TWI666013B - Use of compound for removing hepatotoxicity of Acetaminophen (APAP) medicine - Google Patents
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Abstract
一種化合物用於去除乙醯胺基酚(Acetaminophen，APAP)藥物肝毒性之用途，該化合物係選自於下列群組：Tween 20、微晶纖維素、磷酸氫二鈣、聚氧乙烯23月桂基醚、糖精、甘露醇、聚氧乙烯烷基醚、三氯蔗糖、吡咯烷酮、羥基乙酸澱粉鈉、丙烯酸樹脂S100、羧甲基纖维素鈉、聚氧乙烯聚氧丙烯、薄荷醇、低取代烴丙纖維素、預膠化澱粉、Dextrates NF hydrated、檸檬酸、蓖麻油聚氧乙烯醚、膠態二氧化矽、聚乙二醇單硬脂酸酯、山梨酸、檸檬油、羥丙基纖維素、山梨糖醇、乙鮮舒泛鉀、羥丙甲纖維素酞酸酯、單水合乳糖、麥芽糖糊精、Brij 58、Brij 76、Tween 80、Tween 40、PEG 400、PEG 4000、PEG 2000等。A compound for removing hepatotoxicity of Acetaminophen (APAP) medicine, the compound is selected from the group: Tween 20, microcrystalline cellulose, dicalcium phosphate, polyoxyethylene 23 lauryl Ether, saccharin, mannitol, polyoxyethylene alkyl ether, sucralose, pyrrolidone, sodium starch glycolate, acrylic resin S100, sodium carboxymethyl cellulose, polyoxyethylene polyoxypropylene, menthol, low-substituted hydrocarbons Cellulose, pregelatinized starch, Dexrates NF hydrated, citric acid, castor oil polyoxyethylene ether, colloidal silica, polyethylene glycol monostearate, sorbic acid, lemon oil, hydroxypropyl cellulose , Sorbitol, potassium ethamsulfan, hypromellose phthalate, lactose monohydrate, maltodextrin, Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000, PEG 2000, etc.
Description
本發明係關於一種一種化合物用於去除乙醯胺基酚(Acetaminophen，APAP)藥物肝毒性之用途，特別是利用細胞色素P450 2E1(CYP2E1)酵素抑制劑以降低對乙醯胺基酚之肝毒性等副作用。The present invention relates to the use of a compound for removing hepatotoxicity of Acetaminophen (APAP) drugs, in particular to use a cytochrome P450 2E1 (CYP2E1) enzyme inhibitor to reduce hepatotoxicity to acetaminophen And other side effects.
對乙醯胺基酚(Acetaminophen，俗稱普拿疼)，又稱為paracetamol或N-acetyl-para-aminophenol，簡稱APAP，是市面上使用最普遍的解熱鎮痛劑，每年經常有許多因APAP使用不當而中毒或用於自殺的案例發生。APAP引起的肝臟損傷是造成重症及死亡的最主要因素。已有許多的臨床經驗證明APAP的肝毒性是可以預防的，早期的診斷和即時的給予解毒劑N-acetylcysteine簡稱NAC可有效預防肝毒性的發生。Acetaminophen (commonly known as Panadol), also known as paracetamol or N-acetyl-para-aminophenol, referred to as APAP, is the most commonly used antipyretic and analgesic in the market, and many APAPs are often used improperly every year. Cases of poisoning or suicide occurred. APAP-induced liver damage is the most important cause of severe illness and death. Many clinical experiences have proven that APAP's liver toxicity can be prevented. Early diagnosis and immediate administration of the antidote N-acetylcysteine, referred to as NAC, can effectively prevent the occurrence of liver toxicity.
對乙醯胺基酚服藥過量的早期發現是有必要的，因為在中毒後的8小時內給予解毒劑可達到最佳的預後效果。藥物中毒的早期徵兆包括身體不適、噁心和嘔吐，但有些病人對乙醯胺基酚的血中濃度己達中毒濃度且肝功能也有明顯異常，初期仍無症狀（第一期），肝毒性的徵兆如腹痛、持續嘔吐、黃膽、右上腹疼痛等，是在大量攝取後的24~48小時才會變得比較明顯(第二期)。血清的轉胺酶通常是在服用後的16小時開始上升，伴隨著臨床症狀開始出現。第三期大約是在服用後的3-4天才開始出現，此時肝損傷的程度和預後情形就可以被預估出來。肝毒性的症狀可以從輕微的症狀伴隨著肝功能值上升(AST＞1,000IU/L)到嚴重的猛暴性肝炎併發代謝性酸中毒、黃疸、低血糖、AST＞10,000IU/L、凝血異常及肝腦病變等。第四期會引發寡尿性腎衰竭，嚴重者會導致死亡。Early detection of acetaminophen overdose is necessary because the best prognosis is achieved by giving an antidote within 8 hours of poisoning. The early signs of drug poisoning include physical discomfort, nausea, and vomiting, but some patients have reached toxic levels of acetaminophen in the blood and have significant abnormalities in liver function. They are still asymptomatic at the initial stage (stage 1). Symptoms such as abdominal pain, persistent vomiting, yellow gall bladder, and right upper quadrant pain, etc., become apparent 24 to 48 hours after a large intake (Phase 2). Serum transaminase usually begins to rise 16 hours after taking it, and clinical symptoms begin to appear. The third period only begins to appear about 3-4 days after taking the medicine. At this time, the degree and prognosis of liver damage can be estimated. Symptoms of liver toxicity can range from mild symptoms accompanied by an increase in liver function (AST> 1,000 IU / L) to severe violent hepatitis with metabolic acidosis, jaundice, hypoglycemia, AST> 10,000 IU / L, and abnormal blood coagulation And liver and brain diseases. Stage 4 can cause oliguria renal failure, which can lead to death in severe cases.
有些對乙醯胺基酚中毒的病人其肝臟的傷害很輕，卻有嚴重的腎毒性產生，主要是因APAP直接在腎小管上的細胞色素P-450s (cytochrome P450s，簡稱CYPs)代謝造成腎毒性。但急性腎衰竭也有可能是因急性肝衰竭產生的肝腎症候群，此時可以利用鈉的***分率(fraction excretion of Na，簡寫FeNa)來分辨是原發性腎損傷 (FeNa＞1) 或是肝腎症候群 (FeNa ＜1)。FeNa的計算公式：(Sodiumurinary ÷ Creatinineurinary) ÷ (Sodiumplasma ÷ Creatinineplasma) × 100。Some patients with acetaminophen poisoning have mild liver damage, but have severe nephrotoxicity, mainly due to the metabolism of cytochrome P-450s (cytochrome P450s, CYPs) by APAP directly on the renal tubules. toxicity. However, acute renal failure may also be a liver and kidney syndrome caused by acute liver failure. At this time, the fraction excretion of Na (FeNa) can be used to distinguish between primary renal injury (FeNa> 1) or liver and kidney. Symptoms (FeNa <1). FeNa's calculation formula: (Sodiumurinary ÷ Creatinineurinary) ÷ (Sodiumplasma ÷ Creatinineplasma) × 100.
對乙醯胺基酚口服後1~2小時可達尖峰血中濃度，大部份是從肝臟代謝，90%以上是與glucuronide和sulfate結合形成無毒性的代謝產物。只有小於5%是經由不同的CYPs代謝，包括：CYP2E1、CYP1A2及CYP3A4，其中CYP2E1與CYP1A2是主要代謝酵素。經由這些酵素代謝所產生的代謝產物N-acetyl-p-benzoquinoneimine，簡稱 NAPQI(如圖一)是活性很強的親電物質(electrophile)。在正常情況下，NAPQI會立即和細胞內的glutathione反應形成無毒的硫醇化合物 (mercaptide)。當對乙醯胺基酚使用過量時， 使得細胞內glutathione的消耗速率大於合成速率，細胞內glutathione含量低於正常的30%時，NAPQI便會和細胞內含有cysteine的大分子或核苷酸結合，導致肝細胞損傷。從細胞組織染色可以證明在肝細胞壞死前，NAPQI會和cysteine的硫醇基在肝小葉中心區域形成共價鍵結。The peak blood concentration can be reached within 1 to 2 hours after oral administration of acetaminophen. Most of it is metabolized from the liver, and more than 90% is combined with glucuronide and sulfate to form non-toxic metabolites. Only less than 5% are metabolized by different CYPs, including: CYP2E1, CYP1A2, and CYP3A4, of which CYP2E1 and CYP1A2 are the main metabolic enzymes. The metabolite N-acetyl-p-benzoquinoneimine, abbreviated as NAPQI (Figure 1), produced by the metabolism of these enzymes is a highly active electrophile. Under normal circumstances, NAPQI immediately reacts with intracellular glutathione to form non-toxic mercaptide. When acetaminophen is used in excess, the consumption rate of glutathione in the cell is greater than the synthesis rate. When the content of glutathione in the cell is lower than 30% of normal, NAPQI will bind to the macromolecules or nucleotides containing cysteine in the cell. , Causing damage to liver cells. Cell tissue staining can prove that before hepatocyte necrosis, NAPQI and thiol groups of cysteine form a covalent bond in the central region of the hepatic lobules.
對於有肝臟疾病、酗酒、或是有服用會誘導肝臟細胞色素 P450酵素活性，如: Carbamazepine、Ethanol、Isoniazid、Phenobarbital (possibly other barbiturates)、Phenytoin、Sulfinpyrazone、Sulfonylureas、Rifampin、Primidone等藥物的病人均屬於會引起APAP嚴重肝毒性之高危險群。此時病人若有成人呼吸窘迫症候群、腦水腫、無法控制的出血、感染或多器管衰竭等併發症產生時，就很容易造成死亡。以酒精為例，酒精主要是由肝臟的CYP2E1代謝，其誘發APAP中毒的機轉可分為三個階段，第一階段是酒精和APAP競爭肝臟CYP2E1的接受器，此時NAPQI的濃度會先降低，第二階段是酒精會使得CYP2E1的半衰期從7小時延長到37小時，使得肝臟CYP2E1的含量增加，此時NAPQI的濃度會慢慢回升；第三階段是當酒精戒斷時，肝臟有較多的CYP2E1來代謝對乙醯胺基酚，使得對乙醯胺基酚的毒性代謝產物明顯增加，造成肝細胞損傷。近年來研究證實，利用diallyl sulfide 可有效預防對乙醯胺基酚在小鼠體內所引起之肝毒性傷害，並進一步發現diallyl sulfide具抑制CYP2E1酵素活性作用，推論diallyl sulfide預防對乙醯胺基酚引起之肝毒性的傷害，其保護機制應是透過抑制對乙醯胺基酚產生活性中間產物NAPQI。Patients with liver disease, alcoholism, or those who can induce liver cytochrome P450 enzyme activity, such as: Carbamazepine, Ethanol, Isoniazid, Phenobarbital (possibly other barbiturates), Phynetoin, Sulfinpyrazone, Sulfonylureas, Rifampin, Primidone, etc A high-risk group that can cause severe APAP liver toxicity. At this time, if the patient has complications such as adult respiratory distress syndrome, cerebral edema, uncontrolled bleeding, infection, or multivessel failure, it is easy to cause death. Take alcohol as an example. Alcohol is mainly metabolized by the liver's CYP2E1. The mechanism that induces APAP poisoning can be divided into three stages. The first stage is that alcohol and APAP compete with the liver CYP2E1 receptor. At this time, the concentration of NAPQI will first decrease The second stage is that alcohol will increase the half-life of CYP2E1 from 7 hours to 37 hours, which will increase the content of CYP2E1 in the liver. At this time, the concentration of NAPQI will slowly rise; the third stage is when the alcohol is withdrawn, the liver has more CYP2E1 is used to metabolize acetaminophen, which causes a significant increase in toxic metabolites of acetaminophen, causing liver cell damage. In recent years, studies have confirmed that the use of diallyl sulfide can effectively prevent liver toxicity caused by acetaminophen in mice, and further found that diallyl sulfide can inhibit the enzyme activity of CYP2E1, and deduced that diallyl sulfide prevents acetaminophen The protective mechanism of the hepatotoxicity caused should be through inhibition of the production of the active intermediate NAPQI of acetaminophen.
利用侵入式及非侵入式方法測試大鼠肝功能，以監測肝損害的發展以及篩選肝臟疾病，其中最常使用的方法包含測量血清中之天門冬氨酸轉胺酶(aspartate aminotransferase, AST)、丙氨酸轉胺酶(alanine aminotransferase, ALT)以及鹼性磷酸酶(alkaline phosphatase)數值，以及測量肝細胞產物如：膽紅素(bilirubin)、白蛋白(albumin)，以及利用量測前凝血素時間(prothrombin time)來檢測凝血因子(coagulation factors)等；肝功能定量測試是根據幾乎只經過肝臟代謝之受質在血清中的濃度而定，這些受質的清除是依肝門靜脈、肝動脈血流量以及由肝細胞對這些受質的作用而定，肝臟血流量與提供給肝臟的受質量有關，反之，該受質的清除則決定於肝臟代謝的能力。Use invasive and non-invasive methods to test liver function in rats to monitor the development of liver damage and screen for liver disease. The most commonly used methods include measuring aspartate aminotransferase (AST) in serum, Alanine aminotransferase (ALT) and alkaline phosphatase (alkaline phosphatase) values, and measurement of liver cell products such as: bilirubin, albumin (albumin), and the use of pre-prothrombin Time (prothrombin time) to detect coagulation factors (coagulation factors), etc .; quantitative liver function tests are based on the concentration of serum in the serum almost exclusively through metabolism, the clearance of these substrates is based on hepatic portal vein, hepatic artery blood flow And depending on the effect of hepatocytes on these receptors, liver blood flow is related to the quality of the receptor provided to the liver. Conversely, the clearance of this receptor is determined by the ability of the liver to metabolize.
半乳糖(galactose)是一種具有高萃取率(extraction ratio)、90%在肝臟中代謝的醣類，在肝臟中，半乳糖是由半乳糖激酶(galactokinase)經過差向立體異構化反應(epimerization)，將之轉換成1-磷酸葡萄糖(Glucose-1-phosphate)；半乳糖激酶的作用反應為肝細胞中半乳糖代謝途徑的速率決定步驟(rate-limiting step)。半乳糖的高萃取率使得依賴肝臟血流量及肝臟功能的半乳糖代謝作用成為檢測肝功能最主要的方式，目前並無一定的規則來評估大鼠之殘餘肝功能(residual liver function)，量測一確切化合物(如：半乳糖)之代謝能力，可推測肝臟中一代謝作用之速率決定步驟，亦可能取得殘餘肝功能之代表數質。Galactose is a sugar with a high extraction ratio and 90% metabolism in the liver. In the liver, galactose undergoes epimerization by galactokinase ), Which is converted into glucose 1-phosphate (Glucose-1-phosphate); the action of galactose kinase is a rate-limiting step of the galactose metabolic pathway in liver cells. The high extraction rate of galactose makes galactose metabolism, which is dependent on liver blood flow and liver function, the most important method for detecting liver function. At present, there are no certain rules to evaluate residual liver function in rats. The metabolic capacity of an exact compound (such as galactose) can be speculated to determine the rate of a metabolic action in the liver, and it is also possible to obtain a representative quality of residual liver function.
本案發明人以半乳糖單點法測試慢性肝炎、肝硬化以及肝癌病患，結果顯示半乳糖單點法可精確測出這些肝臟疾病；半乳糖單點法已被成功的應用到測試肝病患者排除如丙嗪(promazine)及抗生素頭孢酮(cefoperazone)等藥物之剩餘肝功能。此外，半乳糖單點法已在美國食品藥物管理局(FDA)所出版的指南(Guidance for Industry)中成為建議採用測試肝功能的方法之一。The inventor of the present case tested patients with chronic hepatitis, cirrhosis, and liver cancer by the galactose single-point method. The results show that the galactose single-point method can accurately detect these liver diseases; the galactose single-point method has been successfully applied to test liver disease patients. Remaining liver function of drugs such as promazine and the antibiotic cefoperazone. In addition, the galactose single spot method has become one of the recommended methods for testing liver function in the Guide for Industry published by the US Food and Drug Administration (FDA).
由此可見，上述習用對乙醯胺基酚仍有諸多缺失，實非一良善之設計者，而亟待加以改良。It can be seen that the above conventional practice still has many shortcomings with respect to acetaminophen, which is not a good designer and needs to be improved urgently.
本案發明人鑑於上述習用對乙醯胺基酚所導致肝毒性等副作用的缺點，乃亟思加以改良創新，並經多年研究後，終於成功研發完成本件一種化合物用於去除乙醯胺基酚(Acetaminophen，APAP)藥物肝毒性之用途。In view of the above-mentioned shortcomings of side effects such as hepatotoxicity caused by acetaminophen, the inventor of the case has been eager to improve and innovate. After years of research, he finally successfully developed a compound for removing acetaminophen ( Use of Acetaminophen (APAP) drug for liver toxicity.
本發明之目的即在於提供一種化合物用以去除對乙醯胺基酚(Acetaminophen, APAP)藥物肝毒性之用途，。The purpose of the present invention is to provide a compound for removing hepatotoxicity of Acetaminophen (APAP) medicine.
為達成前述發明目的，其中該化合物係選自下列群組中之至少一種或其任意組合：聚氧乙烯(20)山梨糖單月桂酸酯(Polyethylene glycol sorbitan monolaurate；Tween 20)、微晶纖維素(Microcrystalline cellulose)、磷酸氫二鈣(Dicalcium phosphate dihydrate)、聚氧乙烯23月桂基醚(Brij 35)、糖精(Saccharin)、甘露醇(Mannitol)、聚氧乙烯烷基醚(Cremophor RH40)、三氯蔗糖(Sucralose)、吡咯烷酮(Crospovidone)、羥基乙酸澱粉鈉(Sodium starch glycolate)、丙烯酸樹脂S100(Eudragit S100)、羧甲基纖维素鈉(Croscarmellose sodium)、聚氧乙烯聚氧丙烯(Pluronic F68)、薄荷醇(Menthol)、低取代烴丙纖維素(Low-substituted hydroxypropyl cellulos)、預膠化澱粉(Pregelatinized starch)、Dextrates NF hydrated、檸檬酸(Citric acid)、蓖麻油聚氧乙烯醚(Cremophor EL)、膠態二氧化矽(Aerosil 200)、聚乙二醇單硬脂酸酯(Myrj 52)、山梨酸(Sorbic acid)、檸檬油(Lemon oil)、羥丙基纖維素(Hydroxypropyl cellulose)、山梨糖醇(Sorbitol)、乙鮮舒泛鉀(Acesulfame potassium)、羥丙甲纖維素酞酸酯(Hydroxypropyl methylcellulose)、單水合乳糖(Lactose monohydrate)、麥芽糖糊精(Maltodextrin)、Brij 58、Brij 76、Tween 80、Tween 40、PEG 400、PEG 4000、PEG 8000、Span 60、苯甲酸鈉(Sodium benzoate)、羥乙甲纖維素酞酸酯(Hydroxy ethylmethylcellulose)、甲纖維素酞酸酯(Methylcellulose)、Span 80、甜蜜素(Sodium cyclamate)、甘油山崳酸酯(Glyceryl behenate)、氧化鐵紅(Oxide red)、甘油單硬脂酸酯(Glycerin monostearate)、Copovidone K28、澱粉醋酸酯(Starch acetate)、硬脂酸鎂(Magnesium stearate)、月桂基硫酸鈉(Sodium lauryl sulfate)、Providone K30、及PEG 2000；以上化合物均為酵素P450抑制劑。In order to achieve the foregoing object of the present invention, the compound is selected from at least one or any combination of the following groups: polyoxyethylene (20) sorbose monolaurate (Polyethylene glycol sorbitan monolaurate; Tween 20), microcrystalline cellulose (Microcrystalline cellulose), Dicalcium phosphate dihydrate, Polyoxyethylene 23 lauryl ether (Brij 35), Saccharin, Mannitol, Polyoxyethylene alkyl ether (Cremophor RH40), three Sucralose, Crospovidone, Sodium starch glycolate, Eudragit S100, Croscarmellose sodium, Pluronic F68 ), Menthol, Low-substituted hydroxypropyl cellulos, Pregelatinized starch, Dexrates NF hydrated, Citric acid, Castor oil polyoxyethylene ether (Cremophor EL), colloidal silicon dioxide (Aerosil 200), polyethylene glycol monostearate (Myrj 52), sorbic acid, lemon oil, hydroxypropyl cellulose Hydroxypropyl cellulose, Sorbitol, Acesulfame potassium, Hydroxypropyl methylcellulose, Lactose monohydrate, Maltodextrin ), Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000, PEG 8000, Span 60, Sodium benzoate, Hydroxy ethylmethylcellulose, methylcellulose phthalate Methylcellulose, Span 80, Sodium cyclamate, Glyceryl behenate, Oxide red, Glycerin monostearate, Copovidone K28, starch acetate Starch acetate, Magnesium stearate, Sodium lauryl sulfate, Provideone K30, and PEG 2000; all of the above compounds are enzyme P450 inhibitors.
為達成前述發明目的，其中該化合物組成係選自下列群組中之至少一種或其任意組合，並限定其組合物之有效使用劑量：聚氧乙烯(20)山梨糖單月桂酸酯(Polyethylene glycol sorbitan monolaurate；Tween 20)其中含量為0.001~55克、微晶纖維素(Microcrystalline cellulose)其中含量為0.001~55克、磷酸氫二鈣(Dicalcium phosphate dihydrate)其中含量為0.001~55克、聚氧乙烯23月桂基醚(Brij 35)其中含量為0.001~55克、糖精(Saccharin)其中含量為0.001~55克、甘露醇(Mannitol)其中含量為0.001~55克、聚氧乙烯烷基醚(Cremophor RH40)其中含量為0.001~55克、三氯蔗糖(Sucralose)其中含量為0.001~55克、吡咯烷酮(Crospovidone)其中含量為0.001~55克、羥基乙酸澱粉鈉(Sodium starch glycolate)其中含量為0.001~55克、丙烯酸樹脂S100 (Eudragit S100)其中含量為0.001~55克、羧甲基纖维素鈉 (Croscarmellose sodium)其中含量為0.001~55克、聚氧乙烯聚氧丙烯 (Pluronic F68) 其中含量為0.001~55克、薄荷醇(Menthol)其中含量為0.001~55克、低取代烴丙纖維素(Low-substituted hydroxypropyl cellulos)其中含量為0.001~55克、預膠化澱粉(Pregelatinized starch)其中含量為0.001~55克、Dextrates NF hydrated其中含量為0.001~55克、檸檬酸(Citric acid)其中含量為0.001~55克、蓖麻油聚氧乙烯醚(Cremophor EL)其中含量為0.001~55克、膠態二氧化矽(Aerosil 200)其中含量為0.001~55克、聚乙二醇單硬脂酸酯(Myrj 52)其中含量為0.001~55克、山梨酸(Sorbic acid)其中含量為0.001~55克、檸檬油(Lemon oil)其中含量為0.001~55克、羥丙基纖維素(Hydroxypropyl cellulose)其中含量為0.001~55克、山梨糖醇(Sorbitol)其中含量為0.001~55克、乙鮮舒泛鉀(Acesulfame potassium)其中含量為0.001~55克、羥丙甲纖維素酞酸酯(Hydroxypropyl methylcellulose)其中含量為0.001~55克、單水合乳糖(Lactose monohydrate)其中含量為0.001~55克、麥芽糖糊精(Maltodextrin)其中含量為0.001~55克、Brij 58其中含量為0.001~55克、Brij 76其中含量為0.001~55克、Tween 80其中含量為0.001~55克、Tween 40其中含量為0.001~55克、PEG 400其中含量為0.001~55克、PEG 4000其中含量為0.001~55克、PEG 8000其中含量為0.001~55克、Span 60其中含量為0.001~55克、苯甲酸鈉(Sodium benzoate)其中含量為0.001~55毫克、羥乙甲纖維素酞酸酯(Hydroxy ethylmethylcellulose)其中含量為0.001~55克、甲纖維素酞酸酯(Methylcellulose)其中含量為0.001~55克、Span 80其中含量為0.001~55克、甜蜜素(Sodium cyclamate)其中含量為0.001~55克、甘油山崳酸酯(Glyceryl behenate)其中含量為0.001~55克、氧化鐵紅(Oxide red)其中含量為0.001~55克、甘油單硬脂酸酯(Glycerin monostearate)其中含量為0.001~55克、Copovidone K28其中含量為0.001~55克、澱粉醋酸酯(Starch acetate)其中含量為0.001~55克、硬脂酸鎂(Magnesium stearate)其中含量為0.001~55克、月桂基硫酸鈉(Sodium lauryl sulfate)其中含量為0.001~55克、Providone K30其中含量為0.001~55克、及PEG 2000其中含量為0.001~55克。In order to achieve the foregoing object of the invention, the composition of the compound is selected from at least one of the following groups or any combination thereof, and the effective dosage of the composition is limited: Polyoxyethylene (20) sorbose monolaurate (Polyethylene glycol) sorbitan monolaurate; Tween 20) with a content of 0.001 to 55 grams, microcrystalline cellulose with a content of 0.001 to 55 grams, dicalcium phosphate dihydrate with a content of 0.001 to 55 grams, polyoxyethylene 23 Lauryl ether (Brij 35) with a content of 0.001 to 55 grams, Saccharin with a content of 0.001 to 55 grams, Mannitol with a content of 0.001 to 55 grams, Polyoxyethylene alkyl ether (Cremophor RH40 ) The content is 0.001 to 55 grams, the content of sucralose is 0.001 to 55 grams, the content of Crospovidone is 0.001 to 55 grams, and the content of sodium starch glycolate is 0.001 to 55 G, acrylic resin S100 (Eudragit S100) in which the content is 0.001 to 55 g, croscarmellose sodium in which the content is 0.001 to 55 g, polyoxyethylene polyoxypropylene (Pl uronic F68) in which the content is 0.001 to 55 g, menthol (Menthol) in which the content is 0.001 to 55 g, Low-substituted hydroxypropyl cellulos in which the content is 0.001 to 55 g, pregelatinized starch ( (Pregelatinized starch) with a content of 0.001 to 55 g, Dexrates NF hydrated with a content of 0.001 to 55 g, Citric acid with a content of 0.001 to 55 g, and castor oil polyoxyethylene ether (Cremophor EL) with a content of 0.001 ~ 55g, colloidal silicon dioxide (Aerosil 200) in which the content is 0.001 ~ 55g, polyethylene glycol monostearate (Myrj 52) in which the content is 0.001 ~ 55g, and sorbic acid (Sorbic acid) in the content 0.001-55 grams, lemon oil (0.001-55 grams), hydroxypropyl cellulose (0.001-55 grams), and sorbitol (0.001-55 grams) Acesulfame potassium content is 0.001 ~ 55 g, Hydroxypropyl methylcellulose content is 0.001 ~ 55 g, Lactose monohydrate content is 0.001 ~ 55g, maltodextrin (Ma ltodextrin) which contains 0.001 ~ 55 grams, Brij 58 contains 0.001 ~ 55 grams, Brij 76 contains 0.001 ~ 55 grams, Tween 80 contains 0.001 ~ 55 grams, Tween 40 contains 0.001 ~ 55 grams, PEG 400 contains 0.001 ~ 55 grams, PEG 4000 contains 0.001 ~ 55 grams, PEG 8000 contains 0.001 ~ 55 grams, Span 60 contains 0.001 ~ 55 grams, Sodium benzoate contains 0.001 ~ 55 mg, Hydroxy ethylmethylcellulose in which the content is 0.001 to 55 grams, Methylcellulose in which the content is 0.001 to 55 grams, and Span 80 in which the content is 0.001 to 55 grams , Sodium cyclamate in which the content is 0.001 to 55 grams, glyceryl behenate in which the content is 0.001 to 55 grams, iron oxide red (Oxide red) in which the content is 0.001 to 55 grams, glycerol is hard Glycerin monostearate contains 0.001 to 55 grams, Copovidone K28 contains 0.001 to 55 grams, Starch acetate contains 0.001 to 55 grams, and Magnesium stearate contains 0.001 to 55 grams, laurel Sulfate (Sodium lauryl sulfate) wherein 0.001 to 55 g, Providone K30 wherein 0.001 to 55 g, wherein the content of PEG 2000 and 0.001 to 55 g.
為達成前述發明目的，其中該化合物係選自甘露醇(Mannitol)併用三氯蔗糖(Sucalose)組合為最佳。In order to achieve the aforementioned object of the invention, the compound is selected from the group consisting of Mannitol and a combination of sucralose (Sucalose).
為達成前述發明目的，其中該化合物與對乙醯胺基酚係經分開、同時或依序地使用。In order to achieve the foregoing object of the invention, the compound is used separately, simultaneously or sequentially from the p-acetamido system.
為達成前述發明目的，其中該化合物係以膠、噴劑、軟錠劑、錠劑、可分散性片劑、膠囊、軟膠囊、丸劑、懸浮液、微球、口腔植入片、乳化針劑或植入劑及藥學上可行之形式投予。In order to achieve the foregoing object of the invention, the compound is a gel, a spray, a soft lozenge, a lozenge, a dispersible tablet, a capsule, a soft capsule, a pill, a suspension, a microsphere, an oral implant, an emulsified injection, or Administration of implants and pharmaceutically acceptable forms.
為達成前述發明目的，其中該化合物被包含於醫藥包、套組或病患包。In order to achieve the aforementioned object of the invention, the compound is contained in a pharmaceutical package, a kit or a patient package.
本案發明人鑑於上述習用對乙醯胺基酚所導致肝毒性等副作用的缺點，乃亟思加以改良創新，並經多年苦心孤詣潛心研究後，終於成功研發完成本件一種化合物用於去除乙醯胺基酚(Acetaminophen，APAP)藥物肝毒性之用途。In view of the above-mentioned shortcomings of side effects such as hepatotoxicity caused by acetaminophen, the inventor of the present case has been eager to improve and innovate. After years of painstaking research, he finally successfully developed a compound for removing acetaminophen. Use of phenol (Acetaminophen, APAP) medicine for liver toxicity.
為達成上述發明目的之無肝副作用之對乙醯胺基酚新複方，本發明首先以對乙醯胺基酚新複方誘導大鼠產生肝毒性為模式，研究合併使用一或其任意組合之CYP2E1抑制劑對大鼠體內對乙醯胺基酚引發之肝毒性的影響；除使用一般肝毒性標記、病理組織切片外，以半乳糖單點法(GSP)進行大鼠的殘餘肝功能之定量量測，進一步評估。In order to achieve the above-mentioned objective of the invention, the new compound of paracetamol without liver side effects, the present invention first uses the new compound of paracetamol to induce hepatotoxicity in rats as a model to study the combined use of one or any combination of CYP2E1 The effect of inhibitors on the hepatotoxicity induced by acetaminophen in rats; in addition to using general liver toxicity markers and pathological tissue sections, the quantitative amount of residual liver function in rats was measured by the galactose single point method (GSP) Test for further evaluation.
本說明書中所述之所有技術性及科學術語，除非另外有所定義，皆為該所屬領域具有通常技藝者可共同瞭解的意義。Unless defined otherwise, all technical and scientific terms described in this specification have the meaning commonly understood by those skilled in the art.
於用於本文中之時，術語“組合物(combination)”，於應用於量兩種或更多種化合物及/或藥劑(於本文中也稱為成分)之時，意欲定義其中有該兩種或更多種化合物/藥劑結合之材料。術語“組合” (“combined”和“combining”)於本文中皆據此詮釋。As used herein, the term "combination", when applied to an amount of two or more compounds and / or pharmaceutical agents (also referred to herein as ingredients), is intended to define where the two A combination of one or more compounds / agents. The terms "combined" and "combining" are interpreted accordingly herein.
醫藥套組(pharmaceutical kits)、醫藥包(pharmaceutical packs)或病患包(patient packs)，其中有兩種或更多種化合物/藥劑經共-包裝或共-呈現(如作為單位劑批的部份者)。Pharmaceutical kits, pharmaceutical packs, or patient packs in which two or more compounds / agents are co-packaged or co-presented (e.g., as part of a unit dose batch) Share).
本發明係以下面的實施例予以示範闡明，但本發明不受下述實施例所限制。本發明所用之藥物、生物材料皆市售易於取得，下列僅為示例可取得之管道。The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. The medicines and biological materials used in the present invention are all commercially available and easily available, and the following are merely examples of available pipelines.
本發明係以下面的實施例予以示範闡明，但本發明不受下述實施例所限制。The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.
實施例一Example one
對乙醯胺基酚合併使用一種或其任意組合安全、藥學上可接受之常用賦型劑於改善藥物肝毒性動物試驗。Paracetamol is used in combination with one or any combination of safe and pharmaceutically acceptable common excipients in animal tests to improve drug hepatotoxicity.
材料與方法Materials and Methods
1. 試驗材料Test material
所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield, OH, USA)，APAP購自Sigma化學公司(St. Louis, MO USA)，半乳糖注射溶液由南光化學製藥股份有限公司製備，係將400克半乳糖(Sigma)溶於1公升含有適當緩衝溶液系統以及等張鹽類之蒸餾水中，供作注射使用。All organic solvents are HPLC grade, purchased from Tedia Co., Ltd. (Fairfield, OH, USA), APAP was purchased from Sigma Chemical Company (St. Louis, MO USA), and the galactose injection solution was prepared by Nanguang Chemical Pharmaceutical Co., Ltd. 400 grams of galactose (Sigma) is dissolved in 1 liter of distilled water containing a suitable buffer solution system and isotonic salts for injection.
2. 試驗動物Experimental animal
體重為175-280公克之雄性SD(Sprague-Dawley)大鼠購自國家實驗動物中心(台灣)，動物實驗係遵照國衛院動物實驗指南進行，所有的大鼠均置於空氣/濕度調節環境下，光照與黑暗各12小時，水及飼料的供給不限，在試驗期間大鼠體重均持續監測，水分照常供給。Male SD (Sprague-Dawley) rats weighing 175-280 grams were purchased from the National Laboratory Animal Center (Taiwan). The animal experiment was performed in accordance with the Animal Experiment Guide of the National Institute of Health. All rats were placed in an air / humidity-conditioned environment. Under the light and dark conditions for 12 hours each, the supply of water and feed was unlimited. During the test, the body weight of rats was continuously monitored, and water was supplied as usual.
3. 試驗處理Test treatment
挑選於體外篩選CYP2E1有效抑制劑，進行APAP不併服或併服抑制劑於改善APAP引起肝毒性之動物試驗。肝毒性試驗動物是以大鼠每公斤體重管餵給予APAP單一劑量1000毫克以產生肝毒性。肝保護實驗動物是以大鼠每公斤體重管餵給予磷酸氫二鈣1.67毫克；或每公斤體重管餵給予甘露醇1.67毫克；或每公斤體重管餵給予薄荷醇1.67毫克；或每公斤體重管餵給予HUEXC041 1.67毫克；或每公斤體重管餵給予甘露醇1.67毫克與三氯蔗糖1.67毫克；或每公斤體重管餵給予甘露醇0.83毫克與三氯蔗糖0.83毫克；或每公斤體重管餵給予甘露醇0.42毫克與三氯蔗糖0.42毫克；或每公斤體重管餵給予甘露醇0.17毫克與三氯蔗糖0.17毫克，併每公斤體重管餵給予APAP單一劑量1000毫克。以測量血漿中的天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)來代表肝發炎指標；於給藥前與給藥後16小時進行測定半乳糖單點法(GSP)，分析定量大鼠剩餘肝功能；同時分析每一組試驗大鼠之肝臟組織病理變化，評估其肝損傷或肝保護的機制。Selected in vitro to screen for effective inhibitors of CYP2E1, to conduct animal tests in which APAP is not taken or taken together to improve APAP-induced liver toxicity. Liver toxicity test animals were given a single dose of 1000 mg of APAP per kilogram of body weight in rats to produce hepatotoxicity. Liver protection experimental animals are: 1.67 mg of dicalcium phosphate per kilogram of tube weight administered to rats; 1.67 mg of mannitol per kilogram of weight tube administered; or 1.67 mg of menthol per kilogram of weight tube; or HUEXC041 1.67 mg fed; or 1.67 mg mannitol and 1.67 mg sucralose per kg of weight tube; 0.83 mg mannitol and 0.83 mg sucralose per kg weight tube; or mannose per kg weight tube 0.42 mg of alcohol and 0.42 mg of sucralose; or 0.17 mg of mannitol and 0.17 mg of sucralose per kilogram of weight tube, and a single dose of 1000 mg of APAP per kilogram of weight tube. Aspartate transaminase (AST) and alanine transaminase (ALT) were measured in plasma to represent indicators of liver inflammation; galactose single-point method (GSP) was performed before and 16 hours after administration ) To analyze and quantify the remaining liver function in rats; at the same time, analyze the pathological changes of liver tissue in each group of experimental rats to assess the mechanism of liver injury or liver protection.
4. 血液樣本Blood sample
處理完畢後，大鼠以***麻醉犧牲，血液由大鼠尾動脈抽取，置於含有EDTA之試管中，血漿(plasma)以13,000g於4˚C離心15分鐘，分離後的血漿分裝到微量小管(Eppendorf tube)中並置於-80˚C中儲存。After the treatment, the rats were sacrificed with ether anesthesia, the blood was drawn from the rat tail artery, placed in a test tube containing EDTA, and the plasma was centrifuged at 13,000g at 415C for 15 minutes. The separated plasma was dispensed into traces. Stored in an Eppendorf tube and stored at -80 ° C.
5. 生化分析5. Biochemical analysis
肝細胞損傷以量測血漿中天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性以進行定量，AST與ALT活性是肝臟毒性常用的指標，係以Synchron LXi 725系統來量測(Beckman Instruments, 美國)。Liver cell injury is measured by measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in plasma for quantification. AST and ALT activities are commonly used indicators of liver toxicity. They are based on the Synchron LXi 725 system. To measure (Beckman Instruments, USA).
6. 光學顯微鏡6. Light microscope
大鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以10%磷酸緩衝液配製之福馬林(phosphate-buffered formalin)固定，隨後脫水並包埋於石蠟(paraffin)中，以5 µm厚度切片，切片樣本以蘇木精(hematoxylin)與伊紅(eosin)染色，並進行肝糖染色試驗(Periodic acid Schiff stain, PAS)，染色後以光學顯微鏡進行組織學觀察。Histological analysis of the liver was performed immediately after the sacrifice of the rats; liver samples were fixed with phosphate-buffered formalin prepared in 10% phosphate buffer, then dehydrated and embedded in paraffin, and sliced at a thickness of 5 µm. The samples were stained with hematoxylin and eosin, and a liver acid staining test (Periodic acid Schiff stain, PAS) was performed. After staining, histological observation was performed with an optical microscope.
7. 肝功能之定量測試7. Quantitative test of liver function
試驗結束後，所有大鼠均進行半乳糖單點法(GSP)測試，大鼠接受在30秒內的快速靜脈注射，注射0.4 g/ml BW半乳糖溶液0.5 g/kg；自注射後5、10、15、30、45以及60分鐘各採血一次，血液樣本取自尾部靜脈；以半乳糖脫氫酶比色法(colorimetric galactose dehydrogenase)量測半乳糖含量，測試濃度範圍為50至1,000 µg/ml，每個濃度的日內差異(within-day variation)係由標準偏差(standard deviation)以及變異係數(coefficient of variation, CV)百分比計算，最大容許的變異係數為10% CV；日間差異(day-to-day variation)則由比較標準檢量線(calibration curves)之斜率及截距來檢驗；半乳糖單點法(GSP)為30秒注射停止後60分鐘時血液中半乳糖濃度。After the end of the test, all rats were subjected to the Galactose Single Point Method (GSP) test. The rats received a rapid intravenous injection within 30 seconds and a 0.4 g / ml BW galactose solution 0.5 g / kg was injected. Blood samples were taken at 10, 15, 30, 45, and 60 minutes each. Blood samples were taken from the tail vein. Galactose content was measured by colorimetric galactose dehydrogenase. The test concentration range was 50 to 1,000 µg / ml, within-day variation of each concentration is calculated from the standard deviation and the percentage of coefficient of variation (CV). The maximum allowable coefficient of variation is 10% CV; day-to-day variation (day- To-day variation) is tested by comparing the slope and intercept of calibration curves; the galactose single point method (GSP) is the concentration of galactose in the blood at 60 minutes after the injection is stopped for 30 seconds.
8. 統計分析8. Statistical analysis
所有的數據皆以平均±標準偏差(SD)表示，試驗結果以單因子變異數分析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用Statistical Package of the Social Science program (Version 13, SPSS Inc.)套裝軟體來計算；隨後使用事後比較(post hoc test)最小差異顯著性(least significant difference)方法做多重比較，以確認族群間的顯著差異；族群平均之顯著差異為p ＜ 0.05。All data are expressed as mean ± standard deviation (SD). The test results are calculated using the single factor analysis of variance (ANOVA) test method to determine whether there is a statistically significant difference. The statistical package of the social science program (Version 13, SPSS) is used. Inc.) software package for calculation; then a post hoc test method of least significant difference was used to make multiple comparisons to confirm the significant differences between the ethnic groups; the significant difference between the ethnic groups was p <0.05.
結果result
1. 生化分析結果Biochemical analysis results
試驗結束時，測量試驗動物的體重及相對肝重量，與正常對照組動物相較之下並無顯著差異；血液生化分析結果如表一所示，除APAP肝毒性組血漿中的天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性明顯高於正常對照組(正常對照組血漿中的AST活性為202±34 IU/L；APAP肝毒性組血漿中的AST活性為499±112 IU/L,p ＜ 0.005；正常對照組血漿中的ALT活性為56±14 IU/L；APAP肝毒性組血漿中的ALT活性為368± 71 IU/L,p ＜ 0.005)，顯示APAP肝毒性組產生生化上的肝損傷；除磷酸氫二鈣試驗組未如預期外，此肝損傷現象可被同時併用如甘露醇、薄荷醇、三氯蔗糖等常用安全賦形劑所改善，所測得肝發炎指標AST、ALT、GSP與代表病理組織切片肝病變嚴重程度Total HAI-score評估均有顯著性地下降，實驗數據如表一所示，其中又以甘露醇併用三氯蔗糖組合最佳，與正常對照組無異。At the end of the test, the body weight and relative liver weight of the test animals were measured, and there was no significant difference compared with the animals in the normal control group; the results of blood biochemical analysis are shown in Table 1, except for aspartic acid in the plasma of the APAP liver toxicity group The transaminase (AST) and alanine transaminase (ALT) activities were significantly higher than the normal control group (the AST activity in the plasma of the normal control group was 202 ± 34 IU / L; the AST activity in the plasma of the APAP liver toxicity group was 499 ± 112 IU / L, p <0.005; ALT activity in the plasma of the normal control group was 56 ± 14 IU / L; ALT activity in the plasma of the APAP liver toxicity group was 368 ± 71 IU / L, p <0.005), showing In the APAP liver toxicity group, biochemical liver damage occurred. Except that the dicalcium phosphate test group was not as expected, this liver damage phenomenon can be improved by using common safety excipients such as mannitol, menthol, and sucralose. The measured liver inflammation indexes AST, ALT, GSP and the total HAI-score assessment of the severity of liver lesions in pathological tissue sections have significantly decreased. The experimental data are shown in Table 1. Among them, mannitol was used in combination with sucralose. The best, no different from the normal control group.
表一、正常對照組、APAP肝毒性組及磷酸氫二鈣、甘露醇、薄荷醇、三氯蔗糖等肝保護試驗組小鼠，於管餵單一劑量試驗後，半乳糖單點法(GSP)分析、天門冬氨酸轉胺酶(AST)、丙氨酸轉胺酶(ALT)活性與病理組織切片Total HAI-score，數值之計算為mean ± SD
2. 組織病理學Histopathology
所改善的結果也反映在相對應的肝臟組織，在經過管餵單一口服劑量1000 mg/kg APAP之大鼠，其體內成功的產生肝毒性，APAP肝毒性組大鼠肝臟組織切片，中央靜脈周圍肝細胞呈現碎裂、空泡化現象明顯，細胞核少，甚至部分肝細胞產生壞死(necrosis)徵狀，相較於正常對照組肝細胞損傷嚴重(如圖二B所示)；相對的，在正常對照組大鼠體內的肝結構則較正常，其肝細胞完整，且排列整齊，無空泡現象(如圖二A所示)；而甘露醇、薄荷醇、三氯蔗糖等肝保護試驗組大鼠肝組織切片結果顯示，肝細胞較完整，細胞核明顯且空泡較少(如圖二C、D、F、G、H所示)，顯示甘露醇、薄荷醇、三氯蔗糖等肝保護試驗組大鼠肝組織較接近正常大鼠肝臟組織，其中以甘露醇併用三氯蔗糖組合保護效果最好，且與劑量呈正相關，劑量愈高保護效果愈佳。The improved results are also reflected in the corresponding liver tissues. In rats fed a single oral dose of 1000 mg / kg APAP, tuberculosis successfully produced hepatotoxicity in vivo, liver tissue sections of rats in the APAP liver toxicity group, and central veins around Hepatocytes show obvious phenomena of fragmentation and vacuole, with few nuclei, and even some hepatocytes develop necrosis symptoms. Compared with normal controls, hepatocytes are severely damaged (as shown in Figure 2B). In contrast, in The liver structure of rats in the normal control group was more normal, and the liver cells were intact and arranged neatly without cavitation (as shown in Figure 2A); while the liver protection test groups such as mannitol, menthol, and sucralose The results of rat liver tissue section showed that the hepatocytes were more intact, the nuclei were obvious and the vacuoles were less (as shown in Figures II, C, D, F, G, and H), showing liver protection such as mannitol, menthol and sucralose The liver tissue of the experimental group was closer to that of normal rats. Among them, the combination of mannitol and sucralose had the best protection effect, and was positively correlated with the dose. The higher the dose, the better the protection effect.
3. 剩餘肝功能之量測3. Measurement of residual liver function
如表一所示，正常對照組與APAP肝毒性組大鼠之半乳糖單點法(GSP)值具有高度的顯著差異(正常對照組大鼠之GSP值為289±38 mg/L；APAP肝毒性組大鼠之GSP值848±123 mg/L,p ＜ 0.005)，此外，磷酸氫二鈣、甘露醇、薄荷醇與三氯蔗糖等肝保護試驗組大鼠之GSP值各為444±60 mg/L、253±29 mg/L、289±20 mg/L、218±31 mg/L，與APAP肝毒性組相較，甘露醇、薄荷醇與三氯蔗糖等肝保護試驗組大鼠之GSP值各與APAP肝毒性組大鼠具有高度的顯著差異(p ＜ 0.005)；單獨施用APAP產生肝毒性大鼠之GSP值明顯增加；然而，在APAP合併施用甘露醇、薄荷醇與三氯蔗糖等賦型劑之肝保護試驗組大鼠則可抵抗這種改變。As shown in Table 1, the galactose single-point method (GSP) values in the normal control group and the APAP liver toxicity group were highly significant (GSP value in normal control group rats was 289 ± 38 mg / L; APAP liver The GSP value of rats in the toxicity group was 848 ± 123 mg / L, p <0.005). In addition, the GSP values of the rats in the liver protection test group such as dicalcium phosphate, mannitol, menthol, and sucralose were 444 ± 60 each. mg / L, 253 ± 29 mg / L, 289 ± 20 mg / L, 218 ± 31 mg / L. Compared with the APAP liver toxicity group, mannitol, menthol and sucralose in rats The GSP values were significantly different from those in the APAP hepatotoxicity group ( p <0.005); the GSP value of hepatotoxic rats produced by APAP alone increased significantly; however, the APAP combined with mannitol, menthol, and sucralose Rats in the liver protection test group with other excipients can resist this change.
實施例二Example two
細胞色素P450 2E1 (CYP2E1)抑制劑之篩選-鼠肝微粒體與人肝微粒體。Screening of cytochrome P450 2E1 (CYP2E1) inhibitors-rat liver microsomes and human liver microsomes.
材料與方法Materials and Methods
1. 試驗材料Test material
本實施例係使用大鼠與人體物種肝臟製備微粒體進行CYP2E1抑制劑之體外篩選，針對55種常見且安全可食用之賦型劑進行細胞色素P450 2E1 (CYP2E1)抑制劑篩選，篩選出對大鼠或人類肝臟之CYP2E1有效抑制劑；該CYP2E1抑制劑篩選原理為：係利用物種肝臟所製備微粒體中CYP2E1與其專一性受質Chlorzoxazone (CZX)反應，加入測試樣品作用後，再偵測CYP2E1代謝物標準品6-OH-CZX (6-Hydroxy-Chlorzoxazone)的生成量，並以對照組(control)的6-OH-CZX生成量為基準，計算測試樣品的CYP 2E1抑制率。In this example, cytochrome P450 2E1 (CYP2E1) inhibitors were screened for in vitro screening of CYP2E1 inhibitors using rat and human species liver microsomes, and 55 common and safe edible excipients were screened. An effective inhibitor of CYP2E1 in mouse or human liver. The screening principle of CYP2E1 inhibitor is to use CYP2E1 in microsomes prepared from the liver of the species to react with its specific receptor Chlorzoxazone (CZX). Add the test sample to detect the CYP2E1 metabolism. The production amount of the standard 6-OH-CZX (6-Hydroxy-Chlorzoxazone) was used to calculate the CYP 2E1 inhibition rate of the test sample based on the 6-OH-CZX production amount of the control group.
各測試樣品均溶於10%甲醇(methanol)或是二次水中，測試不同濃度之賦形劑(66uM, 33uM, 16.5uM；0.167%, 0.08%, 0.042%, w/v)對CYP2E1的抑制率，所測試之賦型劑結果如表二所列。Each test sample was dissolved in 10% methanol or secondary water, and different concentrations of excipients (66uM, 33uM, 16.5uM; 0.167%, 0.08%, 0.042%, w / v) were tested for inhibition of CYP2E1. The results of the tested excipients are shown in Table 2.
本實施例利用大鼠肝臟或人類肝臟微粒體篩選細胞色素CYP2E1抑制劑所需的藥劑如下： （1） CYP2E1：100 mM potassium phosphate (pH 7.4)含有10 mg/mL P450 protein concentration。 （2） Control Protein：10 mg/mL P450 Protein溶於100 mM Potassium Phosphate (pH 7.4)中。 （3） Buffer Solution：0.5 M Potassium Phosphate (pH 7.4)。 （4） Stop Solution：ice-acetonitrile。 （5） Cofactors：含有100 mM NADP+以及10 mM Glucose 6-Phosphate。 （6） Glucose 6-Phosphate Dehydrogenase：2000 units/ml溶于無菌水。 （7） Chlorzoxazone：受質(substrate)，16 mM Chlorzoxazone溶於10% 甲醇(methanol)。 （8） DDTC (Diethyldithiocarbamic acid)：CYP2E1選擇性抑制劑 (陽性對照組)，20 mM DDTC溶於10%甲醇 (methanol)。 （9） NADPH-regenerating System：於3.42 mL中加入530 uL Cofactors、40 uL G6PDH (Glucose 6-Phosphate Dehydrogenase Solution)以及100 uL Control Protein。In this embodiment, the reagents required for screening cytochrome CYP2E1 inhibitors using rat liver or human liver microsomes are as follows: (1) CYP2E1: 100 mM potassium phosphate (pH 7.4) contains 10 mg / mL P450 protein concentration. (2) Control Protein: 10 mg / mL P450 Protein is dissolved in 100 mM Potassium Phosphate (pH 7.4). (3) Buffer Solution: 0.5 M Potassium Phosphate (pH 7.4). (4) Stop Solution: ice-acetonitrile. (5) Cofactors: Contains 100 mM NADP + and 10 mM Glucose 6-Phosphate. (6) Glucose 6-Phosphate Dehydrogenase: 2000 units / ml dissolved in sterile water. (7) Chlorzoxazone: Substrate, 16 mM Chlorzoxazone is dissolved in 10% methanol. (8) DDTC (Diethyldithiocarbamic acid): CYP2E1 selective inhibitor (positive control group), 20 mM DDTC is dissolved in 10% methanol. (9) NADPH-regenerating System: Add 530 uL Cofactors, 40 uL G6PDH (Glucose 6-Phosphate Dehydrogenase Solution) and 100 uL Control Protein to 3.42 mL.
細胞色素P450 2E1 (CYP2E1)抑制劑之篩選Screening for cytochrome P450 2E1 (CYP2E1) inhibitors
使用大鼠肝臟或人類肝臟微粒體進行細胞色素CYP2E1抑制劑篩選的實驗步驟如下所述： （1） 在4o C冰浴環境下，0.1M磷酸緩衝液 (pH 7.4) 包含0.5 mg/mL鼠肝或人肝微粒體、5 mM MgCl2 靜置15分鐘 （2） 此時實驗組加入細胞色素P450 2E1反應基質藥物16 mM Chlorzoxazone 以及欲篩選化合物；對照組以甲醇：無菌水=1：1取代；陽性對照組則以DDTC取代； （3） 最後加入輔酶1 mM NADP+、10 mM G6P與2 IU G6PD。將反應液轉移至37oC水浴預溫 (pre-incubation) 1分鐘，活性測試實驗的反應時間為30分鐘； （4） 反應完後以500 μL acetonitrile終止反應，樣品靜置1分鐘後加入內部標準品 (5 ug/mL 4-hydroxy-tobutamide)，離心後取上層液20 uL，以甲醇: 無菌水作稀釋十倍動作，取5 uL之回溶液注入LC/MS/MS系統進行分析。 （5）結果分析：將LC/MS/MS測得的訊號數值換算成為CYP2E1代謝物標準品6-Hydroxy-Chlorzoxazone生成量(pmol)後，以對照組為基準，即對照組的CYP 2E1抑制率為0%，以下列公式計算各陽性對照組及試驗組的CYP 2E1抑制率：The experimental steps for screening cytochrome CYP2E1 inhibitors using rat liver or human liver microsomes are as follows: (1) In a 4 o C ice bath, 0.1M phosphate buffer (pH 7.4) contains 0.5 mg / mL rat Liver or human liver microsomes, 5 mM MgCl 2 and let stand for 15 minutes (2) At this time, the experimental group was added with cytochrome P450 2E1 reaction matrix drug 16 mM Chlorzoxazone and the compound to be screened; the control group was replaced with methanol: sterile water = 1: 1 ; Positive control group was replaced with DDTC; (3) Coenzyme 1 mM NADP +, 10 mM G6P and 2 IU G6PD were added at the end. Transfer the reaction solution to a 37oC water bath for pre-incubation for 1 minute. The reaction time of the activity test experiment is 30 minutes. (4) After the reaction is completed, stop the reaction with 500 μL acetonitrile. After the sample is left for 1 minute, add the internal standard. (5 ug / mL 4-hydroxy-tobutamide). After centrifugation, take 20 uL of the supernatant, dilute ten times with methanol: sterile water, and inject 5 uL of the solution into the LC / MS / MS system for analysis. (5) Result analysis: After the signal value measured by LC / MS / MS was converted into CYP2E1 metabolite standard 6-Hydroxy-Chlorzoxazone production (pmol), the control group was used as the benchmark, that is, the CYP 2E1 inhibition rate of the control group was 0% , Calculate the CYP 2E1 inhibition rate of each positive control group and test group by the following formula:
結果result
1. 陽性對照組Positive control group
陽性對照組(DDTC)所測出之CYP 2E1抑制率如表二所示，由表可知當DDTC的濃度為100 μM時，CYP 2E1抑制率可達89.2%。The CYP 2E1 inhibition rate measured by the positive control group (DDTC) is shown in Table 2. It can be seen from the table that when the concentration of DDTC is 100 μM, the CYP 2E1 inhibition rate can reach 89.2%.
2. 試驗組CYP 2E1抑制率2. Test group CYP 2E1 inhibition rate
賦型劑於鼠肝微粒體所測出之CYP 2E1抑制率如表二所示，由結果可知各賦型劑於不同濃度(66μM, 33μM, 16.5μM；0.167%, 0.08%, 0.042%, w/v)的條件下，對細胞色素P450 2E1具有不同程度的抑制效果，其中以0.167% Brij 58抑制效果最佳(100.0±0.00%)。The inhibitory rates of CYP 2E1 measured by excipients in rat liver microsomes are shown in Table 2. From the results, it can be seen that each excipient is at different concentrations (66 μM, 33 μM, 16.5 μM; 0.167%, 0.08%, 0.042%, w / v), the inhibitory effect on cytochrome P450 2E1 was different, and the inhibitory effect was 0.167% Brij 58 (100.0 ± 0.00%).
表二、賦形劑於鼠肝微粒體體外篩選CYP 2E1抑制劑之抑制率
賦型劑於人肝微粒體所測出之CYP 2E1抑制率如表三所示，由結果可知各賦型劑於不同濃度(66μM, 33μM, 16.5μM；0.167%, 0.08%, 0.042%, w/v)的條件下，對細胞色素P450 2E1具有不同程度的抑制效果，其中以0.167% Brij 58抑制效果最佳(91.2±1.3%)。The inhibitory rates of CYP 2E1 measured by excipients in human liver microsomes are shown in Table 3. From the results, it can be seen that each excipient is at different concentrations (66 μM, 33 μM, 16.5 μM; 0.167%, 0.08%, 0.042%, w / v), the inhibitory effect on cytochrome P450 2E1 was different, and the inhibitory effect was 0.167% Brij 58 (91.2 ± 1.3%).
表三、賦形劑於人肝微粒體體外篩選CYP 2E1抑制劑之抑制率
用以改善對乙醯胺基酚(Acetaminophen, APAP)藥物肝毒性之無肝副作用之新複方組合，各賦型劑於不同濃度(66μM, 33μM, 16.5μM)的條件下之有效使用劑量範圍分別為：聚氧乙烯(20)山梨糖單月桂酸酯(Polyethylene glycol sorbitan monolaurate；Tween 20)其中含量為0.17~5.5克、微晶纖維素(Microcrystalline cellulose)其中含量為100~1000毫克、磷酸氫二鈣(Dicalcium phosphate dihydrate)其中含量為10~250毫克、聚氧乙烯23月桂基醚(Brij 35)其中含量為100~1000毫克、糖精(Saccharin)其中含量為10~40毫克、甘露醇(Mannitol)其中含量為10~250毫克、聚氧乙烯烷基醚(Cremophor RH40)其中含量為0.17~5.5克、三氯蔗糖(Sucralose)其中含量為10~250毫克、吡咯烷酮(Crospovidone)其中含量為0.17~5.5克、羥基乙酸澱粉鈉(Sodium starch glycolate)其中含量為0.17~5.5克、丙烯酸樹脂S100 (Eudragit S100)其中含量為0.17~5.5克、羧甲基纖维素鈉 (Croscarmellose sodium)其中含量為0.17~5.5克、聚氧乙烯聚氧丙烯 (Pluronic F68) 其中含量為1.4~5.5克、薄荷醇(Menthol)其中含量為8~34毫克、低取代烴丙纖維素(Low-substituted hydroxypropyl cellulos)其中含量為0.19~0.82克、預膠化澱粉(Pregelatinized starch)其中含量為1.7~5.5克、Dextrates NF hydrated其中含量為0.17~5.5克、檸檬酸(Citric acid)其中含量為10~42毫克、蓖麻油聚氧乙烯醚(Cremophor EL)其中含量為1.7~5.5克、膠態二氧化矽(Aerosil 200)其中含量為0.17~5.5克、聚乙二醇單硬脂酸酯(Myrj 52)其中含量為1.4~5.5克、山梨酸(Sorbic acid)其中含量為6~24毫克、檸檬油(Lemon oil)其中含量為0.17~5.5克、羥丙基纖維素(Hydroxypropyl cellulose)其中含量為0.17~5.5克、山梨糖醇(Sorbitol)其中含量為0.17~5.5克、乙鮮舒泛鉀(Acesulfame potassium)其中含量為1.4~5.5克、羥丙甲纖維素酞酸酯(Hydroxypropyl methylcellulose)其中含量為0.17~5.5克、單水合乳糖(Lactose monohydrate)其中含量為6~24毫克、麥芽糖糊精(Maltodextrin)其中含量為0.17~5.5克、Brij 58其中含量為0.17~5.5克、Brij 76其中含量為0.17~5.5克、Tween 80其中含量為0.17~5.5克、Tween 40其中含量為1.4~5.5克、PEG 400其中含量為1.4~5.5克、PEG 4000其中含量為1.4~5.5克、PEG 8000其中含量為1.4~5.5克、Span 60其中含量為1.4~5.5克、苯甲酸鈉(Sodium benzoate)其中含量為2.9~11.9毫克、羥乙甲纖維素酞酸酯(Hydroxy ethylmethylcellulose)其中含量為0.17~5.5克、甲纖維素酞酸酯(Methylcellulose)其中含量為0.17~5.5克、Span 80其中含量為1.4~5.5克、甜蜜素(Sodium cyclamate)其中含量為3.3~13.2毫克、甘油山崳酸酯(Glyceryl behenate)其中含量為17.4~69.9毫克、氧化鐵紅(Oxide red)其中含量為11.3~45.2毫克、甘油單硬脂酸酯(Glycerin monostearate)其中含量為1.4~5.5克、Copovidone K28其中含量為0.17~5.5克、澱粉醋酸酯(Starch acetate)其中含量為0.17~5.5克、硬脂酸鎂(Magnesium stearate)其中含量為9.7~39.0毫克、月桂基硫酸鈉(Sodium lauryl sulfate)其中含量為4.7~19.0毫克、Providone K30其中含量為0.18~0.73毫克、及PEG 2000其中含量為1.4~5.5克。A new compound combination to improve the liver toxicity of Acetaminophen (APAP) drugs without liver side effects. The effective dosage range of each excipient at different concentrations (66μM, 33μM, 16.5μM) is different. It is: Polyoxyethylene (20) sorbose monolaurate (Polyethylene glycol sorbitan monolaurate; Tween 20) in which the content is 0.17 to 5.5 grams, microcrystalline cellulose (microcrystalline cellulose) in which the content is 100 to 1000 mg, hydrogen phosphate di The content of calcium (Dicalcium phosphate dihydrate) is 10-250 mg, the content of polyoxyethylene 23 lauryl ether (Brij 35) is 100-1000 mg, the content of saccharin (Saccharin) is 10-40 mg, and mannitol (Mannitol) The content is 10-250 mg, the content of polyoxyethylene alkyl ether (Cremophor RH40) is 0.17-5.5 g, the content of sucralose (Sucralose) is 10-250 mg, and the content of pyrrolidone (Crospovidone) is 0.17-5.5 G, Sodium starch glycolate, which contains 0.17 to 5.5 g, Acrylic resin S100 (Eudragit S100), which contains 0.17 to 5.5 g, and Croscarmellose sodium Medium content is 0.17 to 5.5 grams, polyoxyethylene polyoxypropylene (Pluronic F68) of which is 1.4 to 5.5 grams, menthol (Menthol) of which is 8 to 34 mg, low-substituted hydroxypropyl cellulose (Low-substituted hydroxypropyl) cellulos) with a content of 0.19 to 0.82 g, Pregelatinized starch with a content of 1.7 to 5.5 g, Dexrates NF hydrated with a content of 0.17 to 5.5 g, and Citric acid with a content of 10 to 42 mg 1, Castor oil polyoxyethylene ether (Cremophor EL) in which the content is 1.7 to 5.5 grams, colloidal silica (Aerosil 200) in which the content is 0.17 to 5.5 grams, polyethylene glycol monostearate (Myrj 52) of which The content is 1.4 to 5.5 g, the content of sorbic acid is 6 to 24 mg, the content of lemon oil is 0.17 to 5.5 g, and the content of hydroxypropyl cellulose is 0.17 to 5.5. G, Sorbitol (0.17 to 5.5 g), Acesulfame potassium (1.4 to 5.5 g), Hydroxypropyl methylcellulose (0.17 to 5.5 g) 5.5 g, Lactose monohydra te) 6 to 24 mg, maltodextrin 0.17 to 5.5 g, Brij 58 0.17 to 5.5 g, Brij 76 0.17 to 5.5 g, Tween 80 0.17 ~ 5.5 g, Tween 40 with 1.4-5.5 g, PEG 400 with 1.4-5.5 g, PEG 4000 with 1.4-5.5 g, PEG 8000 with 1.4-5.5 g, and Span 60 with 1.4 ~ 5.5g, Sodium benzoate content is 2.9 ~ 11.9mg, Hydroxy ethylmethylcellulose content is 0.17 ~ 5.5g, Methylcellulose content is 0.17 ~ 5.5 grams, Span 80 contains 1.4 ~ 5.5 grams, Sodium cyclamate contains 3.3 ~ 13.2 mg, Glyceryl behenate contains 17.4 ~ 69.9 mg, iron oxide red ( Oxide red) with 11.3 ~ 45.2 mg, Glycerin monostearate with 1.4-5.5 g, Copovidone K28 with 0.17-5.5 g, Starch acetate with 0.17 ~ 5.5g, magnesium stearate (Ma gnesium stearate) contains 9.7 to 39.0 milligrams, sodium lauryl sulfate contains 4.7 to 19.0 milligrams, Provideone K30 contains 0.18 to 0.73 milligrams, and PEG 2000 contains 1.4 to 5.5 grams.
本發明所提供之無肝副作用之對乙醯胺基酚新複方，與單獨使用對乙醯胺基酚之試驗結果相互比較時，在生化分析(ALT、AST值)、病理學分析及剩餘肝功能之量測(GSP值)等各方面之分析結果，都有明顯減少使用對乙醯胺基酚所造成的肝毒性副作用的功效。When the new compound of paracetamol without liver side effects provided by the present invention is compared with the test results of paracetamol alone, the biochemical analysis (ALT, AST value), pathological analysis and residual liver Functional analysis (GSP value) and other aspects of the analysis results have significantly reduced the efficacy of hepatotoxic side effects caused by the use of acetaminophen.
上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實施或變更，例如：對乙醯胺基酚、細胞色素P450 2E1抑制劑、細胞色素P450 2E1抑制劑選用之種類、施用濃度及比例，以及等變化之等效性實施例，均應包含於本案之專利範圍中。The above detailed description is a specific description of a feasible embodiment of the present invention, but this embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or change without departing from the technical spirit of the present invention, for example: The equivalent types of selected amino acids, cytochrome P450 2E1 inhibitors, cytochrome P450 2E1 inhibitors, cytochrome P450 2E1 inhibitors, and other equivalent variations should all be included in the scope of patents in this case.
綜上所述，本案不但在對乙醯胺基酚的應用上確屬創新，並以併用常見且安全之常用賦型劑能來確實改善因使用對乙醯胺基酚所造成之肝毒性副作用，應已充分符合新穎性及進步性之法定發明專利要件，爰依法提出申請，懇請 貴局核准本件發明專利申請案，以勵發明。In summary, this case is not only innovative in the application of acetaminophen, but also uses common and safe commonly used excipients to truly improve the hepatotoxic side effects caused by the use of acetaminophen , Should have fully met the novelty and progressive statutory invention patent requirements, apply in accordance with the law, and urge your bureau to approve this invention patent application to encourage invention.
無no
圖一為對乙醯胺基酚(APAP)在肝臟中之代謝途徑圖； 圖二為(A)正常對照組、(B)APAP肝毒性組、(C)磷酸氫二鈣、(D)甘露醇、(E)薄荷醇、(F)三氯蔗糖、(G)甘露醇+三氯蔗糖 (1.67+1.67 mg/kg)與(H)甘露醇+三氯蔗糖 (0.83+0.83 mg/kg)肝保護試驗組，於管餵單一劑量後大鼠肝臟組織切片：(A)正常對照組肝組織型態；(B)在周圍中央靜脈(V)的肝細胞呈現碎裂，且有發炎細胞產生，產生壞死現象及空泡化；相較於APAP肝毒性組，肝保護試驗組大鼠，除磷酸氫二鈣試驗組外，其餘試驗組大鼠中央靜脈周圍肝細胞較為完整，細胞核明顯，且空泡較少(D、E、F、G、H) ，又以(F)、(G)最接近正常大鼠肝臟組織 (H&E染色，200X)。Figure 1 is a diagram of the metabolic pathway of acetaminophen (APAP) in the liver; Figure 2 is (A) the normal control group, (B) the APAP liver toxicity group, (C) dicalcium phosphate, (D) manna Alcohol, (E) menthol, (F) sucralose, (G) mannitol + sucralose (1.67 + 1.67 mg / kg) and (H) mannitol + sucralose (0.83 + 0.83 mg / kg) Liver protection test group. Rat liver tissue sections were given a single dose after tube feeding: (A) liver tissue type of normal control group; (B) hepatocytes in the peripheral central vein (V) showed fragmentation and inflammatory cells were produced Necrosis and vacuolation occurred; compared with APAP liver toxicity group, liver protection test group rats, except for the dicalcium phosphate test group, the rest of the test group around the central vein of the liver cells were more complete, the nucleus was obvious, and Fewer vacuoles (D, E, F, G, H), and (F), (G) are closest to normal rat liver tissue (H & E staining, 200X).
無no
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| WO2012142724A1 (en) * | 2011-04-20 | 2012-10-26 | 国防教育研究基金会 | New low side effect pharmaceutical composition containing antituberculosis drugs |
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