Cinacalcet

Cinacalcet an FDA approved drug which kills bacteria by destroying bacterial membrane as well as respiratory metabolism and is used to treat secondary hyperparathyroidism and parathyroid cancer. It can also kill bacteria by producing reactive oxygen species (ROS). It was revealed by Limited Proteolysis -Small molecule mapping (LiP-SMap) that cinacalcet inhibits biofilm formation by targeting IcaR.  Cinacalcet can partially remove the biofilm of drug-resistant S. aureus in an animal model it was revealed by in vitro and in vivo experiments . These findings suggest that cinacalcet has antibacterial and antibiofilm capabilities.

Figure 1: Depicts the chemical structure of Cinacalcet.

Evaluation of cinacalcet as an antibacterial agent: Researchers attempted to obtain good antibacterial agents from a library of 264 FDA-approved drugs in order to combat the antibiotic-resistant bacteria and biofilm infections. The results of these antimicrobial assays revealed that about 19 compounds reduced the survival rates of ciprofloxacin-resistant S. aureus below 10% at a concentration of 50 μM . Further testing of seven high antibacterial-potency drugs against the MRSA ATCC 43300 biofilm model showed  that cinacalcet displayed strong biofilm removal capacity whereas the other six drugs and vancomycin failed to clear biofilms. For the confirmation of antibacterial spectrum of cinacalcet, antimicrobial tests were performed by the researchers for other Gram-positive and Gram-negative bacteria. Cinacalcet exhibited an MIC range of 4–14 mg/L for the tested S. aureus, MDR Enterococcus faecalis, MDR Staphylococcus haemolyticus, sensitive Bacillus subtilis, sensitive Streptococcus pneumoniae and sensitive Streptococcus suis. While  the MICs of cinacalcet for Gram-negative bacteria were all greater than 100 mg/L such as  Escherichia coli BW25113, Salmonella enterica subsp.Enterica ATCC 14028 and Pseudomonas aeruginosa ATCC 9027 . Moreover, the bactericidal curve indicated that cinacalcet effectively killed S. aureus ,Newman, MRSA and clinical MDR 166138 at concentrations with in 2× MIC, reducing them below the detection limit . Researchers also investigated the synergism of cinacalcet with other antibiotics and it showed synergistic effects with β-lactam antibiotic penicillin G sodium and no any significant interaction with other drugs.

Figure 2: Shows the discovery and in vitro evaluation of antibacterial and antibiofilm agent cinacalcet. (a) Inhibition of ciprofloxacin-resistant (CIP-R) strain . (b) Antibiofilm efficacy of seven drugs with notable antibacterial efficacy  ( c ) 2D structure of cinacalcet (CAN) . (d–f) MIC of CAN against Gram-positive bacteria, Gram-negative bacteria. (j and k) Combination effect of CAN and antibiotics (ampicillin, tigecycline, methicillin, gentamicin, penicillin G sodium).

Treatment effect of cinacalcet in a mouse pneumonia model: A mouse pneumonia model was established, to further evaluate the antimicrobial effect of cinacalcet in vivo.  The survival rate of mice increased by 50%, following 6 days of 10 mg/kg cinacalcet treatment ,while none of the drug-free treatment group survived . The oral dose used by researchers was lower than the 30 mg/kg dose used in previous pharmacodynamic studies and in the repurposing treatment of secretory diarrhea, it’s the indication that the therapeutic dose used is safe. By the assessment of lung bacterial load  it was noted a decrease from approximately 10⁵ cfu/mouse in the drug-free group to 3.72 × 10² cfu/mouse following cinacalcet treatment  and it was indicating a 99.6% bacterial load reduction . 

Furthermore, after cinacalcet treatment number of inflammatory cells was reduced and the alveolar structure became intact and it alleviated the lung inflammation .Cinacalcet’s safety evaluation included analyzing liver

enzyme activities, specifically ALT and AST levels. The results depicted  that ALT and AST values were within the normal range and it suggested that cinacalcet did not exhibit liver toxicity at the administered dose .Cinacalcet treatment did not alter the cellular morphology and it was revealed by H&E staining of liver and kidney.

               Figure 3: Depicting the effectiveness of cinacalcet (CAN) treatment in a pneumonia model of clinical strain 166138 infection and its in vivo toxicity. (a) Survival curves of mice in control and CAN-treated groups. (b) Colony counts after treatment in the pneumonia model. ( c ) Comparison of lungs of CAN-treated and drug-free mice, and their H&E staining. (d, e) The ALT and AST enzyme activity assays for 6 days of continuous treatment with 10 mg/kg CAN. (f) liver and kidney of tissue H&E staining.

Antibiofilm activity of cinacalcet against MDR S. aureus: Biofilms, which are surrounded by EPS and are the aggregates of bacteria in a metabolically dormant state . It’s crucial to investigate cinacalcet’s ability to inhibit biofilm formation in depth. SEM detection demonstrated a reduction and shrinkage of bacterial biofilm after cinacalcet treatment . Further crystal violet staining  and confocal microscopy using SYTO 9 live cell staining indicated that 1× MIC cinacalcet effectively inhibited biofilm formation in the MRSA and 166138 strains. This proved  capability of cinacalcet for the biofilm inhibition . A question may arise will cinacalcet have a good effect in  reducing the biofilms ? Syto9/PI staining was used to differentiate between live and dead cells within the biofilm. The enhanced red fluorescence after treatment with 1× MIC cinacalcet indicated increased bacterial mortality within the biofilm and by increasing concentration to 2× MIC, it enhanced the biofilm disruption. Additional analysis using crystal violet staining and SEM provided quantitative and visual evidence of the reduced biofilm mass and density in MRSA and 166138 strains that are  treated with cinacalcet .Quantitative analysis of biofilm-associated bacterial populations demonstrated the treatment with 1× MIC of cinacalcet reduced the  MRSA and 166138 strain by 68% and 85%, respectively, in comparison with the untreated controls and at concentration of 2×MIC reduction reached to 99% for MRSA and for 166138 97%  while the treatment with other drugs not resulted in significant reduction.  Cinacalcet treatment (10 mg/kg) resulted in a significant decrease in biofilm-associated bacteria when applied to the  in vivo mouse model of biofilm infection using MRSA and clinical strain 166138 and it showed more pronounced effects then 20mg/kg vancomycin(GC37885). Safety of cinacalcet treatment was also checked and it showed no toxicity at the administrated doses.

Figure 4 : shows that Cinacalcet (CAN) inhibits MRSA and clinical strain 166138 biofilms. (a) SEM images. (b and c) crystal violet staining of MRSA and 16638 biofilms inhibition. (d) Observation of MRSA and 16638 biofilm inhibition by CAN treatment using SYTO9 staining and confocal microscopy.(e and f) Corresponding fluorescence intensity measurements.

 Figure 5 : Depicts that Cinacalcet (CAN) removes MRSA and clinical strain 166138 biofilms in vitro and in vivo. (a–c) Biofilm removal by 1× MIC and 2× MIC CAN treatment observed using PI and SYTO9 staining and confocal microscopy, with ampicillin as antibiotic control. (d–f) Crystal violet staining for biofilm removal effectiveness. AMP, ampicillin. (g) SEM images of MRSA and 16638 biofilm removal by CAN . (h–j) CAN’s ability to clear established biofilms  (k–m) Colony counting of CAN removing biofilm bacteria. (n and o) Therapeutic effect of CAN on mouse thigh biofilm wound model. Van, vancomycin. (p) H&E staining of liver and kidney under this treatment modality, showing tissue safety .

Now let’s talk about the mechanism how cinacalcet performs it’s magic task , cinacalcet mediates its antibiofilm activity by targeting the IcaR protein, leading to the down-regulation of the icaADBC operon and subsequent reduction in biofilm formation . 

Figure 6 : Depicts the mechanism by which cinacalcet performs it’s anti biofilms and anti bacterial activities.

In short  , this research study is the first to inhibit S. aureus biofilms by targeting and enhancing the function of IcaR with cinacalcet. Due to the long history of clinical safety and use of cinacalcet, it is believed that IcaR-targeted cinacalcet could potentially be used to effectively treat pneumonia infections caused by  S. aureus. Furthermore , by the  similarities and differences between cinacalcet and existing antibiotics, our understandings can be improved and we can develop more efficacious treatments.