DNA Alkylator
The text indicates that Lomustine (CCNU) is a chloroethylating agent. Its cytotoxicity is correlated with cellular levels of the DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (ATase). [1]

Lomustine inhibits ZR-75-1 and U373 from growing, with IC50 values of 12 μM and 15 μM, respectively. Lomustine inhibits the expression of O6-alkylguanine-DNA alkyltransferase, a DNA repair protein. [1] In both medulloblastoma and normal human epithelial and fibroblast cells, lomustine (420 μM) induces apoptosis via the mitochondrial pathway by lowering the levels of the anti-apoptosis proteins Bcl-2 and Bcl-xl, respectively. In medulloblastoma cells, locustrine causes a cell cycle delay in the G2/M phase; in HFSN1 cells, locustrine upregulates the level of the protein p21 in a p53-independent manner. [2]

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In a panel of seven human tumor cell lines (ZR-75-1, U87MG, U373, LS174T, LOVO, MCF-7, MAWI), the sensitivity (IC50) to a 1-hour exposure of Lomustine (CCNU) showed a reasonable correlation (correlation coefficient r = 0.92) with the cellular ATase content. CCNU was approximately five times more toxic on a molar basis than temozolomide across these cell lines. [1]
Pretreating cells with a non-toxic dose (33 µM) of the ATase inhibitor O⁶-benzylguanine (BG) sensitized cell lines to CCNU, making them up to 6-fold more sensitive. The potentiation was similar in magnitude to that observed with temozolomide. [1]
Control xeroderma pigmentosum (XP) fibroblasts (expressing very low ATase) were more sensitive to the CCNU-related chloroethylating agent mitozolomide than human ATase cDNA-transfected XP cells. [1]

Lomustine may result in irreversible, delayed, cumulative dose-related chronic hepatotoxicity that may be fatal. [4] In cats with spontaneously arising tumors, lomustine may occasionally cause severe hematological toxicity; grade III or IV neutropenia and thrombocytopenia have an incidence of 4.1% and 1.0%, respectively. In cats with spontaneously arising tumors, locustin tends to increase the risk of progressive neutropenia and statistically significant higher response rates. [5]

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In a clinical study of 23 dogs with measurable mast cell tumors (MCT), treatment with Lomustine (CCNU) at a dosage of 90 mg/m² orally every 3 weeks resulted in a measurable response in 8 out of 19 evaluable dogs (42%). One dog achieved a complete response (CR) lasting 440 days. Seven dogs had a partial response (PR) with a median duration of 77 days (mean 109 days, range 21-254 days). Stable disease (SD) was observed in 6 dogs (32%) with a median duration of 78 days (mean 122 days, range 42-347 days). [3]
Of 15 dogs with histologically graded tumors and evaluable response: one Grade I tumor had a PR lasting 254 days; among Grade II tumors, one had a CR (440 days), two had PRs (77 and 166 days), three had SD (42, 60, 125 days); among Grade III tumors, four had PRs (median 62.5 days, range 21-120 days), one had SD (62 days). [3]
No statistically significant association was found between response to CCNU and factors such as age, gender, tumor volume, disease duration prior to treatment, previous response to prednisone, tumor grade, tumor location, or whether the MCT was cutaneous or metastatic. [3]

Cell lines are commonly grown as monolayers in DMEM supplemented with glutamine, penicillin/streptomycin, 10% foetal calf serum, and 25 mm HEPES. Studies on cytotoxicity are conducted in an atmosphere with 5% CO₂ in a medium free of HEPES. In 96-well plates, 750–1000 cells/well are plated, and after an overnight incubation, they are treated for two hours either with or without 33 μM BG. In the same medium, temozolomide or CCNU is then added for an hour, with a final DMSO concentration of no more than 1%. Growth studies reveal that the cells are in log phase growth during the assay period. The cells are then cultured for an additional 7 days in fresh medium before being tested for protein content using the NCI sulphorhodamine assay. Cells are treated consecutively for 24 hours with fresh medium every day as part of the repeated temozolomide dosing schedule. At least two assays are performed.

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Cytotoxicity Assay (Sulphorhodamine B/Protein Content): Human tumor cell lines were grown as monolayers. For cytotoxicity studies, 750-1000 cells/well were plated in 96-well plates. After overnight incubation, cells were pretreated with or without 33 µM O⁶-benzylguanine (BG) for 2 hours. Lomustine (CCNU) was then added to the medium for a 1-hour exposure. The cells were grown in fresh medium for a further 7 days. Cell growth/protein content was then assayed using the sulphorhodamine B assay. Growth studies confirmed cells were in log phase during the assay period. [1]

Mice: During the study, groups of eight B6C3F1 mice each receive PBS or 5-FC as the sole control. A single dose of 30 mg/kg of locustine is given to one group of mice (Lomustine Day 1 + PBS) on day 1, along with six cycles of PBS (800 μL/day, BID for four days in a row every ten days). For four days in a row, the remaining mice are given 5-FC (500 mg/kg/dose, IP, BID) along with either Lomustine on day one (Lomustine (CCNU) Day 1 + 5-FC) or Day 43 (Lomustine (CCNU) Day 43 + 5-FC for maximum benefit. Six cycles of four days on, ten days off, five FC, or five PBS are completed. When the final 5-FC treatment is completed, each experiment comes to an end. Every tissue is gathered and stored for pathology analysis.
Rats: Rat groups (n = 8 per group) are only given PBS or 5-FC as controls during the study. Rats in the Lomustine (CCNU) Day 1 + PBS group are given a single dose of 30 mg/kg of Lomustine on day 1 and six cycles of PBS (8 mL/day, BID). For five days in a row, the remaining rats are given 5-FC (500 mg/kg/dose, IP, BID) plus either Lomustine on Day 1 (Lomustine (CCNU) Day 1 + 5-FC) or Day 22 (Lomustine (CCNU) Day 22 + 5-FC), after which they are given two days off medication. A total of six repetitions of the 5-day on, 2-day off 5-FC or PBS cycle are made[3].

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Canine Clinical Trial for Mast Cell Tumors: Client-owned dogs with measurable mast cell tumors (diagnosed by histology or cytology) were included. Lomustine (CCNU) was administered as a single oral dose at 90 mg/m² of body surface area. The treatment was repeated every 3 weeks. Dogs were examined before, and 7 and 21 days after each treatment. A complete blood count (CBC) was obtained 7 days after the first treatment. If grade 4 neutropenia (see toxicity criteria) or fever (rectal temperature >103°F) was observed, oral antibiotics (trimethoprim-sulfadiazine at 15 mg/kg PO q12h) were prescribed and the CCNU dose was reduced to 70 mg/m² for the remainder of the treatment course. Tumor response was assessed at each examination by measuring tumor dimensions in three planes. [3]

Absorption, Distribution and Excretion
Lomustine is well and rapidly absorbed in the gastrointestinal tract. Following oral administration of radioactive CeeNU (dose range 30 mg/m² to 100 mg/m²), approximately half of the radioactive material is excreted in the urine as degradation products within 24 hours. Lomustine is primarily excreted in the urine as metabolites. Following oral administration of 14C-labeled lomustine, approximately 50% of the radioactive material is excreted within 12 hours, and approximately 75% within 4 days. Lomustine is reported to be widely distributed. Due to its high lipid solubility, lomustine and/or its metabolites can cross the blood-brain barrier and rapidly enter cells. Although intact lomustine is undetectable in cerebrospinal fluid, its active metabolites reach considerably high concentrations within 30 minutes of oral administration. The concentration of metabolites in cerebrospinal fluid has been reported to be 15-50% or higher than the plasma concentration at the same time. Lomustine metabolites are present in breast milk, but at higher concentrations than in maternal plasma. Lomustine is rapidly absorbed from the gastrointestinal tract; it is also absorbed after topical administration. Peak plasma concentrations of the metabolites appear within 1–6 hours after oral administration of lomustine. Following intraperitoneal, intravenous, or oral administration of (14)C-labeled CCNU, it rapidly distributes to various tissues in mice, rats, rabbits, and dogs. Approximately 80% of the label is excreted in urine 24 hours after a single parenteral or oral dose of 50 mg/kg body weight in mice. Metabolites/Metabolites: Liver. Rapidly and completely metabolized, producing active metabolites. CCNU spontaneously decomposes under physiological conditions, releasing alkylated and carbamylated substances. It is cleared from plasma within 5 minutes of oral administration, but the antitumor effects of its metabolites can persist for up to 15 minutes. ...In addition to chemical degradation, CCNU can also be metabolized via microsomal metabolism into six isomeric hydroxylated derivatives, some of which may have different biological properties than CCNU. After oral administration of lomustine, almost all doses are metabolized within one hour. The half-life of lomustine metabolites is biphasic; although the initial plasma half-life is 6 hours, the second phase plasma half-life is 1-2 days, and 15-20% of the metabolites remain in the body even 5 days after lomustine administration. The prolonged plasma concentration is thought to be due to the combined effects of protein binding and enterohepatic circulation of the metabolites. Hepatomegaly. Rapid and complete metabolism, producing active metabolites. Elimination pathway: After oral administration of radioactive CeeNU (dose range 30 mg/m² to 100 mg/m²), approximately half of the radioactive material is excreted in the urine as degradation products within 24 hours.
Half-life: Approximately 94 minutes, but the serum half-life of the metabolites is 16 to 48 hours.
Biological half-life Approximately 94 minutes, but the serum half-life of the metabolites is 16 to 48 hours.
The half-life of lomustine metabolites is biphasic; although the initial plasma half-life is 6 hours, the second phase plasma half-life is 1-2 days, and 15-20% of the metabolites remain in the body even 5 days after lomustine administration.

Toxicity Summary
Lomustine is a highly lipophilic nitrosourea compound that hydrolyzes in vivo to produce active metabolites. These metabolites can lead to alkylation and cross-linking of DNA (O6 position of guanine bases) and RNA, thereby inducing cytotoxicity. Other biological effects include inhibition of DNA synthesis and specificity to certain cell cycle stages. Nitrosoureas generally do not exhibit cross-resistance with other alkylating agents. Because lomustine is a nitrosourea, it may also inhibit some key processes such as carbamylation and cellular protein modification. Hepatotoxicity
A significant proportion of patients receiving anti-tumor regimens containing lomustine experience mild and transient elevations in serum transaminase or alkaline phosphatase levels. These abnormalities are usually transient, asymptomatic, and do not require dose adjustment. While there have been reports of clinically significant liver injury caused by lomustine, this is uncommon. The pattern of elevated serum enzymes has been described as cholestatic, with onset 3 to 4 months after treatment, but beyond this, its clinical characteristics remain unclear. Lomustine is often used in combination with other antitumor drugs, many of which are also hepatotoxic, making the role of lomustine in causing liver injury difficult to assess. Lomustine has not been definitively linked to hepatic sinusoidal obstruction syndrome, but it is generally not used at high doses or for pre-transplant myeloablative therapy, which are common causes of this syndrome. Probability Score: D (Possibly a cause of clinically significant liver injury). Protein Binding Rate 50% Toxicity Data Oral administration in rats: LD50 = 70 mg/kg. Pulmonary toxicity has been reported at cumulative doses generally greater than 1100 mg/m². Only one case reported showed pulmonary toxicity at a cumulative dose of only 600 mg. The onset of toxicity varies widely, ranging from 6 months to 15 years after the start of treatment.

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Interaction
This study investigated the combined effects of the antitumor antibiotics bleomycin and chloroethylnitrosourea in human lymphocytes in vitro. All experiments were performed using 20 μg/mL bleomycin with predefined treatment times. Adding 0.7 and 3.5 μg/mL chloroethylnitrosourea to late G1-S phases significantly increased the percentage of abnormal cells and the production of dicentric and circular chromosomes (5-fold, p < 0.001). In late S-G2 phases, the combined treatment resulted in a significant increase in the number of breaks per cell (p < 0.0001), with more than 12 abnormal cells observed. One possible explanation is the known inhibitory effect of chloroethylnitrosourea on repair, but its purely chromosome-breaking effect still needs to be considered. These results suggest the need to explore chemotherapy regimens with lower combined drug concentrations.
Three patients suddenly developed complete blindness several months after receiving oral CCNU and low-dose whole-brain radiotherapy. The anterior visual system of all patients was located within the radiotherapy field. The patients receiving radiotherapy were: one with frontal lobe glioblastoma, one with small cell lung cancer (for central nervous system prophylaxis), and one with parietal lobe glioblastoma. None of the tumors involved the anterior visual system. Radiotherapy doses ranged from 3000 to 4650 rads, and oral CCNU doses ranged from 300 mg to 1050 mg. Patients 1 and 2 also received other chemotherapy drugs. Patient 3, who received only oral CCNU and cranial radiotherapy, died. Autopsy revealed extensive infiltration of residual high-grade glioma within the brain tissue, along with patchy coagulative necrosis with axonal swelling and dystrophic calcification. Severe demyelination, axonal loss, and vascular hyaline degeneration were observed in the optic chiasm. The synergistic effect of oral CCNU and radiotherapy is believed to have contributed to the blindness.

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Non-human toxicity values
Oral LD50 in rats: 70 mg/kg
Intraperitoneal LD50 in rats: 50, 350 μg/kg
Oral LD50 in female mice: 38 mg/kg body weight
Oral LD50 in male mice: 51 mg/kg
For more non-human toxicity values (complete data) for lomustine (7 in total), please visit the HSDB record page.

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The text states that the dose-limiting toxicity of chloroethylated agents (such as chloroethylnitrosoureas, including lomustine (CCNU)) is severe myelosuppression. [1]

[1]. Br J Cancer . 1993 Jun;67(6):1299-302.

[2]. J Neurooncol . 2008 Apr;87(2):123-32.

[3]. J Vet Intern Med . 1999 Nov-Dec;13(6):601-5.

According to an independent committee of scientific and health experts, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) (lomustine) may be carcinogenic. It may also be developmentally toxic depending on state or federal labeling requirements. Lomustine is an N-nitrosourea with the structure urea, where one nitrogen atom is replaced by a 2-chloroethyl and a nitrosyl group, and the other nitrogen atom is replaced by a cyclohexyl group. It is an alkylating antitumor drug used to treat brain tumors, lung cancer, malignant melanoma, and other solid tumors. It is both an alkylating agent and an antitumor drug. It belongs to the N-nitrosourea class of compounds and is also an organochlorine compound. It is an alkylating agent effective against both hematologic malignancies and solid tumors. Lomustine is an alkylating drug. The mechanism of action of lomustine is alkylating activity. Lomustine is an oral alkylating agent that can be used alone or in combination with other antitumor drugs to treat a variety of malignancies, including Hodgkin's lymphoma, lymphoma, and brain cancer. Lomustine treatment has been associated with mild, transient elevations in serum enzymes and rare cases of clinically significant acute liver injury. Lomustine is a nitrosourea with antitumor activity. Lomustine alkylates and cross-links DNA, thereby inhibiting DNA and RNA synthesis. It also carbamates DNA and proteins, leading to inhibition of DNA and RNA synthesis and disruption of RNA processing. Lomustine is lipophilic and can cross the blood-brain barrier. (NCI04) Lomustine has only been found in individuals who have used or taken the drug. It is an alkylating agent effective against both hematologic malignancies and solid tumors. Lomustine is a highly lipophilic nitrosourea compound that hydrolyzes in vivo to produce active metabolites. These metabolites can lead to alkylation and cross-linking of DNA (at the O6 position of the guanine base) and RNA, thereby inducing cytotoxicity. Other biological effects include inhibition of DNA synthesis and certain cell cycle phase specificity. Nitrosoureas generally do not exhibit cross-resistance with other alkylating agents. Because lomustine belongs to the nitrosourea class of drugs, it may also inhibit some key processes, such as carbamylation and cellular protein modification. It is an alkylating agent effective against both hematologic malignancies and solid tumors. Drug Indications It is used to treat primary and metastatic brain tumors as part of combination chemotherapy, in conjunction with appropriate surgery and/or radiation therapy. It can also be used in combination with other drugs as second-line treatment for refractory or relapsed Hodgkin lymphoma. Mechanism of Action Lomustine is a highly lipophilic nitrosourea compound that hydrolyzes in vivo to produce active metabolites. These metabolites can lead to alkylation and cross-linking of DNA (O6 position of guanine bases) and RNA, thereby inducing cytotoxicity. Other biological effects include inhibition of DNA synthesis and specificity to certain cell cycle stages. Nitrosoureas generally do not exhibit cross-resistance with other alkylating agents. Because lomustine is a nitrosourea, it may also inhibit some key processes, such as carbamylation and cellular protein modification.
Although lomustine is thought to act through alkylation, its mechanism of action is not fully elucidated, and other effects (such as carbamylation and cellular protein modification) may also be involved.
The overall result is considered to be the inhibition of DNA and RNA synthesis.

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Therapeutic Use
Anti-tumor drug, alkylating agent
CeeNU has been shown to be used as a single agent, in combination with other treatment modalities, or in combination with other approved chemotherapy drugs for the following diseases: Brain tumors—including primary and metastatic brain tumors, for patients who have received appropriate surgery and/or radiation therapy. Hodgkin lymphoma—second-line treatment, in combination with other approved drugs for patients who have relapsed during first-line therapy or who have not responded to first-line therapy. /US product label contains/
Anti-tumor drug. Since the early 1970s, this compound has been used in a limited manner to treat Hodgkin lymphoma and various solid tumors. These tumors include primary and metastatic brain tumors, colorectal tumors, and certain lung malignancies. It is usually used in combination with other anti-tumor drugs.
Although the lomustine label indicates it can be used in combination with other drugs as second-line treatment for refractory or relapsed Hodgkin's lymphoma, the preferred treatment for this cancer is currently a combination therapy regimen that includes other drugs. /US product label contains/
For more complete data on the therapeutic uses of lomustine (out of 8), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Warning: CeeNU (lomustine) should be used under the supervision of a qualified physician with experience in the use of chemotherapy drugs for cancer. Bone marrow suppression, particularly thrombocytopenia and leukopenia, can lead to bleeding and serious infections, especially in patients with pre-existing immunodeficiency. This is the most common and serious toxicity of CeeNU. Because the primary toxicity is delayed myelosuppression, blood cell counts should be monitored weekly for at least 6 weeks after administration. At the recommended dose, the dosing interval for CeeNU should not be less than 6 weeks. Bone marrow toxicity of CeeNU is cumulative; therefore, dose adjustments must be considered based on the lowest blood cell count following the previous dose.
Because some metabolites of lomustine are present in breast milk, women taking this medication may not breastfeed.
Delayed pulmonary fibrosis has been reported up to 17 years after treatment in children and adolescents (1-16 years old) who received related nitrosourea drugs in combination with cranial radiotherapy for intracranial tumors. A delayed decline in lung function was observed in all long-term survivors. Nitrosourea-induced pulmonary fibrosis may progress slowly and can lead to death.
Nausea and vomiting occur in 45% to 100% of patients within 45 minutes to 6 hours after oral administration of lomustine. While these symptoms are not severe and usually subside within 24 hours, they may persist for up to 36 hours and are often accompanied by anorexia for 2-3 days. Stomatitis occurs occasionally.
For more complete data on lomustine (25 total), please visit the HSDB records page.

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Pharmacodynamics
Lomustine is a nitrosourea alkylating agent. Lomustine and its metabolites interfere with the function of DNA and RNA. It is not specific to the cell cycle. Cancer develops when certain cells in the body proliferate abnormally and uncontrollably. These cells then spread and destroy neighboring tissues. Lomustine's mechanism of action is to slow this process. It kills cancer cells and prevents them from dividing by damaging DNA (the genetic material within cells).

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Lomustine (CCNU) was used as a reference chloroethylating agent in this study and compared with the methylating agent temozolomide. [1]
The cytotoxicity of both CCNU and temozolomide was correlated with intracellular ATase levels, indicating that damage to O⁶-alkylguanine in DNA is crucial to the cytotoxicity of these two classes of drugs. [1]
ATase repair of O⁶-chloroethylguanine (CCNU-induced damage) can prevent the subsequent formation of cytotoxic DNA interstrand crosslinks. [1]

C9H16CLN3O2

233.69524

233.093

C, 46.26; H, 6.90; Cl, 15.17; N, 17.98; O, 13.69

13010-47-4

3950

Light yellow to yellow solid powder

1.4±0.1 g/cm3

88-90

0.00142mmHg at 25°C

1.583

2.76

1

3

3

15

219

0

O=C(NC1CCCCC1)N(CCCl)N=O

GQYIWUVLTXOXAJ-UHFFFAOYSA-N

InChI=1S/C9H16ClN3O2/c10-6-7-13(12-15)9(14)11-8-4-2-1-3-5-8/h8H,1-7H2,(H,11,14)

1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea

NSC 79037; NSC-79037; NSC79037; Lomustinume. CeeNU; Belustin; Belustine; Cecenu; Citostal; Lomeblastin; Lucostin; Lucostine; Prava; CCNU; RB1509; WR139017

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: ≥ 100 mg/mL (~427.9 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.70 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 (10.70 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 (10.70 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: 30%propylene glycol+ 5%Tween 80+ 65%D5W, pH 4: 10.0mg/ml (42.79mM)

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