RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral RNA. We identified potent lipophilic small interfering RNA (siRNA) conjugates targeting highly conserved regions of SARS-CoV-2 outside of the spike-encoding region capable of achieving ≥3-log viral reduction. Serial passaging studies demonstrated that a two-siRNA combination prevented development of resistance compared to a single siRNA approach. Viral resistance to single siRNA treatment occurred due to emergence of point mutations at critical positions required for siRNA-mediated target binding and cleavage, which led to a loss of siRNA efficacy. With a two-siRNA combination, emergence of mutations within the siRNA binding site was abolished. When delivered intranasally, two-siRNA combination protected Syrian hamsters from weight loss and lung pathology by viral infection upon prophylactic administration but not following onset of infection. Together, the data support potential utility of RNAi as a prophylactic approach with high resistance barrier to counteract SARS-CoV-2 emergent variants and complement vaccination. Most importantly, given that the siRNAs can be rapidly developed from a new pathogen sequence, this strategy has implications as a new type of preventive medicine that may protect against future coronavirus pandemics.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al. 2018, Khojasteh et al. 2019, Khojasteh et al. 2020, Khojasteh et al. 2021, Khojasteh et al. 2022). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation.

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N -acetylga-lactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterol-emia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor – mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conju-gated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. (anti-sense single-strand single

RNA interference is a natural antiviral mechanism that could be harnessed to combat SARS-CoV-2 infection by targeting and destroying the viral genome. We screened lipophilic small-interfering RNA (siRNA) conjugates targeting highly conserved regions of the SARS-CoV-2 genome and identified leads targeting outside of the spike-encoding region capable of achieving ≥3-log viral reduction. Serial passaging studies demonstrated that a two-siRNA combination prevented development of resistance compared to a single-siRNA approach. A two-siRNA combination delivered intranasally protected Syrian hamsters from weight loss and lung pathology by viral infection upon prophylactic administration but not following onset of infection. Together, the data support potential utility of RNAi as a prophylactic approach to limit SARS-CoV-2 infection that may help combat emergent variants, complement existing interventions, or protect populations where vaccines are less effective. Most importantly, this strategy has implications for developing medicines that may be valuable in protecting against future coronavirus pandemics.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2′- O -hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer’s disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

RNA interference (RNAi) therapeutics are a new class of medicines that can address unmet medical needs by silencing disease-causing gene transcripts. While delivery of short interfering RNAs (siRNAs) to hepatocytes has yielded multiple drug approvals, novel delivery solutions are needed to expand the reach of RNAi therapeutics. Here we report that conjugation of 2'- O -hexadecyl (C16) to siRNAs enables efficient silencing in the central nervous system (CNS), eye, and lung of multiple nonclinical species with broad cell type specificity. Intrathecally delivered C16-siRNAs are active across CNS regions and cell types, with sustained silencing for at least three months, which is an especially important outcome considering the challenging dosing route. Similarly, intravitreal and intranasal administration of C16-siRNAs resulted in potent and sustained knockdown in the eye and lung, respectively. Efficient delivery facilitated through C16 conjugation to optimized siRNA designs has enabled candidate selection for investigational human clinical trials assessing therapeutic silencing beyond the liver with infrequent (e.g. bi-annual) dosing.

Serum protein interactions are evaluated during the drug development process since they determine the free drug concentration in blood and thereby can influence the drug's pharmacokinetic and pharmacodynamic properties. While the impact of serum proteins on the disposition of small molecules is well understood, it is not yet well characterized for a new modality, RNA interference therapeutics. When administered systemically, small interfering RNAs (siRNAs) conjugated to the N-acetylgalactosamine (GalNAc) ligand bind to proteins present in circulation. However, it is not known if these protein interactions may impact the GalNAc-conjugated siRNA uptake into hepatocytes mediated through the asialoglycoprotein receptor (ASGPR) and thereby influence the activity of GalNAc-conjugated siRNAs. In this study, we assess the impact of serum proteins on the uptake and activity of GalNAc-conjugated siRNAs in primary human hepatocytes. We found that a significant portion of the GalNAc-conjugated siRNAs is bound to serum proteins. However, ASGPR-mediated uptake and activity of GalNAc-conjugated siRNAs were minimally impacted by the presence of serum relative to their uptake and activity in the absence of serum. Therefore, in contrast to small molecules, serum proteins are expected to have minimal impact on pharmacokinetic and pharmacodynamic properties of GalNAc-conjugated siRNAs.

Givosiran is an N-acetylgalactosamine–conjugated RNA interference therapeutic that targets 5′-aminolevulinate synthase 1 mRNA in the liver and is currently marketed for the treatment of acute hepatic porphyria. Herein, nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion properties of givosiran were characterized. Givosiran was completely absorbed after subcutaneous administration with relatively short plasma elimination half-life (t1/2; less than 4 hours). Plasma exposure increased approximately dose proportionally with no accumulation after repeat doses. Plasma protein binding was concentration dependent across all species tested and was around 90% at clinically relevant concentration in human. Givosiran predominantly distributed to the liver by asialoglycoprotein receptor–mediated uptake, and the t1/2 in the liver was significantly longer (∼1 week). Givosiran was metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species with no human unique metabolites. Givosiran metabolized to form one primary active metabolite with the loss of one nucleotide from the 3′ end of antisense strand, AS(N-1)3′ givosiran, which was equipotent to givosiran. Renal and fecal excretion were minor routes of elimination of givosiran as approximately 10% and 16% of the dose was recovered intact in excreta of rats and monkeys, respectively. Givosiran is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, givosiran has a low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT Nonclinical pharmacokinetics and absorption, distribution, metabolism, and excretion (ADME) properties of givosiran were characterized. Givosiran shows similar pharmacokinetics and ADME properties across rats and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in adequate exposure of givosiran to the target organ (liver). These studies support the interpretation of toxicology studies, help characterize the disposition of givosiran in humans, and support the clinical use of givosiran for the treatment of acute hepatic porphyria.

Aim: Advancements in RNA interference therapeutics have triggered development of improved bioanalytical methods for oligonucleotide metabolite profiling and high-throughput quantification in biological matrices. Results & methodology: HPLC coupled with high-resolution mass spectrometry (LC-HRMS) methods were developed to investigate the metabolism of a REVERSIR (TM) molecule in vivo. Plasma and tissue samples were extracted using solid-phase extraction followed by LC-HRMS analysis for metabolite profiling and quantification. The method was qualified from 10 to 5000 ng/ml (plasma) and 100 to 50000 ng/g (liver and kidney). In rat liver, intra and interday accuracy ranged from 80.9 to 118.5% and 88.4 to 111.9%, respectively, with acceptable precision (<20% CV). Conclusion: The LC-HRMS method can be applied for metabolite profiling and quantification of oligonucleotides in biological matrices.

Small interfering RNAs (siRNAs) represent a new class of medicines that are smaller (∼16,000 Da) than biologic therapeutics (>150,000 Da) but much larger than small molecules (<900 Da). Current regulatory guidance on drug-drug interactions (DDIs) from the European Medicines Agency, Food and Drug Administration, and Pharmaceutical and Medical Devices Agency provides no recommendations for oligonucleotide therapeutics including siRNAs; therefore, small molecule guidance documents have historically been applied. Over ∼10 years, in vitro DDI investigations with siRNAs conjugated to a triantennary N-acetylgalactosamine [(GalNAc)-siRNA] ligand have been conducted during nonclinical drug development to elucidate the potential clinical DDI liability. GalNAc siRNAs were evaluated as substrates, inhibitors, or inducers of major cytochrome P450s (P450s) and as substrates and inhibitors of transporters. Aggregate analysis of these data demonstrates a low potential for DDI against P450s. Zero of five, 10, and seven are inducers, time-dependent inhibitors, or substrates, respectively, and nine of 12 do not inhibit any P450 isoform evaluated. Three GalNAc siRNAs inhibited CYP2C8 at supratherapeutic concentrations, and one mildly inhibited CYP2B6. The lowest K i value of 28 µM is >3000-fold above the therapeutic clinical C max at steady state, and importantly no clinical inhibition was projected. Of four GalNAc siRNAs tested none were substrates for transporters and one caused inhibition of P-glycoprotein, calculated not to be clinically relevant. The pharmacological basis for DDIs, including consideration of the target and/or off-target profiles for GalNAc siRNAs, should be made as part of the overall DDI risk assessment. If modulation of the target protein does not interfere with P450s or transporters, then in vitro or clinical investigations into the DDI potential of the GalNAc siRNAs are not warranted. SIGNIFICANCE STATEMENT: Recommendations for evaluating DDI potential of small molecule drugs are well established; however, guidance for novel modalities, particularly oligonucleotide-based therapeutics are lacking. Given the paucity of published data in this field, in vitro DDI investigations are often conducted. The aggregate analysis of GalNAc-siRNA data reviewed herein demonstrates that, like new biological entities, these oligonucleotide-based therapeutic drugs are unlikely to result in DDIs; therefore, it is recommended that the need for in vitro or clinical investigations similarly be determined on a case-by-case basis. Given the mechanism of siRNA action, special consideration should be made in cases where there may be a pharmacological basis for DDIs.

The objective of this study was to examine the effect of reduced hematocrit and plasma protein levels, which may occur in rats after normal saline replenishment, on the pharmacokinetics of a few model drugs with different RBC (red blood cell) distribution behaviors (i.e., high vs. low). Male Sprague-Dawley rats received either saline or donor blood immediately after each blood sampling following the intravenous administration of model drugs at either 5 or 10 mg/kg. Two drugs with extensive red blood cell distribution, acetazolamide (ACE) and methazolamide (MET), and two additional drugs with negligible RBC distribution, verapamil (VER) and tolbutamide (TOL), were tested. Hematocrit, total plasma protein concentration, and drug concentration were determined after saline or donor blood replenishment. Hematocrit gradually decreased after saline replenishment (30% reduction after 6 h). Consistently, the total plasma protein concentration also decreased after saline replenishment (16% reduction at 6 h). In contrast, hematocrit and total plasma concentration remained unaltered when donor blood was used to replenish the blood volume removed after sampling. Despite the reduction of hematocrit and plasma protein concentration during the course of sampling, the pharmacokinetic profiles of all four drugs tested were similar, regardless of the method of fluid replenishment.

Aim: The electrophoretic mobility shift assay (EMSA) was evaluated as an alternative to ultrafiltration (UF) to assess plasma protein binding (PPB) of small interfering RNAs (siRNA) and antisense oligonucleotides (ASO). Results & methodology: EMSA analysis showed that PPB depended on siRNA and plasma concentration. Conversely, when analyzed by ultrafiltration, siRNA bound the filtration device nonspecifically and PPB remained >98% across physiologically relevant siRNA concentrations. Using EMSA, siRNA exhibited charge-based interactions with plasma proteins, while ASO remained highly bound to plasma proteins or albumin in the presence of 500 mM salt. Conclusion: PPB characteristics of siRNA and ASO can be distinguished using EMSA. Characterization of siRNA PPB by EMSA enhances our knowledge of siRNA absorption, distribution, metabolism and excretion and advanced development of RNA interference therapeutics.

Neutrophil plays a key role in the innate immune system through as the first leukocyte to be migrated to the acute inflammatory region. Lung inflammation is an acute respiratory failure and links closely to excessive neutrophil recruitment and severe mortality. The intranasal LPS introduced mice were shown the over-flowed neutrophils into bronchoalveolar. PLAG is a synthetic monoacetyldiacylglycerol (acetyl-DAG). DAG is a lipid second messenger for PKC activator which is associated with an important for cellular functions. The transmigrated neutrophils in alveolar by LPS stimulation were completely blocked by PLAG administration. LPS-induced cell migration associated factors such as IL-6, MIP-2 and S100A8/9 were decreased by addition of PLAG. STAT3 phosphorylation which related to neutrophil migration was significantly down regulated in PLAG dieted mice. Interestingly, the selectivity of PLAG in the ameliorating of excessive neutrophil migration was verified when compared with PLAG analogues and metabolites. Post treatment of PLAG has also therapeutic effect on LPS-induced lung inflammation. These data could be utilized as a most potential therapeutic agent for lung inflammation through regulation of neutrophil transmigration via blocking of IL-6-STAT3-MIP-2 pathway.

Inappropriate regulation of leukocyte trafficking can lead to impaired neutrophil clearance and increased tissue damage from the accumulation of neutrophil-secreted proteases and reactive oxygen species at the site of inflammation. In this study, we have discovered that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) regulates the activity of signal transducer and activator of transcription 3 (STAT3), which is a master regulator of IL-6 expression. PLAG has been isolated from the antlers of Sika deer (Cervus nippon Temminck), which were known to have immunosuppressive and anti-arthritic activities. PLAG induced a significant decrease of IL-6 production in a macrophage cell line, RAW264.7, and rheumatoid arthritis-fibroblast-like synoviocyte (RA-FLS) via the regulation of STAT3 signaling. IL-6, a multifunctional pro-inflammatory cytokine, plays a critical role in the pathogenesis of joint and systemic inflammation in RA. In collagen-induced arthritis (CIA) mouse model, arthritic symptoms were recapitulated with an increase of IL-6 in the synovium, which was recuperated by the treatment of PLAG to the level comparable with commercial therapeutics (such as Remicade or Methotrexate). When the joint tissues were stained with neutrophil-specific antibodies, PLAG significantly reduced the infiltration of neutrophils into the joint synovium of CIA mouse. Therefore, PLAG inhibits the infiltration of destructive neutrophils into inflammatory sites, and can be utilized as a therapeutic agent for the treatment of sustained inflammation and joint destruction.

Granulocyte colony-stimulating factor (G-CSF) is widely used for preventing neutropenia during chemotherapy. Polyethylene glycol-conjugated granulocyte colony-stimulating factor (PEG-G-CSF, pegfilgrastim) serves the same purpose but has a longer half-life and greater stability than G-CSF. In this study, we investigated whether 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol, acetylated diglyceride (PLAG), augments the therapeutic effect of pegfilgrastim on chemotherapy-induced neutropenia. We compared neutrophil counts in four groups of mice: control mice, gemcitabine-treated mice, gemcitabine/pegfilgrastim-treated mice, and gemcitabine/pegfilgrastim/PLAG-treated mice. PLAG (50 mg/kg) was orally administered every day during the treatment course. CBC analysis showed that the group treated with PLAG experienced a dramatically increased neutrophil counts on the third day following pegfilgrastim treatment. PLAG had no effect on blood cell apoptosis and neutrophil release from bone marrow. Additionally, pegfilgrastim-induced CXCR2 expression in neutrophils was markedly decreased in PLAG-treated animals. These results suggest that PLAG plays a role in inhibiting neutrophil extravasation, giving rise to an increased number of circulating neutrophils when used with pegfilgrastim during gemcitabine treatment. These data support the potential for PLAG to be used with pegfilgrastim to treat or prevent chemotherapy-induced neutropenia by modulating neutrophil transmigration. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (Cervus nippon Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium.

Nonalcoholic fatty liver disease (NAFLD) refers to hepatic pathologies, including simple fatty liver (SFL), nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis, that may progress to hepatocellular carcinoma. These liver disease states may affect the activity and expression levels of drug-metabolizing enzymes, potentially resulting in an alteration in the pharmacokinetics, therapeutic efficacy, and safety of drugs. This study investigated the hepatic cytochrome P450 (CYP) 2B1-modulating effect of a specific NAFLD state in dietary rat models. Sprague-Dawley rats were given a methionine/choline-deficient (MCD) or high-fat (HF) diet to induce NASH and SFL, respectively. The induction of these disease states was confirmed by plasma chemistry and liver histological analysis. Both the protein and mRNA levels of hepatic CYP2B1 were considerably reduced in MCD diet-fed rats; however, they were similar between the HF diet-fed and control rats. Consistently, the enzyme-kinetic and pharmacokinetic parameters for CYP2B1-mediated bupropion metabolism were considerably reduced in MCD diet-fed rats; however, they were also similar between the HF diet-fed and control rats. These results may promote a better understanding of the influence of NAFLD on CYP2B1-mediated metabolism, which could have important implications for the safety and pharmacokinetics of drug substrates for the CYP2B subfamily in patients with NAFLD.

Abstract Chemotherapy-induced neutropenia (CIN) is a common dose-limiting side-effect that often compromises chemotherapy resulting in dose reduction and/or delayed treatment. Chemotherapy-induced myeloid suppression is a prevalent mechanism to describe CIN. However, diapedesis of neutrophils from the blood is also a possible mechanism of CIN. Cytokines, chemokines, and damage- (or danger-) associated molecular pattern molecules (DAMPs) are produced from the damaged tissues by chemotherapy, which accelerates neutrophil transmigration. EC-18 is a synthetic monoacetyldiglyceride (MW=635) currently under development as an oral agent to treat and potentially prevent CIN. Here, we showed that neutrophil transmigration is induced in CIN model, and the efficacy and potential mechanism of EC-18 have been evaluated in mice and various cell models. Gemcitabine (50mg/Kg), cyclophosphamide (100mg/Kg) or tamoxifen (50mg/Kg) was administered daily for 3 days in mice (C57BL/6), followed by oral administrations of EC-18 (50mg/Kg) for 21 days. Blood neutrophils were analyzed by complete blood count (CBC) and fluorescence-activated cell sorting (FACS). All three chemotherapeutic agents significantly reduced absolute neutrophil count (ANC) in the blood; 40.4% decrease in gemcitabine, 19.8% in cyclophosphamide, and 21.8% in tamoxifen, which were recovered by the co-administration of EC-18. Microarray analysis of the lymph nodes from gemcitabine-treated mice revealed that the expression of DAMP molecules, S100A8 and S100A9, was induced by gemcitabine and decreased by the co-treatment of EC-18. Using immune cell lines, the expression of neutrophil transmigratory molecules, such as C3 anaphylatoxin, IL-6 and IL-8, was increased by gemcitabine treatment, and EC-18 blocked the expression of those cytokines in dose-dependent manners. Western blot analysis showed that EC-18 regulated STAT3 and STAT6 signaling pathways to modulate the expression of DAMPs, C3, IL-6, and IL-8. The decrease of C3 and IL-6 levels by EC-18 treatment was also verified in PBMCs from normal adults. In a pilot clinical study (WJON 2015 accepted, ASCO e20697), orally administrated EC-18 (1,000mg/day) alleviated neutropenic phenotypes in patients with pancreatic cancer who had been treated with gemcitabine (1,000mg/m2) and erlotinib (100mg daily). As a supportive oncology agent, EC-18 can maximize the antitumor efficacy of myelo-suppressive chemotherapeutic agents by enabling the treatment to continue or even higher doses to be administered. Disclosures No relevant conflicts of interest to declare.