Pharmacological and clinical studies on purine nucleoside analogs--new anticancer agents.

More recently, three novel purine nucleoside analogs, including clofarabine, nelarabine and immucillin H, have been introduced into clinical trials. These agents have different metabolic properties, novel mechanism of action, and are undergoing phase I-II clinical studies for the treatment of hematopoietic malignancies. Pharmacology and anticancer activity of PNA are the subjects of this review.

Purine nucleoside analogues for the treatment of hematological malignancies: pharmacology and clinical applications.

The purine nucleoside analogues (PNAs), fludarabine (FA), 2-CdA (2-chlorodeoxyadenosine, 2-CdA) and pentostatin (2'-deoxycoformycin, DCF) represent a group of cytotoxic agents with high activity in lymphoid and myeloid malignancies. PNAs share similar chemical structure and mechanism of action. Several mechanisms could be responsible for their cytotoxicity both in proliferating and quiescent cells, such as inhibition of DNA synthesis, inhibition of DNA repair and accumulation of DNA strand breaks. Induction of apoptosis through the mitochondrial pathway, direct binding to apoptosome or modulation of p53 expression all lead to apoptosis, which is the main end-point of PNA action. However, individual PNAs exhibit significant differences, especially in their interaction with enzymes involved in adenosine and deoxyadenosine metabolism. Synergistic interactions between PNAs and other cytotoxic agents (alkylating agents, anthracycline antitumor antibiotics, cytarabine, monoclonal antibodies) have been demonstrated in both preclinical and clinical studies. PNAs are highly effective in chronic lymphoid leukemias and low grade B- and T-cell non-Hodgkin's lymphomas, including Waldenström's macroglobulinemia. DCF and 2-CdA are currently the drugs of choice in hairy cell leukemia. Moreover, clinical studies have confirmed the efficacy of PNAs alone or in combination protocols in the treatment of acute myeloid leukemia and myelodysplastic syndromes. Finally, PNAs, especially FA, play an important role in non-myeloablative conditioning regimens for allogenic stem cell transplantation in high-risk patients. The toxicity profiles of PNAs are similar for all agents and consist mainly of dose-limiting myelotoxicity and prolonged immunosuppression. Three other compounds: clofarabine, nelarabine and immucillin-H are currently being evaluated clinically.

Influence of gemcitabine (2',2'-difluoro-deoxycytidine) and 2-chlorodeoxyadenosine on growth of normal and leukemic cells in vitro.

The aim of the study was to investigate the influence of gemcitabine (2',2'-difluorodeoxycytidine, dFdC) used alone and in combination with 2-chlorodeoxyadenosine (2-CdA) on the colony growth of normal granulocyte-macrophage progenitor cells (CFU-GM) from 15 hematologically healthy donors as well as on CFU-GM from 15 chronic myelogenous leukemia (CML) patients in semisolid cultures in vitro. dFdC and 2-CdA were used either separately or in the following combinations of concentrations: (1) 0.25 nM of dFdC and 2.5 nM of 2-CdA; (2) 0.5 nM of dFdC and 5 nM of 2-CdA; (3) 1 nM of dFdC and 10 nM of 2-CdA; (4) 2 nM of dFdC and 20 nM of 2-CdA. We observed that both dFdC and 2-CdA used separately inhibited the growth of colonies formed by normal and CML CFU-GM cells in a dose-dependent manner. Moreover, the combined therapy with dFdC and 2-CdA caused statistically significant inhibition of the colony growth in both normal and CML CFU-GM cultures. In addition, the inhibition of CML CFU-GM colony formation was greater and statistically significant in the case of the combined therapy using higher concentrations of these drugs as compared with inhibition of the growth of normal CFU-GM colonies. These observations were the basis for the evaluation of the interaction type between dFdC and 2-CdA. We have shown that dFdC used in a combination with 2-CdA acts in an additive way on normal and CML CFU-GM cells.

The influence of amifostine used alone or in combination with 2-chlorodeoxyadenosine on normal and chronic myelogenous leukemia granulocyte-macrophage progenitor cells in vitro.

We evaluated the influence of amifostine used alone or in combination with 2-chlorodeoxyadenosine (2-CdA) on the colony growth of normal and chronic myeloid leukemia (CML) granulocyte-macrophage progenitor cells (CFU-GM) in semisolid culture in vitro. Amifostine at a concentration of 1 mg/ml was either added directly to the culture medium of normal and CML CFU-GM, or mononuclear cells (MNCs) were first preincubated with amifostine at the same concentration, washed in Iscove's modified Dulbecco minimum essential medium (IDMEM) and then added to the culture medium. Amifostine used alone inhibited the growth of CML CFU-GM colonies to a higher degree than those of normal CFU-GM, but the differences were not statistically significant. Amifostine preincubated with MNCs and used together with the highest concentration of 2-CdA significantly inhibited the colony growth of CML CFU-GM as compared to 2-CdA alone (p<0.05). In contrast, the colony growth inhibition of normal CFU-GM was not significantly lower compared to 2-CdA used alone. Our studies suggest that 2-CdA used together with amifostine is more toxic to leukemic CFU-GM than to their normal counterparts.

The interaction of gemcitabine and cytarabine on murine leukemias L1210 or P388 and on human normal and leukemic cell growth in vitro.

BACKGROUND AND OBJECTIVE: Gemcitabine (dFdC) is a new nucleoside antimetabolite of deoxycytidine that resembles cytarabine (Ara-C) in both its structure and metabolism. Little is known about dFdC efficacy in hematologic malignancies, either as a single drug or in combination with other drugs. In this study we have tried to determine whether the cytotoxic effect of Ara-C can be increased by using it in combined therapy with dFdC. DESIGN AND METHODS: In the in vivo part of our study, mice bearing L1210 or P388 leukemia were treated with dFdC and Ara-C. The drugs were administered alone and in combination according to the following schedules: Ara-C and dFdC at the same time, dFdC before Ara-C, and Ara-C before dFdC. The efficacy of the therapy against leukemia (defined as the increase in lifespan, ILS) was assessed as the percentage of the median survival time (MST) of the treated group (T) in relationship to that of the control group (C): ILS=[(MST(C)/MST(T)) -1]x100. In the in vitro part of our study, normal granulocyte-macrophage colony-forming unit (CFU-GM) cells as well as CFU-GM cells obtained from patients with chronic myeloid leukemia (CML) were incubated either with dFdC or Ara-C alone or with adequate concentrations of a combination of these drugs. RESULTS: The in vivo experiment revealed that in both leukemias tested, combined therapy with dFdC given before Ara-C and dFdC given at the same time with Ara-C were more effective than monotherapy with either dFdC or Ara-C. The other treatment schedule (Ara-C before dFdC) did not significantly prolong the survival time of the treated mice bearing L1210 or P388 leukemia as compared with the treatment with dFdC alone. The in vitro experiments showed that dFdC used together with Ara-C acted additively on normal as well as CML CFU-GM cells. Furthermore, the drugs used jointly inhibited the growth of colonies formed by CML CFU-GM cells to a significantly higher degree than normal CFU-GM and the differences were statistically significant in the case of the combination of highest concentrations. INTERPRETATION AND CONCLUSIONS: Gemcitabine increased the activity of Ara-C. As these agents incorporate into DNA blocking chain elongation, and moreover, dFdC influences the cytotoxicity of Ara-C, our results could be explained by the drugs acting at these levels. dFdC used jointly with Ara-C may have an important clinical implication in the treatment of CML and other hematologic malignancies in future.

Pituitary-adrenocortical responses to the chronic administration of granulocyte colony-stimulating factor in rats.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor, but it may play a role in the regulation of the neuroendocrine system activity. Only few data are available about its possible influence on the pituitary gland. We have recently reported an acute stimulatory effect of G-CSF (and of GM-CSF) on adrenocorticotropic hormone (ACTH) secretion in rats in vivo. The purpose of the present study was to evaluate whether chronic administration of G-CSF affects ACTH and corticosterone secretion and growth processes of the rat anterior pituitary gland and adrenal cortex in vivo. We have demonstrated that G-CSF (at a dose of 10.0 microg/kg body weight (BW)) injected s.c. once daily (for 7 days), stimulated both ACTH and corticosterone secretion. Simultaneously, G-CSF treatment did not change the total anterior pituitary cell proliferation as revealed by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). On the other hand, proliferative activity of corticotrophs, detected in the sections of the anterior pituitary using double-labeling. was significantly increased after treatment with G-CSF. Moreover, this growth factor induced an increase in the proliferation ratio in the entire adrenal equatorial section. These findings suggest an involvement of G-CSF in the regulation of pituitary-adrenal axis and support the hypothesis of bidirectional associations between the immune system and the endocrine glands.

Influence of granulocyte-macrophage colony stimulating factor on pituitary-adrenal axis (PAA) in rats in vivo.

We have studied the in vivo influence of granulocyte-macrophage colony stimulating factor (GM-CSF) on blood plasma concentration of adrenocorticotropic hormone (ACTH) and corticosterone in Wistar rats. The administration of 10 micrograms/kg b.w. GM-CSF at 45 (P < 0.01), 90 (P < 0.01) and at 45 (P < 0.001), 90 (P < 0.001) and 180 min (P < 0.001) increased the secretion of ACTH and corticosterone, respectively. Prolonged administration of 10 micrograms/kg b.w. of GM-CSF increased the ACTH (P < 0.001) and corticosterone (P < 0.001) concentration in blood plasma. We have also found that chronic treatment with 10 micrograms/kg b.w. of GM-CSF increased the proliferative activity of corticotrophs (P < 0.05), but it did not significantly change the total cell proliferation in the anterior pituitary gland. Moreover, this cytokine increased cell proliferation of the adrenal cortex (P < 0.001). These experiments suggest that GM-CSF activates the pituitary-adrenal axis and support the hypothesis of bidirectional associations between the immune and neuroendocrine systems.

Serum levels of IL-6 type cytokines and soluble IL-6 receptors in active B-cell chronic lymphocytic leukemia and in cladribine induced remission.

We have investigated the serum concentrations of interleukin-6 (IL-6) and two IL-6 family cytokines-oncostatin M (OSM) and leukemia inhibitory factor (LIF)-in 63 patients with B-cell chronic lymphocytic leukemia (B-CLL) and 17 healthy controls using the enzyme-linked immunosorbent assay (ELISA) method. Simultaneously, we measured the serum levels of the soluble forms of two subunits of the IL-6 receptor complex-ligand binding glycoprotein 80 (sIL-6R) and glycoprotein 130 (sgp130). The cytokines and receptors were evaluated in 25 untreated patients and 38 patients treated with cladribine (2-CdA), as well as in 17 healthy controls. We have correlated the serum levels of these proteins with Rai's clinical stage of the disease, the response to 2-CdA treatment and some hematological parameters. We have also evaluated the correlation of the IL-6 serum level with the concentration of OSM and IL-6 soluble receptors. IL-6 was measurable in 62/63 (98.4%), OSM in 20/25 (80%) of untreated and 14/38 (37.8%) of the treated patients. sIL-6R and sgp130 were detectable in all 63 patients and LIF in none of the CLL patients. IL-6 serum level in untreated patients was not significantly different as compared to its concentration in the control group (P>0.05). However, in the patients treated with 2-CdA the IL-6 level was significantly lower (P<0.02), and the lowest concentration was found in the patients with complete remission (CR; median 1.4pg/ml; P<0.02). The concentration of sIL-6R was significantly higher in untreated (median 61.8 ng/ml) and treated (median 50.1 ng/ml) CLL patients when compared to normal persons (median 41.2 ng/ml; P=0.04; P<0.001, respectively). There was no difference between the sIL-6R levels in the patients with CR and the healthy controls. In non-responders sIL-6R concentration was the highest and similar to its level in the untreated patients. OSM level was higher in the untreated patients (median 1.8pg/ml) than in the normal controls (median 0.0pg/ml; P<0.001) and in the CR patients (median 0.0pg/ml; P<0.03). The serum concentration of sgp130 was similar in the untreated (median 480 pg/ml) and treated (median 470 pg/ml) patients, as well as in the healthy persons (median 420 pg/ml; P>0.05). We have found significant positive correlation between the levels of sIL-6R and the lymphocytes count in CLL patients (p=0.423; P<0.001). In addition, sIL-6R and OSM serum concentrations correlated also with CLL Rai stage. In conclusion, the serum level of IL-6, OSM and sIL-6R, but not LIF and sgp130, are useful indicators of CLL activity.

The influence of recombinant human tumor necrosis factor alpha and its muteins used alone or in combination with 2-chlorodeoxyadenosine on normal and leukemic hematopoiesis in vitro.

We investigated the influence of TNF alpha and its muteins III, V and VI on the colony growth of normal and CML CFU-GM cells as well as on CFU-L clonogenic blasts from AML patients in cultures in vitro. The muteins were constructed using a synthetic cDNA fragment substituting nucleotides coding for the N-terminal amino acids in the chain of native TNF alpha. We observed that TNF alpha and mutein VI inhibited the growth of colonies formed by both types of CFU-GM and CFU-L cells in a greater degree than muteins III and V. In addition, the percentage of colony growth inhibition after the use of mutein VI was the greatest. These observations were the basis for the evaluation of interaction between 2-CdA and TNF alpha and between 2-CdA and mutein VI. We have confirmed that 2-CdA used together with mutein VI acts synergistically on CFU-GM and CFU-L cells, whereas 2-CdA and TNF alpha show the additive effect.

The comparison of 2-chlorodeoxyadenosine (2-Cd A) in combination with interferon alpha (IFN alpha) or interferon gamma (IFN gamma) on granulocyte-macrophage progenitor cells (CFU-GM) and clonogenic blasts in (CFU-L) in vitro cultures.

In the present in vitro study we investigated the influence of 2-chlorodeoxyadenosine (2-CdA) and interferon gamma (IFNgamma) on the clonal growth of granulocyte macrophage progenitor (CFU-GM) cells from 10 normal individuals and from 10 patients with chronic myeloid leukemia (CML) and on CFU-L blasts from 10 patients with acute myeloid leukemia (AML). 2-CdA was added to the culture medium at 20nM/l, 40nM/l and 80nM/l concentrations and IFNgamma at concentrations of 10(2)U/ml, 10(3)U/ml and 10(4)U/ml. Both agents were used alone and in combination with these different concentrations. We observed a decrease in the number of colonies formed by CML CFU-GM as well as by AML CFU-L in a dose-dependent manner. The drugs used alone inhibited to a higher degree the growth of CML than of normal CFU-GM progenitors. 2-CdA and IFN gamma showed the greatest additive effect on the growth of CFU-L blasts at the concentrations of 80nM/l and 10(4)U/ml, respectively. We compared our results with previous in vitro studies in which we have demonstrated the synergistic inhibitory effect of 2-CdA and IFN alpha on both normal and leukemic hematopoiesis. We suggest that the greatest inhibitory effect is observed when 2-CdA and IFN alpha at the highest concentration are added together to the culture of AML CFU-L blasts.

Interaction of 2-chlorodeoxyadenosine in combination either with interferons or recombinant human tumor necrosis factor alpha on myeloid progenitor cells in vitro.

The objective of the present study was to investigate the interactions of 2-chlorodeoxyadenosine (2-CdA) with interferon alpha or gamma (IFN-alpha, IFN-gamma), as well as between 2-CdA and recombinant human tumor necrosis factor alpha (rhTNF-alpha), on the clonal growth of granulocyte-macrophage progenitor cells (CFU-GM) from patients with chronic myeloid leukemia (CML) and on clonogenic leukemia blasts (CFU-L) from acute myeloid leukemia (AML) patients. Progenitor cell culture in semisolid medium in vitro was applied and the percentage of colony growth inhibition was evaluated. The use of 2-CdA either with IFN-alpha or IFN-gamma and 2-CdA with TNF-alpha was found to inhibit, in a dose dependent manner, the growth of colonies formed by hematopoietic precursor cells from CML and AML patients as well as from normal individuals, with the greatest effect being observed after the use of 2-CdA and IFN-alpha at their highest concentrations.

The interaction of 2-chlorodeoxyadenosine (2-CDA) and interferon alpha (IFN-alpha) on normal and myeloid leukemia hematopoiesis in vitro.

The influence of 2-CDA and interferon alpha (IFN-alpha) on the CFU-GM cells from ten normal individuals as well as on the CFU-GM from ten patients with chronic myeloid leukemia (CML) and on clonogenic leukemic blasts (CFU-L) from ten patients with acute myeloid leukemia (AML) in semi-solid cultures in vitro was investigated. The agents were added to the cultures alone and in combinations at concentrations of 20, 40 and 80 nM/l of 2-CDA and 10(2), 10(3) and 10(4) U/ml of IFN-alpha. A dose-dependent growth inhibition of CFU-GM as well as of CFU-L was observed. The inhibitory effect of both drugs used alone was significantly greater on the CFU-GM cells from patients with CML than on cells from normal donors. However, 2-CDA and IFN-alpha used together showed greater additive effect on the CFU-L than on the CFU-GM from normal donors and from CML patients.

Comparison of tin and lead toxic action on erythropoietic system in blood and bone marrow of rabbits.

The aim of this work was to assess and compare morphological changes in blood and bone marrow of rabbits after per os (po) or intraperitoneal (ip) administration of equimolar doses of tin or lead. The experiment was performed on female rabbits that were divided into four groups of six animals each, and received stannous chloride SnCl2 x 2 H2O (Merck) or lead acetate Pb(CH3COO)2 (POCh Gliwice) in equimolar doses (ip--17/microM/kg) or per os (po--85/microM/kg). Group I was administered SnCl2 ip at the dose of 2 mg Sn/kg every day for 3 mo, group II Pb(CH3COO)2 ip at a dose of 3.5 mg Pb/kg every day for 3 wk, group III po SnCl2 (10 mg Sn/kg), and group IV po Pb(CH3COO)2 (17.5 mg Pb/kg), both for 4 mo. The morphological factors hemoglobin (Hb), hematocrit (Ht), erythrocyte (Ercs), and reticulocyte counts, MCV, MCH, MCHC, and erythropoietic system in bone marrow aspirates with sideroblast count, iron concentration, TIBC, and SI were estimated. Tin caused hemolytic anemia depending on abnormal iron utilization. After ip administration of tin, anemia was observed during the whole time of the study, whereas after po exposure, transient anemia was noticed. It has been proven that the mechanism of toxic action of tin on hematopoietic system is similar to the toxic effect of lead.

The influence of recombinant human tumor necrosis factor (rh-TNF alpha) on granulocyte-macrophage progenitor cells (CFU-GM) and clonogenic leukaemic blasts (CFU-L) in cultures in vitro.

We investigated the influence of recombinant human tumor necrosis factor alfa (rh-TNF alpha) on the clonal growth of CFU-GM from 14 normal individuals and clonogenic blasts (CFU-L) from 16 patients with acute myeloid leukaemia (AML) in semi-solid cultures in vitro. Recombinant human TNF was produced by the Center of Molecular and Macromolecular Studies of the Polish Academy of Sciences (Lódz, Poland) as a lyophylized powder. This factor was added to the culture medium at the concentrations of 10, 100, 1000 U/ml. A dose-dependent growth inhibition of CFU-GM and AML CFU-L was observed. The inhibitory effect of rh-TNF alpha was significantly greater on CFU-L than CFU-GM.

The effect of lithium on haematopoiesis of patients with acute myeloid leukaemia.

In 40 patients receiving induction therapy for acute myeloid leukaemia the influence of lithium carbonate on the regeneration of haematopoiesis was investigated. It was observed that lithium treatment decreases chemotherapy-induced suppression of granulo- and megacariopoiesis. The frequency of remissions and the survival time were significantly higher for the patients receiving lithium if compared with the control group. The number of CFU-GM colonies in vitro after 14-th days of incubation was also statistically higher in the lithium treated patients.

The influence of recombinant human tumor necrosis factor-alpha, alone and in combination with cyclophosphamide or methotrexate, on leukemia L1210 and normal hematopoiesis in mice.

We investigated the influence of rh-TNF administered as a single agent or in combination with CY or MTX on the survival time of mice inoculated with lymphoid leukemia L1210 and the effects of similar treatment on normal hematopoiesis in mice. The MST of rh-TNF--treated mice was longer than that of control animals. The longest survivals were observed in mice treated with 250 and 275 micrograms/kg of rh-TNF. Groups of mice receiving a combination of rh-TNF at doses of 225 or 250 micrograms/kg and MTX lived longer than animals treated with these agents separately. We observed the longest survival time of mice treated with combined administration of rh-TNF at a dose of 250 micrograms/kg and CY, but survival time was not significantly prolonged compared with mice receiving only CY. Additional studies were performed to examine the influence of rh-TNF administered as a single agent or in combination with toxic doses of CY or MTX on the number of granulocytes, lymphocytes, erythrocytes with hematocrit values and hemoglobin concentration, and platelets in peripheral blood, and the number of mononuclear cells as well as multipotential stem cells (CFU-GEMM) in bone marrow. Rh-TNF caused dose-dependent suppression of mononuclear cells and multipotential stem cells in bone marrow. The addition of MTX to rh-TNF caused no enhanced suppression of any of the above mentioned hematological parameters. In contrast, the addition of CY to rh-TNF suppressed erythrocytes and hematocrit values, as compared with rh-TNF alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of lithium chloride on granulocyte-macrophage progenitor cells (CFU-GM) and clonogenic leukaemic blasts (CFU-L) in the cultures in vitro.

The influence of lithium chloride on the proliferation of normal granulocyte-macrophage progenitor cells (CFU-GM) and clonogenic blasts (CFU-L) from patients with acute myeloid leukaemia in the semisolid cultures in vitro was investigated. It was observed that while lithium chloride with the concentration of 2 mmol/l increases the number of CFU-GM colonies, it does not increase the number of CFU-L colonies and clusters. Our studies indicate that lithium salts can be used for the treatment of patients with acute myeloid leukaemia after intensive chemotherapy.