Increasing the Robustness of Biopharmaceutical Precipitation Assays - Part II: Recommendations on the use of FaSSIF.

Biopharmaceutical precipitation assays are an important in vitro tool to characterize the precipitation behavior of weakly basic drugs during their transit from the stomach into the small intestine. To mimic the intestinal fluids more closely, biorelevant media like Fasted State Simulated Intestinal Fluid (FaSSIF) and versions thereof are often applied. When applying UV analytics to measure the drug concentration during the transfer experiments, changes in the UV spectrum of the medium have been observed when FaSSIF was stored over a longer period of time or under accelerated conditions. Therefore, this study aimed at evaluating the stability of FaSSIF under various storage conditions. Furthermore, the impact of stressed FaSSIF on the supersaturation and precipitation behavior of ketoconazole was investigated. As a result of this study, it was demonstrated that the FaSSIF powder composition changes during storage, which, in turn, impacts the supersaturation and precipitation behavior of ketoconazole in in vitro transfer studies. Based on the results of this study, we provide recommendations on the application of FaSSIF in biopharmaceutical precipitation assays with the aim to increase reproducibility and enhance data reliability for those compounds where changing FaSSIF composition may impact the supersaturation and precipitation behavior.

Increasing the Robustness of Biopharmaceutical Precipitation Assays - Part I: Derivative UV Spectrophotometric Method Development for in-line Measurements.

In vitro precipitation assays are often applied to support drug and formulation development. Current methods applied to quantify the amount of dissolved drug, in particular (U)HPLC, require time-consuming sample preparation. Furthermore, small precipitates formed during the nucleation phase may not be removed quantitatively by filtration or centrifugation of the sample. Given the drawbacks of standard (U)HPLC analyses during the application in transfer experiments, it was the aim of this work to develop a robust and simple to implement in-line UV spectrophotometric method which accurately reflects the precipitation profile obtained from in vitro transfer assays. Based on the three model compounds cinnarizine, dipyridamole, and ketoconazole, the manuscript describes the development of a design of experiments (DoE) based approach to develop derivative UV spectrophotometric methods accounting for the change in media composition over time due to the dilution of simulated intestinal with simulated gastric fluid. An R script was developed which automatically identifies suitable wavelengths for in-line measurements. As an outcome of this study, a fast, robust, accurate, and specific derivative UV spectrophotometric methodology for measuring the concentration of dissolved drugs in in vitro transfer experiments was successfully developed. This method can flexibly be applied to multi-compartmental precipitation assays.

The effect of buffer species on biorelevant dissolution and precipitation assays - Comparison of phosphate and bicarbonate buffer.

Biorelevant solubility and dissolution testing is an important tool during pharmaceutical development, however, solubility experiments performed using biorelevant media often do not properly match the solubility data observed in human intestinal fluids. Even though the bicarbonate buffer is the predominant buffer system in the small intestine, in vitro assays are commonly performed using non-volatile buffer systems like phosphate and maleate. In the current study, bicarbonate- and phosphate-buffered biorelevant media were applied to solubility, dissolution, and precipitation testing for a broad range of model compounds. It was found that the medium affects primarily the dissolution kinetics. However, with the knowledge of the unique buffering properties of bicarbonate buffer in the diffusion layer, it was not always possible to predict the effect of buffer species on solubility and dissolution when changing from phosphate to bicarbonate buffer. This once again highlights the special role of bicarbonate buffer for simulating the conditions in the human intestinal fluids. Moreover, it is necessary to further investigate the factors which may cause the differences in solubility and dissolution behavior when using phosphate- vs. bicarbonate-buffered biorelevant media.

Improved Prediction of in Vivo Supersaturation and Precipitation of Poorly Soluble Weakly Basic Drugs Using a Biorelevant Bicarbonate Buffer in a Gastrointestinal Transfer Model.

The characterization of intestinal dissolution of poorly soluble drugs represents a key task during the development of both new drug candidates and drug products. The bicarbonate buffer is considered as the most biorelevant buffer for simulating intestinal conditions. However, because of its complex nature, being the volatility of CO2, it has only been rarely used in the past. The aim of this study was to investigate the effect of a biorelevant bicarbonate buffer on intestinal supersaturation and precipitation of poorly soluble drugs using a gastrointestinal (GI) transfer model. Therefore, the results of ketoconazole, pazopanib, and lapatinib transfer model experiments using FaSSIFbicarbonate were compared with the results obtained using standard FaSSIFphosphate. Additionally, the effect of hydroxypropyl methylcellulose acetate succinate (HPMCAS) as a precipitation inhibitor was investigated in both buffer systems and compared to rat pharmacokinetic (PK) studies with and without coadministration of HPMCAS as a precipitation inhibitor. While HPMCAS was found to be an effective precipitation inhibitor for all drugs in FaSSIFphosphate, the effect in FaSSIFbicarbonate was much less pronounced. The PK studies revealed that HPMCAS did not increase the exposure of any of the model compounds significantly, indicating that the transfer model employing bicarbonate-buffered FaSSIF has a better predictive power compared to the model using phosphate-buffered FaSSIF. Hence, the application of a bicarbonate buffer in a transfer model set-up represents a promising approach to increase the predictive power of this in vitrotool and to contribute to the development of drug substances and drug products in a more biorelevant way.

Application of an automated small-scale in vitro transfer model to predict in vivo precipitation inhibition.

The majority of NCEs are weakly basic drugs. Consequently, their solubility is highly pH-dependent, with higher solubility in the acidic stomach and poor solubility in the neutral intestinal environment. The gastric emptying of dissolved drug can lead to the intestinal precipitation of the drug. One option of reducing this process is to formulate the drug together with a precipitation inhibitor (PI). The aim of this study was to investigate the effects of different PIs on the intestinal concentrations of ketoconazole and five orally administered kinase inhibitors (i.e. pazopanib, gefitinib, lapatinib, vemurafenib, and a Merck KGaA research compound, MSC-A) with the aid of a predictive small-scale in vitro transfer model. This screening revealed that HPMCAS and Soluplus® were the most effective PIs. Whereas all other drugs precipitated within several minutes, gefitinib expressed highly variable amorphous precipitation which was confirmed by PXRD. During the transfer model experiments, this intermediate supersaturated state was stabilized using HPMCAS and Soluplus®. The PI screening protocol described herein allows to study the effect of PIs for solubility and potential bioavailability improvement of poorly soluble drugs to support formulation development already in early stages.

Acid-induced degradation of widely used NIR dye DiR causes hypsochromic shift in fluorescence properties.

DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) is one of the most widely used near-infrared dyes for in-vivo imaging due to its favorable optical properties. So far, chemical stability has been taken for granted by most investigators. However, in a recently published imaging study, we found that DiR can exert a hypsochromic shift in fluorescence in-vivo, potentially induced by low pH. This behavior may disturb kinetic measurements and the readout of additional markers fluorescing at lower wavelengths. The present in-vitro experiments were conducted to verify the findings from our in-vivo study and to elucidate the changes of the optical properties of DiR. For this purpose, DiR was incubated in a pH range from pH 2 to neutral pH over 42 days. Fluorescence and absorption measurements as well as mass spectrometry analytics (MS) were conducted to monitor the degradation process of DiR. The protonation effect on DiR optical behavior was estimated using in-silico modelling. For the most acidic sample, a distinct decrease in DIR-fluorescence was noted and several degradation products could be analyzed via MS, confirming the initial hypothesis. Ultimately, scientists should be aware of the possibility of acid-induced DiR degradation, especially when adding a second fluorescence label for dual dye imaging or performing quantitative data analysis.

Automated small-scale in vitro transfer model as screening tool for the prediction of in vivo-dissolution and precipitation of poorly solubles.

Precipitation testing, especially for weakly basic APIs, represents a key parameter in drug substance characterization during early development stages, where the amount of API available is limited. Therefore, it was the aim of this study to develop an automated small-scale in vitro transfer model to characterize the supersaturation and precipitation behavior of two poorly water-soluble drugs. Following automation and scale-down of the standard transfer model, the developed small-scale model was used to assess the impact of gastrointestinal variability, i.e. gastric pH, gastric emptying, and gastrointestinal fluid volumes, on supersaturation and precipitation of two weakly basic model compounds, ketoconazole and a new chemical entity from the research laboratories of Merck KGaA, MSC-A. The experiments revealed that variations in gastrointestinal parameters affected the in vitro behavior of ketoconazole, but not of MSC-A. Elevated gastric pH, as it can result from co-medication with acid-reducing drugs, resulted in lower degrees of supersaturation for both substances. This result is in agreement with the observation that the oral bioavailability of ketoconazole is lowered when proton pump inhibitors are co-administered. The small-scale transfer model presented herein represents a valuable in vitro tool to assess the risk of drug precipitation, additionally covering a broad range of gastrointestinal parameters.

In-line derivative spectroscopy as a promising application to a small-scale in vitro transfer model in biorelevant supersaturation and precipitation testing.

OBJECTIVES: Dissolution testing of poorly soluble and precipitating drugs is of great importance for pharmaceutical industry. As offline HPLC analytics is time-consuming and labour-intensive, the development of suitable in-line analytics to measure drug concentration allows better predictions of drug dissolution and precipitation. The purpose of this study was to develop an in-line derivative spectroscopic method which facilitates drug concentration measurements in suspensions without additional sample preparation. METHODS: Solubility, dissolution and precipitation of ketoconazole were analysed using derivative spectroscopy and HPLC. KEY FINDINGS: Results of solubility and dissolution experiments were highly comparable. Due to higher sampling frequency and lack of sample preparations, supersaturation in a pH-shift experiment was more accurately captured by UV in-line analytics. The application of a prefiltration step and flow-through cuvettes facilitates implementation of in-line derivative spectroscopy into an in vitro transfer model with changing UV-active media and high supersaturation in highly turbid samples. CONCLUSIONS: Although the application of derivative spectroscopy has been described previously, the approach described herein is novel and well-suited for the application in an automated in vitro transfer model. Moreover, it represents a promising tool for drug substance characterisation, candidate selection and formulation development.

Long-Lasting and Fast-Acting in Vivo Efficacious Antiplasmodial Azepanylcarbazole Amino Alcohol.

With ∼429,000 deaths in 2016, malaria remains a major infectious disease where the need to treat the fever symptoms, but also to provide relevant post-treatment prophylaxis, is of major importance. An azepanylcarbazole amino alcohol is disclosed with a long- and fast-acting in vivo antiplasmodial efficacy and meets numerous attributes of a desired post-treatment chemoprophylactic antimalarial agent. The synthesis, the parasitological characterization, and the animal pharmacokinetics and pharmacodynamics of this compound are presented along with a proposed target.

Scaffold hopping approach towards various AFQ-056 analogs as potent metabotropic glutamate receptor 5 negative allosteric modulators.

The metabotropic glutamate receptor subtype 5 has evolved into a promising target for the treatment of various diseases of the central nervous system, such as Fragile X and L-DOPA induced dyskinesia. One of the most advanced clinical compound is Novartis' AFQ-056 (Mavoglurant), which served us as a template for a scaffold hopping approach, generating a structurally diverse set of potent analogs. Both the limited aqueous solubility and the relatively poor metabolic stability of AFQ-056 were improved with hexahydrocyclopenta[c]pyrrole derivative 54a, which proved to be a valuable candidate for further development.

Discovery, synthesis, and structure-activity relationships of 2-aminoquinazoline derivatives as a novel class of metabotropic glutamate receptor 5 negative allosteric modulators.

A virtual screening approach using various in silico methodologies led to the discovery of 2-(m-tolylamino)-7,8-dihydroquinazolin-5(6H)-one (1) as a moderately active negative allosteric modulator (NAM) of the metabotropic glutamate receptor subtype 5 (mGluR5) showing high selectivity against the subtype mGluR1. Modifications of the parent compound by rational design yielded a series of highly potent derivatives which will serve as valuable starting points for further hit-to-lead optimization efforts toward a suitable drug candidate for the treatment of L-DOPA induced dyskinesia.

Multivalent cyclic RGD ligands: influence of linker lengths on receptor binding.

UNLABELLED: Peptides involving the RGD motive (arginine-glycine-aspartic acid) recognize members of the integrin receptor family. Since the receptors are located mainly on the surface of endothelial cells, structural modifications including multimers of c(RGDfE) were recently found to improve the binding avidity for α(v)ß(3) integrin significantly. The multivalent RGD peptides exhibited rather loose linkages partly including oligo(ethylene glycol) spacers (EG(n)) with different chain lengths. Therefore, the dependence of multivalent RGD systems with and without EG(n) linkers were investigated on their binding properties to cultured α(v)ß(3) integrin-expressing U87MG cells. METHODS: We synthesized a series of di-, tri- and tetravalent rigid scaffolds (terephthalic acid, trimesic acid and adamantane-1,3,5,7-tetracarboxylic acid) conjugated to c(RGDyK) ligands, which were linked contiguously or separated by the oligo(ethylene glycol) spacers. The inhibition constants of these c(RGDyK) derivatives were determined by competition assays with (125)I-labeled echistatin. RESULTS: While c(RGDyK) function is a relative weak competitor against [(125)I]echistatin (K(i), 329 ± 18 nM) for α(v)ß(3) integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (K(i), 64 ± 23 nM). This effect was even more pronounced with the RGD trimers (K(i), 40 ± 7 nM) and tetramers (K(i), 26±9 nM). The introduction of EG(n) spacers and the increase of linker lengths proved to be detrimental since more competitors were needed to compete with [(125)I]echistatin. The EG(6) group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer). CONCLUSION: The binding experiments performed with the three forms of multivalent RGD ligands indicate the weakening of competitive potency against [(125)I]echistatin with the introduction of EG(n) spacers. This effect may be related to the decrease of the effective RGD molarity, which becomes most prominent within the tetravalent series.