4 DISCUSSION
In this study, the combination of vancomycin+flucloxacillin caused more kidney damage compared to vancomycin alone and to vancomycin+ imipenem-cilastatin as determined by multiple kidney injury biomarkers and accumulation of vancomycin in the kidney. Imipenem-cilastatin was protective for vancomycin induced kidney injury as shown by lower KIM-1. Thus, this translational rat study reproduces the increased clinical toxicity seen in the CAMERA 2 trial (Legg et al., 2022) with combination therapy (vancomycin+flucloxacillin) which resulted in early termination of the trial. Secondly, these data accord with in pre-clinical models (Becerir, Tokgün, & Yuksel, 2021; He et al., 2021; Hori et al., 2017; Humanes et al., 2015; Im et al., 2017; Kusama et al., 1998; Nakamura et al., 1999; Toyoguchi, Takahashi, Hosoya, Nakagawa, & Watanabe, 1997) that demonstrate protection against kidney injury by cilastatin. It is also notable that previous work in our translational rat models have consistently demonstrated that piperacillin-tazobactam does not worsen vancomycin kidney injury and may in fact be protective (Avedissian, Pais, Liu, Rhodes, & Scheetz, 2019; Chang et al., 2022; Pais, Liu, Avedissian, et al., 2020; Pais, Liu, Zepcan, et al., 2020). Thus, while the exact mechanism is not yet clear, differences in toxicity exist based on the specific type of penicillin used.
The protective findings with cilastatin are notable. Cilastatin is a dehydropeptidase-1 inhibitor (DHP-1), an enzyme produced by the proximal tubules (Kahan, Kropp, Sundelof, & Birnbaum, 1983). It is commercially available in combination with imipenem, a broad spectrum carbapenem beta-lactam antibiotic, with or without relebactam, a beta-lactamase inhibitor, to prevent imipenem-induced nephrotoxicity.
In the present study, cilastatin was purchased in combination with imipenem, to translationally represent the combination that is already FDA approved and available in the hospital settings for treatment of variety of infections. Additionally, it is worthwhile to note that we specifically tested a low allometrically scaled dose of imipenem-cilastatin. A dose of 90 mg kg-1/day in the rat equates to a human dose of 14.5 mg kg-1/day of imipenem-cilastatin or 1 gram daily for a 70 kg patient (”Guidance for Industry. Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. U.S. Department of Health and Human Services. Food and Drug Administration. Center for Drug Evaluation and Research (CDER). ,” 2005), a dose that is 25% of the maximum FDA approved dose (”Primaxin Package insert. https://www.accessdata.fda.gov/drugsatfda_docs/label/2016/050587s074lbl.pdf. Merck 12/2016.,”). Full dose ranging experiments with urinary biomarkers are needed to provide fully quantitative results for percentage of protection available. The results here build upon those in the literature. He et al. (He et al., 2021) found lower SCr and BUN values (as AKI biomarkers) in vancomycin+imipenem-cilastatin/relebactam treated male C57BL/6J mice versus vancomycin treated animals and reported that imipenem-cilastatin/relebactam maintained a nephroprotective effect. This is consistent with absolute amount of urinary biomarker KIM-1 in our studies with imipenem-cilastatin versus vancomycin group. In their study, the investigators used 320 mg kg-1 imipenem-cilastatin/relebactam in mice (which is equivalent to 160 mg kg-1 in the rat), at approximately half the cilastatin dose (45 mg kg-1 of cilastatin component) we were able to reproduce the nephroprotective effect. Humanes et al. (Humanes et al., 2015) assessed effect of cilastatin on vancomycin nephrotoxicity in cultured renal proximal tubular epithelial cells. They reported that cilastatin protects against proximal tubule apoptosis caused by vancomycin. Postulated mechanism of nephroprotection by cilastatin involves megalin and/or p-glycoprotein. Megalin is an endocytic receptor expressed on the apical surface of the proximal tubule cells (Christensen et al., 1995) and mediates the renal uptake of some antibiotics with consequent nephrotoxicity (Kuwahara et al., 2016; Moestrup et al., 1995; Suzuki et al., 2013). Cilastatin competitively binds to megalin and blocks uptake of vancomycin (Hori et al., 2017). A number of studies in mice, rats and in primary porcine proximal tubule cells have demonstrated that cilastatin can decrease cellular uptake of vancomycin and vancomycin concentrations in the kidney (Hori et al., 2017; Humanes et al., 2015; Kusama et al., 1998; Toyoguchi et al., 1997). Hori et al (Hori et al., 2017), assessed nephrotoxicity of vancomycin and other nephrotoxins in proximal tubule epithelial cells and in megalin knock-out mice. Both studies demonstrated that vancomycin binds to megalin and results in kidney damage; while cilastatin binds megalin and reduces damage caused by vancomycin. Others (Im et al., 2017) have suggested that the protection mediated by cilastatin is a function of upregulating p-glycoprotein. Thus, cilastatin may act both through megalin blockade as well as p-glycoprotein upregulation. A study of vancomycin and cilastatin in rabbits demonstrated that vancomycin clearance is higher in rats that received cilastatin than those that received vancomycin alone (Toyoguchi et al., 1997). Both p-glycoprotein upregulation and megalin inhibition both theoretically will increase vancomycin clearance in the urine. Finally, a Wistar albino rat study that used supratherapeutic vancomycin doses between 500-1000 mg kg-1 (which allometrically translate to 80-160 mg kg-1/day in humans) demonstrated that cilastatin protects against decreasing glomerular function (measured by inulin clearance) from vancomycin (Nakamura et al., 1999). Kusama et al. demonstrated that vancomycin accumulates less in kidneys of rats treated with cilastatin (Kusama et al., 1998). Our study is the first to our knowledge to assess the nephroprotective effect of imipenem-cilastatin in a translational rat model using urinary biomarker KIM-1 that has been linked to human outcomes (Scheetz et al., 2021). These results show consistent findings across ex-vivo and in-vivo analyses of multiple species. A small retrospective study in humans (total of 227 patients) found lower rates of nephrotoxicity in patients that received imipenem-cilastatin in combination with vancomycin as opposed to patients in the meropenem (which contains no cilastatin) + vancomycin group (6.2% vs 17.1% respectively) (Hakeam et al., 2019).
Incidence of attributable acute kidney injury (AKI) caused by vancomycin exceeds 10% (Wunderink et al., 2012). Thus, it is essential for clinicians to be aware of drugs (nephroprotectants and nephrotoxins) that can potentiate or alleviate risk of vancomycin AKI. Our rat model confirms that synergistic nephrotoxicity exists, such as observed in CAMERA 2 trial (Legg et al., 2022; J. Liu et al., 2020) due to addition of flucloxacillin to vancomycin; thus we recommend against use of such combination in the hospital settings. Our animal model confirms that imipenem-cilastatin should be explored as a nephroprotectant. However, while cilastatin is available in a clinically approved preparation as imipenem-cilastatin, promotion of antimicrobial resistance will preclude regular use. Thus, the development of a -vancomycin-cilastatin preparation would be desirable, and more information will be needed on the benefits and risks of that combination.
DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR
This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis and Animal Experimentation, and as recommended by funding agencies, publishers and other organizations engaged with supporting research.
AUTHOR CONTRIBUTIONS
MS and GP conceived and designed the study. GP, JL, RM, SM, KV were involved in the acquisition of data. GP, MS analyzed the data. ST, JD, JL, JC, MS interpreted the data;
GP, RR, MS drafted the manuscript. ST, JD, MS revised it critically for important
intellectual content.
All authors have given final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.