Preclinical Pharmacokinetic Studies Research Articles

Simultaneous determination of NTB-3119, a novel anti-tuberculosis agent, and its major metabolites in mouse plasma by LC-MS/MS and its application in preclinical pharmacokinetics study

NTB-3119, a novel benzothiopyranone derivative, has been developed as a potential anti-tuberculosis(TB) drug with strong activity. In this study, three major metabolites of NTB-3119 were firstly identified in vitro and in vivo. A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantitative analysis of NTB-3119 and its major metabolites NTB-3190, NTB-3202 and NTB-3204 in mouse plasma. The plasma samples were processed by protein precipitation with organic solvent. NTB-3119, NTB-3190, NTB-3202, NTB-3204 and NTB-4A (Internal Standard, IS) were separated by a Zorbax-SB C18 column (100 mm × 2.1 mm, 3.5 µm) with a gradient mobile phase of acetonitrile/water at a flow rate of 0.25 mL/min. The analytes were detected by electrospray positive ion mode in Parallel Reaction Monitoring (PRM) mode on a high resolution mass spectrum (HRMS, Thermo Q Executive). The monitored transitions were m/z 456.15632 → 360.06137 for NTB-3119, m/z 426.18214 → 246.01891 for NTB-3190, m/z 472.15124 → 360.06143 for NTB-3202, m/z 442.17706 → 246.01903 for NTB-3204 and m/z 337.13691 → 163.02081 for NTB-4A, respectively. Good linearity was conducted in the range of 5–2000 ng/mL for NTB-3119, NTB-3202 and NTB-3204 as well as 2.5–1000 ng/mL for NTB-3190. The inter- and intra-batch precision (RSD%) were both lower than 13.3 %, with the accuracy ranged from 88.0 % to 108.1 %. The analytes were proved to be stable during all samples storage, preparation and analytic procedures. The validated method was successfully applied to study the pharmacokinetics and bioavailability of NTB-3119 after oral treatment in Balb/c mouse.

Discovery of HZ-L105, a Next Generation of Bcl-2 Inhibitor, Overcomes Bcl-2 Mutation and Exhibits Superior Antitumor Activity

Introduction: Bcl-2, a key regulator of apoptosis pathway, is over-expressed in many hematologic malignancies, which results in blocking of apoptosis and cell survival. Targeting Bcl-2 for the treatment of malignant hematological tumors has been well validated and demonstrated by FDA approval of the first generation Bcl-2 inhibitor Venetoclax for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and acute myeloid leukemia (AML). Although Venetoclax provides significant clinical benefits, there are reports show that after long-term monotherapy patients develop disease progression , often driven by acquisition of Bcl-2 Gly101Val (G101V), Asp103Tyr (D103Y) resistance mutations. Here, we described the discovery of a next generation Bcl-2 inhibitor HZ-L105 as with high potency against Bcl-2, Bcl-2(G101V) and Bcl-2(D103Y) for treatment of Bcl-2 dependent and Venetoclax-resistant hematological cancers. Methods:Biochemical inhibition assays were conducted towards Bcl-2/Bcl-2 G101V/Bcl-2 D103Y/Bcl-xL by time-resolved fluorescence resonance energy transfer (TR-FRET);Cellular proliferation tests of RS4;11 and RS4;11-G101V overexpressed stable line were carried out using MTS assay;Human platelet viability was measured by Cell-titer Glo assay;Cytochrome P450 inhibition assays were performed using fluorometric assay;In vivo pharmacodynamic (PD) studies were performed in RS4;11-derived subcutaneous xenograft SCID mouse model;Preclinical pharmacokinetics (PK) studies were assayed in mice and dogs and parameters were determined by LC-MS. Results: In biochemical assays, HZ-L105 potently inhibited Bcl-2 WT, Bcl-2 G101V and Bcl-2 D103Y with IC50 of 0.54 nM, 6.16 nM and 5.57 nM，while Venetoclax's IC50 of 1.62 nM, 440.6 nM and 279.8 nM, respectively. Therefore, it is expected to overcome the acquisition of Bcl-2 resistance mutations. In cellular assays, HZ-L105 suppressed cell growth not only in RS4;11 but also in RS4;11-G101V stable line (IC50=0.30 nM in RS4;11 and 243.36 nM in RS4;11-G101V), which was much more superior to Venetoclax (IC50= 2.91 nM in RS4;11 and 3360.79 nM in RS4;11-G101V). HZ-L105 was also highly selective against Bcl-2, showing >1000 folds selectivity to Bcl-xL. Furthermore, platelet viability assay in vitro showed HZ-L105 induced hypotoxicity as similarly to Venetoclax, suggesting its good potential to avoid thrombocytopenia due to Bcl-xL inhibition. In the preclinical Pharmacodynamics studies, oral administration of HZ-L105 induced rapid and robust antitumor effect in RS4;11 ALL xenografts. HZ-L105 at a dose of 5 mg/kg demonstrated significantly excellent efficacy than Venetoclax at a dose 15mg/kg in RS4;11-derived xenograft mouse model. In the preclinical pharmacokinetic studies, HZ-L105 showed excellent pharmacokinetic properties in ICR mice and beagle dogs, with long T1/2 and high dose-normalized AUC. HZ-L105 did not reduced hERG tail current (% current blocked 2.87% at 30μM), indicating the compound has a low potential to induce QT prolongation. HZ-L105 has less drug-drug interaction potential, as evidenced by no inhibitory effect on CYPs, especially in CYP2C9 (IC50 >20 μM) compared with Venetoclax (IC50=1.12 μM). Besides, in 14-days oral gavage toxicity study in mice, no compound related deaths occurred at 15, 50 and 300 mg/kg/day, and only leukopenia was observed similarly to Venetoclax. Conclusion: In summary, HZ-L105 as a next generation Bcl-2 inhibitor, showed great potency on wild type and acquisition resistance mutations of Bcl-2 in vitro, excellent oral bioavailability, superior anti-tumor activities on preclinical xenograft model, and safety profiles,. It may provide new thoughts on treatment for a wide range of Bcl-2-dependent and Venetoclax-resistant hematological cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discovery and Preclinical Study of Novel BTK Degrader HZ-Q1060

Introduction: Bruton's tyrosine kinase （BTK） is a clinical validated drug target for therapy of B cell lymphoma and autoimmune diseases. However, same as other kinase inhibitors, acquired drug resistance, which is the result of BTK C481S mutation, has been observed in patients. So reversible BTK inhibitors were developed to overcome acquired drug resistance of covalent BTK inhibitor. While, in order to have enough efficacy, reversible BTK inhibitors required much higher drug exposure which may lead to higher risk of adverse effect. Hence, it is urgent to develop new technique to overcome drug resistance caused by BTK C481S mutation. Proteolysis-targeting chimeras(PROTAC) is promising new strategy for drug development. PROTAC can achieve sustained target degradation in a catalytical mechanism. So comparing with traditional small molecule inhibitor, PROTAC drug has great advantage in solving acquired drug resistance. Therefore, with the aim of solving acquired drug resistance of 1st generation of BTK inhibitor, we identified a novel BTK-PROTAC HZ-Q1060 based on our in-house DaTProD® platform which is setting up to promote PROTAC development. After a series of evaluations, HZ-Q1060 has been validated as a promising pre-clinical candidate for further clinical development. Results:HZ-Q1060 can effectively degrade BTK in cancer cell lines and human PBMC, and the half degradation concentration (DC50) in Mino is less than 0.5nM. At the same time, HZ-Q1060 can also induce the degradation of intracellular mutant BTK-C481S protein.HZ-Q1060 has not observed any inhibition and degradation effect on other kinases except BTK, especially kinases that may be related to potential side effects by inhibiting, such as EGFR and TEC.In vitro cancer cell growth inhibition assay, it completely suppressed the cell proliferation of a panel of lymphoma cell lines such as Mino(IC50=0.11nM) and OCI-ly10(IC50=0.28nM).In the PK/PD experiment in mice, a single oral administration of medium dose can degrade more than 80% of BTK within 6 hours, and the degradation effect can be maintained until 24 hours after administration.In the subcutaneous transplanted tumor model of OCI-ly10 mice, the tumor growth was completely inhibited by 3mg/kg of HZ-Q1060 administered orally every day for 14 days, which was equivalent to that of ibrutinib 10mg/kg.In the preclinical pharmacokinetic studies, HZ-Q1060 showed excellent pharmacokinetic properties in different species of animals, included mice, rats and beagle dogs, with long T1/2 and high dose-normalized AUC.HZ-Q1060 has no significant CYPs inhibitory effect at 10 μM, so it has a low risk of DDI in the future combination therapy.HZ-Q1060 has no inhibitory effect on hERG channel, indicating the compound has a low potential to induce QT prolongation. HZ-Q1060 also shows good safety and tolerance in the preliminary safety evaluation of animals. Conclusion: In summary, HZ-Q1060 is a potent and selective degrader of BTK with good ADME properties and in vivo efficacy, which makes it suitable for further evaluation in clinical development.

Metabolism, pharmacokinetics, and bioavailability of yuanhuacine in rat using LC-MS.

Yuanhuacine is a Daphne-type diterpene ortho-ester and is one of the main active ingredients of genkwa flos. Anticancer activity of yuanhuacine has been well investigated in various tumor cells and animal models, but information on metabolism and pharmacokinetics is limited. The aims of the present study were to investigate the metabolic and pharmacokinetic profiles of yuanhuacine in rat. The metabolic profile of yuanhuacine was obtained from rat plasma, urine, and feces using ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. A total of seven metabolites were detected, and the proposed metabolic pathways involved oxidation and glucuronidation. A simple and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of yuanhuacine in rat plasma. The linear range of yuanhuacine was 1-1000 ng/ml (R2 =0.998). The intra- and inter-precision (coefficient of variation %) of the assay was 3.86-6.18% and 2.65-5.75%, respectively, and the intra- and inter-accuracy (relative error %) was -3.83-4.77% and -3.03-5.11%, respectively. The extraction recovery, matrix effect, stability, and incurred sample reanalysis of yuanhuacine were within acceptable levels. The established method was validated and successfully applied to the preclinical pharmacokinetic study of yuanhuacine. The absolute oral bioavailability of yuanhuacine was calculated as 1.14%, and it reached the maximum plasma concentration of 28.21 ± 2.79 ng/ml in rat plasma at 2h in the oral dosing group. The apparent volume of distribution of intravenous and intragastric administrations was 26.07 ±6.45 and 21.83 ±3.54L/kg, respectively. The half-life of elimination of yuanhuacine was 9.64 ± 1.53 h in the intravenous dosing group.

Preclinical pharmacokinetics, biodistribution, excretion, and plasma protein binding study of 58Fe-labeled hemin by ICP-MS

BackgroundHemin, a stable form of heme iron, is a potential iron supplement for the treatment of iron deficiency. To date, the pharmacokinetics and in vivo ADME properties of hemin are to be elucidated. MethodsIn this study, a rapid, sensitive, and validated inductively coupled plasma mass spectrometry (ICP-MS) method was used in combination with 58Fe stable isotope labeling to systemically investigate the plasma pharmacokinetics, biodistribution, excretion, and plasma binding profiles of hemin in animals. ResultsResults showed that the ICP-MS method is accurate and sensitive enough to quantitatively determine the in vivo disposition process of 58Fe derived from 58Fe-labeled hemin. Following intra-gastric administration, 58Fe was rapidly absorbed in gastrointestinal tract, with Cmax of 41.1 ± 23.1 ng/mL, Tmax of 1.38 ± 0.48 h, and bioavailability of 1.12 ± 0.45 % in beagle dogs. Moreover, 58Fe was distributed to various organs including stomach, small intestine, spleen, and liver, within a few hours after intra-gastric administration in rats. Excretion of 58Fe in rats was predominantly via feces (76.3 ± 15.1 % of dosage), whereas minimally via urine (0.14 ± 0.08 % of dosage). Protein binding study revealed majority of 58Fe in plasma was bound to proteins, with average binding rates of 81.0 % and 92.7 % in human and rat plasma, respectively. ConclusionIn conclusion, the present study validated the work-flow of preclinical pharmacokinetic studies of iron-containing drug candidates with using ICP-MS and stable (trace) isotope labeling strategy. It also provided useful information to support the further development of hemin as a drug/nutrition supplement candidate.

Turning adversity into opportunity: Small extracellular vesicles as nanocarriers for tumor-associated macrophages re-education.

Currently, small extracellular vesicles (sEV) as a nanoscale drug delivery system, are undergoing biotechnological scaling and clinical validation. Nonetheless, preclinical pharmacokinetic studies revealed that sEV are predominantly uptaken by macrophages. Although this "sEV-macrophage" propensity represents a disadvantage in terms of sEV targeting and their bioavailability as nanocarriers, it also represents a strategic advantage for those therapies that involve macrophages. Such is the case of tumor-associated macrophages (TAMs), which can reprogram/repolarize their predominantly immunosuppressive and tumor-supportive phenotype toward an immunostimulatory and anti-tumor phenotype using sEV as nanocarriers of TAMs reprogramming molecules. In this design, sEV represents an advantageous delivery system, providing precision to the therapy by simultaneously matching their tropism to the therapeutic cell target. Here, we review the current knowledge of the role of TAMs in the tumoral microenvironment and the effect generated by the reprogramming of these phagocytic cells fate using sEV. Finally, we discuss how these vesicles can be engineered by different bioengineering techniques to improve their therapeutic cargo loading and preferential uptake by TAMs.

Organoid-derived intestinal epithelial cells are a suitable model for preclinical toxicology and pharmacokinetic studies

SummaryIntestinal organoids are physiologically relevant tools used for cellular models. However, the suitability of organoids to examine biological functions over existing established cell lines lacks sufficient evidence. Cytochrome P450 3A4 (CYP3A4) induction by pregnane X receptor ligands, glucose uptake via sodium/glucose cotransporter 1, and microsomal triglyceride transfer protein-dependent ApoB-48 secretion, which are critical for human intestinal metabolism, were observed in organoid-derived two-dimensional cells but little in Caco-2 cells. CYP3A4 induction evaluation involved a simplified method of establishing organoids that constitutively expressed a reporter gene. Compound screening identified several anticancer drugs with selective activities toward Caco-2 cells, highlighting their characteristics as cancer cells. Another compound screening revealed a decline in N-(4-hydroxyphenyl)retinamide cytotoxicity upon rifampicin treatment in organoid-derived cells, under CYP3A4-induced conditions. This study shows that organoid-derived intestinal epithelial cells (IECs) possess similar physiological properties as intestinal epithelium and can serve as tools for enhancing the prediction of biological activity in humans.

Multiple-reaction monitoring (MRM) LC–MS/MS quantitation of venlafaxine and its O-desmethyl metabolite for a preclinical pharmacokinetic study in rabbits

Preclinical pharmacokinetic (PK) studies in animal models during the formulation development phase give preliminary evidence and near clear picture of the PK behavior of drug and/or its dosage forms before clinical studies on humans and help in the tailoring of the dosage form according to the expected and requisite clinical behavior. The present work reports a first of its kind preclinical PK study on extended-release (ER) solid oral dosage forms of venlafaxine (VEN) in New Zealand White rabbits. The VEN is a highly prescribed and one of the safest and most effective therapeutic agents used in the treatment of different types of depression disorders worldwide. The multiple-reaction monitoring (MRM) LC–MS/MS method developed for this purpose demonstrated enough reliability in simultaneously quantitating VEN and its equipotent metabolite O-desmethylvenlafaxine (ODV) in rabbit plasma. The method described uses solid-phase extraction for sample preparation followed by an ultrafast LC–MS/MS analysis. The chromatographic separation was achieved isocratically with a predominantly polar mobile phase by employing RPLC. The triple quadrupole LC/MS/MS system operated in MRM mode used an ESI probe as an ion source in positive polarity. The validation results are within the permissible limits of US FDA recommendations and acceptance criteria for bioanalytical method validation.

A validated liquid chromatography-tandem mass spectroscopy method for the quantification of tolinapant in human plasma.

Tolinapant (ASTX660), a pan-selective inhibitor of apoptosis protein antagonist with dual cIAP/XIAP activity, was identified as a clinical candidate in preclinical efficacy, pharmacokinetic and safety studies. In order to assess tolinapant in first-in-human Phase I/II clinical trials, a validated bioanalytical method was required to determine plasma pharmacokinetics. Tolinapant and d4-tolinapant were extracted from human plasma using liquid-liquid extraction. Separation chromatography was performed on a Acquity BEH C18 1.7 µM, 50 mm × 2.1mm i.d. column, using a mobile phase of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Mass spectrometry detection was performed by positive turbo ion spray ionisation, in multiple reaction monitoring mode. The method was validated according to the US Food and Drug Administration (FDA) guidelines. The method has a quantifiable linear range of 1-500ng/mL (r 2=0.999). The intra- and inter-day coefficients of variation were <11.4%. Dilution QC samples agreed with prepared concentrations, with a precision of 1.5% and accuracy of 101%. Tolinapant mean recoveries ranged from 85.1-94.4 % with negligible matrix effects. A highly sensitive and selective LC-MS/MS bioanalytical method was developed and validated. The method was successfully applied in Phase 1/2 clinical trials to determine the human pharmacokinetic profile of tolinapant.

Simultaneous determination of novel β-lactamase inhibitor WCK 4234 and Meropenem in dog plasma by LC-MS/MS and its application to preclinical pharmacokinetic study.

A precise and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) bioanalytical method has been developed and validated for the simultaneous quantification of WCK 4234 and meropenem (MEM) in dog plasma. Protein precipitation using acetonitrile was employed as a sample preparation approach. Cefepime was used as an internal standard. The developed method was selective, sensitive (limit of quantification, 0.075 μg/ml for both drugs), accurate (recovery > 90%), precise (CV <10%) and linear (r2 ≥0.99, concentration range 0.075-120 μg/ml for both analytes). The developed method was successfully applied for the determination of both drugs in plasma to assess the pharmacokinetics in beagle dogs. WCK 4234 + MEM in a 1:1 ratio at 15 + 15 and 30 + 30 mg/kg doses were administered by the intravenous route. The mean plasma concentration and area under the concentration-time curve of WCK 4234 ranged from 38.3 to 77.4μg/ml and from 47.8 to 77.1μg h/ml, respectively, and the values for MEM ranged from 52.2 to 115.3μg/ml and 70.5 to 133.6μg h/ml respectively. The elimination half-life of WCK 4234 and MEM was around 0.8h.

Preclinical Pharmacokinetic Studies of a Novel Diuretic Inhibiting Urea Transporters.

Urea transporter (UT) inhibitors are a class of promising novel diuretics that do not cause the imbalance of Na+, K+, Cl−, and other electrolytes. In our previous studies, 25a, a promising diuretic candidate inhibiting UT, was discovered and showed potent diuretic activities in rodents. Here, a sensitive liquid chromatography–tandem mass spectrometry method for the quantitation of 25a in rat plasma, urine, feces, bile, and tissue homogenates was developed and validated to support the preclinical pharmacokinetic studies. The tissue distribution, excretion, and plasma protein binding were investigated in rats. After a single oral dose of 25a at 25, 50, and 100 mg/kg, the drug exposure increased linearly with the dose. The drug accumulation was observed after multiple oral doses compared to a single dose. In the distribution study, 25a exhibited a wide distribution to tissues with high blood perfusion, such as kidney, heart, lung, and spleen, and the lowest distribution in the brain and testis. The accumulative excretion rate of 25a was 0.14%, 3.16%, and 0.018% in urine, feces, and bile, respectively. The plasma protein binding of 25a was approximately 60% in rats and 40% in humans. This is the first study on the preclinical pharmacokinetic profiles of 25a.

A sensitive HILIC-MS/MS method for quantification of theanine in rat plasma and tissues: Application to preclinical pharmacokinetic study

Adolescent depression is a significant public health problem, with the major depressive disorder having been the leading risk factor for suicide and death amongst children and adolescents. For treating depression, antidepressants are used with minimal clinical evidence data and uncertain efficacy in children. L-theanine has anti-depression and other physiological functions. However, few reports are available on the pharmacokinetics of L-theanine, especially in children and adolescents. In this study, a rapid and sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry method was established and validated for the quantification of L-theanine in juvenile rat plasma and tissues. Chromatographic separation was conducted via an Agilent ZORBAX HILIC plus column in gradient elution mode. L-theanine and [2H5]-L-glutamic acid (internal standard) were determined under the multi-reaction monitoring mode transitions of m/z 175.0 → 157.9 and m/z 153.0 → 88.2 in positive ionisation mode, respectively, and completed methodology verification. In addition, 10 and 35 mg kg−1 of L-theanine were given by intragastric administration to determine the brain and plasma pharmacokinetic characteristics in healthy and chronic unpredictable mild stress rats, respectively. The distribution of tissues and the limbic system were measured at the same time. The results showed that juvenile and diseased rats have higher absorption than adult rats, and age, dosage and health status could affect the process of L-theanine in vivo. L-theanine also has a high degree of tissue distribution. This study lays a foundation for the clinical treatment of depression in children and adolescents.

The ultra high sensitivity blood counter: a compact, MRI-compatible, radioactivity counter for pharmacokinetic studies in μl volumes

Quantification of physiological parameters in preclinical pharmacokinetic studies based on nuclear imaging requires the monitoring of arterial radioactivity over time, known as the arterial input function (AIF). Continuous derivation of the AIF in rodent models is very challenging because of the limited blood volume available for sampling. To address this challenge, an Ultra High Sensitivity Blood Counter (UHS-BC) was developed. The device detects beta particles in real-time using silicon photodiodes, custom low-noise electronics, and 3D-printed plastic cartridges to hold standard catheters. Two prototypes were built and characterized in two facilities. Sensitivities up to 39% for 18F and 58% for 11C-based positron emission tomography (PET) tracers were demonstrated. 99mTc and 125I based Single Photon Emission Computed Tomography (SPECT) tracers were detected with greater than 3% and 10% sensitivity, respectively, opening new applications in nuclear imaging and fundamental biology research. Measured energy spectra show all relevant peaks down to a minimum detectable energy of 20 keV. The UHS-BC was shown to be highly reliable, robust towards parasitic background radiation and electromagnetic interference in the PET or MRI environment. The UHS-BC provides reproducible results under various experimental conditions and was demonstrated to be stable over days of continuous operation. Animal experiments showed that the UHS-BC performs accurate AIF measurements using low detection volumes suitable for small animal models in PET, SPECT and PET/MRI investigations. This tool will help to reduce the time and number of animals required for pharmacokinetic studies, thus increasing the throughput of new drug development.

A PBPK Model of Ternary Cyclodextrin Complex of ST-246 Was Built to Achieve a Reasonable IV Infusion Regimen for the Treatment of Human Severe Smallpox.

ST-246 is an oral drug against pathogenic orthopoxvirus infections. An intravenous formulation is required for some critical patients. A ternary complex of ST-246/meglumine/hydroxypropyl-β-cyclodextrin with well-improved solubility was successfully developed in our institute. The aim of this study was to achieve a reasonable intravenous infusion regimen of this novel formulation by a robust PBPK model based on preclinical pharmacokinetic studies. The pharmacokinetics of ST-246 after intravenous injection at different doses in rats, dogs, and monkeys were conducted to obtain clearances. The clearance of humans was generated by using the allometric scaling approach. Tissue distribution of ST-246 was conducted in rats to obtain tissue partition coefficients (K p ). The PBPK model of the rat was first built using in vivo clearance and K p combined with in vitro physicochemical properties, unbound fraction, and cyclodextrin effect parameters of ST-246. Then the PBPK model was transferred to a dog and monkey and validated simultaneously. Finally, pharmacokinetic profiles after IV infusion at different dosages utilizing the human PBPK model were compared to the observed oral PK profile of ST-246 at therapeutic dosage (600 mg). The mechanistic PBPK model described the animal PK behaviors of ST-246 via intravenous injection and infusion with fold errors within 1.2. It appeared that 6h-IV infusion at 5 mg/kg BID produced similar Cmax and AUC as oral administration at 600 mg. A PBPK model of ST-246 was built to achieve a reasonable regimen of IV infusion for the treatment of severe smallpox, which will facilitate the clinical translation of this novel formulation.

Pharmacokinetic Variability in Pre-Clinical Studies: Sample Study with Abiraterone in Rats and Implications for Short-Term Comparative Pharmacokinetic Study Designs.

One of the major concerns for all in vivo experiments is intra- and inter-subject variability, which can be a great source of inaccuracy. The aim of this study is, therefore, to estimate the ability of parallel vs. cross-over design studies in order to describe the relative pharmacokinetic performance of the studied drug formulations. We analyzed the data from a drug development program that examined the performance of innovative abiraterone acetate formulations against the identical reference product in three stages. In stages 1–3, groups A–F were dosed with the reference product once in a parallel manner. Stage 4 was performed to evaluate the intra-individual variability (IIV) by repeated administration of the reference product to the same animals. Although the geometric mean (90% CI) values of abiraterone AUClast in groups A–F were similar to the IIV group (24.36 (23.79–41.00) vs. 26.29 (20.56–47.00) mg/mL·min·g), the results generated in the isolated parallel groups provided imprecise estimates of the true AUClast values ranging from 9.62 to 44.62 mg/mL·min·g due to chance. Notably, in 4 out of 15 possible pair comparisons between the parallel groups, the confidence intervals did not include 100%, which is the true ratio for all comparisons tested after identical formulation administration to all groups. A cross-over design can significantly improve the methodology in short-term comparative pre-clinical pharmacokinetic studies, and can provide more precise and accurate results in comparison to more traditional pre-clinical study designs.

UPLC-MS/MS method for the quantification of MCI-77, a novel sigma-1 receptor ligand, and its application to pharmacokinetic studies

Sigma-1 receptors are involved in pain modulation, particularly in cases of nerve injury and neuropathic pain. High-affinity ligands with improved pharmacokinetic profiles are needed to further investigate the properties of these receptors and their potential as a therapeutic target. The novel compound MCI-77 is one such selective sigma-1 receptor ligand, and the purpose of this study was to characterize its preclinical pharmacokinetic parameters. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify MCI-77 in mouse plasma and brain homogenate. The method was validated for sensitivity, selectivity, linearity, accuracy, precision, stability, and dilution integrity. The method has a linearity range of 2–200 ng/mL, a short run-time of 3.2 min, and requires a low sample volume of 25 µL. A simple protein precipitation procedure was used for compound extraction. Samples were run on an Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm) following a gradient elution method using a mobile phase consisting of water containing 0.1% (v/v) formic acid and acetonitrile. The method was applied to the analysis of plasma and brain homogenate samples from preclinical pharmacokinetic studies in CD-1 mice. MCI-77 exhibited high systemic clearance (8.5 ± 0.3 L/h/kg) and extensive tissue distribution indicated by a high volume of distribution (20.1 ± 0.3 L/kg). The concentration levels were consistently higher in brain samples than in plasma (brain/plasma AUC ratio 2.9), indicating its ability to cross the blood–brain barrier.

In-Vivo and Ex-Vivo Brain Uptake Studies of Peptidomimetic Neurolysin Activators in Healthy and Stroke Animals.

Neurolysin (Nln) is a peptidase that functions to preserve the brain following ischemic stroke by hydrolyzing various neuropeptides. Nln activation has emerged as an attractive drug discovery target for treatment of ischemic stroke. Among first-in-class peptidomimetic Nln activators, we selected three lead compounds (9d, 10c, 11a) for quantitative pharmacokinetic analysis to provide valuable information for subsequent preclinical development. Pharmacokinetic profile of these compounds was studied in healthy and ischemic stroke-induced mice after bolus intravenous administration. Brain concentration and brain uptake clearance (Kin) was calculated from single time point analysis. The inter-relationship between LogP with in-vitro and in-vivo permeability was studied to determine CNS penetration. Brain slice uptake method was used to study tissue binding, whereas P-gp-mediated transport was evaluated to understand the potential brain efflux of these compounds. According to calculated parameters, all three compounds showed a detectable amount in the brain after intravenous administration at 4mg/kg; however, 11a had the highest brain concentration and brain uptake clearance. A strong correlation was documented between in-vitro and in-vivo permeability data. The efflux ratio of 10c was ~6-fold higher compared to 11a and correlated well with its lower Kin value. In experimental stroke animals, the Kin of 11a was significantly higher in ischemic vs. contralateral and intact hemispheres, though it remained below its A50 value required to activate Nln. Collectively, these preclinical pharmacokinetic studies reveal promising BBB permeability of 11a and indicate that it can serve as an excellent lead for developing improved drug-like Nln activators.

LC-MS/MS method for determination of kansuinine a in rat plasma and its application to a rat pharmacokinetic study.

Kansuinine A is a macrocyclic jatrophane diterpene isolated from the plant Euphorbia kansui Liou. It exhibits many pharmacological activities including cytoxic, antitumor, antiallergic and proinflammatory effects. In the present study, a simple and sensitive LC-MS/MS method was established and validated for the determination of kansuinine A in rat plasma. After methanol-mediated protein precipitation, chromatographic separation was achieved on an Acquity BEH C18 column (2.1 × 100mm, 1.7μm) using acetonitrile and 0.1% formic acid in water as mobile phase by gradient elution. Kansuinine A and IS were quantified in negative multiple reaction monitoring mode with ion transitions at m/z 731.1-693.2 for kansuinine A and m/z 723.2-623.1 for IS. The method showed excellent linearity over the range 1-500 ng/ml. The intra- and inter-day precisions (relative standard deviation) were 2.13-4.28 and 3.83-7.67%, respectively, whereas accuracy (relative error) ranged from -4.17 to 3.73%. The extraction recovery, stability and matrix effect met the requirement of the regulations issued by the US Food and Drug Administration. The validated method was successfully applied to the pre-clinical pharmacokinetic study of kansuinine A in rats after oral administration (20 mg/kg) and intravenous administration (2mg/kg). This study provides valuable reference for the further study of E. kansui liou, especially for the drug development and clinical application of kansuinine A.

Inhibition of Vasoactive Intestinal Peptide Signaling with More Potent Inhibitors Augments T-Cell Activation and Prolongs Survival in Leukemic Mice

Introduction: Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide with immunosuppressive effects on T cells. Inhibition of VIP receptor (VIP-R) signaling by VIPhyb, a first-generation VIP-R antagonist, not only enhances T-cell activation and proliferation in vitro but also improves T cell dependent anti-tumor response in mouse models of acute myeloid leukemia (AML) and T lymphoblastic leukemia (Li et al. 2016; Petersen, Li, and Waller 2017). The goal of the project is to develop more potent VIP-R antagonists that generate a significantly more robust anti-tumor response in mouse models of AML, when compared to VIPhyb and validate a screening method to test the efficacy of novel peptides in activating human T cells in vitro. In this study, we report, for the first time, the activity of novel VIP-R antagonists on the activation profile of human T cells.Methods: We utilized in-silico-based modeling to identify 10 novel VIP-R antagonists from a library of 300 peptide sequences predicted to have increased binding affinity to VIP receptors VPAC1 and VPAC2 when compared to VIP or VIPhyb (Table 1). The library was generated from peptide sequences that contain the six charged N-terminal residues of the neurotensin present in VIPhyb with two or more amino acid substitutions within the C-terminal amino acid sequence of VIP. The ability of these peptides was tested in vitro using T cells from multiple healthy human donors activated using anti-CD3 monoclonal antibody coated plates. Activation status was assessed by flow cytometry of CD69, OX40, PD1, Tim3 and Lag3 expression relative to control cultures without added peptides. Potency of the novel antagonists in vivo was tested in a mouse AML model, by treating C1498- bearing mice with subcutaneous administration of VIP, VIPhyb, scrambled peptide (SCRAM1) or the second-generation VIP-R antagonists (labeled as ‘ANT‘) from day 6-12 after tumor implantation.Results: Inhibiting VIP-R signaling in human T cells using second-generation VIP-R antagonists ANT008, ANT308 and ANT195 showed approximately 1.5-to-2-fold increase in CD69, OX40, Tim3, Lag3 and OX40 expression in CD4+ T cells following 24-hour of drug exposure compared to control cultures (Figure 1A). A smaller effect of VIP-R antagonists on activation of CD8+ subsets was observed (Figure 1B). Among the peptides, ANT195 was superior to ANT008 and ANT308 which shows potency even at 1μM compared to 3μM for ANT008 and ANT308. However, significant increase in CD69 expression was observed in both CD4+ and CD8+ T cells in cultures treated with ANT308 (Figure 1 A&B, *p<0.05). Viability of the T cells was not affected by incubation with the queried peptides (Data not shown). These data corresponded to in vivo activity of the novel VIP-R antagonists such as ANT308 and ANT195 which rendered 40% of mice leukemia-free at day 60 compared to only 5% long-term survival with VIPhyb (Figure 2). Another candidate, ANT300, increased median survival time (MST) by up to 47 days compared to MST of 34 days with VIPhyb (Figure 2).Conclusions: Here, we report a simple and robust in vitro method to screen for immune activity potential of novel second-generation VIP-R antagonists using human T cells. Preliminary screen shows VIP-R antagonists augment activation of both CD4+ and CD8+ T cells. Our results indicate that ANT308 and ANT195 are more potent VIP-R antagonists with enhanced activity in vitro (human) and in vivo (mouse) than VIPhyb and ANT008, which demonstrate lower predicted binding affinities to VPAC1 and VPAC2. Our study supports the hypothesis that higher predicted binding affinity to VPAC1 and/or VPAC2 is associated with enhanced activity in stimulating human T cells and promoting anti-leukemia activity in mice. Further mechanistic studies on how inhibition of VIP-R signaling augments T cell activation and function are underway. These novel antagonists can lead to peptide-based immunotherapy for the treatment of various liquid cancers. Clinical development of this novel concept will require appropriate pre-clinical pharmacokinetic and toxicology studies. [Display omitted] DisclosuresWaller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.

Quantification for Antibody-Conjugated Drug in Trastuzumab Emtansine and Application to In Vitro Linker Stability and In Vivo Pharmacokinetic Study in Rat Using an Immuno-Affinity Capture Liquid Chromatography-Mass Spectrometric Method

Trastuzumab emtansine (T-DM1, brand name: Kadcyla®) is the first FDA-approved antibody-drug conjugate (ADC) for metastatic human epidermal growth factor receptor 2 positive (HER2+) breast cancer. It consists of three components: trastuzumab, an anti-HER2 monoclonal antibody, maytansinoid (DM1) as a cytotoxic drug, and maleimidomethyl cyclohexane-1-carboxylate (MCC) as a linker. In particular, the MCC linker is known as a non-cleavable linker and has a feature of being conjugated to DM1 by a covalent thioether bond. In this study, we developed an immuno-affinity capture liquid chromatography-mass spectrometric (LC-MS/MS) assay for quantifying the antibody-conjugated drug (acDrug) component of T-DM1. To quantify acDrug, desulfurated DM1 was prepared using a chemical desulfuration pretreatment and quantified as an acDrug. A quadratic regression (weighted 1/concentration), with equation y = ax2 + bx + c, was used to fit the calibration curves over the concentration range of 17.09~1709.44 ng/mL for the acDrug of T-DM1. The quantification run met the in-house acceptance criteria of ±25% accuracy and precision values for the quality control (QC) samples. In conclusion, an immuno-affinity capture LC-MS/MS assay was successfully developed to quantify acDrug of T-DM1 and applied to evaluate in vitro plasma linker stability and preclinical pharmacokinetic (PK) study in rats. This assay could be helpful when applied to other ADCs with the same linker-cytotoxic drug platform.