Preclinical Pharmacokinetics Research Articles

Pharmacokinetic Studies of Radiopharmaceuticals

Pharmacokinetic (PK) studies of radiopharmaceuticals (RPs) are distinctive because of small concentrations of active ingredients. Whereas methods applicable to non-RP medicinal products cannot be used for RPs, radionuclide methods can provide an insight into radionuclide behaviour. The aim of the study was to compare approaches to pre-clinical PK evaluation of RPs and other medicinal products. The article describes the main aspects of PK studies of RPs and non-RPs, defining similarities and differences in the conduction of these studies. The authors provided examples from their own PK studies of RPs: one investigating the possibility of using 99m Tc–Zoledronic acid to visualise bone pathologies in patients receiving Zometa © ; and the other demonstrating the advantage of orthotopic xenograft models over heterotopic ones. According to the conclusions, methods based on ionising radiation are undeniably superior to other methods used to study the PK of medicinal products (for example, the former offer a possibility of non-invasive detection of active ingredients in the nano and/or pico range). The data obtained during PK studies can be used to characterise both the safety and functional suitability of RPs.

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.

AML-259 Pharmacokinetics and Bioequivalence of Two Formulations of GFH009 Maleate Injection in Sprague Dawley Rats

Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase that regulates the expression of short-lived oncogene products, such as MYC and MCL1, which are key influencers of tumor cell proliferation and survival. CDK9 deregulation has been reported in leukemia and other hematological malignancies. GFH009 is a potent, highly selective, clinical-stage, small molecule that inhibits CDK9. The objective of this study was to characterize the preclinical pharmacokinetic (PK) profiles of two different formulations of GFH009 maleate (pH 4.5 and 6.0). Sprague Dawley (SD) rats were randomized to receive a single dose of one formulation of GFH009 maleate (4 mg/kg) intravenously (IV) at a volume of 5 mL/kg. Blood samples were obtained at pre-dose and pre-specified timepoints post-dose. Plasma concentration of GFH009 was quantitated by a validated LC-MS/MS method, and PK parameters were assessed by non-compartmental analysis approach. Twelve SD rats per group were included. The plasma concentrations of the GFH009 formulations were within 90% to 110% of the nominal concentrations, and the coefficient of variation was <5.0%. The mean plasma concentration peaked at 5 min post-dose (479 ng/mL and 486 ng/mL in groups 1 and 2, respectively) and gradually decreased in a similar profile. The LLOQ was 1.000 ng/mL. In group 1, C0 (mean ± SD) was 586 ± 109 ng/mL, AUClast was 825 ± 73.8 h-ng/mL; AUCInf was 842 ± 75.0 h-ng/mL, clearance (CL) was 4780 ± 429 mL/h/kg, and t1/2 was 5.45 ± 0.649 h. In group 2, C0 was 572 ± 147 ng/mL, AUClast was 924 ± 144 h-ng/mL, AUCInf was 946 ± 145 h-ng/mL, CL was 4310 ± 607 mL/h/kg, and t1/2 was 5.66 ± 0.973 h. Bioequivalence analysis showed that the 90% CI values for C0, AUClast, and AUCInf were within the acceptable range (80-125%). The PK profiles of GFH009 were comparable following single IV administration of two formulations of GFH009 maleate injection in SD rats, which supported application of the pH 6.0 formulation in Phase I trials in patients with relapsed/refractory hematologic conditions, including AML and lymphoma.

Evaluating the efficacy of prototype antiseizure drugs using a preclinical pharmacokinetic approach.

Pharmacokinetics (PK) of a drug drive its exposure, efficacy, and tolerability. A thorough preclinical PK assessment of antiseizure medications (ASMs) is therefore essential to evaluate the clinical potential. We tested protection against evoked seizures of prototype ASMs in conjunction with analysis of plasma and brain PK as a proof-of-principle study to enhance our understanding of drug efficacy and duration of action using rodent seizure models. In vivo seizure protection assays were performed in adult male CF-1 mice and Sprague Dawley rats. Clobazam (CLB), N-desmethyl CLB (NCLB), carbamazepine (CBZ), CBZ-10,11-epoxide (CBZE), sodium valproate (VPA), and levetiracetam (LEV) concentrations were quantified in plasma and brain using liquid chromatography-tandem mass spectrometry. Mean concentrations of each analyte were calculated and used to determine PK parameters via noncompartmental analysis in Phoenix WinNonLin. NCLB concentrations were approximately 10-fold greater than CLB in mice. The antiseizure profile of CLB was partially sustained by NCLB in mice. CLB concentrations were lower in rats than in mice. CBZE plasma exposures were approximately 70% of CBZ in both mice and rats, likely contributing to the antiseizure effect of CBZ. VPA showed a relatively short half-life in both mice and rats, which correlated with a sharp decline in efficacy. LEV had a prolonged brain and plasma half-life, associated with a prolonged duration of action in mice. The study demonstrates the utility of PK analyses for understanding the seizure protection time course in mice and rats. The data indicate that distinct PK profiles of ASMs between mice and rats likely drive differences in drug efficacy between rodent models.

Surface Engineering Promoted Insulin-Sensitizing Activities of Sub-Nanoscale Vanadate Clusters through Regulated Pharmacokinetics and Bioavailability.

The therapeutic application of vanadium compounds is plagued by their poor bioavailability and potential adverse effects. Herein, 1nm polyoxovanadate (POV) clusters are functionalized with alkyl chains of various lengths and studied for the effect of surface engineering on their preclinical pharmacokinetics and typical insulin-sensitizing activity. The concentrations of surface engineered POVs in plasma, urine, and feces are monitored after a single administration to rats. The POVs exhibit a two-compartment profile of in vivo kinetics, and the surface engineering effect plays an important role in renal clearance of the POVs comparable to small molecules. POVs functionalized with long alkyl chains show much shorter elimination half time t1/2β and higher elimination fractions (50%) within 48h than pristine POVs, suggesting favorable elimination kinetics to mitigate the possible side effects of vanadium. Meanwhile, long alkyl chain modification leads to a 76% increment of oral bioavailability in contrast to unmodified POVs. As suggested by glucose tolerance tests and sub-chronic toxicity tests, the above two factors contribute to the enhanced therapeutic efficacy of POVs while mitigating their adverse effects. The surface engineering protocol provides a feasible approach to the optimization of the bioavailability and pharmacokinetic properties of POVs for promoted insulin-sensitizing activities.

Preclinical Pharmacokinetics, Tissue Distribution and in vitro Metabolism of FHND6091, a Novel Oral Proteasome Inhibitor.

IntroductionFHND6091, a novel N-capped dipeptidyl boronic acid proteasome inhibitor with promising pharmacological properties, entirely converted into active form FHND6081 under physiological conditions. The proteasome, a key component of the ubiquitin-proteasome pathway (UPP), has emerged as a validated target of multiple myeloma (MM) therapeutics. FHND6091 is a selective oral proteasome inhibitor that binds irreversibly to the β5 submit of the 20S proteasome and exerts anti-cancer roles.MethodsIn this study, we investigated the metabolic stability, metabolite production, metabolic pathways and plasma protein binding (PPB) of FHND6081 along with its absorption, tissue distribution, excretion (ADME) and pharmacokinetics (PK) in animals.ResultsUltra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) identified a total of nine new metabolites after co-incubation with FHND6091 in hepatocytes from different species. A hypothetical CYP450-metabolic pathway including dehydrogenation, N-dealkylation plus mono-oxygenation and other was proposed. In addition, FHND6081 was highly bound to plasma proteins (>99%); nevertheless, it preferred to partition to red blood cells (B/P ratio: 4.91). The results of microsomal metabolic stability corroborated that FHND6081 was a moderate-clearance compound. In Caco-2 cell experiments, the compound displayed modest permeability suggesting that it may show limited bioavailability via oral routes. Furthermore, FHND6081 was extensively distributed in rats and the highest exposure was achieved in the stomach followed by the small intestine and adrenal gland. Pharmacokinetic studies were done by using Sprague-Dawley (SD) rats, oral absorption was fast and plasma exposure was dose-dependent and oral bioavailability were low. At the same dose, FHND6081 exposure was severalfold higher in whole blood than in plasma, which was consistent with blood cell partitioning. Moreover, only a small fraction of the parent compound was excreted via feces and urine and oxidative metabolites were detected in feces and plasma.ConclusionThe overall preclinical pharmacokinetic profile supported the selection and development of FHND6091 as a clinical candidate.

Preclinical Pharmacokinetics and In Vitro Properties of GS-441524, a Potential Oral Drug Candidate for COVID-19 Treatment.

Preclinical pharmacokinetics (PK) and In Vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62–78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.4 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

Preclinical pharmacology of RA15127343: In vitro and in vivo activity of a novel ultralong-acting basal insulin.

To report the in vitro and in vivo preclinical pharmacokinetic (PK) and pharmacodynamic (PD) properties of RA15127343, a novel ultralong-acting insulin analogue targeting once-weekly administration, in female Göttingen minipigs. In vitro binding and activation of human insulin receptor isoforms (IR-A/IR-B), glucose uptake in rat myocytes, as well as mitogenic activity of RA15127343 were evaluated. In vivo, the PK and PD activities of RA15127343 were assessed in female, normoglycaemic Göttingen minipigs. The half-life (t1/2 ) and time to maximum plasma concentration (Tmax ) of subcutaneously (SC) administered RA15127343 (10/30/45/60 nmol/kg) were estimated. In vivo blood glucose and endogenous plasma C-peptide concentrations after single SC administration (10/30/45/60 nmol/kg) or repeated dosing (15 nmol/kg) were analysed. In comparison to human insulin, RA15127343 showed lower in vitro binding affinity (19.9/6.31 μM vs. 1.10/1.14 nM) and activation (2.054 μM/669.6nM vs. 26.04/18.24 nM) of IR-A/IR-B, lower potency to activate glucose uptake (855.2 vs. 3.37 nM) and lower mitogenic activity (17.92 μM vs. 10.78 nM; proliferation in MCF7 cells). In vivo, the mean t1/2 and Tmax of RA15127343 after SC administration ranged from 48 to 59 and 30 to 39 hours, respectively. Blood glucose and plasma C-peptide concentrations were significantly lower with RA15127343 (single/repeated doses) versus vehicle. RA15127343 showed an ultra-long t1/2 with a slow onset of action. The preclinical pharmacological outcomes suggest RA15127343 could be a potential ultralong-acting insulin analogue with low risk of hypoglycaemia in humans.

Development of Fibroblast Activation Protein Inhibitor-Based Dimeric Radiotracers with Improved Tumor Retention and Antitumor Efficacy.

Fibroblast activation protein (FAP), a fundamental component of the tumor stroma, is overexpressed in cancer-associated fibroblasts (CAFs). As a promising theranostic probe, we evaluated whether the FAP inhibitor (FAPI) dimer (DOTA-2P[FAPI]2) is more effective than its monomeric analogs for FAP-targeted radionuclide therapy. [68Ga]Ga/[177Lu]Lu-DOTA-2P(FAPI)2 were assayed in a stability study, small-animal positron emission tomography (PET) and single-photon emission computed tomography (SPECT), biodistribution, and radionuclide therapy to comprehensively evaluate their preclinical pharmacokinetics. The pharmacokinetics of [68Ga]Ga-DOTA-2P(FAPI)2 and [177Lu]Lu-DOTA-2P(FAPI)2 were determined in FAP-positive hepatocellular carcinoma patient-derived xenografts (PDXs) and HT-1080-FAP cell-derived xenografts (CDXs). [68Ga]Ga-DOTA-2P(FAPI)2 and [177Lu]Lu-DOTA-2P(FAPI)2 were stable in phosphate-buffered saline for 4 h. The tumor retention of [68Ga]Ga-DOTA-2P(FAPI)2 was better than that of [68Ga]Ga-FAPI-46 in HT-1080-FAP CDXs, while healthy organs showed low tracer uptake and fast body clearance. In single-photon emission computed tomography, [177Lu]Lu-DOTA-2P(FAPI)2 showed a higher uptake and longer retention for tumors in both PDXs and CDXs from 1-48 h. [177Lu]Lu-DOTA-2P(FAPI)2 showed the best inhibition of tumor growth in PDXs and CDXs. DOTA-2P(FAPI)2 has increased tumor uptake and retention properties compared to FAPI-46, which significantly improves the use of FAPI-based vectors for PET imaging and radionuclide therapy. [177Lu]Lu-DOTA-2P(FAPI)2 may be safe and effective for the treatment of FAP-positive malignant tumors.

Early Nonclinical and Clinical Development of AG-920, a Repurposed Topical Ocular Anesthetic.

Purpose: To evaluate the preclinical effects, and preclinical and clinical ocular and systemic pharmacokinetics of a new topical, non-preserved ocular anesthetic, AG-920 (articaine ophthalmic solution). Methods: Five studies: one in vitro melanin binding study; three studies in rabbits receiving an ocular dose of AG-920 evaluating corneal sensitivity, ocular tolerability, and systemic exposure to articaine and its inactive metabolite, articainic acid; and one clinical study in 14 healthy adult volunteers receiving an ocular dose of AG-920, with blood samples over 24 h. A liquid chromatography with tandem mass spectrometry (LC-MS-MS) method was used to detect both parent and metabolite with a lower limit of quantitation (LLOQ) of 0.1 and 0.2 ng/mL, respectively. Results: Melanin binding of articaine was up to 7.4%. A decrease in corneal sensitivity was noted for 20 min post-treatment in all active groups, and returned to baseline by 60 min post-dose. No dose-response relationship was observed. Concentrations of articaine in ocular matrices generally peaked early and then decreased over time. Both parent and metabolite were observed in blood at early time points. There were no ocular safety issues with AG-920. Conclusions: These early stage development studies showed that AG-920 was well tolerated in the standard preclinical models and did not cause any toxicity. AG-920 ophthalmic solution elicited a rapid onset and potentially clinically useful duration of corneal anesthesia. The studies supported the clinical evaluation of the 8% strength. Registered with clinicaltrials.gov as NCT04759339.

Preclinical Pharmacokinetic and Pharmacodynamic Investigation of 5'-Methoxynobiletin from Ageratum conyzoides: In vivo and In silico Approaches.

5'-methoxynobiletin (5'-MeONB), a polymethoxyflavone isolated from A. conyzoides, has shown anti-inflammatory property. Nevertheless, the antinociceptive activity and pre-clinical pharmacokinetics (PK) characteristics of 5'-MeONB remain unknown. Considering the anti-inflammatory potential of the 5'-MeONB, this study aimed to investigate the pre-clinical PK behavior of 5'-MeONB, as well as its time course antinociceptive activity. 5'-MeONB plasma concentrations were determined in Wistar rats after intravenous (i.v.) (10mg/kg) and oral (50mg/kg) administration, and in Swiss mice after oral administration (100mg/kg). Plasma samples were deproteinization and 5'-MeONB quantified by a validated UPLC-MS method. Additionally, the antinociceptive activity of 5'-MeONB was evaluated after 15, 30, 60, 180 and 360min following oral administration on the acute nocifensive behavior of mice induced by formalin. 5'-MeONB rats and mice plasma concentration-time profiles were best one-compartment model. After i.v. administration to rats, a short half-life, a high clearance and moderate volume of distribution at steady state were observed. Similar results were obtained after oral administration. The oral bioavailability ranged from 8 to 11%. Additionally, 5'-MeONB exhibited antinociceptive activity in both formalin phases, especially in the inflammatory phase of the model, inhibiting 68% and 91% of neurogenic and inflammatory responses, respectively, after 30min of oral administration. The results described here provide novel insights on 5'-MeONB pharmacokinetics and pharmacodynamic effect, serving as support for future studies to confirm this compound as anti-nociceptive and anti-inflammatory effective agent.

PET imaging of hepatocellular carcinoma by targeting tumor-associated endothelium using [68Ga]Ga-PSMA-617.

Hepatocellular carcinoma (HCC) is a malignant tumor associated with high morbidity and mortality rates. In many non-prostate solid tumors such as HCC, prostate-specific membrane antigens (PSMA) are overexpressed in tumor-associated endothelial cells. Therefore, the aim of this study was to evaluate the performance of [68Ga]Ga-PSMA-617 PET imaging on HCC with different animal models, including cell line-derived xenografts (CDX) and patient-derived xenografts (PDX), and to explore its mechanisms of function. [68Ga]Ga-PSMA-617 was prepared. The expression level of PSMA in two human hepatocellular cancer cells (HepG2 and HuH-7) was evaluated, and the cellular uptakes of [68Ga]Ga-PSMA-617 were assayed. HepG2 and HuH-7 subcutaneous xenograft models, HepG2 orthotopic xenograft models, and four different groups of PDX models were prepared. Preclinical pharmacokinetics and performance of [68Ga]Ga-PSMA-617 were evaluated in different types of HCC xenografts models using small animal PET and biodistribution studies. Low PSMA expression level of HepG2 and HuH-7 cells was observed, and the cellular uptake and blocking study confirmed the non-specificity of the PSMA-targeted probe binding to HepG2 and HuH-7 cells. In the subcutaneous xenograft models, the tumor uptakes at 0.5 h were 0.76 ± 0.12%ID/g (HepG2 tumors) and 0.78 ± 0.08%ID/g (HuH-7 tumors), respectively, which were significantly higher than those of the blocking groups (0.23 ± 0.04%ID/g and 0.20 ± 0.04%ID/g, respectively). In the orthotopic xenograft models, PET images clearly displayed the tumor locations based on the preferential accumulation of [68Ga]Ga-PSMA-617 in tumor tissue versus normal liver tissue, suggesting the possibility of using [68Ga]Ga-PSMA-617 PET imaging to detect primary HCC lesions in deep tissue. In the four different groups of HCC PDX models, PET imaging with [68Ga]Ga-PSMA-617 provided clear tumor uptakes with prominent tumor-to-background contrast, further demonstrating its potential for the clinical imaging of PSMA-positive HCC lesions. The staining of tumor tissue sections with CD31- and PSMA-specific antibodies visualized the tumor-associated blood vessels and PSMA expression on endothelial cells in subcutaneous, orthotopic tissues, and PDX tissues, confirming the imaging with [68Ga]Ga-PSMA-617 might be mediated by targeting tumorassociated endothelium. In this study, in vivo PET on different types of HCC xenograft models illustrated high uptake within tumors, which confirmed that [68Ga]Ga-PSMA-617 PET may be a promising imaging modality for HCC by targeting tumorassociated endothelium.

Abstract 5389: Exploring potential predictive biomarkers for the responsiveness to CTX-712 by in vivo drug sensitivity analysis on PDX models

Abstract Cancer’s remarkable diversity had been understood through a logical framework of six key hallmarks. Additionally, several equally prevalent hallmarks have been identified in recent analyses of cellular phenotypes as the stress phenotypes of cancers. A novel hallmark of cancer RNA deregulation has been newly proposed as another hallmark of cancer within this group of stress phenotypes. However, the development of cancer therapies targeting RNA deregulation has only just begun. Indeed, small molecules that attack the RNA maturation processes and produce aberrant RNA are currently under development. We developed orally available, selective, and a first-in-class pan- CDC-like kinase (CLK) inhibitor, CTX-712, currently in phase 1 clinical trials for advanced, relapsed or refractory malignant cancers. CTX-712 dephosphorylates serine and arginine-rich proteins and induces primarily skipped exon type of splicing changes, resulting in the generation of RNA deregulation stress. In multiple preclinical models, excessive RNA deregulation stress caused cancer cell death and tumor growth inhibition. To explore the patient selection biomarkers for CTX-712, in vivo efficacy survey analysis has been conducted on breast, ovarian, colorectal, pancreatic, gastric and lung cancer models from patient derived xenograft (PDX) library with a detailed clinical information. Besides the efficacy investigation, the preclinical pharmacokinetics (PK) and pharmacodynamics (PD) analysis on tested PDX models were also performed to confirm plasma/tumor PK and PD (target CLK inhibition in xenografted tumors). Importantly, tested PDX models could be divided to sensitive (T/C on the most sensitive model = -71.1) and insensitive (T/C on the least sensitive model = 67.7), and the correlated tendency was observed between the efficacy and the inhibition magnitude of intratumor PD marker, production of exon skipped aberrant mRNA. To clarify the differences of sensitivity among tested PDXs will lead us to identity patient selection biomarkers of CTX-712. Therefore, whole transcriptome RNA sequencing, exome sequencing, and other approaches have been applied to reveal the differences of sensitivity among tested PDXs. Through this study on multiple PDXs, integration of drug responsiveness, and genomic/transcriptomic profiling may provide prediction markers for treatment responsiveness of CTX-712. Citation Format: Daisuke Morishita, Shigehiro Yagishita, Akio Mizutani, Hiroko Yamakawa, Shunsuke Ebara, Akinobu Hamada. Exploring potential predictive biomarkers for the responsiveness to CTX-712 by in vivo drug sensitivity analysis on PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5389.

Abstract 5480: Aristolochic acid as a scaffold for antitumor drug development

Abstract We have synthesized an analog of aristolochic acid (AA) with potent growth inhibitory activity in various human cancer cell lines and report here on mechanistic studies of this potential therapeutic. AA is a natural product and the main active principle of Aristolochia plants, a known source of antitumor agents. In 1964 due to nephrotoxicity, drug development efforts with AA were terminated following Phase I clinical trial in patients with terminal cancer of different origin. Structure activity studies of AA species allowed us to select a structural analog of AA (RJ-34) that lacks the determinants responsible for the renal toxicity and carcinogenicity of the parent compound. Building further on our experience with AA molecules, we developed a synthetic approach that affords efficient high-yield synthesis of RJ-34. Its structure and purity has been confirmed by high-performance liquid chromatography, liquid chromatography coupled with two-stage mass spectrometry and high resolution nucleic magnetic resonance. Sulforhodamine B assay revealed that RJ-34 has growth inhibitory activity in A549 and HCT-15 human cancer cell lines comparable to or stronger than that of paclitaxel or topotecan. At five nanomolar, RJ-34 inhibited DNA synthesis and cut the growth of A549 cells by 50%, leading to cell cycle arrest in G1/M and G2/S, with apparent apoptosis at concentrations greater than 200 nanomolar. Notably, protein synthesis in cultured cells, as evaluated by puromycin labelling, remained unaffected. RJ-34 was screened against the NCI-60 cell line panel - encompassing sixty human cancer cell lines from various tissues - providing clues to its mechanisms of action; namely, that RJ-34 acts through inhibition of DNA topoisomerases I and II and/or DNA intercalation. Follow-up studies using purified calf Topo I and human Topo II alpha and various plasmid substrates confirmed the role of RJ-34 as a DNA intercalator and human Topoisomerase II alpha poison. The pattern of activity of RJ-34 in the NCI-60 panel was distinct from that of AA and aristolactam - another related compound - suggesting that RJ-34 acts by mechanisms distinct from AA. In summary, RJ-34— a non-nephrotoxic analog of aristolochic acid— has a potent growth inhibitory activity in various human cancer cell lines with a mechanism of action different from the parent AA. Moving forward, pre-clinical pharmacokinetics, safety and efficacy studies are necessary to establish the potential of RJ-34 for development as an anticancer therapeutic. Citation Format: Keiji Hashimoto, Rajesh Chennamshetti, Radha Bonala, Francis Johnson, Arthur P. Grollman, Viktoriya S. Sidorenko. Aristolochic acid as a scaffold for antitumor drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5480.

Abstract 5457: QLH11811, a selective 4th-generation EGFR inhibitor for osimertinib-resistant EGFR-mutant NSCLC

Abstract Background: The 3rd-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), like osimertinib, provide marked clinical benefit for EGFR-mutant non-small cell lung cancer (NSCLC) patients with extended overall survival vs former EGFR TKIs (e.g., gefitinib). Approximate 10%-24% of NSCLC patients acquired C797S mutation when treated with osimertinib. Unfortunately, limited treatments are available for patients after osimertinib resistance. QLH11811 is a new generation EGFR TKI designed to target the EGFR with ex19del/L858R/T790M/C797S mutations. Here, we disclosed its preclinical data to support its clinical development in EGFR-mutant NSCLC. Methods: The inhibitory activity of QLH11811 on mutated and wild-type EGFR was tested in engineered cell lines and patient-derived organoid (PDO). The in vivo antitumor activity of QLH11811 was evaluated in the patient-derived xenograft (PDX) model with cis EGFR ex19del/T790M/C797S triple mutations, and the H1975 (cis EGFR L858R/T790M/C797S and cis EGFR ex19del/T790M/C797S), PC-9 (EGFR ex19del), and Ba/F3 (EGFR ex19del/C797S) cell line-derived xenograft (CDX) models. The pharmacokinetic (PK) profile was investigated in animals, and the human PK profile was projected using allometric scaling method. Results: QLH11811 displayed potent anti-proliferation activity against Ba/F3 (EGFR ex19del/T790M/C797S, L858R/T790M/C797S, ex19del/C797S, or L858R/C797S), PC-9 (EGFR ex19del/T790M/C797S), H1975 (EGFR L858R/T790M/C797S, L858R/T790M), H3255 (L858R) and HCC827 (ex19del) with IC50 of 2.6, 3.1, 2.4, 4.1, 51, 50, 27, 21, and 11 nM, respectively. QLH11811 also showed excellent selectivity when compared the above values with its IC50 against Ba/F3 (EGFR wild-type, 61 nM) and A431 (EGFR wild-type, 440nM). QLH11811 demonstrated excellent inhibitory activities against seven osimertinib-resistant PDO models. Daily oral QLH11811 significantly inhibited tumor growth at all doses tested (P &amp;lt;0.001) in the PDX model, the H1975, PC-9, and Ba/F3 CDX models. QLH11811 had good PK profile in mice, rats, dogs, and monkeys, with the absolute bioavailability at 71%, 29%, 80% and 42%. The human PK parameters were obtained by allometric scaling method, and the efficacious dose in human was projected to 103 mg, daily. Conclusion: The in vitro and in vivo preclinical data demonstrated QLH11811 is a highly potent and selective 4th-generation EGFR TKI with activity against the osimertinib-resistant NSCLC with EGFR C797S mutation. The preclinical PK data supported the efficacious dose of QLH11811 in human would be 103 mg. Citation Format: Shansong Zheng, Wei Deng, Qingmei Zheng, Yingying Yang, Na Li, Tan Pang, Xueying Feng, Simon Taylor, Lina Ma, Yaqiong Wu, Ziwei Zhao. QLH11811, a selective 4th-generation EGFR inhibitor for osimertinib-resistant EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5457.

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.

Preclinical Pharmacokinetics and Bioavailability of Oxypeucedanin in Rats after Single Intravenous and Oral Administration.

Oxypeucedanin, a furanocoumarin extracted from many traditional Chinese herbal medicines, has a variety of pharmacological effects. However, the independent pharmacokinetic characteristics and bioavailability of this compound remains elusive. In this study, a rapid, sensitive, and selective method using ultra-high performance liquid chromatography–tandem mass spectrometry (UPLC/MS/MS) was developed for evaluating the intravenous and oral pharmacokinetics of oxypeucedanin. After intravenous administration of oxypeucedanin (2.5, 5, and 10 mg/kg), and intragastric administration of oxypeucedanin (20 mg/kg), blood samples were collected periodically from the tail vein. The plasma concentration-time curves were plotted, and the pharmacokinetic parameters were calculated using a non-compartmental model analysis. After intravenous administration of oxypeucedanin (single dosing at 2.5, 5, and 10 mg/kg) to rats, the pharmacokinetics fit the linear kinetics characteristics, which showed that some parameters including average elimination half-life (T1/2Z of 0.61~0.66 h), mean residence time (MRT of 0.62~0.80 h), apparent volume of distribution (VZ of 4.98~7.50 L/kg), and systemic clearance (CLZ of 5.64~8.55 L/kg/h) are dose-independent and the area under concentration-time curve (AUC) increased in a dose-proportional manner. Single oral administration of oxypeucedanin (20 mg/kg) showed poor and slow absorption with the mean time to reach the peak concentration (Tmax) of 3.38 h, MRT of 5.86 h, T1/2Z of 2.94 h, and a mean absolute bioavailability of 10.26% in rats. These results provide critical information for a better understanding of the pharmacological effect of oxypeucedanin, which will facilitate its research and development.

Translational in vitro and in vivo PKPD modelling for apramycin against Gram-negative lung pathogens to facilitate prediction of human efficacious dose in pneumonia

ObjectivesNew drugs and methods to efficiently fight carbapenem-resistant gram-negative pathogens are sorely needed. In this study, we characterized the preclinical pharmacokinetics (PK) and pharmacodynamics of the clinical stage drug candidate apramycin in time kill and mouse lung infection models. Based on in vitro and in vivo data, we developed a mathematical model to predict human efficacy. MethodsThree pneumonia-inducing gram-negative species Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were studied. Bactericidal kinetics were evaluated with time-kill curves; in vivo PK were studied in healthy and infected mice, with sampling in plasma and epithelial lining fluid after subcutaneous administration; in vivo efficacy was measured in a neutropenic mouse pneumonia model. A pharmacokinetic-pharmacodynamic model, integrating all the data, was developed and simulations were performed. ResultsGood lung penetration of apramycin in epithelial lining fluid (ELF) was shown (area under the curve (AUC)ELF/AUCplasma = 88%). Plasma clearance was 48% lower in lung infected mice compared to healthy mice. For two out of five strains studied, a delay in growth (∼5 h) was observed in vivo but not in vitro. The mathematical model enabled integration of lung PK to drive mouse PK and pharmacodynamics. Simulations predicted that 30 mg/kg of apramycin once daily would result in bacteriostasis in patients. DiscussionApramycin is a candidate for treatment of carbapenem-resistant gram-negative pneumonia as demonstrated in an integrated modeling framework for three bacterial species. We show that mathematical modelling is a useful tool for simultaneous inclusion of multiple data sources, notably plasma and lung in vivo PK and simulation of expected scenarios in a clinical setting, notably lung infections.

Preclinical pharmacokinetics, CYP phenotyping, and tissue distribution study of novel anti-breast cancer candidate S-011-1559

S-011-1559 is a tyrosine-derived novel benzoxazine CDRI molecule targeted to the oestrogen-related receptor (ER-α/β) modulator in breast cancer. To explore the pharmacokinetics of S-011-1559, a selective and sensitive bioanalytical method using LC-MS/MS was established and validated in different biological matrices of female rats. Blood-to-plasma ratio and plasma protein binding (PPB) of S-011-1559 were found to be <1 and >97% in both rats and humans, respectively. The human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) binding was found in the range of > 68 to 45% and >14% respectively. Half-life and intrinsic clearance by microsomal stability study were found to be 28.83 min and 0.05 mL/min/mg in rats, 78.35 min and 0.036 mL/min/mg in humans, respectively. The IC50 value of S-011-1559 against CYP isoforms was revealed to moderately inhibit CYP2D6 by a reversible non-competitive mechanism. Tissue distribution of S-011-1559 on single intravenous injection at 2 mg/kg was found in the order of C lungs > C mammary gland > C spleen > C heart > C kidney > C liver > C brain. The data from the present study provides crucial information about S-011-1559 for further development as a novel potential drug candidate in modulating ER-α/β receptors of lung and breast neoplasia.

Preclinical Safety and Pharmacokinetics of Heat Stable Oxytocin in Sublingual Fast-Dissolving Tablet Formulation.

The work reported here focuses on an evaluation of a novel heat stable formulation of a uterotonic peptide drug oxytocin involving stability testing under elevated temperatures and toxicokinetic response generated by sublingual (SL) administration in rabbits. The formulation was thermotolerant, maintaining the potency of oxytocin in the form of a fast-dissolving tablet at the end of 2-year storage at 30 °C/65% relative humidity with less than 5% loss in oxytocin content based on analytical high performance liquid chromatography (HPLC). The toxicokinetic results in rabbits showed that the fast-dissolving tablet was safe without any reactogenicity or toxicity associated with SL administration or the excipients present in the formulation. The SL route elicited rapid absorption of oxytocin in plasma within 5 min of administration although lower than intramuscular (IM) administration. IM resulted in area under the curve (AUC) values approximately 5 times higher than SL oxytocin. However, due to the limitations encountered during SL administration in an anesthetized rabbit model, the relevance of heat stable oxytocin formulation that has the flexibility to be adapted in different formats may warrant a human clinical study to determine whether therapeutically relevant plasma levels for treating postpartum hemorrhage can be generated via alternate non-injectable routes of administration.