CES1 Metabolic Enzyme

Carboxylesterase 1 (CES1) is the most abundant enzyme in the liver (approximately 1% of liver proteins). CES1 accounts for 80 to 95% of hydrolysis in the liver. It is also found in small amounts in the lungs and brain.
In addition to its important role in the breakdown of xenobiotic compounds, CES1 appears to be involved in endogenous metabolic functions, such as:¹

• Cholesterol esters
• triglycerides
• bioactive lipids

Methylphenidate is primarily metabolized by carboxylesterase 1A1 (CES1) in the liver to ritalinic acid (a-phenyl-2-piperidineacetic acid).² The earlier assumption that MPH is metabolized by hepatic lysozymal enzymes is now outdated.³
CES1 primarily metabolizes L-MPH and, to a lesser extent, d-MPH. As a result, more d-MPH (dexmethylphenidate) remains in the plasma. Most MPH preparations contain a racemic mixture of d- and L-MPH, although only d-MPH is pharmacologically active.⁴ 60 to 80% of the ingested MPH is excreted in the urine as Ritalin acid.⁵

• aromatic hydroxylation to p-hydroxymethylphenidate (p-hydroxy-MPH)⁴, accounting for between 1.5 and 12 percent of the degradation.
• Microsomal oxidation to 6-oxo-methylphenidate (6-oxo-MPH; inactive metabolite)⁴, accounting for up to 2.5% of MPH.
• The percentage of MPH excreted unchanged is reported to range from less than 1%⁵ to 2% in urine and 3% in feces⁴

CES1 activity is highly variable. The factors contributing to this variability were still largely unknown in 2018.⁶
There are significant individual differences in the response to many drugs metabolized by CES1.¹ The expression and activity of CES1 vary widely among humans. Therefore, there may be considerable individual differences in the pharmacokinetics and pharmacodynamics of drugs metabolized by CES1. The bioavailability of MPH in children ranges from 11% to 53%.⁷

A higher CES1 plasma concentration correlated with a lower D-methylphenidate plasma level. In one study, the CES1 plasma protein level accounted for approximately 50% of the variability in D-methylphenidate plasma levels. An individualized dosing strategy based on CES1 measurements could potentially make titrating the dose of D-methylphenidate considerably easier.⁸

Factors that influence degradation by CES1 are either non-genetic or genetic in nature:⁹

1. Non-genetic factors that influence CES1 metabolism¶

• Stage of development
• Gender
• Drug interactions (see substrates, inhibitors, inducers)

2. Genetic factors that influence CES1 metabolism¶

The level of CES1 expression correlates with the degree of methylation of the CpG islands (CGIs) in the CES1 promoter. Melatonin reduces the methylation level of the CES1 promoter by promoting the expression of sirtuin 1 (SIRT1), which mediates the deacetylation of DNA methyltransferase 1 (DNMT1).¹⁰

2.1. CES1 gene variants¶

In the case of a gene variant, a single position in the genetic material is altered (as if a single letter had been swapped). Variants of the CES1 gene influence the rate at which MPH is broken down. However, the available studies tend to show reduced breakdown or a loss of function, which conflicts with the faster breakdown observed in practice in some people with ADHD and the barely observed prolonged effect of MPH.

CES1 gene variants with functional consequences are rare. The main factors influencing CES1 metabolism appear to depend on other factors.⁶

To date, just under 200 variants have been found in the CES1/CES1P1 gene region.¹

• genetic polymorphisms
  • Single-nucleotide polymorphisms (SNPs)
    • More than 2,500 single-nucleotide polymorphisms (SNPs) have been identified in the CES1 gene (NCBI dbSNP). Some SNPs, such as G143E, D269fs, E220G, and L40T, are deleterious to the gene’s enzymatic activity and could alter CES1-mediated drug metabolism. However, these variants account for only a small portion of CES1 variability, leaving the majority unexplained.¹¹
    • G143E (rs71647871)
      • Prevalence: 3.7% among Caucasians¹²
      • G143E carriers required less MPH;^{6 13} in one study, the dose was 28% lower¹⁴ although up to 2.5 times the d-MPH AUC was observed at the same dose of MPH.¹⁵ and one-third of MPH metabolism¹⁶
      • Participants who were heterozygous for the CES1 G143E variant (p.Gly143Glu from rs71647871)
        • metabolize MPH significantly more slowly¹⁷, about half as fast as non-carriers¹⁸
          • Male G143E carriers who consume alcohol are likely to have a higher risk of MPH overexposure
        • p.Gly143Glu (rs71647871) appears to significantly impair the metabolism of:¹
          • Methylphenidate
          • Trandolapril
          • Oseltamivir
      • p.Gly143Glu resulted in a complete loss of the hydrolytic activity of the enzyme carboxylesterase 1 (CES1) toward methylphenidate. This resulted not only in high concentrations but also in elevated hemodynamic readings (blood pressure/pulse).¹⁹²⁰
      • A computer-based modeling study identified rs71647871 as a highly significant covariate in determining interindividual differences in MPH metabolism. rs71647871 GA resulted in a 2.4-fold increase in plasma d-MPH exposure. rs71647871 may be a risk factor for adverse effects of MPH.¹⁷
    • p.Asp260fs results in a complete loss of the hydrolytic activity of the enzyme carboxylesterase 1 (CES1) toward methylphenidate. MPH levels and hemodynamic parameters (blood pressure/pulse) were elevated.¹⁹
    • rs115629050 TG (p.Ala270Ser)
      • An in silico simulation showed significantly reduced MPH degradation, with approximately 68% higher d-MPH plasma exposure compared to the wild-type genotype. For rs115629050 TG, the scores were equal to or greater than those for rs71647871 GA in 6 out of 9 models.¹⁷
      • rs115629050 reduces CES1²¹
      • In vitro, rs115629050 TG had no effect on drug metabolism with respect to angiotensin²²
    • E220G (c.662A>G, rs200707504) is thought to be associated with reduced CES1 activity²³
    • c.428G>A (p.Gly143Glu, rs121912777) is thought to be associated with reduced CES1 activity²³
    • c.780delT (p.Asp260fs, rs71647872) is thought to be associated with reduced CES1 activity²³
    • c56G>T (rs3826190) is thought to be associated with reduced CES1 activity²³
    • c.808G>T (rs115629050) is thought to be associated with reduced CES1 activity²³
    • rs114119971 may be associated with reduced CES1 activity:²⁴
      • The average MPH dose for the 2 (out of 99) people with ADHD was 0.42 mg/kg/day, compared to 0.88 mg/kg/day for individuals without SNV
    • S75N (rs2307240)
      • appears to increase the activity of CES1 in response to clopidogrel²⁵
      • does not appear to affect CES1 activity in relation to methylphenidate in children²⁶
    • rs3815589
      • does not appear to affect CES1 activity in relation to methylphenidate in children²⁷
    • rs2287194
      • does not appear to affect CES1 activity in relation to methylphenidate in children²⁸
    • rs2244613
      • does not appear to affect CES1 activity in relation to methylphenidate in children²⁹
      • which was significantly correlated with sadness as a side effect of immediate release MPH in A/A carriers
      • rs2244613-G was associated with an increased risk of side effects from MPH in patients with concomitant ASA use³⁰
    • rs2002577
      • does not appear to affect CES1 activity in relation to methylphenidate in children³¹
      • tended to correlate with sadness as a side effect of immediate release MPH in G/G carriers
    • rs2307244
      • does not appear to affect CES1 activity in relation to methylphenidate in children³²
    • rs12443580
      • does not appear to affect CES1 activity in relation to methylphenidate in children³³
    • The 75 T/G and 75 G/G polymorphisms appear to be associated with a greater loss of appetite when taking MPH compared to the T/T variant.³⁴
    • Various CES1A2 promoter haplotypes are thought to be associated with increased CES1 expression:¹
      • 47C,
      • 46T
      • 41G
      • 40
      • 37C
      • 34G
      • 32T
• rs2307235-A
  • Increased risk of side effects from MPH in patients with concomitant ASA use³⁰
• rs8192950-T
  • Increased risk of side effects from MPH in patients with concomitant ASA use³⁰
• rs2302722-C
  • reduced risk of side effects from MPH in patients with concomitant ASA use³⁰
  • Number of copies options⁴
    • The various CES1 variants exist in multiple haplotypes and diplotypes. Individuals may carry more than two active copies of CES1 (i.e., two copies of CES1 and one copy of CES1A2 for a total of three copies, or two copies of CES1 and two copies of CES1A2 for a total of four copies).
      • Individuals can carry more than two active copies of CES1

Stevens et al. compiled studies that examined the effects of genetic variants on the efficacy of MPH:⁴

2.2. CES1 haplotypes, hybrid genes¶

A haplotype is a chain of several gene variants on the same chromosome that are inherited together as a package.

There are two known haplotypes of CES1:¹⁴⁶

• The first haplotype (“wild type”) is a hybrid gene consisting of
  • CES1P1
    • CES1P1 (CES1A3) is an inactive, truncated pseudogene. It is located near CES1 on chromosome 16. CES1P1 appears to have arisen through gene exchange.
  • CES1A1 (prototype of CES1)
• The second haplotype is a hybrid gene consisting of
  • CES1A1
  • CES1A2 (a CES1-like variant)
    • A computer-based modeling study found significantly reduced MPH breakdown and approximately 70% higher plasma d-MPH exposure in individuals with two CES1A2 copies compared to the wild-type genotype¹⁷
    • A CES1A2 copy resulted in approximately 22% higher MPH levels¹⁷
    • CES1A2 showed increased degradation with respect to irinotecan³⁵
    • A clinical trial with oseltamivir found that the CES1 diplotype had no effect on metabolism³⁶
    • A study of 99 children regarding MPH found that:²⁴
      • The average MPH dose was 0.79 mg/kg/day.
      • The mean MPH dose by haplotype was
        • CES1A2/CES1A2: 0.92 mg/kg for
        • CES1A2/CES1P1: 0.81 mg/kg
        • CES1P1/CES1P1: 0.78 mg/kg

As a result, some people carry two nearly identical copies of CES1 on the same chromosome.¹
With four copies of CES1, MPH clearance was lowest, and the MPH AUC was approximately 1.5 times higher than in the control group¹⁵
When there were two or three copies of CES1, the reduction in MPH was only slightly less¹⁵

Hybrid gene variants include:

• CES1P1 with CES1
  • higher transcriptional activity than CES1P1
• CES1A2 (another hybrid gene variant derived from CES1 and CES1P1)
  • has 2% of the transcription efficiency of CES1
• CES1A1b
• CES1A1c (CES1VAR)
  • has no noticeable effect on the metabolism of medications⁶

2.3. The number of CES1 gene copies influences MPH degradation¶

The number of CES1 gene copies also influences the rate of MPH breakdown. Contrary to the expectation that a higher copy number would be associated with increased breakdown, one study found reduced breakdown. Individuals with 4 CES1 copies had d-MPH levels (AUC) that were 45% (P = 0.011) and 61% (P = 0.028), respectively, higher d-MPH levels (AUC) than control subjects or individuals with 3 copies of CES1.²⁰

2.4. POR gene variants do not affect CES1 metabolism¶

Unlike with CYP 450 enzymes, the efficacy of CES1 is not influenced by genetic variants of the POR gene (cytochrome P450 oxidoreductase, NADPH P450 oxidoreductase, CPR).

3. CES1 Substrates / CES1 Inhibitors / CES1 Inducers¶

This list—like all information on ADxS.org—is not intended for personal therapeutic use. Although we make every effort to compile all relevant information, the list is nonetheless incomplete. Errors cannot be ruled out. Please consult your doctor or pharmacist.

The lower the IC50, the higher the therapeutic potency of an active ingredient.
The smaller the Ki value, the greater the binding affinity and the smaller the amount of drug needed to inhibit the enzyme’s activity.
If Ki is much greater than the maximum drug concentration to which a patient is exposed under typical administration conditions, it is unlikely that the drug will inhibit the enzyme’s activity.³⁷
The inhibition constant Ki is the inhibitor concentration at which half of the enzymes are inhibited.
Ki reflects binding affinity, while IC50 is more indicative of the functional potency of an inhibitor as a drug. The Ki calculation takes the IC50 into account.
Noncompetitive enzyme inhibition: Ki is approximately equal to IC50
Competitive/noncompetitive inhibition: Ki is approximately half of IC50

3.1. CES1 Substrates¶

CES1 plays a crucial role in the breakdown of various active ingredients.⁹

• 11-Deoxyalisol A (terterpenoid)

• 2-Oxo-clopidogrel (anticoagulant)³⁸

• 25-O-ethylalisol A (triterpenoid)

• Alismanol B (terterpenoid)

• Alismanol D (terterpenoid)

• Alismanol F (terterpenoid)

• Amphetamines (CNS active ingredients)²¹

  • METH
  • although primarily via CYP2D6

• Benzapril (ACE inhibitor, angiotensin receptor neprilysin inhibitor, ARNI)³⁸

• Capecitabine (cancer drug)

• Cholesterol (endogenous compound)

• Ciclesonide (immunosuppressant, adrenal glucocorticoid)³⁸

• Cilazapril (angiotensin receptor-neprilysin inhibitor, ARNI)³⁸

• Clopidogrel (anticoagulant)³⁹

• Dabigatran exilate (anticoagulant)³⁹

• Delapril (angiotensin receptor-neprilysin inhibitor, ARNI)³⁸* Clofibrate (antihyperlipidemic agent)³⁸

• Dimethyl fumarate (MS medication)

• Enalapril (ACE inhibitor, angiotensin receptor neprilysin inhibitor, ARNI)³⁸

• Fenofibrate (antihyperlipidemic drug)³⁸

• Fatty acid ethyl ester (endogenous compound)

• Fosinopril (angiotensin receptor-neprilysin inhibitor, ARNI)³⁸

• Flumazenil (CNS active ingredient)

• Heroin (CNS active ingredient)

• Imidapril (ACE inhibitor, angiotensin receptor-neprilysin inhibitor, ARNI)³⁹³⁸

• Irinotecan (cancer drug)

• Cocaine(CNS active ingredient)

• Lovastatin (antihyperlipidemic agent)³⁸

• Meperidine (CNS active ingredient)

• Methylphenidate (CNS active ingredient)

• Moxeipril (angiotensin receptor-neprilysin inhibitor, ARNI)³⁸

• Mycophenolate mofetil (immunosuppressant)

• Nintedanib (cancer drug)³⁸

• Oseltamivir (antiviral medication)³⁹

• Oxybutynin (anticholinergic; used, among other things, to treat urinary incontinence; antispasmodic)³⁸

• Para-nitrophenyl valerate (pesticide)

• Perindopril³⁸

• Quinapril (ACE inhibitor, angiotensin receptor-neprilysin inhibitor, ARNI)³⁸

• Ramipril (ACE inhibitor, angiotensin receptor-neprilysin inhibitor, ARNI)³⁸

• Rufinamide (CNS active ingredient)

• Sacubitril (angiotensin receptor-neprilysin inhibitor, ARNI; antihypertensive agent)³⁸

• Sarin (chemical weapon)

• Simvastatin (hyperlipidemia medication)³⁸

• Sofosbuvir (antiviral drug)

• Soman (chemical warfare agent)

• Tabun (chemical weapon)

• Telotristat ethyl (tryptophan hydroxylase inhibitor)³⁸

• Telotristat etiprat (cancer drug)

• Temocapril (angiotensin receptor-neprilysin inhibitor, ARNI)³⁸

• Tenofovir alafenamide (antiviral agent)

• Trandolapril (ACE inhibitor, angiotensin receptor-neprilysin inhibitor, ARNI)³⁸

• Trans-permethrin (pesticide)

• Travoprost (prostaglandin analog)³⁸

3.2. CES1 inhibitors¶

These active ingredients inhibit the breakdown of MPH by CES1 and should therefore, if possible, not be combined with MPH. We hypothesize, however, that for ultra-rapid metabolizers, such a combination could be beneficial—provided it is accompanied by particularly close medical supervision.

• 11-Deoxo-glycyrrhetinic acid (IC50: 10.5 µM) (terterpenoid)
• 1,12-Epoxy-5E,8E,14E-eicosatrienoic acid (IC50: 27 µM) (plant-derived fatty acid)
• 15-Deoxy-12,14-prostaglandin J2 (IC50: 12 µM) (plant-derived fatty acid)
• 22(R)-Hydroxycholesterol (unsaturated fatty acid, weak)⁴⁰⁴⁰
• 24(S)-hydroxycholesterol (unsaturated fatty acid, weak)⁴⁰
• 24(S),25-epoxycholesterol (IC50 = 8.1 μM) (unsaturated fatty acid, moderate)⁴⁰
• 25-hydroxycholesterin (unsaturated fatty acid, weak)⁴⁰
• 27-Hydroxycholesterol (27-HC) (IC50 = 33 nM, Kiapp = 10 nM) (unsaturated fatty acid, partially noncompetitive inhibitor)⁴⁰
  • impaired intracellular CES1 activity following treatment of intact THP1 cells
• 3-O-(-Carboxypropionyl)-11-deoxo-glycyrrhetinic acid 30-ethyl ester (IC50: 20.4 µM) (terterpenoid)
• 4,15-Epoxy-5E,8E,11E-eicosatrienoic acid (IC50: 38 µM) (plant-derived fatty acid)
• 7-ketocholesterol (unsaturated fatty acid, weak)⁴⁰
• Alcohol (strong) ⁴¹⁷
  • When taking alcohol and MPH at the same time:⁴¹
    • Does the MPH concentration increase in humans?
    • Alcohol inhibits CES1-mediated MPH degradation by catalyzing the conversion of MPH to ethylphenidate⁷⁴²
      • It appears that more l-ethylphenidate (pharmacologically inactive) is formed than d-ethylphenidate⁴³
      • Ethylphenidate appears to be toxic
      • Ethylphenidate is associated with significantly higher plasma levels of d-MPH and enhanced euphoric effects⁷
      • Ethylphenidate binds to DAT with similar strength, but binds less strongly to NET than MPH⁴³
• Arachidonic acid (strong) (IC50: 2 µM; Ki: 1.7 µM) (Plant-derived fatty acid)⁴⁰
  • the most potent fatty acid inhibitor of recombinant CES1
  • acted through a non-competitive mechanism (Kiapp = 1.7 μM)
• Aripiprazole (high potency) (IC50: 5.7 µM)⁴⁴⁷
• ic acid (triterpenoid), (Ki: 0.64 µM) (potent)³⁸
• Bavachinin (Ki: 0.5 µM) (strong) (plant-derived, phenol)³⁸
• Bakuchiol (plant-based)³⁸
• 4-O-D-(6-galloyl)-benzoic acid glucopyranoside (plant-derived, phenol)³⁸
• Brevifolin (herbal)³⁸
• Cannabidiol (cannabinoid), (Ki: 0.974 µM) (potent)³⁸⁴⁵
  • Nevertheless, CBD (cannabidiol) increased the MPH concentration and AUC only slightly⁴⁶
• cannabinol (cannabinoid), (Ki: 0.263 µM) (potent)³⁸
• , celastrol (triterpenoid), (IC₅₀: 4.43 µM) (potent)³⁸
• Cholesterol (unsaturated fatty acid, weak)⁴⁰
• Corilagina (plant-based)³⁸
• Coryfolin (strong) (Ki: 1.9 µM) (plant-derived, phenol)³⁸
• Corylin (strong) (Ki: 0.7 µM) (plant-based, phenol)³⁸
• Corylifol A (plant-based, phenol)³⁸
• Coryfolin (Ki: 9.4 µM) (plant-derived, phenol)³⁸
• Desmethoxyyangonin (Ki = 25.2 μM)⁴⁷
• Dihydrokavain (Ki = 105.3 μM)⁴⁷
• Dihydromethysticin (Ki = 68.2 μM)⁴⁷
• Dihydrotanshinone (strong) (Ki: 0.39 µM) (Tanshinone)
• Ellagic acid 4-O-D-glucopyranoside (plant-derived, phenol)³⁸
• Euphorbic acid (triterpenoid)
• Euphorbin A (triterpenoid)
• Euphorbin B (triterpenoid)
• Euphorbin C (triterpenoid)
• Fatty acids inhibit CES1⁴⁰
  • particularly unsaturated fatty acids
• Fluoxetine (strong) (IC50: 6.1 µM)⁴⁴⁷
• Gallic acid-4-O–D-(6-O-galloyl)-glucopyranoside (plant-based, phenol)³⁸
• Gallic acid-3-O-D-(6-O-galloyl)-glucopyranoside (plant-derived, phenol, phenol)³⁸
• Gambogic acid³⁸
• Glycyrrhetinic acid (terterpenoid), (IC₅₀: 13 µM) (strong)³⁸
• Isobavachalcone (plant-derived, phenol)³⁸
• Kaempferol (flavonoid), (IC₅₀: 62 µM)³⁸
• Kavain (Ki = 81.6 μM)⁴⁷
• , cryptotanshinone (tanshinone), (Ki: 0.54 µM) (very potent)³⁸
• Kuwanon G (plant-based, phenol)³⁸
• Linoleic acid (strong) (IC50: 9 µM) (plant-derived fatty acid)
• Linolenic acid (IC50: 19 µM) (plant-derived fatty acid)
• , luteolin (flavonoid), (Ki: 5.34 µM) (potent)³⁸
• Methysticin (Ki = 35.2 μM) (kavalactone)⁴⁷
• Miltiron (strong) (Ki: 0.39 µM) (tanshinone)
• Myristic acid (strong) (IC50: 9 µM) (plant-derived fatty acid)
• Myristoleic acid (IC50: 12 µM) (plant-derived fatty acid)
• Naringenin (flavonoid), (IC₅₀: 30 µM)³⁸
• Neobavaisoflavones (strong) (Ki: 5.3 µM) (plant-derived, phenol)³⁸
• Oleic acid (strong) (IC50: 7 µM) (plant-derived fatty acid)
• Oheno (plant-based, phenol)³⁸
• Oleanolic acid (terterpenoid), (IC: 0.28 µM) (strong)³⁸
• Oxysterol⁴⁰
  • Cholesterol metabolite; also inhibits CES1
• Pachyminic acid (triterpenoid), (Ki: 21.7 µM)³⁸
• Paeoveitol B (plant-based, phenol)³⁸
• Palmitic acid (IC50: 25 µM) (plant-derived fatty acid)
• Palmitoleic acid (potent) (IC50: 7 µM) (plant-derived fatty acid)
• Perphenazine (potent) (IC50: 13.9 µM)⁴⁴⁷
• Pyron-2-O–D-(6-galloyl)-glucopyranoside (plant-derived, phenol)³⁸
• Pyron-2-O–D-(2,6-digalloyl)-glucopyranoside (plant-derived, phenol)³⁸
• Pryomeconic acid 3-O–D-glucopyranoside 6-(O-4-hydroxybenzoate) (plant-derived, phenol)³⁸
• Quercetin (flavonoid), (Ki: 33.4 µM)³⁸
• Resveratrol³⁸
• Sanggenon C (plant-based, phenol)³⁸
• Sanggenon D (plant-based, phenol)³⁸
• Scopoletin-7-O–D-(6-galloyl)-glucopyranoside (plant-derived)³⁸
• Sulforaphane³⁸
• Tanshinone I (Tanshinone), (Ki: 26.3 µM)³⁸
• Tanshinon IIA (Tanshinon), (Ki: 6.89 µM) (potent)³⁸
• Tanshinone IIA-sulfonate (Ki: 100 µM) (tanshinone)
• Δ⁹-tetrahydrocannabinol (cannabinoid), (Ki: 0.541 µM) (very potent)³⁸⁴⁵
• Thioridazine (strong) (IC50: 7.0 µM)⁴⁴⁷
• Ursolic acid (triterpenoid), (Ki: 0.24 µM) (potent)³⁸
• Compound 12 (triterpenoid)
• Compound 13 (triterpenoid)
• Yangonin (Ki = 24.9 μM)⁴⁷

3.3. CES1 Inductors¶

A combination of methylphenidate and inducers results in a significant decrease in blood levels of MPH.

• Carmabazine is believed to be an inducer of CES1.⁴⁴
  - Glucose (sugar)⁴⁸
• Phenobarbital (possible)⁴⁴
• Phenytoin (possible)⁴⁴
• Rifampin (possible)⁴⁴
• Sulforaphane compounds (antioxidant)³⁸, sulforaphane (4-methylsulfinylbutyl isothiocyanate; 1-isothiocyanato-4-methylsulfinylbutane) is a dietary and plant-based phytochemical that occurs in plants as a biologically inactive precursor
  • Sulforaphane is a potent CES1 inducer⁴⁹
    - Trinitrobenzenesulfonate (strong)
  • Skin sensitizers, such as those used to test cosmetic products, can increase CES1 levels by up to 20 times.⁵⁰
    - Cinnamaldehyde (strong)
  • Skin sensitizers, such as those used to test cosmetic products, can increase CES1 levels by up to 20 times.⁵⁰