UDP-glucuronosyltransferase, cytochrome P-450 isozyme[1]; RAR, RXR-alpha[2]

It is suggested that formation of more polar metabolites of all-trans-retinoic acid (atRA) via oxidative pathways limits its biological activity. In this report, we investigated the biotransformation of oxidized products of atRA via glucuronidation. For this purpose, we synthesized 4-Hydroxyretinoic acid/4-hydroxy-RA (4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl acetate (4-OH-RAc), and 5,6-epoxy-RA, all of which are major products of atRA oxidation. Glucuronidation of these retinoids by human liver microsomes and human recombinant UDP-glucuronosyltransferases (UGTs) was characterized and compared with the glucuronidation of atRA. The human liver microsomes glucuronidated 4-OH-RA and 4-OH-RAc with 6- and 3-fold higher activity than atRA, respectively. Analysis of the glucuronidation products showed that the hydroxyl-linked glucuronides of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and 5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to those found in human liver microsomes. We therefore postulate that this human isoenzyme, which is expressed in human liver, kidney, and intestine, plays a key role in the biological fate of atRA. We also propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways [1].

Metabolism of retinoic acid to a less active metabolite, 4-Hydroxyretinoic acid, occurs via cytochrome P-450 isozyme(s). Effect of a pharmacological dose of retinoic acid on the level of retinoic acid in skin and on cytochrome P-450 activity was investigated. A cream containing 0.1% retinoic acid or cream alone was applied topically to adult human skin for four days under occlusion. Treated areas were removed by a keratome and a microsomal fraction was isolated from each biopsy. In vitro incubation of 3H-retinoic acid with microsomes from in vivo retinoic acid treated sites resulted in a 4.5-fold increase (P = 0.0001, n = 13) in its transformation to 4-Hydroxyretinoic acid in comparison to in vitro incubations with microsomes from in vivo cream alone treated sites. This cytochrome P-450 mediated activity was oxygen- and NADPH-dependent and was inhibited 68% by 5 microM ketoconazole (P = 0.0035, n = 8) and 51% by carbon monoxide (P = 0.02, n = 6). Cotransfection of individual retinoic acid receptors (RARs) or retinoid X receptor-alpha (RXR-alpha) and a chloramphenicol acetyl transferase (CAT) reporter plasmid containing a retinoic acid responsive element into CV-1 cells was used to determine the ED50 values for stimulation of CAT activity by retinoic acid and its metabolites. Levels of all trans and 13-cis RA in RA-treated tissues were greater than the ED50 values determined for all three RARs with these compounds. Furthermore, the level of all trans RA was greater than the ED50 for RXR-alpha whereas the 4-OH RA level was greater than the ED50 for RAR-beta and RAR-gamma but less than for RAR-alpha and RXR-alpha. These data suggest that there are sufficient amounts of retinoic acid in treated skin to activate gene transcription over both RARs and RXR-alpha [2].

Human Liver Microsomes [1]
The human liver microsomes used in the experiments were from a 56-year-old man who had died of cerebral bleeding (HLM15) and from a 13-year-old girl who died from brain damage (HLM18). These samples were obtained from the University of Groningen, Groningen, The Netherlands. The HLM served as a control for the glucuronidation assays by providing a basis of comparison for recombinant UGTs.

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Human Recombinant UGTs [1]
Human recombinant UGT1A3 was expressed in a mammalian expression system as described previously. UGT2B7 was expressed in human embryonic kidney (HK293) cells as reported previously. Enriched endoplasmic reticulum membrane fractions were prepared as described previously. The membrane fractions were stored at −80 °C in 5 mm HEPES, 0.25m sucrose, 20 mm MgCl2 (pH 7.4). The enzymatic activity of the recombinant UGT proteins was sustained for up to 6 months under these conditions.

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Enzyme Assays [1]
UGT activity was measured with both radioactive and unlabeled forms of atRA and 4-OH-RA as the aglycons with UDP-GlcUA serving as the sugar donor (retinoid structures shown in Fig. 1). All retinoid substrates were prepared in the form of mixed micelles with Brij 58 (0.12%). The Brij 58 micelles both activated the enzyme and solubilized the retinoids. Human liver microsomes and recombinant UGTs (50 μg of protein) were used in the assays. All enzymatic assays were performed under yellow light. The amount of product formed was less than 10% of total substrate added and was linearly proportional to the amount of microsomal protein added. The retinoid derivatives (0.10 mm final concentration) were incubated in 100 mm HEPES-NaOH, pH 7.5, 5 mm MgCl2, 5 mm saccharolactone, and 0.05% Brij 58 in a final volume of 60 μl. The reaction mixture was preincubated with the proteins at room temperature for 10 min before starting the reaction with the addition of either 50 mm UDP-GlcUA for radioactive retinoids (4.17 mmfinal concentration) or 20 mm [14C]-UDP-GlcUA (3.33 mm final concentration) for unlabeled retinoids. The reactions were incubated for 30 min at 37 °C. Reactions were stopped with 20 μl of ethanol, vortexed and placed on ice. For TLC, 60 μl of the reaction mix was applied to the preadsorbent layer of a 19-channeled silica gel TLC plate (Baker Si250-PA (19C); VWR Scientific) after which the plates were dried and developed twice in chloroform-methanol-glacial acetic acid-water (65:25:2:4, v/v). These TLC conditions allowed for separation of carboxyl- and hydroxyl-linked glucuronides. After development, the plates were dried and subjected to autoradiography for 3–7 days at -80 °C.

Metabolism / Metabolites
4-Hydroxyretinoic acid is a known human metabolite of Tretinoin.

[1]. 4-hydroxyretinoic acid, a novel substrate for human liver microsomal UDP-glucuronosyltransferase(s) and recombinant UGT2B7. J Biol Chem. 2000 Mar 10;275(10):6908-14.

[2]. Human skin levels of retinoic acid and cytochrome P-450-derived 4-hydroxyretinoic acid after topical application of retinoic acid in vivo compared to concentrations required to stimulate retinoic acid receptor-mediated transcription in vitro. J Clin Invest. 1992 Oct;90(4):1269-74.

All-trans-4-hydroxyretinoic acid is a retinoid that consists of all-trans-retinoic acid bearing a hydroxy substituent at position 4 on the cyclohexenyl ring. It has a role as a human metabolite. It is a retinoid and a secondary allylic alcohol. It is functionally related to an all-trans-retinoic acid. It is a conjugate acid of an all-trans-4-hydroxyretinoate.
4-Hydroxyretinoic acid has been reported in Homo sapiens with data available.

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The K m for the formation of the carboxyl-linked glucuronide of atRA is in the low micromolar range (1.3–1.5 μm) both for HLM15 and recombinant UGT2B7. The maximal catalytic rates (V max) for atRA, as determined with HLM15 and recombinant UGT2B7, are 764 and 523 pmol glucuronidated × min−1 × mg protein−1, respectively. These correspond to catalytic efficiencies (V max/K m) of 509 μl x min−1 × mg -1 for HLM15 and 402 μl × min−1 × mg–1 for recombinant UGT2B7, revealing significant efficiency of formation of atRA carboxyl-linked glucuronide. The K m values for 4-OH-directed glucuronidation are 273 and 221 μm for HLM15 and recombinant UGT2B7, respectively. Typical V maxvalues were determined to be in the low nanomolar range (2176 and 1709 pmol × mg−1 × min−1 for HLM15 and recombinant 2B7, respectively), leading to a much lower efficiency of formation for the hydroxyl-linked glucuronides, as shown by aV max/K m of 8 μl x min−1 × mg–1 for both HLM15 and recombinant UGT2B7. In general, the presence of the hydroxyl group in the retinoid moiety switches the site of glucuronidation from carboxyl to hydroxyl and reverses the affinity of the UGT(s) involved. The corresponding catalytic efficiencies (V max/K m) were several hundredfold higher for glucuronidation of the carboxyl function of atRA than for glucuronidation of the 4-OH moiety of the hydroxylated retinoid.

Of the UGT isoforms investigated to date, human recombinant UGT2B7 has the highest capacity to glucuronidate atRA and 4-OH-RA. UGT2B7 activities toward atRA and 4-OH-RA are similar to the reported activities in human liver microsomes, suggesting that UGT2B7 plays a key role in metabolizing atRA and 4-OH-RA to the carboxyl-linked RAG and the hydroxyl-linked 4-OH-RAG. UGT2B7 is capable of catalyzing the biosynthesis of the hydroxyl-linked glucuronide when 4-OH-RA is the substrate or the carboxyl-linked glucuronide when atRA is the substrate. Recent studies on glucuronidation of steroid hormones and fatty acids by UGT2B7 have shown that this isoform is actively involved in the formation of both hydroxyl- and carboxyl-linked glucuronides of those lipophilic substrates (30). Taken collectively, retinoids, steroid hormones, and fatty acids are important ligands involved in initiating cellular signaling events. We postulate that UGT2B7 may be involved in controlling intracellular levels of ligands, such as steroids and atRA. If this is the case, it may also be involved in a feedback loop that controls the amounts of ligands available for steroid and retinoid receptors.

In summary, we speculate that atRA induces its own oxidative metabolism via a cytochrome P450, CYP26, mechanism followed by a UGT-dependent mechanism. The hydroxyl-linked glucuronide of 4-OH-RA is the directed product of atRA metabolism by CYP26. Thus, 4-OH glucuronidation of 4-OH-RA terminates the biological activity of atRA, while the carboxyl-linked glucuronide of atRA might be a biologically active compound involved in cellular processes. Thus, CYP26 and UGT2B7 may together play a crucial role in the metabolism and biological fate of atRA. [1]

C20H28O3

316.43

316.204

66592-72-1

6438629

White to yellow solid powder

1.075g/cm3

506.5ºC at 760 mmHg

274.2ºC

1.574

4.573

2

3

5

23

598

0

CC1=C(C(CCC1O)(C)C)/C=C/C(=C/C=C/C(=C/C(=O)O)/C)/C

KGUMXGDKXYTTEY-FRCNGJHJSA-N

InChI=1S/C20H28O3/c1-14(7-6-8-15(2)13-19(22)23)9-10-17-16(3)18(21)11-12-20(17,4)5/h6-10,13,18,21H,11-12H2,1-5H3,(H,22,23)/b8-6+,10-9+,14-7+,15-13+

(2E,4E,6E,8E)-9-(3-hydroxy-2,6,6-trimethylcyclohexen-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid

4-Hydroxyretinoic acid; all-trans-4-hydroxyretinoic acid; 4-hydroxy-Retinoic acid; Retinoic acid, 4-hydroxy-; 4-OH-retinoate; GT84HX78DR; 4-hydroxy-Retinoate; ...; 66592-72-1;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)