Systemic immune response to a CD40 agonist antibody in nonhuman primates.

The cell surface molecule CD40 is a member of the tumor necrosis factor receptor superfamily and is broadly expressed by immune cells including B cells, dendritic cells, and monocytes, as well as other normal cells and some malignant cells. CD40 is constitutively expressed on antigen-presenting cells, and ligation promotes functional maturation, leading to an increase in antigen presentation and cytokine production, and a subsequent increase in the activation of antigen-specific T cells. It is postulated that CD40 agonists can mediate both T cell-dependent and T cell-independent immune mechanisms of tumor regression in mice and patients. In addition, it is believed that CD40 activation also promotes apoptotic death of tumor cells and that the presence of the molecule on the surface of cancer cells is an important factor in the generation of tumor-specific T cell responses that contribute to tumor cell elimination. Notably, CD40 agonistic therapies were evaluated in patients with solid tumors and hematologic malignancies with reported success as a single agent. Preclinical studies have shown that subcutaneous administration of CD40 agonistic antibodies reduces systemic toxicity and elicits a stronger and localized pharmacodynamic response. Two independent studies in cynomolgus macaque (Macaca fascicularis) were performed to further evaluate potentially immunotoxicological effects associated with drug-induced adverse events seen in human subjects. Studies conducted in monkeys showed that when selicrelumab is administered at doses currently used in clinical trial patients, via subcutaneous injection, it is safe and effective at stimulating a systemic immune response.

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Non-proliferative and Proliferative Lesions of the Non-human Primate (M. fascicularis).

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the nonhuman primate used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.

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.

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.

GalNAc Conjugation Attenuates the Cytotoxicity of Antisense Oligonucleotide Drugs in Renal Tubular Cells.

Targeted delivery of antisense oligonucleotide (AON) drugs is a promising strategy to increase their concentration in the desired tissues and cell types while reducing access to other organs. Conjugation of AONs to N-acetylgalactosamine (GalNAc) has been shown to efficiently shift their biodistribution toward the liver via high-affinity binding to the asialoglycoprotein receptor (ASGPR) expressed at the surface of hepatocytes. Nevertheless, GalNAc conjugation does not prevent accumulation of AONs in the kidney cortex, and GalNAc-conjugated AONs might cause kidney toxicities, for example, under conditions of ASGPR saturation. Here, we investigated the nephrotoxicity potential of GalNAc-conjugated AONs by in vitro profiling of AON libraries in renal proximal tubule epithelial cells (PTECs) and in vivo testing of selected candidates. Whereas GalNAc-conjugated AONs appeared generally innocuous to PTECs, some caused mild-to-moderate nephrotoxicity in rats. Interestingly, the in vivo kidney liabilities could be recapitulated in vitro by treating PTECs with the unconjugated (or naked) parental AONs. An in vitro mechanistic study revealed that GalNAc conjugation attenuated AON-induced renal cell toxicity despite intracellular accumulation similar to that of naked AONs and independent of target knockdown. Overall, our in vitro findings reveal ASGPR-independent properties of GalNAc AONs that confer a favorable safety profile at the cellular level, which may variably translate in vivo due to catabolic transformation of circulating AONs.

Scientific and Regulatory Policy Committee Points to Consider: Histopathologic Evaluation in Safety Assessment Studies for PEGylated Pharmaceutical Products.

Colorless, intracytoplasmic vacuoles occur in multiple tissues in animals following repeated administration of polyethylene glycol (PEG)-conjugated molecules. The extent of vacuolation depends on physical characteristics and molecular backbone of the PEG and the dose, product, drug target/pharmacology, and duration of exposure. The collective experience gathered from multiple nonclinical toxicology studies of PEGylated biopharmaceuticals indicates that in general, PEG-related vacuolation is not associated with demonstrable cell and tissue damage or dysfunction and is reversible with sufficient duration of drug-free periods. Existing data are insufficient to predict whether nonclinical animal species differ in their sensitivity to develop PEG-associated vacuoles; however, recent data suggest that there may be species differences. Recent comprehensive reviews have addressed the basic challenges in developing PEGylated pharmaceutical products, including general reference to and description of PEG-associated tissue findings. These manuscripts have identified gaps in our current understanding of PEG-associated vacuolation, including the lack of a widely accepted standardized histological terminology and criteria to record and grade the severity of vacuolation as well as insufficient knowledge regarding the nature of the contents of these vacuoles. The goal of this article is to help address some of the gaps identified above by providing points to consider, including a pictorial review of PEG-associated microscopic findings, when evaluating and reporting the extent, severity, and significance (adversity or lack of adversity) of PEG-associated cytoplasmic vacuolation in safety assessment studies. [Box: see text].

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.

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.

Characterization of post-surgical alterations in the bile duct-cannulated rat.

The bile duct-cannulated (BDC) rat is a standard animal model used in ADME experiments. The aim of this study was to investigate post-surgical alterations that are relevant to ADME investigations in BDC rats compared with sham- and non-operated animals. Water and food intake was reduced in the animals' post-surgery. This led to a lower body weight in operated animals. In BDC animals, aspartate aminotransferase (AST) levels in plasma were transiently elevated and total bile acid levels were reduced. Alpha(1)-acid glycoprotein (AGP) in plasma and the concentration of bile components in bile were elevated. Histopathology showed inflammation in the area of the cannulation between the liver and the small intestine. A microarray-based gene expression and RTq-PCR analysis identified altered expression for several genes involved in drug disposition including the down-regulation of cytochrome P450 enzymes. This led to reduced cytochrome P450 content in the liver and lower metabolic activity in microsomes from BDC and sham-operated rats compared with naïve animals. The results of the study suggest that the post-surgical inflammation leads to physiological changes relevant for drug absorption and disposition. These alterations should be accounted for in the interpretation of ADME studies in BDC animals.

MARCO, a macrophage scavenger receptor highly expressed in rodents, mediates dalcetrapib-induced uptake of lipids by rat and mouse macrophages.

Dalcetrapib (RO4607381/JTT-705), an agent that targets cholesteryl ester transfer protein, is in development for prevention of cardiovascular events. In vitro studies were performed to identify receptors that mediate an off-target effect of dalcetrapib observed in preclinical models: increased lipid uptake into the lamina propria of the small intestine and into mesenteric lymph node macrophages. Uptake of oxidized low-density lipoprotein (LDL) cholesterol or dalcetrapib-treated chylomicrons was quantitated by triglyceride assay or fluorescent labeling in primary macrophages and the cell lines CHO, J774A.1 (mouse macrophages) and THP-1 (human macrophages). Quantitative reverse-transcriptase polymerase chain reaction and immunoblotting measured candidate receptor expression. Lectin-like oxidized LDL receptor (LOX-1) and scavenger receptor type AI (SR-AI) were excluded as candidate receptors based on lack of association between their expression and uptake of dalcetrapib-treated lipids. In J774A.1 cells, uptake of dalcetrapib-treated chylomicrons was increased by LPS and associated with expression of MAcrophage Receptor with COllagenous domain (MARCO). MARCO was expressed at very low levels in human macrophages and was not inducible by LPS. The MARCO receptor may account for the variable species susceptibility towards dalcetrapib-mediated chylomicron uptake by macrophages.