Human PNP (IC50 = 1.19 nM); Mouse PNP (IC50 = 0.48 nM); Rat PNP (IC50 = 1.24 nM); Monkey PNP (IC50 = 0.66 nM); Dog PNP (IC50 = 1.57 nM)[2]

Proliferation of RPMI-8226, MOLT-4, and 5T33MM cells was partially reduced by forodesine (10-30 μM) treatment for 24 and 48 hours [1]. MM cells are unaffected by forodesine (10-30 μM; 24 and 48 hours; RPMI-8226, MOLT-4, and 5T33MM cells); nevertheless, forodesine decreases the percentage of viable cells in MOLT-4 cells by 40%[1]. Forodesine (BCX-1777) suppresses the proliferation of human lymphocytes stimulated by various drugs, mixed lymphocyte reaction (MLR), and interleukin-2 (IL-2). IC50 value of phytohemagglutinin (PHA) is less than 0.1-0.38 μM [2].

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Multiple myeloma (MM) is the second most commonly diagnosed hematological malignancy, characterized by a monoclonal proliferation of malignant cells in the bone marrow. Despite recent advances in treatment strategies, MM remains incurable and new therapeutical targets are needed. Recently forodesine, a purine nucleoside phosphorylase inhibitor, was found to induce apoptosis in leukemic cells of chronic lymphocytic leukemia patients by increasing the dGTP levels. We therefore tested whether forodesine was able to inhibit proliferation and/or induce apoptosis in both murine and human MM cells through a similar pathway. We found that after 48 hours of treatment with forodesine there was a slight dGTP increase in 5T33MM and RPMI-8226 MM cells associated with partial inhibition of proliferation and a limited induction of apoptosis. When investigating the pathways leading to cell cycle arrest and apoptosis, we observed an upregulation of p27, caspase 3, and BIM. We can conclude that forodesine has some effects on MM cells but not as impressive as the known effects in leukemic cells. Forodesine might be however potentiating towards other established cytotoxic drugs in MM [2].

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PNP enzyme inhibition [2]
BCX-1777 inhibited human, mouse, rat, monkey and dog PNP very effectively. The IC50 values ranged from 0.48 to 1.57 nM (Table 1). The maximum inhibitory effect of about 90% to 100% was obtained at 3–10 nM for all these enzymes, indicating that BCX-1777 is a potent inhibitor of human, mouse, rat, monkey and dog PNP.

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Inhibition of IL-2, MLR- and PHA-stimulated lymphoproliferation of human T-cells by BCX-1777 [2]
We examined the ability of BCX-1777 to inhibit IL-2-induced proliferation of human PBLs from normal donors with results illustrated in Fig. 1A. The addition of BCX-1777 produced a dose-dependent inhibition of the proliferative response to IL-2 in the presence of dGuo (10 μM) with an IC50 of about 0.06 μM. In the absence of dGuo, the IC50 for BCX-1777 was greater than 100 μM. dGuo alone at 10 μM showed no inhibition of proliferation of activated lymphocytes. As an in vitro correlate to allograft rejection, we evaluated the ability of BCX-1777 to inhibit the proliferative responses to human PBLs from four donors to alloantigen stimulation in the mixed lymphocyte reaction (Fig. 1B). As shown in Fig. 1B, BCX-1777 produced dose-dependent inhibition of this response with 50% inhibition occurring at 0.05 μM and 90–100% inhibition occurring at 1 μM of BCX-1777. No significant inhibition occurred up to 100 μM of BCX-1777 in the absence of dGuo. The ability of BCX-1777 to inhibit PHA-stimulated proliferation of PBLs from 4 normal donors was also examined (Fig. 1C). BCX-1777 in the presence of 10 μM dGuo demonstrated a dose-dependent inhibition of PHA-stimulated cells with an IC50 of 0.387 μM. In the absence of dGuo, no significant inhibition was observed. Human lymphocytes stimulated with IL-2 and PHA showed a greater than 20-fold increase in thymidine incorporation compared to unstimulated lymphocytes whereas with the MLR, the increase in thymidine incorporation was greater than 10-fold compared to unstimulated lymphocytes.

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Nucleotide pools in T-cells [2]
IL-2 stimulated PBLs in the presence of BCX-1777 and 10 μM dGuo showed an accumulation of dGTP (Table 2). There was approximately a 4.5-fold increase in dGTP at 0.1 μM BCX-1777 and a 7.5-fold increase in dGTP at 1 μM BCX-1777 compared to the control sample. Increase in dGTP parallels the inhibition of proliferation of IL-2 stimulated T-cells. A 4.5-fold increase in dGTP produced 52% inhibition and a 7.5-fold increase in dGTP gave 76% inhibition. Deoxycytidine inhibits the kinase-mediated conversion of dGuo to dGMP. To further evaluate the role of dGTP in T-cell inhibition both proliferation and nucleotide analysis were performed in the presence of BCX-1777, dGuo and deoxycytidine. Deoxycytidine (3 μM) partially reversed the inhibition of T-cell proliferation caused by BCX-1777 and dGuo. Determination of the nucleotide pools of the T-cells incubated with BCX-1777, dGuo and dCyt showed a decrease in dGTP compared to cells incubated with BCX-1777 and dGuo. Higher concentrations (≥10 μM) of deoxycytidine was not used as it caused inhibition of proliferation of T-cells.

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IL-2 stimulated proliferation of mouse splenocytes [2]
To evaluate the specificity of BCX-1777, we evaluated the ability of BCX-1777 to inhibit IL-2-induced proliferation of mouse splenocytes both in the presence and absence of dGuo (10 μM). No significant inhibition of IL-2-induced proliferation of the mouse splenocytes was observed at concentrations as high as 100 μM of BCX-1777 both in the presence and absence of dGuo (10 μM). Unlike human lymphocytes, mouse splenocytes showed no accumulation of dGTP in the presence of BCX-1777 (1 μM) and dGuo (10 μM).

Forodesine (BCX-1777) has excellent oral bioavailability (63%) in mice [2]. Forodesine elevates dGuo to approximately 5 μM at a single dose of 10 mg/kg in mice [2]. In the human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL-SCID) mice model, forodesine can effectively enhance life span by more than 2 times [2].

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Mouse T-cell models [2]
The in vivo efficacy of Forodesine/BCX-1777 was evaluated in the DNFB-induced contact-delayed hypersensitivity mouse ear edema model. The delayed hypersensitivity reaction is mediated by T-cells. BCX-1777 was not effective in reducing ear edema in mice at the 30-mg/kg dose. Cyclosporin (50 mg/kg) was used as a positive control and showed 58% reduction in ear swelling. BCX-1777 was also evaluated in the graft vs. host (GVH)-induced splenomegaly mouse model. Experimental graft vs. host reaction (GVHR) can be induced in adult F1 mice by injecting parental strain T-cells which respond to allogenic MHC antigens and induce symptoms of systemic GVHR. The GVHR is similar to a DTH reaction mediated by T-cells reacting against cellular surface antigens of recipient tissues. Cyclosporin significantly suppressed GVH-induced splenomegaly by 53%, whereas BCX-1777 was ineffective in this mouse T-cell model.

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Hu-PBL SCID mouse model [2]
Mosier et al. reported transplantation of human PBLs into SCID mice to construct hu-PBL SCID mice and suggested it as a useful model for the study of normal human immune function. Recently, Sandhu et al. [19] described a novel protocol for the efficient engraftment of hu-PBLs into SCID mice. The hu-PBLs engrafted into these SCID mice were shown to be functional by the induction of human primary response. The engraftment efficiency of this protocol is so high that almost 100% of the hu-PBL SCID mice succumb to xenogeneic graft vs. host disease (XGVHD) in less than 4 weeks after injection of human PBLs. The hu-PBL engraftment in SCID mice induces severe XGVHD with concomitant weight loss, diarrhea, hunched back and ruffled fur. These mice eventually die due to human lymphocyte infiltration of SCID mice tissues. To determine if Forodesine/BCX-1777 inhibits the proliferation of human T-cells in mice and affects the life span of these mice, the SCID mice were treated with BCX-1777 orally at 20 mg/kg/day (b.i.d.) starting 5 days before engraftment with human PBLs. Pretreatment of the animals with drug was performed to keep the dGuo levels elevated. Dosing continued at the same schedule until the animal died. Human lymphocytes from three donors were engrafted into three groups of SCID mice (n=10). The experimental animals (n=5) were treated with the drug and the control animals (n=5) were treated with vehicle (0.5% CMC). Fig. 5 illustrates a representative profile of mice alive vs. days from one of these experiments. In one experiment, BCX-1777-treated mice lived to an average of 30 days vs. 15 days in the control group. In the other two experiments, BCX-1777-treated mice lived for 39 and 43 days vs. 17 and 20 days, respectively, in the untreated group. Therefore, BCX-1777 prolonged the life span of these mice 2-fold or more in each experiment (Table 3).

Patients with purine nucleoside phosphorylase (PNP) deficiency present a selective T-cell immunodeficiency. Inhibitors of PNP are, therefore, of interest as potential T-cell selective immunosuppressive agents. BCX-1777 is a potent inhibitor of PNP from various species including human, mouse, rat, monkey and dog, with IC50 values ranging from 0.48 to 1.57 nM. BCX-1777, in the presence of 2'-deoxyguanosine (dGuo, 3-10 microM), inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction (MLR) and phytohemagglutinin (PHA) (IC50 values < 0.1-0.38 microM). BCX-1777 is a 10-100-fold more potent inhibitor of human lymphocyte proliferation than other known PNP inhibitors like PD141955 and BCX-34. Nucleotide analysis of human lymphocytes indicate that inhibition of proliferation by BCX-1777 correlates with dGTP levels in the cells[2].

Cell proliferation assay[1]
Cell Types: human RPMI-8226, human MOLT-4 (T-ALL) cells, 5T33MM (multiple myeloma, MM)
Tested Concentrations: 10 μM, 20 μM, 30 μM
Incubation Duration: 24 and 48 Hourly
Experimental Results: After 48 hrs (hours), MOLT-4 cell proliferation was completely blocked, and 5T33MM cell proliferation was diminished by 15%.

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Apoptosis analysis[1]
Cell Types: human RPMI-8226, human MOLT-4 (T-ALL) cells, 5T33MM (multiple myeloma, MM)
Tested Concentrations: 10 μM, 20 μM, 30 μM
Incubation Duration: 24 and 48 hrs (hours)
Experimental Results: Limited induction of apoptosis.

BCX-1777 has excellent oral bioavailability (63%) in mice. At a single dose of 10 mg/kg in mice, BCX-1777 elevates dGuo to approximately 5 microM. BCX-1777 was not effective in mouse T-cell models such as delayed type hypersensitivity (DTH) and splenomegaly because mouse T-cells do not accumulate dGTP as do human T-cells. However, in the human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL-SCID) mouse model, BCX-1777 was effective in prolonging the life span 2-fold or more. This is the first known example of a PNP inhibitor that elevates dGuo in mice similar to the levels observed in PNP-deficient patients. Furthermore, these dGuo levels are also required for in vitro T-cell inhibition by BCX-1777. Thus, BCX-1777 represents a novel class of selective immunosuppressive agents that could have therapeutic utility in various T-cell disorders.[2]

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Oral bioavailability and in vivo pharmacologic activity [2]
Groups of four female mice (Balb/c) received a single oral or intravenous dose of the drug in sterile saline. At various time points, the mice were anesthetized (inhalation anesthesia) and bled through the retro-orbital sinus. For each time point, different sets of mice were used. The blood was centrifuged and plasma was collected and stored at −20°C until analysis. Animals were observed for 48 h for mortality and morbidity. Plasma drug levels were determined using reverse phase HPLC analysis and the quantitation limit was 0.5 μM. Deoxyguanosine levels were determined by reverse-phase HPLC and the quantitation limit for this analysis was 0.75 μM. Oral bioavailability was calculated using non-compartmental model and winnonlin software.

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Mouse T-cell models [2]
Delayed type hypersensitivity (DTH) in mice was performed as described by Braida and Knop and Walsh et al. Briefly, mice were sensitized with 25 μl of 0.5% 2,4-dinitrofluorobenzene (DNFB) in acetone/olive oil (4:1), which was painted onto the shaved abdominal skin on days 0 and 1 (1 h after dosing). Mice were dosed once a day (q.d.) with either vehicle, Forodesine/BCX-1777 (30 mg/kg, orally) or cyclosporin A (50 mg/kg, intraperitoneally) for 6 days. On day 5, the right ears of mice were painted with 20 μl of 0.3% DNFB in acetone/olive oil (4:1), and 24 h later, the thickness of the ears were measured with a vernier caliper.
The graft vs. host (GVH) reaction was evaluated in mice by measuring the degree of splenomegaly induced by the intraperitoneal injection of male hybrid mice with splenocytes from the parental strain (C57BL/6) as described by Roudebush and Bryant. Briefly, spleens were removed aseptically from C57BL/6 mice and dissociated in polypropylene mesh screens. After lysis with an ammonium chloride buffer, splenocytes were washed twice in modified HBSS and were injected i.p. (2×108 cells/ml) in hybrid mice in a volume of 0.5 ml/mouse on day 0. Hybrid mice were dosed with BCX-1777 (30 mg/kg, orally), or cyclosporin A (50 mg/kg, i.p.) for 10 days; controls received HBSS or parental spleen injections at day 0 and were dosed with vehicle either i.p. or orally. Spleens were removed and weighed and the results were recorded as a ratio of spleen weight to body weight.

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Hu-PBL SCID mouse model [2]
The engraftment of human peripheral blood lymphocytes (hu-PBLs) in mice with severe combined immunodeficiency (SCID) was performed as described by Sandhu et al. SCID mice were pretreated 1 day prior to hu-PBL injection with a single dose of anti-ASGM1 antibodies. Anti-ASGM1 consists of rabbit polyclonal antibodies, which recognize murine NK cells, and depletes NK activity. Immediately before hu-PBL engraftment, SCID mice were irradiated. A dose of 3 Gy gamma-radiation was administered from a 137Cs source. Human lymphocytes for engraftment in SCID mice were isolated from buffy coats or from whole blood of volunteer donors. The hu-PBLs were isolated by Ficoll–Hypaque centrifugation and injected i.p. (3.0–5.0×107 PBLs/mouse) into SCID mice under sterile conditions. The experimental animals were pretreated for 5 days with Forodesine/BCX-1777 (20 mg/kg/day, b.i.d.) in 0.5% carboxymethylcellulose (CMC) to elevate the dGuo levels. Control animals were treated with 0.5% CMC. The life span of the control and experimental animals were compared.

Oral bioavailability and in vivo pharmacologic activity [2]
The results of oral and i.v. dosing on plasma levels of BCX-1777 are shown in Fig. 3A. BCX-1777 is absorbed rapidly after oral administration and reaches a Cmax of about 3 μM in half an hour. At 3 h, plasma drug levels were 1.1 μM and at 6 h drug was undetectable. The calculated oral bioavailability of BCX-1777 in mice was 63%.
Increases in plasma dGuo levels were monitored after oral administration of BCX-1777 (10 mg/kg) in mice (Fig. 3B). dGuo increased with time and a Cmax of approximately 5 μM is achieved at 3 h. The level at 6 h is about 2.2 μM. dGuo was not detectable at 24 h. Plasma dGuo levels do not exactly parallel the drug levels. There is a delay in time at which the Cmax of dGuo is achieved (3 h) compared to the time at which the Cmax of drug is achieved (∼30 min). Plasma dGuo and drug levels in mice were monitored 3 h after oral administration of various doses of BCX-1777 (Fig. 4). BCX-1777 produced a dose-related increase in dGuo levels up to the 10 mg/kg dose. Beyond that, there were no further increases in dGuo levels. However, plasma drug levels were elevated with an increase in doses of BCX-1777 up to 100 mg/kg.

[1]. The effects of forodesine in murine and human multiple myeloma cells. Adv Hematol. 2010;2010:131895.

[2]. Purine nucleoside phosphorylase inhibitor BCX-1777 (Immucillin-H)--a novel potent and orally active immunosuppressive agent. Int Immunopharmacol. 2001 Jun;1(6):1199-210.

Immucillin H is a pyrrolopyrimidine and a dihydroxypyrrolidine.
Forodesine is a highly potent, orally active, rationally designed PNP inhibitor that has shown activity in preclinical studies with malignant cells and clinical utility against T-cell acute lymphoblastic leukemia and cutaneous T-cell lymphoma. Additional preliminary findings support its use for the management of some B-cell malignancies.
Forodesine is a transition-state analog inhibitor of purine nucleoside phosphorylase (PNP), with potential antineoplastic activity. Upon administration, forodesine preferentially binds to and inhibits PNP, resulting in the accumulation of deoxyguanosine triphosphate and the subsequent inhibition of the enzyme ribonucleoside diphosphate reductase and DNA synthesis. This agent selectively causes apoptosis in stimulated or malignant T-lymphocytes.

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Drug Indication
Investigated for use/treatment in lymphoma (non-hodgkin's) and leukemia (lymphoid).

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Patients with purine nucleoside phosphorylase (PNP) deficiency present a selective T-cell immunodeficiency. Inhibitors of PNP are, therefore, of interest as potential T-cell selective immunosuppressive agents. BCX-1777 is a potent inhibitor of PNP from various species including human, mouse, rat, monkey and dog, with IC50 values ranging from 0.48 to 1.57 nM. BCX-1777, in the presence of 2'-deoxyguanosine (dGuo, 3-10 microM), inhibits human lymphocyte proliferation activated by various agents such as interleukin-2 (IL-2), mixed lymphocyte reaction (MLR) and phytohemagglutinin (PHA) (IC50 values < 0.1-0.38 microM). BCX-1777 is a 10-100-fold more potent inhibitor of human lymphocyte proliferation than other known PNP inhibitors like PD141955 and BCX-34. Nucleotide analysis of human lymphocytes indicate that inhibition of proliferation by BCX-1777 correlates with dGTP levels in the cells. BCX-1777 has excellent oral bioavailability (63%) in mice. At a single dose of 10 mg/kg in mice, BCX-1777 elevates dGuo to approximately 5 microM. BCX-1777 was not effective in mouse T-cell models such as delayed type hypersensitivity (DTH) and splenomegaly because mouse T-cells do not accumulate dGTP as do human T-cells. However, in the human peripheral blood lymphocyte severe combined immunodeficiency (hu-PBL-SCID) mouse model, BCX-1777 was effective in prolonging the life span 2-fold or more. This is the first known example of a PNP inhibitor that elevates dGuo in mice similar to the levels observed in PNP-deficient patients. Furthermore, these dGuo levels are also required for in vitro T-cell inhibition by BCX-1777. Thus, BCX-1777 represents a novel class of selective immunosuppressive agents that could have therapeutic utility in various T-cell disorders. [2]
In summary, BCX-1777 is a potent inhibitor of PNP enzyme and human T-cell proliferation. BCX-1777 is orally bioavailable in mice and can achieve maximal inhibition of PNP, thus producing elevated dGuo levels. This elevation of dGuo is found in PNP-deficient patients and proven to be necessary for T-cell inhibition. Using the hu-PBL SCID mouse model, in vivo efficacy of BCX-1777 was demonstrated. In view of the in vitro and in vivo data, we conclude that BCX-1777 is a novel, orally active, T-cell selective immunosuppressive agent that could be used for the treatment of T-cell proliferative disorders.[2]

C11H14N4O4

266.25326

266.102

C, 49.62; H, 5.30; N, 21.04; O, 24.04

209799-67-7

Forodesine hydrochloride;284490-13-7

135409409

Light brown to gray solid

2.01

613.5ºC at 760mmHg

324.8ºC

6.61E-16mmHg at 25°C

1.894

-2.3

6

6

2

19

404

4

O=C1C(NC=C2[C@@H]3N[C@H](CO)[C@@H](O)[C@H]3O)=C2NC=N1

IWKXDMQDITUYRK-KUBHLMPHSA-N

InChI=1S/C11H14N4O4/c16-2-5-9(17)10(18)7(15-5)4-1-12-8-6(4)13-3-14-11(8)19/h1,3,5,7,9-10,12,15-18H,2H2,(H,13,14,19)/t5-,7+,9-,10+/m1/s1

7-[(2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidin-2-yl]-3,5-dihydropyrrolo[3,2-d]pyrimidin-4-one

Forodesine; Immucillin H; 209799-67-7; Fodosine; Immucillin-H; mundesine; BCX-1777; 1,4-dideoxy-4-aza-1-(s)-(9-deazahypoxanthin-9-yl)-d-ribitol; Immucillin H; NTR 001; NTR-001; NTR001

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 : ~100 mg/mL (~375.59 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.39 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 (9.39 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 (9.39 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.)