Usefulness of the Beagle Model in the Evaluation of Paracetamol and Ibuprofen Exposure after Oral Administration to Pediatric Populations: An Exploratory Study.

The present study aimed to explore the usefulness of beagle dogs in combination with physiologically based pharmacokinetic (PBPK) modeling in the evaluation of drug exposure after oral administration to pediatric populations at an early stage of pharmaceutical product development. An exploratory, single-dose, crossover bioavailability study in six beagles was performed. A paracetamol suspension and an ibuprofen suspension were coadministered in the fasted-state conditions, under reference-meal fed-state conditions, and under infant-formula fed-state conditions. PBPK models developed with GastroPlus v9.7 were used to inform the extrapolation of beagle data to human infants and children. Beagle-based simulation outcomes were compared with published human-adult-based simulations. For paracetamol, fasted-state conditions and reference-meal fed-state conditions in beagles appeared to provide adequate information for the applied scaling approach. Fasted-state and/or reference-meal fed-state conditions in beagles appeared suitable to simulate the performance of ibuprofen suspension in pediatric populations. Contrary to human-adult-based translations, extrapolations based on beagle data collected under infant-formula fed-state conditions appeared less useful for informing simulations of plasma levels in pediatric populations. Beagle data collected under fasted and/or reference-meal fed-state conditions appeared to be useful in the investigation of pediatric product performance of the two investigated highly permeable and highly soluble drugs in the upper small intestine. The suitability of the beagle as a preclinical model to understand pediatric drug product performance under different dosing conditions deserves further evaluation with a broader spectrum of drugs and drug products and comparisons with pediatric in vivo data.

Osmolality of Excipients for Parenteral Formulation Measured by Freezing Point Depression and Vapor Pressure - A Comparative Analysis.

PURPOSE: To investigate the difference in methods to determine the osmolality in solutions of stabilizers used for long-acting injectable suspensions. METHODS: The osmolality was measured by freezing point depression and vapor pressure for 11 different polymers and surfactants (PEG 3350, 4000, 6000, 8000, 20,000, PVP K12, K17 and K30, poloxamer 188, 388 and 407, HPMC E5, Na-CMC, polysorbate 20 and 80, vitamin E-TPGS, phospholipid, DOSS and SDS) in different concentrations. RESULTS: Independently of the measuring method, an increase in osmolality with increasing concentration was observed for all polymers and surfactants, as would be expected due to the physicochemical origin of the osmolality. No correlation was found between the molecular weight of the polymers and the measured osmolality. The osmolality values were different for PVPs, PEGs, and Na-CMC using the two different measurement methods. The values obtained by the freezing point depression method tended to be similar or higher than the ones provided by vapor pressure, overall showing a significant difference in the osmolality measured by the two investigated methods. CONCLUSIONS: For lower osmolality values (e.g. surfactants), the choice of the measuring method was not critical, both the freezing point depression and vapor pressure could be used. However, when the formulations contained higher concentrations of excipients and/or thermosensitive excipients, the data suggests that the vapor pressure method would be more suited.

In Silico, In Vitro, and In Vivo Evaluation of Precipitation Inhibitors in Supersaturated Lipid-Based Formulations of Venetoclax.

The concept of using precipitation inhibitors (PIs) to sustain supersaturation is well established for amorphous formulations but less in the case of lipid-based formulations (LBF). This study applied a systematic in silico-in vitro-in vivo approach to assess the merits of incorporating PIs in supersaturated LBFs (sLBF) using the model drug venetoclax. sLBFs containing hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), polyvinylpyrrolidone (PVP), PVP-co-vinyl acetate (PVP/VA), Pluronic F108, and Eudragit EPO were assessed in silico calculating a drug-excipient mixing enthalpy, in vitro using a PI solvent shift test, and finally, bioavailability was assessed in vivo in landrace pigs. The estimation of pure interaction enthalpies of the drug and the excipient was deemed useful in determining the most promising PIs for venetoclax. The sLBF alone (i.e., no PI present) displayed a high initial drug concentration in the aqueous phase during in vitro screening. sLBF with Pluronic F108 displayed the highest venetoclax concentration in the aqueous phase and sLBF with Eudragit EPO the lowest. In vivo, the sLBF alone showed the highest bioavailability of 26.3 ± 14.2%. Interestingly, a trend toward a decreasing bioavailability was observed for sLBF containing PIs, with PVP/VA being significantly lower compared to sLBF alone. In conclusion, the ability of a sLBF to generate supersaturated concentrations of venetoclax in vitro was translated into increased absorption in vivo. While in silico and in vitro PI screening suggested benefits in terms of prolonged supersaturation, the addition of a PI did not increase in vivo bioavailability. The findings of this study are of particular relevance to pre-clinical drug development, where the high in vivo exposure of venetoclax was achieved using a sLBF approach, and despite the perceived risk of drug precipitation from a sLBF, including a PI may not be merited in all cases.

Lipid Based Formulations in Hard Gelatin and HPMC Capsules: a Physical Compatibility Study.

PURPOSE: To investigate the compatibility between hard gelatin and HPMC capsules with a range of different isotropic lipid based formulations containing multiple excipients. METHODS: The miscibility was investigated for 350 systems applying five different oils (Labrafac ™ lipophile WL1349, Maisine® CC, Captex 300 EP/NF, olive oil, and Capmul MCM EP/NF), five different surfactans (Labrasol ® ALF, Labrafil M 2125 CS, Kolliphor ® ELP, Kolliphor ® HS 15, Tween 80) and three different cosolvents (propylene glycol, polyethylene glycol 400, and Transcutol ® HP). For the isotropic systems capsule compatibility was investigated in both gelatin and HPMC capsules at 25°C at 40% and 60% relative humidity by examining physical damages to the capsules and weight changes after storage. RESULTS: The miscibility of lipid based vehicles was best when the formulation contained monoglycerides and surfactants with a hydrophilic-lipophilic balance value <12. Gelatin capsules in general resulted in a better compatibility when compared to HPMC capsules for the evaluated formulations. Addition of water to the formulation improved the capsule compatibility for both capsule types. The expected capsule mass change could partly be predicted in binary systems using the provided data of the single excipients weighted for its formulation proportion. CONCLUSIONS: The capsule compatibility was driven by the components incorporated into the formulations, where more was compatible with gelatin than HPMC capsules. Prediction of the mass change from individual excipient contributions can provide a good first estimate if a vehicle is compatible with a capsule, however, this needs to be proved experimentally.

Exploring the Impact of Surfactant Type and Digestion: Highly Digestible Surfactants Improve Oral Bioavailability of Nilotinib.

The scientific rationale for selection of the surfactant type during oral formulation development requires an in-depth understanding of the interplay between surfactant characteristics and biopharmaceutical factors. Currently, however, there is a lack of comprehensive knowledge of how surfactant properties, such as hydrophilic-lipophilic balance (HLB), digestibility, and fatty acid (FA) chain length, translate into in vivo performance. In the present study, the relationship between surfactant properties, in vitro characteristics, and in vivo bioavailability was systematically evaluated. An in vitro lipolysis model was used to study the digestibility of a variety of nonionic surfactants. Eight surfactants and one surfactant mixture were selected for further analysis using the model poorly water-soluble drug nilotinib. In vitro lipolysis of all nilotinib formulations was performed, followed by an in vivo pharmacokinetic evaluation in rats. The in vitro lipolysis studies showed that medium-chain FA-based surfactants were more readily digested compared to long-chain surfactants. The in vivo study demonstrated that a Tween 20 formulation significantly enhanced the absolute bioavailability of nilotinib up to 5.2-fold relative to an aqueous suspension. In general, surfactants that were highly digestible in vitro tended to display higher bioavailability of nilotinib in vivo. The bioavailability may additionally be related to the FA chain length of digestible surfactants with an improved exposure in the case of medium-chain FA-based surfactants. There was no apparent relationship between the HLB value of surfactants and the in vivo bioavailability of nilotinib. The impact of this study's findings suggests that when designing surfactant-based formulations to enhance oral bioavailability of the poorly water-soluble drug nilotinib, highly digestible, medium chain-based surfactants are preferred. Additionally, for low-permeability drugs such as nilotinib, which is subject to efflux by intestinal P-glycoprotein, the biopharmaceutical effects of surfactants merit further consideration.

In Vivo Performance of Innovative Polyelectrolyte Matrices for Hot Melt Extrusion of Amorphous Drug Systems.

Hot melt extrusion of amorphous systems has become a pivotal technology to cope with challenges of poorly water-soluble drugs. Previous research showed that small molecular additives with targeted molecular interactions enabled introduction of a polyelectrolyte matrix into hot melt extrusion that would otherwise not be possible to process due to the unfavorable properties upon heating of the pure polymer. Carboxymethyl cellulose sodium (NaCMC) with lysine or alternatively meglumine led to modified polymeric matrices that showed adequate processability by hot melt extrusion and yielded stable amorphous formulations. The investigated formulations, including fenofibrate as a model drug, were characterized by attenuated total reflectance Fourier transform infrared spectroscopy, differential scanning calorimetry, and viscosity measurements after aqueous dispersion. Further biopharmaceutical assessment started with biorelevant nonsink dissolution testing followed by a pharmacokinetic in vivo study in rats. The in vitro assessment showed superiority of the lysine-containing formulation in the extent of in vitro supersaturation and overall drug release. In accordance with this, the in vivo study also demonstrated increased exposure of the amorphous formulations and in particular for the system containing lysine. In summary, the combination of polyelectrolytes with interacting additives presents a promising opportunity for the formulation of poorly water-soluble drugs.

Novel Biphasic Lipolysis Method To Predict in Vivo Performance of Lipid-Based Formulations.

The absence of an intestinal absorption sink is a significant weakness of standard in vitro lipolysis methods, potentially leading to poor prediction of in vivo performance and an overestimation of drug precipitation. In addition, the majority of the described lipolysis methods only attempt to simulate intestinal conditions, thus overlooking any supersaturation or precipitation of ionizable drugs as they transition from the acidic gastric environment to the more neutral conditions of the intestine. The aim of this study was to develop a novel lipolysis method incorporating a two-stage gastric-to-intestinal transition and an absorptive compartment to reliably predict in vivo performance of lipid-based formulations (LBFs). Drug absorption was mimicked by in situ quantification of drug partitioning into a decanol layer. The method was used to characterize LBFs from four studies described in the literature, involving three model drugs (i.e., nilotinib, fenofibrate, and danazol) where in vivo bioavailability data have previously been reported. The results from the novel biphasic lipolysis method were compared to those of the standard pH-stat method in terms of reliability for predicting the in vivo performance. For three of the studies, the novel biphasic lipolysis method more reliably predicted the in vivo bioavailability compared to the standard pH-stat method. In contrast, the standard pH-stat method was found to produce more predictive results for one study involving a series of LBFs composed of the soybean oil, glyceryl monolinoleate (Maisine CC), Kolliphor EL, and ethanol. This result was surprising and could reflect that increasing concentrations of ethanol (as a cosolvent) in the formulations may have resulted in greater partitioning of the drug into the decanol absorptive compartment. In addition to the improved predictivity for most of the investigated systems, this biphasic lipolysis method also uses in situ analysis and avoids time- and resource-intensive sample analysis steps, thereby facilitating a higher throughput capacity and biorelevant approach for characterization of LBFs.

Chase Dosing of Lipid Formulations to Enhance Oral Bioavailability of Nilotinib in Rats.

PURPOSE: Lipid-based formulations (LBF) have shown oral bioavailability enhancement of lipophilic drugs, but not necessarily in the case of hydrophobic drugs. This study explored the potential of lipid vehicles to improve the bioavailability of the hydrophobic drug nilotinib comparing a chase dosing approach and lipid suspensions. METHODS: Nilotinib in vivo bioavailability in rats was determined after administering an aqueous suspension chase dosed with blank olive oil, Captex 1000, Peceol or Capmul MCM, respectively. Absolute bioavailability was determined (relative to an intravenous formulation). Pharmacokinetic parameters were compared to lipid suspensions. RESULTS: Compared to the lipid suspensions, the chase dosed lipids showed a 2- to 7-fold higher bioavailability. Both long chain chase dosed excipients also significantly increased the bioavailability up to 2-fold compared to the aqueous suspension. Deconvolution of the pharmacokinetic data indicated that chase dosing of nilotinib resulted in prolonged absorption compared to the aqueous suspension. CONCLUSION: Chase dosed LBF enhanced the in vivo bioavailability of nilotinib. Long chain lipids showed superior performance compared to medium chain lipids. Chase dosing appeared to prolong the absorption phase of the drug. Therefore, chase dosing of LBF is favourable compared to lipid suspensions for 'brick dust' molecules such as nilotinib. Graphical Abstract The potential of bio-enabling lipid vehicles, administered via chase dosing and lipid suspensions, has been evaluated as an approach to enhance oral bioavailability of nilotinib.

Transcriptome analysis identifies activated signaling pathways and regulated ABC transporters and solute carriers after hyperosmotic stress in renal MDCK I cells.

Hyperosmolality is found under physiological conditions in the kidneys, whereas hyperosmolality in other tissues may be associated with pathological conditions. In such tissues an association between inflammation and hyperosmolality has been suggested. During hyperosmotic stress, an important phenomenon is upregulation of solute carriers (SLCs). We hypothesize that hyperosmolality affects the expression of many SLCs as well as ABC transporters. Through RNA-sequencing and topological pathway analysis, the cell cycle, the cytokine-cytokine receptor interaction pathway, and the chemokine-signaling pathway were significantly activated in MDCK I cells after hyperosmotic treatment (Δ200 mOsm) with raffinose or NaCl. 9065, 8052 and 5018 genes were significantly regulated by raffinose, NaCl or urea supplementation (500 mOsm), respectively, compared to control (300 mOsm). Cytokines, that have not previously been associated with hyperosmolality, were identified. We further provide an overview of transport proteins that could be of relevance in tissues exposed to hyperosmolality. Especially Slc5a8 was found highly up-regulated.

Supersaturated lipid-based drug delivery systems - exploring impact of lipid composition type and drug properties on supersaturability and physical stability.

Objective: The objective of this study was to systematically investigate the impact of lipid composition on the ability to design supersaturated lipid-based drug delivery systems (sLBDDS) using three model drugs with different physico-chemical properties.Significance: This study expands the list of investigated sLBDDS by using alternative vehicle compositions relative to current literature.Methods and results: Drug supersaturation was thermally-induced based on previously reported methods and was successfully achieved for celecoxib and cinnarizine. For the novel drug, JNJ-2A, a lower supersaturation potential was observed for the tested LBDDS. For celecoxib and cinnarizine, crystalline precipitate was observed for some sLBDDS upon storage at 25 °C/65%RH, particularly for medium chain sLBDDS (celecoxib) and long chain sLBDDS (cinnarizine). The greater risk of precipitation observed for celecoxib and cinnarizine, particularly at higher apparent degree of supersaturation (aDS) may be related to their higher crystallization tendency as determined by differential scanning calorimetry.Conclusions: The potential for supersaturation in LBDDS, and the risk of precipitation, was found to be highly drug dependent. The apparent degree of supersaturation was considered a major factor impacting the ability to maintain drug supersaturation upon storage.

Toward simplified oral lipid-based drug delivery using mono-/di-glycerides as single component excipients.

OBJECTIVE: This study aimed to systematically explore compositional effects for a series of lipid systems, on the in vitro drug solubilization and in vivo bioavailability of three poorly water-soluble drugs with different physico-chemical properties. SIGNIFICANCE: While many lipid-based drug products have successfully reached the market, there is still a level of uncertainty on the design guidelines for such drug products with limited understanding on the influence of composition on in vitro and in vivo performance. METHODS AND RESULTS: Lipid-based drug delivery systems were prepared using either single excipient systems based on partially digested triglycerides (i.e. mono- and/or di-glycerides) or increasingly complex systems by incorporating surfactants and/or triglycerides. These lipid systems were evaluated for both in vitro and in vivo behavior. Results indicated that simple single component long chain lipid systems are more beneficial for the absorption of the weak acid celecoxib and the weak base cinnarizine compared to equivalent single component medium chain lipid systems. Similarly, a two-component system produced by incorporating small amount of hydrophilic surfactant yields similar overall pharmacokinetic effects. The lipid drug delivery systems based on medium chain lipid excipients improved the in vivo exposure of the neutral drug JNJ-2A. The higher in vivo bioavailability of long chain lipid systems compared to medium chain lipid systems was in agreement with in vitro dilution and dispersion studies for celecoxib and cinnarizine. CONCLUSIONS: The present study demonstrated the benefits of using mono-/di-glycerides as single component excipients in LBDDS to streamline formulation screening and improve oral bioavailability for the three tested poorly water-soluble drugs.

Modified Polymer Matrix in Pharmaceutical Hot Melt Extrusion by Molecular Interactions with a Carboxylic Coformer.

Hot melt extrusion (HME) has become an essential technology to cope with an increasing number of poorly soluble drug candidates. However, there is only a limited choice of pharmaceutical polymers for obtaining suitable amorphous solid dispersions (ASD). Considerations of miscibility, stability, and biopharmaceutical performance narrow the selection of excipients, and further technical constraints arise from needed pharmaceutical processing. The present work introduces the concept of molecularly targeted interactions of a coformer with a polymer to design a new matrix for HME. Model systems of dimethylaminoethyl methacrylate copolymer, Eudragit E (EE), and bicarboxylic acids were studied, and pronounced molecular interactions were demonstrated by 1H, 13C NMR, FTIR spectroscopy, as well as by different techniques of microscopic imaging. A difference was shown between new formulations exploiting specifically the targeted molecular interactions and a common drug-polymer formulation. More specifically, a modified matrix with malic acid exhibited a technical extrusion advantage over polymer alone, and there was a benefit of improved physical stability revealed for the drug fenofibrate. This model compound displayed greatly enhanced dissolution kinetics from the ASD formulations. It can be concluded that harnessing molecularly designed polymer modifications by coformers has much potential in solid dispersion technology and in particular regarding HME processing.

New Insights into Using Lipid Based Suspensions for 'Brick Dust' Molecules: Case Study of Nilotinib.

PURPOSE: Lipid suspensions have been shown to be a suitable bio-enabling formulation approach for highly lipophilic or 'grease ball' drug molecules, but studies on 'brick dust' drugs are lacking. This study explored the utility of lipid suspensions for enhancing oral bioavailability of the rather hydrophobic drug nilotinib in vivo in rats. METHODS: Four lipid suspensions were developed containing long chain triglycerides, medium chain triglyceride, long chain monoglycerides and medium chain monoglycerides and in vivo bioavailability was compared to an aqueous suspension. Additionally, in vitro lipolysis and wettability tests were conducted. RESULTS: Nilotinib lipid suspensions did not show a bioavailability increase compared to an aqueous suspension. The bioavailability was lower for triglyceride suspensions, relative to both monoglyceride and an aqueous suspension. The long chain monoglyceride displayed a significantly higher bioavailability relative to triglycerides. In vitro lipolysis results suggested entrapment of nilotinib crystals within poorly dispersible triglycerides, leading to slower nilotinib release and absorption. This was further supported by higher wettability of nilotinib by lipids. CONCLUSION: Monoglycerides improved oral bioavailability of nilotinib in rats, relative to triglycerides. For 'brick dust' drugs formulated as lipid suspensions, poorly dispersible formulations may delay the release of drug crystals from the formulation leading to reduced absorption. Graphical Abstract An aqueous and four lipid suspensions have been evaluated in in vitro and in vivo to gain insights into the potential benefits and limitations of lipid suspensions.

Montmorillonite and Laponite Clay Materials for the Solidification of Lipid-Based Formulations for the Basic Drug Blonanserin: In Vitro and in Vivo Investigations.

Solid-state lipid-based formulations offer great potential for the improved oral delivery of poorly water-soluble drugs. This study investigates the use of the high-surface-area clay materials, montmorillonite and laponite, as solid carriers for lipid-based formulations. The unique cation-exchange properties of clay platelets were exploited to preload the ionizable hydrophobic compound, blonanserin, prior to encapsulating a drug-loaded lipid solution. Thus, solid-state lipid-based formulations with dual-loading capabilities were developed and studied. These formulations were compared with simple clay-based lipid formulations, where blonanserin was loaded in the lipid phase only. The drug release behavior of all clay-based formulations was assessed during in vitro dissolution studies under simulated gastric conditions and in vitro fasting intestinal lipolysis studies. Montmorillonite- and laponite-based lipid formulations significantly reduced blonanserin solubilization relative to a control lipid solution and silica-lipid hybrid particles, owing to incomplete drug release from the clay cation-exchange sites. This phenomenon was replicated during in vivo pharmacokinetic studies, whereby the bioavailability of simple clay-based lipid formulations was decreased relative to controls. Importantly, the solid-state dual-loaded montmorillonite-based lipid formulation provided an optimal pharmacokinetic performance, achieving the same degree of bioavailability enhancement as the control lipid solution. These findings indicate the potential of solid-state dual-loaded clay-based lipid formulations for increasing drug loading levels and enhancing the oral absorption of poorly soluble weak base compounds.

Injectable anti-malarials revisited: discovery and development of new agents to protect against malaria.

Over the last 15 years, the majority of malaria drug discovery and development efforts have focused on new molecules and regimens to treat patients with uncomplicated or severe disease. In addition, a number of new molecular scaffolds have been discovered which block the replication of the parasite in the liver, offering the possibility of new tools for oral prophylaxis or chemoprotection, potentially with once-weekly dosing. However, an intervention which requires less frequent administration than this would be a key tool for the control and elimination of malaria. Recent progress in HIV drug discovery has shown that small molecules can be formulated for injections as native molecules or pro-drugs which provide protection for at least 2 months. Advances in antibody engineering offer an alternative approach whereby a single injection could potentially provide protection for several months. Building on earlier profiles for uncomplicated and severe malaria, a target product profile is proposed here for an injectable medicine providing long-term protection from this disease. As with all of such profiles, factors such as efficacy, cost, safety and tolerability are key, but with the changing disease landscape in Africa, new clinical and regulatory approaches are required to develop prophylactic/chemoprotective medicines. An overall framework for these approaches is suggested here.

Validation of Dissolution Testing with Biorelevant Media: An OrBiTo Study.

Dissolution testing with biorelevant media has become widespread in the pharmaceutical industry as a means of better understanding how drugs and formulations behave in the gastrointestinal tract. Until now, however, there have been few attempts to gauge the reproducibility of results obtained with these methods. The aim of this study was to determine the interlaboratory reproducibility of biorelevant dissolution testing, using the paddle apparatus (USP 2). Thirteen industrial and three academic laboratories participated in this study. All laboratories were provided with standard protocols for running the tests: dissolution in FaSSGF to simulate release in the stomach, dissolution in a single intestinal medium, FaSSIF, to simulate release in the small intestine, and a "transfer" (two-stage) protocol to simulate the concentration profile when conditions are changed from the gastric to the intestinal environment. The test products chosen were commercially available ibuprofen tablets and zafirlukast tablets. The biorelevant dissolution tests showed a high degree of reproducibility among the participating laboratories, even though several different batches of the commercially available medium preparation powder were used. Likewise, results were almost identicalbetween the commercial biorelevant media and those produced in-house. Comparing results to previous ring studies, including those performed with USP calibrator tablets or commercially available pharmaceutical products in a single medium, the results for the biorelevant studies were highly reproducible on an interlaboratory basis. Interlaboratory reproducibility with the two-stage test was also acceptable, although the variability was somewhat greater than with the single medium tests. Biorelevant dissolution testing is highly reproducible among laboratories and can be relied upon for cross-laboratory comparisons.

Investigation of the Intra- and Interlaboratory Reproducibility of a Small Scale Standardized Supersaturation and Precipitation Method.

The high number of poorly water-soluble compounds in drug development has increased the need for enabling formulations to improve oral bioavailability. One frequently applied approach is to induce supersaturation at the absorptive site, e.g., the small intestine, increasing the amount of dissolved compound available for absorption. However, due to the stochastic nature of nucleation, supersaturating drug delivery systems may lead to inter- and intrapersonal variability. The ability to define a feasible range with respect to the supersaturation level is a crucial factor for a successful formulation. Therefore, an in vitro method is needed, from where the ability of a compound to supersaturate can be defined in a reproducible way. Hence, this study investigates the reproducibility of an in vitro small scale standardized supersaturation and precipitation method (SSPM). First an intralaboratory reproducibility study of felodipine was conducted, after which seven partners contributed with data for three model compounds; aprepitant, felodipine, and fenofibrate, to determine the interlaboratory reproducibility of the SSPM. The first part of the SSPM determines the apparent degrees of supersaturation (aDS) to investigate for each compound. Each partner independently determined the maximum possible aDS and induced 100, 87.5, 75, and 50% of their determined maximum possible aDS in the SSPM. The concentration-time profile of the supersaturation and following precipitation was obtained in order to determine the induction time (tind) for detectable precipitation. The data showed that the absolute values of tind and aDS were not directly comparable between partners, however, upon linearization of the data a reproducible rank ordering of the three model compounds was obtained based on the ß-value, which was defined as the slope of the ln(tind) versus ln(aDS)-2 plot. Linear regression of this plot showed that aprepitant had the highest ß-value, 15.1, while felodipine and fenofibrate had comparable ß-values, 4.0 and 4.3, respectively. Of the five partners contributing with full data sets, 80% could obtain the same rank order for the three model compounds using the SSPM (aprepitant > felodipine ≈ fenofibrate). The α-value is dependent on the experimental setup and can be used as a parameter to evaluate the uniformity of the data set. This study indicated that the SSPM was able to obtain the same rank order of the ß-value between partners and, thus, that the SSPM may be used to classify compounds depending on their supersaturation propensity.

Transferrin receptor expression and role in transendothelial transport of transferrin in cultured brain endothelial monolayers.

Receptor-mediated transcytosis of the transferrin receptor has been suggested as a potential transport system to deliver therapeutic molecules into the brain. Recent studies have however shown that therapeutic antibodies, which have been reported to cross the brain endothelium, reach greater brain exposure when the affinity of the antibodies to the transferrin receptor is lowered. The lower affinity of the antibodies to the transferrin receptor facilitates the dissociation from the receptor within the endosomal compartments, which may indicate that the receptor itself does not necessarily move across the endothelial cells by transcytosis. The aim of the present study was to investigate transferrin receptor expression and role in transendothelial transferrin transport in cultured bovine brain endothelial cell monolayers. Transferrin receptor mRNA and protein levels were investigated in endothelial mono-cultures and co-cultures with astrocytes, as well as in freshly isolated brain capillaries using qPCR, immunocytochemistry and Western blotting. Transendothelial transport and luminal association of holo-transferrin was investigated using [125I]holo-transferrin or [59Fe]-transferrin. Transferrin receptor mRNA expression in all cell culture configurations was lower than in freshly isolated capillaries, but the expression slightly increased during six days of culture. The mRNA expression levels were similar in mono-cultures and co-cultures. Immunostaining demonstrated comparable transferrin receptor localization patterns in mono-cultures and co-cultures. The endothelial cells demonstrated an up-regulation of transferrin receptor mRNA after treatment with the iron chelator deferoxamine. The association of [125I]holo-transferrin and [59Fe]-transferrin to the endothelial cells was inhibited by an excess of unlabeled holo-transferrin, indicating receptor mediated association. However, over time the cell associated [59Fe]-label exceeded that of [125I]holo-transferrin, which could indicate release of iron in the endothelial cells and receptor recycling. Luminal-to-abluminal transport of [125I]holo-transferrin across endothelial cell monolayers was low and not inhibited by unlabeled holo-transferrin. This indicated that transendothelial transferrin transport was independent of transferrin receptor-mediated transcytosis.

A Transporter of Ibuprofen is Upregulated in MDCK I Cells under Hyperosmotic Culture Conditions.

Ibuprofen is a widely used drug. It has been identified as an inhibitor of several transporters, but it is not clear if ibuprofen is a substrate of any transporter itself. In the present work, we have characterized a transporter of ibuprofen, which is upregulated by hyperosmotic culture conditions in Madin-Darby canine kidney I (MDCK I) renal cells. [(3)H]-Ibuprofen uptake rate was measured in MDCK I cell cultured under normal (300 mOsm) and hyperosmotic (500 mOsm) conditions. Hyperosmotic conditions were obtained by supplementing urea, NaCl, mannitol, or raffinose to culture medium. The effect of increased osmolarity was investigated for different incubation times. [(3)H]-Ibuprofen uptake in MDCK I cells was upregulated by hyperosmotic culture condition, and was saturable with a Km value of 0.37 ± 0.08 µM and a Vmax of 233.1 ± 17.2 pmol· cm(-2)· min(-1). Racemic [(3)H]-ibuprofen uptake could be inhibited by (R)-(-)- and (S)-(+)-ibuprofen with IC50 values of 19 µM (Log IC50 1.39 ± 0.34) and 0.47 µM (Log IC50 -0.36 ± 0.41), respectively. Furthermore, the [(3)H]-ibuprofen uptake rate was increased by decreased extracellular pH but not dependent on Na(+) or Cl(-) ions. The mRNA of Mct1, -2, -4, and -6 as well as Oat1 and -3 were not upregulated by hyperosmolarity. Our findings present strong evidence for the presence of a yet unknown ibuprofen transporter in MDCK I cells. The transporter was upregulated under hyperosmotic culture conditions, and the present study is therefore a starting point for identification of the molecular correlate and potential impact on ibuprofen disposition.

Pharmacokinetic/Pharmacodynamic Relationship of Gabapentin in a CFA-induced Inflammatory Hyperalgesia Rat Model.

PURPOSE: Gabapentin displays non-linear drug disposition, which complicates dosing for optimal therapeutic effect. Thus, the current study was performed to elucidate the pharmacokinetic/pharmacodynamic (PKPD) relationship of gabapentin's effect on mechanical hypersensitivity in a rat model of CFA-induced inflammatory hyperalgesia. METHODS: A semi-mechanistic population-based PKPD model was developed using nonlinear mixed-effects modelling, based on gabapentin plasma and brain extracellular fluid (ECF) time-concentration data and measurements of CFA-evoked mechanical hyperalgesia following administration of a range of gabapentin doses (oral and intravenous). RESULTS: The plasma/brain ECF concentration-time profiles of gabapentin were adequately described with a two-compartment plasma model with saturable intestinal absorption rate (K m = 44.1 mg/kg, V max = 41.9 mg/h∙kg) and dose-dependent oral bioavailability linked to brain ECF concentration through a transit compartment. Brain ECF concentration was directly linked to a sigmoid E max function describing reversal of hyperalgesia (EC 50, plasma = 16.7 µg/mL, EC 50, brain = 3.3 µg/mL). CONCLUSIONS: The proposed semi-mechanistic population-based PKPD model provides further knowledge into the understanding of gabapentin's non-linear pharmacokinetics and the link between plasma/brain disposition and anti-hyperalgesic effects. The model suggests that intestinal absorption is the primary source of non-linearity and that the investigated rat model provides reasonable predictions of clinically effective plasma concentrations for gabapentin.