Development and Application of a Dissolution-Transfer-Partitioning System (DTPS) for Biopharmaceutical Drug Characterization.

A variety of in vitro dissolution and gastrointestinal transfer models have been developed aiming to predict drug supersaturation and precipitation. Further, biphasic, one-vessel in vitro systems are increasingly applied to simulate drug absorption in vitro. However, to date, there is a lack of combining the two approaches. Therefore, the first aim of this study was to develop a dissolution-transfer-partitioning system (DTPS) and, secondly, to assess its biopredictive power. In the DTPS, simulated gastric and intestinal dissolution vessels are connected via a peristaltic pump. An organic layer is added on top of the intestinal phase, serving as an absorptive compartment. The predictive power of the novel DTPS was assessed to a classical USP II transfer model using a BCS class II weak base with poor aqueous solubility, MSC-A. The classical USP II transfer model overestimated simulated intestinal drug precipitation, especially at higher doses. By applying the DTPS, a clearly improved estimation of drug supersaturation and precipitation and an accurate prediction of the in vivo dose linearity of MSC-A were observed. The DTPS provides a useful tool taking both dissolution and absorption into account. This advanced in vitro tool offers the advantage of streamlining the development process of challenging compounds.

Lipophilic Salts and Lipid-Based Formulations for Bridging the Food Effect Gap of Venetoclax.

Lipid based formulations (LBF) have shown to overcome food dependent bioavailability for some poorly water-soluble drugs. However, the utility of LBFs can be limited by low dose loading due to a low drug solubility in LBF vehicles. This study investigated the solubility and drug loading increases in LBFs using lipophilic counterions to form lipophilic salts of venetoclax. Venetoclax docusate was formed from venetoclax free base and verified by 1H NMR. Formation of stable venetoclax-fatty acid associations with either oleic acid or decanoic acid were attempted, however, the molecular associations were less consistent based on 1H NMR. Venetoclax docusate displayed a up to 6.2-fold higher solubility in self-emulsifying drug delivery systems (SEDDS) when compared to the venetoclax free base solubility resulting in a higher dose loading. A subsequent bioavailability study in landrace pigs demonstrated a 2.5-fold higher bioavailability for the lipophilic salt containing long chain SEDDS compared to the commercially available solid dispersion Venclyxto® in the fasted state. The bioavailability of all lipophilic salt SEDDS in the fasted state was similar to Venclyxto® in the fed state. This study confirmed that lipophilic drug salts increase the dose loading in LBFs and showed that lipophilic salt-SEDDS combinations may be able to overcome bioavailability limitations of drugs with low inherent dose loading in lipid vehicles. Furthermore, the present study demonstrated the utility of a LBF approach, in combination with lipophilic salts, to overcome food dependent variable oral bioavailability of drugs.

Combining species specific in vitro & in silico models to predict in vivo food effect in a preclinical stage - case study of Venetoclax.

The pig has been increasingly used as a reliable preclinical model for assessing and predicting the in vivo bioavailability of different formulation strategies. Nevertheless, differences in the composition between porcine and human intestinal fluids, may impact on the solubility and dissolution behaviour of drugs, in particular BCS II/IV drugs. Recently, a porcine fasted simulated intestinal fluid (FaSSIFp) was developed to mimic the composition in the lumen of landrace pigs under fasted state conditions. In this work, we present the utilization of FaSSIFp to compare solubility against human FaSSIF & FeSSIF and further combine species specific in vitro testing with in silico predictive modelling. Venetoclax was chosen as a model drug, representing a BCS class IV drug, with a reported clinically significant positive food effect, where bioavailability is increased up to approximately five-fold when administered with a high-fat meal. Biorelevant species specific in vitro testing was a promising tool for integrating in vitro data into in silico models, using FaSSIFp resulted in reliable predictions of the plasma concentration profile in fasted pigs, based on a porcine physiologically based absorption model. The porcine physiologically based absorption model was used to prospectively simulate the impact of food on the bioavailability of venetoclax. The use of luminal solubility estimates in combination with dissolution data for venetoclax, measured in species specific simulated fluids, correctly predict the observed pig plasma concentration profile and food effect. Overall, integrating species specific in vitro - in silico models led to accurate prediction of in vivo absorption of venetoclax in a preclinical stage, which can support guidance in early decisions of drug product development. In addition, the study further demonstrated the utility of the pig model to predict the food effects of venetoclax in humans.

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.

Oral biopharmaceutics tools: recent progress from partnership through the Pharmaceutical Education and Research with Regulatory Links collaboration.

OBJECTIVES: To summarise key contributions of the Pharmaceutical Education and Research with Regulatory Links (PEARRL) project (2016-2020) to the optimisation of existing and the development of new biopharmaceutics tools for evaluating the in vivo performance of oral drug products during the development of new drugs and at the regulatory level. KEY FINDINGS: Optimised biopharmaceutics tools: Based on new clinical data, the composition of biorelevant media for simulating the fed state conditions in the stomach was simplified. Strategies on how to incorporate biorelevant in vitro data of bio-enabling drug products into physiologically based pharmacokinetic (PBPK) modelling were proposed. Novel in vitro biopharmaceutics tools: Small-scale two-stage biphasic dissolution and dissolution-permeation setups were developed to facilitate understanding of the supersaturation effects and precipitation risks of orally administered drugs. A porcine fasted state simulated intestinal fluid was developed to improve predictions and interpretation of preclinical results using in vitro dissolution studies. Based on new clinical data, recommendations on the design of in vitro methodologies for evaluating the GI drug transfer process in the fed state were suggested. The optimized design of in vivo studies for investigating food effects: A food effect study protocol in the pig model was established which successfully predicted the food-dependent bioavailability of two model compounds. The effect of simulated infant fed state conditions in healthy adults on the oral absorption of model drugs was evaluated versus the fasted state and the fed state conditions, as defined by regulatory agencies for adults. Using PBPK modelling, the extrapolated fasted and infant fed conditions data appeared to be more useful to describe early drug exposure in infants, while extrapolation of data collected under fed state conditions, as defined by regulators for adults, failed to capture in vivo infant drug absorption. SUMMARY: Substantial progress has been made in developing an advanced suite of biopharmaceutics tools for streamlining drug formulation screening and supporting regulatory applications. These advances in biopharmaceutics were achieved through networking opportunities and research collaborations provided under the H2020 funded PEARRL project.

Exploring porcine gastric and intestinal fluids using microscopic and solubility estimates: Impact of placebo self-emulsifying drug delivery system administration to inform bio-predictive in vitro tools.

Validation and characterisation of in vitro and pre-clinical animal models to support bio-enabling formulation development is of paramount importance. In this work, post-mortem gastric and small intestinal fluids were collected in the fasted, fed state and at five sample-points post administration of a placebo Self-Emulsifying Drug Delivery System (SEDDS) in the fasted state to pigs. Cryo-TEM and Negative Stain-TEM were used for ultrastructure characterisation. Ex vivo solubility of fenofibrate was determined in the fasted-state, fed-state and post-SEDDS administration. Highest observed ex vivo drug solubility in intestinal fluids after SEDDS administration was used for optimising the biorelevant in vitro conditions to determine maximum solubility. Under microscopic evaluation, fasted, fed and SEDDS fluids resulted in different colloidal structures. Drug solubility appeared highest 1 hour post SEDDS administration, corresponding with presence of SEDDS lipid droplets. A 1:200 dispersion of SEDDS in biorelevant media matched the highest observed ex vivo solubility upon SEDDS administration. Overall, impacts of this study include increasing evidence for the pig preclinical model to mimic drug solubility in humans, observations that SEDDS administration may poorly mimic colloidal structures observed under fed state, while microscopic and solubility porcine assessments provided a framework for increasingly bio-predictive in vitro tools.

Characterization of gastrointestinal transit and luminal conditions in pigs using a telemetric motility capsule.

Within preclinical research, the pig has become an important model in regulatory toxicology and pharmacokinetics, to assess oral dosage forms and to compare different formulation strategies. In addition, there are emerging application of the pig model to asses clinical dosing conditions in the fasted and fed state. In this study, the gastrointestinal transit conditions in male landrace pigs were studied with a telemetric motility capsule under fasted and postprandial conditions. The whole gut transit time (WGTT) was determined by administering a SmartPill® capsule to four landrace pigs, under both fasted and fed state conditions in a cross-over study design. Overall, this study found that small intestinal transit in landrace pigs ranged from 2.3 - 4.0 h, and was broadly similar to reported human estimates and was not affected by the intake conditions. Gastric emptying was highly variable and prolonged in landrace pigs ranging from 20 - 233 h and up to 264 h in one specific case. Under dynamic conditions pigs have a low gastric pH comparable to humans, however a high variability under fasted conditions could be observed. The comparison of the data from this study with a recent similar study in beagle dogs revealed major differences between gastric maximum pressures observed in landrace pigs and dogs. In the porcine stomach maximum pressures of up to 402 mbar were observed, which are comparable to reported human data. Intestinal maximum pressures in landrace pigs were in the same range as in humans. Overall, the study provides new insights of gastrointestinal conditions in landrace pigs, which can lead to more accurate interpretation of in vivo results obtained of pharmacokinetic studies in preclinical models. While small intestinal transit conditions, GI pH and pressures were similar to humans, the prolonged gastric emptying observed in pigs need to be considered in assessing the suitability of the pig model for assessing in vivo performance of large non-disintegrated oral drug products.

Supersaturated Lipid-Based Formulations to Enhance the Oral Bioavailability of Venetoclax.

Increasing numbers of beyond Rule-of-Five drugs are emerging from discovery pipelines, generating a need for bio-enabling formulation approaches, such as lipid-based formulations (LBF), to ensure maximal in vivo exposure. However, many drug candidates display insufficient lipid solubility, leading to dose-loading limitations in LBFs. The aim of this study was to explore the potential of supersaturated LBFs (sLBF) for the beyond Rule-of-Five drug venetoclax. Temperature-induced sLBFs of venetoclax were obtained in olive oil, Captex® 1000, Peceol® and Capmul MCM®, respectively. A Peceol®-based sLBF displayed the highest drug loading and was therefore evaluated further. In vitro lipolysis demonstrated that the Peceol®-based sLBF was able to generate higher venetoclax concentrations in the aqueous phase compared to a Peceol®-based suspension and an aqueous suspension. A subsequent bioavailability study in pigs demonstrated for sLBF a 3.8-fold and 2.1-fold higher bioavailability compared to the drug powder and Peceol®-based suspension, respectively. In conclusion, sLBF is a promising bio-enabling formulation approach to enhance in vivo exposure of beyond Rule-of-Five drugs, such as venetoclax. The in vitro lipolysis results correctly predicted a higher exposure of the sLBF in vivo. The findings of this study are of particular relevance to pre-clinical drug development, where maximum exposure is required.

Development and evaluation of a biorelevant medium simulating porcine gastrointestinal fluids.

Simulated human intestinal media, have proved to be a useful biopharmaceutics tool as a dissolution media for predicting in vivo dissolution and pharmacokinetic profile in humans. During drug product development preclinical animal models are also required to assess drug product performance, and there is a need to develop species specific intestinal media to similarly predict in vivo pharmacokinetic profiles in each preclinical model. Pigs, are increasingly being used in preclinical drug development, however to date there is a lack of quantitative information about the composition of porcine gastrointestinal (GI) fluids. As a result, a porcine biorelevant medium has not yet been developed, which is essential to improve interpretation and forecast of preclinical results using biorelevant in vitro dissolution studies. GI fluid samples, were collected from landrace pigs, and characterized. Fasted State Simulated Intestinal Fluid of pigs (FaSSIFp) was developed based on the physiological composition of the GI fluids in terms of pH, buffer capacity, osmolality, surface tension, as well as the bile salt, phospholipid and free fatty acid content. This study demonstrated that FaSSIFp was superior at predicting the solubility of the six model drugs in porcine intestinal fluids (PIF). A markedly high correlation (r2 0.98) was observed between the solubility obtained in PIF and FaSSIFp, whereas poor correlation (r2 0.12) was found for the solubility of the model drugs between human FaSSIF and PIF. This confirms that species specific biorelevant intestinal media are crucial to provide more accurate predictions of pharmacokinetic studies in preclinical models. Additionally, the availability of a species specific intestinal medium offers the potential to improve in vitro-in silico approaches to predict in vivo absorption and to reduce the overall number of animals needed in oral drug product development testing.

Toward the establishment of a standardized pre-clinical porcine model to predict food effects - Case studies on fenofibrate and paracetamol.

A preclinical porcine model that reliably predicts human food effect of fenofibrate was developed. Fenofibrate was administered to pigs as model compound with a positive food effect. Two different types of fed conditions were explored: a FDA style breakfast and a standard pig pellet feed. In order to assess if complete stomach emptying had been achieved under the employed fasting protocol, the amount of gastric and intestinal content was evaluated post-mortem. In addition, the protocol was designed to evaluate gastric emptying in the pre- and postprandial state using paracetamol as a marker. The study confirmed that micronized fenofibrate displayed a positive food effect with a similar fold difference to humans in FDA style fed state. Post-mortem assessment of stomach and intestinal content confirmed significantly lower content in the fasted compared to the pig pellet fed state. In the case of paracetamol, a delayed gastric emptying in the fed state was not observed, which may suggest that the Magenstrasse phenomena reported in humans, may also occur in landrace pigs. The study demonstrated the utility of a food effect protocol in landrace pigs as a pre-clinical approach to predict human food effects and provided new insights into gastric emptying in pigs.

The pig as a preclinical model for predicting oral bioavailability and in vivo performance of pharmaceutical oral dosage forms: a PEARRL review.

OBJECTIVES: In pharmaceutical drug development, preclinical tests in animal models are essential to demonstrate whether the new drug is orally bioavailable and to gain a first insight into in vivo pharmacokinetic parameters that can subsequently be used to predict human values. Despite significant advances in the development of bio-predictive in vitro models and increasing ethical expectations for reducing the number of animals used for research purposes, there is still a need for appropriately selected pre-clinical in vivo testing to provide guidance on the decision to progress to testing in humans. The selection of the appropriate animal models is essential both to maximise the learning that can be obtained from such experiments and to avoid unnecessary testing in a range of species. KEY FINDINGS: The present review, provides an insight into the suitability of the pig model for predicting oral bioavailability in humans, by comparing the conditions in the GIT. It also contains a comparison between the bioavailability of compounds dosed to both humans and pigs, to provide an insight into the relative correlation and examples on why a lack of correlation may be observed. SUMMARY: While there is a general trend towards predicting human bioavailability from pig data, there is considerable variability in the data set, most likely reflecting species specific differences in individual drug metabolism. Nonetheless, the correlation between pigs vs. humans was comparable to that reported for dogs vs. humans. The presented data demonstrate the suitability of the pig as a preclinical model to predict bioavailability in human.

Exploring gastric emptying rate in minipigs: Effect of food type and pre-dosing of metoclopramide.

The present study investigated the gastric emptying rate in Göttingen minipigs pre- and post-prandial and evaluated the effect of metoclopramide on the same parameter, using paracetamol as an absorption marker. The pharmacokinetic evaluation of the obtained plasma concentration data for paracetamol demonstrated that the fastest gastric emptying rate was observed in the animals that were allowed access to normal pig food. There was no significant difference in the stomach emptying rate observed between fasted and fed minipigs, when fed either with a FDA standard breakfast or a nutritional energy drink. Pre-dosing minipigs with metoclopramide (0.2 or 0.4 mg/kg) did not demonstrate any effect on gastric emptying in either the fasted or fed state. The data in the present study demonstrated a relatively prolonged gastric emptying rate in mini-pigs both in the fasted and fed state, hence when conducting pharmacokinetic studies using minipigs, this should be taken into account when planning the plasma sampling time points. Further, as no difference could be observed in gastric emptying rate as a function of food, the data also suggest that the Göttingen mini-pigs seems less suited to predict for food effect studies than other species.