In these cells, lipid formation was inhibited by theobromine doses greater than 25 μM. Theobromine has no effect on the viability of cells. Adipogenesis gene expression, C/EBPα, and PPARγ protein expression were all suppressed by theobromine at dosages greater than 25 μM. These genes' mRNA levels are likewise decreased by theobromine [1].

Compared to the excipient group, the theobromine group had a lower body weight. Moreover, theobromine prevented the growth of perirenal and epididymal adipose tissue weight. Compared to the vehicle group, the theobromine group's average adipocyte area was lower [1]. The theobromine group had lower counts than the other groups (p=0.021 and p=0.055 compared to the reference group (RF) and the cocoa group (CC), respectively) when calculating the number of bacteria per unit fecal weight. pH levels were greater following the theobromine diet than following the RF and CC diets. The lactic acid content in feces (RF group, 4.26±1.54 mM; CC group, 1.96±0.41 mM; theobromine group, 2.69±0.73 mM) was not significantly affected by the experimental diet [2].

Absorption, Distribution and Excretion
The ratio of brain:blood theobromine concentrations decreased continuously from 0.96 at birth to 0.60 in 30-day-old rats. After 24 hr, no organ accumulation of theobromine or its metabolites could be seen in adult animals.
Theobromine is absorbed and distributed rapidly after oral administration to rats and equilibrates freely between plasma and testicular fluid.
Similar kinetic parameters were observed in male and female rabbits when theobromine was administered intravenously or orally at doses of 1 and 5 mg/kg bw, with complete gastrointestinal absorption. A reduction in the absorption rate constant was seen in rabbits when the dose was increased from 10 to 100 mg/kg bw. In spite of delayed gastrointestinal absorption at high doses, probably due to the low solubility of the compound, the absolute bioavailability of theobromine approached 100%. Labelled theobromine was almost completely absorbed after oral administration (1-6 mg/kg); the peak blood level tended to appear later with larger doses.
When theobromine was given as a single oral dose of 15-50 mg/kg bw to male dogs, peak plasma concentrations, with considerable individual variations, were observed within 3 hr. With a higher dose (150 mg/kg bw), the peak plasma concentrations were attained 14-16 hr later, showing delayed intestinal absorption. In rats, plasma protein binding was very low (8-17%) after oral administration of 1-100 mg/kg bw theobromine.
For more Absorption, Distribution and Excretion (Complete) data for 3,7-Dimethylxanthine (17 total), please visit the HSDB record page.

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Metabolism / Metabolites
Pregnancy and increased doses of theobromine were shown to modify theobromine metabolism. At a dose of 50 mg/kg bw, pregnant rabbits excreted more unchanged theobromine in the urine (51% versus 35%). Pregnant rats excreted a higher percentage of a 5 mg/kg dose as unchanged theobromine (53%) than non-pregnant rats (39%); this difference disappeared at the saturation dose (100 mg/kg), when unchanged theobromine corresponded to about 60% of the dose in the urine of both pregnant and non-pregnant animals. Rats given 100 mg/kg excreted more unchanged theobromine than those given 1 mg/kg (73% versus 51%), and showed a corresponding relative decrease in excretion of its uracil metabolite, 6-amino-5-(N-methylformylamino)-1-methyluracil (16% versus 28%).
The compounds identified in bile of phenobarbital-treated rats were 3,7-dimethyluric acid (64-76% of biliary radioactivity), dimethylallantoin (5-8%), 6-amino-5-(N-methylformylamino)- 1-methyluracil (10-17%) and theobromine (8-10%). In 3-methylcholanthrene-treated rats, urinary elimination of unchanged theobromine was reduced from 23-27% to only 2%, while excretion of 6-amino-5-(N-methylformylamino)-1- methyluracil was significantly increased. Only 3,7-dimethyluric acid was produced by liver microsomal incubation in control rats while phenobarbital and 3-methylcholanthrene pretreatment enhanced the biotransformation resulting in the production of all metabolites found in vivo as well as unknown polar compounds.
6-Amino-5-(N-methylformylamino)-1-methyluracil is quantitatively the most important theobromine metabolite in rats, accounting for 20-35% of urinary metabolites. The majority of theobromine-derived radioactivity in the feces of rats could be accounted for by 3,7-dimethyluric acid. The most extensive metabolism of theobromine was observed in rabbits and mice; male mice converted theobromine more extensively into this metabolite than did female mice. In contrast, oxidation of theobromine to 3,7-dimethyluric acid was significantly greater in female than in male rats. Rabbits and dogs metabolized theobromine primarily to 7-methylxanthine and 3-methylxanthine, respectively, and dogs excreted small quantities of an unidentified metabolite.
As a metabolite of caffeine, theobromine has been detected in variable amounts in plasma and urine of humans and different animal species.
For more Metabolism/Metabolites (Complete) data for 3,7-Dimethylxanthine (11 total), please visit the HSDB record page.
Theobromine has known human metabolites that include 3,7-Dimethyluric acid, 7-Methylxanthine, and 3-Methylxanthine.
Theobromine is a known human metabolite of caffeine.

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Biological Half-Life
The mean half-time of theobromine in human serum ranged from 6.1 to 10 hr.
The disposition half-life of theobromine averaged 7.1 +/- 2.1 hours ...
In dogs, an average plasma half-time of 17.5 hr was reported after single oral doses of theobromine ranging from 15 to 150 mg/kg bw. In rabbits, the mean elimination half-time was 4.3-5.6 hr for doses ranging from 1 to 100 mg/kg bw.

Toxicity Summary
IDENTIFICATION AND USE: Theobromine is a white powder. It is an alkaloid found in cocoa and chocolate products. Theobromine is used principally to make caffeine . Formerly, theobromine and its derivatives were used in diuretics, myocardial stimulants, vasodilators and smooth muscle relaxants. Theobromine salts (calcium salicylate, sodium salicylate and sodium acetate) were used previously to dilate coronary arteries at doses of 300 to 600 mg per day. There is no current therapeutic use of theobromine. HUMAN EXPOSURE AND TOXICITY: It has been stated that "in large doses" theobromine may cause nausea and anorexia and that daily intake of 50-100 g cocoa (0.8-1.5 g theobromine) by humans has been associated with sweating, trembling and severe headache. Theobromine responses differed according to dose; it showed limited subjective effects at 250 mg and negative mood effects at higher doses. It also dose-dependently increased heart rate. In other studies, theobromine increased HDL-cholesterol concentrations by 0.16 mmol/L. Furthermore, there was a significant theobromine effect on increasing apolipoprotein A-I and decreasing apolipoprotein B and LDL-cholesterol concentrations. In human lymphocyte cultures, theobromine did not significantly increase the number of sister chromatid exchanges per cell, but, in another experiment using higher doses, the numbers of sister chromatid exchanges per cell were increased in the absence of an exogenous metabolic system. Theobromine induced breaks in human lymphocytes in culture, contrary to the results with rodent cells. ANIMAL STUDIES: High doses 250-300 mg/kg bw (mature animals) and 500 mg/kg bw (immature animals) have been shown to cause complete thymic atrophy in male and female rats. This effect was seen in hamsters only at a level of 850 mg/kg bw and in mice at levels of 1840-1880 mg/kg bw. Theobromine administration in rabbits resulted in marked changes in thymus and testes and the severity of lesions appeared to be related to the amounts of the ingested methylxanthine. Other experiments in rats also found the toxic effects of theobromine on the testis. In rats fed diets containing theobromine (daily doses, 53 or 99 mg/kg bw) on gestation days 6-19, no maternal toxicity was observed. Although no malformation occurred, slight decreases in fetal body weight were observed with the high dose, and a significant increase was seen in the frequency of skeletal variations. In rabbits, decreased fetal body weight and malformations were seen at doses of 125 or 200 mg/kg; the incidence of skeletal variations was increased with 75 mg/kg and over. Theobromine was mutagenic to Escherichia coli under conditions in which a constant growth rate and cell population density were maintained, but it was not mutagenic to Salmonella typhimurium. Theobromine induced mutations in a lower eukaryote, Euglena gracilis. Theobromine did not induce chromosomal aberrations in plants (Vicia faba). It was reported in an abstract that chromosomal aberrations were not observed in Drosophila melanogaster treated with 0.45% theobromine in feeding experiments. Chromosomal aberrations were not induced by theobromine in Chinese hamster cells.

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Interactions
Several concentrations of theobromine (TB) and (-)-epicatechin (EC) were coadministered to rats, and plasma EC and its metabolites were determined using ultra-high-performance liquid chromatography-tandem mass spectrometry. It has been demonstrated that TB increases the absorption of EC in a dose-dependent manner. Cocoa powder had a similar effect, and the mechanism involved is not thought to depend on tight junctions.
Experiments were designed to determine the effects of feeding the methylxanthines caffeine, theobromine, or theophylline to 4- to 6-week-old males rats at a dietary level of 0.5 % for periods ranging from 14 to 75 weeks. In the first two experiments, Osborne-Mendel rats were fed the test substances alone or in combination with sodium nitrite to test the hypothesis that these amines might nitrosate in vivo to produce toxic nitrosamine compounds. The compounds failed to produce neoplastic or preneoplastic lesions, but a significant positive finding was the occurrence of severe bilateral testicular atrophy with aspermatogenesis or oligospermatogenesis in 85-100 % of the rats fed caffeine or theobromine. ...
The effects of allopurinol on the plasma clearance and metabolism of theobromine have been investigated under multiple-dosing conditions. Allopurinol had no effect on the clearance of theobromine, indicating that the elimination of this compound is dependent on enzyme systems other than xanthine oxidase, presumably the hepatic mixed-function oxidases. The excretion of 3-methylxanthine, 6-amino-5-(N-methylformylamino)-1-methyluracil, and unchanged theobromine were similarly unaffected by the allopurinol treatment. Although allopurinol abolished the formation of 7-methyluric acid (7MU) and increased the excretion of 7-methylxanthine (7MX), the metabolic clearance to (7MX + 7MU) was not significantly different with and without allopurinol. It is proposed that the secondary biotransformation of 7MX to 7MU is mediated by xanthine oxidase.
The compounds identified in bile of phenobarbital-treated rats were 3,7-dimethyluric acid (64-76% of biliary radioactivity), dimethylallantoin (5-8%), 6-amino-5-(N-methylformylamino)- 1-methyluracil (10-17%) and theobromine (8-10%). In 3-methylcholanthrene-treated rats, urinary elimination of unchanged theobromine was reduced from 23-27% to only 2%, while excretion of 6-amino-5-(N-methylformylamino)-1- methyluracil was significantly increased. Only 3,7-dimethyluric acid was produced by liver microsomal incubation in control rats while phenobarbital and 3-methylcholanthrene pretreatment enhanced the biotransformation resulting in the production of all metabolites found in vivo as well as unknown polar compounds.

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Non-Human Toxicity Values
LD50 Dog oral 300 mg/kg bw
LD50 Rat (acute) oral 950 mg/kg bw

[1]. Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1.

[2]. Effect of cocoa's theobromine on intestinal microbiota of rats. Mol Nutr Food Res. 2017 Oct;61(10).

Theobromine is an odorless white crystalline powder. Bitter taste. pH (saturated solution in water): 5.5-7. (NTP, 1992)
Theobromine is a dimethylxanthine having the two methyl groups located at positions 3 and 7. A purine alkaloid derived from the cacao plant, it is found in chocolate, as well as in a number of other foods, and is a vasodilator, diuretic and heart stimulator. It has a role as an adenosine receptor antagonist, a food component, a plant metabolite, a human blood serum metabolite, a mouse metabolite, a vasodilator agent and a bronchodilator agent.
Theobromine (3,7-dimethylxanthine) is the principle alkaloid in Theobroma cacao (the cacao bean) and other plants. A xanthine alkaloid that is used as a bronchodilator and as a vasodilator. It has a weaker diuretic activity than theophylline and is also a less powerful stimulant of smooth muscle. It has practically no stimulant effect on the central nervous system. It was formerly used as a diuretic and in the treatment of angina pectoris and hypertension. (From Martindale, The Extra Pharmacopoeia, 30th ed, pp1318-9)
Theobromine has been reported in Camellia sinensis, Ilex perado, and other organisms with data available.
3,7-Dimethylxanthine. The principle alkaloid in Theobroma cacao (the cacao bean) and other plants. A xanthine alkaloid that is used as a bronchodilator and as a vasodilator. It has a weaker diuretic activity than THEOPHYLLINE and is also a less powerful stimulant of smooth muscle. It has practically no stimulant effect on the central nervous system. It was formerly used as a diuretic and in the treatment of angina pectoris and hypertension. (From Martindale, The Extra Pharmacopoeia, 30th ed, pp1318-9)
See also: Paullinia cupana seed (part of).

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Drug Indication
theobromine is used as a vasodilator, a diuretic, and heart stimulant. And similar to caffeine, it may be useful in management of fatigue and orthostatic hypotension.

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Mechanism of Action
Theobromine stimulates medullary, vagal, vasomotor, and respiratory centers, promoting bradycardia, vasoconstriction, and increased respiratory rate. This action was previously believed to be due primarily to increased intracellular cyclic 3′,5′-adenosine monophosphate (cyclic AMP) following inhibition of phosphodiesterase, the enzyme that degrades cyclic AMP. It is now thought that xanthines such as caffeine and theobromine act as antagonist at adenosine-receptors within the plasma membrane of virtually every cell. As adenosine acts as an autocoid, inhibiting the release of neurotransmitters from presynaptic sites but augmenting the actions of norepinephrine or angiotensin, antagonism of adenosine receptors promotes neurotransmitter release. This explains the stimulatory effects of xanthine derivatives such as theobromine and caffeine. Blockade of the adenosine A1 receptor in the heart leads to the accelerated, pronounced "pounding" of the heart upon caffeine intake.

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Therapeutic Uses
Diuretic, bronchodilator, cardiotonic. /Former use/
Formerly, theobromine and its derivatives were used in diuretics, myocardial stimulants, vasodilators and smooth muscle relaxants. Theobromine salts (calcium salicylate, sodium salicylate and sodium acetate) were used previously to dilate coronary arteries at doses of 300 to 600 mg per day. There is no current therapeutic use of theobromine. /Former use/
VET: Diuretic, myocardial stimulant, vasodilator. /Former use/
/EXPL THER/ Cough is a common and protective reflex, but persistent coughing is debilitating and impairs quality of life. Antitussive treatment using opioids is limited by unacceptable side effects, and there is a great need for more effective remedies. The present study demonstrates that theobromine, a methylxanthine derivative present in cocoa, effectively inhibits citric acid-induced cough in guinea-pigs in vivo. Furthermore, in a randomized, double-blind, placebo-controlled study in man, theobromine suppresses capsaicin-induced cough with no adverse effects. We also demonstrate that theobromine directly inhibits capsaicin-induced sensory nerve depolarization of guinea-pig and human vagus nerve suggestive of an inhibitory effect on afferent nerve activation. These data indicate the actions of theobromine appear to be peripherally mediated. We conclude theobromine is a novel and promising treatment, which may form the basis for a new class of antitussive drugs.

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Pharmacodynamics
Theobromine, a xanthine derivative like caffeine and the bronchodilator theophylline, is used as a CNS stimulant, mild diuretic, and respiratory stimulant (in neonates with apnea of prematurity).

C7H8N4O2

180.167

180.064

83-67-0

Theobromine-d6;117490-40-1;Theobromine-d3;65566-69-0

5429

White to off-white solid powder

1.6±0.1 g/cm3

495.5±37.0 °C at 760 mmHg

345-350 °C

253.5±26.5 °C

0.0±1.3 mmHg at 25°C

1.737

-2.08

1

3

0

13

267

0

YAPQBXQYLJRXSA-UHFFFAOYSA-N

InChI=1S/C7H8N4O2/c1-10-3-8-5-4(10)6(12)9-7(13)11(5)2/h3H,1-2H3,(H,9,12,13)

3,7-dimethylpurine-2,6-dione

Santheose; Teobromin; Diurobromine

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 : ~1.1 mg/mL (~6.11 mM)
H2O : ~1.1 mg/mL (~6.11 mM)

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.)