The active component of Euphorbia peplus, ingenol mebutate (also known as ingenol 3-angelate), is a strong PKC activator, with Kis values of 0.3, 0.105, 0.162, 0.376, and 0.171 nM for PKC-α, PKC-β, PKC-γ, PKC-δ, and PKC-ε, in that order. The EC50 of ingenium xylenol methyl butyl ester is 13 ± 2.4 nM (PKC-α), 4.37 ± 0.4 nM (PKC-βI), 10.5 ± 2.2 nM (PKC-βII), 38.6 ± 2.9 nM (PKC-δ) , 1.08 ± 0.01 nM (PKC-ε), 0.9 ± 0.13 nM (PKC-μ), 198 ± 12.5 nM (PKC-α), 69.1 ± 8.2 nM (PKC-βI), 4.6 ± 0.4 nM in WEHI-231 cells (PKC-ε) and 1 nM (PKC-μ), 635 ± 245 nM (PKC-α), 146 ± 35 nM (PKC-βI), 4.7 ± 0.7 nM (PKC-δ), 1.1 in HOP-92 cells ± 0.5 nM (PKC-ε) and 30 nM (PKC-μ) in Colo-205 cells. WEHI-231 cells, HOP-92, and Colo-205 cells are sensitized by ingenol mebutate, with IC50 values of 1.41 ± 0.255 nM, 3.24 ± 2.01 nM, and 11.9 ± 1.307 nM, in that order [1]. PKC-δ-dependent, ingenol mebutate (PEP005; 20 nM) can cause apoptosis in primary AML bone marrow blasts but not in normal myeloblasts [2]. PKCδ is inhibited while PKCα is activated by ingenol mebutate (PEP005). Compared to the parent Colo205-S cells, Colo205-R cells have an IC50 of >10 μM, which makes them more than 300 times more resistant to ingenol methyl butyl ester [3].

Absorption, Distribution and Excretion
Because indomethacin methyl ester is a topical drug, its systemic absorption is less than 0.1 ng/mL. Because indomethacin methyl ester is a topical drug, there is no elimination pathway. Because indomethacin methyl ester is a topical drug, there is no volume of distribution. Because indomethacin methyl ester is a topical drug, there is no clearance. Human plasma clearance and volume of distribution (steady-state) were estimated using simple allometric growth correlation based on body weight. Using a one-compartment model of first-order absorption and elimination kinetics, it was estimated that a daily maximum expected clinical dose of 2 μg/kg administered topically would result in a blood concentration of indomethacin methyl ester below the lower limit of quantitation (LLOQ) of 0.1 ng/mL. The expected steady-state plasma clearance and volume of distribution are approximately 0.22 to 1.01 L/hr/kg and 0.61 L/kg, respectively. The expected absorption rate constant and local bioavailability are 0.0277 h⁻¹ and 0.21%, respectively. For a local dose of 2 μg/kg/day, the expected time to peak plasma concentration (Tmax) is 2 hours, and the expected peak plasma concentration (Cmax) is 0.107 pg/mL. A minimum local dose of 2000 μg/kg/day is required to achieve detectable plasma concentrations in humans. Following intravenous administration, high to very high plasma clearance, moderate to high steady-state volume of distribution, and short half-life were observed in rats, rabbits, dogs, and miniature pigs. In rats, after intravenous injection of 3(H)-ingenol mebutate, the drug-associated radioactive material was uniformly distributed in tissues with no sex difference in affected organs, but female rats showed faster clearance. In vitro studies showed that Ingenol methyl ester and its isomers have high plasma protein binding rates (>99%) in rats, dogs, miniature pigs, and humans. In rats, intravenously administered 3(H)-ingrammidine methyl ester is primarily excreted via the bile route, with minimal urinary excretion. When 0.01%, 0.1%, or 0.05% of PEP005 gel was applied to the skin in rats, miniature pigs, and humans, transdermal absorption was generally low, ranging from 0.04% (miniature pigs) to 8.68% (rats) in different animal species, and from 0.16% to 1.93% in humans. The absorption dose order of 3(H)-ingrammidine methyl ester was: WI rats > SD rats > humans > miniature pigs. In miniature pigs, methyl ester was typically undetectable in the blood after topical application of PEP005 gel, and even when detected, the concentration was only 0.1 ng/mL. In rats, methyl ester was only stably detectable in the blood at doses of 300 μg/kg or higher, at which point its absolute bioavailability was 2% to 4%.
In two studies, a total of 16 subjects with actinic keratosis (AK) were included to assess systemic exposure to 0.05% Picato gel. Subjects applied approximately 1 g of 0.05% Picato gel once daily for two consecutive days to a 100 cm² area on the back of the forearm. In these studies, the concentrations of indomethacin methyl ester and its two metabolites (acyl isomers of indomethacin methyl ester) in the blood were determined. In all subjects' blood samples, the blood concentrations of indomethacin methyl ester and its two metabolites were below the lower limit of quantification (0.1 ng/mL).
Metabolism/Metabolites

Because indomethacin methyl ester is a topical drug and does not inhibit or induce most cytochrome P450 (CYP) enzymes, Picato is not metabolized.
In vitro, the metabolism of indomethacin methyl ester in the blood, skin homogenates, and hepatocytes of rats, dogs, miniature pigs, and humans is similar in nature. Indomethacin methyl ester is relatively stable in blood and skin homogenates, but undergoes extensive metabolism in cryopreserved hepatocytes. In rat, dog, and miniature pig hepatocytes, the primary metabolic pathway is hydrolysis to indomethacin, while in humans, the primary metabolic pathway is hydroxylation of indomethacin methyl ester. In the skin of rats, dogs, miniature pigs, and humans, indomethacin methyl ester is predominantly rearranged to PEP015 (approximately 26% to approximately 31%), with a minor rearrangement to PEP025 (approximately 1% to approximately 2%); the amount hydrolyzed to indomethacin is minimal (0% to 0.81%). However, PEP025 was not detected after local or intravenous injection of indomethacin methyl ester in rats and miniature pigs, and the concentration of PEP015 in the blood was less than 10% of the corresponding indomethacin methyl ester concentration.
Biological Half-Life
Since indomethacin methyl ester is a topical drug, no half-life data are available.

Toxicity Summary
Identification and Uses: Indomethacin methyl ester is a white to pale yellow crystalline powder. It is used as a gel in the form of the drug Picato for the topical treatment of actinic keratosis. Human Exposure and Toxicity: Three pharmacological studies in healthy volunteers demonstrated that indomethacin methyl ester gel has a good topical safety profile, with no evidence of skin sensitization, photosensitivity, or photosensitivity reactions. However, topical overdose may lead to an increased incidence of local skin reactions. Animal Studies: In rats administered intravenously for 28 consecutive days, treatment-related effects included transient tachypnea (dose-independent), somnolence and/or lethargy, and decreased food intake. In mice administered for 7 consecutive days, one animal receiving a dose of 60 μg/kg/day and all animals receiving doses ≥80 μg/kg/day were prematurely euthanized ≤4 days after administration due to dose-related serious signs. In miniature pigs, no deaths occurred after administration of methyl inflamb at 5 μg/kg/day for 4 consecutive days or 3 μg/kg/day for 28 consecutive days. Treatment-related effects were limited to occasional transient behavioral lethargy, vomiting, and a slight decrease in weight gain following administration of a 2.5 μg/kg/day dose. In pregnant rabbits, intravenous administration of methyl inflamb resulted in increased embryo-fetal mortality and an increased incidence of fetal visceral and skeletal variations. In pregnant rats, no treatment-related embryo-fetal toxicity or teratogenic effects were observed after intravenous administration. Methyl inflamb was not found to be mutagenic in in vitro Ames assays, mouse lymphoma assays, or in vivo rat micronucleus assays. In vitro Syrian hamster embryo (SHE) cell transformation assays were positive. A 6-month repeated intravenous administration study involving 154 rats found that one male and one female rat administered a 15 μg/kg dose twice weekly developed renal tubular adenoma and renal tubular hyperplasia. Female rats with renal adenomas also had pituitary adenomas. One male rat developed follicular thyroid carcinoma at euthanasia after a 1-month recovery period. Effects during Pregnancy and Lactation ◉ Overview of Use During Lactation No studies have been conducted on topical use of indomethacin during lactation. However, serum concentrations are undetectable after topical administration, so lactation is not expected to expose the infant to the drug. If the mother needs to use indomethacin, this is not a reason to discontinue breastfeeding. Do not apply indomethacin to the breast or nipples, and ensure that the infant's skin does not come into direct contact with the applied area. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found. ◉ Effects on Lactation and Breast Milk As of the revision date, no relevant published information was found. ◉ Protein Binding Because indomethacin is a topical medication, there is no plasma protein binding data.

[1]. Characterization of the interaction of ingenol 3-angelate with protein kinase C. Cancer Res. 2004 May 1;64(9):3243-55.

[2]. PEP005, a selective small-molecule activator of protein kinase C, has potent antileukemic activity mediated via the delta isoform of PKC. Blood. 2005 Aug 15;106(4):1362-8.

[3]. Epithelial-to-mesenchymal transition and resistance to ingenol 3-angelate, a novel protein kinase C modulator, in colon cancer cells. Cancer Res. 2009 May 15;69(10):4260-9.

Therapeutic Uses
ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure under investigation); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (for providing patient health information) and PubMed (for providing citations and abstracts of academic articles in the medical field). Indomethacin methyl ester is listed in this database. Picardo gel is indicated for the topical treatment of actinic keratosis. /Included in US Product Labelling/
Note: Although antiretroviral therapy (ART) is highly effective in suppressing HIV replication, it cannot eradicate the virus from the body of HIV-infected individuals. A stable latent HIV reservoir can be rapidly established early in HIV infection. Therefore, there is an urgent need for effective strategies to eradicate the HIV reservoir. We report that indomethacin-3-angelic acid ester (PEP005), the only active ingredient in Picato, a previously FDA-approved topical treatment for precancerous actinic keratosis, effectively activates latent HIV in vitro and in vitro with relatively low cytotoxicity. Biochemical analysis shows that PEP005 activates latent HIV by inducing the pS643/S676-PKCδ-8-1κBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone can induce the expression of fully extended and processed HIV RNA in primary CD4+ T cells of HIV-infected individuals receiving antiretroviral therapy (ART). Furthermore, PEP005 exhibits a synergistic effect with the P-TEFb agonist JQ1 in activating latent HIV, with a combined effect 7.5 times greater than PEP005 alone. Conversely, PEP005 inhibits HIV infection in primary CD4+ T cells by downregulating the cell surface expression of HIV co-receptors. This anticancer compound holds promise as a candidate drug for advancing HIV eradication strategies.
Experiment/The purpose of this study was to evaluate the safety of two applications of PEP005 (methyl indomethacin) gel for the treatment of superficial basal cell carcinoma. Efficacy was a secondary endpoint. This randomized, excipient-controlled phase IIa study, conducted in eight private dermatology clinics in Australia, enrolled 60 patients with histologically confirmed superficial basal cell carcinoma (lesion size 4-15 mm). Patients were randomly assigned to either group A (treatment on days 1 and 2) or group B (treatment on days 1 and 8) and received 0.0025%, 0.01%, or 0.05% methyl ester iniquinol gel or excipient gel, respectively. The primary efficacy endpoint was the incidence and severity of adverse events and local skin reactions in groups A and B; lesion clearance at day 85 was a secondary efficacy endpoint. The incidence of adverse events was low. One patient in group A receiving 0.05% methyl ester iniquinol gel experienced severe desquamation/scaling/dryness, with symptoms extending beyond the application site. Non-serious, possibly treatment-related adverse events included erythema spreading beyond the application site, application site pain, and headache, with two patients in each group experiencing these events. Six patients in group A experienced one or more serious local skin reactions. Efficacy appeared to be dose-related, with higher clinical and histological lesion clearance in group A than in group B. Of the 8 patients randomly assigned to the 0.05% methyl ester inglucan gel group in group A, 5 (63%) achieved histological clearance. Two doses of 0.05% methyl ester inglucan gel were safe and effective in patients with superficial basal cell carcinoma.
Drug Warnings

Post-marketing reports of hypersensitivity reactions, including anaphylactic shock and anaphylactic contact dermatitis. If anaphylactic shock or other clinically significant hypersensitivity reactions occur, Picato gel should be discontinued immediately and appropriate medical treatment initiated.
Avoid use in the periorbital area.
Eye problems may occur after contact, including severe eye pain, chemical conjunctivitis, corneal burns, eyelid edema, ptosis, and periorbital edema.
The following adverse reactions have been observed post-marketing with 0.015% and 0.05% Picato (methyl ester) gel: hypersensitivity reactions, anaphylactic contact dermatitis, herpes zoster, chemical conjunctivitis, and corneal burns.
Severe skin reactions may occur at the treatment site after topical application of Picato gel, including erythema, crusting, swelling, blisters/pustules, and erosion/ulceration. Picato gel is not recommended for use until the skin has fully healed from previous medical or surgical treatment.
For more complete data on drug warnings for methyl influoride (12 in total), please visit the HSDB record page.

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Pharmacodynamics
The pharmacodynamic mechanism by which methyl influoride induces cell death in actinic keratosis is unknown.

C25H34O6

430.53

430.235

75567-37-2

Ingenol;30220-46-3

6918670

White to off-white solid powder

1.3±0.1 g/cm3

576.9±50.0 °C at 760 mmHg

191.4±23.6 °C

0.0±3.6 mmHg at 25°C

1.594

5.18

3

6

4

31

926

8

C/C=C(/C)\C(=O)O[C@H]1C(=C[C@@]23[C@@]1([C@@H](C(=C[C@H](C2=O)[C@H]4[C@H](C4(C)C)C[C@H]3C)CO)O)O)C

VDJHFHXMUKFKET-WDUFCVPESA-N

InChI=1S/C25H34O6/c1-7-12(2)22(29)31-21-13(3)10-24-14(4)8-17-18(23(17,5)6)16(20(24)28)9-15(11-26)19(27)25(21,24)30/h7,9-10,14,16-19,21,26-27,30H,8,11H2,1-6H3/b12-7-/t14-,16+,17-,18+,19-,21+,24+,25+/m1/s1

[(1S,4S,5S,6R,9S,10R,12R,14R)-5,6-dihydroxy-7-(hydroxymethyl)-3,11,11,14-tetramethyl-15-oxo-4-tetracyclo[7.5.1.01,5.010,12]pentadeca-2,7-dienyl] (Z)-2-methylbut-2-enoate

PEP 005; PEP-005; PEP005

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)

DMSO : ≥ 100 mg/mL (~232.27 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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