DNA Alkylator

By enhancing the complementary chain of DNA molecules, chlorambucil inhibits the growth of tumor cells by causing an alkalinization-induced cross-reaction. Chlorambucil (0, 2.5, 5, 10 μM) significantly enhanced the expression of DR4 and DR5 mRNA in Raji cells while having a minor blocking impact on them. Rati cells treated with 10 μM chlorambucil and 80 ng/mL of tumor factor-related phosphate ligand (TRAIL) expressed DR4 and DR5 mRNA. Chlorambucil is a DNA alkylating agent at high concentrations and a ligand for nuclear protein synthesis, particularly proteome production, at low concentrations. While long-term treatment maintenance is linked to p53 gene alterations that cause subsequent cancers, increasing doses are linked to a higher frequency of cell sterilization [4].

Ehrlich ascites cancer can be treated with levamisole (5 mg/kg) and chlorambucil (0.2 mg/kg, po) in combination to increase the anti-cancer effect and improve the transparency and anti-cancer rate of the disease. Mice's kidneys and liver are negatively impacted by it [2].

After being broken down by trypsin into a single cell suspension, cultured cells at the log-growth phase are seeded at a density of 1000 cells per well into 96-well plates. The plate is set up in a 37°C, 5% CO₂ chamber. Cells are treated with TRAIL at 0, 20, 40, and 80 ng/mL or chlorambucil at 0, 2.5, 5, and 10 μM for 48 hours after attached growth for 24 hours. After adding 10 μL of CCK-8 reagent to each well, the mixture is incubated for 4 hours at 37°C. Subsequently, a micro-plate reader measures the absorbance values at 450 nm. For every treatment group, six parallel samples are run. Rate of cell proliferation (%) = mean value of control group × 100% / mean value of experimental group[1].

Mice: Swiss female mice are split into five groups at random (20 mice in each group). Group 1 is maintained as the control group. Group 2 is administered 2.5 × 10⁶ Ehrlich ascites carcinoma cells intraperitoneally. Group 3 is given oral treatment with chlorambucil at a dose of 0.2 mg/kg body weight. Group 4 is given oral treatment with levamisole at a dose of 5 mg/kg body weight. Group 5 is given daily oral treatment with a combination of chlorambucil and levamisole, administered through a bent stainless steel stomach tube[2].

Absorption, Distribution and Excretion
Chlorambucil is extensively metabolized in the liver primarily to phenylacetic acid mustard. The pharmacokinetic data suggests that oral chlorambucil undergoes rapid gastrointestinal absorption and plasma clearance and that it is almost completely metabolized, having extremely low urinary excretion.
Chlorambucil is rapidly and completely absorbed from the GI tract. Following single oral doses of 0.6-1.2 mg/kg, peak plasma concentrations of chlorambucil are reached within 1 hour.
In a limited number of patients given a single oral dose of chlorambucil 0.2 mg/kg, an average peak plasma chlorambucil concentration of 492 ng/mL (adjusted to a dose of 12 mg) was reached at about 0.83 hours, and a mean peak plasma concentration of phenylacetic acid mustard (the major metabolite of chlorambucil) of 306 ng/mL (adjusted to a chlorambucil dose of 12 mg) occurred at approximately 1.9 hours. The area under the plasma concentration-time curve (AUC) of phenylacetic acid mustard was about 1.36 times greater than the AUC of chlorambucil.
In a study of 12 patients given single oral doses of 0.2 mg/kg of chlorambucil, the mean dose (12 mg) adjusted (+/-SD) plasma chlorambucil Cmax was 492 +/- 160 ng/mL, the AUC was 883 +/- 329 ng.hr/mL, t1/2 was 1.3 +/- 0.5 hours, and the tmax was 0.83 +/- 0.53 hours. For the major metabolite, phenylacetic acid mustard, the mean dose (12 mg) adjusted (+/- SD) plasma Cmax was 306 +/- 73 ng/mL, the AUC was 1204 +/- 285 ng.h/mL, the t1/2 was 1.8 +/- 0.4 hours, and the tmax was 1.9 +/- 0.7 hours.
Chlorambucil and its metabolites are extensively bound to plasma and tissue proteins. In vitro, chlorambucil is 99% bound to plasma proteins, specifically albumin.
For more Absorption, Distribution and Excretion (Complete) data for CHLORAMBUCIL (12 total), please visit the HSDB record page.

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Metabolism / Metabolites
Chlorambucil undergoes rapid metabolism to phenylacetic acid mustard, the major metabolite, and the combined chlorambucil and phenylacetic acid mustard urinary excretion is extremely low - less than 1% in 24 hours.
Chlorambucil and its major metabolite spontaneously degrade in vivo forming monohydroxy and dihydroxy derivatives.
Chlorambucil is extensively metabolized in rodents by monochloroethylation and by beta oxidation, forming the phenylacetic acid derivative, which also has anticancer activity.
Ten metabolites of chlorambucil were isolated, most of which were phenylacetic acid & benzoic acid derivatives.
Route of Elimination: Chlorambucil is extensively metabolized in the liver primarily to phenylacetic acid mustard. The pharmacokinetic data suggests that oral chlorambucil undergoes rapid gastrointestinal absorption and plasma clearance and that it is almost completely metabolized, having extremely low urinary excretion.
Half Life: 1.5 hours

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Biological Half-Life
1.5 hours
In a study of 12 patients given single oral doses of 0.2 mg/kg of chlorambucil, ... t1/2 was 1.3 +/- 0.5 hours, and the tmax was 0.83 +/- 0.53 hours. For the major metabolite, phenylacetic acid mustard, ... the t1/2 was 1.8 +/- 0.4 hours, and the tmax was 1.9 +/- 0.7 hours.

Toxicity Summary
Alkylating agents work by three different mechanisms: 1) attachment of alkyl groups to DNA bases, resulting in the DNA being fragmented by repair enzymes in their attempts to replace the alkylated bases, preventing DNA synthesis and RNA transcription from the affected DNA, 2) DNA damage via the formation of cross-links (bonds between atoms in the DNA) which prevents DNA from being separated for synthesis or transcription, and 3) the induction of mispairing of the nucleotides leading to mutations.

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Hepatotoxicity
Chlorambucil therapy is associated with a low rate of serum enzyme elevations, but these are generally mild and self limited, not requiring dose adjustment. Rare instances of clinically apparent acute liver injury attributed to chlorambucil have been reported. The onset of symptoms was within 2 to 6 weeks of starting chlorambucil and the typical enzyme pattern was cholestatic. Some cases have had features of hypersensitivity (rash, fever), and liver injury has recurred upon rechallenge. This form of liver injury is rare and resembles the idiosyncratic acute liver injury due to cyclophosphamide. Chlorambucil has not been linked specifically to sinusoidal obstruction syndrome, but it is not used in high doses in neoplastic disease or in conditioning regimens for hematopoietic cell transplantation, situations in which alkylating agents are commonly associated with this complication. Chlorambucil therapy has also been linked to hypersensitivity reactions and severe cutaneous adverse events such as Stevens Johnson syndrome and toxic epidermal necrolysis, both of which can be accompanied by serum enzyme elevations and hepatitis.
Likelihood score: D (possible rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of chlorambucil during breastfeeding. Most sources consider breastfeeding to be contraindicated during maternal cytotoxic antineoplastic drug therapy, especially alkylating agents such as chlorambucil. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Telephone follow-up study was conducted on 74 women who received cancer chemotherapy at one center during the second or third trimester of pregnancy to determine if they were successful at breastfeeding postpartum. Only 34% of the women were able to exclusively breastfeed their infants, and 66% of the women reported experiencing breastfeeding difficulties. This was in comparison to a 91% breastfeeding success rate in 22 other mothers diagnosed during pregnancy, but not treated with chemotherapy. Other statistically significant correlations included: 1. mothers with breastfeeding difficulties had an average of 5.5 cycles of chemotherapy compared with 3.8 cycles among mothers who had no difficulties; and 2. mothers with breastfeeding difficulties received their first cycle of chemotherapy on average 3.4 weeks earlier in pregnancy. None of the patients in the study received chlorambucil.

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Protein Binding
99%

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Interactions
Because normal defense mechanisms may be suppressed by chlorambucil therapy, concurrent use with a live virus vaccine may potentiate the replication of the vaccine virus, may increase the side/adverse effects of the vaccine virus, and/or may decrease the patient's antibody response to the vaccine; immunization of these patients should be undertaken only with extreme caution after careful review of the patient's hematologic status and only with the knowledge and consent of the physician managing the chlorambucil therapy. The interval between discontinuation of medication that cause immunosuppression and restoration of the patient's ability to respond to the vaccine depends on the intensity and type of immunosuppression-causing medications used, the underlying disease, and other factors; estimates vary from 3 months to 1 year. Patients with leukemia in remission should not receive live virus vaccine until at least 3 months after their last chemotherapy. In addition, immunization with oral polio-virus vaccine should be postponed in persons in close contact with the patient, especially family members.
These medications /tricyclic antidepressants and possibly, structurally related compounds such as cyclobenzaprine, haloperidol, loxapine, maprotiline, molindone, monoamine oxidase inhibitors including furazolidone, procarbazine, and selegiline, phenothiazines, pimozide, thioxanthenes/ may lower the seizure threshold and increase the risk of chlorambucil-induced seizures.
Leukopenic and/or thrombocytopenic effects of chlorambucil may be increased with concurrent or recent therapy /with blood dyscrasia-causing medications/ if these medications cause the same effects; dosage adjustment of chlorambucil, if necessary, should be based on blood counts.
Additive bone marrow depression may occur; dosage reduction may be required when two or more bone marrow depressants, including radiation, are used concurrently or consecutively /with chlorambucil/.
For more Interactions (Complete) data for CHLORAMBUCIL (8 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Mouse sc 115 mg/kg
LD50 Mouse ip 30 mg/kg
LD50 Mouse oral 101 mg/kg
LD50 Rat ip 14 mg/kg
LD50 Rat oral 76 mg/kg

[1]. Synergistic effects of chlorambucil and TRAIL on apoptosis and proliferation of Raji cells. Eur Rev Med Pharmacol Sci. 2017 Oct;21(20):4703-4710.

[2]. Biochemical and pathological studies on the effects of levamisole and chlorambucil on Ehrlich ascites carcinoma-bearing mice. Vet Ital. 2011 Jan-Mar;47(1):89-95.

[3]. Chlorambucil-adducts in DNA analyzed at the oligonucleotide level using HPLC-ESI MS. Chem Res Toxicol. 2009;22(8):1435-1446.

[4]. Chlorambucil and malignancy. Ophthalmology. 2010;117(7):1466-1466.e1.

Therapeutic Uses
Antineoplastic Agents, Alkylating; Carcinogens
Chlorambucil is indicated in the treatment of chronic lymphatic (lymphocytic) leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma, and Hodgkin's disease. It is not curative in any of these disorders but may produce clinically useful palliation. /Include in US product label/
Chlorambucil is considered by many clinicians to be the drug of choice for the treatment of (Waldenstrom's) macroglobulinemia. /Not included in US product label/
Chlorambucil has also been used effectively with prednisone in the treatment of children with minimal-change nephrotic syndrome (lipoid nephrosis, idiopathic nephrotic syndrome of childhood) who have frequent relapses, require corticosteroid therapy to maintain remissions, or whose disease is resistant to steroid therapy. In most of these children, chlorambucil and prednisone therapy has induced long-term remissions and decreased the frequency of relapses. Although this type of nephrotic syndrome only occasionally occurs in adults, it is treated similarly. /Not included in US product label/
For more Therapeutic Uses (Complete) data for CHLORAMBUCIL (9 total), please visit the HSDB record page.

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Drug Warnings
/BOXED WARNING/ Chlorambucil can severely suppress bone marrow function. Chlorambucil is a carcinogen in humans. Chlorambucil is probably mutagenic and teratogenic in humans. Chlorambucil produces human infertility.
Chlorambucil is contraindicated in patients with known hypersensitivity to the drug or in patients whose disease was resistant to prior therapy with the drug. The manufacturer states that there may be cross-sensitivity between chlorambucil and other alkylating agents manifested as rash. Chlorambucil should be discontinued promptly in patients who develop skin reactions.
Adverse hematologic effects are the major and dose-limiting effects of chlorambucil. In usual doses, myelosuppression generally occurs gradually, is moderate in severity, and is usually reversible following discontinuance of the drug. Leukopenia, resulting from neutropenia and slowly progressive lymphopenia, occurs in many patients receiving chlorambucil. Thrombocytopenia and anemia may also occur.
Chlorambucil appears to be relatively free of adverse GI effects unless single doses of 20 mg or more are administered. Adverse GI effects include nausea, vomiting, gastric discomfort or abdominal pain, anorexia, and diarrhea. Adverse GI effects are usually mild, last less than 24 hours, and disappear despite continued treatment; however, nausea and weakness have persisted up to 7 days in some patients following a single, high dose of the drug. If necessary, nausea and vomiting may usually be controlled with antiemetics. Oral ulceration has also been reported.
For more Drug Warnings (Complete) data for CHLORAMBUCIL (25 total), please visit the HSDB record page.

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Pharmacodynamics
Chlorambucil is an antineoplastic in the class of alkylating agents that is used to treat various forms of cancer. Alkylating agents are so named because of their ability to add alkyl groups to many electronegative groups under conditions present in cells. They stop tumor growth by cross-linking guanine bases in DNA double-helix strands - directly attacking DNA. This makes the strands unable to uncoil and separate. As this is necessary in DNA replication, the cells can no longer divide. In addition, these drugs add methyl or other alkyl groups onto molecules where they do not belong which in turn inhibits their correct utilization by base pairing and causes a miscoding of DNA. Alkylating agents are cell cycle-nonspecific. Alkylating agents work by three different mechanisms all of which achieve the same end result - disruption of DNA function and cell death.

C14H19CL2NO2

304.21

303.079

C, 55.27; H, 6.30; Cl, 23.31; N, 4.60; O, 10.52

305-03-3

2708

Pale brown to brown solid powder

1.2±0.1 g/cm3

460.1±40.0 °C at 760 mmHg

64ºC

232.1±27.3 °C

0.0±1.2 mmHg at 25°C

1.570

3.1

1

3

9

19

250

0

O=C(O)CCCC1=CC=C(N(CCCl)CCCl)C=C1

JCKYGMPEJWAADB-UHFFFAOYSA-N

InChI=1S/C14H19Cl2NO2/c15-8-10-17(11-9-16)13-6-4-12(5-7-13)2-1-3-14(18)19/h4-7H,1-3,8-11H2,(H,18,19)

4-[4-[bis(2-chloroethyl)amino]phenyl]butanoic acid

CB-1348; WR-139013; CB1348; WR139013; CB 1348; WR 139013; chlorambucilum; chloraminophen; Chlorbutin; chlorbutine; chlorbutinum; chloroambucil; chlorobutin; chlorobutine; Leukersan; Leukoran; Lympholysin; phenylbutyric acid nitrogen mustard; US brand names: Ambochlorin; Amboclorin; Leukeran; Linfolizin. Foreign brand names: Altichlorambucil; Chloraminophene; Linfolysin.

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.22 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 (8.22 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 (8.22 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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 (Please use freshly prepared in vivo formulations for optimal results.)