The Research Of The Cocrystal Of Dipfluzine And Benzoic Acid

Dipfluzine, which was developed by the Hebei Medical University, was apiperazine calcium antagonists.It can induce the cerebrovascular dilateonselectively, reduce thrombosis formation, lower cerebral edema, ameliorateand protect ischemic brain injury in rats, mend memory impairment in micecaused by sodium nitrite significantly.Pharmaceutical cocrystal was the complex of the active pharmaceuticalingredients （API） and cocrystal forms （CCF）, which was formed throughhydrogen bonds and other non-covalent bonding. Cocrystal can improve thephysicochemical properties of API, such as solubility, the dissolution rate, thebioavailability, and the stability.Objective: In order to provide physicochemical parameters forpreparation of drug cocrystal, the dissociation constant and partitioncoefficient of dipfluzine were determined. Used benzoic acid as the CCF anddipfluzine as API, the cocrystals were synthesized. The thermodynamicbehavior of the cocrystal formation was investigated and the physicochemicalproperties of cocrystal were determined, including solubility in water, thedissolution rate in vitro, pharmacokinetics dynamics in rat plasma and thetissue distribution, which aimed to detect the potential of cocrystal inimproving the physicochemical properties of API.Methods:（1） Used dilute hydrochloric acid solution as titrant, the dissociationconstants of dipfluzine was determined by nonlogarithmic potentiotitrationmethod, in which different concentration of dipfluzine were titrated in theethanol/water mixture with different ratio. As a result, the pK_b1was obtained.Curves were drawn with pK_b1as the Y-coordinate versus the proportion ofethanol/water as the abscissa; the pK_b2in pure water was obtained byextrapolating to the location of0%ethanol. Then, curves were drawn with pK_b2as the Y-coordinate versus the concentration of dipfluzine as the abscissa;the pKbin infinite diluted silution was obtained. The pK_aof dipfluzine wascalculated by the relation of K-a*Kb=10¹⁴. The Partition coefficients （logP） ofdipfluzine at different oil/water proportion （1:1,1:3,1:9） were measured byshake flask-UV spectrophotometry method.（2） The cocrystals of dipfluzine and benzoic acid were synthesized bysolvent-assisted grinding and solvent-ultrasonic method. The cocrystals werecharacterized by PXRD, DSC-TG, FT-IR, FT-Raman, THz, ss-NMRtechniques. The concentration of dipfluzine was determined by RP-HPLCmethod. The binding constants K and thermodynamic parameters of cocrystalformation were determined by solubility method, in which the solubilities ofdipfluzine in benzoic acid with different concentration were determined. Theequilibrium solubilities of dipfluzine and cocrystal at different temperaturewere determined by saturated solubility method. Based on the method inpharmacopeia, the dissolution rates of dipfluzine, the physical mixture ofdipfluzine and benzoic acid, and cocrystal in vitro were determined inNaAc-HAc buffer solution of pH4.5by paddle method. The concentration ofdipfluzine was determined by RP-HPLC method.（3） Studies on the pharmacokinetic in rat plasma and tissue distribution:12male SD rats were randomly divided into two groups. The suspensions ofdipfluzine and cocrystal were orally administration to the two groups in asingle dose of40mg/kg, separately. Blood sample were obtained from eachrat at the set points within the eye canthus after intragastric administration andcollected in the heparinized centrifuge plastic tube, respectively. Theconcentration of dipfluzine was determinated by RP-HPLC using liquid-liquidextraction under alkaline conditions after protein precipitation with methanol.The relative bioavailability was calculated and the compartment model wasjudged. Moreover,36male SD rats were randomly divided into two groups,respectively, then suspension of dipfluzine and cocrystal were orallyadministrated to the two groups at a single dose of40mg/kg and equivalentedto40mg/kg, respectively, the rats were sacrificed and taked the tissues at1h, 6h,24h after administration. The concentration of tissue samples weredetermined using liquid-liquid extraction under alkaline conditions afterprotein precipitation with methanol by HPLC.Result:（1） The pK_aof dipfluzine determined by nonlogarithmic potentiotitrationmethod was5.89. The logP at the different oil/water volume ratio of1:1,1:3,1:9of dipfluzine determined by shake-flask method and UV spectrophotomet-ry were0.5900±0.0270,0.8540±0.0352,1.287±0.054, respectively.（2） It was revealed by PXRD, DSC and THz spectra measurement thatthe dipfluzine-benzoic acid cocrystal was formed at a molar ratio of1:2. Thedistinctive THz spectrum showed that the vibrational mode of the co-crystalwas different from those of the starting materials, suggesting that THzspectroscopy was an ideal tool to evaluate co-crystal formation. IR, Ramanand ssNMR spectroscopy verified that O-H···O and O-H···F hydrogen bondswere formed between O-H from benzoic acid and C=O and C-F bonds fromdipfluzine. The apparent binding constant K at25℃,31℃,37℃were4.611×10⁹L²/mol²,3.488×10⁹L²/mol²and1.806×10⁹L²/mol², respectively.The thermodynamic parameters of Δ_rG^θ, Δ_rH^θand Δ_rS^θof cocrystal formationwere-55.31kJ/mol,-59.87kJ/mol, and-15.27J/mol/K at25℃, respectively.The solubility of cocrystal was higher than that of dipfluzine; the equilibriumsolubility of cocrystals at25℃,31℃,37℃in water were161.5,279.6,345.8μg/mL, respectively. In pH4.5NaAc-HAc buffer solutionin, the t1/2ofdipfluzine, the physical mixture of dipfluzine and benzoic acid, and thecocrystal were3.028h,1.405h, and0.48h, respectively. The rate of drugrelease of cocrystal was obviously faster than that of dipfluzine; the drugrelease from cocrystal followed Weibull model.（3） The pharmacokinetics dynamics of the dipfluzine and cocrystalfollowed the one-compartment model. The Cmaxof dipfluzine and cocrystalwere1.054μg/mL and1.393μg/mL, Tmaxwere1.600h and1.119h, the AUCwere5.493μg/mL*h and14.28μg/mL*h, respectively. The relativebioavailability of the cocrystal was260%than that of dipfluzine. The pharmacokinetics dynamics of cocrystal and dipfluzine showed significantdifferences （p<0.05）. Verified by tissue distribution experiment, the dipfluzinemostly distributed in the kidney, small intestine, liver, brain, lung, while,cocrystal mostly distributed in the brain, kidney, liver and lung. Comparedwith dipfluzine, cocrystal gained more distribution in brain and less in smallintestine.Conclusion:（1） The pK_aof dipfluzine was5.89; the ΔpK_abetweendipfluzine and benzoic acid was less than3, which was suitable for cocrystalformation. The oil-water partition coefficient of dipfluzine was greater than0,which showed the low solubility, lipophilic, weakly basic characters ofdipfluzine.（2） The dipfluzine-benzoic acid cocrystal was formed at a molar ratio of1:2. Although the hydrogen bonds were formed in dipfluzine-benzoic acidcocrystal, the complex constants K were evaluated to be10⁹orders ofmagnitude, indicating the complex force was intense. Above results wereobtained from the analysis of the solid state characterization and deducing ofthermodynamic formulas. The studies on solid state characterizations andthermodynamic rules can provide the reference to other API-CCF system.（3） The solubility of the cocrystal was higher than that of dipfluzine by500times; the rate of drug release from cocrystal was faster than that ofdipfluzine by5times; the relative bioavailability was improved to2.6times.The cocrystal resulted more distribution in brain. It indicated that it waspotential for cocrystal to improve the physicochemical properties of API, suchas solubility, the dissolution rate, the bioavailability.