SCIENTIFIC DATA

Cilastatin is an specific inhibitor of Dipeptidase 1 (DPEP1)

Thanks to the research of the scientific founding team of TELARA the MoA of the nephroprotective effect of cilastatin was elucidated and the cholesterol rafts were stablished as one of the main therapeutic targets of non-necrotic death of the proximal cell.

Specifically, it was shown that the pharmacological inhibition of one of the proteins anchored in the raft, Dipeptidase 1 or Dehydropeptidase-I (DPEP1 or DHP-I), by cilastatin blocks the recycling, the endocytosis process and the internalization of the cholesterol rafts, resulting in a reduction or elimination of renal apoptosis induced by toxic agents commonly used in clinical practice (such as chemotherapy agents or antibiotics). 

Thanks to the reversal of the changes in the activation states of the “downstream” regulatory proteins of the apoptosis without the elimination or reduction of the pharmacological power of each drug with respect to its therapeutic targets. This finding was also proven for multiple toxic drugs.

Cilastatin blocks the apoptosis process
Cilastatin specifically protects proximal tubular epithelial cells (PTECs) from death by hindering the internalization of the cholesterol rafts and blocking apoptosis, while it did not offer any type of protection in tumour cell lines of the cervix, breast, colon, bladder, etc. treated with cisplatin (see Fig. 1 below) 1, CD4 lymphocytes treated with cyclosporine or tacrolimus 2, 3 , or in bacteria sensitive to gentamicin or vancomycin 4, 5 ,  giving cilastatin an organ-specific protective role. None of these cells or bacteria had DPEP1 activity detectable in our studies1. 1

Figure 1: Effect of cilastatin on gentamicin-induced nephrotoxicity. Administration of gentamicin and cilastatin (200 μg/mL) to PTECS. Left panel: contrasting phase photographs showing cell morphology. Right panel: Confocal microscopy images of the immunolocalization of active caspase-3. Note the reduced cell death and activation of caspase-3 in the presence of cilastatin.

1 Lazaro A, et al. Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats. Kidney International, 2012; 82(6):652-63

2 Pérez M et al. Inhibition of brush border dipeptidase with cilastatin reduces toxic accumulation of cyclosporin A in kidney proximal tubule epithelial cells. Nephrol Dial Transplant 2004; 19(10): 2445-55.

3 17 Gruss E, et al. Nephroprotective effect of cilastatin in allogenic bone marrow transplantation. Results rom a retrospective analysis. Bone Marrow Transplant 1996; 18: 761–765

4 Jado JC, Humanes B, González-Nicolás MA, Camaño S, Lara JM, Lopez B, Cercenado E, García-Bordas J, Tejedor A, Lazaro A. Nephroprotective Effect of Cilastatin against Gentamicin-Induced Renal Injury In Vitro and In Vivo without Altering Its Bactericidal Efficiency. Antioxidants (Basel). 2020; 9(9):821.

5 Blanca Humanes, Juan Carlos Jado, Sonia Camaño, et al.. Protective effects of cilastatin against vancomycin-induced nephrotoxicity. BioMed Research International, vol. 2015, Article ID 704382, 12 pages, 2015.
Cilastatin reduces the apical entry of drugs into cells by approximately 30% by interfering with the process of pinocytosis or membrane cycling mediated by cholesterol rafts, thus interrupting the apical vesicular transit of PTECs and the endocytosis of the drug transporter proteins present in it such as megalin.  capable of transporting numerous nephrotoxic substances into the cell 5, 6, 7, 8. Cilastatin reduces or cancels several key steps of the auto- and paracrine signaling of the extrinsic apoptosis pathway triggered by many of the toxics. Part of this signaling is triggered by the interaction of the Fas ligand (FasL) with its Fas membrane receptor in the cholesterol rafts where Fas is located.
Cilastatin avoids the internalization via cholesterol rafts of the Fas/FasL complex preventing the extrinsic pathway of apoptosis
Therefore, in PTECs, Fas is externalized in these rafts where it trimerizes by its binding to FasL and is internalized, initiating the activation of the extrinsic pathway of apoptosis, caspases, mitochondrial depolarization, cytochrome C translocation and endonuclease activation that will end in cell death 9 .  Cilastatin avoids the internalization via raft of the Fas/FasL complex into the cell preventing the extrinsic pathway of apoptosis, which in turn causes the reduction of caspase  8, 9 and 3 levels, with a reduction in mitochondrial depolarization, release of cytochrome c into the cytosol and the activity of endonucleases that hydrolyze DNA.  In the presence of cilastatin, all the paracrine amplification of cell death that arises from the raft is blocked, as well as the activation of oxidative and inflammatory molecules that amplify the response and subsequently make AKI chronic.  

Figure 2:   Summary of the postulated protective mechanism of cilastatin against nephrotoxic-induced AKI. Megalin, an endocytic receptor located in the lipid cholesterol rafts on the apical side of the brush border of renal proximal tubular cells, is the main route of entry and accumulation of substances.  In the panel on the left, toxics enter and promote direct tubular damage that affects the mitochondria and the increase in Fas expression that leads (after binding with FasL) to apoptosis and cell death and, finally, to oxidative stress and inflammation, which exacerbate and amplify kidney injury. In the right panel, cilastatin binds to the DPEP1 (DHP-I) membrane of the brush rim in the cholesterol lipid rafts causes a significant reduction in toxicant uptake and nullifies the initial passage of the extrinsic pathway induced by them reducing the activations of caspases 8, 3 and 9,  and oxidative and pro-inflammatory signalling pathways, thereby protecting tubular cells.

6 Lazaro A, Camaño S, Humanes B, Tejedor A. Novel strategies in drug-induced acute kidney injury. In: Pharmacology; Editor. Gallelli L; Chapter 18. Rijeka, Croatia, InTech; 2012. pp. 381–396.

7  Lazaro A, Camano S, Moreno-Gordaliza E, Torres AM, de Lucas C, Humanes B, Lazaro JÁ, Gómez-Gomez MM, Bosca L, Tejedor A. “Cilastatin attenuates cisplatin-induced proximal tubular cell damage”. J Pharmacol Exp Ther. 2010, 334 (2): 419-429.

8 Pérez M et al. Inhibition of brush border dipeptidase with cilastatin reduces toxic accumulation of cyclosporin A in kidney proximal tubule epithelial cells. Nephrol Dial Transplant 2004; 19(10): 2445-55.

9  Lazaro A, Camano S, Moreno-Gordaliza E, Torres AM, de Lucas C, Humanes B, Lazaro JÁ, Gómez-Gomez MM, Bosca L, Tejedor A. “Cilastatin attenuates cisplatin-induced proximal tubular cell damage”. J Pharmacol Exp Ther. 2010, 334 (2): 419-429.

Cilastatin prevents the extrinsic pathway of apoptosis and the amplification of damage caused by inflammatory and oxidative reactions that chronicize and perpetuate kidney damage.

Cilastatin is also effective in a non-toxic AKI such as that caused by sepsis.
In the sepsis process AKI is part of a much larger picture (multi-organ failure) with true distant endocrine effects that amplify the initial injury, affecting more organs and systems with lethality rates greater than 90%. Working on an animal model of cecal ligation and puncture (CLP) that mimics the sepsis observed in the clinic, the same results of renal protection and an additional reduction in the mortality rate were found. Moreover, the protection found in the kidney could be transmitted to organs damaged during the septic process since lungs and retina of septic animals treated with cilastatin had less kidney damage, less lung damage 10, 11 and less damage to the retina. Specifically at the retinal level we could observe a lower glial activation, specifically in the cells of microglia and macroglia (astrocytes and Müller cells).
10 Tejedor A, et al..Cilastatin protection against cyclosporin A-induced nephrotoxicity: clinical evidence. Curr Med Res Opin. 2007; 23(3):505-13.

11 González-Nicolás MÁ, et al. Cilastatin: a potential treatment strategy against COVID-19 that may decrease viral replication and protect from the cytokine storm. Clin Kidney J. 2020; 13(5):903-905.