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.

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.

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.

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.

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.

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.

Synthesis and characterization of long-chain tartaric acid diamides as novel ceramide-like compounds.

Ceramides play a crucial role in the barrier function of the skin as well as in transmembrane signaling. In this study long aliphatic chain tartaric acid diamides able to replace ceramides in an in vitro model of the stratum corneum lipid matrix due to their similar physico-chemical properties were synthesized from diacetoxysuccinic anhydride in four steps. Their pro-apoptotic effect on fibroblast cells was also investigated.

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.

A PAMPA study of the permeability-enhancing effect of new ceramide analogues.

There is a major need in drug discovery for quick, precise, and cost-effective high-throughput screening (HTS) systems in the early stages of drug research. The Parallel Artificial Membrane Permeability Assay (PAMPA) aims at predicting the passive membrane properties of drugs. Since 1998, model membranes have been developed to predict gastro-intestinal absorption or transport through the blood-brain barrier. This paper presents recent results in a project aiming to improve the prediction of transdermal penetration. Using the PAMPA system, we investigated the effect of four newly synthetized ceramide analogues (certramides) on the permeability of three model compounds (ciprofloxacin, nifedipine, and verapamil). The certramides differ in the length of one alkyl chain, while the other alkyl chain and the head group remained the same. A relationship between the membrane concentration of certramides (from 0 to 100%) and the permeability of compounds was found, and the results of different certramides were compared. The strongest effect on permeability was caused by the ceramide analogue CTR(C12-C16). The reproducibility of the experiments and the impact of presence or absence of organic solvents (dodecane and CHCl3) in the membrane were also investigated.

Monte Carlo structure simulations for aqueous 1,4-dioxane solutions.

Monte Carlo simulations in the NpT ensembles have been performed for the structure exploration of aqueous 1,4-dioxane solutions. Three different systems with all-atom dioxane:TIP4P water molar compositions of 2:500 (code:D2), 8:465 (D8), and 17:425 (D17) modeled solutions of 0.22, 0.88, and 1.86 mol/dm3 concentrations, respectively, at T = 298 K and p = 1 atm. The calculated solution densities increase from 0.992 to 1.002 g/cm3 with increasing dioxane concentration and approach the experimentally determined densities within 1%. This close agreement was achieved by utilizing RESP charges fitted to the in-solution IEF-PCM/B3LYP/6-31G* electrostatic potential of dioxane taken in its chair conformation and recently developed C, H steric parameters for ethers for calculations with a 12-6-1 all-atom potential. Solution structure analyses pointed out that the dioxane molecules arrange in the solutions with favorable distances of 4-8 angstroms for the ring symmetry centers. Within this range not only pairs of rings but triangular triads and tetrads have also been observed with center-center distances <8 angstroms. For the D8 system, about 25% of the sampled configurations included such a triad. In the case of the D17 model, two simulations starting from different solution configuration predicted different degrees for the dioxane aggregation in aqueous solution. In the more aggregated structure 3-21 triads are consistently maintained and 1-2 tetrads are formed in 58% of the configurations. Each dioxane oxygen forms about one hydrogen bond, on average, to a water molecule in the 0.22-1.86 molar range. The most likely O(dioxane)...H(water) hydrogen bond distance is 1.75-1.80 angstroms compared to the optimal distance of 1.72 angstroms in the isolated dimer. The optimal dioxane-water interaction energy of -5.65 kcal/mol indicates a remarkable hydrogen-bond acceptor character for dioxane.

Study of equilibrium solubility measurement by saturation shake-flask method using hydrochlorothiazide as model compound.

The experimental conditions that affect equilibrium solubility values measured by the classical saturation shake-flask method have been examined, using hydrochlorothiazide as a model compound. Modifications in temperature, sedimentation time, composition of aqueous buffer and the technique of separation of solid and liquid phases were all found to influence the equilibrium solubility results strongly. However, variations in the amount of solid excess and stirring time were found to have less influence. In the light of these observations, a new, shorter protocol has been developed for measurements of equilibrium solubility, together with recommendations for good analytical practice. The equilibrium solubilities of five other drugs were measured to verify the new protocol.

Physico-chemical characterization of a novel group of dopamine D(3)/D(2) receptor ligands, potential atypical antipsychotic agents.

The fundamental physico-chemical properties such as ionization and lipophilicity of twelve alkyl-aryl-piperidine and aryl-piperazine derivatives have been determined. Compounds are members of a recently identified, new class of potent dopamine D(3)/D(2) receptor ligands as potential atypical antipsychotic agents and were used in the development of a promising drug candidate (RGH-188) being present currently in clinical phase II investigations. The ionization constant (pK(a)) and the partition coefficient in octanol/water (logP(oct)) and cyclohexane/water systems (logP(ch)) were measured by validated analytical methods. Based on the highly precise physico-chemical data the structure-property relationships (SPR) were studied. The effect of the polar and apolar heteroatoms as well as polar and apolar surface areas on the partition in the two solvent systems was investigated by linear regression and multivariate linear regression analyses. Brain/blood concentration ratios (BB values) as a function of time were determined by HPLC analyses on plasma and brain homogenates of Wistar rats. A linear relationship has been found between DeltalogP values (logP(oct)-logP(ch)) and experimental logBB values, verifying that physico-chemical data can predict pharmacokinetic behaviour.

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.

Equilibrium versus kinetic measurements of aqueous solubility, and the ability of compounds to supersaturate in solution--a validation study.

A novel potentiometric procedure has recently been described for rapid measurement of equilibrium aqueous solubility values of organic acids, bases, and ampholytes that form supersaturated solutions. In this procedure, the equilibrium solubility is actively sought by changing the concentration of the neutral form by adding HCl or KOH titrants and monitoring the rate of change of pH due to precipitation or dissolution in a process called Chasing Equilibrium. In this article, we seek to validate the procedure against a shake-flask protocol for solubility determination. A set of 16 small organic compounds, covering a wide range of solubilities was chosen for the study. Interestingly, while 10 compounds in the study were found to chase equilibrium, the others did not. Kinetic solubility data was also collected. It was noted that kinetic solubility was consistently higher than equilibrium solubility for chasers, but correlated well with equilibrium solubility for nonchasers. The ratio of kinetic to equilibrium solubility indicated a compound's ability to form supersaturated solutions.

Physico-chemical profiling of antidepressive sertraline: solubility, ionisation, lipophilicity.

The fundamental physico-chemical parameters of sertraline, a potent selective serotonin reuptake inhibitor and reference compound in the development of new antidepressive agents, were determined. The thermodynamic solubility of the hydrochloride salt (S = 4.24 +/- 0.02 mg/ml) and the free base form (S = 0.002 mg/ml) was measured by the saturation shake-flask method. The co-solvent technique in methanol/water mixtures and the Yasuda-Shedlovsky extrapolation were applied for the determination of the dissociation constant (pKa = 9.16 +/- 0.02). The partition of sertraline was studied in octanol/water and alkane/water systems determining the logPoct and logPch)values by potentiometric and shake-flask methods, respectively. These experimental data were used to interpret the excellent pharmacokinetic properties of the molecule. The high lipophilicity value (logPoct = 4.30 +/- 0.01) of the nonionised form confirms the good absorption and distribution in the body. However, the good brain penetration can better be explained with the lack of polar interactions evidenced here by the zero DeltalogP (logPoct-logPch) value of sertraline.

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.