Study On Azelnidipine Polymorpgs

Azelnidipine, named3-（1-two phenylmethyl-3-aza-cyclobutoxyalkyl）-5-isopropyl-2-amino^-1,4-dihydro-6-methyl-4-（3-nitrophenyl）-3,5-dicarboxylates, which was jointly developed by the Sankyo and Ube Industrialcompany, Japan, and approved in February2003in the Japanese mark_et, andgot into Chinese mark_et in2008. It was a new third-generation long-actingdihydropyridine calcium channel block_ers, which had good oral absorption. Itreached maximum plasma concentration on two^three hours after taking thedrug, and its half-life is19²3hours. Azelnidipine was mainly used for thetreatment of mild or moderate essential hypertension, kidney disorder withhigh blood pressure and severe hypertension in clinical.Azelnidipine belongs to the polymorph drugs having alpha and beta twopolymorphs. Different polymorphs of the drug may show differentphysicochemical properties, such as the dissociation constant, oil-waterdistribution coefficient, solubility, dissolution rate, melting point, density,hardness, appearance, infrared spectroscopy, x-ray diffraction pattern, thermalbehaviours and biological effectiveness, which affect the stability,bioavailability, and efficacy of the drug. Some polymorphs even showineffective or produced toxic characters.Objective: In order to guide the rational design of drug formulations,provide a theoretical reference for clinical drug crystal Screening, thephysicochemical properties of azelnidipine polymorphs （alpha and betapolymorphs）, such as the dissociation constant, oil-water distributioncoefficient, solubility, and dissolution rate, were measured. In order to explorethe differences and thermodynamic changes among the three forms andprovide reference and basis to the drug storage, polymorph transition rule, andsynthesis process of azelnidipine, the solid state characterization and stabilityanalysis of the azelnidipine polymorphs and a pseudopolymorphic form were performed. Furthermore, the pharmacokinetics of azelnidipine polymorphswere investigated, which can provide a theoretical basis for its dosagedevolepment.Method:（1） The dissociation constants of azelnidipine polymorphs alphaand beta were determined by nonlogarithmic potentiotitration method; theoil-water distribution coefficients of azelnidipine polymorphs were measuredby shak_e-flask method. Furthermore,0.1mol·L^-1hydrochloric acid solutionwas used as dissolution medium to obtain the dissolution rate and thecumulative dissolution of this two polymorphs by paddle method.（2） Identifications of the crystal phases, including two polymorphic and apseudopolymorphic crystal forms of azelnidipine, were attempted usingpowder x-ray diffraction （PXRD）, differential scanning calorimetry France（DSC）, infrared spectroscopy France （IR）, Raman spectroscopy France（Raman）, terahertz spectroscopy （THz） and solid state NMR technology（ss-NMR）. The solubility of the two polymorphs of alpha and beta in50%ethanol at25,31,37,42, and49℃was investigated; the values obtainedwere used to calculate the transition temperature and thermodynamicparameters of the transition reaction. Moreover, the kinetics of the solid-statedecomposition was studied using DSC analysis.（3） Studies on validated LC-MS method for determination of azelnidipinepolymorphs in rat plasma and application of pharmacokinetic: Twelve ratswere randomly divided into two groups. Later suspensions of azelnidipinealpha and beta were oral administration to the two groups in a single dose of20mg·kg^-1, separately. Blood sample were obtained from each rat at the setpoints within the eye canthus after intragastric administration and collected inthe heparinized centrifuge plastic tube, respectively. Instead for the tediumliquid-liquid procedure frequently used for the extraction of1,4-dihydropyridines from biologic samples, sample was pretreated only by onestep protein precipitation with acetonitrile （containing0.1%acetic acid）.Determination of plasma samples and internal standard （I.S.） nimodipine wereconducted on a Sapphire C₁₈analytical column （4.6×150mm,5μm） with a mobile phase of acetonitrile-water （containing0.1%acetic acid）（80:20, v/v）and with a flow rate of0.8mL·min^-1. Scan mode was used multiple reactionmonitoring, and the ionization mode was used ESI selective positive iondetection.Results:（1） The dissociation constants pK_bof alpha and beta polymorphswere7.893and8.322, respectively. The oil-water partition coefficients logP ofpolymorphs alpha and beta in n-octyl alcohol-0.1mol·L^-1hydrochloric acidsolution were determined by shak_e-flask method; the values were0.5054and0.4957, respectively.（2） The transition temperature of polymorphic alpha and beta in50%ethanol was50.78℃, and the values of ΔG_α,β^θ, ΔH_α,β^θ, and ΔS_α,β^θat25℃were^-1.18kJ·mol1,^-14.81kJ·mol1, and-45.73J·mol1·K1, respectively. Thetransition temperature of the pseudopolymorphic crystal form with alpha andbeta in50%ethanol were39.47and31.59℃, and the values ofΔGθ, ΔHθ, andΔSθat25℃were^-1.61kJ·mol1,-34.77kJ·mol1,^-111.27J·mol1·K1and-0.43kJ·mol1,^-19.96kJ·mol1,-65.53J·mol1·K1. The kinetics of thesolid-state decomposition showed that the activation energies ofdecomposition of the polymorphs alpha and beta at high temperatures were148.67and151.93kJ·mol1.（3） Plasma concentration of azelnidipine after intragastric administrationof alpha and beta polymorphs was determined. The parameters werecalculated according to the concentration-time curve. After oral administrationof alpha and beta polymorphs, the relevant parameters c_max, t_max, t_1/2, k_e, AUC_0-t,and AUC_0-∞for form α were111.48ng·mL^-1,1.33h,13.00h,0.053,789.19ng·h·mL^-1, and841.23ng·h·mL^-1, respectively, while, for form β, thoseparameters were80.83ng·mL^-1,1.83h,14.20h,0.051,583.22ng·h·mL^-1, and634.28ng·h·mL^-1, respectively. The result of statistics analysis showedsignificant differences （P<0.05） in c_max, t_max, AUC_0-t, and AUC_0-∞. On thecontrary, there were no significant differences in k_eand t_1/2（P>0.05）. As aresult, the AUC of azelnidipine alpha were larger than those of beta and therelative bioavailability was135.32%. Conclusion:（1） The significant differences occurred on the dissociationconstant, oil-water distribution coefficient, solubility, the dissolution rate ofalpha and beta polymorph. The dissolution rate of alpha polymorph wasobviously higher than that of beta polymorph. Although, polymorphs have thesame chemical structure, the difference in molecular conformation could resultin different dissociation behaviors and dissociation constants. The polymorphsof azelnidipine were liposoluble, which favored the transmembrane transport.（2） IR-, Raman-, THz-, and ss-NMR spectra with their assignmentsprovided the indirect indications on molecular arrangements, layer structure,hydrogen-bonding, and-stacking of intermolecular interactions in differentcrystal forms. The two forms of alpha and beta were enantiotropically related,which was proved using thermodynamic analyses, with a transitiontemperature of50.78℃; Form beta was thermodynamically stable below thetransition temperature. The result of pharmacokinetic showed that polymorphalpha could give higher bioavailability than polymorph beta. The differencesof crystal forms affected the rate and extent of absorption of azelnidipine.Alpha and beta polymorphs of azelnidipine were found not to bebioequivalent.（3） In present study, a high selectivity, rapid, accurate, and sensitiveLC-MS method was established using MRM mode for determination ofazelnidipine in plasma samples. The method was sensitive with a lower limitof quantification （LLOQ） of0.8ng·mL^-1and good linearity （r≥0.9987） overthe linear range0.8to160ng·mL^-1. All the methodology data, such asprecision, accuracy, and absolute recovery, were within the required limits.The method was successfully applied to the pharmacokinetic and relativebioavailability studies of azelnidipine polymorphs in rats.（4） The study of drug polymorphs can help to validate the biologicalactivity and bioavailability of the different form in the development of crystaldrugs, and to improve the route of administration, the selection of dosage form,and to optimum the homologue technology, and to elevate quality and stabilityof drugs in every direction. This study provides theory evidences for the clinical experiment research and crystal drug development of azelnidipine.