Triantennary N-acetyl galactosamine (GalNAc, GN3: ), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense ...oligonucleotides (ASOs) 6-10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2'-O-Et-2',4'-bridged nucleic acid) gapmer ASOs, ∼ 60-fold enhancement in potency relative to the parent MOE (2'-O-methoxyethyl RNA) ASO was observed. GN3: -conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3: -ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3: -ASO conjugate was detected in plasma suggesting that GN3: acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.
Abstract
Background
Current treatment of vulvovaginal candidiasis (VVC) is largely limited to azole therapy. Ibrexafungerp is a first-in-class triterpenoid antifungal with broad-spectrum anti-Candida ...fungicidal activity. The objective of this study was to evaluate the efficacy and safety of ibrexafungerp compared with placebo in patients with acute VVC.
Methods
Patients were randomly assigned 2:1 to receive ibrexafungerp (300 mg twice for 1 day) or placebo. The primary endpoint was the percentage of patients with a clinical cure (complete resolution of vulvovaginal signs and symptoms VSS = 0) at test-of-cure (day 11 ± 3). Secondary endpoints included the percentage of patients with mycological eradication, overall success (clinical cure and mycological eradication), clinical improvement (VSS ≤ 1) at test-of-cure, and symptom resolution at follow-up (day 25 ± 4).
Results
Patients receiving ibrexafungerp had significantly higher rates of clinical cure (50.5% 95/188 vs 28.6% 28/98; P = .001), mycological eradication (49.5% 93/188 vs 19.4% 19/98; P < .001), and overall success (36.0% 64/178 vs 12.6% 12/95; P < .001) compared with placebo. Symptom resolution was sustained and further increased with ibrexafungerp compared with placebo (59.6% 112/188 vs 44.9% 44/98; P = .009) at follow-up. Post hoc analysis showed similar rates of clinical cure and clinical improvement at test-of-cure for Black patients (54.8% 40/73 and 63.4% 47/73, respectively) and patients with a body mass index >35 (54.5% 24/44 and 68.2% 30/44, respectively) compared with overall rates. Ibrexafungerp was well tolerated. Adverse events were primarily gastrointestinal and mild in severity.
Conclusions
Ibrexafungerp provides a promising safe and efficacious oral treatment that mechanistically differs from current azole treatment options for acute VVC.
Ibrexafungerp was statistically superior to placebo in reduction of signs and symptoms of acute vulvovaginal candidiasis regardless of the endpoint assessed. This advantage was maintained across subpopulations including African American patients or patients with a body mass index >35.
The comprehensive structure–activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were ...assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc–ASO conjugates exhibited excellent potencies (ED50 0.5–2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc–ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.
Thesis (Ph. D.)--University of California, San Diego, 2009.
Title from first page of PDF file (viewed September 14, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical ...references (p. 86-94).
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Chemical modifications are essential to improve metabolic stability and pharmacokinetic properties of siRNA to enable their systemic delivery. We investigated the effect of combing ...the phosphorothioate (PS) modification with metabolically stable phosphate analog (E)-5′-vinylphosphonate and GalNAc cluster conjugation on the activity of fully 2′-modified siRNA in cell culture and mice. Our data suggest that integrating multiple chemical approaches in one siRNA molecule improved potency 5–10 fold and provide a roadmap for developing more efficient siRNA drugs.
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Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a ...high affinity ligand for the asialoglycoprotein receptor (ASGR)—a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5–10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.
We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in T(m), cell culture and animal experiments and ...compare them to their gap-matched 20-mer 2'-O-methoxyethyl (MOE) and 14-mer 2',4'-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar T(m) and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED(50) 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower T(m) and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animals.
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