Quality by Design (QbD) Approach for a Nanoparticulate Imiquimod Formulation as an Investigational Medicinal Product.

The present article exemplifies the application of the concept of quality by design (QbD) for the systematic development of a nanoparticulate imiquimod (IMQ) emulsion gel formulation as an investigational medicinal product (IMP) for evaluation in an academic phase-I/II clinical trial for the treatment of actinic keratosis (AK) against the comparator Aldara (EudraCT: 2015-002203-28). The design of the QbD elements of a quality target product profile (QTPP) enables the identification of the critical quality attributes (CQAs) of the drug product as the content of IMQ, the particle-size distribution, the pH, the rheological properties, the permeation rate and the chemical, physical and microbiological stability. Critical material attributes (CMAs) and critical process parameters (CPPs) are identified by using a risk-based approach in an Ishikawa diagram and in a risk-estimation matrix. In this study, the identified CPPs of the wet media ball-milling process's milling time and milling speed are evaluated in a central composite design of experiments (DoEs) approach, revealing criticality for both factors for the resulting mean particle size, while only the milling time is significantly affecting the polydispersity. To achieve a mean particle size in the range of 300-400 nm with a minimal PdI, the optimal process conditions are found to be 650 rpm for 135 min. Validating the model reveals a good correlation between the predicted and observed values. Adequate control strategies were implemented for intermediate products as in-process controls (IPCs) and quality control (QC) tests of the identified CQAs. The IPC and QC data from 13 "IMI-Gel" batches manufactured in adherence to good manufacturing practice (GMP) reveal consistent quality with minimal batch-to-batch variability.

UHPLC Enantiomer Resolution for the ɑ/ß-Adrenoceptor Antagonist R/S-Carvedilol and Its Major Active Metabolites on Chiralpak IB N-5.

Carvedilol (CAR), a racemic lipophilic aryloxy propanolamine, acts as a selective α1-adrenoreceptor antagonist and a nonselective ß-adrenoreceptor antagonist. CAR metabolism mainly produces three active metabolites: desmethyl carvedilol (DMC), 4'-hydroxy carvedilol (4'OHC) and 5'-hydroxy carvedilol (5'OHC). The oxidative S-(-)-metabolites contribute to the ß-antagonistic effect, yet not to the α-antagonistic effect to be observed after drug dosage. Therefore, the three ß-adrenoceptor blocking metabolites, which are structurally closely related to the parent CAR, are included into the development of a bioanalytical quantitative method for all major active species relevant with respect to adrenoceptor-blockade. Because of the given pharmacological profile, resolution of the enantiomers of carvedilol, of 4'- and 5'-hydroxy carvedilol as well as of DMC, is mandatory. The current study aims to determine the response surface for the enantiomer separation of the parent CAR as well as the major metabolites on a suitable chiral stationary phase. Design of experiment approach (DoE) was utilized in an initial screening phase followed by central-composite design for delimitation of the response surface for resolution of the four enantiomeric pairs in least run time. The impact of chromatographic variables (composition and percentage of organic modifier(s), buffer type, buffer pH, flow rate) on critical peaks resolution and adjusted retention time was evaluated, in order to select the most significant critical quality attributes. On this basis, a robust UHPLC-UV method was developed and optimized for the simultaneous, enantioselective determination of CAR along with its major active metabolites (4'OHC, 5'OHC, and DMC) on Chiralpak IBN-5. The optimized UHPLC-UV method (which includes metoprolol as the internal standard) was validated according to the ICH M10 guidelines for bioanalytical methods and proven to be linear, precise, accurate, and robust. The validated assay was applied to plasma samples from cardiovascular patients treated with rac-CAR (blood randomly drawn at different times after oral CAR intake). In order to provide more insight into the mechanism of the enantiomer separation of CAR and its metabolites on the CSP, docking experiments were performed. Molecular simulation studies suggest the chiral recognition to be mainly due to different binding poses of enantiomers of the same compound.

Therapy-relevant aberrant expression of MRP3 and BCRP mRNA in TCC-/SCC-bladder cancer tissue of untreated patients.

Multidrug resistance (MDR) is a critical factor, which results in suboptimal outcomes in cancer chemotherapy. One principal mechanism of MDR is the increased expression of ATP-binding cassette (ABC) transporters. Of these, multidrug resistance-associated protein 3 (MRP3) and breast cancer resistance protein (BCRP) confer MDR when overexpressed in cancer cell lines. We measured the mRNA expression of MRP3 and BCRP in primary untreated bladder cancer specimens using reverse transcription-quantitative PCR (RT-qPCR) in comparison to normal bladder tissue. The MRP3 and BCRP expression in the two major histotypes of bladder cancer; transitional cell carcinoma (TCC; urothelial type of bladder cancer) and squamous cell carcinoma (SCC; 'Schistosoma-induced' bladder cancer) were compared. Furthermore, the association between MRP3 and BCRP expression and tumor grade and stage were investigated. MRP3 mRNA expression in bladder cancer specimens was increased ~13-fold on average compared to normal bladder tissue (n=36, P<0.0001). BCRP mRNA expression was decreased in bladder cancer specimens to ~1/5 on average, compared to normal bladder tissue (n=38, P<0.0001). TCC showed significantly increased MRP3 mRNA expression compared to SCC of the bladder (P<0.0001). BCRP mRNA expression was similar in TCC and SCC of the bladder (P=0.1072). The increased MRP3 mRNA expression was not related to bladder tumor grade (P=0.3465) but was, however, significantly higher in superficial than in invasive bladder tumors (P=0.0173). The decreased expression of BCRP was not related to bladder tumor grade (P=0.1808) or stage (P=0.8016). The current data show that bladder cancer is associated with perturbed expression of MRP3 and BCRP. Representing drug resistance factors, determining the expression of these transporters in native tumors may be predictive of the outcome of chemotherapy based-treatment of bladder cancer.

Circadian variations in exsorptive transport: in situ intestinal perfusion data and in vivo relevance.

The circadian timing system (CTS) governs the 24-h rhythm of the organism and, hence, also main pathways responsible for drug pharmacokinetics. P-glycoprotein (P-gp) is a drug transporter that plays a pivotal role in drug absorption, distribution, and elimination, and temporal changes in its activity may affect input, output, activity, and toxicity profile of drugs. In the current study, the influence of different circadian stages on the overall intestinal permeability (P(eff)) of the P-gp substrates talinolol and losartan was evaluated in in situ intestinal perfusion studies in rats. Additionally, in vivo studies in rats were performed by employing the P-gp probe talinolol during the day (nonactive) and night (active) period in rats. Effective intestinal permeabilities of talinolol and losartan were smaller in studies performed during the night (p < .05), indicating that P-gp-dependent intestinal secretion is greater during the nighttime activity span than daytime rest span of the animals. P-gp modulators vinblastine and PSC833 led to a significant decrease of talinolol and losartan exsorption in the intestinal segments as compared with control groups. Strikingly, the permeability-enhancing effect of vinblastine and PSC833 was higher with night perfusions, for both talinolol and losartan. In vivo studies performed with talinolol revealed-consistent with the in situ studies (P(eff) day > night)-a day vs. night difference in the oral availability of talinolol in the group of male rats in terms of the area under the curve (AUC) data (AUC(day) > AUC(night)). The P-gp modulator vinblastine significantly increased talinolol AUC(day) (p < .05), whereas only a weak vinblastine effect was seen in night. According to the in situ data, the functional activity of P-gp was regulated by the CTS in jejunum and ileum, which are major intestinal segments for energy-dependent efflux. In conclusion, circadian rhythms may affect carrier-mediated active efflux and play a role in the absorption process. In addition to daily rhythms in P-gp activity in rat intestine, the in vivo studies indicate that absorption-, distribution-, metabolism-, and elimination-relevant rhythms may be involved in the circadian kinetics of the drug, besides transporter-dependent efflux, such well-known aspects as metabolic or renal clearance or motility. Since this also holds true for a potentially interacting second compound (modulator), modulator effects should be evaluated carefully in transporter related drug-drug interactions.

Baclofen ester and carbamate prodrug candidates: a simultaneous chromatographic assay, resolution optimized with DryLab.

Baclofen exhibits insufficient CNS-availability when dosed systemically. Hence, prodrug candidates (methyl, ethyl, 1-propyl, 2-propyl and butyl 4-(tert-butoxycarbonyl amino)-3-(4-chlorophenyl) butanoate) were synthesized aiming at CNS-levels appropriate for the treatment of spastic disorders. The characterization of some biopharmaceutically highly relevant physicochemical properties (LogP and aqueous solubility) and the evaluation of biophase levels represent one important component of the project. The overall research aim was to generate an HPLC optimized method using DryLab, a simulation software for the optimization of a RP-HPLC method, which was optimized using a simulation software (DryLab), for the simultaneous determination of baclofen and ten synthesized prodrug candidates. The chromatographic resolution predicted and obtained via the simulation is Rs >1.5 for all baclofen derivatives, as well as, with parent baclofen. The method was used to assay the prodrugs and determine their purities, solubility and lipophilicity parameters. The designed analytical method also permits the tracking of the new prodrug candidates' hydrolysis in vitro and in vivo. The determined physicochemical properties indicate for some of the compounds that they might be suitable for CNS-targeting which was exemplified by the detection of significant baclofen levels in rat brain tissues following an i.p. dose of ethyl carbamate (vs. ethyl ester, for which only traces of baclofen were detected).

Expression of RFC/SLC19A1 is associated with tumor type in bladder cancer patients.

Urinary bladder cancer (UBC) ranks ninth in worldwide cancer. In Egypt, the pattern of bladder cancer is unique in that both the transitional and squamous cell types prevail. Despite much research on the topic, it is still difficult to predict tumor progression, optimal therapy and clinical outcome. The reduced folate carrier (RFC/SLC19A1) is the major transport system for folates in mammalian cells and tissues. RFC is also the primary means of cellular uptake for antifolate cancer chemotherapeutic drugs, however, membrane transport of antifolates by RFC is considered as limiting to antitumor activity. The purpose of this study was to compare the mRNA expression level of RFC/SLC19A1 in urothelial and non-urothelial variants of bladder carcinomas. Quantification of RFC mRNA in the mucosa of 41 untreated bladder cancer patients was performed using RT-qPCR. RFC mRNA steady-state levels were ∼9-fold higher (N = 39; P<0.0001) in bladder tumor specimens relative to normal bladder mRNA. RFC upregulation was strongly correlated with tumor type (urothelial vs. non-urothelial; p<0.05) where median RFC mRNA expression was significantly (p<0.05) higher in the urothelial (∼14-fold) compared to the non-urothelial (∼4-fold) variant. This may account for the variation in response to antifolate-containing regimens used in the treatment of either type. RFC mRNA levels were not associated with tumor grade (I, II and III) or stage (muscle-invasive vs. non-muscle invasive) implying that RFC cannot be used for prognostic purposes in bladder carcinomas and its increased expression is an early event in human bladder tumors pathogenesis. Further, RFC can be considered as a potential marker for predicting response to antifolate chemotherapy in urothelial carcinomas.

Bidirectional membrane transport: simulations of transport inhibition in uptake studies explain data obtained with flavonoids.

The purpose of the simulations was to obtain an estimate of concentration-dependent uptake curves when two counteracting transporters are present. On the basis of this experimental data obtained with a pair of ovarian carcinoma cell lines, one of which was not expressing the exsorptive transporter P-glycoprotein and one of which was an MDR1-transfected, P-glycoprotein expressing variant, the kinetics of cellular uptake of the radiolabel (3)H-talinolol were calculated and the inhibitory constants at P-gp were determined for different flavonoids. With respect to the inhibition of P-gp function, among others, naringenin and isoquercitrin were identified as inhibitors, yet estimation of the inhibitory constant was only possible for uptake values corrected for non-P-glycoprotein-mediated processes. It was assumed that an additional inside-directed transporting protein (Carrier B), which is inhibited by the presence of test compounds, uptake of radiolabel was simulated as a function of the concentration of test-compound, with exemplary parameters for the rate constant (k(B)) of the additional Carrier B and the inhibition constants (K(I)-values) for both transporting proteins. The obtained uncorrected experimental data, which showed either inhibition or enhancement of radiolabel uptake as a function of the inhibitor concentration, were appropriately explained by the respective model. The respective model included an exsorptive transporter as well as carrier-mediating facilitated diffusion. It is concluded that flavonoids, such as naringenin and isoquercitrin, inhibit an inside-directed process in addition to their inhibition of P-glycoprotein-mediated exsorption.

In silico modeling of non-linear drug absorption for the P-gp substrate talinolol and of consequences for the resulting pharmacodynamic effect.

PURPOSE: The aim of the present work was to demonstrate P-glycoprotein's involvement in the non-linear talinolol pharmacokinetics using an advanced compartment and transit model (ACAT) and to compare the results predicted from the model to the finding of a phase I dose escalation study with oral talinolol doses increasing from 25 to 400 mg. MATERIALS AND METHODS: Besides minimum input parameters for the compound (pKa(s), solubility at one or more pH's, Peff, doses, formulation, diffusivity), physiological and pharmacokinetic properties, transporter data are included in these predictions. The simulations assumed higher expression levels in lower gastrointestinal regions, in particular in the colon, which is in accordance with the results of intestinal rat perfusion studies and intestinal distribution data from rats, catfishes, micropigs and humans reported in the literature. Optimized values for P-glycoprotein (P-gp) Km and Vmax were used for the final simulation results and for a stochastic virtual trial with 12 patients. RESULTS: Talinolol, a P-gp substrate, exhibits non-linear dose AUC relationship after administration of 25, 50, 100 and 400 mg immediate-release tablets. This dose dependency is due to a decrease of efflux transport caused by saturation of P-gp by talinolol. It was found that oral bioavailability increases after administration of higher doses of talinolol. The predicted bioavailability of the p.o. 25, 50, 100 and 400 mg doses of talinolol was 64, 76, 85, 94%, respectively. Pharmacokinetic parameters (AUC, Cmax) from in silico simulations are within acceptable range comparing with data, observed in vivo. However, the in vitro value of Km for talinolol's interactions with P-gp could not be used in the simulation and still reproduce the observed non-linear dose dependence. For each of the four doses, GastroPlus was used to model pharmacodynamic (PD) response and to optimize the values of CLe, Emax, and EC5o with the effect compartment linked indirectly to the central compartment. For all simulations, EC50 was 114 nM and E0 was 83 bpm. CONCLUSION: Comparison between the results of the in vivo study and the in silico simulations determined the quality and reliability of the in silico predictions and demonstrate the simulation of dose dependent absorption. In contrast to previous simulation work for the non-linear dose dependence of interaction with intestinal transporters or enterocyte metabolism, optimized Km and Vmax values were required to reproduce the clinically observed non-linear dose dependence. The model developed may be useful in the prediction of absorption of other P-gp substrates including pharmacodynamic consequences.

Modulation of drug transport by selected flavonoids: Involvement of P-gp and OCT?

Flavonoids, as a common component of daily nutrition, are a possible source of interference with absorption processes, due to modulation of transporting proteins. In this study, the influence of selected flavonoids (quercetin, isoquercitrin, spiraeoside, rutin, kaempferol, naringenin, naringin, and kaempferol) on the transport of the P-gp substrate [3H]talinolol across Caco-2 cell monolayers was investigated. To elucidate the mechanism behind the interaction observed in this system the potency of the flavonoids to replace [3H]talinolol from its P-gp binding site as well as their activity to inhibit OCT2-mediated [14C]TEA uptake into LLC-PK(1) cells were measured, as P-gp and OCT have been shown to be present in Caco-2 cells. Six of the investigated flavonoids reduced the secretory flux of talinolol across Caco-2 cells (IC50-values: hesperetin

Selective downregulation of the MDR1 gene product in Caco-2 cells by stable transfection to prove its relevance in secretory drug transport.

Considerable interest is focused on overcoming multidrug resistance (MDR) in cancer chemotherapy. The in vitro experiments to characterize P-glycoprotein's (P-gp) function and to decrease its effects have led to a variety of strategies such as addition of competitors or supplementation of the medium with oligonucleotides complementary to the 5'-end of the MDR1-mRNA. For the Caco-2 cell line, an in vitro model for absorption screening, expressing multiple transporters including P-gp, which pumps substances back into the apical solution, P-gp activity might mask other relevant transport proteins' activity. The objective of the present study was to construct a Caco-2 subline with reduced P-gp expression level. Caco-2 cells were transfected by electroporation with two different mammalian expression vectors, and the obtained subclones were investigated at RNA (Northern blotting, RT-PCR), protein (FACS analysis), and functional (transport studies) levels for reduction in P-gp expression. Northern blotting showed that the levels of transcription of the inserted gene were different among the several clones, but those results did not completely correlate with the FACS analysis for P-gp expression. The clones with the strongest reduction in P-gp expression detected by the FACS analysis also showed the lowest secretory fluxes of the P-gp substrate talinolol in transport studies. Repetition of FACS analysis after 7 and 24 months on 20 to 30 passage older subclones still showed reduction in P-gp expression and indicated that they are stably transfected. The new cell lines constructed in the present study provide the possibility to perform in vitro absorption studies in a cell system composed of differentiated enterocytes growing as a monolayer like the normal Caco-2 cell line but with a lower down to almost lacking expression of P-gp.

Lipophilicities of baclofen ester prodrugs correlate with affinities to the ATP-dependent efflux pump P-glycoprotein: relevance for their permeation across the blood-brain barrier?

PURPOSE: Distribution to the effect site is a prerequisite for the therapeutic effect and determined by physicochemical properties and affinities to inside- and outside-directed membrane transporters. Based on the hypothesis that lipophilic esters of the GABA-derivative baclofen have a higher affinity to brain tissue, baclofen esters (methyl, ethyl, 1-propyl, 2-propyl, butyl) were studied regarding their penetration through the blood-brain barrier and their affinities to P-glycoprotein (P-gp). METHODS: Octanol-water distribution coefficients (D) served as lipophilicity parameters. Blood and brain concentrations of baclofen and its methyl ester were determined in vivo in rats following intraperitoneal administration. Affinities to P-gp were evaluated using a radioligand binding assay based on P-gp-overexpressing cells and [3H]-talinolol as radioligand. RESULTS: Log D values for baclofen and ester derivatives were -0.96 (baclofen), 0.48 (methyl), 0.77 (ethyl), 1.31 (1-propyl), 1.27 (2-propyl), and 1.42 (butyl). In-vitro studies yielded negligible affinity of baclofen to P-gp, whereas IC50-values for the esters ranged between 1300 microM (methyl) and 290 microM (2-propyl). Affinity parameters correlated well with the lipophilicity parameters. CONCLUSION: Despite the P-gp affinity, brain concentrations of methyl ester were significantly higher than those of baclofen, however, baclofen levels following administration of the ester were smaller than with baclofen administration indicating only partial hydrolysis.