Safety, pharmacokinetics, and pharmacodynamics of nomlabofusp (CTI-1601) in Friedreich's ataxia.

OBJECTIVE: Current treatments for Friedreich's ataxia, a neurodegenerative disorder characterized by decreased intramitochondrial frataxin, do not address low frataxin concentrations. Nomlabofusp (previously CTI-1601) is a frataxin replacement therapy with a unique mechanism of action that directly addresses this underlying frataxin deficiency. Phase 1 studies assessed the safety, pharmacokinetic, and pharmacodynamic profiles of subcutaneously administered nomlabofusp in adults with Friedreich's ataxia. METHODS: Patients were enrolled in two Phase 1, double-blind, placebo-controlled studies. The single ascending-dose (SAD) study (NCT04176991) evaluated single doses of nomlabofusp (25, 50, 75, or 100 mg) or placebo. The multiple ascending-dose (MAD) study (NCT04519567) evaluated nomlabofusp (25 mg daily for 4 days then every third day, 50 mg daily for 7 days then every 2 days, or 100 mg daily) or placebo for 13 days. RESULTS: Patients aged 19-69 years were enrolled (SAD, N = 28; MAD, N = 27). Nomlabofusp was generally well tolerated through 13 days. Most adverse events were mild and resolved quickly. No serious adverse events or deaths were reported. Peak nomlabofusp plasma concentrations occurred 15 min after subcutaneous administration. Nomlabofusp plasma exposures increased with increasing doses and daily administration and decreased with reduced dosing frequency. Increased frataxin concentrations were observed in buccal cells, skin, and platelets with higher and more frequent nomlabofusp administration. INTERPRETATION: Results from this study support a favorable safety profile for nomlabofusp. Subcutaneous nomlabofusp injections were quickly absorbed; higher doses and daily administration resulted in increased tissue frataxin concentrations. Future studies will evaluate longer-term safety and possible efficacy of nomlabofusp.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

Retinoid X receptor is a nonsilent major contributor to vitamin D receptor-mediated transcriptional activation.

The vitamin D receptor (VDR) belongs to the thyroid hormone/retinoid receptor subfamily of nuclear receptors and functions as a heterodimer with retinoid X receptor (RXR). The RXR-VDR heterodimer, in contrast to other members of the class II nuclear receptor subfamily, is nonpermissive where RXR does not bind its cognate ligand, and therefore its role in VDR-mediated transactivation by liganded RXR-VDR has not been fully characterized. Here, we show a unique facet of the intermolecular RXR-VDR interaction, in which RXR actively participates in vitamin D3-dependent gene transcription. Using helix 3 and helix 12 mutants of VDR and RXR, we provide functional evidence that liganded VDR allosterically modifies RXR from an apo (unliganded)- to a holo (liganded)-receptor conformation, in the absence of RXR ligand. As a result of the proposed allosteric modification of RXR by liganded VDR, the heterodimerized RXR shows the "phantom ligand effect" and thus acquires the capability to recruit coactivators steroid receptor coactivator 1, transcriptional intermediary factor 2, and amplified in breast cancer-1. Finally, using a biochemical approach with purified proteins, we show that RXR augments the 1,25-dihydroxyvitamin D3-dependent recruitment of transcriptional intermediary factor 2 in the context of RXR-VDR heterodimer. These results confirm and extend the previous observations suggesting that RXR is a significant contributor to VDR-mediated gene expression and provide a mechanism by which RXR acts as a major contributor to vitamin D3-dependent transcription.

Ligand modulates VDR-Ser/Thr protein phosphatase interaction and p70S6 kinase phosphorylation in a cell-context-dependent manner.

We have recently shown that in colon cancer cells, Vitamin D receptor (VDR) interacts with the catalytic subunit of Ser/Thr protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. The VDR-PP1c and VDR-PP2Ac interactions were ligand independent in vivo, and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-mediated increase in VDR-associated phosphatase activity resulted in dephosphorylation and inactivation of p70S6 kinase in colon cancer cells. Here, we demonstrate that in myeloid leukemia cells, 1,25(OH)(2)D(3) treatment increased the Thr389 phosphorylation of p70S6 kinase. Accordingly, 1,25(OH)(2)D(3) decreased VDR-associated Ser/Thr protein phosphatase activity by dissociating VDR-PP1c and VDR-PP2Ac interactions. Further, 1,25(OH)(2)D(3) increased the association between VDR and Thr389 phosphorylated p70S6 kinase. Finally, by using non-secosteroidal VDR ligands, we demonstrate a separation between transactivation and p70S6 kinase phosphorylation activities of VDR and show pharmacologically that p70S6 kinase phosphorylation correlates with HL-60 cell differentiation.

Identification of an anabolic selective androgen receptor modulator that actively induces death of androgen-independent prostate cancer cells.

Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously 5α-dihydrotestosterone (DHT)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of DHT on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens.

Discovery of novel cyanamide-based inhibitors of cathepsin C.

The discovery of potent and selective cyanamide-based inhibitors of the cysteine protease cathepsin C is detailed. Optimization of the template with regard to plasma stability led to the identification of compound 17, a potent cathepsin C inhibitor with excellent selectivity over other cathepsins and potent in vivo activity in a cigarette smoke mouse model.

Interaction between the androgen receptor and RNase L mediates a cross-talk between the interferon and androgen signaling pathways.

Signaling by androgens and interferons (IFN) plays an important role in prostate cancer initiation and progression. Using microarray analysis, we describe here a functional cross-talk between dihydrotestosterone and interferon signaling. Glutathione S-transferase pull-down and co-immunoprecipitation experiments reveal that the androgen receptor and the interferon-activated RNase L interact with each other in a ligand-dependent manner. Furthermore, overexpression of wild type RNase L confers IFN sensitivity to a dihydrotestosterone-inducible reporter gene, whereas R462Q-mutated RNase L does not. Based on our data we hypothesize that in 22RV1 cells, activated androgen receptor (AR) contributes to the insensitivity to IFN of the cell. Accordingly, we show that AR knockdown restores responsiveness to IFNgamma. Our findings support a model in which both the activation of AR and the down-regulation of IFN signaling can synergize to promote cell survival and suppress apoptosis. This model provides the molecular basis to understand how mutated RNase L can lead to early onset PCa and illustrates how inflammatory cytokines and nuclear hormone signaling contribute to tumor development.

A vitamin D receptor-Ser/Thr phosphatase-p70 S6 kinase complex and modulation of its enzymatic activities by the ligand.

We provide evidence of a cross-talk between nuclear receptor and Ser/Thr protein phosphatases and show that vitamin D receptor (VDR) interacts with the catalytic subunit of protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. PP1c specifically interacts with VDR but not retinoic acid receptor alpha and retinoid X receptor alpha in yeast. Although VDR-PP1c and VDR-PP2Ac interaction is ligand-independent in vivo, 1alpha,25-dihydroxy-vitamin D(3) induces VDR-associated phosphatase activity. Further, VDR modulation of PP1c/PP2Ac activity results in a rapid and specific dephosphorylation and inactivation of their substrate, p70 S6 kinase (p70(S6k)). Finally, we demonstrate that the endogenous VDR, PP1c or PP2Ac, and p70(S6k) are present in a ternary complex in vivo, and the interaction of p70(S6k) with the VDR-PP complex is modulated by the phosphorylation state of the kinase. Since p70(S6k) is essential for G(1)-S transition, our results provide a molecular basis of 1alpha,25-dihydroxyvitamin D(3)-induced G(1) block in colon cancer cells.

Phylogenesis and regulated expression of the RUNT domain transcription factors RUNX1 and RUNX3.

The RUNX transcription factors are key regulators of lineage specific gene expression in developmental pathways. The mammalian RUNX genes arose early in evolution and maintained extensive structural similarities. Sequence analysis suggested that RUNX3 is the most ancient of the three mammalian genes, consistent with its role in neurogenesis of the monosynaptic reflex arc, the simplest neuronal response circuit, found in Cnidarians, the most primitive animals. All RUNX proteins bind to the same DNA motif and act as activators or repressors of transcription through recruitment of common transcriptional modulators. Nevertheless, analysis of Runx1 and Runx3 expression during embryogenesis revealed that their function is not redundant. In adults both Runx1 and Runx3 are highly expressed in the hematopoietic system. At early embryonic stages we found strong Runx3 expression in dorsal root ganglia neurons, confined to TrkC sensory neurons. In the absence of Runx3, knockout mice develop severe ataxia due to the early death of the TrkC neurons. Other phenotypic defects of Runx3 KO mice including abnormalities in thymopoiesis are also being investigated.

The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons.

The RUNX transcription factors are important regulators of linage-specific gene expression in major developmental pathways. Recently, we demonstrated that Runx3 is highly expressed in developing cranial and dorsal root ganglia (DRGs). Here we report that within the DRGs, Runx3 is specifically expressed in a subset of neurons, the tyrosine kinase receptor C (TrkC) proprioceptive neurons. We show that Runx3-deficient mice develop severe limb ataxia due to disruption of monosynaptic connectivity between intra spinal afferents and motoneurons. We demonstrate that the underlying cause of the defect is a loss of DRG proprioceptive neurons, reflected by a decreased number of TrkC-, parvalbumin- and beta-galactosidase-positive cells. Thus, Runx3 is a neurogenic TrkC neuron-specific transcription factor. In its absence, TrkC neurons in the DRG do not survive long enough to extend their axons toward target cells, resulting in lack of connectivity and ataxia. The data provide new genetic insights into the neurogenesis of DRGs and may help elucidate the molecular mechanisms underlying somatosensory-related ataxia in humans.