GalNAc Research Articles

Progress with RNA Interference for the Treatment of Primary Hyperoxaluria.

Over the last few years, US Food and Drug Administration-approved drugs using RNA interference have come to the market. Many have treated liver-specific diseases utilizing N-acetyl galactosamine conjugation because of its effective delivery and limited off-target effects. The autosomal recessive disorder primary hyperoxaluria, specifically type 1, has benefited from these developments. Primary hyperoxaluria arises from mutations in the enzymes involved in endogenous oxalate synthesis. The severity of disease varies but can result in kidney failure and systemic oxalosis. Until recently, the treatment options were limited and focused primarily on supportive treatments, pyridoxine use in a subset of patients with primary hyperoxaluria type 1, and liver-kidney transplants in those who progressed to kidney failure. Two genes have been targeted with RNA interference; lumasiran targets glycolate oxidase and nedosiran targets lactate dehydrogenase A. Lumasiran was recently approved in the treatment of primary hyperoxaluria type 1 and nedosiran is in the approval process. Unfortunately, despite initial hopes that nedosiran may also be a treatment option for primary hyperoxaluria types 2 and 3, initial data suggest otherwise. The use of RNA interference liver-specific targeting for the treatment of primary hyperoxaluria type 1 will likely transform the natural history of the disease.

In-silico Determination of Insecticidal Potential in Lepidopteron Specific Crystal Toxins with Midgut Alkaline Phosphatase Using Molecular Docking

Alkaline phosphatase (ALP) enzyme plays an important role in binding of lepidopteran specific insecticidal crystal toxins Cry1Ac and Cry1Ab on GPI-anchored membrane receptor. In most of the cases crystal toxin interaction with ALP or Aminopeptidase N (APN) mediated by the terminal N-acetyl galactosamine (GalNAc) moiety. Among toxins Cry1Ac (PDB Id-4ARY) and Cry1Ab (PDB Id - n642) expresses less binding affinity about (-5.79 and -5.62) to GalNAc in ALP or APN region, respectively. The diptera specific crystal toxins Cry4Ba structure resembles to the lepidopteran specific toxins as it mimics like Cry1Aa for binding to ALP receptor by showing least binding affinity about (-6.99). Cry4Ba may be repurposed along with the novel crystal toxins for lepidopteron insects as toxicity over Cry1Ac or Cry1Ab Bt which will effective to bind with ALP or APN for toxin receptor interaction and help to minimize the rate of resistance. The Cry4Ba pyramided in combination with other Cry genes in different crops like cotton and maize to increase crop protection by delaying would be an efficient strategy the insect resistance.

Assembly of a Library of Pel-Oligosaccharides Featuring α-Glucosamine and α-Galactosamine Linkages.

Pseudomonas aeruginosa, a pathogenic Gram-negative bacterium for which currently antibiotic resistance is posing a significant problem and for which no vaccines are available, protects itself by the formation of a biofilm. The Pel polysaccharide, a cationic polymer composed of cis-linked galactosamine (GalN), N-acetyl galactosamine (GalNAc), glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) monosaccharides, is an important constituent of the biofilm. Well-defined Pel oligosaccharides will be valuable tools to probe the biosynthesis machinery of this polysaccharide and may serve as diagnostic tools or be used as components of glycoconjugate vaccines. We here, report on the development of synthetic chemistry to access well-defined Pel-oligosaccharides. The chemistry hinges on the use of di-tert-butylsilylidene protected GalN and GlcN building blocks, which allow for completely cis-selective glycosylation reactions. We show the applicability of the chemistry by the assembly of a matrix of 3 × 6 Pel heptasaccharides, which has been generated from a single set of suitably protected Pel heptasaccharides, in which a single glucosamine residue is incorporated and positioned at different places along the Pel oligo-galactosamine chain.

Assessment of antimicrobial properties of a lectin isolated from <em>Amaranthus cruentus</em> Linn

Lectins are diverse class of proteins which have the ability to bind carbohydrates with considerable specificity and involved in many biological processes like host pathogen interactions, cell targeting, cell- cell communications, cancer metastasis and differentiation. Plants are rich sources of lectins that have great implications in medicinal and therapeutic applications. The antimicrobial property of a lectin isolated from Amaranthus cruentus Linn seeds has been explored in this study. ACL (Amaranthus cruentus Linn lectin) was isolated, purified and characterised by hemagglutination assay with human ABO blood groups and the molecular weight was determined by SDS PAGE. ACL was subjected to evaluation for inhibition of microbial growth by disc diffusion against human pathogenic Gram positive and Gram negative bacterial strains and some fungal strains. MIC and MBC were assessed. The effects of ACL on biofilm forming bacterial strains was assessed using crystal violet assay. The isolated and purified ACL have a molecular weight of 60 KDa. The Hemagglutination activity of ACL was inhibited by N-acetyl galactosamine. ACL exhibited both antibacterial and antifungal activities with considerable zones of inhibition. Biofilm mass reduction was observed in ACL treated bacteria. These findings indicate that ACL is having therapeutic applications with great importance in clinical microbiology.

N-Acetyl Galactosamine Targeting: Paving the Way for Clinical Application of Nucleotide Medicines in Cardiovascular Diseases.

While the promise of oligonucleotide therapeutics, such as (chemically modified) ASO (antisense oligonucleotides) and short interfering RNAs, is undisputed from their introduction onwards, their unfavorable pharmacokinetics and intrinsic capacity to mobilize innate immune responses, were limiting widespread clinical use. However, these major setbacks have been tackled by breakthroughs in chemistry, stability and delivery. When aiming an intervention hepatic targets, such as lipid and sugar metabolism, coagulation, not to mention cancer and virus infection, introduction of N-acetylgalactosamine aided targeting technology has advanced the field profoundly and by now a dozen of N-acetylgalactosamine therapeutics for these indications have been approved for clinical use or have progressed to clinical trial stage 2 to 3 testing. This technology, in combination with major advances in oligonucleotide stability allows safe and durable intervention in targets that were previously deemed undruggable, such as Lp(a) and PCSK9 (proprotein convertase subtilisin/kexin type 9), at high efficacy and specificity, often with as little as 2 doses per year. Their successful use even the most visionary would not have predicted 2 decades ago. Here, we will review the evolution of N-acetylgalactosamine technology. We shall outline their fundamental design principles and merits, and their application for the delivery of oligonucleotide therapeutics to the liver. Finally, we will discuss the perspectives of N-acetylgalactosamine technology and propose directions for future research in receptor targeted delivery of these gene medicines.

EVALUATION OF THE EFFICACY AND SAFETY OF IONIS-TTR-LRX IN PATIENTS WITH TRANSTHYRETIN-MEDIATED AMYLOID CARDIOMYOPATHY: THE CARDIO-TTRANSFORM STUDY

<h3>BACKGROUND</h3> Transthyretin-mediated amyloid cardiomyopathy (ATTR-CM) is a fatal condition caused by misfolding and aggregation of transthyretin (TTR). Despite the treatment with a TTR stabilizer, tafamidis, disease progression still occurs. IONIS-TTR-LRx is an antisense oligonucleotide that inhibits TTR production by the liver. It has the same sequence as inotersen but is conjugated to a triantennary N-acetyl galactosamine (GalNAc) moiety, which allows the use of a lower dose to achieve identical pharmacodynamic results. <h3>METHODS AND RESULTS</h3> CARDIO-TTRansform (ClinicalTrials.gov NCT04136171) is a Phase 3 global, double-blind, randomized, placebo-controlled study assessing the efficacy and safety of IONIS-TTR-LRx in hereditary and wild-type ATTR-CM patients. Approximately 750 patients will be randomized 1:1 to receive either IONIS-TTR-LRx 45 mg or placebo administered by monthly SC injection. Key inclusion criteria include ATTR-CM diagnosis by biopsy or positive PYP/DPD/HMDP scan, interventricular septum thickness >12mm, NT-proBNP >600 pg/mL, NYHA class I-III and 6-minute walk distance (6MWD) ≥150m. Concomitant treatment with tafamidis is allowed. Primary efficacy endpoint is the composite of cardiovascular (CV) mortality and recurrent CV clinical events at the Week 120 study visit using the Andersen-Gill method. <h3>CONCLUSION</h3> The CARDIO-TTRansform trial is a Phase 3 trial designed to evaluate the clinical efficacy and safety of IONIS-TTR-LRx for the treatment of ATTR-CM.

Substrate specificity of Chondroitinase ABC I based on analyses of biochemical reactions and crystal structures in complex with disaccharides

Chondroitinase ABC I (cABC-I) is the enzyme which cleaves the β-1,4 glycosidic linkage of chondroitin sulfate (CS) by β-elimination. To elucidate more accurately the substrate specificity of cABC-I, we evaluated the kinetic parameters of cABC-I and its reactivity with CS isomers displaying less structural heterogeneity as substrates, e.g., approximately 90 percent of disaccharide units in Chondroitin sulfate A (CSA) or Chondroitin sulfate C (CSC) is D-glucuronic acid and 4-O-sulfated N-acetyl galactosamine (GalNAc) (A-unit) or D-glucuronic acid and 6-O-sulfated GalNAc (C-unit), respectively. cABC-I showed the highest reactivity to CSA and CSC among all CS isomers, and the kcat/Km of cABC-I was higher for CSA than for CSC. Next, we determined the crystal structures of cABC-I in complex with CS disaccharides, and analyzed the crystallographic data in combination with molecular docking data. Arg500 interacts with 4-O-sulfated and 6-O-sulfated GalNAc residues. The distance between Arg500 and the 4-O-sulfate group was 0.8 Å shorter than that between Arg500 and the 6-O-sulfated group. Moreover, it is likely that the 6-O-sulfated group is electrostatically repulsed by the nearby Asp490. Thus, we demonstrated that cABC-I has the highest affinity for the CSA richest in 4-O-sulfated GalNAc residues among all CS isomers. Recently, cABC-I was used to treat lumbar disc herniation. The results provide useful information to understand the mechanism of the pharmacological action of cABC-I.

Investigation of chemical and biological properties of an acidic polysaccharide fraction from Pleurotus eous (Berk.) Sacc.

Pleurotus eous, pink oyster mushroom and functional food, is widely cultivated in Southern India. In this study, we successfully attempted, to isolate a new acidic polysaccharide fraction (PEPA-1a) possessing a molecular weight of 8.926 × 104 Da from the fruiting bodies of Pleurotus eous through ion-exchange and gel-filtration chromatographic techniques. Monosaccharide composition comprising glucose, galactose, rhamnose, mannose, and N-acetyl galactosamine were found to have the corresponding mole percentages of 20.25, 56.75, 3.06, 11.07, and 8.86, respectively. The antioxidant activity was assessed through seven in vitro tests, PEPA-1a showed significant antioxidant activity in a dose-dependent way, with EC50 values stretching from 1.08 to 4.91 mg/mL. Analyses of PEPA-1a towards in vitro anti-tumour showed high activity against HT29 (IC50 = 233.50 μg/mL) and PC3 cells (IC50 = 230.80 μg/mL). The anticoagulant activity estimated through APTT, PT and TT tests were found be effective. Also, the congo red analysis revealed the triple-helical structures of carbohydrates. The results indicate that consumption of P. eous polysaccharide may be beneficial for health and will serve as an exceptionally accessible natural source for food and pharmaceutical industries.

P010. Evaluation of the Efficacy and Safety of AKCEA-TTR-LRx (ION-682884) in Patients with Transthyretin-mediated Amyloid Cardiomyopathy: The CARDIO-TTRansform Study

<h3>Background</h3> Transthyretin amyloidosis cardiomyopathy (ATTR-CM) is a fatal condition, leading to heart failure (HF) and ultimately death. ATTR-CM is caused by misfolding and aggregation of transthyretin (TTR), a protein produced by the liver. Depending on the presence or absence of a destabilizing mutation in the TTR gene, the disease can be classified as hereditary ATTR-CM (hATTR-CM) or wild-type ATTR-CM (wtATTR-CM), respectively. Despite the treatment with a TTR stabilizer, tafamidis, recently approved in the United States for the treatment of ATTR-CM, disease progression still occurs. AKCEA-TTR-L_RX (ION-682884) is an antisense oligonucleotide (ASO) that inhibits the production of TTR. It has a similar design and sequence as inotersen (the parent compound), but is conjugated to a triantennary N-acetyl galactosamine (GalNAc3) moiety for selective receptor-mediated delivery to hepatocytes, the principal source of systemically circulating TTR. This delivery approach has yielded an up to 30-fold increase in potency and improved the safety and tolerability profiles of ASOs in human clinical trials. Conjugation of this ligand allows the use of a lower dose to achieve improved pharmacodynamic results. In a phase 1, randomized, placebo-controlled study, AKCEA-TTR-L_RX given at a 45 mg, 60 mg or 90 mg dose, by subcutaneous (SC) injection every four weeks in 36 healthy volunteers achieved a mean pre-steady state reduction in serum TTR of 86%, 91% and 94%, respectively, compared to baseline. The dosage regimen of 45 mg SC every four weeks (27-fold lower exposure vs the inotersen dose in NEURO-TTR trial) was chosen for the pivotal phase 3 study. <h3>Methods</h3> CARDIO-TTRansform (ClinicalTrials.gov NCT04136171) is a Phase 3 global, double-blind, randomized, placebo-controlled study assessing the efficacy and safety of AKCEA-TTR-L _Rx in hATTR-CM or wtATTR-CM patients receiving available background standard of care (SoC) therapy. Approximately 750 patients around the world with a history of HF due to ATTR-CM will be randomized 1:1 to receive either AKCEA-TTR-L_Rx 45 mg or placebo administered by SC injection once every 4 weeks. Key inclusion criteria include diagnosis of ATTR-CM by biopsy or positive PYP/DPD/HMDP scan, interventricular septum thickness >12mm, NT-proBNP > 600 pg/mL, NYHA class I-III and 6-minute walk distance (6MWD) >150 m. Key exclusion criteria include, platelet count < 125 × 10⁹\L and urine protein/creatinine ratio >= 750 mg/g. Concomitant treatment with tafamidis as SoC for ATTR-CM is allowed. The study consists of a 120-week Treatment Period. Primary efficacy endpoint is the composite of cardiovascular (CV) mortality and recurrent CV clinical events at Week 120 study visit using the Andersen-Gill method. Secondary endpoints include the change from baseline in the 6MWD, KCCQ score, CV clinical events, CV death and all-cause of mortality at Week 120. <h3>Conclusions</h3> Despite recent advances, additional efficacious, safe and convenient treatment options for ATTR-CM are needed. The CARDIO-TTRansform trial is a large Phase 3 trial designed to evaluate the clinical efficacy and safety of AKCEA-TTR-LRx compared to placebo in patients with ATTR-CM receiving available SoC therapy.

Interspecies Scaling of Human Clearance and Plasma Trough Exposure for Antisense Oligonucleotides: A Retrospective Analysis of GalNAc3-Conjugated and Unconjugated-Antisense Oligonucleotides.

It is well documented and generally accepted that human clearance (CL) of unconjugated single-strand antisense oligonucleotides (ASOs) can be directly predicted from monkeys by body weight (BW) on a mg/kg dose basis. However, the scaling for triantennary N-acetyl galactosamine (GalNAc3)-conjugated ASOs has not been fully established. In this study, we retrospectively analyzed pharmacokinetic data from 9 GalNAc3-conjugated and 12 unconjugated single-stranded ASOs (ten 2'-methoxyethyl and two 2', 4'-constrained ethyl ASOs) to identify an appropriate allometric scaling factor between the two species. In addition, we compared the trough plasma concentrations (Ctrough, a surrogate for tissue exposure) between monkeys and humans at comparable dose levels, aiming at predicting tissue distribution in humans from monkeys. Overall, the median plasma CL ratios (monkey CL/human CL) were 1.05 and 0.94 when CL was normalized by BW, as compared with 0.33 and 0.29 when CL was normalized by body surface area (BSA) for the 12 unconjugated and 9 GalNAc3-conjugated ASOs, respectively. Similarly, the median Ctrough ratios (Ctrough in monkeys/Ctrough in humans) were 0.96 and 1.71, respectively, when Ctrough was normalized by mg/kg dose as compared with 3.10 and 5.50 when Ctrough was normalized by mg/m2 dose for the same unconjugated and conjugated ASOs, respectively. Equivalent CL and dose-normalized plasma Ctrough between monkeys and humans suggest similar pharmacokinetic profiles and tissue distribution between the two species on a per kilogram BW basis. In conclusion, human CL and plasma Ctrough (a surrogate of tissue distribution) can be directly predicted (1:1 or within twofold) from monkeys by BW on a mg/kg dose basis but these parameters can be under- or over-predicted by BSA on a mg/m2 dose basis. These results provide evidence for single species scaling from monkeys to humans directly and, thus, they can facilitate early human dose prediction in ASO drug development.

Structural Basis of Ligand Selectivity by a Bacterial Adhesin Lectin Involved in Multispecies Biofilm Formation.

Carbohydrate recognition by lectins governs critical host-microbe interactions. MpPA14 (Marinomonas primoryensis PA14 domain) lectin is a domain of a 1.5-MDa adhesin responsible for a symbiotic bacterium-diatom interaction in Antarctica. Here, we show that MpPA14 binds various monosaccharides, with l-fucose and N-acetylglucosamine being the strongest ligands (dissociation constant [Kd ], ∼150 μM). High-resolution structures of MpPA14 with 15 different sugars bound elucidated the molecular basis for the lectin's apparent binding promiscuity but underlying selectivity. MpPA14 mediates strong Ca2+-dependent interactions with the 3,4-diols of l-fucopyranose and glucopyranoses, and it binds other sugars via their specific minor isomers. Thus, MpPA14 only binds polysaccharides like branched glucans and fucoidans with these free end groups. Consistent with our findings, adhesion of MpPA14 to diatom cells was selectively blocked by l-fucose, but not by N-acetyl galactosamine. The MpPA14 lectin homolog present in a Vibrio cholerae adhesin was produced and was shown to have the same sugar binding preferences as MpPA14. The pathogen's lectin was unable to effectively bind the diatom in the presence of fucose, thus demonstrating the antiadhesion strategy of blocking infection via ligand-based antagonists.IMPORTANCE Bacterial adhesins are key virulence factors that are essential for the pathogen-host interaction and biofilm formation that cause most infections. Many of the adhesin-driven cell-cell interactions are mediated by lectins. Our study reveals for the first time the molecular basis underlying the binding selectivity of a common bacterial adhesin lectin from the marine bacterium Marinomonas primoryensis, homologs of which are found in both environmental and pathogenic species. The lectin-ligand interactions illustrated at the atomic level guided the identification of a ligand that serves as an inhibitor to block bacterium-host adhesion. With conventional bactericidal antibiotics losing their potency due to resistance, our work gives critical insight into an antiadhesion strategy to treat bacterial infections.

A RARE CAUSE OF AORTIC STENOSIS IN A SIBLING - MORQUIO SYNDROME - A CASE REPORT

Morquio syndrome is an uncommon variant of autosomal recessive disorder caused by the deficiency of N acetyl galactosamine 6 sulfatase (GALNS) - a lysosomal enzyme, causing dental, cardiac and skeletal abnormalities. A male patient, 29 years of age came with complaints of difficulty in breathing since one month. He was born of a second degree consanguineous marriage and has no mental retardation. On clinical examination he was short in stature, had corneal clouding, widely spaced teeth, genu valgum, joint hypermobility, pectus carinatum and kyphoscoliosis. He had an ejection systolic murmur in aortic area on cardiovascular examination. He has one 26 year old sister with similar physical appearance and cardiac abnormalities. In Echocardiography they both found to have aortic valve calcification with severe aortic stenosis with mild aortic regurgitation. X ray findings of the hand showed shortening of the metacarpal with hypoplasia of the carpal bones, platyspondly of the cervical and thoracic vertebra, genu valgum and coxa valga defect. So he was diagnosed as adult mucopolysaccharoidosis type IV-A (Morquio syndrome) which was confirmed after clinical examination and genetic analysis

New Human Follitropin Preparations: How Glycan Structural Differences May Affect Biochemical and Biological Function and Clinical Effect.

It is well accepted that pituitary follitropin is secreted into the circulation as a mixture of variants, which differ not in primary structure but rather at the level of glycosylation. These glycosidic forms vary in the number of glycosylation sites filled, complexity of glycosidic chains, and sialylation and sulfation. It is generally agreed that high sialylation, 2,3 sialic acid capping of terminal N-acetyl galactosamine or galactose leads to longer circulating half-life, by blocking binding of asialoglycoprotein receptor (ASGPR) in the liver. In contrast, 2,6 sialic acid found in humans does not prevent recognition of galactose and N-acetyl galactosamine by ASGPR. Few studies on clinical outcomes comparing differences in sialylation of follitropin found in commercially available preparations are available. Thus, there is a clear need for a consortium of open data to address this unmet need. Recently, FSH glycosylation, primarily on the β-subunit, which varies as women age, has emerged as a key modifier of follitropin action, with profound biological effects in vivo in animal models. To date, limited information of recombinant follitropin hormone preparations is available. Thus, most of the studies with FSH that is well characterized biochemically have been done in vitro, with engineered non gonadal host cells bearing recombinant receptors or in animal models. Since limited studies in human granulosa cells are available, a question is whether structural differences in glycosylation in commercially available follitropin affects biological function and clinical effect in humans. The presence of fucose, for example, has not been studied greatly even though, in the case of antibody therapy it has been shown to have a large effect on antibody targeting. This review on glycosidic variability of follitropin from the biochemical/structural point of view reflects on this question and presents an assessment in the context of available published data. If clinical differences are to be expected or not, the readers will have a better understanding of the evidence for and limitations of such expectations.

Physiological activity of E.coli engineered to produce butyric acid.

Summary Faecalibacterium prausnitzii (F. prausnitzii) is one of the most abundant bacteria in the human intestine, with its anti‐inflammatory effects establishing it as a major effector in human intestinal health. However, its extreme sensitivity to oxygen makes its cultivation and physiological study difficult. F. prausnitzii produces butyric acid, which is beneficial to human gut health. Butyric acid is a short‐chain fatty acid (SCFA) produced by the fermentation of carbohydrates, such as dietary fibre in the large bowel. The genes encoding butyryl‐CoA dehydrogenase (BCD) and butyryl‐CoA:acetate CoA transferase (BUT) in F. prausnitzii were cloned and expressed in E. coli to determine the effect of butyric acid production on intestinal health using DSS‐induced colitis model mice. The results from the E. coli Nissle 1917 strain, expressing BCD, BUT, or both, showed that BCD was essential, while BUT was dispensable for producing butyric acid. The effects of different carbon sources, such as glucose, N‐acetylglucosamine (NAG), N‐acetylgalactosamine (NAGA), and inulin, were compared with results showing that the optimal carbon sources for butyric acid production were NAG, a major component of mucin in the human intestine, and glucose. Furthermore, the anti‐inflammatory effects of butyric acid production were tested by administering these strains to DSS‐induced colitis model mice. The oral administration of the E. coli Nissle 1917 strain, carrying the expression vector for BCD and BUT (EcN‐BCD‐BUT), was found to prevent DSS‐induced damage. Introduction of the BCD expression vector into E. coli Nissle 1917 led to increased butyric acid production, which improved the strain’s health‐beneficial effects.

Immobilized Carbohydrates for Preparation of 3'-Glycoconjugated Oligonucleotides.

A detailed protocol for preparation 3'-glycoconjugated oligonucleotides is described based on one-pot immobilization of 4,4'-dimethoxytrityl-protected carbohydrates to a solid support followed by on-support peracetylation and automated oligonucleotide assembly. Compared to an appropriate building block approach and post-synthetic manipulation of oligonucleotides, this protocol may simplify the synthesis scheme and increase overall yield of the conjugates. Furthermore, the immobilization to a solid support typically increases the stability of reactants, enabling prolonged storage, and makes subsequent processing convenient. Automated assembly on these carbohydrate-modified supports using conventional phosphoramidite chemistry produces 3'-glycoconjugated oligonucleotides in relatively high yield and purity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 1-O-tert-butyldimethylsilyl-6-O-(4,4'-dimethoxytrityl)-β-D-glucose Basic Protocol 2: Synthesis of 6-O-dimethoxytrityl-2,3,1',3',4',6'-hexa-O-benzoylsucrose Basic Protocol 3: Synthesis of 6″-O-dimethoxytrityl-N-trifluoroacetyl-protected aminoglycosides Basic Protocol 4: Synthesis of 3-O-dimethoxytrityl-propyl β-D-galactopyranoside Basic Protocol 5: Synthesis of trivalent N-acetyl galactosamine cluster Basic Protocol 6: Synthesis of carbohydrate monosuccinates and their immobilization to a solid support Basic Protocol 7: Oligonucleotide synthesis using immobilized carbohydrates.

Abstract 14720: Pre-clinical Safety Assessment of SLN360, a Novel Short Interfering Ribonucleic Acid Targeting LPA

Introduction: SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated siRNA with a promising profile for addressing Lp(a)-related cardiovascular risk. We previously reported the pharmacodynamic effects of SLN360 in female (F) cynomolgus monkeys (cynos) 1 . Here we describe the findings from key pre-clinical safety studies. Methods: SLN360 was tested in vitro in primary human hepatocytes for LPA knockdown and effects on hypothetical off targets identified through in silico screening. An in vivo biodistribution study was performed in rats over 21 days (4 males [M]/4 F per timepoint; single 10 mg/kg s.c. injection). In a GLP safety study, healthy cynos received 5 once weekly s.c. injections of saline control or SLN360 (3 M / 3 F per group, up to 200 mg/kg) followed by an 8-week recovery period (2 M / 2 F in saline and top dose). A standard battery of safety assessments was performed. Results: In vitro, SLN360 reduced LPA expression in primary human hepatocytes with no effects on any hypothetical liver-expressed human off-target genes at concentrations &gt;300-fold the human LPA IC 50 . SLN360 showed a typical rodent GalNAc distribution pattern, with significant levels in liver and kidney (peak 126 or 246 mg/g tissue at 6h respectively; estimated liver half-life of 56 hours). Levels in other organs (including reproductive organs) were &lt;1% of peak liver levels. In cynos, no clinical observations were observed following 5 s.c. doses of SLN360. Liver LPA mRNA levels were significantly reduced by 98% at day 30 and 95% after the 8-week recovery period, while serum Lp(a) was undetectable at both timepoints. Findings were restricted to the liver (increased weight and diffuse hepatocyte hypertrophy) and lymph nodes (vacuolated macrophages) at day 30. These were considered non-adverse due to reversibility after recovery. No dose-related changes in clinical chemistry, hematology, circulatory and ECG parameters, respiratory rate, neurobehavior, plasma cytokines, complement activation or CRP were noted. The NOAEL was defined as 200 mg/kg, &gt;60-fold the active dose in cynos. Conclusions: Overall, the off-target profile, biodistribution and NOAEL of SLN360 indicate suitability for entry into clinical studies. 1 Aleku et al., 2019. Circulation. 2019, 140:A9538.

Abstract 13913: Single Dose Safety, Pharmacokinetics, and Pharmacodynamics of a Potent PCSK9 Synthesis Inhibitor, AZD8233, in Subjects With Elevated Ldl Cholesterol

Background: AZD8233 is a chemically modified 16-mer antisense oligonucleotide linked to N-acetyl galactosamine (GalNAc) for efficient targeting of PCSK9 mRNA in hepatocytes to prevent the synthesis of PCSK9. We are reporting preliminary data from the first in human study. The aim with the study was to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of single doses of AZD8233 in subjects with LDL-C ≥100 mg/dL. Methods: This was a randomized, single-blind, placebo-controlled; single ascending dose study (NCT03593785) of AZD8233 administered subcutaneously (SC) to 56 subjects enrolled in 7 cohorts (6 on active and 2 on placebo in each cohort). Doses studied ranged from 4 to 120 mg. Serial blood samples were collected to assess PK, PCSK9 and LDL-C profiles after administration of AZD8233. PK parameters were calculated using non-compartmental methods. Results: After SC administration, AZD8233 was generally well tolerated, no clinically relevant safety and tolerability findings were observed and no serious adverse events were reported. Peak plasma concentrations were achieved within 1 to 3 hours after dosing. After peaking, plasma concentrations declined biphasically with a terminal half-life of 2 to 3 weeks. A dose dependent decrease in plasma levels of PCSK9 and LDL-C was observed. The largest mean percent reductions from baseline were &gt;90 % for PCSK9 and 70% for LDL-C. PCSK9 and LDL-C levels slowly returned to baseline or close to baseline levels over the 16 weeks post dose follow up period. Conclusions: The safety, PK and PD support further evaluation of AZD8233 in multiple ascending dose studies. The PD data indicate that a high level of PCSK9 and LDL-C reduction can be maintained over a dose interval of once monthly or less frequent at a dose below 100mg.

Anticancer Activity of Lectins from Bauhinia purpurea and Wisteria floribunda on Breast Cancer MCF-7 Cell Lines.

Individual and collaborative efforts are being made worldwide in search of effective chemical or natural drugs with less severe side-effects for treatment of cancer. Due to the specificity and selectivity properties of lectins for saccharides, several plant lectins are known to induce cytotoxicity into tumor cells. To study the antiproliferative activity of two N-acetyl galactosamine specific plant lectins from seeds of Bauhinia purpurea and Wisteria floribunda against MCF-7 Breast cancer cell lines. MTT, lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS), and caspase- 3 assays and flow cytometry for cell cycle analysis were performed. The agglutinins BPL and WFL; 446 μgml-1 (2.2 μM) and 329 μgml-1 (2.8 μM), respectively caused remarkable concentration-dependent antiproliferative effect on MCF-7. The effect was seen to be a consequence of binding of the lectin to the cell surface and triggering S and G2 phase arrest. Apoptosis induced was found to be associated with LDH leakage, cell cycle arrest and ROS generation. The apoptotic signal was observed to be amplified by activation of caspase-3 resulting in cell death. The study provides a base for detailed investigation and further use of lectins in cancer studies.

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 μM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl β-d-galactoside (pNPGal, Kd = 54 ± 6 μM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.

Receptor-Specific Delivery of Peptide Nucleic Acids Conjugated to Three Sequentially Linked N-Acetyl Galactosamine Moieties into Hepatocytes.

Peptide nucleic acids (PNAs) are DNA analogs that bind with high affinity to DNA and RNA in a sequence-specific manner but have poor cell permeability, limiting use as therapeutic agents. The work described here is motivated by recent reports of efficient gene silencing specifically in hepatocytes by small interfering RNAs conjugated to triantennary N-acetyl galactosamine (GalNAc), the ligand recognized by the asialoglycoprotein receptor (ASGPR). PNAs conjugated to either triantennary GalNAc at the N-terminus (the branched architecture) or monomeric GalNAc moieties anchored at Cγ of three consecutive PNA monomers of N-(2-aminoethyl)glycine (aeg) scaffolds (the sequential architecture) were synthesized on the solid phase. These formed duplexes with complementary DNA and RNA as shown by UV and circular dichroism spectroscopy. The fluorescently labeled analogs of GalNAc-conjugated PNAs were internalized by HepG2 cells that express the ASGPR but were not taken up by HEK-293 cells that lack this receptor. The sequential conjugate was internalized about 13-fold more efficiently than the branched conjugate into HepG2 cells, as demonstrated by confocal microscopy. The results presented here highlight the potential significance of the architecture of GalNAc conjugation for efficient uptake by target liver cells and indicate that GalNAc-conjugated PNAs have possible therapeutic applications.