Vitamin C improves the efficacy of TB drugs

Adding vitamin C as a nutritional supplement while treating drug-sensitive tuberculosis patients with first-line TB drugs will boost the efficiency of treatment, a study by Indian researchers shows. The increase in efficiency is not because vitamin C has antibacterial activity, as was suggested by a few researchers in 2013 from in vitro studies, but by doing the complete opposite — inducing dormancy in TB bacteria.

A team led by Prof. Jaya Sivaswami Tyagi from the Department of Biotechnology at AIIMS, New Delhi had first proposed the dormancy-inducing ability of vitamin C in TB bacilli in 2010 and has now reconfirmed it in a comprehensive study published in the journal Redox Biology.

The team found that vitamin C imposes multiple stresses on TB bacteria such as hypoxia, acid stress (where the pH is reduced to around 5.5), oxidative stress (through the generation of H2O2 and reactive oxygen species), reductive stress (due to cessation of aerobic respiration) and metabolic stress. “As a result of these stresses, there is slowing down of metabolism leading to dormancy and further progression to viable but non-culturable (VBNC) state,” says Prof. Tyagi. “Together, these stresses remarkably resemble the host environment that the bacteria would face.”

In the lab, TB bacteria already exposed to vitamin C displayed resistance to two first-line drugs — isoniazid and rifampicin — as it progressed to a dormant state. Unlike these two drugs, pyrazinamide drug is capable of killing TB bacteria even in a dormant state. “The addition of vitamin C increased the population of dormant bacteria and this led to an eight-fold increase in pyrazinamide’s ability to kill the bacteria. There was also a four-fold decrease in the minimum concentration (MIC) of pyrazinamide required to kill the bacteria even in an infection model,” says Dr. Kriti Sikri from the Department of Biotechnology, AIIMS and first author of the paper.

In the presence of pyrazinamide alone nearly 90% of the bacteria survived but when combined with vitamin C, the survival rate in vitro dropped sharply to less than 3%. “Vitamin C induces dormancy and enhances the population of slowly growing bacteria or bacteria that are not growing. And pyrazinamide drug targets these bacteria bringing about sharp reduction in the survival rate,” explains Prof. Tyagi. “The effect of vitamin C combined with TB drugs was reproduced in an intracellular model, which is akin to human infection. So, our findings acquire clinical relevance. When used along with other first-line drugs, vitamin C has the potential to shorten the treatment time.”

Screening novel drugs

Besides improving the efficacy of existing TB regimen, vitamin C can help in producing subclasses of bacteria to test new drugs. For instance, due to hypoxia, the metabolism gets slowed down leading to reductive stress. Lipids are formed as a compensatory mechanism and the breakdown of lipids produces energy. The AIIMS team used novel inhibitors to prevent the breakdown of lipids for energy purpose and this led to the death of TB bacteria.

Similarly, dormancy can be induced by adding vitamin C. Dormant bacteria are able to reduce the effectiveness of isoniazid and rifampicin by pumping them out using efflux pumps. The use of vitamin C can help in producing a population of dormant bacteria which can be used for screening drugs that inhibit efflux pumps, which the researchers were able to do. “We were able to reverse the tolerance and restore the bacterial sensitivity to these drugs by treating the bacteria with either of two efflux pump inhibitors — verapamil and piperine,” Prof. Tyagi says.

Likewise, vitamin C can be used for producing viable but non-culturable (VBNC) TB bacteria. Though VBNC bacteria are present in sputum samples they cannot be cultured and hence never identified leading to misdiagnosis. The VBNC bacteria can always reverse and cause disease. “Unlike reports that 100 days are required to produce VBNC bacteria, our model can generate them in just eight days, which can be used for screening drugs,” Prof. Tyagi says.

When used with first-line drugs, vitamin C can shorten the treatment time

Designing a killer to kill a killer

Unlike normal cells in our body, cancer cells keep on dividing and proliferating without control, forming lumps called tumours. If we find methods to stop such unrestricted growth, we can treat and cure cancer. Anti-cancer drugs are still not specific enough and often damage normal, healthy cells as well, leading to unpleasant side-effects. They also are not able to penetrate the tumour. ‘Surgical Strike’ or cutting off the tumour is not specific enough and tends to damage normal tissues as well. Physical tools such as nanoparticles, coated with ‘magic bullets’, which seek and destroy specific components of cancer cells, are limited by penetrability, clearance from the body after delivery and so forth. What is needed is a killer device that can open up the target cell mass and finish it off, and do so only on cancer cells and not harm healthy ones in the body.

Way back in 1893, Dr William Covey of New York thought up the unusual idea of using whole bacteria (or their extracts) for treating cancer. The argument is this: after all, these microbes enter our cells and wreak damage. He made extracts of microbes such as Mycobacterium bovis (one that causes TB in cattle) and found that tumours actually shrank in size upon treating with such ‘Covey toxins’.

Taming the shrew

This approach is, in effect, the use of one type of killer cells (microbes which cause disease to normal healthy cells, even death, if left untreated) to kill another type of killer cells (cancer). The trouble here is while they kill cancer cells, they may also damage healthy cells of the body; treating the body against such infection allows the cancer to come back. If only we find ways to ‘tame’ such microbes that they do not cause harm to normal cells, but specifically target cancer cells, we may have a winning strategy. While Covey could not do this, we now have ways to do so, thanks to the advances in genetic engineering, molecular and cell biology. The field of using bacterial cells, loaded with anti-cancer molecules, to fight cancer has grown fast in the last 20 years. The killer bacterium chosen by many researchers in the field is Salmonella typhimurium (the one that causes typhoid-like disease in rats, and leads to gastric problems and diarrhea in humans). The molecular biology of this pathogen (let us abbreviate its name as ST) is now well known, and genetically manipulating it is not difficult. A group of researchers at Yale University, Connecticut, USA, found that if we delete the gene called msbB from its genome, its toxicity towards normal cells is vastly reduced. However, although safe, the injection of this gene-deleted strain did not show substantial anti-tumour activity.

The group soon realised that compared to normal cells, tumour cells are far richer in ATP, the energy currency molecule in cells. Given this, if one were to delete the gene called purL (which codes for making the ‘A’ part in ‘ATP’) from ST, the modified strain would need external addition of ATP in order to multiply and grow. And the abundant levels of ATP are the teaser. Thus the purl- deleted ST strain would make a beeline towards the tumour cells, ignoring normal healthy ones. We thus have a second way of herding ST towards cancers.

What if we make a double mutant, that is, generate ST with both the msbB and purL genes deleted, grow and unleash it into the organ afflicted with cancer? This was indeed done about 15 years ago by a group of scientists from the pharmaceutical company called VION, near Yale University. This double mutant strain, termed V20009, was tried against mice with melanomas, and also on mice carrying human tumours grafted on to them. Intravenous injection of VNP20009 inhibited tumour growth anywhere between 57-95%. Plus, only live bacteria showed the anti-tumour effect, meaning that continuous infection by live bacteria is needed to eliminate tumours, and extracts or drugs with limited doses will not do. About the same time, a group from the National Cancer Institute, near Washington, DC, used VNP20009 to treat 24 human patients with secondary skin cancers and found it sufficiently safe for human use.

Making a package deal

Dr. Ravi Bellamkonda of Duke University, Durham, NC, USA went one step further, and decided to use VNP20009 as a carrier or vehicle, and loaded it with the protein called p53 which suppresses tumour growth, and another molecule called azurin which kills cancer cells, and also protects p53 from degradation. That such a twinning of p53 and azurin is useful was earlier shown by Dr. Ananda Chakrabarty of the University of Illinois College of medicine at Chicago.

Dr. Bellamkonda injected this cargo-laden ST on to rats which carried grafts of cancerous brain tumours obtained from humans. This therapy allowed the cancer-bearing rats survive for more than 100 days, compared with barely 26 days for untreated rats.

The beauty in using such cargo-laden double mutant pathogens is that (1) normal and healthy body cells are not affected, (2) they penetrate the body of the cancer cells, (3) this allows delivery of the drugs into the interior of the cancer cell (where conventional drugs find hard to enter), and (4) we can add more cargo, and allow additional cancer-killer drugs while keeping normal cells safe enough.

TB vaccine trial on adults begins in June

In June this year, the Pune-based Serum Institute of India Pvt. Limited will begin a Phase II/III vaccine trial for tuberculosis using a novel, recombinant BCG (bacillus Calmette-Guérin) vaccine.

The double-blind, placebo-controlled, randomised trial will be carried out on 2,000 adults who have been successfully treated (and cured) for TB. While 1,000 adults will receive the vaccine, the remaining volunteers will receive a placebo. A single dose of the vaccine will be administered, and the volunteers will be followed up for a year. The trial will be conducted in 15-17 centres across the country.

The new TB vaccine (VPM1002), which will be tested, is based on the BCG vaccine that is in use. However, it is more powerful and efficacious as it contains a gene, better recognised by the immune system.

Safety test

“Adults who have completed TB treatment will be first screened and enrolled if found eligible 2-4 weeks after completion of TB treatment,” says Dr. Prasad S. Kulkarni, Medical Director at Serum Institute.

“Traces of the drugs may be present in the body for two weeks after completion of the treatment. Since the vaccine contains live, weakened bacteria, the drugs can kill them if given earlier than two weeks after completing the treatment.”

The vaccine will be first administered in 200 volunteers to test its safety. “If there are no safety concerns, the trial will continue in the remaining 1,800 volunteers,” he says.

The safety of the vaccine has already been tested in two Phase I trials — 80 adults in Germany (2009) and 24 in South Africa (2010) — and one Phase 2a trial in South Africa in 2012 in 48 newborns who have not been exposed to HIV. “These trials have confirmed the safety of the vaccine,” says Umesh Shaligram, Director-R&D, Serum Institute.

The results of the Phase 2a trial in newborns in South Africa, published in February this year in Clinical and Vaccine Immunology, has confirmed the safety of the vaccine.

IIT Bombay researchers find a novel target for blocking cancer metastasis

Researchers from IIT Bombay have found a novel pathway that is responsible for the progress of cancer metastasis — spread of cancer cells from its primary site of origin to new areas of the body. The finding holds potential in controlling metastasis to reduce cancer deaths. The study was published in the journal Oncotarget.

Treating metastasis

Surgical removal of primary tumours has long been used as a standard treatment for localised tumours, but treating cancer metastasis remains a formidable challenge. “Cancer stem cells (CSCs) are one cause of cancer metastasis. However, there is no study done so far to examine the impact of biophysical properties of cancer stem cells in cancer metastasis,” says Dr. Rahul Purwar, Assistant Professor at Department of Biosciences & Bioengineering, IIT Bombay.

Contractile dynamics of a tumour cell represents one of the most important biophysical properties and is closely associated with cell spreading and cell adhesion properties of tumour cell. Increased cell contractility in breast cancer can initiate the escape of cancerous cells from their primary sites to distant organs, that is, metastasis.

Dr. Purwar’s investigating team as well as other, earlier researchers have shown a close relationship between cell contractility (ability of cells to contract) and invasiveness in breast cancer cells, ovarian cancer cells and melanoma cells. Increased contractility is correlated with increased migration of cells which helps in metastasis.

However, it remains unknown whether contractile dynamics of CSCs are distinct as compared to the bulk tumour population and contribute in CSC-mediated metastasis.

Robust remission

Study lead author Dr. Purwar explains that “With this study, we identified a distinct pathway which CSCs use to invade the extracellular matrix and metastasise to other organs. Surprisingly, we observed that blockade of this pathway by pharmaceutical drugs completely abolished the invasion of CSCs as well as other tumour cells. Thus, targeting this distinct pathway may lead to the development of robust and long-term remission of cancer metastasis’’.

Cell contractility is regulated by two groups of enzymes including myosin light chain kinase (MLCK) and Rho associated protein kinase (ROCK). The team found that pharmacological targeting of ROCK prevents contractility and cell invasion potential of both CSCs and non-cancer stem cells, and is therefore a novel strategy for the treatment of cancer metastasis.

“Our work provides the first evidence of targeting biophysical properties of cancer stem cells for controlling metastatic cancer. However, further work is required to translate our findings before it goes to clinic,” he says.