PK Studies Research Articles

Abstract 1718: Development of potent estrogen receptor beta agonists for treating glioblastoma

Abstract Background: Glioblastoma (GBM) is the most prevalent and aggressive type of adult brain cancers with low 5-year overall survival rates. Epidemiologic data suggest estrogen may decrease brain tumor growth. The lack of powerful, selective brain permeable estrogen receptor beta (ERβ) ligands to promote its anticancer action is limiting the therapeutic promise of ERβ. The goal of this project is to create a new class of brain permeable ERβ agonists for the treatment of GBM. Method: We have used four different assays to identify potent leads. (1) ERβ and estrogen receptor alpha (ERα) reporter assays, (2) Polar Screen Estrogen Receptor (ER) Competitive Binding Assays, (3) Cell viability assays using GBM cells, and (4) Activity in WT and ERβ-KO GBM cells. CellTiter-Glo Cell Viability and colony formation assays were used to test the efficacy of ERβ agonists. Invasion was measured by matrigel invasion chamber assays and apoptosis was measured by Caspase-Glo® 3/7 and Annexin V assays. Flow cytometry was used to evaluate the cell cycle. Western blotting, RNA-Seq, RT-qPCR, and ERβ KO cells were used in the mechanistic studies. Both orthotopic patient-derived xenografts and GBM cell line-derived (CDX) xenografts were used to test the ERβ agonist's activity in vivo. Results: Indanone and tetralone-oximes or keto-oximes have been hypothesized, produced, and evaluated as new ER agonists. Due to its high action in ERβ reporter assays, specific binding to ERβ in polar screen assays (EC50 91 nM), and potent growth inhibitory activity (IC50 7.4) in GBM cells, CIDD-97 was discovered as a possible hit by screening a library of ~60 compounds. CIDD-97 is significantly more selective for ERβ than ERα (40-fold). Treatment with CIDD-97 markedly reduced U251 cell viability while having little to no effect on U251-ER-KO cells and normal human astrocytes. Further, CIDD-97 treatment decreased the expression of stemness markers in patient derived GSC lines and promoted apoptosis. Additionally, CIDD-ERβ agonist improved TMZ's ability to reduce GBM cell survival. The activation of ERβ target genes in cells treated with CIDD-97 was verified by using RNA-Seq and RTqPCR. PK studies confirmed that CIDD-0149897 has systemic exposure, was found in the brain, and its maximal detectable levels suggested that it has good BBB permeability. Mice tolerated daily intraperitoneal treatment of CIDD-0149897 (50 mg/kg) with a 7-day repeat dosage with no evidence of toxicity. Additionally, when compared to vehicle CIDD-97 (50 mg/kg/i.p.) treatment significantly decreased tumor growth in xenograft models and extended the survival of tumor-bearing mice in orthotopic GSC models. Conclusion: Collectively, these findings pointed to CIDD-97 as a possible ERβ agonist and is easily adaptable to clinical use alongside current chemo- and radiation-therapy regimens, offering GBM patients an additional means of improving survival. Citation Format: Uday P. Pratap, Michael Tidwell, Henriette U. Balinda, Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Stanton McHardy, Andrew Brenner, Ratna K. Vadlamudi. Development of potent estrogen receptor beta agonists for treating glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1718.

Development of Modernized Acinetobacter baumannii Susceptibility Test Interpretive Criteria for Recommended Antimicrobial Agents Using Pharmacometric Approaches.

Acinetobacter baumannii-Acinetobacter calcoaceticus complex (referred to herein as A. baumannii) treatment guidelines contain numerous older antimicrobial agents with susceptibility test interpretive criteria (STIC, also known as susceptibility breakpoints) set using only epidemiological data. We utilized a combination of in vitro surveillance data, preclinical murine thigh and lung infection models, population pharmacokinetics, simulation, and pharmacokinetic/pharmacodynamic (PK/PD) target attainment analyses to evaluate A. baumannii STIC for four commonly recommended antimicrobials from different classes (amikacin, ceftazidime, ciprofloxacin, and minocycline). Antimicrobial in vitro surveillance data were based on 1,647 clinical A. baumannii isolates obtained from 109 centers in the United States and Europe. Among these isolates, 5 were selected for evaluation in murine infection models based on fitness and MIC variability. PK and dose-ranging studies were conducted using neutropenic murine thigh and lung infection models The MIC ranges for the 5 isolates evaluated were as follows: amikacin, 2 to 32 μg/mL; ceftazidime, 4 to 16 μg/mL; ciprofloxacin, 0.12 to 2 μg/mL; minocycline, 0.25 to 4 μg/mL. All organisms grew ≥1.5 log10 CFU in both models in untreated controls. Plasma and epithelial lining fluid (ELF) pharmacokinetics for all drugs were determined in mice using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. For each isolate, 5 dose levels of each drug were tested individually in the thigh and lung infection model. The inoculum ranged from 7.9 to 8.4 and 6.8 to 7.7 log10 CFU/mL for the lung and thigh models, respectively. PK/PD targets associated with net bacterial stasis and 1- and 2-log10 CFU reductions from baseline were identified for each organism/infection model using Hill-type models. Population pharmacokinetic models for each agent were identified from the literature. Using demographic variables for simulated patients with hospital-acquired or ventilator-associated bacterial pneumonia or urinary tract infections (including acute pyelonephritis) who were administered maximal dosing regimens of each agent, estimates of protein binding, and ELF penetration ratios based on data from the literature, free-drug plasma and total-drug concentration-time profiles were generated, and PK/PD indices by MIC were calculated. Percent probabilities of attaining median and randomly assigned PK/PD targets associated with the above-described endpoints were determined. Recommended susceptible breakpoints for each agent were those representing the highest MIC at which the percent probabilities of achieving PK/PD targets associated with a 1-log10 CFU reduction from baseline approached or were ≥90%. The following susceptible breakpoints for A. baumannii were identified: amikacin, ≤8 μg/mL for pneumonia; ceftazidime, ≤32 and ≤8 μg/mL for pneumonia; ciprofloxacin, ≤1 μg/mL; and minocycline, ≤0.5/≤1 μg/mL which correspond to the standard and high minocycline dosing regimens of 200 mg per day and 200 mg every 12 h, respectively. Implementation of appropriate STIC will help clinicians optimally use the above-described agents and improve the likelihood of successful patient outcomes.

Commentary on "The road to reliable peptide assays is paved with good guidelines".

Commentary on "The road to reliable peptide assays is paved with good guidelines".

Defining the therapeutic reference range for cariprazine

IntroductionAccording to the Consensus Guideline, the “therapeutic reference range” (TRR) defines ranges of drug blood concentrations that specify a lower limit below which a drug-induced therapeutic response is unlikely to occur and an upper limit above which tolerability decreases or the therapeutic improvement ceases. The TRR can be obtained from concentration measurements (trough (pre-dose) plasma concentration under steady-state conditions) in studies at therapeutically effective doses.ObjectivesThe aim is to examine the TRR for cariprazine (CAR: 1.5 mg/day to 6 mg/day) in schizophrenia studies.MethodsThe population based TRR for CAR is derived by PK/PD evaluation from phase 2/3 schizophrenia efficacy studies with sparse PK sampling. The population PK simulated TRR is compared to the actually measured values obtained from two PK studies. As the two active metabolites of cariprazine also contribute to the drug effect, plasma exposure is given for Total cariprazine (CAR + DCAR + DDCAR) and the parent drug (CAR).ResultsPK/PD analyses demonstrated an increase in efficacy with increasing exposure. These efficacy results are related to Total cariprazine trough concentrations of ca. 30 nM and 100 nM that determine the lower and upper TRR limits. For the parent drug, the pre-dose mean plasma concentration at 6 mg/day was between 5.7-10 ng/mL in different studies, while at 1.5 mg/day it was 1.9 ng/mL.ConclusionsThe TRR of the trough plasma levels at steady state is ca. 30 – 100 nM for Total cariprazine and ca. 2-10 ng/mL for the parent drug (unchanged drug) for schizophrenia treatment.Disclosure of InterestM. Kapás Employee of: Gedeon Richter Plc., A. Horváth Employee of: Gedeon Richter Plc., D. Djuric Employee of: Gedeon Richter Plc., R. Csehi Employee of: Gedeon Richter Plc., Á. Barabássy Employee of: Gedeon Richter Plc.

Addressing the oxamniquine in vitro-in vivo paradox to facilitate a new generation of anti-schistosome treatments

The antischistosomal drug oxamniquine, OXA, requires activation by a sulfotransferase within the parasitic worm to enable killing. Examination of the pharmacokinetic/pharmacodynamic (PK/PD) relationship for OXA identified an in vitro-in vivo paradox with the maximal clinical plasma concentrations five-to ten-times lower than the efficacious concentration for in vitro schistosomal killing. The parasite resides in the vasculature between the intestine and the liver, and modeling the PK data to determine portal concentrations fits with in vitro studies and explains the required human dose. In silico models were used to predict murine dosing to recapitulate human conditions for OXA portal concentration and time course. Follow-up PK studies verified in mice that a 50–100 mg/kg oral gavage dose of OXA formulated in acetate buffer recapitulates the 20–40 mg/kg dose common in patients. OXA was rapidly cleared through a combination of metabolism and excretion into bile. OXA absorbance and tissue distribution were similar in wild-type and P-gp efflux transporter knockout mice. The incorporation of in vitro efficacy data and portal concentration was demonstrated for an improved OXA-inspired analog that has been shown to kill S. mansoni, S. haematobium, and S. japonicum, whereas OXA is only effective against S. mansoni. Second-generation OXA analogs should optimize both in vitro killing and physiochemical properties to achieve high portal concentration via rapid oral absorption, facilitated by favorable solubility, permeability, and minimal intestinal metabolism.

619. Pharmacokinetic-Pharmacodynamic (PK-PD) Target Attainment Analyses to Support Epetraborole Dose Selection for the Treatment of Patients with Mycobacterium avium Complex (MAC) Lung Disease

619. Pharmacokinetic-Pharmacodynamic (PK-PD) Target Attainment Analyses to Support Epetraborole Dose Selection for the Treatment of Patients with <i>Mycobacterium avium</i> Complex (MAC) Lung Disease

1692. Ceftibuten-Xeruborbactam: In vitro Potency against Enterobacterales in Comparison with Other Oral Beta-lactams (BL) and Beta-lactamase Inhibitor (BLI) Combinations

Abstract Background The broad spectrum BLI xeruborbactam (XER, formerly, QPX7728) can be delivered IV or orally as the isobutyryloxymethyl prodrug. The prodrug form is in clinical development in combination with ceftibuten (BUT) for oral treatment of resistant gram-negative infections. The objective of this study was to compare the in vitro potency of BUT-XER to that of other investigational oral BL/BLI combinations and tebipenem (TBP) in blinded head-to-head testing against extended beta-lactamase (ESBL) producing and carbapenem-resistant Enterobacterales (CRE). Methods A total of 980 Enterobacterales including 515 ESBL and 460 CRE, including 195 KPC-, 97 OXA-48-like- and 168 metallo beta-lactamase (MBL) producers, were susceptibility tested by reference broth microdilution against BUT-XER (at fixed 4 mg/L) and comparator BLs or BL/BLI combinations (tested at their recommended BLI concentrations). MIC testing was conducted blinded to drug identity using CLSI reference microdilution methods and controlled using approved quality control bacterial strains and ranges. Results BLI inhibitor combinations and TBP had excellent potency against ESBL isolates with MIC90 in the ≤ 0.03-0.06 mg/L range for BUT-avibactam (AVI) and BUT-XER and in 0.125-0.25 mg/L range for TBP, BUT-VNTX-6236 (VNRX) and cefpodoxime (CPD)-ETX1317 (ETX) (Table). Against KPC and OXA-48-producers, BUT-XER and BUT-AVI (MIC90 0.25-0.5 mg/L) were 4-fold more potent than BUT-VNRX and CPD-ETX (MIC90 of 1-2 mg/L); TBP was not active against these strains. BUT-XER and CPD-ETX were the most potent agents against MBL-producing strains; however, anti-MBL activity of CPD-ETX was due to intrinsic antibacterial activity of ETX. TPB, BUT-AVI and BUT-VNTX did not have any relevant activity against MBL-producing strains. Conclusion BUT-XER had the broadest antimicrobial profile, with activity against ESBL and all carbapenemase producing isolates. Its potency against ESBL-producing isolates was comparable to the carbapenem TBP. Its in vitro potency and spectrum combined with the potential of once-daily administration based on PK studies warrants further clinical development. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jill Lindley, BS, AbbVie: Grant/Research Support.

The pharmaceutical potential of AR‐65, a novel Alzheimer’s disease drug targeting the immunoproteasome

AbstractBackgroundPreviously, we reported that inhibition of the immunoproteasome (iP), which is upregulated in reactive glial cells near amyloid‐β (Aβ) deposits in the brains of patients with Alzheimer’s disease (AD), leads to improved cognitive function in an AD mouse model of Aβ amyloidosis (Tg2576) via suppression of microglia‐mediated inflammation, independent of Aβ accumulation.MethodHere, we investigated the pharmaceutical properties of our lead iP inhibitor (AR‐65), a macrocyclic peptide epoxyketone, selected based on in vitro and cellulo iP inhibitory activities and cell‐based anti‐inflammatory efficacy. Specifically, the metabolic stability of AR‐65 was assessed using pooled human liver microsomes. In addition, the flux rate of AR‐65 was measured to evaluate the permeability across Caco‐2 monolayers over‐expressing ABCB1 and ABCG2, two major efflux drug transporters present in the human brain.ResultThe macrocyclic AR‐65 displayed improved metabolic stability (6‐fold increased half‐life), permeability, and solubility (in an injectable pharmaceutical buffer system with no cyclodextrin) compared to the FDA‐approved linear peptide epoxyketone proteasome inhibitor, carfilzomib (Kyprolis®). Finally, pilot PK studies performed using ICR mice confirmed that AR‐65 is metabolically more stable than carfilzomib and can cross the blood‐brain barrier to inhibit the brain iP.ConclusionTaken together, the results suggest that AR‐65 may hold great potential to be further investigated in clinical settings.

Development and Validation of Eltrombopag Determination in Human Plasma Blood by HPLC-MS Method

Introduction. "Eltrombopag" is a thrombopoietin receptor agonist (TPO-RA) that is approved for the treatment of immune thrombocytopenic purpura (ITP). According to the literature, very few analytical methods for determining eltrombopag in biological samples have been reported. To study the pharmacokinetics of new formulations of eltrombopag, a sensitive and specific method is required that allows one to accurately determine the concentration of eltrombopag in human blood plasma. Normally, HPLC methods should provide time, accuracy, and sensitivity as a result, it is necessary to develop fast or ultra-fast methods such as LC-MS/MS without any loss in sensitivity or separation efficiency.Aim. We aimed to develop and validate a method for the quantitative determination of eltrombopag levels in human plasma by using HPLC with mass spectrometric detection for performing the analytical part of pharmacokinetic studies.Materials and methods. Eltrombopag levels were determined in human plasma by HPLC with mass spectrometric detection. The samples were prepared using protein deposition.Results and discussion. The method was validated for selectivity, matrix effect, calibration curve, accuracy, precision, the limit of quantification, carry-over effect, and sample stability.Conclusion. The method for the determination of eltrombopag levels in human plasma has been developed and validated by HPLC-MS. The analytical range of eltrombopag levels in human plasma was 10–6750 ng/ml. This method could be used to determine eltrombopag levels in plasma for PK and BE studies.

Markedly Reduced FVIII Recovery Associated with Anti-FVIII Peg Antibodies after BNT162b2 Sars-Cov-2 Vaccination

Background The covalent link of polyethylene glycol (PEG) chains to recombinant factor VIII (FVIII) molecule is one of the most established approaches to produce novel therapeutic FVIII products with extended half-life (EHL). To date, three PEGyated-FVIII molecules, rurioctocog alfa pegol, turoctocog alfa pegol, and damoctocog alfa pegol are available for treatment of hemophilia A patients. From the pivotal clinical studies data, they showed comparable safety profile and pharmacokinetic with a mean half-live 1.3-1.7 times longer than the standard non-PEGylated FVIII. Although the PEG polymer is considered non immunogenic, antibodies against PEG, called pre-existing anti-PEG antibodies (APAs), have been reported in persons never treated with PEGylated drugs owing to PEG exposure during the activities of daily living. These antibodies can impact the clinical efficacy of PEGylated drug therapies by accelerating the blood clearance and changing pharmacokinetic. Recently, small amounts of PEG are also included in the lipid nanoparticles of the mRNA SARS-Cov-2 vaccines, BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna). Aim To describe a poor plasma FVIII recovery after the first infusion of a PEGylated FVIII product in two male patients with severe hemophilia A without inhibitor at about one month from the second dose of the BNT162b2 SARS-Cov-2 vaccination. Method About two months after the first dose of the SARS-Cov-2 vaccine and one month after the second, before switching from a standard half-life to a PEGylated FVIII product, patients underwent a PK study by infusion of 50 IU/kg of turoctocog alfa pegol (patient 1) or damoctocog alfa pegol (patient 2). FVIII activity was measured by Chromogenix Coamatic FVIII (Werfen) in plasma samples collected before and after the PEGylated FVIII infusion. Anti-PEGylated FVIII antibodies were searched by means of a home-made ELISA in plasma samples collected before administration of SARS-Cov-2 vaccine and at PK study. The assay was performed by using as capturing substrates each of the licensed PEGylated FVIII product (rurioctocog alfa pegol, turoctocog alfa pegol, and damoctocog alfa). Moreover, a non-PEGylated standard FVIII (Advate®) was employed as control substrate. Results FVIII coagulant activity measured after the infusion of 50 IU/kg of the PEGylated FVIII product showed a very low plasma recovery, 5% for patient 1 and 4% for patient 2. Anti-PEGylated FVIII antibodies reacting with each PEGylated FVIII products were found in plasma samples collected at PK baseline and after FVIII infusion but not in a sample obtained before the SARS-Cov-2 vaccination. No reactivity was found in vitro against the non-PEGylated standard FVIII product (figure). Conclusions Our finding of antibodies reacting with the PEGylated part of the FVIII molecule induced or boosted by BNT162b2 SARS-Cov-2 vaccination supports the view that the administration of the mRNA vaccine could impair plasma FVIII recovery after infusion of a PEGylated product. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High Potency Vasoactive Intestinal Peptide Receptor Antagonists Induce Durable Antileukemia/Tumor T Cell Memory in Mice with Once Daily Dosing

Introduction: Allogeneic bone marrow transplantation (allo-BMT) is curative for patients with myeloid leukemia (AML) through the activity of cytotoxic effector donor T cells, but high morbidity and mortality of allo-BMT limits the use of this therapy. A long-standing goal is the development of strategies that induce an autologous immune response to leukemic blasts by the patient's T cells. Vasoactive intestinal peptide (VIP) is a neuropeptide with pleiotropic functions, including modulation of immune and inflammatory responses. Previous studies demonstrated that VIP hybrid (VIPhyb), a peptide antagonist of VIP-signaling, induced T cell-dependent anti-leukemic activity in murine allotransplant models and promoted activation of mouse and human T cells (Li et al., 2016, Cancer Res). We developed a series of novel peptide VIP antagonists (VIP-ANT) based upon predicted binding affinity in silico to human VIP receptors VPAC1 and VPAC2. We tested the anti-cancer potential of these new VIP-ANT peptides in murine models of myeloid leukemia and mastocytoma, investigating the optimal dose and schedule of administration, and explored mechanisms underlying anti-tumor memory induced by VIP-ANT peptides. Methods: B6 (CD45.1, or albino) mice were injected intravenously with 1 x 106 C1498 cells (AML), and DBA/2j mice were injected subcutaneously with 5 x 104 P815 cells (myeloid mastocytoma). Leukemia-bearing mice were treated with subcutaneous injections of VIP-ANT or control scrambled peptides. Treatment started six days after injection of cells when C1498 leukemia cells were detectable in the blood, and luciferase+ P815 cells as tumors were palpable. A range of VIP-ANT doses was administered s.c. at 10, 30, or 100 μg. Treatment schedules tested included twice daily for 14 doses, once daily, once every other day, once every three days, and once weekly for 7 doses. Some mice received a single dose at day +6 following leukemia cell injection. Lymphocyte kinetics and leukemia cell burden were analyzed weekly through submandibular peripheral blood collections. Mice without evidence of AML/mastocytoma >60 days post-inoculation received a second injection of a two-fold higher dose of C1498 (2 × 106 cells) or P815 (1 × 105 cells). Results: We tested over 13 new VIP-ANT peptides. Daily or every other day (qod) administration of the novel VIP-ANT peptides to leukemia/tumor-bearing mice led to clearance of C1498 cells from blood or complete regression of P815 in 40 % of mice, leading to significantly improved survival over 60 days. Daily administration of 10 μg of novel VIP-ANT peptides for 7 days to leukemic mice prolonged survival(p<0.0001 VIP-ANT vs scramble, Fig 1A), with the fraction of mice that remained leukemia-free proportional to the predicted binding affinity of the VIP-ANT to human VPAC1 and VPAC2. With increased dose intensity and dose-density of ANT308, the most potent peptide, 40%-80% of mice survived to 70 days (median survival days (MSD) of 60 days) after ANT308 treatment compared with the survival of <20% in mice receiving scrambled peptide (MSD 36 days). Increasing the dose and schedule of ANT308 administration to 100 μg twice-daily led to 65% survival (MSD not reached) versus 40% with daily dosing, 30% with every-other-day dosing, and 0% survival with scrambled-sequence control peptide (MSD 33 days) (p<0.001, Fig 1B). Notably, the affinity of different VIP-ANT peptides to VPAC1 and VPAC2 VIP receptors significantly correlated with the survival of C1498-leukemic mice following prior treatment with the corresponding peptide. Prior treatment of leukemic mice with VIP-ANT induced protective immunological memory, rendering mice resistant to re-challenge with C1498 acute myeloid leukemia (80% survival) and P815 mastocytoma (60% survival) cells, compared with 0% survival (MSD 21 days) among immunologically naïve mice. Conclusion: Blocking VIP-signaling with high-affinity VIP-R peptide antagonists represents a novel pharmacological approach to enhance adaptive T cell responses to leukemia/myeloid sarcoma-associated antigens in murine leukemia/tumor models. We are continuing PK and toxicology studies with the lead VIP-ANT peptides to support the development of phase 1 clinical trials for patients with relapsed AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pre-Clinical Development of a First in Class Half-Life Extended T Cell Engager Targeting ROR-1

Background: ROR1 is a cell surface receptor that is largely absent or expressed at low levels in healthy adult tissues but is expressed at high levels on a wide range of hematological malignancies as well as solid tumours, making it an ideal anti-cancer drug target. NVG-111 is a bispecific T cell engager (TCE) antibody targeting ROR1xCD3 using a tandem scFv format to redirect the cytotoxic activity of T cells to ROR1+ tumour cells. NVG-111 is currently showing efficacy with predictable and manageable toxicity in Phase I/II clinical development for the treatment of Chronic Lymphocytic Leukaemia (CLL) and Mantle Cell Lymphoma (MCL), with the scope to expand into other hematological malignancies and solid tumour indications. These clinical data provide the foundation for the clinical development of a half-life extended (HLE) TCE using the clinically derisked ROR1 and CD3 binders from NVG-111. Objective: Building on the safety and efficacy data emerging from the Phase I/II trial of NVG-111, develop a half-life extended ROR1xCD3 TCE for a more convenient once a week dosing regimen, without compromising the safety, efficacy and CMC attributes of NVG-111. Methods: Utilizing NVG-111 as a core for a ROR1xCD3 HLE-TCE, several formats were designed and evaluated. These incorporated different half-life extending technology solutions by increasing the molecule size and enabling FcRn-mediated recycling. NVG-111 was cloned in-format with an antibody Fc, human serum albumin (HSA), or an HSA-binding antibody into a suitable expression vector and the resultant clones expressed using Expi293 cells. Molecules were then purified using our proprietary downstream purification process. The potency of ROR1 targeting HLE-TCEs were evaluated in cytotoxicity and T cell activation co-culture assays using MCL derived JeKo-1 target cells and T cells from healthy donor PBMCs, with responses measured by flow cytometry. Biophysical properties were evaluated by determining the post-purification expression titer, aggregation profile, and stability at 37°C. Results: Reformatting NVG-111 into an IgG-like bispecific scFv-Fc heterodimer resulted in loss of cytotoxic potency, which may be due to a change in the geometry of binding or poorer target engagement. Fusing NVG-111 directly to an HSA-binding antibody resulted in complete loss of expression. A 3-fold reduction in expression titer was observed for NVG-111 fused directly with HSA, which was also accompanied with a >10-fold reduction in functional activity, loss of binding affinity and reduced stability compared with parental molecule. Fusing NVG-111 to Fc resulted in a molecule that maintained functional potency, with transient expression titer remaining >100mg/L, which is also aligned with the parental molecule. The lead Fc-fused HLE molecule was then further refined with appropriate Fc-silencing technologies, which displayed low levels of aggregation following purification, with a monomer content of >97% (Figure 1). Stability studies performed in formulation buffer and in human serum showed that the Fc-fused HLE molecule was at least as stable as parental NVG-111, retaining binding and no evidence of aggregation or fragmentation. The potency of HLE-TCE with respect to cytotoxicity was influenced by the silencing technologies (Figure 2), with technologies 1 and 2 retaining potency as measured by EC50 to within 2-fold to 5-fold of NVG-111. However, silencing technology 3 displayed a 10-fold lower potency than NVG-111 and was therefore not progressed. Finally, initial PK studies suggest that our selected ROR1 targeting HLE-TCE has a half-life that would support weekly dosing in humans. Conclusion: We have generated a first in class ROR1 targeting half-life extended TCE that maintains the functional and biophysical properties of the clinically promising NVG-111. Our systematic evaluation suggests that careful selection of HLE format is critical for the development of a molecule with good yield, biophysical characteristics and potency. The selected ROR1 HLE-TCE supports weekly administration whilst maintaining potency at sub-nanomolar concentrations, properties which differentiate it from other TCEs in development. IND enabling studies with our 1st in class ROR1 targeting HLE-TCE will conclude in 2023, supporting the initiation of a Phase I clinical trial in ROR1+ hematological malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lintuzumab-Ac225 with Combination with Intensive Chemotherapy Yields High Response Rate and MRD Negativity in R/R AML with Adverse Features

Background: Actimab-A (lintuzumab-Ac225) is a humanized CD33 antibody conjugated to an alpha emitting isotope, Ac225, delivering high energy radiation over a short radius to CD33 expressing AML blasts and requiring no isolation. Preclinical studies also indicate that Actimab-A effectively depletes the anti-apoptotic protein MCL-1, potentially indicating another mechanism of action, which may benefit venetoclax resistant patients (pts). In this study, we hypothesized that low dose Actimab-A after salvage chemotherapy is feasible and improves clearance of residual, resistant AML thereby improving remission rates, and depth of remission. Herein, we report results of our Phase I study, and results of pts enrolled in an expansion cohort to characterize PK. Methods: This study enrolled R/R AML pts deemed fit, with adequate organ function, and with CD33 expression on >25% of leukemic blasts. The primary endpoint of the Phase I study was determination of MTD of Actimab-A in combination with CLAG-M. Secondary endpoints included the composite complete remission (CRc) rate after one induction cycle, the MRD (-) CRc rate determined by multiparametric flow cytometry, median overall survival (OS), and estimated 1- and 2-year OS. Furthermore, we are performing an expansion cohort to characterize PK. Induction consisted of G-CSF, 300mcg/d, given D1-6, cladribine 5mg/m2, given D2-6, cytarabine 2g/m2, given D2-6, and mitoxantrone 10mg/m2, given D2-4. Actimab-A was administered as a single dose on day 8 ±1 day The trial enrolled to 4 cohorts, with Actimab-A at doses ranging from 0.25uCi/kg to 1.0uCi/kg. One induction cycle was administered in all pts. Results: Of the 21 pts evaluable for response, median age was 63 yrs, 52% (n=11) had s-AML/t-AML, 67% (n=14) had adverse cytogenetics, and TP53 mutations were present in 52% (n=11) of pts. Median blast CD33 expression at baseline was 77% (range: 29-100%). Pts received a median 2 lines of therapy, including 57% (n=12) who received prior venetoclax. 57% (n=12) of pts enrolled after relapse post allogeneic HCT (alloHCT) (Table 1). The MTD was determined to be 1.0 uCi/kg, and the RP2D was set at 0.75uCi/kg. Within the 1.0uCi/kg dosing cohort, 2 DLTs were observed in 5 pts; severe mucositis and delayed ANC recovery (>42 days). Among all pts, Grade 3/4 toxicities observed in >10% of pts included febrile neutropenia, infection, rash and hypokalemia. Overall, 52% (n=11) achieved CRc, an additional 14% (n=3) achieved an MLFS, for an ORR (CRc+MLFS) of 67%. MRD negativity by flow was observed in 72% of pts achieving a CRc. At the RP2D, 67% (n=4) achieved CRc, and 75% of these pts achieved MRD negativity by flow. An additional 1 patient achieved MLFS for an ORR=83%. Actimab-A+CLAG-M was active in pts with TP53 mutations (ORR 73%) and in those who received prior venetoclax (ORR 55%) (Table 1). Median OS among all pts was 1.0 year (95% CI, 0.2 to 1.8); 1-year OS was 53% and 2-year OS was 32% (Figure 1). After therapy, 75% (n=6) of pts successfully bridged to alloHCT. Among patients who underwent 1st alloHCT, median OS was 2.5 years (95%CI, 1.0-3.9). PK studies measuring blood radioactivity were performed in 3 pts. Blood Ac225 radioactivity studies revealed median tmax of 0.6 hrs after infusion start. After achieving Cmax, mean half-life of radioactivity was 9.8 hrs. Ac225 radioactivity was below the limit of quantification (BLQ) by 48 hrs in all pts. Conclusions: Actimab-A at 0.75uCi/kg, combined with CLAG-M is feasible and safe, and this combination demonstrates a high response rate and MRD negativity in a high-risk AML population. Pharmacokinetics at the RP2D indicate rapid drug clearance. Furthermore, responses appear durable, particularly among patients who are able to proceed to alloHCT. In conclusion, with no viable options for both R/R TP53 mutated AML and R/R AML previously treated with venetoclax, this regimen represents a potentially important option, and efforts are underway for a confirmatory study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel Chemical Series of Anti-ST2 Small Molecules Suppress Allogeneic T Cells Proliferation While Increasing Treg Cells and Improve Gvhd and Survival In Vivo

Elevation of soluble Stimulation-2 (sST2) is considered a prognostic biomarker in several disease progression, such as cardiac allo-rejection, graft-versus-host disease (GVHD), and is associated with the response to tissue damage that secrets Interleukin-33 (IL-33), an alarmin. sST2 is an alternatively spliced isoform of the Interleukin 1 Receptor-Like 1 (IL1RL1) gene that also encodes the major membrane-bound ST2 (ST2) isoform expressed on tolerogenic immune cells including Foxp3+ regulatory T cells (Tregs), type 2 innate lymphoid cells (ILC2), type 2 helper T cells (Th2), monocytes, M2 macrophages, mast cells, eosinophils and neutrophils. IL-33 is the only known ligand that binds to both sST2 and ST2. Binding of IL-33 to ST2 activates intracellular signaling in ST2 expressing cells whereas sST2 functions as a decoy receptor to sequester the secreted free IL-33. After allogeneic hematopoietic cell transplantation (HCT), elevated sST2 levels are associated with patients with GVHD resistant to steroid therapy and with subsequent non-relapse related death. We hypothesized that therapeutic intervention to release IL-33 from sST2 may modulate the T cells responses for treating GVHD. As proof-of-concept, we have reported studies using either ST2 antibodies (Zhang et. al., Sci. Trans. Med. 2015) or small-molecule ST2 inhibitors (iST2-1 in Ramadan et. al., JCI Insight, 2018) in the murine GVHD models. Herein, we report a novel series of small-molecule ST2 inhibitors from our follow-up development of iST2-1. We show a representative compound, XY-52, effectively reduced sST2 levels, alleviated GVHD and improved survival in a murine GVHD model. Based on our previously discovered iST2-1, we recently developed 1-(furan-2ylmethyl)pyrrolidine-based ST2 Inhibitors and constructed the structure-activity relationship (SAR) of compounds against ST2/IL-33 binding (Yuan et. al., Bioorg. Med. Chem., 2022). That study led to identify 14e (Fig. 1A) that has an IC50 value of 7.77 mM against ST2/IL33 binding determined by the AlphaLISA assay. Despite the improvement of the IC50 value as compared to iST2-1, 14e has deficiency in solubility and metabolic stability (including a similar in vivo half-life as iST2-1) that required further optimization. To improve 14e, we first focused on replacing the furan group (C-ring) of 14e with the goal of eliminating the potential metabolic issue of the furan group. We found replacement of furan by either a pyrrole (ST2-J-100) or an alkylated pyrrole group (ST2-J-106, 107) retained the activity of the inhibitors. We then investigated the replacement of the chemical groups on A and B rings of 14e aiming to improve the solubility of the inhibitors while maintaining their activities. These modifications led to identify a new series of ST2 inhibitors represented by XY-52. In the mixed lymphocyte reaction (MLR) assay, we found XY-52 and analogs (XY-125, XY-146, XY-148, XY-150) inhibited CD4+, CD8+ T cell proliferation while increased Tregs (Fig. 1B). XY-52 has improved in vitro (t1/2= >60m in mouse, 41m in human liver microsome) and in vivo metabolic stability. In vivo PK study showed that XY-52 has higher tissue distribution including spleen, lung, skin, small intestine and liver than iST2-1 or Ruxolitinib (Fig. 1C). XY-52 was then selected to compare with 3c (previously reported) in a clinically relevant minor-mismatched GVHD mouse model (C57BL/6→C3H.SW). Using a 21-day prophylactic regimen of 40mg/kg/day (BID), we found that 3c reduced GVHD score (P=0.02) and extended survival (HR 3.90, P=0.02) in mice. Mice receiving XY-52 treatment had a more profound reduction in GVHD score (P<0.0001) and improvement in survival (HR, 31.22, P<0.0001) (Fig. 1D). Both 3c and XY-52 yielded lower plasma concentrations of sST2 and IFNg compared with the DMSO control (Fig. 1E) at Day 7 and 21 post-HCT. Ex vivo analysis of T cells in the gastrointestinal tract (GI) at day 21 post-HCT showed that XY-52 was more effective than 3c at reducing CD4+ IFNg+, CD8+ IFNγ+, CD4+ IL-17A+ T cells and increasing CD4+ Foxp3+ Tregs frequencies. In summary, XY-52 represents a promising ST2 inhibitor that reduces the plasma sST2 levels, CD4+, CD8+ effector T cells and increases CD4+ Foxp3+ Tregs frequencies in in vitro and in vivo GVHD models. Further improvement in the potency and the PK profile of XY-52 will allow us to develop effective ST2 inhibitors for use as a prophylactic or therapeutic regimen for GVHD therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TMIC-79. MYELOID-DIRECTED TREATMENT USING THE TLR7/8 AGONIST RESIQUIMOD IMPROVES THE SURVIVAL OF MICE WITH MEDULLOBLASTOMA AND ENHANCES EFFICACY OF RADIOTHERAPY

Abstract We show the therapeutic potential of myeloid-directed immunomodulatory treatment for medulloblastoma. Patients with medulloblastoma, the most common malignant pediatric brain tumor, need new treatments, as standard therapy produces disabling neurotoxicities and fails 20% of patients. About 30% of medulloblastomas show hyperactivation of the SHH (Sonic Hedgehog) signaling pathway, and these tumors have large myeloid populations in the tumor microenvironment (TME). We analyzed whether activating toll-like receptors on these myeloid cells would slow tumor growth. Our prior single-cell RNA sequencing studies in both patient samples and mouse models showed that 10% of the cells in SHH medulloblastoma are myeloid cells and that these cells uniquely express TLR7 and TLR8. We treated mice genetically engineered to develop SHH medulloblastomas with the TLR7/8 agonist resiquimod, administered systemically either as free drug or in polyoxazoline nanoparticles (POx-resiquimod), and compared POx-resiquimod+radiotherapy to radiotherapy alone. We found that POx-resiquimod extended the survival time of mice with medulloblastoma, while free drug failed to show benefit. PK studies showed that POx-resiquimod increased tumor drug exposure, consistent with increased efficacy. Mechanistically, POx-resiquimod increases tumor myeloid populations and decreased the fraction of myeloid cells that expressed IGF1. POx-resiquimod plus radiation therapy, moreover, was superior to either POx-resiquimod alone, or radiotherapy alone. Together our data show that the TLR7/8 agonist resiquimod, delivered in nanoparticle formulation, produces a significant anti-tumor effect in SHH medulloblastoma, and increased the efficacy of radiotherapy. As radiotherapy is the mainstay of current medulloblastoma treatment, we propose that TLR7/8-agonist therapy such as resiquimod may be added to current regimens to reduce the radiation dose needed for efficacy, and to increase the fraction of successfully treated patients.

Investigations on In Vivo Pharmacokinetic/Pharmacodynamic Determinants of Fosfomycin in Murine Thigh and Kidney Infection Models.

Background: Amidst the era of widespread resistance, there has been a renewed interest in older antibiotics such as fosfomycin, owing to its activity against certain resistant Gram-negative pathogens, including multidrug-resistant variants expressing extended spectrum β-lactamases or carbapenemases. The goal of the study was to investigate pharmacokinetic/pharmacodynamic (PK/PD) index and PK/PD targets of fosfomycin in murine thigh and kidney infection models, employing clinical isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Methods: Seven isolates of E. coli (one wild-type and six clinical isolates) and five isolates of K. pneumoniae (one wild-type and four clinical isolates) were utilized for in vivo PK/PD studies. Single-dose plasma PK studies were conducted in infected mice by subcutaneous route. PD index was determined from exposure-response analysis employing 24-hr dose fractionation studies in neutropenic murine thigh infection model, while pharmacodynamic targets (PDTs) were derived from both thigh and kidney infection models. Results: Dose fractionation studies demonstrated that in vivo efficacy of fosfomycin best correlated with AUC/MIC for E. coli (R2 = 0.9227) and K. pneumoniae (R2 = 0.8693). The median AUC/MIC linked to 1 log10 kill effects were 346.2 and 745.2 in thigh infection model and 244.1 and 425.4 in kidney infection model for E. coli and K. pneumoniae, respectively. The mice plasma protein binding of fosfomycin was estimated to be 5.4%. Conclusions: The in vivo efficacy of fosfomycin against Enterobacterales was best described by AUC/MIC. The PDTs derived from this study may help define the coverage potential of fosfomycin at the clinical doses approved.

DEVELOPMENT OF PHVS719: AN ORAL EXTENDED-RELEASE BRADYKININ B2 RECEPTOR ANTAGONIST TO PREVENT HEREDITARY ANGIOEDEMA ATTACKS

<h3>Introduction</h3> Hereditary angioedema (HAE) is managed by either of two treatment paradigms: on demand or prophylaxis. PHA121, an orally active bradykinin B2 receptor antagonist, is being developed as two drug products to optimize patient choice. PHVS416, the softgel capsule providing quick absorption for fast onset of symptom relief, is currently in clinical trials for treatment of acute attacks; PHVS719, an extended-release tablet, is being developed for use in prophylaxis. <h3>Methods</h3> To develop a daily oral pill for the prevention of attacks, we developed a PHA121 formulation (PHVS719) providing 24 h plasma exposure. Our strategy relied upon a combination of an optimally designed extended-release formulation and the ability of PHA121 to be absorbed throughout the full gastrointestinal tract. <h3>Results</h3> PHA121 is absorbed following intracolonic administration in rats. A human mass balance study showed only 3% PHA121 excreted in feces and oral bioavailability of 57%. An in vitro dissolution experiment conducted with PHVS719 in conditions mimicking fasted and fed gastrointestinal environments showed a linear dissolution of PHA121 over time. Using PHVS719, a PK study in healthy volunteers demonstrated that a single 40 mg dose provides 24 h plasma exposure above the anticipated therapeutic level. Together these data provide evidence that PHVS719 is optimally suited to serve as a once-daily oral pill. <h3>Conclusion</h3> The active ingredient PHA121 is under clinical investigation in the form of two therapeutic treatment modalities: PHVS416 softgel capsule providing quick absorption for fast symptom relief and PHVS719 once-daily extended-release tablet providing 24 hours coverage to prevent attacks from occurring.

In vitro effect of Withania somnifera, AYUSH-64, and remdesivir on the activity of CYP-450 enzymes: Implications for possible herb−drug interactions in the management of COVID-19

Ayurvedic medicines Withania somnifera Dunal (ashwagandha) and AYUSH-64 have been used for the prevention and management of COVID-19 in India. The present study explores the effect of Ashwagandha and AYUSH-64 on important human CYP enzymes (CYP3A4, CYP2C8, and CYP2D6) to assess their interaction with remdesivir, a drug used for COVID-19 management during the second wave. The study also implies possible herb−drug interactions as ashwagandha and AYUSH-64 are being used for managing various pathological conditions. Aqueous extracts of ashwagandha and AYUSH-64 were characterized using LC-MS/MS. A total of 11 and 24 phytoconstituents were identified putatively from ashwagandha and AYUSH-64 extracts, respectively. In addition, in silico studies revealed good ADME properties of most of the phytoconstituents of these herbal drugs and suggested that some of these might possess CYP-450 inhibitory activity. In vitro CYP-450 studies with human liver microsomes showed moderate inhibition of CYP3A4, 2C8, and 2D6 by remdesivir, while ashwagandha had no inhibitory effect alone or in combination with remdesivir. AYUSH-64 also exhibited a similar trend; however, a moderate inhibitory effect on CYP2C8 was noticed. Thus, ashwagandha seems to be safe to co-administer with the substrates of CYP3A4, CYP2C8, and CYP2D6. However, caution is warranted in prescribing AYUSH-64 along with CYP2C8 substrate drugs. Furthermore, preclinical and clinical PK studies would be helpful for their effective and safer use in the management of various ailments along with other drugs.

Design and conduct considerations for studies in patients with hepatic impairment.

Despite the liver being the primary site for clearance of xenobiotics utilizing a myriad of mechanisms ranging from cytochrome P450 enzyme pathways, glucuronidation, and biliary excretion, there is a dearth of information available as to how the severity of hepatic impairment (HI) can alter drug absorption and disposition (i.e., pharmacokinetics [PK]) as well as their efficacy and safety or pharmacodynamics (PD). In general, regulatory agencies recommend conducting PK studies in subjects with HI when hepatic metabolism/excretion accounts for more than 20% of drug elimination or if the drug has a narrow therapeutic range. In this tutorial, we provide an overview of the global regulatory landscape, clinical measures for hepatic function assessment, methods to stage HI severity, and consequently the impact on labeling. In addition, we provide an in-depth practical guidance for designing and conducting clinical trials for patients with HI and on the application of modeling and simulation strategies in lieu of dedicated trials for dosing recommendations in patients with HI.

DNA-delivered antibody cocktail exhibits improved pharmacokinetics and confers prophylactic protection against SARS-CoV-2

Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics.