The bronchodilator theophylline is combined in a 2:1 ratio with ethylenediamine to form aminophylline. Aminophylline is typically found as a dihydrate, and the ethylenediamine increases solubility. Theophylline is more potent and has a longer half-life than aminophylline. It is most frequently used to treat airway obstruction brought on by COPD or asthma. Off-label, it is employed in nuclear stress testing as a reversal agent. Aminophylline is a phosphodiesterase inhibitor and nonselective antagonist of adenosine receptors. An extracellular messenger found in the body that can control myocardial oxygen requirements is adenosine. It works by blocking atrioventricular node conduction, increasing coronary artery blood flow, slowing heart rate, suppressing cardiac automaticity, and reducing the effects of β-adrenergic on contractility through cellular surface receptors that alter intracellular signaling pathways. Additionally, circulating catecholamines' chronotropic and ionotropic effects are countered by adenosine. All things considered, adenosine lowers the heart's contraction force and rate, which increases blood flow to the heart muscle. This mechanism (meant to protect the heart) may result in atropine-resistant refractory bradyasystole under certain conditions. The effects of adenosine vary with concentration. Aminophylline and other methylxanthines are competitive antagonists of adenosine receptors. Adenosine's cardiac effects are competitively inhibited by aminophylline at the cell surface receptors. It consequently raises contractility and heart rate.

Absorption, Distribution and Excretion
0.3 to 0.7 L/kg
0.29 mL/kg/min [postnatal age 3-15 days]
0.64 mL/kg/min [postnatal age 25-57 days]
1.7 mL/kg/min [ 1-4 years]
1.6 mL/kg/min [4-12 years]
0.9 mL/kg/min [13-15 years]
1.4 mL/kg/min [16-17 years]
0.65 mL/kg/min [Adults (16-60 years), non-smoking asthmatics]
0.41 mL/kg/min [Elderly (>60 years). liver, and renal function]
0.33 mL/kg/min [Acute pulmonary edema]
0.54 mL/kg/min [COPD->60 years, stable non-smoker >1 year]
0.48 mL/kg/min [COPD with cor pulmonale]
1.25 mL/kg/min [Cystic fibrosis (14-28 years)]
0.31 mL/kg/min [Liver disease -cholestasis]
0.35 mL/kg/min [cirrhosis]
0.65 mL/kg/min [acute hepatitis]
0.47 mL/kg/min [Sepsis with multi-organ failure]
0.38 mL/kg/min [hypothyroid]
0.8 mL/kg/min [hyperthyroid]
IV theophylline produces the highest and most rapid serum theophylline concentration. Following a single IV dose of theophylline (as aminophylline) of about 5 mg/kg over 30 minutes to healthy adults, mean peak serum theophylline concentrations of about 10 ug/mL are reached.
In neonates, approximately 50% of the theophylline dose is excreted unchanged in the urine. Beyond the first three months of life, approximately 10% of the theophylline dose is excreted unchanged in the urine. /Theophylline/
When administered IM, theophylline is usually absorbed slowly and incompletely. Rectal suppositories (no longer commercially available in the US) are slowly and erratically absorbed, regardless of whether the suppository base is hydrophilic or lipophilic. /Theophylline/
Dissolution appears to be the rate-limiting step in the absorption of oral theophylline. Under the acidic conditions of the stomach, the theophylline salts and compounds release free theophylline. ... Microcrystalline dosage forms and oral solutions of theophyllines are absorbed more rapidly, but not to a greater extent, than are uncoated tablets. Although the rate of absorption is slower, extended-release preparations (capsules and tablets) of theophylline are generally absorbed to the same extent as uncoated tablets; however, the actual rate of absorption of extended-release preparations may differ. Extended-release preparations of theophyllines have been formulated to release the drug at various rates suitable for dosing every 8-12, 12, or 24 hours; however, the actual dosing frequency for a given patient depends on their individual pharmacokinetic parameters. Since the rate and extent of absorption may differ between various extended-release preparations and sometimes between different dosage sizes of the same preparation, patients should generally be stabilized on a given preparation; substitution of one extended-release preparation for another should generally only be made when the preparations have been shown to be equivalent and/or the patient is evaluated pharmacokinetically during the transition period. Absorption of theophyllines may also be delayed, but is generally not reduced, by the presence of food in the GI tract; however, the effect of food on the absorption of extended-release preparations appears to be variable, and the manufacturer's recommendations for administration of specific preparations should be followed. /Theophyllines/
For more Absorption, Distribution and Excretion (Complete) data for AMINOPHYLLINE (12 total), please visit the HSDB record page.

---

Metabolism / Metabolites
Both the N-demethylation and hydroxylation pathways of theophylline biotransformation are capacity-limited. Due to the wide intersubject variability of the rate of theophylline metabolism, non-linearity of elimination may begin in some patients at serum theophylline concentrations <10 mcg/mL. Since this non-linearity results in more than proportional changes in serum theophylline concentrations with changes in dose, it is advisable to make increases or decreases in dose in small increments in order to achieve desired changes in serum theophylline concentrations. Accurate prediction of dose-dependency of theophylline metabolism in patients a priori is not possible, but patients with very high initial clearance rates (i.e., low steady-state serum theophylline concentrations at above average doses) have the greatest likelihood of experiencing large changes in serum theophylline concentration in response to dosage changes. /Theophylline/
Caffeine and 3-methylxanthine are the only theophylline metabolites with pharmacologic activity. 3-methylxanthine has approximately one tenth the pharmacologic activity of theophylline and serum concentrations in adults with normal renal function are <1 ug/mL. In patients with end-stage renal disease, 3-methylxanthine may accumulate to concentrations that approximate the unmetabolized theophylline concentration. Caffeine concentrations are usually undetectable in adults regardless of renal function. In neonates, caffeine may accumulate to concentrations that approximate the unmetabolized theophylline concentration and thus, exert a pharmacologic effect. /Theophylline/
Theophylline is metabolized by the liver to 1,3-dimethyluric acid, 1-methyluric acid, and 3-methylxanthine. ... Individuals metabolize theophylline at different rates; however, individual metabolism of the drug is generally reproducible. Theophylline and its metabolites are excreted mainly by the kidneys. Renal clearance of the drug, however, contributes only 8-12% of the overall plasma clearance of theophylline. Small amounts of theophylline are excreted in feces unchanged. /Theophylline/
Theophylline is metabolized via the microsomal cytochrome p450 system, primarily by the isozyme CYP1A2. The major pathway is demethylation to 3-methylxanthine in addition to being demethylated or oxidized to other metabolites. Less than 10% of theophylline is excreted in the urine unchanged. /Theophylline/
Following oral dosing, theophylline does not undergo any measurable first-pass elimination. In adults and children beyond one year of age, approximately 90% of the dose is metabolized in the liver. Biotransformation takes place through demethylation to 1-methylxanthine and 3-methylxanthine and hydroxylation to 1,3-dimethyluric acid. 1-methylxanthine is further hydroxylated, by xanthine oxidase, to 1-methyluric acid. About 6% of a theophylline dose is N-methylated to caffeine. Theophylline demethylation to 3-methylxanthine is catalyzed by cytochrome P-450 1A2, while cytochromes P-450 2E1 and P-450 3A3 catalyze the hydroxylation to 1,3-dimethyluric acid. Demethylation to 1-methylxanthine appears to be catalyzed either by cytochrome P-450 1A2 or a closely related cytochrome. In neonates, the N-demethylation pathway is absent while the function of the hydroxylation pathway is markedly deficient. The activity of these pathways slowly increases to maximal levels by one year of age. /Theophylline/

---

Biological Half-Life
7-9 hours
Theophylline clearance rates and half-life values were measured in 15 infants aged three to 23 months, after infusion of aminophylline by the intravenous route for at least 24 hours. ... The mean half-life was 4.4 +/- 2.2 hours. There was a tenfold variability in half-life, suggesting that individualization of theophylline dose is especially important in infants if undertreatment and toxicity are to be avoided.

Toxicity Summary
IDENTIFICATION AND USE: Aminophylline is white or slightly yellowish granules or powder. Aminophylline is prepared from theophylline and aqueous ethylenediamine. It is used as bronchodilator agent. HUMAN EXPOSURE AND TOXICITY: Aminophylline can trigger seizures in patients without known underlying epilepsy or added risk factor for seizure exacerbation in epilepsy. Most of these seizures are difficult to control and are underappreciated compared to other drug toxicities. Despite a long clinical history of aminophylline-induced seizures, relatively little is known about the underlying molecular mechanisms that contribute to methylxanthine-induced seizure generation. Fatalities in adults have generally occurred during or following IV administration of large doses of aminophylline in patients with renal, hepatic, or cardiovascular complications. In other patients, the rapidity of the injection, rather than the dose used, appears to be the more important factor precipitating acute hypotension, seizures, coma, cardiac standstill, ventricular fibrillation, and death. IV aminophylline or theophylline should therefore be given slowly. In children, fatalities usually are a result of overdosage and marked sensitivity to the CNS stimulation of theophylline. There are some reports of aminophylline hypersensitivity reaction and the most cases were delayed type reaction in English literatures. However, most of Japanese cases were immediate type. Acetylation is a main metabolic pathway of ethylenediamine. Most of Japanese have a rapid or intermediate acetylators on the other hand. Caucasian have a 50% likelihood of being slow acetylators. This difference suggest the different incidences of immediate and delayed reaction of aminophylline hypersensitivity reaction in Japanese and Caucasian respectively. Aminophylline treatment might be associated with elevated levels of myocardial enzymes. In vitro, aminophylline protected apoptosis of MRC-5 cells through the inactivation of caspases 3 and 8. ANIMAL STUDIES: Aminophylline (100-250 mg/kg) consistently induced seizures and post-ictal mortality in mice, and conventional anticonvulsants and adenosine agonists were ineffective in antagonizing them. Biochemical assay of brain homogenates showed that aminophylline seizures were associated with enhancements in brain malone dialdehyde and nitric oxide metabolites levels, whereas, superoxide dismutase activity was reduced, and these changes were attenuated after melatonin and L-NAME pretreatment. Aminophylline induced convulsions in rats in a dose-dependent manner, and both incidence of seizure and mortality were maximum at 300 mg/kg and there was significant increase of free radical generation. Pre-treatment with antioxidants showed differential attenuating effects on aminophylline induced free radical generation, but they were very much ineffective in antagonizing aminophylline induced seizures and post-seizure mortality by any appreciable extent. In pregnant rabbits, aminophylline treatment produced no acceleration in general anatomic lung development, as reflected in the ratio of lung air-space capacity to lung tissue weight. Under similar experimental conditions, maternal caffeine treatment had no effect on fetal rabbit lungs. In other experiment, pregnant rabbit does were treated intravenously with aminophylline (6 mg/kg/day) from the twenty-fifth day after the day of mating, and the fetuses were delivered by hysterotomy on the twenty-eighth day. One group of neonates was breathing air, and another group 100% oxygen. Lung mechanics were evaluated in the newborn animals during spontaneous or artificial ventilation, and the lungs were studied histologically with particular reference to the alveolar volume density. Aminophylline-treated litters had greater body weights, an improved survival rate, and an increased amount of phosphatidylglycerol in lung lavage fluid. Respiratory frequency was increased in aminophylline-treated animals breathing air, but data on lung compliance showed no significant difference between treated and control animals. It was concluded that the beneficial effect of aminophylline can be attributed largely to a combination of accelerated fetal growth and improved postnatal regulation of breathing and less to a specific influence on the biochemical and functional maturation of the lung. Aminophylline, exacerbated seizure-induced damage in the developing brain in rats.

---

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Expert opinion considers use of theophylline to be acceptable during breastfeeding. Maternal theophylline use may occasionally cause stimulation and irritability and fretful sleep in infants. Newborn and especially preterm infants are most likely to be affected because of their slow elimination and low serum protein binding of theophylline. There is no need to avoid theophylline products; however, keep maternal serum concentrations in the lower part of the therapeutic range and monitor the infant for signs of theophylline side effects. Infant serum theophylline concentrations can help to determine if signs of agitation are due to theophylline. Avoiding breastfeeding for 2 hours after intravenous or 4 hours after an immediate-release oral theophylline product can decrease the dose received by the breastfed infant. When theophylline is given as an oral sustained-release product, timing of nursing with respect to the dose is of little or no benefit.
◉ Effects in Breastfed Infants
Irritability and fretful sleeping occurred in a 3-day-old breastfed infant on days of maternal aminophylline intake of 200 mg every 6 hours. These effects ceased with discontinuation and recurred on rechallenge over the next 9 months. These effects were probably caused by theophylline in breastmilk. Another five infants reported in this paper showed no adverse reactions after maternal theophylline ingestion. Accumulation of theophylline in infant serum appears most likely in neonates and premature infants because they eliminate theophylline slowly.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.
◉ Summary of Use during Lactation
An expert panel considers use of aminophylline to be acceptable during breastfeeding. Maternal aminophylline use may occasionally cause stimulation and irritability and fretful sleep in infants. Newborn and especially preterm infants are most likely to be affected because of their slow elimination and low serum protein binding of theophylline. There is no need to avoid aminophylline products; however, keep maternal serum theophylline concentrations in the lower part of the therapeutic range and monitor the infant for signs of theophylline side effects. Infant serum theophylline concentrations can help to determine if signs of agitation are due to theophylline. Avoiding breastfeeding for 2 hours after intravenous or 4 hours after an immediate-release oral aminophylline product can decrease the dose received by the breastfed infant.
◉ Effects in Breastfed Infants
Irritability and fretful sleeping occurred in a 3-day-old breastfed infant on days of maternal aminophylline intake of 200 mg every six hours. These effects ceased with discontinuation and recurred on rechallenge over the next 9 months. These effects were probably caused by theophylline in breastmilk. Another five infants reported in this paper showed no adverse reactions after maternal theophylline ingestion. Accumulation of theophylline in infant serum appears most likely in neonates and premature infants because they eliminate theophylline slowly.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

---

Protein Binding
60%

---

Interactions
This study aimed to characterize pharmacodynamic interaction between propofol and aminophylline. Nine beagle dogs were randomly allocated at the propofol rates of 0.75 (group A), 1.00 (group B), and 1.25 (group C) mg/kg/min. During period 1, propofol only was infused, while during period 2, aminophylline only, at the rate of 0.69 (group A), 1.37 (group B), and 2.62 (group C) mg/kg/hr. During periods 3-5, the two drugs were co-administered. The aminophylline infusion rate was 0.69 (period 3), 1.37 (period 4), and 2.62 (period 5) mg/kg/hr. The aminophylline was infused from 0 to 30 h, and the propofol was infused at 24 hr for 20 min. Blood samples and electroencephalograms were obtained at preset intervals. In the linear regression between log-transformed doses of aminophylline and AUC inf, the slope was 0.6976 (95% CI 0.5242-0.8710). Pharmacokinetics of aminophylline was best described by a one-compartment, with enzyme auto-induction, model. Pharmacokinetics and pharmacodynamics of propofol were best described by a three-compartment model and a sigmoid Emax model, respectively. Pharmacodynamic parameter estimates of propofol were: k(e0) = 0.805/min, E0 = 0.76, Emax = 0.398, Ce(50 na) = 2.38 ug/mL (without aminophylline-exposure), C(e50 wa) = 4.49 ug/mL (with aminophylline-exposure), and gamma = 2.21. Propofol becomes less potent when exposed to aminophylline. Pharmacodynamic antagonistic interaction of aminophylline with propofol sedation, may occur, not in a dose-dependent manner, but in an all-or-none response.
There is some evidence from animal studies that concomitant administration of a beta-adrenergic agonist (e.g., isoproterenol) and a theophylline derivative (e.g., aminophylline) may produce increased cardiotoxic effects. Although such an interaction has not been established in humans, a few reports have suggested that such a combination may have the potential for producing cardiac arrhythmias. Further accumulation of clinical data is needed to determine whether this potential interaction exists in humans.
Theophylline interacts with a wide variety of drugs. The interaction may be pharmacodynamic, i.e., alterations in the therapeutic response to theophylline or another drug or occurrence of adverse effects without a change in serum theophylline concentration. More frequently, however, the interaction is pharmacokinetic, i.e., the rate of theophylline clearance is altered by another drug resulting in increased or decreased serum theophylline concentrations. Theophylline only rarely alters the pharmacokinetics of other drugs. The drugs listed in Table have the potential to produce clinically significant pharmacodynamic or pharmacokinetic interactions with theophylline. The information in the "Effect" column of Table assumes that the interacting drug is being added to a steady-state theophylline regimen. If theophylline is being initiated in a patient who is already taking a drug that inhibits theophylline clearance (e.g., cimetidine, erythromycin), the dose of theophylline required to achieve a therapeutic serum theophylline concentration will be smaller. Conversely, if theophylline is being initiated in a patient who is already taking a drug that enhances theophylline clearance (e.g., rifampin), the dose of theophylline required to achieve a therapeutic serum theophylline concentration will be larger. Discontinuation of a concomitant drug that increases theophylline clearance will result in accumulation of theophylline to potentially toxic levels, unless the theophylline dose is appropriately reduced. Discontinuation of a concomitant drug that inhibits theophylline clearance will result in decreased serum theophylline concentrations, unless the theophylline dose is appropriately increased. /Theophylline/
Table: Clinically Significant Drug Interactions with Theophylline [Table#844]

---

Non-Human Toxicity Values
LD50 Mouse iv 146 mg/kg
LD50 Mouse sc 186 mg/kg
LD50 Mouse ip 217 mg/kg
LD50 Mouse oral 150 mg/kg
For more Non-Human Toxicity Values (Complete) data for AMINOPHYLLINE (9 total), please visit the HSDB record page.

[1]. Luteolin, a non-selective competitive inhibitor of phosphodiesterases 1-5, displaced [3H]-rolipram from high-affinity rolipram binding sites and reversed xylazine/ketamine-induced anesthesia. ur J Pharmacol. 2010 Feb 10;627(1-3):269-75.

[2]. Response of blood gas tensions to aminophylline and isoprenaline in patients with asthma. Thorax. 1967 Nov;22(6):543-9.

[3]. Adenosine receptors: development of selective agonists and antagonists. Prog Clin Biol Res. 1987;230:41-63.

Therapeutic Uses
Bronchodilator Agents; Cardiotonic Agents; Phosphodiesterase Inhibitors; Purinergic P1 Receptor Antagonists
/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. Aminophylline is included in the database.
IV theophylline (often as aminophylline) has been used to relieve the periodic apnea and increase arterial blood pH in patients with Cheyne-Stokes respiration. /NOT included in US product labeling/
MEDICATION (VET): Aminophylline is indicated for control of reversible airway constriction, to prevent bronchoconstriction, and as an adjunct with other respiratory disease treatment. The uses are are similar to the indications for theophylline because it is a salt form of theophylline. It is used for inflammatory airway disease in cats (feline asthma), dogs, and horses. In dogs, the uses include collapsing trachea, bronchitis, and other airway disease. It has not been effective for respiratory diseases in cattle.
For more Therapeutic Uses (Complete) data for AMINOPHYLLINE (10 total), please visit the HSDB record page.

---

Drug Warnings
Fatalities in adults have generally occurred during or following IV administration of large doses of aminophylline in patients with renal, hepatic, or cardiovascular complications. In other patients, the rapidity of the injection, rather than the dose used, appears to be the more important factor precipitating acute hypotension, seizures, coma, cardiac standstill, ventricular fibrillation, and death. IV aminophylline or theophylline should therefore be given slowly. In children, fatalities usually are a result of overdosage and marked sensitivity to the CNS stimulation of theophylline.
When administered rectally as suppositories (dosage form no longer commercially available in the US), theophyllines have caused rectal irritation and inflammation. /Theophyllines/
Rapid IV injection of aminophylline may produce dizziness, faintness, lightheadedness, palpitation, syncope, precordial pain, flushing, profound bradycardia, ventricular premature complexes (VPCs, PVCs), severe hypotension, or cardiac arrest. IM injection of aminophylline produces intense local pain and sloughing of tissue ... .
Theophyllines may also produce transiently increased urinary frequency, dehydration, twitching of fingers and hands, tachypnea, and elevated serum AST (SGOT) concentrations. Hypersensitivity reactions characterized by urticaria, generalized pruritus, and angioedema have been reported with aminophylline administration. A contact-type dermatitis, caused by hypersensitivity to the ethylenediamine component of aminophylline, has also been reported. Bone marrow suppression, leukopenia, thrombocytopenia, and hemorrhagic diathesis have also been reported, but their association with theophylline therapy is questionable. Other adverse effects of theophyllines include albuminuria, increased urinary excretion of renal tubular cells and erythrocytes, hyperglycemia, and syndrome of inappropriate secretion of antidiuretic hormone (SIADH). /Theophyllines/
For more Drug Warnings (Complete) data for AMINOPHYLLINE (22 total), please visit the HSDB record page.

---

Pharmacodynamics
Aminophylline is the ethylenediamine salt of theophylline. Theophylline stimulates the CNS, skeletal muscles, and cardiac muscle. It relaxes certain smooth muscles in the bronchi, produces diuresis, and causes an increase in gastric secretion.

C16H24N10O4

420.43

420.198

317-34-0

58-55-9 (free);317-34-0 (EDA);

9433

White to off-white solid powder

454.1ºC at 760mmHg

269-270 °C

4

8

1

30

273

0

FQPFAHBPWDRTLU-UHFFFAOYSA-N

InChI=1S/2C7H8N4O2.C2H8N2/c2*1-10-5-4(8-3-9-5)6(12)11(2)7(10)13;3-1-2-4/h2*3H,1-2H3,(H,8,9);1-4H2

1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione compound with ethane-1,2-diamine (2:1)

Cardophyllin; Aminophyllin; Aminophylline; Theophyllamine; Phyllocontin; Euphyllin; Truphylline; Minomal R 175 mg tab; Minomal R 350 mg tab; Minomal SR 600 mg tab

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

Solubility in Formulation 1: 1.43 mg/mL (3.40 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 14.3 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.

---

Solubility in Formulation 2: ≥ 1.43 mg/mL (3.40 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 14.3 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.

---

View More

Solubility in Formulation 3: ≥ 1.43 mg/mL (3.40 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 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 22 mg/mL (52.33 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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