A Combined In-Vitro and GastroPlus® Modeling to Study the Effect of Intestinal Precipitation on Cinnarizine Plasma Profile in a Fasted State.

Poorly water-soluble weak base molecules such as cinnarizine often exhibit pH-dependent solubility within the gastrointestinal tract. This means that their solubility can be influenced by the pH of the surrounding environment, and this can affect their oral absorption. The differential pH solubility between the fasted-state stomach and intestine is an important consideration when studying the oral absorption of cinnarizine. Cinnarizine has moderate permeability and is known to exhibit supersaturation and precipitation in fasted-state simulated intestinal fluid (FaSSIF), which can significantly impact its oral absorption. The present work is aimed at studying the precipitation behavior of cinnarizine in FaSSIF using biorelevant in vitro tools and GastroPlus® modeling, to identify the factors contributing to the observed variability in clinical plasma profiles. The study found that cinnarizine demonstrated variable precipitation rates under different bile salt concentrations, which could impact the concentration of the drug available for absorption. The results also showed that a precipitation-integrated modeling approach accurately predicted the mean plasma profiles from the clinical studies. The study concluded that intestinal precipitation may be one of the factors contributing to the observed variability in Cmax but not the AUC of cinnarizine. The study further suggests that the integration of experimental precipitation results representing a wider range of FaSSIF conditions would increase the probability of predicting some of the observed variability in clinical results. This is important for biopharmaceutics scientists, as it can help them evaluate the risk of in vivo precipitation impacting drug and/or drug product performance.

Interactions between dipfluzine-based complexes and cytochrome P450 enzymes: Information on salt, cocrystal, and salt cocrystal complexes.

In the new drugs, greater than 90 % of active pharmaceutical ingredients (APIs) or marketed drugs have poor solubility and bioavailability, which restrict the development of pharmaceutical preparations. The use of crystalline molecular complexes (CMCs) involving API and biocompatible precursors to improve solubility has become a shortcut for new drug development. Most of the new drugs registered in CMC form are from postmarketing drugs, and the interaction between these drugs and cytochrome P-450 (CYP) enzymes is well documented. However, it is unclear whether the interactions between CMCs of postmarketing drugs and CYP enzymes should be re-evaluated. To clarify this problem, three dipfluzine (Dip)-based CMCs, including Dip-benzoic acid (BA) cocrystal, Dip-2-hydroxybenzoate (2HB) salt and Dip-4-hydroxybenzoate (4HB) salt-cocrystal, were chosen to investigate the interaction with CYP enzymes. Metabolites of Dip-based CMCs and cocktail probe drugs were measured via LC-MS/MS in the incubation reaction system comprising recombinant CYP enzymes (rCYPs) and human liver microsomes. Dip-based CMCs not only alter Dip-mediated inhibition or activation of CYP enzymes but also change the degree to which rCYPs are involved in Dip metabolism. Specifically, the inhibitory effects of Dip and Dip-HCl were increased compared with Dip-BA and Dip-2HB for CYP1A2; Dip-BA, Dip-2HB and Dip-4HB for CYP3A4; and Dip-BA for CYP2E1. The inhibitory effects of Dip and Dip-HCl were reduced compared with Dip-2HB and Dip-4HB for CYP2C19 and Dip-4HB for CYP2E1. The effects of the alterations of Dip CMCs on the interaction between Dip and CYP enzymes are not attributed to a simple superposition of Dip and the respective precursor and may be due to the presence of interaction forces between Dip and precursor molecules. These results are the first to provide preliminary experimental evidence that CMCs change the interaction between API and CYP enzymes. Moreover, these results further suggest the importance of re-evaluating interactions with CYP enzymes when CMC strategies are used to design new drugs and even for CMCs of postmarketing drugs with known metabolic characteristics.

Small molecules bind human mTOR protein and inhibit mTORC1 specifically.

Inhibition of mTOR activity (mechanistic target of rapamycin) is an anti-cancer therapeutic strategy. mTOR participates in two functional complexes, mTORC1 and mTORC2. Since mTORC1 is specifically activated in multiple tumors, novel molecules that inhibit mTORC1 could be therapeutically important. To identify potentially novel modulators of mTOR pathways, we screened 1600 small molecule human drugs for mTOR protein binding, using novel biolayer interferometry technology. We identified several small molecules that bound to mTOR protein in a dose-dependent manner, on multiple chemical scaffolds. As mTOR participates in two major complexes, mTORC1 and mTORC2, the functional specificities of the binders were measured by S6Kinase and Akt phosphorylation assays. Three novel 'mTOR general' binders were identified, carvedilol, testosterone propionate, and hydroxyprogesterone, which inhibited both mTORC1 and mTORC2. By contrast, the piperazine drug cinnarizine dose-dependently inhibited mTORC1 but not mTORC2, suggesting it as a novel mTORC1-specific inhibitor. Some of cinnarizine's chemical analogs also inhibited mTORC1 specifically, whereas others did not. Thus we report the existence of a novel target for some related piperazines including cinnarizine and hydroxyzine, i.e. specific inhibition of mTORC1 activity. Since mTOR inhibition is a general anti-cancer strategy, and mTORC1 is specifically activated in some tumors, we suggest the piperazine scaffold, including cinnarizine and hydroxyzine, could be proposed for rational therapy in tumors in which mTORC1 is specifically activated. Related piperazines have shown toxicity to cancer cells in vitro as single agents and in combination chemotherapy. Thus piperazine-based mTOR inhibitors could become a novel chemotherapeutic strategy.

Multi-Layer Self-Nanoemulsifying Pellets: an Innovative Drug Delivery System for the Poorly Water-Soluble Drug Cinnarizine.

Beside their solubility limitations, some poorly water-soluble drugs undergo extensive degradation in aqueous and/or lipid-based formulations. Multi-layer self-nanoemulsifying pellets (ML-SNEP) introduce an innovative delivery system based on isolating the drug from the self-nanoemulsifying layer to enhance drug aqueous solubility and minimize degradation. In the current study, various batches of cinnarizine (CN) ML-SNEP were prepared using fluid bed coating and involved a drug-free self-nanoemulsifying layer, protective layer, drug layer, moisture-sealing layer, and/or an anti-adherent layer. Each layer was optimized based on coating outcomes such as coating recovery and mono-pellets%. The optimized ML-SNEP were characterized using scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), in vitro dissolution, and stability studies. The optimized ML-SNEP were free-flowing, well separated with high coating recovery. SEM showed multiple well-defined coating layers. The acidic polyvinylpyrrolidone:CN (4:1) solution presented excellent drug-layering outcomes. DSC and XRD confirmed CN transformation into amorphous state within the drug layer. The isolation between CN and self-nanoemulsifying layer did not adversely affect drug dissolution. CN was able to spontaneously migrate into the micelles arising from the drug-free self-nanoemulsifying layer. ML-SNEP showed superior dissolution compared to Stugeron® tablets at pH 1.2 and 6.8. Particularly, on shifting to pH 6.8, ML-SNEP maintained > 84% CN in solution while Stugeron® tablets showed significant CN precipitation leaving only 7% CN in solution. Furthermore, ML-SNEP (comprising Kollicoat® Smartseal 30D) showed robust stability and maintained > 97% intact CN within the accelerated storage conditions. Accordingly, ML-SNEP offer a novel delivery system that combines both enhanced solubilization and stabilization of unstable poorly soluble drugs.

Integrating virtual screening and biochemical experimental approach to identify potential anti-cancer agents from drug databank.

Chemical libraries constitute a reservoir of pharmacophoric molecules to identify potent anti-cancer agents. Virtual screening of heterocyclic compound library in conjugation with the agonist-competition assay, toxicity-carcinogenicity analysis, and string-based structural searches enabled us to identify several drugs as potential anti-cancer agents targeting protein kinase C (PKC) as a target. Molecular modeling study indicates that Cinnarizine fits well within the PKC C2 domain and exhibits extensive interaction with the protein residues. Molecular dynamics simulation of PKC-Cinnarizine complex at different temperatures (300, 325, 350, 375, and 400[Formula: see text]K) confirms that Cinnarizine fits nicely into the C2 domain and forms a stable complex. The drug Cinnarizine was found to bind PKC with a dissociation constant Kd of [Formula: see text]M. The breast cancer cells stimulated with Cinnarizine causes translocation of PKC-[Formula: see text] to the plasma membrane as revealed by immunoblotting and immunofluorescence studies. Cinnarizine also dose dependently reduced the viability of MDAMB-231 and MCF-7 breast cancer cells with an IC[Formula: see text] of [Formula: see text] and [Formula: see text]g/mL, respectively. It is due to the disturbance of cell cycle of breast cancer cells with reduction of S-phase and accumulation of cells in G1-phase. It disturbs mitochondrial membrane potentials to release cytochrome C into the cytosol and activates caspase-3 to induce apoptosis in cancer cells. The cell death was due to induction of apoptosis involving mitochondrial pathway. Hence, the current study has assigned an additional role to Cinnarizine as an activator of PKC and potentials of the approach to identify new molecules for anti-cancer therapy. Thus, in silico screening along with biochemical experimentation is a robust approach to assign additional roles to the drugs present in the databank for anti-cancer therapy.

NMR-based metabolomic study on the toxicological effects of pesticide, diazinon on adaptation to sea water by endangered Persian sturgeon, Acipenser persicus fingerlings.

NMR-based metabolomics was applied to explore metabolic impacts of diazinon on sea water adaptation of Persian sturgeon fingerlings, Acipenser persicus. Fingerlings were exposed to sub-lethal concentrations of diazinon in freshwater (FW) for 96 h (short-term trial) and 12 days (long-term trial) and then exposed in brackish water (BW) (12 mg L-1 salinity) for 24 h. After 96 h and 12 days exposure in FW, identified metabolites (amino acids, osmolytes, energy metabolites) showed different change-patterns compared to control group (P < 0.05) as follow: (A) short-term trial: higher plasma levels of glucose, lactate (in all diazinon-exposed fish), acetate and acetoacetate (in 0.9 mg L-1diazinon treatment); lower levels of creatine (in all diazinon-exposed fish), trimethylamine-N-oxide, choline, taurine, betaine, N,N-dimethylglycine and almost all amino acids in fish exposed to high concentrations of diazinon (0.54 and 0.9 mg L-1 diazinon). (B) Long-term trial: higher plasma levels of lipid oxidation metabolites and almost all amino acids in fish exposed to 0.54 and 0.9 mg L-1 diazinon; lower levels of creatine, trimethylamine-N-oxide, N,N-dimethylglycine, betaine, choline (in all diazinon-exposed fish), glucose (in 0.54 and 0.9 mg L-1diazinon treatments) and taurine (in 0.9 mg L-1 diazinon treatment). When fish were exposed in BW for 24 h, the plasma levels of osmolytes decreased significantly in almost all experimental groups of short-term and long-term trial (P < 0.05). In short-term trial, the plasma levels of glucose in all groups and lactate in 0.18 and 0.54 mg L-1 diazinon treatments increased after salinity challenge (P < 0.05). However, a significant decrease was observed in lactate levels in 0.9 mg L-1 diazinon treatment (P < 0.05). Also, the plasma levels of amino acids decreased mostly in fish of control group than exposed fish (P < 0.05). The plasma glycerol concentration showed a significant decrease only in fish of 0.54 mg L-1 diazinon treatment (P < 0.05). In long term trial, the energetic metabolites (acetate, acetoacetate, glycerol) showed significant increases mostly in fish exposed to high concentrations of diazinon (P < 0.05). Phosphocreatine was detected only in groups exposed to 0.54 and 0.9 mg L-1 diazinon. Some amino acids decreased in control and diazinon-exposed groups while glycine (in control and 0.18 mg L-1 diazinon treatment), glutamine and alanine (in 0.9 mg L-1 diazinon treatment) elevated significantly after 24 h acclimation in BW (P < 0.05). Our results may help to understand the effects of pesticides on fish osmoregulation from a metabolic approach.

Simultaneous lipolysis/permeation in vitro model, for the estimation of bioavailability of lipid based drug delivery systems.

The simultaneous processes of lipid digestion and absorption together determine the oral bioavailability of drugs incorporated into lipid based drug delivery systems (LBDDS). A number of slightly different protocols for in vitro lipolysis are widely accepted; however, the permeation process has so far not been included into the models due to the harsh conditions of lipid digestion compromising permeation barriers. The present study for the first time combines biomimetic permeation and lipolysis of LBDDS. The focus of the current work was on the functional stability of the barrier - Permeapad® during lipid digestion. Using calcein as a marker molecule the investigations demonstrated that the barrier was able to maintain its permeation properties in the presence of the SNEDDS (self-emulsifying drug delivery system) formulation, the lipolysis medium, and the lipolysis medium while digesting the SNEDDS. Furthermore, the permeation of cinnarizine (CINN) from SNEDDS was demonstrated to be lower, if the formulation as such was applied as compared to the digested formulation. This support the general perception that meaningful in vitro evaluation of lipid based formulations requires consideration of both, the digestion and absorption, i.e. lipolysis and permeation.

High-Throughput Lipolysis in 96-Well Plates for Rapid Screening of Lipid-Based Drug Delivery Systems.

The high-throughput in vitro intestinal lipolysis model (HTP) applicable for rapid and low-scale screening of lipid-based drug delivery systems (LbDDSs) was optimized and adjusted as to be conducted in 96-well plates (HTP-96). Three different LbDDSs (I-III) loaded with danazol or cinnarizine were used as model systems. The distributions of cinnarizine and danazol in the aqueous and precipitated digestion phases generated during lipolysis in HTP-96 were compared with previously published data obtained from HTP. The final HTP-96 setup resulted in the same rank order as the original HTP model with regard to solubilization in the aqueous phase during digestion: LbDDS III > LbDDS II > LbDDS I for danazol and LbDDS III ≈ LbDDS II ≈ LbDDS I for cinnarizine. HTP-96 is a useful model for fast performance assessment of LbDDS in a small scale.

In vivo evaluation of supersaturation/precipitation/re-dissolution behavior of cinnarizine, a lipophilic weak base, in the gastrointestinal tract: the key process of oral absorption.

The aim of this study is to evaluate how supersaturation, precipitation, and re-dissolution processes influence the intestinal absorption of cinnarizine (CNZ), a lipophilic weak base, by monitoring its plasma and luminal concentration-time profile, after oral administration as a HCl solution containing fluorescein isothiocyanate dextran (FD-4), a non-absorbable marker. In the in vitro pH shift experiment, the supersaturation stability was significantly lower when the higher-concentration solution of CNZ (pH1.5) was added to the simulated intestinal fluid. However, although the in vivo bioavailability after oral administration of high and low dose as HCl solutions was greatly improved compared to those as neutral suspensions, the difference in the supersaturation stability was not reflected in the improvement of the in vivo bioavailability. Analysis of CNZ and FD-4 concentrations in each segment of the gastrointestinal tract revealed that most of the CNZ precipitated in the duodenum after gastric emptying, and supersaturation was observed only in the duodenum. Thereafter, the precipitate was rapidly re-dissolved and absorbed in the upper and middle small intestine. The rapid re-dissolution may be caused by smaller particles of the precipitate. In this case, it is considered that the key process for the absorption of CNZ was re-dissolution, not supersaturation. Therefore, different supersaturation stabilities in different doses observed in in vitro precipitation experiment was not reflected to in vivo absorption. These findings may be useful to design efficient supersaturable formulations and to validate and improve current prediction methods.

Utilization of ionotropic gelation technique for bioavailability enhancement of cinnarizine: in-vitro optimization and in-vivo performance in human.

Gastro retentive drug delivery system techniques were adopted to deliver drugs having narrow absorption window from a particular site in the GIT. Therefore, gastro retentive dosage forms were retained in the stomach, thus improving absorption and bioavailability would be improved consequently. In this study, cinnarizine (CNZ) was employed as the model drug. CNZ is a poorly soluble basic drug, suffering from low and erratic bioavailability. This is attributed to its pH-dependant solubility (highly soluble at pH < 4). CNZ is characterized by short half-life (3-6 h). Accordingly, floating CNZ emulsion gel calcium pectinate beads were developed. A mixture design was employed to study the effect of the percent of LM pectin (A), the percent of GMO (B) and the percent of Labrafac Lipophile (C) simultaneously on the percent of drug released and loaded. The optimized floating CNZ emulsion gel calcium pectinate beads and Stugeron® (the marketed reference product) were compared through a pharmacokinetic study carried on healthy human volunteers. Fortunately, simple floating CNZ emulsion gel calcium pectinate beads were prepared with zero-order release profile for 12 h. A promising in-vivo CNZ controlled release dosage form with higher bioavailability, when compared to once daily administration of Stugeron® tablets was achieved.

Instrumentation of Flow-Through USP IV Dissolution Apparatus to Assess Poorly Soluble Basic Drug Products: a Technical Note.

Supersaturation and precipitation are common limitations encountered especially with poorly soluble basic drugs. The aims of this work were to explore the pattern of dissolution and precipitation of poorly soluble basic drugs using a United States Pharmacopoeia (USP) IV dissolution apparatus and to compare it to the widely used USP II dissolution apparatus. In order to investigate the influence of gastric emptying time on bioavailability, tables of two model drugs (dipyridamole 100 mg and cinnarizine 15 mg) were investigated and pH change from 1.2 to 6.8 were achieved after 10, 20 or 30 min using USP II or USP IV dissolution apparatuses. Using USP II, dipyridamole and cinnarizine concentrations dropped instantly as a result of drug precipitation with drug crystals evident in the dissolution vessel. At pH change times of 10, 20 and 30 min, the total amount of dissolved drug was dependent on pH change time. Using USP IV, at a flow rate of 8 ml/min, it was possible to have comparable release to agitation at 50 rpm using USP II suggesting that comparable hydrodynamic forces are possible. No drop in drug percentage occurs as the dissolved fraction was readily emptied from the flow cell, preventing drug accumulation in the dissolution medium. However, a negligible percentage of drug release took place following pH change. In conclusion, the use of the flow-through cell dissolution provided laminar flow, use of realistic fluid volumes and avoided precipitation of dissolved drug fraction in the gastric phase as it is discharged before pH change.

Floating lipid beads for the improvement of bioavailability of poorly soluble basic drugs: in-vitro optimization and in-vivo performance in humans.

The challenge in developing oral drug delivery systems of poorly soluble basic drugs is primarily due to their pH dependent solubility. Cinnarizine (CNZ), a model for a poorly soluble basic drug, has pH dependent solubility; where it dissolves readily at low pH in the stomach and exhibits a very low solubility at pH values greater than 4. It is also characterized by a short half life of 3-6h, which requires frequent daily administration resulting in poor patient compliance. In an attempt to solve these problems, extended release floating lipid beads were formulated. A 2(4) full factorial design was utilized for optimization of the effects of various independent variables; lipid:drug ratio, % Pluronic F-127, % Sterotex, and Gelucire 43/01:Gelucire 50/13 ratio, on the loading efficiency and release of CNZ from the lipid beads. In-vivo pharmacokinetic study of the optimized CNZ-lipid beads compared to Stugeron® (reference standard) was performed in healthy human volunteers. A promising approach for enhancing the bioavailability of the poorly soluble basic drug, CNZ, utilizing novel and simple floating lipid beads was successfully developed. Zero order release profile of CNZ was achieved for 12h. Mean AUC0-24 and AUC0-∞ of the optimized CNZ-loaded lipid beads were 4.23 and 6.04 times that of Stugeron® tablets respectively.

Pharmacokinetic evaluation of dipfluzine and its three metabolites in rat plasma using liquid chromatography-mass spectrometry.

A validated LC-MS/MS method to determine the content of dipfluzine (Dip) and its three metabolites (M1, M2, and M5) simultaneously within rat plasma samples was developed. After a single liquid-liquid extraction, the assay was performed by using a C18 column and positive electrospray ionisation mode (ESI) in the multiple reaction monitoring (MRM) mode with transitions of m/z 417.3→167.3, 251.2→165.2, 199.1→121.3, and 183.2→105.1 for Dip, M1, M2, and M5, respectively. Sulfamethoxazole (SMZ) was used as internal standard (IS). The method was linear ranged from 0.5-518, 0.5-524, 1.0-1036, and 0.5-514 ng/ml for Dip, M1, M2, and M5, respectively and all correlation coefficients were greater than 0.9919. The intra- and inter-day precision values obtained were less than 11.5% and the accuracy was between -3.2 and 9.7% for each analyte. The extraction recoveries of their three concentrations for Dip and its three metabolites were all higher than 71.9%. The technique was successfully applied to a pharmacokinetic study of Dip and its metabolites after a single oral administration of Dip (20 mg/kg) to rats. The results indicated that the metabolite formation was rapid and generated M5 as the predominant metabolite, followed by M1 and M2. The maximum plasma concentrations (Cmax) were 59±7, 37±4, 3±0.2, and 55±5 ng/ml; the time to maximum plasma concentration (Tmax) were 65±12, 95±12, 190±25, and 90±0 min and the areas under the concentration-time curves (AUC0→∞) were 17573±704, 8328±355, 5602±753, and 16101±429 ng min/ml for Dip, M1, M2, and M5, respectively. These results suggested that Dip was extensively metabolized and rapidly absorbed. The half-life (t1/2) of Dip, M1, M2, and M5 were 329±15, 767±75, 2364±434, and 378±36 min, respectively, which indicated that Dip and M5 were eliminated quickly. M2 reached its Tmax later and exhibited a longer t1/2 than the other metabolites, which indicated that there might be some type of flip-flop mechanism at work in the pharmacokinetics of M2.

RS-WebPredictor: a server for predicting CYP-mediated sites of metabolism on drug-like molecules.

SUMMARY: Regioselectivity-WebPredictor (RS-WebPredictor) is a server that predicts isozyme-specific cytochrome P450 (CYP)-mediated sites of metabolism (SOMs) on drug-like molecules. Predictions may be made for the promiscuous 2C9, 2D6 and 3A4 CYP isozymes, as well as CYPs 1A2, 2A6, 2B6, 2C8, 2C19 and 2E1. RS-WebPredictor is the first freely accessible server that predicts the regioselectivity of the last six isozymes. Server execution time is fast, taking on average 2s to encode a submitted molecule and 1s to apply a given model, allowing for high-throughput use in lead optimization projects. AVAILABILITY: RS-WebPredictor is accessible for free use at http://reccr.chem.rpi.edu/Software/RS-WebPredictor/

The effect of administered dose of lipid-based formulations on the in vitro and in vivo performance of cinnarizine as a model poorly water-soluble drug.

The influence of varying the amount of lipid co-administered with the drug on drug solubilisation and absorption is poorly understood. In the current study, the effect of lipid dose on the in vitro drug distribution is compared with the in vivo absorption of cinnarizine (CZ) when formulated using long-chain triacylglyceride (LCT) and medium-chain triacylglycerides (MCT). At a fixed drug-lipid ratio, in the closed in vitro model, the drug concentrations in the aqueous phase increased and decreased for MCT and LCT, respectively, with increasing lipid dose. However, in vivo, the oral bioavailability (F%) of CZ was independent of the quantity of lipid administered for both MCT and LCT, but was higher for LCT (32.1 ± 2.3%) than for MCT (16.6 ± 2.3%). Increasing the quantity of lipid relative to the dose of CZ resulted in an increase in the oral F% when the lipid mass was increased from 125 to 250 mg, but was no greater at 500 mg lipid dose. The results confirm the limitations of the in vitro model but positively indicate that the use of the rat as a pre-clinical model for studying the bioavailability of poorly water-soluble drugs is not compromised by the mass of formulation administered.

Simultaneous determination of a novel diphenylpiperazine calcium channel blocker and its four metabolites in rat liver microsomes by liquid chromatography tandem mass spectrometry.

Dipfluzine hydrochloride (Dip), a novel diphenylpiperazine calcium channel blocker, has revealed the characteristics of a promising candidate for the treatment of cerebral vascular diseases in preclinical studies. Our research identified and quantified Dip and its 4 metabolites (M1, M2, M4 and M5) in rat liver microsomes by liquid chromatography tandem mass spectrometry. The results showed that Dip was firstly metabolized to M1 and M5 by 1- and 4-dealkylation from a piperazine nitrogen, and then the latter was subsequently metabolized to M2 and M4. The concentrations of Dip, M1, M2 and M5 were 557.3 ± 26.3, 854.3 ± 46.0, 2796.7± 126.9, 2473.3 ± 82.6 and 4.0 ± 0.4, 2.4 ± 0.1, 318.2 ± 8.7 and 27.4 ± 1.5 ng/ml in male and female rats, respectively. M4 (404.2 ± 22.2 ng/ml) was detected only in males not in females, suggesting that there is gender difference in the metabolism of Dip.

Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system.

Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK(a) value was associated with a more rapid uptake. The drug-induced displacement of LysoTracker was neither caused by elevation of intra-lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra-lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi-drug resistance by altering subcellular drug distribution.

In vitro gastrointestinal lipolysis of four formulations of piroxicam and cinnarizine with the self emulsifying excipients Labrasol and Gelucire 44/14.

PURPOSE: Labrasol and Gelucire 44/14 are defined admixtures of acylglycerols and PEG esters which are substrates for digestive lipases. METHODS: We investigated their in vitro gastrointestinal lipolysis to understand which compounds are, after digestion, responsible for keeping poorly water-soluble drugs in solution. The precipitation of piroxicam and cinnarizine formulated in these excipients during the gastrointestinal lipolysis was also studied. RESULTS: Monoacylglycerols and PEG monoesters are the largest compounds present at the end of gastric phase whereas PEG-monoesters are the largest compounds after the duodenal phase. The precipitation of piroxicam is mainly due to the gastric lipolysis. In the control experiments performed without digestive lipases, cinnarizine formulated in Labrasol was found to precipitate upon dilution of the gastric medium to form the solution mimicking the duodenal medium. In the presence of gastric lipase, Labrasol was hydrolyzed and the precipitation of cinnarizine was not observed in this case. When the cinnarizine was formulated with Gelucire 44/14 the precipitation was only due to the dilution of the gastric medium. CONCLUSION: Our study highlights the importance of the gastrointestinal lipolysis and the associated phenomena such as the dilution of chyme by biliary and pancreatic secretions in vivo, on the solubilisation of poorly water-soluble drugs formulated with lipid-based excipients.

Improvement of oral bioavailability of flurbiprofen from flurbiprofen/beta-cyclodextrin inclusion complex by action of cinnarizine.

Improvement of the oral bioavailability of flurbiprofen (Flu) after oral administration of flurbiprofen/beta-cyclodextrin inclusion complex (Flu/beta-CD) by the action of cinnarizine (CN) was investigated. Flu and Flu/beta-CD were administered orally to fasted rats at a dose of 20mg/kg as Flu. Thirty minutes after drug administration, CN dissolved in pH 4.0 buffer solution or pH 4.0 buffer solution alone was administered to the rats. The dose of CN was 0.17 mg/kg. Blood samples were taken from rats and Flu concentrations in plasma samples were determined by HPLC. It was found from the comparison of Flu and Flu with CN (Flu+CN) that CN had no effect on plasma concentrations of Flu after oral administration of Flu. The mean plasma levels after oral administration of Flu/beta-CD with CN (Flu/beta-CD+CN) were larger not only than those of Flu and Flu+CN but also than those of Flu/beta-CD. The value of C(max) in Flu/beta-CD+CN was significantly larger than that of Flu/beta-CD. This is considered to be caused by the action of CN as a competing agent. This mechanism was supported by the result of solubility study in which Flu solubility in beta-CD solution decreased with the addition of CN. It was found from these results that CN had strong ability as a competing agent in vivo.

An evaluation of the relative roles of the unstirred water layer and receptor sink in limiting the in-vitro intestinal permeability of drug compounds of varying lipophilicity.

The roles of the unstirred water layer (UWL) and receptor sink on the in-vitro transmembrane permeability of an increasingly lipophilic series of compounds (mannitol (MAN), diazepam (DIA) and cinnarizine (CIN)) have been assessed. Altered carbogen bubbling rates were used as a means to change the UWL thickness and polysorbate-80 (PS-80), bovine serum albumin (BSA) and alpha-1-acid glycoprotein (AAG) were employed to alter sink conditions. After correction for solubilisation, Papp data for MAN, DIA and CIN were consistent across varying donor PS-80 concentrations suggesting that for the drugs examined here, the donor UWL did not limit in-vitro permeability. Similarly, altered bubbling rates and receptor sink conditions had no impact on the permeability of MAN. In contrast, decreasing the size of the receptor UWL or adding solubilising agents to the receptor sink resulted in modest enhancements to the permeability of the more lipophilic probe DIA. For the most lipophilic compound, CIN, very significant changes to measured permeability (>30 fold) were possible, but were most evident only after concomitant changes to both the UWL and sink conditions, suggesting that the effectiveness of enhanced sink conditions were dependent on a decrease in the width of the UWL.