Impaired Itching Perception in Murine Models of Cholestasis Is Supported by Dysregulation of GPBAR1 Signaling.

BACKGROUND & AIMS: In cholestatic syndromes, body accumulation of bile acids is thought to cause itching. However, the mechanisms supporting this effect remain elusive. Recently, GPBAR1 (TGR5) a G-protein coupled receptor has been shown to mediate itching caused by intradermal administration of DCA and LCA. 6α-ethyl-3α, 7α-dihydroxy-24-nor-5ß-cholan-23-ol (BAR502) is a non-bile acid dual ligand for FXR and GPBAR1. METHODS: Cholestasis was induced in wild type and GPBAR1-/- mice by administration of α-naphthyl-isothiocyanate (ANIT) or 17α-ethynylestradiol. RESULTS: In naïve mice skin application of DCA, TLCA, 6-ECDCA, oleanolic and betulinic acid induces a GPBAR1 dependent pruritogenic response that could be desensitized by re-challenging the mice with the same GPBAR1 agonist. In wild type and GPBAR1-/- mice cholestasis induced by ANIT fails to induce spontaneous itching and abrogates scratching behavior caused by intradermal administration of DCA. In this model, co-treatment with BAR502 increases survival, attenuates serum alkaline phosphatase levels and robustly modulates the liver expression of canonical FXR target genes including OSTα, BSEP, SHP and MDR1, without inducing pruritus. Betulinic acid, a selective GPBAR1 ligand, failed to rescue wild type and GPBAR1-/- mice from ANIT cholestasis but did not induced itching. In the 17α-ethynylestradiol model BAR502 attenuates cholestasis and reshapes bile acid pool without inducing itching. CONCLUSIONS: The itching response to intradermal injection of GPBAR1 agonists desensitizes rapidly and is deactivated in models of cholestasis, explain the lack of correlation between bile acids levels and itching severity in cholestatic syndromes. In models of non-obstructive cholestasis, BAR502 attenuates liver injury without causing itching.

Regioselective oxyfunctionalization of unactivated carbons in steroids by a model of cytochrome P-450: osmiumporphyrin complex/tert-butyl hydroperoxide system.

tert-Butyl hydroperoxide catalyzed by (5,10,15,20-tetramesitylporphyrinate) osmium(II) carbonyl [Os(TMP)CO] complex was found to be a highly efficient versatile oxidant for C-H carbons in steroid substrates. When reacted with representative steroids with an estrane, pregnane, 5beta-cholane, or 5alpha-cholestane structure, regioselective oxyfunctionalization and/or oxidative degradation occurred to give a variety of novel and uncommon derivatives in one step.

Biological activity predictions, crystallographic comparison and hydrogen bonding analysis of cholane derivatives.

A total of eighteen molecules of cholane derivatives (I-XVIII) (a series of steroids) have been included to predict their pharmacological effects, specific mechanisms of action, known toxicities, drug-likeness, etc, by using the statistics of multilevel neighbourhoods of atoms (MNA) descriptors for active and inactive fragments. The biological activity spectra for substances have been correlated on SAR base (structure-activity relationships data and knowledge base), which provides the different P(a) (possibility of activity) and P(i) (possibility of inactivity). Most of the probable activities have been characterized by P(a) and P(i) values, which depict that all the molecules have high value of teratogen activity. The Lipinski's thumb rule predicts that all the cholane derivatives have stronger preponderance for "cancer-like-drug" molecules and some of their related analogous have entered in the ANCI (American National Cancer Institute) database. Some selected bond distances and bond angles of interest have been taken into account and deviation of bond distances/bond angles, vis-a-vis the substitutional group and X-H...A intra/intermolecular hydrogen bonds has been discussed in detail. X-H...A intra and intermolecular hydrogen bonds in the molecules have been described with the standard distance and angle cut-off criteria. D-theta and d-theta. scatter plots for intra- and intermolecular interactions are presented for better understanding of packing interactions existing among these derivatives. There exists only one C-H...O intramolecular bifurcated hydrogen bond. while high tendency of intermolecular bifurcated hydrogen bonds based on a defined O-H...O has been observed, in which O atom acts as a prototype donor as well as acceptor. The frequency of occurrence of C-H...O hydrogen bonds is predominant (i.e. 85.7%) in intramolecular interactions, whereas in intermolecular interactions, frequency of occurrence for O-H...O interactions is 62.9%. Solvent-solute/solute-solvent interactions have also been investigated to understand more complicated processes that occur for biomolecules in aqueous solutions. The number of hydrogen donors in each derivative is less than 5, except for molecule XVIII and which has 91.3% of drug-likeness, instead of observed range of 96.5-99.39%.

Self-assembled nanoparticles based on glycol chitosan bearing 5beta-cholanic acid for RGD peptide delivery.

The synthetic peptide bearing Arg-Gly-Asp (RGD) sequence is considered to specifically bind to alpha(v)beta(3) integrin expressed on endothelial cells in the angiogenic blood vessels, which provides a potential to inhibit the tumor growth. As a carrier for the RGD peptide, hydrophobically modified glycol chitosan (HGC) capable of forming nano-sized self-aggregates was prepared by the chemical conjugation of 5beta-cholanic acid to the main backbone of glycol chitosan. The RGD peptide labeled with fluoresein isothiocyanate (FITC-GRGDS) was loaded into self-aggregates in three different conditions: simple mixing, sonication, and solvent evaporation methods. Of different methods applied, solvent evaporation method showed the most promising results for peptide loading, as judged by the yield (>70%) and loading efficiency (>75%). It was found that the presence of FITC-labeled peptides makes the self-aggregates to be compact, possibly due to the role of both hydrophobic FITC and peptides containing carboxylic acids that allow hydrogen bonding and electrostatic interaction with the primary amino groups in the main backbone of glycol chitosan. FITC-labeled peptides were released from self-aggregates in a physiological solution (pH 7.4) for up to 1 day. From the cell adhesion and migration assays, it was demonstrated that FITC labeling of peptides does not significantly deteriorate biological activity of the parent peptide drug (GRGDS). Overall, the self-aggregates loaded with FITC-GRGDS might be useful for monitoring or destroying the angiogenic vessels surrounding the tumor tissue.

Uptake of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate into isolated rat hepatocytes by three transport systems.

Uptake of norcholansulfonate (3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate), an isogeometric analogue of cholate into isolated rat liver hepatocytes occurs only by saturable transport. In order to identify the transport systems involved, uptake of norcholansulfonate was studied using 7 beta-NBD-NCT ({N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl})-2'-aminoethanesulfonate) as a competing substrate. For transport of both bile salt derivatives, which mutually inhibit their mediated transport competitively, the existence of at least three transport systems must be assumed. Uptake studies using the cloned hepatic Na+/cholyltaurine cotransporting polypeptide stably expressed in CHO cells (Chinese hamster ovary cells) showed that both bile salt derivatives were transported and furnished the definite KT values of this single transport system and the ratio of the maximal uptake velocities. On the basis of these data, uptake of both bile salt derivatives into rat hepatocytes and their mutual competitive inhibition could be analyzed for three transport systems. The maximal flux rates J2 and the half-saturation constants KT2 in the presence of Na+ (143 mM) are for norcholansulfonate: J1(Na+ 143) = 1.0 +/- 0.2 nmol/(min . mg protein), KT1(Na+ 143) = 15 +/- 4 microM, J2(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT2(Na+ 143) = 15 +/- 2 microM, J3(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT3(Na+ 143) = 60 +/- 15 microM, and for 7 beta-NBD-NCT J1(Na+ 143) = 0.14 +/- 0.04 nmol/(min.mg protein), KT1(Na+ 143) = 3.1 +/- 0.5 microM, J2(Na+ 143) = 0.014 +/- 0.005 nmol/(min.mg protein), KT2(Na+ 143) = 21 +/- 2 microM, J3(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein), KT3(Na+ 143) = 190 +/- 25 microM. The kinetic parameters are in accordance with the assumptions that the cloned Na+/cholyltaurine cotransporting polypeptide represents transport system 2 and that the kinetically identified additional transport system 1 is either strictly or partially Na(+)-dependent.

3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate: synthesis and suitability for the study of cholate transport.

In order to facilitate the study of transport processes of unconjugated C-24 bile salts, simple syntheses of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate (norcholansulfonate) and 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-[7 beta 5H] cholan-23-sulfonate were devised. The hydrophilic-hydrophobic properties of norcholansulfonate, as determined by its chromatographic behavior as well as by its partition between l-octanol and water, are more similar to those of cholyltaurine than to those of cholate. Self-association of norcholansulfonate in phosphate buffer, pH 7.4, with an ionic strength of 150 mM begins at a concentration of about 1 mM, comparable to that of cholyltaurine and cholate, as determined by spectral changes in fluorescence emissions of {N-[7-(4-nitrobenzo-2-oxa-1, 3-diazol)]-7b-amino-3a, 12a-dihydroxy-5b-cholan-24 - oyl}-2'-aminoethanesulfonate (7 beta-NBD-NCT). The apparent CMC value obtained from solubilization of the dye Orange OT, 8.5 mM, is comparable to that of cholytaurine. 7.5 mM, and lower than that of cholate, 9.5 mM. Norcholansulfonate is readily taken up by rat liver and completely excreted unmetabolized into bile with about the same secretion maximum (Tm) as cholyltaurine. Biliary excretion of norcholansulfonate is inhibited by cholyltaurine, and, vice versa, norcholansulfonate inhibits cholyltaurine secretion. Concerning metabolism and excretion, norcholansulfonate with the sulfonate group in the position where cholate has the carboxylate group should behave as an appropriate cholate analogue in mediated transport processes.

Potential tissue-imaging agents: 23-(trimethyl [117mSn]stannyl)-24-nor-5 alpha-cholan-3 beta-ol.

Tin-117m-labeled 23-(trimethylstannyl)-24-nor-5 alpha-cholan-3 beta-ol (2) has been prepared by reaction of trimethyl [117mSn]tin lithium with 3 beta-acetoxy-23-bromo-24-nor-5 alpha-cholane (1). Tin-117m (2) shows pronounced adrenal uptake (2.5% injected dose) in female rats 1 day after injection. Furthermore, the adrenal to liver (9.1:1) and adrenal to blood (33.7:1) ratios are high after this period. The absorbed radiation dose values from [117mSn]2 to human organs have also been estimated by using rat tissue distribution and excretion data. [117mSn]2 is the first reported tissue-specific organic radiopharmaceutical labeled with this nuclide and may have potential as an adrenal imaging agent.

Tetrahydroxylated bile acids in healthy human newborns.

Urine was collected in four healthy infants, born after 35-39 weeks of gestation. The collections were made for 24 h starting immediately after birth. Bile acids were extracted, separated into conjugate groups, solvolysed, hydrolysed and quantified by gas-liquid chromatography as methyl ester trimethylsilyl ether derivatives. Identification was made by gas-liquid chromatography-mass spectrometry. Cholic acid was the predominating primary bile acid. Non-sulphated tetrahydroxylated bile acids, tentatively identified as 1,3,7,12- and 3,6,7,12-tetrahydroxycholanoic acids, were present in almost the same amounts as cholic acid. A previously unknown tetrahydroxylated cholanoic acid, which might be 2-hydroxylated hyocholic acid, was found in all infants in similar amounts. 3 beta-Hydroxy-5-cholenoic acid was present in the sulphate fraction in the urine from all infants. Lithocholic acid was not found. Small amounts of allo and 6-hydroxylated bile acids were mainly found in the sulphate fraction. Bile acids hydroxylated in the 1-position were found predominantly in the taurine fraction.

Radiation dosimetry of two new tellurium- 123m-labeled adrenal-imaging agents: concise communication.

The absorbed radiation doses to humans from 23-(isopropyl[123mTe]telluro)-24-nor-5 alpha-cholan-3 beta-ol (Te-123m-23-ITC) and 24-(isopropyl[123mTe]telluro)-chol-5-en-3 beta-ol(Te-123m-24-ITC) have been calculated, based on rat biological data, to assess the relative radiation risks to humans from these two new adrenal-imaging agents. The estimated radiation doses to several critical organs have been compared with dose estimates for a variety of other radiolabeled steroids that have been designed as adrenal-imaging agents. Dose estimates to selected organs from Te-123m-23-ITC are as follows (rad/mCi): adrenals 98; ovaries 8.0; liver 1.6. Similar estimated values for Te-123m-24-ITC are: adrenals 210; ovaries 13; liver 2.0. The radiation dose estimates for these two agents are comparable to the calculated radiation doses from 6 beta-[(methyl[75Se]seleno)methyl]-19-nor-cholest-5(10)-en-3 beta-ol (Scintidren) and 19-[131I]iodocholest-5-en-3 beta-ol (NP-59), two agents currently in clinical use for the diagnosis of adrenal disease.

Analysis of fecal bile acids and diet among the Japanese in Hawaii.

Fecal samples of 165 Japanese men in Hawaii, age 43 to 74, were analyzed for bile acid content by their conversion to the methyl ester and the trimethylsilyl ether derivative followed by separation on a gas chromatograph. The arithmetic mean of total bile acids for the 165 specimens was 10.96 mg/g dry weight feces. Each of the following bile acids was detectable in over 77% of the fecal specimens: cholic, deoxycholic, lithocholic, and cholanic acid. The intake of Western foods was not positively correlated with the fecal content of secondary or modified bile acids, even though other workers have observed that these bile acids predominated in persons from Westernized countries. Two of the Japanese foods were negatively correlated with the levels of modified bile acids, which suggested that these foods contributed to a decrease in modified bile acids in fecal specimens. Fecal bile acid measurements appeared to be associated with age, but not with weight, height, or serum cholesterol levels.

Oxidation of cholesterol and cholesterol analogues by mitochondrial preparations of steroid-hormone-producing tissue.

The rate of oxidation of cholesterol and its analogues to pregnenolone (3beta-hydroxypregn-5-en-20-one) by various mitochondrial preparations was measured. Sterols with the cholest-5-en-3beta-ol ring system and saturated side chains of different lengths were converted into pregnenolone rat rates similar to that of cholesterol. This marked lack of mitochondrial specificity towards the steroid side chains is in direct contrast with the rat liver microsomal cholesterol 7alpha-hydroxylase, which has a high specificity for the side chain. Steroids that retain the ring system, but contain hydroxyl groups at various points in the side chain, are converted into pregnenolone at rates three to eight times higher than in cholesterol. The results are discussed with reference to current ideas on the mechanism of the side-chain cleavage of cholesterol. The results are discussed with reference to current ideas on the mechanism of the side-chain cleavage of cholesterol.

Reduction of 3-oxosteroids in human liver microsomes.

The 3alpha- and 3beta-reduction of the following steroids was studied in human liver microsomes: 5alpha-androstane-3,17-dione, 17beta-hydroxy-5alpha-androstan-3-one, 5alpha-pregnane-3,20-dione, 5beta-pregnane-3,20-dione, 3-oxo-5beta-cholanoic acid and 7alpha-hydroxy-5alpha-cholestan-3-one. With NADH as cofactor there was a preferential 3alpha-reduction of the C19- and C21-3-oxo-steroids and a preferential 3beta-reduction of the C24- and C27-3-oxo-steroids. Substitution of NADH with NADPH influenced reduction of the substrates in different ways, indicating the presence of several 3alpha- and 3beta-hydroxysteroid dehydrogenases with different substrate specificity and specificity towards NADH and NADPH. Only small or insignificant sex differences could be observed in the reductions studied.

Microbiological degradation of bile acids. The preparation of some hypothetical metabolites involved in cholic acid degradation.

1. To identify the intermediates involved in the degradation of cholic acid, the further degradation of (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IVa) by Arthrobacter simplex was attempted. The organism could not utilize this acid but some hypothetical intermediate metabolities of compound (IVa) were prepared for later use as reference compounds. 2. The nor homologue (IIIa) and the dinor homologue (IIIb) of compound (IVa) were prepared by exposure of 3-oxo-24-nor-5beta-cholan-23-oic acid (I) and (20S)-3beta-hydroxy-5-pregnene-20-carboxylic acid (II) to A. simplex respectively. These compounds correspond to the respective metabolites produced by the shortening of the valeric acid side chain of compound (IVa) in a manner analogous to the conventional fatty acid alpha- and beta-oxidation mechanisms. Their structures were confirmed by partial synthesis. 3. The following authentic samples of reduction products of the oxodicarboxylic acids (IIIa), (IIIb) and (IVa) were also synthesized as hypothetical metabolities: (4R)-4-[3aalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]valeric acid (Vb) and its nor homologue (VIIa) and dinor homologue (IXa);(4R)-4-[3Aaalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]-pentan-1-ol (Vc); and their respective 5beta epimers (Ve), (VIIc), (IXc) and (Vf). 4. In connexion with the non-utilization of compound (IVa) by A. simplex, the possibility that not all the metabolites formed from cholic acid by a certain micro-organism can be utilized by the same organism is considered.