PKC ( EC50 = 11.7 nM ); NF-κB; SphK; protein kinase C (PKC)

PMA induces Thy-1 up-regulation, raises Thy-1 mRNA and protein levels in endothelial cells, and prevents the formation of capillary-like tubes and endothelial cell migration.[1]

PMA first increases endothelial cell migration in the zebrafish model, then it activates the PKC-δ/Syk/NF-κB-mediated pathway to up-regulate Thy-1, which in turn prevents endothelial cell migration.[1]

Researchers previously showed that overexpression of Thy-1 inhibited and knock-down of Thy-1 enhanced endothelial cell migration. Here, Researchers used phorbol-12-myristate-13-acetate (PMA) as an inducer for Thy-1 expression to investigate molecular mechanisms underlying Thy-1 up-regulation. The data showed that increased levels of Thy-1 mRNA and protein in endothelial cells were observed at 14-18 hours and 20-28 hours after PMA treatment, respectively. Treatment with PMA for 32 hours induced Thy-1 up-regulation and inhibited capillary-like tube formation and endothelial cell migration. These effects were abolished by Röttlerin (a PKC-δ inhibitor), but not Gö6976 (a PKC-α/β inhibitor). Moreover, pre-treatment with Bay 61-3606 (a Syk inhibitor) or Bay 11-7082 (a NF-κB inhibitor) abolished the PMA-induced Thy-1 up-regulation and migration inhibition in endothelial cells. Using the zebrafish model, Researchers showed that PMA up-regulated Thy-1 and inhibited angiogenesis through the PKC-δ-mediated pathway. Surprisingly, they found that short-term (8-10 hours) PMA treatment enhanced endothelial cell migration. However, this effect was not observed in PMA-treated Thy-1-overexpressed endothelial cells. Taken together, our results suggest that PMA initially enhanced endothelial cell migration, subsequently activating the PKC-δ/Syk/NF-κB-mediated pathway to up-regulate Thy-1, which in turn inhibited endothelial cell migration. Our results also suggest that Thy-1 might play a role in termination of angiogenesis.[7]

Monolayer cultured αT3-1 and LβT-2 cells are grown in DMEM in a humidified incubator with 5% CO2 at 37°C. Serum starvation lasts 16 hours when 0.1% FCS is added to the same medium. Then, for the duration specified, GnRH and PMA are added. αT3-1 cells can be transfected temporarily using either jetPRIME or ExGen 500, whereas LβT2 cells can only be transfected using the jetPRIME transfection reagent. In experiments involving dominant-negative (DN) PKCs, 1.5 μg of p38α-GFP or 3 μg of the DN-PKCs constructs are transfected into αT3-1 cells (in 6 cm plates) in combination with pCDNA3, the control vector. Transfections of LβT2 cells are carried out (in 10 cm plates) using 4 μg of p38α-GFP in combination with 9 μg of either the DN-PKCs constructs or the control vector, pCDNA3. The cells are serum starved (0.1% FCS) for 16 hours approximately 30 hours after transfection. They are then stimulated with GnRH or PMA, twice washed with ice-cold PBS, treated with the lysis buffer, and then subjected to one freeze-thaw cycle. After harvesting the cells, the supernatants are taken for immunoprecipitation experiments and centrifuged at 15,000 x g for 15 minutes at 4°C.[7]

Rats: Male Wistar rats weighing between 250 and 280 grams are used in all experiments. Seven groups of fifteen thirty-five Wistar rats are randomly assigned. (1) A 0.9% normal saline injection is administered to rats in the sham group (n = 21); (2) A 0.9% normal saline injection is administered to rats in the IR group (n = 21) 30 minutes prior to middle cerebral artery occlusion (MCAO); (3) A lateral cerebral ventricle injection of CBX (5 μg/mL×10 μL) is administered to rats in the Carbenoxolone (CBX) group (n = 21) 30 minutes prior to MCAO; (4) Rats in the Sch-6783 group (n = 21) receive a lateral cerebral ventricle injection of DZX (2 mM×30 μL) 30 minutes before MCAO; (5) Rats in the 5-HD group (n = 21) receive a lateral cerebral ventricle injection of 5-HD (100 mM×10 μL); (6) The rats in the DZX + Ro group (n = 15) receive a lateral cerebral ventricle injection of DZX, and after 10 min, Ro-31-8425 (400 μg/kg) is injected 15 minutes before MCAO; (7) Rats in the 5-HD+PMA group (n = 15) receive an intraperitoneal injection of PMA (200 μg/kg) following the injection of 5-HD and DZX.

Absorption, Distribution and Excretion
...Mouse skin localization expt...determined that at 3-6 hr after skin application /with tritiated PMA/ the keratin layer just above basal cells was highly labeled, & sebaceous glands & hair follicles were moderately labeled. After 48 hr there was still some labeling in sebaceous glands & hair follicles. Half-life of...promoter was close to 24 hr.

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Metabolism / Metabolites
...the major pathway in the metabolism of TPA is the hydrolysis of the two ester groups, ... in the rodent skin model all hydrolytic products lack tumor promoting activity, the major toxicological effect of TPA. The metabolic hydrolysis requires the activity of esterases, the activity of which differs between tissues and species. ... both ester groups of TPA can be hydrolysed in mouse skin and in cultured cells, giving rise to the monoesters 12-tetradecanoylphorbol and phorbol-13-acetate, as well as the product of complete hydrolysis, i.e. phorbol. Reduction of the keto group at C-3 was identified as a further metabolic pathway in mouse skin. ... Noteworthy, no other metabolites were detected in the microsomal incubations, suggesting that cytochrome 450-mediated oxidative metabolism is not involved in TPA metabolism. Ester group hydrolysis was also the only metabolic reaction observed in various cultured cells ... .
... the hydrolysis of TPA paralleled the loss of activity for induction of ornithine decarboxylase (ODC). As ODC is a marker for tumor promotion, these findings suggest that all three hydrolytic metabolites of TPA (the two monoesters and phorbol) are devoid of tumor promoting activity. Marked differences in the rate of hydrolysis of TPA and a structural analogue, phorbol-12,13-didecanoate (PDD) were observed between cultured fibroblasts from various animal species, suggesting that the hydrolytic metabolism of phorbol diesters depends on the cell type and on the chemical structure of the diester ... .
... the metabolism of radiolabeled TPA /was studied/ in the back skin of mice in vivo. In addition to hydrolytic metabolites, several novel lipophilic metabolites were detected and identified as TPA esterified with long chain fatty acids at the C-20 hydroxyl group. These TPA-20-acylates appeared to be devoid of tumor promoting activity but were partly hydrolysed back to TPA in mouse skin ... .
The few in vitro metabolism studies of TPA involving human cells indicate that many human cell lines in culture do not metabolize TPA to an appreciable extent ... .

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Biological Half-Life
... after /mouse/ skin application /with tritiated PMA/... Half-life of ... promoter was close to 24 hr.
A terminal half-life of 11 +/- 3.9 hours was calculated (from five infusions in four patients) ... .

Toxicity Summary
IDENTIFICATION AND USE: TPA is a colorless powder. It is used in cancer research to study mechanisms of tumor promotion, to screen for potential inhibiting agents, and as positive control for tumor promoting agents. It has been tested as experimental medication for the treatment of leukemias and lymphomas and other types of cancer. HUMAN EXPOSURE AND TOXICITY: TPA is a human platelet aggregating agent. TPA was used in clinical trials in humans suffering from recurrent malignancies, particularly hematological malignancies including severe forms of leukemia. The objective of this trial was the use of TPA as an agent to induce, at low doses,apoptosis and cell differentiation. The TPA application was based on current protocols for cytostatic agents, and involved 35 patients given a low dose constant rate infusion over a defined period. Various patients developed severe side effects following the treatment, such as transient fatigue, anemia, neutropenia and thrombocytopenia, mild dyspnea, nausea fever, rigor and cardiovascular effects with syncope and hypotension, but only one patient exhibited a tumor response, consisting in a reduction in mass dimensions. TPA is routinely used in human cell studies in vitro. ANIMAL STUDIES: TPA has been recognized as a tumor promoter in a mouse skin bioassay and in the mouse forestomach as well as in in vitro cell proliferation assays. However, there was no evidence for tumor-initiating properties of TPA. Tumor initiation and promotion was investigated in the epithelium of the forestomach of mice treated intragastrically with a single dose of 7,12-dimethylbenz [a]anthracene(DMBA) at 50 mg TPA/kg bw followed by repeated dosing (twice per week) for 35 weeks of TPA at 10 mg/kg bw. Forty-five out of 50 mice which received this treatment had tumors (papillomas) in the forestomach. There were no forestomach tumors noted for mice in the untreated control and the TPA-only groups, although in the DMBA-only group, papillomas were observed in the forestomach of 10 mice. TPA was not demonstrated to be a genotoxicant. Clastogenic, mutagenic and sister chromatid exchange-inducing effects of TPA have been shown in some experimental systems but are mediated by secondary products (possibly from arachidonic acid) formed by the cell, only under culture conditions with low antioxidant content in culture media and sera, in response to the tumor promoter. When tested in whole rat embryo culture, TPA exposure led to reduced prosencephalon, growth retardation and incomplete axial rotation in the body. An abundance of embryonic E-cadherin mRNA was found after culture. TPA is routinely used in animal cell studies in vitro.

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Interactions
To determine effect of interval between initiation & promotion & the effect of aging of mice in two-stage carcinogenesis, 20 ug 7,12-dimethylbenz(a)anthracene (initiator) was applied once only & 2.5 ug PMA was applied 3 times/wk to dorsal skin of 5 groups of female ICR/ha Swiss mice. For groups 1, 2, 3, 4 & 5, age (in wk) at primary treatment (initiator) was 6, 44, 56, 6 & 6 respectively; interval (in wk) to secondary treatment (promotor) was 2, 2, 2, 36 & 56 respectively; number of mice/group were 120, 20, 50, 35 & 35 respectively; % mice with papillomas was 100, 100, 56, 90 & 57 respectively; % mice with squamous carcinoma was 50, 30, 6, 25 & 11 respectively. Appropriate control groups consisted of one agent only given at various time intervals. Results show that skin carcinomas are induced whether interval between initiation & promotion is 2, 36 or 56 wk. Carcinoma incidences are significantly lower in groups 3 & 5 where secondary treatment was started when animals were 58 & 62 wk old. /From table/
Because endogenous proteases may play role in mechanism of action of tumor promotors, 3 known protease inhibitors were tested for ... inhibitory effects in two-stage carcinogenesis. Protease inhibitors ... tosyl chloromethyl ketone, tosyl phenylalanine chloromethyl ketone, & tosyl arginine methyl ester ... were applied to mouse ears after initiation with single dose of 7,12-dimethylbenz(a)anthracene followed by promotion with ... PMA. Inhibitors were applied 3 times/wk immediately after application of promoting agent. Protease inhibitors delayed appearance of 1st tumors, changed general pattern of rate of tumor appearance, & caused some decr in tumor incidences.
Low dosages of sulfur mustard, bis(beta-chloroethyl)sulfide completely inhibited two-stage carcinogenesis in mouse skin. 30 Female ICR/ha mice per group received either control applications or different combinations of initiator, promotor & inhibitor test compd for 400 days. 7,12-Dimethylbenz(a)anthracene (DMBA), 20 ug/0.1 mL acetone, applied once only was used as initiator. PMA, the promotor, was applied at 2.5 ug/0.1 mL acetone 3 times/wk. BCS, bis(beta-chloroethyl)sulfide, at 20 ug/0.1 mL acetone was applied beginning 14 days after initiator. DMBA + PMA + BCS (2 times/wk) produced papillomas in 1 mouse with 1st tumor occurring at 90 days; DMBA + PMA + BCS (3 times/wk) produced papillomas in 2 mice occurring at 209 days; DMBA + PMA only produced papillomas in 27 mice & squamous cell carcinomas in 16 occurring at 40 days; PMA alone produced papillomas in 4 mice occurring at 218 days; DMBA + BCS (2 times/wk) produced papillomas in 1 mouse at 385 days; DMBA + BCS (3 times/wk) produced no papillomas; BCS alone (3 times/wk) produced papillomas in 1 mouse at 323 days; DMBA + acetone only produced papillomas in 1 mouse at 219 days; no papillomas were observed in control group admin acetone alone or in group which did not receive either of the test compd. /From table/
The aim of the present study was to determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyldithiocarbamate (DDTC) alone or in combination on human pancreatic cancer cells cultured in vitro and grown as xenograft tumors in nude mice. Pancreatic cancer cells were treated with either DDTC or TPA alone, or in combination and the number of viable cells was then determined by trypan blue ecxlusion assay and the number of apoptotic cells was determined by morphological assessment by staining the cells with propidium iodide and examining them under a fluorescence microscope. Treatment with DDTC or TPA alone inhibited the growth and promoted the apoptosis of pancreatic cancer cells in a concentration-dependent manner. These effects were more prominent following treatment with TPA in combination with DDTC than following treatment with either agent alone in PANC-1 cells in monolayer cultures and in 3 dimensional (3D) cultures. The potent effects of the combination treatment on PANC-1 cells were associated with the inhibition of nuclear factor-kappaB (NF-kappaB) activation and the decreased expression of Bcl-2 induced by DDTC, as shown by NF-kappaB-dependent reporter gene expression assay and western blot analysis. Furthermore, treatment of nude mice with DDTC + TPA strongly inhibited the growth of PANC-1 xenograft tumors. The results of the present study indicate that the administration of TPA and DDTC in combination may be an effective strategy for inhibiting the growth of pancreatic cancer.
For more Interactions (Complete) data for 12-O-TETRADECANOYLPHORBOL-13-ACETATE (16 total), please visit the HSDB record page.

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Human Toxicity Values: LD50 Mouse iv 309 ug/kg

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Adverse Effects: Reproductive Toxin - A chemical that is toxic to the reproductive system, including defects in the progeny and injury to male or female reproductive function. Reproductive toxicity includes developmental effects. See Guidelines for Reproductive Toxicity Risk Assessment.

[1]. Autocrine and paracrine roles of sphingosine-1-phosphate. TRENDS in Endocrinology and Metabolism. Volume 18, Issue 8, October 2007, Pages 300-307. https://doi.org/10.1016/j.tem.2007.07.005

[2]. MPTP-Induced Depletion in Basolateral Amygdala via Decrease of D2R Activation Suppresses GABAA Receptors Expression and LTD Induction Leading to Anxiety-Like Behaviors. Front Mol Neurosci. 2017 Aug 7;10:247.

[3]. Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. J Leukoc Biol. 1996;59(4):555-561.

[4]. Differential roles of PKC isoforms (PKCs) and Ca2+ in GnRH and phorbol 12-myristate 13-acetate (PMA) stimulation of p38MAPK phosphorylation in immortalized gonadotrope cells. Mol Cell Endocrinol. 2017 Jan 5;439:141-154.

[5]. The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium. PLoS One. 2018;13(3):e0193601.

[6]. Mechanism of Mitochondrial Connexin43's Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury. Int J Mol Sci. 2016 May 5;17(5). pii: E679.

[7]. PMA inhibits endothelial cell migration through activating the PKC-δ/Syk/NF-κB-mediated up-regulation of Thy-1. Sci Rep. 2018 Nov 2;8(1):16247.

12-o-tetradecanoylphorbol-13-acetate appears as white crystals. (NTP, 1992)
Phorbol 13-acetate 12-myristate is a phorbol ester that is phorbol in which the hydroxy groups at the cyclopropane ring juction (position 13) and the adjacent carbon (position 12) have been converted into the corresponding acetate and myristate esters. It is a major active constituent of the seed oil of Croton tiglium. It has been used as a tumour promoting agent for skin carcinogenesis in rodents and is associated with increased cell proliferation of malignant cells. However its function is controversial since a decrease in cell proliferation has also been observed in several cancer cell types. It has a role as a protein kinase C agonist, an antineoplastic agent, a reactive oxygen species generator, a plant metabolite, a mitogen, a carcinogenic agent and an apoptosis inducer. It is an acetate ester, a tetradecanoate ester, a diester, a tertiary alpha-hydroxy ketone and a phorbol ester.
Phorbol 12-myristate 13-acetate diester is an inducer of neutrophil extracellular traps (NETs).
Phorbol 12-myristate 13-acetate has been reported in Iris tectorum, Phormidium tenue, and other organisms with data available.
Tetradecanoylphorbol Acetate is a phorbol ester with potential antineoplastic effects. Tetradecanoylphorbol acetate (TPA) induces maturation and differentiation of hematopoietic cell lines, including leukemic cells. This agent may induce gene expression and protein kinase C (PKC) activity. In addition to potential antineoplastic effects, TPA may exhibit tumor promoting activity. (NCI04)
Phorbol Ester is polycyclic compound isolated from croton oil in which two hydroxyl groups on neighboring carbon atoms are esterified to fatty acids. The commonest of these derivatives is phorbol myristoyl acetate (PMA). Potent co carcinogens or tumor promotors, they are diacyl glycerol analogs and activate protein kinase C irreversibly.
A phorbol ester found in CROTON OIL with very effective tumor promoting activity. It stimulates the synthesis of both DNA and RNA.

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Mechanism of Action
The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a differential role on the regulation of the cell cycle in a variety of tumor cells. The mechanism between TPA and the cell cycle in breast cancer is not fully understood. Therefore, we investigated the regulatory mechanism of TPA on control of the cell cycle of breast cancer cells. Our results showed that TPA increased the level of p21 expression in MCF-7 cells with wild-type p53 and MDA-MB-231 cells with mutant p53 in a dose-dependent manner. In contrast, TPA decreased the expression of p53 in MCF-7 cells, but did not affect MDA-MB-231 cells. We next examined the regulatory mechanism of TPA on p21 and p53 expression. Our results showed that the TPA-induced up-regulation of p21 and down-regulation of p53 was reversed by UO126 (a MEK1/2 inhibitor), but not by SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor), although TPA increased the phosphorylation of ERK and JNK in MCF-7 cells. In addition, the TPA-induced arrest of the G2/M phase was also recovered by UO126 treatment. To confirm the expression of p21 through the MEK/ERK pathway, cells were transfected with constitutively active (CA)-MEK adenovirus. Our results showed that the expression of p21 was significantly increased by CA-MEK overexpression. Taken together, we suggest that TPA reciprocally regulates the level of p21 and p53 expression via a MEK/ERK-dependent pathway. The up-regulation of p21 in response to TPA is mediated through a p53-independent mechanism in breast cancer cells.

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Therapeutic Uses
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. 12-O-Tetradecanoylphorbol-13-acetate is included in the database.
/EXPL THER/ Phorbol esters activate protein kinase C and modulate a variety of downstream cell signaling pathways. 12-O-tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester that induces differentiation or apoptosis in a variety of cell lines at low concentrations. A phase I dose escalation trial of TPA was undertaken for patients with relapsed or refractory malignancies. The starting dose was 0.063 mg/sq m and most patients were treated with an intravenous infusion of TPA on days 1-5 and 8-12 followed by a 2-week rest period prior to retreatment. Thirty-five patients were treated. A biological assay was used to monitor levels of TPA-like activity in the blood after treatment. Serious adverse events included individual episodes of gross hematuria, a grand mal seizure, syncope, and hypotension. Many patients had transient fatigue, mild dyspnea, fever, rigors, and muscular aches shortly after the infusion. Dose-limiting toxicities included syncope and hypotension at a dose of 0.188 mg/sq m. Only a single patient had evidence of tumor response. These studies establish 0.125 mg/sq m as the maximally tolerated dose when TPA is administered on this schedule.

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We examined the role of PKCs and Ca2+ in GnRH-stimulated p38MAPK phosphorylation in the gonadotrope derived αT3-1 and LβT2 cell lines. GnRH induced a slow and rapid increase in p38MAPK phosphorylation in αT3-1 and LβT2 cells respectively, while PMA gave a slow response. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs), has revealed differential role for PKCα, PKCβII, PKCδ and PKCε in p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in p38MAPK phosphorylation may be explained by differential localization of the PKCs. Basal, GnRH- and PMA- stimulation of p38MAPK phosphorylation in αT3-1 cells is mediated by Ca2+ influx via voltage-gated Ca2+ channels and Ca2+ mobilization, while in the differentiated LβT2 gonadotrope cells it is mediated only by Ca2+ mobilization. p38MAPK resides in the cell membrane and is relocated to the nucleus by GnRH (∼5 min). Thus, we have identified the PKCs and the Ca2+ pools involved in GnRH stimulated p38MAPK phosphorylation.[4]

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THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. However, the conditions used for differentiation, particularly the concentration of PMA and the duration of treatment, vary widely. Here we compare several differentiation conditions and compare the ability of THP-1 macrophages to interact with the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The results show that THP-1 macrophages differentiated in high concentrations of PMA rapidly died following infection whereas those differentiated in low concentrations of PMA survived and were able to control the intracellular bacteria similar to primary human macrophages.[5]

C36H56O8

616.8251

616.397

C, 70.10; H, 9.15; O, 20.75

16561-29-8

27924

White to off-white solid powder

1.2±0.1 g/cm3

698.1±55.0 °C at 760 mmHg

162 °F (NTP, 1992)
50-70 °C (melting pt-freezing pt)

208.1±25.0 °C

0.0±5.0 mmHg at 25°C

1.553

7.71

3

8

17

44

1150

8

O(C(C([H])([H])[H])=O)[C@@]12[C@@]([H])([C@@]([H])(C([H])([H])[H])[C@@]3([C@]4([H])C([H])=C(C([H])([H])[H])C([C@]4(C([H])([H])C(C([H])([H])O[H])=C([H])[C@@]3([H])[C@]1([H])C2(C([H])([H])[H])C([H])([H])[H])O[H])=O)O[H])OC(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])=O

PHEDXBVPIONUQT-RGYGYFBISA-N

InChI=1S/C36H56O8/c1-7-8-9-10-11-12-13-14-15-16-17-18-29(39)43-32-24(3)35(42)27(30-33(5,6)36(30,32)44-25(4)38)20-26(22-37)21-34(41)28(35)19-23(2)31(34)40/h19-20,24,27-28,30,32,37,41-42H,7-18,21-22H2,1-6H3/t24-,27+,28-,30-,32-,34-,35-,36-/m1/s1

[(1S,2S,6R,10S,11R,13S,14R,15R)-13-acetyloxy-1,6-dihydroxy-8-(hydroxymethyl)-4,12,12,15-tetramethyl-5-oxo-14-tetracyclo[8.5.0.02,6.011,13]pentadeca-3,8-dienyl] tetradecanoate

TPA; NSC262244; PD616; PMA; Phorbol myristate acetate; NSC 262244; Phorbol 12-myristate 13-acetate; 16561-29-8; phorbol-12-myristate-13-acetate; 12-O-Tetradecanoylphorbol-13-acetate; 12-O-Tetradecanoylphorbol 13-acetate; Tetradecanoylphorbol acetate; Phorbol ester; Factor A1; NSC-262244; PD-616; RP-323; PD 616; RP 323; PMA; RP323

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~162.1 mM)
Ethanol: ~100 mg/mL (~162.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.05 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 (4.05 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (4.05 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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Solubility in Formulation 4: ≥ 2.5 mg/mL (4.05 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: 2.5 mg/mL (4.05 mM) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH 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 6: ≥ 2.5 mg/mL (4.05 mM) (saturation unknown) in 10% EtOH + 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 EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 5%DMSO + Corn oil: 5.0mg/ml (8.11mM)

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