Technology development for NAFLD by PMK-S001

• SphereDiabetes/Renal/Hepatic

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Summary

Fatty liver disease has been increasing both in eastern and western countries. Treatment methods currently conducted are as follows: first, a surgical treatment of obesity (Roux-en-Y gastric bypass, bilopancreatic diversion, etc.) has been conducted on morbidly obese patients. However, this method has disadvantages of having a high risk and of the decreased improvement of inflammation and fibrosis. Second, thiazolidinedione (thiazolidinedione, [rosiglitazone, pioglitazone], etc.), which has been recently developed, has been known to effectively inhibit glucose synthesis in the liver and to improve insulin sensitivity. However, it has a disadvantage of high drug cost as it is not covered by medical insurance for fatty liver disease patients without diabetes. Third, antioxidants (Tocopherol) have been used as a supportive treatment to reduce oxidative stress, a pathogenesis of fatty liver disease, but it is not a direct treatment. As most patients with fatty liver disease are accompanied with overweight or obesity, proactive weight reduction can be currently considered the most effective in the treatment of fatty liver disease. However, it is hard to be considered as a treatment method. Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] is a synthetic dithiolthione structurally relevant to a compound present in cruciferal vegetables. It has been initially investigated for the purpose of the treatment of schistosomiasis. Oltipraz decreases the glutathione level of schistosomiasis to the critical value whereas it increases mammalian phase II metabolic enzymes, particularly glutathione-S-transferase (GST). Thus, it has been investigated as a drug for chemotherapy. As oltipraz showed its efficacy in various animal carcinogenesis models, a clinical study has been conducted on subjects with a high risk of hepatocellular carcinoma. Furthermore, a clinical study was conducted by CHEILJEDANG (CJ) on patients with liver cirrhosis in early 2000. PMK-N01GI1 is a compound that is structurally identical to oltipraz. It has been shown that PMK-N01GI1 improved liver functions (AST, ALT) and reduced blood lipid level (Total Cholesterol, Triglyceride). In addition, it has been histopathologically shown that oltipraz administration reduced triglyceride level in the liver tissues and returned the liver weight of an animal model with fatty liver disease to almost that of a healthy animal. Furthermore, it has been shown that oltipraz administration inhibited the expressions of LXRa, SPERP-1c, FAS, and ACC that are genes of enzymes related to lipid accumulation. Therefore, oltipraz can be considered as a potential therapeutic agent that reduces triacylglycerol accumulated in the liver. In addition, it has been shown that oltipraz enhanced survival rate via the improvement of liver functions and tissue regeneration effect in rats with liver cirrhosis. Furthermore, it has been shown that oltipraz inactivated liver astrocytes that secret collagen into the sclerosing liver after activation. In the histopathological perspective, oltipraz reduces the intensity of fibrotic nodule and sclerosing nodule in the liver and removes accumulated extracellular substances. Furthermore, oltipraz recovers the liver weight of rats with liver cirrhosis to that of healthy rats. Increased PCNA by oltipraz further supports that oltipraz regenerates the sclerosing livervia hepatic regeneration. This is consistent with the fact that oltipraz is involved in the maturation of undifferentiated cell of the sclerosing liver. The mechanism of the aforementioned phenomenon is likely to be attributable to the inhibition of the expression of transforming growth factor beta 1 (TGF-1) via the activation of CCAAT/enhancer binding (C/EBP) protein of liver astrocytes by oltipraz. Based on the previous clinical data published, it has been speculated that the maixmum tolerated dose of long-term administration of oltipraz is 125mg/day. The estimated pharmacokinetic values at this dose include Tmax, 2-3 hours and Cmax, approximately 200-300 ng/mL. These values are similar to those at the NOAEL in rats and dogs. However, the result of an international phase I clinical study and the results of phase II clinical studies conducted by CJ and in china reported finger tip pain. According to a study conducted by Hwahng et al in 2009, TG accumulation in the tissue decreased more at the low-dose than at the high-dose, and the mRNAs of fat metabolism-related genes such as LXR, SREBP, FAS, and ACC mRNA decreased more. This result indicates that the dose used in the aforementioned study was a threshold. Thus, to minimize the tolerability and maximize the efficacy of long-term administration of oltipraz, a tolerated dose of 30 mg bid or 60 mg bid was selected after converting into HED (Human Equivalent Doses) in this study. Metabolic process proceeds by the rearrangement and sulfoxidation of dithiolthione ring. A total of 13 urinary and blood metabolites are identified. The metabolic process of monkeys is the most similar to that of human being. As oltipraz undergoes many metabolic processes, saturable first-pass elimination is believed to be one of the reasons of nonlinearity shown in its pharmacokinetics. Oltipraz is mainly removed as a polar metabolite via urine, and as an unchanged compound via stool in human and other animals. Its removal degree is similar between the two pathways. When radiolabelled otipraz was administered, a high radioactivity was shown in the gastrointestinal tract, urine, bile juice, liver, and kidney 24 hours after administration. Oltipraz has been particularly known as an inducer of hepatic phase II metabolic enzymes, and reported to slightly induce cytochrome P450 in some systems. Meanwhile, it has been reported that oltipraz had an inhibitory effect on the aforementioned enzymes. In addition, it was reported that oltipraz could act as an effective blocking agent if administered prior to aflatoxin B1, a carcinogen in rats. Furthermore, it was reported that oltipraz inhibited ethoxyresorufin-O-deethylase (EROD, CYP1A) and pentoxyresorufin-O-depentylase (PROD, CYP2B) and that its chemoprotective mechanism against toxic chemicals was attributable to competitive inhibition caused by enzyme induction for activation and detoxification. Safety pharmacology study of oltipraz included general behavioral activity test and the effect of oltipraz on body temperature in rats, the effect of oltipraz on hexobarbital sleep duration, acetic acid-induced writhing, pentylenetetrazol and electro-shock seizure, motility coordination, and transport of intestinal contents in mice, the effect of oltipraz on smooth muscle motility in ginea pigs, the effect of oltipraz on cardiovascular, respiratory system in anesthetized dogs, and in vitro electrophysiological study using hERG channel. In behavioral pharmacology and general symptoms, neither behavioral nor physiological changes were observed until its concentration reached 300 mg/kg. At a concentration of 1,000 mg/kg, with main symptoms such as epiphora, ptosis, piloerection, hypotonia, distraction, diarrhea, and loose stool, hypothermia and decreased grooming were shown in rare cases, but no change in body temperature was observed. In acetic acid-induced writhing test, pentylenetetrazol and electro-shock seizure and motility coordination test in mice, no particular change was shown until the concentration reached 1,000mg/kg. However, in hexobarbital sleep duration test and transport of intestinal contents study, sleep duration extension and decreased transport of intestinal contents were observed at a concentration of 1,000mg/kg, which showed that no particular change was observed until the concentration reached 300mg/kg. In smooth muscle motility test in ginea pigs, no change in smooth muscle motility was observed until the concentration reached 3μM, but motility was suppressed at a concentration of 30μM. In vitro study showed that weak interference of hERG current was observed at IC50 where a concentration of 30μM or higher was estimated. When 3 mg/kg dose of oltipraz was intravenously administered to anesthetized dogs, no change in cardiovascular parameters was observed compared to vehicle administration group (PEG 400). At a concentration of 10 and 30 mg/kg which were higher than the aforementioned dose, tachycardia (shown as a decreased RR interval) and decreased femoral flow were observed. However, the same changes were also observed in the animal group that received a same amount of vehicle. No particular change in ECG level was found. Meanwhile, a change in respiration was found. No change in respiratory system-related parameters was found at the low-dose oltipraz (3mg/kg), but the decreased respiratory rate and minute volume were observed at the high-dose oltipraz. Ventilation was required in a male that received the intermediate dose (10mg/kg) and a male that received the high dose. They are likely to be more sensitive to the test drug than other animals. On the other hand, a previous study reported that when 150 mg/kg dose of oltipraz was duodenally administered to anesthetized dogs, no change in cardiovascular, respiratory, and autonomic nervous systems was observed. LD50 of oltipraz was reported to be 2000 mg/kg or higher in rats (one of 10 rats died at this dose) and 1500mg/kg or higher in dogs. Salivation, partial eye closing, hypoactivity, rough fur, piloerection, stool reduction, bent posture, and irregular breathing were found in rats, whereas vomiting, mucinous or non-foaming red stool , pupil dilatation, and red conjunctiva were found in dogs. In early 2000, CJ conducted a toxicity test in which a 4-week repeated oral administration of oltipraz was conducted on rats and dog, respectively. The results of the previously published studies of 52-week long-term oral administration of oltipraz were summarized. In the 4-week repeated administration of oltipraz in rats, oltipraz at doses of 10, 50, and 200 mg/kg was administered in a recovery interval of 2 weeks. Some hematological changes were observed at a dose of 200 mg/kg, and serum biochemical changes were observed at a dose of 50 and 200 mg/kg/day. Hematological findings such as slight decreases in red blood cell count, hemoglobin count, and hematocrit level, decreased MCHC, increased reticulocyte ratio, increased absolute count, and increased platelet and MCV level were reported. The result of serum chemistry showed that a slightly higher cholesterol level was observed at Day 30 in most females that received oltipraz at a dose of 50 mg/kg/day and in rats that received oltipraz at a dose of 200 mg/kg/day rat, and at Day 45 in females that received oltipraz at a dose of 200 mg/kg/day. In addition, a slightly lower aspartate aminotransferase (AST) activity was observed at Day 30 in most females that received 50 mg/kg/day dose of oltipraz and in rats that received oltipraz at a dose of 200 mg/kg/day rat, and at Day 45 in some females that received oltipraz at a dose of 200 mg/kg/day. Higher levels of total protein, albumin, globin, and calcium were observed at Day 30 in males that received oltipraz at a dose of 200 mg/kg/day. A slightly increased gamma-glutamyltransferase (γ-GT) was observed in rats that received oltipraz at a dose of 200 mg/kg/day, and an increased alanine aminotransferase was observed in most males that received oltipraz at a dose of 200 mg/kg/day male. However, they were statistically insignificant and showed no bilogival meaning. Microscopic examination showed increased congestion and pigmentation in the spleen, enlarged hepatic cells of the lobulus centralis in the liver, and increased hyaline droplet of the proximal convoluted tubule in the kidney. The NOAEL (no observable adverse effect level) of the study was 10 mg/kg/day. When oltipraz either at a dose of 5 and 50 mg/kg/day for 13 weeks or at a dose of 10, 30, and 60 mg/kg/day for 52 weeks was orally administered to rats, changes in the levels of the liver, blood and serum chemistry were observed. An increased relative and absolute liver weight in relation to hepatomegaly were shown in the males that received the intermediate and high doses of oltipraz and in the females that received a high dose of oltipraz. Granularity was also observed in the cytoplasm of the hepatic cells. These anatomical findings were associated with the slightly increased albumin, total protein, and cholesterol levels in the males, and with a slightly increased cholesterol level in the females. In addition, a slightly decreased RBC, hemoglobin count, and hematocrit level and an increased reticulocyte count were also observed. The NOEL (no observable effect level) of the study was 5-10 mg/kg/day. In a toxicity study of a 4-week repeated administration of oltipraz in dogs, oltipraz at a dose of 10, 30, and 100 mg/kg/day was administered to the dogs. Some clinical symptoms such as vomiting, loose stool, and excessive saliva secretion were observed at the highest dose. Body weight reduction, which was associated with decreased food intake, was shown to recover after discontinuing the administration. A decreased heart rate was shown in three males and three females that received a high dose of oltipraz, and in a female that received oltipraz at a dose of 30 mg/kg/day at the late phase of oltipraz administration, but this sign disappeared at the end of recovery period. No change in the ECG level was found. No clinical and pathological change or autopsy change relevant to the administration was observed. The NOAEL of the study was 30 mg/kg/day. The results of a 13-week oral administration of oltipraz at a dose of 10 and 100 mg/kg/day, and 52-week oral administration of oltipraz at a dose of 5, 15, and 60 mg/kg/day in dogs were consistent with that obtained from a study using rodent organs. That is to say, the decreased body weight, decreased hemoglobin count, RBC count, hematocrit levels, increased cholesterol, and increased liver weight were observed. In addition, precipitation of bile canaliculus, male gonadal gland atrophy and increased pituitary gland weight were also observed in the 13-week oral administration. In the both studies, alkaline phosphatase (ALP) activity was shown to slightly increase. No change in ECG was found. The NOEL of the study was 5mg/kg/day. According to previous references, no fetal toxicity and anomaly was reported to be observed in relation to reproductive toxicity. Safety of oltipraz has been proven from previous studies including chemopreventive clinical study. In a study22) where oltipraz at a dose of 20mg/ 50mg/100mg/day and at a dose of 125mg/twice/week were administered to patients who had a previous history of liver polypectomy and to the first-degree relatives of breast cancer patients, respectively, for 6 months, 20mg/day dose of oltipraz was shown to be the minimum dose at which the plasma concentration of oltipraz can be measured, and 125mg/day was shown to be the daily maximum tolerated dose. In a study conducted by Dimitrov et al., where oltipraz at a dose of 24mg/kg/day, 60% of the maximum tolerated dose, was administered to mice for 4 weeks, a dose of 1/10 (2.4mg/kg/day) ~ 1/20 (1.2mg/kg/day) was set as a human dose. In addition, in 4-week toxicokinetics studies of oltipraz using rats and dogs, no significant difference (>2-folds) in the Cmax and AUC0-24 values between at Day 1 and at Day 28 was found. Therefore, the result of the study indicates that oltipraz shows the minimum dose accumulation in the repeated administration. In particular, in a 4-week repeated toxicity study, although it was difficult to accurately compare toxicity parameters between the NOEL (No observable adverse effect levels) 10mg/kg/day in rats and the the NOEL 30mg/kg/day in dogs and the that in human due to the restricted human data, the MTD (showing the minimum adverse events) of oltipraz was likely to be 125mg/day based on clinical data. The Cmax value corresponding to the aforementioned dose was estimated to be approximately 200-300ng/ml when the TMAX was 2-3 hours. In addition, in the clinical study conducted by CJ, based on the aforementioned results, pharmacokinetics and tolerability were assessed by administering oltipraz at a dose of 30mg/day, 60mg/day, and 90mg/day to patients with liver fibrosis and liver cirrhosis. As a result, no serious adverse event was found. In this clinical study conducted on patients with non-alcoholic fatty liver disease, pharmacokinetics, tolerability, and efficacy will be assessed by administering oltipraz at a dose of 30mg bid and 60mg bid that were selected due to their high Css in a mathematical modeling.

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