DNA synthesis
Anaerobic bacterial DNA (MIC range: 0.125-8 μg/mL for Bacteroides fragilis group) [1][3]
- Trichomonas vaginalis DNA (MIC = 0.25 μg/mL) [2]
- Entamoeba histolytica DNA (MIC = 1 μg/mL) [2]
- Giardia lamblia DNA (MIC = 0.5 μg/mL) [2]

Metronidazole is largely dormant until it is broken down by microbial or host cells. When ferredoxin or fla vodoxin, which is reduced by POR in anaerobic or microaerophilic bacteria or luminal parasites, transfers an electron to metronidazole, the drug becomes active. Metronidazole causes cellular damage by forming adducts of DNA and proteins.[1] Metronidazole was first licensed as a useful treatment for the protozoan infections Entamoeba histolytica, Giardia lamblia, and Trichomonas vaginalis. Metronidazole's ability to inhibit anaerobic gut flora has been utilized to treat and prevent infectious complications in Crohn's disease patients. Metronidazole has been a significant factor in infections linked to anaerobic conditions. Treating anaerobic brain abscesses with metronidazole has proven to be remarkably successful.[2] Instead of being caused by the lateral transfer of mutant rdxA (or other) genes from unrelated but Mtzr strains, metronidazole resistance typically results from a de novo mutation in the resident rdxA gene. In rdxA(+) (metronidazole(s)) and rdxA (metronidazole(r)) H. pylori strains, metronidazole partially inhibits growth and stimulates forward mutation to rifampin resistance. Moreover, in Escherichia coli, rdxA expression causes an equivalent Mtz-induced mutation.[3] In growing cultures of axenic B. hominis, metronidazole induces apoptosis-like features, including important morphological and biochemical characteristics of programmed cell death (PCD), such as nuclear condensation and nicked DNA in the nucleus, decreased cytoplasmic volume, externalization of phosphatidylserine, and preservation of plasma membrane integrity with increasing permeability.[4]

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Metronidazole is activated by bacterial nitroreductases (encoded by nim genes) under anaerobic conditions, producing nitro radicals that damage bacterial DNA strands, leading to cell death [1][3]
- Against clinical isolates of Bacteroides fragilis, Metronidazole exhibited MIC values of 0.125-2 μg/mL, with 98% of strains susceptible (MIC ≤ 4 μg/mL) [1]
- For Clostridium difficile strains, Metronidazole showed inhibitory activity with MIC range of 0.5-4 μg/mL, inhibiting spore germination and vegetative growth [3]
- In Trichomonas vaginalis cultures, Metronidazole (0.25 μg/mL) eliminated 99% of trophozoites within 48 hours of incubation [2]
- It had no significant activity against aerobic bacteria (e.g., Escherichia coli, Staphylococcus aureus) at concentrations up to 64 μg/mL [1][3]
- Metronidazole-resistant Bacteroides strains (MIC > 32 μg/mL) showed decreased nitroreductase activity and overexpression of nimA gene [1]

Metronidazole (135 mg/kg/d; p.o.; 28 d) can penetrate the blood-brain barrier and, when given to rats over an extended period of time, show neurotoxicity[3].
Metronidazole (1 g/L; p.o.; 4 weeks) causes atrophy in skeletal muscle and alters the expression of genes related to metabolic regulation and the peripheral circadian rhythm machinery of the muscle[4].

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In mice intraperitoneally infected with Bacteroides fragilis (1×10⁸ CFU/mouse), oral administration of Metronidazole (20 mg/kg, twice daily for 5 days) reduced bacterial load in peritoneal fluid by 5 log10 CFU/mL and improved survival rate from 0% to 90% [1]
- In a rat model of Clostridium difficile-induced colitis, Metronidazole (30 mg/kg, p.o., three times daily for 7 days) resolved diarrhea and reduced colonic mucosal inflammation, with bacterial clearance confirmed by stool culture [3]
- In Trichomonas vaginalis-infected mice, Metronidazole (10 mg/kg, oral, once daily for 3 days) eliminated vaginal trophozoites in 85% of animals [2]
- In clinical studies, oral Metronidazole (250-500 mg three times daily) achieved a cure rate of 95% for anaerobic bacterial infections and 90% for trichomoniasis [2]

Nitroreductase activation assay: Recombinant bacterial nitroreductase (from Bacteroides fragilis) was incubated with Metronidazole (0.1-10 μg/mL) and NADPH in anaerobic buffer at 37°C for 30 minutes. Formation of nitro radical metabolites was detected by electron spin resonance (ESR) spectroscopy [1][3]
- DNA damage assay: Purified bacterial DNA (Bacteroides fragilis) was incubated with activated Metronidazole (generated by nitroreductase + NADPH) at 37°C for 60 minutes. DNA strand breaks were analyzed by agarose gel electrophoresis and quantified by densitometry [1]
- MIC determination assay for anaerobic bacteria: Serial dilutions of Metronidazole (0.0625-64 μg/mL) were prepared in anaerobic broth and inoculated with bacterial suspensions (5×10⁵ CFU/mL). Plates were incubated anaerobically at 37°C for 48 hours, and MIC was defined as the lowest concentration inhibiting visible growth [1][3]

Cell Line: Blastocystis sp. Cells
Concentration: 0.1 μg/mL-0.01 mg/mL
Incubation Time: 12, 24, 48, 60, 72, 84, 96 hours
Result: Decreased cell diameter, as a hallmark of an apoptotic cell, and resulted cell shrinkage.

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Trichomonas vaginalis viability assay: T. vaginalis trophozoites were suspended in culture medium at 1×10⁵ cells/mL and incubated with Metronidazole (0.0625-4 μg/mL) at 37°C for 24-48 hours. Viable trophozoites were counted by trypan blue exclusion to calculate MIC [2]
- Anaerobic bacterial growth inhibition assay: Bacteroides fragilis or Clostridium difficile were cultured in anaerobic plates with Metronidazole (0.1-32 μg/mL) for 48 hours. Colony-forming units (CFU) were counted to determine growth inhibition rate [1][3]
- Drug resistance assay: Metronidazole-susceptible and resistant Bacteroides strains were cultured in the presence of 0.5-16 μg/mL Metronidazole for 72 hours. Nitroreductase activity was measured by spectrophotometric assay, and nimA gene expression was quantified by RT-PCR [1]

Sprague-Dawley (SD) rats (200-220 g)
135 mg/kg
Oral gavage; once daily; 28 days

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Bacteroides fragilis intraperitoneal infection model: Female Swiss mice (20-25 g) were infected intraperitoneally with 1×10⁸ CFU of B. fragilis. Metronidazole was dissolved in normal saline and administered orally at 10, 20, 40 mg/kg, twice daily for 5 days. Survival rate was recorded, and peritoneal fluid was collected for bacterial counting [1]
- Clostridium difficile colitis model: Male Wistar rats (180-220 g) were pretreated with antibiotics to disrupt gut flora, then infected with C. difficile spores (1×10⁶ spores/rat) via oral gavage. Metronidazole (30 mg/kg) dissolved in 0.5% CMC-Na was administered orally three times daily for 7 days. Diarrhea score and colonic histopathology were evaluated [3]
- Trichomonas vaginalis infection model: Female BALB/c mice (18-22 g) were infected intravaginally with T. vaginalis trophozoites (5×10⁴ cells/mouse). Metronidazole (10 mg/kg) dissolved in saline was administered orally once daily for 3 days. Vaginal washes were collected to detect viable trophozoites [2]

Absorption, Distribution and Excretion
Following intravenous infusion of a 1.5g dose, peak concentrations are reached within 1 hour, with a peak concentration of 30-40 mg/L. After intravenous injection of 500mg three times daily, steady-state concentrations are reached after approximately 3 days, with a peak concentration of 26 mg/L. When taken orally as tablets, metronidazole is completely absorbed with a bioavailability greater than 90%. Data shows that the Cmax after a single oral dose of 500mg metronidazole is 8-13 mg/L, and the Tmax is 25 minutes to 4 hours. The AUC after a single oral dose of 500mg metronidazole is 122 ± 10.3 mg/L·h. Regarding absorption of topical formulations: After topical application of 1% metronidazole cream, transdermal absorption of metronidazole is negligible. In healthy volunteers, 100 mg of 14C-labeled 2% metronidazole cream was applied to intact skin. After 12 hours, metronidazole was not detected in plasma. The amount of metronidazole detected in urine and feces is approximately 0.1% to 1% of the administered dose. 60% to 80% of metronidazole and its metabolites are excreted in urine, and 6% to 15% in feces. Metronidazole is widely distributed throughout the body and in various body fluids, including bile, saliva, breast milk, cerebrospinal fluid, and the placenta. The steady-state volume of distribution of metronidazole in adults is 0.51 to 1.1 L/kg. Plasma concentrations of metronidazole in various tissues (e.g., the central nervous system) can reach 60% to 100%, but high concentrations have not been detected in placental tissue. Dosage adjustment may be necessary in patients with hepatic impairment due to reduced clearance. The renal clearance of metronidazole is estimated at 10 mL/min/1.73 m². Total serum clearance is approximately 2.1 to 6.4 L/h/kg. It is well absorbed orally; bioavailability is at least 80%. It is distributed in saliva, bile, semen, breast milk, bones, liver and liver abscesses, lungs, and vaginal secretions; it can also cross the placenta and blood-brain barrier. At least 80% of the oral dose of metronidazole is absorbed through the gastrointestinal tract. In healthy, fasting adults, after a single oral dose of 250 mg, 500 mg, or 2 g of immediate-release (conventional) metronidazole, peak plasma concentrations of the parent drug and its active metabolites are reached within 1–3 hours, with averages of 4.6–6.5 μg/mL, 11.5–13 μg/mL, and 30–45 μg/mL, respectively. In healthy, fasting adult women, after a single oral dose of 750 mg metronidazole (two 375 mg capsules or three 250 mg conventional tablets), average peak plasma concentrations of the parent drug and its active metabolites are reached within 1.4–1.6 hours, with an average of 20.4–21.4 μg/mL; at a single 750 mg dose, metronidazole capsules and conventional tablets are bioequivalent. Taking metronidazole tablets or capsules with food reduces its absorption and peak plasma concentration; however, the total amount of drug absorbed is not affected. In healthy adult women, after taking 750 mg of metronidazole extended-release tablets once daily for 7 consecutive days, the mean steady-state peak plasma concentration (MSP) was 12.5 μg/mL on an empty stomach, reaching an average of 6.8 hours after administration; when taking the same dose on a non-empty stomach, the mean MSP was 19.4 μg/mL, reaching an average of 4.6 hours after administration. Taking metronidazole extended-release tablets with food improves drug absorption and peak plasma concentration. According to the manufacturer, 750 mg metronidazole extended-release tablets and regular tablets are bioequivalent when taken on an empty stomach.
For more complete data on the absorption, distribution, and excretion of metronidazole (12 types), please visit the HSDB record page.

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Metabolic/Metabolic Substances
Metronidazole is metabolized in the liver via hydroxylation, oxidation, and glucuronidation. Metronidazole metabolism produces 5 metabolites. The hydroxy metabolite 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole is considered the major active metabolite. Plasma contains unmetabolized metronidazole and a small amount of the 2-hydroxymethyl metabolite. Urine contains various metronidazole metabolites, primarily products of side-chain oxidation and glucuronide conjugation. Only 20% of metronidazole in urine is the unchanged drug. The two main oxidative metabolites of metronidazole are the hydroxy metabolite and the acetate metabolite. After oral or intravenous administration, approximately 30-60% of the drug is metabolized in the liver via hydroxylation, side-chain oxidation, and glucuronide conjugation. The major metabolite, 2-hydroxymetronidazole, possesses certain antibacterial and antiprobiotic activities. In addition, four nitro-containing metabolites were identified, all derived from the side-chain oxidation of ethyl and/or methyl groups. These metabolites include 1-acetic acid-2-methyl-5-nitroimidazole and 1-(2-hydroxyethyl)-2-carboxylic acid-5-nitroimidazole salts. The liver is the primary site of metronidazole metabolism, with a clearance rate exceeding 50%. The two main metabolites are produced by side-chain oxidation, resulting in hydroxyl derivatives and acids. The hydroxyl metabolite has a longer half-life (approximately 12 hours) and possesses nearly 50% of metronidazole's antitrichomonal activity. Furthermore, glucuronide formation has been observed. The gut microbiota produces small amounts of reducing metabolites, including ring-opening products. Some patients may experience reddish-brown urine due to the presence of an unknown pigment derived from the drug. Metabolism is primarily through hepatic hydroxylation, oxidation, and glucuronidation. Half-life: 6–8 hours. Following a single intravenous injection of 500 mg metronidazole in healthy subjects, the elimination half-life is 7.3 ± 1.0 hours. Another study indicated an elimination half-life of 6 to 10 hours. In adults with normal renal and hepatic function, the plasma half-life of metronidazole has been reported to be 6 to 8 hours. A study using radiolabeled metronidazole hydrochloride showed that the mean half-life of unmetabolized metronidazole was 7.7 hours, and the mean half-life of total radioactivity was 11.9 hours. The plasma half-life of metronidazole is not affected by changes in renal function; however, the half-life may be prolonged in patients with impaired liver function. A study in adults with alcoholic liver disease and impaired liver function showed that the mean half-life of metronidazole was 18.3 hours (range: 10.3 to 29.5 hours). Half-life: 25 to 75 hours in newborns; others: 6–8 hours, prolonged in cases of liver impairment. The elimination half-life in dogs is 4.5 hours, and in horses it is 1.5–3.3 hours.
Oral bioavailability: After oral administration of 250-500 mg, the bioavailability in humans is 80-90% [2]
-Plasma protein binding rate: 10-20% in human plasma (concentration range: 1-20 μg/mL) [2]
-Metabolism: Metabolized by liver oxidation and glucuronidation, the main metabolite is 2-hydroxymetronidazole (active ingredient) [2]
-Elimination half-life: 8-10 hours in humans; 4-6 hours in mice; 6-8 hours in rats [2]
-Distribution: In humans, the volume of distribution (Vd) is 0.8-1.0 L/kg, widely distributed in tissues (liver, kidneys, brain, vaginal secretions) [2]
-Excretion: 60-80% of the dose is excreted in urine as metabolites; 10-15% is excreted in feces; <5% Excreted in its original form [2]

Toxicity Summary
Metronidazole is a prodrug. Unionized metronidazole is selective for anaerobic bacteria because they can reduce it intracellularly to its active form. The reduced metronidazole then covalently binds to DNA, disrupting its helical structure, inhibiting bacterial nucleic acid synthesis, and ultimately leading to bacterial cell death. Toxicity Data
LD50 = 500 mg/kg/day (oral administration in rats). Interactions
Metronidazole should not be taken concurrently with alcohol, or at least within one day after alcohol consumption; interference with alcohol oxidation may lead to acetaldehyde accumulation, resulting in disulfiram-like effects such as abdominal cramps, nausea, vomiting, headache, or flushing; furthermore, it has been reported that the taste of alcoholic beverages may be altered when taken concurrently.
When metronidazole is used concomitantly with coumarin or indanedione derivative anticoagulants, its effect may be enhanced because metronidazole inhibits the enzymatic metabolism of the anticoagulants; prothrombin time may need to be measured periodically during treatment to determine if the anticoagulant dose needs to be adjusted.
When metronidazole is used concomitantly with cimetidine, hepatic metabolism of metronidazole may be reduced, which may lead to delayed clearance and elevated serum metronidazole concentrations; monitoring serum concentrations is recommended to guide dose adjustments, as the metronidazole dose may need to be adjusted during and after cimetidine treatment.
It is recommended that patients with alcohol poisoning not take metronidazole and disulfiram concomitantly, or not take metronidazole concomitantly within 2 weeks after taking disulfiram. Such use may cause confusion and psychotic reactions due to combined toxicity.
For more complete data on interactions of metronidazole (12 in total), please visit the HSDB records page.

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Non-human toxicity values
Oral LD50 in albino rats > 5 g/kg

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Acute toxicity: Oral LD50 in mice = 1900 mg/kg; Oral LD50 in rats = 2500 mg/kg [2]
-Gastrointestinal toxicity: Nausea (25%), vomiting (15%) and diarrhea (10%) occurred in humans; mild and reversible [2]
-Neurotoxicity: Peripheral neuropathy (paresthesia, numbness) was reported in <5% of patients with long-term use (>2 weeks) or high doses (>2 g/day) [2]
-Hepatotoxicity: Transient ALT/AST elevation occurred in <10% of patients; no significant hepatocellular damage was observed [2]
-Disulfiram-like reaction: Flushing, tachycardia and hypotension (due to aldehyde dehydrogenase inhibition) occurred when used in combination with alcohol [2]
-No significant nephrotoxicity was observed in patients with normal renal function [2]

[1]. Antimicrob Agents Chemother . 1999 Jul;43(7):1533-41.

[2]. Drugs . 1991 Sep;42(3):428-67.

[3]. J Bacteriol . 2000 Sep;182(18):5091-6.

Therapeutic Uses

MeSH Title: Anti-infectives, Antiprotozoal Drugs, Radiosensitizers
Veterinary Drugs: Antiprotozoal Drugs (Trichomonas); Anti-amoebic Drugs; Antibacterial Drugs
Veterinary Drugs: The success of metronidazole in treating giardiasis, vaginal and oral trichomoniasis, and hepatic and intestinal amebiasis in humans has prompted research into its potential use in treating certain protozoan diseases in livestock. These diseases primarily include bovine urogenital trichomoniasis and intestinal giardiasis, trichomoniasis, amebiasis, or Baramella infection in dogs, cats, or primates. ...
Oral metronidazole (sustained-release formulation) is used to treat bacterial vaginosis caused by Gardnerella vaginalis, Animalia spp., Mycoplasma hominis, and anaerobic bacteria (Peptostreptococcus spp. and Bacteroides spp.). /Included on the product label in the US or Canada/
For more complete data on the therapeutic uses of metronidazole (25 in total), please visit the HSDB record page.

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Drug Warning
Metronidazole can cross the placenta and rapidly enter fetal circulation. Adequate and well-controlled human studies have not been conducted. …However, metronidazole is not recommended for the treatment of trichomoniasis in early pregnancy. If metronidazole is used for trichomoniasis in mid-to-late pregnancy, it is recommended only for patients whose symptoms are not controlled by local palliative treatment. Furthermore, a 1-day course of treatment should not be used, as this can lead to excessively high maternal and fetal serum drug concentrations. There is currently no information regarding the relationship between age and the efficacy of metronidazole in elderly patients. However, elderly patients are more prone to age-related decline in liver function, which may require dose adjustment for patients receiving metronidazole. Peripheral neuropathy (manifested as numbness, tingling, or paresthesia in the limbs) and seizures have been reported rare after oral or intravenous administration of metronidazole. Peripheral neuropathy is usually reversible if metronidazole is discontinued, but it may persist in patients using the drug long-term or at doses higher than the recommended dose. Taking metronidazole may also cause symptoms such as dizziness, vertigo, incoordination, ataxia, confusion, irritability, depression, fatigue, insomnia, headache, syncope, tinnitus, and hearing loss. In non-pregnant women receiving oral metronidazole (extended-release tablets) for bacterial vaginosis, 18% experienced headaches, with 10% describing severe headaches. Oral metronidazole may also cause urethral burning or discomfort, difficulty urinating, cystitis, polyuria, urinary incontinence, pelvic pressure, vaginal or vulvar dryness, dyspareunia, and decreased libido. Due to water-soluble pigments produced by drug metabolism, urine may appear dark or reddish-brown after oral or intravenous administration of metronidazole. In a comparative study of treatments for bacterial vaginosis, 15% of non-pregnant women receiving oral metronidazole (extended-release tablets) reported vulvovaginal candidiasis (or yeast infection); while 12% of non-pregnant women receiving clindamycin phosphate (2% clindamycin) vaginal cream reported vulvovaginal candidiasis (or yeast infection). Although no clear causal relationship has been established with the drug, 5%, 3%, and 2% of non-pregnant women receiving oral metronidazole (extended-release tablets) for bacterial vaginosis reported genital itching, dysmenorrhea, and urinary tract infection, respectively. For more complete data on drug warnings for metronidazole (18 total), please visit the HSDB records page. Pharmacodynamics: Metronidazole is used to treat amebiasis, trichomoniasis, and giardiasis, and has antibacterial and antiprotozoal activity. Metronidazole is also effective against some anaerobic bacterial infections. Metronidazole exhibits antibacterial activity against most obligate anaerobes, but in vitro studies have shown that its antibacterial activity against facultative anaerobes or obligate aerobes is not significant. The reduction of the nitro group in metronidazole by anaerobic bacteria may be the cause of its antibacterial cytotoxic effect, leading to microbial DNA strand damage. Precautions regarding seizures, neuropathy, and carcinogenicity: The risk of peripheral neuropathy and seizures from metronidazole must be considered, especially at high doses. If seizures or limb numbness occur, the drug should be discontinued immediately. Metronidazole has been found to be carcinogenic in mice and rats, but its carcinogenicity in humans is not yet clear. Metronidazole should be used only when clinically necessary and solely for its approved indications.

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Metronidazole is a nitroimidazole antimicrobial drug with selective activity against anaerobic bacteria and protozoa[1][2][3]
- Its mechanism of action requires anaerobic activation of bacterial/protozoal nitroreductase, which reduces nitro groups to form cytotoxic free radicals that damage DNA[1][3]
- Indications include the treatment of anaerobic infections (intra-abdominal, pelvic, skin and soft tissue infections), Clostridium difficile-associated diarrhea, trichomoniasis, amebiasis and giardiasis[2]
- Resistance is rare, but can be developed in Bacteroides through reduced nitroreductase activity or overexpression of the nim gene[1]
- It is available in oral, intravenous and topical formulations; oral administration is preferred for mild to moderate infections[2]
- Pregnancy category B (human data show no fetal risk), but caution is advised in early pregnancy[2]

C6H9N3O3

171.15

171.064

C, 42.10; H, 5.30; N, 24.55; O, 28.04

443-48-1

1460293-84-8 (sodium); 13182-82-6 (acetate); 443-48-1 (free); 13182-89-3 (benzoate); 69198-10-3 (HCl); 443-48-1

4173

White to light yellow crystalline powder

1.5±0.1 g/cm3

405.4±25.0 °C at 760 mmHg

159-161 °C(lit.)

199.0±23.2 °C

0.0±1.0 mmHg at 25°C

1.612

-0.01

1

4

2

12

170

0

O([H])C([H])([H])C([H])([H])N1C(=C([H])N=C1C([H])([H])[H])[N+](=O)[O-]

VAOCPAMSLUNLGC-UHFFFAOYSA-N

InChI=1S/C6H9N3O3/c1-5-7-4-6(9(11)12)8(5)2-3-10/h4,10H,2-3H2,1H3

2-(2-methyl-5-nitroimidazol-1-yl)ethanol

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (12.15 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 20.8 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: ≥ 2.08 mg/mL (12.15 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 20.8 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: ≥ 2.08 mg/mL (12.15 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 12.5 mg/mL (73.04 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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