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P Langguth

Publications and source records attributed to P Langguth.

At least 19 recordsLinked to original sources
PubMed2005

Cyclosporine A (CsA) affects the pharmacodynamics and pharmacokinetics of the atypical antipsychotic amisulpride probably via inhibition of P-glycoprotein (P-gp).

The importance of P-glycoprotein (P-gp) in the pharmacokinetics of amisulpride and the effects of a P-gp inhibitor cyclosporine A (CsA) was investigated both, in vitro and in vivo. In vitro and in vivo results indicated amisulpride as a substrate of P-gp. Amisulpride was not metabolized by rat liver microsomes. Open field behavior showed time dependent abolishment in locomotion by amisulpride (50 mg kg(-1)). Co-administration of CsA (50 mg kg(-1)) resulted in a higher and significantly longer antipsychotic effect (24 h after drug administration). Accordingly, the area under concentration-time curve in serum and brain was higher in CsA co-treated rats (13.5 vs. 29.8 micromol h l(-1) for serum and 2.16 vs 2.98 micromol h l(-1) for brain tissue) while renal clearance was not affected. These results pointed to a pharmacokinetic drug interaction between CsA and amisulpride most likely caused by inhibition of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Nanosuspension formulations for low-soluble drugs: pharmacokinetic evaluation using spironolactone as model compound.

Various particle sizes of spironolactone as a model low solubility drug were formulated to yield micro-and nanosuspensions of the type solid lipid nanoparticles and DissoCubes. Seven oral and one i.v. formulations were tested in an in vivo pharmacokinetic study in rats with the aim of characterizing the bioavailability of spironolactone on the basis of its metabolites canrenone and 7-alpha-thiomethylspirolactone. In addition, a dose escalation study was carried out using nonmicronized spironolactone suspension as well as a nanosuspension type DissoCubes. On the basis of AUC as well as Cmax ratios, three groups of formulations were distinguished. The biggest improvement was seen with a solid lipid nanoparticle formulation yielding a 5.7-fold increase in AUC for canrenone and a similar improvement based on the Cmax metric, followed by a group of three formulations containing nanosized, micronized, and coarse drug material and surfactant. The DissoCubes nanosuspension yielded highly significant improvements in bioavailability averaging 3.3-fold in AUC and 3.0-fold in terms of Cmax for canrenone. The third class encompasses all other formulations, which showed very little to no improvement in bioavailability. The results show that the particle size minimization was not the major determining factor in the bioavailability improvement. Rather, the type of surfactant used as stabilizer in the formulations was of greater importance. Improvement in drug solubility in the intestine as well as in dissolution rate of spironolactone are the most likely mechanisms responsible for the observed effect, although additional mechanisms such as permeability enhancement may also be involved.

Administration, Oral↗
PubMed2004

In vivo evaluation of a novel pH- and time-based multiunit colonic drug delivery system.

BACKGROUND: Targeted drug delivery to the colon is important for topical treatment of inflammatory bowel diseases. Established targeting systems predominantly focus on either pH- or time-dependent release, or bacterial degradation. AIM: To perform a three-phase, crossover design trial evaluating a novel combined pH- and time-based multiunit delivery system. METHODS: Twelve healthy male volunteers each received 200 mg of caffeine as either uncoated immediate release tablets, coated pellets with pH-dependent rapid release (EUDRAGIT FS 30D), and pellets with pH- and time-based release (inner layer EUDRAGIT RL/RS 30D; outer layer EUDRAGIT FS 30D). Orocecal transit time was measured using lactose-[13C]ureide. Serum concentrations of caffeine were measured by high-performance liquid chromatography. RESULTS: In contrast to the uncoated tablet, both coated systems reached the ileocecal region almost at the same time (3.19 +/- 0.71 and 3.33 +/- 0.81 h). Serum caffeine profiles were significantly prolonged for the pH and time delivery system compared with the pH-only based system (median tmax 12.0 vs. 5.5 h; P < 0.001). This was further reflected by a lower Cmax value and a lower area under the curve within 24 h after application. CONCLUSION: Compared with the conventional delivery systems, drug release from the new dosage form may offer a new dimension for the oral treatment of mid to distal ulcerative colitis.

Administration, Oral↗
PubMed2003

Presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters.

Psoriasis is a chronic inflammatory skin disease. Its treatment is based on the inhibition of proliferation of epidermal cells and interference in the inflammatory process. A new systemic antipsoriasis drug, which consists of dimethylfumarate and ethylhydrogenfumarate in the form of their calcium, magnesium and zinc salts has been introduced in Europe with successful results. In the present study, a homologous series of mono- and diesters of fumaric acid has been studied with respect to the sites and kinetics of presystemic ester degradation using pancreas extract, intestinal perfusate, intestinal homogenate and liver S9 fraction. In addition, intestinal permeability has been determined using isolated intestinal mucosa as well as Caco-2 cell monolayers, in order to obtain estimates of the fraction of the dose absorbed for these compounds. Relationships between the physicochemical properties of the fumaric acid esters and their biological responses were investigated. The uncharged diester dimethylfumarate displayed a high presystemic metabolic lability in all metabolism models. It also showed the highest permeability in the Caco-2 cell model. However, in permeation experiments with intestinal mucosa in Ussing-type chambers, no undegraded DMF was found on the receiver side, indicating complete metabolism in the intestinal tissue. The intestinal permeability of the monoesters methyl hydrogen fumarate, ethyl hydrogen fumarate, n-propylhydrogen fumarate and n-pentyl hydrogen fumarate increased with an increase in their lipophilicity, however, their presystemic metabolism rates likewise increased with increasing ester chain length. It is concluded that for fumarates, an increase in intestinal permeability of the more lipophilic derivatives is counterbalanced by an increase in first-pass extraction.

Animals↗
PubMed2001

Intestinal drug efflux: formulation and food effects.

The intestine, primarily regarded as an absorptive organ, is also prepared for the elimination of certain organic acids, bases and neutral compounds depending on their affinity to intestinal carrier systems. Several of the transport systems known to mediate efflux in the major clearing organs--liver and kidney--are also expressed in the intestine. Examples of secretory transporters in the intestine are P-glycoprotein, members of the multidrug resistance associated protein family, breast cancer resistance protein, organic cation transporters and members of the organic anion polypeptide family. In this communication, the P-glycoprotein mediated intestinal secretion of talinolol, a model compound showing metabolic stability, has been investigated in the jejunum, ileum and colon of rat intestine by single-pass perfusion. A model has been developed which demonstrates an increase in carrier-mediated secretion in the order jejunum<ileum<colon. Furthermore, the potency of common excipients in peroral drug products towards inhibition of P-gp mediated secretion has been investigated using a radioligand-binding assay and transport studies in Caco-2 cell monolayers. Finally, evidence is provided which demonstrates that constituents of grapefruit juice not only may influence intestinal drug metabolism, but can also interfere with secretory transport systems, leading to a new and yet undescribed mechanism in drug-food interactions.

Animals↗
PubMed2001

Grapefruit juice enhances intestinal absorption of the P-glycoprotein substrate talinolol.

Grapefruit juice (GFJ) is known to affect the pharmacokinetics of various drugs, presumably mainly via inhibition of oxidative metabolism. In order to evaluate the effect of GFJ on P-glycoprotein-related transport processes, measurements of transport characteristics through Caco-2 monolayers and in vivo drug absorption studies were performed with the transported, yet not metabolized model compound talinolol. Apical-to-basolateral talinolol transport in the Caco-2 model at 1 mM racemate concentration was increased almost 3-fold when GFJ was present (S-talinolol P(eff): 0.16 x 10(-6) vs. 0.61 x 10(-6) cm/s without vs. with GFJ; R-talinolol P(eff): 0.19 x 10(-6) vs. 0.71 x 10(-6) cm/s without vs. with GFJ). In vivo in rats, doubled maximum plasma concentrations, enhanced AUC values (C(max) of S-talinolol: control, 77.5 ng/ml vs. GFJ, 163.6 ng/ml; C(max) of R-talinolol: control, 79.5 ng/ml vs. GFJ, 163.0 ng/ml; AUC of S-talinolol: control, 19.3 microg ml(-1)min vs. GFJ, 29.9 microg ml(-1)min; AUC of R-talinolol: control, 22.2 microg ml(-1)min vs. GFJ, 30.1 microg ml(-1)min), and decreased apparent oral clearances were found for both talinolol enantiomers when GFJ was administered together with a racemic 10 mg/kg b.w. p.o. dose. Furthermore, GFJ tended to accelerate the rate of talinolol input, but did not significantly affect terminal talinolol half-lives. It is concluded that inhibition of intestinal secretion may contribute to bioavailability enhancement upon GFJ intake.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Pretreatment with potent P-glycoprotein ligands may increase intestinal secretion in rats.

The expression of P-glycoprotein is induced in cell cultures upon exposure to various inducers. Therefore, the aim of the present study was to evaluate the in-vivo relevance of this observation, i.e. the influence of chronic pretreatments with selected drugs -- all of which are ligands to P-glycoprotein (P-gp) as demonstrated in radioligand binding studies and all of which have some or a considerable effect on P-gp expression in Caco-2 cells -- on the effective intestinal permeabilities of the model compound talinolol in rats employing in-situ single-pass intestinal perfusion of three different gut segments. Talinolol was selected, because it shows high selectivity for one of the exsorptive transporters (P-gp) and its intestinal permeability is very sensitive to changes in exsorption when the perfusate concentration is low. Prior to the induction study the perfusion model was optimized regarding the type and concentration of a competitive inhibitor which may be used to block the exsorption-related permeability reduction (through intestinal exsorption) during an ongoing perfusion and would permit an intra-individual comparison of the effective permeability without and with blockade of exsorption. While repetitive verapamil and talinolol dosing had no statistically significant exsorption-inducing effect, vinblastine and rifampicin pretreatments resulted in decreased intestinal talinolol permeabilities in the three tested gut segments, duodenum, jejunum, and colon [e.g., S-talinolol in jejunum: control, 2.50 x 10(-4) cm/s; vinblastine induction, 1.48 x 10(-4) cm/s (P<0.05); rifampicin induction, 1.51 x 10(-4) cm/s (P<0.05)]. Addition of an efficient secretion inhibitor (vinblastine) to the perfusate permitted the determination of the impact of inhibitable secretory processes on the total effective permeabilities and an estimation of passive permeability in the respective individual. The inhibitable permeability fractions were higher for vinblastine than for any other pretreatment and the difference from control pretreatment was statistically significant for all intestinal segments (duodenum, 61.8%; jejunum, 63.1%; colon, 43,7%; S-talinolol). Statistically significant differences were also detected for rifampicin in the perfused duodenum and jejunum (33.1 and 27.5% increase in inhibitable fraction, respectively, for S-talinolol). These differences are explained by a significant induction of outside-directed transport in the intestinal enterocytes by vinblastine and rifampicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Pharmacokinetic behaviour of sublingually administered 8-methoxypsoralen for PUVA therapy.

BACKGROUND: Conventional oral PUVA therapy is hampered by large inter- and intraindividual variations in the bioavailability of 8-methoxypsoralen (8-MOP), caused by its low solubility in the gastrointestinal juices and large interindividual differences in hepatic metabolism rate (hepatic first pass). AIMS: New galenic formulations of 8-MOP based on solid dispersions, suspensions, and saturated solutions containing penetration enhancers were developed for sublingual administration, a drug delivery route which avoids the hepatic first pass metabolism. METHODS: Solubility properties of 8-MOP were tested in 22 potential penetration enhancers and solubilizers. Following preliminary in vivo tests of 13 sublingual 8-MOP formulations, five were administered to groups of volunteers at a nominal dose of 0.6 mg/kg body weight: two solid dispersions based on PEG 1540 (with and without Xylitol); a solution in Labrasol (glyceryl and PEG-8 caprylate/caprate), PEG 400, Transcutol (ethoxydiglycol) (1:1:1); a micronized suspension in sorbitol, water, ethanol, propylene glycol (ca. 3:1:1:5 w/v); and Oxsoralen capsules. Pharmacokinetic behaviour of 8-MOP was examined in serum; samples were analysed by HPLC. Photosensitivity was measured in seven subjects. RESULTS: The peak of maximum 8-MOP concentration in blood was sharp, rapid and reproducible: tmax of 8-MOP in blood averaged 23+/-3 min, independent of the particular formulation. Cmax was higher when 8-MOP was presented in dissolved form (solution and capsule formulations, 85+/-29 and 85+/-35 ng/ml, respectively) and lowest with the suspension (42+/-15 ng/ml). Photosensitivity peaked reproducibly at 45 min. post dosing. CONCLUSIONS: Sublingual PUVA therapy is suitable for patients with skin types I and II, in particular patients who are less suitable candidates for standard PUVA therapy (due to hepatic, renal, or cardiac insufficiency) or who have experienced side effects with standard PUVA.

Administration, Sublingual↗
PubMed2001

Compound mixtures in Caco-2 cell permeability screens as a means to increase screening capacity.

The purpose of this investigation was a study of simultaneous permeability measurement using compound mixtures (cassette dosing) as an alternative to single compound evaluation in order to increase the capacity of screens for intestinal drug permeability. Drug transport across Caco-2 monolayers was studied, both in the apical to basolateral and the basolateral to apical direction. The apparent permeability coefficients for ten compounds displaying different intestinal transport mechanisms were determined, first as single compounds and then as components of a mixture. Seven beta-adrenoceptor antagonists and baclofen were analysed simultaneously using reversed phase HPLC with UV detection, D-glucose and mannitol were measured by scintillation counting. The results indicated that the Papp from the mixture as donor phase correlated well with that of the single compounds and merely small changes in the Papp of each compound were observed between the single compound and mixture experiments. This minor variation resulted in a change in rank-order of the poorly permeable compounds in the mixture, however, without affecting their association with the permeability class according to the biopharmaceutics classification system (BCS). It can be concluded that the use of compound mixtures is a suitable method for improving the capacity in permeability screens. Further improvement of the throughput may be expected upon automatisation of permeability measurements using robotics combined with increased selectivity using LC-MS analysis.

Adrenergic beta-Antagonists↗
PubMed2000

Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.

PURPOSE: The objective of this study was a systematic characterization and evaluation of cell culture models based on mixtures of Caco-2/HT29-MTX co-cultures for their use in screening for drug absorption and intestinal permeability in comparison to the properties of the respective mono-cultures. METHODS: Co-cultures of Caco-2 cells (absorptive-type) and HT29-MTX cells (goblet-type) were set up. Three different co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10, 70/30, and 50/50) were grown on permeable filter supports, and monolayers were used for permeability studies with model compounds for paracellular absorption (atenolol, furosemide, H334/75, mannitol, terbutaline), transcellular absorption (antipyrine, ketoprofen, metoprolol, piroxicam), carrier-mediated absorption (D-glucose, Gly-Pro, and L-phenylalanine) as well as substrates for carrier-mediated secretion via P-glycoprotein (cimetidine and talinolol). Electrophysiological and microscopic controls were performed to characterize the cell cultures. RESULTS: For compounds undergoing passive intestinal absorption permeabilities were generally higher in co-cultures than in Caco-2 monolayers, yielding highest values in pure HT29-MTX monolayers. This difference was most obvious for compounds transported via the paracellular pathway, where HT29-MTX cells may be up to 30 times more permeable than Caco-2 cells, whereas for lipophilic and highly permeable compounds, the difference in permeability values was less obvious. For drugs undergoing intestinal secretion mediated by P-glycoprotein, co-cultivation of Caco-2 cells with HT29-MTX cells led to increased apical to basolateral permeability which was decreased in the opposite direction, consistent with the fact that HT29-MTX cells do not express P-glycoprotein. When a carrier-mediated absorption mechanism is involved, the permeabilities observed were lower than the values reported for human small intestine and co-cultivation of HT29-MTX cells with Caco-2 cells resulted in even lower values as compared to the plain Caco-2 cultures. CONCLUSIONS: Co-cultures of HT29-MTX and Caco-2 cells offer the opportunity of modifying the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp. Thus, in special cases, they allow more flexibility in adapting the in vitro system to the in vivo situation as compared to the monocultures. Another advantage is the obvious robustness of the method with respect to the reproducibility of the results. A problem remaining, however, is the quantitative expression of carriers involved in intestinal uptake of many nutrients and drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Affinities at the verapamil binding site of MDR1-encoded P-glycoprotein: drugs and analogs, stereoisomers and metabolites.

OBJECTIVES: The multidrug transporter P-glycoprotein (P-gp) appears to play a significant role in drug absorption and disposition. Hence, it is of interest to evaluate structure-affinity relationships for the purpose of making predictions. METHODS: The affinity to P-gp of related molecular structures from various groups of drugs was determined using competitive radioligand-binding assays. Structural analogs, stereoisomers and metabolites of verapamil-type calcium antagonists, beta-adrenoceptor antagonists as well as omeprazole, omeprazole enantiomers and the sulfone metabolite of omeprazole were investigated. RESULTS: Whereas some stereoselectivity was detected for the high-affinity P-gp substrates verapamil and carvedilol, little or no differences were observed in the case of other beta-blockers. One of the 4 labetalol stereoisomers, the R,R-isomer dilevalol, had an IC50 value half that of labetalol. CONCLUSIONS: Metabolites of verapamil, gallopamil, carvedilol and omeprazole are characterized by having higher IC50 values (lower P-gp affinity) than the respective parent compounds. Only the acebutolol metabolite, diacetolol, had a P-gp affinity comparable to that of the parent compound.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Role of P-glycoprotein-mediated secretion in absorptive drug permeability: An approach using passive membrane permeability and affinity to P-glycoprotein.

It has been shown in vivo and in vitro that P-glycoprotein (P-gp) may be able to influence the permeability of its substrates across biological membranes. However, the quantitative contribution of the secretion process mediated by P-gp on the overall permeability of membranes has not been determined yet. In particular, observations need to be clarified in which substrates showing high affinity to P-glycoprotein, e.g., verapamil, apparently do not seem to be greatly influenced by P-gp in their permeability and consequently also with respect to their extent of GI-absorption after oral administration, whereas weaker substrates of P-gp, e.g., talinolol, have clearly shown P-gp-related absorption phenomena such as nonlinear intestinal permeability and bioavailability. Experiments with Caco-2 cell monolayers and mathematical simulations based on a mechanistic permeation model should aid in clarifying the underlying mechanism for these observations and quantifying the influence of passive membrane permeability and affinity to P-gp to the overall transmembrane drug flux. In addition, the concentration range of drug at which P-glycoprotein-mediated transport across the biological membrane is relevant should be examined. The permeability of various drugs in Caco-2 monolayers was determined experimentally and modeled using a combination of passive absorptive membrane permeability and a Michaelis-Menten-type transport process in the secretory direction. The passive permeabilities were experimentally obtained for the apical and basolateral membrane by efflux experiments using Caco-2 monolayers in the presence of a P-gp inhibitor. The Michaelis-Menten parameters were determined by a newly developed radioligand-binding assay for the quantification of drug affinity to P-gp. The model was able to accurately simulate the permeability of P-glycoprotein substrates, with differing passive membrane permeabilities and P-glycoprotein affinities. Using the outlined approach, permeability vs donor-concentration profiles were calculated, and the relative contribution of passive and active transport processes to the overall membrane permeability was evaluated. A model is presented to quantitatively describe and predict direction-dependent drug fluxes in Caco-2 monolayers by knowing the affinity of a compound to the exsorptive transporter P-gp and its passive membrane permeability. It was shown that a combination of high P-gp affinity with good passive membrane permeability, e.g., in the case of verapamil, will readily compensate for the P-gp-mediated reduction of intestinal permeability, resulting in a narrow range in which the permeability depends on the apical drug concentration. On the other hand, the permeability of compounds with low passive membrane permeability (e. g., talinolol) might be affected over a wide concentration range despite low affinity to P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Characterization of binding properties to human P-glycoprotein: development of a [3H]verapamil radioligand-binding assay.

Interaction with the exsorptive transporter P-glycoprotein (P-gp) is a possible source of peculiarities in drug pharmacokinetics, including dose-dependent absorption, drug-drug interactions, intestinal secretion, and limited permeability of the blood-brain barrier. Among the established in vitro methods of the analysis of drug interactions with P-gp, none directly quantifies the affinity of ligands with P-gp. Instead, they measure the result of a membrane permeation and a receptor-binding process; this may lead to difficulties in the interpretation of results. An assay for quantification of drug affinity to the transporter is presented on the basis of the radioligand-binding assay principle. This has the advantage of directly quantifying the interaction between drugs and P-gp. Because of the reversible and competitive interaction of numerous substrates with P-gp, a radioligand-binding assay was developed by taking [3H]verapamil and [3H]vinblastine as radioligands and the human intestinal Caco-2 cells, overexpressed with P-gp by culturing in the presence of vinblastine or transfecting with multidrug resistance gene MDR-1 as receptor preparation. The assay was performed in 96-well plates and has the potential to be used as a high-throughput method. A clear induction of the expression of P-gp was demonstrated in the Caco-2 cells grown in the presence of vinblastine, as well as in the transfected cells, although to a lesser extent. Both radioligands were shown to bind to P-gp. Verapamil was the radioligand of choice for further investigations due to its lower nonspecific binding to the transporter preparation. Kinetics as well as specificity of the binding of verapamil to the P-gp preparation were demonstrated. A two-affinity model was found to adequately describe the data derived from saturation as well as from competition experiments, in accordance with previous findings on two exsorption sites for P-gp. The binding properties of [3H]verapamil and [3H]vinblastine to a P-gp preparation derived from induced Caco-2 cells are described. The concentration-dependent displacement of the radioligand by nonlabeled substrates for P-gp should be a suitable principle for the determination of drug affinity to the respective binding sites at the human intestinal multidrug transporter P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

P-Glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels.

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody. Previous drug exposure of the cells showed that verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid (MKA) decreased the P-gp expression level as compared to the control. Permeation studies consolidated the theory that P-gp is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to basolateral could be measured. Incubation of the cell monolayer with MRK 16 reduced the secretion process to the apical side, but did not influence [3H]mannitol flux. Caco-2 cells seem to be a suitable cell line model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Chemical degradation kinetics of recombinant hirudin (HV1) in aqueous solution: effect of pH.

PURPOSE: To gain information on the chemical stability pattern and the kinetics of the degradation of recombinant hirudin variant HV1 (rHir), a thrombin-specific inhibitor protein of 65 amino acids, in aqueous solution as a function of pH. METHODS: Stability of rHir was monitored at 50 degrees C in the framework of a classical pH-stability study in aqueous buffers pH 1-9.5. Two capillary electrophoresis (CE) protocols were used: one for the kinetics of succinimide formation at Asp53-Gly54 (C-terminal tail) and Asp33-Gly34 (loop section), the other for the kinetics of rHir degradation. To check for potential effects of conformational changes by thermal denaturation, circular dichroism (CD) measurements were performed between 25 and 80 degrees C. RESULTS: Throughout the pH range studied no effect of thermal denaturation on rHir confirmation at 50 degrees C was observed. rHir was most stable at a neutral pH whereas, at slightly acidic pH, an intermediate stability plateau was found. Both, strongly acidic and alkaline conditions led to fast rHir degradation. Depending on the pH of degradation, rHir was found to degrade in various combinations of multiple parallel and sequential degradation patterns. Special focus was on succinimide formation at Asp53-Gly54 (C-terminal tail) and Asp33-Gly34 (loop) and on the potential of isoAsp formation in position 53 and 33. CONCLUSIONS: Chemical rHir stability in the intermediate pH range depends strongly on succinimide formation. At slightly acidic conditions succinimides represent the major degradation product (up to 40%). Around neutral pH succinimides react further, presumably by isoAsp formation, and concentrations remain low. Relative preference of succinimide formation in the C-terminal tail domain versus the loop domain is explained by higher backbone flexibility in the tail.

Amino Acid Sequence↗
PubMed1998

Radioligand-binding assay employing P-glycoprotein-overexpressing cells: testing drug affinities to the secretory intestinal multidrug transporter.

PURPOSE: To develop a rapid and reliable system for affinity determination of conventional as well as newly synthesized compounds to P-gp. METHODS: The principles of radioligand-binding assay were adapted to the human intestinal P-gp. Acceptor protein was obtained from the human carcinoma cell line Caco-2, where overexpression of P-gp was induced by growing cells in the presence of the cytostatic drug vinblastine. 3H-Verapamil was chosen as radioligand. RESULTS: The saturability and specificity of 3H-verapamil as the radioligand for the binding to P-gp was demonstrated. From concentration dependence of displacement of the radioligand by various non-labeled ligands for P-gp, affinity constants to P-gp binding sites were calculated. The binding results obtained were in agreement with those published earlier where influx and efflux experiments with cell monolayers had been conducted in order to functionally characterize the P-gp -drug interaction. CONCLUSIONS: A radioligand-binding assay on the basis of P-gp overexpressing Caco-2 cells has been developed. The method might be suitable for high-throughput screening of drug interaction with human P-gp. It will allow modeling of the interaction of drugs with the human multidrug transporter and has also the potential to serve as a high-throughput screening tool to detect compounds prone to P-gp mediated intestinal secretion and potential P-gp related drug/drug interactions in drug discovery and early development.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

First-pass metabolism of peptide drugs in rat perfused liver.

To elucidate the extent and mechanisms of the first-pass metabolism of peptide drugs in the liver after oral administration, a liver perfusion study was performed in rats using metkephamid, a stable analogue of methionine enkephalin, and thyrotropin-releasing hormone (TRH), as model peptides. The fraction of intact metkephamid recovered after single-pass constant perfusion through rat liver reached steady-state very quickly, and it was concluded that metkephamid was hydrolysed enzymatically at the surface of hepatocytes or endothelial cells of microvessels, or both, rather than being taken up by hepatocytes. The fraction of metkephamid recovered intact was approximately 40% under protein-free conditions but increased to 70-75% on addition of bovine serum albumin (BSA) to the perfusate. The fraction of metkephamid bound to BSA was approximately 50% under these conditions, implying that only the free fraction of metkephamid in the plasma was metabolized in the liver. Calculations based on the tube model showed that approximately 30-35% of metkephamid absorbed from the intestine undergoes first-pass metabolism before entering the systemic circulation in-vivo. In contrast, the fraction of TRH metabolized in the liver was less than 10%, indicating a remarkably low contribution of first-pass metabolism to the bioavailability of TRH. These results show that hepatic first-pass metabolism of metkephamid contributes to its low systemic bioavailability. After intestinal absorption free metkephamid is rapidly hydrolysed on the surface of hepatocytes or endothelial cells, rather than being taken up by hepatocytes. This information has important implications in the oral delivery of many kinds of peptide.

Administration, Oral↗