Partition of N-monodemethylated phenothiazine drugs to phosphatidylcholine bilayer vesicles studied by second-derivative spectrophotometry

The partition coefficients (Kps) of three N-monodemethylated metabolites of phenothiazines (chlorpromazine (CPZ), triflupromazine, and promazine) between phosphatidylcholine (PC) vesicles (SUV) and water were determined to evaluate their affinity to biomembranes by second-derivative spectrophotometry without any separation procedures. The second derivative spectra showed distinct derivative isosbestic points confirming the entire elimination of the residual background signal effects of the SUV, which were observed in the absorption spectra. From the relationship between PC SUV concentration and the derivative intensity change (DeltaD) of each metabolite induced by its interaction with the PC bilayer, the Kp values were calculated and could be obtained with the R.S.D. of below 10% (n = 5) proving an accuracy of the derivative method. The obtained Kp values were similar to those of the parent drugs (the relative differences were within 15%), although the partition coefficient of N-monodemethylated CPZ measured in an octanol/buffer system was reported to be about 1/18 of CPZ. The results obtained from the PC liposome/buffer system do not contradict with psychoactivity and brain accumulation of N-monodemethylated metabolites of phenothiazines, in contrast to the results of the octanol/buffer system.     

Determination of lipid bilayer/water partition coefficient of new phenothiazines using the second derivative of absorption spectra method.

The partition coefficients (K(p)) between lipid bilayer of phosphatidylcholine (PC) vesicles and buffer for five new phenothiazines were determined using the second derivatives of ultraviolet absorption spectra. The lambda(max) of absorption band for each of the investigated phenothiazine derivatives (PDs) was shifted to the longer wavelengths in the presence of PC vesicles with increasing of lipid concentration. As a result of light scattering in liposome suspension no isosbestic point could be observed in absorption spectra. However, the background signal could be eliminated using the method of second derivative of absorption spectra. In the second derivative of absorption spectra two isosbestic points were observed. Changes of intensity (Delta D) of second derivative of absorption spectra at the lambda(max) (wavelength of absorption maximum for drug in buffer) caused by the increase in lipid concentration were measured for set of phenothiazine derivatives. K(p) for these drugs were calculated from the relationship between Delta D and lipid concentration. The K(p) values for all studied phenothiazine derivatives are in the order of magnitude of 10(5) and they increase about 1.7-fold when length of the alkyl phenothiazine chain was enhanced by addition of the each next one (-CH(2)) group. Substitution of -H atom by -CF(3) group at position 2 of phenothiazine ring results in 3.5-fold increase in K(p) values.     

Determination of partition coefficients of diazepam and flurazepam between phosphatidylcholine bilayer vesicles and water by second derivative spectrophotometric method

Second derivative spectrophotometry allowed the establishment of a simple and accurate method for the determination of partition coefficients of benzodiazepine drugs in a liposome/water system. The absorption spectra of diazepam (DZ) and flurazepam (FZ) in phosphatidylcholine (egg yolk) bilayer vesicle suspensions showed small spectral changes depending on the concentration of phosphatidylcholine vesicles. However, the intense background signals caused by the light scattering of the phosphatidylcholine vesicles made it difficult to yield a correct base line, thus the quantitative spectral data could not be obtained. In the second derivative spectra, the spectral changes were enhanced and three derivative isosbestic points were observed for each drug indicating the entire elimination of the residual background signal effects. The derivative intensity change of each drug (DeltaD) induced by its interaction with phosphatidylcholine bilayers was measured at a specific wavelength. From the relationship between the DeltaD value and the lipid concentration, the molar partition coefficients (K(p)s) of DZ and FZ were calculated and obtained with a good precision of R.S.D below 10%. The fractions of the partitioned DZ and FZ calculated by using the obtained K(p) values agreed well with the experimental values. The results prove that the derivative method can be usefully and easily applied to the determination of partition coefficients of benzodiazepines in the liposomes/water system without any separation procedures.     

Determination of conformer-specific partition coefficients in octanol/water systems

The first conformer-specific experimental partition coefficients are presented for octanol/water, the most widespread solvent system to predict lipophilicity of drugs. Rotamer populations in octanol and water were elucidated from 1H NMR vicinal coupling constants and were combined with classical partition coefficients to obtain the conformer-specific ones. Feasibility of the determination of conformer-specific partition coefficients is exemplified on amphetamine and clenbuterol, two flexible drug molecules. Partition capacities of the amphetamine rotamers have been proven to be essentially equal. The conformers of clenbuterol, however, have been found to be greatly different in partition properties, which could be interpreted in terms of intramolecular interactions between the vicinal polar sites and the solvent-accessibility of the groups. The conformers could be put into order of their membrane-influx and -outflow propensities. Deviations between experimental and calculated log P values could also be interpreted in view of the species-specific partition coefficients.     

沙美特罗和布地奈德的脂质体/水分配系数测定及影响因素考察

目的测定沙美特罗和布地奈德的脂质体/水分配系数(P_L/w)，考察影响两种药物P_L/w的因素，揭示药物与磷脂双分子层的作用机制。方法用平衡透析法测定两种药物在不同脂质体组成和不同介质中的P_L/w。结果两种药物P_L/w随脂质体中胆固醇含量及磷脂饱和程度增加而减小；脂质体表面负电荷、介质pH值及离子强度增加，沙美特罗的P_L/w增加。脂质体表面电荷、介质pH值及离子强度对布地奈德的P_L/w影响较小。结论药物P_L/w受药物性质、脂质体磷脂种类、饱和程度、胆固醇含量和表面电荷以及介质pH和离子强度等因素影响，在测定时应选择与生物膜环境相近的P_L/w测定条件，以反映药物在生物膜中的分配。     

Impurity profiling in bulk pharmaceutical batches using 19F NMR spectroscopy and distinction between monomeric and dimeric impurities by NMR-based diffusion measurements

The impurity profile of production batches of fluorine-containing drugs can be characterised efficiently using 19F NMR spectroscopy. This yields the number and proportions of impurities in the bulk drug to a level of approximately equal 0.1 mole% in a few minutes of NMR experiment time. The approach has been exemplified using a partially purified batch of the steroidal product fluticasone propionate, the impurities in which include a number of dimeric species. Further distinction between the monomer and dimer impurities has been achieved through high resolution chemical shift-resolved NMR measurement of molecular diffusion coefficients on the intact mixture using 19F NMR spectroscopy. The ability of NMR-based diffusion coefficient determination to distinguish between monomeric and dimeric substances was validated using a standard mixture of authentic materials containing both monomers and dimers.     

Solubilisation of dipalmitoylphosphatidylcholine bilayers by sodium taurocholate: A model to study the stability of liposomes in the gastrointestinal tract and their mechanism of interaction with a model bile salt

In order to better understand the mechanism of destabilization of liposomes used as drug carriers for oral administration by bile salts, the insertion and partition of sodium taurocholate (TC) into small unilamellar vesicles (SUV) and multilayers (ML) of dipalmitoylphosphatidylcholine (DPPC) were examined by continuous turbidity analysis and DSC. Optical density was recorded during the progressive solubilisation of DPPC SUV and ML into DPPC/TC mixed micelles by varying the rate of TC addition and the temperature. The results show that the insertion and diffusion of TC in the DPPC membrane is a slow process influenced by the polymorphism of the lipid, independently of its organisation. This dynamic study mimics physiological phenomena of the digestion of liposomes. In the gastrointestinal tract, DPPC SUV would be more resistant to TC than egg phosphatidylcholine (EPC) SUV [K. Andrieux, L. Forte, S. Lesieur, M. Paternostre, M. Ollivon, C. Grabielle-Madelmont, Insertion and partition of sodium taurocholate into egg phosphatidylcholine vesicles, Pharm. Res. 21 (2004) 1505-1516] because of the lower insertion of TC into DPPC bilayer at 37 °C at low TC concentration in the medium (fasted conditions). At high TC concentration (postprandially or after lipid absorption), the use of DPPC to prepare liposomes will delay or reduce the liberation of a drug encapsulated into liposomes in the gastrointestinal tract. As a conclusion, the addition of DPPC appears an attractive strategy to formulate orally administered liposomes.     

Protective effect caused by the exopolymer excreted by Pseudoalteromonas antarctica NF(3) on liposomes against the action of octyl glucoside

The capacity of the glycoprotein (GP) excreted by Pseudoalteromonas antarctica NF(3), to protect phosphatidylcholine (PC) liposomes against the action of octyl glucoside (OG) was studied in detail. Increasing amounts of GP assembled with liposomes resulted for the same interaction step in a linear increase in the effective surfactant to PC molar ratios (Re) and in a linear fall in the surfactant partitioning between bilayer and the aqueous phase (partition coefficients K). Thus, the higher the proportion of GP assembled with liposomes the lower the surfactant ability to alter the permeability of vesicles and the lower its affinity with these bilayer structures. In addition, increasing GP proportions resulted in a progressive increase of the free surfactant concentration (S(W)) needed to produce the same alterations in liposomes. The fact that S(W) was always lower than the surfactant critical micelle concentration indicates that the interaction was mainly ruled by the action of surfactant monomers, regardless of the amount of assembled GP.     

Evaluation of in vitro percutaneous absorption of lorazepam and clonazepam from hydro-alcoholic gel formulations

Clonazepam and lorazepam are two anxiolytics, antidepressant agents, having suitable features for transdermal delivery. The objectives of this study were to evaluate the in vitro percutaneous absorption of these drugs through excised human skin (stratum corneum and epidermis, SCE) and to determine their in vitro permeation behavior from a series of hydro-alcoholic gel formulations containing various enhancing agents. The best permeation profile was obtained for both drugs applying them together with Azone in combination with propylene glycol (PG): these enhancers were able to increase the clonazepam and lorazepam percutaneous fluxes at steady-state about threefold, compared to the free enhancer formulations (Control). To explain the mechanism of the used promoters, the benzodiazepine diffusion and partitioning coefficients from the gel containing the enhancers were calculated. The results indicated that the Azone in combination with PG could act by increasing the benzodiazepine diffusion coefficients, Transcutol increased only the SC/vehicle partition coefficients, limonene in combination with PG appeared to increase both partition and diffusion coefficients moderately, while PG did not increase both the parameters. Furthermore, to evaluate the potential application of tested benzodiazepine formulations containing Azone in combination with PG using the flux values from the in vitro experiments, the corresponding steady-state plasma concentrations (C(SS)) were calculated. The obtained calculated C(SS) values are within the lorazepam therapeutic range and suggest that transdermal delivery of this drug could be regarded as feasible.     

Transport across 1,9-decadiene precisely mimics the chemical selectivity of the barrier domain in egg lecithin bilayers

The barrier domain solubility-diffusion theory of lipid bilayer permeability relates the permeability coefficient (P(m)) to the solute's partition coefficient (PC(barrier/w)) and diffusion coefficient (D(barrier)) in the ordered chain region of the bilayer that serves as the barrier region for polar permeants. To select the best solvent to mimic the barrier domain, permeability coefficients across a layer of 1,9-decadiene were compared with permeability coefficients from bilayer transport. Rate constants for transport, k, of alpha-methyl substituted analogues of p-toluic and p-methylhippuric acid were measured across a layer of 1,9-decadiene embedded in a PTFE filter membrane placed between two aqueous solutions in side-by-side diffusion cells. Permeability coefficients (P(1,9-decadiene)) were normalized to that obtained for p-toluic acid, which was included in donor solutions. The correlation of log(P(bilayer)) versus log(P(1,9-decadiene)) was linear with a slope of 0.99 +/- 0.02 SD, indicating that 1,9-decadiene precisely mimics the egg lecithin bilayer barrier domain in its chemical selectivity. Using the decadiene membrane transport method to indirectly estimate partition coefficients for similarly sized permeants extended the range of measurable values beyond those readily attainable by the traditional shake-flask method, allowing measurement of 1,9-decadiene/water PCs as low as 3 x 10(-7).     

Gas-liquid chromatographic determination and pharmacological studies of six clinically-used local anesthetics.

A gas-liquid chromatographic method was developed for the simultaneous assay of six local anesthetics, including amethocaine, bupivacaine, etidocaine, lignocaine, mepivacaine and prilocaine, in biological samples. These drugs and internal standard (clomipramine) in basified samples were extracted into 5 ml n-hexane, and the extract was analyzed by a temperature programming method (the column temperature was kept at 210 degrees C for 5 min, then raised to 280 degrees C at the rate of 10 degrees C/min) using a 3% W/W SP 2250 glass column (2 m x 2 mm i.d.) connected to a nitrogen sensitive detector. The injector and detector temperature were maintained at 300 degrees C. The pKa values, partition coefficients (K) and buccal absorptions of six local anesthetics were determined. The results showed that the onset of action and duration could be shown to be dependent on the pKa values and partition coefficients, respectively. A relatively good positive correlation (r = 0.991) was observed between the percentage of buccal absorption at pH 7.4 and the logarithms of K in n-hexane-S?rensen buffer system, and hence the more lipid soluble the local anesthetic, the greater the buccal absorption. The buccal absorption test supplemented by the n-hexane-buffer partition coefficient could be used as indicators for the ability of the anesthetics to penetrate biological barriers. The lipid penetration of the drugs, and thus their pharmacological action, is also influenced by the pKa values of the anesthetics.     

Improved techniques for the photometric determination of partition coefficients, with particular reference to the chloroform-ammonia solvent system

Accurate partition coefficients K = Corg/Caq in chloroform-ammonia can be obtained by measuring the absorbance of the aqueous layer, replenishing with fresh aqueous solvent, and remeasuring the absorption after reequilibration. Provided the solute has a reasonably strong UV absorption, only 0.1-5 mg of material is required. Neither the amount, the extinction coefficient, nor (in most cases) the purity of the substrate material need be known. In general, K values ranging from 100 to 0.01 can be measured with a precision of greater than or equal to 10%. A detailed analysis of the absorbance and volumetric error sources permits the optimum combination of experimental conditions such as volume ratios, cell lengths, and absorbance (either direct or differential, or with scale expansion) to be predicted a priori. Quantitative estimates for all primary error sources, such as photometric precision, baseline error, stray light, and volumetric errors were also experimentally determined. The theoretical error model was tested by determining K for aniline under widely ranging experimental conditions and by comparing the experimental standard deviations with those calculated from the theoretical model. The agreement was found to be satisfactory. The method described appears to be of particular usefulness for the determination of extraction or chromatographic parameters of basic drugs.     

Kinetic model to predict the absorption of nasally applied drugs from in vitro transcellular permeability of drugs

The purpose of this study is to propose a kinetic model to predict the absorption of nasally applied drugs from their permeability to the Caco-2 monolayer (P(Caco-2)). Since a drug applied to the nose in an in vivo physiologic condition is translocated to the gastrointestinal (GI) tract by coordinated beats of cilia (mucociliary clearance, MC), the drug undergoes absorption both from the nasal cavity and from the GI tract. The detailed MC of the rat was examined, using inulin as a marker of the applied solution. Inulin disappeared monoexponentially from the nasal cavity, indicating that the MC can be assumed to follow first-order kinetics. From the disappearance of inulin, the first order rate constant for MC (k(MC)) was calculated as 0.0145 min(-1). In the proposed kinetic model, the fractional absorption of the drug following nasal application is predicted as the sum of F(NC) (fractional absorption from the nasal cavity) and F(GI) (fractional absorption from the GI tract), both of which are estimated indirectly from P(Caco-2). F(NC) is calculated according to the equation, k(a)/(k(a)+k(MC)), where k(a) is the absorption rate constant. Nasal drug absorption is assumed to follow first order kinetics. The k(a) of four drugs was initially calculated from k(MC) and their F(NC); thereafter, the linear relationship between k(a) and P(Caco-2), from which k(a) is predicted, was determined. F(GI) is calculated as F(p.o.)(1-F(NC)), where F(p.o.) is fractional absorption after oral administration. F(p.o.) was predicted from the previously determined sigmoid curve between F(p.o.) and P(Caco-2). The proposed kinetic model is the first estimation system for nasal drug absorption based on drug disposition after nasal application and is useful for the development of nasal dosage forms.     

19F and 1H magnetic resonance strategies for metabolic studies on fluorinated xenobiotics: application to flurbiprofen [2-(2-fluoro-4-biphenylyl)propionic acid

Strategies for the use of 1H and 19F nuclear magnetic resonance (NMR) spectroscopy as an aid to the study of the metabolic fate of fluorinated drugs are discussed with reference to the application of these methods to flurbiprofen metabolism in man. 1H and 19F NMR analysis of untreated urine enabled the detection of two major and eight minor metabolites of the drug. The two major metabolites were identified using a combination of NMR spectroscopy, solid-phase extraction chromatography with 19F and 1H NMR detection and chemical hydrolysis to a flurbiprofen glucuronide and the glucuronide of the 4-hydroxy metabolite. 1H-19F 2D shift correlated spectroscopy and spin-echo difference experiments are discussed in relation to their use in the structural identification of drug metabolites.     

Ionization constants by curve fitting: determination of partition and distribution coefficients of acids and bases and their ions

A convenient procedure has been developed for the determination of partition and distribution coefficients. The method involves the potentiometric titration of the compound, first in water and then in a rapidly stirred mixture of water and octanol. An automatic titrator is used, and the data is collected and analyzed by curve fitting on a microcomputer with 64 K of memory. The method is rapid and accurate for compounds with pKa values between 4 and 10. Partition coefficients can be measured for monoprotic and diprotic acids and bases. The partition coefficients of the neutral compound and its ion(s) can be determined by varying the ratio of octanol to water. Distribution coefficients calculated over a wide range of pH values are presented graphically as "distribution profiles". It is shown that subtraction of the titration curve of solvent alone from that of the compound in the solvent offers advantages for pKa determination by curve fitting for compounds of low aqueous solubility.     

Prediction of adipose tissue: plasma partition coefficients for structurally unrelated drugs

Tissue:plasma (P(t:p)) partition coefficients (PCs) are important parameters describing tissue distribution of drugs. The ultimate goal in early drug discovery is to develop and validate in silico methods for predicting a priori the P(t:p) for each new drug candidate. In this context, tissue composition-based equations have recently been developed and validated for predicting a priori the non-adipose and adipose P(t:p) for neutral organic solvents and pollutants. For ionizable drugs that bind to different degrees to common plasma proteins, only their non-adipose P(t:p) values have been predicted with these equations. The only compound-dependent input parameters for these equations are the lipophilicity parameter, such as olive oil-water PC (K(vo:w)) or n-octanol-water PC (P(o:w)), and/or unbound fraction in plasma (fu(p)) determined under in vitro conditions. Tissue composition-based equations could potentially also be used to predict adipose tissue-plasma PCs (P(at:p)) for ionized drugs. The main objective of the present study was to modify these equations for predicting in vivo P(at:p) (white fat) for 14 structurally unrelated ionized drugs that bind substantially to plasma macromolecules in rats, rabbits, or humans. The second objective was to verify whether K(vo:w) or P(o:w) provides more accurate predictions of in vivo P(at:p) (i.e., to verify whether olive oil or n-octanol is the better surrogate for lipids in adipose tissue). The second objective was supported by comparing in vitro data on P(at:p) with those on olive oil-plasma PC (K(vo:p)) for five drugs. Furthermore, in vivo P(at:p) was not only predicted from K(vo:w) and P(o:w) of the non-ionized species, but also from K*(vo:w) and P*(o:w), taking into account the ionized species in addition. The P(at:p) predicted from K*(vo:w), P*(o:w), and P(o:w) differ from the in vivo P(at:p) by an average factor of 1.17 (SD = 0.44, r = 0.95), 15.0 (SD = 15.7, r = 0.59), and 40.7 (SD = 57.2, r = 0.33), respectively. The in vitro values of K(vo:p) differ from those of P(at:p) by an average factor of 0.86 (SD = 0.16, r = 0.99, n = 5). The results demonstrate that (i) the equation using only data on fu(p) as input and olive oil as lipophilicity surrogate is able to provide accurate predictions of in vivo P(at:p), and (ii) olive oil is a better surrogate of the adipose tissue lipids than n-octanol. The present study is an innovative method for predicting in vivo fat partitioning of drugs in mammals.     

Investigation of drug partition property in artificial sebum

Targeted delivery of a therapeutic agent into the hair and sebaceous follicles greatly depends on the extent of drug partitioning/diffusion in the sebum. The objective of the present research was to develop a method to determine the sebum partition coefficient in order to facilitate the selection of sebum-targeted drug candidates. Partition coefficients of model drugs with different chemical structures and 4-hydroxybenzoate series compounds were measured in artificial sebum/water (K(sebum)) and human stratum corneum/water (K(sc)) at 37 degrees C. The relationship was evaluated between logK(sebum), logK(sc) and clogP. The results of the partition coefficient studies indicate that the K(sebum) of some drugs was significantly higher than the K(sc), whereas some drugs showed lower or similar K(sebum) when compared with K(sc). Overall, a relatively poor correlation was observed between logK(sebum), logK(sc) and clog P. However, a linear relationship exists between logK(sebum) and clog P in the 4-hydroxybenzoate series compounds, indicating that K(sebum) depends on the lipophilicity and chemical structure of the compounds. The results of the present study demonstrate that K(sebum) is different from K(sc) and calculated P and is likely to be a critical parameter reflecting drug delivery into hair and sebaceous follicles.     

High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes

This study reports on a novel, high-throughput assay, designed to predict passive, transcellular permeability in early drug discovery. The assay is carried out in 96-well microtiterplates and measures the ability of compounds to diffuse from a donor to an acceptor compartment which are separated by a 9-10 microm hexadecane liquid layer. A set of 32 well-characterized, chemically diverse drugs was used to validate the method. The permeability values derived from the flux factors between donor and acceptor compartments show a good correlation with gastrointestinal absorption in humans. For comparison, correlations based on experimental or calculated octanol/water distribution coefficients (log D(o/w,6.8)) were significantly lower. In addition, this simple and robust assay allows determination of pH permeability profiles, critical information to predict gastrointestinal absorption of ionizable drugs and difficult to obtain from cell culture experiments. Correction for the unstirred water layer effect allows to differentiate between effective and intrinsic membrane permeability and opens up the dynamic range of the method. In addition, alkane/water partition coefficients can be derived from intrinsic membrane permeabilities, making this assay the first high-throughput method able to measure alkane/water log P in the microtiterplate format.     

Spectral Studies on the Molecular Interaction of Anticancer Drug Mitoxantrone with CTAB Micelles

The interaction of anticancer drug mitoxantrone with cationic surfactant cetyltrimethylammonium bromide (CTAB) has been investigated by absorption spectroscopy as a function of surfactant concentration ranging from the premicellar to postmicellar region at pH 7.4 and 10. Interaction of mitoxantrone with CTAB micelles induces a bathochromic shift of both absorption maxima and spectral data showed that the micellization reduces the dimerization process and mitoxantrone is bound into micelles in the monomeric form. Binding constant and partition coefficient were estimated using the red shifts of the absorption maxima in the presence of surfactant. From the resulting binding constants for mitoxantrone–surfactant interactions, it was concluded that the hydrophobic interactions have a great effect on the binding of mitoxantrone to CTAB micelles. Also, by comparing the partition coefficients obtained using pseudo-phase model, the hydrophobic interactions have a major role in the distribution of mitoxantrone between micelle–water phases. Gibbs free energy of binding and distribution of mitoxantrone between the bulk aqueous medium and surfactant micelles were calculated.