Coenzyme Q10의 劑形別 物理化學的 特性과 藥物動態學的 파라미터

최치호 충북대학교 2009 국내석사

배경 코엔자임 Q10은 항산화성을 가지고 있으며 전자전달계에 중요한 역할을 하는 물질로 알려져 있다. 또한 낮은 수용성과 bioavailability를 가진다. 본 실험은 코엔자임 Q10 의 다양한 약제학적 분석을 위해 실행되었다. 오일 솔루션, 에멀젼 제제, 리포좀 제제의 세 가지 제형으로 나누어 실험을 실행하였다. 방법 오일 솔루션은 따뜻한 미네랄 오일에 녹였고 에멀젼 제제는 DW와 Phospholipon 85G (85G)와 Phospholipon 80H (80H)를 ultra-homomixer. 를 이용하여 만들었고 리포좀 제제는 Phospholipon과 콜레스테롤을 이용하여 film method로 만들었다. Physicochemical properties는 리포좀 제제의 입자크기와 봉입률 그리고 각 제형별 빛, 온도, ㏗ 등에 대한 안정성을 실험하였다. Pharmacokinetic parameters 는 각 제형을 Sprague-Dawey rat에 경구투여한 후 혈액을 취하여 HPLC로 코엔자임 Q10의 농도를 구하였다. 결과 리포좀 제형의 입자 크기는 289.7 and 2019.3 ㎚ 이며 봉입률은 85G와 80H가 각각 46 % and 52 % 로 나타났다. 에멀젼 제제와 리포좀 제제는 20,000 Lux 세기의 빛에 노출시켜 광안정성을 실행한 결과 안정성이 증가하는 결과를 볼 수 있었다. 코엔자임 Q10 의 ㏗ 와 온도별 안정성 측정에는 별다른 유효성을 찾아볼 수 없었다. 에멀젼 제형의 AUC 오일 솔루션에 비하여 85G와 80H를 사용한 제형에서 각각 170% 와30% 의 증가를 나타내었다. 특히85G를 사용한 에멀젼 제형의 Cmax 는 오일 솔루션에 비하여 40% 가 증가하는 결과를 얻었다. 그러나 리포좀 제형의 경구투여 시 AUC 와 Cmax 이 감소하였고 특히 80H를 사용한 리포좀 제형에서 더욱 심한 감소를 보였다. 결론 코엔자임 Q10 의 낮은 수용성과 bioavailability를 고려해 본다면 본 실험 제형 중 에멀젼 제형이 가장 이상적이라는 할 수 있다. Purpose of this study was to investigate physicochemical properties and pharmacokinetic parameters of coenzyme Q10 (CoQ10) in various formulations. Three different formulations (oil solution, emulsion, liposomal formulations) containing CoQ10 were prepared. Oil solution was prepared simply by dissolving CoQ10 into warm mineral oil, and emulsion was prepared by emulsification of the drug with Phospholipon 85G (85G) or Phospholipon 80H (80H) using homomixer. Liposomal formulation was prepared by traditional film method using the Phospholipons and cholesterol. Physicochemical properties were evaluated in terms of particle size, entrapment efficiency and stability (light, temperature, ㏗). Pharmacokinetic parameters were studied after oral administration of each formulation to Sprague-Dawey rats, and measurement of CoQ10 was performed by HPLC. Result showed that particle size of the liposome was 289.7 and 2019.3 ㎚ for the liposomes prepared with 85G and 80H, respectively. Entrapment efficiency was 46 % and 52 % in the corresponding order, respectively. Stability of CoQ10 against light (20,000 Lux) was significantly improved in both formulations (emulsion and liposomal formulations) compared to oil solution. Stability of CoQ10 at various temperature and ㏗ showed no difference among the all formulations tested. AUC of emulsion formulations was significantly improved compared to oil solution (170% and 30% increase in 85G and 80H formulations, respectively). Cmax of the emulsion formulation prepared with 85G significantly increased (about 40%) compared to oil solution. However, AUC and Cmax of the liposomal formulations were decreased, especially in the one prepared with 80H.

크로톤 오일로 유발시킨 쥐의 직장 염증에 대한 웅담 또는 그 구성 성분의 치유효과

유동호 충북대학교 2009 국내석사

웅담은 동양, 특히 중국에서 간 손상이나 염증 치료를 위한 약물로서 많이 처방되어온 약물이다. 최근 수 십 년간 개개의 담즙산에 대한 연구가 많이 진행되어 왔으며 그로 인해 암, 간 손상, 담석증 등에 대한 효과가 입증되었다. 특히 최근의 몇 몇 보고에 의하면 Ursodeoxycholic acid 같은 특정 담즙산의 경우 간과 관련된 질환 뿐 아니라 몸 안의 모든 염증에 효과가 있다는 것이 알려졌다. 이런 담즙산의 항염 효과를 더 알아보기 위해 웅담과 그 주요 구성 담즙산인 Ursodeoxycholic acid (UDCA), Chenodeoxycholic acid (CDCA) 가 크로톤 오일로 인위적으로 유발시킨 쥐의 직장염증에 어떠한 효과가 있는지 알아보았다. 웅담에 함유되어 있는 각각의 UDCA 와 CDCA 는 HPLC 를 이용하여 정량하였다. 쥐의 직장염증의 유발은 크로톤 오일 용액을 적신 면봉을 쥐의 직장에 직접 삽입하는 방법으로 하였다. 항염 효과를 알아보기 위한 약물의 경구투여는 UDCA, CDCA 그리고 웅담을 각각 50 mg/kg 의 용량으로 염증 유발 후 1시간부터 8시간 간격으로 2회 투여하였다. 또한 약물의 국소투여는 웅담을 10 mg/kg 의 용량으로 염증 유발 후 1시간 후에 1회 직장에 액상좌제의 형태로 직접 투여하였다. 약물을 투여한 쥐들은 염증 유발 후 4시간 또는 24시간 후에 평가하였다. 염증상태의 평가는 외관상의 충혈, 직장의 무게, 직장의 두께 그리고 직장 조직의 현미경 사진 으로 평가하였다. 담즙산의 항염효과를 알아보기 위해 In vitro 시험으로 NO assay 도 수행하였다. 사용된 웅담 1mg 에는 UDCA 가 39.26 ㎍/ml, CDCA 가 47.11 ㎍/ml 가 각각 함유되어 있는 것으로 확인 되었다. 웅담의 투여는 경구투여나 국소투여 모두에서 효과적으로 염증을 감소시키는 것으로 확인 되었다. 그에 반에 UDCA 나 CDCA 의 투여는 염증 완화에 효과적이지 않은 것으로 확인 되었다. CDCA 나 UDCA 와 비교했을 때 웅담의 투여는 염증의 진행을 억제하고 염증을 치유하는 효과가 매우 뛰어난 것으로 생각된다. 이는 웅담에 함유된 다른 어떤 물질에 의한 것 이거나 몇몇 물질들의 상호작용에 의한 것으로 생각된다. 활성성분이나 작용 메카니즘의 규명을 위해 이에 대한 많은 연구가 필요하다. Bear biles has been used as therapeutics of cerebral and coronary thrombosis, convulsion, hepatitis, jaundice and abscess in traditional oriental medicine. In recent decades, it was studied many reports about the effects of bile acids on cancer, cholestasis, and liver injury. In this study, we investigated the anti-inflammatory effects of whole bear bile (WBB) and its two major components, CDCA and UDCA on rectal inflammation in rats. Bile acids in WBB was quantitatively analyzed by HPLC method. Rectal inflammation was induced to the male Sprague-Dawley rats by insertion of croton oil-absorbed cotton tips. WBB, UDCA or CDCA was administrated by orally at one hour after induction of rectal inflammation to rats. WBB also was administered by intra-rectal to rats. Rats were sacrificed 4 or 24 hours after induction of rectal inflammation. The evaluation was performed by measurement of weight and thickness of rectum and histopathologic examination of rectal tissue. Furthermore, we examined the inhibition effect of NO production of WBB, UDCA or CDCA in LPS-stimulated RAW 264.7 cells. The contents of UDCA and CDCA of WBB were 39.26 ㎍/㎎ and 47.11 ㎍/㎎, respectively. WBB treated group significantly reduced the weight and thickness of rectum compared with UDCA or CDCA treated group. The inhibition of NO production of WBB, UDCA and CDCA in LPS-stimulated RAW 264.7 cells was much higher than the control. And WBB treatment suppressed the induction of NO synthase in rectum homogenate. This results suggest that the anti-inflammatory effect of WBB could be related to the combination effect of UDCA, CDCA and other bile acids of WBB by inhibiting suppressed the induction of NO synthase and inhibition of NO production. Further study are need to be performed about other bear bile components and their interactions.

Xanthiside Hydrate의 결정구조

임병휘 충북대학교 2009 국내석사

단결정 X선 회절법을 이용하여 Xanthium strumarium에서 얻은 천연물질인 xanthiside hydrate (C17H23O8NS·H2O)의 결정구조를 밝혔다. 메탄올 용액에서 단결정을 얻었으며 결정은 단사정계에 속한다. 격자상수는 a=9.950(1), b=26.279(2), c=7.288(1)Å, 이며 측정한 밀도는 Dm = 1.46g/cm3, 계산된 밀도는 Dx = 1.462 g/cm3 이었으며 공간군은 P212121이고, unit cell에는 4개의 분자들이 존재하였다. Intensity data는 CuKα선을 이용한 ω-2θ법으로 모아졌다. 구조는 직접법으로 해석하였으며 최소자승법에 의한 정밀화로 1745개의 독립회절 반점에 대해 최종 R값은 0.063이었다. 결정내의 분자는 두 개의 intermolecular hydrogen bonds를 형성하여 안정화 하고 있었으며. 그 이외의 분자내부결합은 반데르발스 힘으로 이루어지고 있었다. The three-dimensional structure of a natural product, xanthiside hydrate (C17H23O8NS·H2O) from Xanthium strumarium was determined by single crystal X-ray diffraction analysis. The compound was recrystallized from a methanol solution into orthorhombic crystal system, with a=9.950(1), b=26.279(2), c=7.288(1)Å, Dm = 1.46, Dx = 1.462 g/cm3, space group P212121, and Z=4. The intensity data were collected by ω-2θ scan technique with CuKα radiation. The structure was solved by direct method and refined by least-squares procedure to the final R value of 0.063 for 1745 observed reflections with I≥2σ(I). There are two intermolecular hydrogen bonds which stabilize the molecular packing of xanthiside hydrate in the crystal. The other intermolecular contacts are kept by normal vander Waals` forces.

수종의 암세포주에 대한 조직배양 산삼 부정근의 항암효과

이태웅 충북대학교 대학원 2006 국내석사

Prephenate Dehydrogenase의 결정 구조

도남혁 충북대학교 2009 국내석사

Chorismate mutase(CM) is an enzyme that catalyzes the chemical reaction for the conversion of chorismate to prephenate in the pathway to the production of phenylalanine and tyrosine, also known as the shikimate pathway. This is achieved by conversion of chorismate to prephenate, which is then acted upon subsequently by prephenate dehydrogenase(PDH) and prephenate dehydratase(PDT) to form 4-hydroxyphenylpyruvate and phenylpyruvate. At this moment, the enzyme PDH catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. The shikimate pathway is present only in bacteria, fungi and higher plants and hence PDH exists only in them. This makes PDH an attractive target for developing drugs and herbicides. To explore the crystal structure, at first, PDH protein was overexpressed in E.coli BL21. It was cultured overnight at 20℃ after inject IPTG 0.3mM and purified by Ni-NTA agarose resin, thrombin cleavage and superdex200 gel filtration column. PDH was concentrated to 8mg/ml and crystallized using the hanging drop method at 20℃. The best crystal was obtained at Li2SO4 230mM, Polyethylene glycol 4000 10%, pH7.5 Tris-HCl 100mM reservoir solution, and 2.1Å X-ray diffraction data set was collected from this crystal. X-ray diffraction data was processed with the CCP4, COOT programs and finally analyzed the crystal structure of PDH from S.mutans

임포틴 베타와 스네일 핵단백질 복합체의 정제 및 결정화

신윤식 충북대학교 2009 국내석사

The transport of macromolecules through NPC(Nuclear pore complex) into the nucleus is generally regulated by specific members of the importins. Importins recognize Nuclear localization signal (NLS) of their substrate in the cytoplasm and transport them through NPC into the nucleus. In the nucleoplasm, RanGTP binds to this importins-substrate complex, including the release of importins. But snail has other pathway. Snail, a DNA-binding zinc finger protein, functions as a transcriptional repressor for genes including E-cadherin during development and the acquisition of tumor cell invasiveness. Human Snail is a 264-amino acid nuclear protein with an amino-terminal basic amino acid-rich domain(SNAG domain) and a carboxyl-terminal DNA-binding domain (zinc finger domain). Using an in vitro transport system, the nuclear import of Snail was reconstituted by importin (karyopherin)β in the presence of Ran and NTF2. Snail binds directly to importin β in a zinc finger domain-dependent manner. These results indicate that zinc finger domain of Snail functions as a nuclear localization signal(NLS) and Snail can be transported into the nucleus in an importin β-mediated manner. Importin β(mouse) and Snail(human) were expressed in E.coli BL21 at 20℃ overnight using pET-30a vector and pGEX-2T vector, respectively. But Snail could not be expressed well and was unstable. And Snail could not bind to Importin β efficiently. So we cloned Zinc finger domain only and we exchanged pGEX-TEV vector. As a result, it was expressed well and bound to Importin β well. So we have continued research with Snail Zinc finger domain. Two proteins were mixed for making complex and purified by phenyl sepharose and glutathione sepharose. Tev protease was added to protein complex for cleavaging GST Tag. Final Protein complex was purified by gel filtration through a Superdex 200 column with buffer containing pH7.4 tris-HCl 20mM, 100mM NaCl, 10mM Mercaptoethanol. Cocrystallization of Importin β-snail Zn complex was tried. Crystals of Importin β-snail Zn complex were grown at 20℃ by hanging drop method. The reservoir solution consisted of PEG3350 7.5%, pH6.5 MES 100mM, Tween 80 1 CMC and PEG3350 7.5%, pH6.5 MES 100mM, Tween 20 1 CMC.

Streptozotocin 유발 당뇨 쥐에 대한 야콘 괴경 추출물과 그 성분인 Chlorogenic Acid의 혈당강하효과

양재식 충북대학교 2009 국내석사

Smallanthus sonchifolius (Yacon, Asteraceae) was originally cultivated in South America and used in traditional medicine by Andean inhabitants. Yacon is known to be potentially beneficial for the management of diabetes. Yacon is composed of fructooligosaccharides, proteins, minerals and phenolic compounds. The aim of study is to investigate hypoglycemic effect of yacon tuber extract (YTE) and its constituent, chlorogenic acid (CGA), in streptozotocin(STZ)-induced diabetic rats. In this study, a HPLC method was developed for simultaneous determination of major active phenolic components, CGA and caffeic acid in YTE. We investigated the hypoglycemic effect of YTE and CGA in STZ-induced diabetic rats and studied glucose tolerance test (GTT). The effect of orally administered multiple dose of YTE and CGA on plasma biochemical parameters was examined using diabetic rats. We also measured free radical scavenging activity by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and determined nitric oxide (NO) production. The stability test of pharmaceutical formulations (tablet and suspension) of YTE was studied under accelerated condition. The oral administration of YTE (200mg/kg) and CGA (10mg/kg) for 6 weeks produced significant hypoglycemic effect on STZ-induced diabetic rats. YTE and CGA-treated groups showed significant decrease in plasma glucose surge during the GTT. Total cholesterol (TC) and triglyceride (TG) concentrations were significantly decreased by 33 % and 49 %, respectively in YTE-treated rats. TC and TG concentrations were also significantly decreased by 26 % and 41 %, respectively in CGA-treated rats. In the DPPH assay, free radical scavenging activity of CGA was similar to vitamin E, a positive control in the assay. YTE and CGA were found to prevent NO production. CGA concentration in yacon tuber tablet and suspension remained 92 % and 79 %, respectively after 6 months of the accelerated stability test (40 ± 2 ℃, 75 ± 5 % RH). This study suggests that YTE and its constituent, CGA, may be an useful option for management of hyperglycemia and diabetic nephropathy. Smallanthus sonchifolius (야콘, Asteraceae)는 남아메리카에서 자생하는 식물로서 지역 원주민에 의해 전통 약물로 이용되어져 왔다. 야콘은 당뇨병에 이로운 식이로서의 알려져 있다. 야콘은 물, 프럭토올리고당, 단백질, 미네랄, 폴리페놀 등으로 이루어져 있다. 이번 연구에서 야콘 괴경 추출물의 주요 활성 폴리페놀 성분으로 chlorogenic acid (CGA)와 caffeic acid 가 있음을 HPLC를 통하여 발견하였다. Streptozotocin (STZ)으로 유발된 당뇨 쥐에서의 야콘 괴경 추출물과 CGA의 혈당강하효과를 연구하였고 내당능 시험도 수행하였다. 야콘 괴경 추출물과 CGA의 반복투여로 인한 당뇨 쥐에서의 생화학적 지표들에 대해서도 조사하였다. 항산화 효과와 nitric oxide (NO) 생성에 미치는 영향을 측정하고 야콘 괴경 추출물의 두 가지 제형에서의 안정성 시험을 수행하였다. 야콘 괴경 추출물 200 mg/kg 과 CGA 10 mg/kg 의 6주간의 투여에서 STZ 유발 당뇨 쥐에서의 유의한 혈당강하효과를 얻을 수 있었다. 내당능 시험에서도 야콘 괴경 추출물과 CGA 모두 내당능이 있었다. 야콘 괴경 추출물 투여군에서 total cholesterol (TC)과 triglyceride (TG)의 농도가 각각 33 % 와 49 % 로 유의성 있게 감소하였다. CGA 투여군에서는 TC와 TG의 농도가 각각 26 % 와 41 % 로 유의성 있게 감소하였다. CGA는 양성대조군인 vitamin E와 비교하여 비슷한 정도의 항산화 효과를 나타내었고 야콘 괴경 추출물과 CGA 모두 NO 생성을 억제하는 효과를 보였다. 안정성 시험을 6개월간 시행한 결과 야콘 괴경 추출물의 정제와 액제에 남아있는 CGA의 양은 각각 92 % 와 79 %로 나타났다. 이번 연구를 통해 CGA를 함유한 야콘 괴경 추출물은 hyperglycemia 와 diabetic nephropathy 등에 유용한 치료 물질로서 생각된다.

흰쥐 및 비글견에서 CoenzymeQ10의 약물동태학적 해석

조병석 충북대학교 2009 국내석사

Coenzyme Q10 is an important mitochondrial redox component and the only endogenously produced lipid-soluble antioxidant. It shows potential clinical functions in cardiovascular disease and neuroregenerative disease. Coenzyme Q10 is used as a dietary supplement as daily health care. The aim of our study was to investigated pharmacokinetic parameter about the absorption, distribution, metabolism and elimination (ADME) of Coenzyme Q10 after its intravenous injection and oral administration. Coenzyme Q10 disappeared from the blood circulation during 120 min after i.v injection (0.5 mg/kg). The half-lives at the distribution phase (t1/2) and post-distribution phase were 16.9 min and 176 min, respectively. The absoption rate of Coenzyme Q10 was slow and bioavailability was low (2.4%) after the oral administration. The Tmax and Cmax were 4.67 hr and 0.665 ㎍/㎖, respectively. In the bile, Coenzyme Q10 was rapidly eliminated during 120 min. On the other hand, Coenzyme Q10 was not detected in the urine during 72 hr. Coenzyme Q10 was distributed almost into rat liver. Coenzyme Q10 shows very low absorption ability at oral administration. Coenzyme Q10 was not detected in the urine and it most excreted through the bile. Especially, Coenzyme Q10 was extensively distributed in the liver than other organs. In Beagle dogs, Coenzyme Q10 disappeared from the blood circulation during 30 min after i.v injection (0.6 mg/kg). The half-lives at the distribution phase (t1/2) and post-distribution phase were 1.17 min and 403 min, respectively. After the oral administration, the Tmax and Cmax were 7.5 hr and 0.179 ㎍/㎖, respectively. Bioavailability was very low (2.44%).

표면전하가 CoenzymeQ10 liposome의 물리화학적 성질과 약물동력학에 미치는 영향

김시훈 충북대학교 2009 국내석사

실험목적. 표면전하가 CoenzymeQ10 (CoQ10)을 함유하는 liposome의 물리화학적성질과 약묵동력학에 어떤 영향을 주는지 조사하기 위하여 연구를 진행하였다. 실험방법. Liposome은 Phospholipon 85G (PL)와 cholesterol 그리고 CoQ10으로 만들어졌다. Dioleoyl-trimethylammonium-propane (DOTAP)은 양이온성지질로서, dimyristoyl glycerophosphate (DMPA)은 음이온성 지질로서 사용되었다. Liposome의 물리화학적성질의 연구는 크기를 측정하고 봉입효율을 계산하며 안정성을 시험하는 방법으로 진행되었다. 안정성 시험은 각각의 온도, 빛, pH의 조건에서 실행되었다. 약물동력학실험은 쥐에게 CoQ10의 liposome 제형을 경구투여한 후, 혈장속의 CoQ10의 농도를 HPLC로 분석하는 방법으로 진행되었다. 결과. 세가지 liposome의 크기는 1 μm보다 작았다. 봉입효율의 측정결과 anionic liposome은 약 68%, neutral liposome과 cationic liposome은 약 41 %의 효율은 기록했다. 전하를 가진 liposome은 온도의 조건에서 안정성이 neutral liposome에 비해 약간 더 좋았으나 큰 차이는 없었다. 빛에 대한 안정성은 neutral liposome이 전하를 가진 liposome에 비하여 약간 더 좋았으나 역시 큰 차이는 없었다. PH에서의 안정성시험에서도 표면전하의 효과를 찾아볼 수 없었다. 전하를 가진 liposome에 비하여, CoQ10이 neutral liposome으로서 쥐에게 경구투여 되었을 때, AUC와 Cmax에서의 큰 증가를 확인할 수 있었다 결론. 안정성 시험에서 표면전하의 효과는 크게 없었다. liposome의 크기는 neutral liposome이 가장 컸으며, 봉입효율은 anionic liposome이 가장 컸다. 약물동력학 실험에서, neutral liposome이 약물전달시스템을 고려했을 때, 가장 적합한 liposome제형이라고 생각된다. Purpose. To investigate the effect of surface charge on the physicochemical properties and pharmacokinetic parameters of liposomes containing Coenzyme Q(CoQ10). Method. Three different liposomes (neutral, cationic, and anionic) containing CoQ10 were prepared using Phospholipon 85G (PL) and cholesterol. Dioleoyl-trimethylammonium-propane (DOTAP) or dimyristoyl glycerophosphate (DMPA) were also added for charge-bearing liposomes in addition to PL and cholesterol. Physicochemical properties were evaluated in terms of particle size, entrapment efficiency, and stability (light, temperature, pH). Pharmacokinetic parameters were studied after oral administration of the liposomal formulations to Sprague-Dawley rats, and measurement of CoQ10 was performed by HPLC. Result. Particle size of the three liposomes was less than a micron. Entrapment efficiency was about 46% in anionic liposome while it was about 28% in both neutral and cationic liposomes. Charged liposomes were more stable than neutral liposome at temperature condition but there was significant difference. Stability of CoQ10 against light was slightly improved in neutral liposome comparison with cationic and anionic liposome. However, there was no significant charge effect among them. Stability of CoQ10 in the liposomal formulation did not differ in pH tested. When CoQ10 was administered to the rats as neutral liposome, there were significant increases in AUC and Cmax compared to cationic and anionic liposome. Conclusion. Charged liposomes of CoQ10 slightly improved stability of CoQ10 against temperature but there was no significant surface charge effect. Neutral liposome had the largest size and anionic liposome had the largest entrapment efficiency among them. In pharmacokinetic test, neutral liposome had significant increases of AUC and Cmax compared to charged liposomes.

11-Hydroxyaclacinomycin X의 종간 스케일링에 있어서 단백결합의 역할

최규석 충북대학교 2009 국내석사

11-Hydroxyaclacinomycin X (HAMX) is isolated from S. galiaeus 3113 (pMC213) with main anti-cancer effects. The pharmacokinetics of HAMX, which has the inhibitory effects on DNA synthesis as well as topoisomerase like other anthracyclines, was investigated in mice, rat, rabbits and beagle dogs after i.v. bolus administration. To support its pharmacokinetic study which was essential for the pre-clinical research for new drug development, the total body clearance (CLt), apparent volume of distribution at steady state (Vdss) and terminal half-life (t1/2) were calculated. CLt, Vdss and t1/2 were 73.7 ml/mim/kg, 18.1 L/kg and 219 min in mice, 64.0 ml/min/kg, 10.9 L/kg and 227 min in rats, 47.2 ml/min/kg, 21.3 L/kg and 206 min in rabbits, and 27.4 ml/min/kg, 2.24 L/kg and 118 min in dogs, respectively. Based on animal data, CLt, Vdss and t1/2 of HAMX was predicted in humans using simple allometry and invariant time methods. The corresponding allometric equations were CLt = 45.896W0.8452, CLt × MLP = 31.175W1.1405, Vdss = 10140W0.8653 and t1/2 = 178.43W-0.0883. These allometric equations were extrapolated to predict CLt, Vdss and t1/2 in human based on 70 kg body weight. Plasma concentration of HAMX was also predicted by several species-invariant time transformations using equivalent time, Kallynochron, Apolysichron and Dienerichron. The human pharmacokinetic parameters of CLt, Vdss and t1/2 predicted by the simple allometry and various species-invariant time methods were 17∼42 ml/min/kg, 6∼19 L/kg and 112.5∼399 min, respectively. Plasma and cytosol protein binding of drug is an important factor that influences its pharmacokinetic parameters. To predict human CLt based on the unbound drug concentration in plasma and cytosol (fu), protein binding of the drug was assessed in vitro at five concentration levels (1, 5, 10, 50, 100 μg/ml) in plasma and cytosol using equilibrium dialysis and followed by HPLC analysis. The method was optimized against some influencing factors during both experimental procedures. And it was validated to be specific, sensitive, accurate, precise and of satisfactory recovery. The free fraction in plasma (fu) was 0.104 in mouse, 0.081 in rat, 0.049 in rabbit, 0.012 in dog, 0.011 in human, respectively. It exhibited a difference from mouse to human for the plasma and cytosol protein binding of 11-Hydroxyaclacinomycin X. Unbound clearance (CLt, u) was 707.68 ml/min/kg in mouse, 790.12 ml/min/kg in rat, 963.26 ml/min/kg in rabbit, 2258.33 ml/min/kg in dog, respectively. Based on animal data, the unbound clearance of HAMX was predicted in humans. The results indicated that HAMX data obtained from animals could be utilized to generate preliminary estimates of the pharmacokinetic parameters in humans. These parameter can serve as guidelines for better planning of clinical studise.