Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans.

BACKGROUND AND AIMS: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. APPROACH AND RESULTS: Fah-/- , Rag2-/- , and Il2rg-/- knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. CONCLUSIONS: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.

Discovery of a novel series of guanidinebenzoates as gut-restricted enteropeptidase and trypsin dual inhibitors for the treatment of metabolic syndrome.

A novel series of guanidinebenzoate enteropeptidase and trypsin dual inhibitors has been discovered and SAR studies were conducted. Optimization was focused on improving properties for gut restriction, including increased aqueous solubility, lower cellular permeability, and reduced oral bioavailability. Lead compounds were identified with efficacy in a mouse fecal protein excretion study.

Pharmacokinetics and metabolism of LNP023 in rats by liquid chromatography combined with electrospray ionization-tandem mass spectrometry.

In this study, a simple and sensitive LC-tandem mass spectrometric method was developed and validated for the determination of LNP023 in rat plasma. The plasma sample was precipitated with acetonitrile and then separated on an ACQUITY HSS T3 column (50 mm × 2.1 mm, 1.8 µm) using 0.1% formic acid in water and acetonitrile as the mobile phase. The MS detection was performed in positive multiple-reaction monitoring mode with precursor-to-product ion transitions of m/z 423.3 → 174.1 and m/z 435.3 → 367.1 for LNP023 and olaparib (internal standard), respectively. The developed assay was validated in the linear range of 0.1-1000 ng/mL with correlation coefficient (r) greater than 0.9992. The validation parameters were all within the acceptable limits. The validated method has been successfully used to investigate the pharmacokinetics of LNP023 in rat plasma, and our results indicated that LNP023 showed low clearance and high bioavailability (62.2%). Furthermore, four minor metabolites from rat plasma were detected and identified by LC combined with high-resolution mass spectrometry. The metabolic pathways were O-deethylation (M1), hydroxylation (M4), oxidation (M3), and acyl-glucuronidation (M2).

Targeted Delivery of Soluble Guanylate Cyclase (sGC) Activator Cinaciguat to Renal Mesangial Cells via Virus-Mimetic Nanoparticles Potentiates Anti-Fibrotic Effects by cGMP-Mediated Suppression of the TGF-ß Pathway.

Diabetic nephropathy (DN) ranks among the most detrimental long-term effects of diabetes, affecting more than 30% of all patients. Within the diseased kidney, intraglomerular mesangial cells play a key role in facilitating the pro-fibrotic turnover of extracellular matrix components and a progredient glomerular hyperproliferation. These pathological effects are in part caused by an impaired functionality of soluble guanylate cyclase (sGC) and a consequentially reduced synthesis of anti-fibrotic messenger 3',5'-cyclic guanosine monophosphate (cGMP). Bay 58-2667 (cinaciguat) is able to re-activate defective sGC; however, the drug suffers from poor bioavailability and its systemic administration is linked to adverse events such as severe hypotension, which can hamper the therapeutic effect. In this study, cinaciguat was therefore efficiently encapsulated into virus-mimetic nanoparticles (NPs) that are able to specifically target renal mesangial cells and therefore increase the intracellular drug accumulation. NP-assisted drug delivery thereby increased in vitro potency of cinaciguat-induced sGC stabilization and activation, as well as the related downstream signaling 4- to 5-fold. Additionally, administration of drug-loaded NPs provided a considerable suppression of the non-canonical transforming growth factor ß (TGF-ß) signaling pathway and the resulting pro-fibrotic remodeling by 50-100%, making the system a promising tool for a more refined therapy of DN and other related kidney pathologies.

Comprehensive characterization and resolution of discrepant spectrophotometric bilirubin results in patients on eltrombopag therapy.

Background Eltrombopag is a thrombopoietin receptor agonist used for the treatment of thrombocytopenic conditions. It can cause pH-dependent discoloration of plasma/serum. Eltrombopag is potentially hepatotoxic. It can affect the assessment of hyperbilirubinemia because of its (i) absorbance at ~450 nm (bilirubin), (ii) absorbance at ~550 nm (diazo-bilirubin) and (iii) it can cause yellowish discoloration of the eyes at normal circulating bilirubin levels. Methods We collected 66 samples from patients on a range of eltrombopag dosages up to 150 mg daily. Bilirubin was measured using multiple routine spectrophotometric analyzers, the Doumas reference method and high-performance liquid chromatography (HPLC). Plasma/serum eltrombopag concentrations were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spike-in and admixture experiments delineated the effects of eltrombopag and its metabolites. Results Forty-nine of 52 samples from patients on ≥50 mg daily eltrombopag therapy showed significantly discrepant inter-analyzer total bilirubin results, a difference up to 64 µmol/L (3.7 mg/dL). In one sample, total bilirubin varied from 8 to 65 µmol/L (0.4-3.8 mg/dL) by different routine analyzers, with direct bilirubin ≤4 µmol/L (0.2 mg/dL). There was a positive correlation between total bilirubin difference and plasma eltrombopag concentration (r = 0.679), and spike-in experiments demonstrated that Beckman AU and Doumas reference methods were susceptible to positive interference. HPLC can quantify bilirubin after separating eltrombopag, and results suggest different analyzers are affected to varying degrees by eltrombopag and its metabolites. Conclusions Eltrombopag and its metabolites can cause positive interference to the spectrophotometric measurements of total bilirubin. Accurate measurements of total bilirubin may improve our understanding of the prevalence of hyperbilirubinemia in patients on eltrombopag therapy.

Nine absorbed components pharmacokinetic of raw and processed Moutan Cortex in normal and blood-heat and hemorrhage syndrome model rats.

Raw Moutan Cortex (RMC) and Processed Moutan Cortex (PMC) have a long history of use in China and other Asian countries. In this study, a rapid and accurate ultra-high-pressure liquid chromatography coupled with diode array detector (UHPLC-DAD) method was developed and validated for the simultaneous determination of nine absorbed compounds of RMC/PMC. After extraction by protein precipitation with methanol from plasma, the analytes were separated on an Acquity UPLC® BEH Shield RP18 column (2.1 × 100 mm, 1.7 µm, Waters, USA). Acetonitrile (A) and 0.1% (v/v) formic acid in water (B) were selected as the mobile phase to perform gradient elution. The linearity of nine analytes was >0.9915. The intra- and inter-assay precision (RSD) values were within 11.18%, and accuracy ranged from 91.32 to 101.29%. Suitable stability, matrix effect and extraction recoveries were also obtained. The validated method was applied to compare the pharmacokinetics of RMC and PMC in Blood-Heat and Hemorrhage Syndrome Model and normal rats. The results revealed that processing and the pathological state could influence the pharmacokinetic characteristics of compounds in RMC/PMC. The study willbe useful for further studies on pharmacokinetics and clinical application of raw and processed Moutan Cortex.

Novel spectrofluorimetric quantification of alogliptin benzoate in biofluids exploiting its interaction with 4-chloro-7-nitrobenzofurazan.

The direct determination of alogliptin benzoate (ALO) using fluorescence has not yet been accomplished because ALO cannot fluoresce naturally. Accordingly, it should be derivatized first with a fluorogenic reagent to enhance the sensitivity required for its bioanalysis. This method is the first spectrofluorimetric assay for ALO quantification exploiting the nucleophilic nature of its amino group to react with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer at pH 8.5 to produce a strong fluorescent compound that is excited at and emits at wavelengths 470 and 527 nm, respectively. Experimental variables concerning the conditions of reaction and fluorogenic intensity were carefully investigated and optimized. Linearity was from 1-250 ng ml-1 with a lower detection limit of 0.29 ng ml-1 and a lower quantification limit of 0.88 ng ml-1 . Validation of the current study was accomplished with mean per cent recovery of 100.62 ± 1.59 in tablets and 99.86 ± 0.82 in human plasma. Furthermore, the current method has been utilized in the bioanalysis of ALO in real rat plasma after oral administration with a simple specimen preparation. The developed method has proven to be a promising alternative method for ALO analysis in bioequivalence studies.

Class IIb HDAC Inhibition Enhances the Inhibitory Effect of Am80, a Synthetic Retinoid, in Prostate Cancer.

Combination therapy is often an effective strategy to treat cancer. In this study, we examined the growth-inhibitory effects of Am80 (tamibarotene), a specific retinoic acid receptor (RAR) α/ß agonist, in combination with a histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), or a DNA methyl transferase (DNMT) inhibitor, 5-aza-2'-deoxycytidine, on androgen receptor (AR)-positive and AR-negative prostate cancer cell lines (LNCaP and PC-3, respectively). We found that the combination therapy of SAHA and Am80 showed an enhanced growth-inhibitory effect on LNCaP cells. Further studies with various HDAC isotype-selective inhibitors showed that SAHA and KD5170 (a selective class I and II HDAC inhibitor) each increased the RARα protein level in LNCaP cells. Our results indicate that the target of the enhancing effect belongs to the Class IIb HDACs, especially HDAC6. Dual targeting of Class IIb HDAC and RARα may be a candidate therapeutic strategy for prostate cancer.

Effect of pharmacogenetic markers of vitamin D pathway on deferasirox pharmacokinetics in children.

OBJECTIVES: Patients with ß-thalassemia major have extremely low vitamin D levels, owing to reduced intestinal absorption, subicteric tint, and/or iron-induced higher pigmentation. We investigated whether some polymorphisms within the VDR, CYP24A1, CYP27B1, and GC genes could play a role in deferasirox pharmacokinetics in a cohort of pediatric patients. PATIENTS AND METHODS: Eighteen children with ß-thalassemia were enrolled. Drug plasma concentrations at the end of dosing interval (Ctrough) and after 0, 2, 4, 6, and 24 h of drug administration were measured by a HPLC-UV method. Allelic discrimination for VDR (TaqI, FokI, BsmI, Cdx2, and ApaI), CYP24A1 (22776, 3999 and 8620), CYP27B1 (2838 and -1260), and GC (1296) single nucleotide polymorphisms was performed by real-time PCR. RESULTS: CYP24A1 8620 AG/GG group negatively predicted Ctrough in regression analysis (P=0.012). ApaI AA genotype resulted as a negative predictor of Ctrough (P=0.025) and area under the concentration curve (P=0.007); FoKI CC genotype remained as area under the concentration curve positive predictor (P=0.008) and TC/CC group as half-life (t1/2) (P=0.003) and volume of distribution (Vd) (P=0.011) negative one; TaqI TC/CC was retained as a negative predictor of drug maximum concentration (Cmax) (P=0.004). Moreover, GC 1296 TG/GG seemed able to predict lower time to reach drug maximum concentration (Tmax) (P=0.033). CONCLUSION: Our preliminary experience suggested the potential usefulness of vitamin D pharmacogenetic to better understand deferasirox interindividual variability, also in pediatric patients.

A randomized trial to study the comparative efficacy of phenylbutyrate and benzoate on nitrogen excretion and ureagenesis in healthy volunteers.

PURPOSE: Benzoate and phenylbutyrate are widely used in the treatment of urea cycle disorders, but detailed studies on pharmacokinetics and comparative efficacy on nitrogen excretion are lacking. METHODS: We conducted a randomized, three-arm, crossover trial in healthy volunteers to study pharmacokinetics and comparative efficacy of phenylbutyrate (NaPB; 7.15 g•m-2BSA•day-1), benzoate (NaBz; 5.5 g•m-2BSA•day-1), and a combination of two medications (MIX arm; 3.575 g NaPB and 2.75 g NaBz•m-2BSA•day-1) on nitrogen excretion. Stable isotopes were used to study effects on urea production and dietary nitrogen disposal. RESULTS: The conjugation efficacy for both phenylbutyrate and benzoate was 65%; conjugation was superior at the lower dose used in the MIX arm. Whereas NaPB and MIX treatments were more effective at excreting nitrogen than NaBz, nitrogen excretion as a drug conjugate was similar between phenylbutyrate and MIX arms. Nitrogen excreted per USD was higher with combination therapy compared with NaPB. CONCLUSION: Phenylbutyrate was more effective than benzoate at disposing nitrogen. Increasing phenylbutyrate dose may not result in higher nitrogen excretion due to decreased conjugation efficiency at higher doses. Combinatorial therapy with phenylbutyrate and benzoate has the potential to significantly decrease treatment cost without compromising the nitrogen disposal efficacy.

Clinical safety, tolerability, pharmacokinetics and effects on urinary electrolyte excretion of AZD9977, a novel, selective mineralocorticoid receptor modulator.

AIMS: AZD9977 is the first mineralocorticoid receptor modulator in clinical development exerting similar organ protection as eplerenone with minimal urinary electrolyte effects in preclinical studies. The aim was to perform the initial clinical assessment of AZD9977. METHODS: A first-in-human trial explored doses from 5 to 1200 mg. To study effects on urinary electrolyte excretion an additional randomized placebo controlled cross-over four-period clinical trial was performed. Twenty-three healthy volunteers were administered fludrocortisone alone or in combination with AZD9977, eplerenone or both. AZD9977/eplerenone combination was given to assess if AZD9977 can attenuate eplerenone induced natriuresis. RESULTS: AZD9977 at doses from 5 to 1200 mg was safe and well tolerated and pharmacokinetics were compatible with further development. AZD9977 exhibited similar effects on urinary ln [Na+ ]/[K+ ] as eplerenone when using fludrocortisone as mineralocorticoid receptor agonist, and the combination had an additive effect on ln [Na+ K+ ]. CONCLUSIONS: The results in man contradict the results in rodent models driven by aldosterone, in which AZD9977 has minimal electrolyte effects. Future clinical studies with AZD9977 should be performed in presence of endogenous or exogenous aldosterone to assess potential benefit of AZD9977 in patients.

Design and synthesis of a potent, highly selective, orally bioavailable, retinoic acid receptor alpha agonist.

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARß, and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARß (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.

Conversion and pharmacokinetics profiles of a novel pro-drug of 3-n-butylphthalide, potassium 2-(1-hydroxypentyl)-benzoate, in rats and dogs.

Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB) is a novel pro-drug of 3-n-butylphthalide (dl-NBP) that is used to treat ischemic stroke. Currently, dl-PHPB is in phase II-III clinical trials in China. In this study, we investigated the conversion and pharmacokinetics profiles of dl-PHPB in vitro and in vivo. The conversion of dl-PHPB to dl-NBP was pH- and calcium-dependent, and paraoxonase was identified as a major enzyme for the conversion in rat plasma. The pharmacokinetics, tissue distribution and excretion of dl-PHPB were studied and compared with equal-molar doses of dl-NBP in rats and dogs. The in vivo studies showed that dl-PHPB could be quickly and completely converted to dl-NBP. The plasma concentration-time course of converted dl-NBP after intravenous dl-PHPB administration was nearly the same as that after equal-molar dl-NBP. The Cmax and AUC of dl-NBP after oral dl-PHPB administration in rats and dogs were higher by 60% and 170%, respectively, than those after oral dl-NBP administration. Analysis of the tissue distribution of dl-PHPB revealed that converted dl-NBP was primarily distributed in fat, the brain and the stomach. In the brain, the levels of dl-NBP were relatively higher after dl-PHPB treatment by orally than after treatment with equal-molar dl-NBP. Approximately 3%-4% of dl-NBP was excreted within 72 h after dosing with dl-PHPB or dl-NBP, but no dl-PHPB was detected in urine or feces excrements. Our results demonstrate that the conversion of dl-PHPB is fast after oral or intravenous administration. Furthermore, the bioavailability of dl-PHPB was obviously better than that of dl-NBP.

Therapeutic Doses of Eltrombopag do not Inhibit Hepatic BCRP in Healthy Volunteers: Intravenous Ceftriaxone as a Model.

PURPOSE: Ceftriaxone elimination occurs through breast cancer resistance transporter (BCRP) and multidrug resistance-associated protein 2 (MRP-2) which are expressed on the canalicular membrane of hepatocytes. Eltrombopag, a thrombopoetin receptor agonist used in the treatment of immune thrombocytopenic purpura, is reported in in vitro studies as an inhibitor of intestinal BCRP but not an inhibitor of hepatic BCRP. Thus, the present study evaluates the effect of therapeutic doses of eltrombopag on the clinical pharmacokinetics of intravenous ceftriaxone. METHODS: Healthy adult (n=12) were treated with oral doses of eltrombopag (0, 25 or 50 mg) 28 and 4 h prior to intravenous ceftriaxone administration (1g). Serial blood samples were collected up to 48 h after ceftriaxone administration and plasma samples were analysed by LC-MS/MS using 50 µL aliquots (total concentration) and 100 µL (unbound concentration). RESULTS: A method to analyze total and unbound ceftriaxone in plasma using LC-MS/MS was developed and validated with linearity from 1 to 200 µg/mL. Both methods are sensitive, precise and accurate with coefficients of variation less than 15% in the study of inter- and intra-assay precision and accuracy. Ceftriaxone pharmacokinetics in healthy adults were described using a bicompartmental model, with a mean clearance of 0.96 L/h (CI95% 0.71-1.20) and AUC0-∞of 1106 mg.h/mL (CI95% 811-1400) for volunteers that received only ceftriaxone; clearance of 0.95 L/h (CI95% 0.77-1.13) and AUC0-∞ of 1083 mg.h/mL (CI95% 876-1290) for volunteers that received ceftriaxone plus 25 mg of eltrombopag and clearance of 0.96 L/h (CI95% 0.74-1.19) and AUC0-∞ of 1072 mg.h/mL (CI95% 872-1273) for volunteers that received ceftriaxone plus 50 mg of eltrombopag. CONCLUSIONS: The results do not support the existence of a clinical pharmacokinetic drug interaction involving hepatic BCRP in human subjects receiving intravenous ceftriaxone and oral eltrombopag. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Human metabolism and kinetics of tri-(2-ethylhexyl) trimellitate (TEHTM) after oral administration.

Tri-(2-ethylhexyl) trimellitate (TEHTM) is a plasticizer for PVC material and is used for medical devices as an alternative to di-(2-ethylhexyl) phthalate. As plasticizers are known to migrate easily into contact liquids, exposure of patients to TEHTM is highly probable. In the present study, human metabolism pathways of TEHTM and its elimination kinetics were investigated. For that purpose, four healthy volunteers were orally exposed to a single dose of TEHTM. TEHTM and its postulated primary metabolites were investigated in blood samples (up to 48 h after exposure), and in urine samples (collected until 72 h after exposure) using liquid chromatography tandem mass spectrometry (LC-MS/MS). TEHTM was found to be regioselectively hydrolyzed to its diesters di-2-(ethylhexyl) trimellitates (1,2-DEHTM, 2,4-DEHTM) with maximum blood concentrations at 3-h post-exposure, and to its monoester isomers mono-2-(ethylhexyl) trimellitates (1-MEHTM, 2-MEHTM) with peak blood concentrations 5-h post-exposure. For the elimination of investigated urinary metabolites, biphasic elimination kinetics was observed. The most dominant urinary biomarker was found to be 2-MEHTM (2-mono-(2-ethylhexyl) trimellitate), followed by several specific secondary metabolites. All in all, approximately 5.8% of the orally administered dose was recovered in urine over a period of 72 h, indicating a comparatively low resorption rate of TEHTM in humans in combination with an apparently rather slow metabolism and excretion rate. In fact, TEHTM and selected metabolites were still detectable in blood and urine 48-h and 72-h post-exposure, respectively. This study is the first to elucidate TEHTM metabolism pathways in humans and to identify metabolites of TEHTM in blood and urine by usage of especially designed human biomonitoring methods. Powerful tools for exposure monitoring and risk assessment of TEHTM are therewith available for future research.

Pharmacokinetics and antinociceptive effects of the soluble epoxide hydrolase inhibitor t-TUCB in horses with experimentally induced radiocarpal synovitis.

This study determined the pharmacokinetics, antinociceptive, and anti-inflammatory effects of the soluble epoxide hydrolase (sEH) inhibitor t-TUCB (trans-4-{4-[3-(4-Trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid) in horses with lipopolysaccharide (LPS)-induced radiocarpal synovitis. A total of seven adult healthy mares (n = 4-6/treatment) were administered 3 µg LPS into one radiocarpal joint and t-TUCB intravenously (i.v.) at 0 (control), 0.03, 0.1, 0.3, and 1 mg/kg in a blinded, randomized, crossover design with at least 3 weeks washout between. Two investigators independently assigned pain scores (at rest, walk and trot) and lameness scores before and up to 48 hr after t-TUCB/LPS. Responses to touching the joint skin to assess tactile allodynia, plasma, and synovial fluid (SF) t-TUCB concentrations were determined before and up to 48 hr after t-TUCB/LPS. Blood and SF were collected for clinical laboratory evaluations before and up to 48 hr after t-TUCB/LPS. Areas under the curves of pain and lameness scores were calculated and compared between control and treatments. Data were analyzed using repeated measures ANOVA with Dunnett or Bonferroni post-test. p < .05 was considered significant. Data are mean ± SEM. Compared to control, pain, lameness, and tactile allodynia were significantly lower with 1 mg/kg t-TUCB, but not the other doses. For 0.1, 0.3, and 1 mg/kg t-TUCB treatments, plasma terminal half-lives were 13 ± 3, 13 ± 0.5, and 24 ± 5 hr, and clearances were 68 ± 15, 48 ± 5, and 14 ± 1 ml hr-1  kg-1 . The 1 mg/kg t-TUCB reached the SF at high concentrations. There were no important anti-inflammatory effects. In conclusion, sEH inhibition with t-TUCB may provide analgesia in horses with inflammatory joint pain.

Pharmacokinetic and Metabolism Studies of Curculigoside C by UPLC-MS/MS and UPLC-QTOF-MS.

Pharmacokinetic and metabolism studies were carried out on curculigoside C (CC), a natural product with good antioxidant and neuroprotective effects, with the purpose of investigating the effects of the hydroxyl group at C-3' in curculigoside. A rapid and sensitive method with UPLC-MS was developed and fully validated for the first time in the pharmacokinetic analysis for quantification of CC in rat plasma. The assay was linear (R² > 0.9984) over the concentration range of 1⁻2500 ng/mL, with the lower limit of quantification (LLOQ) being 1 ng/mL. The intra-day and inter-day precision (expressed as relative standard deviation, RSD) ranged from 4.10% to 5.51% and 5.24% to 6.81%, respectively. The accuracy (relative error, RE) ranged from -3.28% to 0.56% and -5.83% to -1.44%, respectively. The recoveries ranged from 92.14% to 95.22%. This method was then applied to a pharmacokinetic study of rats after intragastric administration of 15, 30 and 60 mg/kg CC. The results revealed that CC exhibited rapid oral absorption (Tmax = 0.106 h, 0.111 h, and 0.111 h, respectively), high elimination (t1/2 = 2.022 h, 2.061 h, and 2.048 h, respectively) and low absolute bioavailability (2.01, 2.13, and 2.39%, respectively). Furthermore, an investigation on the metabolism of CC was performed by UPLC-QTOF-MSE. Twelve metabolites of CC from plasma, bile, urine and faeces of rats were confirmed. The main metabolic pathways of CC, which involve dehydration, glucosylation, desaturation, formylation, cysteine conjugation, demethylation and sulfonation, were profiled. In conclusion, this research has developed a sensitive quantitative method and demonstrated the metabolism of CC in vivo.

Tamibarotene-Loaded PLGA Microspheres for Intratumoral Injection Administration: Preparation and Evaluation.

Tamibarotene (Am80) has good curative effect on advanced hepatocellular carcinoma (HCC). To improve the therapeutic efficacy furtherly, we prepared tamibarotene-loaded PLGA microspheres (Am80-PLGA-MS) for intratumoral injection. Firstly, Am80-PLGA-MS were prepared by emulsion-solvent evaporation method. Subsequently, microspheres were characterized by particle size analysis, drug loading (DL), and entrapment efficiency (EE). Finally, the drug release characteristics in vitro, pharmacokinetic, and pharmacodynamics were studied separately. According to results obtained, microspheres were spherical with a uniform particle size 7.04 ± 0.03 µm and its EE and DL were 82.23 ± 0.74 and 11.74 ± 0.11%, respectively. In vitro, Am80-PLGA-MS can release drug for 14 days and its release behavior was fitted with the Higuchi equation. In pharmacokinetic studies, the t1/2ß, MRT, and AUC of microspheres were 15.43-fold, 8.62-fold, and 9.98-fold those of Am80 solution, respectively, which revealed that the utilization of drug was improved obviously. The pharmacodynamics studies showed that the tumor doubling time, growth inhibition rate, and specific growth rate of tumor of Am80-PLGA-MS were 1.34 times, 2.63 times, and 0.72 times those of drug solution, respectively, indicating that the inhibitory effect on tumor by the microspheres was significantly improved. In summary, Am80-PLGA-MS are promising carrier to enhance the inhibitory effect on tumor, which will provide significantly clinical value for treatment of HCC.

Multicentre, randomised phase III study of the efficacy and safety of eltrombopag in Chinese patients with chronic immune thrombocytopenia.

Eltrombopag, a thrombopoietin receptor agonist, raises platelet counts and reduces bleeding in patients with immune thrombocytopenia (ITP). In Chinese patients, eltrombopag was evaluated at an initial dose of 25 mg, vs. 50 mg for non-Asians, because the plasma exposure of eltrombopag is higher in East Asians. A multicentre, double-blind, randomised, placebo-controlled, 8-week, phase III study enrolled 155 patients with chronic, previously treated ITP. Dosage could be adjusted (25-75 mg/day) to maintain platelet counts 50-250 × 109 /l. The primary efficacy endpoint was the proportion of patients with a platelet count ≥50 × 109 /l after Day 42. Pharmacokinetics and pharmacodynamics of eltrombopag were analysed in an open-label extension. After Day 42, 57·7% of eltrombopag-treated and 6·0% of placebo-treated patients achieved platelet counts ≥50 × 109 /l. Odds of achieving a platelet count ≥50 × 109 /l were 26·08 times greater with eltrombopag than placebo (P < 0·001). Compared with placebo, time to response and duration of response were better with eltrombopag (P < 0·001) and the odds of any bleeding were reduced by 72% (P = 0·001). Tolerability, pharmacokinetics, and pharmacokinetics/pharmacodynamics were similar to previous findings in East Asian patients. In conclusion, in Chinese patients with chronic ITP, eltrombopag 25 mg once daily, elevated platelet counts to a safe range and reduced bleeding.

Comprehensive Evaluation of the Binding of Lipocalin-Type Prostaglandin D Synthase to Poorly Water-Soluble Drugs.

Low water solubility of candidate drug compounds is a major problem in pharmaceutical research and development. We developed a novel drug delivery system (DDS) for poorly water-soluble drugs using lipocalin-type prostaglandin D synthase (L-PGDS), which belongs to the lipocalin superfamily and binds a large variety of hydrophobic molecules. In this study, we comprehensively evaluated the capability of L-PGDS to bind and solubilize various poorly water-soluble drugs using structure-based docking. Docking simulations of 2892 commercially available approved drugs indicated that L-PGDS shows higher binding affinities for various drugs compared with 2-hydroxypropyl-ß-cyclodextrin. Five drugs selected from the top 100 with the highest binding affinities for L-PGDS exhibited very low solubility in PBS (pH 7.4). However, in the presence of 1 mM L-PGDS, the apparent solubility of all drugs improved markedly, from 19.5- to 166-fold. Calorimetric experiments on two drugs, telmisartan and imatinib, revealed that L-PGDS forms a 1:2 complex with each drug, with dissociation constants of 0.4-40.0 µM. Kinetic simulations of drug dissolution with L-PGDS indicated that the difference in free energy change (ΔΔG) between the insoluble state and the L-PGDS-bound state are within the range from -10 to +5 kJ mol-1. The ΔΔG value is a critical factor in evaluating whether a poorly water-soluble drug can be solubilized by L-PGDS. Collectively, these results demonstrate that in silico docking is a promising approach for identifying drug molecules suitable for the L-PGDS-based DDS.