Creating a Roadmap to Quantitative Systems Pharmacology-Informed Rare Disease Drug Development: A Workshop Report.

One of the goals of the Accelerating Rare Disease Cures (ARC) program in the Center for Drug Evaluation and Research (CDER) at the US Food and Drug Administration (FDA) is the development and use of regulatory and scientific tools, including drug/disease modeling, dose selection, and translational medicine tools. To facilitate achieving this goal, the FDA in collaboration with the University of Maryland Center of Excellence in Regulatory Science and Innovation (M-CERSI) hosted a virtual public workshop on May 11, 2023, entitled "Creating a Roadmap to Quantitative Systems Pharmacology-Informed Rare Disease Drug Development." This workshop engaged scientists from pharmaceutical companies, academic institutes, and the FDA to discuss the potential utility of quantitative systems pharmacology (QSP) in rare disease drug development and identify potential challenges and solutions to facilitate its use. Here, we report the main findings from this workshop, highlight the key takeaways, and propose a roadmap to facilitate the use of QSP in rare disease drug development.

In Vitro-In Vivo Correlation of Buprenorphine Transdermal Systems Under Normal and Elevated Skin Temperature.

PURPOSE: Application of external heat using a heating pad over buprenorphine transdermal system, Butrans® has been shown to increase systemic levels of buprenorphine in human volunteers. The purpose of this study was to perform in vitro permeation studies at normal as well as elevated temperature conditions to evaluate the correlation of in vitro data with the existing in vivo data. METHODS: In vitro permeation tests (IVPT) were performed on human skin from four donors. The IVPT study design was harmonized to a previously published clinical study design and skin temperature was maintained at either 32 ± 1 °C or 42 ± 1 °C to mimic normal and elevated skin temperature conditions, respectively. RESULTS: IVPT studies on human skin were able to demonstrate heat induced enhancement in flux and cumulative amount of drug permeated from Butrans® which was reasonably consistent with the corresponding enhancement observed in vivo. Level A in vitro-in vivo correlation (IVIVC) was established using unit impulse response (UIR) based deconvolution method for both baseline and heat arms of the study. The percent prediction error (%PE) calculated for AUC and Cmax values was less than 20%. CONCLUSIONS: The studies indicated that IVPT studies performed under the same conditions as those of interest in vivo may be useful for comparative evaluation of the effect of external heat on transdermal delivery system (TDS). Further research may be warranted to evaluate factors, beyond cutaneous bioavailability (BA) assessed using an IVPT study, that can influence plasma exposure in vivo for a given drug product.

Advanced harmonization techniques result in accurate establishment of in vitro-in vivo correlations for oxybenzone from four complex dermal formulations with reapplication.

Due to high variability during clinical pharmacokinetic (PK) evaluation, the prediction of in vivo exposure from in vitro absorption testing of topical semisolid and liquid dermal products has historically proven difficult. Since absorption from unoccluded formulations can be influenced by environmental factors such as temperature and humidity, maximal effort must be placed on the harmonization of experimental parameters between in vitro and in vivo testing conditions to establish accurate in vitro/in vivo correlations (IVIVC). Using four different sunscreen formulations as a model, we performed in vitro permeation testing (IVPT) studies with excised human skin and maintained strict harmonization techniques to control application time, occlusion, temperature, and humidity during in vivo human serum PK evaluation. The goal was to investigate if increased control over experimental parameters would result in decreased inter-subject variability of common topical formulations leading to acceptable IVIVC establishment. Using a deconvolution-based approach, excellent point-to-point (Level A correlation) IVIVC for the entire 12-h study duration was achieved for all four sunscreen formulations with < 10% prediction error of both area under the curve (AUC) and peak concentration (Cmax) estimation. The low variability of in vivo absorption data presents a proof-of-concept protocol design for testing of complex semisolid and liquid topical formulations applied over a large surface area with reapplication in a reliable manner. This work also presents the opportunity for expanded development of testing for the impact of altered temperature and humidity conditions on product absorption in vivo with a high degree of precision.

Understanding Formulation and Temperature Effects on Dermal Transport Kinetics by IVPT and Multiphysics Simulation.

PURPOSE: It is often unclear how complex topical product formulation factors influence the transport kinetics through skin tissue layers, because of multiple confounding attributes. Environmental factors such as temperature effect are also poorly understood. In vitro permeation testing (IVPT) is frequently used to evaluate drug absorption across skin, but the flux results from these studies are from a combination of mechanistic processes. METHOD: Two different commercially available formulations of oxybenzone-containing sunscreen cream and continuous spray were evaluated by IVPT in human skin. Temperature influence between typical skin surface temperature (32°C) and an elevated 37°C was also assessed. Furthermore, a multiphysics-based simulation model was developed and utilized to compute the flux of modeled formulations. RESULTS: Drug transport kinetics differed significantly between the two drug products. Flux was greatly influenced by the environmental temperature. The multiphysical simulation results could reproduce the experimental observations. The computation further indicated that the drug diffusion coefficient plays a dominant role in drug transport kinetics, influenced by the water content which is also affected by temperature. CONCLUSION: The in vitro testing and bottom-up simulation shed insight into the mechanism of dermal absorption kinetics from dissimilar topical products.

Investigator Impact on Reproducibility of Drug Bioavailability in Stratum Corneum Sampling by Tape Stripping.

PURPOSE: Skin sampling by tape stripping measures the local bioavailability of topical drug products in the stratum corneum (SC). The goal of the current study was to evaluate the impact of different investigators in studies that utilize a tape stripping protocol designed to minimize investigator variability. METHODS: Two open-label clinical studies compared two lidocaine patches and a diclofenac patch and solution in twelve healthy volunteers. The mass of drug was determined in SC samples collected on tape strips at three time points following product removal in duplicate by two investigators. Investigator results were compared with each other and with results for the diclofenac solution measured by another laboratory using a similar protocol. RESULTS: For drug mass, the geometric mean ratio comparing two investigators is within the acceptable bioequivalence interval for most measurement times and drug products. Drug uptake into the SC from the diclofenac solution was not statistically different from that determined in another laboratory. The average flux from the SC over the clearance intervals for the four drug products correspond well with flux measurements from in vitro permeation tests. CONCLUSIONS: Results from different investigators are reproducible within the limitations of measurement variability, which can be managed by increasing volunteer numbers.

Evaluation of in vitro/in vivo correlations for three fentanyl transdermal delivery systems using in vitro skin permeation testing and human pharmacokinetic studies under the influence of transient heat application.

The value of developing an in vitro/in vivo correlation (IVIVC) is substantial in biopharmaceutical drug development because once the model is developed and validated, an in vitro method may be used to efficiently assess and predict drug product performance in vivo. In this study, three bioequivalent, matrix-type, fentanyl transdermal delivery systems (TDS) were evaluated in vitro using an in vitro permeation test (IVPT) and dermatomed human skin, and in vivo in human pharmacokinetic (PK) studies under harmonized study designs to evaluate IVIVC. The study designs included 1 h of transient heat application (42 ± 2°C) at either 11 h or 18 h after TDS application to concurrently investigate the influence of heat on drug bioavailability from TDS and the feasibility of IVPT to predict the effects of heat on TDS in vivo. Level A (point-to-point) and Level C (single point) IVIVCs were evaluated by using PK-based mathematical equations and building IVIVC models between in vitro fraction of drug permeation and in vivo fraction of drug absorption. The study results showed that the three differently formulated fentanyl TDS have comparable (p > 0.05) heat effects both in vitro and in vivo. In addition, the predicted steady-state concentration (Css) from in vitro flux data and the observed Css in vivo showed no significant differences (p > 0.05). However, the effects of heat on enhancement of fentanyl bioavailability observed in vivo were found to be greater compared to those observed in vitro for all three drug products, resulting in a weak prediction of the impact of heat on bioavailability from the in vitro data. The results from the current work suggest that while IVPT can be a useful tool to evaluate the performance of fentanyl TDS in vivo with a relatively good predictability at a normal temperature condition and to compare the effect of heat on drug delivery from differently formulated TDS, additional testing measures would enhance the ability to predict the heat effects in vivo with a lower prediction error.

Effect of Controlled Heat Application on Topical Diclofenac Formulations Evaluated by In Vitro Permeation Tests (IVPT) Using Porcine and Human Skin.

PURPOSE: Heat therapy is widely used for pain relief and may unintentionally be used in conjunction with pain relieving topical formulations. The purpose of this study was to evaluate the influence of heat on the permeation of diclofenac through porcine and human skin, comparing four marketed products. METHODS: In vitro permeation tests (IVPT) were performed on porcine skin from a single miniature pig and human skin from three donors. Skin temperature was maintained at either 32 ± 1°C or 42 ± 1°C to mimic normal and elevated skin temperature conditions, respectively. RESULTS: IVPT studies on porcine and human skin were able to demonstrate heat-induced enhancement in flux and cumulative amount of drug permeated from the four diclofenac products. The pivotal data showed the most significant heat-induced enhancement for the solution, followed by the patch and two gels in decreasing order of significance based on p values. Diclofenac solution showed the highest flux and cumulative amount permeated at both baseline and elevated skin temperature compared to the patch and gels. CONCLUSIONS: The studies demonstrated that exposure to heat can alter drug permeation from topical formulations, but the increased levels are not expected to lead to systemic concentrations that are of concern. Formulation design and excipients can influence drug permeation at elevated skin temperature.

Minimally invasive technique for measuring transdermal glucose with a fluorescent biosensor.

There is a need for blood glucose monitoring techniques that eliminate the painful and invasive nature of current methods, while maintaining the reliability and accuracy of established medical technology. This research aims to ultimately address these shortcomings in critically ill pediatric patients. Presented in this work is an alternative, minimally invasive technique that uses microneedles (MN) for the collection of transdermal glucose (TG). Due to their comparable skin properties, diffusion studies were performed on full thickness Yucatan miniature pig skin mounted to an in-line diffusion flow cell and on different skin sites of human subjects. Collected TG samples were measured with a L255C mutant of the E. coli glucose-binding protein (GBP) with an attached fluorescent probe. The binding constant (Kd = 0.67 µM) revealed the micromolar sensitivity and high selectivity of the his-tagged GBP biosensor for glucose, making it suitable for TG measurements. In both the animal and human models, skin permeability and TG diffusion across the skin increased with MN application. For intact and MN-treated human skin, a significant positive linear correlation (r > 0.95, p < 0.01) existed between TG and BG. The micromolar sensitivity of GBP minimized the volume required for interstitial fluid glucose analysis allowing MN application time (30 s) to be shortened compared to other studies. This time reduction can help in eliminating skin irritation issues and improving practical use of the technique by caregivers in the hospital. In addition, the his-tagged optical biosensor used in this work can be immobilized and used with a portable sensing fluorometer device at the point of care (POC) making this minimally invasive technology more ideal for use in the pediatric intensive care unit. Graphical abstract ᅟ.

The Sensitivity of In Vitro Permeation Tests to Chemical Penetration Enhancer Concentration Changes in Fentanyl Transdermal Delivery Systems.

Chemical penetration enhancers (CPEs) are frequently incorporated into transdermal delivery systems (TDSs) to improve drug delivery and to reduce the required drug load in formulations. However, the minimum detectable effect of formulation changes to CPE-containing TDSs using in vitro permeation tests (IVPT), a widely used method to characterize permeation of topically applied drug products, remains unclear. The objective of the current exploratory study was to investigate the sensitivity of IVPT in assessing permeation changes with CPE concentration modifications and subsequently the feasibility of IVPT's use for support of quality control related to relative CPE concentration variation in a given formulation. A series of drug-in-adhesive (DIA) fentanyl TDSs with different amounts of CPEs were prepared, and IVPT studies utilizing porcine and human skin were performed. Although IVPT could discern TDSs with different amounts of CPE by significant differences in flux profiles, maximum flux (Jmax) values, and total permeation amounts, the magnitudes of the CPE increment needed to see such significant differences were very high (43-300%) indicating that IVPT may have limitations in detecting small changes in CPE amounts in some TDSs. Possible reasons for such limitations include formulation polymer and/or other excipients, type of CPE, variability associated with IVPT, skin type used, and disrupted stratum corneum (SC) barrier effects caused by CPEs.

Unique treatment potential of cannabidiol for the prevention of relapse to drug use: preclinical proof of principle.

Cannabidiol (CBD), the major non-psychoactive constituent of Cannabis sativa, has received attention for therapeutic potential in treating neurologic and psychiatric disorders. Recently, CBD has also been explored for potential in treating drug addiction. Substance use disorders are chronically relapsing conditions and relapse risk persists for multiple reasons including craving induced by drug contexts, susceptibility to stress, elevated anxiety, and impaired impulse control. Here, we evaluated the "anti-relapse" potential of a transdermal CBD preparation in animal models of drug seeking, anxiety and impulsivity. Rats with alcohol or cocaine self-administration histories received transdermal CBD at 24 h intervals for 7 days and were tested for context and stress-induced reinstatement, as well as experimental anxiety on the elevated plus maze. Effects on impulsive behavior were established using a delay-discounting task following recovery from a 7-day dependence-inducing alcohol intoxication regimen. CBD attenuated context-induced and stress-induced drug seeking without tolerance, sedative effects, or interference with normal motivated behavior. Following treatment termination, reinstatement remained attenuated up to ≈5 months although plasma and brain CBD levels remained detectable only for 3 days. CBD also reduced experimental anxiety and prevented the development of high impulsivity in rats with an alcohol dependence history. The results provide proof of principle supporting potential of CBD in relapse prevention along two dimensions: beneficial actions across several vulnerability states and long-lasting effects with only brief treatment. The findings also inform the ongoing medical marijuana debate concerning medical benefits of non-psychoactive cannabinoids and their promise for development and use as therapeutics.

In vitro-in vivo correlations for nicotine transdermal delivery systems evaluated by both in vitro skin permeation (IVPT) and in vivo serum pharmacokinetics under the influence of transient heat application.

The in vitro permeation test (IVPT) has been widely used to characterize the bioavailability (BA) of compounds applied on the skin. In this study, we performed IVPT studies using excised human skin (in vitro) and harmonized in vivo human serum pharmacokinetic (PK) studies to evaluate the potential in vitro-in vivo correlation (IVIVC) of nicotine BA from two, matrix-type, nicotine transdermal delivery systems (TDS). The study designs used for both in vitro and in vivo studies included 1h of transient heat (42±2°C) application during early or late time periods post-dosing. The goal was to evaluate whether any IVIVC observed would be evident even under conditions of heat exposure, in order to investigate further whether IVPT may have the potential to serve as a possible surrogate method to evaluate the in vivo effects of heat on the bioavailability of a drug delivered from a TDS. The study results have demonstrated that the BA of nicotine characterized by the IVPT studies correlated with and was predictive of the in vivo BA of nicotine from the respective TDS, evaluated under the matched study designs and conditions. The comparisons of single parameters such as steady-state concentration, heat-induced increase in partial AUCs and post-treatment residual content of nicotine in TDS from the in vitro and in vivo data sets showed no significant differences (p≥0.05). In addition, a good point-to-point IVIVC (Level A correlation) for the entire study duration was achieved by predicting in vivo concentrations of nicotine using two approaches: Approach I requiring only an in vitro data set and Approach II involving deconvolution and convolution steps. The results of our work suggest that a well designed IVPT study with adequate controls can be a useful tool to evaluate the relative effects of heat on the BA of nicotine from TDS with different formulations.

Binge Alcohol Exposure Transiently Changes the Endocannabinoid System: A Potential Target to Prevent Alcohol-Induced Neurodegeneration.

Excessive alcohol consumption leads to neurodegeneration, which contributes to cognitive decline that is associated with alcohol use disorders (AUDs). The endocannabinoid system has been implicated in the development of AUDs, but little is known about how the neurotoxic effects of alcohol impact the endocannabinoid system. Therefore, the current study investigated the effects of neurotoxic, binge-like alcohol exposure on components of the endocannabinoid system and related N-acylethanolamines (NAEs), and then evaluated the efficacy of fatty acid amide hydrolase (FAAH) inhibition on attenuating alcohol-induced neurodegeneration. Male rats were administered alcohol according to a binge model, which resulted in a transient decrease in [³H]-CP-55,940 binding in the entorhinal cortex and hippocampus following two days, but not four days, of treatment. Furthermore, binge alcohol treatment did not change the tissue content of the three NAEs quantified, including the endocannabinoid and anandamide. In a separate study, the FAAH inhibitor, URB597 was administered to rats during alcohol treatment and neuroprotection was assessed by FluoroJade B (FJB) staining. The administration of URB597 during binge treatment did not significantly reduce FJB+ cells in the entorhinal cortex or hippocampus, however, a follow up "target engagement" study found that NAE augmentation by URB597 was impaired in alcohol intoxicated rats. Thus, potential alcohol induced alterations in URB597 pharmacodynamics may have contributed to the lack of neuroprotection by FAAH inhibition.

LC-MS determination of fentanyl in human serum and application to a fentanyl transdermal delivery pharmacokinetic study.

AIM: Fentanyl is an opioid agonist used for acute and chronic pain management. In this report, a highly sensitive and simple LC-MS/MS method using Hydrophilic Interaction Chromatography (HILIC) column was validated and used for fentanyl quantification in human serum. RESULTS: The isocratic mobile phase was composed of acetonitrile: 10 mM ammonium formate buffer (pH = 3.2; 90:10, v/v). The assay was linear over a concentration range of 10-10,000 pg/ml. The accuracy of the validation method ranged from 93.2 to 107%, and the precision was within 6.4%. Fentanyl was stable during short- and long-term storage. CONCLUSION: The assay has been successfully applied to serum samples obtained from healthy subjects of a fentanyl transdermal pharmacokinetic study.

On the Road to Development of an in Vitro Permeation Test (IVPT) Model to Compare Heat Effects on Transdermal Delivery Systems: Exploratory Studies with Nicotine and Fentanyl.

PURPOSE: At elevated temperatures, the rate of drug release and skin permeation from transdermal delivery systems (TDS) may be higher than at a normal skin temperature. The aim of this study was to compare the effect of heat on the transdermal delivery of two model drugs, nicotine and fentanyl, from matrix-type TDSs with different formulations, using in vitro permeation tests (IVPT). METHODS: IVPT experiments using pig skin were performed on two nicotine and three fentanyl TDSs. Both continuous and transient heat exposures were investigated by applying heat either for the maximum recommended TDS wear duration or for short duration. RESULTS: Continuous heat exposure for the two nicotine TDSs resulted in different effects, showing a prolonged heat effect for one product but not the other. The Jmax enhancement ratio due to the continuous heat effect was comparable between the two nicotine TDS, but significantly different (p < 0.05) among the three fentanyl TDSs. The Jmax enhancement ratios due to transient heat exposure were significantly different for the two nicotine TDSs, but not for the three fentanyl TDSs. Furthermore, the transient heat exposure affected the clearance of drug from the skin depot after TDS removal differently for two drugs, with fentanyl exhibiting a longer heat effect. CONCLUSIONS: This exploratory work suggests that an IVPT study may be able to discriminate differences in transdermal drug delivery when different TDS are exposed to elevated temperatures. However, the clinical significance of IVPT heat effects studies should be further explored by conducting in vivo clinical studies with similar study designs.

Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable Gas Vesicle Nanoparticles.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant protein organelles produced by the extremophilic microbe Halobacterium sp. NRC-1 and are being developed as bioengineerable and biocompatible antigen and drug-delivery systems (DDS). Dynamic light scattering measurements of purified GVNP suspensions showed a mean diameter of 245 nm. In vitro diffusion studies using Yucatan miniature pig skin showed GVNP permeation to be enhanced after MN-treatment compared to untreated skin. GVNPs were found to be nontoxic to mammalian cells (human kidney and rat mycocardial myoblasts). These findings support the use of GVNPs as DDS for intradermal/transdermal permeation of protein- and peptide-based drugs.

Norelgestromin/ethinyl estradiol intravenous infusion formulation optimization, stability and compatibility testing: A case study to overcome polysorbate 80 interference in chromatographic analysis.

Norelgestromin/ethinyl estradiol is a progestin/estrogen combination hormonal contraceptive indicated for the prevention of pregnancy in women. The very poor solubility and wettability of these drugs, along with their high potency (adsorption issues), give rise to difficulties in designing intravenous (IV) formulations to assess absolute bioavailability of products containing both drugs. The purpose of this study was to develop an IV formulation, evaluate its stability under different conditions and evaluate its compatibility with IV sets for potential use in absolute bioavailability studies in humans. Also, a selective high-performance liquid chromatography (HPLC) method for quantification of ethinyl estradiol and norelgestromin in polysorbate 80 matrix was developed and validated. Norelgestromin/ethinyl estradiol IV solution was prepared using sterile water for injection with 2.5% ethanol and 2.5% polysorbate 80 as a cosolvent/surfactant system to obtain a final drug solution of 25µg ethinyl estradiol and 252µg norelgestromin from a concentrated stock drug solution. The stabilities of the concentrated stock and IV solutions were assessed after storing them in the refrigerator (3.7±0.6°C) and at room temperature (19.5±0.5°C), respectively. Additional studies were conducted to examine the stability of the IV solution using an Alarias(®) low sorbing IV administration set with and without an inline filter. The solution was allowed to drip at 1mL/min over a 60min period. Samples were obtained at the beginning, middle and end of the 60min duration. The chemical stability was evaluated for up to 10 days. Norelgestromin and ethinyl estradiol concentration, purity, and degradant levels were determined using the HPLC method. The norelgestromin/ethinyl estradiol IV formulation met the chemical stability criteria when tested on day 1 through day 9 (216h). Norelgestromin concentrations assayed in stock and IV solutions were in the range of 90.0-98.5% and 90.9-98.8% after 9 days, respectively. As for ethinyl estradiol, the assayed concentrations were in the range of 91.8-100.9% and 92.7-100.8% for the stock and IV solutions, respectively. The administration set was found to be compatible with both drugs; the assayed concentrations were in the range of 99.2-100.3% for norelgestromin and 96.3-102.7% for ethinyl estradiol, but the inline filter showed some adsorption of ethinyl estradiol; where the assayed concentrations were in the range of 98.1-99.8% for norelgestromin and 95.9-97.4% for ethinyl estradiol. The present study provided evidence supporting the suitability of an intravenous formulation for norelgestromin/ethinyl estradiol using ethanol/polysorbate 80 as a cosolvent/surfactant system. Both IV and concentrated stock solutions when stored at room temperature and refrigeration, respectively, were found to be chemically stable up to 9 days. These results indicated that this formulation is chemically stable and can be used over the time period tested. This IV formulation can be used to evaluate the absolute bioavailability of products containing norelgestromin and ethinyl estradiol provided that microbial testing of the IV formulation is performed.

A fully validated LC-MS/MS method for simultaneous determination of nicotine and its metabolite cotinine in human serum and its application to a pharmacokinetic study after using nicotine transdermal delivery systems with standard heat application in adult smokers.

A sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of nicotine and its main metabolite cotinine in human serum samples. Liquid-liquid extraction using ethyl acetate was employed for serum sample extractions. Chromatographic separation was achieved on Phenomenex Luna(®) HILIC column (150 mm x 3.0mm, 5 µm). Isocratic elution was performed using acetonitrile:100mM ammonium formate buffer (pH=3.2) (90:10, v/v) as the mobile phase, at a flow rate of 0.4 mL/min. Tandem mass spectrometric detection was employed at positive electrospray ionization in MRM mode for the determination of both nicotine and cotinine and their stable isotope labeled internal standards. Analysis was carried out in 8 min over a concentration range of 0.26-52.5 ng/mL and 7.0-1500 ng/mL for nicotine and cotinine, respectively. The assay was validated according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained; the accuracy ranged between 93.39% and 105.73% for nicotine and between 93.04% and 107.26% for cotinine. No significant matrix effect was observed. Stability assays indicated both nicotine and cotinine were stable during sample storage, preparation and analytical procedures. The method was successfully applied to biological samples obtained from a pharmacokinetic study conducted in adult smokers to investigate heat effect on nicotine and cotinine serum levels after nicotine transdermal delivery system (TDS) application.

Quantification of anandamide, oleoylethanolamide and palmitoylethanolamide in rodent brain tissue using high performance liquid chromatography-electrospray mass spectroscopy.

Reported concentrations for endocannabinoids and related lipids in biological tissues can vary greatly; therefore, methods used to quantify these compounds need to be validated. This report describes a method to quantify anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) from rodent brain tissue. Analytes were extracted using acetonitrile without further sample clean up, resolved on a C18 reverse-phase column using a gradient mobile phase and detected using electrospray ionization in positive selected ion monitoring mode on a single quadrupole mass spectrometer. The method produced high recovery rates for AEA, OEA and PEA, ranging from 98.1% to 106.2%, 98.5% to 102.2% and 85.4% to 89.5%, respectively. The method resulted in adequate sensitivity with a lower limit of quantification for AEA, OEA and PEA of 1.4 ng/mL, 0.6 ng/mL and 0.5 ng/mL, respectively. The method was reproducible as intraday and interday accuracies and precisions were under 15%. This method was suitable for quantifying AEA, OEA and PEA from rat brain following pharmacological inhibition of fatty acid amide hydrolase.

Synthesis and in vitro stability of amino acid prodrugs of 6-ß-naltrexol for microneedle-enhanced transdermal delivery.

A small library of amino acid ester prodrugs of 6-ß-naltrexol (NTXOL, 1) was prepared in order to investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino acid ester prodrugs were synthesized (6a-f). 6b, 6d, and 6 e were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. The lead compound (6 e) exhibited the most rapid bioconversion to NTXOL in human plasma (t1/2 = 2.2 ± 0.1h).

Fluvastatin as a micropore lifetime enhancer for sustained delivery across microneedle-treated skin.

Microneedles (MNs), a physical skin permeation enhancement technique, facilitate drug delivery across the skin, thus enhancing the number of drugs that can be delivered transdermally in therapeutically relevant concentrations. The micropores created in the skin by MNs reseal because of normal healing processes of the skin, thus limiting the duration of the drug delivery window. Pore lifetime enhancement strategies can increase the effectiveness of MNs as a drug delivery mechanism by prolonging the delivery window. Fluvastatin (FLU), a HMGCoA reductase inhibitor, was used in this study to enhance the pore lifetime by inhibiting the synthesis of cholesterol, a major component of the stratum corneum lipids. The study showed that using FLU as a pretreatment it is possible to enhance the pore lifetime of MN-treated skin and thus allow for sustained drug delivery. The skin recovered within a 30-45-min time period following the removal of occlusion, and there was no significant irritation observed due to the treatment compared to the control sites. Thus, it can be concluded that localized skin treatment with FLU can be used to extend micropore lifetime and deliver drugs for up to 7 days across MN-treated skin.