Pretomanid (also known as PA-824; PA824), a bicyclic nitroimidazolepyran analog, is a novel, potent and selective anti-tuberculosis (TB) drug approved in 2019 for treating multi-drug-resistant tuberculosis with an MIC of less than 2.8 μM. It is generally used together with bedaquiline and linezolid. PA-824 has been found to exhibit bactericidal activity against replicating bacilli and non-replicating bacilli under hypoxic or prolonged culture conditions in a dose dependent fashion through two possible mechanisms, which include PA-824 induced inhibition of ketomycolate synthesis and PA-824 mediated donation of nitric oxide during enzymatic nitro-reduction.

Physicochemical Properties

Molecular Formula	C14H12F3N3O5
Molecular Weight	359.26
Exact Mass	359.072
Elemental Analysis	C, 46.80; H, 3.37; F, 15.86; N, 11.70; O, 22.27
CAS #	187235-37-6
Related CAS #	Pretomanid-d4;1346617-34-2
PubChem CID	456199
Appearance	Typically exists as off-white to yellow solids at room temperature
Density	1.6±0.1 g/cm3
Boiling Point	462.3±55.0 °C at 760 mmHg
Melting Point	150 °C
Flash Point	233.4±31.5 °C
Vapour Pressure	0.0±1.1 mmHg at 25°C
Index of Refraction	1.589
LogP	2.7
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	4
Heavy Atom Count	25
Complexity	468
Defined Atom Stereocenter Count	1
SMILES	FC(OC1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])O[C@]1([H])C([H])([H])OC2=NC(=C([H])N2C1([H])[H])[N+](=O)[O-])(F)F
InChi Key	ZLHZLMOSPGACSZ-NSHDSACASA-N
InChi Code	InChI=1S/C14H12F3N3O5/c15-14(16,17)25-10-3-1-9(2-4-10)7-23-11-5-19-6-12(20(21)22)18-13(19)24-8-11/h1-4,6,11H,5,7-8H2/t11-/m0/s1
Chemical Name	6S)-2-nitro-6-[[4-(trifluoromethoxy)phenyl]methoxy]-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
Synonyms	PA-824; Pretomanid; PA 824; 187235-37-6; (S)-2-Nitro-6-((4-(trifluoromethoxy)benzyl)oxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine; (S)-PA 824; UNII-2XOI31YC4N; PA824;
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Tuberculosis
ln Vitro	PA-824 exhibits the high activity against multidrug-resistant clinical isolates from Asia (India and South Korea) and from throughout the United States (MIC < 1 μg/ml) and is equally active against the drug-sensitive and multidrug-resistant isolates of M. tuberculosis (MICs range, 0.039 to 0.531 μg/ml). A recent study shows that single-nucleotide polymorphisms of PA-824 resistance genes (fgd1 [Rv0407] and ddn [Rv3547]) dont significantly affect the PA-824 MICs (≤ 0.25 μg/ml).
ln Vivo	In the rapid tuberculosis mouse model, PA-824 shows significant anti-microbial activity in a dose-dependent manner: at 50 mg/kg, PA-824 in MC produces a more than 1-log reduction of the CFU in the lungs; at 100 mg/kg it produces about a 2-log reduction, and at 300 mg/kg it produces a 3-log reduction. Furthermore, long-term treatment of PA-824 at 100 mg/kg in cyclodextrin/lecithin also leads to the reduction of the bacterial load below 500 CFU in the lungs and spleen. PA-824 exhibits time-dependent anti-microbial activity in a murine model of tuberculosis with a maximal observed bactericidal effect of 0.1 log CFU/day over 24 days. PA-824 is one of two nitroimidazoles in phase II clinical trials to treat tuberculosis. In mice, it has dose-dependent early bactericidal and sterilizing activity. In humans with tuberculosis, PA-824 demonstrated early bactericidal activity (EBA) at doses ranging from 200 to 1,200 mg per day, but no dose-response effect was observed. To better understand the relationship between drug exposure and effect, we performed a dose fractionation study in mice. Dose-ranging pharmacokinetic data were used to simulate drug exposure profiles. Beginning 2 weeks after aerosol infection with Mycobacterium tuberculosis, total PA-824 doses from 144 to 4,608 mg/kg were administered as 3, 4, 8, 12, 24, or 48 divided doses over 24 days. Lung CFU counts after treatment were strongly correlated with the free drug T(>MIC) (R(2) = 0.87) and correlated with the free drug AUC/MIC (R(2) = 0.60), but not with the free drug C(max)/MIC (R(2) = 0.17), where T(>MIC) is the cumulative percentage of the dosing interval that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions and AUC is the area under the concentration-time curve. When the data set was limited to regimens with dosing intervals of ≤72 h, both the T(>MIC) and the AUC/MIC values fit the data well. Free drug T(>MIC) of 22, 48, and 77% were associated with bacteriostasis, a 1-log kill, and a 1.59-log kill (or 80% of the maximum observed effect), respectively. Human pharmacodynamic simulations based on phase I data predict 200 mg/day produces free drug T(>MIC) values near the target for maximal observed bactericidal effect. The results support the recently demonstrated an EBA of 200 mg/day and the lack of a dose-response between 200 and 1,200 mg/day. T(>MIC), in conjunction with AUC/MIC, is the parameter on which dose optimization of PA-824 should be based.[3] Leprosy responds very slowly to the current multidrug therapy, and hence there is a need for novel drugs with potent bactericidal activity. PA-824 is a 4-nitroimidazo-oxazine that is currently undergoing phase I clinical trials for the treatment of tuberculosis. The activity of PA-824 against Mycobacterium leprae was tested and compared with that of rifampin in axenic cultures, macrophages, and two different animal models. Our results conclusively demonstrate that PA-824 has no effect on the viability of M. leprae in all three models, consistent with the lack of the nitroimidazo-oxazine-specific nitroreductase, encoded by Rv3547 in the M. leprae genome, which is essential for activation of this molecule.[5]
Enzyme Assay	Pretomanid (PA-824) is a small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis; the MIC values of PA-824 against a panel of MTB pan-sensitive and rifampin mono-resistant clinical isolates ranged from 0.015 to 0.25 ug/ml. IC50 value: 0.015 to 0.25 ug/ml (MICs). Microdilution MIC plate assay.[1] A method described by Wallace et al. was used to determine the MICs by a microdilution plate assay by using M. tuberculosis H37Rv. INH was dissolved in sterile, double-distilled water at a stock concentration of 500 μg/ml. Pretomanid (PA-824) was dissolved in 100% dimethyl sulfoxide to a stock concentration of 100 μg/ml. A 1:2 dilution series of both compounds was made in a separate 96-well microtiter plate by using the same diluents. The interior 60 wells of a 96-well round-bottom microtiter assay plate were seeded with 98 μl of bacterial suspension (as described above). Two microliters of each drug was transferred to the assay plate wells containing bacteria. The final concentrations of INH in the wells ranged from 10.0 to 0.039 μg/ml; the final concentrations of Pretomanid (PA-824) ranged from 2.0 μg/ml to 8.0 pg/ml. The assay plates were incubated at 37°C for at least 21 days and were observed every 3 to 4 days to evaluate changes in growth. Inhibition of growth was determined both by visual examination and with a spectrophotometer at an OD600. Determination of the MIC.[3] The MIC was determined by the agar proportion method. Middlebrook 7H11 agar supplemented with 10% OADC and containing serial 2-fold concentrations of Pretomanid (PA-824) ranging from 0.007 to 2.0 μg/ml were inoculated with 0.5 ml of serial 100-fold dilutions of a log-phase broth culture of M. tuberculosis H37Rv with an optical density at 600 nm corresponding to ∼108 CFU/ml. Drug-free and isoniazid-containing plates served as negative and positive controls, respectively. CFU were counted after 21 days incubation at 37°C with 5% ambient CO2. The MIC was defined as the lowest concentration at which the CFU count on drug-containing plates was <1% of the CFU count on drug-free plates. Mycobacterial strains and growth conditions.[2] A total of 65 Mycobacterium strains (62 clinical isolates and 3 ATCC reference strains), including M. tuberculosis, M. africanum, M. bovis, M. caprae, M. pinnipedii, M. microti, and “M. canettii” strains, were used in this study (Table 1). All strains belonged to a reference collection comprising all major phylogenetic lineages of the MTBC and had been described earlier. Most of these were pansusceptible to standard antituberculosis drugs. Furthermore, three well-characterized Pretomanid (PA-824)-resistant control strains (H37Rv-T3, H37Rv-5A1, and H37Rv-14A1) were included. Strains used for DNA isolation and MIC determination were cultivated on Löwenstein-Jensen agar slants. Drug susceptibility testing.[2] Pretomanid (PA-824) drug susceptibility testing was performed in the supranational reference laboratory in Borstel, Germany, using the modified proportion method in the Bactec MGIT 960 system. The PA-824 concentrations used were 1, 0.5, 0.25, 0.125, 0.0625, and 0.0312 μg/ml. The M. canettii strains and the PA-824-resistant positive controls were additionally tested at concentrations of 32, 16, 8, 4, and 2 μg/ml.
Cell Assay	A method is used to determine the MICs by a microdilution plate assay by using M. tuberculosis H37Rv. INH is dissolved in sterile, double-distilled water at a stock concentration of 500 μg/ml. PA-824 is dissolved in 100% dimethyl sulfoxide (DMSO) to a stock concentration of 100 μg/ml. A 1:2 dilution series of both compounds is made in a separate 96-well microtiter plate by using the same diluents. The interior 60 wells of a 96-well round-bottom microtiter assay plate are seeded with 98 μl of bacterial suspension. Two microliters of each drug is transferred to the assay plate wells containing bacteria. The final concentrations of INH in the wells range from 10.0 to 0.039 μg/mL; the final concentrations of PA-824 range from 2.0 μg/mL to 8.0 pg/mL. The assay plates are incubated at 37 °C for at least 21 days and are observed every 3 to 4 days to evaluate changes in growth. Inhibition of growth is determined both by visual examination and with a spectrophotometer at an OD600.
Animal Protocol	Drug preparation for in vivo models.[1] RIF was dissolved in 100% DMSO, with subsequent dilution in sterile water prior to administration. The final concentration of DMSO in the drug preparation was 5%. INH, PZA, STR, GAT, and MXF were dissolved in water. Pretomanid (PA-824) was formulated either in 0.5% methylcellulose or in cyclodextrin/lecithin (CM2). The CM2 formulation for Pretomanid (PA-824) was developed by PathoGenesis, and that formulation was exactly copied for the in vivo experiments. Briefly, for the preparation of the 100-mg/kg dose, 10 mg of PA-824 was added to 1 ml of a 10% solution of hydroxypropyl-β-cyclodextrin, and the mixture was stirred gently for 24 h at room temperature. The resulting suspension was sonicated with a Vibra Cell probe sonicator (model VC-130; Sonics and Materials, Inc., Newtown, CT) for 10 min at 25% amplitude. Frozen lecithin was added at a final concentration of 10%; the suspension was stirred for 10 min at room temperature, cooled in an ice-water bath, and sonicated at 30% amplitude for 15 min while the solution temperature was kept at less than 50°C. For the preparation of the lower and higher doses (50 and 300 mg/kg of PA-824, respectively, in CM2), the amount of drug was adjusted. The concentration of cyclodextrin/lecithin remained the same as that described above, as was the volume administered to each mouse (200 μl). Rapid in vivo screen.[1] Eight- to 10-week-old female specific-pathogen-free C57BL/6-Ifngtm1ts mice (gamma interferon gene-disrupted [GKO] mice) were purchased from Jackson Laboratories, Bar Harbor, ME. The mice were infected via a low-dose aerosol exposure to M. tuberculosis Erdman in a Middlebrook aerosol generation device, and the short-course mouse model was performed as described previously. One day postinfection, three mice were killed to verify the uptake of 50 to 100 CFU of bacteria per mouse. Following infection, the mice were randomly divided into 11 treatment groups. Negative control mice remained untreated. Positive control mice received INH (at 25 mg/kg of body weight), RIF (at 20 mg/kg), or MXF (at 100 or 300 mg/kg). Six groups received Pretomanid (PA-824) formulated in either MC or CM2 (at 50, 100, or 300 mg/kg). Each treatment group consisted of five mice. Treatment was started 19 days postinfection and continued for nine consecutive days. Three infected mice were killed at the start of treatment as pretreatment controls. Drugs were administered daily by oral gavage. Long-term in vivo screen.[1] Six- to 8-week-old female specific-pathogen-free immunocompetent C57BL/6 mice were infected via a low-dose aerosol exposure to M. tuberculosis Erdman as described before. Two successive aerosol runs were performed with 90 mice in each round. One day postinfection, three mice from each run were killed to verify the uptake of 50 to 100 CFU of bacteria per mouse. Following infection, the mice were randomly divided into 10 treatment groups. Negative control mice remained untreated. Positive control mice received INH (at 25 mg/kg of body weight), GAT (at 100 mg/kg), or MXF (at 100 mg/kg). The other treatment groups received Pretomanid (PA-824) (at 100 mg/kg) in the CM2 formulation. Each group consisted of five to six mice at each time point. Treatment was started 3 weeks postinfection and continued for 12 weeks. Five infected mice were killed at the start of treatment as pretreatment controls. Drugs were administered 5 days per week by oral gavage. To determine drug efficacies at intermediate time points, a group of mice from each treatment group was killed at weeks 2, 6, and 12 after the start of treatment. Dose-ranging PK of Pretomanid (PA-824).[3] All procedures involving animals were approved by the institutional animal care and use committee. Single-dose pharmacokinetics (PK) of Pretomanid (PA-824) in serum were evaluated in uninfected 6-week-old female BALB/c mice after oral administration of 3, 10, 18, 30, 54, 96, 162, 243, 486, 729, and 1,458 mg/kg doses. Multidose PK were also determined for 6-, 9.6-, 28.8-, 96-, and 192-mg/kg doses administered once daily for 5 days, with serum sampling performed after the fifth dose. In a second multidose PK study, mice received PA-824 at 192 mg/kg every 6 days, with serum sampling performed after the third dose. Mice had access to food and water ad libitum. PA-824 was administered by esophageal gavage. Three mice from each group were sacrificed at 0.5, 1, 2, 4, 8, 16, 24, 36, 48, 72, 96, and 120 h after the last dose. Mice were anesthetized with isoflurane and exsanguinated by cardiac puncture. Blood was collected in microcentrifuge tubes and left at room temperature for 30 min before being centrifuged to harvest the serum. Serum samples were frozen at −80°C before the concentration of PA-824 was determined by a validated high-pressure liquid chromatography (HPLC) method. Briefly, the concentration of PA-824 was determined with a system consisting of a ThermoFinnegan P4000 HPLC pump with a model AS1000 fixed-volume autosampler, a model UV2000 UV detector, a Gateway series e computer, and the Chromquest HPLC data management system. The plasma standard concentration curve for PA-824 ranged from 0.20 to 50 μg/ml. The absolute recovery of PA-824 from plasma was 88.2%. The overall precision of the validation assay across all standards was 0.67 to 5.38%.[3] Serum concentration data were entered into a WinNonlin worksheet and analyzed by using standard noncompartmental and compartmental techniques in order to determine the relevant PK parameters for simulations. The serum concentration-time profile of each dosing regimen described in the dose fractionation protocol (Table 1) was modeled over a 6-day period to estimate the Cmax, the AUC0-144, and the T>MIC(0-144h) for each regimen for free Pretomanid (PA-824) concentrations, assuming 92.5% serum protein binding based on 93% protein binding in serum obtained from mice at the Tmax after a 25-mg/kg dose and up to 90% protein binding at a concentration of 3.6 μg/ml in spiked mouse serum, as determined by equilibrium dialysis (TB Alliance, data on file). The AUC0-24 was calculated by dividing the AUC0-144 by 6. Formulated either in 0.5% methylcellulose (MC) or in cyclodextrin/lecithin (CM2); ≤300 mg/kg; p.o. Gamma interferon gene-disrupted (GKO) mice are infected via a low-dose aerosol exposure to M. tuberculosis Erdman.
ADME/Pharmacokinetics	Absorption, Distribution and Excretion This drug is absorbed in the gastrointestinal tract. The steady-state Cmax of pretomanid was estimated to be 1.7 μg/mL after a single 200mg oral dose. In a separate pharmacokinetic modeling study, the Cmax of a 200mg dose was 1.1 μg/ml. Tmax in a study of healthy subjects in the fed or unfed state was achieved within 4 to 5 hours. The AUC in the same study was found to be about 28.1 μg•hr/mL in the fasted state and about 51.6 μg•hr/mL in the fed state, showing higher absorption when taken with high-calorie and high-fat food. Healthy adult male volunteers were administered a 1,100 mg oral dose of radiolabeled pretomanid in one pharmacokinetic study. An average of about 53% of the radioactive dose was found to be excreted in the urine. Approximately 38% was measured mainly as metabolites in the feces. A estimated 1% of the radiolabeled dose was measured as unchanged drug in the urine. A pharmacokinetic modeling study estimated the volume of distribution at 130 ± 5L. A pharmacokinetic study in healthy volunteers determined a volume of distribution of about 180 ± 51.3L in fasted state and 97.0 ± 17.2L in the fed state. The clearance of pretomanid in a pharmacokinetic simulation study has been estimated at 4.8 ± 0.2 liters/h. According to the FDA label, the clearance of a single 200 mg oral dose of pretomanid is estimated to be 7.6 liters/h in the fasted state, and 3.9 liters/h in the fed state. Metabolism / Metabolites Various reductive and oxidative pathways are responsible for pretomanid metabolism, with no single major metabolic pathway identified. According to in vitro studies, CYP3A4 is responsible for a 20% contribution to the metabolism of pretomanid. Biological Half-Life The elimination half-life was determined to be 16.9-17.4 hours in a pharmacokinetic study of healthy subjects. An FDA briefing document reports a half-life of 18 hours. Dose-ranging PK of PA-824 in mice. [3] Single doses of PA-824 up to 1,456 mg/kg were well tolerated, with no adverse effects observed. Similarly, no untoward effect was observed in the multidose PK studies. The time to reach Cmax (Tmax) in serum was 4.0 h. The elimination half-life was 4 to 6 h. PA-824 concentrations increased in a dose-proportional fashion over the dose range from 10 to 243 mg/kg (Fig. 1). At doses of >486 mg/kg, the serum concentration-time profile suggested more complex PK behavior, possibly due to saturation of oral absorption. Also, late secondary and tertiary peaks at 24 and 48 h suggested precipitation and subsequent redissolution of PA-824 in the gastrointestinal tract, with diurnal variation. Hence, with the exception of 384 mg/kg administered every 6 days, individual dosing regimens in the dose fractionation study did not exceed 288 mg/kg. This dose range easily encompasses the achievable range of serum concentrations in humans with current oral formulations
Toxicity/Toxicokinetics	Hepatotoxicity Liver test abnormalities occur in 30% of patients treated with multiple drug regimens that include pretomanid. These abnormalities are usually asymptomatic, mild-to-moderate in severity and self-limited in duration. In many instances, it is difficult to determine which of the antituberculosis medications account for the abnormalities, but regular monitoring of liver tests is recommended during triple therapy with pretomanid, bedaquiline and linezolid. Clinically apparent liver injury has been reported with pretomanid-based therapies, but largely in regimens that included moxifloxacin or pyrazinamide or both. The clinical features, course and outcome of these cases has not been well defined. In the pivotal study of pretomanid combined with bedaquiline and linezolid in 109 adults with drug resistant pulmonary tuberculosis, 12 patients (11%) developed ALT levels above 3 times the upper limit of normal (ULN) of whom two also developed mild jaundice (bilirubin above twice but less than 3 times ULN) arising during month two of therapy. Both patients had mild nausea accompanying the liver test abnormalities, and the abnormalities resolved in both with temporary interruption in treatment, followed by re-initiation using lower dose linezolid without change in the pretomanid dose. In this pivotal trial, most adverse events were attributed to linezolid. Likelihood score: D (possible cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the use of pretomanid during breastfeeding, although the estimated dose for a breastfed infant is low. If pretomanid is required by the mother, it is not a reason to discontinue breastfeeding, but until more data become available, an alternate drug may be preferred, especially while nursing a newborn or preterm infant. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding The plasma protein binding of pretomanid is about 86.4%.
References	[1]. Antimicrob Agents Chemother.2005 Jun;49(6):2294-301 [2]. Antimicrob Agents Chemother.2011 Dec;55(12):5718-22 [3]. Antimicrob Agents Chemother.2011 Jan;55(1):239-45. [4].Nature. 2000 Jun 22;405(6789):962-6. [5].Antimicrob Agents Chemother. 2006 Oct;50(10):3350-4.
Additional Infomation	Pharmacodynamics Pretomanid kills the actively replicating bacteria causing tuberculosis, known as Mycobacterium tuberculosis, and shortens the duration of treatment in patients who suffer from resistant forms of pulmonary TB by killing dormant bacteria. In rodent models of tuberculosis infection, pretomanid administered in a regimen with bedaquiline and linezolid caused a significant reduction in pulmonary bacterial cell counts. A decrease in the frequency of TB relapses at 2 and 3 months after treatment was observed after the administration of this regimen, when compared to the administration of a 2-drug regimen. Successful outcomes have been recorded for patients with XDR and MDR following a clinical trial of the pretomanid regimen, demonstrating a 90% cure rate after 6 months. **A note on cardiac QT prolongation, hepatotoxicity, and myelosuppression** This drug has the propensity to caused cardiac QT interval prolongation and significant hepatotoxicity, as well as myelosuppression. Caution must be observed during the administration of this drug.

Solubility Data

Solubility (In Vitro)

DMSO:72 mg/mL (200.4 mM) Water:<1 mg/mL Ethanol:16 mg/mL (44.5 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2.08 mg/mL (5.79 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 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. Solubility in Formulation 2: 2.08 mg/mL (5.79 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. Solubility in Formulation 3: 2.08 mg/mL (5.79 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. 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. Solubility in Formulation 4: 0.5% methylcellulose: 30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.7835 mL	13.9175 mL	27.8350 mL
	5 mM	0.5567 mL	2.7835 mL	5.5670 mL
	10 mM	0.2783 mL	1.3917 mL	2.7835 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.