Schematic of EVOC probe consumption

EVOC Probes

Exogenous Volatile Organic Compound (EVOC®) Probes for targeted assessment of biological pathways.

Sources of VOCs

Exogenous Vs Endogenous VOCs
Figure 1. Sources of VOCs are endogenous and exogenous.

Endogenous volatile organic compounds (VOCs) are produced as a result of metabolic processes within the body, meaning that underlying changes in metabolic activity, including those from your gut microbiome, can produce patterns of VOCs characteristic of specific diseases. As disease has an immediate effect on metabolism, the pattern of VOCs exhaled will change, making Breath Biopsy® an excellent tool with the potential to enable earlier disease diagnosis and precision medicine applications.

Over 1,000 VOCs have been detected in breath, making it a rich source of information regarding the overall health status of individuals. However, it can be challenging to identify and validate breath biomarkers associated with a specific disease using an untargeted approach, and indeed suitable endogenous VOC biomarkers may not always be identifiable or present.

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EVOC Probes

We are developing our EVOC Probe method as a reliable and high-sensitivity alternative to untargeted bioamrker discovery. This approach offers the advantage that compounds not normally found at significant levels in breath can be introduced into the body (individually or as a cocktail), to explore how they are absorbed, metabolized or excreted.

The concept involves exogenous VOC (EVOC) Probes, which are safe compounds that when administered undergo metabolism in the body and are excreted via breath, offering a readout of metabolism by enzymes and organs. This approach could permit high levels of compounds to be administered, substantially improving signal-to-noise ratios compared to signals from endogenous sources and thus greatly improving reliability of detection.

EVOC Exogenous VOCs Diagram
Figure 2. Use of Exogenous VOC (EVOC) Probes to provide clear biomarkers in breath.

Through this approach we are exploring the development of research tests for applications where naturally occurring VOCs alone may be insufficient, and that could be used to run smaller, focused and likely faster clinical trials for us and our customers. Our initial focus is in liverdiseases where we aim to target specific enzymes in the liver (e.g. CYP450 family), however EVOC Probes also hold potential in a wide range of additional applications.

How could targeted VOC probes advance biomarker research?

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Example research applications of EVOC Probes

We are currently focusing on the development of EVOC Probe-based Breath Biopsy applications to support research taking place in academic, clinical and pharmaceutical settings. Three early areas of focus are research in:

a. Monitoring liver function

The liver is crucial to the body’s healthy operation. It is responsible for an array of biological functions including the uptake of toxic substances from the blood to render them harmless and the metabolism of drugs to their active forms and their breakdown for excretion from the body.

When the liver is not functioning properly due to infection, transplant, alcohol abuse, genetic disease, or other reasons, the impact on the health of an individual can be enormous. Liver disease remains a major cause of death, with 844 million people having chronic liver diseases (CLDs) globally, with a mortality rate of 2 million deaths per year.

It is therefore important that clinicians be able to reliably measure liver function to detect and  monitor the progression of disease or the impact of therapy. A range of diagnostic tests currently exist, including for aspartate aminotransferase, alkaline phosphatase, albumin, and bilirubin levels in blood. Each, however, produces results that can originate from a range of physiological inputs and so are used in concert with clinical presentation to suggest further investigations.  

In contrast, a Breath Biopsy assay using EVOC Probes has the potential to generate a clear and unambiguous measurement of liver function and potentially to distinguish among different types of liver disorders by measuring the activity of key liver enzymes.

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b. Drug metabolism

Approximately 40% of drugs do not work as expected when prescribed, leading to adverse events or to the drug not providing the therapeutic benefit expected. This is largely due to drug metabolism, the biologic processing of drugs from prodrugs into their active forms and the breaking down of drugs for excretion. When this process goes wrong, a drug can build up in the body to toxic levels or the active form never reach sufficient levels to have the desired therapeutic effect.

Drug Metabolism Graph
Figure 3. When a drug is metabolized too slowly or quickly, it can lead to it being toxic or not having the therapeutic effect expected.

The vast majority of small molecule drugs are metabolized by a class of enzymes called the cytochrome P450s, of which four are responsible for approximately 70% of activity, including metabolizing most of the most highly prescribed medications across all indications including Lipitor (cardiovascular), Zoloft (mental health), and Tramadol (pain).

Drug and CYP targets table 1
Table 1. The table indicates the Cytochrome P450 enzyme family that metabolizes each drug, alongside disease indications and yearly prescriptions.

Knowledge of a patient’s drug metabolism status allows clinicians to make more informed decisions as to which drugs to prescribe and at what dose. As a result, the pharmacogenomics market, testing a person’s genes to look for polymorphisms that predict how that individual would metabolize a drug, is growing rapidly. The problem with this approach is that genes are only one measure of risk, but not a complete measure of how a patient would actually metabolize a drug. For example, genotyping cannot account for diet, smoking, or other drugs a patient may be taking. A potentially more holistic approach is through pharmacobreathomics* - testing drug metabolism status by phenotype through Breath Biopsy.

*In pharmacobreathomics, the profile of VOCs on breath is used to study how a drug interacts with the body. This new field combines pharmacology (the science of drugs) and breathomics (the study of VOCs in breath) to develop precision medicine tools that aim to deliver the right drug, to the right patient, at the right time.

Grapefruit Juice Graph 900 v1
Figure 4. Eucapyptol washout curves before and after CYP3A4 inhibition by grapefruit juice. As CYP3A4 is inhibited, less of the substrate Eucalyptol is metabolized and so is seen at higher levels in breath.

We are exploring the use of  EVOC Probes that are metabolized by the same P450 enzymes as common medications and that could be used to anticipate the rate of drug processing and breakdown, this would produce a phenotypic profile that, if sufficiently accurate, could be used to modulate drug treatments for more effective results.


c. Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH)

Nonalcoholic fatty liver disease (NAFLD) is a condition in which excess fat is stored in the liver but with alcohol abuse not being the underlying cause. There are two subtypes of NAFLD: simple fatty liver and nonalcoholic steatohepatitis (NASH). Simple fatty liver is the more benign form in which liver fat is present but there is little or no inflammation and typically does not progress to cause liver damage or complications. In approximately 25% of cases however, NAFLD develops into NASH where inflammation and liver cell damage can cause fibrosis, or scarring, of the liver, and can lead to cirrhosis or liver cancer.

NASH Prevalence World Map
Figure 5. The global prevalence of NAFLD(3)

The global burden for these diseases is enormous, with the global prevalence of NAFLD at approximately 25% and NASH at up to 7%. There are currently 48 NASH drugs in clinical trials, 14 at Phase 1, 30 at Phase 2 and four at Phase 3(4). Gold standard testing for the diagnosis of these diseases involves a range of highly invasive, expensive and time consuming tests. Therefore there is a need for a reliable and non-invasive method to monitor progression of disease, identify patient subpopulations for risk profiling, and to help optimize treatment regimes.

As with other forms of liver disease, EVOC Probes offer a promising alternative approach which can be used to investigate whether breath analysis can be deployed as a novel non-invasive approach to monitor NAFLD and NASH disease progression.

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Development of EVOC Probes

Our approach to EVOC Probe development is one based on the underlying biology, understanding disease pathology and taking a close look at enzymatic pathways that underlie it. Through this approach we have identified potential probes that relevant pathways.

EVOC Probe Development Flow
Figure 6. The development pathway of EVOC Probes

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References

1. Marcellin P, Kutala BK. Liver diseases: A major, neglected global public health problem requiring urgent actions and large-scale screening. Liver Int. 38(Suppl. 1):2–6. (2018) https://doi.org/10.1111/liv.13682

2. Fernández del Río, R. et al. Volatile Biomarkers in Breath Associated With Liver Cirrhosis — Comparisons of Pre- and Post-liver Transplant Breath Samples. EBioMedicine, Volume 2, Issue 9, 1243 - 1250. (2015) - https://doi.org/10.1016/j.ebiom.2015.07.027

3. Adapted from Younossi, Z. M. The epidemiology of nonalcoholic steatohepatitis. Clinical Liver Disease, 11: 92-94. (2018) - https://aasldpubs.onlinelibrary.wiley.com/doi/full/10.1002/cld.710

4. www.clinicaltrials.gov

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