The Angers CT Score is a Risk Factor for the Failure of the Conservative Management of Adhesive Small Bowel Obstruction: A Prospective Observational Multicentric Study.

BACKGROUND: Identifying the 30% of adhesive small bowel obstructions (aSBO) for which conservative management will require surgery is essential. The association between the previously described radiological score and failure of the conservative management of aSBO remains to be confirmed in a large prospective multicentric cohort. Our aim was to assess the risk factors of failure of the conservative management of aSBO considering the radiological score. MATERIAL AND METHODS: This prospective observational study took place in 15 French centers over 3 months. Consecutive patients experiencing aSBO with no early surgery were included. The six radiological features from the Angers radiological computed tomography (CT) score were noted (beak sign, closed loop, focal or diffuse intraperitoneal liquid, focal or diffuse mesenteric haziness, focal or diffuse mesenteric liquid, and diameter of the most dilated small bowel loop > 40 mm). RESULTS: Two hundred and seventy nine patients with aSBO were screened. Sixty patients (21.5%) underwent early surgery, and 219 (78.5%) had primary conservative management. In the end, 218 patients were included in the analysis of the risk factors for conservative treatment failure. Among them, 162 (74.3%) had had successful management while for 56 (25.7%) management had failed. In multivariate analysis, a history of surgery was not a significant risk factor for the failure of conservative treatment (OR = 0.11; 95%CI = 0-1.23). A previous episode of aSBO was protective against the failure of conservative treatment (OR = 0.36; 95%CI = 0.15-0.85) and an Angers CT score ≥ 5 as the only individual risk factor (OR = 2.39; 95%CI = 1.01-5.69). CONCLUSION: The radiological score of aSBO is a promising tool in improving the management of aSBO patients. A first episode of aSBO and/or a radiological score ≥5 should lead physicians to consider early surgical management.

Safety, Tissue Distribution, and Metabolism of LNA-Containing Antisense Oligonucleotides in Rats.

Antisense oligonucleotides (ASOs) are chemically modified nucleic acids with therapeutic potential, some of which have been approved for marketing. We performed a study in rats to investigate mechanisms of toxicity after administration of 3 tool locked nucleic acid (LNA)-containing ASOs with differing established safety profiles. Four male rats per group were dosed once, 3, or 6 times subcutaneously, with 7 days between dosing, and sacrificed 3 days after the last dose. These ASOs were either unconjugated (naked) or conjugated with N-acetylgalactosamine for hepatocyte-targeted delivery. The main readouts were in-life monitoring, clinical and anatomic pathology, exposure assessment and metabolite identification in liver and kidney by liquid chromatography coupled to tandem mass spectrometry, ASO detection in liver and kidney by immunohistochemistry, in situ hybridization, immune electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry imaging. The highly toxic compounds showed the greatest amount of metabolites and a low degree of tissue accumulation. This study reveals different patterns of cell death associated with toxicity in liver (apoptosis and necrosis) and kidney (necrosis only) and provides new ultrastructural insights on the tissue accumulation of ASOs. We observed that the immunostimulatory properties of ASOs can be either primary from sequence-dependent properties or secondary to cell necrosis.

Antisense-mediated reduction of proprotein convertase subtilisin/kexin type 9 (PCSK9): a first-in-human randomized, placebo-controlled trial.

AIMS: LDL-receptor expression is inhibited by the protease proprotein convertase subtilisin/kexin type 9 (PCSK9), which is considered a pharmacological target to reduce LDL-C concentrations in hypercholesterolaemic patients. We performed a first-in-human trial with SPC5001, a locked nucleic acid antisense inhibitor of PCSK9. METHODS: In this randomized, placebo-controlled trial, 24 healthy volunteers received three weekly subcutaneous administrations of SPC5001 (0.5, 1.5 or 5 mg kg(-1)) or placebo (SPC5001 : placebo ratio 6 : 2). End points were safety/tolerability, pharmacokinetics and efficacy of SPC5001. RESULTS: SPC5001 plasma exposure (AUC(0,24 h)) increased more than dose-proportionally. At 5 mg kg(-1), SPC5001 decreased target protein PCSK9 (day 15 to day 35: -49% vs. placebo, P < 0.0001), resulting in a reduction in LDL-C concentrations (maximal estimated difference at day 28 compared with placebo -0.72 mmol l(-1), 95% confidence interval - 1.24, -0.16 mmol l(-1); P < 0.01). SPC5001 treatment (5 mg kg(-1)) also decreased ApoB (P = 0.04) and increased ApoA1 (P = 0.05). SPC5001 administration dose-dependently induced mild to moderate injection site reactions in 44% of the subjects, and transient increases in serum creatinine of ≥20 µmol l(-1) (15%) over baseline with signs of renal tubular toxicity in four out of six subjects at the highest dose level. One subject developed biopsy-proven acute tubular necrosis. CONCLUSIONS: SPC5001 treatment dose-dependently inhibited PCSK9 and decreased LDL-C concentrations, demonstrating human proof-of-pharmacology. However, SPC5001 caused mild to moderate injection site reactions and renal tubular toxicity, and clinical development of SPC5001 was terminated. Our findings underline the need for better understanding of the molecular mechanisms behind the side effects of compounds such as SPC5001, and for sensitive and relevant renal toxicity monitoring in future oligonucleotide studies.

Acute kidney injury during therapy with an antisense oligonucleotide directed against PCSK9.

Antisense oligonucleotides have been explored widely in clinical trials and generally are considered to be nontoxic for the kidney, even at high concentrations. We report a case of toxic acute tubular injury in a healthy 56-year-old female volunteer after a pharmacologically active dose of a locked nucleic acid antisense oligonucleotide was administered. The patient received 3 weekly subcutaneous doses of experimental drug SPC5001, an antisense oligonucleotide directed against PCSK9 (proprotein convertase subtilisin/kexin type 9) that is under investigation as an agent to reduce low-density lipoprotein cholesterol levels. Five days after the last dose, the patient's serum creatinine level increased from 0.81 mg/dL at baseline (corresponding to an estimated glomerular filtration rate [eGFR] of 78 mL/min/1.73 m(2)) to 2.67 mg/dL (eGFR, 20 mL/min/1.73 m(2)), and this increase coincided with the presence of white blood cells, granular casts, and minimal hematuria on urine microscopy. The patient's serum creatinine level peaked at 3.81 mg/dL (eGFR, 13 mL/min/1.73 m(2)) 1 week after the last oligonucleotide dose. Kidney biopsy showed multifocal tubular necrosis and signs of oligonucleotide accumulation. Upon conservative treatment, the patient's serum creatinine level gradually decreased and reached her baseline level 44 days after the last oligonucleotide was administered. The patient recovered fully and kidney function was normal at every follow-up visit.

Platelet Activation by Antisense Oligonucleotides (ASOs) in the Göttingen Minipig, including an Evaluation of Glycoprotein VI (GPVI) and Platelet Factor 4 (PF4) Ontogeny.

Antisense oligonucleotide (ASO) is a therapeutic modality that enables selective modulation of undruggable protein targets. However, dose- and sequence-dependent platelet count reductions have been reported in nonclinical studies and clinical trials. The adult Göttingen minipig is an acknowledged nonclinical model for ASO safety testing, and the juvenile Göttingen minipig has been recently proposed for the safety testing of pediatric medicines. This study assessed the effects of various ASO sequences and modifications on Göttingen minipig platelets using in vitro platelet activation and aggregometry assays. The underlying mechanism was investigated further to characterize this animal model for ASO safety testing. In addition, the protein abundance of glycoprotein VI (GPVI) and platelet factor 4 (PF4) was investigated in the adult and juvenile minipigs. Our data on direct platelet activation and aggregation by ASOs in adult minipigs are remarkably comparable to human data. Additionally, PS ASOs bind to platelet collagen receptor GPVI and directly activate minipig platelets in vitro, mirroring the findings in human blood samples. This further corroborates the use of the Göttingen minipig for ASO safety testing. Moreover, the differential abundance of GPVI and PF4 in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatric patients.

Outcomes of the European Federation of Pharmaceutical Industries and Associations Oligonucleotide Working Group Survey on Nonclinical Practices and Regulatory Expectations for Therapeutic Oligonucleotide Safety Assessment.

The Oligonucleotide Working Group of the European Federation of Pharmaceutical Industries and Associations (EFPIA) conducted a survey of companies to understand the trends in nonclinical practices and regulatory expectations for oligonucleotide drug safety assessment. Twenty-two companies of different types, with varying oligonucleotide experience levels in the field, participated. The survey identified key regulatory challenges and areas of perceived health authority (HA) concern regarding nonclinical safety strategies for oligonucleotides, such as the choice of toxicology species, approaches to dose setting in toxicity studies, dose scaling from animals to humans, the implementation (and regulatory acceptability) of lean packages, and methods for dealing with impurities and human-specific off-targets. The perceived oligonucleotide experience of HAs and the relevance of guidance to oligonucleotide development were also assessed. The results showed a general lack of consensus on nonclinical safety assessment approaches being used for this growing class of medicines and highlight the need for continuing collaboration between sponsors and HAs to better define best practices.

Safety Testing of an Antisense Oligonucleotide Intended for Pediatric Indications in the Juvenile Göttingen Minipig, including an Evaluation of the Ontogeny of Key Nucleases.

The adult Göttingen Minipig is an acknowledged model for safety assessment of antisense oligonucleotide (ASO) drugs developed for adult indications. To assess whether the juvenile Göttingen Minipig is also a suitable nonclinical model for pediatric safety assessment of ASOs, we performed an 8-week repeat-dose toxicity study in different age groups of minipigs ranging from 1 to 50 days of age. The animals received a weekly dose of a phosphorothioated locked-nucleic-acid-based ASO that was assessed previously for toxicity in adult minipigs. The endpoints included toxicokinetic parameters, in-life monitoring, clinical pathology, and histopathology. Additionally, the ontogeny of key nucleases involved in ASO metabolism and pharmacologic activity was investigated using quantitative polymerase chain reaction and nuclease activity assays. Similar clinical chemistry and toxicity findings were observed; however, differences in plasma and tissue exposures as well as pharmacologic activity were seen in the juvenile minipigs when compared with the adult data. The ontogeny study revealed a differential nuclease expression and activity, which could affect the metabolic pathway and pharmacologic effect of ASOs in different tissues and age groups. These data indicate that the juvenile Göttingen Minipig is a promising nonclinical model for safety assessment of ASOs intended to treat disease in the human pediatric population.

Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia.

Single-stranded oligonucleotides (ON) comprise a promising therapeutic platform that enables selective modulation of currently undruggable targets. The development of novel ON drug candidates has demonstrated excellent efficacy, but in certain cases also some safety liabilities were reported. Among them are events of thrombocytopenia, which have recently been evident in late stage trials with ON drugs. The underlying mechanisms are poorly understood and the risk for ON candidates causing such events cannot be sufficiently assessed pre-clinically. We investigated potential thrombocytopenia risk factors of ONs and implemented a set of in vitro assays to assess these risks. Our findings support previous observations that phosphorothioate (PS)-ONs can bind to platelet proteins such as platelet collagen receptor glycoprotein VI (GPVI) and activate human platelets in vitro to various extents. We also show that these PS-ONs can bind to platelet factor 4 (PF4). Binding to platelet proteins and subsequent activation correlates with ON length and connected to this, the number of PS in the backbone of the molecule. Moreover, we demonstrate that locked nucleic acid (LNA) ribosyl modifications in the wings of the PS-ONs strongly suppress binding to GPVI and PF4, paralleled by markedly reduced platelet activation. In addition, we provide evidence that PS-ONs do not directly affect hematopoietic cell differentiation in culture but at higher concentrations show a pro-inflammatory potential, which might contribute to platelet activation. Overall, our data confirm that certain molecular attributes of ONs are associated with a higher risk for thrombocytopenia. We propose that applying the in vitro assays discussed here during the lead optimization phase may aid in deprioritizing ONs with a potential to induce thrombocytopenia.

From the Cover: The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides.

Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.

Inhibition of EGF Uptake by Nephrotoxic Antisense Drugs In Vitro and Implications for Preclinical Safety Profiling.

Antisense oligonucleotide (AON) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies. Advances in improving AON affinity and stability by incorporation of high affinity nucleotides, such as locked nucleic acids (LNA), have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule. In an attempt to predict and understand the mechanisms of LNA-AON-induced renal tubular toxicity, we established human cell models that recapitulate in vivo behavior of pre-clinically and clinically unfavorable LNA-AON drug candidates. We identified elevation of extracellular epidermal growth factor (EGF) as a robust and sensitive in vitro biomarker of LNA-AON-induced cytotoxicity in human kidney tubule epithelial cells. We report the time-dependent negative regulation of EGF uptake and EGF receptor (EGFR) signaling by toxic but not innocuous LNA-AONs and revealed the importance of EGFR signaling in LNA-AON-mediated decrease in cellular activity. The robust EGF-based in vitro safety profiling of LNA-AON drug candidates presented here, together with a better understanding of the underlying molecular mechanisms, constitutes a significant step toward developing safer antisense therapeutics.

Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.

Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development.

A low-cost and simple imaging technique of the anterior and posterior segments: eye fundus, ciliary bodies, iridocorneal angle.

PURPOSE: The authors recently used topical endoscopy to image the mouse eye fundus. Here, they widened the field of application for this ophthalmologic tool, imaging both the posterior and the anterior eye segments in larger animals commonly encountered in research laboratories and veterinary clinics. METHODS: Pupils were dilated, and local anesthetic and gel were applied to the animal cornea. The endoscopic probe was placed in contact with the cornea of conscious rats, sedated cats and dogs, anesthetized sheep, and nonhuman primates. RESULTS: High-resolution digital images of the eye fundus were obtained in all investigated animals using the endoscopic probe along the eye axis. Arteriovenous filling time was monitored with fluorescein angiography in pigmented rats. The retinal periphery and ciliary bodies could be visualized with the probe placed at an oblique angle. The probe was inclined further to observe the iridocorneal angle such that the pectinate ligaments could be seen at high resolution in cats. The authors used the probe on eyes with retinal detachment, luxation of a cataractous lens, and pigment infiltration in the iridocorneal angle, demonstrating its potential use in eye diseases. CONCLUSIONS: This topical endoscopic technique provides a unique tool for single eye examinations. The authors obtained a circular view of the anterior (iridocorneal angle) and the posterior (fundus) eye segments from all animal species studied. This technique is inexpensive and easy to use. It can be easily moved to the eye of the patient who cannot move to stand in front of classic apparatus, offering new opportunities in ophthalmology.