Patent Application
20110319360
The present invention is generally directed to the treatment of hematologic malignancies, such as Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin's Lymphomas (NHL), and more particularly to a method of inducing apoptosis/cell death in leukemia cells using a purine nucleoside analogue.
Chronic lymphocytic leukemia (CLL) is an incurable and common adult B-cell malignancy. While highly effective chemo-immunotherapy regimens exist for the first-line setting, treatment options for relapsed or refractory CLL are limited. Additionally, therapy for CLL is usually accompanied by treatment-induced immuno- and myelosuppression (References 1 and 2). Purine analogue-based treatments in particular are associated with long-term T-cell depletion and increased risk of opportunistic infections, limiting the number of treatment cycles able to be administered (References 1-4). Therefore, identifying alternate treatment options without accompanying hematologic toxicities has clinical relevance.
CLL cells can produce tumor necrosis factor (TNF), a cytokine that can improve viability and promote proliferation of CLL cells in vitro (References 5 and 6). TNF is likely a clinically relevant target in CLL, given that elevated plasma TNF levels are correlated with poor prognosis (Reference 7). These observations have led to the hypothesis that blocking TNF in CLL patients would be efficacious.
We have assessed the effect of the purine nucleoside analogue LMP-420 (2-amino-6-chloro-9-[5(dihydroxyboryl)-pentyl] purine; shown in
The present disclosure is directed to various aspects of the present invention.
One aspect of the present invention includes a new use of a purine nucleoside analogue, 2-amino-6-chloro-9-[5(dihydroxyboryl)-pentyl], purine, as an anticancer agent.
Another aspect of the present invention includes a purine nucleoside analogue, which has potent cytotoxic and anti-proliferative effects on chronic lymphocytic leukemia cells and minimal or negligible toxicity for normal hematopoietic cells.
Another aspect of the present invention includes a new use of a purine nucleoside analogue, 2-amino-6-chloro-9-[5(dihydroxyboryl)-pentyl] purine, as an agent for inducing apoptosis or cell death in a leukemia cell, particularly a chronic lymphocytic leukemia cell.
Another aspect of the present invention includes a new use of a purine nucleoside analogue, 2-amino-6-chloro-9-[5(dihydroxyboryl)-pentyl] purine, as an agent for enhancing the cytotoxic activity of fludarabine in inducing apoptosis or cell death in a leukemia cell, particularly a chronic lymphocytic leukemia cell.
Another aspect of the present invention includes a new use of a purine nucleoside analogue, 2-amino-6-chloro-9-[5(dihydroxyboryl)-pentyl] purine, as an agent for inhibiting proliferation of leukemia cells, particularly chronic lymphocytic leukemia cells.
Another aspect of the present invention includes a new use of a purine nucleoside analogue, 2-amino-6-chloro-9-[5(dihydroxyboryl)-pentyl] purine, as an agent for treating hematologic malignancies, such as Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin's Lymphomas (NHC).
Another aspect of the present invention includes a method of inducing apoptosis or cell death in a leukemia cell, which includes subjecting a leukemia cell to a purine nucleoside analogue.
Another aspect of the present invention includes a method of enhancing the cytotoxic activity of fludarabine in inducing apoptosis or cell death in a leukemia cell, which includes subjecting a leukemia cell to a purine nucleoside analogue and fludarabine.
Another aspect of the present invention includes a method of inhibiting proliferation of a leukemia cell, which includes subjecting a leukemia cell to a purine nucleoside analogue.
Another aspect of the present invention includes a method of treating leukemia, which includes administering to a subject in need thereof an effective amount of a purine nucleoside analogue.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
One of the above and other aspects, novel features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiment(s) invention, as illustrated in the drawings, in which:
a-b illustrate cytotoxicity of LMP-420 for CLL cells;
a-b illustrate potentiation of fludarabine cytotoxicity by LMP-420;
a-b illustrate apoptotic effects of LMP-420 and fludarabine on normal B- and T-lymphocytes;
B-cell chronic lymphocytic leukemia (CLL) is characterized by accumulation of malignant cells, which are supported in the microenvironment by cell-cell interactions and soluble cytokines such as tumor necrosis factor (TNF). We evaluated the effect of the small molecule TNF inhibitor LMP-420 on primary CLL cells. The mean concentration of LMP-420 required to induce 50% cytotoxicity (ED50) at 72 hours was 245 nM. LMP-420 induced time- and dose-dependent apoptosis, as demonstrated by annexin V staining, caspase activation, and DNA fragmentation. These changes were associated with decreased expression of anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl-2. CLL cells from patients with poor prognostic indicators exhibited LMP-420 sensitivity equal to that of cells from patients with favorable prognostic indicators. In addition, LMP-420 potentiated the cytotoxic effect of fludarabine for CLL cells and inhibited in vitro proliferation of stimulated CLL cells. Gene expression profiling indicated that the mechanism of action of LMP-420 may involve suppression of NF-kappa B and immune response pathways in CLL cells. LMP-420 had minimal effects on normal peripheral blood mononuclear cell, B- and T-cell function, and normal hematopoietic colony formation. Our data support efficacious and therapeutic uses of LMP-420 for CLL with negligible hematologic toxicities.
CLL patients from the Duke University and Durham VA Medical Centers were enrolled in research protocols to collect clinical data and blood. Institutional review boards (IRBs) at both institutions approved these protocols, and patients signed informed consent prior to phlebotomy, in accordance with the Declaration of Helsinki. Clinical data were obtained by chart review.
On the same day that heparin-anticoagulated venous blood was collected from participants, CLL cells were purified by methods described previously (Reference 11). Briefly, CLL cells were isolated from whole blood using the RosetteSep® B-cell enrichment cocktail (Stem Cell Technologies, Vancouver, BC, Canada) together with ficoll-Hypaque density gradient centrifugation. This method yielded CLL cell purity of greater than 95% CD5+CD19+ B-cells.
Various prognostic markers including IgVH mutation status, CD38 and ZAP70 expression, and interphase cytogenetics were measured as described previously (Reference 11). Poor risk cytogenetic subgroups are defined as 17p13 deletion or 11q22 deletion, and favorable cytogenetic subgroups are defined as those that are normal, 13q14 deletion, or trisomy 12.
For toxicity studies involving normal blood cells, normal volunteers were enrolled in an IRB-approved protocol for blood collection. PBMCs were isolated from heparin-anticoagulated blood by ficoll-Hypaque density gradient centrifugation.
LMP-420 was synthesized to 96-98% purity by Scynexis Inc. (Durham, N.C.) under a Material Transfer Agreement between Duke University and LeukoMed, Inc. (Raleigh, N.C.) and stored as a 25 mM stock solution in 5% sorbitol, pH 8.5-9.0, at 4° C. Chlorambucil, cladribine, bendamustine, and fludarabine phosphate (fludarabine) were purchased from Sigma-Aldrich, St. Louis, Mo. 4-hydroperoxycyclophosphamide (4-HC) was synthesized by Eno Research & Development (Hillsborough, N.C.). Bendamustine and 4-HC were prepared fresh in sterile distilled water for each experiment.
2.5×105 CLL cells were incubated in triplicate or quadruplicate in a 96-well tissue culture plate (Costar, Corning, N.Y.) with serial dilutions of LMP-420, fludarabine, and/or recombinant TNF-alpha (R&D Systems, Minneapolis, Minn.) in Hybridoma serum free media (SFM) (GIBCO/Invitrogen, Carlsbad, Calif.) containing 10% heat-inactivated fetal bovine serum (FBS) (Sigma) at 37° C. with 5% CO2. After 72 hours, 20 μl CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Madison, Wis.) MTS (3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium) reagent was added to each well and the absorbance was read at 490 nM on a Thermomax microplate reader (Molecular Devices, Sunnyvale, Calif.). We calculated fractional cytotoxicity as performed previously (Reference 12), comparing absorbance at 490 nm from treated cells to control cells incubated with media alone.
The concentration effective at killing 50% of CLL cells compared to media alone (ED50) was calculated when 50% fractional cytotoxicity occurred within the range of serial dilutions tested (Reference 12). If the ED50 fell below or above this range, it was set to either the lowest or the highest concentration tested, respectively, for the purposes of statistical analyses and graphical representation.
Cells were cultured in either of two different media: Hybridoma SFM with 10% FBS or RPMI 1640 medium (Sigma) supplemented with 5% heat-inactivated human AB serum (Sigma), 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine, 25 mM HEPES (N2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 100 μM MEM non-essential amino acids, and 1 mM sodium pyruvate (GIBCO). CLL cell sensitivity to LMP-420 was not affected by culture media, as determined by direct comparison of the apoptotic effects observed in both media for seven samples. After drug activity in the presence of human AB serum was confirmed, further studies were performed in Hybridoma SFM with 10% FBS.
CLL cells were cultured at 2×106 cells/ml in a volume of 100 μl/well in a U-bottom 96-well tissue culture plate (BD Biosciences, San Jose, Calif.) with either vehicle control or with drug. After 24, 48, or 72 hours of incubation, cells were analyzed for apoptosis by three different methods. For the annexin-based assay, 100 μl of Guava Nexin Reagent (Millipore, Billerica, Mass.) was added for 20 min. Cells were analyzed for annexin V-PE and 7-AAD staining with flow cytometric analysis by the Guava EasyCyte™ Plus system (Millipore). To facilitate comparison of samples with different levels of spontaneous apoptosis, drug-specific apoptosis is reported. Drug-specific apoptosis excludes background spontaneous apoptosis and was calculated as follows: 100%×(drug-induced apoptosis−spontaneous apoptosis)/(100−spontaneous apoptosis) (Reference 13).
For the second method, apoptosis-associated activation of caspases 3 and 7 was measured by Caspase-Glo® 3/7 Assay, according to the manufacturer's protocol (Promega). Luminescence was measured by Thermo Luminoskan Ascent microplate reader (Thermo Scientific, Waltham, Mass.). For the third method, cell lysates and cell culture supernatants were assayed for histone-associated DNA fragments using the Roche Cell Death Detection ELISAPlus kit (Roche Applied Science, Indianapolis, Ind.) according to the manufacturer's protocol. Nucleosome enrichment was calculated as Absorbance405 drug-treated/Absorbance405 control sample.
CLL cells were cultured at 2×106 cells/ml in Hybridoma SFM with 10% FBS in 6-well tissue culture plates (BD Biosciences). After 72 hours treatment with vehicle control or drug, cells were harvested and washed twice with 8 ml Hanks Balanced Salt Solution (HBSS). Staining with antibodies was performed at 4° C. For cell surface staining of CD40, FITC-conjugated CD40 antibody (BD Pharmingen 555588) or FITC-conjugated IgG1 kappa isotype control (BD Pharmingen 555748) were used. For internal staining, cells were fixed at 4×107 cells/ml in 250 μl Cytofix/Cytoperm™ solution (BD Biosciences) for 20 min at 4° C. Washing and staining were then performed in Perm/Wash buffer (BD Biosciences), with 30 min incubation for both primary and secondary antibodies. Internal staining was performed with the following antibodies: Bcl-2 (SC-509), Bcl-xL (SC-8392), Mcl-1 (SC-819) (Santa Cruz Biotechnology, Santa Cruz, Calif.), isotype control antibodies (Sigma), PE-labeled donkey anti-mouse F(ab′)2 (eBioscience, San Diego, Calif.), and FITC-labeled goat anti-rabbit IgG (ABD Serotec, Raleigh, N.C.). Samples were analyzed by the Guava EasyCyte™ Plus system. Expression was measured as geometric mean fluorescence intensity, subtracting out isotype control staining.
PBMCs were cultured at 106 cells/ml in a 12-well tissue culture plate. Cells were treated for 72 hours with vehicle control or drug for 72 hours, then harvested as above for staining. PBMCs were resuspended at 2×105 cells/tube in 100 μl buffer (HBSS, 0.5% bovine serum albumin, 0.1% sodium azide) and incubated for 30 min with CD3-FITC or CD19-FITC antibodies according to the manufacturer's protocol (BD Biosciences). Cells were then incubated with PE-annexin V and 7-AAD (BD Biosciences) in annexin V binding buffer (BD Biosciences) for 15 minutes prior to analysis of apoptosis on the Guava EasyCyte™ Plus system.
[3H]-thymidine Proliferation Assay
CLL cells (2×105 cells/well) were cultured in quadruplicate in a volume of 200 μl in 96-well tissue culture plates for 96 hours with 1 μM CpG-oligodeoxynucleotides (ODN) DSP30 (Reference 14) (Midland Certified Reagent Company, Midland, Tex.) and 100 units/ml IL-2 (R&D Systems) in the presence or absence of drug. As controls, cells were treated with media without DSP30/IL-2. Cells were incubated with 1 μCi/well [methyl-3H]-thymidine (specific activity 6.7 Ci/mmol, PerkinElmer, Waltham, Mass., USA) for 14-18 hours prior to harvesting onto glass-fiber filters using a semi-automated cell harvester. [3H]-thymidine incorporation was measured as counts per minute (cpm) using a Tri-Carb 2100 timed-resolved liquid scintillation counter (PerkinElmer). The same protocol was utilized for proliferation assays with normal PBMCs, with the exception that growth was stimulated by treatment with 10 μg/ml Phytohemagglutinin-P (PHA, Sigma), 5 μg/ml concanavalin A (ConA, Sigma), 2 μg/ml pokeweed mitogen (PWM, Sigma), 25 ng/ml muromonab-CD3 (α-CD3, Ortho Pharmaceuticals, Raritan, N.J.), or 50 ng/ml phorbol myristate acetate (PMA, Sigma) plus 1 μg/ml ionomycin (iono, Sigma) for 96 hours. Proliferation is reported as percent of control (drug-treated cpm/control-treated cpm×100%).
CD34+ hematopoietic progenitor cells were isolated from PBMCs from two healthy donors using the CD34 Microbead Kit (Miltenyi Biotec, Auburn, Calif.). 103 CD34+-enriched cells were cultured in a 35 mm culture plate (1000 cells/plate) with or without LMP-420 or fludarabine in 1.1 ml Human Methylcellulose Complete Media (R&D Systems) according to the manufacturer's protocol. Plates were incubated for 14 days at 37° C. in humidified 5% CO2. Each treatment group was tested in triplicate. Colonies on plates were scored by two independent observers, blinded to the treatment groups, by microscopic evaluation under 10× and 20× power. Colonies were categorized as being of either erythroid or myeloid lineage.
This assay was performed by MDS Pharma Services. Human recombinant HEK-293 cells over-expressing the human hERG receptor were incubated for 60 min at 25° C. with 1.5 nM [3H] Astemizole in the presence or absence of the indicated concentrations of LMP-420. Nonspecific binding (<10%) was determined by binding of radioligand in 10 μM Astemizole. All samples were determined in duplicate.
CLL cells (2.5×107 cells) were cultured in Hybridoma SFM with 10% FBS with or without 2 μM LMP-420 in 100×20 mm tissue culture dishes (BD Falcon). After 24 hours, cells were harvested, washed twice with cold Dulbecco's Phosphate-Buffered Saline (PBS, Sigma), pelleted, and stored at −80° C. Total RNA was extracted from cell pellets using Qiashredder and RNeasy Mini columns (Qiagen, Valencia, Calif.). RNA concentration and quality were assessed using a Nanodrop spectrophotometer (Thermo Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif.).
RNA samples were prepared and hybridized to U133Plus 2.0 GeneChips (Affymetrix, Santa Clara, Calif.) according to the manufacturer's instructions. All analyses were performed in a minimal information about a microarray experiment (MIAME) compliant fashion. Genomic data are archived in the Gene Expression Omnibus (GEO #GSE20211). Raw data were processed using the Robust Multi-array Average (RMA) algorithm, and underwent subsequent normalization using ComBat to eliminate batch effect (Reference 15). Probe expression from control and LMP-420-treated cells were compared using the Wilcoxon signed-rank test, with a p-value cut-off of less than 0.001 and a Benjamini-Hochberg false discovery rate of <0.05. These analyses were performed using R version 2.7.1.
Cellular pathway induction or repression by LMP-420 treatment was assessed using gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway terms, using Gene Annotation Tool to Help Explain Relationships (GATHER) (Reference 16). Terms with Bayes factors greater than or equal to three were considered significantly enriched. Predictions of genomic signatures of pathway deregulation were evaluated with the binreg algorithm using Matlab (MathWorks, Novi, Mich.) (Reference 17). Pathway signatures for TNF and interferon-alpha (IFN-α) were generated using datasets found at GEO repository numbers GSE2638/2639 and GSE3920, respectively. Binreg parameters used to generate these signatures are listed in Table 4 (below).
Continuous variables were compared using the non-parametric Wilcoxon rank-sum test. Paired Wilcoxon signed-rank testing was performed when appropriate. The Friedman test with Dunn's post-test was used to test for two-way repeated measures analysis among multiple treatment groups. A significance level of less than 0.05 was considered statistically significant.
Ninety-four consecutively collected CLL samples were used for experiments testing the effect of LMP-420 on CLL cells. The characteristics of the CLL samples used are outlined in Table 1 (below). The majority of samples were obtained from patients with early stage disease who had not required therapy prior to sample collection. However, some samples were obtained from patients with advanced disease and from those with poor prognostic markers such as unmutated IgVH (27%), high CD38 expression (16%), poor risk interphase cytogenetics (11%), and short lymphocyte doubling time (9% of subjects with doubling time of less than 1 year).
The capacity of LMP-420 to induce cytotoxicity in CLL cells was assessed using the MTS assay.
As seen in
To further characterize the cytotoxic effect of LMP-420 on CLL cells, apoptosis was assessed in a second series of patients.
In these 43 CLL patient samples incubated with drug for 24, 48, or 72 hours, LMP-420 induced statistically significant apoptosis in a time-dependent manner (p<0.0001, Wilcoxon signed-rank test). Incubation with 5,000 nM LMP-420 resulted in median drug-specific apoptosis of 15%, 28%, and 32% at 24, 48, and 72 hours, respectively (
There was a statistically significant dose-dependent increase in apoptosis in a series of 50 CLL samples incubated with 50, 500, or 5,000 nM LMP-420 for 72 hours (
A more pronounced cytotoxic effect was observed by MTS assay than by annexin V staining at 72 hours. To assess the extent to which CLL cells had undergone prior apoptosis and lysis (“secondary necrosis,” Reference 18), we quantified nucleosomes in the cell culture supernatant and in cell lysate at 72 hours (
The apoptotic effect of LMP-420 was associated with decreased levels of the anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl-2 at 72 hours (
LMP-420 Cytotoxicity for CLL Cells from Patients in Poor Prognostic Groups
Drug response was analyzed for CLL samples categorized according to patient characteristics. Cytotoxicity and level of drug-specific apoptosis observed with LMP-420 treatment did not correlate with Rai stage, prior treatment, or cell surface expression of Zap70 or CD38.
However, unmutated IgVH status was associated with significantly greater LMP-420-induced apoptosis than mutated IgVH status (p<0.01, Wilcoxon rank-sum test). The median percent drug-specific apoptosis after 72-hour incubation with 5,000 nM LMP-420 was 34% for the mutated IgVH subgroup (n=30) and 59% for the unmutated IgVH subgroup (n=10). No significant difference in ED50 by IgVH status was observed by MTS assay in a partially overlapping series of patients (p=0.5, Wilcoxon rank-sum test).
Samples from patients with unfavorable cytogenetic profiles were also examined by apoptosis and cytotoxicity assays. 5,000 nM LMP-420 induced apoptosis in patients with 11 q22 deletion or complex cytogenetics (including 11q22 or 17p13 deletion), with ranges from 13-58% (n=3). CLL cell samples with poor risk cytogenetic aberrations had a median MTS ED50 of 332 nM (n=5, range 92 to >1000 nM), while favorable risk cytogenetic aberrations had a median MTS ED50 of 162 nM (n=27, range 27 to 2522 nM) (p=0.3, Wilcoxon rank-sum test).
To determine if LMP-420 treatment can inhibit the proliferation of leukemic cells that may be responsible for maintaining the larger population, CLL cells were stimulated to divide in vitro (References 14 and 19).
LMP-420 inhibits proliferation of CLL cells Incubation with LMP-420 for two hours prior to stimulation suppressed proliferation in a dose-dependent manner (
Fludarabine is an integral component of many CLL therapeutic regimens. Consequently, LMP-420 was evaluated to determine if it could potentiate the anti-CLL activity of fludarabine in vitro.
We initially hypothesized that LMP-420 induced cytotoxicity and suppressed proliferation of CLL cells by inhibiting TNF production and its subsequent autocrine effects. To determine if this was the case, CLL cells were incubated with serial dilutions of LMP-420 in combination with 1 ng/mL or 25 ng/mL recombinant human TNF. Addition of exogenous TNF did not abrogate the cytotoxicity of LMP-420 at 72 hours (n=9, data not shown). These results were confirmed with apoptosis assays treating cells with 50 ng/mL TNF in combination with LMP-420 (n=4, data not shown). Thus, the mechanism by which LMP-420 is toxic to CLL cells is not likely due to the suppression of TNF production.
To study the mechanism of action of LMP-420 further, gene expression profiling was performed on CLL cells from thirteen patients after a 24-hour incubation with LMP-420 or media alone.
Expression of 763 gene probes was increased, and expression of 633 gene probes was suppressed by treatment with 2 μM LMP-420 (
Gene expression effects were further assessed by applying genomic signatures of TNF and IFN-α pathway deregulation to the gene expression data. We found a significant reduction in the TNF and IFN-α pathway signature predictions for the LMP-420-treated cells compared to control cells (p=0.027 and 0.003, respectively, Wilcoxon signed-rank test). These genomic alterations in cytokine pathways confirm the results obtained by analyzing gene annotations.
As seen in the gene lists in
To determine whether the cytotoxic effect of LMP-420 is specific to leukemia cells, PBMCs from healthy donors were incubated with a range of doses of LMP-420 for 72 hours. The median ED50 of LMP-420 on normal PBMCs was not reached at concentrations of up to 512 μM in the MTS assay. The therapeutic index (PBMC ED50/CLL ED50) is therefore greater than 2000, based on the mean ED50 of 245 nM observed in CLL samples in this study. For comparison, the toxicity of several chemotherapeutics currently used in the treatment of CLL was examined, using drug concentrations reported in the literature and observed in MTS assays performed in our laboratory. Using the lowest reported ED50 values for CLL cells, the therapeutic index for each of these drugs is below 30 (Table 2).
To assess the apoptotic effect of LMP-420 on normal B- and T-lymphocyte subsets, normal PBMCs incubated with LMP-420 for 72 hours were stained with annexin V and CD19 or CD3.
As noted in
In contrast to normal B and T cells,
Currently-accepted CLL chemotherapeutics are associated with immuno- and myelosuppression, placing patients at risk for infectious complications during treatment (References 2 and 20). To mimic the proliferative response of immune cells to foreign agents, normal PBMCs from healthy donors (n=3-5) were stimulated for 96 hours with B- and T-cell mitogens in the presence of LMP-420 or other drugs. The doses required to inhibit proliferation by 50% (IC50) in stimulated PBMCs ranged from less than 1 μM to greater than 90 μM (Table 3). The IC50 values for LMP-420 were 100- to 250-fold higher than the mean ED50 of LMP-420 in CLL cells. In contrast, the IC50 values for the other chemotherapeutics tested were no greater than 15-fold above the concentrations of these agents that caused in vitro cytotoxicity to CLL cells (refer to Table 2 for CLL ED50 values). Thus, LMP-420 has dramatically less effect on the proliferation of normal PBMCs at therapeutic CLL cell doses than these cytotoxic chemotherapy agents.
Colony formation assays were performed to examine the effect of LMP-420 on colony formation by hematopoietic progenitor cells isolated from healthy donors (n=2). Erythroid and myeloid colony counts were compared among plates cultured in the presence or absence of various doses of LMP-420 or fludarabine. The mean IC50s of LMP-420 for normal erythroid and myeloid colony formation were 10 and 25 μM, respectively, while those for fludarabine were 5 and 1 μM, respectively. Thus, the IC50s for LMP-420 suppression of both colony types were greater than 40-fold above than the mean cytotoxic ED50 against CLL cells. By contrast, the mean IC50s of fludarabine for suppressing erythroid and myeloid colony formation occurred at concentrations four-fold above and equal to the CLL ED50 for fludarabine, respectively. These results demonstrate the relative specificity in toxicity of LMP-420 for CLL cells compared to normal hematopoietic cells.
Lack of Effect of LMP-420 on the Human Potassium Channel hERG.
We have demonstrated that LMP-420 has potent cytotoxic and anti-proliferative effects on primary CLL cells in vitro. Incubation with LMP-420 resulted in time- and dose-dependent loss of viability through primarily an apoptotic mechanism. Normal PBMCs were not significantly affected by LMP-420 at concentrations well above the mean ED50 for CLL cells. LMP-420 had neither the immuno- nor the myelosuppressive properties of fludarabine, but still potentiated the cytotoxic effects of this agent. LMP-420 does not bind to the human hERG (human Ether-α-go-go-Related-Gene) cardiac ion channel. This makes it unlikely that the agent will cause significant cardiac problems in humans. Our data support efficacious and therapeutic uses of LMP-420 for CLL, either alone or in combination with other agents. Combination chemo-immunotherapy with the fludarabine, cyclophosphamide, and rituximab (FCR) regimen is currently the front-line standard of care (References 4 and 21). Co-morbid conditions, advanced age, and infectious complications reduce the number of patients eligible for this treatment and the number of cycles that patients are able to receive (References 2 and 4). Given these barriers to treatment and the lack of full effectiveness of current therapies, better treatments with improved toxicity profiles are needed.
At least one major advantage of LMP-420 over conventional chemotherapy is its specificity for CLL cells over normal cells. Our results demonstrate that LMP-420 induces cytotoxicity and inhibits proliferation in CLL cells but not in normal PBMCs. By contrast, fludarabine concentrations that are toxic to CLL cells also cause greater than 50% apoptosis of normal lymphocytes and suppress mitogen-stimulated proliferation of normal PBMCs. Our prior studies in rodents showed that LMP-420 is non-toxic in vivo at doses achieving a peak plasma level greater than 10 μM. These results suggest that LMP-420 will be a good treatment for CLL therapeutic with low toxicity relative to currently-approved chemotherapies for CLL.
It is important to note that LMP-420 is cytotoxic to malignant cells from CLL patients with a broad range of clinical characteristics. Regardless of Rai stage, ZAP-70 or CD38 positivity, IgVH mutation status, or previous treatment, CLL cells are sensitive to the apoptotic effect of LMP-420. Likewise, apoptosis induced by 5,000 nM LMP-420 was 13 to 58% in samples with unfavorable cytogenetics. There was no significant difference in cytotoxicity between samples with poor-risk (n=5) or favorable-risk (n=27) cytogenetics (ED50 332 vs. 162 nM, p=0.3). Although our analysis of certain subgroups is limited by sample size, we believe that LMP-420 will be useful for the treatment of poor-risk CLL patients for whom current therapeutic options are limited.
LMP-420 induces CLL cell-specific cytotoxicity via apoptotic mechanisms. This may be linked to its ability to alter the cytokine milieu and modulate expression of cell surface receptors involved in interactions with supporting cells in the microenvironment. LMP-420 potently inhibits macrophage and lymphocyte production of TNF and interferon-gamma (Reference 8—unpublished data). Both cytokines can inhibit CLL cell apoptosis, and TNF can also stimulate CLL cell proliferation (References 6 and 22-24). LMP-420 decreases endothelial cell expression of CD40 (Reference 25), a factor implicated in CLL cell survival and proliferation through interactions with CD40 ligand (CD40L) in the microenvironment (References 26 and 27). Likewise, we found significantly decreased cell surface expression of CD40 in CLL cells after treatment with LMP-420. CD40 down-regulation could impact cell survival in our culture conditions by decreasing interactions with CD40L-expressing CLL cells (Reference 28) or the small percentage of T cells potentially remaining after purification.
CLL is thought to be a malignancy with a primary defect in apoptosis, with elevations of Mcl-1 and Bcl-2 contributing to apoptosis resistance (References 29 and 30). Prior research has shown that siRNA suppression of Mcl-1 expression is sufficient to induce apoptosis in CLL cells (Reference 31). We observed decreased expression of the key anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl-2 in CLL cells after treatment with LMP-420.
Recent studies have indicated that CLL progression involves not only decreased apoptosis, but also in vivo proliferation of a small, but significant subset of the CLL clone (Reference 32 and 33). Higher CLL cell birth rates in vivo are associated with more aggressive disease. Therefore, targeting these proliferating CLL cells has therapeutic importance (Reference 20 and 34). We assessed the effect of LMP-420 on CLL cells that were stimulated in vitro to proliferate with the CpG-ODN DSP30 and IL-2 (Reference 14 and 35). We believe that this stimulated proliferation may reflect the in vivo proliferative compartment, because both higher proportions of proliferating CLL cells in vivo and proliferative response to DSP30 in vitro are associated with progressive disease (Reference 36). LMP-420 significantly inhibited proliferation of in vitro-stimulated patient samples. This demonstrates that LMP-420 has deleterious effects on both resting and proliferating CLL cells. Inhibition of CLL cell proliferation by LMP-420 occurred at concentrations well below those affecting normal PBMC proliferation in response to mitogens or alloantigens. Given this novel specificity for CLL lymphocytes, LMP-420 will be an important adjunct to currently available CLL treatments. It is recommended that LMP-420 be administered intravenously, orally, or subcutaneously at a dosage of 0.1 to 50 mg/kg body weight, when treating a CLL patient.
We initially hypothesized that LMP-420 would be toxic to CLL cells via inhibition of autocrine TNF production. However, our results indicate that the mechanism of action of LMP-420 is more complex, with gene expression profiling suggesting it involves the inhibition of NF-kappa B, toll-like receptor signaling pathways, and cytokine-cytokine receptor interactions. For example, LMP-420 induced the expression of RhoH and SQSTM1, both known to suppress the NF-kappa B pathway (References 37 and 38). Likewise, LMP-420 suppressed the expression of CD40, TICAM2 (TRAM), and TNFSF10 (TRAIL), all of which activate the NF-kappa B pathway (References 39-41). These results provide more explanation for the CLL cell cytotoxic activities of LMP-420, given that constitutive and inducible NE-KB activity support CLL cell survival, and inhibitors of this pathway enhance CLL cell death (References 42 and 43).
One would observe from the above that the present invention demonstrates that LMP-420 is cytotoxic to primary CLL cells from patients with low- or high-risk prognostic factors. Furthermore, the drug potentiates the activity of fludarabine and exhibits a high degree of selectivity for leukemic B cells as compared to normal blood cells. LMP-420 also has the practical benefits of being inexpensive to synthesize, highly stable, well-tolerated in vivo, and orally bioavailable (Reference 44). In addition, LMP-420 has a favorable toxicity profile and efficacy both as a single agent and in combination with fludarabine.
†4-hydroperoxycyclophosphamide (4-HC) is a stable precursor to the cyclophosphamide metabolite 4-hydroxycyclophosphamide, which is active in vitro.
While this invention has been described as having preferred sequences, ranges, steps, materials, structures, features, components, or designs, it is understood that it is capable of further modifications, uses and/or adaptations of the invention following in general the principle of the invention, and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains, and as may be applied to the central features hereinbefore set forth, and fall within the scope of the invention.
The following references, and those cited or discussed herein, are hereby incorporated herein in their entirety by reference.
The work leading to the present invention was funded in part by the U.S. Government (NIH and the Department of Veterans Affairs), and the Leukemia/Lymphoma Society. The U.S. Government therefore has certain rights in the invention.
