Short Communication: Renal Tubular Vacuolation in Animals Treated with Polyethylene-Glycol-Conjugated Proteins

Abstract

During toxicologic evaluation of a dimeric PEG-linked protein, tumor necrosis factor binding protein (TNF-bp), vacuolation of renal cortical tubular epithelium was seen in male and female Sprague-Dawley rats (200-300 g) given i.v. doses of 40, 20, or 10 mg/kg every other day for 3 months. Tubular lesions in rats treated with 20 or 40 mg/kg for 3 months were only partially reversible after a 2-month recovery period. Despite the presence of marked vacuolation, there were no changes in BUN, creatinine, urinalysis parameters, urinary NAG, urinary B2-microglobulin, or fractional sodium excretion. Single i.v. doses > or = 20 mg/kg TNF-bp caused similar but milder changes. However, equivalent doses of PEG alone or the non-PEG-linked TNF-bp did not cause light microscopic evidence of vacuolation. Treatment of rats with another PEG-linked protein of similar molecular weight resulted in similar changes. Immunostaining for TNF-bp revealed positivity in the apical cytoplasm of renal tubular epithelium within 1 h of i.v. dosing. Immunostaining of kidneys from chronically dosed rats indicated that protein was present in some vacuoles as long as dosing continued; however, kidneys from animals on a reversibility study had vacuoles but no immunostaining for TNF-bp. These results, along with a study that showed more severe lesions with PEG-linked proteins of lower molecular weight and minimal if any lesions with PEG-linked proteins > 70 kDa, suggest that TNF-bp is filtered through the glomerulus and that the protein with attached PEG is reabsorbed by the proximal tubules. Vacuolation may be a result of fluid distension of lysosomes due to the hygroscopic nature of PEG. These studies demonstrated that PEG-linked proteins have the capacity to induce renal tubular vacuolation at high doses. However, the change was not associated with alteration of clinical pathology or functional markers.

PEGylated Biopharmaceuticals

“…PEG has been localized within the proximal convoluted tubules of the kidney (Bendele et al 1998;Webster, Didier et al 2007;Rudmann et al 2013), which is consistent with renal excretion followed by reuptake via mechanisms (such as pinocytosis) that are common to other solutes that are freely filtered at the level of the glomerulus.…”

“…The predominant mode of cellular uptake (nonspecific vs. drug target mediated) and tissue distribution of PEGylated biopharmaceuticals and/or their free PEG catabolites likely depends on many factors, including dose, dose frequency and duration of treatment, the product's pharmacological activity, distribution of relevant receptors, potential immunogenicity, overall molecular weight, PEG molecular weight, and types of clearance/ removal mechanisms for drug or PEG Ikada 1994, 1995;Webster, Didier et al 2007;Webster, Elliott et al 2009;Baumann et al 2014;Bendele et al 1998). The frequent presence of PEG and PEG-associated vacuoles in Kupffer cells and macrophages as reported in the BioSafe survey (Tables 1, 2 and Table S2 in the Online Appendix) suggests that mechanisms of cellular uptake unique to phagocytic cells are major determinants of tissue reactions to high molecular weight PEG.…”

“…IHC studies using anti-PEG antibodies have also demonstrated that PEG-related cytoplasmic immunoreactivity occurs in cells that were not vacuolated in hematoxylin and eosin histological stain (H&E) sections (survey results for company 6, compound 2; Bendele et al 1998;Rudmann et al 2013) and, although the presence of vacuoles in toxicology studies of PEG or PEGylated therapeutics generally correlates with PEG dose, the vacuoles are not always immunoreactive for PEG (Bendele et al 1998). This may reflect different sensitivity and specificity of available reagents, and/or duration of fixation prior to conduct of IHC, but also suggests that morphologic observations of vacuolation may not reflect PEG tissue burden.…”

PK/PD modelling of comb-shaped PEGylated salmon calcitonin conjugates of differing molecular weights

“…In theory, the PEG teeth should break away from the methacrylate backbone due to esterase action, thereby permitting renal filtration and this is the basis of ongoing study. It is notable that while PEG itself has Generally Regarded As Safe (GRAS) status as a "non-active" excipient, there are historical reports that it can induce epithelial vacuolisation in rat renal cortical tubules following chronic repeated parenteral administration of high doses of large MW PEGs [31]. An initial toxicology study carried out in rats using a commercially available form of the comb-shaped polymer (PolyPEG®, Warwick Effect Polymers, UK), did not find any evidence of vacuolisation in renal epithelia following repeated dosing of up to 200mg to rats [32].…”

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

“…Various forms of long-acting GH have since been generated: those currently in clinical development include LB03002 (microparticle suspension), NNC126-0083 (PEGylation), NNC0195-0092 (reversible albumin-binding GH derivative), MOD-4023 (CTP modification), ACP-001 (prodrug strategy), Albutropin (albumin fusion), and VRS-317 (XTEN fusion), and have been extensively reviewed elsewhere (5). However, challenges associated with heterogeneity, stability, immunogenicity, toxicity, and/or compromised potency could potentially limit their clinical utility (3,(7)(8)(9)(10).…”