Classifications

• • 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
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/243—Platinum; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• This invention relates to various methods for using squalamine. Squalamine, having
• Fig. 1 is an aminosterol which has been isolated from the liver of the dogfish shark, Squalus acanthias. This aminosterol is the subject of U.S. Patent No.
• squalamine e.g., as a sodium/proton exchanger (isoform 3), or NHE3, inhibiting agent and as an agent for inhibiting the growth of endothelial cells
• squalamine synthesis techniques are also used.
• CNS central nervous system
• metastatic tumors e.g., lung, breast, melanomas
• about 35,000 are metastatic tumors (e.g., lung, breast, melanomas) and about
• gliomas i.e. , cancers of the brain
• gliomas are the third leading cause of death from cancer in persons between the ages of 15 and 34.
• Radiation treatment which is known to work on CNS tumors is radiation, and it can prolong survival. Radiation treatment, however, has many undesirable side effects. It can damage the normal tissue of the
• Radiation also can cause the patient to be sick (e.g. ,
• tumor angiogenesis i.e., blood vessel formation
• CNS tumors are among the most angiogenic of all
• the tumor is viable, but not growing, and it is unable to spread. Over time, however, angiogenesis begins and the tumor converts to a "vascular"
• phase In this phase, perfusion replaces diffusion as the growth mechanism, and tumor growth
• Mitotic cells cluster around new blood vessels and metastases occur in the vascular phase (i.e. , the vascular phase
• Lung cancer kills more Americans annually than the next four most frequently diagnosed neoplasms
• NSCLC non-small cell lung cancer
• breast cancer hormone therapy or combinations of the various methods.
• breast cancer hormone therapy or combinations of the various methods.
• breast tumors Like other solid tumors, breast
• Antiangiogenic agents are particularly useful in the treatment of resistance by the treated tumor.
• CNS tumors are treated; in another aspect, lung tumors
• breast tumors are treated; and in yet another, breast tumors are treated.
• squalamine is used in combination with conventional cancer treatments to treat tumors.
• the tumor is treated by administering an
• the cytotoxic chemical compound used in the first treatment procedure is a conventional cancer treating agent.
• Preferable agents include a nitrosourea,
• BCNU nitrosourea
• Another preferred cytotoxic agent is cisplatin, and yet another is cyclophosphamide.
• the cytotoxic chemical compound administered in the first treatment step may be administered by any conventional technique used in the art (e.g. , oral, subcutaneously, intralymphatically, intraperitoneally, intravenously, or intramuscularly).
• any conventional technique used in the art e.g. , oral, subcutaneously, intralymphatically, intraperitoneally, intravenously, or intramuscularly.
• the cytotoxic chemical compound preferably BCNU, cisplatin, or
• cyclophosphamide is administered intravenously.
• squalamine can be administered by any conventional administration method known in the art, such as those mentioned above.
• Intravenous administration of squalamine one or two times a day are used in this invention.
• the first treatment procedure with the cytotoxic chemical compound may take place prior to the second treatment procedure (using squalamine), after the second treatment
• the first treatment procedure may be completed before the second treatment procedure is initiated (or vice versa).
• the first treatment procedure is a one time
• a cytotoxic chemical compound e.g. , BCNU, cisplatin, or
• the first treatment procedure is a radiation treatment, which may be one or more conventional radiation
• timing of the radiation treatment procedure with respect to the squalamine treatment regimen can be determined by those skilled in the art through routine experimentation in order to
• Fig. 1 shows the general structural formula of squalamine
• Fig. 2 shows a general overview of the angiogenesis process
• Fig. 3 is a drawing used to illustrate the sodium hydrogen exchanger (NHE) process
• Fig. 4 illustrates the effects of conventional amilorides on inhibiting various isoforms of mammalian NHEs
• Figs. 5a and 5b illustrate the effect of squalamine on NHE isoform 3 (NHE3) and NHE1 inhibition, respectively;
• Figs. 6a to 6c show the results of a pharmacokinetic study relating to squalamine
• Fig. 7 illustrates squalamine distribution in various tissues after i.v. administration
• Fig. 8 shows an angiogenesis index using squalamine as determined in the rabbit corneal micropocket assay
• Fig. 9 shows the inhibitory effect of squalamine on growth of endothelial cells as compared to tumor cell lines
• Fig. 10 illustrates survival test results using squalamine in a glioma lethality study
• Fig. 11 shows the survival of mice carrying human MX-1 breast tumor xenografts
• Fig. 12 depicts the inhibition of a human lung adenocarcinoma (H460) in a mouse
• Fig. 13 illustrates the number of lung metastases following various chemotherapeutic treatment procedures in mice with subcutaneous implanted Lewis lung carcinomas.
• Squalamine has been recognized to have angiogenesis inhibiting activity, i.e., it inhibits the formation of blood vessels. Therefore, it is believed that squalamine, as an antiangiogenic agent, will be effective in treating certain diseases or ailments which depend on neovascularization. For example, squalamine may be used for treating such disparate
• squalamine can selectively inhibit certain sodium/proton exchangers (also
• NHEs or “proton pumps” in this application.
• squalamine may be used for treating proliferation or activation dependent conditions which rely on the
• NHE3 ischemic reprofiision injury
• squalamine in combination with conventional cancer treating agents, e.g. , cytotoxic chemical compounds and radiation treatments, will decrease the size and growth of tumors. Even more significantly, applicants have found that the combination decreases the cancer treating agents, e.g. , cytotoxic chemical compounds and radiation treatments.
• a cytotoxic chemical compound is used in a first tumor treatment procedure, and squalamine is used in a second rumor treatment
• the first and second treatments may be administered in any time sequence or even simultaneously.
• two or more cytotoxic chemical agents may be administered simultaneously or sequentially in the first treatment process.
• the cytotoxic chemical compound(s) used in the first treatment procedure may be any cytotoxic chemical compound(s) used in the first treatment procedure.
• BCNU which is also known as “carmustine” or "1,3-
• Cyclophosphamide also is known as N,N-Bis-(2- chloroethyl)-N'-(3-hydroxypropyl)phosphordiamidic acid cyclic ester monohydrate.
• Adriamycin also is known as doxorubicin.
• Paclitaxel is available under the tradename "Taxol. " Various derivatives of paclitaxel may be used in accordance with the invention, such as taxotere or other related taxanes. Cisplatin, another of the cytotoxic chemical compounds which may be used in accordance with
• the invention also is known as cis-Diamminedichloroplatinum.
• cytotoxic agents that could be used in the process of the invention.
• chemotherapeutic agents that can be used with squalamine in the process of the invention include methotrexate, thiotepa, mitoxantrone, vincristine,
• vinblastine etoposide, ifosfamide, bleomycin, procarbazine, chlorambucil, fludarabine,
• the first and/or second treatments may be administered by any suitable technique, such as oral, “s.q. , “ “i.p. , “ “i.m.,” “i.l. , “ or i.v. " In this application, the terms “s.q.,” “i.p., "i.m.,” “i.l. , “ or i.v. " In this application, the terms “s.q.,” “i.p., "
• intraperitoneal administration of squalamine or other substances intramuscular administration of squalamine or other substances, intralymphatic administration of squalamine or other substances, and intravenous administration of squalamine or other
• BCNU is delivered to a patient first as a one time intravenous dosage, and thereafter squalamine is injected s.q. twice daily.
• cyclophosphamide is the cytotoxic agent.
• cisplatin is the cytotoxic
• the cytotoxic chemical compound and the squalamine may be delivered simultaneously by a common pharmaceutical carrier (e.g., one injection including both
• the squalamine treatment procedure in accordance with the invention also may be used with radiation treatment (e.g., cobalt or X-ray treatment) as the first treatment procedure.
• radiation treatment e.g., cobalt or X-ray treatment
• the first treatment procedure is a radiation treatment
• the second treatment procedure is squalamine administration.
• the tumor also may be treated with one or more cytotoxic chemical compounds in a third treatment procedure.
• Squalamine has been demonstrated to be useful as an antiangiogenic agent, i.e. , squalamine inhibits angiogenesis.
• Angiogenesis the process of forming new blood vessels
• Angiogenesis also is essential for the progression of many pathological processes, such as diabetic retinopathy, inflammation, and malignancy (tumor development).
• squalamine may be used for treating various ailments and
• Angiogenesis is a multiple step process which is schematically illustrated in Fig. 2.
• endothelial cells must become activated, for example, by attaching a growth factor such as vascular endothelial growth factor ("VEGF”) or basic-fibroblast growth factor (“b-FGF”).
• VEGF vascular endothelial growth factor
• b-FGF basic-fibroblast growth factor
• Certain compounds which are believed to be antiangiogenic compounds e.g. , matrix
• metalloproteinase inhibitors such as minocycline, SU101 or marimistat
• angiogenesis inhibitors for a discussion of matrix metalloproteinase inhibitors, please refer to those described above.
• matrix metalloproteinase inhibitors please refer to those described above.
• squalamine acts at a very early stage in the process by inhibiting
• the growth factor signal is aborted in the presence of squalamine.
• squalamine was applied to capillary beds of young chick embryos that were 2-3 days old.
• the capillary bed also was observed after sixty minutes. At this time, it was noted that
• microcapillary blood vessels i.e. , the microvascular bed
• the NHE antiporter of cells may be activated in different ways. For example,
• insoluble fibronectin activates the NHE antiporter by clustering and immobilizing Integrin
• the NHE antiporter When activated, the NHE antiporter induces cell growth by regulating the pH of the
• the chloride-bicarbonate exchanger and NHE are complementary pH
• the chloride-bicarbonate exchanger makes the cell become more alkaline, while NHE contributes to the control of hydrogen ion concentration in the cell.
• the NHE When the NHE is inhibited, the cells become acidic (lower pH) and growth stops. This does not mean
• the cell dies; it means only that the cell enters a quiescent state (i.e. , it does not divide).
• the cell If the cell returns to a normal pH, growth may resume.
• the NHE is activated, the cell becomes more alkaline (higher pH), it pumps out protons, and growth proceeds.
• various modulatory factors e.g. , serum components, secondary messengers, etc.
• portion of the cytoplasmic region of NHE activates the antiporter, while interaction with
• Sodium-proton pumps are responsive to different growth stimuli which activate the pump.
• the proton pump may be activated by attachment of growth factors (e.g., VEGF and b-FGF) to the cell. Additionally, as shown in
• NHE activation and alkalinization of the cell also can cause NHE activation and alkalinization of the cell. Attachment of these stimuli to the extracellular matrix activates the NHE antiporter of the cell and induces cell growth.
• NHE mammalian isoforms of NHE exist, and each has a distinct tissue distribution. Nonetheless, all act in the same manner.
• NHEl is the antiporter found in all tissues.
• NHE2 and NHE3 are more restrictive in their tissue distribution.
• NHE activity can be measured under various different cellular conditions. Acid loading a cell activates all of the antiporters and permits
• NHE activity also can be measured after growth factor stimulation of
• the NHE activity can be measured when the cell is in an unstimulated state, because the antiporters, even if unstimulated, continue to function at a slow, but non ⁇ zero rate. In each of these cellular conditions, NHE activity usually is measured in the
• NHE3 in particular is relatively resistant to inhibition by the amilorides.
• NHEl activity was measured in unstimulated melanoma cells, applicants found that squalamine substantially down
• NHE deficient fibroblast cells PS 120 transfected with an individual human NHE gene were loaded with a pH sensitive dye 2'7'-bis(2-carboxyethyl)-5,6-
• BCECF carboxy fluorescein
• LDH leakage is a non-specific marker of cytotoxicity, it was concluded that squalamine does not have a general cytotoxic effect.
• This NHE3 inhibiting activity of squalamine has been mapped to the 76 C-terminal amino acids on the NHE3 molecule. If the 76 C-terminal amino acids of rabbit NHE3 are
• squalamine has been found to have virtually no effect on the activity of the molecule, while the molecule remains active as a sodium/hydrogen exchanger.
• the 76 C-terminal amino acids of NHE3 are the site of inhibition by squalamine. It is believed that the squalamine effect on these accessory proteins of NHE3 is tied to an inhibitory
• NHE3 e.g. , amiloride, amiloride analogues, genestein, calmodulin, and protein kinase C
• Such inhibitors affect only the absolute number of protons that can be secreted by the cell (i.e., "V ma ,”), if one looks at the kinetic characteristics of the inhibition.
• squalamine inhibits NHE with nonallosteric kinetics (i.e., nonclassical allosteric
• NHE3 is important in maintaining homeostasis of the unstimulated cell.
• the applicants further believe that prevention of cellular activation by squalamine, especially
• transport proteins which control cell volume and shape may be a squalamine
• BBMV membrane vesicle
• FIGs. 6a to 6c illustrate the test results where squalamine was
• FIG. 7 illustrates the
• squalamine is contained in most of the tissues, with most of the squalamine concentrating in
• rabbit corneal micropocket Assay In determining whether a compound is antiangiogenic, the rabbit corneal micropocket
• assay is an accepted standard test. In this test, an incision is made in one rabbit cornea, and a
• the stimulus is placed in the incision.
• the stimulus is used to induce blood vessel formation in the normally avascular corneal region.
• a solid tumor in a polymeric matrix can be placed in the cornea as the stimulus because the tumor will release a number of angiogenic
• the tumor-derived angiogenic growth factors stimulate the endothelial cells at the scleral junction in the eye to initiate blood vessel growth toward the stimulus.
• a second polymer pellet e.g., an ethylene/vinyl acetate copolymer is placed between the scleral junction and the stimulus. This polymer pellet is
• a negative control test pellet either empty (a negative control test pellet), or it contains a compound whose antiangiogenic characteristics are to be tested.
• the polymer pellet is used to provide a controlled release of the material to be studied. Because of the avascular cornea background in the rabbit cornea,
• VX2 rabbit carcinoma was implanted in 26 rabbit eyes, in the normally avascular
• the loaded polymer pellets were placed in 13 of the corneas to provide a sustained local release of squalamine. Polymer blanks were provided in the remaining 13 eyes as a control. In this manner, one eye of each rabbit served as the squalamine test eye and the other
• squalamine was found to be a potent inhibitor of tumor induced capillary formation. Fewer blood vessels were observed in the cornea treated with squalamine as compared to the control cornea, and these vessels were generally shorter than the vessels in
• the untreated control corneas had many vessels in and adjacent to the tumor.
• the tumors in the squalamine-treated corneas were still viable (i.e., the tumors were
• Fig. 8 shows a graphical representation of the results of the rabbit cornea micropocket
• the vessel length (“L vesse , ”) was then measured in each cornea.
• the vessel length is the length of the longest vessel measured from the cornea-scleral junction to the distal edge of the
• FIG. 8 shows the mean Angiogenesis Index for each group of corneas (squalamine treated
• squalamine was very inhibitory to the growth of new blood vessels.
• squalamine in and of itself, was responsible for any inflammatory response in the cornea.
• Several non-bioresorbable ethylene/vinyl acetate copolymer pellets were loaded with different concentrations of squalamine, namely, 2%, 10% , and 20% squalamine, by weight.
• pellets were then placed in rabbit corneas which did not include an angiogenic stimulus.
• squalamine may be a potent antiangiogenic agent that inhibits neovascularization. Recognizing
• malignant gliomas are the most common form of cancerous
• Malignant gliomas are characterized by their ability to induce the normally quiescent brain and/or CNS endothelial cells into a highly proliferative and
• the gliomas express vascular endothelial growth factor ("VEGF”) and other growth factor (VEGF).
• VEGF vascular endothelial growth factor
• VEGF growth factors which stimulate inducible receptors on CNS endothelial cells in a paracrine manner
• the VEGF originates from the tumor cell and stimulates the endothelial cells.
• the CNS endothelial cells subsequently initiate angiogenic invasion and thus provide
• endothelial cells Applicants used endothelial cells because such cells are involved in the early steps of angiogenesis, as described above in conjunction with Fig. 2. Specifically, tumor angiogenesis is a series of sequential and overlapping steps. First, the endothelial cells
• retinal endothelial cells (b) 9L and C6 rat glioma cells; (c) human H80 glioma cells; and (d)
• VX2 rabbit carcinoma cells (the same type as the tumors implanted in the rabbit corneal micropocket assay test described above).
• the endothelial mitogen which was used in this analysis was VEGF at a concentration of 20 ng/ml.
• the cells were allowed to attach overnight to tissue culture plates containing an
• bovine retinal endothelial cells were grown and treated in an identical manner to the other cell lines, except that the growth of these cells was measured after the addition of 20 ng/ml of human recombinant VEGF to the cells prior to the squalamine treatment.
• Percentage endothelial cell growth inhibition was determined by the following equation:
• FIG. 9 shows the growth of the various cell lines as a percentage of the growth in the control groups for in vitro administration of
• squalamine at 30 ⁇ g/ml after 1 , 2, and 3 days.
• growth is reduced for the VEGF-stimulated endothelial cells specifically, while the growth in the other cell lines (H80, C6, and VX2) is not dramatically affected.
• squalamine is believed to be well suited for reducing or diminishing the neovasculature induced by tumors for use in tumor specific antiangiogenic therapy.
• squalamine In addition to inhibiting VEGF-stimulated growth of endothelial cells, squalamine also has been found to interfere with growth stimulation in human brain capillary endothelial cells
• squalamine has an inhibitory effect on several.
• squalamine 20 mg/kg/day of squalamine (i.p.) was initiated in one group of rats.
• the control animals (“vehicle control” in Fig. 10) were given the carrier vehicle only (no squalamine), and the other animals were treated with squalamine ("Squalamine" in Fig.
• BCNU which is a conventional
• squalamine is an upstream inhibitor of the angiogenesis process by inhibiting the activation of endothelial cells after growth factor interaction. Because of its
• B.I.D. means that the component is administered twice a day (10 mg/kg given at two different times each day).
• the tumor size was measured directly.
• Table 5 summarizes the results.
• the tumor volumes shown in Table 5 represent the mean tumor volumes for each treatment group for those animals that survived to the end of the experiment.
• Table 5 illustrates the advantageous results achieved when treating tumors with the
• cytotoxic chemical compounds can slow or halt the spread of brain cancers.
• the human MX-1 breast cancer line has previously been used to document in vivo
• Squalamine was examined as adjunctive therapy following a single 200 mg/kg dose of cyclophosphamide.
• the cyclophosphamide was injected on day 14 following implantation of the tumor, at a time when the tumors measured 65-125 ⁇ L
• the cyclophosphamide caused partial regression in all animals and complete regression in a small fraction of the animals.
• squalamine has synergistic activity in combination with cisplatin (e.g. , Fig. 12).
• experimental lung cancer model design involves subcutaneous injection of 5 x IO 6 tumor cells
• Intralipid as a vehicle began the following day
• mice receiving squalamine alone started receiving the aminosterol on the same day
• the murine Lewis lung adenocarcinoma was implanted subcutaneously in the hind-leg
• mice Male C57BL/6 mice and allowed t ⁇ grow for one week. Groups of mice were then left
• the mode of administration of squalamine may be selected to suit the particular
• Modes of administration generally include, but are not limited to,
• transdermal intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, inhalation,
• the squalamine compounds may be
• epithelial or mucocutaneous linings e.g., oral mucosa, rectal, and
• intestinal mucosa etc.
• it may be administered together with other biologically active agents
• Administration may be local or systemic.
• the present invention also provides pharmaceutical compositions which include
• compositions include a therapeutically effective amount of a compound selected from the group consisting of a compound selected from the group consisting of a compound selected from the group consisting of a compound selected from the group consisting of a compound selected from the group consisting of a compound selected from the group consisting of a compound selected from the group consisting of a compound selected from the group consisting of a compound selected from the group consisting of a compound selected from the group consisting of a compound, such as an active ingredient.
• squalamine and a pharmaceutically acceptable carrier or excipient.
• a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffered saline, dextrose, water, oil in
• water microemulsions such as Intralipid, glycerol, and ethanol, and combinations thereof.
• formulation of the pharmaceutical composition should be selected to suit the mode of administration.
• the pharmaceutical composition if desired, also may contain effective amounts of
• the pharmaceutical composition may include wetting or emulsifying agents, or pH buffering agents.
• the pharmaceutical composition may include
• compositions may be in any suitable form, such as a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
• the composition also may be formulated as a suppository, with traditional binders and carriers, such as triglycerides.
• Oral formulations
• mannitol may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch,
• magnesium stearate sodium saccharine, cellulose, magnesium carbonate, etc.
• a therapeutic compound of the invention e.g. , encapsulation in liposomes, microparticles, enteric coated
• the pharmaceutical composition is formulated in accordance with routine procedures to provide a composition adapted for intravenous administration to humans.
• compositions for intravenous administration are solutions in 5 % dextrose and sterile
• the pharmaceutical composition also may include a
• solubilizing agent and a local anesthetic to ameliorate pain at the site of an injection.
• the ingredients of the pharmaceutical composition are supplied either separately or
• unit dosage form for example, as a dry lyophilized powder or water-free
• composition is to be administered by a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• infusion it may be dispensed with an infusion bottle containing sterile pharmaceutical grade water, dextrose, saline, or other pharmaceutically acceptable carriers.
• an ampoule of sterile water or saline is administered by injection, an ampoule of sterile water or saline
• the amount of the therapeutic compound (i.e. , active ingredient) which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques known to those
• Suitable dosages for intravenous administration are generally about 1 microgram to 40
• Suitable dosage ranges for intranasal administration are generally about 0.01 mg/kg body weight to 20 mg/kg body
• Suitable dosages for oral administration are generally about 500 micrograms to 800 milligrams per kilogram body weight, and preferably about 1-200 mg/kg body weight.
• Suppositories generally contain, as the active ingredient, 0.5 to 10% by weight of squalamine. Oral formulations preferably contain 10% to 95% active ingredient.
• squalamine as an antiangiogenic or cytotoxic agent or in cancer therapies,
• exemplary dosages are from about 0.01 mg/kg body weight to about 100 mg/kg body weight.
• Preferred dosages are from 0.1 to 40 mg/kg body weight.
• the invention also may include a pharmaceutical pack or kit including one or more

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