Dissolution testing of modified release products with biorelevant media: An OrBiTo ring study using the USP apparatus III and IV.

During the OrBiTo project, our knowledge on the gastrointestinal environment has improved substantially and biorelevant media composition have been refined. The aim of this study was to propose optimized biorelevant testing conditions for modified release products, to evaluate the reproducibility of the optimized compendial apparatus III (USP apparatus III) and compendial apparatus IV (USP apparatus IV, open-loop mode) dissolution methods and to evaluate the usefulness of these methods to forecast the direction of food effects, if any, based on the results of two «ring¼ studies and by using two model modified release (MR) products, Ciproxin / Cipro XR and COREG CR. Six OrBiTo partners participated in each of the ring studies. All laboratories were provided with standard protocols, pure drug substance, and dose units. For the USP apparatus III, the dissolution methods applied to Ciproxin / Cipro XR, a monolithic MR product of an active pharmaceutical ingredient (API) with moderate aqueous solubility, were robust with low intra- and inter-laboratory data variability. Data from all partners were in line on a qualitative basis with food effect data in humans. For the USP apparatus IV, the dissolution methods applied to COREG CR, a multiparticulate, pH dependent, MR product of an API with low and pH dependent solubility led to high intra- and inter- laboratory data variability. Data from all partners were in line, on a qualitative basis, with the previously observed food effects in humans.

An in silico approach to determine challenges in the bioavailability of ciprofloxacin, a poorly soluble weak base with borderline solubility and permeability characteristics.

Ciprofloxacin is administered as the hydrochloride salt in immediate release formulations for the treatment of various infectious diseases in different patient populations. Due to its weakly basic properties and poor solubility, the in vivo behaviour of this compound could be influenced by both physicochemical and physiological factors. The first aim of this study was to investigate the behaviour of ciprofloxacin (Ciprobay® 500 mg tablets) in the human gastro-intestinal tract with in vitro dissolution, transfer and two-stage experiments. Ciprobay® IR tablets dissolved completely in FaSSGF-V2, but dissolution was incomplete in FaSSIF-V2 and in an achlorhydric medium (FaSSGF-achlorhydric) and slow precipitation was observed in all three media. Ciprofloxacin did not precipitate in the transfer model but in the two-stage test, a simplified version of the transfer model, some precipitation was detected. In the second part of this study the data obtained in the in vitro transfer experiment were integrated into a Physiologically Based Pharmacokinetic (PBPK) Model. Based on the in vitro results, it was concluded that precipitation of ciprofloxacin would be unlikely in vivo. When precipitation was assumed to be negligible in the PBPK model, good predictions of plasma concentration time profiles provided by Bayer Pharma AG and obtained from the open literature were attained. Parameter Sensitivity Analysis (PSA) was conducted on several parameters which may influence the in vivo behaviour of ciprofloxacin. It was shown that precipitation in the gastro-intestinal tract in humans after a dose of 500 mg is not a determinant of the PK profile. PSA further suggested that ciprofloxacin behaves in vivo as a BCS Class I drug according to the Biopharmaceutics Classification System (BCS), even though on the basis of available solubility and permeability data the compound has been categorised as a BCS II/IV drug. These findings illustrate the importance of coupling in vitro results with in silico PBPK models to better understand the in vivo behaviour of weakly basic drugs. The PBPK model of ciprofloxacin, which was set up for healthy volunteers, was also modified to predict the in vivo behaviour of ciprofloxacin in several different patient populations. It was shown on the basis of these simulations that the plasma concentration time profile may be less influenced by disease state than previously expected.

Advantage of the Dissolution/Permeation System for Estimating Oral Absorption of Drug Candidates in the Drug Discovery Stage.

In order to increase the success rate in the development of oral drugs, an in vitro method, which can accurately estimate human oral absorption of a large variety of compounds from solid formulations, is required in the drug discovery stage. A dissolution/permeation (D/P) system is an in vitro system that simultaneously evaluates dissolution and permeation processes of drugs administered orally. In this study, we have investigated the advantages of a D/P system for use in the provisional estimation of human oral absorption of a drug (absorbed fraction, Fa) by applying it in its solid state. The D/P system mounted with a Madin-Darby canine kidney (MDCK) II cell monolayer was used to simultaneously evaluate the dissolved and the permeated amounts (% of dose) of 48 marketed drugs. Slightly modified, fasted-state simulated intestinal fluid (FaSSIFmod, 8 mL) was used as the apical medium of the D/P system. Each test drug was applied to the apical side of the D/P system as a suspension at one-hundredth of the clinical dose. The apparent permeability coefficient across the MDCK II cell monolayer was estimated in a buffer solution (pH 6.5). The octanol/water distribution coefficient (Log D) was measured at pH 6.5 by a flask shaking method. Transport medium (TM, pH 6.5), a buffer solution removing lecithin and taurocholate from FaSSIFmod, was used to determine the solubility at 24 h after applying drugs. The solubility in TM was used as a free drug concentration in FaSSIFmod. A good correlation was obtained between observed human Fa and the permeated amount in the D/P system. When the sigmoidal curve was obtained by the curve fitting to the data, the determination coefficient was R(2) = 0.79 and the 95% interval of the predicted Fa values was about ±24% for all drugs tested in the present study. For comparison, the permeated amount was calculated by multiplying the permeability of each drug (in vitro Papp) by the solubility in FaSSIFmod. However, the calculated permeated amount showed a lower correlation with the observed human Fa compared to the observed permeated amount in the D/P system. The ratio of the observed permeated amount to the calculated permeated amount was in inverse proportion to the ratio of solubility in FaSSIFmod to solubility in TM. This finding suggests the necessity of determining the free fraction of the dissolved drug in the gastrointestinal (GI) tract when calculating the human Fa. In the case of the D/P system, since applied drugs dissolved in FaSSIFmod first, and then only the free fraction permeated the membrane, an accurate estimation of the human Fa was possible from only the observed permeated amount. This in vitro system is expected to contribute to the selection of better compounds for oral use during the lead- and formulation-optimization processes in the drug discovery stage.

Novel comb-shaped PEG modification enhances the osteoclastic inhibitory effect and bone delivery of osteoprotegerin after intravenous administration in ovariectomized rats.

PURPOSE: Recombinant osteoprotegerin (OPG) has been proven to be useful for treating various bone disorders such as osteoporosis. To improve its in vivo pharmacological effect, OPG was conjugated to novel comb-shaped co-polymers of polyethylene glycol (PEG) allylmethylether and maleamic acid (poly(PEG), 5 kDa). Biodistribution and bioactivity were evaluated. METHODS: OPG was conjugated via lysine to poly(PEG) and to linear PEG (0.5 kDa and 5 kDa). Poly(PEG)-OPG was compared with linear PEG0.5k-OPG and PEG5k-OPG in terms of in vitro and in vivo efficacy and bone distribution. RESULTS: The in vitro receptor binding study showed that poly(PEG)-OPG could be the most bioactive among the three PEG-OPG derivatives. Pharmacokinetic studies in ovariectomized (OVX) rats showed that serum half-life and AUC of poly(PEG)-OPG were comparable with those of linear PEG-OPG derivatives. For in vivo pharmacological effect, poly(PEG)-OPG showed the strongest inhibitory effect on bone resorption activity in OVX rats. Poly(PEG)-OPG demonstrated enhanced bone marrow distribution with higher selectivity than linear PEG5k-OPG. CONCLUSION: Poly(PEG) modification could provide longer residence time in serum and higher bone-marrow specific delivery of OPG, leading to a higher in vivo pharmacological effect.

In vitro evaluation of the potential for drug-induced toxicity based on (35)S-labeled glutathione adduct formation and daily dose.

BACKGROUND: Drug-induced toxicity such as idiosyncratic drug toxicity is believed to be reduced when either reactive metabolite formation or exposure to a drug is minimized. The objective of the present study was therefore to clarify the relationship between the daily doses, the formation rates of reactive metabolite adduct with (35)S-glutathione (RM-GS) and the safety profiles of compounds. RESULTS: The RM-GS formation rates for 113 test compounds were determined by incubation with human liver microsomes, and the test compounds were classified into three categories of safe, warning and withdrawn/black box warning. A total of 23 out of 28 withdrawn/black box warning drugs showed both a RM-GS formation rate of over 1 pmol/30 min/mg protein and a dose of over 100 mg. CONCLUSION: These results suggest that when compounds are plotted in this region, the compounds would have a relatively high idiosyncratic drug toxicity potential.

Distribution of KAI-9803, a novel δ-protein kinase C inhibitor, after intravenous administration to rats.

KAI-9803 is composed of a selective δ-protein kinase C (δPKC) inhibitor peptide derived from the δV1-1 portion of δPKC (termed "cargo peptide"), conjugated reversibly to the cell-penetrating peptide 11-amino acid, arginine-rich sequence of the HIV type 1 transactivator protein (TAT47₋57; termed "carrier peptide") via a disulfide bond. KAI-9803 administration at the end of ischemia has been found to reduce cardiac damage caused by ischemia-reperfusion in a rat model of acute myocardial infarction. In the study presented here, we examined the TAT47₋57-mediated distribution of KAI-9803 in rats after a single intravenous bolus administration (1 mg/kg). ¹4C-KAI-9803 was rapidly delivered to many tissues, including the heart (1.21 µg eq/g tissue), while being quickly cleared from the systemic circulation. The microautoradiography analysis showed that ¹4C-KAI-9803 was effectively delivered into various cells, including cardiac myocytes and cardiac endothelial cells within 1 min after dosing. The tissue distribution of ¹²5I-labeled KAI-9803 was compared to that of ¹²5I-labeled cargo peptide; this comparison demonstrated that the distribution of KAI-9803 to tissues such as the liver, kidney, and heart was facilitated by the reversible conjugation to TAT47₋57. In an in vitro cardiomyocyte study, the extent of ¹²5I-KAI-9803 internalization was greater at 37°C than that at 4°C, whereas the internalization of the ¹²5I-cargo peptide at 37°C was not observed, indicating that the uptake of ¹²5I-KAI-9803 into the cardiomyocytes was mediated by the TAT47₋57 carrier. Our studies demonstrated that after a single intravenous administration, KAI-9803 can be delivered into the target cells in the liver, kidney, and heart by a TAT47₋57-mediated mechanism.

Combination of GSH trapping and time-dependent inhibition assays as a predictive method of drugs generating highly reactive metabolites.

Covalent binding (CB) of reactive metabolites (RMs) is potentially involved in severe adverse drug reactions. Because the CB assay is of low throughput and costly, a qualitative trapping assay using agents such as [(35)S]GSH is often performed in the early stages of drug discovery. However, trapping methods alone cannot replace the CB assay. We hypothesized that the time-dependent inhibition (TDI) assay might be complementary to the [(35)S]GSH trapping assay in detecting RMs. We performed CB assays, [(35)S]GSH trapping assays, and TDI assays for 42 structurally diverse compounds. First, we showed that the [(35)S]GSH trapping assay alone does not correlate with the extent of CB. Four compounds that the [(35)S]GSH trapping assay failed to detect but that showed high extent of CB were inactivators of the enzyme in the TDI assay. There was a tendency for compounds judged as positive in the TDI assay to show a high degree of CB irrespective of the result of the [(35)S]GSH trapping assay. Finally, to combine parameters from the two assays, we introduced intrinsic clearance to describe the formation of RMs (CL(int, RMs)). The Spearman rank correlation coefficient between the extent of CB and CL(int, RMs) was 0.77 (p < 0.0001), which was better than that for the formation rates of [(35)S]GSH adducts. Therefore, we demonstrated that a combination of the [(35)S]GSH trapping and TDI assays is an effective method for detecting compounds potentially capable of generating highly reactive metabolites in the early stages of drug discovery.

Use of an intravenous microdose of 14C-labeled drug and accelerator mass spectrometry to measure absolute oral bioavailability in dogs; cross-comparison of assay methods by accelerator mass spectrometry and liquid chromatography-tandem mass spectrometry.

A technique utilizing simultaneous intravenous microdosing of (14)C-labeled drug with oral dosing of non-labeled drug for measurement of absolute bioavailability was evaluated using R-142086 in male dogs. Plasma concentrations of R-142086 were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and those of (14)C-R-142086 were measured by accelerator mass spectrometry (AMS). The absence of metabolites in the plasma and urine was confirmed by a single radioactive peak of the parent compound in the chromatogram after intravenous microdosing of (14)C-R-142086 (1.5 microg/kg). Although plasma concentrations of R-142086 determined by LC-MS/MS were approximately 20% higher than those of (14)C-R-142086 as determined by AMS, there was excellent correlation (r=0.994) between both concentrations after intravenous dosing of (14)C-R-142086 (0.3 mg/kg). The oral bioavailability of R-142086 at 1 mg/kg obtained by simultaneous intravenous microdosing of (14)C-R-142086 was 16.1%, this being slightly higher than the value (12.5%) obtained by separate intravenous dosing of R-142086 (0.3 mg/kg). In conclusion, on utilizing simultaneous intravenous microdosing of (14)C-labeled drug in conjunction with AMS analysis, absolute bioavailability could be approximately measured in dogs, but without total accuracy. Bioavailability in humans may possibly be approximately measured at an earlier stage and at a lower cost.

Appearance of double peaks in plasma concentration-time profile after oral administration depends on gastric emptying profile and weight function.

PURPOSE: Mechanism for double-peak occurrence in plasma concentration profile after oral administration of drugs is controversial, although irregular gastric emptying would be an important factor. The objective of this study was to assess the effect of gastric emptying and a weight function, i.e. pharmacokinetics after reaching the systemic circulation, on the double-peak appearance in plasma concentration profiles. MATERIALS AND METHODS: Alprazolam, which generates irregular gastric emptying, was orally co-administered with theophylline to rats, and the plasma concentration profiles or absorption rates were compared between the two drugs. Both drugs are highly absorbable, but alprazolam is rapidly eliminated from plasma, while the elimination of theophylline is very slow. RESULTS: Oral administration of alprazolam generated the irregular gastric emptying profiles, resulting in multiple peaks in the absorption rate profiles of both drugs. The double peaks in the absorption rate profiles led to the double peaks in plasma concentration profiles for alprazolam, but not necessarily for theophylline. Simulation study clearly indicated that the slower elimination from plasma made the first peak less recognizable. CONCLUSIONS: The irregular gastric emptying could be a main reason for the double peaks in plasma concentration profiles. However, the frequency of double-peak occurrence depends on the weight function, particularly the elimination rate, of each drug.