Alisol A 24-acetate protects oxygen-glucose deprivation-induced brain microvascular endothelial cells against apoptosis through miR-92a-3p inhibition by targeting the B-cell lymphoma-2 gene.

CONTEXT: Alisol A 24-acetate has been used to treat vascular diseases. However, the underlying mechanisms still remain unclear. OBJECTIVE: The present study evaluated the antiapoptotic effect of alisol A 24-acetate on brain microvascular endothelial cells (BMECs) and explored the underlying mechanisms. MATERIALS AND METHODS: BMECs were injured through oxygen -glucose deprivation (OGD) after alisol A 24-acetate treatment. Cell viability and half-maximal inhibitory concentration (IC50) were measured using CCK-8, whereas inflammatory factors and oxidative stress indicators were measured using enzyme linked immunosorbent assay. Cell invasion and wound healing assays were detected. Cell apoptosis was assessed using flow cytometry. B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X (Bax) expression were analyzed using Western blotting. Dual-luciferase assay was applied to detect target genes of miR-92a-3p. RESULT: Alisol A 24-acetate had an IC50 of 98.53 mg/L and inhibited cell viability at concentrations over 50mg/L. OGD induced apoptosis and promoted miR-92a-3p overexpression in BMECs. However, alisol A 24-acetate treatment suppressed inflammation, improved migration and invasion abilities, increased Bcl-2 expression, inhibited Bax expression, and repressed apoptosis and miR92a-3p overexpression in OGD-induced BMECs. MiR-92a-3p overexpression promoted cell apoptosis and suppressed Bcl-2 expression, whereas its inhibitor reversed the tendency. Alisol A 24-acetate treatment relieved the effects of miR-92a-3p overexpression. Dual-luciferase assay confirmed that miR-92a-3p negatively regulated the Bcl-2 expression. CONCLUSIONS: These findings suggest that alisol A 24-acetate exerts antiapoptotic effects on OGD-induced BMECs through miR-92a-3p inhibition by targeting the Bcl-2 gene, indicating its potential for BMECs protection and as a novel therapeutic agent for the treatment of cerebrovascular disease.

Pharmacokinetics and tissue distribution of five major triterpenoids after oral administration of Rhizoma Alismatis extract to rats using ultra high-performance liquid chromatography-tandem mass spectrometry.

Rhizoma Alismatis (RA) was wildly used for treatment of dysuria, pyelonephritis, hyperlipidemia, enteritis diarrhea, diabetes, inflammation, and cancer. Triterpenoids are the major active components of RA, and its extract is mainly composed of alisol A (ALA), alisol B (ALB), alisol C 23-acetate (ALC-23A), alisol A 24-acetate (ALA-24A), and alisol B 23-acetate (ALB-23A). In this study, a simple, reliable, and sensitive ultra high-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-MS/MS) method was created and validated for the quantification of the five major triterpenoids in rat plasma and various tissues biosamples (including intestine, stomach, liver, kidney, fat, muscle, brain, heart, lung, spleen, and testes). The plasma and tissues biosamples were pretreated by direct precipitation deproteinization method with acetonitrile. 17α-Hydroxyprogesterone was used as internal standard (IS). The chromatography was performed on a Phenomenex C8 column (30×2.00mm, 1.8µm) at room temperature with gradient elution. Compounds were quantified by selected multi-reactions monitoring (SRM) scanning with positive electric spray ionization mode. The linearity of detection for each triterpene was respectively from 1 to 1000ng/mL for ALC-23A and ALA, from 4 to 4000ng/mL for ALA-24A, from 10 to 10,000ng/mL for ALB, and from 2 to 2000ng/mL for ALB-23B (r>0.99) with low quantification limits of 1-10ng/mL for all analytes. All of the other validation parameters were also in an acceptable range. The validated UHPLC-MS/MS method subsequently applied for the pharmacokinetic and tissue distribution studies of RA extract. After orally given 100mg/kg of RA extract, ALA was the most exposed component, followed by ALB and ALA-24A. Whereas significant gender difference was observed for ALB, ALA, and ALA-24A between female and male rats. The AUC(0-∞) of ALA, ALB, and ALA-24A in female rats were approximately 2-5 fold larger than that in male rats. These triterpenoids also displayed approximately 1.5-2 times longer half-life (t1/2) in female rats. Appearant Km, Vmax and Clint of ALA, ALB, and ALA-24A were calculated by substrate depletion approach, rat P450 CYP3A2 plays an important role in the metabolism of ALA, ALB, and ALA-24A, which is an important factor leading to the different exprosures of ALA, ALB, and ALA-24A between the male rats and the female rats. Furthermore, results from tissue distribution in male rats showed that the main tissue depots of five triterpenoids were the stomach/intestine, followed by the liver, brain, and fat. However, ALA was still measured in the kidney after a long elimination time. ALB and ALB-23B exhibited lower elimination rate in the testis. These results provide a fundamental support for further pharmacological development and clinical safety application of RA.

Safety and efficacy of olesoxime in patients with type 2 or non-ambulatory type 3 spinal muscular atrophy: a randomised, double-blind, placebo-controlled phase 2 trial.

BACKGROUND: Spinal muscular atrophy (SMA) is a progressive motor neuron disease causing loss of motor function and reduced life expectancy, for which limited treatment is available. We investigated the safety and efficacy of olesoxime in patients with type 2 or non-ambulatory type 3 SMA. METHODS: This randomised, double-blind, placebo-controlled, phase 2 study was done in 22 neuromuscular care centres in Belgium, France, Germany, Italy, Netherlands, Poland, and the UK. Safety and efficacy of olesoxime were assessed in patients aged 3-25 years with genetically confirmed type 2 or non-ambulatory type 3 SMA. A centralised, computerised randomisation process allocated patients (2:1 with stratification by SMA type and centre) to receive olesoxime (10 mg/kg per day) in an oral liquid suspension or placebo for 24 months. Patients, investigators assessing outcomes, and sponsor study personnel were masked to treatment assignment. The primary outcome measure was change from baseline compared with 24 months between the two treatment groups in functional domains 1 and 2 of the Motor Function Measure (MFM D1 + D2) assessed in the full analysis population. A shorter, 20-item version of the MFM, which was specifically adapted for young children, was used to assess patients younger than 6 years. Safety was assessed in all patients who received one or more doses of the study drug. The trial is registered with ClinicalTrials.gov, number NCT01302600. FINDINGS: The trial was done between Nov 18, 2010, and Oct 9, 2013. Of 198 patients screened, 165 were randomly assigned to olesoxime (n=108) or placebo (n=57). Five patients in the olesoxime group were not included in the primary outcome analysis because of an absence of post-baseline assessments. The change from baseline to month 24 on the primary outcome measure was 0·18 for olesoxime and -1·82 for placebo (treatment difference 2·00 points, 96% CI -0·25 to 4·25, p=0·0676). Olesoxime seemed to be safe and generally well tolerated, with an adverse event profile similar to placebo. The most frequent adverse events in the olesoxime group were pyrexia (n=34), cough (n=32), nasopharyngitis (n=25), and vomiting (n=25). There were two patient deaths (one in each group), but these were not deemed to be related to the study treatment. INTERPRETATION: Olesoxime was safe at the doses studied, for the duration of the trial. Although the primary endpoint was not met, secondary endpoints and sensitivity analyses suggest that olesoxime might maintain motor function in patients with type 2 or type 3 SMA over a period of 24 months. Based on these results, olesoxime might provide meaningful clinical benefits for patients with SMA and, given its mode of action, might be used in combination with other drugs targeting other mechanisms of disease, although additional evidence is needed. FUNDING: AFM Téléthon and Trophos SA.

Effective cytotoxic activity of OSW-1 on colon cancer by inducing apoptosis in vitro and in vivo.

As a natural compound, Ornithogalum caudatum Ait is primarily used as an anti-inflammatory and antitumor agent in Chinese folk medicine. In 1992, OSW-1 was isolated from this compound, which is a new member of cholestane saponin family. In numerous recent studies, OSW-1 has been shown to have powerful cytotoxic anticancer effects against various malignant cells. However, the therapeutic efficacy of OSW-1 on colon cancer and the underlying mechanism are not understood. To explore the mechanism underlying OSW-1 in antitumor therapy, a therapeutic function analysis of OSW-1 on colon cancer was performed in vitro and in vivo. It was shown that with low toxicity on normal colonic cells, OSW-1 suppresses colon cancer cells in vitro and this inhibition was via the intrinsic apoptotic pathway, which increased cellular calcium, changed mitochondrial membrane potential, disrupted mitochondrial morphology, and led to the release of cytochrome c and the activation of caspase-3. Furthermore, in a nude mouse model, OSW-1 had a powerful effect on suppressing colon tumor proliferation without significant side effects through the apoptosis pathway. Taken together, these results demonstrate that OSW-1 is a potential drug for colon cancer treatment.

Cholest-4-en-3-one attenuates TGF-ß responsiveness by inducing TGF-ß receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells.

PURPOSE: The transforming growth factor-beta (TGF-ß) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-ß signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro. METHODS: The inhibitory effects of cholest-4-en-3-one on TGF-ß-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-ß receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation. RESULTS: Cholest-4-en-3-one attenuated TGF-ß signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-ß-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-ß responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-ß receptors and facilitating rapid degradation of TGF-ß and thus suppressing TGF-ß-induced signaling. CONCLUSIONS: Our results suggest that cholest-4-en-3-one inhibits TGF-ß signaling may be due, in part to the translocation of TGF-ß receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-ß deficiency.

Protective Effects of Alisol B 23-Acetate Via Farnesoid X Receptor-Mediated Regulation of Transporters and Enzymes in Estrogen-Induced Cholestatic Liver Injury in Mice.

PURPOSE: To investigate protective effects of alisol B 23-acetate (AB23A) against hepatotoxity and cholestasis induced by 17α-ethinylestradiol (EE) in association with farnesoid X receptor (FXR) activation in vivo and in vitro. METHODS: The cholestatic liver injury model was established by subcutaneous injections of EE in C57BL/6 mice. Serum biomarkers, bile flow assay and H&E staining were used to identify the amelioration of cholestasis after AB23A treatment. Mice primary hepatocytes culture, gene silencing experiment, real-time PCR and Western blot assay were used to elucidate the mechanisms underlying AB23A hepatoprotection. RESULTS: AB23A treatment protected against liver injury induced by EE through increasing hepatic efflux and reducing uptake of bile acid via an induction in efflux transporters (Bsep and Mrp2) and an inhibition in hepatic uptake transporter (Ntcp) expression. AB23A also reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, and increased bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrated that the changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo and were abrogated after FXR was silenced in vitro. CONCLUSIONS: AB23A produces protective effects against EE-induced cholestasis, due to FXR-mediated gene regulation.

Enhanced distribution and anti-tumor activity of ergosta-4,6,8(14),22-tetraen-3-one by polyethylene glycol liposomalization.

Ergosta-4,6,8(14),22-tetraen-3-one (ergone) was isolated from P. umbellatus, which has been demonstrated to possess a variety of pharmacological activities in vivo and in vitro. The purpose of this study was to evaluate the potent ergone formulations for cancer chemotherapy, the liposomal formulations were less toxic and provide longer systemic circulation time were selected as candidates of nanocarriers for ergone. The effect of modification polyethylene glycol (PEG) on the pharmacokinetics of liposome showed that the retaining time of ergone in blood circulation was prolonged by modified PEG. Moreover, the results of pharmacokinetic analysis showed that of PEG liposome was about 2.8 times higher than that of free PEG liposome after intravenous injection into normal rats due to the lower distribution into the reticuloendothelial system tissues. Since PEG liposome was able to stably encapsulate ergone in blood, area under plasma concentration-time curve of ergone was also extensively enhanced after intravenous dosing of ergone-PEG liposome into normal rats. In the in vivo studies utilizing solid tumor-bearing mice, it was confirmed that ergone-PEG liposome delivered remarkably larger amount of ergone to tumor tissue and provided more significant anti-tumor activity than free ergone. In conclusion, PEG liposome was an effective delivery formulation to achieve increased ergone release in tumor and therapeutic efficacy.

Olesoxime protects embryonic cortical neurons from camptothecin intoxication by a mechanism distinct from BDNF.

BACKGROUND AND PURPOSE: Olesoxime is a small cholesterol-oxime promoting rat embryonic motor neurons survival in the absence of trophic factors. Because olesoxime can substitute for neurotrophic factors in many situations, and to gain further understanding of its mechanism of action, we wondered if it could prevent neuronal death induced by camptothecin (CPT) and compared its effects with those of brain-derived neurotrophic factor (BDNF). EXPERIMENTAL APPROACH: E17 rat embryonic cortical neurons were treated with olesoxime, BDNF or vehicle and intoxicated with CPT. Caspase-dependent and caspase-independent death pathways along with pro-survival pathways activation were explored. KEY RESULTS: As previously reported for BDNF, olesoxime dose-dependently delayed CPT-induced cell death. Both compounds acted downstream of p53 activation preventing cytochrome c release and caspases activation. When caspase activation was blocked, both olesoxime and BDNF provided additional neuroprotective effect, potentially through the prevention of apoptosis-inducing factor release from mitochondria. While BDNF activates both the PI3K/Akt and the ERK pathway, olesoxime induced only a late activation of the ERK pathways, which did not seem to play a major role in its neuroprotection against CPT. Rather, our results favour preserved mitochondrial membrane integrity by olesoxime. CONCLUSIONS AND IMPLICATIONS: Albeit different, olesoxime and BDNF mechanisms for neuroprotection converge to preserve mitochondrial function. These findings emphasize the importance of targeting the mitochondria in the process of neurodegeneration. Importantly olesoxime, by mimicking neurotrophin pro-survival activities without impacting PI3K/Akt and ERK signalling, may have greater therapeutic potential in many diseases where neurotrophins were considered as a therapeutic solution.

Folate-functionalized nanoparticles for controlled ergosta-4,6,8(14),22-tetraen-3-one delivery.

To improve the therapeutic effect of ergosta-4,6,8(14),22-tetraen-3-one (ergone), a folate-decorated ergone-bovine serum albumin nanoparticles (abbreviated FA-ergone-BSANPs) was prepared. The properties were extensively studied by Zetasizer Nano Particle Size Analyzer and TEM, which indicated the prepared nanoparticles were spherical in shape and uniform in size with a zeta potential of -23.8 mV. The drug-loading capacity also has been determined with drug loading content of 2.73% and encapsulation efficiency of 61.8%. In vitro release studies proved the much slow drug release from the nanoparticles during circulating in the blood stream and the increase of drug release at the target sites. The FA-ergone-BSANPs showed enhanced cellular uptake, increased targeting capacity, and increased cytotoxicity against KB cells over-expressing folate receptor (FR), which indicated that its potent cell-killing activity is specific for cells that express the FR. In vivo experiment also confirmed that FA-ergone-BSANPs represent a FR-targeted chemotherapeutic that can produce potent activity against FR-positive tumors. In conclusion, this report has a great significance in pharmacology and clinical medicine as well as methodology. Further detailed dose-optimization studies will be required for better understanding in vivo pharmacokinetic and bio-distribution behaviors.

Effect of ergosta-4,6,8(14),22-tetraen-3-one (ergone) on adenine-induced chronic renal failure rat: a serum metabonomic study based on ultra performance liquid chromatography/high-sensitivity mass spectrometry coupled with MassLynx i-FIT algorithm.

BACKGROUND: Ergosta-4,6,8(14),22-tetraen-3-one (ergone) has been proven to prevent the progression of renal injury and the subsequent renal fibrosis. We investigated the therapeutic effects and mechanism of ergone on a chronic renal failure model of rats induced by adenine. METHODS: A serum metabonomic method based on the UPLC Q-TOF/MS was undertaken to explore the excretion pattern of low molecular mass metabolites. RESULTS: Coupled with blood biochemistry and kidney histopathology results, the significant difference in metabolic profiling between adenine-induced chronic renal failure group and the ergone treated group by using pattern recognition analysis indicated that changes in global serum metabolites occurred. Some significantly changed metabolites like lysophosphatidylcholines, adenine, dopamine, creatinine, aspartic acid and phenylalanine have been found and identified. These biochemical changes in serum metabolites are related to the perturbations of amino acid metabolism and lecithin metabolism, which may be helpful to further understand the chronic renal failure and therapeutic mechanisms of ergone. CONCLUSION: The work shows that the metabonomic method is a valuable tool for studying the essence of chronic kidney disease and therapeutic effect mechanism of preclinical or clinical drug.

Olesoxime (cholest-4-en-3-one, oxime): analgesic and neuroprotective effects in a rat model of painful peripheral neuropathy produced by the chemotherapeutic agent, paclitaxel.

Olesoxime is a small cholesterol-like molecule that was discovered in a screening program aimed at finding treatment for amyotrophic lateral sclerosis and other diseases where motor neurons degenerate. In addition to its neuroprotective and pro-regenerative effects on motor neurons in vitro and in vivo, it has been shown to have analgesic effects in rat models of painful peripheral neuropathy due to vincristine and diabetes. We used a rat model of painful peripheral neuropathy produced by the chemotherapeutic agent, paclitaxel, to determine whether olesoxime could reverse established neuropathic pain. In addition, we determined whether giving olesoxime during the exposure to paclitaxel could prevent the development of the neuropathic pain syndrome and the accompanying degeneration of the terminal arbors of sensory fibers in the epidermis. Olesoxime significantly reduced established mechano-allodynia and mechano-hyperalgesia. There was no indication of tolerance to the effect during five days of dosing and the analgesia persisted for 5-10 days after the last injection. Giving olesoxime during the exposure to paclitaxel significantly and permanently reduced the severity of mechano-allodynia and mechano-hyperalgesia and significantly reduced the amount of sensory terminal arbor degeneration. Olesoxime targets mitochondrial proteins and its effects are consistent with the mitotoxicity hypothesis for paclitaxel-evoked painful peripheral neuropathy. We conclude that olesoxime may be useful clinically for both the prevention and treatment of paclitaxel-evoked painful peripheral neuropathy.

Dietary 5-campestenone (campest-5-en-3-one) enhances fatty acid oxidation in perfused rat liver.

The effect of dietary 5-campestenone (campest-5-en-3-one), a chemical modification product of a naturally-occurring plant sterol, campesterol, on lipid metabolism was examined using a rat liver perfusion system. Male Sprague-Dawley rats weighing about 140 g were fed a diet supplemented with or without 0.2% 5-campestenone for 14 d. 5-Campestenone feeding resulted in a marked reduction in the concentrations of serum lipids, such as triacylglycerol (TG), cholesterol, phospholipid, and free fatty acid, without influencing food intake or growth. Then, isolated livers from both groups were perfused for 4 h in the presence of an exogenous linoelaidic acid substrate. Dietary 5-campestenone markedly elevated hepatic ketone body production, while cumulative secretions of TG, cholesterol, and phospholipid by the livers of rats fed 5-campestenone were all significantly lowered as compared to those fed without the compound: the extent of the reduction was more prominent in the secretion of TG than other lipid components. In addition, the reduction of TG secretion was concomitantly accompanied by the reduced incorporation of both exogenous and endogenous fatty acids into this lipid molecule. These results suggest that dietary 5-campestenone exerts its hypotriglyceridemic effect, at least, in part through an enhanced metabolism of endogenous and exogenous fatty acids to oxidation at the expense of esterification in rat liver.

Effect of 5-Campestenone (24-methylcholest-5-en-3-one) on Zucker diabetic fatty rats as a type 2 diabetes mellitus model.

We examined the therapeutic effects of dietary exposure to 5-campestenone (24-methylcholest-5-en-3-one), an enone derivative of campesterol, in Zucker diabetic fatty (ZDF) rats, an animal model of type 2 diabetes mellitus. Dietary 0.6 % exposure to 5-campestenone caused marked reduction in hemoglobin A1c (HbA1c), plasma total cholesterol, triglycerides and non esterified fatty acid (NEFA). In particular, plasma triglyceride levels were reduced in the 0.6 % 5-campestenone-fed group to about 25 % of that in the control group. In the oral glucose tolerance test (OGTT) at three and seven weeks after the beginning of treatment, 5-campestenone limited the rise of blood glucose levels by oral administration of glucose dose-dependently. Amounts of adipose tissue in the retroperitoneum and periepididymal area as well as abdominal subcutaneous fat were significantly decreased in animals fed 0.6 % 5-campestenone. The blood leptin concentration on the final day of feeding was significantly in animals administered 5-campestenone. No obvious anomaly due to consumption of 5-campestenone was detected in necropsy or clinical observations.

Inhibitors of sterol synthesis. Effects of a new fluorinated analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one in rats.

3 beta-Hydroxy-25,26,26,26,27,27,27-heptafluoro-5 alpha-cholest-8(14)-en-15-one (VII), an analog of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I) in which conversion to 26- and 25-oxygenated metabolites is blocked by the F7-substitution, was administered to male Sprague-Dawley rats at levels of from 0.025 to 0.15% by weight in a ground chow diet. Administration of VII resulted in lowering of the levels of serum cholesterol at dosages as low as 0.025% by weight in diet. In marked contrast to I, VII had little or no effect on food consumption. Whereas administration of I at a level of 0.1% by weight in diet resulted in a cessation of growth, VII, at approximately the same molar concentration in diet, had only slight or no effect on changes in total body weight. Significant levels of 25,26,26,26,27,27,27-heptafluorocholesterol (VIII) were observed in serum and liver, indicating the conversion of VII to VIII. Characterization of VIII in liver was based upon the results of gas chromatography, low and high resolution mass spectral studies, infrared spectroscopy, and 1H and 13C nuclear magnetic resonance spectroscopy. The levels of VIII in serum appeared to be related to dosage and duration of administration of VII.

Inhibitors of sterol synthesis. Metabolism of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one after oral administration to a nonhuman primate.

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one is a potent inhibitor of cholesterol biosynthesis which has significant hypocholesterolemic activity upon oral administration to rodents and nonhuman primates. In the present study the metabolism of the 15-ketosterol has been investigated after the oral administration of a mixture of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one and [4-14C]cholesterol to 8 baboons. Blood samples were obtained at 4, 8, 12, 16, and 24 h after administration of the labeled sterols. Clear differences in the time courses of the levels of 3H and 14C in plasma were observed. 3H in plasma showed maximum values at 4 to 8 h, whereas maximum values for the levels of 14C were observed much later. 3H in plasma was shown to be primarily in the form of its metabolites, i.e. esters of the 15-ketosterol, cholesterol, and cholesteryl esters. The levels of the 15-ketosterol and of each of these metabolites showed different changes with time. The labeled cholesterol (and the cholesterol moiety of the cholesteryl esters), formed from the [2,4-3H]-15-ketosterol, was characterized by chromatography and by purification by way of its dibromide derivative. At 24 h after the administration of the labeled sterols, the distribution of 3H in plasma lipoprotein fractions paralleled that of 14C, with most of the 3H and 14C in high density lipoprotiens (HDL) and low density lipoproteins (LDL). Almost all of the 3H in HDL and in LDL was found as cholesterol, cholesteryl esters and esters of the 15-ketosterol. The distribution of 3H in HDL and in LDL of the free 15-ketosterol, esters of the 15-ketosterol, cholesterol, and cholesteryl esters was similar to that of plasma, thereby indicating no unusual concentration of any of the 3H labeled components in HDL or LDL.

Inhibitors of sterol synthesis. Studies of the distribution and metabolism of 5 alpha-[2,4-3H]cholest-8(14)-en-3 beta-ol-15-one after intragastric administration to rats.

5 alpha-[2,4-3H]Cholest-8(14)-en-3 beta-ol-15-one was administered to a series of male Sprague-Dawley rats by intragastric intubation in the form of an emulsion in a mixture of triolein, sodium taurocholate, bovine serum albumin, and glucose. [4-14C]Cholesterol was similarly administered to a second series of rats. The distribution of 3H and 14C was studied at 12 and 48 h after the administration of the sterols. The results demonstrated that the 15-ketosterol is absorbed and metabolized to material with the chromatographic properties of fatty acid esters of the 15-ketosterol, to cholesterol, and to fatty acid esters of cholesterol. The [3H]cholesterol formed from the 15-ketosterol was characterized by its behavior on silicic acid-Super Cel column chromatography, by the chromatographic behavior of its acetate derivative on alumina-AgNO3 column chromatography, and by purification by way of its dibromide derivative without significant change in specific activity. The general distribution of 3H was similar to that of 14C. No unusual concentration of 3H in any of the organs studied was observed.

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one. In vivo conversion to cholesterol upon oral administration to a nonhuman primate.

The metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I), a potent inhibitor of cholesterol synthesis with marked hypocholesteremic activity, has been studied in a nonhuman primate. A mixture of [2,4-3H]-I and [4-14C]-cholesterol was administered to a male baboon in the form of a feedball. Blood was samples at 4, 8, 12, 16, and 24 hr. Detailed analyses of the plasma lipids indicated very rapid absorption of I (relative to cholesterol) and metabolism to cholesterol, cholesteryl esters, and esters of I. The labeled cholesterol was characterized by chromatographic techniques and by purification by way of its dibromide derivative. The levels of 3H in plasma associated with I, esters of I, cholesterol, and cholesteryl esters each showed a different time course. By 24 hr after the administration of [2,4-3H]-I, most of the 3H in plasma was associated with cholesterol and cholesteryl esters. The levels of total 3H and 14C in plasma at various times after the administration of the mixture of [2,4-3H]-I and [4-14C]-cholesterol differed markedly with 3H showing a maximum value at 4 hr and 14C showing a maximum value at 24 hr.