The Distribution Kinetics of Topical 14C‐Sulfur Mustard in Rabbit Ocular Tissues and the Effect of Acetylcysteine

Sulfur mustard (2,2‐dichlorodiethyl sulfide; HD), is a potent alkylating agent which in liquid or vapor form is capable of causing severe injuries to skin and respiratory tract, and was shown to cause short‐ and long‐term ocular injuries. N‐Acetylcysteine (NAC) may act as a mucolytic agent, changing the “wetting” and scavenging properties of the cornea and thus the adhesion of HD. Moreover, NAC is a scavenger of HD, an antioxidant and a glutathione precursor, which was shown to reduce HD toxicity in various systems. The ocular distribution of ¹⁴C, after topical application of liquid ¹⁴C‐sulfur mustard (¹⁴C‐HD) to the rabbit cornea, and the role of NAC in reducing HD retention and toxicity are presented in this study. Groups of rabbits were exposed to 0.4 µL of liquid ¹⁴C‐HD, placed at the center of the cornea, with or without NAC treatment. Fifty µL NAC (10% aqueous solution) was topically applied, 10 minutes before and 10 minutes after HD exposure. Three time points were evaluated: 1, 6, and 24 hr after HD exposure, six rabbits per time point. Evaluation consisted of clinical observation, measurement of biochemical parameters in aqueous humor (AQ), counting radioactivity concentration in ocular tissues, and histology of corneal sections. One hour after corneal exposure to liquid ¹⁴C‐HD, approximately 2% of total applied radioactivity was recovered. The highest ¹⁴C concentration was found in the cornea, followed by the tarsal section of eyelid, aqueous humor, nictitating membrane, and the frontal sclera (including conjunctiva). The rate of radioactivity decrease varied from one ocular tissue to the other, the highest rate was found in aqueous and vitreous humors, also in accordance with their higher turnover rates. The NAC treatment reduced the radioactivity in most ocular tissues. The HD exposure caused typical clinical and histological signs of HD intoxication, and increased the aqueous protein and prostaglandin (PGE) content. The NAC treatment lowered eyelid edema but had no effect on AQ protein or PGE content; however, there was some aggravating effect of the NAC treatment on corneal epithelial cells, seen at 1 and 6 hr after exposure.     

Protective Effect of Gabapentin on N-methyl-D-aspartate–Induced Excitotoxicity in Rat Hippocampal CA1 Neurons

Gabapentin was developed as an anticonvulsant, but has also been used to alleviate hyperalgesia in neuropathic pain. In this study, the protective effect of gabapentin against N-methyl-D-aspartate (NMDA)-induced excitotoxicity in rat hippocampal CA1 neurons was investigated. Pre-treatment with gabapentin reduced the degree of neuronal damage induced by NMDA exposure in cultured hippocampal slices. Patch-clamp studies revealed that gabapentin significantly inhibited the NMDA receptor–activated ion current in dissociated hippocampal CA1 neurons, resulting in suppression of glutamate-induced neuronal injury. These results show that gabapentin may exert protective effects against glutamate-induced neuronal injury at least in part by inhibiting the NMDA receptor–activated ion current.     

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.     

In Vivo Antitumor Activity of a Novel Orally Bioavailable Ifosfamide Nanostructured Lipid Carrier Against Dalton’s Ascitic Lymphoma

Purpose

Ifosfamide is a widely used antineoplastic drug belonging to the alkylating agents. It is difficult to develop a conventional oral dosage form as it degrades in acidic media, with its rate of degradation depending on the pH of the solution. Thereby, ifosfamide is available either as a powder which is reconstituted with diluents for injection or as injectable solution. The present work has attempted to improve oral bioavailability of ifosfamide by incorporating in nanostructured lipid carriers (NLCs). The purpose of this research was to study whether the oral bioavailability of ifosfamide could be improved by administering ifosfamide-loaded NLCs.

Method

Ifosfamide NLCs were developed using chitosan cross-linking with sodium alginate by solvent diffusion technique. Bioavailability studies were conducted in albino rats after oral administration of ifosfamide suspension and NLC. In vivo antitumor activity of ifosfamide nanostructured lipid carrier against Dalton’s ascitic lymphoma (DAL) was also investigated.

Results

The results showed that the relative bioavailability of ifosfamide from NLC was increased by sixfold higher than that of ifosfamide suspension. Administration of ifosfamide NLC significantly decreased the tumor volume and viable cell count. Nonviable cell count was significantly higher in ifosfamide NLC-treated animals when compared with DAL control animals. Further, the median survival time was increased to 35 % on administration of ifosfamide NLC. Ifosfamide NLC significantly decreased biochemical parameters, increased the RBC count and Hb content, and reduced the WBC count as well.

Conclusions

The results of this study show no significant difference in the antitumor efficacy of ifosfamide when administered as injection and nanoformulation. The obtained results are indicative of NLCs as potential carriers for improving the oral bioavailability and acid instability problems. The new nanoformulation would be interchangeable with the standard formulation.     

In Vitro–In Vivo Correlations of Scalable PLGA-Risperidone Implants for the Treatment of Schizophrenia

Purpose  

Nonadherence to antipsychotic medications is a major obstacle preventing optimal outcomes for patients with schizophrenia. Extended release systems exist in the form of depot injections, but these formulations exhibit several disadvantages. To address these concerns, we previously demonstrated proof of concept for an antipsychotic implant containing risperidone and the biodegradable polymer poly(lactic-co-glycolic) acid (PLGA).     

Correction: In Vitro—In Vivo Extrapolation of Hepatic Clearance in Preclinical Species

Pharmaceutical Research -     

In Vitro–In Vivo Extrapolation Method to Predict Human Renal Clearance of Drugs

Renal clearance is a key determinant of the elimination of drugs. To date, only few in vitro–in vivo extrapolation (IVIVE) approaches have been described to predict the renal organ clearance as the net result of glomerular filtration, tubular secretion, and tubular reabsorption. In this study, we measured in LLC-PK₁ cells the transport of 20 compounds that cover all four classes of the Biopharmaceutical Drug Disposition System. These data were incorporated into a novel kidney model to predict all renal clearance processes in human. We showed that filtration and secretion were main contributors to the renal organ clearance for all compounds, whereas reabsorption was predominant for compounds assigned to classes 1 and 2. Our results suggest that anionic drugs were not significantly secreted in LLC-PK₁ cells, resulting in under-predicted clearances. When all study compounds were included a high overall correlation between the reported and predicted renal organ clearances was obtained (R² = 0.83). The prediction accuracy in terms of percentage within twofold and threefold error was 70% and 95%, respectively. In conclusion, our novel IVIVE method allowed to predict the human renal organ clearance and the contribution of each underlying process.     

In Vitro–In Vivo Correlation on Delivery of Drug Candidates to Articular Cartilage

PURPOSE: In the treatment of osteoarthritis (OA), some of the therapeutic approaches require delivery of drug(s) to the diseased cartilage. Presence of adequate drug levels in the cartilage is one of the important criteria in selection and ranking of lead compounds. The purpose of this study was to investigate the correlation in cartilage compound levels between in vitro experiments and in vivo animal studies. MATERIALS AND METHODS: Bovine cartilage samples were incubated with test compounds of various concentrations in a culture medium, in the absence or presence of 25 mg/ml of serum albumin which served as an artificial synovial fluid (SF). The test compounds were also dosed to rabbits, the animal model used for efficacy studies, over a six-week treatment period. Test article concentrations in plasma, SF, and cartilage were determined by LC/MS/MS analysis. RESULTS AND CONCLUSIONS: A correlation in cartilage drug concentration was observed between in vitro and in vivo studies. Plasma protein binding and the test article's affinity to cartilage were the major determining factors for drug delivery to cartilage in vivo.     

Pharmacokinetic-Pharmacodynamic Analysis of the EEG Effect of Alfentanil in Rats Followingβ-Funaltrexamine-Induced μOpioid Receptor “Knockdown”In Vivo

Purpose. The objective of this investigation was to determine theinfluence of pre-treatment with the irreversible -opioid receptorantagonist -funaltrexamine (-FNA) on thepharmacokinetic-pharmacodynamic (PK/PD) relationship of alfentanil in rats. Methods. The PK/PD correlation of alfentanil (2 mg.kg^–1intravenously in 20 min) was determined in chronically instrumented ratsusing amplitudes in the 0.5–4.5 Hz frequency band of the EEG aspharmacodynamic endpoint. -FNA was administered intravenously(10 mg.kg^–1) either 35 min or 24 h prior to the PK/PD experiments. Results. Pre-treatment with -FNA had no influence on thepharmacokinetics of alfentanil. The in vivo concentration-EEG effectrelationships, however, were steeper and shifted towards higher concentrationswith no difference between the 35-min and the 24-h pre-treatmentgroups. Analysis of the data on basis of the operational model agonismrevealed that the observed changes could be explained by a 70–80%reduction in alfentanil efficacy in -FNA pre-treated rats. This isconsistent with results from an in vitro receptor bioassay showing a40–60% reduction in the number of specific -opioid binding sites inthe brain. Conclusions. This investigation confirms the validity of a previouslypostulated mechanism-based PK/PD model for the effect of syntheticopiates in rats.     

Inhibition of Agitation‐Induced Aggregation of an IgG‐Antibody by Hydroxypropyl‐β‐Cyclodextrin

In order to provide an alternative to nonionic surfactants as excipients for protein formulations, cyclodextrin‐derivatives (CDs) were examined for their potential to inhibit agitation‐induced aggregation of an IgG in aqueous solution. Loss of monomeric protein and protein aggregation were monitored throughout the agitation experiments by size exclusion chromatography. Hydroxypropyl‐β‐cyclodextrin (HPβCD) completely suppressed IgG‐aggregation at a remarkably low concentration (2.5 mM) and in contrast to other CDs it also did not negatively affect IgG‐stability during storage in nonagitated solution. Further agitation experiments demonstrated the superiority of HPβCD to other common excipients in protein formulation, such as sugars and sugar alcohols or polysorbate 80 in low concentrations. Spectroscopic (fluorescence spectroscopy and Fourier transform infrared spectroscopy), thermodynamic (microcalorimetry), and physical investigations (surface tension measurements) were carried out to elucidate the mechanism of stabilization of the IgG. In contrast to other studies with HPβCD, protein stabilization could not be attributed to direct interaction between hydrophobic amino acids on the IgG and this excipient. Competition with the protein for the air–water interface appears to be the dominating mechanism of stabilization. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1193–1206, 2010     

Comparative In Silico–In Vivo Evaluation of ASGP-R Ligands for Hepatic Targeting of Curcumin Gantrez Nanoparticles

The present study aims to design hepatic targeted curcumin (CUR) nanoparticles using Gantrez (GZ) as a polymer. Three carbohydrate-based hepatocyte asialoglycoprotein receptor (ASGP-R) ligands were selected for the study, namely kappa carrageenan (KC), arabinogalactan (AG), and pullulan (P). AG and KC are galactose based while P is a glucose-based polymer. CUR-GZ nanoparticles were prepared by nanoprecipitation and anchored with the ligands by nonspecific adsorption onto preformed nanoparticles. The change in zeta potential values confirmed adsorption of the ligands. Docking simulation was evaluated as a tool to predict ligand ASGP-R interactions, using grid-based ligand docking with energies (Glide). Monomers and dimers were used as representative units of polymer for docking analysis. The binding of ASGP-R was validated using d-galactose as monomer. The interaction of the ligands with the receptor was evaluated based on Glide scores and E_model values, both for monomers and dimers. The data of the docking study based on Glide scores and E_model values suggested higher affinity of AG and P to the ASGP-R, compared to KC. At 1 h, following intravenous administration of the nanoparticles to rats, the in vivo hepatic accumulation in the order CUR-GZAG > CUR-GZKC > CUR-GZP correlated with the docking data based on Glide scores. However, at the end of 6 h, pullulan exhibited maximum hepatic accumulation and arabinogalactan minimum accumulation (p < 0.05). Nevertheless, as predicted by docking analysis, arabinogalactan and pullulan revealed maximum hepatic accumulation. Docking analysis using dimers as representative stereochemical units of polymers provides a good indication of ligand receptor affinity. Docking analysis provides a useful tool for the preliminary screening of ligands for hepatic targeting.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-013-9474-6) contains supplementary material, which is available to authorized users.Key words: arabinogalactan, docking, hepatic targeting, kappa carrageenan, pullulan     

Preparation and In Vivo. Antitumor Activity of κ-Carrageenan Oligosaccharides

Abstract

Carrageenan oligosaccharides were produced from the final products of commercial extractions of gelling carrageenan and thickening carrageenan, which were specifically depolymerized by κ-carrageenase extracted from the marine bacterium Cytophaga. MCA-2. The weight-average molecular weights of the two oligosaccharides produced were 681 and 798 Da. The sulfate contents were assayed as 17.2% and 21.8%. Antitumor activities of the oligosaccharides were tested on Sarcoma 180 tumor transplanted in mice. The carrageenan oligosaccharide from the thickening carrageenan product showed much higher tumor inhibition (70.8%) and catalase activity at a dosage of 100 mg kg^?1 compared with the control group. The gelling and thickening carrageenan oligosaccarides could also significantly increase the weight of immune organs such as the thymus, which suggests that the antitumor mechanism of the carrageenan oligosaccharides may be initiated via organ-mediated defense reactions.     

Applications of In Vitro–In Vivo Correlations in Generic Drug Development: Case Studies

In vitro–in vivo correlation (IVIVC) is a predictive mathematical model describing the relationship between an in vitro property and a relevant in vivo response. The main objective of an IVIVC is to serve as a surrogate for human bioequivalence (BE) studies, which may reduce the number of BE studies performed during the initial approval process as well as with certain scale-up and postapproval changes. The US Food and Drug Administration (FDA) published a regulatory guidance related to development, evaluation, and applications of IVIVC for extended-release (ER) oral dosage forms in September 1997. Despite the publication of this guidance, the deficiencies related to IVIVC are still identified by the Division of Bioequivalence in the process of Abbreviated New Drug Application (ANDA) review. Thus, the main objective of this article is to present the most commonly occurring deficiencies associated with IVIVCs via selected case studies from the ANDAs for oral ER drug products only. We searched internal FDA databases from January 1996 to December 2014 to identify the ANDAs for proposed generic oral ER drug products containing IVIVC. Only 14 ANDA submissions had IVIVC data, and most were not acceptable. Only one ANDA submission included adequate information related to IVIVC data enabling the completion of BE review within first review cycle. It is hoped that awareness of the deficiencies presented in our article would help the generic drug applicants to submit complete and appropriate information related to IVIVC data, ultimately, resulting in a more timely approval of ANDAs.KEY WORDS: bioequivalence, extended-release drug products, generics, IVIVC, SUPAC     

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.     

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.     

Azithromycin Hydrates—Implications of Processing‐Induced Phase Transformations

According to label claims, in commercial solid dosage forms, azithromycin (AZI) exists either as a monohydrate (MH) or as a dihydrate (DH). Although these two forms are known to be relatively stable in the solid state, AZI sesquihydrate (SH) was observed in a drug product. This was believed to be a consequence of a processing‐induced phase transformation. Our goal was to prepare and characterize AZI SH and map its solid‐state transition pathways with other AZI phases. When dehydrated at temperatures <80°C, DH yielded an isomorphic dehydrate, whereas dehydration at ≥80°C yielded SH. Heating SH to 100°C or holding at 0% RH at room temperature, yielded an amorphous product through an intermediate isomorphic dehydrate, isostructural to SH. On the other hand, dehydration of MH (at ≥60°C) resulted in amorphization with no intermediate crystalline anhydrate. Diagnostic XRD peaks of AH, MH, SH, and DH enabled their unambiguous identification. However, the presence of crystalline excipients hindered active pharmaceutical ingredient characterization in drug product. Pattern subtraction method was used to selectively remove the contribution of the crystalline excipients to the overall diffraction pattern, thereby facilitating the physical characterization of AZI in the drug product. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3095–3106, 2014     

In Vitro and in Vivo Transport of Zidovudine (AZT) Across the Blood–Brain Barrier and the Effect of Transport Inhibitors

The transport of the antiviral nucleoside analogue zidovudine (3-azido-3-deoxythymidine; AZT) into the central nervous system (CNS) was characterized in vitro and in vivo. The in vitro model consisted of primary cultures of isolated bovine capillary endothelial cells. The transport rate of AZT across the monolayer, expressed as endothelial permeability P, was determined following luminal and abluminal administration. P did not differ between the two administration sites (luminal, 1.65 ± 0.44 cm/min/10³; abluminal, 1.63 ± 0.28 cm/min/10³). The transport of AZT across the endothelial cell monolayer was found to be concentration independent in the range between 0.4 and 50 µg/mL. AZT transport was not affected by pre-treatment of the cells with either metabolic inhibitors (DODG and DODG/NaN₃) or probenecid. This suggests that AZT passes the monolayer mainly by passive diffusion. The in vivo transport of AZT across the blood–brain barrier and the blood–CSF barrier was studied in male Wistar rats after coadministration of potential inhibitors of active transport of AZT: probenecid (organic anion transport) and thymidine (nucleoside transport). Intracerebroventricular and intravenous coadministration of probenecid caused a significant (P < 0.001) increase in the CSF/plasma concentration ratio compared to the control phase, indicating that the organic anion carrier is involved in AZT transport from CSF to blood. Since there was no effect of probenecid on the transport of AZT in vitro, it is suggested that this carrier is located at the choroid plexus. Coadministration of thymidine did not affect the CSF/plasma concentration ratio, suggesting that a nucleoside carrier system is not involved in AZT transport into or out of the CNS.     

The Influence of In Vivo Metabolic Modifications on ADMET Properties of Green Tea Catechins–In Silico Analysis

The health effects of green tea are associated with catechins: (?)-epigallocatechin-3-O-gallate (EGCG), (?)-epigallocatechin, (?)-epicatechin-3-O-gallate, and (?)-epicatechin. An understanding of compound absorption, distribution, metabolism, excretion, and toxicity characteristics is essential for explaining its biological activities. Herein, absorption, distribution, metabolism, excretion, and toxicity properties of in vivo detected metabolites of green tea catechins (GTCs) have been analyzed in silico. The influence of metabolic transformations on absorption, distribution, metabolism, and excretion profiles of GTCs corresponds to the effects of size, charge, and lipophilicity, as already observed for other small molecules. Mutagenic, carcinogenic, or liver toxic effects were predicted only for a few metabolites. Similar to galloylated GTCs EGCG and (--)-epicatechin-3-O-gallate, the sulfo-conjugates were predicted to bind at the warfarin binding site. The low free plasma concentration of these derivatives may be consequential to their serum albumin binding. The activity cliff detected for methylated conjugates of EGCG indicates that GTCs' pro-oxidative activity in bound state comes primarily from free hydroxyl groups of the pyrogallol ring B.     

Influence of Drug Property and Product Design on In Vitro–In Vivo Correlation of Complex Modified-Release Dosage Forms

The present study examines how drug's inherent properties and product design influence the evaluation and applications of in vitro–in vivo correlation (IVIVC) for modified-release (MR) dosage forms consisting of extended-release (ER) and immediate-release (IR) components with bimodal drug release. Three analgesic drugs were used as model compounds, and simulations of in vivo pharmacokinetic profiles were conducted using different release rates of the ER component and various IR percentages. Plasma concentration–time profiles exhibiting a wide range of t_max and maximum observed plasma concentration (C_max) were obtained from superposition of the simulated IR and ER profiles based on a linear IVIVC. It was found that depending on the drug and dosage form design, direct use of the superposed IR and ER data for IVIVC modeling and prediction may (1) be acceptable within errors, (2) become unreliable and less meaningful because of the confounding effect from the non-negligible IR contribution to C_max, or (3) be meaningless because of the insensitivity of C_max to release rate change of the ER component. Therefore, understanding the drug, design and drug release characteristics of the product is essential for assessing the validity, accuracy, and reliability of IVIVC of complex MR products obtained via directly modeling of in vivo data.