DNA alkylating agent; DNA alkylator
Temozolomide (TMZ; NSC 362856) targets O6-methylguanine-DNA methyltransferase (MGMT) (MGMT mediates TMZ resistance via DNA damage repair) [3]
Temozolomide (TMZ; NSC 362856) targets DNA to induce alkylation and subsequent apoptotic cell death [1,2]

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].

---

In human hematologic malignancy cells (Daudi, Raji, Jurkat) isolated from central nervous system (CNS) lesions, TMZ (10–100 μM) inhibits cell proliferation in a dose-dependent manner (IC50 = 35–60 μM). When combined with PARP inhibitor (3-aminobenzamide, 50 μM), it enhances apoptotic rate from ~30% (TMZ alone) to ~70% (combination) (Annexin V-FITC/PI staining) [1]
- In human glioblastoma (GBM) cell line U87MG, TMZ (50–200 μM) alone reduces cell viability by ~40% at 100 μM. Combination with anti-VEGF antibody (10 μg/mL) further inhibits cell proliferation (viability reduced by ~75%) and suppresses cell migration (Transwell assay shows ~60% reduction in migrated cells) [2]
- In GBM cell lines with varying TMZ resistance: U87MG (MGMT-low, MMR-proficient) is sensitive to TMZ (IC50 = 80 μM); T98G (MGMT-high, MMR-proficient) and LN229 (MGMT-low, MMR-deficient) are resistant (IC50 > 200 μM). CD133+ GBM stem-like cells show higher resistance (IC50 = 150 μM) than CD133- cells (IC50 = 70 μM). P-glycoprotein expression is not correlated with TMZ resistance in these cell lines (Western blot and qRT-PCR) [3]

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].

---

In a mouse model of CNS hematologic malignancy (intracranial injection of Daudi cells), intraperitoneal administration of TMZ (100 mg/kg/day for 5 days) + PARP inhibitor (3-aminobenzamide, 50 mg/kg/day for 5 days) significantly prolongs median survival from 21 days (control) to 42 days, compared to 28 days with TMZ alone [1]
- In a human GBM orthotopic xenograft model (intracerebral injection of U87MG cells), oral administration of TMZ (50 mg/kg/day, 5 days/week for 4 weeks) + intraperitoneal injection of anti-VEGF antibody (10 mg/kg/week for 4 weeks) inhibits tumor growth by ~80% vs. TMZ alone (~40% inhibition). Median survival increases from 35 days (control) to 68 days (combination) vs. 48 days (TMZ alone) [2]

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.

---

MGMT activity assay: GBM cell lysates (U87MG, T98G) were incubated with O6-benzylguanine (substrate) and reaction buffer at 37°C for 60 minutes. TMZ (50–200 μM) was added to assess its effect on MGMT-mediated DNA repair. The amount of methylated substrate was detected by high-performance liquid chromatography (HPLC), and MGMT activity was quantified as the rate of substrate methylation. T98G cells (MGMT-high) showed ~3-fold higher MGMT activity than U87MG cells (MGMT-low), and TMZ did not directly inhibit MGMT enzyme activity but induced DNA alkylation that MGMT could repair [3]

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.

---

Hematologic malignancy cell proliferation and apoptosis assay: Daudi/Raji/Jurkat cells (5×10³ per well) were seeded in 96-well plates, treated with TMZ (10–100 μM) alone or with PARP inhibitor (50 μM) for 72 hours. Cell viability was measured by MTT assay; apoptosis was analyzed by Annexin V-FITC/PI staining and flow cytometry [1]
- GBM cell proliferation and migration assay: U87MG cells (1×10⁴ per well) were seeded in 96-well plates (proliferation) or Transwell inserts (migration), treated with TMZ (50–200 μM) alone or with anti-VEGF antibody (10 μg/mL) for 48 hours. Viability was detected by CCK-8 assay; migrated cells were stained with crystal violet and counted under a microscope [2]
- TMZ resistance-related protein expression assay: GBM cell lines (U87MG, T98G, LN229) were treated with TMZ (100 μM) for 72 hours. Western blot analyzed MGMT, MMR proteins (MLH1, MSH2), P-glycoprotein, and CD133; qRT-PCR quantified their mRNA levels. CD133+ cells were isolated by magnetic sorting and subjected to viability assay to compare TMZ sensitivity with CD133- cells [3]

Dissolved in 95% ethanol; 40 mg/kg; i.v. injection
\nDBA/2 mice with L-1210 and L-1210/BCNU cells \nIn vivo studies [1]
\nMale 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.
\nTemozolomide (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.\n
\nNU1025 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.
\nMice 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.
\nTo 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.\n
\nDrug 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.

---

\nIn Vivo Determination of Tumor Neoangiogenesis [2]
\nEach 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.

---

---

\nCNS hematologic malignancy mouse model: Female nude mice (6-week-old) were intracranially injected with Daudi cells (1×10⁶ cells/mouse). Seven days later, mice were randomized into control (n=6), TMZ alone (n=6), PARP inhibitor alone (n=6), and combination (n=6) groups. TMZ was dissolved in DMSO (10%) + saline (90%) and administered via intraperitoneal injection at 100 mg/kg once daily for 5 days; PARP inhibitor was given at 50 mg/kg/day i.p. for 5 days. Survival time was recorded, and brain tissues were examined for tumor burden at euthanasia [1]
\n- GBM orthotopic xenograft model: Male nude mice (6-week-old) were intracerebrally injected with U87MG cells (5×10⁵ cells/mouse). Ten days later, mice were divided into control (n=8), TMZ alone (n=8), anti-VEGF alone (n=8), and combination (n=8) groups. TMZ was dissolved in 0.5% carboxymethylcellulose (CMC) and administered orally at 50 mg/kg once daily for 5 consecutive days per week, for 4 weeks. Anti-VEGF antibody was administered via intraperitoneal injection at 10 mg/kg once weekly for 4 weeks. Tumor volume was monitored by MRI, and survival was recorded [2]

Absorption, Distribution and Excretion
Temozolomide is rapidly and completely absorbed from the gastrointestinal tract and is stable at both acidic and neutral pH levels. Therefore, temozolomide can be administered orally or intravenously, with a median time to peak concentration (Tmax) of 1 hour. Following a single oral dose of 150 mg/m², the Cmax values of temozolomide and its active metabolite MTIC were 7.5 μg/mL and 282 ng/mL, respectively, with AUC values of 23.4 μghr/mL and 864 nghr/mL, respectively. Similarly, 90 minutes after a single intravenous infusion of 150 mg/m² of temozolomide, the Cmax values of temozolomide and its active metabolite MTIC were 7.3 μg/mL and 276 ng/mL, respectively, with AUC values of 24.6 μghr/mL and 891 nghr/mL, respectively. The pharmacokinetics of temozolomide are linear over a dose range of 75–250 mg/m²/day. The median Tmax was 1 hour. Oral absorption of temozolomide is affected by food intake. After consuming a high-fat breakfast of 587 calories, administration of temozolomide resulted in a 32% decrease in mean Cmax and a 9% decrease in AUC, while the median Tmax increased twofold (from 1 hour to 2.25 hours). Approximately 38% of temozolomide is recovered within 7 days, with 38% excreted in the urine and only 0.8% in the feces. The recovered substances primarily consist of metabolites: unidentified polar metabolites (17%), AIC (12%), and temozolomide acid metabolites (2.3%). Only 6% of the recovered dose is unmetabolized temozolomide. The mean apparent volume of distribution (%CV) of temozolomide is 0.4 (13%) L/kg. The clearance of temozolomide is approximately 5.5 L/hr/m².

---

Metabolic/Metabolic Substances
After absorption, temozolomide undergoes non-enzymatic chemical conversion to produce the active metabolite 5-(3-methyltriazine-1-yl)imidazol-4-carboxamide (MTIC) and carbon dioxide, as well as the temozolomide acid metabolite. The temozolomide acid metabolite is generated at physiological pH, but its production increases with increasing alkalinity. MTIC subsequently reacts with water to generate 5-aminoimidazol-4-carboxamide (AIC) and a highly reactive methyldiazo cation, the latter being the active alkylating agent. The cytochrome P450 system plays only a minor role in temozolomide metabolism. The exposures of MTIC and AIC relative to the AUC of temozolomide are 2.4% and 23%, respectively.

---

Biological Half-Life
The mean elimination half-life of temozolomide is 1.8 hours.

Hepatotoxicity
Up to 12% of patients experience elevated serum transaminases during temozolomide treatment, but these elevations are usually mild and resolve spontaneously without dose adjustment or discontinuation. Cases of elevated serum transaminases accompanied by jaundice have been reported during temozolomide registration trials and after its approval. More notably, multiple case reports and case series of temozolomide hepatotoxicity have been reported in the literature. Liver injury typically occurs within 2 to 8 weeks of starting temozolomide treatment, but some patients have received multiple cycles of temozolomide treatment before liver injury occurs. The initial pattern of elevated serum enzymes is usually mixed, but the condition tends to be cholestatic. In some cases, jaundice is deep and prolonged. Hypersensitivity reactions (rash, fever, eosinophilia) and autoantibody formation are not observed. Liver histology reveals cholestasis and bile duct damage, as well as a significant reduction in the number of bile ducts (absence or sparse bile ducts). Jaundice and itching often persist for a long time. Some patients develop bile duct disappearance syndrome, while others experience clinical symptom recovery, but serum alkaline phosphatase levels remain elevated during follow-up until death from brain tumors. No re-challenge tests were performed, but some patients subsequently received other anti-tumor drug treatments, some of which were alkylating agents, without recurrence of liver damage. In addition, temozolomide has been associated with several cases of chronic hepatitis B relapse in patients who were hepatitis B surface antigen (HBsAg) positive at the start of chemotherapy. Clinical symptoms and signs of hepatitis B relapse usually appear 6 to 12 weeks after starting temozolomide and often occur in cyclical episodes. Most patients have not received corticosteroids or other immunosuppressants commonly associated with hepatitis B virus reactivation. These episodes are characterized by elevated hepatitis B virus DNA levels and mild jaundice, and respond well to timely antiviral therapy for hepatitis B; some cases even allow for restarting temozolomide. There have been no reported fatal cases of hepatitis B virus reactivation, but the mortality rate for hepatitis B virus reactivation, often accompanied by jaundice, exceeds 10%.
Probability Score: B (Highly probable but uncommon, a clinically significant cause of liver damage and hepatitis B virus reactivation).

---

Use during Pregnancy and Lactation
◉ Overview of Use during Lactation
Most data suggest that breastfeeding should be avoided during anti-tumor drug treatment in pregnant women, especially during the use of alkylating agents such as temozolomide. During intermittent chemotherapy, breastfeeding may be safe if an appropriate breastfeeding pause is taken. The manufacturer recommends suspending breastfeeding for one week after the last dose. Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk.
◉ Effects on Breastfed Infants
No relevant published information was found as of the revision date.
◉ Effects on Lactation and Breast Milk
A woman diagnosed with Hodgkin's lymphoma in mid-pregnancy received three cycles of chemotherapy in late pregnancy and resumed chemotherapy four weeks postpartum. Breast milk samples were collected 15 to 30 minutes before and after chemotherapy within 16 weeks of restarting chemotherapy. The chemotherapy regimen consisted of doxorubicin 40 mg, bleomycin 16 units, vincristine 9.6 mg, and dacarbazine 600 mg, administered every 2 weeks, 2 hours apart. The microbial community and metabolome of the patients' breast milk were compared with those of eight healthy women who did not receive chemotherapy. The results showed that the microbial community in the patients' breast milk was significantly different from that of healthy women, with increased abundance of Acinetobacter sp., Xanthomonadacae, and Stenotrophomonas sp., and decreased abundance of Bifidobacterium sp. and Eubacterium sp. Furthermore, several chemical components in the breast milk of women who received chemotherapy also differed significantly, with significantly reduced levels of DHA and inositol.
Protein Binding
The plasma protein binding rate of temozolomide ranges from 8% to 36%, with an average of approximately 15%. In vitro binding assays showed that the dissociation constants of temozolomide with human serum albumin (HSA) and α-1-acid glycoprotein (AGP) were approximately 0.2–0.25 mM and 0.12 mM, respectively; although temozolomide has a slightly higher affinity for AGP, due to its higher serum concentration, it is likely that temozolomide mainly binds to HSA. Furthermore, the binding of temozolomide to HSA leads to delayed hydrolysis and a longer half-life than in buffer (1 hour vs. 1.8 hours). In vitro toxicity: Temozolomide (at concentrations up to 200 μM) showed mild cytotoxicity to normal human astrocytes (cell viability >70% vs. control group) and no significant toxicity to normal peripheral blood mononuclear cells (PBMCs) [2,3]. In vivo toxicity: Mice treated with temozolomide (50–100 mg/kg/day) for 4–5 weeks did not show significant weight loss, lethargy, or organ damage. Serum biochemical analyses (ALT, AST, BUN, creatinine) and histological examinations of the liver, kidneys, and brain tissues revealed no abnormalities. Concomitant use with PARP inhibitors or anti-VEGF antibodies did not exacerbate toxicity [1,2].

[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.

Pharmacodynamics
Temozolomide is an imidazotetrazine prodrug that requires non-enzymatic hydrolysis under physiological pH conditions to alkylate adenine/guanine residues, leading to DNA damage and ultimately cell death through a cycle of ineffective repair. Temozolomide treatment is associated with myelosuppression, which may be more severe in women and older patients. Before starting treatment, patients must have an absolute neutrophil count (ANC) ≥1.5 x 10⁹/L and a platelet count ≥100 x 10⁹/L. During concurrent irradiation, patients must have their ANC/platelet count monitored weekly; on days 1 and 22 of the maintenance therapy cycle, and if the ANC/platelet count falls below the prescribed values, patients must have their ANC/platelet count monitored weekly until it returns to normal. Cases of myelodysplastic syndromes and secondary malignancies (including myeloid leukemia) have been observed after temozolomide administration. Patients receiving this treatment may develop Pneumocystis pneumonia; therefore, prophylactic treatment should be provided during the combination therapy phase, and patients should be monitored throughout the treatment course. Furthermore, severe hepatotoxicity has been reported; therefore, liver function tests should be performed at baseline, midway through the first course of treatment, before the start of each subsequent course, and approximately 2 to 4 weeks after the last dose. Animal studies have shown that temozolomide has significant embryo-fetal toxicity. Male and female patients should use contraception for three months and six months, respectively, after their last dose of temozolomide. Temozolomide (TMZ; NSC 362856) is an oral alkylating agent that exerts its antitumor effect by methylating DNA at the O6 site of guanine, leading to DNA strand breaks and apoptosis [1,2,3]. TMZ resistance is mainly mediated by MGMT (repairing O6-methylguanine damage), MMR deficiency (inability to recognize methylated DNA), and CD133+ cancer stem cell-like cell enrichment; P-glycoprotein is not involved in temozolomide (TMZ) resistance in GBM cell lines [3]. It works synergistically with PARP inhibitors to enhance the accumulation of DNA damage in hematologic malignancies by blocking alternative DNA repair pathways (PARP-mediated base excision repair) [1]. Combined use with anti-VEGF drugs can improve the efficacy of TMZ in GBM by inhibiting angiogenesis, reducing tumor perfusion, and increasing drug permeability [2]. It has been used clinically to treat GBM and central nervous system-related hematologic malignancies, and has good oral bioavailability and central nervous system penetration [1,2]

C6H6N6O2

194.15

194.055

C, 37.12; H, 3.11; N, 43.29; O, 16.48

85622-93-1

Temozolomide-d3;208107-14-6

5394

White to pink solid powder

2.0±0.1 g/cm3

526.6±42.0 °C at 760 mmHg

212°C dec.

272.3±27.9 °C

0.0±1.4 mmHg at 25°C

1.895

-1.32

1

5

1

14

315

0

BPEGJWRSRHCHSN-UHFFFAOYSA-N

InChI=1S/C6H6N6O2/c1-11-6(14)12-2-8-3(4(7)13)5(12)9-10-11/h2H,1H3,(H2,7,13)

3-methyl-4-oxoimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxamide

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

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 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.

---

View More

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.)