Limonene Metabolism and CYP2C19 Genetic Variants

Overseen By Eric Gross, MD/PhD

Age: 18 - 65

Sex: Any

Trial Phase: Academic

Sponsor: Stanford University

Must not be taking: CYP2C19 substrates, inhibitors

Disqualifiers: Pregnancy, Cardiovascular disease, Veganism, others

No Placebo Group All trial participants will receive the active study treatment (no placebo)

Approved in 2 Jurisdictions This treatment is already approved in other countries

Compensation: Varies

Number of Visits: 1

Duration: 1 day

20 Participants Needed

What You Need to Know Before You Apply

What is the purpose of this trial?

This trial aims to develop a simple test to identify genetic differences affecting how the body processes certain drugs. Specifically, it examines how the body metabolizes limonene, a compound from orange peels, to detect variants in the CYP2C19 gene. Participants will consume limonene and provide breath samples, helping researchers understand how genetic differences impact drug processing. People of East Asian descent who haven't experienced recent chest pain or shortness of breath and aren't allergic to citrus might be a good fit. As an unphased trial, this study offers participants the chance to contribute to groundbreaking research that could personalize drug treatments in the future.

Do I need to stop my current medications for the trial?

The trial excludes people taking certain medications that affect CYP2C19, so you may need to stop those specific medications. It's best to check with the trial team to see if your current medications are affected.

What prior data suggests that this non-invasive screening tool for CYP2C19 genetic variants is safe?

Research has shown that limonene, a compound in citrus peels, is generally safe for humans. The liver uses special proteins to break down limonene, helping the body process it safely at certain levels.

Studies have not reported any serious side effects from limonene. However, genetic differences may cause individuals to process it differently, affecting their response. Despite this, no major safety concerns have emerged.

Since this trial is in the "Not Applicable" phase, it likely focuses on understanding how the body processes limonene rather than testing its safety. This suggests that limonene is considered safe enough for such studies. ^{1 2 3 4 5}

Why are researchers excited about this trial?

Researchers are excited about this trial because it explores how genetic differences in the CYP2C19 enzyme affect the metabolism of limonene, a compound found in orange peels. Unlike standard treatments that may not consider genetic variations, this trial aims to uncover personalized insights into how people metabolize limonene based on their genetic makeup. This could pave the way for more tailored approaches to using natural compounds in medicine, potentially offering more effective and individualized treatment strategies. By understanding these genetic interactions, researchers hope to enhance the precision of treatments for conditions where limonene might be beneficial.

What evidence suggests that this method is effective for developing a non-invasive screening tool for CYP2C19 genetic variants?

Research shows that enzymes like CYP2C19 break down limonene, a substance in citrus peels, in the body. Genetic differences can cause this enzyme to function differently among individuals. Some studies suggest these genetic variations can alter drug processing in the body. This trial will compare two groups: one with participants having the wild-type CYP2C19 and another with participants having the CYP2C19*2 and/or CYP2C19*3 variants. Although another enzyme, CYP2C9, might also assist in breaking down limonene, CYP2C19 remains crucial for understanding the genetic impact on this process. Studying limonene breakdown could help develop a tool to identify these genetic differences. ^{3 5 6 7 8}

Are You a Good Fit for This Trial?

This trial is for men and women of East Asian ethnicity, aged between 18 and 45 years. It aims to find out if a simple test can be made to check genetic differences affecting how the body handles certain substances.

Inclusion Criteria

I am either male or female.

Ethnicities: East Asian

I am between 19 and 44 years old.

Exclusion Criteria

Previous allergic reaction to limonene, citrus fruits, bovine gelatin, glycerin

Veganism

I have a history of stroke or heart disease.

Timeline for a Trial Participant

Screening

Participants are screened for eligibility to participate in the trial

1-2 weeks

Treatment

Participants consume 1 oz water mixed with 500mg limonene and provide breath samples over 2 hours

1 day

1 visit (in-person)

Follow-up

Participants are monitored for safety and effectiveness after treatment

1 week

What Are the Treatments Tested in This Trial?

Interventions

• Limonene

Trial Overview The study is testing whether limonene, a common substance found in citrus oils, can help develop a non-invasive tool to screen for genetic variants that affect drug metabolism.

How Is the Trial Designed?

Group I: Wildtype CYP2C19 Experimental Treatment 1 Intervention

The group of participants has been genotyped to have wild-type CYP2C19. The participants will consume 1 oz water mixed with 500mg limonene (Jarrow Formula orange peel extract) and be asked to swish 5 times and swallow. All participants will then provide breath samples over the course of 2 hours. The breath sample outcomes will be compared to those of the CYP2C19\*2 and/or CYP2C19\*3 study subjects.

Group II: CYP2C19*2 and CYP2C19*3 Variants Experimental Treatment 1 Intervention

The group of participants has been genotyped to have the CYP2C19\*2 and/or CYP2C19\*3 variant during the first part of the study. The participants will consume 1 oz water mixed with 500mg limonene (Jarrow Formula orange peel extract) and be asked to swish 5 times and swallow. All participants will then provide breath samples over the course of 2 hours. The breath sample outcomes will be compared to those of the wild-type CYP2C19 study subjects.

Limonene is already approved in European Union, United States for the following indications:

Approved in European Union as Limonene for:

• Flavoring agent
• Fragrance in perfumery and personal care products
• Botanical insecticide
• Organic herbicide
• Cleaning agent

Approved in United States as Limonene for:

• Flavoring agent
• Fragrance in perfumery and personal care products
• Botanical insecticide
• Organic herbicide
• Cleaning agent

Who Is Running the Clinical Trial?

Stanford University

Lead Sponsor

Trials

2,527

Recruited

17,430,000+

Published Research Related to This Trial

Polymorphisms in drug-metabolizing enzymes, particularly CYP2D6 and CYP2C19, can significantly affect how individuals metabolize medications, leading to variations in drug efficacy and safety.

Understanding a patient's metabolic profile (poor, extensive, or ultrarapid metabolizers) can guide personalized dosing strategies to avoid toxicity and improve treatment outcomes.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

Update: genetic polymorphism of drug metabolizing enzymes in humans. Tanaka, E.[2019]

(+)-Limonene causes renal toxicity specifically in male rats, which is linked to the male-specific enzyme CYP2C11 that metabolizes limonene differently than in females and other species.

The study found significant species-related differences in how various animals metabolize limonene, particularly in converting (+)-carveol to (+)-carvone, but it remains uncertain if these differences explain the observed renal toxicity.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

Species differences in the metabolism of (+)- and (-)-limonenes and their metabolites, carveols and carvones, by cytochrome P450 enzymes in liver microsomes of mice, rats, guinea pigs, rabbits, dogs, monkeys, and humans. Shimada, T., Shindo, M., Miyazawa, M.[2019]

A specific genetic variation (SNP 4, IVS8-109A>T) in the CYP2C9 gene is significantly associated with higher metabolic rates of the drug losartan in healthy Swedish individuals, indicating its potential role in ultra-rapid drug metabolism.

No significant relationships were found between other SNPs (1, 2, and 3) and CYP2C9 activity, suggesting that SNP 4 is a key factor in determining metabolic rates, while variations in the CYP2C9 gene did not affect metabolism in Korean subjects.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

Search for the molecular basis of ultra-rapid CYP2C9-catalysed metabolism: relationship between SNP IVS8-109A>T and the losartan metabolism phenotype in Swedes. Hatta, FH., Teh, LK., Helldén, A., et al.[2022]

This study demonstrates a proof-of-concept for using breath tests to assess drug metabolism, specifically showing how the enzyme CYP2C9 metabolizes diclofenac into its active form, which could lead to personalized medicine approaches.

The research also reveals that limonene, a common food ingredient, inhibits the metabolism of diclofenac by CYP2C9, indicating that dietary components can affect drug efficacy and metabolism, which is important for patient treatment plans.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

Establishing a cell-based screening workflow for determining the efficiency of CYP2C9 metabolism: moving towards the use of breath volatiles in personalised medicine. Lochmann, F., Nikolajevic, A., Stock, V., et al.[2023]

The study analyzed three genetic variants of the CYP2C19 enzyme (CYP2C19*23, CYP2C19*24, and CYP2C19*25) found in the Chinese population, revealing that all variants exhibited very low enzyme activity (6.1% or less in intrinsic clearance) towards the drugs omeprazole and 3-cyano-7-ethoxycoumarin.

Inhibition studies showed that these variants had enhanced binding to certain inhibitors, suggesting that structural changes in the enzyme's active site affect how well it interacts with different drugs, highlighting the importance of both genetic factors and the chemical properties of the drugs in determining enzyme activity.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

The Molecular and Enzyme Kinetic Basis for Altered Activity of Three Cytochrome P450 2C19 Variants Found in the Chinese Population. Dong, AN., Ahemad, N., Pan, Y., et al.[2022]

D-limonene is recognized as safe for consumption and has low toxicity in humans, with studies showing no mutagenic or carcinogenic risks, even after long-term use.

In addition to its safety, d-limonene has demonstrated potential therapeutic benefits, including the ability to dissolve cholesterol gallstones and provide relief from heartburn and gastroesophageal reflux, as well as showing chemopreventive activity against certain cancers.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

D-Limonene: safety and clinical applications. Sun, J.[2022]

Genetic variations in drug-metabolizing enzymes, particularly CYP2D6, can lead to significant differences in how individuals metabolize medications, affecting drug efficacy and safety.

CYP2D6 activity can vary widely among individuals, from complete absence to ultra-rapid metabolism, which highlights the importance of considering genetic testing for personalized medicine approaches in drug therapy.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

Pharmacogenetics: the molecular genetics of CYP2D6 dependent drug metabolism. Wong, JY., Seah, ES., Lee, EJ.[2022]

In a pilot study with seven healthy volunteers, ingestion of 100 mg/kg of d-limonene showed no significant toxicity, indicating it is safe for human consumption at this dose.

The study identified several metabolites of limonene in human plasma, suggesting that limonene is metabolized similarly in humans and rats, which supports its potential as an effective chemotherapeutic agent for treating cancers.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

Human metabolism of the experimental cancer therapeutic agent d-limonene. Crowell, PL., Elson, CE., Bailey, HH., et al.[2018]

D-Limonene, a compound found in citrus oils, has been shown to block tumor formation in lab animals by preventing the activation of cancer-causing substances and enhancing the detoxification of harmful metabolites.

In studies, D-Limonene maintained liver glutathione levels during paracetamol-induced stress, suggesting it may help protect the liver from damage, although it did not affect certain liver enzyme activities related to drug metabolism.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

Effects of D-limonene on hepatic microsomal monooxygenase activity and paracetamol-induced glutathione depletion in mouse. Reicks, MM., Crankshaw, D.[2019]

d-Limonene, a common ingredient in cosmetics and food, has low toxicity based on animal studies, with a calculated margin of exposure indicating minimal risk for human use.

While d-limonene is generally safe, it can cause skin irritation in some individuals, highlighting the need for regulation in cosmetic products to ensure safety.

NCBI (Pubmed)

pubmed.ncbi.nlm.nih.gov

Safety evaluation and risk assessment of d-Limonene. Kim, YW., Kim, MJ., Chung, BY., et al.[2022]

Citations

1. pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov/articles/PMC10899532/

From genes to drugs: CYP2C19 and pharmacogenetics in ... This review summarizes the most recent information on how genetic variants in CYP2C19 affect drug metabolism and therapeutic outcomes.

2. researchgate.net researchgate.net/publication/11415780_Metabolism_of_-_and_--Limonenes_to_Respective_Carveols_and_Perillyl_Alcohols_by_CYP2C9_and_CYP2C19_in_Human_Liver_Microsomes

Limonenes to Respective Carveols and Perillyl Alcohols by ... CYP2C9 may be more important than CYP2C19 in catalyzing limonene oxidations in human liver microsomes, since levels of the former protein are more abundant than ...

3. platform.opentargets.org platform.opentargets.org/target/ENSG00000165841

CYP2C19 profile page Genetic variants in CYP2C19 that have been associated with drug response ... Subcellular location data for CYP2C19. Source: UniProt, HPA. HPA main ...

4. biorxiv.org biorxiv.org/content/10.1101/2023.09.07.555071v2.full.pdf

The linear and non-linear effects of CYP2C19 metaboliser ... Forty-eight CpG probes were significantly associated with both linear and quadratic terms of CYP2C19 metaboliser status (PBonferroni < 0.05).

5. en.wikipedia.org en.wikipedia.org/wiki/CYP2C19

CYP2C19 There are many common genetic variations that affect the expression of the CYP2C19 gene, which in turn influences the enzyme activity in the metabolic pathways ...

6. ncbi.nlm.nih.gov ncbi.nlm.nih.gov/gene/1557

CYP2C19 cytochrome P450 family 2 subfamily C member ... These findings suggest that using CYP2C19 genotype to guide P2Y12 inhibitor selection is feasible.

7. dmd.aspetjournals.org dmd.aspetjournals.org/article/S0090-9556(24)04443-X/abstract

Limonenes to Respective Carveols and Perillyl Alcohols by ... These results suggest that both (+)- and (−)-limonene enantiomers are oxidized at 6- and 7-positions by CYP2C9 and CYP2C19 in human liver microsomes.

8. pubmed.ncbi.nlm.nih.gov pubmed.ncbi.nlm.nih.gov/33082528/

Global distribution of CYP2C19 risk phenotypes affecting ... Genetic variability of CYP2C19 may affect safety or efficacy of many clinically important medications as outlined in the clinical ...