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

Trial Summary

What is the purpose of this trial?

This trial tests a way to identify people with certain genetic traits by measuring a substance in their breath after drinking orange peel extract.

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.

Is limonene safe for human use?

Limonene, commonly found in citrus oils, is generally recognized as safe for use in foods and has low toxicity in humans. It may cause skin irritation in some people, but other serious risks are not reported. Studies show it does not pose a cancer risk to humans, although it can cause kidney issues in male rats, which is not relevant to humans. ^{1 2 3 4 5}

How does the drug limonene differ from other treatments for its condition?

Limonene is unique because it is metabolized by the CYP2C9 enzyme, which can vary greatly between individuals due to genetic differences, potentially affecting how the drug works for each person. This variability in metabolism makes limonene a candidate for personalized medicine approaches, where treatment can be tailored based on a person's specific genetic makeup. ^{6 7 8 9 10}

Eligibility Criteria

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

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

Treatment Details

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.

Participant Groups

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+

Findings from Research

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]

(+)-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]

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]

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]

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]

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]

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]

References

1. United States pubmed.ncbi.nlm.nih.gov

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

2. England 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. [2019]

3. Germany pubmed.ncbi.nlm.nih.gov

Human metabolism of the experimental cancer therapeutic agent d-limonene. [2018]

4. England pubmed.ncbi.nlm.nih.gov

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

5. England pubmed.ncbi.nlm.nih.gov

Safety evaluation and risk assessment of d-Limonene. [2022]

6. Germany 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. [2022]

7. England pubmed.ncbi.nlm.nih.gov

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

8. Singapore pubmed.ncbi.nlm.nih.gov

Pharmacogenetics: the molecular genetics of CYP2D6 dependent drug metabolism. [2022]

9. England 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. [2023]

10. United Arab Emirates 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. [2022]