Multi-Compartmental Dissolution Method, an Efficient Tool for the Development of Enhanced Bioavailability Formulations Containing Poorly Soluble Acidic Drugs.

The purpose of this study was to investigate the applicability of the Gastrointestinal Simulator (GIS), a multi-compartmental dissolution model, to predict the in vivo performance of Biopharmaceutics Classification System (BCS) Class IIa compounds. As the bioavailability enhancement of poorly soluble drugs requires a thorough understanding of the desired formulation, the appropriate in vitro modelling of the absorption mechanism is essential. Four immediate release ibuprofen 200 mg formulations were tested in the GIS using fasted biorelevant media. In addition to the free acid form, ibuprofen was present as sodium and lysine salts in tablets and as a solution in soft-gelatin capsules. In the case of rapid-dissolving formulations, the dissolution results indicated supersaturation in the gastric compartment, which affected the resulting concentrations in the duodenum and the jejunum as well. In addition, a Level A in vitro-in vivo correlation (IVIVC) model was established using published in vivo data, and then the plasma concentration profiles of each formulation were simulated. The predicted pharmacokinetic parameters were consistent with the statistical output of the published clinical study. In conclusion, the GIS method was found to be superior compared to the traditional USP method. In the future, the method can be useful for formulation technologists to find the optimal technique to enhance the bioavailability of poorly soluble acidic drugs.

The Effect of the Particle Size Reduction on the Biorelevant Solubility and Dissolution of Poorly Soluble Drugs with Different Acid-Base Character.

Particle size reduction is a commonly used process to improve the solubility and the dissolution of drug formulations. The solubility of a drug in the gastrointestinal tract is a crucial parameter, because it can greatly influence the bioavailability. This work provides a comprehensive investigation of the effect of the particle size, pH, biorelevant media and polymers (PVA and PVPK-25) on the solubility and dissolution of drug formulations using three model compounds with different acid-base characteristics (papaverine hydrochloride, furosemide and niflumic acid). It was demonstrated that micronization does not change the equilibrium solubility of a drug, but it results in a faster dissolution. In contrast, nanonization can improve the equilibrium solubility of a drug, but the selection of the appropriate excipient used for nanonization is essential, because out of the two used polymers, only the PVPK-25 had an increasing effect on the solubility. This phenomenon can be explained by the molecular structure of the excipients. Based on laser diffraction measurements, PVPK-25 could also inhibit the aggregation of the particles more effectively than PVA, but none of the polymers could hold the nanonized samples in the submicron range until the end of the measurements.

Understanding the pH Dependence of Supersaturation State-A Case Study of Telmisartan.

Creating supersaturating drug delivery systems to overcome the poor aqueous solubility of active ingredients became a frequent choice for formulation scientists. Supersaturation as a solution phenomenon is, however, still challenging to understand, and therefore many recent publications focus on this topic. This work aimed to investigate and better understand the pH dependence of supersaturation of telmisartan (TEL) at a molecular level and find a connection between the physicochemical properties of the active pharmaceutical ingredient (API) and the ability to form supersaturated solutions of the API. Therefore, the main focus of the work was the pH-dependent thermodynamic and kinetic solubility of the model API, TEL. Based on kinetic solubility results, TEL was observed to form a supersaturated solution only in the pH range 3-8. The experimental thermodynamic solubility-pH profile shows a slight deviation from the theoretical Henderson-Hasselbalch curve, which indicates the presence of zwitterionic aggregates in the solution. Based on pKa values and the refined solubility constants and distribution of macrospecies, the pH range where high supersaturation-capacity is observed is the same where the zwitterionic form of TEL is present. The existence of zwitterionic aggregation was confirmed experimentally in the pH range of 3 to 8 by mass spectrometry.

In vitro and in silico evaluation of Ononis isoflavonoids as molecules targeting the central nervous system.

Isoflavonoids with various structural elements show a promising potential effect on central nervous system activities. Despite their favorable medicinal properties, the pharmacokinetic characteristics of this thoroughly investigated group of natural phenolics have only been described to a limited extent. Regarding the lack of information about the BBB permeability of isoflavones, isoflavanones, and pterocarpans found in Ononis species, the aim of our study was to investigate their physico-chemical properties influencing their absorption and distribution. Furthermore, we aimed to characterize the possible MAO-B inhibiting features of Ononis isoflavonoids in silico. Octanol-water partitioning and BBB-PAMPA permeability of formononetin, calycosin D, onogenin, sativanone, medicarpin and maackiain were assessed for the first time in our study. The log P values ranged from 2.21 to 3.03 and log D7.4 values from 2.48 to 3.03, respectively, indicating optimal polarity for BBB permeation. The results of PAMPA-BBB expressed as log Pe values fell between -5.60 and -4.45, predicting their good permeation capability as well. The effective permeability values showed structure-dependent differences, indicating that the pterocarpan type skeleton was the most preferred type, followed by isoflavanones, then isoflavones. The methoxy or methylenedioxy substitution of the same skeleton did not influence the permeability significantly, contrary to an additional hydroxyl group. Membrane retention showed a similar structure dependent pattern to that of effective permeability, ranging from 16% to 70%. For the identification of volumes of chemical space related to particular biological activities the ChemGPS-NP framework was used. The MAO-B inhibitory potency and selectivity were also predicted and validated. Based on our results, MAO-B inhibitory potency could be predicted with good precision, but in the case of selectivity, only the direction could be concluded (favors MAO-B or MAO-A), not the magnitude. Our finding reflects that Ononis isoflavonoid aglycones show an excellent fit with the suggested parameters for BBB permeability and this is the first study to confirm the highly favorable position of these natural products for MAO-B inhibition.

Flux-Based Formulation Development-A Proof of Concept Study.

The work aimed to develop the Absorption Driven Drug Formulation (ADDF) concept, which is a new approach in formulation development to ensure that the drug product meets the expected absorption rate. The concept is built on the solubility-permeability interplay and the rate of supersaturation as the driving force of absorption. This paper presents the first case study using the ADDF concept where not only dissolution and solubility but also permeation of the drug is considered in every step of the formulation development. For that reason, parallel artificial membrane permeability assay (PAMPA) was used for excipient selection, small volume dissolution-permeation apparatus was used for testing amorphous solid dispersions (ASDs), and large volume dissolution-permeation tests were carried out to characterize the final dosage forms. The API-excipient interaction studies on PAMPA indicated differences when different fillers or surfactants were studied. These differences were then confirmed with small volume dissolution-permeation assays where the addition of Tween 80 to the ASDs decreased the flux dramatically. Also, the early indication of sorbitol's advantage over mannitol by PAMPA has been confirmed in the investigation of the final dosage forms by large-scale dissolution-permeation tests. This difference between the fillers was observed in vivo as well. The presented case study demonstrated that the ADDF concept opens a new perspective in generic formulation development using fast and cost-effective flux-based screening methods in order to meet the bioequivalence criteria. Graphical Abstract.

Towards a Better Understanding of the Post-Gastric Behavior of Enteric-Coated Formulations.

PURPOSE: The aim of our work was to develop a biorelevant dissolution method for a better understanding of the in vivo performance of delayed-release tablet formulations. METHODS: The typical pH profile and residence times in the stomach and small intestine were determined in fasted conditions based on the published results of swallowable monitoring devices. Then, a multi-stage pH shift dissolution method was developed by adding different amounts of phosphate-based buffer solutions to the initial hydrochloric acid solution. Because of the highly variable in vivo residence times in the stomach, two alternatives of the method were applied, modeling rapid and slow gastric emptying as well. This approach provided an opportunity to study the effect of the acidic treatment on post gastric release. Six enteric-coated low-dose acetylsalicylic acid (ASA) formulations including the reference Aspirin Protect were tested as a model compound. Moreover, the thickness of the coating of each formulation was investigated by scanning electron microscope. RESULTS: Comparing the in vitro results to the known properties of the formulations, the new method was found to be more discriminative than the USP dissolution method. Ingredients affecting the in vitro dissolution, and thus probably the in vivo performance, were identified in both the tablet core and the coating of the tested formulations. The limited available in vivo data also indicated an increased predictivity. CONCLUSION: Overall, the presented method may be an efficient tool to support the development of enteric coated generic formulations.

Use of an In Vitro Skin Parallel Artificial Membrane Assay (Skin-PAMPA) as a Screening Tool to Compare Transdermal Permeability of Model Compound 4-Phenylethyl-Resorcinol Dissolved in Different Solvents.

Absorption through the skin of topically applied chemicals is relevant for both formulation development and safety assessment, especially in the early stages of development. However, the supply of human skin is limited, and the traditional in vitro methods are of low throughput. As an alternative, an artificial membrane-based Skin Parallel Artificial Membrane Permeability Assay (Skin-PAMPA) has been developed to mimic the permeability through the stratum corneum. In this study, this assay was used to measure the permeability of a model compound, 4-phenylethyl-resorcinol (PER), dissolved in 13 different solvents that are commonly used in cosmetic formulation development. The study was performed at concentrations close to the saturated solution of PER in each solvent to investigate the maximum thermodynamic potential of the solvents. The permeability of PER in selected solvents was also measured on ex vivo pig skin for comparison. Pig ear skin is an accepted alternative model of human skin. The permeability coefficient, which is independent of the concentration of the applied solution, showed a good correlation (R2 = 0.844) between the Skin-PAMPA and the pig skin permeation data. Our results support the use of the Skin-PAMPA to screen the suitability of different solvents for non-polar compounds at an early stage of formulation development.

Revisit of solubility of oxytetracycline polymorphs. An old story in the light of new results.

In the literature the therapeutic nonequivalence of oxytetracycline hydrochloride (OTCH) capsules and tablets was attributed to the different aqueous solubility of polymorphs without their comprehensive study. Our aim was to reveal the effects of polymorphism on equilibrium solubility, dissolution kinetics and the supersaturation of two OTCH polymorphs (stable Form A and metastable Form B).The equilibrium solubility was measured in biorelevant pH range 4-7.4 by the standardized saturation shake-flask method. We also studied the solubility in SGF at pH 1.2 and the effect of the pH change from 1.2 to 5.0 on solubility. The dissolution was studied using real-time concentration monitoring with an ATR probe attached to a UV spectrophotometer (µDISS Profiler). A wide spectrum of solid phase analysis methods (SEM, IR, XRPD, Raman) was applied for characterization of polymorphs and to identify which form is present at the equilibrium solubility. Identical equilibrium solubility values were obtained at the same pHs in region 4.0-7.4 using the two polymorphs as starting materials. The XRPD analysis of the isolated solid phases proved that both polymorphic forms were converted to dihydrate form. In situ monitoring of the dissolution at pH 5.0 showed immediate dissolution, no difference in supersaturation, and short equilibration time for both forms indicating the immediate conversion. In SGF (pH 1.2) Form B dissolved better than Form A and showed significantly different dissolution kinetic and stability. A long-lasting, false chain-citation stating that Form B dissolves 28x better in water than Form A, was cut by the present study (i) revealing that the cited data was measured in IPA not in water, and (ii) proving that only the intrinsic solubility of OTC dihydrate can be measured in water due to conversion of polymorphs under the experimental conditions of solubility measurement. However this conversion is inhibited below pH 1.5, so the differences in solubility and dissolution kinetic found at pH 1.2 may contribute to the interpretation of the different serum-levels reported at solid formulations.

Prediction of Bioequivalence and Food Effect Using Flux- and Solubility-Based Methods.

In this work, two different approaches have been developed to predict the food effect and the bioequivalence of marketed itraconazole (ITRA) formulations. Kinetic solubility and simultaneous dissolution-permeation tests of three (ITRA) formulations (Sporanox capsules and solution and SUBA-ITRA capsules) were carried out in simulated fasted and fed states. Fraction of dose absorbed ratios estimating food effect and bioequivalence were calculated based on these results and were compared to the in vivo study results published by Medicines Agencies. The comparison demonstrated that kinetic solubility and flux values could be used as input parameters for biopharmaceutics modeling and simulations to estimate food effect and bioequivalence. Both prediction methods were able to determine a slightly negative food effect in the case of the Sporanox solution and also a pronounced positive food effect for the Sporanox capsule. Superior bioavailability was predicted when the Sporanox solution was compared to the Sporanox capsule (in agreement with in vivo data).

Effect of Formulation Additives on Drug Transport through Size-Exclusion Membranes.

The aim of this research was to investigate the driving force of membrane transport through size-exclusion membranes and to provide a concentration-based mathematical description of it to evaluate whether it can be an alternative for lipophilic membranes in the formulation development of amorphous solid dispersions. Carvedilol, an antihypertensive drug, was chosen and formulated using solvent-based electrospinning to overcome the poor water solubility of the drug. Vinylpyrrolidone-vinyl acetate copolymer (PVPVA64) and Soluplus were used to create two different amorphous solid dispersions of the API. The load-dependent effect of the additives on dissolution and permeation through regenerated cellulose membrane was observed by a side-by-side diffusion cell, µFLUX. The solubilizing effect of the polymers was studied by carrying out thermodynamic solubility assays. The supersaturation ratio (SSR, defined as the ratio of dissolved amount of the drug to its thermodynamic solubility measured in exactly the same medium) was found to be the driving force of membrane transport in the case of size-exclusion membranes. Although the transport through lipophilic and size-exclusion membranes is mechanistically different, in both cases, the driving force of membrane transport in the presence of polymer additives was found to be the same. This finding may enable the use of size-exclusion membranes as an alternative to lipid membranes in formulation development of amorphous solid dispersions.

Synthesis and characterization of amino acid substituted sunitinib analogues for the treatment of AML.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.

Equilibrium solubility measurement of compounds with low dissolution rate by Higuchi's Facilitated Dissolution Method. A validation study.

Incubation time plays a critical role in the accurate measurement of equilibrium solubility of compounds. Substances which dissolve very slowly generally need long incubation times (days or weeks) to reach equilibrium. However, long times may pose several problems, such as decomposition of solute, molding of buffer, and drifting of pH. Higuchi in 1979 proposed the Facilitated Dissolution Method (FDM) to dramatically reduce incubation time. It employs a small volume of water-immiscible organic solvent to partly solubilize the sample and thereby increase the surface area available for dissolution. The method has been used only rarely. In this study we performed a systematic validation of FDM using progesterone as model compound. The reference solubility value, 7.95±0.21µg/mL (p<0.05, n=5), was determined in Britton-Robinson buffer solution (pH7.4) at 25.0°C by the standardized protocol of Saturation Shake-Flask (SSF) method. Also, the solubility was measured by the FDM approach under varied experimental conditions (e.g., type and volume of organic solvent, time of agitation, and amount of solid excess), and compared to the reference value. It was demonstrated that the small amount of organic solvent used in the FDM does not impact the measured solubility, compared to the reference value. Additionally, four compounds of low dissolution rate (dexamethasone, digoxin, haloperidol and cosalane) were used to demonstrate that FDM can reduce the long equilibration time to the standardized 24h (6h stirring and 18h sedimentation). The time dependence of solubility equilibrium was measured by SSF, and the results were compared with those obtained by FDM. Our study, based on >200 solubility experiments, supports the validity of Higuchi's method. In this study we propose a standardized protocol for the FDM, where 1% v/v of organic solvent is used. Octane (or isooctane) was found to be suitable for highly hydrophobic compounds. Alternatively, octanol or 1,2-dichloroethane can be used for less lipophilic compounds.

Effect of solubility enhancement on nasal absorption of meloxicam.

Besides the opioids the standard management of the World Health Organization suggests NSAIDs (non-steroidal anti-inflammatory drugs) alone or in combination to enhance analgesia in malignant and non-malignant pain therapy. The applicability of NSAIDs in a nasal formulation is a new approach in pharmaceutical technology. In order to enhance the nasal absorption of meloxicam (MX) as an NSAID, its salt form, meloxicam potassium monohydrate (MXP), registered by Egis Plc., was investigated in comparison with MX. The physico-chemical properties of the drugs (structural analysis, solubility and dissolution rate) and the mucoadhesivity of nasal formulations were controlled. In vitro and in vivo studies were carried out to determine the nasal applicability of MXP as a drug candidate in pain therapy. It can be concluded that MX and MXP demonstrated the same equilibrium solubility at the pH5.60 of the nasal mucosa (0.017mg/ml); nonetheless, MXP indicated faster dissolution and a higher permeability through the synthetic membrane. The animal studies justified the short Tmax value (15min) and the high AUC of MXP, which is important in acute pain therapy. It can be assumed that the low mucoadhesivity of MXP spray did not increase the residence time in the nasal cavity, and the elimination from the nasal mucosa was therefore faster than in the case of MX. Further experiments are necessary to prove the therapeutic relevance of this MXP-containing innovative intranasal formulation.

Investigation of the Efficacy of Transdermal Penetration Enhancers Through the Use of Human Skin and a Skin Mimic Artificial Membrane.

The aim of this study was to investigate the behavior of promising penetration enhancers through the use of 2 different skin test systems. Hydrogel-based transdermal formulations were developed with ibuprofen as a nonsteroidal anti-inflammatory drug. Transcutol and sucrose esters were used as biocompatible penetration enhancers. The permeability measurements were performed with ex vivo Franz diffusion cell methods and a newly developed Skin Parallel Artificial Membrane Permeability Assays (PAMPA) model. Franz diffusion measurement is commonly used as a research tool in studies of diffusion through synthetic membranes in vitro or penetration through ex vivo human skin, whereas Skin PAMPA involves recently published artificial membrane-based technology for the fast prediction of skin penetration. It is a 96-well plate-based model with optimized artificial membrane structure containing free fatty acid, cholesterol, and synthetic ceramide analog compounds to mimic the stratum corneum barrier function. Transdermal preparations containing 2.64% of different sucrose esters and/or Transcutol and a constant (5%) of ibuprofen were investigated to determine the effects of these penetration enhancers. The study demonstrated the good correlation of the permeability data obtained through use of human skin membrane and the in vitro Skin PAMPA system. The Skin PAMPA artificial membrane serves as quick and relatively deep tool in the early stages of transdermal delivery systems, through which the enhancing efficacy of excipients can be screened so as to facilitate the choice of effective penetration components.

Permeability test for transdermal and local therapeutic patches using Skin PAMPA method.

Using the skin as absorption site presents unique advantages that have facilitated the progression of transdermal drug delivery in the past decades. Efforts in drug research have been devoted to find a quick and reproducible model for predicting the skin permeation of molecules. The Parallel Artificial Membrane Permeability Assay (PAMPA) has been extended for prediction of transdermal permeation by developing a model with completely artificial membrane, which can mimic the permeation through the stratum corneum. The present study aims to extend the Skin PAMPA method for testing transdermal and local therapeutic patches. The original method was modified and seven commercially available transdermal and local therapeutic patches with four different active pharmaceutical ingredients (nicotine, fentanyl, rivastigmine and ketoprofen) were studied. Data were compared to the declared delivery rates that are indicated by the manufacturers. Ex vivo permeation study was also performed in order to compare the permeated amount of the released drugs obtained by the two methods. The flux across the artificial membrane as well as the human skin (ex vivo) has been calculated and compared to the in vivo flux deduced from the labelled delivery rate and the active area of the patches. The results suggest that Skin PAMPA system can serve as a useful tool for evaluation and classification of the transdermal patches.

PAMPA study of the temperature effect on permeability.

The purpose of this work was to investigate the temperature dependence of permeability measured by PAMPA method. The effective permeability (logPe) of seven drugs representing diverse structures and different acid-base properties was determined on three membrane models (GIT, BBB, Skin). The incubation temperature was varied in the range of 15-55 °C with ten degree steps. The intrinsic permeability (logP0) of the compounds is in linear relation with temperature (T). The slope of the logP0=aT+b regression equation is a good measure of the temperature effect on permeability. Results show intensive and significant temperature dependence of permeability influenced by the properties of the compounds and also by the selected PAMPA model. The Skin PAMPA(™) proved to be the most sensitive on temperature alteration, though GIT and BBB PAMPA results were also affected. The compound with acid function showed the lowest temperature dependence, while the permeability of bases increased considerably in response to the increasing temperature. The importance of human-relevant incubation conditions at in vitro assays is concluded for the better in vivo prediction.

Biorelevant solubility of poorly soluble drugs: rivaroxaban, furosemide, papaverine and niflumic acid.

In this work the biorelevant solubility of four drugs representing different acid-base property, wide range of lipohilicity and low aqueous solubility was studied. The equilibrium solubility of rivaroxaban (non-ionizable), furosemide (acid), papaverine (base) and niflumic acid (ampholyte) was determined in simulated gastric fluid (SGF pH 1.2), in simulated intestinal fluid fasted state (FaSSIF pH 6.5) and fed state (FeSSIF pH 5.0) and their corresponding blank buffers at a temperature of 37 °C using saturation shake-flask method. The concentration was measured by optimized HPLC analysis. The solubilizing effect of bile acid/lipid micelles as additive components of biorelevent media (BRM) is expressed with the solubility ratio (SR: SBRM/Sblank buffer) and the food effect was estimated from SFeSSIF/SFaSSIF coefficient. It was revealed that ionization plays primarily role in solubility of compounds which undergo ionization in BRM. The solubilizing effect in FaSSIF was marginal for the neutral compound (rivaroxaban) and for molecules are anionic at pH 6.5 (furosemide and niflumic acid). The higher concentration of solubilizing agents in FeSSIF improved the solubility of papaverine carrying positive charge and niflumic acid being partially zwitterionic at pH 5.0.

Predicting the exposure and antibacterial activity of fluoroquinolones based on physicochemical properties.

The purposes of this study are to evaluate if the PAMPA (Parallel Artificial Membrane Permeability Assay) permeability and the true partition coefficient could be useful for predicting AUC and MIC data of a group of antibacterial fluoroquinolones (FQs). The protonation macro- and microconstants, the n-octanol/water partition coefficients at isoelectric pHs, and the PAMPA permeability of 11 selected FQs were determined, and used to calculate the true partition coefficient, the interactivity parameter between the acidic and basic group, and the apparent intrinsic permeability. It has been shown that the apparent intrinsic permeability correlates well with the AUC in human, whereas the true partition coefficient and the interactivity parameter correlate with 1/MIC values on two Gram-positive bacteria, namely Streptococcus pneumonia and Staphylococcus aureus (methicillin-susceptible). The AUC/MIC ratios predicted from these correlations have shown to be in good agreement with the literature values. It is envisaged that the models described in this study could be useful in the development of new FQs by enabling an early prediction of AUC/MIC ratios based on physicochemical properties.

Skin-PAMPA: a new method for fast prediction of skin penetration.

The goal of this study was to develop a quick, reliable, and cost-effective permeability model for predicting transdermal penetration of compounds. The Parallel Artificial Membrane Permeability Assay (PAMPA) was chosen for this purpose, as it already has been successfully used for estimating passive gastrointestinal absorption and blood-brain barrier permeability. To match the permeability of the rate-limiting barrier in human skin, synthetic certramides, which are analogs of the ceramides present in the stratum corneum, were selected for the skin-PAMPA model. The final skin-PAMPA membrane lipid mixture (certramide, free fatty acid, and cholesterol) was selected and optimized based on data from three different human skin databases and the final model was found to correlate well to all of the databases. The reproducibility of the skin-PAMPA model was investigated and compared to that of other PAMPA models. The homogeneity of the filter-impregnated lipid mixture membrane was confirmed with Raman microscopy. It was shown that skin-PAMPA is a quick and cost-effective research tool that can serve as a useful model of skin penetration in pharmaceutical and cosmetic research.