WO1998044923A1

WO1998044923A1 - Anti-neoplastic effects of actinonin

Info

Publication number: WO1998044923A1
Application number: PCT/US1998/006363
Authority: WO
Inventors: David Scheinberg; Yang Xu
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 1997-04-10
Filing date: 1998-04-01
Publication date: 1998-10-15

Abstract

The present invention provides a method of treating a neoplastic cell comprising administering a pharmacologically effective dose of actinonin to said cell. Preferably, actinonin can be used to treat neoplastic cell such as lymphomas, leukemias, carinomas, sarcomas, and other pathological states in humans involving lymphocytes such as infections and auto-immune disorders.

Description

ANTI-NEOPLASTIC EFFECTS OF ACTINONIN

BACKGROUND OF THE INVENTION

Field of the Invention The present invention relates generally to the fields of immunology and cancer treatment. More specifically, th e present invention relates to anti-neoplastic effects of actinonin.

Description of the Related Art CD13/Aminopeptidase-N (EC 3.4.11.2) is a ubiquitous cell surface zinc aminopeptidase involved in down regulation of regulatory peptide signals (1). Recently, aminopeptidase N has been shown to be the major receptor for the enteropathogenic coronavirus TGEV (2) and for human coronavirus 229E (3), and to be involved in tumor-cell invasion (4, 5). Human aminopeptidase N is identical to the myeloid differentiation antigen CD13 (6, 7), found on HL60 leukemia cells (7, 8), myeloid and monocytic cells and most myeloblastic leukemias, as well as o n cells and tissues outside th e hematopoietic system including fibroblasts, intestinal epithelium, renal tubular epithelium and synaptic membrane s of the central nervous system (1). Aminopeptidase N occurs a s a homodimer and the molecule is a 150-kDa, transmembrane glycoprotein with an intracellular amino terminus (1). F23, a n anti-human CD13/aminopeptidase N monoclonal antibody, is able to completely block the active site of the enzyme (9).

Bestatin, a CD 13/aminopeptidase N inhibitor, h a s been examined in preclinical and clinical studies. Bestatin inhibited lymph node metastasis of P388 leukemia in mice ( 10), and was used in clinical trials in malignant skin tumors (11), i n head and neck cancer (12), in esophageal cancer (13), and i n gynecologic tumors (14). High doses of bestatin resulted in th e significant inhibition of preexisting experimental an d spontaneous metastasis in mice (15).

Actinonin, (3- [ [ l -2-(hydroxymethyl)-pyroli-dinyl] carbony]-2-methyl-propyl]carbamoyl]octanohydroxaminic acid, a naturally occurring antibiotic derivative of L-prolinol and a potent CD 13/aminopeptidase N inhibitor, is obtained from th e culture filtrates of a Streptomyces species classified a s Streptomyces Cutter C/2 NCIB 8845 (16). Actinonin has b een shown to be generally active against Gram-positive bacteria. The action of the antibiotic involves disruption of RNA synthesis in bacteria. In vivo , it has no apparent toxicity to mice in doses up to 400 mg/kg body weight (16). Actinonin is eight times more potent than bestatin (9).

The prior art is deficient in the lack of th e demonstration of anti-tumor and cytostatic activity of actinonin in vivo and effective means of treating tumors using actinonin. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention discloses an anti-cancer agent, actinonin, which has anti-proliferative effects on hum an leukemia and lymphoma cells in vitro and on syngeneic leukemia in vivo. In one embodiment of the present invention, there is provided a method of treating a neoplastic cell comprising administering a pharmacologically effective dose of actinonin to the cell. Preferably, actinonin can be used to treat neoplastic cell such as lymphomas, leukemias, and carcinomas, other pathological states in humans involving lymphocytes such as infections and auto-immune disorders. Generally, actinonin has anti-proliferative activity on tumors both in vitro and in vivo by inducing cell growth arrest at Gl and inducing cell apoptosis.

In one embodiment of the present invention, there is provided a method of treating an individual having a lymphoma, comprising the step of administering a therapeutically effective dose of actinonin to the individual.

In one embodiment of the present invention, there is provided a method of treating an individual having a leukemia, comprising the step of administering a therapeutically effective dose of actinonin to the individual.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well a s others which will become clear, are attained and can b e understood in detail, more particular descriptions of th e invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in th e appended drawings. These drawings form a part of th e specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention an d therefore are not to be considered limiting in their scope. Figure 1 shows cytotoxicity and inhibition of protein synthesis in cell lines by actinonin. Figure 1 A show s inhibition of protein synthesis in cells by actinonin. Cell lines

4

(5x 10 cells/ml) were incubated for 5 days at 37°C in th e presence of actinonin. Levels of protein synthesis were determined by a 5 hour incorporation of tritiated leucine into trichloroacetic acid-precipitable protein. Figure IB shows cell

4 viability determined by trypan blue exclusion. Cell lines (5x 10 cells/ml) were incubated for 5 days at 37°C in the presence of actinonin. Trypan blue was added, and live and dead cells were enumerated.

Figure 2 shows the effects of actinonin on the cell cycle. Aliquots of HL60 cells were collected at different times after actinonin treatment (10 μg/ml) . Figure 3 shows effects of actinonin on AKR leukemia cells in vivo. Figure 3 A shows survival curves of AKR mice after transplantation of AKR leukemia cells. AKR

6 leukemia cells (2x 10 ) were transplanted into AKR mice. Three days after the transplantation, the mice were treated with actinonin intraperitoneally, daily for 5 days. Figure 3B shows treatment of AKR leukemia cells in vivo by actinonin. AKR mice were transplanted subcutaneously with 2x l 0⁶ AKR leukemia cells. After the third day, mice were treated intraperitoneally with 100 μg actinonin per mouse, daily for 3 days, then treated with an additional injections of 100 μg actinonin (one injection of 100 μg actinonin every other day for three times). At th e times indicated on the X axis, tumor surface area (mm²) w a s measured.

DETAILED DESCRIPTION OF THE INVENTION

Actinonin, an antibiotic and CD 13/aminopeptidase-N inhibitor, has been shown to be cytotoxic to tumor cell lines in vitro . The present invention details the anti-proliferative effects of actinonin on human and murine leukemia and lymphoma cells. Also disclosed is a method of treating an individual with actinonin.

Thus, the present invention is directed a method of treating a neoplastic cell comprising administering a pharmacologically effective dose of actinonin to the cell. Representative examples of neoplastic cells include lymphomas , leukemias, carinomas, sarcomas, and other pathological states in humans involving lymphocytes such as infections an d auto-immune disorders. Actinonin can be used against a neoplastic cell in a human or animal, for example, inducing cell growth arrest and apoptosis. Actinonin can be used to treat a neoplastic cell in vitro. Preferably, actinonin is administered to an individual at the concentration of about 1 mg/kg to about 100 mg/kg, in single or repeated doses.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1

Materials Actinonin, amastatin and bestatin were purchased from Sigma (St. Louis, MO). Monoclonal antibodies 4B4, OKT9, Leu- 11 a, Leu- 15, Leu-Ml , MY4, control IgGl and IgG2a w ere purchased from Coulter (Hialeah, FL) or Becton Dickinson (San Jose, CA). Fluoresceinated, affinity-purified goat antiserum to mouse immunoglobulins (GAM-FITC) were purchased from Kirkegaard & Perry (Gaithersburg, MD). Monoclonal antibodies F23, TA99, JD12 and M195 were produced in Memorial Sloan- Kettering Cancer Center. Recombinant human IL-3 w a s obtained from Amgen (Thousand Oaks, CA). Stock vials of human IL-3 was stored at 4°C. For each experiment, all factors were diluted in serum-containing medium on the day of use. EXAMPLE 2

Cell Separation Techniques

Bone marrow cells were obtained from healthy volunteers after informed consent. The mononuclear cells w ere isolated by centrifugation on Ficoll-Hypaque gradients ( 1 .077 g/ml; Pharmacia Fine Chemicals, Piscataway, NJ), washed twice in phosphate-buffered saline (PBS), and suspended in Iscove' s modified Dulbecco's medium (IMDM) containing 10% fetal calf serum (FCS; Hyclone, Logan, UT) supplemented with penicillin ( 100 U/ml; GIBCO, Grand Island, NY), streptomycin (100 μg/ml ; GIBCO), and 3 mg/ml glutamine (GIBCO). These cells were u s e d as target cell populations for the colony forming unit- granulocyte macrophage (CFU-GM) progenitor cell assay.

EXAMPLE 3 Colony Forming Unit-Granulocyte Macrophage (CFU-GM)

Low-density ( l x l O⁵ cells/ml) or CD34+ (5x l 0³ cells/ml) bone marrow cells were cultured in 35-mm tissue culture dishes (Corning, Corning, NY) in McCoy's modified as say medium containing 0.3% agar piFCO, Detroit, MI) and 10% FCS (17). Cultures were stimulated by the addition of 100 n g/ml IL-3.

EXAMPLE 4

Animals

Five-week-old female AKR mice were purchased from Jackson Laboratory (Bar Harbor, ME). All bedding material was sterilized before use; the cages were covered with an air filter and maintained in isolation cabinets. Animal handling and experiments were performed in a sterile atmosphere using a laminar flow hood following institutional care guidelines.

EXAMPLE 5

Cell Lines and Culture Conditions AKR leukemia cells were obtained from the spleen of old AKR mice who spontaneously developed leukemia at 1 0 months. HL60 (acute myeloid leukemia, CD13 positive), NB4 (acute promyelocytic leukemia, CD13 positive, from Dr. M. Lanotte of the Louis Pasteur Institute, Paris, France), RAJI an d DAUDI (B lineage Burkitt's lymphomas, CD13 negative) w ere maintained in culture using RPMI 1640 supplemented with 10% Serum Plus (JRH Biosciences, Lenexa, KS) and 10% heat inactivated fetal calf serum (Intergen, Purchase, NY) at 37°C i n a humidified atmosphere of 5% CO2 air. Cell viability w a s always higher than 90% and cells were free of mycoplasma contamination.

EXAMPLE 6

Transplantation of AKR Leukemia Cells into AKR Mice an d Therapy

A 0.1 ml aliquot containing 2x l 0⁶ AKR leukemia cells from suspension culture was transplanted subcutaneously into AKR mice. Tumors grew subcutaneously and the cutaneous tumor size was measured as a cross product to derive surface area. To protect animals, mice were considered "dead" and sacrificed when the tumor surface are a reached more than 400 mm². The test animals were treated intraperitoneally with actinonin in a final volume of 0.1 ml. Control mice were treated with 0.1 ml saline.

EXAMPLE 7

Flow Cytometry Assays

Cells were washed and resuspended in 2% rabbit serum (Pel Freeze, Rogers, AK) to reduce nonspecific binding. 5x l 0⁵ cells in a final volume of 0.1 ml were incubated one hour on ice in the presence of primary antibody. Cells were w ashed twice, incubated 30 min on ice with secondary fluorescein isothiocyanate (FITC) labeled antibody (goat anti-mouse immunoglobulin) (Kirkegaard & Perry, Gaithersburg, MD), washed twice, and fixed with 0.5% paraformaldehyde. FITC fluorescence intensity was measured on an EPICS Profile II flow cytometer (Coulter, Hialeah, FL).

EXAMPLE 8

Inhibition of Tritiated Thymidine or Leucine Incorporation

An aliquot containing 200 μl of cells were washed and incubated at 37°C in 96 well plates in the presence or absence of actinonin. After an incubation time of 3 to 7 days, 50 μl of 10 μCi/ml of tritiated thymidine or leucine (Du Pont-New England Nuclear, Wilmington, DE) was added to each well and allowed to incorporate for 5 to 6 hours. Trichloroacetic acid was added at a final concentration of 10% to precipitate protein for [³H]leucine incorporation experiments. Cells were harvested using a semiautomatic harvester (Skatron, Norway) and read in a scintillation counter LS 6000 IC (Beckman, Fullerton, CA).

EXAMPLE 9

Flow Cytometric Analysis of CDl lb and BCL-2 Proteins

Cells were incubated for 1 hour on ice with phycoerythrin-conjugated anti-CDl lb monoclonal antibody (Becton Dickinson, San Jose, CA) and then washed with phosphate-buffered saline (PBS) and fixed in 2% paraformaldehyde for 10 min, and then exposed to 0.1% Triton- X100 for 10 min. After washing with PBS and blocked with 1% human AB serum, cells were incubated with FITC-conjugated anti-BCL-2 monoclonal antibody (DAKO A/S, Denmark) on ice for 30 min and then analyzed on an EPICS Profile II flow cytometer (Coulter, Hialeah, FL). Ten thousand events were counted for each sample. Mean peak fluorescence intensity (MPF) for a n isotype-matched control antibody was set at 1.

EXAMPLE 10

Cell Cycle and Apoptosis Analysis by Flow Cytometry

Cells were collected and fixed in 1.5% paraformaldehyde/PBS for 15 min. After washing with PBS, th e cells were resuspended in 70% ice-cold ethanol and kept at -

20°C for up to 5 days. Analysis for cell cycle distribution an d apoptosis was performed according to the instructions in th e APOPTAG kit (ONCOR, Gaithersburg, MD). Stained cells w ere analyzed on a FACScan flow cytometer (Becton Dickinson, S an Jose, CA); evidence of apoptosis and percentage of cells in each phase of the cell cycle were analyzed by the CellFIT and PC- LYSIS software (Becton Dickinson, San Jose, CA).

EXAMPLE 11

Actinonin Inhibited Growth of Leukemia Cells in vitro.

Actinonin was tested for its ability to kill CD 13 positive and CD 13 negative cells. Activity and cytotoxicity were determined by inhibition of incorporation of [³H]leucine into protein and by trypan blue exclusion. Dose-response curves were generated by testing the inhibitory effects of actinonin o n the protein synthesis of NB4 and HL60 cells (CD13 positive) o r RAJI (CD 13 negative) in culture (see Figure 1A). In these in vitro studies, 2-5 μg/ml of actinonin was required to inhibit protein synthesis by 50% in CD13 positive and negative cells.

The cytotoxicity of actinonin was initially determined by trypan blue exclusion (Figure IB and Table 1 ) . The IC5₀ (the concentration of actinonin required to kill 50% of cells) ranged from 2 to 5 μg/ml, comparable to the concentration of actinonin required to inhibit 50% of protein synthesis. The similar dose-response curves for cytotoxicity and protein synthesis inhibition on cells that expressed or did not express CD 13/aminopeptidase N suggested that the mechanism of cytotoxicity did not necessarily involve inhibition of CD 13/aminopeptidase N by actinonin. Treatment of NB4 an d HL60 cells for 4 days with 100 μg/ml monoclonal antibody F23, which blocks substrate binding to CD 13/aminopeptidase N an d its activity (18), had no effect on cell viability (91% alive) i n comparison to no treatment (90% alive) or an isotype matched control antibody TA99 (91% alive) (data not shown). I n addition, cytotoxicity of actinonin was not abrogated b y pretreatment of cells with F23 antibody, which blocks actinonin binding to its active site (20). These data together showed th at the inhibition of cell growth by actinonin is not through th e inactivation of cell surface enzyme.

TABLE 1

Effect of actinonin on cell viability determined by trypan blue exclusion in vitro. Cell Type IC _, (μ /m\) (Mean ± SO)*

HL60 cells 2 ± 0.5

NB4 cells 5 ± 1.0

DAUDI cells 2 ± 0.5

RAJI cells 4 ± 1.0 AKR cells 3 + 0.6

*IC₅₀ is the concentration of actinonin required to kill 50% of cells. EXAMPLE 12

Cell Cycle Gl Arrest and Apoptosis in Leukemia Cells durin g Actinonin Treatment Cell cycle distribution of leukemia cells showed small changes early after treatment with 10 μg/ml actinonin. After 24 hours of exposure to actinonin the number of HL60 cells arrested in Gl phase increased and the percentage of cells in S phase decreased (Figure 2). A similar effect was seen i n NB4 (Gl phase increased by 24%) and RAJI (Gl phase increased by 37%) after 24 hours of treatment with 10 μg/ml actinonin (not shown). This Gl arrest is in accordance with the growth inhibition observed after 2 days of exposure to actinonin (not shown). 96 hours following exposure to actinonin at 10 μg/ml, 20-35% of HL60 and NB4 cells showed apoptosis, whereas only 10% of RAJI had apoptosis after treatment (Table 2).

TABLE 2

Effect of actinonin (10 μg/ml) on apoptosis in cells at 96 hours.

HL60 NB4 RAJI

Control 2.9% 9.1% 4.3% Actinonin 37.9% 29.4% 13.6% EXAMPLE 13

Effects of Actinonin on AKR Leukemia Cells in vivo

The significant anti-proliferative effects of actinonin in vitro prompted an analysis of the effects of actinonin in vivo against leukemia or lymphoma cells. In order to avoid th e problems associated with a xenograft model, a syngeneic leukemia/lymphoma model in AKR mice was used to better approximate actual human use. AKR cells are inhibited in vitro by actinonin with an IC5₀ of about 3 μg/ml (Table 1). In the s e experiments, AKR mice were injected subcutaneously with

2,000,000 AKR leukemia cells (day 0) and treated intraperitoneally with injections of actinonin beginning at day 3.

The effect of actinonin on tumor growth an d survival rates of AKR mice after transplantation of AKR leukemia cells was investigated. After transplantation, mice were treated with a total of five injections of actinonin (one injection, daily for 5 days). As compared to controls, actinonin increased the mean survival time of mice by nearly twofold b y reducing the rate of tumor growth. Reduction in tumor growth rates and prolongation of survival was actinonin dose-related (Figure 3A). Toxicity due to actinonin was not observed. Actinonin doses up to 8000 μg per mouse (400 mg/Kg) were tolerated without apparent toxicity (16). In a second trial, mice were treated with 100 μg actinonin daily for 3 days beginning at day 3 after transplantation, then treated with an additional three injections of actinonin (every other day). On day 17, the control mice showed tumors with a mean surface area of 287 ± 95 mm². I n contrast, no tumors were found in mice in the actinonin treated group (Figure 3B). These results indicate that actinonin h a s significant anti-tumor effects on AKR leukemia in vivo. I n contrast, actinonin did not have any significant growth inhibiting properties for subcutaneously implanted RAJI lymphoma in nude mice (data not shown) over the same dose range as was effective in the AKR model.

EXAMPLE 14

Effects of Actinonin on Human Bone Marrow Cell Growth ex vivo The effects of actinonin, amastatin and bestatin w ere evaluated on normal human bone marrow colony forming unit- granulocyte macrophage (CFU-GM) (Table 3). Actinonin decreased 16-56% colony formation in a dose dependent manner. Bestatin (10 μg/ml) at doses designed to approximate its dose level in clinical trials (19) also decreased normal hum an bone marrow CFU-GM by 64-70%. This suggested that actinonin may have some myelosuppressive activities at high doses. F23 (anti-active site of CD13) and M195 (anti-CD33) both of which bind to bone marrow progenitors had no effect on human bone marrow CFU-GM on Day 7 and 14 (data not shown). These data further support the contention that the cy to toxic effects of actinonin are not mediated through CD13/aminopeptidase N. TABLE 3

Response of low-density bone marrow cells to actinonin. amastatin and bestatin no IL-3 with IL-3: Number of colonies

Drugs (doses) Day 7 Day 14 (% of inhibition (% of inhibition)

Control (saline) 0 177 ± 28 100 ± 4

Actinonin (10 μg/ml) 0 99 + 18 (44%) 44 ± 8 (56%) Actinonin (5 μg/ml) 0 123 ± 10 (31%) 49 ± 7 (51%)

Actinonin (0.5 μg/ml) 0 148 ± 6 (16%) 74 ± 7 (26%)

Amastatin (5 μg/ml) 0 157 ± 1 (11%) 82 ±- l 1 ( 18 %)

Bestatin (10 μg/ml) 0 63 ± 27 (64%) 30 + 7 (70%)

Data represent the mean + standard deviation for four plates scored on day 7 and day 14. For these studies, l x l O⁵ low-density bone marrow cells were cultured for 7 and 14 days, respectively with IL-3 100 ng/ml.

EXAMPLE 15

Effects of Actinonin on BCL-2 Expression

BCL-2 expression was studied in preliminary experiments designed to screen for mechanisms or pathw ays involved in the anti-proliferative effects of actinonin. BCL-2 expression, observed by intracellular flow cytometry in about 20% of HL60 cells and 60% of NB4 cells, showed no significant changes after treatment with actinonin (Table 4). BCL-2 expression, however, was decreased by 80% in RAJI cells after treatment with actinonin.

TABLE 4

Effects of actinonin (10 μg/ml) on Bcl-2 expression in cell lines at 96 hours HL60 NB4 RAJI

Control 20.4%/1 .7 58.8 %/5.3 35.5 %/3.1

Actinonin 18.5 %/2.0 57.3 %/5.1 8.0%/l . l

Data represent percentage of positive cells/MPF (mean peak fluorescence) intensity, i.e., protein density per cell.

EXAMPLE 16

Effects of Actinonin on Selected Cell Surface Protein Expression

The effects of actinonin were evaluated on selected cell surface protein expression to screen for mechanisms o r pathways involved in the anti-proliferative effects of actinonin (Table 5). Cell surface proteins (CDl lb, 13, 15, 29, 33, HLA-A) significantly decreased in NB4 cells after treatment with actinonin, consistent with the toxic effects of actinonin. TABLE 5

Selected cell surface protein expression in NB4 cells in vitro after treatment with actinonin (10 μg/ml) for 48 hours.

Monoclonal Antibodies - Actinonin + Actinonin

Control (IgG2a) -11 -11

Control (IgGl) -11 -11

Anti-HLA class I (JD12) 99.8/13.3

63.8/7.5

Anti-CD13 (F23) 100/225.3

99.1/44.0

Anti-CD29 (4B4) 100/46.8

90.9/10.8

Anti-CD33 (M195) 99.6/12.6

22.1/3.3

Anti-CD71 (OKT9) 97.7/11.3

79.1/11.4

Anti-CD 16 (Leu- 11a) -/l -/l

Anti-CDllb (Leu-15) 64.8/2.35

6.2/1.43

Anti-CD 14 (MY4) -/l 5.0/1

Anti-CD 15 (Leu-Ml) 99.5/129.088.6/25.0

Data represent percentage of positive cells / MPF*.

*MPF is mean peak fluorescence intensity (protein density per cell). Discussion

Actinonin, a naturally occurring derivative of L- prolinol, is a potent inhibitor of CD 13/aminopeptidase-N (APN). The present invention shows 1) the anti-proliferative effects of actinonin on human leukemias and lymphoma cells in vitro, 2 ) the induction of growth arrest and apoptosis in target cells, 3 ) the anti-tumor effects of actinonin on AKR leukemias in AKR mice and 4) that these effects do not appear to be mediated through inhibition of CD13/APN.

Actinonin had significant anti-proliferative effects on human leukemia cells of various derivations. The cytotoxicity of actinonin was directly determined by trypan

3 blue analysis and [ Hjleucine incorporation. The IC₅₀ was about 2 to 5 μg/ml (Table 1). However, the IC₅₀ for other CD13/APN inhibitors, amastatin and bestatin, was above 100 μg/ml (data not shown). Actinonin not only inhibited growth of CD13 positive cells (NB4 or HL60 cells) but also CD13 negative cells (RAJI or DAUDI cells) (Table 1), suggesting that the effect is no t mediated by CD13/APN. Experiments with monoclonal antibody (mAb) F23 also suggested that the effect was not mediated b y CD13/APN as cell viability was not changed by treatment with mAb F23 in comparison to no treatment or treatment with a n isotype matched control antibody. In addition, the inhibitory effect of actinonin on CD13 positive cells was not blocked b y pretreatment with the anti-CD13/APN mAb F23 (20). Since actinonin binds to F23 epitopes and is blocked by prior incubation with mAb F23 (18) and mAb F23 is not inhibitory of cell growth, it was concluded that actinonin induced cell death in human leukemia cells is not likely to be associated with binding and inhibition of cell surface CD13/APN. The effect of actinonin in vitro was mediated at least partly through Gl arrest and apoptosis. After 10 μg/ml actinonin treatment for 96 hours, 20-35% of NB4 and HL60 cells showed evidence of apoptosis (Table 2). 1 , 10-phenanthroline, which inhibits APN activity by chelating the zinc ion (3, 21), also induces apoptosis in HL60 cells (22). Actinonin binds to zinc domains of CD13/APN as determined by competition assays (18). This may suggest that the zinc binding motif of important intracellular enzymes may account for apoptosis, but cell surface CD13/APN does not appear to be associated with actinonin action. In addition, there is no increase in th e percentage of CDl lb in NB4, HL60 or RAJI cells (data not shown) after actinonin treatment, suggesting that there is no differentiating activity induced by actinonin.

Actinonin was also cytotoxic to RAJI cells, without a n increase in apoptosis, in preliminary experiments designed to screen for other mechanisms or pathways involved in the anti- proliferative effects in these cells, BCL-2 expression w a s studied. BCL-2 expression, observed by intracellular flow cytometry in about 20% of HL60 cells and 60% of NB4 cells, showed no significant changes after treatment with actinonin (Table 4). BCL-2 expression, however, was decreased by 80% in RAJI cells after treatment with actinonin. Other cell surface proteins (CDl lb, 13, 15 , 29, 33, HLA-A) were significantly decreased in NB4 cells after treatment with actinonin, consistent with the toxic effects of actinonin (Table 5).

Bestatin, another APN inhibitor, has shown anti- tumor therapeutic effects in several clinical trials (23). In a multi-institutional study, 101 patients with acute nonlymphocytic leukemia (ANLL) were randomized to receive bestatin or control. The bestatin group achieved a statistically significant prolongation of both the remission duration an d survival in patients aged 50 to 65 years (24). It also normalized the CD4/CD8 ratio in peripheral blood and maintained th e immune homeostasis in cancer patients (25, 26). This may b e beneficial to AIDS patients with lymphoma. Recently, bestatin also showed direct inhibition of the growth of hum an choriocarcinoma in nude mice in a dose dependent manner (27) . A recent randomized study in patients with non-Hodgkins lymphoma after autologous bone marrow transplant who w ere treated with bestatin showed increases in NK activity i n pokeweed mitogen and phytohemagglutinin (PHA) responses of lymphocytes, in CD4 T cell and B cell numbers (26). Results with bastatin prompted the investigation whether a more potent APN inhibitor, actinonin, could inhibit the growth of syngeneic AKR leukemia cells in a mouse model in vivo. Actinonin showed dose dependent anti-tumor effects on AKR leukemia in vivo. There was a significant effect at doses of 1 00 μg per mice (5 mg/kg). Prolonged treatment appeared to further improve the activity against the leukemias. Since actinonin shows no apparent toxicity to mice in doses up to 400 mg/kg in mice (about 8 mg per mouse) (16), and no apparent toxicity was seen in the experiments conducted here, the drug may be considered safe in this mouse model at the se doses. In contrast, little anti-tumor activity was seen in a nu d e mouse model. One explanation for this may be related to th e hypothesis that these inhibitors work via a nonspecific immune augmentation in vivo (26, 28) which may not be possible in nude mice. Alternatively, there may simply be differences i n the biology of the cells that account for the lack of effects with RAJI cells. In conclusion, the data showed that actinonin induces Gl phase arrest and apoptosis in human leukemia a n d lymphoma cells; moreover, actinonin can treat AKR leukemia i n AKR mice with minimal toxicity. The site of action does not appear to be via inhibition of CD13/APN.

The following references were cited herein.

1 . Shipp MA, et al., Blood 82: 1052- 1070; 1993.

2. Delmas B, et al., Nature 357:417-420; 1992.

3. Yeager CL, et al., Nature 357: 420-422; 1992. 4. Menrad A, et al., Cancer Res 53: 1450-1455; 1993.

5. Saiki I, et al., Int J Cancer 54: 137-143; 1993.

6. Olsen J, et al., FEBS Lett 238: 307-314; 1988.

7. Look AT, et al., J Clin Invest 83: 1299-1307; 1989.

8. Griffin JD, et al., J Clin Invest 68: 932-941 ; 1981. 9. Xu Y, et al., Biochem Biophys Res Commun 208: 664-674 ; 1995.

10. Tsuruo T, et al., J Antibiot (Tokyo) 34, 1206-1209; 1981.

1 1 . Ikeda S, et al., In: Umezawa H (ed), Small Molecular Immunomodifiers of Microbial Origin, pp. 143 - 158. Elmsford, NY: Pergamon Press ( 1981 ) .

12. Inuyama Y, et al., In: Umezawa H (ed), Small Molecular

Immunomodifiers of Microbial Origin, pp. 179- 1 86. Elmsford, NY: Pergamon Press (1981). 1 3. Isono K, et al., In: Umezawa H (ed), Small Molecular

Immunomodifiers of Microbial Origin, pp. 1 87-200. Elmsford, NY: Pergamon Press ( 1981).

14. Akiya K, et al., In: Umezawa H (ed), Small Molecular Immunomodifiers of Microbial Origin, pp. 201 -210. Elmsford, NY: Pergamon Press (1981)

15. Abe F, et al., Cancer Immunol Immunother 29: 23 1 -236 ; 1989.

16. Gordon JJ, et al., Nature 195: 701-702; 1962.

17. Gabrilove JL, et al., Blood 83: 907-910; 1994. 1 8. Xu Y, et al., Exp Hematol 25: 521-529; 1997.

19. Ota K, et al., Biotherapy. 4: 205-214; 1992.

20. Xu Y, et al., Proc Am Assoc Cancer Res 38: 1368; 1997.

21 . Vallee BL, et al., Biochemistry 29: 5647-5659; 1990.

22. Byrnes RW., Arch Biochem Biophys 332: 70-78; 1996. 23. Urabe A, et al., Ann Hematol. 67: 63-66; 1993.

24. Ota K, et al., Cancer Immunol Immunother. 23: 5 - 10 ; 1986.

25. Takeuchi T., J Cancer Res Clin Oncol. 121 : 505-510; 1995.

26. Talmadge JE, et al., Proc Am Assoc Cancer Res 37: 3340 ; 1 996

27. Inoi K, et al., Anticancer Res. 15: 2081- 2087; 1995

28. Talmadge JE, et al., Cancer Res 46: 4505-4510; 1986

Any patents or publications mentioned in this specification are indicative of the levels of those skilled in th e art to which the invention pertains. These patents and publications are herein incorporated by reference to the s ame extent as if each individual publication was specifically an d individually indicated to be incorporated by reference.

One skilled in the art will readily appreciate that th e present invention is well adapted to carry out the objects an d obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with th e methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended a s limitations on the scope of the invention. Changes therein an d other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined b y the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1 . A method of treating a neoplastic cell comprising the step of administering a pharmacologically effective dose of actinonin to said cell.

2. The method of claim 1, wherein said neoplastic cell is selected from the group consisting of lymphomas, leukemias, carinomas, sarcomas, and other pathological states in humans involving lymphocytes.

3. The method of claim 1, wherein said neoplastic cell is in a human.

4. The method of treating of claim 1, wherein said neoplastic cell is in vitro.

5. The method of claim 1, wherein actinonin is administered to an individual at the concentration of about 0. 1 mg/kg to about 100 mg/kg.

6. The method of claim 1, wherein said actinonin induces cell growth arrest and apoptosis.

7. A method of treating an individual having a lymphoma, comprising the step of administering a therapeutically effective dose of actinonin to said individual.

8. The method of claim 7, wherein actinonin is administered to said individual at the concentration of about 0. 1 mg/kg to about 100 mg/kg.

9. A method of treating an individual having a leukemia, comprising the step of administering a therapeutically effective dose of actinonin to said individual.

10. The method of claim 9, wherein actinonin is administered to said individual at the concentration of about 0. 1 mg/kg to about 100 mg/kg.