Chemical, Analytical and Pharmacokinetic Characterisation of RO7304898, an API Consisting of Two Rapidly Interconverting Diastereoisomers.

PURPOSE: Exploration of the chemical, analytical and pharmacokinetic properties of the API, RO7304898, an allosteric EGFR inhibitor, intended to be developed as a mixture of two rapidly interconverting diastereoisomers with composition ratio of approximately 1:1. METHODS: Assessment of diastereoisomer stereochemistry, interconversion rates, binding to EGFR protein, metabolic stability and in vivo PK in Wistar-Han rats was conducted. RESULTS: The two diastereoisomers of the API undergo fast interconversion at physiologically relevant pH and direct EGFR binding studies revealed diastereoisomer B to be the active moiety. Pharmacokinetic studies in rat revealed a low-moderate total plasma clearance of the API along with similar plasma concentration-time profiles for diastereoisomers A and B, and the diastereoisomeric ratio reached stable equilibrium favoring formation of the potent diastereoisomer B. In in vitro incubations, the API was metabolically stable in plasma and hepatocyte suspension incubations in all species tested except that of rat hepatocytes. Additionally, only small species differences in the A:B composition were observed in vitro with the potent diastereoisomer B being the predominant form. CONCLUSIONS: We demonstrated that the API, a mixture of two diastereoisomers; A (impotent) and B (potent), undergoes rapid interconversion which is faster than the apparent distribution and elimination rates of the individual diastereoisomers in vivo in rat, serving to diminish concerns that separate diastereoisomer effects may occur in subsequent pharmacologic and pivotal toxicological studies. Whilst vigilant monitoring of the diastereoisomeric ratio will need to be continued, this data adds confidence on the development pathway for this API to the clinic.

Development of a Unified Dissolution and Precipitation Model and Its Use for the Prediction of Oral Drug Absorption.

Drug absorption is a complex process involving dissolution and precipitation, along with other kinetic processes. The purpose of this work was to (1) establish an in vitro methodology to study dissolution and precipitation in early stages of drug development where low compound consumption and high throughput are necessary, (2) develop a mathematical model for a mechanistic explanation of generated in vitro dissolution and precipitation data, and (3) extrapolate in vitro data to in vivo situations using physiologically based models to predict oral drug absorption. Small-scale pH-shift studies were performed in biorelevant media to monitor the precipitation of a set of poorly soluble weak bases. After developing a dissolution-precipitation model from this data, it was integrated into a simplified, physiologically based absorption model to predict clinical pharmacokinetic profiles. The model helped explain the consequences of supersaturation behavior of compounds. The predicted human pharmacokinetic profiles closely aligned with the observed clinical data. In summary, we describe a novel approach combining experimental dissolution/precipitation methodology with a mechanistic model for the prediction of human drug absorption kinetics. The approach unifies the dissolution and precipitation theories and enables accurate predictions of in vivo oral absorption by means of physiologically based modeling.

Oral Absorption from Surfactant-Based Drug Formulations: The Impact of Molecularly Dissolved Drug on Bioavailability.

Enabling drug formulations are often required to ensure sufficient absorption after oral administration of poorly soluble drugs. While these formulations typically increase the apparent solubility of the drug, it is widely acknowledged that only molecularly dissolved, i.e., free fraction of the drug, is prone for direct absorption, while colloid-associated drug does not permeate to the same extent. In the present study, we aimed at comparing the effect of molecularly and apparently (i.e., the sum of molecularly and colloid-associated drug) dissolved drug concentrations on the oral absorption of a poorly water-soluble drug compound, Alectinib. Mixtures of Alectinib and respectively 50 %, 25 %, 12.5 %, and 3 % sodium lauryl sulfate (SLS) relative to the dose were prepared and small-scale dissolution tests were performed under simulated fed and fasted state conditions. Both the molecularly and apparently dissolved drug concentrations were assessed in parallel using microdialysis and centrifugation/filtration sampling, respectively. The data served as the basis for an in vitro-in vivo correlation (IVIVC) and as input for a GastroPlusTM physiologically-based biopharmaceutics model (PBBM). It was shown that with increasing the content of SLS the apparently dissolved drug in FeSSIF and FaSSIF increased to a linear extent and thus, the predicted in vivo performance of the 50 % SLS formulation, based on apparently dissolved drug, would outperform all other formulations. Against common expectation, however, the free (molecularly dissolved) drug concentrations were found to vary with SLS concentrations as well, yet to a minor extent. A systematic comparison of solubilized and free drug dissolution patterns at different SLS contents of the formulations and prandial states allowed for interesting insights into the complex dissolution-/supersaturation-, micellization-, and precipitation-behavior of the formulations. When comparing the in vitro datasets with human pharmacokinetic data from a bioequivalence study, it was shown that the use of molecularly dissolved drug resulted in an improved IVIVC. By incorporating the in vitro dissolution datasets into the GastroPlusTM PBBM, the apparently dissolved drug concentrations resulted in both, a remarkable overprediction of plasma concentrations as well as a misprediction of the influence of SLS on systemic exposure. In contrast, by using the molecularly dissolved drug (i.e., free fraction) as the model input, the predicted plasma concentration-time profiles were in excellent agreement with observed data for all formulations under both fed and fasted conditions. By combining an advanced in vitro assessment with PBBM, the present study confirmed that only the molecularly dissolved drug, and not the colloid-associated drug, is available for direct absorption.

Using molecularly dissolved drug concentrations in PBBMs improves the prediction of oral absorption from supersaturating formulations.

Predicting the absorption of drugs from enabling formulations is still challenging due to the limited capabilities of standard physiologically based biopharmaceutics models (PBBMs) to capture complex absorption processes. Amongst others, it is often assumed that both, molecularly and apparently dissolved drug in the gastrointestinal lumen are prone to absorption. A recently introduced method for measuring concentrations of molecularly dissolved drug in a dynamic in vitro dissolution setup using microdialysis has opened new opportunities to test this hypothesis and refine mechanistic PBBM approaches. In the present study, we compared results of PBBMs that used either molecularly or apparently dissolved concentrations in the simulated gastrointestinal lumen as input parameters. The in vitro dissolution data from three supersaturating formulations of Posaconazole (PCZ) were used as model input. The modeling outcome was verified using PCZ concentration vs. time profiles measured in human intestinal aspirates and in the blood plasma. When using apparently dissolved drug concentrations (i.e., the sum of colloid-associated and molecularly dissolved drug) the simulated systemic plasma exposures were overpredicted, most pronouncedly with the ASD-based tablet. However, if the concentrations of molecularly dissolved drug were used as input values, the PBBM resulted in accurate prediction of systemic exposures for all three PCZ formulations. The present study impressively demonstrated the value of considering molecularly dissolved drug concentrations as input value for PBBMs of supersaturating drug formulations.

Combining in vitro dissolution/permeation with microdialysis sampling: Capabilities and limitations for biopharmaceutical assessments of supersaturating drug formulations.

Many novel small drug molecules are poorly water-soluble and thus, enabling drug formulations may be required to ensure sufficient absorption upon oral administration. Biopharmaceutical assessment and absorption prediction of enabling formulations, however, remains challenging. Combined in vitro dissolution/permeation (D/P) assays have gained increasing interest since they may provide a more realistic formulation ranking based on the drug permeation profiles from different formulations as compared to conventional dissolution, which captures both readily permeable and not readily permeable fractions of "dissolved" drug. Moreover, the combined in vitro D/P assays allow to better predict intestinal supersaturation and precipitation processes as compared to simple dissolution setups due to the effect of an absorptive sink. Microdialysis on the other hand has proven useful to determine molecularly dissolved drug in colloidal dispersions, thus allowing for a deeper mechanistic insight into the mechanism of drug release from supersaturating formulations. Here, microdialysis sampling from the donor compartment was used in combination with the dissolution/permeation (D/P) tool PermeaLoop™ to study commercial supersaturating drug formulations of the poorly soluble and weakly basic drug Posaconazole (PCZ). An amorphous solid dispersion (ASD)-based tablet, as well as a crystalline suspension in acidified and neutral dilution medium, respectively, were tested. Microdialysis sampling allowed for differentiation between molecularly dissolved and micellar drug concentration, as expected, but, surprisingly, it was found that the presence of the microdialysis probe affected the precipitation behavior of a crystalline suspension within the two-stage D/P setup, simulating the oral administration of the acidified PCZ (Noxafil®) suspension: the extent and duration of supersaturation in the donor decreased significantly, which also affected permeation. Similarly, for the ASD-based tablet, a less pronounced supersaturation was observed during the first 120 min of the experiment. Hence, in this case, the formulation ranking and the prediction of intestinal supersaturation in the in vitro D/P assay became less predictive as compared to a conventional PermeaLoop™ study without microdialysis sampling. It was concluded that valuable mechanistic insights into the molecularly dissolved drug profiles over time can be obtained by microdialysis. However, since the presence of the probe may affect the degree of supersaturation and precipitation, a conventional D/P assay (without microdialysis sampling) is preferred for formulation ranking of supersaturating drug formulations.

Preclinical Characterization of a Next-Generation Brain Permeable, Paradox Breaker BRAF Inhibitor.

PURPOSE: Disease progression in BRAF V600E/K positive melanomas to approved BRAF/MEK inhibitor therapies is associated with the development of resistance mediated by RAF dimer inducing mechanisms. Moreover, progressing disease after BRAFi/MEKi frequently involves brain metastasis. Here we present the development of a novel BRAF inhibitor (Compound Ia) designed to address the limitations of available BRAFi/MEKi. EXPERIMENTAL DESIGN: The novel, brain penetrant, paradox breaker BRAFi is comprehensively characterized in vitro, ex vivo, and in several preclinical in vivo models of melanoma mimicking peripheral disease, brain metastatic disease, and acquired resistance to first-generation BRAFi. RESULTS: Compound Ia manifested elevated potency and selectivity, which triggered cytotoxic activity restricted to BRAF-mutated models and did not induce RAF paradoxical activation. In comparison to approved BRAFi at clinical relevant doses, this novel agent showed a substantially improved activity in a number of diverse BRAF V600E models. In addition, as a single agent, it outperformed a currently approved BRAFi/MEKi combination in a model of acquired resistance to clinically available BRAFi. Compound Ia presents high central nervous system (CNS) penetration and triggered evident superiority over approved BRAFi in a macro-metastatic and in a disseminated micro-metastatic brain model. Potent inhibition of MAPK by Compound Ia was also demonstrated in patient-derived tumor samples. CONCLUSIONS: The novel BRAFi demonstrates preclinically the potential to outperform available targeted therapies for the treatment of BRAF-mutant tumors, thus supporting its clinical investigation.

Fundamental aspects of DMPK optimization of targeted protein degraders.

Targeted protein degraders are an emerging modality. Their properties fall outside the traditional small-molecule property space and are in the 'beyond rule of 5' space. Consequently, drug discovery programs focused on developing orally bioavailable degraders are expected to face complex drug metabolism and pharmacokinetics (DMPK) challenges compared with traditional small molecules. Nevertheless, little information is available on the DMPK optimization of oral degraders. Therefore, in this review, we discuss our experience of these DMPK optimization challenges and present methodologies and strategies to overcome the hurdles dealing with this new small-molecule modality, specifically in developing oral degraders to treat cancer.

Interactions between iron(III)-hydroxide polymaltose complex and commonly used drugs / simulations and in vitro studies.

Under physiological conditions, ferric ions are essentially insoluble because of the formation of polynuclear hydroxo-bridged complexes. Ferrous ions are more soluble but may produce hydroxyl radicals on reaction with hydrogen peroxide. Chelation of ferric and ferrous ions with organic ligands may prevent these undesirable reactions. Alternatively, iron(III)-hydroxide/oxide can be stabilized and solubilized by tight interactions with carbohydrates. The data presented in this work show that, because of its physicochemical properties, the iron(III)-hydroxide polymaltose complex (IPC, Maltofer) does not interact with the active ingredients of commonly used drugs such as acetylsalicylic acid (CAS 50-78-2), tetracycline hydrochloride (CAS 64-75-5), calcium hydrogen-phosphate (CAS 7757-93-9), methyl-L-dopa sesquihydrate (CAS 41372-08-1), and magnesium-L-aspartate hydrochloride (CAS 28184-71-6). In contrast, as confirmed by calculations using thermodynamic parameters, FeCl3 x 6H2O (CAS 10025-77-1) can form different types of complexes with these substances. Moreover, the data show that under aerobic conditions high concentrations of ascorbic acid (CAS 50-81-7) can lead to mobilization of iron from IPC and, thus, support the observation that orange juice slightly increases the uptake of iron from IPC.