The In Vitro, In Vivo, and PBPK Evaluation of a Novel Lung-Targeted Cardiac-Safe Hydroxychloroquine Inhalation Aerogel.

Hydroxychloroquine (HCQ) was repurposed for COVID-19 treatment. Subtherapeutic HCQ lung levels and cardiac toxicity of oral HCQ were overcome by intratracheal (IT) administration of lower HCQ doses. The crosslinker-free supercritical fluid technology (SFT) produces aerogels and impregnates them with drugs in their amorphous form with efficient controlled release. Mechanistic physiologically based pharmacokinetic (PBPK) modeling can predict the lung's epithelial lining fluid (ELF) drug levels. This study aimed to develop a novel HCQ SFT formulation for IT administration to achieve maximal ELF levels and minimal cardiac toxicity. HCQ SFT formulation was prepared and evaluated for physicochemical, in vitro release, pharmacokinetics, and cardiac toxicity. Finally, the rat HCQ ELF concentrations were predicted using PBPK modeling. HCQ was amorphous after loading into the chitosan-alginate nanoporous microparticles (22.7±7.6 µm). The formulation showed a zero-order release, with only 40% released over 30 min compared to 94% for raw HCQ. The formulation had a tapped density of 0.28 g/cm3 and a loading efficiency of 35.3±1.3%. The IT administration of SFT HCQ at 1 mg/kg resulted in 23.7-fold higher bioavailability, fourfold longer MRT, and eightfold faster absorption but lower CK-MB and LDH levels than oral raw HCQ at 4 mg/kg. The PBPK model predicted 6 h of therapeutic ELF levels for IT SFT HCQ and a 100-fold higher ELF-to-heart concentration ratio than oral HCQ. Our findings support the feasibility of lung-targeted and more effective SFT HCQ IT administration for COVID-19 compared to oral HCQ with less cardiac toxicity. Graphical abstract.

Encapsulation of morin in lipid core/PLGA shell nanoparticles significantly enhances its anti-inflammatory activity and oral bioavailability.

Morin (3,5,7,2',4'-pentahydroxyflavone; MR) is a bioactive plant polyphenol whose therapeutic efficacy is hindered by its poor biopharmaceutical properties. The purpose of this study was to develop a nanoparticle (NP) formulation to enhance the bioactivity and oral bioavailability of MR. The nanoprecipitation technique was employed to encapsulate MR in lipid-cored poly(lactide-co-glycolide) (PLGA) NPs. The optimal NPs were about 200 nm in size with an almost neutral surface charge and a loading efficiency of 82%. The NPs exhibited sustained release of MR within 24 h. In vitro antioxidant assays showed that MR encapsulation did not affect its antioxidant activity. On the other hand, anti-inflammatory assays in lipopolysaccharide-stimulated macrophages revealed a superior anti-inflammatory activity of MR NPs compared to free MR. Furthermore, oral administration of MR NPs to mice at a single dose of 20 mg/kg MR achieved a 5.6-fold enhancement in bioavailability and a prolongation of plasma half-life from 0.13 to 0.98 h. The results of this study present a promising NP formulation for MR which can enhance its oral bioavailability and bioactivity for the treatment of different diseases such as inflammation.

Safety, Tolerability, and Pharmacokinetics of Nebulized Hydroxychloroquine: A Pilot Study in Healthy Volunteers.

Background: Early in the coronavirus disease 2019 (COVID-19) pandemic, hydroxychloroquine (HCQ) drew substantial attention as a potential COVID-19 treatment based on its antiviral and immunomodulatory effects in vitro. However, HCQ showed a lack of efficacy in vivo, and different groups of researchers attributed this failure to the insufficient drug concentration in the lung following oral administration (HCQ is only available in the market in the tablet form). Delivering HCQ by inhalation represents a more efficient route of administration to increase HCQ exposure in the lungs while minimizing systemic toxicity. In this pilot study, the safety, tolerability, and pharmacokinetics of HCQ nebulizer solution were evaluated in healthy volunteers. Methods: Twelve healthy participants were included in this study and were administered 2 mL of HCQ01 solution (equivalent to 25 mg of HCQ sulfate) through Aerogen® Solo, a vibrating mesh nebulizer. Local tolerability and systemic safety were assessed by forced expiratory volume in the first and second electrocardiograms, clinical laboratory results (e.g., hematology, biochemistry, and urinalysis), vital signs, and physical examinations. Thirteen blood samples were collected to determine HCQ01 systemic exposure before and until 6 hours after inhalation. Results: The inhalation of HCQ01 was well tolerated in all participants. The mean value of Cmax for the 12 participants was 9.66 ng/mL. Tmax occurred at around 4.8 minutes after inhalation and rapidly decreased thereafter. The reported systemic exposure was very low with a mean value of 5.28 (0.6-15.6) ng·h/mL. Conclusion: The low systemic concentrations of HCQ01 of 9.66 ng/mL reported by our study compared with 1 µg/mL previously predicted after 200 mg BID oral administration, and the safety and tolerability of HCQ01 administered as a single dose through nebulization, support the assessment of its efficacy, safety, and tolerability in further studies for the treatment of COVID-19.

12th GCC Closed Forum: critical reagents; oligonucleotides; CoA; method transfer; HRMS; flow cytometry; regulatory findings; stability and immunogenicity.

The 12th GCC Closed Forum was held in Philadelphia, PA, USA, on 9 April 2018. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: critical reagents; oligonucleotides; certificates of analysis; method transfer; high resolution mass spectrometry; flow cytometry; recent regulatory findings and case studies involving stability and nonclinical immunogenicity. Conclusions and consensus from discussions of these topics are included in this article.

Evaluation of patients' real-world post-dispensing use and storage environments of tiotropium bromide Respimat® soft mist inhaler on its in vitro dose delivery and lung deposition.

BACKGROUND: Oral inhalation is the main drug delivery route for treating obstructive lung conditions. Thus, many inhaler devices with various design and pharmaceutical formulation have been introduced. The fine particle dose (FPD) and mass median aerodynamic diameter (MMAD ≤ 5 µm) of the aerosol delivered dose (DD) dictate the therapeutically effective peripheral lung deposition. This study evaluated the in vitro aerosol emission performance of tiotropium bromide emitted from Spiriva® Respimat® soft mist inhalers (R) after living under patients' real-world, post-dispensing handling environments. METHODS: This was a two-stage investigation. In the first clinical stage, research ethical approval was obtained to enrol patients already been using R for at least 3 months. Those who signed consent were given both new R to use and temperature and relative humidity (RH) handheld, portable data loggers to keep in the vicinity of the given R. The participants returned the given R and data loggers after 2 weeks. Patient recruitment took place in Amman, Jordan, during the summer (RS) and winter (RW). Subsequently, in the second laboratory stage, other R were strictly stored at an average of 21.0 °C and 46.9% RH as control (RC). The Next Generation Impactor (NGI) was used to evaluate the RS, RW and RC. The NGI was operated at a flow rate of 30 L/min. RESULTS: The RS were exposed to an average (range) 23.6 °C (18.2-37.5 °C) and 43.8% RH (21.4-60.0% RH) that were statistically comparable (p > 0.05) to that of the RW; 17.3 °C (13.2-26.7 °C) and 52.8% RH (26.3-69.1% RH). The RW and RC retention environments were statistically different (p < 0.05), whilst the RS and RC had comparable (p > 0.05) conditions. No significant differences (p > 0.05) were found in the tiotropium bromide DD (2.39 vs 2.43 µg), FPD (0.88 vs 0.90 µg) and MMAD (5.1 vs 4.98 µm) between the RS and RW, respectively. Compared to the RC inhalers, both the RS and RW devices had significantly higher FPD and relatively smaller tiotropium bromide particles. CONCLUSIONS: Using the R under the fluctuating summer and winter environments of our patients would not affect its overall tiotropium bromide emission performance. The significant increase in the respirable mass of the RS and RW might be offset by the increase in particles <1 µm particularly in patients with poor inhaler technique.

Delivered Lung Dose and Aerodynamic Particle Size Distribution of Salbutamol Pressurized Metered Dose Inhaler After Living Under Patients' Realistic Retention Environments.

Background: The impact of inhalers' postdispensing, real-life temperature and relative humidity (RH) environments on their delivered dose (DD) and aerodynamic particle size distribution (APSD) is usually overlooked. This work evaluated the salbutamol DD and APSD of Ventolin® Evohaler® (V) inhalers already been used and stored by respiratory patients. Methods: Adult patients, prescribed V for ≥3 months before study enrollment, were dispensed both new V to use and portable, handheld electronic temperature and RH data loggers to keep close to the given V before returning them both after 2-3 weeks. Patients' enrollment took place during summer (VS) and winter (VW) seasons. The returned V was then in vitro evaluated using the Next Generation Impactor, and compared with control V (VC) counterparts stored under 21°C and 46% RH. Results: The VS survived in fluctuating habitats of 21.2°C-40.4°C and 16.2%-63.2% RH, which significantly (p < 0.05) decreased the salbutamol DD from 80.4 to 70.5 µg compared with VC. This 12.3% DD reduction was accompanied with a decrease in the fine particle dose from 26.2 to 20.4 µg (p < 0.05), and an increase in the mass median aerodynamic diameter from 2.3 to 2.5 µm (p < 0.05). The VW and VC had equivalent DD and APSD. Conclusion: Patients using V are expected to receive smaller lung doses during the hot summer season compared with intentionally well-kept VC. To have equivalent lung deposition, V users should be advised to retain their inhalers around 20°C with minimal daily environmental fluctuations during summer times.

The impact of patients' real-life environmental temperature and humidity use conditions of tiotropium dry powder inhaler on its aerosol emission characteristics.

INTRODUCTION: Many factors can affect dry powder inhalers' (DPIs) aerosol emission and lung deposition. The fluctuation of environmental temperature and relative humidity (RH) that inhalers experience in realistic daily use has not been extensively evaluated. This work aimed to evaluate the delivered dose (DD) and aerodynamic particle size distribution (APSD) of tiotropium Handihaler DPI (H) after exposure to patients' real-life use environments. METHODS: Ethical approval was obtained to enrol adult patients already using H. Patients who gave written consent were given new H to use and HygroLog temperature and RH data loggers to keep in the vicinity of the given inhaler. The H and HygroLog were returned after 2 weeks. Patient recruitment was done during the summer (HS) and winter (HW). As control, other HC were stored as per the leaflet storage instructions. The Next Generation Impactor was used to evaluate the inhalers. RESULTS: The HC were stored under an average of 21.0 °C and 46.9% RH. The patients' HS and HW lived in an average (range) temperature (°C) 23.2 (18.3-38.2) and 17.8 (13.5-24.6), respectively, and RH 50.8% (24.3-65.3%) and 50.4% (30.6-72.4%), respectively. All H groups had comparable environments (p > 0.05). The HC, HS and HW gave similar tiotropium DD (µg) 7.60, 8.01 and 7.61, respectively (p > 0.05). Moreover, the fine particle dose µg (median diameter (µm)) were HC 2.41 (3.84), HS 2.55 (3.81) and HW 2.37 (3.83) (p > 0.05). CONCLUSIONS: The aerosol emission behaviour of tiotropium Handihaler was tolerant to real-life retention environments of patients in Amman, Jordan.

Recommendations for classification of commercial LBA kits for biomarkers in drug development from the GCC for bioanalysis.

Over the last decade, the use of biomarker data has become integral to drug development. Biomarkers are not only utilized for internal decision-making by sponsors; they are increasingly utilized to make critical decisions for drug safety and efficacy. As the regulatory agencies are routinely making decisions based on biomarker data, there has been significant scrutiny on the validation of biomarker methods. Contract research organizations regularly use commercially available immunoassay kits to validate biomarker methods. However, adaptation of such kits in a regulated environment presents significant challenges and was one of the key topics discussed during the 12th Global Contract Research Organization Council for Bioanalysis (GCC) meeting. This White Paper reports the GCC members' opinion on the challenges facing the industry and the GCC recommendations on the classification of commercial kits that can be a win-win for commercial kit vendors and end users.

11th GCC Closed Forum: cumulative stability; matrix stability; immunogenicity assays; laboratory manuals; biosimilars; chiral methods; hybrid LBA/LCMS assays; fit-for-purpose validation; China Food and Drug Administration bioanalytical method validation.

The 11th Global CRO Council Closed Forum was held in Universal City, CA, USA on 3 April 2017. Representatives from international CRO members offering bioanalytical services were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The second CRO-Pharma Scientific Interchange Meeting was held on 7 April 2017, which included Pharma representatives' sharing perspectives on the topics discussed earlier in the week with the CRO members. The issues discussed at the meetings included cumulative stability evaluations, matrix stability evaluations, the 2016 US FDA Immunogenicity Guidance and recent and unexpected FDA Form 483s on immunogenicity assays, the bioanalytical laboratory's role in writing PK sample collection instructions, biosimilars, CRO perspectives on the use of chiral versus achiral methods, hybrid LBA/LCMS assays, applications of fit-for-purpose validation and, at the Global CRO Council Closed Forum only, the status and trend of current regulated bioanalytical practice in China under CFDA's new BMV policy. Conclusions from discussions of these topics at both meetings are included in this report.

Bioequivalent pharmacokinetics for generic and originator hepatitis C direct-acting antivirals.

Mass production of low-cost, generic direct-acting antivirals (DAAs) will be required to achieve targets of eliminating hepatitis C (HCV) by 2030. The pharmaceutical companies Gilead and Bristol-Myers Squibb have granted voluntary licences (VLs) to generic companies to mass produce the DAAs sofosbuvir and daclatasvir at low cost. However, generic manufacturers need to demonstrate bioequivalent pharmacokinetics for their DAAs, compared to the originator versions, to fulfil World Health Organization standards for prequalification. The aim of this study was to determine whether generic forms of sofosbuvir and daclatasvir had bioequivalent pharmacokinetics to the originator versions. Generic companies were contacted for results of bioequivalence studies with sofosbuvir and daclatasvir, two of the most widely used DAAs in the developing world. Data on maximum concentration (Cmax) and area under the curve (AUC) were compiled from five generic companies. Pre-specified limits for the 90% confidence intervals were 80-125% of the originator pharmacokinetic concentrations for AUC, and 69-145% for Cmax. The pharmacokinetics of generic sofosbuvir and daclatasvir were shown to be bioequivalent to the originator versions for all five generic companies. This is a crucial step towards securing prequalification of the manufacture of these drugs from these companies. WHO prequalification of bioequivalent generic DAAs could then permit their export to eligible countries for mass-treatment programmes. Mass-treatment with low-cost generic HCV DAAs is the most promising method to achieve the ambitious World Health Organization targets for HCV elimination by 2030.

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud.

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.

Discovery of new human epidermal growth factor receptor-2 (HER2) inhibitors for potential use as anticancer agents via ligand-based pharmacophore modeling.

To discover potential antitumor agents directed toward human epidermal growth factor receptor-2HER2/ErbB2 overexpression in cancer, we have explored the pharmacophoric space of 115 HER2/ErbB2 inhibitors. This identified 240 pharmacophores which were subsequently clustered into 20 groups and cluster centers were used as 3D-pharmacophoric descriptors in QSAR analysis with 2D-physicochemical descriptors to select the optimal combination. We were obliged to use ligand efficiency as the response variable because the logarithmic transformation of bioactivities failed to access self-consistent QSAR models. Two binding pharmacophore models emerged in the optimal QSAR equation, suggesting the existence of distinct binding modes accessible to ligands within the HER2/ErbB2 binding pocket. The QSAR equation and its associated pharmacophore models were employed to screen the National Cancer Institute (NCI) and Drug Bank databases to search for new, promising, and structurally diverse HER2 inhibitory leads. Inhibitory activities were tested against HER2-overexpressing SKOV3 Ovarian cancer cell line and MCF-7 which express low levels of HER2. In silico mining identified 80 inhibitors out of which four HER2 selective compounds inhibited the growth of SKOV3 cells with IC50 values < 5µM and with virtually no effect in MCF-7 cells. These lead compounds are excellent candidates for further optimization.

Indacaterol determination in human urine: validation of a liquid-liquid extraction and liquid chromatography-tandem mass spectrometry analytical method.

BACKGROUND: Indacaterol is a novel once-a-day inhaled ultra-long-acting ß2-agonist. Quantitative bioanalysis supports pharmacokinetic and clinical research. The aim of the current work was to validate an in-house developed high performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) analytical method for indacaterol determination in human urine samples. METHODS: A liquid-liquid extraction method has been developed to extract indacaterol from human urine samples using ethyl acetate. Indacaterol dry extract was reconstituted with 200 µL of the mobile phase (acidified water:methanol (30:70, v/v)) of which 5 µL was needed for the HPLC-MS/MS analysis. Indacaterol was eluted on a reversed C18 stationary phase with an isocratic mobile phase at a flow of 1 mL/min. Formoterol was the internal standard (IS). The MS/MS detection was employed with a turbo-ion spray ionization in the positive ion mode. A consensus of the international Guidelines for Bioanalytical Method Validation was followed. RESULTS: Indacaterol was detected at a mass to charge ratio (m/z) of 393.3 and its MS/MS daughter at 173.2. The retention times of indacaterol and IS were 1.60 and 1.20 min, respectively. Validated calibration curves were linear over a range of 0.075-100 ng/mL with correlation coefficients (r)≥0.990. The curves' regression weighting factor was 1/x. Method specificity was established in six different human urine batches. No matrix interference was observed. The intra- and inter-batch precision and accuracy within±20% (at lower limit) and±15% (other quality control (QC) levels) were confirmed. The indacaterol mean recovery (precision) percentages at Low, Mid, and High QC levels were 93.5 (3.84), 89.8 (2.15), and 92.2 (2.17), respectively. Short-term, long-term, freeze-thaw, and auto-sampler stability results were accepted. CONCLUSIONS: A specific, accurate and precise HPLC-MS/MS method has been validated for indacaterol quantification in human urine. This simple method is reproducible and robust to support future, indacaterol-related pharmacokinetic, bioequivalence and clinical studies.

Ligand-based designing, in silico screening, and biological evaluation of new potent fructose-1,6-bisphosphatase (FBPase) inhibitors.

Fructose-1,6-bisphosphatase - hereafter abbreviated as FBPase has been recently implicated in diabetes prompting several attempts to discover and optimize new FBPase inhibitors. Toward this end we explored the pharmacophoric space of 136 FBPase inhibitors using three diverse sets of inhibitors. This identified 520 pharmacophores that were subsequently clustered into 104 groups. Cluster centers were evaluated by receiver operating characteristic (ROC) curves analysis and correlation with bioactivities of collected compounds. Pharmacophore model Hypo1/7 illustrated the best combination of classification power (ROC-AUC) and correlation with bioactivity. Two other pharmacophores (Hypo2/1 and Hypo2/6) were found to be mergeable and their combined model (Hypo2-1/2-6) illustrated excellent ROC performance. We employed Hypo1/7 and Hypo2-1/2-6 models to screen the National Cancer Institute (NCI) list of compounds. In silico mining identified 18 FBPase inhibitors out of which six were of sub-micromolar IC(50) values.

Ethnopharmacological survey of medicinal plants in Jordan, Mujib Nature Reserve and surrounding area.

AIM OF THE STUDY: Medicinal plants are an important element of indigenous systems in Jordan. These resources are usually regarded as part of a culture's traditional knowledge. Therefore, the aim of this study is to collect information from local population concerning the use of medicinal plants of the Mujib region; identify the most important medicinal plants used; determine the relative importance of the species surveyed and calculate the informant consensus factor (F(ic)) in relation to medicinal plant use. MATERIALS AND METHODS: Qualitative tools were used for data collection and to record the interviewee's personal information and topics related to the medicinal use of specific plants. The collected data were used to calculate the F(ic) and the plant use values. RESULTS AND CONCLUSIONS: Fifty-eight plants were identified to be still in use in traditional practice in Mujib. Our results showed that the highest use values were recorded for the species Artemisia sieberi Bess. and Silybum marianum (L.) Gaertn., while the highest F(ic) was cited for digestive problems. Anthropologically, women were the primary gatherers while healers were reported to be both females, predominantly, and males; yet, herbalists are deficient in this local community.

Ethnopharmacological survey of medicinal herbs in Jordan, the Ajloun Heights region.

The study of local knowledge about natural resources is becoming increasingly important in defining strategies and actions for conservation of medicinal plants. This study therefore sought to collect information from local population concerning the use of Ajloun Heights region medicinal plants; identify the most important species used; determine the relative importance of the species surveyed and calculate the informant consensus factor (ICF) in relation to medicinal plant use. Data collection relied predominantly on qualitative tools to record the interviewee's personal information and topics related to the medicinal use of specific plants. Our results revealed that 46 plant species grown in the study region are still in use in traditional medicine for the treatment of various diseases. Most of the locals interviewed dealt with well-known safe medicinal plants such as Achillea falcata, Matricaria aurea, Majorana syriaca, Allium sativum and Allium cepa. The use of moderately unsafe or toxic plants was noted to be practiced by practitioners and herbalists rather than the locals. These plants include Ecballium elaterium, Euphorbia hierosolymitana, Mandragora autumnalis and Citrullus colocynthis. Kidney problems scored the highest ICF while Crocus hyemalis was the plant of highest use value. Searching the literature evidenced some concordance with the solicited plant uses mentioned by the informants.

Variable omeprazole kinetics in healthy Jordanian adults.

OBJECTIVES: The objective of this study was to examine the pharmacokinetics of orally administered omeprazole in healthy adult Jordanian men. METHOD: Plasma concentrations of omeprazole were measured over a 12 h period after administration of a single oral dose of 40 mg omeprazole (Losec), AstraZeneca, UK). Subjects were healthy adult Jordanian men age 18-38 (24 +/- 4, mean +/- SD). The pharmacokinetic parameters were derived from the plasma concentration-time profiles for AUC(0-t), AUC(0-inf), C(max), t(max), t(1/2e) and K(e). RESULTS: The pharmacokinetic of omeprazole were scattered over a wide range. The median AUC(0-inf) was 784.86 +/- 1182.88 (ng.h/ml), and the median C(max) was 521 +/- 354 (ng/ml) (median +/- SD). In general, most subjects showed normal distribution (approximately 90%). Some subjects (10%) did show very high AUC and C(max) compared with the reported AUC and C(max) levels. These subjects had higher half-lives and lower rates of elimination. CONCLUSION: Significant difference in the pharmacokinetics of omeprazole after a single dose administration was noted. Approximately 10% of the study group showed very high omeprazole plasma levels and AUCs. Differences in the pharmacokinetics might be due to differences in the genetic make-up of subjects.