An anti-TNF-glucocorticoid receptor modulator antibody-drug conjugate is efficacious against immune-mediated inflammatory diseases.

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.

Targeting the Tyrosine Kinase 2 (TYK2) Pseudokinase Domain: Discovery of the Selective TYK2 Inhibitor ABBV-712.

Tyrosine kinase 2 (TYK2) is a nonreceptor tyrosine kinase that belongs to the JAK family also comprising JAK1, JAK2, and JAK3. TYK2 is an attractive target for various autoimmune diseases as it regulates signal transduction downstream of IL-23 and IL-12 receptors. Selective TYK2 inhibition offers a differentiated clinical profile compared to currently approved JAK inhibitors. However, selectivity for TYK2 versus other JAK family members has been difficult to achieve with small molecules that inhibit the catalytically active kinase domain. Successful targeting of the TYK2 pseudokinase domain as a strategy to achieve isoform selectivity was recently exemplified with deucravacitinib. Described herein is the optimization of selective TYK2 inhibitors targeting the pseudokinase domain, resulting in the discovery of the clinical candidate ABBV-712 (21).

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.

Increasing the Reuse of Protein Non-Naïve Nonhuman Primates in Pharmaceutical Drug Discovery and Development: An Overview and Industry Position on the Challenges and Benefits.

The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper's content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.

In vitro and in vivo characterization of the JAK1 selectivity of upadacitinib (ABT-494).

BACKGROUND: Anti-cytokine therapies such as adalimumab, tocilizumab, and the small molecule JAK inhibitor tofacitinib have proven that cytokines and their subsequent downstream signaling processes are important in the pathogenesis of rheumatoid arthritis. Tofacitinib, a pan-JAK inhibitor, is the first approved JAK inhibitor for the treatment of RA and has been shown to be effective in managing disease. However, in phase 2 dose-ranging studies tofacitinib was associated with dose-limiting tolerability and safety issues such as anemia. Upadacitinib (ABT-494) is a selective JAK1 inhibitor that was engineered to address the hypothesis that greater JAK1 selectivity over other JAK family members will translate into a more favorable benefit:risk profile. Upadacitinib selectively targets JAK1 dependent disease drivers such as IL-6 and IFNγ, while reducing effects on reticulocytes and natural killer (NK) cells, which potentially contributed to the tolerability issues of tofacitinib. METHODS: Structure-based hypotheses were used to design the JAK1 selective inhibitor upadacitinib. JAK family selectivity was defined with in vitro assays including biochemical assessments, engineered cell lines, and cytokine stimulation. In vivo selectivity was defined by the efficacy of upadacitinib and tofacitinib in a rat adjuvant induced arthritis model, activity on reticulocyte deployment, and effect on circulating NK cells. The translation of the preclinical JAK1 selectivity was assessed in healthy volunteers using ex vivo stimulation with JAK-dependent cytokines. RESULTS: Here, we show the structural basis for the JAK1 selectivity of upadacitinib, along with the in vitro JAK family selectivity profile and subsequent in vivo physiological consequences. Upadacitinib is ~ 60 fold selective for JAK1 over JAK2, and > 100 fold selective over JAK3 in cellular assays. While both upadacitinib and tofacitinib demonstrated efficacy in a rat model of arthritis, the increased selectivity of upadacitinib for JAK1 resulted in a reduced effect on reticulocyte deployment and NK cell depletion relative to efficacy. Ex vivo pharmacodynamic data obtained from Phase I healthy volunteers confirmed the JAK1 selectivity of upadactinib in a clinical setting. CONCLUSIONS: The data presented here highlight the JAK1 selectivity of upadacinitinib and supports its use as an effective therapy for the treatment of RA with the potential for an improved benefit:risk profile.

Systemic administration of an Fcgamma-Fc(epsilon)-fusion protein in house dust mite sensitive nonhuman primates.

Crosslinking Fc(epsilon)RI and FcgammaRIIB receptors inhibits mast cell and basophil activation, decreasing mediator release. In this study, a fusion protein incorporating human Fcgamma and Fc(epsilon) domains, hGE2, was shown to inhibit degranulation of human mast cells and basophils, and to exhibit efficacy in a nonhuman primate model of allergic asthma. hGE2 increased the provocative concentration of dust mite aeroallergen that induced an early phase asthmatic response. The treatment effect lasted up to 4 weeks and was associated with reduction in the number of circulating basophils and decreased expression of Fc(epsilon)RI on repopulating basophils. Repeat hGE2 dosing induced production of serum antibodies against human Fcgamma and Fc(epsilon) domains and acute anaphylaxis-like reactions. Immune serum induced histamine release from human IgE or hGE2-treated cord blood-derived mast cells and basophils in vitro. These results indicate that repeat administration with hGE2 induced an antibody response to the human molecule that resulted in activation rather than inhibition of allergic responses.

Nasal drug administration: potential for targeted central nervous system delivery.

Nasal administration as a means of delivering therapeutic agents preferentially to the brain has gained significant recent interest. While some substrates appear to be delivered directly to the brain via this route, the mechanisms governing overall brain uptake and exposure remain unclear. Some substrates utilize the olfactory nerve tract and gain direct access to the brain, thus bypassing the blood-brain barrier (BBB). However, most agents of pharmacologic interest likely gain access to the brain via the olfactory epithelium, which represents a more direct route of uptake. While the traditional BBB is not present at the interface between nasal epithelium and brain, P-glycoprotein (and potentially other barrier transporters) is expressed at this interface. In addition, work in this laboratory has demonstrated that P-glycoprotein throughout the brain can be modulated with nasal administration of appropriate inhibitors. The potential for targeted central nervous system delivery via this route is discussed.

Pharmacokinetics of substrate uptake and distribution in murine brain after nasal instillation.

PURPOSE: This study was conducted to develop a physiologically relevant mathematical model for describing brain uptake and disposition of nasally administered substrates. METHODS: [14C]-antipyrine, [14C]-diazepam, [3H]-sucrose, or [3H]-verapamil was administered nasally to CF-1 mice. P-glycoprotein (P-gp)-deficient mice also received [3H]-verapamil to probe the influence of P-gp on uptake/distribution. Mice were sacrificed at selected intervals, and 20 serial 300-microm coronal brain sections were obtained to determine radioactivity. A series of compartmental pharmacokinetic models was developed and fit to concentration vs. time/distance data. RESULTS: After nasal instillation, substrate concentration was highest in the olfactory bulb and decreased with distance. In the absence of transport-mediated flux, peak brain exposure occurred at 6 h. A catenary pharmacokinetic model with slice-specific brain-to-blood efflux rate constants and slice-to-slice diffusivity factors was capable of fitting the data. P-gp limited fractional absorption of [3H]-verapamil via efflux from the nasal cavity and olfactory epithelium. P-gp also increased the rate constants associated with [3H]-verapamil efflux 1.5- to 190-fold, depending on brain region. P-gp limited [3H]-verapamil uptake from the nasal cavity into brain and facilitated removal of [3H]-verapamil from brain during rostral-to-caudal distribution. CONCLUSIONS: Taken together, the data and associated modeling provide a comprehensive assessment of the influence of P-gp on brain uptake and disposition of nasally administered substrates.

Functional evidence for P-glycoprotein at the nose-brain barrier.

PURPOSE: Experiments were performed to assess the brain distribution of [3H]-verapamil, including the influence of delivery route of inhibitor and substrate (nasal vs. systemic) on brain distribution. The anatomic location of P-glycoprotein (P-gp) at the nose-brain barrier also was investigated. METHODS: Separate groups of mice were pretreated with rifampin or vehicle nasally or intravenously. [3H]-verapamil was administered either nasally or via in situ brain perfusion, and dose-response profiles were constructed for P-gp inhibition. Localization of P-gp in freshly obtained brain slices and olfactory tissue was evaluated by confocal microscopy. RESULTS: Rifampin inhibited the P-gp-mediated efflux of [3H]-verapamil, regardless of delivery route (Imax = 62 +/- 6%). The ED50 for enhancement of [3H]-verapamil uptake by nasal rifampin was approximately 400-fold lower than for intravenous rifampin (0.16 vs. 65 mg/kg, respectively). Microscopy showed that P-gp was located in endothelial cells that line the olfactory bulb and within the olfactory epithelium. CONCLUSIONS: Nasal delivery of rifampin enhanced brain uptake of [3H]-verapamil. The magnitude of transport inhibition was dependent on the dose and route of the inhibitor, the time after administration of the inhibitor, and the specific brain region examined. P-gp is localized to both the olfactory epithelium and the endothelial cells that surround the olfactory bulb.

Use of loperamide as a phenotypic probe of mdr1a status in CF-1 mice.

PURPOSE: A subpopulation of the CF-1 mouse strain (approximately 25%) lacks P-gp expression, and consequently, increased brain penetration of many substrates is observed in these animals. Mice lacking the mdr1a gene represent an important research tool to study the potential effects of P-gp on CNS substrate disposition. METHODS: Adult CF-1 mice were used in all experiments. Loperamide-induced antinociception was determined by the hotplate latency test at 0.25, 2, and 4 h post-dose. At the conclusion of the pharmacodynamic experiment(s), trunk blood and brain tissue were collected and analyzed by high-performance liquid chromatography-mass spectrometry (LC-MS/MS). Mice were also genotyped for their mdrla status via RT-PCR. RESULTS: All mice with three consecutive effects of maximum hotplate latency (60 s) showed considerable opioid-like behavior in addition to antinociception. Mice without three consecutive effects of maximum hotplate latency (< or = 30 s) showed no opioid-like behavior. The loperamide brain-to-serum ratio in mice identified as P-gp-deficient was 65-fold higher compared to the P-gp-competent animals (10.1 +/- 1.0 vs. 0.155 +/- 0.018). All animals identified as phenotypically P-gp-competent based on the hotplate assay evidenced the mdrla(+/+) genotype. CONCLUSION: This assay appears to offer a rapid and unambiguous measure via a relatively non-invasive, simple technique to identify P-gp status in the CF-1 subpopulation of mice.

Drug transport at the blood-brain barrier and the choroid plexus.

The blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) represent the main interfaces between the central nervous system (CNS) and the peripheral circulation. Drug exposure to the CNS is dependent on a variety of factors, including the physical barrier presented by the BBB and the BCSFB and the affinity of the substrate for specific transport systems located at both of these interfaces. It is the aggregate effect of these factors that ultimately determines the total CNS exposure, and thus pharmacological efficacy, of a drug or drug candidate. This review discusses the anatomical and biochemical barriers presented to solute access to the CNS. In particular, the important role played by various efflux transporters in the overall barrier function is considered in detail, as current literature suggests that efflux transport likely represents a key determinant of overall CNS exposure for many substrates. Finally, it is important to consider not only the net delivery of the agent to the CNS, but also the ability of the agent to access the relevant target site within the CNS. Potential approaches to increasing both net CNS and target-site exposure, when such exposure is dictated by efflux transport, are considered.

Variable modulation of opioid brain uptake by P-glycoprotein in mice.

The efflux transporter P-glycoprotein (P-gp) is an important component of the blood-brain barrier (BBB) that limits accumulation of many compounds in brain. Some opioids have been shown to interact with P-gp in vitro and in vivo. Genetic or chemical disruption of P-gp has been shown to enhance the antinociceptive and/or toxic effects of some opioids, although the extent of this phenomenon has yet to be understood. The purpose of this study was to assess quantitatively the influence of mdr1a P-gp on initial brain uptake of chemically diverse opioids in mice. The brain uptake of opioids selective for the mu (fentanyl, loperamide, meperidine, methadone, and morphine), delta (deltorphin II, DPDPE, naltrindole, SNC 121) and kappa (bremazocine and U-69593) receptor subtypes was determined in P-gp-competent (wild-type) and P-gp-deficient [mdr1a(-/-)] mice with an in situ brain perfusion model. BBB permeability of the opioids varied by several orders of magnitude in both mouse strains. The difference in brain uptake between P-gp-competent and P-gp-deficient mice ranged from no detectable effect (meperidine) to >/=8-fold increase in uptake (DPDPE, loperamide, and SNC 121). In addition, loperamide efflux at the BBB was inhibited by quinidine. These results demonstrate that P-gp modulation of opioid brain uptake varies substantially within this class of compounds, regardless of receptor subtype. P-gp-mediated efflux of opioids at the BBB may influence the onset, magnitude, and duration of analgesic response. The variable influence of P-gp on opioid brain distribution may be an important issue in the context of pharmacologic pain control and drug interactions.

P-Glycoprotein attenuates brain uptake of substrates after nasal instillation.

PURPOSE: Previous literature has suggested the absence of an effective barrier between the nasal mucosa and the brain for compounds administered via the nasal route. These experiments were conducted to elucidate the role of the blood-brain barrier efflux transporter P-glycoprotein (P-gp) in attenuating delivery of P-gp substrates to the brain after nasal administration in mice. METHODS: Brain uptake of several radiolabeled P-gp substrates, was measured in P-gp-deficient and P-gp-competent mice following nasal instillation. Additional experiments were performed to assess the potential for enhancing brain uptake by inhibiting P-gp with intranasal rifampin. RESULTS: All substrates examined were measurable in brain tissue within 2 min. Substrate accumulation in P-gp-deficient mice was higher than in P-gp-competent animals; the degree to which P-gp attenuated brain uptake after nasal administration was similar to that during in situ brain perfusion. Co-administration of rifampin enhanced brain uptake of relevant substrates, and resulted in complete elimination of P-gp-mediated transport for 3H- verapamil. CONCLUSIONS: P-gp attenuates brain accumulation of intranasally-administered P-gp substrates. Thus, biochemical components of the blood-brain barrier, such as efflux transporters may influence brain penetration after nasal administration. Co-administration of a P-gp inhibitor enhances the brain uptake of relevant substrates, suggesting that the transporter barrier functions may be reversible.