Evaluation of Vitamin C as a potential anti-tuberculosis drug in Guinea pig model of tuberculosis

Misbah Un Nisa

12056100018

Tuberculosis; definition and symptoms

Tuberculosis is a second largest infectious disease caused by Mycobacterium tuberculosis that usually effects lungs although other organs may also be involved.

The symptoms of active disease include mild fever, headache, chills, night sweats, fatigue, coughing up blood, chest pain etc

Transmission usually takes place through the airborne spread of droplet nuclei produced by patients with infectious TB.

TB as global health threat

• In 2012, there were an estimated 8.6 million cases of TB

• 1.3 million people died from the disease

• India accounts for the 26% of total cases

• Largest no. of incident cases of TB in 2012 were reported from India & the largest increase in MDR TB cases between 2011 & 2012 were reported from India

• Leading killer of people living with HIV/AIDSGlobal TB report 2013, WHO

Combating strategies for TBA. Vaccines:• Currently only available vaccine, BCG, is in use for non pulmonary TB

B. Therapeautic strategy:• The currently recommended treatment for new cases of drug-susceptible

TB is a regimen with two months of four first line drugs: isoniazid, rifampin, pyrazinamide & ethambutol, followed by continuation phase for four to eight months with isoniazid & rifampin.

• For patients with MDR-TB/XDR TB, second line drugs which include injectable amino glycosides capreomycin, kanamycin and amikacin; and fluoroquinone antibiotics are used

• For patients with TDR TB no drugs are available as yet

What is MDR, XDR, TDR-TB?

• A form of disease caused by bacilli resistant to at least isoniazid & rifampin is called MDR TB

• XDR TB is a form of disease in which MDR TB is compounded by additional resistance to the most powerful second line drugs : fluoroquinolones and at least one of the injectable drugs amaikacin, kanamycin and capreomycin

• TDR TB is the form of TB in which the bacilli are resistant to all the available anti-TB drugs

Is there a need for new drugs?

• The anti-TB drugs currently used in first-line treatments are around 50 years old.

• The fact that 8.6 million new TB cases and 1.3 million TB deaths were reported in 2012 clearly suggests the urgent need of new strategies for TB treatment

• There is a demand for discovery of new drug regimens which should:

1. Shorten treatment duration

2. Target MDR-TB & XDR-TB strains

3. Simplify treatment

4. Be co-administered safely with HIV medications

Origin of the project

• In a recent study by Catherine et al Vit C has shown potent in vitro activity against both drug susceptible and drug resistant strains of M. tuberculosis

• The study we plan to undertake is to evaluate Vit C in preclinical animal models as anti-TB drug

Exploring Vitamin C as a potent anti-tuberculosis drug

Exploring Vitamin C as a potent anti-tuberculosis drug

• Recent research on vitamin C has shown it to sterilize cultures of drug susceptible and drug resistant strains of M.Tuberculosis.

• Vit C is non mutagenic, non toxic & one of the essential components of body

• Vitamin C drives Fenton Reaction and results in production of hydroxyl radicals which cause DNA damage and thus cell death.

• Bactericidal activity of vitamin C is dependent on – High ferrous ion levels.– Production of reactive oxygen species.

• Recent research on vitamin C has shown it to sterilize cultures of drug susceptible and drug resistant strains of M.Tuberculosis.

• Vit C is non mutagenic, non toxic & one of the essential components of body

• Vitamin C drives Fenton Reaction and results in production of hydroxyl radicals which cause DNA damage and thus cell death.

• Bactericidal activity of vitamin C is dependent on – High ferrous ion levels.– Production of reactive oxygen species. (Catherine et al, 2013)

Reaction Reaction

Mechanism of actionMechanism of action

Hypothesis

Since Vit C has shown potent in vitro activity

against both drug susceptible and drug resistant strains of M. tuberculosis, therefore we hypothesize that Vit C bears a great potential for TB treatment keeping in view bioavailability of Vit C upon oral dosing in humans and other animals.

Specific objectives

1. To evaluate Vit C in vitro against drug susceptible, MDR and XDR strains of M.tb with or without existing anti-TB drugs

2. To determine if activity of Vit C is driven by concentration or time or both under in vitro conditions

3. To evaluate pharmacokinetics of Vit C in guinea pigs over a range of doses

4. To evaluate mono therapy of vit C against acute as well as chronic models of guinea pig TB

5. To evaluate vit C in combination with existing anti-TB drugs in guinea pig model of TB

Methodology

M.tb H37Rv

Middlebrook 7H9 medium

(2 weeks)

Vit C with or without existing drugsMIC/ MBC

determination

Repeat with MDR strains of TB

Vit C in vitro study

Concentration/time dependence?

M.Tb H37Rv

Vit C (MIC & > MIC)

0-14 Days

Determine if activity is determined by conc./ time/ both

PK studies in Guinea pigs

Inject guinea pigs with different

doses of vit C

Draw blood at different

time points

HPLC analysis for PK of vit C

•Infect groups of Guinea pigs with 105CFU/ml & 108 CFU/ml resp.

•Sacrifice after a day for determination of basal bacterial load

•Start treatment after 28 / 14 days resp. with different doses of Vit C based on MIC/PK studies for 4 weeks

•Sacrifice at day 28/14 resp. for CFU enumeration

•Repeat Vit C groups with different combinations of drugs

Vit C evaluation in Guinea pigs

Significance of the project

This study enlightens the possible benefits of adding vit C to anti-TB regimen & suggests that the development of drugs that generate high oxidative burst could be of great use in TB treatment.

Total budget estimateItem Budget in Lakhs (INR) Total INR in

lakhs

Ist year 2nd year 3rd year

A RECURRING

Salaries 3.12 3.12 3.12 9.36

Consumables 5.0 5.0 5.0 15.0

Travel 0.5 0.5 0.5 1.5

Overhead charges @ 10%

0.862 0.862 0.862 2.586

SUBTOTAL 9.482 9.482 9.482 28.446

B NON RECURRING

Equipment (BSL-2-cabinet,centifuge,spectrophotometer etc)

37.0 37.0

GRAND TOTAL 65.446

Thank you