Development of minimal physiologically-based pharmacokinetic-pharmacodynamic models for characterizing cellular kinetics of CAR T cells following local deliveries in mice.

Chimeric antigen receptor (CAR) T cell therapies have revolutionized the treatment of hematologic malignancies and have potentials for solid tumor treatment. To overcome limited CAR T cell infiltration to solid tumors, local delivery of CAR T cells is a practical strategy that has shown promising therapeutic outcome and safety profile in the clinic. It is of great interest to understand the impact of dosing routes on CAR T cell distribution, subsequent proliferation and tumor killing in a quantitative manner to identify key factors that contribute to CAR T efficacy and safety. In this study, we established mouse minimal physiologically-based pharmacokinetic (mPBPK) models combined with pharmacodynamic (PD) components to delineate CAR T cell distribution, proliferation, tumor growth, and tumor cell killing in the cases of pleural and liver tumors. The pleural tumor model reasonably captured published CAR T cellular kinetic and tumor growth profiles in mice. The mPBPK-PD simulation of a liver tumor mouse model showed a substantial increase in initial tumor infiltration and earlier CAR T cell proliferation with local hepatic artery delivery compared to portal vein and intravenous (i.v.) injections whereas portal vein injection showed little difference from i.v. administration, suggesting the importance of having the injection site close to tumor for maximal effect of non-systemic administration. Blood flow rate in the liver tumor was found to be a sensitive parameter for cellular kinetics and efficacy, indicating a potential role of tumor vascularization in the efficacy of CAR T cell therapies.

A Reduction in B, T, and Natural Killer Cells Expressing CD38 by TAK-079 Inhibits the Induction and Progression of Collagen-Induced Arthritis in Cynomolgus Monkeys.

Ectoenzyme CD38 is increased on lymphocytes in response to an antigenic challenge and it is hypothesized that targeting these activated lymphocytes could ameliorate pathologic activities in autoimmune diseases. The cynomolgus monkey is an appropriate model for assessing potential effects of targeting CD38 in humans because these species exhibit similar expression profiles. TAK-079 is a human monoclonal antibody (IgG1 λ ) that binds to CD38 and lyses bound cells by complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. TAK-079 binds to monkey CD38 with an affinity at EC50 4.5 nM, and the potential activity of TAK-079 was investigated in a monkey collagen-induced arthritis model of autoimmune disease. Prophylactic administration of TAK-079 (3 mg/kg i.v. weekly) was well tolerated and prevented arthritis development compared with vehicle-treated control animals, which exhibited progressive disease with radiographic damage and worsening clinical scores over the study course. Therapeutic treatment of arthritic monkeys with TAK-079 (3 mg/kg i.v. weekly) was also well tolerated and reduced disease progression and symptoms. Arthritis scores and joint swelling were significantly lower than the vehicle control, accompanied by decreases in blood levels of C-reactive protein, alkaline phosphatase, and natural killer, B, and T cells. Histopathology, morphometry, and radiology revealed significantly less joint damage in animals exposed prophylactically to TAK-079 treatment compared with vehicle-treated animals and significantly less damage in animals treated therapeutically with TAK-079 or dexamethasone (0.1 mg/kg oral gavage daily), illustrating potential disease-modifying activity. In conclusion, these data indicate that depletion of CD38-expressing cells could be a therapeutic mechanism for treating autoimmune diseases. SIGNIFICANCE STATEMENT: This study demonstrates that targeting CD38-expressing leukocytes with a cytolytic antibody can ameliorate autoimmune disease in cynomolgus monkeys. The study gives a unique perspective into this therapeutic strategy because the three other anti-CD38 cytolytic antibodies in clinical development (daratumumab, isatuximab, and MOR202) cannot be tested in similar models because they do not crossreact with CD38 expressed by new world primates.

Comparison of uptake transporter functions in hepatocytes in different species to determine the optimal model for evaluating drug transporter activities in humans.

A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis-Menten constant (Km,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17ß-glucuronide and metformin. The Km,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PSinf,act) while no difference in %PSinf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug-drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.

Preclinical absorption, distribution, metabolism, excretion and pharmacokinetics of a novel selective inhibitor of breast cancer resistance protein (BCRP).

1. Breast cancer resistance protein (BCRP) plays an important role in drug absorption, distribution and excretion. It is challenging to evaluate BCRP functions in preclinical models because commonly used BCRP inhibitors are nonspecific or unstable in animal plasma. 2. In this work, in vitro absorption, distribution, metabolism and elimination (ADME) assays and pharmacokinetic (PK) experiments in Bcrp knockout (KO) (Abcg2-/-) and wild-type (WT) FVB mice and Wistar rats were conducted to characterize the preclinical properties of a novel selective BCRP inhibitor (ML753286, a Ko143 analog). 3. ML753286 is a potent inhibitor for BCRP, but not for P-glycoprotein (P-gp), organic anion-transporting polypeptide (OATP) or major cytochrome P450s (CYPs). It has high permeability, but is not an efflux transporter substrate. ML753286 has low to medium clearance in rodent and human liver S9 fractions, and is stable in plasma cross species. Bcrp inhibition affects oral absorption and clearance of sulfasalazine in rodents. A single dose of ML753286 at 50-300 mg/kg orally, and at 20 mg/kg intravenously or 25 mg/kg orally inhibits Bcrp functions in mice and rats, respectively. 4. These findings confirm that ML753286 is a useful selective inhibitor to evaluate BCRP/Bcrp activity in vitro and in rodent model systems.

Efflux transporter breast cancer resistance protein dominantly expresses on the membrane of red blood cells, hinders partitioning of its substrates into the cells, and alters drug-drug interaction profiles.

1. Red blood cell (RBC) partitioning is important in determining pharmacokinetic and pharmacodynamic properties of a compound; however, active transport across RBC membranes is not well understood, particularly without transporter-related cell membrane proteomics data. 2. In this study, we quantified breast cancer resistance protein (BCRP/Bcrp) and MDR1/P-glycoprotein (P-gp) protein expression in RBCs from humans, monkeys, dogs, rats and mice using nanoLC/MS/MS, and evaluated their effect on RBC partitioning and plasma exposure of their substrates. BCRP-specific substrate Cpd-1 and MDR1-specific substrate Cpd-2 were characterized using Caco-2 Transwell® system and then administered to Bcrp or P-gp knockout mice. 3. The quantification revealed BCRP/Bcrp but not MDR1/P-gp to be highly expressed on RBC membranes. The knockout mouse study indicated BCRP/Bcrp pumps the substrate out of RBCs, lowering its partitioning and thus preventing binding to intracellular targets. This result was supported by a Cpd-1 and Bcrp inhibitor ML753286 drug-drug interaction (DDI) study in mice. Because of enhanced partitioning of Cpd-1 into RBCs after BCRP/Bcrp inhibition, Cpd-1 plasma concentration changed much less extent with genetic or chemical knockout of Bcrp albeit marked blood concentration increase, suggesting less DDI effect. 4. This finding is fundamentally meaningful to RBC partitioning, pharmacokinetics and DDI studies of BCRP-specific substrates.

Quantification of Four Efflux Drug Transporters in Liver and Kidney Across Species Using Targeted Quantitative Proteomics by Isotope Dilution NanoLC-MS/MS.

PURPOSE: The expression levels of several efflux drug transporters in the liver and kidney were evaluated across species to address potential roles of the transporters in species dependent excretion of drugs and their metabolites. METHODS: Four efflux transporters, namely MDR1/P-gp, BCRP/Bcrp, MRP2/Mrp2 and MRP3/Mrp3 in liver and kidney in three preclinical species and humans were quantified using targeted quantitative proteomics by isotope dilution nanoLC-MS/MS. RESULTS: In liver, the level of P-gp was highest in monkey and lowest in rat. The concentration of BCRP/Bcrp was highest in dog followed by monkey. MRP2/Mrp2 level was highest in monkey and rat, whereas MRP3/Mrp3 levels were similar in human, monkey and dog. In the kidney, the concentrations of MDR1/P-gp in human and monkey were roughly 2 to 3-fold higher than in rat and dog. In rat, BCRP/Bcrp concentrations were substantially higher than in any of the other species. MRP2/Mrp2 concentrations were similar across species, whereas expression of MRP3/Mrp3 was highest in rat. CONCLUSION: Overall, the results indicated that the pattern of hepatic and renal expression of the transporters was quite species dependent. This information should be helpful in the estimation of transport mediated drug and metabolites excretion in liver and kidney across species.

Synthesis of a new inhibitor of breast cancer resistance protein with significantly improved pharmacokinetic profiles.

The design, synthesis, in vitro inhibitory potency, and pharmacokinetic (PK) profiles of Ko143 analogs are described. Compared to commonly used Ko143, the new breast cancer resistance protein (BCRP) inhibitor (compound A) showed the same potency and a significantly improved PK profile in rats (lower clearance [1.54L/h/kg] and higher bioavailability [123%]). Ko143 on the other hand suffers from poor bioavailability. Compared to Ko143, compound A would be a useful probe for delineating the role of BCRP during in vivo studies in animals.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Application of receiver operating characteristic analysis to refine the prediction of potential digoxin drug interactions.

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 ≥ 0.03 and [I2]/IC50 ≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values.

PK/PD and Bioanalytical Considerations of AAV-Based Gene Therapies: an IQ Consortium Industry Position Paper.

Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.

Drug efflux transporters and multidrug resistance in acute leukemia: therapeutic impact and novel approaches to mediation.

Multidrug resistance (MDR), which is mediated by multiple drug efflux ATP-binding cassette (ABC) transporters, is a critical issue in the treatment of acute leukemia, with permeability glycoprotein (P-gp), multidrug resistance-associated protein 1, and breast cancer resistance protein (i.e., ABCG2) consistently being shown to be key effectors of MDR in cell line studies. Studies have demonstrated that intrinsic MDR can arise as a result of specific gene expression profiles and that drug-induced overexpression of P-gp and other MDR proteins can result in acquired resistance, with multiple ABC transporters having been shown to be overexpressed in cell lines selected for resistance to multiple drugs used to treat acute leukemia. Furthermore, numerous anticancer drugs, including agents commonly used for the treatment of acute leukemia (e.g., doxorubicin, vincristine, mitoxantrone, and methotrexate), have been shown to be P-gp substrates or to be susceptible to efflux mediated by other MDR proteins, and multiple clinical studies have demonstrated associations between P-gp or other MDR protein expression and responses to therapy or survival rates in acute leukemia. Here we review the importance of MDR in cancer, with a focus on acute leukemia, and we highlight the need for rapid accurate assessment of MDR status for optimal treatment selection. We also address the latest research on overcoming MDR, from inhibition of P-gp and other MDR proteins through various approaches (including direct antagonism and gene silencing) to the design of novel agents or novel delivery systems for existing therapeutic agents, to evade cellular efflux.

Predicting Approximate Clinically Effective Doses in Oncology Using Preclinical Efficacy and Body Surface Area Conversion: A Retrospective Analysis.

The correlation between efficacious doses in human tumor-xenograft mouse models and the human clinical doses of approved oncology agents was assessed using published preclinical data and recommended clinical doses. For 90 approved small molecule anti-cancer drugs, body surface area (BSA) corrected mouse efficacious doses were strongly predictive of human clinical dose ranges with 85.6% of the predictions falling within three-fold (3×) of the recommended clinical doses and 63.3% within 2×. These results suggest that BSA conversion is a useful tool for estimating human doses of small molecule oncology agents from mouse xenograft models from the early discovery stage. However, the BSA based dose conversion poorly predicts for the intravenous antibody and antibody drug conjugate anti-cancer drugs. For antibody-based drugs, five out of 30 (16.7%) predicted doses were within 3× of the recommended clinical dose. The body weight-based dose projection was modestly predictive with 66.7% of drugs predicted within 3× of the recommended clinical dose. The correlation was slightly better in ADCs (77.7% in 3×). The application and limitations of such simple dose estimation methods in the early discovery stage and in the design of clinical trials are also discussed in this retrospective analysis.

The Perspective of DMPK on Recombinant Adeno-Associated Virus-Based Gene Therapy: Past Learning, Current Support, and Future Contribution.

Given the recent success of gene therapy modalities and the growing number of cell and gene-based therapies in clinical development across many different therapeutic areas, it is evident that this evolving field holds great promise for the unmet medical needs of patients. The recent approvals of Luxturna® and Zolgensma® prove that recombinant adeno-associated virus (rAAV)-based gene therapy is a transformative modality that enables curative treatment for genetic disorders. Over the last decade, Takeda has accumulated significant experience with rAAV-based gene therapies, especially in the early stage of development. In this review, based on the learnings from Takeda and publicly available information, we aim to provide a guiding perspective on Drug Metabolism and Pharmacokinetics (DMPK) substantial role in advancing therapeutic gene therapy modalities from nonclinical research to clinical development, in particular the characterization of gene therapy product biodistribution, elimination (shedding), immunogenicity assessment, multiple platform bioanalytical assays, and first-in-human (FIH) dose projection strategies. Graphical abstract.

Recent advances in the translation of drug metabolism and pharmacokinetics science for drug discovery and development.

Drug metabolism and pharmacokinetics (DMPK) is an important branch of pharmaceutical sciences. The nature of ADME (absorption, distribution, metabolism, excretion) and PK (pharmacokinetics) inquiries during drug discovery and development has evolved in recent years from being largely descriptive to seeking a more quantitative and mechanistic understanding of the fate of drug candidates in biological systems. Tremendous progress has been made in the past decade, not only in the characterization of physiochemical properties of drugs that influence their ADME, target organ exposure, and toxicity, but also in the identification of design principles that can minimize drug-drug interaction (DDI) potentials and reduce the attritions. The importance of membrane transporters in drug disposition, efficacy, and safety, as well as the interplay with metabolic processes, has been increasingly recognized. Dramatic increases in investments on new modalities beyond traditional small and large molecule drugs, such as peptides, oligonucleotides, and antibody-drug conjugates, necessitated further innovations in bioanalytical and experimental tools for the characterization of their ADME properties. In this review, we highlight some of the most notable advances in the last decade, and provide future perspectives on potential major breakthroughs and innovations in the translation of DMPK science in various stages of drug discovery and development.

Sinusoidal Organic Anion-Transporting Polypeptide 1B1/1B3 and Bile Canalicular Multidrug Resistance-Associated Protein 2 Play an Essential Role in the Hepatobiliary Disposition of a Synthetic Cyclic Dinucleotide (STING Agonist).

The liver is central to the elimination of many drugs from the body involving multiple processes and understanding of these processes is important to quantitively assess hepatic clearance of drugs. The synthetic STING (STimulator of INterferon Genes protein) agonist is a new class of drugs currently being evaluated in clinical trials as a potential anticancer therapy. In this study, we used ML00960317 (synthetic STING agonist) to investigate the hepatobiliary disposition of this novel molecular entity. A bile-duct cannulated (BDC) rat study indicated that biliary excretion is the major route of elimination for ML00960317 (84% of parent dose in bile). The human biliary clearance using in vitro sandwich cultured human hepatocyte model predicted significant biliary excretion of ML00960317 (biliary excretion index (BEI) of 47%). Moreover, the transport studies using transporter expressing cell lines, hepatocytes, and membrane vesicles indicated that ML00960317 is a robust substrate of OATP1B1, OATP1B3, and MRP2. Using relative expression factor approach, the combined contribution of OATP1B1 (fraction transported (ft) = 0.62) and OATP1B3 (ft = 0.31) was found to be 93% of the active uptake clearance of ML00960317 into the liver. Furthermore, OATP1B1 and OATP1B3-mediated uptake of ML00960317 was inhibited by rifampicin with IC50 of 6.5 and 2.3 µM, respectively indicating an in vivo DDI risk (R value of 1.5 and 2.5 for OATP1B1 and OATP1B3, respectively). These results highlighted an important role of OATP1B1, OATP1B3, and MRP2 in the hepatobiliary disposition of ML00960317. These pathways may act as rate-determining steps in the hepatic clearance of ML00960317 thus presenting clinical DDI risk.

Immunogenicity assessment of AAV-based gene therapies: An IQ consortium industry white paper.

Immunogenicity has imposed a challenge to efficacy and safety evaluation of adeno-associated virus (AAV) vector-based gene therapies. Mild to severe adverse events observed in clinical development have been implicated with host immune responses against AAV gene therapies, resulting in comprehensive evaluation of immunogenicity during nonclinical and clinical studies mandated by health authorities. Immunogenicity of AAV gene therapies is complex due to the number of risk factors associated with product components and pre-existing immunity in human subjects. Different clinical mitigation strategies have been employed to alleviate treatment-induced or -boosted immunogenicity in order to achieve desired efficacy, reduce toxicity, or treat more patients who are seropositive to AAV vectors. In this review, the immunogenicity risk assessment, manifestation of immunogenicity and its impact in nonclinical and clinical studies, and various clinical mitigation strategies are summarized. Last, we present bioanalytical strategies, methodologies, and assay validation applied to appropriately monitor immunogenicity in AAV gene therapy-treated subjects.

Inhibition of hepatic organic anion-transporting polypeptide by RNA interference in sandwich-cultured human hepatocytes: an in vitro model to assess transporter-mediated drug-drug interactions.

Organic anion-transporting polypeptides (OATPs), members of the SLCO/SLC21 family, mediate the transport of various endo- and xenobiotics. In human liver, OATP1B1, 1B3, and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However, there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study, a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction, microarray and immunoblotting analyses, along with uptake assays, illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30% of the total uptake of cerivastatin into human hepatocytes, it caused a 50% reduction in cerivastatin metabolism, which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs.

Preclinical Antitumor Activity and Biodistribution of a Novel Anti-GCC Antibody-Drug Conjugate in Patient-derived Xenografts.

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody-dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Mechanistic study of potentiation of chemotherapy by a haloenol lactone derivative in vitro.

PURPOSE: The objective of this study was to understand the biochemical mechanisms by which a haloenol lactone (HEL) derivative potentiates cisplatin-induced cytotoxicity in vitro. HEL was originally designed and synthesized as a site-directed inactivator of glutathione S-transferase pi isozyme (GST-pi). Over-expression of GST-pi has been found to be associated with chemotherapy resistance. METHODS: A concentration-dependent GST inhibition was assessed after UOK130 cells were exposed to HEL at concentrations of 10 and 20 muM. Potentiated cytotoxicity was evaluated by treatment of UOK130 cells with a selection of alkylating agents in the presence or absence of HEL. Intracellular glutathione (GSH) was determined after exposure to HEL. Protective effect of GSH was examined by co-treatment with GSH ester in UOK130 cells exposed with a combination of cisplatin and HEL. Multiple resistance-associated protein (MRP) 1-3 activity was assayed by determining the rate of (3)H-LTC(4) and (3)H-E(2)17betaG through the MRPs into recombinant membrane vesicles. RESULTS: Exposure of HEL at 10 and 20 muM caused 28 and 41% of inhibition of cellular GST activity. Cytotoxicity of cisplatin, chlorambucil, and melphalan was enhanced 1.8-2.7-fold by HEL at 10 muM. No significant protection effect by GSH ester exposure was observed on cisplatin toxicity co-treated with HEL. HEL was found to inhibit MRP1, MRP2, and MRP3 with IC(50) of 1.30, 28.2, and 3.66 muM, respectively. CONCLUSION: Haloenol lactone showed inhibitory effect on GST-pi and MRP1-3 (selective inhibition of MRP1 and MRP3), and it was also found to deplete intracellular GSH.

CYP Suppression in Human Hepatocytes by Monomethyl Auristatin E, the Payload in Brentuximab Vedotin (Adcetris®), is Associated with Microtubule Disruption.

BACKGROUND AND OBJECTIVES: Monomethyl auristatin E (MMAE), the toxin linked to CD30-specific monoclonal antibody of Adcetris® (brentuximab vedotin), is a potent anti-microtubule agent. Brentuximab vedotin has been approved for the treatment of relapsed or refractory Hodgkin lymphoma and anaplastic large cell lymphoma. Cytochrome P450 (CYP) induction assessment of MMAE was conducted in human hepatocytes to assess DDI potentials and its translation to clinic. METHODS: MMAE was incubated at 1-1000 nM with cultured primary human hepatocytes for 72 h, and CYP1A2, CYP2B6, and CYP3A4 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction and CYP-specific probe substrate by liquid chromatography coupled with mass spectrometry, along with microtubule disruption by immunofluorescence staining using anti-ß-tubulin antibody and imaging. RESULTS: MMAE up to 10 nM had no significant effect on CYP1A2, CYP2B6, and CYP3A4 mRNA expression and activity, whereas at higher concentrations of 100- and 1000-nM MMAE, the CYP mRNA expression and activity were diminished substantially. Further investigation showed that the degree of CYP suppression was paralleled by that of microtubule disruption by MMAE, as measured by increase in the number of ß-tubulin-positive aggregates. At the clinical dose, the concentration of MMAE was 7 nM which did not show any significant CYP suppression or microtubule disruption in hepatocytes. CONCLUSIONS: MMAE was not a CYP inducer in human hepatocytes. However, it caused a concentration-dependent CYP mRNA suppression and activity. The CYP suppression was associated with microtubule disruption, supporting the reports that intact microtubule architecture is required for CYP regulations. The absence of CYP suppression and microtubule disruption in vitro at the clinical plasma concentrations of MMAE (< 10 nM) explains the lack of pharmacokinetic drug interaction between brentuximab vedotin and midazolam, a sensitive CYP3A substrate, reported in patients.