Methadone Metabolism and Drug-Drug Interactions: In Vitro and In Vivo Literature Review

Methadone is utilized for the treatment of individuals with opiate dependence. Methadone undergoes N-demethylation by multiple cytochrome P450 (CYP) enzymes including CYP3A4, CYP2B6, CYP2C19, CYP2D6, CYP2C9, and CYP2C8. In vivo, polymorphism effects on methadone systemic exposure have been noted for CYP2B6, CYP3A4, and CYP2D6. Clinical drug interaction studies with antiviral drugs in methadone maintenance treatment patients yield varying results on methadone pharmacokinetics and pharmacodynamics. In general, CYP inhibitors altered methadone exposure with no adverse effects. CYP inducers generally decreased methadone exposure with some reports of withdrawal symptoms in the subjects. Interaction studies with antiviral drug combinations yielding differing results depend on the enzyme(s) affected. For certain antiviral medicines which are dual inhibitor(s) and inducer(s) for CYP enzymes, their effect on methadone pharmacokinetics can change with time since the effect of induction is usually delayed compared to the effect of inhibition.     

Potential of Cationic-Polymeric Nanoparticles for Oral Delivery of Naringenin: In Vitro and In Vivo Investigations

The objective of the study was to improve the bioavailability and anticancer potential of naringenin (NRG) by developing a drug-loaded polymeric nanodelivery system. NRG-loaded eudragit E100 nanoparticle (NRG-EE100-NPs) system was developed and physicochemically characterized. In vivo pharmacokinetic and in vitro cytotoxicity abilities of the NRG-EE100-NPs were investigated. In vivo anticancer activity was evaluated in murine BALB/c mice-bearing colorectal tumor. The NRG-EE100-NPs had an optimum mean particle size (430.42 ± 5.78 nm), polydispersity index (0.283 ± 0.089) with percent entrapment efficiency (68.83 ± 3.45%). The NRG-EE100-NPs demonstrated significant higher bioavailability (～96-fold; p <0.05) as well as cytotoxicity (～16-fold; p <0.001) as compared to free NRG. Furthermore, NRG-EE100-NPs indicated significant tumor suppression (p <0.01) subsequently improvement in survival rate compared to free NRG in vivo. Thus, the physicochemical properties and colorectal cancer efficacy of NRG were improved by successful encapsulating in cationic-polymeric nanoparticle system.     

In Vitro and In Vivo Correlation of Hepatic Fraction of Metabolism by P450 in Dogs

1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases–mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUC_ABT/AUC_veh ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.     

Diosmin Nanocrystal–Loaded Wafers for Treatment of Diabetic Ulcer: In Vitro and In Vivo Evaluation

This work aimed at loading of diosmin nanocrystals into alginate-based wafers for treatment of highly exuding diabetic ulcer in rats using topical route of administration. For this purpose, different formulation variables and preparation techniques to enhance the flexibility and adhesion properties of the prepared sodium alginate (SA) wafers were carried out. The prepared wafers were characterized regarding hydration capacity, bioadhesion, scanning electron microscope, and Fourier-transform infrared spectroscopy. Efficacy of treating diabetic ulcer was studied using diabetic-induced rat model using streptozotocin. Results obtained showed that using SA:gelatin with 1.5%/1.5% w/w gave acceptable wafers with a sustained release of diosmin over 8 h. A complete re-epithelialization, well-organized dermal layers, well-formed granulation tissue, and mature collagen bundles were observed in treated rats. It was concluded that combination of gelatin with SA provided an excellent wafer as a promising medicated wound dressing holding diosmin nanocrystals while maintaining its stability.     

The Effect of Drug Content Reduction on the In Vitro and In Vivo Properties of Levonorgestrel-Releasing Intravaginal Rings

Intravaginal rings (IVRs) are an option for continuous administration of drugs in women. However, a considerable amount of excess drug often remains in the ring upon removal. The current study focuses on comparing 2 IVRs releasing levonorgestrel (LNG). Both formulations were designed to release 40 μg of LNG daily, however, with a significant difference in the total amount of drug (10.6 vs. 176.9 mg). Numerical simulations and in vitro release rate testing were utilized in designing the IVRs and confirming the similarity of drug release. Moreover, a pharmacokinetic (PK) study was performed in 13 healthy Japanese women to investigate both formulations during the intended wearing period of 28 days. The primary PK metrics was the average concentration of LNG in plasma at defined time points under stable conditions. Statistical evaluation of the ratio of the main PK metrics indicated values almost in the bioequivalence range. Furthermore, drug content determinations for used and unused IVRs were analyzed for confirming the expected drug delivery in vivo. In summary, it was shown that with proper design, even major differences in the total drug content of IVR formulations might not result in significant effects in the in vitro and in vivo release properties.     

Formulation Development,Optimization, and In Vitro–In Vivo Characterization of Natamycin-Loaded PEGylated Nano-Lipid Carriers for Ocular Applications

The present study aimed at formulating and optimizing natamycin (NT)-loaded polyethylene glycosylated nano-lipid carriers (NT-PEG-NLCs) using Box-Behnken design and investigating their potential in ocular applications. Response surface methodology computations and plots for optimization were performed using Design-Expert^® software to obtain optimum values for response variables based on the criteria of desirability. Optimized NT-PEG-NLCs had predicted values for the dependent variables which are not significantly different from the experimental values. NT-PEG-NLCs were characterized for their physicochemical parameters; NT's rate of permeation and flux across rabbit cornea was evaluated, in vitro, and ocular tissue distribution was assessed in rabbits, in vivo. NT-PEG-NLCs were found to have optimum particle size (<300 nm), narrow polydispersity index, and high NT entrapment and NT content. In vitro transcorneal permeability and flux of NT from NT-PEG-NLCs was significantly higher than that of Natacyn^®. NT-PEG-NLC (0.3%) showed improved delivery of NT across the intact cornea and provided concentrations statistically similar to the marketed suspension (5%) in inner ocular tissues, in vivo, indicating that it could be a potential alternative to the conventional suspension during the course of fungal keratitis therapy.     

Optimization of Nanostructured Lipid Carriers of Lurasidone Hydrochloride Using Box-Behnken Design for Brain Targeting: In Vitro and In Vivo Studies

Intranasal nanostructured lipid carrier (NLC) of lurasidone hydrochloride (LRD) for brain delivery was prepared by the solvent evaporation method. The effects of independent variables, X1-lipid concentration, X-2 surfactant, and X-3 sonication times on dependent variables, Y1-particle size, Y-2 polydispersity index, and Y-3% entrapment efficiency were determined using Box-Behnken design. Optimized LRD-NLC was selected from the Box-Behnken design and evaluated for their morphological, physiological, nasal diffusion, and in vivo distribution in the brain after intranasal administration. Particle size, polydispersity index, and entrapment efficiency of optimized LRD-NLC were found to be 207.4 ± 1.5 nm, 0.392 ± 0.15, and 92.12 ± 1.0%, respectively. Transmission electron microscopy and scanning electron microscopy was used to determine the particle size and surface morphology of LRD-NLC. The prepared LRD-NLC follows biphasic in vitro drug release. Prepared NLC showed a 2-fold increase in LRD concentration in the brain when compared with the drug solution following intranasal administration. Results showed that intranasal route can be a good and efficient approach for delivering the drug directly to the brain and enhancing the drug efficacy in the brain for the management of schizophrenia and a good alternative to oral drug delivery.     

Adaptation of Quality by Design-Based Development of Isradipine Nanostructured–Lipid Carrier and Its Evaluation for In Vitro Gut Permeation and In Vivo Solubilization Fate

The present study demonstrated the systematic adaptation of quality by design-integrated approach for the development of novel nanostructured lipid carrier (NLC) of an anti-hypertensive drug isradipine (ISD) to address the inherent challenges such as low solubility and low oral bioavailability. Plackett-Burman design was used for preliminary screening of significant process and formulation variables (p <0.05), which were further processed using Box-Behnken design for the attainment of optimization goal that is, mean particle size (85.7 ± 7.3 nm), drug entrapment efficiency (87.4 ± 3.29%), and in vitro drug release characteristics (92.89 ± 5.47%). The optimized ISD-NLC formulation also demonstrated well-dispersed uniform-shaped particles (polydispersity index 0.207 ± 0.029), high gastrointestinal fluid stability (zeta potential ?10.17 ± 0.59 mV), and higher in vitro gut permeation (21.69 ± 2.38 μg/cm² of ISD-NLC as compared to 11.23 ± 1.74 μg/cm² in ISD suspension). Furthermore, lipolysis studies were performed for the purpose of in vivo fate, and significantly higher drug content of ISD from ISD-NLC in aqueous phase was found (72.34 ± 4.62%) as compared to drug suspension (3.01 ± 0.91%). Relative bioavailability of ISD-NLC and ISD suspension was increased by 4.2-fold and 1.78-fold in the absence and presence of cycloheximide which is a lymphatic uptake inhibitor revealing lymphatic uptake of ISD-NLC in bioavailability improvement. Hence, systematic adaptation of quality by design integrated approach improved gut permeation and potential solubilizaton fate (dynamic lipolysis) of ISD-NLC, which further improved the lymphatic uptake and biodistribution of drug thereby promisingits in vivo prospect and clinical efficacy.     

Segmental-Dependent Intestinal Drug Permeability: Development and Model Validation of In Silico Predictions Guided by In Vivo Permeability Values

The goal of this work was to develop an in silico model that allows predicting segmental-dependent permeability throughout the small intestine (SI). In vivo permeability of 11 model drugs in 3 SI segments (jejunum, mid-SI, ileum) was studied in rats, creating a data set that reflects the conditions throughout the SI. Then, a predictive model was developed, combining physicochemical drug properties influencing the underlying mechanism of passive permeability: Log p, polar surface area, M_W, H-bond count, and Log f_u, with microenvironmental SI conditions. Excellent correlation was evident between the predicted and experimental data (R² = 0.914), with similar predictability in each SI segment. Log p and Log f_u were identified as the major determinants of permeability, with similar contribution. Total H-bond count was also a significant determinant, followed by polar surface area and M_W. Leaving out any of the model parameters decreased its predictability. The model was validated against 5 external drugs, with excellent predictability. Notably, the model was able to predict the segmental-dependent permeability of all drugs showing this trend experimentally. Model predictability was better in the high-permeability versus low-permeability range. Overall, our approach of constructing a straightforward in silico model allowed reliable predictions of segmental-dependent intestinal permeability, providing new insights into relative effects of drug-related factors and gastrointestinal environment on permeability.     

Developing Quantitative In Vitro–In Vivo Correlation for Fenofibrate Immediate-Release Formulations With the Biphasic Dissolution-Partition Test Method

This study is to evaluate 3 fenofibrate (FEN) formulations including Fournier® 200 mg capsule, Lipidil® 145 mg tablet, and a clinical HME 160 mg tablet by an in vitro biphasic method. Key experimental parameters were evaluated including the selection of biorelevant media, the United States Pharmacopeia IV flow rate, and the United States Pharmacopeia paddle speed. Varying the hydrodynamic condition resulted in a significant impact on FEN concentration time profiles in both aqueous and octanol phases for these formulations. In vivo pharmacokinetic profiles of the HME tablet, the Lipidil tablet, and Fournier capsule under the fasting and low-fat fed states are reported. Their corresponding absorption-time profiles were obtained through deconvolution by the Wagner-Nelson method. When fed state simulated intestinal fluid version 2 was used, the partitioned FEN amount–time profiles in octanol from the 3 formulations under an appropriate hydrodynamic condition exhibited a good agreement with their in vivo absorbed amount–time profiles, permitting a quantitative in vitro–in vivo correlation. When fasted state simulated intestinal fluid version 2 was used, partitioned FEN amounts into octanol from these formulations are significantly lower than those from in vivo data. Although no food effect was observed for both HME and Lipidil tablets, the positive food effect of the Fournier capsules significantly overestimated by the biphasic test.     

Contribution of Human Liver and Intestinal Carboxylesterases to the Hydrolysis of Selexipag In Vitro

In liver microsomes, selexipag (NS-304; ACT-293987) mainly undergoes hydrolytic removal of the sulfonamide moiety by carboxylesterase 1 (CES1) to yield the pharmacologically active metabolite MRE-269 (ACT-333679). However, it is not known how much CES in the liver and intestine contributes to the hydrolysis of selexipag or how selexipag is metabolized in the intestine, including by hydrolysis. To obtain a better understanding of selexipag metabolism in humans, we determined the percentage contribution of CES1 and carboxylesterase 2 (CES2) to the hydrolysis of selexipag and 7 of its analogs with different sulfonamide moieties and evaluated its nonhydrolytic metabolism in human liver microsomes and human intestinal microsomes (HIMS). For selexipag, the percentage contributions of CES1 and CES2 in human liver microsomes were 77.0% and 9.99%, respectively, while the percentage contribution of CES2 in HIMS was 100%. In HIMS, the rate of hydrolysis of selexipag was the lowest among the compounds tested, and no difference between the presence and absence of nicotinamide adenine dinucleotide phosphate was noted. We infer from these results that selexipag is likely to be hydrolyzed by CES2 as well as CES1, and only selexipag itself and the MRE-269 produced by hydrolysis in the intestine would be absorbed after oral administration.     

In Vitro–In Vivo Evaluation of an Oral Ghost Drug Delivery Device for the Delivery of Salmon Calcitonin

An orally administered site-specific Oral Ghost Drug Delivery (OGDD) device was developed and evaluated for the administration of salmon calcitonin. In vitro drug release studies have been undertaken using biorelevant media and aspirated gastrointestinal fluid from a large white pig in addition to characterization of a formulated trimethyl chitosan blend formulated and prepared into a loaded mini-pellet system. In vivo drug release analysis in a large white pig model has further been undertaken on the OGDD device and a commercial intramuscular injection to ascertain the release properties of the OGDD device in an animal model in comparison with the currently used treatment option for the administration of salmon calcitonin. Results of this study have detailed the success of the prepared system during both in vitro and in vivo analyses with the OGDD providing a greater control of release of salmon calcitonin when compared to the commercial product.     

In vitro and in vivo effects of cadmium on cholinesterases in Nile tilapia fingerlings: implications for biomonitoring aquatic pollution

Effects of cadmium on in vitro and in vivo cholinesterase (ChE) activities of brain and muscle tissues of Oreochromis niloticus fingerlings were evaluated, considering its potential use in biomonitoring tropical water pollution. Results show that in vitro ChE activities were depressed significantly by millimolar concentration ranges of Cd²⁺. The IC₅₀ values of Cd²⁺ on in vitro ChE activity in brain and muscle tissues were 1.56 and 4.31 mM, respectively. Exposure of fish to environmentally relevant concentrations of Cd²⁺ (5–30 μg l^−l) for 28 days evoked only a transient inhibition (21–34%) of in vivo ChE activities. Prior exposure and co-exposure of fish to 15 μg l⁻¹ of Cd²⁺ enhanced the extent of inhibition of ChE levels induced by the organophosphorous insecticide chlorpyrifos. As high concentrations of cadmium have the potential to depress ChE activities, monitoring of metal levels in water bodies with suspected high levels of metal inputs is necessary to accurately interpret the fish ChE inhibition data in relation to insecticide contaminations.     

In vitro–In Vivo Correlations: Tricks and Traps

In vitro-in vivo correlation (IVIVC) is a biopharmaceutical tool recommended to be used in development of formulation. When validated, it can speed up development of formulation, be used to fix dissolution limits and also as surrogate of in vivo study. However, as do all tools, it presents limitations and traps. The aim of the present paper is to investigate five common traps which could limit either the setting or use of IVIVC (1) using mean or individual values; (2) correction of absolute bioavailability; (3) correction of lag time and time scaling; (4) flip-flop model; and (5) predictability corrections.     

Biotherapeutic Antibody Subunit LC-MS and Peptide Mapping LC-MS Measurements to Study Possible Biotransformation and Critical Quality Attributes In Vivo

Biotransformation monitoring involves tracking drug modification occurring during in-life studies. Critical Quality Attribute monitoring from forced degraded drug material or in-life sample sets can provide an in-depth assessment of product quality for support in early- or late-stage drug development. For Critical Quality Attribute analysis, biotherapeutic monoclonal antibody (mAb) subunit analysis and peptide mapping liquid chromatography–mass spectrometry (LC-MS) approaches are used, although typically from an in vitro setting (e.g., formulation buffer) not involving biological samples or material. Here, samples from a high-dose rat study (in vivo) are subjected to analysis by ligand binding assay, mAb subunit LC-MS, and peptide mapping by LC-MS. Taken together, data from the 3 analytical approaches provide information regarding drug concentration in circulation, biotransformation, and biotherapeutic drug product quality. The concept of a multitier workflow for preclinical or clinical sample sets can be applied to other biotherapeutic mAb products such as bispecific mAbs, fusions proteins, or antibody-drug conjugates.     

Metabolic disorders and cardiovascular consequences of HIV infection and antiretroviral therapy

Metabolic disturbances associated with HIV disease have become an important factor in patient management and have important implications for long-term outcomes, both in regards to mortality and healthcare burden. Recent research has implicated both HIV infection itself and specific antiretroviral therapies in the development of these disorders. This review examines recent findings from research into insulin and glucose dysregulation, serum lipid abnormalities, adipose tissue and derangements in bone metabolism. This review then describes the cardiovascular consequences and management of these metabolic disorders, and summarizes current thinking on the pathogenesis and effects of antiretroviral therapy. Finally, the review raises some questions regarding ongoing challenges and unmet needs in this field of research.     

Incorporation With Dendrimer-Like Biopolymer Leads to Improved Soluble Amount and In Vitro Anticancer Efficacy of Paclitaxel

The aim of this study was to explore the solubilization efficacy of octenylsuccinate hydroxypropyl phytoglycogen (OHPP), an amphiphilic dendrimer-like biopolymer, in improving the soluble amount and efficacy of paclitaxel (PTX), a potent anticancer active pharmaceutical ingredient. PTX was incorporated with OHPP in the form of solid dispersion (PTX-OHPP SD), which was characterized for its PTX crystallinity, interactions between PTX and OHPP, PTX soluble amount and dissolution profile, and in vitro inhibitory efficacy against cancer cell lines. The incorporation with OHPP led to the amorphous state of PTX. FTIR spectra showed potential hydrogen bonding between PTX and OHPP, and hydrophobic interaction could play an important role in the association of PTX molecules with OHPP particulates. The soluble amount of PTX with PTX-OHPP SD was >14,000 times that of PTX alone. During dissolution, over 70% PTX dissolved in 5 min and this soluble amount was maintained for at least 180 min. With the 3 cancer cell lines tested, the PTX of PTX-OHPP SD showed a much lower IC₅₀ than the dimethyl sulfoxide–assisted PTX solutions. Based on the result, the mechanism of using OHPP as a highly potent solubilizer for PTX was discussed.     

Changes of electrocardiogram and hemodynamics in response to dipyridamole: In vivo comparative analyses using anesthetized beagle dogs and microminipigs

Microminipigs are expected as a novel animal model for cardiovascular pharmacological experiments. Since inherent vulnerability of coronary circulation of microminipigs has not been characterized, we performed dipyridamole-stress test to both microminipigs and beagle dogs, and compared the results. Dipyridamole in doses of 0.056 and 0.56 mg/kg were intravenously infused over 10 min (n = 4 for each animal). Dipyridamole decreased the systolic/diastolic blood pressures and double product in dogs as well as in microminipigs; but it did not significantly alter the heart rate or the global balance between the myocardial oxygen demand and supply in either animal. While organic coronary arterial stenosis was not detected in either animal, dogs have well-developed epicardial intracoronary networks unlike microminipigs. Like in humans, dipyridamole did not affect the ST segment of microminipigs, whereas it substantially depressed that in dogs. The results indicate the onset of subendocardial ischemia by dipyridamole in dogs may be partly associated with their well-developed native coronary collateral channels. Microminipigs would be more useful to evaluate the drugs which may affect the coronary circulation in the pre-clinical study than dogs.     

InVitro,Ex Vivo,and In Vivo Evaluation of a Dual pH/Redox Responsive Nanoliposomal Sludge for Transdermal Drug Delivery

A dual pH/redox responsive copper-glyglycine-prednisolone succinate–loaded nanoliposomal (NL) sludge was successfully synthesized and optimized using a Box-Behnken design of experiments. Preformulation design variables indicated that relative ratios of phospholipids, considerably influences NL size, thus altering the degree of drug loading in the formulation. In vitro evaluation further confirmed optimum release kinetics of the NL sludge, corresponding closely to ex vivo permeation studies, demonstrating effective transdermal delivery of prednisone succinate (PS) through a pig skin model, which closely resembles human skin anatomy. The pH/redox stimuli responsiveness of the NL sludge further demonstrated superior properties in vivo using a Sprague-Dawley rat model. The NL sludge displayed the greatest release of PS within 24 h of evaluation, falling within the acceptable therapeutic range of PS dose efficiency. In vivo results further displayed the greatest absorption of PS under inflammatory induced conditions, thus confirming the unique pH/redox responsive properties of the NL sludge. It was thus confirmed that the copper-glyglycine-prednisolone succinate–loaded NL sludge has significant potential for application in chronic inflammatory conditions such as tumor necrosis factor receptor–associated periodic syndrome (TRAPS), designed to release an effective dose of corticosteroid, as a transdermal drug delivery formulation, for effective therapeutic efficacy.     

Bioaccumulation and physiological effects of mercury in Pteris vittata and Nephrolepis exaltata

Anatomical, histochemical and biochemical approaches were used to study mercury uptake and phytotoxicity as well as anti-oxidative responses in two species of ferns [Chinese brake fern (Pteris vittata) and Boston fern (Nephrolepis exaltata)], grown in a hydroponic system. The roots of both cultivars accumulated large amounts of mercury, but exhibited limited mercury translocation to shoots. Mercury exposure led to more pronounced phytotoxicity accompanied by stronger oxidative stress in the shoots of P. vittata than in N. exaltata. N. exaltata established a more effective anti-oxidative system against mercury-induced oxidative stress than did P. vittata. The activity of anti-oxidative enzymes (superoxide dismutase, catalase and glutathione reductase) increased. The reduced ascorbate (ASA) and oxidized ascorbate (DHA) are regulated. Mercury exposure led to an increase in the concentration of glutathione (GSH) in both fern species. The present study suggests that N. exaltata is more tolerant to mercury exposure than P. vittata, which has been also reported to be more tolerant to arsenic exposure. N. exaltata may thus have potential for phytostabilization of soils or phytofiltration of waste water contaminated with mercury.