Metabolism of simvastatin by Streptomyces griseolus CYP105A1 and its variants for the production of human simvastatin metabolites.

Potential Role of the Vitamin D Receptor in Control of Cholesterol Levels

In vitro inhibition of simvastatin metabolism in rat and human liver by naringenin

Statins, hydroxyl-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are cholesterol-lowering drugs that are majorly used to treat hypercholesterolaemia and dyslipidaemia implicated in the pathogenesis of coronary heart disease and atherosclerosis [1]. They are generally considered safe drugs, but there are a number of reports of skeletal muscle damage associated with their use [2]. The myotoxicity ranges from a mild clinical syndrome consisting of benign myalgia to rare but life-threating rhabdomyolysis [3]. These side-effects can impact on quality of life and compliance, and in extreme cases lead to death [4]. Because millions of people in the world are currently taking statins every day, it is an urgent task to uncover the mechanism by which statins lead to side effects [5]. This thesis includes two published papers and one still in preparation. Our first paper presents a comparison between three different statins on the market: simvastatin, atorvastatin and rosuvastatin. Since there are differences among statins in terms of their efficacy and toxicity, we aimed to analyze the different molecular mechanisms that may contribute to the diverse grade of toxicity between simvastatin, atorvastatin and rosuvastatin. Simvastatin and atorvastatin appear to have a higher than average risk of myotoxicity contributing to the highest number of cases of rhabdomyolysis among statins [6] [7]. On the contrary rosuvastatin, the most hydrophilic statin, appears to have a reduced myotoxicity [8] [7]. C2C12 myotubes were exposed to 10 µM or 50 µM simvastatin, rosuvastatin or atorvastatin for 24 hours. We demonstrated that myotubes were more susceptible to simvastatin and atorvastatin than to rosuvastatin treatment. Therefore, difference between rosuvastatin and atorvastatin or simvastatin could point to possible mechanisms of toxicity. The cytotoxicity of simvastatin and atorvastatin was associated with a drastic and dose-dependent impairment of AKT signaling cascade that led to inhibition of the protein synthesis, increase of the protein degradation and promotion of apoptosis. Conversely, rosuvastatin blocked AKT signaling only at high concentration and to a lesser extent compared with the other two statins. The reduced effect on cytotoxicity and AKT signaling inhibition in C2C12 myotubes treated with rosuvastatin was accompanied with normal protein synthesis and absence of protein degradation and apoptosis. These results provide evidence that an impairment of AKT signaling pathways might be a causative factor in statin-induced myotoxicity. Our second paper expands on these previous results by showing that the myotoxicity, and with it, the impairment of AKT signaling, can be prevented by the addition of IGF-1. IGF-1 is well known for exerting an anabolic effect on skeletal muscle [9] by activating IGF-1/AKT pathway [10]. Therefore we investigated whether IGF-1 could antagonize the myotoxicity induced by statins. Myotubes were exposed to 10 µM simvastatin and/or 20 ng/ml IGF-1 for 18 hours. Simvastatin-induced myotoxicity was completely antagonized by IGF-1. Moreover, the protective effect of IGF-1 was mediated by the activation of IGF-1/AKT pathway that led to a suppression of atrophic markers and apoptosis, and simultaneously triggered pro-synthetic pathways. These studies provide new insight into the prevention of statin toxicity and may herald new discoveries for the treatment of statin-induced myalgia. The final paper takes the work of the previous two papers and places it into a novel system: the cardiac muscle. Statins are primarily prescribed to cure and prevent cardiovascular disease. Thus, cardiac side-effects may be masked by falsely attributing them to the underlying disease. In this paper, we investigated on the effect of simvastatin in cardiomyocyte in vitro and in vivo. We treated H9c2 rat cardiomyocytes with 10 µM and 100 µM simvastatin for 24 hours. H9c2 cells showed a reduction in the mitochondrial membrane potential and energetic impairment linked to mitochondrial dysfunction. Consequently, the cellular ATP level was decreased. This decrease led to the activation of AMPK, nuclear translocation of FoxO3, upregulation of atrogin-1 and initiation of apoptosis. We confirmed these results in vivo. We demonstrated that the treatment of mice with simvastatin 5 mg/kg/day for 21 days impaired the activity of several enzyme complexes of the electron transport chain in cardiomyocytes and increased mRNA expression of atrogin-1 and markers of apoptosis. This is the first study that shows energetic impairment linked to atrophy and apoptosis induced by statins in the heart, and warrants further investigation to assess statin safety in susceptible patients.

Simvastatin is a lactone prodrug whose hydroxy acid (SVA) is a potent competitive inhibitor of HMG-CoA reductase. Disposition and metabolism of the drug were investigated in male rats after administration of [14C] simvastatin.1. After oral dosing of [14C] simvastatin at doses of 3, 10 and 30mg/kg, plasma radioactivity increased proportionally to administered dose with peak plasma concentration at 2hr and declined with half-lives of 3.8 ?? 5.2hr.2. Radioactivity was rapidly and widely distributed to tissues, reaching maximum levels at 2hr in many of the tissues. The radioactivity was high in the gastrointestinal tract and liver, secondary in the kidney. At 96hr, the level in each tissue was very low when compared with the maximum value. Similar distribution characteristics were observed by whole body autoradiography. 3. Excretion of radioactivity was 8.1% and 83.3% of the dose in urine and feces, respectively, within 96hr after oral administration. In bile-duct cannulated rats, radioactivity excreted into bile and urine represented 45.3% and 3.9% of the dose, respectively. Gastrointestinal absorption of radioactivity was estimated to be about 50% During the enterohepatic circulation study, 31% of the radioactivity excreted in bile was reabsorbed.4. Hydroxy acid of simvastatin(SVA) was highly bound to serum protein(>90%). After oral administration, the extent of binding of radioactivity to rat plasma protein was 33 ?? 37 %, and red-blood cells to plasma concentration ratio was 0.34 ?? 0.53.5. Simvastatin was extensively metabolized in rats after oral dosing, and systemic bioavailability estimated on the basis of HMG-CoA reductase inhibitor equivalents was 4%.6. Plasma radioactivity was comprised mainly of 2, 2-dimethylbutyric acid (DMB), 2, 2-dimethyl-3-hydroxybutyric acid (DMHB) and SVA. In the liver, SVA was the main metabolite. In bile, 3'-hydroxy metabolite of SVA, 6'-caboxylic acid of SVA and a number of unknown metabolites were present. DMHB, DMB glucuronide and the glycine conjugate of DMB were the three major species found in the urine. Simvastatin was difficult to detect in the tissues and excreta examined.

Introduction: Simvastatin is a semisynthesis statin. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme of cholesterol synthesis, in AMPK (AMP-activated protein kinase) signaling pathway. Simvastatin is able to cross blood brain barrier more than the other statins, due to its lipophilic nature. There is controversy about the effect of simvastatin on Alzheimer’s disease (AD). For example, simvastatin can induce AD through insulin signaling pathway but can ameliorate AD via MAPK (Mitogen-Activated Protein Kinase) signaling pathway. In this study, we report the synthesis of a conjugated form of simvastatin with citicoline, to block negative effect of simvastatin on insulin signaling pathway and increase positive effect of simvastatin on MAPK signaling pathway and chitosan as a linker between these two drugs. Methods and Results: for simvastatin-n-succinyl chitosan-citicoline synthesis, chitosan reacted with succinic acid to form n-succinyl chitosan. Then simvastatin connected to n-succinyl chitosan via acetylation reaction. After 24 hours citicoline was added to reaction media. H-NMR and FT-IR were done to examine whether the conjugation reaction has been done or not. Characterization and morphology tests have been done on reaction result. H-NMRresults approved the synthesis of drug-polymer. FT-IR results showed both amide and ester peaks. Maximum absorptions (λ max ) of all primary chemicals were seen in UV visible spectroscopy results of conjugated form.SEM result showed that the conjugated form has nanoparticulate structure in size range of 100-300 nanometers. X-RD result showed a peak under 25 theta. Another characterization test wasRBC hemolysis with six different concentrations, in which normal saline was negative control and Triton was positive control. Conclusions: Conjugation of lipophilic simvastatin with hydrophilic citicoline to improve AD can be done with helping of a polymer which is rich in carboxylic acid. Key words: Alzheimer’s disease, simvastatin, chitosan, insulin signaling pathway, MAPK signaling pathway. Grants: This study was part of a Pharm.D thesis supported by Shiraz University of Medical Sciences (SUMS; Grant no 11710)

Simvastatin is frequently administered to diabetic patients with hypercholesterolemia. The aim of the study was to investigate the pharmacokinetics of simvastatin and its hydrolysate simvastatin acid in a rat model of type 2 diabetes. Diabetes was induced in 4-week-old rats by a treatment of high-fat diet combined with streptozotocin. After the rats received a single dose of simvastatin (20 mg/kg, po, or 2 mg/kg, iv), the plasma concentrations of simvastatin and simvastatin acid were determined. Simvastatin metabolism and cytochrome P4503A (Cyp3a) activity were assessed in hepatic microsomes, and its uptake was studied in freshly isolated hepatocytes. The expression of Cyp3a1, organic anion transporting polypeptide 2 (Oatp2), multidrug resistance-associated protein 2 (Mrp2) and breast cancer resistance protein (Bcrp) in livers was measured using qRT-PCR. After oral or intravenous administration, the plasma concentrations and areas under concentrations of simvastatin and simvastatin acid were markedly decreased in diabetic rats. Both simvastatin metabolism and Cyp3a activity were markedly increased in hepatocytes of diabetic rats, accompanied by increased expression of hepatic Cyp3a1 mRNA. Furthermore, the uptake of simvastatin by hepatocytes of diabetic rats was markedly increased, which was associated with increased expression of the influx transporter Oatp2, and decreased expression of the efflux transporters Mrp2 and Bcrp. Diabetes enhances the metabolism of simvastatin and simvastatin acid in rats via up-regulating hepatic Cyp3a activity and expression and increasing hepatic uptake.

Conversion processes of organic compounds using biocatalyst generally have high regio- and stereo-selectivity, and are becoming increasingly important for efficient production of chemicals. In addition, biocatalysis is less hazardous, less polluting and less energy-consuming than the conventional chemical method. We report the highly efficient bioconversion system using actinomycete Rhodococcus erythropolis to produce active form of vitamin D3 currently used as a pharmaceutical. The improvement of performance of the enzyme used for the bioconversion has been achieved by the combination of evolutionary engineering and structure-based methods. Accordingly, the practical production efficiency of active form of vitamin D3 has been substantially increased. In addition, we have succeeded in significant improvement of cellular permeability of vitamin D3 by using nisin-treated cells, and have developed a new platform for vitamin D3 hydroxylation process.

Neuroprotective role of vitamin D3 in colchicine-induced Alzheimer’s disease in rats

In vitro inhibition of simvastatin metabolism, a HMG-CoA reductase inhibitor in human and rat liver by bergamottin, a component of grapefruit juice

MDZ is a validated CYP3A biomarker but disadvantages make it a suboptimal probe. The FDA has suggested the use of SV as a CYP3A biomarker to assess drug interactions (DIs). SV is a P-glycoprotein (PGP) substrate and its metabolism is mainly via CYP3A with CYP2C8 having a minor role. The aim of this study was to compare SV with MDZ before and after INHIB and INDUC of CYP3A enzyme activity in a fixed sequential, open label, 3-way crossover study. 19 adults (9M/10F, mean age 38.2±8.7 yr) randomly received single doses of oral MDZ 0.075mg/kg and SV 40mg, 1 to 3d apart, during 3 phases [BAS, INHIB with ketoconazole 400mg x 10d, & INDUC with rifampin 600mg x 9d]. The washout period between phases was 1 & 2 wk after BAS & INHIB, respectively. Area under the plasma concentration time curve (AUC) for MDZ & SV was calculated by a noncompartmental model using WinNonlin®. Log transformed AUCs were evaluated by Pearson correlation (r). Mean increases in AUC from BAS to INHIB were 883%±304% (MDZ) & 1335%±747% (SV). Mean decreases from BAS to INDUC were 87%±4% (MDZ) & 89%±11% (SV). Correlation of AUCs for MDZ vs SV at BAS was r=0.56 (p=0.01); during INHIB, r=0.43 (p=0.07); during INDUC, r=0.36 (p=0.15). SV metabolism may not be influenced only by altered CYP3A activity but by altered CYP2C8 and/or PGP activity during INHIB and INDUC. SV is not a useful biomarker compared to the validated probe MDZ for studying CYP3A DIs due to its lack of CYP3A specificity. Clinical Pharmacology & Therapeutics (2004) 75, P96–P96; doi: 10.1016/j.clpt.2003.11.365

Statins are widely used in primary and secondary prevention of atherosclerotic cardiovascular disease. Despite being generally considered as safe for use, current clinical evidence regarding the neurological effects of statins is conflicting. Although a number of large-scale studies suggest that statins are largely neuroprotective, case reports suggest a possible association between statins and adverse cognitive effects in the form of cognitive decline or memory impairment. There are numerous limitations surrounding existing in vitro and in vivo studies investigating statins' central nervous system (CNS) effects, including a lack of comparison between the different statin compounds. Consequently, a thorough understanding of statins' mechanistic effects within the CNS has not been possible to date. The overall aim of this project was to determine the mechanisms underlying the effects of multiple structurally and pharmacologically diverse statins in neuroinflammation and subsequent neurodegeneration. This was achieved through a combination of in vitro and in vivo studies. [...]

Pancreatic ductal adenocarcinoma (PDA) is a disease with an exceptionally poor prognosis, high therapy resistance and poor effective therapeutic options. Advances in therapeutic treatments are urgently required. Cancer stem-like cells (CSCs), capable of unlimited self-renewal, have been proposed as a mechanism for cancer growth, therapy resistance and metastasis, involving PDA. Besides a function in normal tissue development, Sonic hedgehog (Shh) is highly expressed at all stages of human PDA. Recent data demonstrate that the expression of Shh is highly upregulated in CSCs and regulates them. Simvastatin, which is widely prescribed as cholesterol-lowering drug, was shown to inhibit tumor growth, metastasis and cancer-specific mortality in some studies, but the available data are not consistent. Most importantly, the hypothesis of my thesis, namely that simvastatin attacks pancreatic CSCs by inhibition of Sonic hedgehog signaling was never examined before. In my thesis, I evaluated the effect of simvastatin on 3 established and 1 primary PDA cell lines, as wells as non-malignant pancreas cells and mesenchymal stromal cells from human bone marrow. Results from cell viability assays show that simvastatin significantly reduces viability even at low concentration in CSC-enriched cell lines. I observed synergetic effects on pancreatic cancer cells upon combination of simvastatin with gemcitabine in vitro. Colony and spheroid assays indicated that simvastatin inhibits self-renewal, with an even stronger effect upon combination with gemcitabine. In addition, simvastatin significantly induced the differentiation potential. My results obtained with pancreatic cancer xenografts transplanted on the CAM of fertilized chicken eggs show that simvastatin inhibits tumor growth and metastasis in vivo. Importantly，no pronounced toxic side effects of simvastatin to non-malignant cells or chick embryos occurred. My further experiments suggest that the underlying mechanism of simvastatin is associated with inhibition of cholesterol biosynthesis, which is essential for post-translational modification and thereby Sonic hedgehog activation. Simvastatin alone or combined with gemcitabine diminished the expression of Shh and related proteins Smo, Gli1 and activated the expression of the Gli-inhibitor Sufu. Likewise, the siRNA-mediated inhibition of Shh expression mimicked the simvastatin effect, whereas it´s overexpression prevented it. I confirmed these in vitro and in vivo findings in tissue of patients who did (n=34) or did not (n=34) receive simvastatin prior to surgery. Thus, the expression of Shh, its downstream signaling protein Gli1, along with the levels of progression markers Vimentin, CXCR4 and c-Met were lower in PDA tissues from patients with statin medication. Therefore, I conclude that simvastatin, as a robust, cost-effective and well-tolerated drug for prevention of hypercholesterolemia, may also be suited to prevent PDA and to improve the efficacy of standard therapy in patients suffering from PDA.

17528 Background: Primarily monocyte-derived cytokines and soluble interleukin-2 receptor (sIL-2R) form a group of proinflammatory cytokines and mononuclear cell activaton respectively which are significantly elevated in patients with CML-chronic phase (CML-CP) or CML-blastic crisis (CML-BC).C-reactive protein (CRP) correlates with IL-6. IM is a tyrosine kinase inhibitor that specifically acts intracellularly by blocking the ATP-binding site of the kinase domain of Bcr/abl in CML. Acquired drug resistance in CML-CP patients treated with IM may occur by upregulation of P-glycoprotein (P-gp) drug efflux with P-gp+ leukemic cells and P-gp dependent decline of intracellular IM levels with retained phosphorylation Bcr/abl pattern and loss of IM effect on apoptosis and cellular proliferation. Modulation of P-gp with HMG-CoA reductase inhibitor (SV), a potent inhibitor of P-gp transport, readily overcame IM resistance. Methods: We present two cases of CML: CML-CP and CML-BC with evidence of IM resistance. After one year of IM therapy an 86 yo female with CML-CP stage II, developed reactivation of Bcr/abl signaling due to excretion of IM from the cell transmembrane transporters consistent with increased proinflammatory cytokines, sIL-2R, CRP, acute coronary syndrome but normal neopterin.Results: Modulation of proinflammatory cytokines with SV, IM dose adjustments, serial measurements of Bcr-abl/abl ratio didnot exceed 0.02% on three occasions or 0.05% on two occasions after changing from 0.001% to 0.003% and now after two years of IM plus SV, real-time Bcr-abl/abl ratio is 0.000 (a 5 log reduction).sIL-2R levels normalized from 31,709.55 pg/ml to 5,345.4 pg/ml (Normal 1770–9753 pg/ml). CRP decreased to 6.23 mg/dl from 16 mg/dl. Conclusions: IM an intracellular drug demonstrating high activity against Bcr/abl gene positive CML-CP or CML-BC patients may develop resistance and loss of cellular proliferation via upregulated P-gp-mediated drug efflux mechanism. Modulation of P-gp by HMG-CoA reductase (statins) inhibitor simvastatin, a potent inhibitor of P-gp, may allow intracellular levels of IM to restore its cytotoxicity and overcome resistance mechanism. No significant financial relationships to disclose.

Background Statins has prominent roles in regulating lipids, anti-inflammation, autoxidation and protecting vascular endothelial cells.Sartans can promote cell growth and the expression of cytokines.Since the pleiotropic effects of statins and sartans on a variety of cell types, it is inferred that the two medicines can delay retinal aging. Objective This study was to explore the anti-aging effect of simvastatin and telmisartan on the physiological aging of retina. Methods Sixty-six three-month-old healthy SD rats were selected in this study, and 6 of them served as the youth group and the right eyeballs were immediately enucleated.The other rats were raised until 9-month-old in the same conditions and then randomly divided into the simvastatin group, telmisartan group and the control group with 20 rats for each group.The simvastatin of 5 mg/kg and telmisartan of 8 mg/kg were given by intragastric administration once a day in the simvastatin group and the telmisartan group until 17-month-old, and the equal amount of normal saline was used in the control group in the same way.The number of survival rats was 12 in the simvastatin group, 10 in the telmisartan group and 8 in the control group.The right eyes were enucleated after heart perfusion of 4% paraformaldehyde solution for the preparation of retinal paraffin sections.Retinal thickness was measured by pathological examination, and the expressions of the retinal neuron markers, including Thy-1, protein kinase C-α (PKC-α), opsin and rhodopsin, were detected by immunofluorescence technique to evaluate the morphology of retinal ganglion cells (RGCs), bipolar cells as well as the thickness of the outer segment of photoreceptors. Results The retinal structure was clear in the rats of the youth group.However, the RGCs arrangement and inner segment (IS) and outer segment (OS) structure were abnormal in the simvastatin group, the telmisartan group and the control group.Compared with the rats of the youth group, the thickness of outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and the total thickness of the aging rats were decreased, and the IS/OS thickness was increased in the simvastatin group and the telmisartan group (all at P<0.01). Thy-1 stain showed that the number of RGCs was reduced in the simvastatin group, telmisartan group and the control group compared with the youth group, and that in the simvastatin group was increased in comparison with the control group (all at P<0.01). PKC-α stain exhibited that the density of bipolar cells was increased but the axon terminal bouton was declined in the simvastatin group, telmisartan group and the control group compared with the youth group, and the axon terminal bouton was declined in the simvastatin group compared with the youth group and the control group (all at P=0.000). Opsin and rhodopsin stains displayed that the OS thickness was increased in the simvastatin group, telmisartan group and the control group compared with the youth group, and that in the telmisartan group was reduced in comparison with the control group (all at P<0.01). Conclusions As SD rat aging, retinal thickness is gradually attenuated and the number of RGCs is gradually declined.Although the density of bipolar cells seem to be unchanged, their synaptic connections are decreased and the OS is thicken.Simvastatin and telmisartan can delay retinal senescence by protecting retinal neurons against aging and thinning thickened OS. Key words: Aging/physiology; Retina/physiopathology; Aged; Eye proteins/drug effect; Neurons/structure; Statins; Sartans; Animal, rats

Objective To explore the low-dose simvastatin (5 mg)on vascular endothelial function in hypertensives and safety of low-dose.Methods 204 hypertensives were randomly divided into 3 groups: conventional control group( conventional antihypertensive treatment alone without oral simvastatin),5 mg group (simvastatin 5 mg/d),20 mg group( simvastatin 20 mg/d)respectively 68 cases were treated for 12 weeks,and blood lipid,hepatic and rena function,muscle enzymes,blood cells were detected before treatment and two weeks after treatment,during the the patients with adverse drug reaction of simvastatin will withdraw from the study,Before and after treatment brachial artery endothelial FMD ( flow-mediated dilation) and NMD ( nitroglycerin-mediated dilation) were detected using color Doppler ultrasound,the changes in level of lipid of 3 groups were observed before and after treatment and changes in endothelial function and lipid levels in each group before and after treatment and incidence of adverse reactions of simvastatin each group were compared.Results ①The incidence of adverse drug reactions of simvastatin in 5 mg and 20 mg group Group were respectively 4.41%,25% and the incidence of adverse drug reactions of simvastatin in 5 ng was was significantly lower than in 20 mg group ( P ＜ 0.05 ).②The level of TC,LDL-C after treatment in 20 mg group than before treatment decreased significantly(P ＜0.05);FMD in 5 mg and 20 mg groups were significantly improved pre-treatment(P ＜0.05),Changes in the value of FMD were no difference in 5 mg and 20 mg groups( P ＞ 0.05).Conclusion The incidence of adverse drug reactions in Low-dose simvastatin is fewer,both low-dose and conventional-dose simvastatin can significantly improve vascular endothelial function in hypertensives.These patients which need long-term use of statins and whose lipid levels are not too high or low may use low doses statins drugs,which could play a role improving vascular endothelial function but not a role of regulation of lipid levels and reduce incidence of adverse cardiovascular and cerebrovascular events,ultimately reduce the complications of hypertension. Key words: Low-dose simvastatin; Hypertension; Vascular endothelial function; Security; Serum lipid