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.