296 - Drug Repurposing for Neonatal Neuroprotection: Unanticipated Pitfalls of Generic Drug Formulations

Publication Number: 4291.296

John D. Barks, University of Michigan Medical School, Ann Arbor, MI, United States; Yiqing Liu, Pediatrics, Ann Arbor, MI, United States; Julie Sturza, University of Michigan Medical School, Ann Arbor, MI, United States; Faye Silverstein, Univ Michigan, Ann Arbor, MI, United States; Manjunath Pai, University of Michigan Pharmacokinetics Core Laboratory, Ann Arbor, MI, United States; Hemmen Sabir, German Center for Neurodegenerative Diseases, Bonn, Nordrhein-Westfalen, Germany

Background: Azithromycin (AZ) neuroprotection was reported in adult mouse focal stroke and spinal cord injury in 2015-16. AZ neuroprotection in neonatal [postnatal day 7 (P7) and P10] rat unilateral cerebral hypoxia-ischemia (HI) was reported by Lab#1 in 2019. However, in a 2023 report (conducted in Lab#2), comparing neuroprotective efficacy among >20 repurposed drugs in the P7 rat HI model, AZ neuroprotection was not confirmed. Generic AZ for injection from hospital pharmacies was used in both neonatal studies, but from different manufacturers in the 2 labs.

Objective: We hypothesize that neuroprotective efficacy in neonatal rat HI varies among generic azithromycin formulations.

Design/Methods: P7 rats underwent unilateral carotid artery ligation (Lab#1: right; Lab#2: left) followed by 90 min in 8% O2; this elicits moderate ipsilateral damage and contralateral sensorimotor deficits. AZ 45 mg/kg (or saline) was injected intraperitoneal 2h after HI, with 22.5 mg/kg (or saline) at 24 and 48h post-HI. Littermates were divided into 3 groups: generic AZ from 2 different sources, vs saline-only. In total, Lab#1 tested AZ from 4 sources (designated AZ1, AZ2, AZ3, AZ4) and Lab#2 tested AZ from 2 sources: AZ1, AZ2. Severity of ipsilateral brain injury was quantified on P21 using the calculated ipsilateral Percent Intact hemisphere tissue (range 0-100, death scored as 0), based on either hemisphere weights (Lab#1) or hemisphere areas (Lab#2). %Intact values were pooled from the 2 labs. In Lab#1 rats also underwent P21 forepaw placement and grip-strength testing, and a 30-point Composite Score that accounted for pathology, function and death was calculated. In both labs surgeons and animal outcome evaluators were blinded to treatment. Group differences were analyzed by Mixed Models.

Results: In both labs ipsilateral %Intact was higher with AZ1 vs. AZ2 (p=0.0004, Mixed Model with Tukey-Kramer correction); only AZ1 statistically differed from controls (p=0.0037), indicating neuroprotection (see Fig. 1). In Lab#1 Composite Score was evaluated for all 4 AZ formulations. AZ1 and AZ4 had the highest mean Composite Scores vs saline controls (see Fig. 2); AZ2 and AZ3 scores were intermediate, but higher than controls. Blood and brain AZ concentrations at 2 and 24h after the same AZ dose did not differ between AZ1 and AZ2 (Fig. 3).

Conclusion(s): Although all 4 AZ formulations, from different countries, fulfilled regulatory criteria for antibiotic use, their off-target neuroprotective efficacy varied significantly. Potential reasons for this unexpected pitfall of drug repurposing remain to be explored.

Figure 1: Variable Neuroprotection Among Different Azithromycin (AZ) Formulations - Neuropathology:
P7 rats underwent 90 min HI followed by injections of AZ 45 mg/kg 2h after HI and 22.5 mg/kg at 24 and 48h after HI (or saline placebo injections (NS)). Azithromycin formulations AZ1 and AZ2 were tested in both labs; formulations AZ3 and AZ4 were only tested in Lab#1. The neuropathology endpoint at P21 was %Intact ipsilateral hemisphere, calculated from bilateral hemisphere weights (Lab#1) or bilateral hemisphere areas (Lab#2), calculated as 100*Ipsilateral/Contralateral. A higher %Intact signifies less damage; deaths were scored 0. Rats treated with AZ1 had significantly less damage than controls or rats treated with AZ2 (Mixed Models, accounting for clustering within litters).

Figure 2: Variable Neuroprotection Among Different Azithromycin (AZ) Formulations - Composite Outcome:
In experiments conducted in Lab#1, P7 rats underwent 90 min HI followed by injections of AZ 45 mg/kg 2h after HI and 22.5 mg/kg at 24 and 48h after HI (or saline (NS)). All 4 AZ formulations were tested in multiple litters, but only two AZ formulations (plus NS controls) were used in any given litter. At P21, bilateral vibrissae-stimulated forepaw placing in 10 trials/rat (normal rats score 10/10) and bilateral forepaw grip traction strength contralateral/ipsilateral ratio in 3 trials/rat (normal ratio ~1) were measured before neuropathology quantitation. Composite Score was calculated by adding: [left forepaw vibrissae score] + [(L/R forepaw grip ratio) x 10] + [(%Intact right hemisphere)/10]. Normal rats score 30; deaths prior to P21 are scored 0. Group scores were compared by Mixed Models, accounting for clustering within litters. Rats treated with all AZ formulations scored higher than controls, but scores for AZ1 and AZ4 were significantly higher than for AZ2 or AZ3 (*p < 0.05, ***p < 0.001, ****p < 0.0001).

Figure 3: Blood and Brain Azithromycin Concentrations with Different Azithromycin Formulations:
In experiments conducted in Lab#1, P7 rats underwent 90 min HI followed by injections of AZ 45 mg/kg 1h after HI; animals received either AZ1 of AZ2. Blood and bilateral hemisphere samples were assayed by LC-MS/MS for AZ concentration at 2 or 24h post-dose. There was no difference in blood or tissue AZ concentration between the two formulations.