Use of Ltb4 Inhibitors for the Treatment of B-Cell Leukemias and Lymphomas

Abstract

The invention relates to the use of an inhibitor of the biosynthesis and/or function of LTB4 for the manufacture of a medicament for the treatment of B-cell chronic lymphocytic leukemia (B-CLL), B-prolymphocytic leukemia (B-PLL) or B-cell lymphoma. Preferably, the inhibitor of the biosynthesis and/or function of LTB4 is the inhibitor of 5-LO BWA4C or the inhibitor of FLAP MK-886.

Description

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the level of biosynthesis of LTB₄ by B-CLL cells under various conditions. B-CLL cells (10×10⁶) were:

incubated for five minutes at 37° C. with calcium ionophore A23187 (final concentration 1 μM);

incubated for five minutes at 37° C. with arachidonic acid (AA) (final concentration 40 μM);

incubated for five minutes at 37° C. with A23187 (1 μM) plus arachidonic acid (40 μM);

sonicated and subsequently incubated for five minutes at 37° C. with ATP (1 mM), calcium chloride (2 mM) and arachidonic acid (40 μM); or

pre-incubated (intact cells) with diamide (100 μM) for two minutes, followed by stimulation with A23187 (1 μM) and arachidonic acid (40 μM).

Values given in FIG. 1 are mean±S.D. of six independent experiments.

FIG. 2 depicts the expression of BLTR1 on human leukocytes. The expression BLTR1 was analysed in various leukocytes by FACS. The specific leukocytes were:

A) PMNL;

B) peripheral CD8⁺T-cells;

C) peripheral CD4⁺T-cells;

D) normal peripheral B-cells;

E) B-CLL cells; and

F) B-PLL cells.

In all of the graphs depicted in FIG. 2, the large panel shows expression of BLTR1 and the cell specific antigen, whereas the small panel shows results with negative control antibodies. The figure depicts one typical experiment out of six except for B-PLL (two experiments).

FIG. 3 depicts the effects of leukotriene biosynthesis inhibitors on CD40L-induced thymidine incorporation in B-CLL cells. B-CLL cells (2×10⁵) were co-cultured with either irradiated L cells alone (L), irradiated CD40L-L cells or irradiated CD40L-L cells plus indicated inhibitor for 96 hr. When inhibitors were used, B-CLL cells were pre-treated with the inhibitor for 30 min prior co-culturing with L cells or CD40L-L cells. The inhibitors used were:

A) MK886 (10⁻⁶ to 10⁻⁹ M (10⁻⁶ M was only used in three experiments); or

B) BWA4C (10⁻⁷ to 10⁻⁹ M),

with or without LTB₄ (10⁻⁷ M) for 96 hrs in triplicates. The control result reported in the Figure represents B-CLL cells co-cultured with irradiated CD40L-L cells alone. ³H-thymidine (1 μCi) was present for the final eight hours. Activation of B-CLL cells with CD40L-L treatment led to between 3580 and 15369 cpm (³H-thymidine) incorporation (control) in different experiments. This was set as 100% in each experiment. The results show the mean±S.D from eight separate experiments (B-CLL cells from two patients were analyzed two times). Student's t-test was used to calculate statistics i.e. control vs. control plus indicated compound(s) (** P<0.01, *** P<0.001).

FIG. 4 depicts the effects of leukotriene biosynthesis inhibitors on the expression of CD23, CD54 and CD150 in CD40L activated B-CLL. Purified B-CLL cells were co-cultured with either L cells or CD40L-L cells in the absence or presence of MK886 (10⁻⁷ M), BWA4C (10⁻⁷ M), and/or LTB₄ (10⁻⁷ M) for 96 hrs. When inhibitors were used, B-CLL cells were pre-treated with the inhibitor for 30 min prior to co-culturing with L cells or CD40L-L cells. B-CLL cells were collected and analysed by FACS with antibodies against CD23, CD54 or CD150. The figure depicts one typical experiment out of six. In order to more clearly demonstrate the different degree of expression of indicated antigen in the various samples, the inserted dotted line represents the expression of the indicated antigen in B-CLL cells stimulated with CD40L-L alone.

Biological Tests

Materials and Methods

Reagents and Cell Lines:

The calcium ionophore A23187 was purchased from Calbiochem-Behring (La Jolla, Calif., U.S.A.). HPLC solvents were obtained from Rathburn chemicals (Walkerburn, U.K.) and the synthetic standards of LTB₄ and prostaglandin (PG) B. were from Biomol (Plymouth meeting, Pa., U.S.A.). BWA4C was a kind gift from Lawrie G Garland, Wellcome Research Laboratories, UK and MK-886 from Jilly F. Evans, Merck Frosst Centre for Therapeutic Research, CA. Azodicarboxylic acid bis(dimethylamide) (diamide) was purchased from Sigma (Stockholm, SE). Mouse fibroblastic L cells transfected with the human CD40L (CD40L⁺L cells) were used for activation and untransfected L cells (CD40L⁻) as control (see J. Exp. Med. 182, 1265 (1995)).

Isolation of Cells:

B-cells were isolated from patients suffering with B-CLL or B-prolymphocytic leukemia (B-PLL) who had not received chemotherapy within during the previous six weeks (see Table 1 below).

TABLE 1
Clinical data on patients with B-CLL.
Patient				Survival	Sample
No.	Sex	Age	Rai	(Months)	(Months from diagnosis)
1	F	51	0	234+	232
2	F	67	0	114+	48
3	M	72	I	62+	57
4	M	66	III	91+	88
5	M	63	I	101+	90
6	F	68	0	37+	35
(Patient data and Rai stadium at diagnosis. Survival is measured as months from diagnosis (+ means that patients are still alive). Patients 3 and 6 have never received treatment. The other patients have received several courses of therapy with one to six different regiments.)

Peripheral blood samples were obtained after informed consent and with local ethics committee approval. Blood samples were Ficoll-Isopaque purified and washed twice in phosphate buffered saline (PBS). After that, cells were either frozen in PBS with 50% human AB serum and 10% dimethylsulfoxide or analyzed fresh. Frozen cell samples were thawed and washed in ice cold fetal calf serum and subsequently in PBS before analysis. Cells from two patients were used twice, both freshly isolated cells and after freezing with similar results. However, similar results were obtained (data not shown). The purity of the isolated cells was estimated by flow cytometric analysis (with FACS Calibur, Becton Dickinson, Mountain View, Calif.). Morphological analysis was performed after staining with May-Grunewald/Giemsa solution. The purity of B-CLL and B-PLL cells was >98%.

Incubation of Intact B-CLL cells:

10×10⁶ cells were suspended in 1 mL PBS and pre-incubated for two minutes with/without azodicarboxylic acid bis(dimethylamide), abbreviated diamide, (100 μM) prior to stimulation with arachidonic acid (40 μM) and/or calcium ionophore A23187 (1 μM). The cells were stimulated for five minutes at 37° C. and the incubations were terminated with 1 mL methanol.

Incubation of Sonicated B-CLL Cells:

10×10⁶ cells were resuspended in 1 ml calcium-free PBS including EDTA (2 mM) and sonicated 3×5 s. The cells were pre-incubated for two minutes in the presence of ATP (1 mM) prior to addition of calcium chloride (2 mM) and arachidonic acid (40 μM). The reaction was terminated with 1 mL methanol after five minutes of incubation at 37° C.

Analysis of Leukotrienes:

After addition of 0.5 ml PBS and the internal standard PGB₁ (100 pmol) to the samples, the cells were centrifuged (2500 rpm, 5 min). The supernatant was subsequently subjected to solid phase extraction on Chromabond C₁₈ columns (200 mg, Macherey & Nagel). The methanol eluate was analysed on Waters Alliance 2695 reverse phase HPLC and detected with Waters PDA 996. Methanol:water:trifiuoroacetic acid (70:30:0.007, v/v) was used as mobile phase at a flow rate of 1.2 mL/min. Qualitative analysis was performed by comparison of retention times of synthetic standards and quantitative determinations were performed by computerized integration of the area of eluted peaks.

Expression of BLT1:

Fresh blood samples from normal donors and fresh samples from patients were Ficoll-Isopaque separated and washed in PBS. For analysis of whole blood leukocytes (including granulocytes) from healthy donors, cells were washed in PBS and lysed with FACS lysing solution (Becton Dickinson) is and washed in PBS. Frozen patient samples (B-CLL and B-PLL) were thawed (as described above) and washed in PBS. After resuspending cells in 100 μL PBS, antibodies were added according to manufacturer's instructions and incubated at room temperature for 10 minutes. The cells were washed in 2 mL PBS and fixed in 1% paraformaldehyde, before analysis with FACS Calibur (Becton Dickinson) using the CeliQuest software.

In this study all the antibodies used for flow cytometry were directly conjugated with either fluorescein isothionine (FITC), phycoerythrin (Pe) or peridinin chlorophyll protein (PerCP).

The BLT1 antibody 7B1 FITC was raised in-house (see: Biochem. Biophys. Res. Commun. 279, 520 (2000)). IgG1-FITC, IgG1-Pe, IgG1 Percp, CD4-Pe, CD5-Pe, CD8-Percp, CD14-FITC, CD14-Pe, CD19-FITC, CD19-Pe, CD20-Percp, CD22-Pe, CD33-FITC, CD33-Pe, IgG2a-FITC (Becton Dickinson).

DNA Synthesis:

Purified B-CLL cells were cultured in RPMI 1640 medium, supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin and incubated at 3720 C. in an atmosphere of 5% CO₂. 2×10⁵ of B-CLL cells were seeded in 200 μL medium in 96-well plates. B-CLL cells were pretreated with MK-886 (a specific FLAP inhibitor) (10⁻⁶ to 10⁻⁹ M) or BWA4C (a specific 5-LO inhibitor) (10⁻⁷ to 10⁻⁹ M) for 30 min, before co-culturing with irradiated (15,000 Rad) CD40L expressing L (CD40L-L) cells or control L (L) cells in the presence of inhibitors. LTB₄ (10⁻⁷ M) was present in the indicated cultures. Each sample was represented by triplicates. 1 μCi ³H-thymidine was present in the wells for the final eight 15 hours of the 96 hr cultures. The cells were harvested onto glass fibre filter and radioactivity was measured in a liquid scintillation counter.

Flow Cytometry Analysis of CD23, CD54 and CD150 Expression:

Cultured (described above) B-CLL cells were collected (without the plastic attached L cells) and used for FACS detection. Surface marker expression was detected by indirect immunofluorescence. One million cells/sample were washed in cold PBS containing 1% FCS and 0.1% sodium azide and then exposed to the relevant antibodies. The cells were washed and incubated with the RPE conjugated secondary antibody. All incubations were done at 4° C.

Samples were run on a Becton Dickinson FACScan flow cytometer (Becton Dickinson, Mountain View, Calif.). The CellQuest software (Becton Dickinson) was used both for acquisition and analysis of the samples. Ten thousand events were collected on a FACScan flow cytometer, and the results were analysed using CellQuest (Becton Dickinson) software.

Only the viable cells were considered for analysis based on their light scatter (FSC/SSC) characteristics. The following antibodies were used: MAb MHM-6 (anti-CD23, from Dr. M. Rowe, University of Wales, Cardiff, Wales, UK), MAb LB-2 (anti-CD54, from E. A. Clark, University of Washington, Seattle, Wash.), MAb IPO-3 (anti-SLAM, kind gift from S. Sidorenko, Acad. of Science of Ukraine, Kiev, Ukraine) and RPE conjugated rabbit anti-mouse Ig F(ab′)₂ (Dako, Copenhagen, Denmark) were used as secondary antibody.

Results

Biosynthesis of Leukotrienes in B-CLL cells:

The capacity of B-CLL cells to produce leukotrienes was investigated. The cells were challenged with either calcium ionophore A23187, arachidonic acid or calcium ionophore A23187 plus arachidonic acid. No cell clones produced detectable amounts of leukotrienes after challenge with either calcium ionophore A23187 or arachidonic acid only. Activation of the cells with calcium ionophore A23187 and arachidonic acid led to the formation of LTB₄ (mean 2.6±0.8 pmol/10⁶ cells). Preincubation of intact cells with the thiol-reactive agent diamide, prior to addition of calcium ionophore and arachidonic acid, led to a markedly increased production of LTB₄ (mean 33.5±1.2 pmol/10⁶ cells) in comparison to untreated intact cells (FIG. 1). These results are in agreement with earlier reports (see Proc. Natl. Acad. Sci. USA 89, 3521 (1992) and Eur. J. Biochem. 242, 90 (1996)). Similar amounts of LTB₄ (mean 34.8±1.7 pmol/10⁶ cells) were produced in broken-cell preparation, incubated with arachidonic acid. There was no obvious correlation between the capacity to produce leukotrienes and the clinical stage of the disease (data not shown). Taken together, the results demonstrated that all investigated B-CLL clones had the capacity to produce LTB₄ and that all B-CLL clones contained substantial amounts of 5-lipoxygenase which could be activated under certain conditions.

BLT1 Expression:

Peripheral blood leukocytes from healthy donors were analysed with FACS for the expression of BLTR1. Gates for granulocytes, lymphocytes and monocytes were set on the basis of forward and side scatter. Virtually all cells gated as granulocytes (and CD33 positive) expressed BLT1 (FIG. 2a). Cells in the monocyte gate (CD14 positive) showed the same pattern of BLT1 expression (data not shown). In the lymphocyte gate, no expression of BTL1 was observed on peripheral non-activated CD4⁺- or CD8⁺-positive T-lymphocytes (FIGS. 2b and 2c). These results are in agreement with the observation that naive non-activated mouse T lymphocytes do not express BLT1 (see Nat. Immunol. 4, 982 (2003)). In contrast, 30-50% of CD19, CD20 and CD22 expressing peripheral B-lymphocytes stained positively for BLT1 (FIG. 2d). The BLT1 expression on peripheral B-lymphocytes was weaker than on granulocytes and monocytes and showed a pattern of gradually increased expression within the peripheral B-lymphocyte population. Similar results have recently been reported (see Int. Immunopharmacol. 3, 1467 (2003)).

B-cells from five patients with B-CLL and two with B-prolymphocytic leukemia (B-PLL) were analysed with FACS for BLT1 expression. BLT1 expression analysed with FACS varied from about 15% to 85% in 5 B-CLL clones (average 42%) (FIG. 2e). In the B-PLL group, the average expression of BLT1 was 74% in the two investigated clones. (FIG. 2f).

Effects of Leukotriene Synthesis Inhibitors on DNA Synthesis in B-CLL Cells:

In order to elucidate if leukotrienes are of importance for proliferation of B-CLL, the cells were cultivated in the presence of leukotriene biosynthesis inhibitors. B-CLL cells were co-cultured with CD40L expressing L cells or control L cells for 96 hr in the absence or presence of MK-886 (a specific FLAP inhibitor) or BWA4C (a specific 5-lipoxygenase inhibitor). CD40-CD40L interactions activated B-CLL cells and resulted in an increased DNA synthesis, measured as ³H-thymidin incorporation during the final eight hours of four days cultures (FIG. 3). MK-886, at a concentration of 100 nM, markedly inhibited DNA synthesis induced by CD40-ligand stimulation (FIG. 3A). Due to the relatively high binding of MK-886 to serum proteins (see Can. J. Physiol. Pharmacol. 67, 456 (1989)), the effect of 1 μM MK-886 on DNA synthesis was also investigated in certain experiments. This concentration of the inhibitor only caused a little more pronounced inhibition of DNA synthesis. The inhibitory action of 1 μM and 100 nM MK-886 on thymidine incorporation was 46 and 38%, respectively. Leukotriene B₄ (final concentration 150 nM) did not amplify CD40-induced thymidine incorporation. However, exogenously added LTB₄ (150 nM) almost completely reversed the inhibitory effect of MK-886 on thymidine incorporation. The specific 5-lipoxygenase inhibitor BWA4C was an even more potent inhibitor than MK-886 to block DNA synthesis (FIG. 3B). A significant inhibitory effect of BWA4C on thymidine incorporation was observed at 10 nM. In line with the results with MK-866, exogenous addition of LTB₄ (150 nM) almost completely reversed the inhibitory action of 100 nM BWA4C on thymidine incorporation (FIG. 3B). The cell survival after four days cultivation was about 80% in all B-CLL cultures stimulated with CD40L-L, both in the absence or presence of inhibitor or LTB₄ (data not shown). Taken together, these specific inhibitors of leukotriene synthesis caused at low concentrations a pronounced inhibition of DNA synthesis, which could be reversed by exogenous addition of LTB₄.

Effects of Leukotriene Biosynthesis Inhibitors and LTB₄ on CD23, CD54 and CD150 Expression in B-CLL Cells:

The expression of CD23 is a marker of activation of B-cells. CD54 (ICAM-1) is an important adhesive molecule expressed to various extents on many B-CLL clones. CD150 is an antigen involved in the bidirectional stimulation of T- and B-cells and is upregulated on activated B-cells. FACS analysis demonstrated that CD40-CD40L interactions caused an increased expression of all three antigens (FIG. 4). MK-886 and BWA4C, at a concentration of 100 nM, markedly counteracted this CD40-induced increased expression of CD23, CD54 and CD150. Leukotriene B₄ did not cause any significant effect alone on the expression of the investigated is antigens. However, addition of 150 nM LTB₄ almost completely reversed the inhibitory effect of the inhibitors on antigen expression (FIG. 4). These results show that LTB₄ is involved in the expression of these antigens, which are associated with activation and tissue infiltration of B-CLL cells.

Claims

1-12. (canceled)

13. A method of treating B-CLL, B-PLL or B-cell lymphoma, which method comprises administering an inhibitor of the biosynthesis and/or function of LTB4 to a patient in need of such treatment.

14. A method as claimed in claim 13, wherein the inhibitor of the function of LTB4 is an antagonist of the BLT) receptor.

15. A method as claimed in claim 13, wherein the inhibitor of the biosynthesis of LTB4 is an inhibitor of 5-LO, and inhibitor of FLAP and/or an inhibitor of LTA4 hydrolase.

16. A method as claimed in claim 13, wherein the method comprises administering to the patient an inhibitor of 5-LO or an inhibitor of FLAP.

17. A method as claimed in claim 16, wherein the inhibitor of 5-LO is BWA4C, or the inhibitor of FLAP is MK-886.

18. A method as claimed in claim 13, wherein the inhibitor of the biosynthesis and/or function of LTB4 is the sole cancer chemotherapeutic agent administered to the patient.

19. A method as claimed in claim 18, wherein the inhibitor of the function of LTB4 is an antagonist of the BLT1 receptor.

20. A method as claimed in claim 18, wherein the inhibitor of the biosynthesis of LTB4 is an inhibitor of 5-LO, and inhibitor of FLAP and/or an inhibitor of LTA4 hydrolase.

21. A method as claimed in claim 18, wherein the method comprises administering to the patient an inhibitor of 5-LO or an inhibitor of FLAP.

22. A method as claimed in claim 21, wherein the inhibitor of 5-LO is BWA4C, or the inhibitor of FLAP is MK-886.

23. A method of treating B-CLL, B-PLL or B-cell lymphoma, which method comprises administering an inhibitor of the biosynthesis and/or function of LTB4 to a patient in need of such treatment, which patient is administered a cancer chemotherapeutic agent having a different mechanism of action.

24. A method as claimed in claim 13, wherein the patient is human.

25. A combination product comprising: (A) an inhibitor of the biosynthesis and/or function of LTB4, or a pharmaceutically-acceptable derivative thereof; and(B) a cancer chemotherapeutic agent having a different mechanism of action, or a pharmaceutically acceptable derivative thereof,wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier.