RESUMO
Accurate prediction of the phospholipidosis-induction risk of drugs at early stages is important in drug development. So far, discrimination models for predicting the induction risk of cationic drugs have been proposed, but it is still challenging to accurately predict the risk of cationic drugs with intermediate hydrophobicity (logP). In this study, we introduced a parameter (Δlogk40) reflecting not only hydrophobic interaction but also interactions with the polar headgroup between cationic drugs and phospholipids, obtained with liquid chromatography using an immobilized artificial membrane column. The parameter was used along with other physicochemical properties as features to construct discrimination models. Linear discriminant analysis, the modified Mahalanobis discriminant analysis, support vector machine, and random forest were employed for model construction. The results showed that all discrimination models exhibited good predictive performance, with the modified Mahalanobis discriminant analysis and random forest providing the best results for cationic drugs, suggesting that the usefulness of the parameter reflecting complex interactions between cationic drugs and immobilized artificial membrane for constructing discrimination models to predict the induction risk. Furthermore, by applying the parameter as a feature in constructing discrimination models, we demonstrated an improvement in the predictive performance for drugs with intermediate hydrophobicity.
RESUMO
Cationic amphiphilic drugs (CADs) can induce the hyperaccumulation of phospholipids in cells and tissues. This side effect, which is known as drug-induced phospholipidosis, is sometimes problematic in the development and clinical use of CADs. It is known that CADs generally interact with phospholipids via both hydrophobic and acid-base interactions, and CADs with the larger affinity to phospholipid exhibit the larger induction risk. To develop a chromatographic assay system to predict the phospholipidosis-inducing potential with considering the acid-base interaction between CAD and phosphate group of phospholipid, hydrophilic interaction chromatographic (HILIC) methods were tested in this study. First, a PC HILIC column with phosphocholine groups on a packed material was used. The acid-base or other hydrophilic interactions to the stationary phase differed among basic drugs, and retention to the PC HILIC column did not accurately reflect the induction potential of phospholipidosis. As an alternative HILIC approach, the elution of CADs with the phosphate buffer from an amide column was tested. The elution effect, which is expressed as ratio of retention factors between different phosphate content in the mobile phase, closely correlated with the induction potential. Using the elution effect and retention factor to a reversed-phase HPLC column, the phospholipidosis-inducing drugs were clearly discriminated from the non-inducers. These results suggest that the proposed chromatographic approach can screen phospholipidosis-inducing drugs.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Lipidoses/induzido quimicamente , Preparações Farmacêuticas/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipidoses/metabolismo , Preparações Farmacêuticas/químicaRESUMO
We developed a high-performance liquid chromatography-tandem mass spectrometric method for phospholipid biomarker discovery and applied it to a cell-based assay system for the screening of phospholipidosis-inducing drugs. We studied the compositions of phospholipid molecules exceeding 100 species in cultured cells and found a characteristic alteration in the composition by treatment with cationic amphiphilic drugs possessing phospholipidosis-inducing potency. The compositions of phosphatidylinositol in RAW264 cells were significantly affected by the drug treatment. Similar alterations were also found in THP-1 cells. These phenomena were not observed when cells were treated with warfarin, which does not have phospholipidosis-inducing potency. Structural analysis of the altered phosphatidylinositols by a product ion scan revealed the presence of certain fatty acyl chains. Based on our findings, we proposed a prediction parameter (PP) for phospholipid accumulation calculated from the relative compositions of phosphatidylinositol species. As the dosage of imipramine (a cationic amphiphilic drug) increased, both the PP and cellular phospholipid content increased. Our results suggest that PP has potency as a biomarker for phospholipid accumulation in cells treated with drugs.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Imipramina/farmacologia , Fosfatidilinositóis/análise , Fosfatidilinositóis/química , Espectrometria de Massas em Tandem/métodos , Animais , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Lipidoses , Camundongos , Modelos Biológicos , Fosfolipídeos/análise , Fosfolipídeos/química , Análise de Componente PrincipalRESUMO
Cationic amphiphilic drugs (CADs) inhibit phospholipases competitively/uncompetitively. It has also been reported that CADs spontaneously accumulate in acidic organelles and increase their luminal pH, which may lead to deactivation of phospholipid-metabolising enzymes, causing cellular phospholipid accumulation. Recently, however, contradictory results have also been reported in that the luminal pH is not increased by CAD treatment. In this study, we examined whether the lysosomal/late endosomal acidic pH was maintained by vacuolar ATPase (v-ATPase) after treatment with chlorpromazine (CPZ) as a model CAD. The activity of lysosomal protease after CPZ treatment was also measured. Oregon Green-dextran-tetramethylrhodamine conjugate was employed to determine the luminal pH of the lysosomes/late endosomes in RAW264 cells. The luminal pH remained acidic after treatment with CPZ for 23 h, and the lysosomal protease activity was not decreased by 5-min CPZ treatment. Co-treatment with CPZ and bafilomycin A1 (v-ATPase inhibitor) raised the luminal pH. These results suggest that the lysosomal/late endosomal pH is not affected by a 23-h CPZ treatment. In addition, lysosomal enzymes presumably maintain their activity when CPZ accumulates. Our results imply that the pH homeostasis in lysosomes/late endosomes is strictly maintained even after a longer treatment with CADs.
Assuntos
Clorpromazina/farmacologia , Endossomos/enzimologia , Lisossomos/enzimologia , Fosfolipídeos/metabolismo , Tensoativos/farmacologia , Animais , Linhagem Celular , Clorpromazina/metabolismo , Endossomos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Macrolídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Tensoativos/metabolismo , ATPases Vacuolares Próton-TranslocadorasRESUMO
Capillary electrophoresis (CE) methods for transferrin analysis are widely used in clinical laboratories, but complement C3 peaks often overlap with carbohydrate-deficient transferrin peaks. In this study, we discovered that the electrophoretic mobility of the complement C3 peak could be controlled by adding carboxymethylcellulose (CMC) and dextran sulfate (DS) to the background electrolyte solution, probably because of adsorption of the polyanions to the proteins. The improved capillary electrophoresis method was developed using spermine, CMC, and DS as the background electrolyte solution additives. The carbohydrate-deficient transferrin concentrations determined using this system were in good agreement with those determined by HPLC, with acceptable reproducibility. These results suggest that this method has the potential to be developed into a new clinical test.
Assuntos
Complemento C3/metabolismo , Eletroforese Capilar/métodos , Transferrina/análogos & derivados , Adsorção , Carboximetilcelulose Sódica/química , Cromatografia Líquida de Alta Pressão/métodos , Sulfato de Dextrana/química , Eletrólitos/química , Humanos , Polieletrólitos , Polímeros/química , Reprodutibilidade dos Testes , Espermina/química , Transferrina/análiseRESUMO
Bis(monoacylglycero)phosphate (BMP) is localized in acidic organelles such as late endosomes or lysosomes. It has been reported that BMP levels increase under phospholipidosis induced by cationic amphiphilic drugs. In the present study, the effect of BMP on the binding of propranolol (PRO) to phospholipid liposomes under acidic conditions was investigated. Binding experiments were conducted by high-performance frontal analysis/capillary electrophoresis. PRO showed nonspecific binding to BMP-containing liposomes (BMP:phosphatidylcholine = 1:4), when numbers of bound drug molecules per lipid molecule (r) ranged 0.01-0.06. Total binding affinity increased depending on the BMP content. Binding affinity was decreased by low ionic strength, or by substitution of BMP with diacylglycerol, suggesting that electrostatic interactions were involved. The binding-enhancement effect of BMP was almost equivalent to that of phosphatidylglycerol, and slightly larger than that of phosphatidylserine. An acidic environment (pH 5.0) decreased total binding affinity to BMP-containing liposomes. This could be explained by the pH-partition theory (i.e., the loss in affinity was caused by a decrease in the neutral form of the drug accessible to the membrane core). These results suggest that PRO binding is enhanced by BMP in late endosomes or lysosomes, whereas an acidic environment weakens such binding.
Assuntos
Eletroforese Capilar/métodos , Lipossomos/química , Lisofosfolipídeos/química , Monoglicerídeos/química , Fosfolipídeos/química , Propranolol/química , Concentração de Íons de Hidrogênio , Lipossomos/metabolismo , Lisofosfolipídeos/metabolismo , Monoglicerídeos/metabolismo , Concentração Osmolar , Tamanho da Partícula , Fosfolipídeos/metabolismo , Propranolol/metabolismoRESUMO
An in vitro method to predict phospholipidosis-inducing potential of cationic amphiphilic drugs (CADs) was developed using biochemical and physicochemical assays. The following parameters were applied to principal component analysis, as well as physicochemical parameters: pK(a) and clogP; dissociation constant of CADs from phospholipid, inhibition of enzymatic phospholipid degradation, and metabolic stability of CADs. In the score plot, phospholipidosis-inducing drugs (amiodarone, propranolol, imipramine, chloroquine) were plotted locally forming the subspace for positive CADs; while non-inducing drugs (chlorpromazine, chloramphenicol, disopyramide, lidocaine) were placed scattering out of the subspace, allowing a clear discrimination between both classes of CADs. CADs that often produce false results by conventional physicochemical or cell-based assay methods were accurately determined by our method. Basic and lipophilic disopyramide could be accurately predicted as a nonphospholipidogenic drug. Moreover, chlorpromazine, which is often falsely predicted as a phospholipidosis-inducing drug by in vitro methods, could be accurately determined. Because this method uses the pharmacokinetic parameters pK(a), clogP, and metabolic stability, which are usually obtained in the early stages of drug development, the method newly requires only the two parameters, binding to phospholipid, and inhibition of lipid degradation enzyme. Therefore, this method provides a cost-effective approach to predict phospholipidosis-inducing potential of a drug.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Lipidoses/induzido quimicamente , Análise Multivariada , Análise de Componente Principal , Cátions , Difenilexatrieno , Corantes Fluorescentes , Humanos , Microssomos/enzimologia , Inibidores de Fosfolipase A2 , Ligação ProteicaRESUMO
High-throughput characterization of drug-drug interactions in plasma protein binding was demonstrated by using a surface plasmon resonance (SPR) biosensor. The method used in this study enabled the discrimination between the two modes of binding inhibition, direct competition and negative allosteric effect, which was difficult in conventional SPR approaches. Two theoretical equations were used representing SPR binding response for directly competitive binding or for independent binding. The experimental binding data for human serum albumin was processed by non-linear least squared regression of the equations. By this approach, drug-drug interactions were classified into three modes, direct competition, independent binding, and allosteric interaction, which were almost consistent with previous reports. In addition, dissociation constants were also estimated roughly for direct competition and for independent binding. The analytical throughput was almost as high as in the previous reports; three minutes per injection. This method is a powerful tool for the characterization of drug-drug interaction at an early stage of new drug development.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Sanguíneas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Sítios de Ligação , Interações Medicamentosas , Humanos , Naproxeno/metabolismo , Fenilbutazona/metabolismo , Ligação Proteica , Varfarina/metabolismoRESUMO
On-line capillary isoelectric focusing-mass spectrometry (cIEF-MS) was applied to determine concentrations of peptides and proteins using angiotensin II and human tetrasialo-transferrin as the model samples. The concentration of the carrier ampholyte was optimized for both resolution and ion intensity. cIEF-MS employing 1% Pharmalyte 3-10 and a sheath liquid containing water/methanol/acetic acid (50/49/1) resolved angiotensin I and II (5 microM each, DeltapI=0.2) at an Rs value of 2.29. The determined concentration of angiotensin II (0.1-5 microM) well correlated (R=0.999) with that obtained by the conventional RP-HPLC method. The limit of detection was 0.22 microM, which was about 10 times lower than that by UV detection (2 microM). The repeatability and accuracy were <15 and <11%, respectively. cIEF-MS was also applied to determine human tetrasialo-transferrin concentration. The good linearity (R2=0.998) was also observed between the transferrin concentration (0.5-1.2 g/L) and peak area ratio (IS; beta-lactoglobulin B) with acceptable accuracy (<1.9%) and repeatability ( approximately 10% at 1g/L).
Assuntos
Sistemas On-Line , Peptídeos/análise , Proteínas/análise , Animais , Bovinos , Eletroforese Capilar/métodos , Humanos , Focalização Isoelétrica/métodos , Espectrometria de Massas/métodos , OvinosRESUMO
The conversion of soluble, nontoxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. Therefore, extensive studies have been carried out on the mechanisms involved in Abeta aggregation and the characterization of Abeta aggregates formed in aqueous solutions mimicking biological fluids. On the other hand, several investigators pointed out that membranes play an important role in Abeta aggregation. However, it remains unclear whether Abeta aggregates formed in solution and membranes are identical and whether the former can bind to membranes. In this study, using a dye-labeled Abeta-(1-40) as well as native Abeta-(1-40), the properties of Abeta aggregates formed in buffer and raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin were compared. Fourier transform infrared spectroscopic measurements suggested that Abeta aggregates formed in buffer and in membranes have different beta-sheet structures. Fluorescence experiments revealed that Abeta aggregated in buffer did not show any affinity for membranes.
Assuntos
Peptídeos beta-Amiloides/química , Lipossomos/química , Doença de Alzheimer/etiologia , Colesterol , Dimerização , Gangliosídeo G(M1) , Humanos , Modelos Biológicos , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , EsfingomielinasRESUMO
Drug-plasma protein binding analysis is indispensable for drug development and clinical use. However, conventional methods for binding analyses were not suitable for small amounts of proteins because of large sample requirements. On the other hand, high-performance frontal analysis/capillary electrophoresis (HPFA/CE) consumes very small sample volumes, and is useful for ligand-binding study of small amounts of proteins. In this study, HPFA/CE was used in a drug-binding study of alpha 1-acid glycoprotein (AGP) subtypes in which plasma concentrations change dynamically to elucidate the effects of structural variation on drug binding. Binding study on desialyrated AGP revealed that (S)-enantiomer selectivity in propranolol-AGP binding was caused by sialic acid residues, while neither sialic acid nor galactose caused the enantioselectivity of verapamil binding to AGP. Biantennary glycans slightly suppressed disopyramide binding to AGP, whereas the glycans did not have any influence on propranolol and verapamil binding. Disopyramide and verapamil were selectively bound to the A variant rather than the F1S variant. The A variant showed larger enantioselective binding to disopyramide, but not to verapamil.
Assuntos
Eletroforese Capilar/métodos , Orosomucoide/metabolismo , Sítios de Ligação , Disopiramida/metabolismo , Variação Genética , Humanos , Orosomucoide/química , Orosomucoide/genética , Polissacarídeos , Ligação Proteica , Estereoisomerismo , Verapamil/metabolismoRESUMO
A high-performance frontal analysis-capillary electrophoresis (HPFA-CE) method was applied to investigate the effect of pH on the drug binding properties of genetic variants of human alpha1-acid glycoprotein (AGP), A variant and a mixture of F1S variants. The unbound concentrations of a model basic drug, disopyramide (DP), in A variant solutions and in F1S variant solutions were measured by HPFA-CE to evaluate binding constants at pH 4.0, 5.0, 6.0 and 7.4. The binding between DP and A variant was gradually weakened by acidification of background buffer (from pH 7.4 to 4.0), while the binding between DP and FIS variants decreased at first (from pH 7.4 to 6.0), and then gained (from pH 6.0 to 4.0). Consequently, DP was more strongly bound to A variant than to FIS variants at pH 7.4, while at pH 4.0 DP was more strongly bound to F1S variants. At any pH (S)-DP was bound more strongly than (R)-DP, and the enantioselectivity of A variant was significantly higher than that of F1S variants. Electrophoretic mobilities of the AGP genetic variants decreased along with a decrease in pH. Fluorescent emission of these genetic variants indicated a distinct conformational change between pH 5.0 and 4.0. However, there was no significant difference in the electrophoretic mobility and the fluorescent emission spectrum between these variants at any pH. On the other hand, circular dichroism analyses revealed that beta-sheet content in FIS variants diminished as pH decreased, while that in A variant increased. These results suggest that the conformational change induced by acidification of background buffer differs between these genetic variants, and this causes the difference in DP bindability.
Assuntos
Antiarrítmicos/química , Disopiramida/química , Orosomucoide/genética , Eletroforese Capilar/métodos , Humanos , Concentração de Íons de Hidrogênio , Orosomucoide/químicaRESUMO
The binding study between basic drugs ((S)-verapamil (VER) and (S)-propranolol (PRO)) and phospholipid liposomes was performed by using high-performance frontal analysis/capillary electrophoresis (HPFA/CE) in order to investigate the effect of oxidative modification of low-density lipoprotein (LDL) upon drug-binding affinity from molecule-based viewpoint. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC, 16:0, 18:1), 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC, 16:0, 18:2), dilauloyl-phosphatidylcholine (DLaPC, 12:0, 12:0), 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (POPG, 16:0, 18:1), and 1-palmitoyl-sn-glycero-3-phosphocholine (monoPPC, 16:0) were used to prepare the model liposomes. At physiological pH (pH 7.4), the model liposome prepared from POPG+POPC had negative net charges, while the total net charge of the other model liposomes (POPC liposome, PLPC liposome, DLaPC liposome, and monoPPC+POPC liposome) was zero. The drug and the model liposome mixed solutions were subjected to HPFA/CE, and the total binding affinities (nK) were calculated. The nK values of VER and PRO to POPG+POPC liposome were more than six and 10 times higher than those of other liposomes, respectively. On the other hand, the nK values of the model drugs to POPC liposome, PLPC liposome, DLaPC liposome and monoPPC+POPC liposome showed small differences less than twice. These results indicate that the electrostatic interaction plays an important effect on drug-liposome binding, and suggest that the increase in the negative charge of LDL phospholipids gives more significant effect on the drug-binding affinity of the basic drugs than the acyl-chain structure.
Assuntos
Lipoproteínas LDL/metabolismo , Preparações Farmacêuticas/metabolismo , Fosfolipídeos/fisiologia , Tecnologia Farmacêutica/métodos , Eletroforese Capilar/métodos , Preparações Farmacêuticas/química , Fosfolipídeos/metabolismo , Ligação Proteica/fisiologiaRESUMO
Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.
Assuntos
Proteínas Sanguíneas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Mandélicos/metabolismo , Humanos , Ligação ProteicaRESUMO
Plasma protein binding of N-desethyloxybytynin (DEOXY), a major active metabolite of oxybutynin (OXY), was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of DEOXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. DEOXY is bound in human plasma strongly and enantioselectively. The unbound drug fraction in human plasma samples containing 5 microM (R)- or (S)-DEOXY was 1.19 +/- 0.001 and 2.33 +/- 0.044%, respectively. AGP plays the dominant role in this strong and enantioselective plasma protein binding of DEOXY. The total binding affinity (nK) of (R)-DEOXY and (S)-DEOXY to AGP was 2.97 x 10(7) and 1.31 x 10(7) M(-1), respectively, while the nK values of (R)-DEOXY and (S)-DEOXY to HSA were 7.77 x 10(3) and 8.44 x 10(3) M(-1), respectively. While the nK value of (S)-DEOXY is weaker than that of (S)-OXY (1.53 x 10(7) M(-1)), the nK value of (R)-DEOXY is 4.33 times stronger than that of (R)-OXY (6.86 x I0(6) M(-1)). This suggests that the elimination of an ethyl group weakens the binding affinity of the (S)-isomer because of the decrease in hydrophobicity, while the binding affinity of the (R)-isomer is enhanced by the decrease in steric hindrance. The total binding affinity of DEOXY to HSA is much lower than that of DEOXY-AGP binding as well as OXY-HSA binding (2.64 x 10(4) and 2.19 x 10(4) M(-1) for (R)-OXY and (S)-OXY, respectively). The study on competitive binding between OXY and DEOXY indicated that DEOXY enantiomers and OXY enantiomers are all bound competitively at the same binding site of AGP molecule.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácidos Mandélicos/metabolismo , Humanos , EstereoisomerismoRESUMO
High performance frontal analysis coupled with capillary electrophoresis (HPFA/CE) was applied to the ultramicroanalysis of enantioselective binding of drug to plasma lipoproteins. A small volume (ca. 80 nl) of (R)- or (S)-propranolol (PRO, 25-150 microM) and human high-density lipoprotein (HDL, 2.63 g/l) or human low-density lipoprotein (LDL, 4.37 g/l) mixed solution, which was in the state of binding equilibrium, was introduced hydrodynamically into a non-coated fused silica capillary. Positively charged unbound PRO enantiomers migrated toward cathodic end much faster than negatively charged lipoproteins and the bound form. Once unbound PRO migrated apart from lipoprotein, the bound PRO was quickly released from the lipoprotein to maintain the binding equilibrium. Thus, PRO migrated as a zone in the capillary, giving a peak with a plateau region, where the concentration is the same as the unbound PRO concentration in the original sample solution. The unbound PRO concentration calculated form the plateau height agreed with that determined by a conventional ultrafiltration method used as a reference method. It was found that the bindings of PRO to HDL and PRO to LDL were not enantioselective, while the total binding affinity of PRO to LDL (4.01 x 10(5) per M) was 17 times higher than that of PRO-HDL binding (2.38 x 10(4) per M).