EC Pharmacology and Toxicology: Evaluation of UNI-494 in Acute Kidney Injury Treatment Efficacy When Administered After Ischemia-Reperfusion in a Rat Model

Satya Medicherla^1*, Guru Reddy² and Shalabh Gupta³

¹Preclinical Pharmacology, Unicycive Therapeutics, Inc., Los Altos, CA, United States
²Preclinical Research and Development, Unicycive Therapeutics, Inc., Los Altos, CA, United States
³Chairman of the Board of Directors and CEO, Unicycive Therapeutics, Inc., Los Altos, CA, United States

^*Corresponding Author: Satya Medicherla, Preclinical Pharmacology, Unicycive Therapeutics, Inc., Los Altos, CA, United States.

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Abstract

Background

There are no FDA approved drugs for the treatment of acute kidney injury (AKI). Inflammation- and reactive oxygen species-driven mitochondrial permeability transition pore (mPTP) opening causes mitochondrial dysfunction/swelling and cell death. This is implicated in acute diseases including AKI originating from ischemia-reperfusion (I/R) injury or delayed graft function. Additionally, unresolved inflammation exacerbates sustained mPTP opening and induces fibrotic lesions, evident in chronic kidney diseases. Nicotinamide adenine dinucleotide can suppress the frequency and duration of mPTP opening. UNI-494, a novel nicotinamide ester derivative and a selective mitochondrial ATP-sensitive potassium channel activator, reverses mitochondrial dysfunction by closing the mPTP. We present results from a study evaluating UNI-494’s efficacy in treating AKI in an I/R AKI rat model.

Methods

48 Sprague-Dawley rats were randomly assigned to 4 groups: one sham group and three I/R models. Rats were anesthetized, the right kidney was removed, and I/R was induced in the left kidney. I/R models received either no treatment or UNI-494 IV at 1 hour or 4 hours after ischemia. Blood and urinary samples were collected for AKI markers. The clamped left kidney was collected for tubular injury scores.

Results

UNI-494 at 10 mg/kg/IV reduced AKI markers and tubular injury scores in an AKI rat model compared to vehicle treated I/R group.

Conclusions

A single IV dose of UNI-494 at 10 mg/kg at 1 or 4 hours after ischemia significantly reduced the kidney functional markers and tubular injury scores. The data suggest UNI-494 slows down/reverses the progression of AKI in its established state.

Keywords

Acute Kidney Injury; UNI-494; Ischemia-Reperfusion; Acute Kidney Injury Treatment; Efficacy

Methods

Experimental animals

Jugular vein cannulated Sprague Dawley rats were purchased from Charles River Laboratories (Hollister, CA) to have body weights of approximately 250 – 275g at the start of the study. Following arrival, animals were singly housed in cages with micro-filtered tops using SaniChip bedding 7990A (Harlan Teklad, Hayward, CA), except as noted below for metabolic caging. Animal room controls were set to maintain temperature and relative humidity at 20°C ± 1°C and 50% ± 20%, respectively, with at least 10 air changes/hour. Housing rooms were on a 12-h:12-h light/dark cycle. Animals were provided with ad libitum access to water (via water bottles) and to a standard chow (2018 Teklad Global 18% Protein Rodent Diet; Harlan Teklad). Animals were acclimated on site for at least 7 days before initiation of the study.

Study design

48 jugular vein cannulated Sprague-Dawley rats were randomly assigned to 4 groups (n = 10+2). One group was the Sham group and the other three groups were established as the I/R models. I/R models received either no treatment (vehicle), 10 mg/kg UNI-494 IV at 1 hour after ischemia, or 10 mg/kg UNI-494 IV at 4 hours after ischemia (Figure 1). The vehicle for IV formulation consisted of Sterile Water for Injection (WFI), which was obtained by the Testing Facility from a commercial vendor.

Figure 1: UNI-494 I/R study design-therapeutic dosing

Study procedures

Rats were anesthetized and the abdomen was opened at the midline. The right kidney was removed (right nephrectomy). Ischemic injury was induced in the left kidney by placing a small clip (Sugita standard aneurysm clip, holding force 145g; Mizuho Ikakogyo, Tokyo, Japan) on the renal vascular pedicle for 30 minutes. Removal of the clip was defined as the start of reperfusion (“0 hours”). Sham operations were performed in a similar manner, except that the right kidney was not removed, and the renal vessels were not clamped. At 0 hours (or the equivalent for sham-operated animals), each rat was moved to an individual metabolic cage for the collection of urine. UNI-494 was administered at 1 or 4 hours after ischemia with established renal injury confirmed by elevated serum creatinine (sCr).

Study assessments

After 24h of reperfusion, animals were anesthetized, subjected to terminal cardiocentesis, and euthanized without recovery. Collections were performed and processed as follows.

A volume of blood sufficient to provide 5 mL of serum was collected into serum separator tubes. Following centrifugation, the resulting serum from each animal was aliquoted to individual tubes (5 x 1 mL). The tubes then were flash-frozen on dry ice and stored at -20°C pending analysis. Subsequently, serum levels of Cr, neutrophil gelatinase-associated lipocalin (sNGAL), and blood urea nitrogen (sBUN) were determined in the non-acetic acid-stabilized samples using instruments/kits according to the respective manufacturer’s instructions.

The volumes and pH levels of the 24-h urine samples were recorded. The urine samples were aliquoted to individual tubes, flashfrozen on dry ice, and stored at -20°C pending analysis. Subsequently, urine levels of creatinine (uCr), albumin (uALB), and NGAL (uNGAL) were determined using kits according to the respective manufacturer’s instructions. The uCr and uALB values were used to calculate the uALB:uCre ratio (uACR).

At necropsy, the left kidney of each animal was collected and fixed overnight in 10% neutral buffered formalin at room temperature, then stored in 70% ethanol at room temperature. Fixed tissues were processed, sectioned, and stained with hematoxylin and eosin. The resulting slides were transferred to the Sponsor-designated facility for histopathological evaluation, including scoring of tubular damage (necrosis, dilation, and casts), scoring of inflammation, and inspection for other renal changes (especially mitochondrial changes). Tissues were assessed for tubular injury using Jablonski., et al. (1983) proximal convoluted tubule scoring method (0 = No change present; 1 = Minimal; 2 = Mild; 3 = Moderate; 4 = Marked) [12]. Representative images of histopathological changes were derived from images captured with 3DHistech Pannoramic digital scanner.

Statistical analysis

Data were analyzed with a one-way ANOVA followed by Dunnett’s multiple comparison tests. A threshold of p < 0.05 was used to determine a significant difference between groups. Statistical Package for the Social Sciences (SPSS) was used for all statistical analyses.

Results

Kidney function analyses

Renal injury was established by confirming elevation of sCr by 1 hour and 4 hours after ischemia in the UNI-494 groups (Figure 2). At 24 hours, I/R induced significant increases of sCr, sBUN, uACR, uNGAL, and tubular injury scores in the vehicle treated I/R group compared to the no I/R sham group (p < 0.05, ANOVA with Dunnett’s Multiple Comparisons Test) (Figure 2 and 3). A 10 mg/kg/IV UNI-494 dose consistently reduced AKI markers compared to the vehicle treated I/R group (p < 0.05 for sCR and sBUN at 1 and 4 hours, p <0.05 for uACR and uNGAL at 4 hours) (Figure 2). Mean sCr for I/R groups administered UNI-494 at 1 and 4 hours were 1.8 and 1.6 mg/dL, respectively, compared to 3.3 mg/dL in the vehicle treated I/R group (Figure 2). Mean sBUN for I/R groups administered UNI-494 at 1 and 4 hours were 73.6 and 55.4 mg/dL, respectively, compared to 109.4 mg/dL in the vehicle treated I/R group. Mean sNGAL for I/R groups administered UNI-494 at 1 and 4 hours were 2140 and 1587 μg/dL, respectively, compared to 4961 μg/dL in the vehicle treated I/R group. Mean uALB for I/R groups administered UNI-494 at 1 and 4 hours were 148 and 81 μg/mL, respectively, compared to 283 μg/mL in the vehicle treated I/R group. Mean uACR for I/R groups administered UNI-494 at 1 and 4 hours were 0.54 and 0.26 mg/dL, respectively, compared to 0.81 mg/dL in the vehicle treated I/R group (Figure 2). Mean uNGAL for I/R groups administered UNI-494 at 1 and 4 hours were 4845 and 4272 μg/mL, respectively, compared to 6428 μg/mL in the vehicle treated I/R group (Figure 2).

Figure 2: Mean (SE) serum creatinine, serum BUN, urinary ACR, and urinary NGAL between treatment groups – IV.

A 10 mg/kg/IV UNI-494 dose also reduced proximal tubular necrosis/injury and decreased tubular injury scores compared to the vehicle treated I/R group (p < 0.05, ANOVA with Dunnett’s Multiple Comparison Test) (Figure 3).

Figure 3: Histological image (Arrows point to proximal tubules) – Therapeutic

Safety

There were no mortalities during any phase of the study. All the rats in the study survived to the scheduled euthanasia time point. No clinical observations were noted during any phase of the study.