Large- and Small-Animal Studies of Safety, Pharmacokinetics (PK), and Biodistribution of Inflammasome-Targeting Nanoligomer in the Brain and Other Target Organs

Abstract

The human immune system is the key line of defense in the body against infections and disease and is critical for our survival. However, immune malfunction or misrecognition of healthy cells and tissue, termed autoimmune disease, is implicated in more than 80 disease conditions and multiple other secondary pathologies. While pan-immunosuppressive therapies like steroids offer some relief for systemic inflammation for some organs, many patients never achieve remission and such drugs do not cross the blood-brain barrier making them ineffective for tackling neuroinflammation. Especially in the brain, unintended activation of microglia and astrocytes is hypothesized to be directly or indirectly responsible for several neurodegenerative diseases including Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Parkinson’s Disease (PD), and Alzheimer’s Disease (AD). Recent studies have also shown that targeting inflammasome and specific immune targets can be beneficial for a range of neurodegenerative diseases. Further, our previous studies have shown targeting NF-κB and NLRP3 through brain penetrant Nanoligomer cocktail SB_NI_112 (abbreviated to NI112) can be therapeutic for several neurodegenerative diseases. Here we show safety-toxicity studies, followed by pharmacokinetics (PK) and biodistribution in small-(mice) and large-animal (dog) studies of this inflammasome-targeting Nanoligomer cocktail NI 112 which downregulates NF-κB and NLRP3. We conducted studies using four different routes of administration: intravenous (IV), subcutaneous (SQ), intraperitoneal (IP), and intranasal (IN), and identified the drug concentration over time using inductively coupled plasma mass spectrometry (ICP-MS) in the blood serum, the brain (including different brain regions), and other target organs like liver, kidney, and colon. Our results indicate the Nanoligomer cocktail has a strong safety profile, has a consistent peak concentration in blood serum, and shows high biodistribution (F) and delivery across all routes of administration. Further analysis of multiexponential serum concentration with time shows while the bound/unbound ratio is highly dependent on the route of administration, species (dog/mice), and dosage, the bound serum to Nanoligomer concentration in the brain is consistent at ∼30%. Moreover, different dosing in dogs through IV and SQ routes also show predictability in bound serum concentration, and constant ratio when comparing large- and small-animal data (dog and mice). We hypothesize the constant ratio is determined by target protein amount, and PK parameters such as area under the curve (AUC) and can be used for more accurate interspecies scaling/dose determination. These results provide an excellent platform for translating and predicting therapeutic dosage between routes of administration in the same species, and a good prediction of scaling between species to attain similar target inhibition using high affinity and specificity of Nanoligomer binding the target.

Brain-Penetrant NF-κB and NLRP3 Targeting Nanoligomers are Therapeutic in Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s Disease (AD) Human Organoid and Mouse Models

“…We have shown that NI112 reduces pro-inflammatory astrocytes/microglia even in prion-treated mice, 61 agingassociated neuropathology and AD, 64 and EAE mice 63 (models of severe neurodegeneration), and that this is associated with cognitive improvements. Moreover, the high binding specificity (K D ~5 nM 60,62,72 ), minimal off-targeting, 60,62,65 high safety profile, 65 lack of any immunogenic response or accumulation in first pass organs, 65 absence of any observable histological damage to organs even for long (>15-20 weeks) treatments, [60][61][62]65 and facile delivery to the brain to counter neuroinflammation, 58,61,63,65 make Nanoligomers and specifically the NI112 cocktail a promising, safe and targeted approach to mitigate neuropathological inflammation, as well as microglial and astrocytic activation.…”

“…40,[53][54][55] Inflammasome inhibition has been intensely investigated as a novel target, as well as a common cure for a range of neurodegenerative and autoimmune diseases, 56,57 and using NF-κB and NLRP3 mRNA targeting and translation-inhibiting Nanoligomer combination (NI112) is a safe, effective, and targeted approach. [58][59][60][61][62][63][64][65] However, even other inflammasome-targeting smallmolecule therapeutics (e.g., MCC950) are limited by: 1) potential off-target effects; [66][67][68] and 2) the fact that they target only NF-κB or NLRP3 alone. NI112, an RNA therapeutic that crosses the blood-brain barrier, addresses problems with pan immunosuppressive therapies (such as steroids), and the cocktail potently inhibits neuroinflammation.…”

Inflammasome-Inhibiting Nanoligomers are Neuroprotective Against Space-Induced Pathology in Healthy and Diseased 3D Human Motor and Pre-Frontal Cortex Brain Organoids

“…[37][38][39][40][41][42] Nanoligomers utilize peptide nucleic acids (PNAs) as the sequence-specific binding component that demonstrate strong hybridization and specificity to their RNA (or DNA) sequence targets, 43 and exhibit no known enzymatic cleavage, leading to increased stability in human blood serum and mammalian cellular extracts. 44 Given the low K D , 35,36,43 high binding specificity, minimal off-targeting, 35,36 lack of any immunogenic response or accumulation in first pass organs, 34 absence of any observable histological damage to organs even for long (>15-20 weeks) treatments, [34][35][36]40 and facile delivery to the brain to counter neuroinflammation, 34,[37][38][39][40][41][42] we have demonstrated that Nanoligomers are a safe and targeted approach to screen for therapeutic targets in organoids and rodents for multiple neurodegenerative diseases aboard the International Space Station (ISS).…”

“…Nanoligomer-based High-Throughput Screening is a Safe and Targeted Approach. Nanoligomer™ therapeutic platform generates safe, targeted, 34 and highly-specific (K D 3.37 nM 35,36 ) RNA-targeting molecules for facile delivery to targeted organs and both up-and downregulation of desired protein through translational and transcriptional regulation. [37][38][39][40][41][42] Nanoligomers utilize peptide nucleic acids (PNAs) as the sequence-specific binding component that demonstrate strong hybridization and specificity to their RNA (or DNA) sequence targets, 43 and exhibit no known enzymatic cleavage, leading to increased stability in human blood serum and mammalian cellular extracts.…”