Absorption, Distribution and Excretion
Caffeine is rapidly absorbed after oral or parenteral administration, reaching peak plasma concentrations within 30 minutes to 2 hours. After oral administration, the onset of action is 45 to 1 hour. Food may delay caffeine absorption. The peak plasma concentration range of caffeine is 6–10 mg/L. The absolute bioavailability in newborns is unknown, but it is approximately 100% in adults. The main metabolites of caffeine are excreted in the urine. Due to the significant reabsorption of caffeine in the renal tubules, approximately 0.5% to 2% of the dose is excreted in the urine. Caffeine can rapidly cross the blood-brain barrier. It is soluble in both water and lipids and is distributed throughout the body. Caffeine concentrations in the cerebrospinal fluid of preterm infants are similar to those in plasma. The average volume of distribution of caffeine in infants is 0.8–0.9 L/kg, and in adults it is 0.6 L/kg.
Caffeine clearance varies from person to person, but averages approximately 0.078 L/kg/h (1.3 mL/min/kg).
Globally, many fetuses and infants are exposed to these substances through maternal consumption of coffee and other beverages containing methylxanthines. Methylxanthines (caffeine, theophylline, and aminophylline) are also commonly used to treat apnea in preterm infants. …Methylxanthines readily cross the placental barrier and enter all tissues, therefore, if the effector system matures, they can have an effect on the fetus/neonatal at any time during pregnancy or after birth. …
Oral caffeine and caffeine citrate are well absorbed. Oral caffeine may be absorbed faster than intramuscular caffeine and sodium benzoate. Rectal administration (using caffeine suppositories) may result in slow and unstable absorption. …After oral administration of 100 mg of caffeine (in coffee form), peak plasma concentrations are reached at approximately 1.5–1.8 μg/mL after 50–75 minutes.
After oral administration of 10 mg/kg caffeine to preterm infants, the peak plasma caffeine concentration ranges from 6 to 10 mg/L, with a mean time to peak concentration (Tmax) of 30 minutes to 2 hours. Formula feeding does not affect Tmax.
Caffeine rapidly distributes to body tissues and easily crosses the placenta and blood-brain barrier. The caffeine concentration in the cerebrospinal fluid of preterm infants is close to that in plasma. The mean volume of distribution of caffeine in infants (0.8-0.9 L/kg) is slightly higher than that in adults (0.6 L/kg). …Studies have shown that caffeine is distributed in breast milk, with a concentration ratio of 0.5-0.76 to serum.
For more information on the absorption, distribution, and excretion (complete) of caffeine (11 items in total), please visit the HSDB record page.

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Metabolism/Metabolites
Caffeine is primarily metabolized in the liver by the cytochrome CYP1A2 enzyme. Caffeine metabolites include paraxanthine, theobromine, and theophylline. The first step in caffeine metabolism is demethylation, producing paraxanthine (a major metabolite), followed by theobromine and theophylline, both minor metabolites. These are excreted in urine as urate after further metabolism. Xanthine oxidase and N-acetyltransferase 2 (NAT2) are also involved in caffeine metabolism. Caffeine is primarily metabolized via the cytochrome P-450 (CYP) enzyme system, particularly isoenzyme 1A2. Therefore, caffeine may interact with drugs metabolized by CYP1A2 or drugs that induce or inhibit this isoenzyme. In adults, caffeine is rapidly metabolized in the liver to 1-methyluric acid, 1-methylxanthine, and 7-methylxanthine. Interconversion between caffeine and theophylline has been reported in premature infants… In vivo and in vitro studies have shown that the activity of hepatic microsomal enzymes that metabolize caffeine gradually increases during neonatal development. In beagle puppies, changes in caffeine clearance depend on the maturation rate of caffeine-7-demethylase. Caffeine is eliminated in animals via hepatic biotransformation into dimethylxanthine, dimethyluric acid, monomethyluric acid, and uracil derivatives; significant quantitative differences exist in the formation and elimination of metabolites among rats, mice, and Chinese hamsters. These differences are more pronounced in monkeys, where caffeine is almost entirely metabolized to theophylline. … Several species-dependent metabolites have been identified. Trimethylallantoin was first discovered in rats. A paraxanthine derivative was found in mice and identified as 3-β-D-glucuronide of paraxanthine. Trace amounts of methylurea and sulfur-containing derivatives in urine are produced by the gut microbiota. In contrast, the acetylated uracil derivative 5-acetamido-6-formylamino-3-methyluracil is one of the most important caffeine metabolites in humans, but has not been found in rodents or other animal species. Other uracil derivatives produced by the metabolism of caffeine, theobromine, and paraxanthine in rats have been found in human urine. In rats, hepatic demethylation of caffeine decreases with age, leading to a significantly prolonged elimination half-life in older rats. Caffeine metabolism in animals and humans is relatively similar in nature… The main metabolic pathway is 8-position demethylation and hydroxylation, generating the corresponding uracil and uric acid derivatives. However, there are some quantitative differences in the metabolic profile. In humans, 3-methyl demethylation is particularly important, generating paraxanthine, especially its metabolites from subsequent metabolic steps. The main metabolites of caffeine in human urine include 1-methyluric acid, 1-methylxanthine, 5-acetamido-6-formylamino-3-methyluracil (not found in rats and mice), 1,7-dimethyluric acid, and paraxanthine. In rats and mice, caffeine is primarily metabolized via theobromine and theophylline. The main urinary metabolites include 1,3-dimethyluracil, paraxanthine, trimethyluric acid, theophylline, and theobromine. Caffeine metabolism slows during pregnancy, leading to elevated serum concentrations.
Known metabolites of caffeine in the human body include theobromine, theophylline, 1,3,7-trimethyluric acid, and paraxanthine.
Cytochrome P450 1A2 (CYP 1A2) in the liver is involved in the biotransformation of caffeine. Approximately 80% of the caffeine dose is metabolized to paraxanthine (1,7-dimethylxanthine), 10% to theobromine (3,7-dimethylxanthine), and 4% to theophylline (1,3-dimethylxanthine).
Elimination pathway: Due to the immature development of liver and kidney function, the elimination rate of caffeine in infants and young children is much slower than in adults.
Half-life: 3 to 7 hours in adults, 65 to 130 hours in newborns.
Biological half-life
For adults of moderate size or children over 9 years of age, the half-life of caffeine is approximately 5 hours. Many factors and conditions can affect the half-life of caffeine. The half-life in smokers can be shortened by up to 50%. The half-life of caffeine in pregnant women can be prolonged to 15 hours or longer, especially in late pregnancy. The half-life of caffeine in newborns is prolonged, approximately 8 hours in full-term infants and approximately 100 hours in preterm infants, possibly due to decreased metabolic capacity. Liver disease or CYP1A2 inhibitors can prolong the half-life of caffeine. The elimination half-life in adults is 2.5–4.5 hours; [Reference 1] The plasma half-life (t1/2) of caffeine in adults is 3–5 hours. One study showed that if a pregnant woman took caffeine before delivery, the average half-life of the newborn was prolonged to 80 hours. The average half-life/T1/2 of caffeine in infants and the proportion of caffeine excreted unchanged in urine are negatively correlated with gestational age/post-conception age. The half-life of caffeine in newborns is approximately 3-4 days…
The half-life of caffeine in rats and mice is 0.7-1.0 hours, in rabbits 1-1.6 hours, in monkeys 3-5 hours, in dogs 4-4.3 hours, and in baboons 11-12 hours.
The authors studied 17 premature infants treated with caffeine and used high-performance liquid chromatography to determine the concentrations of caffeine and theophylline metabolites in their plasma. Half-lives were calculated using a computer analysis with the least squares method. The patients' mean gestational age was 29.7 ± 1.9 weeks (mean ± standard deviation), and they were studied at 20.7 ± 6.6 days postnatal time (mean ± standard deviation). The half-life of caffeine was 52.03 +/- 23.87 hours (mean +/- standard deviation), and the half-life of theophylline was 77.04 +/- 65.01 hours (mean +/- standard deviation).

Toxicity Summary
Caffeine stimulates the medulla oblongata, vagus nerve, vasomotor center, and respiratory center, leading to bradycardia, vasoconstriction, and increased respiratory rate. Previously, this effect was thought to be primarily due to increased intracellular cAMP levels resulting from inhibition of phosphodiesterase (an enzyme that degrades cyclic adenosine monophosphate). It is now believed that caffeine and other xanthine compounds act as antagonists of adenosine receptors on the plasma membranes of almost all cells. Since adenosine, as an endogenous substance, inhibits the release of neurotransmitters at the presynaptic site but enhances the effects of norepinephrine or angiotensin, the antagonism of adenosine receptors promotes neurotransmitter release. This explains the stimulant effect of caffeine. Blockade of cardiac adenosine A1 receptors leads to a significant increase in heart rate after caffeine intake.

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Toxicity Data
LD50: 127 mg/kg (oral, mouse) (A308)Interactions
When caffeine and disulfiram are administered concurrently…the total plasma clearance of caffeine is significantly reduced, and the elimination half-life is prolonged.
…The independent effects of cocaine and caffeine on cardiovascular dynamics have been documented, but to our knowledge, the combined effects of the two on intact cardiovascular hemodynamics remain to be investigated. We performed surgery on 18 dogs, inserting cardiac catheters into their right and left ventricles. The experiments were conducted after the dogs recovered from anesthesia. In the first phase (30 experiments on 8 dogs), the dose was determined using dose-response curves. In the second and third phases, we performed 28 experiments on an additional 10 dogs. Researchers administered cocaine intravenously followed by caffeine and vice versa to investigate its effects on hemodynamics and coronary flow reserve. Phase 1: Determining the doses of cocaine (2 mg/kg) and caffeine (5 mg/kg). Phase 2: Cocaine increases heart rate, blood pressure, and dP/dt, but significantly decreases coronary flow reserve (CFR). Administering caffeine after cocaine administration attenuates these effects (dP/dt decreased from 5066 ± 110 mmHg/s to 4910 ± 104 mmHg/s; pThe effects of caffeine and phenylpropanolamine, two widely used drugs, are achieved by activating the central and sympathetic nervous systems. It has been reported that the combined use of the two drugs can cause severe, life-threatening, and even fatal hypertension. This study aimed to investigate the possible pharmacokinetic interactions between phenylpropanolamine and caffeine. Sixteen healthy subjects received combination therapy with caffeine, phenylpropanolamine, and placebo. In subjects receiving combination therapy with 400 mg caffeine and 75 mg phenylpropanolamine, the mean (± standard error) peak plasma caffeine concentration was 8.0 ± 2.2 μg/mL, which was significantly higher than that after taking 400 mg caffeine alone (2.1 ± 0.3 μg/mL; t[24] = 2.4; p < 0.01). The frequency of physical side effects was higher after phenylpropanolamine-caffeine combination therapy compared to either drug alone or placebo. The increase in systolic and diastolic blood pressure after combination therapy was greater than that after taking either drug alone. Since certain drugs significantly increase caffeine levels in the body when used in combination with caffeine, these data suggest that phenylpropanolamine may enhance caffeine absorption or inhibit its elimination, which may explain the increased reported side effects after co-administration with caffeine. For more complete data on caffeine interactions (out of 21), please visit the HSDB records page. Non-human toxicity values: Rat intraperitoneal LD50 260 mg/kg; Rat oral LD50 192 mg/kg; Rat rectal LD50 300 mg/kg; Rat intravenous LD50 105 mg/kg. For more complete data on caffeine non-human toxicity values (out of 13), please visit the HSDB records page.

Therapeutic Uses
Central nervous system stimulant; phosphodiesterase inhibitor; purinergic P1 receptor antagonist. Caffeine can be taken orally as a mild central nervous system stimulant to help fatigued patients stay awake and restore mental alertness. Preterm infant apnea. Caffeine citrate can be used for short-term (10-12 days) treatment of apnea in preterm infants with a gestational age of 28 to less than 33 weeks. Caffeine is designated as an orphan drug by the U.S. Food and Drug Administration (FDA) for the treatment of sleep apnea in preterm infants. Caffeine, in combination with ergotamine tartrate, is used to relieve vascular headaches, such as migraines and cluster headaches (histamine headaches). For more complete data on the therapeutic uses of caffeine (11 in total), please visit the HSDB record page. Drug Warnings Because studies have shown that caffeine may promote peptic ulcers, patients with a history of peptic ulcer disease should use this medication with caution. Because caffeine may have arrhythmic effects, it is generally recommended that patients with symptomatic arrhythmias and/or palpitations, as well as those recovering from an acute myocardial infarction, avoid caffeine intake for the first few days to weeks. Before initiating treatment with caffeine citrate, baseline serum caffeine levels should be measured in newborns who have previously received theophylline, as preterm infants metabolize theophylline into caffeine. Similarly, because caffeine readily crosses the placenta, baseline serum caffeine levels should also be measured in infants born to mothers who consumed caffeine prenatally. Serious toxic reactions have been reported when serum caffeine concentrations exceed 50 μg/mL. /Caffeine Citrate/ Cases of hypoglycemia and hyperglycemia have been reported in clinical trials reported in the literature; therefore, regular monitoring of blood glucose levels in newborns receiving caffeine citrate may be necessary. Caffeine Citrate: In a placebo-controlled trial of caffeine citrate for apnea in preterm infants conducted in the United States, necrotizing enterocolitis occurred in 6 of the 85 newborns, with 3 of them dying. Of these 6 newborns, 5 had been randomly assigned to receive caffeine citrate treatment or had been exposed to the drug. Literature reports suggest a possible association between methylxanthine use and the development of necrotizing enterocolitis, but a causal relationship has not been established. Therefore, as with all preterm infants, patients receiving caffeine citrate treatment should be closely monitored for the development of necrotizing enterocolitis. Caffeine Citrate
For more complete data on drug warnings for caffeine (25 in total), please visit the HSDB record page.

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Pharmacodynamics
Caffeine stimulates the central nervous system (CNS), increasing alertness and sometimes causing restlessness. It relaxes smooth muscle, stimulates myocardial contraction, and enhances motor performance. Caffeine promotes gastric acid secretion and increases gastrointestinal motility. It is often used in combination with analgesics and ergot alkaloids to relieve symptoms of migraines and other types of headaches. Finally, caffeine also has a mild diuretic effect.

C8H10N4O2

194.19060087204

194.08

58-08-2

2519

White, prismatic crystals
Hexagonal prisms
White, fleecy masses or long, flexible, silky crystals

460 °F (NTP, 1992)
235-237
236.2 °C
238 °C
235 °C

-0.1

0

3

0

14

293

0

RYYVLZVUVIJVGH-UHFFFAOYSA-N

InChI=1S/C8H10N4O2/c1-10-4-9-6-5(10)7(13)12(3)8(14)11(6)2/h4H,1-3H3

1,3,7-trimethylpurine-2,6-dione

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.)