CPA (Endoxan; Baxter) and IFO (Holoxan; Baxter) were provided by Gustave Roussy Cancer Campus Grand Paris. Preactivated analog of IFO, G-IFO, was synthesized with 99% purity as previously described in.16 (link) For in vivo studies, CPA and IFO were dissolved in NaCl 0.9% or DMSO/Tween 80/NaCl 0.9% (5/5/90, v/v/v). G-IFO was dissolved in DMSO/Tween 80/NaCl 0.9% (5/5/90, v/v/v). Monoclonal anti-CD4 (GK1.5), anti-CD8α (53–6.72), anti-PD-1 (RMP1-14) and their isotype control rIgG2a (2A3) for in vivo experiments were purchased from BioXCell (West Lebanon, New Hampshire, USA) and dissolved in phosphate-buffered saline. mAbs used for flow cytometry and immunohistochemistry analysis are described in table 1 .
Holoxan
Manufactured by Baxter
Sourced in United States, Germany
Holoxan is a laboratory equipment product designed for use in research and scientific applications. It serves as a core function for various laboratory processes, providing essential capabilities to support research activities. The detailed technical specifications and intended usage of Holoxan are not included in this concise, unbiased, and factual description.
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4 protocols using holoxan
1
Preparation and Administration of Alkylating Agents
2
Xenograft Tumor Regression by Combination Chemotherapy
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Mice were randomized to receive different drugs when xenografts were approximately 150 mm3. Eight mice for the experimental group were used. The treatment dose and schedule for each drug were selected from literature (Table 1 ). After dilution in sterile water (doxorubicin (Adriblastina, Pfizer Italia) and ifosfamide (Holoxan (Baxter, Deerfield, IL, USA)), saline solution (gemcitabine (Gemzar, Eli Lilly Italia)) or 0.5% carboxymethylcellulose and 0.1% Tween 80 (EPZ-011989, kindly provided by Epizyme (Cambridge, MA, USA)), drugs were administered as follows: doxorubicin was delivered intravenously (i.v.) every 7 days, three times (q7d × 3); ifosfamide was administered intraperitoneally (i.p.) 3 days/week, 2 times, after a 2-week rest (qd × 3 q 2w). The same doses and schedules were used when the two drugs were administered as single agents and in combination. gemcitabine was administered i.v. every 4 days, four times (q4d × 4); EPZ-011989 was delivered orally (p.o.) twice a day for 28 consecutive days (2qd × 28) (Table 1 ).
Drug treatment activity was assessed in terms of TV inhibition percentage (TVI%) in treated versus control mice, expressed as TVI% = 100−[(mean TV treated/mean TV control) × 100]. Treatment toxicity was determined in terms of body weight loss and lethal toxicity.
Drug treatment activity was assessed in terms of TV inhibition percentage (TVI%) in treated versus control mice, expressed as TVI% = 100−[(mean TV treated/mean TV control) × 100]. Treatment toxicity was determined in terms of body weight loss and lethal toxicity.
3
Holoxan and N-Acetyl-L-Cysteine Administration
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holoxan (sterile ifosfamide, C 7 H 15 C l2 N 2 O 2 P) vial (Baxter oncology GmbH, Germany) is a synthetic analog of cyclophosphamide contains 1 g holoxan in a dry powder form. The content of each vial was freshly dissolved in sterile saline solution (0.9% NaCl) immediately before injection. holoxan was administered at 50 mg/kg body weight, daily for five consecutive days. The dose of holoxan (50 mg kg À1 ) is given to the rats as it is the recommended dose administered to patients with cancer daily for 5 days each cycle; [21] [22] [23] [24] N-Acetyl-Lcysteine (Sigma-Aldrich St. Louis, MO), its molecular formula is C 5 H 9 NO 3 S was freshly dissolved in normal sterile saline (0.9% NaCl) prior to injection. Female rats were injected (i.p.) with N-acetyl-L-cysteine at 160 mg/ kg/day; 25 96 h (4 days) before holoxan injection and continued with holoxan injection (50 mg/kg/day) for 5 consecutive days before mating.
4
Ifosfamide Pharmacokinetics in Rodents
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Animals in group 3 received five intraperitoneal doses of 50 mg/kg b.w. of ifosfamide (Holoxan, Baxter). In group 4, placebo treatment with saline instead of IF was used. Each individual was weighed before each administration of the drug, in order to determine a precise dose. During the time of administration of subsequent doses, animals were housed in collective, unisex cages. After administration of the last dose of IF or saline, animals were placed for 24 hours in individual metabolic cages, for determination of the same parameters as those measured in groups 1 and 2. Further procedures were the same as those applied to animals in groups 1 and 2, described above.
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