Stereoselective determination of chiral drugs containing carboxyl or Amino Group

Abstract

제 1부 : 카르복실기를 가진 약물인 세티리진의 광학이성체 분리, 약물동태, 약효에 관한 연구와 레보플록사신 제제 중 광학순도측정 광학활성의약품은 광학이성체에 따라서 약물동태, 약효, 독성 등이 다를 수 있다. 미국식약청에서는 의약품의 입체화학의 중요성을 인식하여 1992년에 가이드라인을 제정하였으며 1997년 이래로 ICH 가이드라인에도 광학활성의약품에 관한 품질관리방안이 포함되어 있어 전세계적으로 광학활성의약품의 중요성을 인식하고 있는 실정이다. 따라서, 광학활성의약품의 개발 및 관리기술에 관한 연구의 일환으로 정책실현의 모델약물로 카르복실기를 가지는 항히스타민제인 세티리진과 항균제인 레보플록사신을 가지고 연구하였다. 광학이성체 혼합물질로 판매되는 약물의 단일이성체로의 개발여부를 검토하고 가이드라인 제정의 기초자료로 활용하고자 1개의 키랄 센터를 가지는 세티리진을 가지고 연구하였다. 세티리진은 알러지성비염에 사용되는 제2세대 항히스타민제로서 최근 개발되었으며 약효가 우수하고 부작용이 적어서 주목되는 약물이다. 광학이성체 분리는 알파1-글리코프로테인 칼럼을 사용하고 유기용매-인산염완충액을 이동상으로하여 측정파장 230nm에서 액체크로마토그라프로 수행하였다. 유기용매와 인산염완충액의 비율과 농도를 달리하여 최적분리조건을 확립하고, prep. 칼럼을 이용하여 광학순도가 99.9%인 (+)-세티리진과 (-)-세티리진을 제조하였고, 두 물질이 이성체임을 확인하였다. 약물동태 연구를 위해 쥐의 혈장과 사람의 뇨에서 시간대별로 취하여 세티리진 광학이성체 양을 정량한 결과 유의할만한 차이를 나타내지 않았다. 또한, 분리된 광학이성체를 가지고 RBL 2H3 세포를 이용하여 항히스타민 약효를 연구한 결과 (-)-세티리진이 더 강력하였다. 단일이성체로 개발된 신약의 광학순도 연구에 관한 식품의약품안전청 가이드라인 제정의 기초자료로 활용하고자 레보플록사신을 가지고 연구하였다. 레보플록사신은 오플록사신의 (-)-이성체로서 (+)-이성체보다 항균력이 강력하여 최근 개발된 약물로서 선별합성법에 특허가 걸려 있어 국내제약회사에서도 경쟁적으로 합성법을 연구하는 항균제이다. 본 연구에서는 국내 시판되는 제제의 광학순도를 조사한 결과 우수한 광학순도를 가진 물질임을 확인하였다. 위 연구를 통하여 광학이성체 혼합물질의약품의 광학이성체의 손쉬운 정성 및 정량분석법 확립, 약물동태, 약효연구를 통하여 식품의약품안전청에서 필요로 하는 광학활성의약품의 관리를 위한 기술개발 및 규격제정의 기초자료로 활용하고자 하였다. 또한 부작용이 적고 우수한 광학활성의약품 신약개발 분야에서 선도적 역할로 모범을 보임으로써 제약산업의 미래지향적 연구개발을 촉진하고자 하였다

주요어 : 광학이성체분리, 세티리진, 알파1-글리코프로테인, 약물동태, 약효, RBL 2H3 cell, 레보플록사신, 광학순도

제 2부 : 컴퓨터 프로그램을 이용한 아민기를 가진 광학활성의약품의 키랄CBH 칼럼에서의 분리능 예측에 관한 연구 광학이성체혼합물질의약품의 광학이성체 분리는 시간과 노력이 많이 소요되는 작업이다. 따라서, 분자의 3차원적 구조로부터 광학이성체혼합물질의약품의 분리능을 예측할 수 있다면, 광학활성의약품의 개발 및 관리기술 개발에 획기적인 도구가 될 것이다. 본 연구에서는 11개의 아민기를 가지고 유사한 구조적 특성을 가지는 약물을 모델로 선택하고 키랄CBH칼럼을 이용하여 연구하였다. 먼저 키랄CBH칼럼에서의 이동상의 파라미터, 유기용매의 종류와 양, 이동상의 pH, 칼럼온도, 이동상의 이온강도를 변경하여 각 화합물의 광학이성체 분리 메커니즘에 대하여 연구하였다. 유기용매를 이소프로판올에서 아세토니트릴로 변경하고, 함량을 높였을 때 화합물의 분리능이 높아졌다. 인산염완충액의 pH와 칼럼 온도는 화합물의 분리능에 큰 영향을 미쳤다. 또한, 인산염완충액의 최적 이온강도는 10 mmol이었다. 컴퓨터 프로그램을 이용한 아민기를 가진 광학활성의약품의 키랄CBH 칼럼에서의 분리능 예측에 관한 연구를 하였다. 컴퓨터 프로그램은 QSAR/CoMFA 소프트웨어를 이용하였고, 계산에는 최소좌승법을 이용하였다. 정량적 지용성-분리능 관계 연구에서는 상관관계가 발견되지 않았다. 정량적 CoMFA-분리능 관계 연구에서는 유의할만한 상관관계가 발견되었으며, 10 mmol/l 인산염완충액-이소프로판올 (97 : 3)의 이동상 조건에서 CoMFA 값이 130이상인 화합물은 CBH 칼럼으로 완전한 광학이성체의 분리가 가능할 것으로 사료된다. 분리능에 미치는 정전기적, 입체적 인자를 고려할 때 유사하였으며, 정전기적인자의 비율이 더 높았다. 본 연구의 결과는 분자의 3차원적 구조로부터 광학이성체혼합물질의약품의 분리능을 예측이 가능하였으며 광학활성의약품의 개발 및 관리기술 개발에 유용한 도구가 될 것이다.

주요어 : 키랄CBH칼럼, 분리능 예측, 아민기를 가진 광학활성의약품, QSAR, CoMFA, 최소좌승법 ; PARTⅠ: Studies on Chiral separation, Pharmacokinetics and Pharmacological Activities of Cetirizine Enantiomers and Optical Purity Determination of Levofloxacin Preparations Containing Carboxyl Group. Numerous drugs are chiral because they possess one or more chiral centers. Enantiomers may differ in their pharmacokinetic, pharmacological and toxicological properties. However, the significance of stereochemistry of drugs in their therapeutic uses has received relatively little attention until recently. The US authorities (FDA) issued a guideline on stereoisomeric drugs in 1992, and the European agency describes tests for new drug substances which are optically active in an ICH (International Conference of Harmonization) Guideline from 1997. Therefore, we have studied chiral separation method development, pharmacokinetics, pharmacology of cetirizine enantiomers and checked optical purity of levofloxacin preparations, in order to collect necessary data in preparation of a KFDA guideline on stereoisomeric drugs. Cetirizine is a non sedating H1-receptor antagonist used in patients with urticaria and allergic rhinitis. It is known that each enantiomer of the racemic mixtures of some chiral antihistaminic drugs, such as chlorpheniramine, neobenodine etc., showed characteristic stereoselective activity over racemates in therapeutic uses. We have developed chiral separation methods using HPLC in order to study the differences in pharmacokinetics and pharmacological activities between (+)-cetirizine and (-)-cetirizine in rat plasma and human urine. A chiral stationary phase a1-acidglycoprotein, AGP-CSP, was used to separate the enantiomers. The pH of phosphate buffer as well as the content of organic modifier in the mobile phase markedly affected the chromatographic behavior of (+)- and (-)-cetirizine in the process. A mobile phase of 10 mmol/l phosphate buffer (pH 7.0)-acetonitrile (95:5, v/v) was used for the assays. The ultraviolet absorption was monitored at 230nm, and roxatidine was employed as an internal standard for quantification. Observed enantioselectivity (a) was 2.0. The AGP-CSP was also used on a preparative scale to isolate the enantiomers with an optical purity (enantiomeric excess) of greater than 99%. An analysis was also carried out for the cetirizine enantiomers in rat plasma to study the differences between the members of enantiomers in pharmacokinetics. Both (+)- and (-)-cetirizine were obtained from separation using a reversed phase column of AGP, and were detected in the range of 2.5-200 mg/ml in plasma. The detection limit for cetirizine enantiomers was 800 ng/ml in plasma. Although there were no recognizable differences in pharmacokinetics between these particular enantiomers in rat, the method appears to be useful for their pharmacokinetic studies in general. Also a pharmacokinetic study in human urine sample was conducted with the help of 5 healthy volunteers who were administered with a single oral dose of racemic cetirizine (20 mg). The ratios of peak area provided by the cetirizine enantiomers were linear (r>0.997) over a concentration range of 2.5-200 mg/ml. The peak of the excreted cetirizine enantiomers appeared in urine sample within 1-2 hrs. The excreted level of (+)-cetirizine was slightly higher than (-)-cetirizine but the difference was not statistically significant. However, this method appears to be applicable for enantioselective pharmacokinetic studies of racemic drugs. We compared the pharmacological activities of (+)-cetirizine and (-)-cetirizine using RBL 2H3 cell employing a radioimmuno assay (RIA). The antiallergic drug, cetirizine, inhibits the mediator release from a rat basophilic leukemia (RBL 2H3) cell line, which is frequently used as a mast cell model. By investigating inhibitory activities of (+)- and (-)- cetirizine in RBL-2H3 cells on the mediator release, we aimed to evaluate the effect of their structual characteristics on the antihistamine activity. The study on RBL-2H3 cell has clearly demonstrated that the (-)- cetirizine is significantly more potent than the (+)- or the racemic cetirizine, although there was no difference in pharmacokinetics between (+)- and (-)- cetirizine in rats. Levofloxacin is the (-)-isomer of ofloxacin. Ofloxacin was originally marketed as a racemate; i.e., a mixture of the optical isomers in equal ratios. And (-)-isomer of ofloxacin was more potent than the (+)-isomer in antibacterial activity. Therefore, levofloxacin has been developed as a single isomeric preparation by racemic switch. In this study, we prepared levofloxacin through the chiral separation of the ofloxacin enantiomers using mobile phase additives, derivatization reagents and a chiral stationary phase. A few conditions were tried for this purpose: firstly a novapak C18 column using 1 mmol/l cupric sulfate-methanol (90 : 9) as a mobile phase with UV detection ; secondly a novapak C18 column using 0.2 mmol/l phosphoric acid (pH 1.85)-acetonitrile (80 : 20) as a mobile phase with fluorescence detection after derivatization with diphenylphosphinylchloride and L-leucinamide; thirdly a bovineserumalbumin column using 0.2 mmol/l phosphate buffer (pH 8.0)-methanol (97 : 3) as a mobile phase with UV detection. All of the conditions mentioned above were judged to provide quite satisfactory results in terms of the optical purity of levofloxacin. With the ease and reproducibility of the method developed, we suggest that our findings be adopted in KFDA guidelines on chiral separation of stereoisomeric drugs containing carboxyl groups.

Keywords : chiral separation, cetirizine, a1-acidglycoprotein, pharmacokinetics, pharmacological activity, RBL 2H3 cell, levofloxacin, optical purity

PART Ⅱ: Prediction of Chiral Separation of Drugs with Amine Moiety on Chiral CBH Column using Partial Least Square Method Enantioseparation of chiral drugs are sometimes laborious and time-consuming and the chiral stationary phase is very expensive. Therefore, if the chiral separation mechanism of CSP were known and the prediction of chiral separation of the drug were possible by their 3-dimensional molecular structure, it should be certain that it will be a very useful tool in studying the chiral separation of drugs. In this study, eleven of the most popular b-blockers and some other drugs which have very similar structures with amine moiety were chosen as model drugs. In order to study the mechanism leading to chiral separation of some drugs with amine moiety, the influence on enantioselective retention of several mobile phase parameters; e. g., types and content of organic modifier, i.e., 2-propanol and acetonitrile, concentration of organic modifier, mobile phase buffer pH and column temperature were studied using chiral CBH columns. Changing the organic solvents from isopropanol to acetonitrile gave higher values of both a and Rs for drugs with amine moiety in the mobile phase of 10 mmol/l phosphate buffer (pH 7.0) : organic modifier (97 : 3, v/v). And both a and Rs increased with decreasing the content of organic modifier. As the pH of the phosphate buffer changes, chromatographic parameters were markedly effected in several cases. Especially, betaxolol and pindolol weren t separated at pH 7.0, but as the pH was decreased to 6.0 and 5.0, chromatographic parameters were markedly increased. In the mobile phase of 10 mmol/l phosphate buffer (pH 7.0) : organic modifier (97 : 3, v/v), optimum column temperature was 40℃ but in the mobile phase of 10 mmol/l phosphate buffer (pH 5.0) : organic modifier (97:3, v/v), optimum column temperature was 25℃. Optimum buffer strength was 10 mmol/l in the phosphate buffer. Secondly in order to predict the chiral separation behavior in chiral CBH column of several drugs with amine moiety, we used QSAR/CoMFA software. When the Quantitative Lipophilicity-Resolution (Separation factor) Relationship was studied, no relationship was found. From the results of the Quantitative CoMFA-Resolution (Separation factor) Relationship study using partial least square method, prediction of the chiral separation of drugs with amine moiety was possible by the characteristics of their three dimensional molecular structure. As a result of the above study in the mobile phase of 10 mmol/l phosphate buffer ( pH 6.0) - isopropanol (97 : 3), if the CoMFA is above 130, we can predict potentially complete enantioseparation of drug with amine moiety. As far as the mechanism for the chiral separation of drugs with amine moiety using chiral CBH column is concerned, proportion of electrostatic (blue/red) and steric factor (green/yellow) in their molecular structures were similar but the effect of the electrostatic factor was a little bit higher than the steric factor. When results are considered, this method appears to be a very useful tool for the prediction of drugs with amine moiety in chiral CBH column before the experimental operation was actually performed. Also, this study seem to be useful in understanding the mechanism of enantioseparation in some chiral columns.