Krisztina Takács-Novák

Potentiometric pKa determination of water-insoluble compounds: validation study in methanol/water mixtures

Abstract The apparent ionization constants (psKa) of 25 molecules, based on 431 separate potentiometric titrations, were determined in methanol/water mixtures of 15–65 wt% methanol content. The Yasuda-Shedlovsky extrapolation (psKa+log[H2O]=a/ϵ+b) was used to obtain the pKa values in zero methanol%. In the case of water-soluble drugs the extrapolated data were in very good agreement with pKa values measured in aqueous solutions under the same experimental conditions (average deviation=0.05). The water-insoluble molecules showed acceptable accordance with spectroscopically measured or with literature pKa values. Concentration dependence between 1–5 mM was not observed while the range of extrapolation (water-rich: R=15–35 wt%, or methanol-rich: R=40–65 wt%) significantly influenced the accuracy of pKa values. Remarkable changes in the solvation structure of weak acids in methanol-rich mixtures (>35 wt%) were suggested from analyzing the slopes of Yasuda-Shedlovsky equations. Recommendations for the proper application of a mixed-solvent procedure in order to gain the most reliable aqueous pKa values are suggested.

Lipophilicity of antibacterial fluoroquinolones

Abstract The octanol/water partition coefficients of nine antibacterial fluoroquinolones and nalidixic and oxolinic acids were investigated. The pH-partition profile of amphoteric fluoroquinolones obtained between pH 4 and 10 showed maximum partitioning at the isoelectric point. From the two microspecies (zwitterionic and nonionic forms) which exist predominantly at this pH, the nonionic form is assumed to be transferred into the octanol phase. A relationship is derived between the apparent and true partition coefficients, valid for ampholyte compounds capable of forming zwitterions and having nonionic microspecies present in significant amounts. On the bases of true partition coefficients, three groups of examined fluoroquinolones are distinguished: lipophilic compounds (e.g., pefloxacin and amifloxacin), molecules of intermediate lipophilicity (such as ciprofloxacin and ofloxacin, etc.) and hydrophilic derivatives (e.g., norfloxacin and lomefloxacin, etc.). The influence of structural modification on the lipophilicity of these drugs is discussed.

Interlaboratory study of log P determination by shake-flask and potentiometric methods

The pKa and log P values of 23 structurally diverse compounds, including well known drugs and two pharmacons under development, were determined by potentiometry. Also, the log P data were measured by the shake-flask method. Many of the samples were investigated at both of the participating laboratories in order to evaluate the reproducibility of the pH-metric log P technique. The interlaboratory evaluation of pKa and log P data obtained by potentiometry showed excellent agreement (average delta pKa = +/-0.02 and delta log P = +/-0.07). The log P values obtained by the two different methods, ranging from -1.84 to 5.80 (nearly eight orders of magnitude), were in very good concordance, as shown by the linear regression analysis: log PpH-metric = 0.9794 log Pshake-flask -0.0397 (r = 0.9987, s = +/-0.091, F = 8153). The advantages of potentiometric log P determination are discussed.

Multiwavelength spectrophotometric determination of acid dissociation constants part V: microconstants and tautomeric ratios of diprotic amphoteric drugs.

The acid-base equilibria of several diprotic amphoteric drugs, namely, niflumic acid, norfloxacin, piroxicam, pyridoxine and 2-methyl-4-oxo-3H-quinazoline-3-acetic acid have been characterized in terms of microconstants and tautomeric ratios. A multiwavelength spectrophotometric (WApH) titration method for determination of acid dissociation constants (pKa values) of ionizable compounds developed previously was applied for this purpose. Microspeciation was investigated by three approaches: (1) selective monitoring of ionizable group by spectrophotometry, (2) deductive method and (3) k(z) method for determination of tautomeric ratio from co-solvent mixtures. The formulation for (3) has been derived and found to invoke fewer assumptions than a reported procedure (K. Takács-Novák, A. Avdeef, K.J Box, B. Podányi, G. Szász, J. Pharm. Biomed. Anal., 12 (1994) 1369-1377). It has been shown that the WApH technique, for such types of ampholytes, is able to deduce the microconstants and tautomeric ratios which are in good agreement with literature data.

Lipophilicity of amphoteric molecules expressed by the true partition coefficient

The octanol/water partition coefficients of six amphoteric drugs were investigated. Nitrazepam, albendazole and sulfadimidine are ordinary ampholytes, while pyridoxine, niflumic acid and terbutaline belong to the zwitterionic amphoteric compounds. The pH-partition profile of compounds showed maximum (parabolic) curve. Analyses of the UV spectra in aqueous and octanol phases at different pH values after partitioning equilibrium had been achieved proved the transfer of the neutral species into the octanol phase. The true partition coefficients were calculated from log Papp values using macroprotonation constants for ordinary ampholytes and microprotonation constants in the case of zwitterionic molecules. The results emphasize that only the true partition coefficient closely represents the intrinsic lipophilicity of zwitterionic amphoteric compounds.

Determination of protonation macro- and microconstants and octanol/water partition coefficient of the antiinflammatory drug niflumic acid

The drug niflumic acid is an amphoteric substance with overlapping pKa values. The acid-base chemistry of the molecule has been characterized in terms of protonation macroconstants (with reference to stoichiometric ionizations) and microconstants (with reference to ionizations of individual species). The proton-binding sites were assigned using 1H and 13C NMR spectroscopy. Due to the very poor water solubility of niflumic acid, the aqueous pKa values were determined from the apparent ionization constants in methanol-water solutions of various proportions by extrapolation to zero co-solvent using the Yasuda-Shedlovsky procedure. The kz tautomerization microconstant of the equilibrium unionized form<-->zwitterionic form was determined from mixtures of organic solvent (dioxane or methanol) with aqueous buffer (at the pH of isoelectric point) by UV spectroscopy, and used for calculation of the other protonation microconstants. The zwitterionic form of the molecule predominates over the uncharged form, the concentration being maximal at the isoelectric pH. The apparent partition coefficients (Papp) of niflumic acid were measured in octanol/water solution by the shake-flask method over a wide pH range. The lipophilicity profile (logPapp vs pH) shows a parabolic shape near its maximum at the isoelectric point. A relationship derived between Papp, PXH0(micropartition coefficient of the uncharged microspecies) and PX-(partition coefficient of the anion) is valid for amphoteric drugs, in cases where the partition of the unionized form and the ion-pair partition of anion can be confirmed. The logP values of microspecies indicate the high lipophilicity of niflumic acid, which is consistent with its good skin penetration and absorption.

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.

Ion-pair partition of quaternary ammonium drugs: The influence of counter ions of different lipophilicity, size, and flexibility

AbstractPurpose. The ion-pair partition of quaternary ammonium (QA) pharmacons with organic counter ions of different lipophilicity, size, shape and flexibility was studied to elucidate relationships between ion-pair formation and chemical structure. Methods. The apparent partition coefficient (P′) of 4 QAs was measured in octanol/pH 7.4 phosphate buffer system by the shake-flask method as a function of molar excess of ten counter ions (Y), namely: mesylate (MES), acetate (AC), pyruvate (PYRU), nicotinate (NIC), hydrogenfumarate (HFUM), hydrogenmaleate (HMAL), p-toluenesulfonate (PTS), caproate (CPR), deoxycholate (DOC) and prostaglandin E1 anion (PGE1). Results. Based on 118 of highly precise logP′ values (SD< 0.05), the intrinsic lipophilicity (without external counter ions) and the ion-pair partition of QAs (with different counter ions) were characterized. Linear correlation was found between the logP′ of ion-pairs and the size of the counter ions described by the solvent accessible surface area (SASA). The lipophilicity increasing effect of the counter ions were quantified and the following order was established: DOC ~ PGE1≫ CPR ~ PTS ≫ NIC ~ HMAL ≫ PYRU ~ AC ~ MES ~ HFUM. Analyzing the lipophilicity/molar ratio (QA:Y) profile, the differences in the ion-pair formation were shown and attributed to the differences in the flexibility/rigidity and size both of QA and Y. Conclusions. Since the largest (in average, 300 ×) lipophilicity enhancement was found by the influence of DOC and PGE1 and considerable (on average 40×) increase was observed by CPR and PTS, it was concluded that bile acids and prostaglandin anions may play a significant role in the ion-pair transport of quaternary ammonium drugs and caproic acid and p-toluenesulfonic acid may be useful salt forming agents to improve the pharmacokinetics of hydrophilic drugs.

Study of pH-dependent solubility of organic bases. Revisit of Henderson-Hasselbalch relationship

In this paper the pH-equilibrium solubility profiles of six organic drugs are presented. The equilibrium solubility values were determined using the saturation shake-flask and the Chasing Equilibrium Solubility (CheqSol) methods. Results obtained by the two methods are in good agreement. The aim of the present work was to study the validity of the Henderson-Hasselbalch (HH) relationship in the case of structurally diverse weak bases. The significance of pH control and the effect of the salt form (e.g., fumarate) was also investigated. In the case of monoprotic bases, namely papaverine, promethazine, propafenone and ticlopidine the experimental solubility data precisely follow the HH equation until the limit of salt solubility. The common ion effect on salt solubility was found to be significant at low pHs. Deviation from the HH equation in the case of dibasic quetiapine hydrogen fumarate and the ampholyte desvenlafaxine hydrogen fumarate can be easily interpreted with the formation of different salt compositions. It was concluded that precise pH control is essential in shake-flask solubility measurements. It is also critical that the pK(a) value and the intrinsic solubility are accurately determined when the HH relationship is used to predict the pH-dependent aqueous solubility of drugs.

Acid-base properties and proton-speciation of vancomycin

Abstract The protonation-deprotonation equilibria of the glycopolypeptide antibiotic vancomycin are characterized in terms of protonation macroconstants, isoelectric point and diagram of pH-dependent species distribution. Vancomycin contains two basic and four acidic groups. Of these, dissociation of the C-terminal carboxyl group takes place separately at low pH, whereas proton binding of the two amino sites and deprotonation of the three phenolic hydroxyl groups occur between pH 5 and 13, in a highly overlapping fashion. Our study indicates for an isoelectric point of 8.30 the vancomycin molecule and an average charge of + 0.67, with the predominant existence of penta- and tetraprotonated species at physiological pH.