Pharmacokinetic Studies In Monkeys Research Articles

Nanobody-based microfluidic human Fc assay for preclinical plasma quantification of IgG1/1.1 and IgG1-Fc-conjugates

BackgroundTherapeutic antibodies and Fc-conjugates are becoming increasingly popular for disease management and accurate and sensitive pharmacokinetic measurements are critical in lead candidate selection in pre-clinical drug discovery. Methods and study designHuman Fc-specific intact monoclonal antibodies, polyclonal antibodies, Fab fragments, aptamers, affibodies and nanobodies were screened for potential as biotinylated capture moieties in a microfluidic assay. Test compounds were Bevacizumab, Rituximab, Infliximab as well as an in-house IgG1.1 and an IgG1-drug conjugate. ResultsCapture molecules were tested for specificity in plasma matrices from beagle dog, rat, mouse, pig, rhesus monkey and cynomolgus monkey. We find that the llama nanobody provides the best selectivity across across species. The assay usability were verified in cynomolgus monkey pharmacokinetic studies of in-house IgG1.1 and IgG1-fusion molecules. ConclusionThe presented generic nanobody-based assay may find relevance in preclinical testing of future human Fc-containing drug conjugates devoid of Fab fragments and intact monoclonal antibodies.

Role of Epoxide Hydrolases and Cytochrome P450s on Metabolism of KZR-616, a First-in-Class Selective Inhibitor of the Immunoproteasome.

KZR-616 is an irreversible tripeptide epoxyketone-based selective inhibitor of the human immunoproteasome. Inhibition of the immunoproteasome results in anti-inflammatory activity in vitro and based on promising therapeutic activity in animal models of rheumatoid arthritis and systemic lupus erythematosus KZR-616 is being developed for potential treatment of multiple autoimmune and inflammatory diseases. The presence of a ketoepoxide pharmacophore presents unique challenges in the study of drug metabolism during lead optimization and clinical candidate profiling. This study presents a thorough and systematic in vitro and cell-based enzymatic metabolism and kinetic investigation to identify the major enzymes involved in the metabolism and elimination of KZR-616. Upon exposure to liver microsomes in the absence of NADPH, KZR-616 and its analogs were converted to their inactive diol derivatives with varying degrees of stability. Diol formation was also shown to be the major metabolite in pharmacokinetic studies in monkeys and correlated with in vitro stability results for individual compounds. Further study in intact hepatocytes revealed that KZR-616 metabolism was sensitive to an inhibitor of microsomal epoxide hydrolase (mEH) but not inhibitors of cytochrome P450 (P450) or soluble epoxide hydrolase (sEH). Primary human hepatocytes were determined to be the most robust source of mEH activity for study in vitro. These findings also suggest that the exposure of KZR-616 in vivo is unlikely to be affected by coadministration of inhibitors or inducers of P450 and sEH. SIGNIFICANCE STATEMENT: This work presents a thorough and systematic investigation of metabolism and kinetics of KZR-616 and related analogs in in vitro and cell-based enzymatic systems. Information gained could be useful in assessing novel covalent proteasome inhibitors during lead compound optimization. These studies also demonstrate a robust source in vitro test system that correlated with in vivo pharmacokinetics for KZR-616 and two additional tripeptide epoxyketones.

Abstract 913: FLT3-targeting TriTACs are T cell engagers for treatment of acute myeloid leukemia

Abstract FLT3 (fms related receptor tyrosine kinase 3) is a cell surface protein expressed in hematopoietic stem and progenitor cell populations1. FLT3 RNA is overexpressed in greater than 95% of acute myeloid leukemia (AML) samples2, and FLT3 mutations are found in 25% to 35% of AML samples1. In 2020 in the United States, an estimated 19,940 new AML cases and 11,180 AML-related deaths are expected3, exemplifying that better therapies are needed to treat this disease. Given the frequency of FLT3 expression in AML and unmet medical need for patients, FLT3 represents an attractive target for T cell-redirecting immunotherapy in AML. FLT3-targeting Tri-specific T cell Activating Constructs (FLT3 TriTACs) are designed to simultaneously engage FLT3 on a target AML cell and CD3 on a T cell, resulting in T cell activation, proliferation, and lysis of the AML cell. FLT3 TriTACs are ~53 kDa molecules that consist of three binding domains: an N-terminal single domain antibody (sdAb) that binds to human FLT3, a middle sdAb that binds to human serum albumin (HSA) to extend the half-life, and a C-terminal single chain Fv (scFv) that binds to CD3ϵ of the T cell receptor (TCR). Biophysical analyses of these molecules indicate they are stable with high monomer content. FLT3 TriTACs bind to human and cynomolgus monkey FLT3, CD3ϵ and albumin with similar affinities. Flow cytometry analysis of T cells from various normal donors and a panel of FLT3-positive and FLT3-negative tumor cell lines confirmed binding of FLT3 TriTACs to their targets expressed on the cell surface. FLT3 TriTACs induce potent killing of FLT3-expressing AML cell lines in vitro. In co-cultures of T cells from normal human donors, target tumor cells, and HSA, these TriTACs mediated dose-dependent and FLT3-dependent cytotoxicity. Cell lines engineered to express cynomolgus FLT3 were also readily killed by T cells with FLT3 TriTACs. Preclinical evaluation of FLT3 TriTACs in vitro, in mouse tumor models, and cynomolgus monkey pharmacokinetic studies will be presented.

Quantitation and modeling of post-translational modifications in a therapeutic monoclonal antibody from single- and multiple-dose monkey pharmacokinetic studies using mass spectrometry.

Post-translational modifications (PTMs) of therapeutic monoclonal antibodies (mAbs) are important product quality attributes (PQAs) that can potentially impact drug stability, safety, and efficacy. The PTMs of a mAb may change remarkably in the bloodstream after drug administration compared to in vitro conditions. Thus, monitoring in vivo PTM changes of mAbs helps evaluate the criticality of PQAs during the product risk assessment. In addition, quantitation of the subject exposures to PTM variants helps assess the impact of PTMs on the safety and efficacy of therapeutic mAbs. Here, we developed an immunocapture-liquid chromatography/mass spectrometry (LC/MS) method to quantify in vivo PTM changes a therapeutic mAb overtime in single- and multiple-dose monkey pharmacokinetic (PK) studies. We also built mathematical models to predict the in vivo serum concentrations of PQAs, the subject exposures to PQAs, and the relative abundance of PQAs in single- and multiple-dose regimens. The model predictions are in good agreement with the experimental results. The immunocapture-LC/MS method and mathematical models enable bioanalytical chemists to quantitatively assess the criticality of PQAs during drug development.

Early formulation development of CKD-519, a new CETP inhibitor, for phase 1 clinical study based on in vitro and in vivo evaluation

CKD-519, a potent cholesteryl ester transfer protein (CETP) inhibitor, is a clinical candidate being developed for the treatment of dyslipidemia. It is considered a Biopharmaceutical Classification System II compound with low solubility and high permeability. The objective of this study was to develop early formulations focusing on the dissolution rate of the compound to achieve dose-dependent exposure. High performance formulation strategies including solid dispersion (SD) and a self-microemulsifying drug delivery system (SMEDDS) were investigated and their in vivo and in vitro correlations were also evaluated in monkeys along with dose optimization in human volunteers. The SD granules were prepared in a fluid bed granulator using microcrystalline cellulose and mannitol as carriers. Poloxamer 407 and Eudragit E PO were each found to be a suitable solubilizing agent and polymer for the improvement of the CKD-519 dissolution rate. Pharmacokinetic studies in monkeys showed that the SD tablets exhibited better absorption than the SMEDDS in a dose-dependent manner from 1.5 mg to 100 mg. The mannitol-based SD tablet formulations were bioequivalent. However, pharmacokinetics studies in humans showed that the dose was saturable above 100 mg of CKD-519. This study was performed to determine how to develop early formulations for clinical studies and to identify rational formulation development strategies for CKD-519 to establish the pharmaceutical proof-of-concept in humans.

Application of an electrochemiluminescence assay for quantification of E6011, an antifractalkine monoclonal antibody, to pharmacokinetic studies in monkeys and humans.

E6011, a humanized antifractalkine monoclonal antibody, is under development for the treatment of various inflammatory diseases, such as rheumatoid arthritis. A reproducible assay method has been developed for the determination of E6011 in monkey and human serum by electrochemiluminescence (ECL) assay. E6011 in serum was captured by fractalkine and detected by ruthenium-labeled rabbit anti-E6011 Fab polyclonal antibodies for ECL detection. E6011 in serum was quantifiable from 0.02 and 0.1μg/mL in monkey and human serum, respectively, with minimum required dilution of 500. The method was then validated in accordance with bioanalytical guidelines. Accuracy and precision of quality control samples at five concentrations in intra- and interbatch reproducibility demonstrated that relative error and relative standard deviation were within acceptable criteria. Recovery of E6011 was 92.9%-121.7% and 85.0%-109.3% in humans and monkeys. Dilution integrity, no prozone effects, and no impacts by antigen were also ensured. Parallelism was also confirmed using incurred clinical sample analysis. Various types of stability were assessed, which confirmed that E6011 in serum was stable for 367 and 735days in monkey and human sera, respectively, under frozen conditions. The developed method was successfully applied supporting pharmacokinetic studies in monkeys and humans.

Discovery and Assessment of Atropisomers of (±)-Lesinurad.

(+)- and (-)-Lesinurad were isolated as atropisomers from racemic lesinurad for the first time. No interconversion was observed between the two atropisomers under various conditions tested. The two atropisomers showed significant differences in hURAT1 highly expressed HEK293 cell-based inhibition assays, monkey pharmacokinetic studies, and in vitro human recombinant CYP2C9 stability studies. It was speculated that (+)-lesinurad might offer a better hyperuricemia/gout therapy than (-)-lesinurad or the racemate.

Abstract 3217: MGD013, a bispecific PD-1 x LAG-3 Dual-Affinity Re-Targeting (DART®) protein with T-cell immunomodulatory activity for cancer treatment

Abstract Introduction: The combination of monoclonal antibodies (mAbs) that targets the immune checkpoint molecules CTLA-4 and PD-1 has shown clinical benefit beyond that observed with either mAb alone. This finding has prompted exploring whether such an approach could be applied within the context of additional combinations of checkpoint molecules, such as PD-1 and lymphocyte activation gene-3 (LAG-3). Animal tumor models have validated combining anti-PD-1 with anti-LAG-3 mAbs in eliciting synergistic tumor-eradicating immunity; expression of PD-1 and LAG-3 on exhausted T cells and tumor infiltrating lymphocytes (TILs) further supports their dual-targeting. We have developed a bispecific DART® protein that targets PD-1 and LAG-3, aimed at inducing potent antitumor immunity through simultaneous blockade of non-redundant checkpoint pathways intrinsic to exhausted T cells. Methods: mAbs against PD-1 and LAG-3 were generated and selected for DART conversion based on binding, biophysical and functional blocking against their respective receptor/ligand axes and functional activity in re-activation of prior superantigen-stimulated T cells or in antigen-specific recall assays. Results: Lead PD-1 and LAG-3 mAbs demonstrating favorable functional properties were selected for humanization. Immunohistochemistry confirmed that the lead LAG-3 and PD-1 mAbs display restricted lymphocyte expression in human tissues and overlapping expression in TILs. The humanized mAbs were assembled into MGD013, an Fc-bearing PD-1 x LAG-3 DART protein that demonstrated favorable biophysical and manufacturability properties. MGD013 bound specifically with high affinity to PD-1 and LAG-3 as well as to target-expressing cell lines and chronically-activated T cells. MGD013 blocked PD-1/PD-L1, PD-1/PD-L2 and LAG-3/HLA (MHC-II) interactions and PD-1 signaling. Further functional characterization of MGD013 revealed enhanced cytokine secretion in response to antigenic re-challenge of previously stimulated T cells compared to that observed upon independent blockade of either the PD-1 or LAG-3 pathways alone. Furthermore, under the above experimental conditions, MGD013 mediated greater cytokine secretion than that observed with the combination of equivalent (equimolar) levels of replicas of the approved PD-1 mAb, nivolumab, and the LAG-3 mAb, 25F7, which is currently undergoing clinical testing. Finally, cynomolgus monkey pharmacokinetic studies demonstrated a prolonged circulating half-life consistent with that of an Fc-bearing molecule. Conclusions: MGD013 blocks both PD-1 and LAG-3 pathways, resulting in enhanced T-cell responses compared to single or combination mAb blockade. Together with favorable cynomolgus monkey PK, these studies support further clinical development of MGD013. Citation Format: Ross LaMotte-Mohs, Kalpana Shah, Doug Smith, Sergey Gorlatov, Valentina Ciccarone, James Tamura, Hua Li, Jill Rillema, Monica Licea, Leilei He, Farha Vasanwala, Wei Chen, Xiao-Tao Yao, Francine Chen, Jennifer Brown, Jeffrey Nordstrom, Scott Koenig, Ezio Bonvini, Syd Johnson, Paul Moore. MGD013, a bispecific PD-1 x LAG-3 Dual-Affinity Re-Targeting (DART®) protein with T-cell immunomodulatory activity for cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3217.

Long-Term Methamphetamine Administration in the Vervet Monkey Models Aspects of a Human Exposure: Brain Neurotoxicity and Behavioral Profiles

Methamphetamine (METH)-associated alterations in the human striatal dopamine (DA) system have been identified with positron emission tomography (PET) imaging and post-mortem studies but have not been well correlated with behavioral changes or cumulative METH intake. Animal studies that model some aspects of human long-term METH abuse can establish dose-dependency profiles of both behavioral changes and potential brain neurotoxicities for identifying consequences of particular cumulative exposures. Based on parameters from human and our monkey pharmacokinetic studies, we modeled a prevalent human METH exposure of daily multiple doses in socially housed vervet monkeys. METH doses were escalated over 33 weeks, with final dosages resulting in estimated peak plasma METH concentrations of 1-3 microM, a range measured in human abusers. With larger METH doses, progressive increases in abnormal behavior and decreases in social behavior were observed on 'injection' days. Anxiety increased on 'no injection' days while aggression decreased throughout the study. Thereafter, during 3 weeks abstinence, differences in baseline vs post-METH behaviors were not observed. Post-mortem analysis of METH brains showed 20% lower striatal DA content while autoradiography studies of precommissural striatum showed 35% lower [3H]WIN35428 binding to the DA transporter. No statistically significant changes were detected for [3H]dihydrotetrabenazine binding to the vesicular monoamine transporter (METH-lower by 10%) or for [3H]SCH 23390 and [3H]raclopride binding to DA D1 and D2 receptors, respectively. Collectively, this long-term, escalating dose METH exposure modeling a human abuse pattern, not associated with high-dose binges, resulted in dose-dependent behavioral effects and caused persistent changes in presynaptic striatal DA system integrity.

Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening.

Granulocyte-colony stimulating factor (G-CSF) is used worldwide to prevent neutropenia caused by high-dose chemotherapy. It has limited stability, strict formulation and storage requirements, and because of poor oral absorption must be administered by injection (typically daily). Thus, there is significant interest in developing analogs with improved pharmacological properties. We used our ultrahigh throughput computational screening method to improve the physicochemical characteristics of G-CSF. Improving these properties can make a molecule more robust, enhance its shelf life, or make it more amenable to alternate delivery systems and formulations. It can also affect clinically important features such as pharmacokinetics. Residues in the buried core were selected for optimization to minimize changes to the surface, thereby maintaining the active site and limiting the designed protein's potential for antigenicity. Using a structure that was homology modeled from bovine G-CSF, core designs of 25-34 residues were completed, corresponding to 10(21)-10(28) sequences screened. The optimal sequence from each design was selected for biophysical characterization and experimental testing; each had 10-14 mutations. The designed proteins showed enhanced thermal stabilities of up to 13 degrees C, displayed five-to 10-fold improvements in shelf life, and were biologically active in cell proliferation assays and in a neutropenic mouse model. Pharmacokinetic studies in monkeys showed that subcutaneous injection of the designed analogs results in greater systemic exposure, probably attributable to improved absorption from the subcutaneous compartment. These results show that our computational method can be used to develop improved pharmaceuticals and illustrate its utility as a powerful protein design tool.