Adenosine deaminase (Ki = 2.5 pM)
Adenosine deaminase inhibitor (induces T-cell apoptosis through adenosine deaminase inhibition) [1]

Both pentostatin and ECP result in T-cell and host DC depletion and a shift of the remaining DC and T-cell population to a tolerogenic DC2 and T-regulatory population leading to the low rate of GVHD observed by Miller et al with a regimen combining ECP, pentostatin and 600 cGy TBI for HLA-identical and non-identical (5/6) allogeneic HCT.[1]

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The aim of this study was to evaluate the anti-trypanosomal effect of treatment with 3'-deoxyadenosine (cordycepin) combined with deoxycoformycin (pentostatin: inhibitor of the enzyme adenosine deaminase) in vitro by using mice experimentally infected with Trypanosoma evansi. In vitro, a dose-dependent trypanocidal effect of cordycepin was observed against the parasite[2].

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ECP product showed reduced alloreactivity and NK cell function after UVA activation. [1]

Pentostatin (2 mg/kg) combined with cordycepin (2 mg/kg) was 100% effective against Trypanosoma evansi-infected mice. Increased levels of some biochemical parameters, especially liver enzymes, were accompanied by histological lesions of the liver and kidneys. Pentostatin alone has no effect on infected groups. All dogs developed granulocytopenia with granulocyte counts <500 cells/μL starting on day 4. Thrombocytopenia (<20,000 platelets/μL) begins on day 7 after HCT, with a nadir of 3000 to 14000 platelets/μL [1].

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In the in vivo trials, the two drugs were used individually and in combination of different doses. The drugs when used individually had no curative effect on infected mice. However, the combination of cordycepin (2 mg kg-1) and pentostatin (2 mg kg-1) was 100% effective in the T. evansi-infected groups. There was an increase in levels of some biochemical parameters, especially on liver enzymes, which were accompanied by histological lesions in the liver and kidneys. Based on these results we conclude that treatment using the combination of 3'-deoxyadenosine with deoxycoformycin has a curative effect on mice infected with T. evansi. However, the therapeutic protocol tested led to liver and kidney damage, manifested by hepatotoxicity and nephrotoxicity[2].

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Extracorporeal photopheresis (ECP) and the purine analog pentostatin exert potent immunomodulatory effects. We evaluated the use of these treatment modalities to prevent GVHD in a canine model of unrelated dog leukocyte Ag-mismatched hematopoietic cell transplantation, after conditioning with 920 cGy TBI. We have shown previously in this model that 36/40 dogs given MTX alone as postgrafting immunosuppression engrafted and that 25 of 40 dogs had severe GVHD and median survival of 21 days. In the current study, nine dogs received conditioning with 920 cGy TBI and postgrafting MTX either with ECP on days -2 to -1 alone (n=5) or ECP on days -6 and -5 combined with two doses of pentostatin (days -4 to -3) (n=4). Seven of nine dogs achieved engraftment. Six dogs developed severe acute GVHD (four in the group with ECP alone and two with pentostatin and ECP). We failed to demonstrate a positive impact of ECP and pentostatin for the prevention of GVHD compared with historical control dogs.[1]

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In a canine model of DLA-nonidentical hematopoietic cell transplantation, pentostatin was administered intravenously at 4 mg/m² on days –4 and –3 before transplantation, combined with ECP and 920 cGy TBI, followed by postgrafting MTX. All dogs developed severe acute GVHD, and the combination did not prevent GVHD compared to historical controls. [1]

Mixed leukocyte cultures (MLC) and natural killer (NK) cell cytotoxicity assay [1]
Mixed leukocyte cultures were used to assess the dogs’ cellular immune function before and after ECP as described previously. To evaluate NK cell activity before and after ECP, chromium release assays were performed as described previously.

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Chimerism analysis [1]
Donor and host cell chimerism were evaluated using a polymerase chain reaction (PCR) based assay of polymorphic (CA)n dinucleotide repeats with primers specific for informative microsatellite markers. Genomic DNA of the cells of interest was extracted, and PCR was performed under conditions described previously. The technique used enables to detect between 2.5% to 97.5% donor cell chimerism.

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Detection of apoptosis by Annexin V (Ax)/PI staining [1]
Apoptosis of cells exposed to ECP was assessed by flow cytometry with the use of Annexin V binding, which allows detection of phosphatidylserine on the cell surface of apoptotic cells. Briefly, after overnight incubation at 37 °C in 5% humidified atmosphere, cells were harvested, lysed, washed with PBS and incubated with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s manual. Cells were analyzed by flow cytometry by means of CellQuest Analysis software. A minimum of 10 000 events were counted per sample. Cells positive for Annexin V but negative for PI are in early apoptosis, cells double positive for Annexin V and PI are in late apoptosis. Results are reported as a percentage of annexin V-FITC positive cells.

DLA-nonidentical marrow grafts [1]
All recipient dogs were conditioned for transplantation by 920 cGy TBI at 7 cGy/minute using a linear accelerator. Dogs in group A1 received ECP administered on days −2 and −1 with TBI on day 0 and dogs in group A2 received ECP on days −6 and −5, intravenous (IV) infusion of pentostatin at a dose of 4mg/m2 on days −4 and −3, and TBI on day 0 (Table 1). Donor marrow cells from DLA-nonidentical donors were aspirated under general anesthesia through needles inserted into humeri and femora and stored in heparinized tissue culture medium at 4°C for no more than 6 hours.22 Within 4 hours of TBI, harvested marrow cells were infused IV into recipients at a median dose of 2.9 (range, 1.9 to 6.1) ×108 total nucleated cells (TNC)/kg. The day of marrow grafting was designated as day 0. In addition to marrow graft, recipients were given IV infusions of peripheral blood buffy coat cells obtained by leukapheresis from the marrow donor on days 1 and 2, at a median dose of 2.3 (range, 1.2 to 6.9) ×108 TNC/kg to ensure consistent hematopoietic engraftment. MTX, at a dose of 0.4 mg/kg intravenously was used as postgrafting immunosuppression and administered on days +1, +3, +6 and +11, then weekly thereafter until day 102.

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Pentostatin was administered intravenously at a dose of 4 mg/m² on days –4 and –3 before transplantation in dogs receiving DLA-nonidentical marrow grafts after 920 cGy TBI. ECP was performed on days –6 and –5, and postgrafting immunosuppression included MTX. Dogs were monitored for engraftment, GVHD, and survival. [1]

Absorption, Distribution and Excretion
Not absorbed orally, it crosses the blood-brain barrier.
In humans, approximately 90% of a single intravenous infusion of 4 mg/m² pentostatin is excreted unchanged in the urine and/or as its metabolites 5 minutes later, as determined by adenosine deaminase inhibitory activity.
68 mL/min/m²
In a small number of patients with advanced, refractory cancers, plasma pentostatin concentrations were approximately 3.2–9.7 ng/ml after daily direct intravenous administration of 0.25 mg/kg pentostatin for 4 or 5 days. Plasma concentrations appear to increase linearly with dose; in a study of leukemia patients, the mean plasma pentostatin concentrations measured 1 hour after intravenous infusion of 0.25 or 1 mg/kg pentostatin 30 minutes later were approximately 0.4 or 1.26 μg/ml, respectively. No significant correlation has been found between the mean or absolute concentrations of plasma adenosine or deoxyadenosine and the therapeutic or toxic effects of pentostatin; however, limited data suggest that the drug response may be correlated with the ratio of deoxyadenosine triphosphate (DAP) to adenosine triphosphate (ATP) in lymphoblasts. Furthermore, elevated plasma DAP levels have been reported to parallel the accumulation of DAP in erythrocytes and lymphoblasts, and toxicity appears to be correlated with the DAP/ATP ratio in erythrocytes. Animal studies have shown that pentostatin rapidly distributes to all body tissues, but the degree of drug accumulation in different tissues appears to vary by species. Following intraperitoneal injection in mice, the highest drug concentrations were found in the kidneys, liver, and spleen. Following intravenous injection in dogs, tissue concentrations of pentostatin were directly proportional to tissue adenosine deaminase activity, with the highest concentrations found in the lungs, spleen, pancreas, heart, liver, and jejunum. Pentostatin is reported to enter erythrocytes via the same facilitated transport system as other nucleoside analogs or through simple diffusion; although the efflux of the drug from cells is not yet clear, the time course of pentostatin's action (e.g., adenosine deaminase inhibition) varies in different cell types (e.g., lymphocytes, erythrocytes). Limited animal and human data suggest that pentostatin is relatively poorly distributed in cerebrospinal fluid (CSF), with peak CSF concentrations averaging about 10% of the corresponding plasma concentrations. In a 6-year-old leukemia patient who received intravenous injections of 0.25 mg/kg pentostatin daily for 3 consecutive days, the concentrations in serum and CSF (via lumbar puncture) were approximately 147 ng/ml and 19 ng/ml, respectively, four hours after the first dose, measured by enzyme inhibition titration; one hour after the third dose, the corresponding serum and CSF concentrations were approximately 241 ng/ml and 35 ng/ml, respectively. For more complete data on the absorption, distribution, and excretion of tebustatin (7 types), please visit the HSDB record page. Metabolism/Metabolites: Primarily metabolized in the liver, but only a small amount is metabolized. Elimination route: In humans, approximately 90% of the dose of a single intravenous infusion of 4 mg/m² tebustatin is excreted in the urine as unchanged tebustatin and/or metabolites, as determined by adenosine deaminase inhibitory activity assay. Half-life: 5.7 hours (range 2.6 to 16 hours). Biological half-life: 5.7 hours (range 2.6 to 16 hours). In healthy individuals, the distribution half-life and terminal elimination half-life of a single intravenous injection of 4 mg/m² tebustatin (within 5 minutes) have been reported to be 11 minutes and 5.7 hours, respectively. In a multiple-dose study in a small number of patients, after 36 cycles of tebustatin (4 mg/m² IV), the mean distribution half-life and terminal elimination half-life were reported to be 9.6 minutes (range: 3.1–48.5 minutes) and 4.9 hours, respectively. In other studies in a small number of patients with advanced cancer, after a single IV injection of 0.1 or 0.25 mg/kg tebustatin, the mean distribution half-life was 17–85 minutes, and the mean terminal elimination half-life was 2.6–15 hours. In patients with renal impairment (creatinine clearance less than 60 ml/min), the mean half-life of tebustatin was approximately 18 hours.

Toxicity Summary
Pentostatin is a potent adenosine deaminase (ADA) transition state inhibitor, with its activity most pronounced in lymphatic system cells. ADA activity is higher in T cells than in B cells, and higher in T-cell malignancies than in B-cell malignancies. Cytotoxicity resulting from inhibition of adenosine or deoxyadenosine catabolism is thought to be caused by elevated intracellular dATP levels, which can block DNA synthesis by inhibiting ribonucleotide reductase. Intracellular activation leads to the incorporation of dATP into DNA as pseudopurine bases. Furthermore, pentostatin incorporation into RNA also produces cytotoxic effects. Cytotoxicity exhibits cell cycle phase specificity (S phase).

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Toxicity Data>
Mice (intravenous): LD50 122 mg/kg
LD50=128 mg/kg (mice)
Interactions>
Limited data suggest that combination therapy with pentosatine (4 mg/m² every 2 weeks) and fludarabine (primarily 10 mg/m² daily for 4 consecutive days, in 28-day cycles) may be associated with severe and/or fatal pulmonary toxicity (e.g., pneumonia). In one study, 4 out of 6 patients with refractory chronic lymphocytic leukemia receiving combination therapy with pentosatine and allopurinol reported such toxicities.
Although both pentosatine and allopurinol treatments have been associated with the occurrence of rash, limited evidence suggests that concomitant use of these two drugs in patients with refractory hairy cell leukemia does not increase the incidence of rash compared to pentosatine alone. However, other toxicities, including renal or hepatic dysfunction, have been observed in a small number of patients receiving co-treatment with pentostatin and allopurinol. …One patient was reported to have developed fatal hypersensitivity vasculitis while receiving co-treatment with pentostatin and allopurinol; however, a causal relationship with these drugs has not been established. Pentostatin inhibits the degradation of vidarabine and enhances its cytotoxicity in cell cultures and experimentally induced animal models of leukemia. Furthermore, limited data from acute leukemia patients suggest that co-treatment with these drugs may result in higher plasma vidarabine concentrations and/or a longer half-life, and greater toxicity compared to pentostatin alone. Although there have been reports of improvement and/or remission in a small number of patients with acute T-cell lymphoblastic leukemia receiving co-treatment with vidarabine and pentostatin.

[1]. Extracorporeal photopheresis combined with pentostatin in the conditioning regimen for canine hematopoietic cell transplantation does not prevent GVHD. Bone Marrow Transplant. 2014 Sep;49(9):1198-204.

[2]. Cordycepin (3'-deoxyadenosine) pentostatin (deoxycoformycin) combination treatment of mice experimentally infected with Trypanosoma evansi. Parasitology. 2013 Apr;140(5):663-71.

Therapeutic Uses
Antibiotics; Antitumor Drugs; Enzyme Inhibitors; Immunosuppressants Antibiotics, Antitumor Drugs; Enzyme Inhibitors; Immunosuppressants Antitumor Drugs Pentostatin is used to treat hairy cell leukemia (leukemic reticuloendothelial hyperplasia) that has not responded well to interferon-alpha therapy or whose disease has progressed. Pentostatin has been designated an orphan drug by the U.S. Food and Drug Administration (FDA) for the treatment of this disease. Pentostatin can achieve clinically significant tumor regression or disease stabilization (complete or partial remission) in approximately 80–100% of patients with hairy cell leukemia, including previously untreated patients (e.g., patients who have not received splenectomy or other treatments) and patients who have not responded to splenectomy and/or other medications (e.g., interferon, antitumor drugs) (e.g., patients whose disease has progressed). In clinical studies of patients with interferon-alpha refractory hairy cell leukemia, complete remission with pentostatin was generally defined as: clearance of hairy cells in peripheral blood and bone marrow; normalization of organ and lymphadenopathy; and restoration of hemoglobin concentration to at least 12 g/dL, platelet count to at least 100,000/mm³, and granulocyte count to at least 1500/mm³. Partial remission was defined as a reduction of more than 50% in the number of hairy cells in peripheral blood and bone marrow, and a reduction of more than 50% in organ and lymphadenopathy; the hematological parameters for partial remission were the same as those for complete remission. In a limited number of patients receiving pentostatin 4 mg/m² intravenously every two weeks for 3 months, an overall complete remission rate of 58% and a partial remission rate of 28% were reported; patients in remission continued treatment for 3–9 months. The median duration of remission in these patients was reported to be 4.7 months (range: 2.9–24.1 months). According to reports, in two clinical studies of patients with hairy cell leukemia, the median duration of response to pentostatin treatment exceeded 7.7 months and 15.2 months, respectively, with approximately 15-20% of patients experiencing initial remission relapsing. For patients whose disease has progressed after splenectomy, pentostatin is often considered an alternative to interferon-alpha or a second-line treatment option for interferon-refractory disease due to the greater experience with interferon-alpha to date. However, the merits of these two drugs or other therapies remain to be determined. For more complete data on the therapeutic uses of pentostatin (out of 10), please visit the HSDB record page.
Drug Warnings
Pentostatin is a toxic drug with a low therapeutic index; a therapeutic response is unlikely to occur without the presence of toxicity. This drug must be used under the continuous supervision of a physician experienced in cytotoxic drug therapy. Most (but not all) adverse reactions to pentostatin are reversible if detected promptly. If a serious adverse reaction occurs during pentostatin treatment, the drug should be discontinued or the dose reduced, and appropriate measures should be taken. If pentostatin needs to be restarted, caution should be exercised, and the need for continued use should be carefully considered, with an awareness of the possibility of relapse into toxicity. Patients with poor performance status appear to be more prone to pentostatin toxicity; therefore, this drug should only be used if the expected benefit outweighs the potential risks. Hematological function must be monitored frequently and carefully during and after pentostatin treatment, especially in the first few cycles in patients at high risk of bone marrow suppression (e.g., patients with hairy cell leukemia). Initiating pentostatin treatment in these patients may result in severe bone marrow suppression. If severe granulocytopenia persists beyond the initial cycle of pentostatin treatment, the patient should be examined, including a bone marrow examination, to determine their disease status. Furthermore, for patients with this type of leukemia, the presence of hairy cells in peripheral blood should be monitored regularly to assess the patient's response to treatment. Bone marrow aspiration and biopsy may be necessary every 2 to 3 months. Patients receiving pentostatin treatment should be closely monitored for signs of non-hematological (e.g., neurological) toxicity. If serious adverse reactions occur, treatment should be discontinued and appropriate corrective measures should be taken as directed. If a patient presents with evidence of neurotoxicity, pentostatin treatment should be temporarily discontinued or stopped. For more complete data on drug warnings for pentostatin (11 in total), please visit the HSDB records page. Pharmacodynamics: Pentostatin is an antitumor antimetabolite used to treat various types of leukemia, including acute non-lymphocytic leukemia and hairy cell leukemia. Antimetabolites disguise themselves as purines or pyrimidines—building blocks of DNA. They prevent these substances from being incorporated into DNA during the “S” phase of the cell cycle, thus inhibiting normal cell development and division. It is a 6-thiopurine analogue of the naturally occurring purine bases hypoxanthine and guanine. Intracellular activation leads to its incorporation into DNA as a pseudopurine base. Furthermore, it can be incorporated into RNA, resulting in cytotoxic effects. Cytotoxicity is cell cycle phase-specific (S phase). Pentostatin is a purine analogue that induces T cell apoptosis by inhibiting adenosine deaminase. It has been used in pretreatment protocols for hematopoietic stem cell transplantation to deplete host T cells and prevent graft rejection and graft-versus-host disease (GVHD). In this study, pentostatin combined with ECP did not prevent severe acute GVHD in a canine DLA-mismatched transplantation model. [1]

C11H16N4O4

268.2691

268.117

C, 49.25; H, 6.01; N, 20.88; O, 23.86

53910-25-1

439693

White crystals from methanol/water
White to off-white solid

1.8±0.1 g/cm3

673.1±65.0 °C at 760 mmHg

220-225ºC

360.9±34.3 °C

0.0±2.2 mmHg at 25°C

1.793

-2.16

4

6

2

19

356

4

O1[C@]([H])(C([H])([H])O[H])[C@]([H])(C([H])([H])[C@]1([H])N1C([H])=NC2[C@@]([H])(C([H])([H])N=C([H])N([H])C1=2)O[H])O[H]

FPVKHBSQESCIEP-KDXUFGMBSA-N

InChI=1S/C11H16N4O4/c16-3-8-6(17)1-9(19-8)15-5-14-10-7(18)2-12-4-13-11(10)15/h4-9,16-18H,1-3H2,(H,12,13)/t6-,7+,8+,9-/m0/s1

(R)-3-((2S,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol

Deoxycoformycin; CI825; CI-825; Deoxycoformycin; Nipent; 53910-25-1; 2'-Deoxycoformycin; PD-ADI; Pentostatina; Pentostatine; CI 825; PD81565; PD-81565; PD 81565; covidarabine; deoxycoformycin; pentostatine. brand name: Nipent.

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)

H2O : ~100 mg/mL (~372.76 mM)
DMSO : ≥ 50 mg/mL (~186.38 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.75 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.75 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.75 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 of corn oil and mix evenly.

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