(392a) Graduate Student Award Session: Galactosylated Dendrimer Targets Hepatocytes and Improves Therapeutic Outcomes in a Severe Model of Acetaminophen Poisoning

Authors: 
Porterfield, J. E., Johns Hopkins University School of Medicine
Sharma, R., Johns Hopkins University School of Medicine
Sharma, A., Johns Hopkins University School of Medicine
Jimenez, A., Johns Hopkins Medical Institute
Norwood, S., Johns Hopkins University
Kannan, S., Johns Hopkins University School of Medicine
Kannan, R., Johns Hopkins University School of Medicine
Introduction: Hepatocytes comprise about 80% of the liver by mass, yet have been historically difficult to target with nanoparticles due to their role in removing circulating toxins and either breaking them down or expelling them into bile. This is a hurdle to the development of new therapies for many diseases where the hepatocytes are the loci of disease such as hepatitis, cirrhosis, and acetaminophen poisoning. While considered safe at standard doses, the over-the-counter painkiller, acetaminophen, is highly toxic to the liver in high doses due to the formation of NAPQI, which is a side product of acetaminophen metabolism formed due to N-hydroxylation of the acetaminophen by cytochrome P-450. NAPQI can conjugate to glutathione endogenous to hepatocytes; however, glutathione is quickly depleted in cases of overdose, leading to the build up of NAPQI that forms protein adducts, binds to mitochondria, and induces apoptosis. The standard-of-care for acetaminophen overdose is the intravenous administration of a 1300 mg/kg dose of N-acetyl cysteine (NAC) over 72 hours. When patients present in the clinic too late or with too high of an overdose, NAC is ineffective and the only viable treatment is a liver transplant, with acetaminophen poisoning responsible for over 20% of liver transplants in the United States [1].

Hydroxyl-terminated polyamidoamide (PAMAM) dendrimers are a highly safe and versatile nanocarrier shown to be adept at targeting inflammation in a multitude of animal models of disorders of the brain and eye [2-4]. We have shown previously that conjugating biocompatible sugar to the dendrimer surface modifies its cellular binding and transport kinetics allowing the targeting of different organs and cell populations [5]. We hypothesized that conjugating galactose to the dendrimer surface would allow it to target hepatocytes, which naturally take up galactose for digestion via asialoglycoprotein receptor 1 (ASGPR1). Due to the ability of dendrimer to perfuse freely through both healthy and diseased tissue, we will be able to deliver NAC in a targeted manner to hepatocytes, through dendrimer conjugation, to enlarge the therapeutic window and minimize the need for liver transplants.

Methods: Dendrimer-galactose (D-Gal) was synthesized and characterized, in a manner similar to dendrimer-mannose as published [5]. In vitro tests were performed in HEPG2 cells cultured in EMEM supplemented with 10% FBS at passages <10. In vivo tests were performed using a C%&BL/6 model of severe acetaminophen poisoning. In short, animals were starved for 15-16 hours before intraperitoneal injection of 700 mg/kg of acetaminophen in saline with 10% DMSO. Food was returned two hours later and animals were monitored for weight loss and signs of distress. All dendrimers were administered in sterile saline via tail vein injection with all pharmacokinetic and imaging studies being administered 55 mg/kg of Cy5-labeled dendrimer. In addition to survival, treated and untreated animals were sacrificed and the same time point for metabolite and cytokine analysis as well as histological analysis.

Results: D-Gal binds more effectively to ASGPR1 than unmodified PAMAM dendrimer (D-OH) as shown in cellular uptake studies involving competitive galactose binding where D-OH enters HEPG2 cells and also has a lower KD as is shown by in vitro binding studies. This translates to an uptake of ~60 micrograms of D-Gal per gram of tissue in the healthy liver by 4 hours after injection that persists until 48 hours after injection. Non-targeted dendrimer is present at levels 50-100 fold lower than D-Gal, and does not localize in hepatocytes. In the acetaminophen poisoning model uptake decreases to ~20 micrograms of D-Gal per gram of tissue. Confocal imaging and flow cytometry showed that the majority of dendrimer signal was specific to hepatocytes. We then conjugated NAC to some of the remaining hydroxyl groups on the dendrimer surface (Gal-D-NAC) with stability and release of the conjugate tested in vitro. After undergoing acetaminophen poisoning, mice treated with Gal-D-NAC survived longer than their untreated and free NAC treated counterparts while also showing decreased necrosis through histology and reduced expressions of markers of hepatic damage such as ALT, AST, and inflammatory cytokine presence in both the liver tissue and the circulating serum.

Conclusion: Galactosylation of hydroxyl terminated PAMAM dendrimers shifts their biodistribution from a kidney based clearance, towards hepatocyte-mediated uptake in the liver due to their interaction with asialoglycoprotein receptor 1. This new carrier for hepatic therapies can still perfuse through the liver and target hepatocytes in a model of severe acetaminophen poisoning with hepatic necrosis. By conjugating a payload of N-acetyl cysteine to the galactosylated dendrimer we are able to improve both phenotypic and molecular therapeutic outcomes, including survival. The application of this technology could significantly extend the therapeutic window for patients with acetaminophen overdoses and reduce the burden on liver transplants.

This work was funded by grants from the National Institute of Biomedical Imaging and Bioengineering (5R01EB018306) and the National Institute of Child Health and Human Development (5R01HD076901).

References:

[1] Yoon E, Babar A, Choudhary M, Kutner M, Pyrsopoulos N. Acetaminophen-Induced Hepatotoxicity: a Comprehensive Update. J Clin Transl Hepatol. 2016;4(2):131–142. doi:10.14218/JCTH.2015.00052

[2] Nance E, Kambhampati SP, Smith ES, et al. Dendrimer-mediated delivery of N-acetyl cysteine to microglia in a mouse model of Rett syndrome [published correction appears in J Neuroinflammation. 2018 Jan 12;15(1):14]. J Neuroinflammation. 2017;14(1):252. Published 2017 Dec 19. doi:10.1186/s12974-017-1004-5

[3] Kannan S, Dai H, Navath RS, et al. Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model. Sci Transl Med. 2012;4(130):130ra46. doi:10.1126/scitranslmed.3003162

[4] Kambhampati SP, Clunies-Ross AJ, Bhutto I, et al. Systemic and Intravitreal Delivery of Dendrimers to Activated Microglia/Macrophage in Ischemia/Reperfusion Mouse Retina. Invest Ophthalmol Vis Sci. 2015;56(8):4413–4424. doi:10.1167/iovs.14-16250

[5] Sharma A, Porterfield J, Smith E, Sharma R, Kannan S, Kannan RM. Effect of mannose targeting of hydroxyl PAMAM dendrimers on cellular and organ biodistribution in a neonatal brain injury model. J Control Rel. 2018; 283:175-189. doi: 10.1016/j.jconrel.2018.06.003