODUCTION: The kidney is a major target for toxicity elicited by pharmaceuticals and environmental pollutants. Standard testing which often does not investigate underlying mechanisms has proven not to be an adequate hazard assessment approach. As such, there is an opportunity for the application of computational approaches that utilize multiscale data based on the Adverse Outcome Pathway (AOP) paradigm, coupled with an understanding of the chemistry underpinning the molecular initiating event (MIE) to provide a deep understanding of how structural fragments of molecules relate to specific mechanisms of nephrotoxicity. Aims covered: The aim of this investigation was to review the current scientific landscape related to computational methods, including mechanistic data, AOPs, publicly available knowledge bases and current in silico models, for the assessment of pharmaceuticals and other chemicals with regard to their potential to elicit nephrotoxicity. A list of over 250 nephrotoxicants enriched with, where possible, mechanistic and AOP-derived understanding was compiled. Expert opinion: Whilst little mechanistic evidence has been translated into AOPs, this review identified a number of data sources of in vitro, in vivo, and human data that may assist in the development of in silico models which in turn may shed light on the interrelationships between nephrotoxicity mechanisms.

Dermal bioaccessibility of flame retardants from indoor dust and the influence of topically applied cosmetics.

Despite extensive literature on their potential adverse health effects, there is a lack of information on human dermal exposure to organic flame retardant chemicals (FRs). This study applies an in vitro physiologically based extraction test to provide new insights into the dermal bioaccessibility of various FRs from indoor dust to synthetic sweat/sebum mixture (SSSM). The bioaccessible fractions of α-, ß- and γ-hexabromocyclododecane (HBCD) and tetrabromobisphenol A (TBBPA) to 1:1 (sweat/sebum) mixture were 41%, 47%, 50% and 40%, respectively. For Tris-2-chloroethyl phosphate (TCEP), tris (1-chloro-2-propyl) phosphate (TCIPP) and tris-1,3-dichloropropyl phosphate (TDCIPP), bioaccessible fractions were 10%, 17% and 19%. Composition of the SSSM and compound-specific physicochemical properties were the major factors influencing the bioaccessibility of target FRs. Except for TBBPA, the presence of cosmetics (moisturising cream, sunscreen lotion, body spray and shower gel) had a significant effect (P<0.05) on the bioaccessibility of the studied FRs. The presence of cosmetics decreased the bioaccessibility of HBCDs from indoor dust, whereas shower gel and sunscreen lotion enhanced the bioaccessibility of target PFRs. Our bioaccessibility data were applied to estimate the internal exposure of UK adults and toddlers to the target FRs via dermal contact with dust. Our worst-case scenario exposure estimates fell far below available health-based limit values for TCEP, TCIPP and TDCIPP. However, future research may erode the margin of safety for these chemicals.

Human dermal absorption of chlorinated organophosphate flame retardants; implications for human exposure.

Tris-2-chloroethyl phosphate (TCEP), tris (1-chloro-2-propyl) phosphate (TCIPP) and tris-1,3-dichloropropyl phosphate (TDCIPP) are organophosphate flame retardants (PFRs) widely applied in a plethora of consumer products despite their carcinogenic potential. Human dermal absorption of these PFRs is investigated for the first time using human ex vivo skin and EPISKIN™ models. Results of human ex vivo skin experiments revealed 28%, 25% and 13% absorption of the applied dose (500 ng/cm(2), finite dose) of TCEP, TCIPP and TDCIPP, respectively after 24h exposure. The EPISKIN™ model showed enhanced permeability values (i.e. weaker barrier), that were respectively 16%, 11% and 9% for TCEP, TCIPP and TDCIPP compared to human ex vivo skin. However, this difference was not significant (P>0.05). Estimated permeability constants (Kp, cm/h) showed a significant negative correlation with log Kow for the studied contaminants. The effect of hand-washing on dermal absorption of PFRs was investigated. Washing reduced overall dermal absorption, albeit to varying degrees depending on the physicochemical properties of the target PFRs. Moreover, slight variations of the absorbed dose were observed upon changing the dosing solution from acetone to 20% Tween 80 in water, indicating the potential influence of the dose vehicle on the dermal absorption of PFRs. Finally, estimated dermal uptake of the studied PFRs via contact with indoor dust was higher in UK toddlers (median ΣPFRs=36 ng/kg bw day) than adults (median ΣPFRs=4 ng/kg bw day). More research is required to fully elucidate the toxicological implications of such exposure.

Evaluation of 3D-human skin equivalents for assessment of human dermal absorption of some brominated flame retardants.

Ethical and technical difficulties inherent to studies in human tissues are impeding assessment of the dermal bioavailability of brominated flame retardants (BFRs). This is further complicated by increasing restrictions on the use of animals in toxicity testing, and the uncertainties associated with extrapolating data from animal studies to humans due to inter-species variations. To overcome these difficulties, we evaluate 3D-human skin equivalents (3D-HSE) as a novel in vitro alternative to human and animal testing for assessment of dermal absorption of BFRs. The percutaneous penetration of hexabromocyclododecanes (HBCD) and tetrabromobisphenol-A (TBBP-A) through two commercially available 3D-HSE models was studied and compared to data obtained for human ex vivo skin according to a standard protocol. No statistically significant differences were observed between the results obtained using 3D-HSE and human ex vivo skin at two exposure levels. The absorbed dose was low (less than 7%) and was significantly correlated with log Kow of the tested BFR. Permeability coefficient values showed increasing dermal resistance to the penetration of γ-HBCD>ß-HBCD>α-HBCD>TBBPA. The estimated long lag times (>30 min) suggests that frequent hand washing may reduce human exposure to HBCDs and TBBPA via dermal contact.

Effect of Bromine Substitution on Human Dermal Absorption of Polybrominated Diphenyl Ethers.

Human dermal absorption of eight mono- to deca-brominated diphenyl ethers (PBDEs) was investigated for the first time using EPISKIN human skin equivalent tissue. Using a standard in vitro protocol, EPISKIN tissues mounted in specially designed diffusion cells were exposed to the target PBDEs for 24 h. Estimated steady-state flux (Jss) and permeation coefficients (Papp) across the skin increased with decreasing bromine substitution from BDE-153 (Papp = 4.0 × 10(-4) cm/h) to BDE-1 (Papp = 1.1 × 10(-2) cm/h). This was accompanied by an increase in the time required to traverse the skin tissue into the receptor fluid (lag time) from 0.25 h for BDE-1 to 1.26 h for BDE-153. Papp values for the studied PBDEs were correlated significantly (P < 0.05) with physicochemical parameters like water solubility and log KOW. While less brominated congeners achieved faster dermal penetration, higher PBDEs displayed greater accumulation within the skin tissue. The PBDEs thus accumulated represent a contaminant depot from which they may be slowly released to the systemic circulation over a prolonged period. Maximal percutaneous penetration was observed for BDE-1 (∼ 30% of the applied 500 ng/cm(2) dose). Interestingly, BDE-183 and BDE-209 showed very low dermal absorption, exemplified by a failure to reach the steady state within the 24 h exposure period that was studied.

High-resolution mass spectrometry provides novel insights into products of human metabolism of organophosphate and brominated flame retardants.

The high resolution, accurate mass, and fast scanning features of the Orbitrap(TM) mass spectrometer, combined with the separation power of ultrahigh-performance liquid chromatography were applied for the first time to study the metabolic profiles of several organic flame retardants (FRs) present in indoor dust. To mimic real-life exposure, in vitro cultured HepG2 human hepatocyte cell lines were exposed simultaneously to various FRs in an indoor dust extract for 24 h. Target parent FRs, hexabromocyclododecanes (α-, ß-, and γ-HBCDs), tris-2-chloroethyl phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCIPP), and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), were separated in a single run for the first time using alternating positive and negative heated ESI source. Further metabolite separation and identification was achieved using full scan (70,000 full width at half maximum (FWHM)), accurate mass (up to 1 ppm) spectrometry. Structural confirmation was performed via all ion fragmentation (AIF) spectra using the optional higher collisional dissociation (HCD) cell and MS/MS analysis. First insights into human metabolism of HBCDs revealed several hydroxylated and debrominated phase I metabolites, in addition to conjugated phase II glucuronides. Furthermore, various hydroxylated, oxidized, and conjugated metabolites of chlorinated phosphorous FRs were identified, leading to the suggestion of α-oxidation as a significant metabolic pathway for these compounds.

Evaluation of in vitro vs. in vivo methods for assessment of dermal absorption of organic flame retardants: a review.

There is a growing interest to study human dermal exposure to a large number of chemicals, whether in the indoor or outdoor environment. Such studies are essential to predict the systemic exposure to xenobiotic chemicals for risk assessment purposes and to comply with various regulatory guidelines. However, very little is currently known about human dermal exposure to persistent organic pollutants. While recent pharmacokinetic studies have highlighted the importance of dermal contact as a pathway of human exposure to brominated flame retardants, risk assessment studies had to apply assumed values for percutaneous penetration of various flame retardants (FRs) due to complete absence of specific experimental data on their human dermal bioavailability. Therefore, this article discusses the current state-of-knowledge on the significance of dermal contact as a pathway of human exposure to FRs. The available literature on in vivo and in vitro methods for assessment of dermal absorption of FRs in human and laboratory animals is critically reviewed. Finally, a novel approach for studying human dermal absorption of FRs using in vitro three-dimensional (3D) human skin equivalent models is presented and the challenges facing future dermal absorption studies on FRs are highlighted.

LC-MS/MS-ESI method for simultaneous quantitation of metformin and repaglinidie in rat plasma and its application to pharmacokinetic study in rats.

A highly sensitive and specific LC-MS/MS-ESI method has been developed for simultaneous quantification of metformin (MFN) and repaglinide (RGN) in rat plasma (50 µL) using phenacetin as an internal standard (IS). Simple protein precipitation was used to extract MFN and RGN from rat plasma. The chromatographic resolution of MFN, RGN and IS was achieved with a mobile phase consisting of 0.2% formic acid in water-acetonitrile (1:1, v/v) with a time program flow gradient on a Chromolith RP-18e column. The total chromatographic run time was 3.5 min and the elution of MFN, RGN and IS occurred at 1.64, 2.21 and 2.15 min, respectively. A linear response function was established for the range of concentrations 0.855-394 and 0.021-21.7 ng/mL for MFN and RGN, respectively. The intra- and inter-day precision values for MFN and RGN met the acceptance as per FDA guidelines. MFN and RGN were stable in battery of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. The developed assay was applied to a pharmacokinetic study in rats.

Development and validation of a highly sensitive LC-MS/MS-ESI method for the determination of bicalutamide in mouse plasma: application to a pharmacokinetic study.

A highly sensitive, rapid assay method has been developed and validated for the estimation of bicalutamide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative-ion mode. The assay procedure involves extraction of bicalutamide and tolbutamide (internal standard, IS) from mouse plasma with a simple protein precipitation method. Chromatographic separation was achieved using an isocratic mobile phase (0.2% formic acid:acetonitrile, 35:65, v/v) at a flow rate of 0.5 mL/min on an Atlantis dC18 column (maintained at 40 ± 1°C) with a total run time of 3.0 min. The MS/MS ion transitions monitored were m/z 428.9 → 254.7 for bicalutamide and m/z 269.0 → 169.6 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 1.04 ng/mL and the linearity range extended from 1.04 to 1877 ng/mL. The intra- and inter-day precisions were in the ranges of 0.49-4.68 and 2.62-4.15, respectively.

Development and validation of an LC-MS/MS-ESI method for the determination of lorglumide, a CCK-1 antagonist in mouse plasma: application to a pharmacokinetic study.

A highly sensitive, rapid assay method was developed and validated for the estimation of lorglumide in mouse plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in positive-ion mode. The assay procedure involves extraction of lorglumide and phenacetin (internal standard, IS) from mouse plasma with simple protein precipitation. Chromatographic separation was achieved using an isocratic mobile (0.2% formic acid solution-acetonitrile, 20:80, v/v) at a flow-rate of 0.5 mL/min on an Atlantis dC18 column maintained at 40 °C with a total run time of 4.0 min. The MS/MS ion transitions monitored were 459.2 → 158.4 for lorglumide and 180.1 → 110.1 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.42 ng/mL and the linearity range extended from 0.42 to 500 ng/mL. The intra- and inter-day precisions were in the ranges of 1.47-10.9 and 3.56-7.53, respectively.