DNA alkylator
Alkylating agent (induces DNA damage by cross-linking DNAs and DNA and proteins) . [2]

Busulfan suppresses the frequency of cobblestone area-forming cells but does not significantly raise the rate of apoptosis in hematopoietic stem cell progenitors and similar cells. By an apoptosis-independent mechanism, busulfan inhibits the hematopoietic function of HSC progenitors and cells alike. In a time-dependent manner, busulfan causes bone marrow hematopoietic cell senescence, which is linked to an upregulation of p16^Ink4a and p19^Arf expression.[1] Normal human diploid WI38 fibroblasts are exposed to busulfan, an alkylating agent that damages DNA by cross-linking DNAs and DNA and proteins. This agent causes senescence through the extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK) cascade, which is independent of the p53-DNA damage pathway. Busulfan causes a temporary decrease in GSH but a persistent rise in ROS generation.[2] By reducing the expression of PCNA in testicular cells, busulfan-induced hypophosphorylation of Rb stops spermatogonial stem cells from undergoing apoptosis.[3]

---

Treatment of normal human diploid WI38 fibroblasts with busulfan (120 µM for 24 hours) induced cellular senescence, characterized by permanent cell cycle arrest. This was evidenced by a drastic decrease in BrdU incorporation (3.6% vs 45.1% in control) and a significant increase in senescence-associated β-galactosidase (SA-β-gal) staining (75.4% vs 9.7% in control) at day 11 post-treatment. [2]
Busulfan-induced senescence in WI38 cells was mediated primarily through the extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK) pathway, and not through the p53-DNA damage pathway. Inhibition of Erk or p38 with specific inhibitors (PD98059 or SB203580) abrogated the senescence phenotype, while inhibition of p53 (with α-PFT) or JNK had no significant effect. [2]
Busulfan treatment caused a rapid (within 30 min) and transient depletion (approximately 50% reduction) of intracellular glutathione (GSH) in WI38 cells, followed by a continuous increase in reactive oxygen species (ROS) production that lasted for at least 11 days. The initial ROS increase correlated with GSH depletion, but the sustained ROS production was attributed to elevated activity of NADPH oxidase, as it was suppressed by inhibitors diphenylene iodonium (DPI) and apocynin. [2]
Pre-treatment with the antioxidant N-acetyl-cysteine (NAC) replenished intracellular GSH, inhibited busulfan-induced ROS production, attenuated the activation of the Erk-p38 MAPK pathway, and significantly reduced the induction of senescence in WI38 cells. [2]

Busulfan-treated mice show a significant decrease in testis weight and an increase in apoptosis. In order to maximize the number of apoptotic cells and minimize the number of necrotic cells, 40 mg/kg body weight of busulfan is administered.[3] Using limiting dilution analysis, busulfan conditioning and radiation produce HSC detection sensitivity that is comparable in NOD/SCID mice.[4] Mice transplanted with busulfan exhibit incomplete and sluggish lymphoid engraftment. Mice that receive busulfan (20 mg/kg to 100 mg/kg) exhibit dose-dependent reconstitution of congenic lymphoid tissue.[5]

---

Administration of graded single doses of busulfan (10, 20, 35, 50, 80, 100 mg/kg) as a pre-transplant conditioning regimen to C57BL/6 Ly-5.2 recipient mice, followed by congenic Ly-5.1 hematopoietic cell transplantation (HCT), resulted in dose-dependent lymphoid reconstitution by donor cells in peripheral blood, lymph nodes, and spleen. [5]
At a low dose of 10 mg/kg busulfan, lymphoid engraftment was slow and incomplete, with only 6-11% donor (Ly-5.1) lymphocytes in various tissues at day 30, stabilizing at 40-46% by 180 days post-HCT. [5]
Higher doses (20-100 mg/kg) provided more robust and dose-dependent reconstitution. At 30 days post-HCT, donor lymphocyte percentages were: 43-54% (20 mg/kg), 66-71% (50-80 mg/kg), and 77-85% (100 mg/kg). By 60 days, these increased to: 57-68% (20 mg/kg), 72-79% (35 mg/kg), and 75-90% (≥50 mg/kg). [5]
In mice given at least 20 mg/kg busulfan, donor lymphocytes exceeded 90-95% in all lymphoid tissues by 90-120 days after HCT, achieving virtually complete and sustained lymphoid reconstitution comparable to that seen after total body irradiation (TBI, 900 rad). [5]

Induction of cellular senescence is a common response of a normal cell to a DNA-damaging agent, which may contribute to cancer chemotherapy- and ionizing radiation-induced normal tissue injury. The induction has been largely attributed to the activation of p53. However, the results from the present study suggest that busulfan (BU), an alkylating agent that causes DNA damage by cross-linking DNAs and DNA and proteins, induces senescence in normal human diploid WI38 fibroblasts through the extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK) cascade independent of the p53-DNA damage pathway. The induction of WI38 cell senescence is initiated by a transient depletion of intracellular glutathione (GSH) and followed by a continuous increase in reactive oxygen species (ROS) production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which leads to the activation of the Erk and p38 MAPK pathway. Incubation of WI38 cells with N-acetylcysteine (NAC) replenishes intracellular GSH, abrogates the increased production of ROS, ameliorates Erk and p38 MAPK activation, and attenuates senescence induction by BU. Thus, inhibition of senescence induction using a potent antioxidant or specific inhibitor of the Erk and p38 MAPK pathway has the potential to be developed as a mechanism-based strategy to ameliorate cancer therapy-induced normal tissue damage.[2]

---

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spermatocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited[3].

Cell Line: WI38 cells
Concentration: 120 μM
Incubation Time: 24 hours
Result: Incited a moderate p53 activation, but strong Erk, p38, and JNK phosphorylation, in a time-dependent manner.
Elicited an immediate up-regulation of p21 expression, which subsided by day 11.

---

Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in >95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P < 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P < 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture, 33% and 72% of hematopoietic cells survived IR- and BU-induced damage, respectively, as compared with control cells, but they could not form colony forming units-granulocyte macrophages. Moreover, these surviving cells expressed an increased level of senescence-associated beta-galactosidase, p16(Ink4a), and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis, whereas BU does so mainly by inducing premature senescence. In addition, induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly, the induction of hematopoietic cell senescence by IR, but not by BU, was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner, whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway.[1]

---

Cell Culture and Senescence Induction: Human diploid WI38 fibroblasts were cultured in MEM medium supplemented with fetal bovine serum and antibiotics. For senescence induction, cells at about 70% confluence were exposed to busulfan (120 µM) for 24 hours. After treatment, the drug was removed by washing with PBS, and cells were re-cultured in fresh medium for various durations up to 11 days. [2]
Senescence-associated β-galactosidase (SA-β-gal) Staining: SA-β-gal activity was determined using a commercial staining kit. Senescent cells were identified as blue-stained cells under light microscopy. A minimum of 1,000 cells were counted in random fields to determine the percentage of SA-β-gal positive cells. [2]
BrdU Incorporation Assay: DNA synthesis was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA. Cells were incubated with BrdU, then fixed and stained with an anti-BrdU antibody and Hoechst 33342 for nuclear counterstaining. Fluorescent images were captured, and the percentage of BrdU-positive nuclei among total cells (minimum 1000 cells counted) was calculated. [2]
Reactive Oxygen Species (ROS) Detection: After treatment, cells were harvested, incubated with the fluorescent probe dihydrorhodamine (DHR, 1 µg/ml) for 30 minutes at 37°C in the dark. DHR is oxidized by ROS to fluorescent rhodamine 123 (R123). The mean fluorescent intensity of R123 in at least 20,000 cells was measured by flow cytometry. To identify the source of ROS, cells were pre-incubated with various oxidase inhibitors (DPI, apocynin, rotenone, allopurinol, indomethacin, ketoconazole) before DHR staining. [2]
Glutathione (GSH) Detection: Cells were incubated with the non-fluorescent probe monochlorobimane (mBCI, 40 µM) for 30 minutes at 37°C in the dark. mBCI forms a stable fluorescent adduct with GSH in a reaction catalyzed by glutathione S-transferases. The fluorescent intensity of the GSH-bimane adduct was measured using a fluorescent plate reader. [2]
Western Blot Analysis: Cell lysates were prepared and analyzed by Western blotting to detect the phosphorylation (activation) status of p53, Erk, p38, and JNK, as well as the expression levels of p21 and p16 proteins. β-actin was used as a loading control. [2]
Inhibitor Studies: To assess pathway involvement, cells were pre-treated for 30 minutes with specific inhibitors: α-PFT (p53 inhibitor, 20 µM), PD98059 (Erk inhibitor, 50 µM), SB203580 (p38 inhibitor, 1 µM), or SP600125 (JNK inhibitor, 20 µM) prior to and during busulfan exposure, and the inhibitors were maintained in the culture medium (refreshed every 3 days) until analysis for senescence markers. [2]

ICR male mice ranging in age from 8 to 12 weeks (30-40 g)
40 mg/kg (in sesame oil)
IP; single dose

---

Human hematopoietic stem cell (HSC) xenotransplantation in NOD/SCID mice requires recipient conditioning, classically achieved by sublethal irradiation. Pretreatment with immunosuppressive and alkylating agents has been reported, but has not been rigorously tested against standard irradiation protocols. Here, we report that treatment of mice with a single dose (35 mg/kg) of Busilvex, an injectable form of busulfan, enables equivalent engraftment compared to 3.5 Gy irradiation. Mice treated with two doses of 25 mg/kg to reduce busulfan toxicity showed increased chimerism. Busulfan conditioning and irradiation resulted in comparable sensitivity of HSC detection as evaluated by limiting dilution analysis.[4]

---

Syngeneic Hematopoietic Cell Transplantation (HCT) Model: Nine-day-old C57BL/6 mice (Ly-5.2 immunophenotype) received a single intraperitoneal injection of busulfan at graded doses ranging from 10 mg/kg to 100 mg/kg. The drug was dissolved in 1.0 g/dL carboxymethylcellulose, administered at a volume of 0.03 mL per gram of mouse body weight. A control group received total body irradiation (TBI, 900 rad from a cesium-137 source). [5]
Twenty-four hours after busulfan administration or TBI, the recipient mice underwent HCT. They received an intraperitoneal transplant of bone marrow and spleen cells collected from congenic donor mice (C57BL/6 Ly-5.1). [5]
Analysis: The kinetics and extent of lymphoid repopulation by donor-derived cells were assessed at various time points (e.g., 30, 60, 90, 120, 180 days) post-transplant. Lymphocytes from peripheral blood, lymph nodes, and spleens of recipient mice were analyzed by flow cytometry using monoclonal antibodies specific for the donor Ly-5.1 allele to quantify the percentage of donor cells. [5]

Absorption, Distribution and Excretion
Busulfan is completely absorbed from the gastrointestinal tract. It is a small molecule, highly lipophilic drug that can cross the blood-brain barrier. The absolute bioavailability of a single intravenous bolus of 2 mg busulfan in adults is 80% ± 20%. In children (1.5–6 years), the absolute bioavailability is 68% ± 31%. The area under the curve (AUC) after a single oral dose is 130 ng•hr/mL. Peak plasma concentration is 30 ng/mL after oral administration (dose normalized to 2 mg). Peak plasma concentration is reached in 0.9 hours after dose normalization to 4 mg. After administration of 14C-labeled busulfan in humans, approximately 30% of the radioactive material is excreted in the urine within 48 hours; the amount recovered in feces is negligible. Within 24 hours, less than 2% of the administered dose is excreted unchanged in the urine. Elimination of busulfan is independent of renal function.
2.52 ml/min/kg [0.8 mg/kg every six hours, 16 infusions over four days]
Busulfan's pharmacokinetic distribution differs between children and adults. Children have lower mean bioavailability than adults; oral bioavailability of busulfan varies considerably among individuals, especially in children. A pharmacokinetic study of children receiving intravenous busulfan (0.8 or 1 mg/kg based on actual body weight) reported an estimated volume of distribution of 0.64 L/kg (11% inter-patient variability).
Busulfan is a small molecule and highly lipophilic drug that readily crosses the blood-brain barrier. Busulfan concentrations in cerebrospinal fluid (CSF) are approximately equal to concurrent plasma concentrations. It is currently unknown whether the drug is distributed into breast milk.
In adult patients who received single oral doses of 2, 4, or 6 mg busulfan over several consecutive days, the peak plasma concentration and area under the concentration-time curve (AUC) of the drug exhibited linear kinetic characteristics; the mean peak plasma concentration (normalized to a 2 mg dose) was observed to be approximately 30 ng/mL. In a study involving 12 patients who received single oral doses of 4–8 mg busulfan, the reported mean peak plasma concentration (normalized to a 4 mg dose) was approximately 68 ng/mL; the time to peak concentration was approximately 0.9 hours.
Following oral administration, busulfan is rapidly and completely absorbed from the gastrointestinal tract. The effect of food on the bioavailability of busulfan is unknown.
For more complete data on the absorption, distribution, and excretion of busulfan (11 studies in total), please visit the HSDB record page.

---

Metabolism/Metabolites
Bustulfan is extensively metabolized in the liver. Busulfan is primarily metabolized by binding to glutathione, a process that can occur both spontaneously and by catalysis by glutathione S-transferase (GST). GSTA1 is the main GST isoenzyme that promotes busulfan metabolism. Other GST isoenzymes involved in metabolism include GSTM1 and GSTP1. At least 12 metabolites have been identified, including tetrahydrothiophene, tetrahydrothiophene-12-oxide, sulfolane, and 3-hydroxysulfolane. These metabolites are not cytotoxic.
After intraperitoneal injection of 2:3-(14)C-Myleran in rats, rabbits, and mice, 60% of the urinary radioactive material was found to be in the form of 3-hydroxytetrahydrothiophene-1,1-dioxide (a sulfone). This suggests that Myleran reacts in vivo with cysteine or cysteine residues to form a cyclic sulfonium ion, which is subsequently cleaved to form tetrahydrothiophene, oxidized to 1,1-dioxide, and bio-hydroxylated to form a 3-hydroxy compound. In rats and mice, following intraperitoneal injection of a single dose of Myleran-(35)S (10 mg/kg body weight, dissolved in peanut oil), 50-60% of the drug was excreted within 24 to 48 hours, primarily as mesylate; small amounts of unmetabolized Myleran and two unidentified components were present in the urine. In rabbit urine, mesylate was the only metabolite found. The metabolic pathways of busulfan have been investigated in rats and humans using substances labeled with 14C and 35S. In humans, as in rats, almost all the radioactivity in 35S-labeled busulfan was excreted in the urine as 35S-mesylate. Studies have shown that the formation of mesylate in rats is not due to the simple hydrolysis of busulfan to 1,4-butanediol, as only about 4% of 2,3-14C-busulfan is excreted as carbon dioxide, while 2,3-14C-1,4-butanediol is almost completely converted to carbon dioxide. In mice, the main reaction of busulfan is the alkylation of thiol groups (especially cysteine and cysteine-containing compounds) to form cyclic sulfonium compounds, which are precursors to 3-hydroxytetrahydrothiophene-1,1-dioxide, the main urinary metabolite of the 4-carbon moiety. This effect is known as the “desulfurization” effect of busulfan, which may alter the function of certain sulfur-containing amino acids, peptides, and proteins; however, it is unclear whether this effect makes a significant contribution to the cytotoxicity of busulfan. Five male Sprague-Dawley rats were given intraperitoneal injection of (14)C-labeled busulfan (15 mg/kg). After 72 hours, the recovery rate of (14)C in urine was approximately 70% of the total dose, while the fecal excretion was in the range of 1.5-2%. The pattern of busulfan urinary metabolites was analyzed by high performance liquid chromatography (HPLC) combined with radioactivity detection. At least eight radioactive components were isolated. Three major metabolite peaks were identified by gas chromatography-mass spectrometry (GC/MS) and nuclear magnetic resonance spectroscopy (NMR): 3-hydroxysulfolane (accounting for 39% of the total radioactivity in urine), tetrahydrothiophene-1-oxide (20%), and sulfolane (13%). In addition, busulfan (6%) and tetrahydrofuran (2%) were also identified. A sulfonium ion-glutathione conjugate was presumed to be another metabolite, but it could not be separated due to its extreme instability. However, another compound was observed. This metabolite was co-eluted with the sulfonium ion generated by the reaction of busulfan with N-acetyl-L-cysteine, and hydrolyzed to form tetrahydrothiophene. Finally, in vitro cytotoxicity assays were performed on busulfan and its three major metabolites in Chinese V79 hamster cells. Only busulfan induced cytotoxicity, indicating that in vivo cytotoxicity is mediated by the parent compound, as expected. Busulfan is extensively metabolized in the liver. It is primarily metabolized via binding to glutathione, a process that can occur both spontaneously and by glutathione S-transferase (GST). GSTA1 is the major GST isoenzyme that promotes busulfan metabolism. Other GST isoenzymes involved in metabolism include GSTM1 and GSTP1. At least 12 metabolites have been identified, including tetrahydrothiophene, tetrahydrothiophene-12-oxide, sulfolane, and 3-hydroxysulfolane. These metabolites do not possess cytotoxic activity. Elimination pathway: Following administration of 14C-labeled busulfan to humans, approximately 30% of the radioactive material is excreted in the urine within 48 hours; the amount recovered in feces is negligible. Less than 2% of the administered dose is excreted unchanged in the urine within 24 hours. Elimination of busulfan is not related to renal function.
Half-life: 2.6 hours
Biological half-life
2.6 hours
The terminal half-life in children aged 6 months to 17 years is 2.26 to 2.52 hours.
The elimination half-life of oral busulfan in adults is approximately 2.6 hours.
The mean half-life of busulfan after intravenous administration is 2.83 to 3.90 hours… The mean half-life of oral busulfan is 3.87 hours.
The main metabolic pathway of busulfan is through the binding of glutathione S-transferase to glutathione (GSH).
[2]
Bustard
inhibits thioredoxin reductase, an enzyme involved in cellular redox regulation. [2]

Toxicity Summary
Busulfan is an alkylating agent whose molecular structure contains two unstable methanesulfonic acid groups, each attached to one end of a four-carbon alkyl chain. Upon hydrolysis of busulfan, the methanesulfonic acid groups are released, producing carbocations. These carbocations alkylate DNA, interfering with DNA replication and RNA transcription, ultimately leading to nucleic acid dysfunction. Specifically, its alkylation mechanism produces guanine-adenine intrachain crosslinks. This is achieved via an SN2 reaction, where the more nucleophilic guanine N7 atom attacks the carbon atom adjacent to the methanesulfonic acid leaving group. This damage cannot be repaired by cellular mechanisms, thus leading to apoptosis. Interactions Compared to patients who did not receive itraconazole, patients treated with itraconazole showed up to a 25% reduction in busulfan clearance. Concomitant use of itraconazole may lead to increased busulfan exposure in some patients, resulting in toxic plasma concentrations. Fluconazole has no effect on busulfan clearance. Pretreatment with phenytoin sodium increased the clearance of both cyclophosphamide and busulfan in patients receiving combination therapy, potentially leading to decreased plasma concentrations of both drugs. However, even with cyclophosphamide alone, busulfan clearance may be reduced, possibly due to competition for glutathione. Busulfan-induced pulmonary toxicity may be additive with other cytotoxic drugs. In one study, approximately 330 patients with chronic myeloid leukemia receiving combination therapy with busulfan and thioguanine developed portal hypertension and esophageal varices, along with abnormal liver function. Follow-up liver biopsies were performed on four of these patients, all showing nodular regenerative hyperplasia. The duration of combination therapy ranged from 6 to 45 months prior to the development of esophageal varices. Based on current data analysis, no cases of hepatotoxicity were observed in the busulfan-only group. Long-term continuous use of thioguanine and busulfan should be approached with caution.
Busulfan may produce additive myelosuppression when used in combination with other myelosuppressive drugs.
For more complete data on interactions of busulfan (8 items in total), please visit the HSDB record page.

---

Non-human toxicity values
Rat intravenous LD50: 14-25 mg/kg
Mouse oral LD50: 120 mg/kg

---

Therapeutic use of busulfan is associated with a variety of normal tissue damage (side effects), including myelosuppression, chronic pulmonary fibrosis, hepatic veno-occlusive disease (VOD), and hemorrhagic cystitis. [2]
At the cellular level, the key mechanism leading to its toxicity is the induction of oxidative stress and subsequent cellular senescence in normal cells such as WI38 fibroblasts. This involves the rapid depletion of intracellular GSH and the sustained production of ROS, primarily through the activation of NADPH oxidase. [2]

[1]. Cancer Res . 2003 Sep 1;63(17):5414-9.

[2]. Free Radic Biol Med . 2007 Jun 15;42(12):1858-65.

[3]. FEBS Lett . 2004 Sep 24;575(1-3):41-51.

[4]. Haematologica . 2006 Oct;91(10):1384.

[5]. Blood . 1991 Dec 15;78(12):3312-6.

[6]. Eur J Clin Pharmacol . 2012 Jun;68(6):923-35.

Therapeutic Uses
Busulfan, in combination with cyclophosphamide, is used as a pre-treatment regimen before allogeneic hematopoietic stem cell transplantation in patients with chronic myeloid leukemia (CML) and has been designated an orphan drug by the U.S. Food and Drug Administration (FDA) for the treatment of this disease. /VET/ is an anti-tumor drug used as adjuvant therapy for acute myeloid leukemia in small animals. Busulfan is an alkylating agent with myeloscavenging effects, inhibiting non-dividing bone marrow cells and potentially non-dividing malignant cells. Its use in the treatment of hematologic malignancies is widespread, especially for patients with chronic myeloid leukemia and other myeloproliferative disorders. For pediatric patients, Busilvex in combination with cyclophosphamide (BuCy4) or melphalan (BuMel) can be used as a pre-treatment regimen before routine hematopoietic stem cell transplantation. Drug Warnings Myleran is a potent drug. Unless chronic myeloid leukemia has been definitively diagnosed and the attending physician has the expertise to assess the response to chemotherapy, this medication should not be used. Merida can induce severe myelosuppression. If any abnormal signs of myelosuppression (manifested as an abnormal decrease in blood cell counts) appear, the dose should be reduced immediately or the medication discontinued. If the bone marrow status is uncertain, a bone marrow examination should be performed. Life-threatening hepatic venous occlusion has occurred in patients receiving busulfan (often in combination with cyclophosphamide or other antitumor drugs as part of pre-transplant myeloablative therapy). The manufacturer states that a clear causal relationship between busulfan and hepatic venous occlusion has not been established. In allogeneic transplantation clinical trials, 8% (5/61) of patients receiving intravenous busulfan were diagnosed with hepatic venous occlusion by clinical and laboratory examinations, of whom 40% (2/5) died. The overall mortality rate for hepatic venous occlusion in the entire study population was 3%. A retrospective analysis found that 3 out of 5 patients diagnosed with hepatic venous occlusion met Jones' diagnostic criteria. In randomized controlled trials, the incidence of hepatic venous occlusive disease (HVOC) was 7.7%–12% in patients receiving high-dose oral busulfan as part of their pre-transplant conditioning regimen. Interstitial pneumonia and pulmonary fibrosis have also been reported in patients receiving high-dose oral busulfan as part of their allogeneic pre-transplant conditioning regimen; these diseases, though rare, are often fatal. One patient receiving intravenous busulfan was diagnosed with nonspecific interstitial fibrosis by lung biopsy and subsequently died of respiratory failure. In patients receiving oral busulfan, pancytopenia usually occurs when hematological status is not adequately monitored and when the drug is not discontinued in a timely manner due to a significant or rapid decrease in white blood cell or platelet counts. Although individual variability in drug response does not appear to be a major influencing factor, some patients may be particularly sensitive to busulfan and experience sudden onset of neutropenia or thrombocytopenia. Busulfan-induced cytopenia may last longer than that caused by other alkylating agents; although recovery may take 1 month to 2 years, its toxicity is potentially reversible, and patients should be actively supported during any period of severe cytopenia. Some patients may develop myelofibrosis or chronic aplastic anemia, possibly due to busulfan toxicity. Rare, sometimes irreversible, aplastic anemia has been reported in patients receiving oral busulfan treatment. Aplastic anemia usually occurs after taking high doses or long-term use of regular doses. For more complete data on busulfan (42 total), please visit the HSDB records page.
Pharmacodynamics
Busulfan is an alkylating agent used to treat various cancers. Alkylating agents are named for their ability to add alkyl groups to numerous electronegative groups under specific intracellular conditions. They inhibit tumor growth by cross-linking guanine bases in the DNA double helix—directly attacking the DNA. This prevents the DNA strand from unwinding and separating. Since DNA replication requires unwinding and separation, cells cannot divide. Furthermore, these drugs can add methyl or other alkyl groups to molecules that shouldn't be present, leading to DNA mismatches. Alkylating agents are not cell cycle specific and have three mechanisms of action, but the end result is the same—damage to DNA function and cell death. Overexpression of the glutathione S-transferase MGST2 is thought to confer resistance to busulfan. However, the role of MGST2 in busulfan metabolism is unclear. Busulfan is a commonly used alkylating agent for the treatment of chronic myeloid leukemia and for myeloablative therapy before bone marrow transplantation. [2] Unlike some DNA damaging agents, busulfan can induce normal cell senescence through a p53-independent mechanism that relies on the Erk-p38 MAPK pathway, the upstream activation of which depends on reactive oxygen species (ROS). [2] This study suggests that antioxidant therapy (e.g., using N-acetylcysteine) or specific inhibition of the Erk-p38 MAPK pathway may be a potential strategy to improve busulfan-induced damage to normal tissues by preventing senescence. [2]

C6H14O6S2

246.3018

246.023

C, 29.26; H, 5.73; O, 38.98; S, 26.04

55-98-1

55-98-1 (Busulfan); 299-75-2 (Treosulfan); 52-24-4 (Thiotepa; Girostan; AI3-24916; NSC-6396)

2478

White to khaki solid powder

1.4±0.1 g/cm3

464.0±28.0 °C at 760 mmHg

114-117 °C(lit.)

234.4±24.0 °C

0.0±1.1 mmHg at 25°C

1.471

-0.52

0

6

7

14

294

0

S(C([H])([H])[H])(=O)(=O)OC([H])([H])C([H])([H])C([H])([H])C([H])([H])OS(C([H])([H])[H])(=O)=O

COVZYZSDYWQREU-UHFFFAOYSA-N

InChI=1S/C6H14O6S2/c1-13(7,8)11-5-3-4-6-12-14(2,9)10/h3-6H2,1-2H3

4-methylsulfonyloxybutyl methanesulfonate

Busulfex; Mitosan; Myleran; Mielucin; Misulban; Misulfan; BU; BUS; Myleran; Busulphan; Leucosulfan; Sulphabutin; Busulfex; Myelosan; CB2041; GT41; WR19508

2905591000

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: 63~125 mg/mL (255.8~507.5 mM)
Methanol: ~1 mg/mL (~4.1 mM)
H2O: ~1 mg/mL (~4.1 mM)

Solubility in Formulation 1: 6.25 mg/mL (25.38 mM) in 15% Cremophor EL + 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (8.44 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (8.44 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (8.44 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

---

Solubility in Formulation 5: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 3.15mg/ml (12.79mM)

---

Solubility in Formulation 6: 3.12 mg/mL (12.67 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

---

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