Enzymatic prodrug degradation in the fasted and fed small intestine: In vitro studies and interindividual variability in human aspirates.

The aim of the current study was (1) to develop an automation-based protocol for in vitro assessment of enzymatic drug stability at fasted- and fed-state intestinal conditions, (2) to characterize the inter-individual variability of drug degradation in fasted- and fed-state human intestinal fluids, and (3) to compare the obtained in vitro results to drug degradation in human intestinal fluids by taking variability into account. In human intestinal fluids, drug degradation displayed large inter-individual variability, with coefficients of variance generally ranging between 30 and 70 %. The effect of food on the inter-individual variability was highly dependent on the type of drug. The increase of pH in the range between 5.0 and 7.0 significantly accelerated the degradation rate of the studied drugs both in the in vitro and ex vivo experiments. In contrast, the increase of bile salt and phospholipid concentrations in the in vitro screen decreased strongly the degradation rate of the hydrophobic drugs. The developed automated in vitro screen mimicked relatively well the ex vivo degradation of all drugs in the fasted state, whereas in the fed state the degradation of only one of the drugs was adequately reproduced.

Exploring precipitation inhibitors to improve in vivo absorption of cinnarizine from supersaturated lipid-based drug delivery systems.

Supersaturated lipid-based drug delivery systems are increasingly being explored as a bio-enabling formulation approach, particularly in preclinical evaluation of poorlywater-soluble drugs. While increasing the drug load through thermally-induced supersaturation resulted in enhanced in vivo exposure for some drugs, for others, such as cinnarizine, supersaturated lipid-based systems have not been found beneficial to increase the in vivo bioavailability. We hypothesized that incorporation of precipitation inhibitors to reduce drug precipitation may address this limitation. Therefore, pharmacokinetic profiles of cinnarizine supersaturated lipid-based drug delivery systems with or without precipitation inhibitors were compared. Five precipitation inhibitors were selected for investigation based on a high throughput screening of twenty-one excipients. In vivo results showed that addition of 5% precipitation inhibitors to long chain monoglyceride (LCM) or medium chain monoglyceride (MCM) formulations showed a general trend of increases in cinnarizine bioavailability, albeit only statistically significantly increased for Poloxamer 407 + LCM system (i.e. 2.7-fold increase in AUC0-24h compared to LCM without precipitation inhibitors). It appeared that precipitation inhibitors mitigated the risk of in vivo precipitation of cinnarizine from sLBDDS and overall, bioavailability was comparable to that previously reported for cinnarizine after dosing of non-supersaturated lipid systems. In summary, for drugs which are prone to precipitation from supersaturated lipid-based drug delivery systems, such as cinnarizine, inclusion of precipitation inhibitors mitigates this risk and provides the opportunity to maximize exposure which is ideally suited in early efficacy and toxicology evaluation.

Using Potentiometric Free Drug Sensors to Determine the Free Concentration of Ionizable Drugs in Colloidal Systems.

The present study investigates the use of free drug sensors (FDS) to measure free ionized drug concentrations in colloidal systems, including micellar solutions, emulsions, and lipid formulations during in vitro lipolysis. Diphenhydramine hydrochloride (DPH) and loperamide hydrochloride (LOP) were selected as model drugs. Self-diffusion nuclear magnetic resonance studies were performed and confirmed the entrapment of drugs in micelles in Brij 35 and sodium taurodeoxycholate (TDC)/phosphatidylcholine (PC) micellar solutions. The FDS measurements indicated that with a constant level of drug, the percentage of free DPH and LOP decreased from 84% to 57% and from 51% to 18%, respectively, as the concentration of Brij 35 was increased from 4.7 to 22 mM; and from 99% to 46% and from 100% to 21%, respectively, as the concentration of TDC/PC was increased from 0.49/0.04 to 8.85/0.78 mM. During the in vitro lipolysis of a lipid formulation, free drug concentration decreased with lipolysis time. The percentage of free DPH was higher than for LOP in the same colloidal system because DPH is less lipophilic than LOP. The study showed that FDS can be used to monitor the free drug concentration in colloidal systems with fast response, no sample treatment and simple data analysis.

A label-free potentiometric sensor principle for the detection of antibody-antigen interactions.

We report here on a new potentiometric biosensing principle for the detection of antibody-antigen interactions at the sensing membrane surface without the need to add a label or a reporter ion to the sample solution. This is accomplished by establishing a steady-state outward flux of a marker ion from the membrane into the contacting solution. The immunobinding event at the sensing surface retards the marker ion, which results in its accumulation at the membrane surface and hence in a potential response. The ion-selective membranes were surface-modified with an antibody against respiratory syncytial virus using click chemistry between biotin molecules functionalized with a triple bond and an azide group on the modified poly (vinyl chloride) group of the membrane. The bioassay sensor was then built up with streptavidin and subsequent biotinylated antibody. A quaternary ammonium ion served as the marker ion. The observed potential was found to be modulated by the presence of respiratory syncytial virus bound on the membrane surface. The sensing architecture was confirmed with quartz crystal microbalance studies, and stir effects confirmed the kinetic nature of the marker release from the membrane. The sensitivity of the model sensor was compared to that of a commercially available point-of-care test, with promising results.

Online monitoring of dissolution tests using dedicated potentiometric sensors in biorelevant media.

The performance of the Ion-Selective Electrode (ISE) for in vitro dissolution testing using biorelevant media was evaluated in this study. In vitro dissolution was carried out using USP apparatus 2 (paddle method) with classical and with updated biorelevant media to simulate the pre- and postprandial states. The ISE was used as an analytical stand-alone system and in combination with a single-point HPLC-UV measurement. A modified method enabling the use of the ISE for very poorly soluble substances is also proposed. In terms of f(2)-factor, the results acquired using the ISE for the drug diphenhydramine-HCl were found to be very similar to the results obtained by manual sampling followed by HPLC-UV analysis. In Fed State Simulated Gastric Fluid (FeSSGF), a medium containing 50% milk, the ISE is more practical since the need to separate proteins from the analyte prior to HPLC-UV analysis is eliminated. Further work will be needed to establish ISE methodology for Fed State Simulated Intestinal Fluid (FeSSIF) media. In summary, the ISE has promise as an analytical tool for research and development applications.

Unique potentiometric detection systems for HPLC determination of some steroids in human urine.

Isocratic HPLC with potentiometric detection is used for the determination of some 17-ketosteroids (17-KS), e.g., androsterone, dehydroepiandrosterone and estrone, and their respective sulfated conjugates (17-KSS). Glassy carbon or composite electrodes containing a mixture of graphite and poly(vinyl chloride), PVC, were used as substrate electrodes. These substrates were covered either by montmorillonite or potassium tetrakis(p-chlorophenyl) borate containing PVC-based rubber phase membranes. The neutral 17-KS compounds were derivatized with Girard's reagent P (GP) to obtain cationic pyridinium acetohydrazones prior to the HPLC/potentiometric detection assay. No side reactions were observed, and the GP itself was not interfering. The method yielded accurate and reproducible results and was applicable to samples containing down to micromolar concentrations. Next, the 17-KSS compounds, acting as anionic charged molecules, were determined directly in human urine samples with the HPLC/potentiometry combination without preliminary derivatization. For this purpose, a new anion-sensitive potentiometric electrode was developed using a macrocyclic polyamine containing, PVC-based, rubber phase membrane. The three 17-KSS compounds were also determined accurately down to micromolar concentrations. Especially, the main androgen metabolites as dehydroepiandrosterone sulfate and androsterone sulfate could be selectively determined with a developed potentiometric sensor in human urine samples without time-consuming cleanup and preconcentration step.

In situ dissolution testing using potentiometric sensors.

Potentiometric sensors can be used to determine the amount of API dissolved in the dissolution medium in function of time by measuring directly in the dissolution vessel of a Paddle (USP type 2) and Basket (USP type 1) apparatus. The prototype potentiometric sensor instrumentation showed very promising results for a selection of APIs with different physico-chemical properties. The applicability, benefits and limitations of the prototype were explored. The applicability of the measurement technique strongly depends on the log(P) of the API. Here, it is shown that measurements can easily be performed for APIs with a log(P)>4. Electrode performance however decreases with decreasing logP of the APIs due to decreased drug selectivity in comparison to the excipients and ionic strength of the applied dissolution medium. The potentiometric sensors are shown to be insensitive towards undissolved particles and air bubbles as opposed to UV spectrometric measurement where these can lead to severe light scattering. For the tested APIs, the obtained dissolution profiles are very reproducible and show a low variation compared to the measurements using manual sampling and UV or HPLC analysis. The measurements demonstrate that potentiometric sensors are a very promising technology that can become a standard for in situ dissolution measurements.

Response of DNA fragments to potentiometric sensors studied using HPLC.

Potentiometric sensors are studied as viable candidates for the construction of high throughput DNA arrays. For preliminary investigations, such sensors were used in an HPLC setup in the present work. This avoided errors due to ionic contaminants or additives in the commercial samples. The oligonucleotides dT(10), dT(20) and dT(30) were used as test substances. The potentiometric sensors were of the coated wire type, containing PVC, DOP, MTDDACl and a synthetic podand urea receptor. The HPLC system consisted of a reversed phase column eluted with a phosphate buffer, triethylammoniumacetate (TEAA), and an acetonitrile gradient. Molar responses and sensitivities increased with increasing chain length of oligonucleotides, yielding detection limits as low as 10(-6)M (dT(30), injected concentration). The slopes of the calibration graphs were at least 23 mV/decade (dT(10)), which was much higher than expected. The results are discussed in view of the potential use of this sensor type in high throughput microarrays.

Development of in situ ion selective sensors for dissolution.

The dissolution of formulations of the drugs dapoxetine, paliperidone, cinnarizine, tetrazepam, mebeverine, loperamide, galantamine and ibuprofen was studied by an in-line potentiometric measurement system. The transpose of a Nikolskii-Eisenman type function performed the conversion of potential to percentage of dissolution. A novel gradient membrane electrode was developed especially for dissolution, varying continuously in composition from an ionically conducting rubber phase to an electronically conducting solid state PVC/graphite composite. The gradient part had a thickness of 200 microm. The electrodes life span exceeded 6 months. An ion exchange procedure was used to prepare them for one specific drug. This enabled us to use one universal electrode built to measure a wide array of drugs. The system parameters such as accuracy, reproducibility and linearity were presented with the data obtained for the drug dapoxetine. In dissolution, accurate measurements were possible from 10(-9) to 10(-3) M concentrations, for high log P drugs. The effect of t(90) response times on the measurement error was estimated. The t(90) response times of the electrodes were concentration dependent, and varied between 50 and 10 s for, respectively, 10(-6) and 10(-3) M concentrations. Potential drift was studied in detail. The measurements performed with these electrodes showed an accuracy of 1%, and inter- and intra electrode variabilities of 0.6 and 1.7%, respectively. The electrodes were successfully applied in colloidal media containing suspended matter, typically formed during dissolution of tablets. The advantages and pitfalls of potentiometry over the presently used techniques for dissolution testing are discussed.

Coated wire potentiometric detection for capillary electrophoresis studied using organic amines, drugs, and biogenic amines.

Capillary electrophoresis was coupled successfully and reliably to potentiometric sensors, which are based on an ionically conductive rubber phase coating, applied on a 250 microm diameter metal substrate. The membrane components included potassium tetrakis(p-chlorophenyl)borate (TCPB), bis(2-ethylhexyl)sebacate (DOS), and high molecular mass poly(vinyl chloride) (PVC). Potentiometry reveals a very sensitive CE detection mode, with sub-micromolar detection limits for amines and the randomly chosen drugs quinine, clozapine, cocaine, heroine, noscapine, papaverine, and ritodrine. The lowest detection limit, 1 x 10(-8) M injected concentration, was obtained for the quaternary ammonium compound tetrahexylammonium chloride. The more polar lower aliphatic amines and the biogenic amines dopamine, adrenaline, and cadaverine have much higher detection limits. The detection limits are log P dependent. Addition of a commercially available calixarene molecule or a synthetic macrocyclic amphiphilic receptor molecule to the electrode coatings enhanced the sensitivity respectively for the lower aliphatic amines and for the biogenic amines. A transpose of the Nikolskii-Eisenman-type function was suggested and used to convert the signal of the detector to a concentration-dependent signal.