In vitro-in vivo correlation and bioavailability studies of captopril from novel controlled release donut shaped tablet

A controlled release formulation of captopril which was coated and fabricated into a donut shaped tablet formulation, was investigated in rabbit for pharmacokinetic and in vitro-in vivo correlation studies. Coated donut shaped tablets were prepared and in vitro release was studied in simulated gastric fluid at three different RPMs. New Zealand albino male rabbits have been used as animal model for in vivo study. A sensitive and simple HPLC method was developed for the determination of captopril content in rabbit plasma. In vitro release studies showed that release patterns followed zero order for around 4 h. Single oral administration of coated donut shaped tablets in rabbit illustrated retained availability of captopril to the injected drug. Captopril content could pursue the same release pattern over the same time course in in vivo study. The in vivo-in vitro correlation coefficients obtained from point-to-point analysis were greater than 99% between concentrations at certain time points obtained from release study in simulated gastric fluid at different RPMs and HPLC analysis of rabbit's plasma. From the in vitro-in vivo correlation prediction it was evident that the coated donut shaped tablet is a good device for controlled delivery of captopril.     

Corticosteroid solubility and lipid polarity control release from solid lipid nanoparticles

Solid lipid nanoparticles (SLN) show promise as a drug delivery system for skin administration. The solid state of the lipid particle enables efficient drug encapsulation and controlled drug release. The present study addresses the influence of lipid composition and drug substance lipid solubility on the in vitro release profile of corticosteroids from SLN for topical administration. Firstly, the effect of lipid composition on the lipid solubility and in vitro release of betamethasone-17-valerate (BMV) was determined by varying the lipid monoglyceride content and the chain length of the fatty acid moiety. Secondly, the effect of drug substance physicochemical properties was determined by studying five different corticosteroid derivatives with different lipophilicity. A high concentration of monoglyceride in SLN increased the amount of BMV released. The corticosteroid release rate depended on the drug substance lipophilicity and it was clear that the release profiles depended on drug partitioning to the aqueous phase as indicated by zero order kinetics. The results emphasize that the corticosteroid solubility in the lipid phase greatly influence drug distribution in the lipid particles and release properties. Thus knowledge of drug substance solubility and lipid polarity contributes to optimize SLN release properties.     

Feasibility, stability and release performance of a time-dependent insulin delivery system intended for oral colon release

The aim of the present work was to evaluate the viability of a time-dependent delivery platform (Chronotopic^TM) in preparing an insulin-based system intended for oral colon delivery. The main objectives were to assess the influence of the manufacturing process and storage conditions on the protein stability. Insulin-loaded cores were manufactured by direct compression and were subsequently coated with hydroxypropyl methylcellulose (HPMC) in a top-spray fluid bed up to increasing weight gains, namely 20%, 60% and 100%. In order to evaluate the impact the operating conditions may have on the protein integrity, insulin and its main degradation products (A21-desamido insulin - A21, Other Insulin-Related Compounds - OIRCs, and High-Molecular Weight Proteins - HMWPs) were assayed on samples collected after each process step by chromatographic methods. Furthermore, long-term (4 °C) and accelerated (25 °C-60% RH) stability studies were carried out on tablet cores and coated systems by assessing insulin, A21, OIRC and HMWP percentages throughout a one-year storage period. In addition, the in vitro release behaviour was investigated during the same study period. The overall results indicated that the manufacturing process is not detrimental for insulin integrity and that 4 °C storage temperature alters neither the protein content nor the release performances of the device.It was therefore concluded that insulin-containing systems intended for oral colon delivery can be obtained by the Chronotopic^TM technology.     

Physicochemical Characterization and Cytotoxicity Screening of a Novel Colloidal Nanogold-Based Phenytoin Conjugate

A novel, colloidal nanogold-based drug delivery system for phenytoin, a well-known anti-epileptic drug with an enhanced efflux via P-glycoprotein, has been proposed in this study. The vital physical properties that would aid in predicting the biological interaction of this system were profiled using various techniques such as UV-Vis, DLS, and TEM in corroboration with theoretical calculations. It was significant to note that the binding of phenytoin to colloidal nanogold was strongly pH-dependent with the optimum at pH 5.5 and a consistently reproducible spectral shift. Analysis of the conjugate by FTIR revealed that the imide functional group of phenytoin mediated a dative coordinate bond to colloidal nanogold at the optimum pH. The amount of the drug bound to the gold was quantified to be 85.8±2.5% (w/v) by HPLC. Hypothetically, the surface charge of the conjugate could possibly imply charge-mediated uptake across the cell membrane. Further, the novel conjugate was screened for its cytotoxicity in two cell lines and the dosage range was identified. Subsequent development, thorough evaluations in suitable model systems, and the potential for bioimaging to track the payload would validate our hypothesis on the conjugate for better intracellular retention at the site of action, and thereby achieve the targeted delivery.     

Diisocyanate mediated polyether modified gelatin drug carrier for controlled release

Gelatin is an extensively studied biopolymer hydrogel drug carrier due to its biocompatibility, biodegradability and non-toxicity of its biodegraded products formed in vivo. But with the pristine gelatin it is difficult to achieve a controlled and desirable drug release characteristics due to its structural and thermal lability and high solubility in aqueous biofluids. Hence it is necessary to modify its solubility and structural stability in biofluids to achieve controlled release features with improved drug efficacy and broader carrier applications. In the present explorations an effort is made in this direction by cross linking gelatin to different extents using hitherto not studied isocyanate terminated poly(ether) as a macrocrosslinker prepared from poly(ethylene glycol) and isophorone diisocyanate in dimethyl sulfoxide. The crosslinked samples were analyzed for structure by Fourier transform-infrared spectroscopy, thermal behavior through thermogravimetric analysis and differential scanning calorimetry. The cross linked gelatins were biodegradable, insoluble and swellable in biofluids. They were evaluated as a carrier for in vitro drug delivery taking theophylline as a model drug used in asthma therapy. The crosslinking of gelatin decreased the drug release rate by 10–20% depending upon the extent of crosslinking. The modeled drug release characteristics revealed an anomalous transport mechanism. The release rates for ampicillin sodium, 5-fluorouracil and theophylline drugs in a typical crosslinked gelatin carrier were found to depend on the solubility and hydrophobicity of the drugs, and the pH of the fluid. The observed results indicated that this material can prove its mettle as a viable carrier matrix in drug delivery applications.     

Composite microparticles with in vivo reduction of the burst release effect

The aim of this study was to develop microparticles containing nanoparticles (composite microparticles) for prolonged drug delivery with reduced burst effect in vitro and in vivo. Such composite microparticles were prepared with hydrophobic and biodegradable polymers [poly(ε-caprolactone), poly(lactic-co-glycolic) acid]. Ibuprofen was chosen as the model drug, and microparticles were prepared by the extraction technique with ethyl acetate as the solvent. Nanoparticles and microparticles and an ibuprofen solution (Pedea^®) were administered subcutaneously at the dose of 1 mg of ibuprofen per kg to overnight-fasted rats (male Wistar). Composite microparticles showed prolonged ibuprofen release and less burst effect when compared to simple microparticles (without nanoparticles inside) or nanoparticles both in vitro (PBS buffer) and in vivo. Moreover, ibuprofen was still detected in the plasma after 96 h with composite microparticles. Consequently, it has been demonstrated that composite microparticles were able to reduce burst release and prolong the release of ibuprofen for a long period of time.     

Sodium alginate based mucoadhesive system for gatifloxacin and its in vitro antibacterial activity

The objective of this study was to formulate sodium alginate based ophthalmic mucoadhesive system of gatifloxacin and its in vitro antibacterial potential on pathogenic microorganisms, Staphylococcus aureus and Escherichia coli. Sodium carboxymethylcellulose (NaCMC) was added to the formulations to enhance the gel bioadhesion properties. The prepared formulations were evaluated for their in vitro drug release, gelation behaviour, rheological behavior, and mucoadhesion force. All formulations in non-physiological and physiological condition showed pseudo plastic behavior. Increase in the concentration of sodium alginate and sodium CMC enhanced the mucoadhesive force significantly. In vitro release of gatifloxacin from the system in simulated tear fluid (STF, pH a 7.4), was influenced significantly by the properties and concentration of sodium alginate, NaCMC. Significant reduction in total bacterial count was observed between control and treatment groups with both the test organisms.     

Mechanistic studies on release of large and small molecules from biodegradable SiO2

Both small and large biologically active agents were encapsulated into biodegradable sol-gel derived SiO2. Both fast and slowly-eroding SiO2 matrices were prepared. Propranolol represented a small molecule and a model protein, BSA (bovine serum albumin) the larger one. The release mechanisms were studied using two different dissolution media representing extreme cases with respect to the matrix erosion, free dissolution of the SiO2 matrix in sink conditions and a dissolution medium saturated with respect to the matrix. The utilisation of the two different dissolution media as such provided information on the general release mechanisms and power law-based mathematical models supported the propranolol release results. A modified power law is suggested, where both the initially released amount and time are included. BSA was not released without matrix erosion and propranolol release was mainly diffusion-controlled, although the matrix dissolution was needed for R=3 monoliths due to closed pores. It is also shown that for R=30 microparticles propranolol release was partly matrix erosion controlled.     

Cd-induced apoptosis was mediated by the release of Ca from intracellular Ca storage

Previous studies found that cadmium (Cd) could induce apoptosis via interfering with the intracellular calcium (Ca) ions homeostasis. But the detailed mechanisms remain poorly understood. In the present study two cell lines (normal human liver cell HL-7702, and tumor cell Raji cell) were exposed to Cd along or co-incubated with ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and 1,2-bis (2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), respectively. After the incubation, the apoptosis and intracellular Ca²⁺ ([Ca²⁺]_i) were measured. Excessive apoptosis was observed both in HL-7702 and Raji cells treated with Cd. Significant elevation of [Ca²⁺]_i was also detected in the cells with higher levels of apoptosis. EGTA (the extracellular Ca²⁺ chelator) decreased Cd-elicited [Ca²⁺]_i (22% in HL-7702 and 41% in Raji cells; p < 0.05) significantly except for apoptosis. However, BAMTA-AM (the [Ca²⁺]_i chelator) attenuated the Cd-elevated [Ca²⁺]_i (78% in HL-7702 and 59% in Raji cells; p < 0.05) and inhibited Cd-induced apoptosis significantly (p < 0.05). These results suggest that (1) Ca²⁺ was primarily generated intracellularly and only a small portion was generated extracellularly; (2) Cd-induced apoptosis was mediated by the release of Ca²⁺ from intracellular Ca storage but not an influx of extracellular Ca²⁺.     

Starch-based coatings for colon-specific delivery. Part II: Physicochemical properties and in vitro drug release from high amylose maize starch films

This work reports an investigation into free-film properties of a high amylose maize starch-based film coating that has been used in the preparation of formulations for drug delivery to the colon (WO 2008/012573 A1) and relates these properties to in vitro drug release from pellets.Maize starch/ethylcellulose free films were prepared and characterised by scanning electron microscopy (SEM), light microscopy, modulated differential scanning calorimetry (mDSC), Fourier-transform infrared (FT-IR), X-ray and % swelling in aqueous fluids with pH conditions similar to the stomach and small intestine. 5-ASA release from film-coated pellets was tested in enzyme free simulated gastric fluid and phosphate buffer pH 7.2. Selected formulations were further assessed in simulated gastric and intestinal fluids containing pepsin and pancreatin, respectively.The free films prepared were smooth and homogeneous in their appearance. The two polymers are immiscible, and neither mDSC nor FT-IR could detect interactions between them. Films made from high amylose starches were found to have a considerably lower swelling ability than high amylopectin-based films, and they suppressed drug release in the enzyme free media successfully.5-ASA release from pellets coated with mixtures of high amylose starches (Hylon^® VII, Hylon^® V or LAPS) and Surelease^® in a ratio of 1 to 2 w/w was found to be minimal in simulated gastric and intestinal fluids. This suggests that these mixed films provide starch domains that are resistant to the enzymes present in the upper GI tract and thus can potentially be used in the preparation of colon-specific delivery devices. Starches with a minimum amylose content of 56% such as the starches used in this study (Hylon^® VII and Hylon^® V) are preferred, and although pure amylose can also be used this is not essential.     

Cyclosporine A treated in vitro models induce cholestasis response through comparison of phenotype-directed gene expression analysis of in vivo Cyclosporine A-induced cholestasis

In vitro models for hepatotoxicity testing are a necessity for advancement of toxicological research. Assessing the in vitro response requires in vivo validated gene sets reflective of the hepatotoxic phenotype. Cholestasis, the impairment of bile flow, is induced in C57BL/6J mice treated with cyclosporine A (CsA) to identify phenotype reflective gene sets. CsA treatment through oral gavage for 25 days induced cholestasis, as confirmed by histopathology and serum chemistry. Over 1, 4, and 11 days of CsA exposure gradual increases in serum markers were correlated to gene expression. This phenotype-directed analysis identified gene sets specific to the onset and progression of cholestasis, such as PPAR related processes and drug metabolism, by circumventing other effects of CsA, such as immunosuppression, found in dose*time group analysis. In vivo gene sets are enriched in publicly available data sets of CsA-treated HepaRG and primary mouse hepatocytes. However, genes identified within these gene sets did not overlap between in vivo and in vitro. In vitro regulated genes represent the initial response to cholestasis, whereas in vivo genes represent the later adaptive response. We conclude that the applicability of in vitro models for hepatotoxicity testing fully depends on a solid in vivo phenotype anchored analysis.     

Preparation of a novel floating ring capsule-type dosage form for stomach specific delivery

Study objectives were to develop a unique floating ring capsule dosage form which combines gastric soluble and insoluble portions, and to evaluate its suitability for stomach specific drug delivery. New floating ring capsules were developed using different polymers and were compared for various parameters. The formulation with HPMC and sodium CMC has better floating properties. The effects of polymers concentration on drug release were studies by in vitro release studies. The interaction studies of combined drug with polymers were determined using FT-IR spectroscopy. The entrapped air within the gel barrier and lower densities of HPMC and sodium CMC resulted in better floating behavior. Steady slow gel formations showed prolonged drug release. The in vitro release rates were generally found to be faster with low concentration of carbopol showing release within 2 h, while formulations containing high amount of HPMC showed release in 8 h. In particular, the higher concentration of HPMC formulation shows the best drug release performance. A very low change in peak shift was observed only with sodium alginate formulations. Further, FT-IR measurements confirmed the absence of any chemical interactions. Results indicate that new floating ring capsule is a promise dosage form for stomach specific delivery.     

In vitro cytokine release from human peripheral blood mononuclear cells in the assessment of the immunotoxic potential of chemicals

Alternative methods to the use of animals in testing of chemicals are needed. We investigated if the immunotoxic potential of 12 dietary toxicants could be predicted from effects on cytokine release from human peripheral blood mononuclear cells (PBMC) after in vitro exposure. Nine cytokines were selected to reflect different types of immune responses. The toxicants were classified as immunotoxic or non-immunotoxic substances according to the published in vivo data.Isolated human PBMC were exposed for 20 h to three concentrations of each of the 12 substances in the presence of human liver S9 fraction. After further incubation of PBMC in fresh medium containing the mitogen phytohemagglutinin (PHA, 10 μg/ml) for 48 h, release of the nine selected cytokines into the supernatant as well as cell proliferation were measured by Luminex technology™ and the BrdU incorporation assay, respectively.All 12 substances investigated affected the release of one or more cytokines, and each of the substances showed different cytokine release patterns. Within the limitations of the study design, the present study suggests that the effect of the substances on mitogen-induced cytokine release from PBMC cannot predict their immunotoxic potential, but may be useful in mechanistic studies.     

Less is more: reducing the reliance on animal models for nausea and vomiting research

Animals have been used as experimental models for centuries and their use has enabled researchers to make significant advances in many areas of human health and disease. However, this is not always the case and there are limitations in using animal models as surrogates for humans, which have hampered the development of efficacious therapeutics for some pathologies. Scientific limitations, together with ethical concerns, legislative changes and the current economic climate are driving researchers to look for and develop alternative non-animal research tools. Technological advances in tissue engineering, ‘omics’ approaches and in silico modelling for example, are enabling scientists to conduct their research without using animals in a broad range of disciplines, including complex multi-system reflexes such as nausea and vomiting.     

An investigation into effects of <Emphasis Type="Italic">In Vitro</Emphasis> Test condition on the release properties of theophylline from HPMC matrices using factorial design

The purpose of this study was to investigate the effect of various in vitro test conditions, on the release properties of theophylline (TP) from aminophylline (AP) matrices based on different hydroxypropylmethylcellulose (HPMC) ratio and viscosity grades. The General full factorial experimental design 3 × 3 × 3 was used, based on three independent variables: applied in vitro test (X1), HPMC/drug ratio (X2) and polymer viscosity grade (X3). The drug release percent at 2h (Y_2h), 4h (Y_4h) and 8 h (Y_8h) and time for 50% of TP release from matrices (Y_T50%) were response variables. Three in vitro tests were used: Test 1 and Test 4 (Theophylline Extended-release Capsules, USP 30) and Half-change method. According to factorial design analyses, in vitro test was the most significant factor influencing mechanism and amount of drug release. For Half Change method erosion was the predominant mechanism indicating Case — II transport, while for Test 1 the release mechanism were followed by both diffusion and erosion. The lowest release exponent n values, obtained from Ritger-Pepass equation, for Test 4 indicate diffusion process inclining from Fickian diffusion to Anomalous transport. Therefore, it is in the stage of development, useful to consider the influence of various in vitro test conditions on the formulation, in order to choose an optimal test for the purpose of future drug release examination.     

Percutaneous penetration and distribution of VX using in vitro pig or human excised skin validation of demeton-S-methyl as adequate simulant for VX skin permeation investigations

The organophosphorus (OP) chemical warfare V agent O-ethyl-S-[2(di-isopropylamino)ethyl] methyl phosphonothioate (VX), is a highly toxic compound which mainly penetrates the body via percutaneous pathways. Hence, the following prerequisite: to ascertain compound absorption and percutaneous profile distribution with a view to further assessing the efficacy of topical skin protectants. We first selected the most appropriate receptor fluid to carry out in vitro VX absorption experiments, namely: Hanks's Balanced Salt Solution (HBSS). After a 24-h topical exposure time lapse, we measured altogether the percentage of applied dose unabsorbed and absorbed, penetration rate, lag time, permeability coefficient (K(p)), and dose of VXeq present in skin. To such an end, we used full-thickness and split-thickness pig-ear or human abdominal skin membranes. Further, we scrutinised the potential use of two specific molecules as suitable surrogates for VX percutaneous penetration analyses: thus, we compared the present VX toxicokinetic parameters to earlier findings from our research unit, with respect to OP insecticides demethon-S-methyl (DSM) and paraoxon (POX). Within the framework of our study, we wish to highlight the following evidence: (a) pig-ear skin proves a relevant model to predict in vitro human abdominal skin, taking into account a 2-fold higher skin permeability to VXeq; (b) both full or split-thickness skin membranes may be used indiscriminately to gauge penetration rate and absorbed dose; (c) DSM applied on full-thickness pig-ear skin is the most relevant model to mimic the in vitro VX absorption through full-thickness skin model.     

Use of conventional surfactant media as surrogates for FaSSIF in simulating in vivo dissolution of BCS class II drugs

The usefulness of selected conventional surfactant media to enhance dissolution of BCS class II drugs similarly to fasted state simulated intestinal fluid (FaSSIF) and to predict the absorption of drugs in vivo was evaluated. Dissolution behavior of danazol (Danol®), spironolactone (Spiridon®) and N74 (phase I compound) was compared between FaSSIF, containing physiological levels of sodium taurocholate (STC) and lecithin, and dissolution media containing various concentrations of anionic surfactant, sodium lauryl sulfate (SLS) or non-ionic surfactant, polysorbate (Tween) 80. Although these media differed largely in their solubilization ability, micelle size, diffusivity and surface tension, similar dissolution enhancing levels were achieved between FaSSIF and drug-specific concentrations of conventional surfactants. The dissolution enhancement was shown, however, to be important only for danazol and N74, molecules that are characterized by high hydrophobicity. An in vivo pharmacokinetic dog study was carried out with N74. Comparison of observed plasma profiles with simulated profiles obtained using compartmental absorption and transit model (CAT) indicated that 0.1% SLS medium was the best to predict in vivo plasma profiles and pharmacokinetic parameters (C_max and AUC). This study demonstrates the potential of substituting FaSSIF with more simple and cost-effective conventional surfactant media. Use of in vivo prognostic amounts of synthetic surfactants in dissolution testing could largely assist in industrial drug development as well as in quality control purposes.     

Applicability of in vitro tests for skin irritation and corrosion to regulatory classification schemes: Substantiating test strategies with data from routine studies

Skin corrosion or irritation refers to the production of irreversible or reversible damage to the skin following the application of a test substance, respectively. Traditionally, hazard assessments are conducted using the in vivo Draize skin test, but recently in vitro tests using reconstructed human epidermis (RhE) models have gained regulatory acceptance. In this study, skin corrosion (SCT) and irritation tests (SIT) using a RhE model were implemented to reduce the number of in vivo tests required by regulatory bodies. One hundred and thirty-four materials were tested from a wide range of substance classes included 46 agrochemical formulations. Results were assessed according to UN GHS, EU-CLP, ANVISA and US EPA classification schemes. There was high correlation between the two in vitro tests. Assessment of the SCT sensitivity was not possible due to the limited number of corrosives in the data set; SCT specificity and accuracy were 89% for all classification systems. Accuracy (63–76%) and sensitivity (53–67%) were low in the SIT. Specificity and concordance for agrochemical formulations alone in both the SCT and SIT were comparable to the values for the complete data set (SCT: 91% vs. 89% specificity, 91% vs. 89% accuracy and SIT: 64–88% vs. 70–85% specificity, 56–75% vs. 63–76% accuracy).