A mechanistic investigation of thrombotic microangiopathy associated with IV abuse of Opana ER.

Since 2012, a number of case reports have described the occurrence of thrombotic microangiopathy (TMA) following IV abuse of extended-release oxymorphone hydrochloride (Opana ER), an oral opioid for long-term treatment of chronic pain. Here, we present unique clinical features of 3 patients and investigate IV exposure to the tablet's inert ingredients as a possible causal mechanism. Guinea pigs were used as an animal model to understand the hematopathologic and nephrotoxic potential of the inert ingredient mixture (termed here as PEO+) which primarily contains high-molecular-weight polyethylene oxide (HMW PEO). Microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury were found in a group of 3 patients following recent injection of adulterated extended-release oxymorphone tablets. Varying degrees of cardiac involvement and retinal ischemia occurred, with TMA evident on kidney biopsy. A TMA-like state also developed in guinea pigs IV administered PEO+. Acute tubular and glomerular renal injury was accompanied by nonheme iron deposition and hypoxia-inducible factor-1α upregulation in the renal cortex. Similar outcomes were observed following dosing with HMW PEO alone. IV exposure to the inert ingredients in reformulated extended-release oxymorphone can elicit TMA. Although prescription opioid abuse shows geographic variation, all physicians should be highly inquisitive of IV drug abuse when presented with cases of TMA.

In vitro test methods for evaluating high molecular weight polyethylene oxide polymer induced hemolytic and thrombotic potential.

To combat opioid abuse, the U.S. Food and Drug Administration (FDA) released a comprehensive action plan to address opioid addiction, abuse, and overdose that included increasing the prevalence of abuse-deterrent formulations (ADFs) in opioid tablets. Polyethylene oxide (PEO) has been widely used as an excipient to deter abuse via nasal insufflation. However, changes in abuse patterns have led to unexpected shifts in abuse from the nasal route to intravenous injection. Case reports identify adverse effects similar to thrombotic thrombocytopenic purpura (TTP) syndrome following the intravenous (IV) abuse of opioids containing PEO excipient. Increased risk of IV opioid ADF abuse compared to clinical benefit of the drug led to the removal of one opioid product from the market in 2017. Because many generic drugs containing PEO are still in development, there is interest in assessing safety consistent with generic drug regulation and unintended uses. Currently, there are no guidelines or in vitro assessment tools to characterize the safety of PEO excipients taken via intravenous injection. To create a more robust excipient safety evaluation tool and to study the mechanistic basis of HMW PEO-induced TMA, a dynamic in vitro test system involving blood flow through a needle model has been developed.

Determining critical overlap concentration of polyethylene oxide to support excipient safety assessment of opioid products.

Intravenous administration of abuse-deterrent opioid products poses high safety risks, in part due to the presence of high molecular weight polymeric excipients. Previous in vivo studies in animal models have shown that the higher molecular weight (Mw) polymeric excipients like polyethylene oxide (PEO) were directly linked to such adverse responses as intravenous hemolysis and kidney damage. PEO polymers have been widely used in abuse-deterrent formulations (ADF) of opioid products, adding to concerns over the general safety of the opioid category due to the unknown safety risk from abuse via unintended routes. The current study focused on the determination of the critical overlap concentration (c*) at various PEO molecular weights to aid in explaining differences in observed adverse responses from previous animal studies on the intravenous administration of PEO solutions. Adverse in vivo responses may be related to the viscoelastic regime of the polymer solution, which depends not only on Mw but also on concentration. Having a localized polymer concentration in the blood above the c*, i.e., the transition from the dilute to semi-dilute entangled viscoelastic regime, may influence the flow behavior and interactions of cells in the blood. The relationship of c* to this combination of physical, chemical, and rheological effects is a possible driving force behind adverse in vivo responses.

Polyethylene Oxide Molecular Size Determines the Severity of Atypical Thrombotic Microangiopathy in a Guinea Pig Model of Acute Intravenous Exposure.

In 2017, Opana ER was voluntarily removed from the U.S. market based on concerns that its risks outweighed its therapeutic benefits. The data that supported this conclusion were based on postmarketing evaluation that demonstrated increased intravenous abuse associated outbreaks of HIV, hepatitis C, and uniquely, a thrombotic thrombocytopenic purpura (TTP)-like syndrome. In 2017, the cause was mechanistically linked to intravenous exposure of the high-molecular weight polyethylene oxide (PEO), an excipient component of the drug product. However, it was unknown how differing PEO preparations might alter this response in vivo. Knowing the likelihood of a PEO driven atypical thrombotic microangiopathy with hemolytic uremic syndrome (TMA-HUS), this study was specifically designed with the primary objective focused on understanding the impact of PEO molecular weight on TMA-HUS in a guinea pig model of acute repeat PEO (1, 4, and 7 MDa) dosing. Results from this analysis suggest that repeated dosing with PEO 4 and 7 MDa, but not 1 MDa induced a marked intravascular hemolysis with schistocytes, mild anemia, thrombocytopenia, hemoglobinuria, and kidney injury, consistent with observations of a TMA-HUS-like syndrome. Nonetheless, observations of tissue microthrombi, complement or altered von Willebrand factor involvement were not observed, which would be consistent with a definitive TMA. Further, only 7 MDa PEO dosing was associated with marked renal hypoxia. Taken together, this study defines renal injury risk with PEO formulations >1 MDa that is driven by a robust intravascular hemolysis and potentially, tissue hypoxia.

Down selection of polymerized bovine hemoglobins for use as oxygen releasing therapeutics in a guinea pig model.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being developed as resuscitative fluids for use in multiple medical applications and in lieu of blood transfusion. However, cardiovascular, central nervous system, and renal adverse events have largely impeded progress. This has prompted a need to evaluate novel down selection approaches for HBOCs prior to in-depth preclinical and clinical safety testing. In the present study, polymerized bovine Hbs (PolybHbs) were prepared with increasing ratios of glutaraldehyde to bovine Hb (10:1, 20:1, 30:1, and 40:1). The optimal PolybHb candidate selection was based on a priori determined in vivo response to include a long circulating PolybHb with no measurable renal exposure, minimal cardiovascular response, limited oxidation to metHb in vitro, or in circulation and absence of acute end organ toxicity. Guinea pigs were dosed via a 50% blood for PolybHb exchange transfusion. Data suggested that the 30:1 preparation exhibited maximum circulatory exposure (AUC(0)(-∞)) with the lowest level of oxidation (plasma metHb formation) and minimal (< 10%) blood pressure elevation. Additionally, the 30:1 preparation was absent renal iron deposition as well as abnormal glomerular/tubular histopathology or serum creatinine elevation. Clearance pathways predominantly followed those consistent with endogenous Hb clearance based pathways. Therefore, data confirmed the ability to select a single PolybHb from a small library of HBOCs based on a priori determined characteristics. Moreover, the approach to down selection described could be applied to enhance the early predictability of human safety for this class of biological therapeutics to optimize for specific indications.

Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells.

Embryonic stem (ES) cells have the ability to self-replicate and differentiate into cells from all three germ layers, holding great promise for tissue regeneration applications. However, controlling the differentiation of ES cells and obtaining homogenous cell populations still remains a challenge. We hypothesize that a supportive three-dimensional (3D) environment provides ES cell-derived cells an environment that more closely mimics chondrogenesis in vivo. In the present study, the chondrogenic differentiation capability of ES cell-derived embryoid bodies (EBs) encapsulated in poly(ethylene glycol)-based (PEG) hydrogels was examined and compared with the chondrogenic potential of EBs in conventional monolayer culture. PEG hydrogel-encapsulated EBs and EBs in monolayer were cultured in vitro for up to 17 days in chondrogenic differentiation medium in the presence of transforming growth factor (TGF)-beta1 or bone morphogenic protein-2. Gene expression and protein analyses indicated that EB-PEG hydrogel culture upregulated cartilage-relevant markers compared with a monolayer environment and induction of chondrocytic phenotype was stimulated with TGF-beta1. Histology of EBs in PEG hydrogel culture with TGF-beta1 demonstrated basophilic extracellular matrix deposition characteristic of neocartilage. These findings suggest that EB-PEG hydrogel culture, with an appropriate growth factor, may provide a suitable environment for chondrogenic differentiation of intact ES cell-derived EBs.