Clinical Value Of Serum Levels Of 11-Oxygenated Metabolites Of Testosterone In Women With Polycystic Ovary Syndrome

Context/Objective Recent data suggested that 11-oxygenated androgens may be the preponderant circulating androgens in women with PCOS. However, the pathophysiological significance of these hormones remains unclear. The aim of this study was to evaluate the relationships between serum 11-OH testosterone (11-OHT) and 11-Keto testosterone (11-KetoT) and clinical and biochemical hyperandrogenism, as well as the metabolic parameters, in women with PCOS. Subjects One hundred and twenty-three women with PCOS, diagnosed according to the Rotterdam criteria, and 38 healthy controls. Design The main classic and 11-oxygenated androgens were measured by LC-MS/MS and direct equilibrium dialysis. Insulin sensitivity was assessed by hyperinsulinemic euglycemic clamp. Results Serum 11-oxygenated androgens were higher in women with PCOS than in controls. Elevated levels of 11-OHT and 11-KetoT were found in 28.5% and 30.1% of PCOS women, respectively, whereas free testosterone (FT) was increased in 61.0% of them. Serum 11-oxygenated androgens showed a limited performance in recognizing women with classically-defined hyperandrogenism. Unlike FT, 11-oxygenated androgens did not show significant relationships with anthropometric and metabolic parameters, except for a direct association with insulin sensitivity. In multivariable analysis, 11-OHT and 11-KetoT, directly, and FT, inversely, remained significant independent predictors of insulin sensitivity. Conclusions Serum levels of 11-oxygenated androgens are higher in women with PCOS than in controls. However, these hormones show a poor performance in recognizing women with hyperandrogenism, as currently defined. The relationships of these androgens with insulin sensitivity strongly differ from that of FT, suggesting a different role of classic and 11-oxygenated androgens in the pathophysiology of PCOS.

Protein Binding of a Novel Platinum-Based Anticancer Agent BP-C1 Containing a Lignin-Derived Polymeric Ligand

Platinum (Pt) antineoplastic agents remain indispensable for the treatment of oncological disease. Pt-based drugs are mainly used in the therapy of ovarian cancer and non-small-cell lung carcinoma. A novel platinum-containing antineoplastic agent BP-C1 is a complex of diamminoplatinum with an oxygen-donor polymeric ligand of benzene-polycarboxylic acids, isolated from natural lignin. The aim of the study was to investigate ex vivo protein binding of BP-C1. Protein binding of BP-C1 was tested using equilibrium dialysis. Pooled blood plasma was used in the study. Control solutions contained the same dosages of BP-C1 in PBS (pH 7.2). Plasma and control solutions were submitted to equilibrium dialysis across a vertical 8 kDa cut-off membrane for 4 h at 37 °C under gentle shaking. Platinum was quantified in dialysis and retained fractions using inductively coupled plasma mass spectrometry after microwave digestion. The dialysis system was tested and validated; this showed no protein saturation with platinum. A medium degree of binding of platinum to macromolecular species of ca. 60% was observed. The study showed the maintenance of a high fraction of free BP-C1 in the bloodstream, facilitating its pharmacological activity.

DDRE-08. A BIOANALYTICAL LC-MS/MS ASSAY TO QUANTIFY TOTAL AND UNBOUND LY3214996, ABEMACICLIB AND ITS ACTIVE METABOLITES IN HUMAN PLASMA, CEREBROSPINAL FLUID AND HUMAN GLIOBLASTOMA

BACKGROUND Here, we report on our development and validation of a sensitive and rapid LC-MS/MS method for the determination of total and unbound concentrations of ERK inhibitor LY3214996, CDK4/6 inhibitor abemaciclib and its M2 and M20 active metabolites in human plasma, cerebrospinal fluid and human glioblastoma tissue. METHODS Analytes were extracted from patient plasma, CSF and glioblastoma homogenate samples by protein precipitation with methanol. Four levels of quality controls were used during validation. The method was validated according to FDA guidelines and CAP/CLIA regulations. Equilibrium dialysis was performed to determine unbound fraction of four analytes in plasma and brain tissue. The detection was performed on Sciex QTRAP 6500+ mass spectrometer in positive electrospray ionization mode. RESULTS The method was validated over a concentration range from 0.2-500 nmol/L for all four analytes. The analytical separation was optimized on Phenomenex Kinetex™ F5 column with total run time of 3.8 min using gradient elution. For all the analytes the maximum coefficient of variation for intra- and inter-day precision was 12.0% and the accuracy was within 90.2-119.7% in all matrixes. The analytes are stable in plasma and brain homogenate for at least 19 hours and 6 hours at room temperature (RT), respectively. Stability of stock and working solutions was demonstrated for at least 15 hours (RT) and 28 days (2-8°C). The unbound fraction of the analytes in pooled human plasma and brain were determined to be 0.371 and 0.065 for LY3214996, 0.026 and 0.013 for abemaciclib, 0.052 and 0.008 for M2, 0.024 and 0.021 for M20, respectively. CONCLUSIONS A bioanalytical method to quantify LY3214996, abemaciclib and its M2 and M20 metabolites is successfully developed and validated. The method is currently applied to evaluate plasma pharmacokinetics and CNS penetration of LY3214996 and abemaciclib in recurrent glioblastoma patients in an ongoing Phase 0 clinical trial (NCT04391595).

EXTH-09. TUMOR PHARMACOKINETICS, PHARMACODYNAMICS AND RADIATION SENSITIZATION IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA TREATED WITH THE AURORA KINASE A INHIBITOR LY3295668

Cell-cycle deregulation is at the crux of all malignancies, including glioblastoma (GBM). Aurora Kinase A (AURKA) plays a central role in G2/M transition and faithful chromosome segregation. In this study, we evaluated the pharmacokinetics, pharmacodynamics, and radiation sensitization properties of LY3295668, a highly specific AURKA inhibitor, in orthotopic patient-derived xenograft (PDX) models of GBM. Mice with intracranial tumors were randomized to 50 mg/kg LY3295668 PO BID x 4 days vs. placebo. LY3295668 levels in plasma and contrast-enhancing tumor tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unbound fractions were determined by equilibrium dialysis. Immunohistochemistry was performed to assess levels of pAURKA (T288), phospho-Histone H3 (pHH3), and cleaved caspase 3 (CC3). For survival studies, mice with intracranial tumors were randomized to four cohorts – vehicle, radiotherapy, LY3295668 monotherapy, and LY3295668 plus radiotherapy. The median unbound concentration of LY3295668 was 270.88 nmol/L and 22.33 nmol/kg in plasma and tumor tissue, respectively – significantly higher than the biochemical IC50 of LY3295668 for AURKA inhibition (0.8 nM). A decrease in pHH3(+) cells (0.8% vs. 6.4%, p=0.036) indicated drug-induced mitotic arrest and was accompanied by an increase in CC3(+) cells (6.4% vs. 8.0%, p=0.67). Combination of LY3295668 with radiotherapy prolonged survival compared to either therapy alone in orthotopic GBM PDX models. LY3295668 is well tolerated, achieves pharmacologically-relevant unbound concentrations in GBM PDX models, and is associated with significant target modulation. Preclinical combination of LY3295668 with radiation therapy leads to synergistic effects and supports future clinical study of this multimodal strategy in glioblastoma patients.

EXTH-24. TUMOR PHARMACOKINETICS, PHARMACODYNAMICS AND RADIATION SENSITIZATION IN PATIENT-DERIVED XENOGRAFT MODELS OF GLIOBLASTOMA TREATED WITH THE SECOND-GENERATION HDAC INHIBITOR, QUISINOSTAT

BACKGROUND Class I Histone deacetylases (HDACs) are highly expressed in glioblastoma (GBM) and are considered promising therapeutic targets for cancer treatment. Quisinostat is a class I HDAC inhibitor with high specificity for HDAC1 and 2. In this study, we evaluated the pharmacokinetics (PK), pharmacodynamics (PD), and radiation sensitization properties of quisinostat in orthotopic and flank human glioma models of GBM. METHODS In vitro drug-uptake and PK-PD correlation experiments were conducted in patient-derived glioma stem cell (GSC) lines. Pilot PK studies were performed in mice to optimize the dosing regimen and administration route (subcutaneous (s.q.), intraperitoneal (i.p.) and oral gavage). Athymic mice with intracranial or flank tumors were treated with the most optimized treatment schedule to evaluate plasma PK profile and brain/tumor distribution of quisinostat. The drug levels in plasma, normal brain, and tumor-tissue were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unbound fractions were determined by equilibrium dialysis. Western blot analysis and immunocytochemistry were performed to assess changes in acetylated histone, Ki67 (proliferation) and cleaved caspase 3 (cell death) levels. Tumor-bearing mice were randomized to four cohorts – vehicle, radiotherapy, quisinostat monotherapy, and quisinostat plus radiotherapy. RESULTS Quisinostat exhibited potent growth inhibition in multiple GSC lines (IC50 ~60 nM), radiosensitization, increased histone acetylation, and decreased expression of key stem cell markers. Significant toxicity was observed at 10 mg/kg s.q. dosing while the same dosing via i.p. route was tolerated relatively well. The unbound concentration of quisinostat was 19 nmol/L, 0.03 nmol/kg and 0.03 nmol/kg in plasma, tumor, and brain tissue, respectively, at 2 hours post-dose. Pharmacodynamic changes were observed in flank but not in intracranial tumors. CONCLUSION Our data indicate low free quisinostat levels in the brain primarily due to drug instability.

Buffer composition affects rose bengal dialysis rate through cellulose membrane

Due to its fluorescent and phototoxic properties, rose bengal (RB) is used in photodynamic therapy. To improve the delivery of RB to its site of action, the application of nanocarrier systems has been proposed. The most promising approach includes the use of pH-responsive nanoparticles. To evaluate the pattern of drug release in different buffers, equilibrium dialysis is commonly used. Here, we used water and two buffers to determine the impact of solvent composition on the aggregation and dialysis rate of RB through a cellulose membrane. The results show that buffer composition does not influence the fluorescent properties of RB. However, the presence of additional ions causes a change in diffusion rate that is most probably linked to the size of RB aggregates.

Potential and practical implementation of bioactive polyvinylsuccinate derivatives

A review of the works reflecting the authors' research on the synthesis and study of the physico-chemical properties of polymer biologically active substances (BAS) is presented. The research results are analyzed in relation to the specific biological activity of polymers in order to establish the possibility of predicting their prolonged bioactive effect based on the values of the constants of formation, stability, dissociation and other characteristics of complexes of ionogenic polymers and BAS using methods of potentiometric titration, conductometry, viscometry, thermal analysis methods, equilibrium dialysis, biological tests.

Distribution, metabolism, excretion and toxicokinetics of vitexin in rats and dogs

Background: Vitexin is the main bioactive compound of hawthorn (Crataegus pinnatifida), a famous traditional Chinese medicine, and vitexin for injection is currently in phase I clinical trial in China. Objective: This investigation systematically evaluated the metabolism and toxicokinetics of vitexin in rats and dogs. Methods: Rats and beagle dogs were administrated different doses of vitexin, and then the plasma concentration, tissue distribution, excretion, metabolism, pharmacokinetics and plasma protein binding were investigated. Results : The elimination half-life (t1/2) values in rats after a single intravenous dose of 3, 15 and 75 mg/kg were estimated as 43.53±10.82, 22.86±4.23, and 21.17±8.64 min, and the values of the area under the plasma concentration-time curve (AUC0→∞) were 329.34±144.07, 974.79±177.27, and 5251.49±786.98 mg•min/L, respectively. The plasma protein binding rate in rats was determined as about 65% by equilibrium dialysis after 72 hr. After 24 hr of intravenous administration, 16.30%, 3.47% and 9.72% of the given dose were excreted in urine, feces and bile, respectively. The metabolites of the vitexin were hydrolyzed via deglycosylation. The pharmacokinetics of dogs after intravenous administration revealed t1/2, AUC0-∞ and mean residence time (MRT0-∞) values of 20.43±6.37 min, 227.96±26.68 mg•min/L and 17.12±4.33 min, respectively. The no-observed-adverse-effect level (NOAEL) was 50 mg/kg body weight/day. There was no significant accumulation effect at 8 or 20 mg/kg/day in dogs over 92 days of repeated administration. For the 50 mg/kg/day dose group, the exposure (AUC, Cmax) decreased significantly with prolonged administration. This trend suggests that repeated administration accelerates vitexin metabolism. Conclusion: The absorption of vitexin following routine oral administration was very low. To improve the bioavailability of vitexin, the development of an injectable formulation would be a suitable alternative choice.

Solid-phase microextraction for assessment of plasma protein binding, a complement to rapid equilibrium dialysis

Aim: Determination of plasma protein binding ( PPB) is considered vital for better understanding of pharmacokinetic and pharmacodynamic activities of drugs due to the role of free concentration in pharmacological response. Methodology & results: Solid-phase microextraction (SPME) was investigated for measurement of PPB from biological matrices and compared with a gold standard approach (rapid equilibrium dialysis [RED]). Discussion & conclusion: SPME-derived values of PPB correlated well with literature values, and those determined by RED. Respectively, average protein binding across three concentrations by RED and SPME was 33.1 and 31.7% for metoprolol, 89.0 and 86.6% for propranolol and 99.2 and 99.0% for diclofenac. This study generates some evidence for SPME as an alternative platform for the determination of PPB.

Insulin-Mediated Substrate Utilization in Women with the Different Phenotypes of PCOS: Role of Androgens

Context/Objective Few studies have explored in vivo insulin action on substrate utilization in women with PCOS. In particular, no data are available in women with different PCOS phenotypes. The aim of the study was to evaluate insulin action on glucose (Gox) and lipid (Lox) oxidation, non-oxidative glucose metabolism (Gnonox), and serum free fatty acid (FFA) in different PCOS phenotypes. Subjects One hundred and eighty-seven non-diabetic women with PCOS, diagnosed according to the Rotterdam criteria. Data from a historical sample of 20 healthy women were used as reference values. Design Whole body substrate utilization data were obtained by the hyperinsulinemic euglycemic clamp associated with indirect calorimetry. Serum androgens were assessed by liquid chromatography-mass spectrometry and equilibrium dialysis. Results During hyperinsulinemia, the increase of Gox (Δ Gox), Gnonox, as well as the suppression of Lox (Δ Lox) and serum FFA (Δ% FFA) were altered in each PCOS phenotype. Moreover, Gnonox and Δ% FFA were lower in women with the classic phenotype than in those with the ovulatory or the normoandrogenic phenotypes, and Δ Gox was lower in women with the classic than in those with the ovulatory phenotype. In multivariate analysis fat mass and free testosterone were independent predictors of Δ Gox, Gnonox and Δ% FFA, whereas only fat mass predicted Δ Lox. Conclusions In women with PCOS, regardless of their phenotypes, insulin-mediated substrate utilization is impaired. This phenomenon is greater in subjects with the classic phenotype. Free testosterone plays an independent role on insulin action abnormalities on glucose and lipid metabolism.