Final Progress Report

Proposal No. IBD-0233
Principal Investigator:  Michael T. Collins, D.V.M., Ph.D.
Applicant Organization:  University of Wisconsin, Madison (U.S.A.)
Project Title:  Evidence-based selection of antibiotics for treatment of Crohn's disease
Period of Award:  January 1, 2008 – February 28, 2009

Project aims

  1. Measure the MIC and MBC of 10 conventional antibiotics individually against MAP.
     
  2. Measure the MIC and MBC of 10 novel proprietary drugs in the ethambutol compound family from Sequella against MAP.
     
  3. Measure the effect of including 6MP and AZA on the MIC and MBC values of 10 antibiotics and 10 novel ethambutol derivatives against MAP.
     
  4. Measure the MIC and MBC of the most efficacious drugs in combinations of two or three both alone and in the presence of 6MP or AZA. 
Accomplishments towards meeting those aims
 
  1. Aim #1 was accomplished in hll. The MICs and MBCs for 10 strains of M a. paratuberculosis against 11 drugs were established and published. Additionally, the stability of these drugs in the culture medium used for the in vitro assay was established and published separately as.
     
  2. Aim #2 was accomplished in full. The MICs and MBCs for 10 novel compounds provided by Sequella were established and reported to the company. One compound in particular, a capuramycin derivative, was highly active against M. a. paratuberculosis and several other related compounds showed modest activity. Publication of these results is pending.
     
  3. Aim #3 was accomplished in full for combinations of 6-MP (the active derivative of AZA) with conventional antibiotics. Insufficient quantities of the Sequella compounds were available to permit their analysis in combination with 6-MP.
     
  4. Aim #4 was accomplished in full. The interaction of the most efficacious antibiotics against M. a. paratuberculosis with and without 6-MP were evaluated and a manuscript describing the study has been submitted for publication.
List of significant results
 
  1. The stability of antimicrobial drugs in the MGIT paraTB culture medium used for in vitro antimicrobial susceptibility testing (AST) was established in this study. We now know that if a drug does not inhibit M. a. paratuberculosis growth in vitro it is due to the organism's drug resistance, not drug degradation in the medium. This finding provides for the first time a crucial basis for in vitro AST data interpretation. The results were published in the International Journal of Antimicrobial Agents.
     
  2. The novel MGIT method for AST of M. a. paratuberculosis was validated by comparison to two different conventional and accepted methods for AST of mycobacteria. The drug susceptibility profile based on MICs and MBCs was found to be generally similar to that of other M. avium subspecies. This is the first comprehensive report of drug susceptibility data for a significant number of human-origin M. a. paratuberculosis strains against a wide array of antimicrobials. Other important nuances of the findings are described in the publication in the Journal of Antimicrobial Chemotherapy.
     
  3. One of the 10 novel compounds provided by the commercial partner Sequella was highly active against M a. paratuberculosis and several others showed modest activity. Among the capuramycin analogues SQ641 showed the best activity, inhibiting 6 strains at MICs ≤ µg/mL and 3 strains at 2 µg/mL. RKS2244 inhibited four strains at MICs ≤ 4 µg/mL, four at 8 µg/mL and one at 16 µg/mL. Due to insufficient amounts, RKS2243 and RKS2137 could be tested only against 3 and 4 strains and showed MICs in the range 4 - 8 and 4 - 16 µg/mL respectively. SQ641 was bactericidal to most MAP strains. MBCs ranged between 2xMIC and 8xMIC for seven strains. RKS2244 also was found bactericidal to MAP strains with MBCs ranging between 1xMIC to 8xMIC.

    Drug interaction studies were limited due to the sparse quantities of these novel compounds. As a primary screen, two MAP strains (Linda and Dominic) were tested with 2 drug combinations containing SQ641 and ethambutol, rifampicin, RKS2244, azithromycin, clarithromycin, amikacin, ciprofloxacin or 6-mercaptopurine (6-MP). SQ641 was synergistic with rifampicin against both the strains and with ethambutol against one strain. The compound did not show any interaction with RKS2244 or any other drug tested. Further, SQ64 1 synergism with rifampicin/ethambutol was confirmed in two more strains- both the combinations were synergistic to MAP UCF4 and only SQ41 -RIF combination was synergistic to MAP UCF8. Three drug combinations: SQ641 was tested in three different combinations against strain UCF8: SQ641+RIF+EMB; SQ641+RIF+RKS2244; SQ41+EMB+RKS2244. Both the RIF containing combinations were synergistic. Publication of these results in cooperation with Sequella scientists is pending.
     
  4. Synergism was observed between 6-MP and azithromycin (7 isolates), clarithromycin, rifarnpin, rifabutin (4 isolates each) and ethambutol(2 isolates). 6-MP was not antagonistic with any of the antibacterial agents tested. Among the two- and three-antibacterial combinations tested, the clarithromycin-rifampin combination was synergistic against 4 isolates, while all other combinations showed no interaction. This study suggests that 6-MP may be potentially synergistic with macrolides and rifamycin derivatives against M. a. paratuberculosis. Activity of clarithromycin against MAP seems to be enhanced by rifampin. These data provide a scientific basis for therapy of Crohn's disease using these drugs. This is the first such report of drug interactions in vitro using standardized methods for drug interaction analysis. The results have been submitted for publication in the Journal of Antimicrobial Chemotherapy.
Lay summary of the progress report
 
Mycobacterium avium subspecies paratuberculosis (MAP) has been implicated as a cause or complicating infection in some patients with Crohn's disease. Treatment of MAP infections for a subset of Crohn's disease patients therefore may be warranted. However, laboratory data (in vitro tests) on which drugs are most effective against MAP is lacking. This project set out to define the best drugs for treatment of MAP infections in humans and to evaluate combinations of antibiotics and use of antibiotics in combination with azathioprine (AZA) or its derivative, 6-MP, against 10 isolates of MAP.
 
The first phase of the work on this project required validation of a novel laboratory method that held promise for efficiently providing reliable and quantifiable results. . Specifically the stability of the drugs in the test medium needed to be confirmed. The new method was compared to two conventional approaches to determine the rate of drug degradation, if any, in the trial media.
 
After validating our laboratory methods, we established the effective concentration of eleven antibiotics against MAP, referred to at the minimal inhibitory concentrations (MICs). Using antibiotics individually, we found that MAP are more resistant to isoniazid than a closely related pathogen, M. avium, but more susceptible to amikacin and ciprofloxacin. Similar to M avium, all MAP isolates tested were found susceptible to azithromycin and clarithromycin making these two antibiotics the first choice drugs for treatment of MAP infections.
 
Drugs for therapy of Crohn's disease can potentially interact with each other. The interactions can be helpful (synergistic) or harmful (antagonistic) to the therapy. Using the most effective drugs against MAP and the commonly used anti-inflammatory drug used in Crohn's patients (6- MP; essentially the same as AZA) we evaluated drug interactions on MAP growth in standardized laboratory assays. Synergism was observed between 6-MP and azithromycin (7 MAP isolates), clarithromycin, rifarnpin, rifabutin (4 MAP isolates each) and ethambutol (2 MAP isolates). 6-MP was not antagonistic with any of the antibiotics tested. Among the two- and three-antibacterial combinations tested, the clarithromycin-rifampin combination was synergistic against 4 MAP isolates, while all other combinations showed no interaction. This laboratory data suggests that 6-MP may be potentially synergistic with azithromycin, clarithromycin and the rifamycin family of drugs against MAP. Activity of clarithromycin against MAP seems to be enhanced by rifampin. These findings give medical doctors a scientific basis for selection of drugs should they and their patients elect to try treatment of MAP infections. The findings also illustrate that not all MAP strains behave the same in response to antibiotics making it impossible to predict clinical outcomes with certainty.
 
Lastly, novel drugs developed for treatment of human tuberculosis, provided by a private company called Sequella, were tested against MAP using the same methods as for the traditional antibiotics. We found one highly active and 3 other moderately active new drugs. These drugs should synergistic activity with selected conventional antibiotics. This will provide an impetus for Sequella to expand their drug discovery research to include both the cause of tuberculosis, M. tuberculosis, and MAP with the possibility of one day having more potent antibiotics for treatment of MAP infections of humans.
Last updated 06/23/2010