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
Rapidly and well absorbed from the gastrointestinal tract.
Approximately 70% of an oral dose is excreted in the urine, mainly by glomerular filtration within 24 hours
Pyrazinamide is well absorbed from the gastrointestinal tract, and it is widely distributed throughout the body. The oral administration of 500 mg produces plasma concentrations of about 9-12 ug/ml at two hours and 7 ug/ml at 8 hours.
Pyrazinamide is well absorbed from the GI tract. Following a single 500 mg oral dose in healthy adults, peak plasma concentrations of pyrazinamide ranging from 9-12 ug/ml are attained within 2 hours; plasma concentrations of the drug average 7 ug/ml at 8 hours and 2 ug/ml at 24 hours. Plasma concentrations following doses of 20-25 mg/kg reportedly range from 3-50 ug/ml. Plasma concentrations of pyrazinoic acid, the major active metabolite of pyrazinamide, generally are greater than those of the parent drug and peak within 4-8 hours after an oral dose of the drug.
In a single-dose study in healthy fasting males, the extent of absorption (as measured by area under the plasma concentration-time curve) of isoniazid, rifampin, or pyrazinamide in dosages of 250, 6O0, or 1500 mg, respectively, was similar whether the drugs were administered individually as capsules (rifampin) and tablets (isoniazid and pyrazinamide) or as a fixed combination containing isoniazid 50 mg, rifampin 120 mg, and pyrazinamide 300 mg per tablet.
Pyrazinamide is widely distributed into body tissues and fluids including the liver, lungs, and CSF. In a limited number of adults with tuberculous meningitis, mean serum and CSF concentrations of pyrazinamide 2 hours after an oral dose of approximately 41 mg/kg were 52 and 39 ug/ml, respectively. Within 5 hours after an oral dose, CSF concentrations of pyrazinamide are reported to be approximately equal to concurrent plasma concentrations of the drug. Plasma protein binding of pyrazinamide (determined by ultrafiltration) in a limited number of healthy men averaged approximately 17% at a pyrazinamide concentration of 20 ug/ml. It is not known if pyrazinamide crosses the placenta. It is not known if pyrazinamide is distributed into milk.
For more Absorption, Distribution and Excretion (Complete) data for PYRAZINAMIDE (12 total), please visit the HSDB record page.

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Metabolism / Metabolites
Hepatic.
Pyrazinamide is hydrolyzed to pyrazinoic acid and subsequently hydroxylated to 5-hydroxypyrazinoic acid, the major excretory product.
The major metabolic pathway of pyrazinamide is conversion to pyrazinoic acid followed by subsequent conversion to hydroxypyrazinoic acid, a reaction catalyzed by xanthine oxidase.
Eight healthy volunteers were treated with a single dose of pyrazinamide 35 mg/kg. The aim of the study was to evaluate the pharmacokinetic profile of the product and of its metabolites. Urine and blood samples were collected till the 60th h. The kinetics of pyrazinamide could be characterized as follows: CPmax = 50.1 micrograms/ml, tmax less than 1 h, t1/2 alpha = 3.2 h, t1/2 beta = 23 h, U(0-60 h) = 1.6% of the dose administered. The kinetics of the main metabolite, the pyrazinoic acid, gave the following values: CPmax = 66.6 micrograms/ml, tmax = 4 h, t1/2 beta = 12.3 h, U(0-60 h) = 37.5%, of the administered dose.
All available data support the idea that the PZA metabolite pyrazinoic acid (PA) is the active compound against M. tuberculosis. ... Caffeine, which is widely used as a drug and is a common constituent of most diets, shares with PZA the same metabolic enzyme, xanthine oxidase (XO).

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Biological Half-Life
9-10 hours (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 of pyrazinamide is 9-10 hours in patients with normal renal and hepatic function. The plasma half-life of the drug may be prolonged in patients with impaired renal or hepatic function.

Hepatotoxicity
Combination therapy for tuberculosis using pyrazinamide is commonly associated with transient and asymptomatic elevations in the serum aminotransferase levels. These elevations are usually less than five times the upper limit of the normal range. Because pyrazinamide is used only in combination with other antituberculosis medications, its contributions to serum enzyme elevations is not completely clear, but it is frequently incriminated in transient serum enzyme elevations. Clinically apparent liver disease with symptoms and jaundice also occur with pyrazinamide therapy and it is often considered the culprit in causing liver injury in the face of double or triple antituberculosis therapy (Case 1). Indeed, the use of a short, 2 month course of combination therapy with rifampin and pyrazinamide for latent tuberculosis was abandoned because of the frequency of severe liver injury with this regimen that was occasionally fatal (Case 2). The onset of injury due to pyrazinamide is generally after 4 to 8 weeks and occasionally becomes apparent only after the pyrazinamide is stopped. The pattern of liver enzyme elevations is typically hepatocellular and the clinical syndrome resembles acute viral hepatitis, much like isoniazid hepatotoxicity. Features of hypersensitivity (rash, fever and eosinophilia) are uncommon as are autoantibody formation. Liver biopsy demonstrates changes typical of acute hepatitis with portal and lobular inflammation, hepatocellular necrosis and variable degrees of cholestasis.
Likelihood score: A (well known cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Limited information indicates that maternal pyrazinamide therapy produces potentially substantial levels in milk. Exclusively breastfed infants should be monitored for rare cases of jaundice, hepatitis and arthralgia if this drug is used during lactation. The amount of pyrazinamide in milk is probably insufficient to treat tuberculosis in the breastfed infant, but if both the mother and breastfed infant are receiving the drug, infant serum levels could be monitored to assess for excessive infant serum concentrations. The Centers for Disease Control and Prevention and other professional organizations state that breastfeeding should not be discouraged in women taking pyrazinamide.
◉ Effects in Breastfed Infants
Pyrazinamide was used as part of multi-drug regimens to treat 2 pregnant women with multidrug-resistant tuberculosis throughout pregnancy and postpartum. Their two infants were breastfed (extent and duration not stated). At age 1.25 and 5.1 years, the children were developing normally.
Two mothers in Türkiye were diagnosed with tuberculosis at the 30th and 34th weeks of pregnancy. They immediately started isoniazid 300 mg, rifampin 600 mg, pyridoxine 50 mg daily for 6 months, plus pyrazinamide 25 mg/kg and ethambutol 25 mg/kg daily for 2 months. Both mothers breastfed their infants (extent not stated). Their infants were given isoniazid 5 mg/kg daily for 3 months prophylactically. Tuberculin skin tests were negative after 3 months and neither infant had tuberculosis at 1 year of age. No adverse effects of the drugs were mentioned.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
~10% (bound to plasma proteins)

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Interactions
Pyrazinamide may increase serum uric acid concentrations and decrease the efficacy of gout therapy; dosage adjustments of these medications /allopurinol, colchicine, probenecid, sulfinpyrazone/ may be necessary to control hyperuricemia and gout when antigout medications are used concurrently with pyrazinamide.
Concurrent use with pyrazinamide may decrease the serum concentrations of cyclosporine, possibly leading to inadequate immunosuppression; cyclosporine serum concentrations should be monitored.
A 35 year old black Somalian woman with miliary tuberculosis developed hepatotoxicity after a few days of treatment with isoniazid, rifampicin, pyrazinamide, and ethambutol. After withdrawal of all drugs the liver profile returned to normal and remained so after challenge with isoniazid. Hepatotoxicity recurred when rifampicin was added, but it was well tolerated when reintroduced without isoniazid.
The utility of pyrazinamide (PZA) in the short-course antituberculous treatment is well established. All available data support the idea that the PZA metabolite pyrazinoic acid (PA) is the active compound against M. tuberculosis. This situation warranted a deeper investigation of possible interactions with respect to its metabolic disposition. Caffeine, which is widely used as a drug and is a common constituent of most diets, shares with PZA the same metabolic enzyme, xanthine oxidase (XO). This study investigated if, and in what manner, concomitant administration of caffeine affects PZA metabolism. PZA and caffeine, in various doses (PZA=50 or 100 mg kg(-1) and caffeine= 0, 50, 100, and 150 mg kg(-1)), were administered to female Sprague-Dawley rats. PZA and its three main metabolites were quantified in 24 h urine samples by reversed phase-HPLC Concomitant administration of 100 mg kg(-1) caffeine and 50 mg kg(-1) PZA increased from the excretion (p<0.05) of the most water-soluble and the least toxic PZA metabolite 5-hydroxypyrazinoic acid (5-OH-PA) from 66.18+/-10.87 to 94.56+/-8.65 micromol/24 h. This effect was more pronounced when 100 mg kg(-1) of PZA was administered increasing excretion of 5-OH-PA from 113.28+/-70 to 173.23+/-17.82 micromol/24 h. These results show that the metabolic disposition of PZA is affected by concomitant caffeine intake.
For more Interactions (Complete) data for PYRAZINAMIDE (9 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LDLo Rat oral 3 g/kg
LDLo Mouse oral 3 g/kg
LD50 Mouse intraperitoneal 1680 mg/kg
LD50 Mouse subcutaneous 2793 mg/kg

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LiverTox Summary
Pyrazinamide is a first line antituberculosis medication, but is used only in combination with other antituberculosis medications such as isoniazid or rifampin. Pyrazinamide is associated with transient and asymptomatic elevations in serum aminotransferase levels and is a well known cause of clinically apparent, acute liver injury that 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
Antitubercular Agents
Pyrazinamide is indicated in combination with other antimycobacterial drugs, in the treatment of tuberculosis. Pyrazinamide is effective only against mycobacteria. /Included in US product labeling/
Rifampin, isoniazid, and pyrazinamide combination is indicated in the initial phase of the short-course treatment of all forms of tuberculosis. During this phase, which should last 2 months, rifampin, isoniazid, and pyrazinamide combination should be administered on a daily, continuous basis. Additional medications are indicated if multidrug-resistant tuberculosis is suspected. /Included in US product labeling/
Pyrazinamide has become an important component of short-term (6 month) multiple-drug therapy of tuberculosis.

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Drug Warnings
Patients hypersensitive to ethionamide, isoniazid, niacin (nicotinic acid), or other chemically related medications may be hypersensitive to this medication also.
Pyrazinamide should be used only when close observation of the patient is possible. Serum AST (SGOT), ALT (SGPT), and uric acid concentrations should be determined prior to and every 2-4 weeks during pyrazinamide therapy. If signs of hepatic damage occur, pyrazinamide should be discontinued.
Hepatotoxicity is the most commonly reported adverse effect, with elevated transaminase levels being the earliest indication of toxicity...Pyrazinamide decreases the tubular excretion of uric acid, which may induce an acute gouty arthritis. Other adverse reactions include nausea, vomiting, dysuria, malaise, fever and skin rashes.
The drug inhibits excretion of urate, resulting in hyperuricemia in nearly all patients; acute episodes of gout have occurred. Other untoward effects that have been observed with pyrazinamide are arthralgias, anorexia, nausea and vomiting, dysuria, malaise and fever.
For more Drug Warnings (Complete) data for PYRAZINAMIDE (19 total), please visit the HSDB record page.

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Pharmacodynamics
Pyrazinamide kills or stops the growth of certain bacteria that cause tuberculosis (TB). It is used with other drugs to treat tuberculosis. It is a highly specific agent and is active only against Mycobacterium tuberculosis. In vitro and in vivo, the drug is active only at a slightly acid pH. Pyrazinamie gets activated to Pyrazinoic acid in the bacilli where it interferes with fatty acid synthase FAS I. This interferes with the bacteriums ability to synthesize new fatty acids, required for growth and replication.

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PZA is a prodrug that is converted to the active form pyrazinoic acid (POA) by pyrazinamidase (PZase)/nicotinamidase, encoded by the pncA gene in M. tuberculosis.

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While both susceptible M. tuberculosis and other nonsusceptible mycobacteria have PZases to convert PZA to POA, the specificity of PZA for M. tuberculosis appears to be conferred at the stage of POA efflux, which is much weaker in M. tuberculosis than in the nonsusceptible M. smegmatis. It is noteworthy that the two types of PZA resistance, the acquired PZA resistance found in susceptible M. tuberculosis and the intrinsic PZA resistance found in nontuberculous mycobacteria, are caused by very different mechanisms. Acquired PZA resistance in susceptible M. tuberculosis is caused by mutations in the pncA gene which render the organisms unable to convert the prodrug PZA to bactericidal POA. In contrast, the intrinsic PZA resistance in M. smegmatis, and probably in many other nontuberculous mycobacteria, is due to a much more active POA efflux mechanism which does not allow accumulation of POA in the cells.[1]

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Pyrazinamide is a white powder. Sublimes from 318 °F. (NTP, 1992)
Pyrazinecarboxamide is a monocarboxylic acid amide resulting from the formal condensation of the carboxy group of pyrazinoic acid (pyrazine-2-carboxylic acid) with ammonia. A prodrug for pyrazinoic acid, pyrazinecarboxamide is used as part of multidrug regimens for the treatment of tuberculosis. It has a role as an antitubercular agent and a prodrug. It is a member of pyrazines, a N-acylammonia and a monocarboxylic acid amide. ChEBI Pyrazinamide is an antibacterial prescription medicine approved by the U.S. Food and Drug Administration (FDA) for the treatment of active tuberculosis (TB). (Active TB is also called TB disease.) ChEBI TB can be an opportunistic infection (OI) of HIV.

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A pyrazine that is used therapeutically as an antitubercular agent. ChEBI Pyrazinamide is an Antimycobacterial. ChEBI Pyrazinamide is a first line antituberculosis medication, but is used only in combination with other antituberculosis medications such as isoniazid or rifampin. Pyrazinamide is associated with transient and asymptomatic elevations in serum aminotransferase levels and is a well known cause of clinically apparent, acute liver injury that can be severe and even fatal. ChEBIChEBI Pyrazinamide is a synthetic pyrazinoic acid amide derivative with bactericidal property. Pyrazinamide is particularly active against slowly multiplying intracellular bacilli (unaffected by other drugs) by an unknown mechanism of action. Its bactericidal action is dependent upon the presence of bacterial pyrazinamidase, which removes the amide group to produce active pyrazinoic acid. Pyrazinamide is an important component of multidrug therapy for tuberculosis. (NCI04) ChEBI PYRAZINAMIDE is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 1971 and is indicated for tuberculosis and pulmonary tuberculosis and has 6 investigational indications. ChEBIChEBI Pyrazinecarboxamide is a metabolite found in or produced by Saccharomyces cerevisiae. ChEBI A pyrazine that is used therapeutically as an antitubercular agent.

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.)