Beneficial herb-drug interactions of EGb 761 with donepezil and its enhanced quality control via in vitro-in vivo correlation development

Ph.D.Background:EGb 761, the standard extract of Ginkgo biloba, contains seven marker components, namely quercetin, kaempferol, isorhamnetin, bilobalide, ginkgolides A, B and C, and has been associated with beneficial effects on neurodegeneration disorders. Donepezil (DPZ), an acetylcholinesterase (AChE) inhibitor, is a frontline therapeutic agent for the treatment of mild to moderately severe Alzheimer’s disease (AD). Both the anti- AChE activities and brain uptake of DPZ and its three major metabolites, namely 6-O-desmethyl DPZ (6-DDPZ), 5-O-desmethyl DPZ (5-DDPZ) and DPZ N-oxide, have never been clearly investigated. Previous clinical studies have suggested enhanced therapeutic efficacy and fewer side effects after co-administration of DPZ with EGb 761 in AD patients, however, mechanisms for such beneficial interactions are not known. Given the potential beneficial effects of EGb 761 on cholinergic transmission and oxidative stress, it is hypothesized that co-administration of EGb 761 and DPZ could complement each other on their mechanisms of action.. In addition, based on the evidenced effectiveness of EGb 761, improvement on the quality control of its preparations such as tablets should be obtained via identification of biorelevant dissolution medium to correlate better with its in vivo performance. We hypothesize that the concept of in vitro-in vivo correlation (IVIVC) for western drugs could be applicable to the quality control of medicinal product such as EGb 761 tablet.Purpose:The current study was proposed aiming to 1) investigate in vitro anti-AChE activities and in vivo brain uptake of DPZ and its three metabolites; 2) comprehensively evaluate the pharmacokinetics and pharmacodynamics interactions between DPZ and EGb 761; and 3) identify in vivo relevant dissolution media for the marker components in EGb 761 tablet for its quality control improvement.Method:In vitro Ellman’s method was first used to verify and compare the anti-AChE activities of DPZ and its three metabolites followed by investigation and comparison of pharmacokinetics and brain distribution after DPZ oral administration at 2.4 mg/kg in both healthy and scopolamine induced rats.Pharmacodynamics interactions between DPZ and EGb 761 was first evaluated by Morris water maze tests followed by ex vivo evaluation of biomarkers of AChE, superoxide dismutase and malondialdehyde (MDA) in the brain of rats from different treatment groups (n=10/group) including healthy rats treated with vehicle (G1) and scopolamine-induced rats treated with vehicle (G2), low/high-dose DPZ (G3/G4, 0.5/1.0 mg/kg, p.o., q.d.), low/high-dose DPZ (0.5/1.0 mg/kg, p.o., q.d.) in combination with EGb 761 (8.27 mg/kg, p.o., t.i.d.) (G5/G6) and EGb 761 (G7, 8.27 mg/kg, p.o., t.i.d.) for 2 weeks. Meanwhile their pharmacokinetics interactions were further investigated in another set of scopolamine-induced rats (G3-G7, n=10/group) receiving the same treatment. Moreover, impact of EGb 761 orally absorbable components on DPZ brain penetration was evaluated with hCMEC/D3 cell monolayer model.To enhance the quality control of EGb 761 tablet, dissolution profiles of bilobalide, ginkgolides A and B were obtained in seven selected dissolution media via United States Pharmacopeia Apparatus 2. In vivo absorption profiles of these components were then obtained by numerical deconvolution of their reported human plasma pharmacokinetics profiles. Correlation between the dissolution profile and the corresponding absorption profile was established for each component in each dissolution medium. Finally, the in vivo relevant medium for each component was selected based on the adjusted correlation coefficients or values of Akaike Information Criterion and Schwarz’s Bayesian Criterion.Results:DPZ and its three metabolites demonstrated anti-AChE activities with the IC₅₀ in the order of DPZ (7.20×10⁻² μM), 6-DDPZ (1.14×10⁻¹ μM), 5-DDPZ (4.03×10⁻¹ μM) and DPZ N-oxide (1.61 μM). After oral administration in rats, DPZ and its three active metabolites all evenly distributed in the brain. Due to scopolamine-induced suppression of gastrointestinal motility, significantly reduced plasma Cₘₐₓ and AUC₀-->∞ of DPZ together with prolonged plasma Tₘₐₓ and decreased brain uptakes for both DPZ and its three active metabolites were observed after oral administration of DPZ in scopolamine-induced rats . In this amnestic model, it was also noticed that rats with co-treatment of DPZ and EGb 761 had significantly improved cognitive function in water maze test with increased brain levels of SOD and decreased brain levels of AChE and MDA than that with DPZ or EGb 761 only. Despite such beneficial pharmacodynamics outcomes, two-week co-administration did not affect the plasma pharmacokinetics and brain uptake of DPZ or bilobalide, which was further verified in hCMEC/D3 monolayer model with unchanged permeability coefficient. Based on our established IVIVC for bilobalide, ginkgolides A and B from EGb761 tablet, 0.1 M hydrochloric acid, fasted state simulated gastric fluid, fasted state simulated intestinal fluid version 2 were identified to be their corresponding best in vivo relevant dissolution media.Conclusions:Our current study for the first time demonstrated the anti-AChE activities and brain uptake of the three DPZ metabolites. Co-administration of DPZ and EGb 761 exerted better anti-amnestic effect via further enhanced pro-cholinergic and antioxidative effects of DPZ or EGb 761 in scopolamine-induced cognitive impairment rat without alteration in their systemic/brain exposure. Moreover, we for the first time applied the concept of IVIVC for western drugs to herbal medicinal products like EGb 761 tablet and successfully identified the in vivo relevant dissolution media of its three bioavailable marker components for its quality control improvement.研究背景：銀杏葉的標準提取物EGb 761包含七種標記成分，即槲皮素，山奈酚，異鼠李素，白果內酯，銀杏內酯A，B和C，並且對治療神經退行性疾病展現極其有效作用。目前為止，乙酰膽鹼酯酶抑製劑多奈哌齊是治療輕度至中度嚴重的阿爾茨海默氏病的一線藥物。多奈哌齊和它的三種主要代謝產物的乙酰膽鹼酯酶抑制活性和腦攝取均有待研究。儘管之前的臨床研究表明阿爾茨海默氏病的患者同時服用多奈哌齊與EGb 761的治療效果比單獨服用多奈哌齊好且副作用少，其作用機理卻需要進一步確定。基於EGb 761對膽鹼能傳遞和氧化應激的有益作用，它與多奈哌齊同時使用可以在作用機理上相互補充從而發揮出有益作用。此外，基於已證實的EGb 761的有效性，我們認為可以借鑒西藥的體外-體內相關性的概念，通過鑒定與體內相關的溶出介質來改善EGb 761片劑的質量控制。研究目的：本課題旨在1）研究多奈哌齊及其三種代謝產物的體外乙酰膽鹼酯酶抑製活性和腦攝取；2）綜合評價EGb 761和多奈哌齊的藥代動力學和藥效學的相互作用及其機理；3）通過比較EGb 761中各標記成分在不同溶出介質中溶出行為與其體內吸收行為的相關性鑒定出與體內相關的溶出介質，從而進一步提高EGb 761片劑的質量控制。研究方法：我們首先採用Ellman方法來驗證和比較多奈哌齊及其三種代謝產物的體外乙酰膽鹼酯酶抑制活性。接著還比較了健康大鼠和東莨菪鹼誘導大鼠單次口服多奈哌齊(2.4毫克/千克)後多奈哌齊及其三種代謝產物的血漿藥代動力學和腦分佈。我們通過對七組老鼠（每組十隻）的莫里斯水迷宮實驗和標誌物检测（乙酰膽鹼酯酶活性，超氧化物歧化酶活性和丙二醛水平）評估了EGb 761與多奈哌齊的藥效學相互作用。實驗包括一組健康大鼠連續兩週多次口服空白溶劑（G1）和六組東莨菪鹼誘導大鼠連續兩週多次分別口服空白溶劑（G2），低/高劑量多奈哌齊（G3/G4，0.5/1.0毫克/千克，每日一次），低/高劑量多奈哌齊（0.5毫克/千克，每日一次）合用EGb 761（8.27毫克/千克，每日三次）（G5/G6），和EGb 761（G7，8.27毫克/千克，每日三次）。同時，我們設計另一套實驗進一步研究了EGb 761和多奈哌齊之間的藥代動力學相互作用，其中東莨菪鹼誘導大鼠連續两周多次口服的組別與上述藥效學研究設計相同（G3-G7，每組10只）。另外，我們還在hCMEC/D3單層細胞模型上評估了EGb 761可吸收成份對多奈哌齊的腦滲透的影響。為了進一步改進EGb 761片劑質量控制的方法，我們首先使用《美國藥典》的槳法得到白果內酯，銀杏內酯A和B在七種不同溶出介质中的溶出曲線。然後，我們將每種標記成分在不同溶出介質中的溶出曲線與其通過反捲積的方法獲得的體內吸收曲線相關聯。最後，通過比較調整後相關性係數或赤池信息量規、施瓦茨貝葉斯準則來最終鑑定每種標記成分與體內相關性最好的溶出介質。研究結果：多奈哌齊及其三種代謝產物均具有乙酰膽鹼酯酶抑制活性，其抑制百分之五十乙酰膽鹼酯酶所需要的濃度從小到大依次為多奈哌齊（7.20×10⁻²μM），6-O-去甲基多奈哌齊（1.14×10⁻¹μM），5-O-去甲基多奈哌齊（4.03×10⁻¹μM）和多奈哌齊N-氧化物（1.61μM）。大鼠中口服多奈哌齊後，多奈哌齊及其三種活性代謝物均勻地分佈在大腦中。由於東莨菪鹼可以抑制胃腸蠕动，與健康大鼠相比，東莨菪鹼誘導大鼠除了血漿中多奈哌齊的Cₘₐₓ和AUC₀-->∞分別顯著降低了大約52.0％和30.1％，而且其多奈哌齊及其三種活性代謝產物的血漿Tₘₐₓ顯著延長，脑摄取也顯著減少。與多奈哌齊或EGb 761組單獨治療相比，多奈哌齊和EGb 761聯合治療組大鼠的记忆能力更好，表現在它們的聯合治療不僅可以顯著降低大鼠大腦中乙酰膽鹼酯酶的活性和丙二醛的水平，還可以顯著提高大腦中超氧化物歧化酶的活性。與此同時，與多奈哌齊或EGb 761單獨給藥相比，東莨菪鹼誘導大鼠同時服用與多奈哌齊和EGb 761後，多奈哌齊和EGb 761可吸收成分的血漿藥代動力學和腦攝取則幾乎沒有改變。通過hCMEC/D3單層細胞模型測得的多奈哌齊的滲透係數不會受到EGb 761可吸收成分的影響。基於我們建立的針對EGb 761片中白果內酯，銀杏內酯A，銀杏內酯B的IVIVC模型，0.1摩爾/升的鹽酸，禁食狀態下模擬胃液以及禁食狀態下模擬腸液被分別鑒定為它們各自與體內吸收相關性最好的溶出介質。研究結論：我們當前的研究首次證明了多奈哌齊三種代謝物的乙酰膽鹼酯酶抑制活性和腦攝取。同時服用多奈哌齊和EGb 761不僅不會改變多奈哌齊和EGb 761中標記成分的血漿藥代動力學和大腦攝取而且對東莨菪鹼誘導認知障礙的抗遺忘效果優於多奈哌齊或EGb 761單獨使用。進一步機理研究表明多奈哌齊和EGb 761合用可以進一步增強多奈哌齊或EGb 761的促膽鹼能和抗氧化作用。此外，我們首次將西藥體內外相關性的概念應用於諸如EGb 761之類的植物藥產品，並成功鑑定了其三種可吸收的標記成分的體內相關性溶出介質以改善對其片劑的質量控制。Zhao, Jiajia.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 185-214).Abstracts also in Chinese.Title from PDF title page (viewed on March 23, 2022)