Givosiran: A Review in Acute Hepatic Porphyria.

Givosiran (Givlaari®) is an δ-aminolevulinic acid synthase 1 (ALAS1)-directed small interfering RNA (siRNA) approved for the treatment of acute hepatic porphyria (AHP). In the phase 3 ENVISION trial, givosiran significantly reduced the annualized rate of composite porphyria attacks (i.e. attacks requiring hospitalization, urgent healthcare visit or intravenous hemin administration at home) compared with placebo in patients with recurrent acute intermittent porphyria (the most common type of AHP) attacks. Givosiran also improved several other outcomes, including hemin use and pain (the cardinal symptom of AHP). While generally well tolerated with an acceptable safety profile, the drug may increase the risk of hepatic and kidney adverse events. Givosiran offers the convenience of once-monthly subcutaneous administration. Available evidence indicates that givosiran is an important newer therapeutic option for patients with AHP and severe recurrent attacks.

The Evolution of Hemophilia Care: Clinical and Laboratory Advances, Opportunities, and Challenges.

Hemophilia A (HA) and B (HB) are X-linked bleeding disorders caused by mutations in the F8 or F9 gene that result in the absence, or reduced activity, of the corresponding clotting factor. The severity of bleeding and related complications is proportional to the amount of residual circulating functional factor. The development of a safe and effective hemophilia treatment lasted several decades and has been mainly based on clotting factor replacement. Advances in the engineering and manufacturing of clotting concentrates have led to the widespread availability of extended half-life products that reduced the number of intravenous infusions needed to achieve adequate trough levels. The recent development of new nonfactor replacement treatments and biotechnology techniques has offered therapeutic alternatives for hemophilia patients with and without inhibitors. These are characterized by an easier route of administration, low immunogenicity, and, regarding gene therapy and cell-based treatments, potential long-term protection from bleeding after a single treatment course. In this review, we analyze recent progresses in the management of hemophilia and discuss opportunities and challenges.

Purification and Assays of Tachylectin-2.

Tachylectin-2, a 27-kDa protein consisting of a five-bladed ß-propeller structure, is purified by three steps of chromatography, including dextran sulfate-Sepharose CL-6B, CM-Sepharose CL-6B, and Mono S. Three isolectins of tachylectin-2 including tachylectin-2a, -2b, and -2c are purified. These isolectins exhibit hemagglutinating activity against human A-type erythrocytes in a Ca2+-independent manner with tachylectin-2b showing the highest activity. Tachylectin-2b specifically agglutinates Staphylococcus saprophyticus KD. The tachylectin-2b-mediated hemagglutination is inhibited in the presence of GlcNAc and GalNAc. The association constants for GlcNAc and GalNAc are Ka = 1.95 × 104 M-1 and Ka = 1.11 × 103 M-1, respectively. Ultracentrifugation analysis shows that tachylectin-2b is present in monomer form in solution.

Givosiran: First Approval.

Givosiran (Givlaari™) is an aminolevulinate synthase 1 (ALAS1)-directed small interfering RNA (siRNA) covalently linked to a ligand to enable specific delivery of the siRNA to hepatocytes. This results in downregulation of ALAS1 mRNA and prevents accumulation of neurotoxic δ-aminolevulinic acid and porphobilinogen levels that are associated with acute porphyria attacks. Givosiran is being developed by Alnylam Pharmaceuticals for the treatment of acute hepatic porphyria (AHP). In November 2019, givosiran was approved in the USA for the treatment of adults with AHP based on the positive results from the multinational, phase III ENVISION trial. In the EU, givosiran received a positive opinion in January 2020 for the treatment of AHP in adults and adolescents aged 12 years and older. This article summarizes the milestones in the development of givosiran leading to this first approval for the treatment of adults with AHP.

Pharmacokinetics and Pharmacodynamics of the Small Interfering Ribonucleic Acid, Givosiran, in Patients With Acute Hepatic Porphyria.

Givosiran is a small interfering ribonucleic acid agent that was recently approved in the United States for the treatment of acute hepatic porphyria (AHP). This phase I study evaluated the safety, pharmacokinetic, and pharmacodynamic profile of subcutaneously (SC) administered givosiran in patients with acute intermittent porphyria, the most common AHP type. Givosiran was rapidly absorbed from the SC injection site with peak plasma concentrations achieved within 0.5-5 hours followed by elimination with a short half-life of 4-10 hours. Plasma exposures of AS(N-1)3' givosiran, an active metabolite with equal potency as givosiran, was 35%-75%. Givosiran treatment resulted in a rapid and dose-dependent reduction in urinary aminolevulinic acid (ALA) and porphobilinogen (PBG) towards the upper limit of normal (ULN) in AHP patients. Greater and more sustained reductions in ALA and PBG were achieved with once monthly dosing compared with once quarterly dosing. After monthly dosing, trough ALA levels were reduced to below the ULN, approximately 95% reduction from baseline, at both the 2.5 and 5.0 mg/kg doses.

Nonclinical Safety Profile of Revusiran, a 1st-Generation GalNAc-siRNA Conjugate for Treatment of Hereditary Transthyretin-Mediated Amyloidosis.

Revusiran is a 1st-generation short interfering RNA targeting transthyretin conjugated to an N-acetylgalactosamine ligand to facilitate delivery to hepatocytes via uptake by the asialoglycoprotein receptors. Revusiran, in development for the treatment of hereditary transthyretin-mediated amyloidosis, was discontinued after an imbalance in deaths in the "ENDEAVOUR" phase 3 clinical trial. Nonclinical safety assessments included safety pharmacology, acute and repeat-dose toxicity, genotoxicity, and carcinogenicity. There were no effects on cardiovascular or respiratory function in monkeys after single doses of up to 100 mg/kg. No neurological effects were noted in monkeys in repeat-dose studies up to 300 mg/kg. Revusiran was well tolerated in repeat-dose mouse (weekly doses) and rat and monkey (five daily doses followed by weekly doses) toxicity studies. The no observed adverse effect level (NOAEL) in rats was 30 mg/kg based on reversible microscopic changes in liver that were accompanied by correlating elevations in clinical chemistry at higher doses. Dose-limiting toxicity was absent in monkeys, and the NOAEL was 200 mg/kg. There was no evidence of genotoxicity in vitro or in vivo at limit doses or carcinogenicity in a 2-year study in rats at doses up to 100 mg/kg. Overall, these results demonstrate that revusiran had a favorable nonclinical safety profile.

Ligand Conjugated Multimeric siRNAs Enable Enhanced Uptake and Multiplexed Gene Silencing.

Small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) ligands have been used to treat disease in patients. However, conjugates with other ligands deliver siRNA less efficiently, limiting the development of new targeted therapies. Most approaches to enhancing the potency of such conjugates have concentrated on increasing ligand effectiveness and/or the chemical stability of the siRNA drug. One complementary and unexplored alternative is to increase the number of siRNAs delivered per ligand. An ideal system would be a single chemical entity capable of delivering multiple copies of an oligonucleotide drug and/or several such drugs simultaneously. Here we report that siRNAs can be stably linked together under neutral aqueous conditions to form chemically defined siRNA "multimers," and that these multimers can be delivered in vivo by a GalNAc ligand. Conjugates containing multiple copies of the same siRNA showed enhanced activity per unit of ligand, whereas siRNAs targeting different genes linked to a single ligand facilitated multigene silencing in vivo; this is the first demonstration of silencing several genes simultaneously in vivo using ligand-directed multimeric siRNA. Multimeric oligonucleotides represent a powerful and practical new approach to improve intracellular conjugate delivery.

In Vitro Drug-Drug Interaction Evaluation of GalNAc Conjugated siRNAs Against CYP450 Enzymes and Transporters.

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.

Synthetically glycosylated antigens induce antigen-specific tolerance and prevent the onset of diabetes.

Homeostatic antigen presentation by hepatic antigen-presenting cells, which results in tolerogenic T-cell education, could be exploited to induce antigen-specific immunological tolerance. Here we show that antigens modified with polymeric forms of either N-acetylgalactosamine or N-acetylglucosamine target hepatic antigen-presenting cells, increase their antigen presentation and induce antigen-specific tolerance, as indicated by CD4+ and CD8+ T-cell deletion and anergy. These synthetically glycosylated antigens also expanded functional regulatory T cells, which are necessary for the durable suppression of antigen-specific immune responses. In an adoptive-transfer mouse model of type-1 diabetes, treatment with the glycosylated autoantigens prevented T-cell-mediated diabetes, expanded antigen-specific regulatory T cells and resulted in lasting tolerance to a subsequent challenge with activated diabetogenic T cells. Glycosylated autoantigens targeted to hepatic antigen-presenting cells might enable therapies that promote immune tolerance in patients with autoimmune diseases.

Current and innovative emerging therapies for porphyrias with hepatic involvement.

Porphyrias are rare inherited disorders caused by specific enzyme dysfunctions in the haem synthesis pathway, which result in abnormal accumulation of specific pathway intermediates. The symptoms depend upon the chemical characteristics of these substances. Porphyrins are photoreactive and cause photocutaneous lesions on sunlight-exposed areas, whereas accumulation of porphyrin precursors is related to acute neurovisceral attacks. Current therapies are suboptimal and mostly address symptoms rather than underlying disease mechanisms. Advances in the understanding of the molecular bases and pathogenesis of porphyrias have paved the way for the development of new therapeutic strategies. In this Clinical Trial Watch we summarise the basic principles of these emerging approaches and what is currently known about their application to porphyrias of hepatic origin or with hepatic involvement.

Targeted delivery of atorvastatin via asialoglycoprotein receptor (ASGPR).

Targeted drug delivery platforms can increase the concentration of drugs in specific cell populations, reduce adverse effects, and hence improve the therapeutic effect of drugs. Herein, we designed two conjugates by installing the targeting ligand GalNAc (N-acetylgalactosamine) onto atorvastatin (AT). Compared to the parent drug, these two conjugates, termed G2-AT and G2-K-AT, showed increased hepatic cellular uptake. Moreover, both conjugates were able to release atorvastatin, and consequently showed dramatic inhibition of ß-hydroxy-ß-methylglutaryl-CoA (HMG-CoA) reductase and increased LDL receptors on cell surface.

N-Acetyl-d-galactosamine prevents soya bean agglutinin-induced intestinal barrier dysfunction in intestinal porcine epithelial cells.

Soya bean agglutinin (SBA) is a glycoprotein and the main anti-nutritional component in most soya bean feedstuffs. It is mainly a non-fibre carbohydrate-based protein and represents about 10% of soya bean-based anti-nutritional effects. In this study, we sought to determine the effects of N-Acetyl-D-galactosamine (GalNAc or D-GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC-J2) cells. The IPEC-J2 cells were pre-cultured with 0, 0.125 × 10-4 , 0.25 × 10-4 , 0.5 × 10-4 , 1.0 × 10-4 and 2.0 × 10-4  mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre-incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans-epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre-treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin-3 were lower in the SBA-treated groups without pre-treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin-3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre-treated groups, occludin and claudin-3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC-J2s.

Evaluation of the effect of 2'-O-methyl, fluoro hexitol, bicyclo and Morpholino nucleic acid modifications on potency of GalNAc conjugated antisense oligonucleotides in mice.

The potency of antisense oligonucleotide (ASO) drugs has significantly improved in the clinic after exploiting asialoglycoprotein receptor (ASGR) mediated delivery to hepatocytes. To further this technology, we evaluated the structure-activity relationships of oligonucleotide chemistry on in vivo potency of GalNAc-conjugated Gapmer ASOs. GalNAc conjugation improved potency of ASOs containing 2'-O-methyl (2'-O-Me), 3'-fluoro hexitol nucleic acid (FHNA), locked nucleic acid (LNA), and constrained ethyl bicyclo nucleic acid (cEt BNA) 10-20-fold compared to unconjugated ASOs. We further demonstrate that GalNAc conjugation improves activity of 2'-O-(2-methoxyethyl) (2'-O-MOE) and Morpholino ASOs designed to correct splicing of survival motor neuron (SMN2) pre-mRNA in liver after subcutaneous administration. GalNAc modification thus represents a viable strategy for enhancing potency of ASO with diverse nucleic acid modifications and mechanisms of action for targets expressed in hepatocytes.

The Nonclinical Safety Profile of GalNAc-conjugated RNAi Therapeutics in Subacute Studies.

Short interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) are the most clinically advanced oligonucleotide-based platforms. A number of N-acetylgalactosamine (GalNAc)-conjugated siRNAs (GalNAc-siRNAs), also referred to as RNA interference (RNAi) therapeutics, are currently in various stages of development, though none is yet approved. While the safety of ASOs has been the subject of extensive review, the nonclinical safety profiles of GalNAc-siRNAs have not been reported. With the exception of sequence differences that confer target RNA specificity, GalNAc-siRNAs are largely chemically uniform, containing limited number of phosphorothioate linkages, and 2'-O-methyl and 2'-deoxy-2'-fluoro ribose modifications. Here, we present the outcomes of short-term (3-5 week) rat and monkey weekly repeat-dose toxicology studies of six Enhanced Stabilization Chemistry GalNAc-siRNAs currently in clinical development. In nonclinical studies at supratherapeutic doses, these molecules share similar safety signals, with histologic findings in the organ of pharmacodynamic effect (liver), the organ of elimination (kidney), and the reticuloendothelial system (lymph nodes). The majority of these changes are nonadverse, partially to completely reversible, correlate well with pharmacokinetic parameters and tissue distribution, and often reflect drug accumulation. Furthermore, all GalNAc-siRNAs tested to date have been negative in genotoxicity and safety pharmacology studies.

Role of N-acetyl galactosamine-4-SO4, a ligand of CD206 in HSV-induced mouse model of Behçet's disease.

CD206 is a macrophage mannose receptor involved in variety of autoimmune and inflammatory diseases. This study aimed to identify the pathogenic role of CD206 in a herpes simplex virus (HSV) induced Behçet's disease (BD) mouse model. CD206 positive cells were detected in peripheral blood mononuclear cells and quantified by flow cytometry. Levels of cytokines were measured by ELISA. CD206 was found to be down-regulated both in vitro (10-6M) and in vivo (200µg/mouse) after treatment with N-acetylgalactosamine (GalNAc), a ligand for CD206. The down-regulation of CD206 was correlated with improvement in BD symptoms. Colchicine (2µg/mouse) or pentoxifylline (400µg/mouse) treated mice displayed improvement in BD symptoms with fewer CD206 positive cells. The prevalence of CD206-positive cells differed between ligand-responsive and non-responsive BD mice. Inhibition of CD206 was associated with down-regulated serum level of interleukin-17 in GalNAc-treated BD mice. These results suggest that the expression of CD206 is correlated with HSV-induced BD symptoms in mice, implicating that CD206 might have a pathogenic role in BD.

L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins.

Overexpression of P-glycoprotein (P-gp, drug transporter) in neoplastic cells is the most frequently observed molecular cause of multidrug resistance. Here, we show that the overexpression of P-gp in L1210 cells leads to resistance to tunicamycin and benzyl 2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAc-α-O-benzyl). Tunicamycin induces both glycosylation depression and ubiquitination improvement of P-gp. However, the latter is not associated with large increases in molecular mass as evidence for polyubiquitination. Therefore, P-gp continues in maturation to an active membrane efflux pump rather than proteasomal degradation. P-gp-positive L1210 cells contain a higher quantity of ubiquitin associated with cell surface proteins than their P-gp-negative counterparts. Thus, P-gp-positive cells use ubiquitin signaling for correct protein folding to a higher extent than P-gp-negative cells. Elevation of protein ubiquitination after tunicamycin treatment in these cells leads to protein folding rather than protein degradation, resulting at least in the partial lack of cell sensitivity to tunicamycin in L1210 cells after P-gp expression. In contrast to tunicamycin, to understand why P-gp-positive cells are resistant to GalNAc-α-O-benzyl, further research is needed.

Association of Cardiac Galectin-3 Expression, Myocarditis, and Fibrosis in Chronic Chagas Disease Cardiomyopathy.

Chronic Chagas disease cardiomyopathy, caused by Trypanosoma cruzi infection, is a major cause of heart failure in Latin America. Galectin-3 (Gal-3) has been linked to cardiac remodeling and poor prognosis in heart failure of different etiologies. Herein, we investigated the involvement of Gal-3 in the disease pathogenesis and its role as a target for disease intervention. Gal-3 expression in mouse hearts was evaluated during T. cruzi infection by confocal microscopy and flow cytometry analysis, showing a high expression in macrophages, T cells, and fibroblasts. In vitro studies using Gal-3 knockdown in cardiac fibroblasts demonstrated that Gal-3 regulates cell survival, proliferation, and type I collagen synthesis. In vivo blockade of Gal-3 with N-acetyl-d-lactosamine in T. cruzi-infected mice led to a significant reduction of cardiac fibrosis and inflammation in the heart. Moreover, a modulation in the expression of proinflammatory genes in the heart was observed. Finally, histological analysis in human heart samples obtained from subjects with Chagas disease who underwent heart transplantation showed the expression of Gal-3 in areas of inflammation, similar to the mouse model. Our results indicate that Gal-3 plays a role in the pathogenesis of experimental chronic Chagas disease, favoring inflammation and fibrogenesis. Moreover, by demonstrating Gal-3 expression in human hearts, our finding reinforces that this protein could be a novel target for drug development for Chagas cardiomyopathy.

Glycoengineering of Esterase Activity through Metabolic Flux-Based Modulation of Sialic Acid.

This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity. By contrast, hexosamine analogues not targeted to sialic acid biosynthesis (e.g., butanoylated GlcNAc or GalNAc) had minimal impact. Measurement of mRNA and protein confirmed that esterase activity was controlled through glycosylation and not through transcription or translation. Azide-modified ManNAc analogues widely used in MGE also enhanced esterase activity and provided a way to enrich targeted glycoengineered proteins (such as CES2), thereby providing unambiguous evidence that the compounds were converted to sialosides and installed into the glycan structures of esterases as intended. Overall, this study provides a pioneering example of the modulation of intracellular enzyme activity through MGE, which expands the value of this technology from its current status as a labeling strategy and modulator of cell surface biological events.

Exposure to siRNA-GalNAc Conjugates in Systems of the Standard Test Battery for Genotoxicity.

Registration of pharmaceuticals requires an assessment of their genotoxic potential using in vitro and in vivo tests outlined in the International Conference on Harmonisation (ICH) guidance S2(R1). We have evaluated numerous siRNA-N-acetylgalactosamine (GalNAc) conjugates containing phosphorothioate linkages and various combinations of 2'-fluoro and 2'-O-methyl ribose modifications of multiple nucleotides in the ICH battery of assays, all of which have uniformly yielded negative results. To verify these negative genotoxicity results, in this study we confirm test article exposure using toolkit small interfering RNAs (siRNAs) representative of those in the clinic. In the Ames test, the highest uptake of the siRNA-GalNAc conjugates occurred at 1 h postdose in all bacterial strains independent of siRNA sequence or chemistry (up to ∼14,000 siRNA molecules per cell), followed by metabolic degradation of the parent siRNA at 6, 24, and 48 h postdose. siRNA-GalNAc conjugates were internalized by bacteria as assessed by protection from the addition of nucleases to the culture media following uptake and by the requirement of cell lysis for detection of the siRNA. In the in vitro chromosome aberration assay, uptake was observed in Chinese hamster ovary cells (up to ∼5,500 siRNA molecules per cell at 21 h postdose) and in CD3+ human peripheral blood lymphocytes (up to ∼500 siRNA molecules per cell at 21 h postdose). In the in vivo micronucleus assay in rat bone marrow, exposure to parent siRNA was 100-350 µg of antisense strand per gram of protein at 24 and 48 h postlimit dose of 2 g/kg. Loss of terminal nucleotides was detected in bone marrow by mass spectrometry, indicating exposure to monomer metabolites as well. Negative genotoxicity results were also confirmed in an in vitro double-strand DNA break assay in HeLa and HepG2 cells where exposure was maximized using transfection reagents. Thus negative genotoxicity assay results for siRNA-GalNAc conjugates were valid and not the result of poor or no intracellular exposure.