Antitubercular antibiotic

Pyrazinamide is inert against growing Mycobacterium TB, except in settings with an acidic pH, and shows considerable efficacy in vivo. Acidic pH increases the intracellular build-up of POA, the active derivative of PZA, in Mycobacterium TB. It is believed that POA interferes with Mycobacterium tuberculosis's membrane energetics at acidic pH levels and impairs membrane transport functions [1].

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Pyrazinamide (PZA) is an important antituberculosis drug. Unlike most antibacterial agents, PZA, despite its remarkable in vivo activity, has no activity against Mycobacterium tuberculosis in vitro except at an acidic pH. M. tuberculosis is uniquely susceptible to PZA, but other mycobacteria as well as nonmycobacteria are intrinsically resistant. The role of acidic pH in PZA action and the basis for the unique PZA susceptibility of M. tuberculosis are unknown. We found that in M. tuberculosis, acidic pH enhanced the intracellular accumulation of pyrazinoic acid (POA), the active derivative of PZA, after conversion of PZA by pyrazinamidase. In contrast, at neutral or alkaline pH, POA was mainly found outside M. tuberculosis cells. PZA-resistant M. tuberculosis complex organisms did not convert PZA into POA. Unlike M. tuberculosis, intrinsically PZA-resistant M. smegmatis converted PZA into POA, but it did not accumulate POA even at an acidic pH, due to a very active POA efflux mechanism. We propose that a deficient POA efflux mechanism underlies the unique susceptibility of M. tuberculosis to PZA and that the natural PZA resistance of M. smegmatis is due to a highly active efflux pump. These findings may have implications with regard to the design of new antimycobacterial drugs.[1]

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Pyrazinamide (PZA) is a unique antituberculosis (anti-TB) drug that plays a key role in shortening TB therapy. PZA kills nonreplicating persisters that other TB drugs fail to kill, which makes it an essential drug for inclusion in any drug combinations for treating drug-susceptible and drug-resistant TB such as multidrug-resistant TB. PZA acts differently from common antibiotics by inhibiting multiple targets such as energy production, trans-translation, and perhaps pantothenate/coenzyme A required for persister survival. Resistance to PZA is mostly caused by mutations in the pncA gene encoding pyrazinamidase, which is involved in conversion of the prodrug PZA to the active form pyrazinoic acid. Mutations in the drug target ribosomal protein S1 (RpsA) are also found in some PZA-resistant strains. The recent finding that panD mutations are found in some PZA-resistant strains without pncA or rpsA mutations may suggest a third PZA resistance gene and a potential new target of PZA. Current phenotype-based PZA susceptibility testing is not reliable due to false resistance; sequencing of the pncA gene represents a more rapid, cost-effective, and reliable molecular test for PZA susceptibility testing and should be used for guiding improved treatment of multidrug-resistant and extensively multidrug-resistant TB. Finally, the story of PZA has important implications for not only TB therapy but also chemotherapy in general. PZA serves as a model prototype persister drug and hopefully a "tipping point" that inspires new efforts at developing a new type of antibiotic or drug that targets nonreplicating persisters for improved treatment of not only TB but also other persistent bacterial infections. [2]

The inclusion of PZA/Pyrazinamide with INH and RIF forms the basis for our current short-course chemotherapy based on the work by McDermott and colleagues in a mouse model of TB infection.. More recent efforts to find optimal drug combinations with new drug candidates for shortening TB treatment in the mouse model suggest that PZA is the only drug that cannot be replaced without compromising treatment efficacy. The common clinically used weak acids aspirin and ibuprofen enhanced the activity of PZA in the mouse model of TB infection. In vivo, PZA has high sterilizing activity against persisters in an acidic environment that is present during inflammation, which is responsible for its ability to shorten TB therapy. [2]

PZA/Pyrazinamide accumulation and conversion in PZA-susceptible and -resistant M. tuberculosis complex organisms.[1]
Two- to 3-week-old M. tuberculosis H37Ra and M. bovis BCG cultures, grown in Sauton’s medium, were harvested and then washed with Sauton’s medium, and the cell pellets were resuspended in Sauton’s medium (pH 6.6) at 5 × 109 cells/ml. [14C]PZA was added to these cell suspensions to a concentration of 1 μCi/ml, and the cell mixtures were incubated at 37°C. At different time points, 50-μl portions were removed and washed with Sauton’s medium by filtration on 0.45-μm-pore-size nitrocellulose filters by the use of a vacuum pump. The amount of radioactivity associated with the bacterial cells was determined by scintillation counting.

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Effect of reserpine and valinomycin on accumulation of POA in M. smegmatis and M. tuberculosis.[1]
[14C]PZA/Pyrazinamide was added to a concentrated bacterial suspension (5 × 109 cells/ml) in 7H9 liquid medium at pH 6.6 to a final concentration of 2 μCi/ml. A sublethal concentration of reserpine (20 μM) was added to the M. smegmatis cells after they had been incubated with [14C]PZA for 1 min, allowing a substantial amount of PZA to be taken up by the cells and converted to POA. At various times after the addition of reserpine, 50-μl portions were removed and spotted onto 0.45-μm-pore-size nitrocellulose membranes under a vacuum. Because washing the M. smegmatis cells with buffer tends to remove [14C]POA associated with the cells, the supernatant was removed by vacuum filtration without washing. The membrane area where the cell suspension was spotted was cut out, and the radioactivity was determined. The effect of valinomycin (1 μM; sublethal concentration) was determined in the same manner, using 10 mM potassium. The effect of reserpine and valinomycin on [14C]POA accumulation in M. tuberculosis was examined in a similar manner with the following modifications. Reserpine (50 μM) and valinomycin (1 μM) were added 2 h after addition of [14C]PZA to allow sufficient conversion of PZA to POA. Portions (50 μl) of suspension were filtered, and the radioactive cells on the membrane were washed twice with 2 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 0.1 M LiCl.

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Effect of pH on [14C]PZA/Pyrazinamide conversion and [14C]POA accumulation in M. tuberculosis.[1]
Late-log-phase M. tuberculosis H37Ra cultures (2 to 3 week old), grown in 7H9 liquid medium supplemented with albumin-dextrose-catalase, were centrifuged, and the cells were resuspended to a density of about 5 × 109/ml in 7H9 medium adjusted to various pH values. [14C]PZA was added to a concentration of 2.5 μCi/ml. Following incubation at 37°C for about 16 h, both the supernatant fluids and bacterial lysates, prepared by sonication of concentrated bacterial cells washed with phosphate-buffered saline, were analyzed by TLC followed by autoradiography (see below). To test the effect of pH on POA accumulation in the M. tuberculosis cells, [14C]POA was added to the bacterial suspensions, at various pH values, at a concentration of 1 μCi/ml. At various times, 50-μl portions were removed, filtered through 0.45-μm-pore-size nitrocellulose membranes, and washed with 0.1 M potassium phosphate buffer (pH 7.0) containing 0.1 M LiCl. In this as well as other experiments, the radioactivity associated with cells was measured by scintillation counting. The intracellular concentration of POA was calculated by assuming that 1 mg of dry cells is equivalent to 3 μl of internal water

Absorption, Distribution and Excretion
Pyrazinamide is rapidly and well absorbed from the gastrointestinal tract. Approximately 70% of the oral dose is excreted in the urine, primarily through glomerular filtration, and is eliminated within 24 hours. Pyrazinamide is well absorbed from the gastrointestinal tract and widely distributed throughout the body. After an oral dose of 500 mg, the plasma concentration is approximately 9-12 μg/mL at 2 hours and approximately 7 μg/mL at 8 hours. Pyrazinamide is well absorbed from the gastrointestinal tract. In healthy adults, after a single oral dose of 500 mg, peak plasma pyrazinamide concentrations are reached within 2 hours, ranging from 9-12 μg/mL; the average plasma concentration at 8 hours is 7 μg/mL, and the average concentration at 24 hours is 2 μg/mL. It has been reported that after a dose of 20-25 mg/kg, plasma concentrations range from 3-50 μg/mL. The plasma concentration of pyrazinic acid, the main active metabolite of pyrazinamide, is generally higher than that of its parent drug and reaches peak concentration within 4-8 hours after oral administration. In a single-dose study in healthy, fasting men, the absorption rates of isoniazid, rifampin, and pyrazinamide (measured by the area under the plasma concentration-time curve) were similar at doses of 250 mg, 600 mg, or 1500 mg, regardless of whether the drugs were administered alone in capsule (rifampin) or tablets (isoniazid and pyrazinamide), or in fixed-dose combination formulations containing 50 mg isoniazid, 120 mg rifampin, and 300 mg pyrazinamide per tablet. Pyrazinamide is widely distributed in body tissues and fluids, including the liver, lungs, and cerebrospinal fluid. In a small number of adult patients with tuberculous meningitis, the mean serum and cerebrospinal fluid concentrations of pyrazinamide were 52 μg/ml and 39 μg/ml, respectively, two hours after oral administration of approximately 41 mg/kg pyrazinamide. It has been reported that five hours after oral administration, the concentration of pyrazinamide in cerebrospinal fluid is approximately equal to the plasma concentration at the same time. In a small number of healthy men, the plasma protein binding rate of pyrazinamide (as determined by ultrafiltration) averaged approximately 17% at a concentration of 20 μg/ml. It is currently unknown whether pyrazinamide crosses the placenta. It is also unknown whether pyrazinamide is excreted into breast milk.
For more complete data on the absorption, distribution, and excretion of pyrazinamides (12 in total), please visit the HSDB record page.

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Metabolism/Metabolites
Hepatic metabolism.
Pyrazinamide is hydrolyzed to pyrazinoic acid, which is subsequently hydroxylated to 5-hydroxypyrazinoic acid, the major excretion product.
The main metabolic pathway of pyrazinamide is its conversion to pyrazinoic acid, followed by conversion to hydroxypyrazinoic acid, a reaction catalyzed by xanthine oxidase.
Eight healthy volunteers received a single dose of pyrazinamide at 35 mg/kg. This study aimed to evaluate the pharmacokinetic characteristics of this product and its metabolites. Urine and blood samples were collected up to 60 hours later. The pharmacokinetic characteristics of pyrazinamide are as follows: CPmax = 50.1 μg/ml, tmax < 1 h, t1/2α = 3.2 h, t1/2β = 23 h, U(0-60 h) = 1.6% of the administered dose. The pharmacokinetic characteristics of its major metabolite, pyrazinic acid, are as follows: CPmax = 66.6 μg/ml, tmax = 4 h, t1/2β = 12.3 h, U(0-60 h) = 37.5% of the administered dose. All existing data support the view that pyrazinamide metabolite pyrazinic acid (PA) is the active ingredient against Mycobacterium tuberculosis. Caffeine, a widely used drug and a common component of most diets, shares the same metabolic enzyme as pyrazinamide (PZA)—xanthine oxidase (XO).

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Biological Half-Life
9-10 hours (under normal conditions)
The plasma half-life is 9-10 hours in patients with normal renal function.
The half-life of pyrazinamide is 23 hours. …The elimination half-life is 10 to 16 hours.
The plasma half-life is 9-10 hours in patients with normal renal and hepatic function. In patients with impaired renal or hepatic function, the plasma half-life of this drug may be prolonged.

Hepatotoxicity
The use of pyrazinamide in combination therapy for tuberculosis often results in transient and asymptomatic elevations in serum transaminase levels. These elevations are typically less than five times the upper limit of normal. Because pyrazinamide is used only in combination with other anti-tuberculosis drugs, its effect on serum enzyme elevations is not fully understood, but it is often considered a cause of transient serum enzyme elevations. Pyrazinamide treatment can also cause clinically apparent liver disease, accompanied by symptoms and jaundice; therefore, in cases of dual or triple anti-tuberculosis therapy, pyrazinamide is often considered a culprit for liver injury (Case 1). In fact, this therapy has been discontinued because short-term (2 months) combination therapy of rifampin and pyrazinamide for latent tuberculosis often leads to severe, even life-threatening, liver damage (Case 2). Pyrazinamide-induced liver injury usually appears 4 to 8 weeks after administration, sometimes only after discontinuation. The pattern of liver enzyme elevation is typically hepatocellular, with clinical presentations resembling acute viral hepatitis, very similar to isoniazid hepatotoxicity. Hypersensitivity reactions (rash, fever, and eosinophilia) and autoantibody formation are uncommon. Liver biopsy shows typical changes of acute hepatitis, including portal vein and hepatic lobule inflammation, hepatocellular necrosis, and varying degrees of cholestasis. Probability score: A (Known cause of clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Limited information suggests that pyrazinamide use in pregnant women may result in significantly elevated drug concentrations in breast milk. If a breastfeeding woman is taking this medication, exclusively breastfed infants should be monitored for rare cases of jaundice, hepatitis, and arthralgia. The amount of pyrazinamide in breast milk may be insufficient to treat tuberculosis in breastfed infants, but if both the mother and breastfed infant are taking this medication, serum drug concentrations in the infant should be monitored to assess for excessively high infant serum concentrations. The Centers for Disease Control and Prevention (CDC) and other professional agencies advise that women taking pyrazinamide should not discontinue breastfeeding.
◉ Effects on Breastfed Infants
Pyrazinamide was used as part of a multidrug combination therapy to treat two pregnant women with multidrug-resistant tuberculosis throughout their pregnancies and postpartum. Both of their infants were breastfed (the extent and duration of breastfeeding were not specified). The two children developed normally at 1 year and 25 months and 5 years and 1 month, respectively.
Two mothers in Turkey were diagnosed with tuberculosis at 30 and 34 weeks of gestation, respectively. They immediately began taking isoniazid 300 mg, rifampin 600 mg, and pyridoxine 50 mg once daily for 6 months; simultaneously, they also took pyrazinamide 25 mg/kg and ethambutol 25 mg/kg once daily for 2 months. Both mothers breastfed (the extent of breastfeeding was not specified). Their infants received prophylactic treatment with isoniazid 5 mg/kg once daily for 3 months. After 3 months, the tuberculin skin test was negative, and neither infant had tuberculosis at 1 year of age. No adverse drug reactions were mentioned.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding rate
~10% (bound to plasma proteins)

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Drug interactions
Pyrazinamide may increase serum uric acid concentrations and reduce the efficacy of gout treatment; when pyrazinamide is used concomitantly with antigout medications, the dosage of drugs such as allopurinol, colchicine, probenecid, and sulfinpyrazone may need to be adjusted to control hyperuricemia and gout.
Concomitant use with pyrazinamide may decrease serum cyclosporine concentrations, potentially leading to insufficient immunosuppression; serum cyclosporine concentrations should be monitored.
A 35-year-old Somali woman with miliary tuberculosis developed hepatotoxicity several days after treatment with isoniazid, rifampin, pyrazinamide, and ethambutol. Liver function returned to normal after discontinuation of all medications and remained normal upon re-administration of isoniazid. Hepatotoxicity recurred after the addition of rifampin, but was well tolerated after re-administration of rifampin (without isoniazid). The use of pyrazinamide (PZA) in short-term anti-tuberculosis therapy is well-established. All existing data support the view that the PZA metabolite pyrazinic acid (PA) is the active ingredient against Mycobacterium tuberculosis. Further investigation is needed regarding potential interactions in its metabolic distribution. Caffeine, a widely used drug and a common component of most diets, shares the same metabolic enzyme as pyrazinamide (PZA)—xanthine oxidase (XO). This study aimed to investigate whether and how concomitant caffeine administration affects the metabolism of PZA. Female Sprague-Dawley rats were administered different doses of PZA and caffeine (PZA doses of 50 or 100 mg/kg, and caffeine doses of 0, 50, 100, and 150 mg/kg). Pyrazinamide (PZA) and its three major metabolites in 24-hour urine samples were quantitatively analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). Concomitant administration of 100 mg/kg caffeine and 50 mg/kg PZA significantly increased (p<0.05) the excretion of 5-hydroxypyrazinic acid (5-OH-PA), the most water-soluble and least toxic PZA metabolite, from 66.18 ± 10.87 μmol/24 h to 94.56 ± 8.65 μmol/24 h. This effect was more pronounced with administration of 100 mg/kg PZA, increasing the excretion of 5-OH-PA from 113.28 ± 70 μmol/24 h to 173.23 ± 17.82 μmol/24 h. These results indicate that the metabolic distribution of pyrazinamide is influenced by concurrent caffeine intake. For more complete data on interactions of pyrazinamides (9 in total), please visit the HSDB record page.

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Non-human toxicity values
Rats: Oral LDLo 3 g/kg
Mice: Oral LDLo 3 g/kg
Mice: Intraperitoneal LD50 1680 mg/kg
Mice: Subcutaneous LD50 2793 mg/kg
Hepatotoxicity Summary
Pyrazinamide is a first-line anti-tuberculosis drug, but it must be used in combination with other anti-tuberculosis drugs (such as isoniazid or rifampin). Pyrazinamide can cause a transient and asymptomatic increase in serum transaminase levels, which is a known cause of clinically apparent acute liver injury, which can be severe and even fatal.

[1]. Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. J Bacteriol. 1999 Apr;181(7):2044-9.

[2]. Mechanisms of Pyrazinamide Action and Resistance. Microbiol Spectr. 2014 Aug;2(4):MGM2-0023-2013.

Therapeutic Uses

Anti-tuberculosis Drug
Pyrazinamide is used in combination with other anti-mycobacterial drugs to treat tuberculosis. Pyrazinamide is effective only against mycobacteria. /US Product Label Includes/
The combination of rifampin, isoniazid, and pyrazinamide is suitable for the initial phase of a short course of treatment for all types of tuberculosis. During this phase (which should last 2 months), the combination of rifampin, isoniazid, and pyrazinamide should be taken daily. If multidrug-resistant tuberculosis is suspected, other drugs are required. /US Product Label Includes/
Pyrazinamide has become an important component of short-term (6 months) multidrug combination therapy for tuberculosis.
Drug Warnings
Patients with a hypersensitivity to ethionamide, isoniazid, nicotinic acid (Nicotinic acid), or other chemically related drugs may also be hypersensitive to this drug.
Pyrazinamide should only be used when the patient can be closely monitored. Serum AST (SGOT), ALT (SGPT), and uric acid levels should be monitored before starting pyrazinamide treatment and every 2–4 weeks during treatment. If signs of liver damage occur, pyrazinamide should be discontinued. Hepatotoxicity is the most frequently reported adverse reaction, with elevated transaminase levels being the earliest sign of toxicity… Pyrazinamide reduces renal tubular excretion of uric acid, which may induce acute gouty arthritis. Other adverse reactions include nausea, vomiting, dysuria, malaise, fever, and rash. This drug inhibits the excretion of urate, leading to hyperuricemia in almost all patients; acute gout attacks have been reported. Other adverse reactions of pyrazinamide include arthralgia, anorexia, nausea and vomiting, dysuria, malaise, and fever. For more complete data on drug warnings for pyrazinamide (19 in total), please visit the HSDB record page. Pharmacodynamics: Pyrazinamide kills or inhibits the growth of certain bacteria that cause tuberculosis (TB). It is used in combination with other drugs to treat tuberculosis. It is a highly specific drug, effective only against Mycobacterium tuberculosis. In vitro and in vivo, this drug is effective only under weakly acidic pH conditions. Pyrazinamide is activated in Mycobacterium tuberculosis to pyrazinoic acid, interfering with the fatty acid synthase FAS I. This interferes with the bacteria's ability to synthesize new fatty acids required for growth and replication. Pyrazinamide (PZA) is a prodrug that is converted to its active form, pyrazinoic acid (POA), in Mycobacterium tuberculosis by pyrazinamidinase (PZase)/nicotinamidease encoded by the pncA gene. While both PZA-sensitive and PZA-insensitive Mycobacterium tuberculosis possess PZase, which can convert PZA to POA, the specificity of PZA for Mycobacterium tuberculosis appears to be determined during the POA efflux phase, with Mycobacterium tuberculosis exhibiting a much weaker POA efflux capacity than the insensitive Mycobacterium smegma. Notably, the two types of pyrazinamide (PZA) resistance—acquired PZA resistance in susceptible Mycobacterium tuberculosis and intrinsic PZA resistance in non-tuberculous mycobacteria—are caused by distinctly different mechanisms. Acquired PZA resistance in susceptible Mycobacterium tuberculosis is caused by a mutation in the pncA gene that prevents the bacteria from converting the prodrug PZA into the bactericidal pyrazinamide (POA). In contrast, intrinsic PZA resistance in Mycobacterium smegmatis and possibly many other nontuberculous mycobacteria is due to a more active POA efflux mechanism that prevents POA from accumulating in the cell. [1]

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Pyrazinamide is a white powder. It sublimes at 318°F (150°C). (NTP, 1992) Pyrazinamide is a monocarboxylic acid amide formed by the condensation of the carboxyl group of pyrazinic acid (pyrazine-2-carboxylic acid) with amino groups. As a prodrug of pyrazinic acid, pyrazinamide is often used in multidrug combination therapy regimens for tuberculosis. It is both an anti-tuberculosis drug and a prodrug. Pyrazinamide belongs to the pyrazine class of compounds and is an N-acylamino monocarboxylic acid amide. Pyrazinamide is a prescription antibacterial drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of active tuberculosis (TB). (Active TB is also known as tuberculosis.) TB can be an opportunistic infection of HIV. Pyrazinamide is a pyrazine-based drug used to treat TB. Pyrazinamide is an antimycobacterial drug. Pyrazinamide is a first-line anti-TB drug but must be used in combination with other anti-TB drugs, such as isoniazid or rifampin. Pyrazinamide can cause a transient and asymptomatic increase in serum transaminase levels and is a known cause of clinically significant acute liver injury, which can be severe and even fatal. Pyrazinamide is a synthetic pyrazinamide derivative with bactericidal activity. Pyrazinamide is particularly effective against slowly multiplying intracellular Mycobacterium tuberculosis (unaffected by other drugs), but its mechanism of action is not fully understood. Its bactericidal effect depends on the presence of bacterial pyrazinamidinase, which removes the amide group to produce reactive pyrazinic acid. Pyrazinamide is an important component of multidrug combination therapy for TB. (NCI04) ChEBIP pyrazinamide is a small molecule drug, with its clinical trial phase up to Phase IV (covering all indications). It was first approved in 1971 for the treatment of tuberculosis and pulmonary tuberculosis, and has six investigational indications. ChEBIP pyrazinamide is a metabolite found or produced in Saccharomyces cerevisiae. ChEBIA pyrazine is an anti-tuberculosis drug used to treat tuberculosis.

C5H5N3O

123.1127

123.043

C, 48.78; H, 4.09; N, 34.13; O, 13.00

98-96-4

Pyrazinamide-d3;1432059-16-9

1046

White to off-white solid powder

1.4±0.1 g/cm3

273.3±43.0 °C at 760 mmHg

189-191 °C(lit.)

119.1±28.2 °C

0.0±0.6 mmHg at 25°C

1.648

-0.83

1

3

1

9

115

0

C1=CN=C(C=N1)C(=O)N

IPEHBUMCGVEMRF-UHFFFAOYSA-N

InChI=1S/C5H5N3O/c6-5(9)4-3-7-1-2-8-4/h1-3H,(H2,6,9)

pyrazine-2-carboxamide

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 : ≥ 50 mg/mL (~406.14 mM)
H2O : ~6.67 mg/mL (~54.18 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (20.31 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (20.31 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (20.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 27.5 mg/mL (223.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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