WO1995024199A2 - Use of compounds which inhibit phosphatidic acid formation for the manufacture of a medicament for the treatment of cancer - Google Patents
Use of compounds which inhibit phosphatidic acid formation for the manufacture of a medicament for the treatment of cancer Download PDFInfo
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- WO1995024199A2 WO1995024199A2 PCT/US1995/003081 US9503081W WO9524199A2 WO 1995024199 A2 WO1995024199 A2 WO 1995024199A2 US 9503081 W US9503081 W US 9503081W WO 9524199 A2 WO9524199 A2 WO 9524199A2
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- dimethylxanthine
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- hydroxyundecyl
- dodecylamino
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
Definitions
- the present invention provides a method for treating a wide variety of cancers using a novel approach to involve abnormal cell signaling mechanisms that are active in a transformed cellular phenotype.
- the novel approach is based upon the discovery that certain phospholipid-based cell signaling enzymes are a common event of oncogenic mutations that result in abnormal and continuous stimulation of a phospholipid signaling pathway. Several inhibitors of this cell signaling mechanism are provided. Background of the Invention
- Cancer is a disease generally characterized by growth and spread of malignant tumors. Tumors result from rapid cell growth not controlled by the body's normal regulatory mechanisms. A tumor is considered malignant, and therefore a cancer, when it demonstrates unrestrained growth and a capacity to invade remote areas of the body. The exact causes of cancer are still largely unknown. However, recent discoveries have focused attention on a class of related genes that appear to control cancer cell growth and differentiation. These genes are termed "oncogenes" and are expressed in a variety of human cancers. When ' mutated, oncogenes encode for proteins, which appear to be involved in the process by which tumor cells respond to growth factors and other extracellular stimuli, and cause the cells to grow and proliferate in an unregulated "malignant" state. The malignant nature of cancer is its relentless progression throughout the body (a process termed metastasis), invading and destroying normal organs and leading to the death of the patient.
- metastasis a process termed metastasis
- Cancer cells secrete enzymes called metalloproteases which "breakdown" blood vessel walls and allow the cancer cells to enter the bloodstream and form tumors elsewhere. This process is termed proteolysis.
- metalloproteases which "breakdown" blood vessel walls and allow the cancer cells to enter the bloodstream and form tumors elsewhere. This process is termed proteolysis.
- Type IV collagenase One of these enzymes, called Type IV collagenase, is associated with tumors that metastasize or spread. Attachment of tumor cells to blood vessel walls and to normal organs occurs through tumor cell receptors (or adhesion receptors) called integrins. Attachment appears to be necessary to allow tumors to reside in different organs in the body.
- Cancer is a disease generally characterized by the growth and spread of malignant tumors. Tumors result from rapid cell growth and are not controlled by the body's normal regulatory mechanisms. A tumor is considered malignant, and therefore a cancer, when it demonstrates unrestrained growth and a capacity to invade remote areas of the body.
- An additional feature of cancer cells is their ability to secrete certain proteins, such as bFGF that stimulates the development of new blood vessels (a process termed angiogenesis or neovascularization). By doing so, as cancerous tumors enlarge, they develop new blood vessels to bring them a sufficient supply of nutrients for growth.
- the steps in tumor cell growth and metastasis are shown in Figure 1.
- the 5 step cancer cascade includes all the components necessary for successful growth and spread (metastases) of tumor cells to distant sites: proliferation (e.g., growth), proteolysis, migration, adhesion and angiogenesis.
- Neovascularization is critical for the growth for tumors and is important in a variety of angiogenic diseases, such as diabetic retinopathy, arthritis, psoriasis and haemangiomas. More than 70% of cancer patients die from metastatic dissemination of the initial tumor. Tumor neovascularization is the crucial process for survival of a primary tumor and for metastatic dissemination. Angiostatic steroids and heparin with anti-angiogenic agents such as protamin have been used as therapies to suppress tumor growth. These therapeutic approaches have serious limitations, because when the dose of heparin exceeded an optimum level for inhibition of angiogenesis, both tumor growth and angiogenesis were stimulated. Also, high doses of cortisone that are required for antiangiogenesis led to immunosuppression. Acquisition of an angiogenic phenotype marked a transition from hyperplasia to neoplasia (Folkman et al., Nature, 339:58-60 1989).
- PA Phosphatidic acid
- PI- PLC ⁇ phosphatidylinositol-directed phospholipase C ⁇
- ras-GAP ras-deactivating protein
- 1 ,2-sn-dipalmitoyl PA which is derived largely from PC (Song et al., Biochem. J. 294:711-717, 1993), is not effective in inhibiting ras-GAP, but is effective in inhibiting rho-GAP (Tsai et al., Mol. Cell. Biol. 9:5260-5264, 1989).
- PA's with varying provenance are produced simultaneously in response to the same stimulus.
- An example of this is the PDGF effect on Balb/3T3 fibroblasts. Kiss (Kiss,
- Phospholipase D activity in this system, as well as PA mass, are associated with mitogenesis (Boarder, Trends in Pharm. Sci. 15, 57-62, 1994; and Boarder, Trends in Pharm. Sci. 15, 57-62, 1994; and Fukami et al., J. Biol. Chem. 267:10988-10993, 1992).
- 1,2-dilinoleoyl PA production is a marker of activation of LPAAT (Kanoh et al., Cellular Signaling 5:495-503, 1993), whereas the latter PA species with arachidonate in the sn-2 position is associated with activation of PI-PLC ⁇ and DG kinase.
- Activation of phospholipase D does not always produce PA (or phosphatidylethanol in the presence of ethanol), but may produce either bis (diacylglycerol) phosphate (bis[PA]), bis (monoacylglycerol) phosphate (lyso(bis)PA), or closely-related species derived from phosphatidylglycerol. This is observed for a variety of glycerol forms containing free hydroxyl (-OH) moieties (Tettenborn et al., Biochem. Biophys. Res. Comm. 155:249-255, 1988; and Guillemain et al., Amer. J. Physiol. 266:C692-C699, 1994). Hence, lipid metabolism centering on PA and its relatives as a signaling molecule is apparently complex and structure- dependent.
- the present invention provides a method for treating cancer and preventing metastatic spread of cancer cells in a patient having cancer, comprising administering an effective amount of a compound that inhibits formation of phosphatidic acid (PA4) species within cancer cells.
- the method for treating and preventing metastatic spread of cancer cells in a patient having cancer comprises administering an effective amount of a compound that inhibits PC- PLD ⁇ (phosphotidylcholine phospholipase D Type beta).
- compounds that both inhibit formation of PA4 species within cancer cells and inhibit PC-PLD ⁇ activity include, for example, l-(l l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, and 1-(11-N- octylaminoundecyl)-3,7-dimethylxanthine.
- the PA4 species include, for example, PC-derived PA's having a myristylated and palmitoyl sn-1 and sn-2 side chains without alkyl, ether or vinyl ether side chains.
- Such PA4's include, for example, those PA's seen in mass spectroscopy (Fab negative) having molecular weights of 619, 627, 643-649, 677, 703, 707 and 587.
- PA4 species include lyso (bis) PA species having arachidonate and polydocososaenate sn- 2 and sn-1' species, and lyso (bis) PA sn-2 arachidonoyl and sn-2 eicosatrienoyl species.
- the present invention provides a rational drug development approach to finding multiple-active compounds that are useful for the treatment of a wide variety of cancers, including, decreasing tumor cell growth by blocking oncogene induced events, blocking autocrine or paracrine growth factor stimulation of tumor cells, decreasing metastatic potential by blocking metalloprotease production, decreasing tumor adhesion to normal organs by blocking adhesion receptors, and decreasing the ability of tumors to induce nutrient carrying blood vessel formation by blocking bFGF or other tumor-dependent growth factor signaling.
- Compounds with this spectrum of activities are useful for treating and preventing the metastatic growth of cancer cells and are illustrated herein.
- Such compounds i.e., small molecule pharmaceuticals
- the illustrated compounds appear in vitro to be non-toxic to normal cells at concentrations which are lethal to cancer cells.
- the present invention is further based upon the pioneering discovery that certain PA species are released in response to pro-inflammatory stimuli mediated by, by example, PDGF, EGF, FGF and VEGF, and that the increase in the PA species can be inhibited by addition of certain compounds. Therefore, this invention has resulted in the discovery of a new class of compounds useful for treating or preventing the progression of a large group of diseases mediated by such pro-inflammatory cytokines and treatable by inhibition of intracellular signaling of such pro-inflammatory cytokines.
- PA inhibition in response to inflammatory stimulus is useful for treating diseases associated with increased proliferation in response to PDGF, VEGF, EGF, or FGF or other heparin-binding growth factors such as Her2,3,4/regulin, IGF-1 or 2.
- a number of intracellular signaling events take place following PDGF, EGF, FGF or VEGF binding to their respective cell surface receptors. All of the receptors in this class of cytokines possess intrinsic tyrosine phosphorylation activity. Shortly after binding, the receptors are phosphorylated at various sites in their intracellular domain by intrinsic tyrosine kinase activity of the receptor. This leads to the creation of additional binding sites for intracellular proteins.
- PLC- ⁇ -1 phospholipase C- ⁇ -1
- GAP GTPase activating protein
- PBkinase phosphatidylinositol 3 kinase
- pp60c- src pp60c- src
- p62c-ye.s pp50-fyn
- Nek a kd species
- CRB2 CRB2 as well as a 120 kd and a 64 kd species.
- Some of the proteins that associate with the receptor are signal transduction enzymes.
- PLC- ⁇ -1 is a specific phosphodiesterase that produces diacylgycerol (DAG) and inositol triphosphate, two second messengers that activate a serine/threonine specific protein kinase protein kinase C (PKC) and increase intracellular calcium levels.
- DAG diacylgycerol
- PLC protein kinase C
- PBkinase is a lipid kinase that phosphorylates the D3 position of phosphatidylinositol phosphatidylinositol-4-phosphate, or PI 4,5,P2.
- Figure 1 illustrates a graph of illustrated PA species, including PAj, PA2, PA3 and PA4 species (note Fab negative mass spec, numbers) of NCI-H460 tumor cells before and after treatment with 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (4 ⁇ M).
- Figure 2 illustrates the effect of 1 -( 11 -dodecylamino- 10-hydroxyundecyl)-3,7- dimethylxanthine on spontaneous PA levels in two kinds of cancer cell lines.
- HT-29 and MCF-7 cells were treated with different concentrations of 1-(11 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine and the content of different PA determined by HPLC analysis.
- HT-29 colon carcinoma cells and MCF7 breast cancer cells were grown to 80% confluence, followed by fixation in ice-cold methanol, extraction of lipids, and separation of lipid species by HPLC. Under these conditions, each tumor cell line was found to spontaneously produce large amounts of differently migrating PA species, especially PAj species (migrating Rf 4-7) and PA2 (Rf 9-11).
- the cells were grown in the presence of 1-(1 1- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine at different concentrations, followed by a reexamination of lipids.
- 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine was found to suppress spontaneous production of these PAj and PA2 species at the concentrations indicated.
- Figures 3 and 4 show anti-proliferative ( Figure 3) and anti-clonogenicity ( Figure 4) of 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine of HT-29 cells.
- HT-29 cells (1 x 10 ⁇ cells/35 mm dish) were plated in McCoy's medium with 10% serum and incubated overnight. Concentrations of 3 and 6 ⁇ M 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7- dimethylxanthine was added and viable cell counts made at the times shown.
- treated and control cells 300/plate were plated and allowed to grow colonies.
- Figure 4 are the means of 3 plates.
- Figures 5 and 6 illustrate cytotoxicity and concentration dependence of 1 -( 11 - dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine against 3LL cells (Lewis lung carcinoma).
- 3LL cells (3 x 10 ⁇ cells/well) were plated and incubated overnight in RPMI medium containing 10% serum.
- l-( 11 -dodecylamino- 10-hydroxyundecyl)-3 ,7- dimethylxanthine was added at different concentrations and the cell number was determined at various time points by a vital dye uptake method.
- the values shown are triplicate of wells.
- Figure 7 shows that 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine, even at much higher concentrations than shown having cytotoxicity to tumor cells, lacks cytotoxic activity in normal human bone marrow stromal cells.
- Human bone marrow stromal cells (1 x l ⁇ 4 cells/well) were plated in 96 well plates in McCoy's medium with serum and incubated overnight. Different dilutions of 1 -( 11 -dodecylamino- 10-hydroxyundecyl)-3 ,7- dimethylxanthine were added and viable cell counts made by vital dye uptake.
- Figure 8 illustrates a densitometric analysis of matrix metalloproteinase expression measured from gelatin Zymogram gel with 1-(1 1 -dodecylamino- 10-hydroxyundecyl)-3,7- dimethylxanthine.
- THP-1 human leukemia cells 1-2 x 10 ⁇ /35 mm dish
- 1 -( 11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine was added at 2.5 ⁇ M.
- TNF ⁇ 0.5 ng/ml
- the supernatants from control and treated plates were collected and protease activity was determined in gelatin gels (zymogram) after electrophoretic separation of proteins.
- Figures 9 and 10 show the effects of 1-(11 -dodecylamino- 10-hydro yundecyl)-3,7- dimethylxanthine on matrigel invasion and viability in 3LL cells.
- 3LL cells (4.5 x 10 ⁇ cells/well) were plated into the inner membrane of Matrigel chambers. Different concentrations of 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine were added to the chamber and incubated for 48 hours at 37 °C. The cells on top of the membrane were removed and the cells that migrated to the bottom were stained with Diff Quick Solutions and scored for relative invasion. The effect of 1-(11 -dodecylamino- 10-hydroxyundecyl)-3 ,7- dimethylxanthine on viability of 3LL cells at different concentrations was determined separately.
- Figure 11 illustrates the effect of 1-(11 -dodecylamino- 10-hydroxyundecyl)-3 ,7- dimethylxanthine on PDGF-BB (platelet-derived growth factor) induced proliferation of Balb/3T3 cells as a predictive assay of angiogenesis.
- 3T3 cells (3 x 10 3 cells/well were plated in DMEM with 10% serum in 96 well plates and incubated for 48 hours. Different dilutions of 1 -( 11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine were added and incubated for 1 hour.
- PDGF 50 ng/ml
- tritiated thymidine 1 ⁇ Ci/ml
- Cell proliferation was measured by measuring tritiated thymidine incorporation with each sample run in quadruplicate.
- FIG 12 illustrates vascular endothelial growth factor (VEGF) induced proliferation of human umbilical vein endothelial cells (HUVEC) as an assay for adhesion.
- HUVECs were plated in EBM medium with serum and allowed to grow for 4 days. Different dilutions of 1- (11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine were added to the plates, followed by VEGF (50 ng/ml) along with tritiated thymidine (1 ⁇ Ci/ml). Proliferation was measured in quadruplicate and these data show the effect of 1-(11 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine to inhibit adhesion.
- VEGF vascular endothelial growth factor
- Figure 13 shows the effect of 1-(11 -dodecylamino- 10-hydroxyundecyl)-3 ,7- dimethylxanthine on THP-1 adherence to LL-lbeta stimulated HUVECs.
- HUVECs (4 x 10 3 cells/well) were plated in RPMI medium with 10% serum and incubated for 48 hours. Different concentrations of 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine were added and incubated for 1 hour; IL-lbeta (15 ng/ml) was added and incubated for 6 hrs.
- FIG. 14 illustrates the effect of 1-(11 -dodecylamino- 10-hy droxyundecyl)-3, 7- dimethylxanthine on VCAM-1 surface expression of TNF ⁇ stimulated HUVECs. This assay is a predictive model of adhesion. HUVECs grew to 90% confluence in 6 well plates in RPMI medium with 10% serum.
- TNF ⁇ (20 ng/ml) was added and the cells were incubated for 5 hours. The cells were collected and the amount of VCAM-1 determined by indirect immunostaining followed by FACS (fluorescence activated cell sorter) analysis. Mean fluorescent intensity of TNF-stimulated cells was normalized to 100% with drug-treated samples expressed as a percent of control.
- Figure 15 illustrates the effect of 1-(11 -dodecylamino- 10-hy droxyundecyl)-3, 7- dimethylxanthine on ICAM-1 surface expression of TNF ⁇ stimulated HUVECs.
- This assay is a predictive model of adhesion. The procedures followed were the same as in Figure 14 except that the cells were stained with an ICAM-1 antibody.
- Figure 16 illustrates the T cell and B cell response assays of the mice treated with 1- (1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine.
- Spleens from the treated mice were made into single cell suspensions in RPMI medium supplemented with 10% serum and placed (200,000 cells/well) in flat-bottomed 96 well plates.
- Anti-CD3 or a mixture of an anti- mu/IL-4 were added to the wells at final concentrations of 1 ⁇ g/ml and 1 ⁇ g/ml/12.5 ng/ml, respectively.
- Appropriate positive and negative controls were set up on each plate and all samples were assayed in quadruplicate. The plates were incubated for 2 days and proliferation was measured by tritiated thymidine incorporation.
- Figure 17 illustrates an in vivo experiment showing that 1-(11 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine can arrest growth of Lewis Lung Carcinoma in mice.
- BDFI mice were injected (s.c.) with 1 x 10 ⁇ 3LL cells on day 0 and then treated with 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (20 mg/kg i.p.), cyclophosphamide (20 mg/kg) or vehicle on alternate days beginning on day 7.
- the animals were sacrificed on day 20 and the lung tumors dissected and weighed.
- Figure 17 illustrates that 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine showed superior results over an existing cancer chemotherapeutic agent.
- Figure 18 illustrates platelet and neutrophil counts of the sacrificed mice in the experiment in Figure 17. In addition, platelet and neutrophil counts in the mice were not altered from vehicle, indicating that the bone marrow was not a side of toxicity for 1-(11- dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine.
- Figure 19 illustrates mass spectroscopy tracings of HPLC-isolated PA and liso(bis)PA fractions from bone marrow stromal cells.
- Figure 20 illustrates mass spectroscopy tracings of HPLC-isolated PA and liso(bis)PA fractions from NCI H460 tumor cells.
- Figure 21 illustrates mass spectrometric (FAB-PI) analysis of HPLC-isolated PC fractions from NCI H460 tumor cells.
- HPLC-derived PC fractions from NCI H460 cells in the presence (21 hrs.) and absence of 10 ⁇ M l-( 11 -dodecylamino- 10-hydroxyundecyl)-3 ,7- dimethylxanthine were isolated, collected, dried under N2, and stored at -70° C under argon.
- the present invention was made as a result of discovering an entire new approach and therapeutic target of cancer therapy.
- a prototype inhibitor compound 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, has been discovered that acts by inhibiting cellular accumulation of specific PA species and was a likely inhibitor of PC directed PLD ⁇ .
- the present invention provides a method for treating and preventing the spread of cancers comprising treating a patient with a tumor with an effective amount of a compound that reduces cellular accumulation of PA4 species.
- PA 1 species include LPAAT (lysophosphatidic acid acyl transferase)-derived PA's, such as dilinoleoyl (C18 unsaturated) PA's.
- PA2 species include PE
- PA species including alkyl, and alkeneyl (saturated) sn-1 and sn-2 PA species (e.g., mass spectroscopy Fab negative 717, 737, 749, 773, 767 and 753).
- PA3 species include PiG (glycan phosphatidylinostitol) directed PLD species, including l-O- myristylated, myristylated, and myristylated palmitoyl PA species.
- PiGPLD is activated by the oncogene v-src.
- PA4 species include PC (phosphotidylcholine) directed PLD ⁇ including many myristylated PA species. According to the terminology employed herein, PC-PLD ⁇ is distinguished from PC-PLD ⁇ . PC-PLD ⁇ is stimulated by sphingosine- 1 -phosphate, resulting in production of different PA species.
- Inhibition of PA generation leads to inhibition of selective biologic effects transduced by specific receptor-ligand interactions.
- Inhibition of PA does not inhibit normal homeostatic functions; high concentrations of these inhibitors are non-toxic to cells in vitro and have no apparent toxicities when given to animals at doses yielding plasma concentrations far in excess of their ⁇ 's for biologic effects.
- Table 1 lists the designated PA species produced in response to receptor-ligand interactions and the respective class of inhibitor and prototypic compound.
- a compound selectively blocks one or more components of the biologic signal transduced by the ligand (e.g., proliferation or upregulation of cell surface receptors)
- the ligand e.g., proliferation or upregulation of cell surface receptors
- activation of other signaling pathways such as phosphorylation of receptors, or src-homologous proteins or activation of PLC ⁇ are unaffected by compounds which inhibit PA production.
- Post receptor events such as MAP kinase activation, calcium flux, upregulation of nuclear protooncogenes, or proliferation signaled by ligands that do not use the PA pathway are similarly unaffected by these compounds.
- PA4 inhibitors exemplified by 1-(1 l-dodecylamino-10-hydroxyundecy ⁇ )-3,7- dimethylxanthine, have a broad spectrum of activities which indicate they have novel anti- cancer therapeutic properties.
- 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine was cytotoxic to all solid tumor cell lines tested with LCso's (the concentration at which 50% of cells die) of approximately 4 ⁇ M (micromolar) with an 8 to 18 hour exposure to the compound.
- PA4 inhibitors exemplified by 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine
- PA4 inhibitors are unique in that they inhibit several major steps in tumor progression.
- Compounds of this class are cytotoxic and growth inhibitors to tumors. They also inhibit metastasis and angiogenesis.
- l-(l l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine was a potent inhibitor of endothelial cell proliferation stimulated by PDGF, acidic or basic FGF and VEGF.
- the 50% inhibitory concentrations (Kilo's) with each of these ligands is ⁇ 500 nM.
- 1-(1 1- dodecylamino- 10-hy droxyundecyl)-3,7-dimethylxanthine also completely inhibited expression of the 92 kd, type IV collagenase (MMP9) associated with tumor invasion at a concentration of 2.5 ⁇ M by direct assays of gelatin gels.
- MMP9 type IV collagenase
- the functional effect of inhibition of collagenase is evidenced by the ability of 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine to suppress tumor invasion into matrigel at concentrations of ⁇ 0.5 ⁇ M.
- the Cmax was approximately 2 ⁇ M and at 20 mg/kg was extrapolated to be approximately 4 ⁇ M.
- dosed at 20 mg/kg i.p. every other day for 14 days there were no apparent adverse effects on clinical observation, and on gross examination there were no apparent organ toxicities at time of autopsy. No significant weight loss was observed in treated animals. In addition there was no suppression of the neutrophil count, platelet count or of bone marrow or splenic cellularity. In treated animals, T and B cell mediated immune responsiveness was intact.
- the compound also inhibited the primary tumor growth of Lewis lung carcinoma by 45% when established tumors were treated with the compound at 20 mg/kg QOD for 10 days, while cyclophosphamide at 20 mg/kg QD (a common cancer treatment agent) had no effect on the tumor growth.
- the following table shows solid tumor cell cytotoxicity of 1-(11 -dodecylamino- 10- hy droxyundecyl)-3,7-dimethylxanthine for different tumor cell lines.
- the data is expressed as the lethal concentration of drug wherein 50% of the cells have been killed by drug exposure
- Tumor cells were plated (3.5 x 10 3 cells/well) into individual wells of 96 well plates in complete medium with 10% fetal bovine serum. Cells were allowed to grow overnight at 37 °C in a 5% C0 2 incubator. 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7- dimethylxanthine, at various concentrations, was added and incubated at 37 °C. At various time points the medium with 1-(11 -dodecylamino- 10-hy droxyundecyl)-3, 7- dimethylxanthine was replaced with fresh medium and incubated at 37 °C for 24 hours. The LC 50 was determined based on the viable cell number as determined by BCECF, a vital dye stain. Appropriate medium controls were set up, and all test samples and controls were run in triplicate.
- Table 5 shows data from normal (not transformed) cells and their cytotoxicity to 1- ( 1 1 -dodecylamino- 10-hydroxyundecyl)-3 ,7-dimethylxanthine
- Mac-11 a murine IL-3 dependent cell line
- human bone marrow stromal cells were plated in 96 well plates in medium containing serum and various concentrations of 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine. After 4 hours incubation for Mac- 11, or 24 hours for human stromal cells, the LC50 values were determined based on the number of viable cells in 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine exposed samples compared to controls as determined by a vital dye (BCECF).
- BCECF vital dye
- Mouse bone marrow cells obtained from femurs were incubated with various concentrations of 1-(1 1 -dodecylamino- 10-hy droxyundecyl)-3,7-dimethylxanthine for 8 hours. Following incubation, the cells were washed and plated for colony forming unit-granulocyte macrophage (CFU-GM) growth with spleen conditioned medium as the source of colony stimulating factor (CSF). After 7 days of incubation at 37 °C, the colonies formed were counted, and the LC50 was determined. Quadruplicate samples were set up for each compound dilution and control.
- CFU-GM colony forming unit-granulocyte macrophage
- CSF colony stimulating factor
- Drug resistance (using 1 -( 11 -dodecylamino- 10-hydroxyundecyl)-3 ,7-dimethylxanthine) was determined. Approximately 2 x 10 ⁇ ras transformed hamster 3T3 cells, or human NCI- H460 large cell lung tumor cells, were plated into medium containing 20 ⁇ M 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine. After 2 weeks the colonies from both groups were counted, and frequencies of resistance were found to be approximately 10 ⁇ 6.
- 3,7-dimethylxanthine is a low frequency event (i.e., 10"6), thus attempts to develop resistant clones at incremental concentrations (“step wise selection") would prove near impossible.
- similar methods have been successfully employed in the past (Rice et al., Proc. Natl. Acad Sci, USA 83: 5978-5982, 1986 and Rice et al., Proc. Natl. Acad. Sci, USA 84: 9261-9264, 1987) using methotrexate and doxorubicin in generating drug resistant mutants with amplification of either the DHFR gene or the p-glycoprotein gene.
- Lyso(bis)PA species containing arachidonate and other long acyl chains increase in short Rf PA fractions (Rf 6-10 min.), but later (longer Rf) fractions which also contain these lyso(bis)PA species show little significant change.
- NCI H460 cells stimulated with serum demonstrate a more diffuse response in PA synthesis compared to marrow stromal cells.
- arachidonoyl and long-acyl-chain docosapolyenoyl species were synthesized, but substantial concentrations of sn-2-linoleoyl and alkylUinoleoyl species were found.
- the disaturated species 1 ,2-dipalmitoyl, 1- myristoyl 2-stearoyl, and 1-palmitoyl 2-stearoyl PA were found to be increased after stimulation with serum. These latter PA species were present to a greater degree in NCI H460 cells as compared to marrow stromal cells. The advantages of mass spectrometric analysis of HPLC-peaks which migrate similarly is demonstrated by this finding.
- 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine increased PA and lyso(bis)PA mass in NCI H460 cells.
- 1-(11 -dodecylamino- 10-hy droxyundecyl)-3, 7- dimethylxanthine abrogated an increase in linoleoyl, alkyl-linoleoyl, and disaturated PA species, while increasing arachidonoyl and heavy-chain docosapolyenoyl PA species.
- 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine increased arachidonoyl-containing lyso-(bis)PA species in marrow stromal cells.
- 1-(11 -dodecylamino- 10-hy droxyundecyl)-3,7-dimethylxanthine increased PA and PA- related species which contain arachidonate and docosapolyenoates in all cells examined, but decreased the proportion of or abrogated PA species which contain linoleate, alkyl-linoleate, or are disaturated.
- the presence of a disaturated-acyl-specific PC -PLD is supported by data that 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine had a marked effect on preventing hydrolysis of these disaturated sub-species of PC.
- mice An in vivo study in mice was conductedwhere B16 melanoma cells were injected iv through a tail vein on day 0 and 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7- dimethylxanthine was administered i.p. at 10 mg/kg QD or 20 mg/kg QOC starting days 1-14. The mice were sacrificed on day 15 and the lungs were dissected and fixed in formalin, n addition bone marrow toxicity was evaluated by measuring neutrophil and platelet counts in the mice on day 15. These data are shown graphically below the lungs.
- the objective of this study was to demonstrate anti-metastatic activity of 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine against B 16 melanoma in an experimental metastasis model.
- B16 melanoma cells (1 x 10-> per mouse) were inoculated intravenously (i.v.) into tail vein on day 0.
- 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7- dimethylxanthine was administered i.p. at 10 mg/kg, once daily or 20 mg/kg, QOD starting day 1-14. The animals were sacrificed on day 15, and the lungs were dissected and fixed in formalin.
- the study objective was to demonstrate anti-tumor activity and lack of hematopoietic toxicity of 1-(11 -dodecylamino- 10-hy droxyundecyl)-3,7-dimethylxanthine in established tumors in mice.
- BDFI mice from Charles River Laboratories
- Animals were treated with 1-(11 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine (20 mg/kg i.p.) on alternate days starting on day 7.
- the animals were sacrificed on day 20, and the tumors were dissected and weighed. Peripheral blood was also collected at autopsy, and the platelets and neutrophil counts were determined.
- a widely used anti-tumor agent, cyclophosphamide was used as a positive control.
- Human bone marrow stromal cells were plated (10,000 cells per well) in McCoy's medium, supplemented with 12.5% FBS, 12.5% horse serum, 6% juice (1% pen.-strep., 1% glutamine, 1% vitamins, 0.8% essential amino acids, 1% sodium pyruvate, 1% sodium bicarbonate, 0.4% non-essential amino acids, 0.036% hydrocortisone) and fibroblast growth factor (FGF), at a final concentration of 10 ng/ml. Plates containing both cell lines were incubated overnight at 37 °C.
- FGF fibroblast growth factor
- BCECF [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester] diluted in phosphate buffer solution (PBS), was added at 10 ⁇ g/ml, and plates were incubated for 30 minutes at 37 °C. BCECF dye was removed from each well and replaced with PBS. Plates were read at 530 nm on a Millipore fluorescence plate reader. BCECF fluorescence has been shown to correlate linearly with viable cell numbers in the range of 500 - 50,000 cells per well. Average and standard deviations were calculated from BCECF fluorescence units of each well.
- the samples were harvested by first collecting the cell supernatant from each well, then the cells were removed by trypsin. Each well treated with trypsin was added back to the corresponding supernatant sample and the cells were spun down. Each sample was re-suspended in 1 ml McCoy's 5A+10% FBS, and both live and dead cells were counted by trypan blue exclusion procedures. The cell suspensions of each sample are saved and replated at 300 cells per well of a 35 mm dish. The 35 mm dish samples were incubated for 7 days at 37 °C to allow for colony formation. Following this incubation the media was removed and the colonies were fixed with 10% formaldehyde for 1 - 2 hours.
- the Matrigel Invasion chambers consisting of the inner membrane filter and the outer wells, were incubated for 48 hours at 37 °C. After incubation for 2 days, the cells on top of the filter that had not migrated through the inner membrane were removed using a cotton swab and washing the filter with medium. The filter membranes were stained with all three Diff Quick solutions, rinsed and allowed to air dry. Each membrane was removed from the inner chamber and mounted on slides (right side up). Photographs represent the field of each slide, showing those cells that have migrated through the inner membrane and adhered to the surface of the outer membrane. CFU-GM Assay
- Femoral cells were removed using a syringe containing RPMI supplemented with 10% FBS and a 20 gauge needle. Cell were counted, and marrow cellularity was calculated per femur. For the in vivo studies, the cells were plated, colonies assessed 7 days later and CFU- GM per femur calculated. In the in vitro studies, the cells were incubated at one million cells per ml in the presence or absence of 1-(11 -dodecylamino- 10-hy droxyundecyl)-3, 7- dimethylxanthine and Vinblastine for 8 hours. Following incubation the cells were spun down and the supernatant discarded.
- CFU-GM Colony Forming Unit-Granulocyte Macrophage
- Spleens were obtained from mice treated with and without 1-(11 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine, and a single cell suspension was prepared in RPMI medium supplemented with 10% FBS. Two hundred thousand cells were plated into individual wells of flat bottom 96 well plates. Anti-CD3, or a mixture of a Anti-mu/EL-4, were added to the appropriate wells at a final concentration of 1 ⁇ g/ml and 10 ⁇ g/ml/12.5 ng/ml respectively. Appropriate positive and negative controls were set up on each plate, and all samples, along with the controls, were set in quadruplicate. The plates were incubated for two days at 37 °C.
- HUVECs Two days prior to the adhesion experiment, 4,000 human umbilical vein endothelial cells HUVECs were plated into individual wells of round bottom 96 well plates in RPMI media supplemented with 10% FBS. 1 -(11 -dodecylamino- 10-hydroxyundecyl)-3,7- dimethylxanthine at varying concentrations was added to wells containing cells and plates were incubated for 1 hour at 37 °C. Following this pre-incubation, HUVECs were stimulated with human TNF alpha or IL-1 beta at a final concentration of 20 ng/ml and 15 ng/ml respectively. The plates were then incubated for 6 hours at 37 °C.
- THP- 1 cells Human monocytic leukemia cells maintained in exponential growth were prestained with BCECF dye for 15 minutes at 37 °C.
- BCECF dye for 15 minutes at 37 °C.
- the plates were inverted and spun at 850 rpm to remove any additional cells.
- the plates were washed once with warm PBS to remove any additional non-adherent cells from the sides of the wells.
- PBS was added to each well and the plates were read at 485 nm/530 nm on a Millipore fluorescence plate reader. Average and standard deviations were calculated from the BCECF fluorescence units of each well.
- HUVECs were plated into individual wells of flat bottom 96 well plates in Endothelial Basal Media (EBM) supplemented with 5% FBS and endothelial growth factors. After this initial incubation at 37 °C, the media was removed and each well was washed once with PBS. EBM without growth factors and containing 0.5% fetal bovine serum, was added to wells for a 24 hour rest period. 1-(11 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine at varying concentrations was added to wells containing cells, and plates were incubated for 1 hour at 37 °C.
- EBM Endothelial Basal Media
- VEGF tritiated thymidine
- Murine Balb/3T3 cells were maintained in exponential growth. Cells were removed from flasks with EDTA. Three thousand cells were plated into individual wells of flat bottom 96 well plates in DMEM supplemented with 10% fetal bovine serum. After incubating for 48 hours at 37 °C the media was removed, and each well was washed once with PBS. DMEM containing 0.2% FBS was added to wells for a 24 hour rest period. 1-(11 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine at varying concentrations was added to wells containing cells, and plates were incubated for 1 hour at 37 °C.
- PDGF-BB was added to the wells at a final concentration of 50 ng/ml, along with 1 ⁇ Ci of 3 H-TdR, and incubated for 18 - 24 hours.
- Appropriate positive and negative controls were set up on each plate, and all test samples and controls were run in quadruplicate.
- the plates were then harvested, and the incorporation of 3 H-TdR was determined in a liquid scintillation counter. Average and standard deviations were calculated from the CPM of each well.
- Zymogram MMP Assay THP-1 cells were plated at 1 - 2 million cells per well of a 6 well plate in RPMI medium supplemented with 0.5% bovine calf serum (CS).
- the remaining supernatant was removed and the cells were re ⁇ suspended in 0.5 ml RPMI + 0.5% CS. Each cell sample was counted using trypan blue exclusion, counting both live and dead cells. The supernatant samples were diluted 1 : 1 with 2X sample buffer, and each sample was loaded on to a Zymogram pre-poured gel from Novex. The Zymogram gel was run at 200 volts for approximately 1 hour. The gel was removed and incubated for 30 minutes at room temperature in Triton X-100.
- the gel was incubated overnight in a buffer containing 12.5 ml of 1.0 M Tris buffer pH 8.0, 2.5 ml 1.0M CaCl2, 1.0 ml 0.5 mM ZnSO4 and 500 ml H2O. The following day the gel was fixed for 2 hours in a solution of 50% methanol, 10% glacial acetic acid and 40% H2O. The gel was stained with coomassie blue at room temperature, for approximately 4 hours and de-stained overnight. A picture was taken of the gel, and the gel was analyzed using an image analyzer. TNF and IL-1 Induced VCAM Surface Expression
- PA2 species are selected from the group consisting of 1-o-octadecanyl 2-oleoyl PA (687), 1-oleoyl 2-linoleoyl PA (697 or 698), 1-o-octadecanyl 2-linoleoyl PA (681), l-o- octadecanyl-9,12-dienyl 2-linoleoyl PA (679), 1-myristoyl 2-oleoyl PA (645), 1 -o-myristoyl 2- stearoyl PA (633), 1 ,2-sn-dilinoleoyl PA (695), 1-oleoyl 2-linoleoyl PA (697), 1-stearoyl 2- oleoyl PA (701), 1-o-oleoyl 2-20:4 PA (707), 1-o-linoleoyl 2-20:4 PA (705), 1 -o-linoleoyl 2- 20:5 PA (703), and combinations thereof
- (X)j - (core moiety) wherein j is an integer from one to three, the core moiety comprises at least one five to seven- membered ring or an open chain analog of such a ring group and X is a racemate mixture or R or S enantiomer of:
- *C is a chiral carbon atom
- n is an integer from one to four; one or more carbon atoms of (CH2) n av be substituted by a keto or hydroxy group
- m is an integer from one to fourteen
- R ⁇ and R2 are hydrogen, a straight or branched chain alkane or alkene of up to twelve carbon atoms in length, or -(CH2) W R5, w being an integer from two to fourteen and R5 being a mono-, di- or tri-substituted or unsubstituted aryl group, substituents on R5 being selected from the group consisting of hydroxy, chloro, fluoro, bromo, or C ⁇ .g alkoxy; or jointly, R] and R2 form a substituted or unsubstituted, saturated or unsaturated heterocyclic group having from four to eight carbon atoms, N being a hetero atom; and R3 is hydrogen or C1-.3: or
- R4 is a hydrogen, a straight or branched chain alkane or alkene of up to eight carbon atoms in length, -(CH2) W R5, w being an integer from two to fourteen and R5 being a mono-, di- or tri-substituted or unsubstituted aryl group, substituents on R5 being selected from the group consisting of hydroxy, chloro, fluoro, bromo, or alkoxy Cj.g alkoxy, or a substituted or unsubstituted, saturated or unsaturated heterocyclic group having from four to eight carbon atoms, N being a hetero atom; r and s are independently integers from one to four; the sum (r + s) is not greater than five; t is an integer from one to fourteen; and one or more carbon atoms of (CH2) S br (CH2) may be substituted by a keto or hydroxy group, or
- X is independently a resolved enantiomer ⁇ -1 secondary alcohol-substituted alkyl (C5_g) substantially free of the other enantiomer, or X is a branched -(CF ⁇ -CHRg- (CH2)b"R7 > wherein a is an integer from about 4 to about 12, b is an integer from 0 to 4, Rg is an enantiomer (R or S) or racemic mixture (Cj.g) alkyl or alkenyl, and R7 is a hydroxy, keto, cyano, chloro, iodo, flouro, or chloro group.
- the core moiety has from one to three, five to six-membered ring structures in a predominantly planar configuration.
- the amino alcohol substituent (X) is bonded to a ring nitrogen if one exists.
- the core moiety may be selected from the group consisting of substituted or unsubstituted barbituric acid; benzamide; benzene; cyclohexanedione; cyclopentanedione; delta-lactam; flutarimide; glutarimide; homophthalimide; imidazole amide; isocarbostyrile; lumazine; napthlalene; pthalimide; piperidine; pyridine; pyrimidine; pyrrole amide; quinazolinedione; quinazolinone; quinolone; recorsinol; succinimide; thymine; triazine; uracil or xanthine.
- Preferred cores include substituted or unsubstituted xanthine, more preferably halogen-substituted xanthine.
- Exemplary preferred cores include, but are not limited to: 1,3-cyclohexanedione, 1,3- cyclopentanedione; 1 ,3-dihydroxynaphthalene; 1-methyllumazine; methylbarbituric acid; 3,3- dimethylflutarimide; 2-hydroxypyridine; methyldihydroxypyrazolopyrimidine (preferably, 1,3- dimethyldihydroxypyrazolo[4,3-d] pyrimidine); methylpyrrolopyrimidine (preferably, 1- methylpyrrolo [2,3-d] pyrimidine); 2-pyrrole amides; 3-pyrrole amides; 1,2,3,4- tetrahydroisoquinolone; 1 -methyl-2,4( 1 H,3H)-quinazolinedione ( 1 -methylbenzoyleneurea
- the compound are a resolved R or S (preferably R) enantiomer of an ⁇ -1 alcohol of a straight chain alkyl (C5_g) substituted at the 1-position of 3,7-disubstituted xanthine.
- the amino alcohol substituent (X) is bonded to a ring nitrogen if one exists.
- the core moiety is selected from the group consisting of xanthine, halogen-substituted xanthines, 3,7- dimethylxanthine, 3-methylxanthine, 3-methyl-7-methylpivaloylxanthine, 8-amino-3- methylxanthine, 7-methylhypoxanthine, 1-methyluracil, 1 -methylthymine, 1-methyl -5,6- dihydrouracil, glutarimides, phthalimide, l-methyl-2,4(lH,3H)-quinazolinedione (1- methylbenzoyleneurea), 6-aminouracil, homophthalimide, succinimide, 1 ,3-cyclohexanedione, resorcinol, 1 ,3-dihydroxynaphthalene, 1,3-cyclopentanedione, 1,3- dimethyldihydroxypyrazolo[4,3-d] pyrimidine, 5-substit
- the core moiety is a substituted xanthine, such as a 3,7 dimethylxanthine.
- the core moiety can also include a non-cyclic group. Examples of non-cyclic core groups include open chain analogs of glutarimide, carboxilic acid, a hydroxyl group, sulfone, sulfonate, and the like.
- Examples of compounds with demonstrated activity include compounds selected from the group consisting of R-l-(5-hydroxyhexyl)-3,7-dimethylxanthine, N-(l l-octylamino-10-hydroxyundecyl)-homophthalimide, N-(l l-octylamino-10- hydroxyundecyl)-3-methylxanthine, N-(l 1-octylamino- 10-hy droxyundecyl)-2-piperdone, 3- ( 11 -octylamino- 10-hy droxyundecyl)- 1 -methyluracil, 3-( 11 -octy lamino- 10-hydroxyundecyl)- 1 - methyldihydrouracil, l-(9-decylamino-8-hydroxynonyl)-3,7-dimethylxanthine, l-(9- dodecylamino-8-hydroxynon
- the core moiety is a member selected from the group consisting of substituted or unsubstituted: barbituric acid; benzamide; benzene; biphenyl; cyclohexanedione; cyclopentanedione; delta-lactam; flutarimide; glutarimide; homophthalimide; imidazole amide; isocarbostyrile; lumazine; napthlalene; pteridine; pthalimide; piperidine; pyridine; pyrimidine; pyrrole amide; quinazolinedione; quinazolinone; quinolone; recorsinol; stilbene; succinimide; theobromine; thymine; triazine; tricyclododecane; uracil and xanthine. Specific compounds are listed below.
- the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
- a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof is administered to a patient in an amount sufficient to be cytotoxic to tumor cells and to prevent metastatic spread and growth of cancer cells.
- the route of administration of the illustrated compound is not critical but is usually oral or parenteral, preferably oral.
- parenteral includes intravenous, intramuscular, subcutaneous, intranasal, intrarectal, transdermal, opthalmic, intravaginal or intraperitoneal administration.
- the subcutaneous and intramuscular forms of parenteral administration are generally preferred.
- the daily parenteral dosage regimen will preferably be from about 0.01 mg/kg to about 25 mg/kg of total body weight, most preferably from about 0.1 mg/kg to about 4 mg/kg.
- each parenteral dosage unit will contain the active ingredient in an amount of from about 0.1 mg to about 400 mg.
- the compounds are generally active when given orally and can be formulated as liquids, for example, syrups, suspensions or emulsions, tablets, capsules and lozenges.
- a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent.
- a suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agent.
- a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
- a composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule.
- a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example, aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
- suitable pharmaceutical carrier(s) for example, aqueous gums, celluloses, silicates or oils
- the daily oral dosage regimen will preferably be from about 0.01 mg/kg to about 40 mg/kg of total body weight.
- each oral dosage unit will contain the active ingredient in an amount of from about 0.1 mg to about 1000 mg.
- the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment (i.e., the number of doses of a compound or a pharmaceutically acceptable salt or hydrate or solvate thereof given per day and duration of therapy) can be ascertained by those skilled in the art using conventional course of treatment determination tests.
- This example illustrates HPLC-derived PA and PA-related fractions from marrow stromal cells in the presence and absence of 1-(11 -dodecylamino- 10-hy droxyundecyl)-3, 7- dimethylxanthine were isolated, collected, dried under N 2 , and stored at -70° C under argon.
- the lung cancer cell line NCI H460 and a non-transformed, primary marrow stromal cell line were used in these studies.
- the cells were grown in RPMI 1640 containing 10% fetal bovine serum (FBS). Before the FBS stimulation to initiate proliferation, the cells were starved in media containing only 0.5% FBS overnight.
- FBS fetal bovine serum
- the compound tested 1-(1 1 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine at 10 ⁇ M was added 30 min. before the addition of the 20% FBS for stimulation. Cell stimulation was stopped by removal of media and the addition of cold methanol 18 hrs. after serum addition. Triplicate samples had the cells scraped from plates and transferred to stoppered glass centrifuge tubes. A volume of chloroform was added to complete the ratio of one volume aqueous material to 19 volumes of 2: 1 chloroform: methanol.
- Lipids in the samples were extracted by a modification of the procedure of Folch et al., 1957 , then separated and quantitated by normal phase HPLC using a gradient of hexane:2- propanol (3:4 v/v) and water in a gradient from 1 to 10% of the total volume and using ultraviolet detection at 217 and 206 nm.
- Fractions of the column effluent were analyzed by fast atom bombardment mass spectrometry (FAB-MS) for identification of sub-species within each lipid class.
- FAB/MS spectra were acquired using a VG 70 SEQ tandem hybrid instrument of
- EBqQ geometry (VG Analytical, Altrincham, UK).
- the instrument was equipped with a standard unheated VG FAB ion source and a standard saddle-field gun (Ion Tech Ltd., Middlesex, UK) that produced a beam of xenon atoms at 8 KeV and 1 mA.
- the mass spectrometer was adjusted to a resolving power of 1000 and spectra were obtained at 8 kV and at a scan speed of 10 sec/decade.
- 2-hydroxyethyl disulfide (2-HEDS) was used as matrix in the positive ion mode FAB/MS (FAB-PI), and triethanolamine was used as a matrix in the negative ion mode FAB/MS (FAB-NI).
- PA species observed in FAB-NI were native species coupled with lyso-PA breakdown M-H/z ions, and ranged between 550 and 820 mass units in FAB-NI. Because of differential protonation of the phosphate group of PA species in acidic target matrices, most PA species were detectable on FAB-PI as a larger range of ions, with most positive ions in clusters 2-4 mass units heavier than corresponding negative ions.
- HPLC methods were capable of separating some lyso(bis)PA species from PA, and mass spectrometry of HPLC fractions allowed analysis of lyso(bis)PA sub-species. Other lyso(bis)PA species migrated with PA and were identified only by mass spectrometry. Lyso(bis)PA demonstrated a significant overlap with PA species in weights, as it did in charge distribution. The range of detected lyso(bis)PA species separated on this system began at 715- 722 M-H/z and extended to hemi(bis) PA species (with 3 acyl groups, sn-1, sn-2, and sn-1' or 2') detected at 985- 1 100 M-H/z .
- Peaks of interest were analyzed using FAB-NI and FAB-PI mass spectrometry. Peaks were analyzed from the 21 hr. incubation time point for the marrow stromal and NCI H460 cells.
- Figure 19 illustrates fractions taken from the primary non-transformed human cell line, bone marrow stromal cells, after 21 hrs. of incubation with and without 10 ⁇ M 1-(11 -dodecylamino- 10-hy droxyundecyl)- 3,7-dimethylxanthine.
- Figures 19A-D represent marrow stromal cells in the presence of serum alone
- Figure 19A'-D' represent marrow stromal cells in the presence of serum and 10 ⁇ M 1- (1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine.
- the mass spectrograms have been normalized to each other, so that relative masses from analogous peaks (e.g., Figures 19A and 28 A') may be directly compared. Peaks are shown in descending order of total mass accumulation:
- Figures 19 A/A' represent HPLC peak A5, B/B' peak A4, C/C peak A2, and D/D' peak A7-13.
- 567/569, 595/597/599, 603, and 615 are relatively constant ions, and regarded to be matrix ion/adducts and/or breakdown product adducts previously seen in PA fractions analyzed by FAB mass spectrometry.
- FIG 19A shows significant mixed PA and lyso(bis)PA masses in peak A5 of marrow stromal cells stimulated with serum for 21 hrs.
- Peak A5 contained M-H/z 699 (1,2-dioleoyl PA), M-H/z 782 (1-eicosanoyl (C20:0), 2-docosatrienoyl (C22:3) PA; also this ion is consistent with l-O-octadecanyl/l'-arachidonoyl lyso(bis)PA), and a cluster at M-H/z 808-812 (l-O-octadecanyl/l'-docosatrienoyl(C22:3), /l'-docosatetraenoyl(C22:4), and IV- docosapentaenoyl (C22:5) lyso(bis)PA).
- M-H/z 855-858 (1-eicosanoyl, l'-docosapentaenoyl (C22:5)/l'-docosatetraenoyl (C22:4) lyso(bis) PA) and M-H/z 883/884 (1-docosanoyl (22:0), l'-docosapentaenoyl (C22:5) lyso(bis)PA) are present in lesser amounts.
- the A4 peak also demonstrated similar features of interest.
- Figure 19B there were substantial amounts of palmitoyl/oleoyl, 1-O-hexadecanyl oleoyl, and 1-O-tetradecanyl PA species, as well as 1-O-octadecanyl 2-stearoyl PA (M-H/z 689).
- M-H/z 637 was an apparent matrix adduct of a breakdown product, and was found equally in both serum-treated and 1-(11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine-treated cells.
- Figure 19C shows mass spectrometric tracings from peak A2, in which 217 nm- determined mass decreased in the presence of 10 ⁇ M 1-(11 -dodecylamino- 10- hydroxyundecyl)-3,7-dimethylxanthine.
- the best represented PA species include the previously observed M-H/z 630-631 (1-O-tetradecanyl 2-oleoyl PA) and M-H/z 673/675 (1- palmitoyl 2-oleoyl/2-stearoyl PA), with appearance of a new species, 647 (1,2-dipalmitoyl PA).
- Figure 19D shows the mass spec, data for peak A7-13 for marrow stromal cells. There was little change in either total mass observed or molecular species detected, both with serum ( Figure 19D) and in the presence of 10 ⁇ M 1-(11 -dodecylamino- 10-hy droxyundecyl)- 3,7-dimethylxanthine ( Figure 19D'). Lyso(bis)PA species were found in limited quantities except for M-H/z 885/886 (1-O-docosanoyl 1 '-docosadienoyl (C22:4) lyso(bis)PA).
- M-H/z 766 (1-palmitoleoyl/ l'- arachidonoyl lyso(bis)PA) and a decrease in M-H/z 788 (l-O-eicosanoyl/l'-oleoyl).
- serum appears to stimulate a variety of (a) palmitoyl and stearoyl 2-polyunsaturated (n>2), (b) hexadecanyl- and tetradecanyl 2-oleoyl and 2-polyunsaturated, and (c) 1 -saturated 2-arachidonoyl PA species in marrow stromal cells.
- arachidonoyl and docosapolyenoyl lyso(bis)PA species that appeared with serum. These species were not inhibited by 1-(1 1 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine, and arachidonoyl- PA and lyso(bis)PA species were stimulated by 1-(11 -dodecylamino- 10-hy droxyundecyl)-3, 7- dimethylxanthine. These fractions were then subjected to fast-atom bombardment mass spectrometry
- PA 699 2-dioleoyl PA
- Example 2 This example illustrates the HPLC and mass spectroscopy analysis performed in Example 1, except with lung NCI H260 tumor cells. NCI H460 cells were stimulated with serum in the presence and absence of 10 ⁇ M 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7- dimethylxanthine.
- HPLC peaks were collected and analyzed by FAB-PI and FAB-NI mass spectrometry, and were normalized using adduct peaks M-H/z 567/569 and 615, and analyzed in descending order of absolute mass (with the greatest increase in mass analyzed first): Peak A4 in Figure 20A(i),A(ii), and A', peak A2 in Figure 20B/B', peak A5 in Figure 20C/C, and peak A7-13 in Figure 20D & E/D' & E'.
- Peaks A4 and A5 showed mass accretion with 1-(11- dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, whereas peak 2 (which had significant total mass) and peak A 7-13 were essentially stable.
- Species identified by mass spec, analysis for NCI H460 cells are detailed in Table 9.
- Figures 20A(i) and A(ii) illustrate the advantages of using mass spectrometry to analyze HPLC peaks, in combination with acyl chain analysis. The PA species content of this peak was in great contrast to the content of the A5 and A4 peaks from marrow stromal cells seen in Figures 19A,B/A,B'.
- Figures 20A(i) and A(ii) were normalized to 567/569, as was Figure 20A', which illustrates the contrasting subspecies masses.
- Figures 20A(i) and (ii) were both included because they demonstrate slightly different M-H/z peaks despite being taken from the same broad HPLC Rf area.
- the 21-hr. NCI H460 A4 peak demonstrated significant amounts of linoleoyl-containing PA species (M-H/z 671, 695, 697, 699, 705), as well as conspicuous amounts of 1 ,2-sn-dipalmitoyl PA/1-myristoyl 2-stearoyl PA (M-H/z 647).
- 1- stearoyl 2-arachidonoyl PA (M-H/z 723) was present, as are the longer acyl-chain species M- H/z 742, 749, and 766.
- Lyso(bis)PA species M-H z 771 and 773 (1 -oleoyl/ l'-linoleoyl and 1- oleoyl/l'-oleoyl lyso(bis)PA) were also present, as was an alkyl C18/C22 cluster at M-H/z 808-810.
- lyso(bis)PA species M-H/z 801, 849, and 879 were also present, with persistence of M-H/z 771 (in smaller amounts).
- a noteworthy feature of these lyso(bis)PA species is their marked linoleate content, which appeared to correlate with suppression of linoleoyl-containing PA species.
- the UV-determined mass in HPLC peak A4 has increased, a substantial number of PA species containing linoleate have been suppressed.
- HPLC peak A5 contained significant amounts of PA species with masses of M-H/z 647, M-H/z 695/697/699, M-H/z 721/723 (1 -stearoyl, 2-20:3/2- arachidonoyl), M-H/z 739 (1 -O-octadecanyl 2-docosadienoyl (C22:2)), M-H/z 742 (1,2-sn- diarachidonoyl), and M-H/z 754 (1-stearoyl 2-docosatrienoyl (C22:3)).
- Lyso(bis)PA species M-H/z 781 (l-O-hexadecanyl/l'-docosapentaenoyl), 798 (l-linolenoyl/l'-eicosanyl), 818-819 (1 -oleoyl/ 1 '-docosahexaenoyl (22:6)), and 827 (1 -oleoyl/ l'-docosadienoyl (C22:2)) were also present.
- lyso(bis)PA species M-H/z 853 (1-eicosanoyl/l '-docosatetraenoyl (C22:4) or 1-docosanoyl/l '-arachidonoyl lyso(bis)PA, especially given the significant 303 peak found in the linked mass scans: data not shown) had appeared.
- Peak A7-13 which represented a lyso(bis)PA fraction, demonstrated stable mass in the presence of 10 ⁇ M 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine.
- Figures 20E and E' were expanded scale plots of Figures 20D and D', which show significant change in species content induced by 1-(1 l-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine.
- Figures 20D and E illustrate a significant concentration of oleate and arachidonate-containing lyso(bis)PA species, which were confirmed by the linked scans of acyl chains with masses of 281 and 303 seen in Figure 20D. These species were consistent with those identified in marrow stromal cells in Figures 19D/D' and Table 8.
- HPLC-derived PA and PA-related fractions from NCI H460 cells in the presence and absence of 1 -(11 -dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine were isolated, collected, dried under N2, and stored at -70° C under argon. These fractions were then subjected to fast-atom bombardment mass spectrometry (FAB-MS) using negatively charged triethanolamine (TEM) matrices, which caused the expulsion of negatively-charged ions (FAB-NI).
- FAB-NI-detected ions from isolated PA and lyso(bis)PA are illustrated in Figure 20, and the identity of each ion is detailed in Table 9 below.
- PA 649 1 -O-'en-tetradecanyl 2-eicosapentaenoyl
- This example illustrates the mass spectrometric determination of changes in phosphatidylcholine (PC) species in the NCI H460 tumor cell line induced by 1-(11- dodecylamino- 10-hydroxyundecyl)-3,7-dimethylxanthine.
- PC phosphatidylcholine
- PA species were conspicuously suppressed by pre-incubation with 1-(11 -dodecylamino- 10- hy droxyundecyl)-3,7-dimethy lxanthine.
- the commonest location for possible precursors of these PA species particularly given the paucity of palmitoyl CoA acyltransferases or palmitoyl transacylases in the majority of cellular membranes, were PC species in the plasma membrane.
- PE fractions contain relatively few fully saturated species, and hence were less likely to serve as precursors for disaturated PA species.
- a phospholipase D activity specific for saturated acyl chains and directed against PC has been associated with the oncogene v-src.
- M+H/z 704-707 represent 1 -myristoyl, 2-palmitoyl/2-palmitoleoyl PC, 718-719 represents l-O- tetradecanyl 2-linoleoyl PC, 735 represents 1,2-sn-dipalmitoyl and 1 -myristoyl 2-stearoyl PC, 759-761 represent 1 -palmitoyl, 2-oleoyl/2-linoleoyl PC, and 787-789 represent 1 -stearoyl, 2- stearoyl/2-oleoyl/2-linoleoyl PC.
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EP95912888A EP0708645A1 (en) | 1994-03-08 | 1995-03-08 | Use of compounds which inhibit phosphatidic acid formation for the manufacture of a medicament for the treatment of cancer |
CA002166324A CA2166324A1 (en) | 1994-03-08 | 1995-03-08 | Use of compounds which inhibit phosphatidic acid formation for the manufacture of a medicament for the treatment of cancer |
AU19901/95A AU1990195A (en) | 1994-03-08 | 1995-03-08 | Use of compounds which inhibit phosphatidic acid formation for the manufacture of a medicament for the treatment of cancer |
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US20876594A | 1994-03-08 | 1994-03-08 | |
US08/208,765 | 1994-03-08 |
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WO1995024199A3 WO1995024199A3 (en) | 1995-11-23 |
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EP (1) | EP0708645A1 (en) |
AU (1) | AU1990195A (en) |
CA (1) | CA2166324A1 (en) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6833373B1 (en) | 1998-12-23 | 2004-12-21 | G.D. Searle & Co. | Method of using an integrin antagonist and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia |
US6858598B1 (en) | 1998-12-23 | 2005-02-22 | G. D. Searle & Co. | Method of using a matrix metalloproteinase inhibitor and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992021344A2 (en) * | 1991-05-24 | 1992-12-10 | Fred Hutchinson Cancer Research Center | Modulation of cellular response to external stimuli |
US5288721A (en) * | 1992-09-22 | 1994-02-22 | Cell Therapeutics, Inc. | Substituted epoxyalkyl xanthines |
WO1994022863A1 (en) * | 1993-03-31 | 1994-10-13 | Cell Therapeutics, Inc. | Second messenger cell signaling inhibitors |
-
1995
- 1995-03-08 CA CA002166324A patent/CA2166324A1/en not_active Abandoned
- 1995-03-08 EP EP95912888A patent/EP0708645A1/en not_active Withdrawn
- 1995-03-08 AU AU19901/95A patent/AU1990195A/en not_active Abandoned
- 1995-03-08 WO PCT/US1995/003081 patent/WO1995024199A2/en not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992021344A2 (en) * | 1991-05-24 | 1992-12-10 | Fred Hutchinson Cancer Research Center | Modulation of cellular response to external stimuli |
US5288721A (en) * | 1992-09-22 | 1994-02-22 | Cell Therapeutics, Inc. | Substituted epoxyalkyl xanthines |
WO1994022863A1 (en) * | 1993-03-31 | 1994-10-13 | Cell Therapeutics, Inc. | Second messenger cell signaling inhibitors |
Non-Patent Citations (3)
Title |
---|
Cancerlit Abstract No. 95604126 & Proc. An nu. Meet.Am.Assoc. Cancer Res., vol. 35, A2441, March 1994 * |
CIRCULATORY SHOCK, vol.44, no.1, pages 14 - 29 STUART BURSTEN ET AL. 'Potential role for phosphatidic acid in mediating inflammatory responses to TNF-alpha and IL-1beta' * |
Proc. Annu. Meet. Am.Assoc. Cancer Res., v ol. 35, Abstract no. A401, March 1994 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6833373B1 (en) | 1998-12-23 | 2004-12-21 | G.D. Searle & Co. | Method of using an integrin antagonist and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia |
US6858598B1 (en) | 1998-12-23 | 2005-02-22 | G. D. Searle & Co. | Method of using a matrix metalloproteinase inhibitor and one or more antineoplastic agents as a combination therapy in the treatment of neoplasia |
Also Published As
Publication number | Publication date |
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AU1990195A (en) | 1995-09-25 |
EP0708645A1 (en) | 1996-05-01 |
WO1995024199A3 (en) | 1995-11-23 |
CA2166324A1 (en) | 1995-09-14 |
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