A validated surrogate analyte UPLC-MS/MS assay for quantitation of TUDCA, TCDCA, UDCA and CDCA in rat plasma: Application in a pharmacokinetic study of cultured bear bile powder.

Bear bile is a valuable medicinal material used in traditional Chinese medicine for over 2000 years. However, developing a substitute has become necessary because of protection measures for this endangered species. The ingredients of in vitro cultured bear bile powder (CBBP) include tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA, and it has pharmacological properties that are similar to those of natural bear bile powder (NBBP). In this study, the pharmacokinetic parameters of both CBBP and NBBP were measured in rats with a new surrogate analyte LC-MS method using stable isotopes as surrogate analytes (D4-TUDCA, D4-TCDCA, D4-UDCA and D4-CDCA) with response factors validated in authentic matrix (plasma) for simultaneously monitoring the authentic analytes (TUDCA, TCDCA, UDCA and CDCA). The method validation was satisfactory for the linear regression (r, 0.9975-0.9994), precision (RSD intra-day, 0.72-9.35%; inter-day, 3.82-9.02%), accuracy (RE, -12.42-5.67%) and matrix effect (95.53-99.80%), along with analyte recovery (95.90-98.82%) and stability (89.48-101.81%) of surrogate analytes, and precision (RSD intra-day, 1.06- 11.51%; inter-day, 2.23- 11.38%), accuracy (RE, -7.40-10.76%) and stability (87.37-111.70%) of authentic analytes. We successfully applied this method to evaluate the pharmacokinetics of CBBP and NBBP in rats, which revealed the critical in vivo properties of both bear bile preparations.

Population Pharmacodynamics of Amphotericin B Deoxycholate for Disseminated Infection Caused by Talaromyces marneffei.

Amphotericin B deoxycholate (DAmB) is a first-line agent for the initial treatment of talaromycosis. However, little is known about the population pharmacokinetics and pharmacodynamics of DAmB for talaromycosis. Pharmacokinetic data were obtained from 78 patients; among them, 55 patients had serial fungal CFU counts in blood also available for analysis. A population pharmacokinetic-pharmacodynamic model was fitted to the data. The relationships between the area under the concentration-time curve (AUC)/MIC and the time to blood culture sterilization and the time to death were investigated. There was only modest pharmacokinetic variability in the average AUC, with a mean ± standard deviation of 11.51 ± 3.39 mg·h/liter. The maximal rate of drug-induced kill was 0.133 log10 CFU/ml/h, and the plasma concentration of the DAmB that induced the half-maximal rate of kill was 0.02 mg/liter. Fifty percent of patients sterilized their bloodstreams by 83.16 h (range, 13 to 264 h). A higher initial fungal burden was associated with a longer time to sterilization (hazard ratio [HR], 0.51; 95% confidence interval [CI], 0.36 to 0.70; P < 0.001). There was a weak relationship between AUC/MIC and the time to sterilization, although this did not reach statistical significance (HR, 1.03; 95% CI, 1.00 to 1.06, P = 0.091). Furthermore, there was no relationship between the AUC/MIC and time to death (HR, 0.97; 95% CI, 0.88 to 1.08; P = 0.607) or early fungicidal activity {slope = log[(0.500 - 0.003·(AUC/MIC)]; P = 0.319} adjusted for the initial fungal burden. The population pharmacokinetics of DAmB are surprisingly consistent. The time to sterilization of the bloodstream may be a useful pharmacodynamic endpoint for future studies. (This study has been registered at the ISRCTN registry under no. ISRCTN59144167.).

Elution of High Dose Amphotericin B Deoxycholate From Polymethylmethacrylate.

Fungal periprosthetic joint infections are rare, devastating complications of arthroplasty. There is conflicting evidence as to the efficacy of amphotericin B elution from cement spacers. The purpose of this study was to determine whether concentrations of amphotericin B released from bone cement over time would be efficacious in treating a periprosthetic infection. A continuous flow chamber was used to evaluate the in vitro release of amphotericin from cement beads containing 7.5% amphotericin. Following polymerization, 3.3% of the initially loaded amphotericin B was detected. The peak mean concentration eluted from the bone cement was 0.33 µg/mL at 8 hours. The AUC0-24 was 2.79 µg/mL/h; 0.20% of the amphotericin B was released. In conclusion, amphotericin B is released from bone cement at a clinically useful concentration.

Mechanism of enhanced antiosteoporosis effect of circinal-icaritin by self-assembled nanomicelles in vivo with suet oil and sodium deoxycholate.

BACKGROUND: Circinal-icaritin (CIT), one new active aglycone of Epimedium, can exert a beneficial effect on osteoporotic bone. However, its low bioavailability limits its clinical efficacy for the treatment of osteoporosis. MATERIALS AND METHODS: In this paper, suet oil (SO) was used to improve the oral bioavailability of CIT and enhance its antiosteoporosis effect and absorption. After oral administration of CIT together with SO, the CIT and SO self-assembled into nanomicelles under the action of sodium deoxycholate (DOC) by bile secretion. The antiosteoporosis effects of the CIT-SO-DOC nanomicelles were evaluated in osteoporotic rats by bone mineral density, serum biochemical markers, bone microarchitecture, bone biomechanical properties, and related protein and gene expressions. We examined the bioavailability of CIT and its nanomicelles in vivo, and subsequently the nanomicelles were verified using transmission electron microscopy. Finally, we evaluated absorption across a rat intestinal perfusion model. RESULTS: Compared with CIT, in the CIT-SO groups, protein and messenger ribonucleic acid expressions of osteoprotegerin were increased, while expressions of receptor activator of nuclear factor-κB ligand in bone tissue were decreased; bone-turnover markers in serum of hydroxyproline, alkaline phosphatase, tartrate-resistant acid phosphatase 5b, and receptor activator of nuclear factor-κB ligand levels were decreased, while osteoprotegerin and osteocalcin levels were increased; and trabecular bone mass, microarchitecture, and bone biomechanical strength were enhanced. The relative bioavailabilities of CIT-SO high dosage, CIT-SO medium dosage, and CIT-SO low dosage (area under concentration-time curve [AUC]0-∞) compared with that of raw CIT high dosage, CIT medium dosage, and CIT low dosage (AUC0-∞) were 127%, 121%, and 134%, respectively. The average particle size of CIT-DOC was significantly decreased after adding SO (P<0.01), and the intestinal permeability coefficients of CIT-SO-DOC nanomicelles in the duodenum, jejunum, ileum, and colon were all significantly improved (P<0.01). CONCLUSION: The increased antiosteoporosis effects and bioavailability of CIT-SO-DOC self-assembled nanomicelles were due to an increase in absorption of CIT by reducing the particle sizes of CIT. SO may be a practical oral carrier for antiosteoporosis drugs with low bioavailability.

Prevention effect of orally active heparin conjugate on cancer-associated thrombosis.

Thrombogenesis is a major cause of morbidity and mortality in cancer patients. Prophylaxis with low-molecular-weight heparin (LMWH) is recommended for cancer patients, but requires non-parenteral delivery methods for long-term treatments. In this study, we sought to generate a new oligomeric-bile acid conjugate of LMWH that can be used for oral delivery. We first synthesized a tetramer of deoxycholic acid (tetraDOCA), which was site-specifically conjugated at the end saccharide of LMWH. When LMWH-tetraDOCA conjugate (LHe-tetraD) was orally administered at a dose of 5 mg/kg in ICR mice, the maximum anti-factor Xa level was increased up to 0.62±0.05 IU/mL without any evidence of liver toxicity, gastrointestinal damage, or thrombocytopenia. The cancer-associated thrombosis was induced in tumor-bearing mice by local heat application, and the fibrin deposition in tumors was evaluated. The oral administration of LHe-tetraD (either a single dose or multiple daily doses for up to 10 days) in mice substantially abolished the coagulation-dependent tropism of fibrinogen in the heated tumors and significantly decreased hemorrhage, compared to the mice treated with saline or subcutaneous injection of LMWH. Thus, the anticoagulation effect of oral LHe-tetraD invokes the benefits of oral delivery and promises to provide an effective and convenient treatment for cancer patients at risk of thrombosis.

Orally active desulfated low molecular weight heparin and deoxycholic acid conjugate, 6ODS-LHbD, suppresses neovascularization and bone destruction in arthritis.

The regulation of angiogenesis is an interesting area to consider for novel therapeutic approaches to rheumatoid arthritis (RA). Chemically modified heparins have been developed as possible candidates for angiogenesis inhibitor; however, they have a major clinical drawback in exhibiting poor oral bioavailability. Here, orally absorbable O-desulfated low molecular weight heparin (ODS-LMWH) derivatives were newly synthesized by conjugating 2-O- or 6-O-desulfated LMWH with deoxycholic acid (DOCA) or bisDOCA (a dimer of DOCA), and their physicochemical properties, antiangiogenic potency and pharmacokinetic profiles were assessed. After selecting the best candidate among those derivatives, its therapeutic efficacy on arthritis was investigated in a murine collagen antibody-induced arthritis (CAIA) model. ODS-LMWH derivatives significantly inhibited the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs) and basic fibroblast growth factor (bFGF)-induced angiogenesis in the Matrigel plug assay. Among all the compounds, 6ODS-LHbD showed the highest oral bioavailability in rats (19.3%). In the CAIA mouse model, 6ODS-LHbD (10 mg/kg, p.o., S.I.D.) significantly inhibited neovascularization in the joint, the increase of hind-paw thickness, and the structural damage in the bone. Therefore, 6ODS-LHbD would be a promising candidate for an orally active drug for the treatment of RA.

Pharmacokinetic evaluation of an oral tablet form of low-molecular-weight heparin and deoxycholic acid conjugate as a novel oral anticoagulant.

This study was designed to develop a solid oral dosage form of deoxycholic acid (DOCA)-conjugated low-molecular-weight heparin (LMWH) and to evaluate its oral absorption, distribution, and metabolic stability for the prospect of providing an orally bioavailable LMWH. The LMWH derivative (LHD) was synthesised and then formulated with solubilisers and other pharmaceutical excipients to form a solid tablet. Its absorption and distribution after oral administration were evaluated in mice, rats, and monkeys. The in vitro metabolic stability of LHD was examined by liver microsome assays. More than 80% of LHD was released from the tablet within 60 minutes, guaranteeing rapid tablet disintegration after oral administration. Oral bioavailability of LHD in mice, rats and monkeys were 16.1 ± 3.0, 15.6 ± 6.1, and 15.8 ± 2.5%, respectively. After the oral administration of 131I-tyramine-LHD, most of the absorbed drug remained in the blood circulation and was eliminated mainly through the kidneys. LHD was hardly metabolised by the liver microsomes and showed a stable metabolic pattern similar to that of LMWH. In a rat thrombosis model, 10 mg/kg of orally administered LHD reduced thrombus formation by 60.8%, which was comparable to the anti-thrombotic effect of the subcutaneously injected LMWH (100 IU/kg). Solid tablets of LHD exhibited high oral absorption and statistically significant therapeutic effects in preventing venous thromboembolism. Accordingly, LHD tablets are expected to satisfy the unmet medical need for an oral heparin-based anticoagulant as an alternative to injectable heparin and oral warfarin.

Pharmacodynamic activity of amphotericin B deoxycholate is associated with peak plasma concentrations in a neutropenic murine model of invasive pulmonary aspergillosis.

We conducted a dose fractionation study of neutropenic, corticosteroid-immunosuppressed mice to characterize the pharmacodynamic/pharmacokinetic (PK/PD) parameter most closely associated with amphotericin B (AMB) efficacy in the treatment of invasive pulmonary aspergillosis. Pharmacokinetic parameter estimates were determined by a nonparametric population pharmacokinetic analysis of plasma drug concentrations following single intraperitoneal doses (0.25, 1.0, and 3.0 mg/kg of body weight) of amphotericin B deoxycholate. Three dosage groups (0.5, 0.75, and 1.0 mg/kg) fractionated into three dosing intervals (every 8 h [q8h], q24h, or q72h) were tested to discriminate between the PK/PD parameters (the ratio of maximum concentration of drug in serum [Cmax]/MIC, the ratio of area under the concentration-time curve/MIC, and percentage of time above MIC) most closely associated with AMB efficacy over a range of clinically achievable exposures in humans. The efficacy of each regimen was determined by quantitative PCR and survival. Reductions in pulmonary fungal burden and improvements in survival were maximized at the highest peak plasma concentrations in each of the dosage groups. Reductions in pulmonary fungal burden and increased survival were most closely associated with Cmax/MIC, with maximal activity occurring as the Cmax/MIC approached 2.4. In our model, Cmax/MIC is the PK/PD parameter most closely associated with efficacy in the treatment of invasive pulmonary aspergillosis. These data predict that less frequently administered, higher dosages of AMB would optimize efficacy.

Oral delivery of chemical conjugates of heparin and deoxycholic acid in aqueous formulation.

INTRODUCTION: Heparin, one of the most potent anticoagulants widely used for the treatment and prevention of deep vein thrombosis (DVT) and pulmonary embolism (PE), is currently available to patients only by parental administration. In this study, we propose a new oral delivery system of heparin by conjugating it with deoxycholic acid which reformulated by adding dimethyl sulfoxide to increase its bioavailability. MATERIALS AND METHODS: The chemical conjugates (LMWH-DOCA) of low molecular weight heparin (4.5 kDa) with deoxycholic acid (DOCA) were synthesized by controlling the conjugation ratio. The absorption of LMWH-DOCA after its oral administration was measured by anti-FXa assay according to the conjugation ratio of DOCA, concentration of DMSO solution and dose of LMWH-DOCA, respectively. Furthermore, the incidences of mucosal damage by LMWH-DOCA in 10% DMSO solution were evaluated using H&E staining and SEM. RESULTS: Three kinds of LMWH-DOCA were synthesized according to the DOCA conjugation ratios of LD1, LD2 and LD3, whose anticoagulant activities were 89, 86 and 85 IU/mg, respectively, and the activity of LMWH was 97 IU/mg. LMWH-DOCA was completely dissolved in 10% DMSO solution, and its bioavailability in the oral dose was significantly increased (17.6% for LD2 in 10% DMSO solution) without causing any damages in intestinal tissues. CONCLUSIONS: The chemical conjugate of heparin and DOCA in the soluble state could be efficiently absorbed in the intestine. Therefore, we propose this system as a new strategy of oral heparin delivery for the treatment of patients who are at high risk to DVT and PE.

Physicochemical characteristics and in vivo deposition of liposome-encapsulated tea catechins by topical and intratumor administrations.

Tea polyphenols, including (+)-catechin, (-)-epicatechin, and (-)-epigallocatechin-3-gallate (EGCG), have been shown to possess potent antioxidant and anticancer activities. The aim of this study was to evaluate the possibility of using liposomes for the local delivery, including skin and tumor deposition, of these polyphenols. Liposomes containing egg phosphatidylcholine, cholesterol, or anionic species were prepared by a solvent evaporation method and then were subjected to a probe sonicator. The size, zeta potential and entrapment efficiency of these liposomal formulations were determined to provide correlations with results from a subsequent in vivo study. The release rate study showed that inclusion of an anionic species, such as deoxycholic acid (DA) or dicetyl phosphate (DP), increased the permeability of the lipid bilayers, leading to the rapid release of these formulations. No significant increase in skin deposition of catechins was observed after topical application of liposomes. On the other hand, a greater amount of catechins were delivered into the solid tumor by liposomes than by the aqueous solution. The drug release rate and vesicle size of liposomes may influence drug deposition in tumor tissues. The isomers, (+)-catechin and (-)-epicatechin, showed different physicochemical properties in liposomes and for local deposition in the skin and tumor. Finally, the presence of gallic acid ester in the structure of EGCG significantly increased the tissue uptake of catechins.

Effect of mixed micelle formulations including terpenes on the transdermal delivery of diclofenac.

The significant inhibitory action of diclofenac formulated in mixed micelles of lecithin with cholate or deoxycholate was observed on the rat hind paw edema induced by carrageenan. In the primary stage, mixed micelle formulation of deoxycholate was more effective compared with that of cholate. However, in the final term, the inhibitory action was similar in both formulations. In a previous study, the flux of diclofenac was greater in the mixed micelle formulation of deoxycholate compared with that of cholate. It was suggested that the permeation rate of diclofenac through skin was proportional to the pharmacological activity. The hind paw edema was quickly inhibited when cyclic monoterpene such as d-limonene or l-menthol was included in the formulations. All the micelle formulations significantly decreased the value of AUC estimated the hind paw thickness-time profile. This suggests that the micelle formulation of cholate in addition to deoxycholate showed significant anti-inflammatory activity to hind paw edema of rats. Incorporation of d-limonene or l-menthol was more effective on the decrease of AUC. A pharmacological study revealed that micelle formulations were able to reduce the skin irritation of chemicals.

Efficacies of amphotericin B-desoxycholate (Fungizone), liposomal amphotericin B (AmBisome) and fluconazole in the treatment of systemic candidosis in immunocompetent and leucopenic mice.

The in-vitro activities of amphotericin B-desoxycholate (AmB-DOC), liposomal amphotericin B (L-AmB) and fluconazole were determined against a single strain of Candida albicans. In addition, the efficacies of these agents in the treatment of systematic candidosis in both immunocompetent and leucopenic mice were compared. The minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of AmB-DOC and L-AmB were similar, but on the basis of time-kill studies, the fungicidal activity of L-AmB was significantly less than that of AmB-DOC. In immunocompetent mice, the dosage of AmB-DOC was limited by toxicity, resulting in a maximum tolerated dosage (MTD) of 0.4 mg/kg/day during treatment for 5 days; L-AMB was less toxic, the MTD being 7 mg/kg/day following a treatment period of the same duration. Both AmB-DOC and L-AmB led to significant reductions in the numbers of C. albicans in the kidneys of these mice and prevented relapse of infection after completion of treatment. Fluconazole in dosages of 0.4 and 64 mg/kg/day resulted in initial reductions in the numbers of cfu but failed to prevent relapse. In leucopenic mice, treatment for 5 days with AmB-DOC in a dosage of 0.3 mg/kg/day resulted in survival of the animals and a significant reduction in the numbers of cfu in the liver, spleen and lungs. However, there was no reduction in the number of cfu in the kidneys and this led to relapse of infection once therapy was terminated. Fluconazole in a dosage of 64 mg/kg/day produced effects which were similar to those of AmB-DOC; prolonged treatment with fluconazole for 18 days did not improve the efficacy of this agent. Only treatment with high-dosage (7 mg/kg/day) L-AmB was effective in significantly reducing the numbers of cfu of C. albicans in the kidneys and other organs of leucopenic mice, as well as preventing relapse, even in severely infected animals.

Metabolism of 3,7-dioxo-5 beta-cholanoic acid in the biliary fistula rodents and rabbits.

Intestinal absorption and metabolism of 3,7-dioxo-5 beta-cholanoic acid, were studied in the bile fistula rats, hamsters, guinea-pigs and rabbits. The influence of dose (1 and 100 mg/kg) on the absorption and the metabolism was also estimated. The dioxo bile acid was absorbed efficiently from the intestine and quickly excreted into bile in these animals. Large dose did not retard the absorption rate and showed a significant choleretic effect for a few hours. Species differences were observed in the metabolism of this compound. In hamsters and guinea-pigs, most of the metabolites in the bile were conjugated with either taurine or glycine. The proportion of bile acids amidated with glycine was greater with the large dose. In rats, the biliary metabolites were conjugated with taurine, but not with glycine, whereas in rabbits, glycine conjugates were the only recovered metabolites. Unconjugated metabolites were also detected in the bile of the rodents, and the proportion of them rose to 17-29% after the administration of 100 mg/kg quantities. A small part of unchanged 3,7-dioxo-5 beta-cholanoic acid was excreted into the bile as both the conjugated and unconjugated forms in these animals. The greater part of this compound administered was metabolized to 7-ketolithocholic acid, chenodeoxycholic acid and ursodeoxycholic acid. In hamsters and guinea-pigs, chenodeoxycholic acid was a greater metabolite of this compound than ursodeoxycholic acid, while in rats and rabbits, the amount of ursodeoxycholic acid exceeded that of chenodeoxycholic acid. In rats, the resulting dihydroxy bile acids were further metabolized to alpha- and beta-muricholic acids.