EVOC Probes

Exogenous Volatile Organic Compound (EVOC) Probes enable targeted stimulation 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 the end product of metabolic processes within the body, meaning that underlying changes in metabolic activity, including that 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 for precision medicine applications.

There are over 1,000 VOCs present in breath, making them 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

As opposed to untargeted biomarker discovery, Breath Biopsy® has 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.

Owlstone Medical’s Exogenous VOC (EVOC) Probes are safe compounds, largely food additives, that when administered orally undergo metabolism in the body and are excreted via breath, offering a readout of metabolism by enzymes and organs. This approach enables high levels of compounds to be administered, such that fold changes in levels are much more visible, with substantially improved signal-to-noise ratios over those that come from endogenous sources.

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 to run smaller, focused and likely faster clinical trials for us and our customers. Our initial focus is in liver function and health, and drug metabolism testing where we are able to target specific enzymes in the liver (e.g. CYP450 family), however they also hold the potential in a wide range of additional applications.

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Terpenes: An Example of EVOC Probes

Terpenes are a large class of hydrocarbon compounds which are then acted upon by a range of enzymes. As an example of the use of EVOC Probes, an EVOC Probe (a capsule containing several terpenes) was administered and breath collected and analyzed over a period of 8 hours in a ‘washout study’. As shown in Figure 3, ingestion of the capsule resulted in a marked increase in concentration of terpenes in breath after 30 minutes, compared to baseline levels while no increase was observed for endogenous compounds not contained in the EVOC Probe, such as acetone and isoprene. Levels of terpenes then decrease over time, returning to baseline levels by 6 - 8 hours after capsule administration.

Peppermint Graph Capsule 900
Figure 3. Washout curves of acetone, isoprene, and different terpenes/terpenoids from one healthy subject at baseline (Ctrl) and after ingestion of the EVOC Probe. Washout curve following ingestion of a peppermint capsule. Breath samples from repeated collects from one individual over 8 hours (16 timepoints) were analyzed. Four replicate VOC sample tubes were collected and analyzed at each time point.

From the washout experiment, three time points were selected (pre-ingestion control, peak at 45 minutes and plateau at 3 hours) for inclusion in a longitudinal study where the washout experiment was repeated multiple times over 5 weeks. In the longitudinal study, fold changes for all compounds are presented for the peak and plateau time points, relative to the corresponding pre-ingestion control baseline (Figure 4). Breath metabolites acetone and isoprene show only small differences between peak and plateau fold change as expected, however large fold changes are observed for the
peppermint-related VOCs such as α-pinene, β-pinene, although high variability is observed for peak data points.

As seen in Figure 4, as few as three timepoints can be sufficient to observe differences in the levels of breath metabolites following administration of an EVOC Probe, with samples collected to establish baseline, and at set times (which can vary based on the individual EVOC Probe) to measure peak levels and when washout will have substantially occurred. This profile, when compared with a broader population, can then be used to determine the relative metabolic response of that individual to the EVOC Probe.

Peppermint Boxplots 900
Figure 4. Longitudinal study repeating the washout experiment nine times over five weeks in a single individual. Breath samples from pre-ingestion control, peak (45 min after administration of EVOC Probe) and 3 hours after EVOC Probe were analyzed in each washout experiment. Two replicate VOC sample tubes were collected and analyzed at each time point. Fold changes in breath levels of acetone, isoprene and different terpenes compared with baseline are shown.

Examples of longitudinal measurements of breath VOCs in the scientific literature

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Example research applications of EVOC Probes

Owlstone Medical is focused at the current time on the development of EVOC probe-based Breath Biopsy applications to support of research taking place in academic, clinical and pharmaceutical settings.  Three early areas of focus are research in:

a. Monitoring Liver function using EVOC Probes

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 from Owlstone Medical utilizing 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. This is done by measuring the levels of both the substrate in the EVOC Probe and its metabolites. Higher than expected levels of the substrate and lower levels of downstream metabolites in breath would suggest impaired liver function.


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 5. 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% including 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 6. 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.

Owlstone Medical is exploring the development of a test that uses EVOC probes that are metabolized by the same P450 enzymes that process medications, and so by measuring VOC levels and their rate of processing and breakdown, a reliable phenotypic profile that is more predictive than genotype alone can be built.


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 7. 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%. As an indication of need in this space, 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 expensive and time consuming tests followed by liver biopsy for confirmation, which is invasive and carries substantial risk for the patient. 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.

ReCIVA is a reliable and reproducible way to capture VOC biomarkers in breath samples

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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. We then work to pair potential compounds to them, identifying potential probes that would interact with those pathways and validating by confirming route of administration, distribution kinetics within the body, intra- and inter-individual variability, likelihood of secretion in breath, and finally determining dosage.

EVOC Probe Development Flow
Figure 8. The development pathway of EVOC probes

We are currently focused on liver health and function, using the following EVOC Probes.

EVOC Probes development table
Table 2. The table shows current EVOC Probes and the specific enzymes that process them in the liver.

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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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