Quantitative evaluation of trastuzumab deruxtecan pharmacokinetics and pharmacodynamics in mouse models of varying degrees of HER2 expression.

Trastuzumab deruxtecan (T-DXd; DS-8201; ENHERTU®) is a human epithelial growth factor receptor 2 (HER2)-directed antibody drug conjugate (ADC) with demonstrated antitumor activity against a range of tumor types. Aiming to understand the relationship between antigen expression and downstream efficacy outcomes, T-DXd was administered in tumor-bearing mice carrying NCI-N87, Capan-1, JIMT-1, and MDA-MB-468 xenografts, characterized by varying HER2 levels. Plasma pharmacokinetics (PK) of total antibody, T-DXd, and released DXd and tumor concentrations of released DXd were evaluated, in addition to monitoring γΗ2AX and pRAD50 pharmacodynamic (PD) response. A positive relationship was observed between released DXd concentrations in tumor and HER2 expression, with NCI-N87 xenografts characterized by the highest exposures compared to the remaining cell lines. γΗ2AX and pRAD50 demonstrated a sustained increase over several days occurring with a time delay relative to tumoral-released DXd concentrations. In vitro investigations of cell-based DXd disposition facilitated the characterization of DXd kinetics across tumor cells. These outputs were incorporated into a mechanistic mathematical model, utilized to describe PK/PD trends. The model captured plasma PK across dosing arms as well as tumor PK in NCI-N87, Capan-1, and MDA-MB-468 models; tumor concentrations in JIMT-1 xenografts required additional parameter adjustments reflective of complex receptor dynamics. γΗ2AX longitudinal trends were well characterized via a unified PD model implemented across xenografts demonstrating the robustness of measured PD trends. This work supports the application of a mechanistic model as a quantitative tool, reliably projecting tumor payload concentrations upon T-DXd administration, as the first step towards preclinical-to-clinical translation.

Discovery, Optimization, and Biological Evaluation of Arylpyridones as Cbl-b Inhibitors.

Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.

A PKPD Case Study: Achieving Clinically Relevant Exposures of AZD5991 in Oncology Mouse Models.

Capturing human equivalent drug exposures preclinically is a key challenge in the translational process. Motivated by the need to recapitulate the pharmacokinetic (PK) profile of the clinical stage Mcl-1 inhibitor AZD5991 in mice, we describe the methodology used to develop a refined mathematical model relating clinically relevant concentration profiles to efficacy. Administration routes were explored to achieve target exposures matching the clinical exposure of AZD5991. Intravenous infusion using vascular access button (VAB) technology was found to best reproduce clinical target exposures of AZD5991 in mice. Exposure-efficacy relationships were evaluated, demonstrating that dissimilar PK profiles result in differences in target engagement and efficacy outcomes. Thus, these data underscore the importance of accurately ascribing key PK metrics in the translational process to enable clinically meaningful predictions of efficacy.

ADME and DMPK considerations for the discovery and development of antibody drug conjugates (ADCs).

 The therapeutic concept of antibody drug conjugates (ADCs) is to selectively target tumour cells with small molecule cytotoxic drugs to maximise cell kill benefit and minimise healthy tissue toxicity.An ADC generally consists of an antibody that targets a protein on the surface of tumour cells chemically linked to a warhead small molecule cytotoxic drug.To deliver the warhead to the tumour cell, the antibody must bind to the target protein and in general be internalised into the cell. Following internalisation, the cytotoxic agent can be released in the endosomal or lysosomal compartment (via different mechanisms). Diffusion or transport out of the endosome or lysosome allows the cytotoxic drug to express its cell-killing pharmacology. Alternatively, some ADCs (e.g. EDB-ADCs) rely on extracellular cleavage releasing membrane permeable warheads.One potentially important aspect of the ADC mechanism is the 'bystander effect' whereby the cytotoxic drug released in the targeted cell can diffuse out of that cell and into other (non-target expressing) tumour cells to exert its cytotoxic effect. This is important as solid tumours tend to be heterogeneous and not all cells in a tumour will express the targeted protein.The combination of large and small molecule aspects in an ADC poses significant challenges to the disposition scientist in describing the ADME properties of the entire molecule.This article will review the ADC landscape and the ADME properties of successful ADCs, with the aim of outlining best practice and providing a perspective of how the field can further facilitate the discovery and development of these important therapeutic modalities.

Function and Expression of Bile Salt Export Pump in Suspension Human Hepatocytes.

The mechanistic understanding of bile salt disposition is not well established in suspension human hepatocytes (SHH) because of the limited information on the expression and function of bile salt export protein (BSEP) in this system. We investigated the transport function of BSEP in SHH using a method involving in situ biosynthesis of bile salts from their precursor bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). Our data indicated that glycine- and taurine-conjugated CA and CDCA were generated efficiently and transported out of hepatocytes in a concentration- and time-dependent manner. We also observed that the membrane protein abundance of BSEP was similar between SHH and sandwich-cultured human hepatocytes. Furthermore, known cholestatic agents significantly inhibited G-CA and G-CDCA efflux in SHH. Interestingly, cyclosporine A, troglitazone, itraconazole, loratadine, and lovastatin inhibited G-CA efflux more potently than G-CDCA efflux (3- to 5-fold). Because of these significant differential effects on G-CA and G-CDCA efflux inhibition, we determined the IC50 values of troglitazone for G-CA (9.9 µM) and for G-CDCA (43.1 µM) efflux. The observed discrepancy in the IC50 was attributed to the fact that troglitazone also inhibits organic anion transporting polypeptides and Na+/taurocholate cotransporting polypeptide in addition to BSEP. The hepatocyte uptake study suggested that both active uptake and passive diffusion contribute to the liver uptake of CA, whereas CDCA primarily undergoes passive diffusion into the liver. In summary, these data demonstrated the expression and function of BSEP and its major role in transport of bile salts in cryopreserved SHH. SIGNIFICANCE STATEMENT: BSEP transport function and protein abundance was evident in SHH in the present study. The membrane abundance of BSEP protein was similar between SHH and sandwich-cultured human hepatocytes. The study also illustrated the major role of BSEP relative to basolateral MRP3 and MRP4 in transport of bile salts in SHH. Understanding of BSEP function in SHH may bolster the utility of this platform in mechanistic understanding of bile salt disposition and potentially in the assessment of drugs for BSEP inhibition.

Impact of ritonavir, atazanavir and their combination on the CYP3A4 induction potential of efavirenz in primary human hepatocytes.

Currently used combinations of anti-HIV drugs, known as Highly Active Antiretroviral Therapy (HAART), have considerably reduced the mortality in patients with AIDS. However, HAART medications such as efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) often cause adverse drug-drug interactions (DDIs) that result from changes in the expression and activity of drug metabolizing enzymes. Since EFV is most commonly used with ATV and RTV, the known CYP inhibitors, we evaluated the effects of combinations of these agents on the CYP3A4 induction by EFV. We determined the induction of CYP3A4 by EFV, RTV, ATV, EFV+RTV, EFV+ATV, EFV+RTV+ATV and rifampicin (RIF) employing primary human hepatocytes from 3 donors. Also, concentration dependent activation of human Pregnane X-receptor (hPXR) which is key transcriptional regulator of CYP3A4 by EFV, RIF and RTV was estimated in transiently transfected LS180 cells. CYP3A4 activity (testosterone-6beta-hydroxylation) was induced by EFV (3 fold) and RIF (4 fold), but was significantly suppressed in the presence of RTV and ATV. All treatments significantly induced the CYP3A4 transcripts (3-25 fold) as quantitated by RT-PCR. hPXR activation data in LS180 cells were consistent with the induction of transcripts and the estimated EC(50) values were 0.87 microM, 0.44 microM and 3.7 microM for RIF, RTV and EFV, respectively. However, in primary hepatocytes the net effect was suppression of EFV mediated CYP3A4 induction by RTV and ATV. This observation corresponds to the clinical observations of attenuated CYP3A4 induction by EFV induction in the presence of RTV and other protease inhibitors (PIs). Our results underscore the limitation of transcriptional activation assays in predicting the net outcome for compounds that exhibit complex interactions resulting from induction and inhibition of CYP enzymes.

Comparison of immortalized Fa2N-4 cells and human hepatocytes as in vitro models for cytochrome P450 induction.

Fa2N-4 cells have been proposed as a tool to identify CYP3A4 inducers. To evaluate whether Fa2N-4 cells are a reliable surrogate for cryopreserved human hepatocytes, we assessed the basal mRNA expression of 64 drug disposition genes in Fa2N-4 cells. Significant differences were found in the expression of major drug-metabolizing enzymes, nuclear receptors, and transporters between both cell types. Importantly, the expression of constitutive androstane receptor (CAR) and several hepatic uptake transporters was significantly lower (>50-fold) in Fa2N-4 cells, whereas the expression of pregnane X-receptor (PXR) and aryl hydrocarbon receptor (AhR) was similar between Fa2N-4 cells and human hepatocytes. By using an optimized induction assay for Fa2N-4 cells, CYP3A4 induction by rifampicin, the prototypical PXR activator, increased from 1.5- to 7-fold at the level of functional activity. With nine selected compounds, which are known inducers of CYP3A4 either via activation of PXR, CAR, or both, we evaluated CYP3A4 and CYP2B6 mRNA induction using Fa2N-4 cells and human hepatocytes. No response was observed in Fa2N-4 cells treated with the selective CAR activators 6-(4-chlorophenyl)imidazo[2,1-b][1,3]-thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and artemisinin. CYP3A4 and CYP2B6 induction in Fa2N-4 cells were also low for phenytoin, phenobarbital, and efavirenz, which are dual activators of PXR/CAR. This finding was in agreement with the lack of expression of CAR. The EC(50) value for rifampicin-mediated CYP3A4 induction was 10-fold higher than that in human hepatocytes. This result could be attributed to the low expression of hepatic organic anion-transporting polypeptides OATP1B1 and OATP1B3 in Fa2N-4 cells. In summary, our findings identify limitations of Fa2N-4 cells as a predictive induction model.

Cytochrome P450 enzymes and transporters induced by anti-human immunodeficiency virus protease inhibitors in human hepatocytes: implications for predicting clinical drug interactions.

Although many of the clinically significant drug interactions of the anti-human immunodeficiency virus (HIV) protease inhibitors (PIs) can be explained by their propensity to inactivate CYP3A enzymes, paradoxically these drugs cause (or lack) interactions with CYP3A substrates that cannot be explained by this mechanism (e.g., alprazolam). To better understand these paradoxical interactions (or lack thereof), we determined the cytochromes P450 and transporters induced by various concentrations (0-25 microM) of two PIs, ritonavir and nelfinavir, and rifampin (positive control) in primary human hepatocytes. At 10 microM, ritonavir and nelfinavir suppressed CYP3A4 activity but induced its transcripts and protein expression (19- and 12- and 12- and 6-fold, respectively; a >2-fold change over control was interpreted as induction). At 10 microM, rifampin induced CYP3A4 transcripts, CYP3A protein, and activity by 23-, 12-, and 13-fold, respectively. The induction by rifampin of CYP3A activity was significantly correlated with its induction of CYP3A4 transcripts (r = 0.96, p < 0.05) and CYP3A protein (r = 0.89, p < 0.05). All three drugs (10 microM) induced CYP2B6 activity by 2- to 4-fold, CYP2C8 and 2C9 activity by 2- to 4-fold and the transcripts of CYP2B6, 2C8, and 2C9 by >3-, 5-, and 3-fold, respectively. CYP2C19 and 1A2 activity and transcripts were modestly induced (2-fold), whereas, as expected, CYP2D6 was not induced by any of the drugs. Of the transporters studied, protease inhibitors moderately induced multidrug resistance 1 (ABCB1) and multidrug resistance-associated protein (ABCC1) transcripts but had no or minimal effect on the transcripts of breast cancer resistance protein (ABCG2), organic anion-transporting peptide (OATP) 1B1 (SLCO1B1), or OATP1B3 (SLCO1B3). On the basis of these data, we concluded that many of the paradoxical drug interactions (or lack thereof) with the PIs are metabolismrather than transporter-based and are due to induction of CYP2B6 and 2C enzymes.

Dose-dependent induction of cytochrome P450 (CYP) 3A4 and activation of pregnane X receptor by topiramate.

PURPOSE: In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription. METHODS: Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 microM) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 microM). The rate of testosterone 6beta-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. RESULTS: Compared with controls, TPM (50-500 microM)-treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 microM) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50-500 microM) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 microM) caused a 6-fold activation. CONCLUSIONS: The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used.