DNA Alkylator
Alkylating agent . [1]

Treosulfan is an agent that alkylates. Treosulfan exhibits nearly 100% cytotoxicity at 100 μg/mL on a number of cancer cell lines, including Panc-1, Miapaca-2, and Capan-2 cells, with IC50s of 3.6 μg/mL, 1.8 μg/mL, and 2.1 μg/mL, respectively. When combined with LY 188011, treosulfan (0.1-100 μg/mL) shows increased activity against cancer cells. On the other hand, Treosulfan (1, 2.5, and 5 μg/ml) in combination with 5-FU (0.1, 0.25, and 0.5 μg/ml) exhibits antagonistic effects on Miapaca-2 cells at all doses and on Panc-1 cells at intermediate and high concentrations[1]. Treosulfan (800 µg/mL) significantly lowers erythrocyte forward scatter and raises the proportion of ROS, [Ca²⁺]_i, and annexin-V-binding cells. When extracellular Ca²⁺ is removed, Treosulfan's effect on annexin-V binding is negated[2].

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Treosulfan demonstrated potent, dose-dependent cytotoxicity against three human pancreatic ductal carcinoma cell lines (Panc-1, MIA PaCa-2, and Capan-2) after 72 hours of exposure, as measured by Alamar Blue assay. At 100 μg/ml, it caused nearly 100% cytotoxicity. The IC50 values were 3.6 μg/ml for Panc-1, 1.8 μg/ml for MIA PaCa-2, and 2.1 μg/ml for Capan-2 cells. [1]
Flow cytometry analysis (Annexin V/7-AAD staining) confirmed that treatment with 10-100 μg/ml treosulfan induced a strong dose-dependent increase in late apoptotic and necrotic cell populations. Trypan blue exclusion also confirmed dose-dependent cell killing. [1]
The combination of treosulfan and gemcitabine showed strong synergistic cytotoxic effects against Panc-1 and MIA PaCa-2 cell lines, independent of the sequence of drug administration (simultaneous or sequential with a 12-hour interval). Combination Index (CI) values for Panc-1 ranged from 0.17 to 0.68 across all tested doses, indicating synergy. For MIA PaCa-2, synergy (CI 0.66-0.74) was observed at middle and high concentrations. [1]
The combination of treosulfan and irradiation (1-10 Gy) produced synergistic to additive cytotoxic effects in Panc-1 and MIA PaCa-2 cells, with CI values ranging from 0.7 to 1.1. This synergism was also independent of the application sequence. [1]
The combination of treosulfan and 5-fluorouracil (5-FU) showed antagonistic effects in MIA PaCa-2 cells at all tested doses (CI 1.16-1.28) and in Panc-1 cells at intermediate to high concentrations (CI 1.6-2.1). [1]

Treosulfan (1.5 g/kg/day) causes mice to rapidly undergo myeloablation and lose all of their splenic B and T cells. Treosulfan (1.5 g/kg/day) briefly increases the production of olny interleukin-2 in the spleen cells without clearly having a major impact on the synthesis of tumor necrosis factor-α and/or IFN-γ in mice[3].

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Treatment of BALB/c mice with treosulfan (1.5 g/kg/day for 3 consecutive days) induced a rapid, strong, and persistent myeloablation. Colony-forming unit granulocyte-macrophage (CFU-GM) counts in bone marrow reached their nadir on day 1 after the last dose and remained at this low level until the end of the observation period (day 12). This myeloablative effect was comparable to that of busulfan and more durable than that of cyclophosphamide. [3]
Treatment with treosulfan caused a rapid and pronounced depletion of both B cells (CD19+) and T cells (CD3+) in the spleen. The nadir (12.5% of control for B cells, 25% for T cells) was sustained from day 1 to day 12 post-treatment. This depletion was stronger and more durable than that induced by cyclophosphamide or busulfan. Both CD4+ and CD8+ T-cell subsets were equally depleted. [3]
Analysis of cytokine production in splenic T cells after in vitro stimulation with PMA/ionomycin showed that treosulfan treatment induced a transient increase in the percentage of IL-2 producing cells from day 1 to day 3, which then decreased to 50% of control levels from day 6 to day 12. The percentage of TNF-α producing cells was not significantly changed compared to controls, while the percentage of IFN-γ producing cells was generally decreased from day 1 to day 12. [3]

In tissue culture plates with 96 wells, the cells are grown in 100 μL volume per well and plated at 1×10⁴ cells/mL for cytotoxicity assays. After allowing the cells to adhere for a full night, they are cultured with varying concentrations of either Treosulfan alone or in conjunction with LY 188011. The drug combination is introduced either sequentially—the second drug is added 12 hours after the first—or simultaneously to the cell cultures. Alamar Blue® solution is added to the wells following a 72-hour incubation period before an additional overnight incubation. Next, absorbance is determined using a spectrophotometer, and drug cytotoxicity and cell proliferation are computed. Additionally, in certain experiments, trypan blue exclusion is used to determine proliferation and cytotoxicity, and an improved Neubauer hemocytometer is used to count cells. Cell viability is evaluated by staining the cells with 7-amino-actinomycin D (final concentration 200 μg/mL) and Annexin-V, followed by flow cytometry analysis using an FACS Scan flow cytometer[1].

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Cytotoxicity Assay (Alamar Blue): Cells (Panc-1, MIA PaCa-2, Capan-2) were seeded in 96-well plates at 1x10^4 cells/ml (100 μl/well) and allowed to adhere overnight. They were then incubated with various concentrations of treosulfan alone or in combination with other agents (gemcitabine, 5-FU) for 72 hours. For combination studies, drugs were added either simultaneously or sequentially (the second drug added 12 hours after the first). After incubation, Alamar Blue solution was added, and plates were incubated overnight. Absorbance was measured using a spectrophotometer, and cell proliferation/cytotoxicity was calculated. [1]
Cell Viability Assessment (Flow Cytometry): To differentiate between inhibition of proliferation and cell death, cells treated with treosulfan were stained with Annexin-V and 7-amino-actinomycin D (7-AAD) and analyzed using a flow cytometer to identify apoptotic and necrotic cell populations. [1]
Cell Viability Assessment (Trypan Blue Exclusion): Cell viability was also assessed using trypan blue exclusion followed by cell counting with a hemocytometer. [1]

Mice: At 10 to 12 weeks of age, female BALB/c mice weighed about 20 g. Standard pelleted feed and unlimited water are provided to the animals. They are kept in a climate-controlled room with a 12-hour light/dark cycle. There are four groups that they are split up into: one group gets treated with liposomal NCI C01592 (37 mg/kg/day) for four days straight; another group receives NSC-26271 (0.1 g/kg/day) for two days straight; a control group does not receive any treatment. To sustain the animals' survival in the absence of bone marrow support, sublethal doses of NSC-26271, NCI C01592, and treosulfan are administered. Days 1, 3, 6, 9, and 12 following the final treatment dose are dedicated to animal sacrifice, during which the femurs and spleen are extracted. Two control and six treated animals are included at each time point [3].

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Myeloablative and Immunosuppressive Study in Mice: Female BALB/c mice (10-12 weeks old) were divided into groups. The treosulfan treatment group received intraperitoneal injections of treosulfan at a dose of 1.5 g/kg/day for 3 consecutive days. This dose was sublethal, allowing survival without bone marrow support. Control groups received either cyclophosphamide (0.1 g/kg/day for 2 days), liposomal busulfan (37 mg/kg/day for 4 days), or no treatment. Animals were sacrificed on days 1, 3, 6, 9, and 12 after the last drug dose. Bone marrow (from femurs) and spleens were collected for clonogenic assays and immunological analyses (flow cytometry, cytokine analysis, MLR). [3]

Absorption, Distribution and Excretion
In a pharmacological study of the bioavailability of trichomoniasis capsules, patients with recurrent ovarian cancer received alternating oral and intravenous (iv) trichomoniasis capsules at daily doses of 1.5 or 2.0 g for 5 to 8 days. …The bioavailability ratio (f) for oral versus intravenous administration was calculated to be 0.97 ± 0.18 (mean ± standard deviation) using oral AUC = 82.1 ± 39.4 ug/ml hr and intravenous AUC = 85.4 ± 30.3 ug/ml hr. The peak plasma concentration (cmax) after intravenous administration (29 ± 14 μg/ml vs 65 ± 23 μg/ml) was significantly higher than that after oral administration (P < 0.01), and the tmax after oral administration was 1.5 ± 0.34 hours. The terminal half-life of trichomoniasis capsules is approximately 1.8 hours. Within 24 hours, the average urinary excretion of the parent compound was approximately 15% (range 6-26%) of the total dose administered. …A feasible and reliable oral formulation of treosver could lay the foundation for long-term low-dose outpatient treatment in patients with malignant tumors. In clinical high-dose chemotherapy regimens, plasma concentrations of treosver can exceed 500 μg/ml. [1]

dogtLDLotintravenoust 222 mg/kgt Gastrointestinal: Other changes; Blood: Leukopenia; Blood: Other changes Cancer Chemotherapy Report, Part 2, 2(203), 1965
monkeytLDLotintravenoust 222 mg/kgt Blood: Leukopenia; Blood: Agranulocytosis; Blood: Other changes Cancer Chemotherapy Report, Part 2, 2(203), 1965

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Interactions
L-Butylthionine-[S,R]-sulfonylimine has minimal effect on the toxicity of doxorubicin, ACNU (1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea, nimustine), and vincristine. L-Butylthionine-[S,R]-sulfonylimine failed to alter the toxicity of teniposide or cytarabine. L-Butylthionine-[S,R]-sulfonylimine significantly enhanced sensitivity to the alkylating agent trioxurfan in both cell lines through viability assays, in situ DNA end-labeling, and quantitative DNA fragmentation analysis. Trioxurfan is believed to mediate toxicity through the formation of reactive epoxides. PMID: 9484802

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Antidote and Emergency Treatment
Basic Treatment: Maintain an open airway. Suction if necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via a non-invasive ventilation mask at a flow rate of 10 to 15 L/min. Monitor for pulmonary edema and treat as necessary… Monitor for shock and treat as necessary… Anticipate seizures and treat as necessary… If eyes are contaminated, flush immediately with water. During transport, continuously flush each eye with saline… Do not use emetics. If swallowed, rinse mouth; if the patient is able to swallow, has a strong gag reflex, and does not drool, dilute with 5 mL/kg body weight to 200 mL of water… After decontamination, cover burns with a dry, sterile dressing… (Class A and B poisoning)
Advanced treatment: For patients with impaired consciousness, severe pulmonary edema, or respiratory arrest, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation using a bag-valve-mask may be effective. Monitor heart rhythm and treat arrhythmias as needed… Establish intravenous access using 5% glucose solution (SRP: maintain patency of the intravenous access at the minimum flow rate). If signs of hypovolemia appear, use lactated Ringer's solution. Watch for signs of fluid overload. Consider medical treatment for pulmonary edema… Infuse fluids with caution in cases of hypotension with signs of hypovolemia. Watch for signs of fluid overload… Use diazepam (Valium) to treat seizures… Use promecaine hydrochloride to assist eye irrigation… /Toxins A and B/

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Human Toxicity Excerpt.
.. 22 patients (15 with ovarian cancer and 7 with other cancers/lymphomas), with a median age of 48 years, received 28 cycles of high-dose treatment. Triosulfan was infused at escalating doses (from 20 to 56 g/m²) over 2 hours, and pharmacokinetic parameters were analyzed. At a dose of 56 g/m², three out of six patients experienced dose-limiting toxicities: three patients experienced grade III/IV diarrhea; one patient experienced grade III mucositis/stomatitis; one patient experienced toxic epidermal necrolysis; and one patient experienced grade III acidosis. Other mild side effects included erythema, pain, fatigue, and nausea/vomiting. Two patients died within four weeks of treatment, one due to rapid tumor progression and the other due to fungal infection. The plasma half-life, volume of distribution, and renal clearance of treosylvaline were dose-independent, while the area under the curve increased linearly up to 56 g/m². The maximum tolerated dose of high-dose treosylvaline was 47 g/m². Fractionated dosing or continuous infusion regimens are recommended for future high-dose trials. Given its antitumor activity and limited organ toxicity, incorporating high-dose treosylvaline into combination therapy with autologous peripheral blood stem cell transplantation appears worthwhile. Venous blood was collected from healthy volunteers and cancer patients at different time points before and after treatment with a single cytotoxic drug. Untreated cells were exposed to different concentrations of the drug in culture media. Chlorpheniramine, trooseltamivir, and cyclophosphamide (activated by liver microsomes) significantly increased the number of sister chromatid exchanges (SCEs) in vitro and in patient lymphocytes. Methotrexate and 5-fluorouracil had no effect, while bleomycin slightly increased the number of SCEs in vitro. Although in vitro dose-response relationships indicate which drugs increase the frequency of SCEs in vivo, the magnitude of the response is often overestimated. When patients receive drug treatment, the frequency of SCEs initially increases and then decreases over time. Although this complicates the quantitative relationship between dose and damage, sister chromatid exchanges (SCEs) may be helpful in monitoring the effects of alkylating agents on normal tissues. PMID:6891648

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Excerpt on Non-Human Toxicity
This study investigated the cytotoxicity and mutagenicity of the human carcinogen sulfanilamide and its hydrolysis product dl-1,2:3,4-diepoxybutane (DEB) in Chinese hamster ovary cells AS52. Sulfanilamide (0.1–1.0 mM) was toxic and mutagenic to the gpt locus, exhibiting strong pH dependence. dl-1,2:3,4-diepoxybutane was cytotoxic and mutagenic at much lower doses (0.025 mM), but these effects were not affected by pH. The results indicate that the toxicity and mutagenicity of trithionine are mediated by its hydrolysis product diethylamine (DEB), and the conversion of trithionine to DEB is highly pH dependent. PMID:8419160
This study tested the ability of two human carcinogens, 4-aminobiphenyl (4AB) and trithiophene (Treo), to induce micronuclei in bone marrow and peripheral blood cells using single, double, and triple exposure protocols in male B6C3F1 mice. Both compounds were detected positively. The increase in the incidence of polychromatic erythrocyte micronuclei was significantly greater in the double and triple exposure protocols than in the single exposure protocol. The results for Treo in peripheral blood were consistent with those usually observed compared to bone marrow, but with a 24-hour delay. However, the results for 4AB in peripheral blood were different from expectations. The incidence of MN-PCE was significantly higher in the peripheral blood of animals exposed to 4AB than in the bone marrow observed in the double and triple exposure protocols. Furthermore, the percentage of PCE also increased over time at a dose level of 60 mg/kg. Based on these studies, we conclude that the stepwise scoring protocol is likely the optimal protocol for rodent micronucleus testing, which includes a three-exposure protocol with one bone marrow sampling (24 hours after the last administration) and two peripheral blood samplings (24 and 48 hours after the first administration). This approach is cost-effective, reduces the number of animals required, and provides maximum sensitivity.

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Treosulfan induces erythrocyte apoptosis, the suicide death of erythrocytes, characterized by cell contraction and exposure of phosphatidylserine on the cell surface. [2]
Treosulfan stimulation of erythrocyte apoptosis is considered one of the potential mechanisms leading to common anemia in patients treated with this drug. The concentration required for in vitro stimulation of erythrocyte apoptosis (800 μg/ml) is within the range of concentrations achieved in vivo during clinical application. [2]
Excessive stimulation of erythrocyte apoptosis may lead to erythrocyte adhesion to the blood vessel wall and stimulate blood clotting, which may promote vascular occlusion and thrombosis. These conditions have been observed in the treatment of treosver. [2]

[1]. Synergistic cytotoxic activity of treosulfan and LY 188011 in pancreatic cancer cell lines. Anticancer Res. 2014 Apr;34(4):1779-84.

[2]. Programmed erythrocyte death following in vitro Treosulfan treatment. Cell Physiol Biochem. 2015;35(4):1372-80.

[3]. Myeloablative and immunosuppressive properties of treosulfan in mice. Exp Hematol. 2006 Jan;34(1):115-21.

Triosulfan is carcinogenic under California labor law. Triosulfan is an odorless, white crystalline powder. (NTP, 1992) Triosulfan is a mesylate. Triosulfan is being studied in allogeneic hematopoietic stem cell transplantation. Triosulfan has also been used to treat childhood acute lymphoblastic leukemia. Triosulfan is a prodrug of a bifunctional sulfonate alkylating agent with myeloscavenging, immunosuppressive, and antitumor activities. Under physiological conditions, triosulfan is non-enzymatically converted to L-diepoxybutane via a monoepoxide intermediate. The monoepoxide intermediate and L-diepoxybutane alkylate guanine residues in DNA, causing interstrand crosslinks, leading to DNA fragmentation and apoptosis. At dose escalation, the drug also exhibits myeloscavenging and immunosuppressive activities.

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Drug Indications
Ovastat in combination with fludarabine is indicated for adult patients and pediatric patients with malignant or non-malignant disease for more than one month as a pretreatment regimen before allogeneic hematopoietic stem cell transplantation (alloHSCT).
Pretreatment before hematopoietic progenitor cell transplantation
Mechanism of Action
The antitumor drug Ovastat (L-threitol 1,4-dimethylsulfonate, Ovastat) is a prodrug of an epoxide compound that, under physiological conditions, is non-enzymatically converted to L-diepoxidebutane via the corresponding monoepoxide. This study supports the hypothesis that this conversion of Ovastat is necessary for its in vitro cytotoxicity. Alkylation and interstrand crosslinking of plasmid DNA were observed after Ovastat treatment, but this phenomenon was also generated via the epoxide. Alkylation occurred on guanine bases, with sequence selectivity similar to other alkylating agents (e.g., nitrogen mustard). In Ovastat-treated K562 cells, crosslinking formation was slow, peaking at approximately 24 hours. Incubation of K562 cells with pre-formed epoxides resulted in faster and more efficient DNA cross-linking.

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Efficacy and Safety
Efficacy was evaluated in the MC-FludT.14/L Phase II trial (NCT00822393). This was a randomized, active-controlled trial comparing the efficacy of triosulfan versus busulfan in combination with fludarabine as an allogeneic transplant conditioning regimen. Eligible patients included adult patients aged 18 to 70 years with AML or MDS, a Karnofsky score ≥ 60%, and an age ≥ 50 years or a Hematopoietic Transplant Comorbidity Index (HCTCI) score > 2. A total of 570 patients were randomized to the triosulfan group (n=280) or the busulfan group (n=290).
The primary efficacy endpoint was overall survival (OS), defined as the time from randomization to death from any cause. In the randomized population, the hazard ratio for overall survival (stratified by donor type and risk group) compared to busulfan was 0.67 (95% CI: 0.51, 0.90); 0.73 (95% CI: 0.51, 1.06) in patients with acute myeloid leukemia (AML); and 0.64 (95% CI: 0.40, 1.02) in patients with myelodysplastic syndromes (MDS). The most common adverse reactions (≥20%) were musculoskeletal pain, stomatitis, fever, nausea, edema, infection, and vomiting. Selected grade 3 or 4 non-hematological laboratory abnormalities included elevated GGT, elevated bilirubin, elevated ALT, elevated AST, and elevated creatinine.
Recommended Dosage
The recommended dose of treosylvaline is 10 g/m² once daily, administered on days -4, -3, and -2; combined with fludarabine 30 mg/m² once daily, administered on days -6, -5, -4, -3, and -2; and allogeneic hematopoietic stem cell infusion on day 0.

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Treosylvaline is an alkylating agent known to have clinical activity against ovarian cancer and other solid tumors. This study is the first to report its activity against pancreatic cancer cell lines. [1]
The synergistic effect of treosylvaline with gemcitabine and radiotherapy, independent of the order of administration, warrants further investigation into its application in the treatment of pancreatic cancer. [1]

C6H14O8S2

278.30056

278.013

C, 25.89; H, 5.07; O, 45.99; S, 23.04

299-75-2

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

9882105

White to off-white solid powder

1.6±0.1 g/cm3

607.0±55.0 °C at 760 mmHg

216 °F (NTP, 1992)

320.9±31.5 °C

0.0±3.9 mmHg at 25°C

1.518

-1.64

2

8

7

16

345

2

O[C@H]([C@@H](O)COS(C)(=O)=O)COS(C)(=O)=O

YCPOZVAOBBQLRI-WDSKDSINSA-N

InChI=1S/C6H14O8S2/c1-15(9,10)13-3-5(7)6(8)4-14-16(2,11)12/h5-8H,3-4H2,1-2H3/t5-,6-/m0/s1

[(2S,3S)-2,3-dihydroxy-4-methylsulfonyloxybutyl] methanesulfonate

NSC-39069; Treosulfan; NSC 39069; Treosulphan; Ovastat; Dihydroxybusulfan; threosulphan; Treosulfano; Treosulfanum; NSC39069; (2S,3S)-2,3-Dihydroxybutane-1,4diyl dimethanesulfonate

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: ~56 mg/mL (~201.2 mM)
Water: ~56 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.98 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 (8.98 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 (8.98 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: 16.67 mg/mL (59.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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