Temozolomide (Methazolastone; CCRG81045; NSC 362856; SCH 52365; MB39831; and RP46161) is an orally bioavailable and brain penetrant DNA alkylating agent / damage inducer used as an anticancer drug. Methazolastone causes formation of DNA alkali-labile sites which are present in similar amounts and repaired at a similar rate in L-1210 and L-1210/BCNU cell lines. In L-1210 but not in L-1210/BCNU methazolastone induces an arrest of cells in SL-G2-M phases. Methazolastone sensitivity of both chemo-sensitive and resistant cells (D54-R and U87-R) is enhanced significantly under hyperoxia.

Physicochemical Properties

Molecular Formula	C6H6N6O2
Molecular Weight	194.15
Exact Mass	194.055
Elemental Analysis	C, 37.12; H, 3.11; N, 43.29; O, 16.48
CAS #	85622-93-1
Related CAS #	Temozolomide-d3;208107-14-6
PubChem CID	5394
Appearance	White to pink solid powder
Density	2.0±0.1 g/cm3
Boiling Point	526.6±42.0 °C at 760 mmHg
Melting Point	212°C dec.
Flash Point	272.3±27.9 °C
Vapour Pressure	0.0±1.4 mmHg at 25°C
Index of Refraction	1.895
LogP	-1.32
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	1
Heavy Atom Count	14
Complexity	315
Defined Atom Stereocenter Count	0
InChi Key	BPEGJWRSRHCHSN-UHFFFAOYSA-N
InChi Code	InChI=1S/C6H6N6O2/c1-11-6(14)12-2-8-3(4(7)13)5(12)9-10-11/h2H,1H3,(H2,7,13)
Chemical Name	3-methyl-4-oxoimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxamide
Synonyms	CCRG81045, NSC362856; NSC 362856; CCRG 81045; NSC-362856; CCRG-81045; SCH-52365; SCH52365; 85622-93-1; Methazolastone; Temozolamide; 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxamide; Sch 52365; SCH 52365; MB39831; MB-39831; MB 39831; RP46161; RP 46161; R-P46161; CCRG81045; TMZ. US trade names: Methazolastone; Temodar. Foreign brand name: Temodal
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 Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
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	DNA alkylating agent; DNA alkylator
ln Vitro	Temozolomide (TZM) is a methylating drug prescribed for metastatic melanoma and malignant glioma that is able to pass the blood-brain barrier. Temozolomide works well against tumor cells that have a functional mismatch repair system (MR) and low amounts of O6-alkylguanine DNA alkyltransferase (OGAT) [1]. Cell lines with low IC50 values (<50 μM), such as A172 (14.1±1.1 μM) and LN229 cells (14.5±1.1 μM), and those with high IC50 values (>100 μM), such as SF268 (147.2±2.1 μM) and SK-N-SH cells (234.6±2.3 μM), were found to have varying temozolomide (TZM) IC50 values in different cell lines ranging from 14.1 to 234.6 μM [3]. Background: The use of Temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Methods: Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2'-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Results: Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. Conclusions: These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients[3].
ln Vivo	Temozolomide (TZM) as a single drug did not significantly increase median survival time (MST) compared with controls. Notably, intracranial injection of NU1025 before administration of 100 or 200 mg/kg Temozolomide significantly increased longevity in the control or Temozolomide-only group. When temozolomide was divided, the lifespan extension (ILS) obtained with this regimen was higher than that observed when NU1025 was combined with a single injection of temozolomide (statistical comparison of survival curves: NU1025 intracranial injection + temozolomide 100 mg/kg×2 vs NU1025 + temozolomide 200 mg /kg; P=0.023)[1]. Temozolomide (TZM) is a DNA-methylating agent that has recently been introduced into various clinical trials for treatment of solid or hematologic neoplasias, including brain lymphomas. In the current study, we have investigated whether the antitumor activity of TZM could be selectively enhanced at the central nervous system (CNS) site by intracerebral injection of a poly(ADP-ribose) polymerase (PARP) inhibitor. Mice were injected intracranially with lymphoma cells. The PARP inhibitor NU1025 (1 mg/animal) was delivered intracerebrally, whereas TZM was given as a single or a fractionated dose of 200 mg/kg by intraperitoneal administration. Results indicated that this drug combination significantly enhanced the survival of tumor-bearing mice and that this fractionated modality of treatment was the most effective schedule. Increased survival time was related to a marked reduction of tumor growth, as evidenced by histologic studies. Treatment with TZM alone was ineffective. This is the first report exploring in vivo the combination of TZM with PARP inhibitor for intracerebral neoplasias. [1] Temozolomide , a proautophagic and proapoptotic drug, decreased the expression levels of HIF-1alpha, ID-1, ID-2, and cMyc in the glioma models investigated, all of which playing major roles in angiogenesis and the switch to hypoxic metabolism. These changes could be, at least partly, responsible for the impairment of angiogenesis observed in vitro and in vivo. Moreover, combining bevacizumab with temozolomide increased the survival of glioma-bearing mice in comparison to each compound administered alone. Conclusions: In addition to the numerous mechanisms of action already identified for temozolomide, we report here that it also exerts antitumor effects by impairing angiogenic processes. We further emphasize that bevacizumab, which is an antiangiogenic drug with a different mechanism of action, could be useful in combination with temozolomide to increase the latter's therapeutic benefit in glioma patients[2].
Enzyme Assay	Methylation-specific PCR analysis [3] DNA was extracted from culture cells using the QIAamp DNA Mini Kit in accordance with the manufacturer's standard recommendations. Thus, 2 μg of DNA from each cell line was denatured, modified, and purified using the EpiTect Bisulfite kit. The MGMT promoter CpG islands methylation status of different cell lines was based on chemical modification of unmethylated cytosine with bisulfite to uracil. Methylation-specific PCRs (MSP) were performed using specific primers for either methylated or unmethylated DNA in the MGMT promoter. Primer sequences for MGMT were 5'-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3' (forward primer) and 5'-AACTCCACACTCTTCCAAAAACAAAACA-3' (reverse primer) for the unmethylated (UM) reaction and 5'-TTTCGACGTTCTAGGTTTTCGC-3' (forward primer) and 5'-GCACTCTTCCGAAAACGAAACG-3' (reverse primer) for the methylated (M) reaction. Agarose electrophoresis visualization by ethidium bromide and UV illumination was performed after PCR. High-resolution MGMT methylation analysis [3] The high-resolution MGMT methylation analysis of bisulfite samples was performed using high-sensitive SYBR® Green at the Center for Genomics and Oncological Research. The reaction was conducted using an Eco Real-Time PCR System and data were analyzed using the Eco Real-Time PCR System v4.0 software. Methylated EpiTect Control DNA, methylated and unmethylated EpiTect Control DNA, were used for the methylation curve, with methylated-unmethylated ratios of 0, 0.25, 0.5, 0.75, and 1. All samples and the methylation curve were analyzed using a pair of primers for the specific region.
Cell Assay	In vitro studies [1] The murine lymphoma cell line L5178Y of DBA/2 (H-2d/H-2d) origin was cultured in RPMI-1640 containing 10% fetal calf serum and antibiotics. Inhibition of PARP was obtained by treating cells (105 cells/mL) with 8-hydroxy-2-methylquinazolin-4[3H]-1, at a concentration (25 μM) that abrogates PARP activity. Cells were then exposed to Temozolomide (TZM) (7.5-125 μM) and were cultured for 3 days. Cell growth was evaluated by counting viable cells in quadruplicate, and apoptosis was assessed by flow cytometry analysis of DNA content.13 Long-term survival was analyzed by colony-formation assay. In Vitro Overall Growth Determination [2] Overall cell growth was assessed using the 3-[4,5-dimethylthiazol-2yl]-diphenyltetrazolium bromide (MTT) colorimetric assay, as detailed elsewhere. All determinations were carried out in sextuplicate. Control conditions consisted of endothelial cells cultured with endothelial cell growth medium EGM-2 MV BulletKit. Treatments were as follows: conditioned media from U373 GBM cells left untreated or treated with 100 °M Temozolomide /TMZ for 72 hours were collected. The MTT test was performed on the two HUVEC primary cultures in the presence of these 100% conditioned media, a mixture of conditioned medium, and HUVEC EGM-2 MV BulletKit medium in various proportions ranging from 90% U373 conditioned medium + 10% endothelial cell medium to 10% U373 conditioned medium + 90% endothelial cell culture medium. As U373 cells are cultured in minimum essential medium supplemented with 5% FCS, minimum essential medium + 5% FCS-treated cells were included as an internal control. In Vitro Determination of HUVEC Capillary Networking [2] When cultured on Matrigel, HUVECs form capillary-like networks [31]. An amount of 800 µl of cold Matrigel was allowed to set at 37°C for 10 minutes in a 3-cm Petri dish. The HUVECs growing as primocultures in 25-mm2 flasks were trypsinized, counted, and resuspended in the following culture media: control medium was composed of 90% untreated U373 conditioned medium mixed with 10% EGM medium; treated medium was composed of 90% conditioned medium of Temozolomide /TMZ-treated U373 cells mixed with 10% EGM medium. U373 conditioned media were prepared as detailed above. A total of 250,000 HUVECs were seeded onto the matrix for each experiment conducted in duplicate. Formation of capillary networks was observed during 24 hours by means of a computer-assisted stereomicroscope. In vitro drug treatments [3] Temozolomide treatment of all tumor cell lines comprised a double cycle (3 days of drug exposure followed by 3 days without drug) using the previously determined IC50 dose. Cell lines exposed to the first and second Temozolomide /TMZ cycle (named -1C and -2C, respectively) were subsequently subjected to further studies at the IC50 for Temozolomide /TMZ. 5-Aza was used in de-methylation studies at a concentration of a 30 μM for A172 and LN229 and 10 μM for SF268 and SK-N-SH. In addition, SF268 and SK-N-SH cell lines were exposed to 30 μM O6-BG prior to TMZ treatment. Cytotoxicity assays [3] Cell lines exposed to Temozolomide /TMZ (with or without 5-Aza or O6-BG pre-treatment) were grown in 24-well plates under standard culture conditions for 6 days. Cytotoxicity was determined using the sulphorhodamine-B (SRB) method. Briefly, the cells were fixed with 10% trichloroacetic acid for 20 min at 4°C then washed three times with water. After 24 hours, cells were stained for 30 min at room temperature with 0.4% SRB dissolved in 1% acetic acid and then washed three times with 1% acetic acid. The plates were air-dried and the dye solubilized with 300 ml/well of 10 mM Tris base (pH 10.5) for 10 min on a shaker. The optical density of each well was measured spectrophotometrically using a Titertek multiscan colorimeter at 492 nm.
Animal Protocol	In vivo studies [1] Male B6D2F1 (C57BL/6 × DBA/2) mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) in 0.9% NaCl solution (10 mL/kg intraperitoneally). L5178Y cells (104 in 0.03 mL RPMI-1640) were then injected intracranially, through the center-middle area of the frontal bone to a 2-mm depth, using a 0.1-mL glass microsyringe and a 27-gauge disposable needle. To evaluate tumor cell growth, brains were fixed in 10% phosphate-buffered formaldehyde, and histologic sections (5 μm) were cut along the axial plane, stained with hematoxylin-eosin, and analyzed by light microscopy. Temozolomide (TZM) was dissolved in dimethyl-sulfoxide (40 mg/mL), diluted in saline (5 mg/mL), and administered intraperitoneally on day 2 after tumor injection at 100 mg/kg or 200 mg/kg, doses commonly used for in vivo preclinical studies. Because cytotoxicity induced by TZM and PARP inhibitors can be improved by fractionated modality of treatment, in selected groups a total dose of 200 mg/kg TZM was divided in 2 doses of 100 mg/kg given on days 2 and 3. NU1025 was dissolved in polyethylene glycol-400 (40% in saline) and was injected intracranially at the maximal deliverable dose (1 mg/mouse, 0.03 mL) or, in selected groups, intraperitoneally (0.3 mL) on day 2 after tumor challenge, 1 hour before Temozolomide (TZM) administration. Control mice were injected with drug vehicles. Mice were monitored for mortality for 90 days. Median survival time (MST) was determined, and percentage of increase in lifespan (ILS) was calculated as [MST (days) of treated mice/MST (days) of control mice] −1] × 100. Efficacy of treatments was evaluated by comparing survival curves between treated and control groups. To assess the ability of different treatments to reduce tumor growth, histologic examination of the brains was performed using additional animals not considered for analysis of survival. Mice were killed at different time points after tumor challenge, selected within the MST range of untreated tumor-bearing animals. Areas of tumor infiltration were measured by histomorphometry, using an automated image analyzer system. Drug toxicity was evaluated by treating intact mice (10/group) with the compounds under investigation or with vehicles only. Weights and survival times of the mice were recorded for 3 weeks. Animal care was in compliance with international guidelines. In Vivo Determination of Tumor Neoangiogenesis [2] Each mouse receiving a GBM orthotopic xenograft underwent euthanasia (in a CO2 atmosphere during 5 min) for ethical reasons when it had lost 20% of its body weight compared to the day of tumor grafting. The brain was removed from the skull, fixed in buffered formalin for 5 days, embedded in paraffin, and then cut into 5-µm-thick sections. Resulting histology slides were stained with hematoxylin and eosin for blood vessel counts. To quantify the level of angiogenesis, a grid was used to determine the surface area of blood vessels in brain sections as reported previously. Antiangiogenic effects were analyzed in two distinct GBM models U373 (Figure 1C) and Hs683 (Figure 1D) with and without treatment with TMZ. The types of blood vessels taken into account are illustrated in Figure 2A. A minimum of five fields at a Gx400 magnification were analyzed per histologic slide and two slides were analyzed per tumor. Thus, a minimum total of 10 histologic fields per tumor were analyzed. Dissolved in 95% ethanol; 40 mg/kg; i.v. injection DBA/2 mice with L-1210 and L-1210/BCNU cells
ADME/Pharmacokinetics	Absorption, Distribution and Excretion Temozolomide is rapidly and completely absorbed in the gastrointestinal tract and is stable at both acidic and neutral pH. Therefore, temozolomide may be administered both orally and intravenously with a median Tmax of one hour. Following a single oral dose of 150 mg/m2, temozolomide and its active MTIC metabolite had Cmax values of 7.5 μg/mL and 282 ng/mL and AUC values of 23.4 μg\*hr/mL and 864 ng\*hr/mL, respectively. Similarly, following a single 90-minute IV infusion of 150 mg/m2, temozolide and its active MTIC metabolite had Cmax values of 7.3 μg/mL and 276 ng/mL and AUC values of 24.6 μg\*hr/mL and 891 ng\*hr/mL, respectively. Temozolomide kinetics are linear over the range of 75-250 mg/m2/day. The median Tmax is 1 hour Oral temozolomide absorption is affected by food. Administration following a high-fat breakfast of 587 calories caused the mean Cmax and AUC to decrease by 32% and 9%, respectively, and the median Tmax to increase by 2-fold (from 1-2.25 hours). Roughly 38% of administered temozolomide can be recovered over seven days, with 38% in the urine and only 0.8% in the feces. The recovered material comprises mainly metabolites: unidentified polar metabolites (17%), AIC (12%), and the temozolomide acid metabolite (2.3%). Only 6% of the recovered dose represents unchanged temozolomide. Temozolomide has a mean apparent volume of distribution (%CV) of 0.4 (13%) L/kg. Temozolomide has a clearance of approximately 5.5 L/hr/m2. Metabolism / Metabolites After absorption, temozolomide undergoes nonenzymatic chemical conversion to the active metabolite 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC) plus carbon dioxide and to a temozolomide acid metabolite, which occurs at physiological pH but is enhanced with increasing alkalinity. MTIC subsequently reacts with water to produce 5-aminoimidazole-4-carboxamide (AIC) and a highly reactive methyl diazonium cation, the active alkylating species. The cytochrome P450 system plays only a minor role in temozolomide metabolism. Relative to the AUC of temozolomide, the exposure to MTIC and AIC is 2.4% and 23%, respectively. Biological Half-Life Temozolomide has a mean elimination half-life of 1.8 hours.
Toxicity/Toxicokinetics	Hepatotoxicity Serum aminotransferase elevations occur during temozolomide therapy in up to 12% of patients, but these elevations are usually mild and self-limited, not requiring dose adjustment or drug discontinuation. An instance of serum aminotransferase elevation with jaundice was reported in the registration trials of temozolomide and subsequent to its approval. More strikingly, multiple single case reports and several case series of temozolomide hepatotoxicity have been reported in the literature. The onset of injury was typically within 2 to 8 weeks of starting temozolomide but several patients had received multiple courses before the onset of liver injury. The pattern of serum enzyme elevations was usually mixed initially, but the disease tended to be cholestatic. In several instances, jaundice was deep and prolonged. Features of hypersensitivity (rash, fever, eosinophilia) and autoantibody formation were not present. Liver histology demonstrated cholestasis and bile duct injury and a striking decrease in bile ducts (bile duct loss or paucity). Jaundice and pruritus tended to be prolonged and some patients developed vanishing bile duct syndrome, while others recovered clinically but had persistent serum alkaline phosphatase elevations during follow up and to the time of death from the brain tumor. Rechallenge was not done, but several patients subsequently received other antineoplastic agents, some of which were alkylating agents without recurrence of liver injury. In addition, temozolomide has been associated with several cases of reactivation of chronic hepatitis B in patients who were hepatitis B surface antigen (HBsAg) positive at the start of chemotherapy. Clinical symptoms and signs of a flare of hepatitis B arose 6 to 12 weeks after starting temozolomide frequently in a cyclic pattern. Most patients had not received corticosteroids or other immunosuppressive agents that are more traditionally associated with reactivation. The episodes are marked by rises in HBV DNA levels and mild jaundice and responded to prompt antiviral therapy for hepatitis B which allowed for restarting of temozolomide in some cases. Fatal cases of reactivation have not been reported, but in general hepatitis B reactivation with jaundice has a mortality rate in excess of 10%. Likelihood score: B (highly likely but uncommon cause of clinically apparent liver injury and reactivation of hepatitis B). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Most sources consider breastfeeding to be contraindicated during maternal antineoplastic drug therapy, especially alkylating agents such as temozolamide. It might be possible to breastfeed safely during intermittent therapy with an appropriate period of breastfeeding abstinence. The manufacturer recommends withholding breastfeeding for 1 week after the last dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk A woman diagnosed with Hodgkin's lymphoma during the second trimester of pregnancy received 3 rounds of chemotherapy during the third trimester of pregnancy and resumed chemotherapy 4 weeks postpartum. Milk samples were collected 15 to 30 minutes before and after chemotherapy for 16 weeks after restarting. The regimen consisted of doxorubicin 40 mg, bleomycin 16 units, vinblastine 9.6 mg and dacarbazine 600 mg, all given over a 2-hour period every 2 weeks. The microbial population and metabolic profile of her milk were compared to those of 8 healthy women who were not receiving chemotherapy. The breastmilk microbial population in the patient was markedly different from that of the healthy women, with increases in Acinetobacter sp., Xanthomonadacae and Stenotrophomonas sp. and decreases in Bifidobacterium sp. and Eubacterium sp. Marked differences were also found among numerous chemical components in the breastmilk of the treated woman, most notably DHA and inositol were decreased. Protein Binding Temozolomide plasma protein binding varies from 8-36%, with an average of around 15%. _In vitro_ binding experiments revealed approximate dissociation constants of 0.2-0.25 and 0.12 mM for temozolomide with human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP), respectively; despite the slightly higher affinity for AGP, it is likely that temozolomide is predominantly bound to HSA due to its higher serum concentration. In addition, temozolomide binding to HSA results in delayed hydrolysis and a longer half-life than in buffer (1 versus 1.8 hours).
References	[1]. Combined treatment with temozolomide and poly(ADP-ribose) polymerase inhibitor enhances survival of mice bearing hematologic malignancy at the central nervous system site. Blood. 2002 Mar 15;99(6):2241-4. [2]. Combining Anti-Human VEGF with temozolomide increases the antitumor efficacy of temozolomide in a human glioblastoma orthotopic xenograft model. Neoplasia. 2008 Dec;10(12):1383-92. [3]. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression. PLoS One. 2015 Oct 8;10(10):e0140131.
Additional Infomation	Pharmacodynamics Temozolomide is a prodrug of the imidazotetrazine class that requires nonenzymatic hydrolysis at physiological pH _in vivo_ to perform alkylation of adenine/guanine residues, leading to DNA damage through futile repair cycles and eventual cell death. Temozolomide treatment is associated with myelosuppression, which is likely to be more severe in females and geriatric patients. Patients must have an ANC of ≥1.5 x 109/L and a platelet count of ≥100 x 109/L before starting therapy and must be monitored weekly during the concomitant radiotherapy phase, on days one and 22 of maintenance cycles, and weekly at any point where the ANC/platelet count falls below the specified values until recovery. Cases of myelodysplastic syndrome and secondary malignancies, including myeloid leukemia, have been observed following temozolomide administration. Pneumocystis pneumonia may occur in patients undergoing treatment, and prophylaxis should be provided for patients in the concomitant phase of therapy with monitoring at all stages. Severe hepatotoxicity has also been reported, and liver testing should be performed at baseline, midway through the first cycle, before each subsequent cycle, and approximately two to four weeks after the last dose. Animal studies suggest that temozolomide has significant embryo-fetal toxicity; male and female patients should practice contraception up to three and six months following the last dose of temozolomide, respectively.

Solubility Data

Solubility (In Vitro)

DMSO:38 mg/mL (195.7 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.25 mg/mL (6.44 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 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1.25 mg/mL (6.44 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 12.5 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: ≥ 1.25 mg/mL (6.44 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 5% DMSO +30% PEG 300 +ddH2O: 2mg/mL Solubility in Formulation 5: 9.09 mg/mL (46.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	5.1507 mL	25.7533 mL	51.5066 mL
	5 mM	1.0301 mL	5.1507 mL	10.3013 mL
	10 mM	0.5151 mL	2.5753 mL	5.1507 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.