Author ORCID Identifier

https://orcid.org/0000-0001-8447-3881

Semester

Spring

Date of Graduation

2023

Document Type

Thesis

Degree Type

MS

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Werner J Geldenhuys

Committee Member

John B. Barnett

Committee Member

Ivan Martinez

Abstract

Osteoarticular tuberculosis (OAT) is a bone degenerative disease that results in bone erosion, joint effusion, joint swelling, and sometimes, nerve compression. OAT occurs from the hematogenous dissemination of Mycobacterium tuberculosis (Mtb) bacilli spreading from the primary site of infection to the bones and joints; the primary site of infection tends to be the lungs as pulmonary tuberculosis infections are the most common. OAT can occur as the result of an active or latent infection. Latent infections result in OAT when bacilli are able to escape granulomas. The incidence of OAT is not as high as pulmonary infections, making up about 10- 15% of all extrapulmonary tuberculosis infections. Currently, the only treatment available for OAT is the standard regimen of care for treating tuberculosis; this includes 4-6 months of antibiotic treatment. Antibiotic treatment can be extended up to 12+ months for extrapulmonary TB. This does not resolve bone degeneracies or deformities that occur as a result of OAT. The novel compound, ELP-004, reduces bone erosion in mouse models of arthritis. To determine the effectiveness of ELP-004 in the treatment of OAT, a tuberculosis experimentation model was set up in vitro and in vivo. The in vivo model used RAW264.7 cells, a mouse monocyte/macrophage cell line commonly used to study osteoclastogenesis. RAW264.7 cells were exposed to extract from the H37Rv strain of Mtb did not result in TRAP positive syncytial formation; however, exposure to this TB extract still resulted in multinucleation. Cells exposed to Mtb extract also could not mobilize calcium matrices, and qPCR analysis revealed that osteoclast formation markers were absent. Although osteoclast-like cells could not be formed with this model, treatment with ELP- 004 resulted in a knockdown of syncytial cells, showing promise for its use in patients. Work on the in vitro model ceased to continue and a reevaluation of the model is necessary. Since ELP-004 was able to knockdown multinucleation in vitro, it was important to determine if the drug could be used in combination with current tuberculosis treatments. Using two first-line antibiotics, rifampin and isoniazid, the metabolism of combination therapy was assessed using cytochrome P450 enzymes (CYP). Cytochrome P450 enzymes are responsible for the metabolism of 90% of all drugs and xenobiotics. They are located primarily in the liver and are responsible for phase I metabolism. Of the CYPs tested, only Cyp1a2 and Cyp2b10 had increased mRNA expression. Using a western blot to confirm protein expression, only CYP2B10 showed increased protein expression. With this data, it can be concluded that CYP2B6, the human equivalent of CYP2B10, is the primary induced metabolic pathway for the combination therapy. These data combined show promise for using ELP-004 to treat bone erosion from a tuberculosis infection. The ability of ELP- 004 to inhibit increased osteoclastogenesis in an arthritis model can be expanded to prevent enhanced osteoclastogenesis in OAT.

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