Design of experiment-oriented development and validation of novel bioanalytical reverse phase high performance liquid chromatography method of palbociclib in rat plasma and tissues, and its application in pharmacokinetic studies.

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0 ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r2 ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies.

Quantification of KRAS inhibitor sotorasib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Sotorasib is a KRAS inhibitor with promising anticancer activity in phase I clinical studies. This compound is currently under further clinical evaluation as monotherapy and combination therapy against solid tumors. In this study, a liquid chromatography-tandem mass spectrometric method to quantify sotorasib in mouse plasma and eight tissue-related matrices (brain, liver, spleen, kidney, small intestine, small intestine content, lung, and testis homogenates) was developed and validated. Protein precipitation using acetonitrile was utilized in 96-well format to extract sotorasib and erlotinib (internal standard) from mouse plasma and tissue homogenates. Separation of the analytes was performed on an Acquity UPLC® BEH C18 column by gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.6 ml/min. Sotorasib was detected by a triple quadrupole mass spectrometer with positive electrospray ionization in selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml of sotorasib was achieved during the validation. Accuracy values were in the range of 90.7-111.4%, and precision values (intra- and interday) were between 1.7% and 9.2% for all tested levels in all investigated matrices. The method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of sotorasib in female wild-type mice.

Chemogenetics Reveal an Anterior Cingulate-Thalamic Pathway for Attending to Task-Relevant Information.

In a changing environment, organisms need to decide when to select items that resemble previously rewarded stimuli and when it is best to switch to other stimulus types. Here, we used chemogenetic techniques to provide causal evidence that activity in the rodent anterior cingulate cortex and its efferents to the anterior thalamic nuclei modulate the ability to attend to reliable predictors of important outcomes. Rats completed an attentional set-shifting paradigm that first measures the ability to master serial discriminations involving a constant stimulus dimension that reliably predicts reinforcement (intradimensional-shift), followed by the ability to shift attention to a previously irrelevant class of stimuli when reinforcement contingencies change (extradimensional-shift). Chemogenetic disruption of the anterior cingulate cortex (Experiment 1) as well as selective disruption of anterior cingulate efferents to the anterior thalamic nuclei (Experiment 2) impaired intradimensional learning but facilitated 2 sets of extradimensional-shifts. This pattern of results signals the loss of a corticothalamic system for cognitive control that preferentially processes stimuli resembling those previously associated with reward. Previous studies highlight a separate medial prefrontal system that promotes the converse pattern, that is, switching to hitherto inconsistent predictors of reward when contingencies change. Competition between these 2 systems regulates cognitive flexibility and choice.

Concentrations of aripiprazole and dehydroaripiprazole in hair segments from deceased individuals with mental disorders.

Segmental hair analysis provides information regarding previous long-term drug exposure, which is useful in the evaluation of cause of death for individuals with mental disorders. The aim was to analyze postmortem concentrations of the antipsychotic drug aripiprazole and its active metabolite dehydroaripiprazole in hair segments from individuals with known aripiprazole intake. Hair samples were collected during autopsy. Each sample was segmented into one to six 1cm segments, depending on the length of the hair shaft. Pulverized hair was extracted and analyzed using a previously published ultra-high-performance liquid chromatography-tandem mass spectrometric method. The 10th-90th percentile of aripiprazole concentrations in all hair segments (n=78) from 17 individuals were 0.024ng/mg-11ng/mg with a median of 2.3ng/mg, and the 10th-90th percentile concentrations of dehydroaripiprazole were 0.020ng/mg-11ng/mg, with a median of 2.6ng/mg, in all hair segments (n=71). The metabolite-to-parent drug ratios ranged from 0.21 to 1.5, with a median of 0.72. The administered doses were calculated for each individual based on aripiprazole prescription data and pharmacy pickups, giving dose estimates of 1mg-32mg daily. A positive significant correlation was observed between concentrations in hair and blood, whereas no trends were observed between the concentrations in hair and the estimated doses. Besides aripiprazole, other antipsychotic drugs were found in several hair segments, indicating a high degree of polypharmacy among all subjects. The present study establishes concentrations of aripiprazole and dehydroaripiprazole in hair segments from 17 deceased individuals with long-term aripiprazole use. In addition, hair analysis demonstrates the possibility of evaluating polypharmacy.

LC-MS/MS Estimation of the Anti-Cancer Agent Tandutinib Levels in Human Liver Microsomes: Metabolic Stability Evaluation Assay.

PURPOSE: Tandutinib (MLN518 or CT 53518) (TND) is a novel, oral, small-molecule inhibitor of type III receptor tyrosine kinases utilized for the treatment of acute myeloid leukemia (AML). MATERIALS AND METHODS: In silico prediction of hepatic drug metabolism for TND was determined using the StarDrop® WhichP450™ module to confirm its metabolic liability. Second, an efficient and accurate LC-MS/MS method was established for TND quantification to evaluate metabolic stability. TND and entrectinib (ENC) (internal standard; IS) were resolved using an isocratic elution system with a reversed stationary phase (C8 column). RESULTS: The established LC-MS/MS method exhibited linearity (5-500 ng/mL) with r2 ≥0.9999 in the human liver microsomes matrix. The method sensitivity was indicated by the limit of quantification (3.8 ng/mL), and reproducibility was revealed by inter- and intraday precision and accuracy (below 10.5%). TND metabolic stability estimation was calculated using intrinsic clearance (22.03 µL/min/mg) and in vitro half-life (29.0 min) values. CONCLUSION: TND exhibited a moderate extraction ratio indicative of good bioavailability. According to the literature, the approach developed in the present study is the first established LC-MS/MS method for assessing TND metabolic stability.

Mass balance, metabolic disposition, and pharmacokinetics of [14C]ensartinib, a novel potent anaplastic lymphoma kinase (ALK) inhibitor, in healthy subjects following oral administration.

PURPOSE: Ensartinib is a novel, potent and highly selective inhibitor of anaplastic lymphoma kinase (ALK) that has promising clinical activity and low toxicity in patients with ALK-positive non-small cell lung cancer. This study was conducted to investigate the pharmacokinetics, metabolism and excretion of ensartinib following a single 200 mg/100 µCi oral dose of radiolabeled ensartinib to healthy subjects. METHODS: Six healthy male subjects were enrolled and administrated an oral suspension in a fasted state. Blood, urine and feces were collected. Radioactivity concentrations were measured by liquid scintillation counting and plasma concentrations of ensartinib by liquid chromatography-tandem mass spectrometry. Both techniques were applied for metabolite profiling and characterization. RESULTS: The mean total recovery was 101.21% of the radiolabeled dose with 91.00% and 10.21% excreted in feces and urine, respectively. Unchanged ensartinib was the predominant drug-related component in urine and feces, representing 4.39% and 38.12% of the administered dose, respectively. Unchanged ensartinib and its metabolite M465 were the major circulating components, accounting for the same 27.45% of the plasma total radioactivity (AUC0-24h pool), while other circulating metabolites were minor, accounting for less than 10%. Mean Cmax, AUC0-∞, T1/2 and Tmax values for ensartinib in plasma were 185 ng/mL, 3827 h ng/mL, 18.3 h and 3.25 h, respectively. The total radioactivity in plasma was cleared with terminal half-life of 27.2 h. Treatment with ensartinib was well tolerated, and no serious adverse events were reported. CONCLUSION: It was well tolerated in the six healthy male subjects following a single oral administration of 200 mg/100 µCi dose of ensartinib. Besides unchanged ensartinib, metabolite of M465 was the predominant circulating drug-related component. The drug was primarily eliminated in feces. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03804541.

The phenomenon of para-Fluorophenylpiperazine (pFPP) in combination with the synthetic cannabinoid AMB-FUBINACA in seized plant material in New Zealand.

New psychoactive substances have emerged as a vast and diverse group of illicit drugs over the past decade, with synthetic cannabinoids comprising the largest of the categories. Commonly, a single synthetic cannabinoid is applied to plant material, creating a product that is designed to be smoked by the user. The clandestine preparation process can result in an unevenly distributed product, with varying concentration within and between plant materials. This investigation describes the novel co-detection of the synthetic cannabinoid AMB-FUBINACA, with the piperazine para-fluorophenylpiperazine (pFPP), in a number of plant material samples analysed in New Zealand in 2017. Of 157 samples of plant material containing AMB-FUBINACA, pFPP was detected in 55 of them. A range of pFPP concentrations was observed between the plant material samples, as well as intra-batch variation. The presence of both drugs may be designed to enhance, prolong or balance the psychoactive effects caused from smoking the plant material. However the intended purpose has not been verified. This is the first reported combination of a synthetic cannabinoid and a piperazine in plant material.

In vitro metabolism of olaparib in liver microsomes by liquid chromatography/electrospray ionization high-resolution mass spectrometry.

RATIONALES: Olaparib is a Poly (ADP-ribose) Polymerase (PARP) inhibitor which has been developed as an anti-cancer agent. The purpose of this study was to characterize the metabolites of olaparib from liver microsomes and to reveal the interspecies differences between animals and humans. METHODS: Olaparib (20 µM) was incubated with different species of liver microsomes at 37°C for 1 h in the presence of NADPH. The incubation samples were analyzed by liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) operated in positive ion mode. The metabolites were characterized by accurate masses, MS2 spectra and retention times. RESULTS: A total of 12 metabolites were detected and the structures of the metabolites were characterized based on their accurate masses, fragment ions and retention times. Four metabolites, i.e., M1, M10, M11 and M12, were unambiguously identified by using reference standards. The metabolic pathways of olaparib included hydroxylation, bis-hydroxylation, hydrolysis, dealkylation, dehydrogenation, and alcohol oxidation. CONCLUSIONS: Compared with animal species, no human-specific metabolite was found in HLM. Dog also had a closer metabolic profile to humans. This study will be helpful for a better understanding of the species difference in pharmacokinetics/pharmacodynamics.

An Activatable Cancer-Targeted Hydrogen Peroxide Probe for Photoacoustic and Fluorescence Imaging.

Reactive oxygen species play an important role in cancer, however, their promiscuous reactivity, low abundance, and short-lived nature limit our ability to study them in real time in living subjects with conventional noninvasive imaging methods. Photoacoustic imaging is an emerging modality for in vivo visualization of molecular processes with deep tissue penetration and high spatiotemporal resolution. Here, we describe the design and synthesis of a targeted, activatable probe for photoacoustic imaging, which is responsive to one of the major and abundant reactive oxygen species, hydrogen peroxide (H2O2). This bifunctional probe, which is also detectable with fluorescence imaging, is composed of a heptamethine carbocyanine dye scaffold for signal generation, a 2-deoxyglucose cancer localization moiety, and a boronic ester functionality that specifically detects and reacts to H2O2. The optical properties of the probe were characterized using absorption, fluorescence, and photoacoustic measurements; upon addition of pathophysiologic H2O2 concentrations, a clear increase in fluorescence and red-shift of the absorption and photoacoustic spectra were observed. Studies performed in vitro showed no significant toxicity and specific uptake of the probe into the cytosol in breast cancer cell lines. Importantly, intravenous injection of the probe led to targeted uptake and accumulation in solid tumors, which enabled noninvasive photoacoustic and fluorescence imaging of H2O2. In conclusion, the reported probe shows promise for the in vivo visualization of hydrogen peroxide. SIGNIFICANCE: This study presents the first activatable and cancer-targeted hydrogen peroxide probe for photoacoustic molecular imaging, paving the way for visualization of hydrogen peroxide at high spatiotemporal resolution in living subjects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5407/F1.large.jpg.

Single Chain Cationic Polymer Dot as a Fluorescent Probe for Cell Imaging and Selective Determination of Hepatocellular Carcinoma Cells.

This letter describes formation of single chain cationic polymer dots (Pdots) made of poly[1,4-dimethyl-1-(3-((2,4,5-trimethylthiophen-3-yl)oxy)propyl)piperazin-1-ium bromide] conjugated polyelectrolyte (CPE). The single chain Pdot formation relies on a simple process which is a rapid nanophase separation between CPE solution of ethylene glycol and water. Pdots show narrow monodisperse size distribution with a 3.6 nm in diameter exhibiting high brightness and excellent colloidal and optical stability. It has been demonstrated that photoluminescent Pdots provide selective nuclear translocation to hepatocellular carcinoma cells as compared to healthy liver cells. The Pdot labeling effectively discriminates cancer cells in the coculture media. Pdots hold great promise as a luminescent probe to diagnose cancer cells in histology and may guide surgeons during operations to precisely separate out cancerous tissue due to augmented fluorescence brightness.

An available strategy based on accurate mass by ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry technology to characterization of metabolic profile of palbociclib in rat urine, feces and bile.

Palbociclib (named PD 0332991) is a novel highly selective cyclin-dependent kinase 4 and 6 (CDK 4/6) inhibitor, which has been approved by the Food and Drug Administration (FDA) for the treatment of hormone-receptor-positive advanced breast cancer. This present study developed a comprehensive strategy to investigate the metabolic profile of palbociclib in rat urine, feces and bile samples based on an ultra high performance liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR MS). A total of 29 metabolites, including 18 phase I metabolites and 11 phase II metabolites, were detected and identified. The metabolic pathways included hydroxylation, oxidation, dehydrogenation, N-dealkylation, carbonylation, oxidative deamination, acetylation, glucuronidation, sulphate conjugation as well as the crossover of multiple metabolic pathways in vivo, and 16 of these metabolites were proposed for the first time. This study showed an insight into the metabolism of palbociclib in vivo, which may provide relevant chemical information for subsequent studies in the future.

Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics in mice.

Muscarinic Designer Receptors Exclusively Activated by Designer Drugs (DREADD) gated by clozapine-N-oxide (CNO) allow selective G-protein cascade activation in genetically specified cell-types in vivo. Here we compare the pharmacokinetics, off-target effects and efficacy of CNO, clozapine (CLZ) and compound 21 (Cmpd-21) at the inhibitory DREADD human Gi-coupled M4 muscarinic receptor (hM4Di). The half maximal effective concentration (EC50) of CLZ was substantially lower (0.42 nM) than CNO (8.1 nM); Cmpd-21 was intermediate (2.95 nM). CNO was back-converted to CLZ in mice, and CLZ accumulated in brain tissue. However, CNO itself also entered the brain, and free cerebrospinal fluid (CSF) levels were within the range to activate hM4Di directly, while free (CSF) CLZ levels remained below the detection limit. Furthermore, directly injected CLZ was strongly converted to its pharmacologically active metabolite, norclozapine. Cmpd-21 showed a superior brain penetration and long-lasting presence. Although we identified a wide range of CNO and Cmpd-21 off-targets, there was hardly any nonspecific behavioural effects among the parameters assessed by the 5-choice-serial-reaction-time task. Our results suggest that CNO (3-5 mg/kg) and Cmpd-21 (0.4-1 mg/kg) are suitable DREADD agonists, effective at latest 15 min after intraperitoneal application, but both require between-subject controls for unspecific effects.

UHPLC-MS/MS method for analysis of sobuzoxane, its active form ICRF-154 and metabolite EDTA-diamide and its application to bioactivation study.

Sobuzoxane (MST-16) is an approved anticancer agent, a pro-drug of bisdioxopiperazine analog ICRF-154. Due to the structural similarity of ICRF-154 to dexrazoxane (ICRF-187), MST-16 deserves attention as a cardioprotective drug. This study presents for the first time UHPLC-MS/MS assay of MST-16, ICRF-154 and its metabolite (EDTA-diamide) in cell culture medium, buffer, plasma and cardiac cells and provides data on MST-16 bioactivation under conditions relevant to investigation of cardioprotection of this drug. The analysis of these compounds that differ considerably in their lipophilicity was achieved on the Zorbax SB-Aq column using a mixture of aqueous ammonium formate and methanol as a mobile phase. The biological samples were either diluted or precipitated with methanol, which was followed by acidification for the assay of MST-16. The method was validated for determination of all compounds in the biological materials. The application of the method for analysis of samples from in vitro experiments provided important findings, namely, that (1) MST-16 is quickly decomposed in biological environments, (2) the cardiac cells actively metabolize MST-16, and (3) MST-16 readily penetrates into the cardiac cells and is converted into ICRF-154 and EDTA-diamide. These data are useful for the in-depth examination of the cardioprotective potential of this drug.

Multi-Steps Fragmentation-Ion Trap Mass Spectrometry Coupled to Liquid Chromatography Diode Array System for Investigation of Olaparib Related Substances.

A high-performance liquid chromatography-diode array-mass spectrometric (LC-DAD-MS) method was developed and validated to investigate the related substances of olaparib (OLA) in bulk form. OLA was exposed to acid⁻base hydrolysis, boiling, oxidation with hydrogen peroxide, and UV light followed by LC-DAD-MS analysis. OLA and OLA-related substances were simultaneously and quantitatively monitored by DAD at 278 nm and triple quadrupole mass spectrometry (QQQ-MS). The investigated compounds were auto-scanned by an ion trap MS which applied positive and negative modes separately. The fragmentation pathway was confirmed by applying multi-steps fragmentation to identify the resulted cleaved ions and their parent ion. OLA was found to be sensitive to the alkaline hydrolysis and less sensitive to UV light. Two major hydrolytic degradation products, including the protonated molar ions m/z 299 and m/z 367, were identified. Three potential impurities were also characterized. The LC-MS limit of detection (LOD) and limit of quantification (LOQ) were 0.01 and 0.05 ng/µL, respectively. The quantitative results obtained by LC-DAD was comparable with that of LC-QQQ-MS. The proposed method shows good intra-day and inter-day precision with relative standard deviation (RSD) <2%.

Segmental Hair Analysis-Interpretation of the Time of Drug Intake in Two Patients Undergoing Drug Treatment.

The present study involved segmental testing of hair in two clinical cases with known dosage histories. Hair analysis confirmed the first patient's exposure to the prescribed sertraline and citalopram for several months. Citalopram was generally distributed along the hair shaft in accordance with the drug ingestion period. By contrast, "false" positive results were observed for sertraline in distal hair segments, corresponding to a period of no sertraline exposure, which may indicate incorporation from sweat or sebum, which transport the drugs along the hair surface. The second patient received various drugs during her treatment for brain cancer. Metoclopramide, morphine, oxazepam, paracetamol, sumatriptan, tramadol, and zopiclone, which had been part of the therapy, were all detected in the proximal hair segment. The results of these two cases indicated that results-especially concerning the time of drug intake-must be interpreted with caution and allow for the possibility of incorporation from sweat or sebum.

A quantitative LC-MS/MS method for determination of a small molecule agonist of EphA2 in mouse plasma and brain tissue.

Compound 27 {1, 12-bis[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]dodecane-1,12-dione} is a novel small molecule agonist of EphA2 receptor tyrosine kinase. It showed much improved activity for the activation of EphA2 receptor compared with the parental compound doxazosin. To support further pharmacological and toxicological studies of the compound, a method using liquid chromatography and electrospray ionization tandem mass spectrometry (LC-MS/MS) has been developed for the quantification of this compound. Liquid-liquid extraction was used to extract the compound from mouse plasma and brain tissue homogenate. Reverse-phase chromatography with gradient elution was performed to separate compound 27 from the endogenous molecules in the matrix, followed by MS detection using positive ion multiple reaction monitoring mode. Multiple reaction monitoring transitions m/z 387.3 → 290.1 and m/z 384.1 → 247.1 were selected for monitoring compound 27 and internal standard prazosin, respectively. The linear calibration range was 2-200 ng/mL with the intra- and inter-day precision and accuracy within the acceptable range. This method was successfully applied to the quantitative analysis of compound 27 in mouse plasma and brain tissue with different drug administration routes.

Validated stability indicating assay method of olaparib: LC-ESI-Q-TOF-MS/MS and NMR studies for characterization of its new hydrolytic and oxidative forced degradation products.

Olaparib (OLA) is a poly ADP ribose polymerase (PARP) enzyme inhibitor used to treat prostate, ovarian and breast cancer. The drug substance OLA was subjected to forced degradation as per ICH prescribed guidelines. It was degraded in hydrolytic (acidic and basic) and oxidative stress conditions and yielded four degradation products (DPs) while it remained stable in neutral hydrolytic, dry heat and photolytic stress conditions. A stability indicating assay method was developed to separate OLA and its DPs using InertSustain C18 column (250 × 4.6 mm, 5 µm) with a gradient mobile phase of 10 mM ammonium acetate (pH 4.5) and acetonitrile (ACN) at a flow rate of 1 mL min-1. The characterization of DPs was carried out by using liquid chromatography-electrospray ionization-quadrupole-time of flight tandem mass spectrometry (LC-ESI-Q-TOF-MS/MS). Major degradation products (DP-1 and DP-2) were isolated by using preparative HPLC and structures were further confirmed by using NMR spectroscopy. All the obtained DPs were new and not reported previously. The developed chromatographic method was validated as per ICH Q2 (R1) guideline and USP general chapter on method validation.

A Fluorescent Probe with Improved Water Solubility Permits the Analysis of Protein S-Depalmitoylation Activity in Live Cells.

S-Palmitoylation is an abundant lipid post-translational modification that is dynamically installed on and removed from target proteins to regulate their activity and cellular localization. A dearth of tools for studying the activities and regulation of protein S-depalmitoylases, thioesterase "erasers" of protein cysteine S-palmitoylation, has contributed to an incomplete understanding of the role of dynamic S-palmitoylation in regulating proteome lipidation. Recently, we developed "depalmitoylation probes" (DPPs), small molecule probes that become fluorescent upon S-depalmitoylase enzymatic activity. To be suitable for application in live cells, the first-generation DPPs relied on a shorter lipid substrate (C8 vs naturally occurring C16), which enhanced solubility and cell permeability. However, the use of an unnatural lipid substrate on the probes potentially limits the utility of the approach. Herein, we present a new member of the DPP family, DPP-5, which features an anionic carboxylate functional group that increases the probe water solubility. The enhanced water solubility of DPP-5 permits the use of a natural, palmitoylated substrate (C16), rather than a surrogate lipid. We show that DPP-5 is capable of monitoring endogenous S-depalmitoylases in live mammalian cells and that it can reveal changes in S-depalmitoylation levels due to lipid stress. DPP-5 should prove to be a useful new tool for probing the regulation of proteome lipidation through dynamic S-depalmitoylation.

Optimization and validation of RP-HPLC method for simultaneous estimation of palbociclib and letrozole.

A simple, rapid, and robust RP-HPLC method have been developed and validated to measure palbociclib (PB) and letrozole (LT) at single wavelength (254 nm). A isocratic elution of samples performed on Intersil C8 (4.6 mm × 250 mm particle size 5 µm) column with mobile phase consisting 0.02 M sodium dihydrogen phosphate buffer (pH 5.5): acetonitrile: methanol (80:10:10 v/v/v) delivered at flow rate 1.0 mL min-1. A good linear response was achieved over the range of 5-50 µg mL-1. The LODs for PB and LT were found to be 0.098 and 0.0821 µg mL-1, while the LOQs for PB and LT were 0.381-0.315 µg mL-1, respectively. The method was quantitatively evaluated in terms of system suitability test, linearity, precision, accuracy (recovery) and robustness as per standard guidelines. The method is simple, convenient and suitable for the analysis of PB and LT in bulk drug.

Combination of palbociclib with enzalutamide shows in vitro activity in RB proficient and androgen receptor positive triple negative breast cancer cells.

OBJECTIVES: Triple negative breast cancer (TNBC) lacks specific drug targets and remains challenging. Palbociclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor is approved for metastatic estrogen receptor (ER)-positive and human epithermal growth factor 2 (HER2)-negative breast cancer. The nature of cell cycle inhibition by palbociclib suggests its potential in TNBC cells. Retinoblastoma (RB, a known substrate of CDK4/6) pathway deregulation is a frequent occurrence in TNBC and studies have revealed that pharmacological CDK4/6 inhibition induces a cooperative cytostatic effect with doxorubicin in RB-proficient TNBC models. In addition, recent studies reported that anti-androgen therapy shows preclinical efficacy in androgen-receptor (AR)-positive TNBC cells. Here we examined the effect of palbociclib in combination with an anti-androgen enzalutamide in TNBC cells. METHOD: MDA-MB-453, BT-549, MDA-MB-231 and MDA-MB-468 TNBC cell lines were used for in vitro studies. Protein expressions were assessed by Western blot analysis. Cytostatic effect was examined by MTT assay. Cell cycle and apoptosis were examined by flow cytometry. RESULTS: Palbociclib showed inhibitory effect in RB-proficient TNBC cells, and enzalutamide inhibited cell viability in AR-positive TNBC cells. Enzalutamide treatment could enhance the palbociclib-induced cytostatic effect in AR-positive/RB-proficient TNBC cells. In addition, palbociclib-mediated G1 arrest in AR-positive/RB-proficient TNBC cells was attenuated by RB knockdown. CONCLUSION: Our study provided a preclinical rationale in selecting patients who might have therapeutic benefit from combining CDK4/6 inhibitors with AR antagonists.