EP1119361A2 - Traitement des carcinomes a base de squalamine combinee a d'autres agents anticancereux - Google Patents

Traitement des carcinomes a base de squalamine combinee a d'autres agents anticancereux

Info

Publication number
EP1119361A2
EP1119361A2 EP99952905A EP99952905A EP1119361A2 EP 1119361 A2 EP1119361 A2 EP 1119361A2 EP 99952905 A EP99952905 A EP 99952905A EP 99952905 A EP99952905 A EP 99952905A EP 1119361 A2 EP1119361 A2 EP 1119361A2
Authority
EP
European Patent Office
Prior art keywords
tumor
squalamine
treatment procedure
treatment
chemical compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99952905A
Other languages
German (de)
English (en)
Other versions
EP1119361A4 (fr
Inventor
Michael Zasloff
Jon Williams
Mitchell H. Sokoloff
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genaera Corp
Original Assignee
Magainin Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Magainin Pharmaceuticals Inc filed Critical Magainin Pharmaceuticals Inc
Publication of EP1119361A2 publication Critical patent/EP1119361A2/fr
Publication of EP1119361A4 publication Critical patent/EP1119361A4/fr
Withdrawn legal-status Critical Current

Links

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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/28—Antiandrogens

Definitions

  • Squalamine having the structure illustrated in 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. 5,192,756 to Zasloff, et al, which patent is entirely incorporated herein by reference.
  • U.S. Patent Nos. 5,733,899 and 5,721,226 describe the use of squalamine as an antiangiogenic agent. These U.S. patents are entirely inco ⁇ orated herein by reference. Additional uses of 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) and squalamine synthesis techniques are disclosed in U.S. Patent No. 5,792,635. This U.S. patent is also entirely inco ⁇ orated herein by reference. II. INFORMATION RELATING TO THIS INVENTION
  • CNS central nervous system
  • metastatic tumors e.g., lung, breast, melanomas
  • primary tumors mostly astrocytomas
  • Astrocytomas, along with other malignant gliomas are the third leading cause of death from_ cancer in persons between the ages of 15 and 34.
  • Treatment options for a patient with a CNS tumor are very limited. Currently, surgery is the treatment of choice. Surgery provides a definite diagnosis, relieves the mass bulkiness of the tumor, and extends survival of the patient.
  • the only post-surgery adjuvant 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 patient, including the brain tissue. Radiation also can cause the patient to be sick (e.g., nausea) and/or to temporarily lose their hair.
  • the other common post-surgery adjuvant cancer treatment, chemotherapy is relatively ineffective against CNS tumors. Specifically, chemotherapy against CNS tumors with nitrosoureas is not curative. Many other cancer treating agents have been studied and tested, but generally they have a minimal effect on extending survival.
  • the current prognosis for persons with CNS tumors is not good.
  • the median survival term for patients with malignant astrocytomas having surgery and no adjuvant treatment is about 14 weeks. Radiation therapy after surgery extends the median to about 36 weeks.
  • the current two year survival rate for all forms of treatment is less than 10%.
  • CNS tumors are among the most angiogenic of all human tumors.
  • a diffusion mechanism i.e., the cells receive their nutrition, etc. by diffusion into the cell.
  • angiogenesis begins and the tumor converts to a "vascular" phase.
  • Lung cancer kills more Americans annually than the next four most frequently diagnosed neoplasms combined.
  • Estimates for 1994 indicate more than 170,000 new cases of lung cancer and approximately 150,000 deaths (Boring et al.; CA Cancer J. Clin. 1994, 44: 7- 26).
  • Approximately 80% of primary lung tumors are of the non-small cell variety, which includes squamous cell and large cell carcinomas, as well as adenocarcinomas.
  • NSCLC non-small cell lung cancer
  • Early stage tumors are potentially curable with surgery, chemotherapy, or radiotherapy, and late stage patients usually receive chemotherapy or best supportive care.
  • Intermediate stage or locally advanced NSCLC which comprises 25% to 30% of all cases of NSCLC, is more typically treated with multimodality therapy.
  • This is a stage of tumor development when angiogenesis is a very important factor. New blood vessels are needed to support further tumor growth and for the development of metastases. Therefore, this stage is amenable to treatment with antiangiogenic agents to prevent the development of new blood vessels.
  • the efficacy of this therapy can be further improved by the combination of the antiangiogenic therapy with cytotoxic chemotherapy or radiation therapy to eliminate existing tumor.
  • breast cancer also presents treatment difficulties using known agents.
  • the incidence of breast cancer in the United States has been rising at a rate of about 2%/year since 1980, and the American Cancer Society estimated that 182,000 cases of invasive breast cancer were diagnosed in 1995.
  • Breast cancer is usually treated with surgery, radiotherapy, chemotherapy, hormone therapy, or combinations of the various methods.
  • breast cancer requires the development of new blood vessels to support its growth beyond a certain size, and at that stage in its development, it will be amenable to treatment with antiangiogenic agents.
  • a major reason for the failure of cancer chemotherapy in breast cancer is the development of resistance to the cytotoxic drugs. Combination therapy using drugs with different mechanisms of action is an accepted method of treatment which prevents development of resistance by the treated tumor.
  • Antiangiogenic agents are particularly useful in combination therapy because they are not likely to cause resistance development since they do not act on the tumor, but on normal host tissue.
  • Prostate cancer is another cancer for which new therapies are needed.
  • Prostate cancer is another cancer for which new therapies are needed.
  • b-FGF basic f ⁇ roblast growth factor, also known as FGF-2
  • VEGF vascular endothelial growth factor
  • the outcome of a patient with prostate cancer largely depends upon the tumor's capacity for unhindered growth, local invasion, and the establishment of distant metastasis.
  • anti-angiogenic agents may effectively inhibit the growth and metastasis of such tumors.
  • antiangiogenic agents may be most effective at this stage, particularly agents that are most potent on freshly sprouting, young blood vessels, thereby preventing neovascularity and repressing further tumor growth and metastasis.
  • Ovarian cancer is the most serious gynecologic tumor type. Over 50% of all cancer-related gynecologic deaths are attributable to ovarian cancers, of which 80-90% are epithelial-derived tumors. In 1997 there were 26,700 new cases of ovarian cancer and more than 14,000 deaths. There is a clear genetic component to ovarian cancer. Newly developed detection methods have shown strong correlations between breast cancer, ovarian cancer and expression of genetic markers that include BRCA1 or mutant oncogenes such as aberrant forms of erbB-2 or c-Myc. Another good marker for ovarian cancer of clinical utility is the circulating analyte CA125, for which serum levels generally reflect the state of cancer progression. CA125 is often monitored in ovarian cancer, although it is not a validated marker. There is also a hormonal influence on cancer risk in ovarian cancer, as is seen in breast cancer
  • ovarian cancer patients present with localized disease.
  • Early stage ovarian cancer is treatable with some combination of surgery, radiation and chemotherapy; the 5 year survival rate for localized ovarian cancer is greater than 80%. However, the 5 year survival rate for metastatic stage III or IV ovarian cancer is less than 20%. It is these advanced patients, those for whom the ovarian tumors escape the ovarian capsule and invade intraperitoneal surfaces, who require the most aggressive therapy yet benefit only marginally with chemotherapy.
  • the first line chemotherapeutic treatment for ovarian cancers has been the use of platinum-based regimens for over a decade.
  • Hepatocellular carcinoma (HCC) or hepatoma is the most common liver cancer tumor and is a tumor type that is closely _ associated with chronic hepatitis B or hepatitis C infection.
  • HCC Hepatocellular carcinoma
  • the high frequency of hepatitis viral infections observed in Asia and Africa make these regions the sites of the largest number of liver cancers, most of which are HCC tumors.
  • viral infection is considered essential for predisposition to HCC, infection alone is not the only contributing factor to hepatocellular transformation and proliferation.
  • liver cancers The prognosis for liver cancers is universally poor with the minor exception of those tumors which are only locally invasive and are located favorably for curative surgical removal.
  • limited efficacy has been seen with interferon therapy, polyprenoic acid (vitamin A derivative), 5-fluorouracil or cryoablation.
  • No meaningful combination chemotherapy has proven itself in human trials for HCC to date, but the urgent clinical need continues to drive experimental evaluation of various therapeutic approaches.
  • a recent report (LX Qin et al, Ann Acad Med Singapore 28,147- 51 (1999)) suggests that angiogenesis inhibitors can inhibit hepatoma tumor growth in mouse xenograft tumor models and may be candidates for the control of recurrence and metastasis after HCC resection.
  • neuroblastoma which is of neuroendocrine origin.Other solid tumors among children are Wilms' tumor, rhabdomyosarcoma and retinoblastoma. Neuroblastomas represent 9% of all childhood cancers, but 15%o of pediatric cancer deaths. Many pediatric tumors have a genetic basis; for example, it is estimated that perhaps 20% of neuroblastomas are genetic in nature.
  • Neuroblastomas most commonly are seen in the abdomen, many of these being found in the adrenal gland.
  • the median age at diagnosis for neuroblastoma patients isJ2 years.
  • Surgery can be curative in early stage neuroblastomas, but most children present with metastatic disease. Even in children with minimal residual disease following surgery, recurrences are seen in more than half of all patients.
  • Clinical trials have emphasized the positive value of using post-surgical chemotherapy with or without radiation therapy.
  • Neoadjuvant chemotherapy is also commonly used with neuroblastoma patients, but it is more common to use chemotherapy in the adjuvant setting.
  • Neuroblastoma is a particularly difficult tumor to treat, and platinum-based regimens are frequently used in first or second-line treatments.
  • chemotherapeutic regimens for pediatric patients are highly aggressive, often involving megatherapy with 4-6 cytotoxic agents.
  • Neuroblastoma tumors represent an interesting opportunity for antiangiogenic therapy as neuroblastomas are highly vascular, grow quickly and metastasize rapidly.
  • Antiangiogenic therapy for neuroblastoma may allow new aggressive combination chemotherapy treatments involving cytostatic agents since they are anticipated to provide minimal additional toxic side effects and should not diminish the efficacy of the cytotoxic agents to which they are matched.
  • the invention can be applied to any responsive tumor, in particularly preferred aspects of the invention, the tumors treated are found in the CNS, lung, breast, ovary, liver, neuroendocrine and prostate tissues.
  • squalamine is used in combination with conventional cancer treatments to treat tumors.
  • conventional cancer treatments include the use of cytotoxic agents as well as anti-hormonal agents.
  • the tumor is treated by administering an effective amount of a cytotoxic chemical compound or a combination of cytotoxic compounds in a first treatment procedure, and an effective amount of squalamine is administered in a second treatment procedure.
  • the cytotoxic chemical compound used in the first treatment procedure is a conventional cancer treating agent.
  • Preferable agents include a nitrosourea, cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, topotecan and irinotecan, carmustine, estramustine, paclitaxel and its derivatives, and cisplatin, carboplatin, iproplatin and related platinum compounds.
  • These conventional cancer treating agents are well known to those skilled in this art. Note, M.C. Wiemann and Paul Calabresi, "Pharmacology of Antineoplastic Agents," Medical Oncology. Chapter 10, edited by Paul Calabresi, et.
  • BCNU nitrosourea
  • Another preferred cytotoxic agent is a platinum compound such as carboplatin, iproplatin or cisplatin, and yet another is cyclophosphamide.
  • Other conventional cytotoxic chemical compounds such as those disclosed in Medical Oncology. supra., can be used without departing from the invention.
  • the tumor is treated by first inhibiting hormones that affect the tumor and then administering an effective amount of squalamine in a second treatment procedure.
  • the hormones may be specifically inhibited.
  • the hormone inhibition may result from orchiectomy, i.e., the removal of one or both testes. Orchiectomy may result from surgery or from the administration of chemical agents such as LHRH (luteinizing hormone releasing hormone) agonists and/or anti-androgens (such as flutamide, biclutamide, nilutamide or luprolide).
  • LHRH leukinizing hormone releasing hormone
  • anti-androgens such as flutamide, biclutamide, nilutamide or luprolide
  • the cytotoxic and anti-hormonal chemical compounds administered in the first treatment step may be administered by any conventional technique used in the art (i.e., oral, subcutaneously, intralymphatically, intraperitoneally, intravenously, or intramuscularly).
  • the cytotoxic chemical compound preferably BCNU, cisplatin, or cyclophosphamide
  • squalamine can be administered by any conventional administration method known in the art, such as those mentioned above.
  • Subcutaneous injections of squalamine one or two times a day are used in one embodiment of this invention.
  • Intravenous administration of squalamine one or two times a day are used in another embodiment of the present invention.
  • the first treatment procedure with the cytotoxic chemical or anti-hormonal compound may take place prior to the second treatment procedure (using squalamine), after the second treatment procedure, or at the same time as the second treatment procedure. Furthermore, 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 intravenous administration of a cytotoxic chemical or anti-hormonal compound (i.e., BCNU, cisplatin, or cyclophosphamide), and the second treatment procedure involves daily subcutaneous injections of squalamine.
  • a cytotoxic chemical or anti-hormonal compound i.e., BCNU, cisplatin, or cyclophosphamide
  • the invention encompasses the use of squalamine together with cytotoxic compounds or antihormonal compounds or the use of two or more of these compounds with squalamine.
  • the invention also encompasses the use of squalamine together with a cytostatic agent or the use of these two treatment modalities with a cytotoxic compound.
  • a cytostatic agent is any chemical compound which is capable of arresting the growth of tumor cells or normal stromal cells in a tumor but which is not toxic at pharmacologically active concentrations.
  • a pharmacologically active concentration of a cytostatic agent used in the first treatment procedure may be any know cell growth modulator, but it is preferably the calcium pump inhibitor carboxyamidotriazole.
  • the first treatment procedure is a radiation treatment, which may be one or more conventional radiation modalities, using a conventional radiation treatment regimen known to those skilled in the art.
  • the tumor is exposed to radiation in this first treatment procedure.
  • an effective amount of squalamine is administered to treat the tumor. Appropriate 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 provide effective tumor treatment.
  • the tumor also may be treated with one or more cytoxic chemical or anti-hormonal compounds in a third treatment procedure.
  • the tumor also may be treated with one or more cytostatic chemical compounds in a third treatment procedure.
  • the cytostatic agent used in the third threatment procedure may be any known cell growth modulator which is not cytotoxic, but it is preferably the calcium pump inhibitor carboxymidotriazole. It is additionally envisioned in the present embodiment of the invention that tumors treated with radiation, squalamine and a cell growth modulator may also be treated with one or more cytotoxic chemical compounds in a fourth treatment procedure.
  • the invention also relates to the use of both cytotoxic chemical and/or anti-hormonal compounds in addition to radiation, or any other combination of treatment methods.
  • 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, 6b and 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 with a rat 9L glioma introduced into the brains of healthy rats;
  • Fig. 11 shows the survival of mice carrying human MX-1 breast tumor xenografts and treated with squalamine subsequent to cyclophosphamide treatment
  • Fig. 12 depicts the inhibition of a human lung adenocarcinoma (H460) in a mouse xenograft-combination therapy study with squalamine and cisplatin;
  • Fig. 13 illustrates the number of lung metastases following various chemotherapeutic treatment procedures in mice with subcutaneous implanted Lewis lung carcinomas.
  • Fig.14a and 14 b illustrate the effect of squalamine, VEGF, and a combination of VEGF and squalamine treatment on LNCaP human prostate cell growth;
  • Fig. 14b illustrates the effects of squalamine, VEGF, and a combination of VEGF and squalamine treatment an C4-2 human prostate cell growth.
  • [ 3 H] -thymidine inco ⁇ oration assays were utilized.
  • Figs. 15a and 15b illustrate the effect of squalamine on PSA levels (a) and tumor growth (b) in control athymic mice post-orchiectomy.
  • Figs. 16a and 16b illustrate the effect of squalamine on PSA levels (a) and tumor growth (b) in athymic mice that underwent orchiectomy and subsequent treatment with squalamine.
  • 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 conditions as solid tumor cancers, macular degeneration, diabetic retinopathy, psoriasis, or rheumatoid arthritis, all of which require a separate and new blood flow.
  • squalamine can selectively inhibit certain sodium/proton exchangers (also called “NHEs” or “proton pumps” in this application).
  • NHEs sodium/proton exchangers
  • Several different isoforms of NHE are known to exist in mammals (i.e., NHE1, NHE2, NHE3, NHE4, and NHE5). Squalamine has been found to specifically inhibit NHE3 and not NHE1 or NHE2. Accordingly, squalamine may be used for treating proliferation or activation dependent conditions which rely on the function of NHE3, such as cancer, viral diseases, and ischemic reprofusion injury. Further studies with squalamine and NHE have demonstrated that squalamine acts on a very specific portion of the NHE3, namely the 76 carboxyl-terminal amino acids of the molecule. If this portion of the NHE3 molecule is removed, squalamine has virtually no effect on the activity of the molecule, even though the molecule is still active as a sodium/proton exchanger.
  • squalamine in combination with conventional cancer treating agents, i.e., cytotoxic chemical and anti-hormonal compounds and radiation treatments, will decrease the size and growth of tumors. Even more significantly, applicants have found that the combination decreases the growth rate of highly proliferative CNS tumors, lung tumors, breast tumors, ovarian tumors, liver tumors, neuroendocrine tumors and prostate tumors and can confer survival advantages.
  • conventional cancer treating agents i.e., cytotoxic chemical and anti-hormonal compounds and radiation treatments
  • either a cytotoxic chemical compound or an anti-hormonal agent is used in a first tumor treatment procedure, and squalamine is used in a second tumor treatment procedure.
  • the first and second treatments may be administered in any time sequence or even simultaneously.
  • two or more cytotoxic chemical and/or anti-hormonal 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 conventional agent, but it is preferably one of the following agents: a nitrosourea, cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, topotecan, irinotecan, carmustine, estramustine, paclitaxel and its derivatives, and cisplatin carboplatin, iproplatin and related platinum compounds.
  • These materials are conventional cancer treating agents which are known to those skilled in this art, as set forth in Medical Oncology, supra.
  • BCNU which is also known as “carmustine” or " 1 ,3- Bis(2-chloroethyl)-l -nitrosourea.”
  • Cyclophosphamide also is known as N,N-Bis-(2- chloroethyl)-N'-(3-hydroxypropyl)phosphordiamidic acid cyclic ester monohydrate.
  • Doxorubicin also is known as adriamycin.
  • topoisomerase inhibitors include irinotecan [7-ethyl-10-[4-(l- piperidino)-l-piperidino]carbonyloxycamptothecin], also known as CPT 11, and topotecan [9-dimethylaminomethyl- 10-hydroxy-camptothecin] .
  • 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.
  • the anti-hormonal agent used in the first treatment procedure may be any conventional agent, but it is preferably an androgen inhibiting agent.
  • preferred androgen inhibiting agents are LHRH (luteinizing hormone releasing hormone) agonists and anti-androgens such as flutamide, biclutamide, nilutamide, and luprolide. These agents are preferred for the treatment of prostate tumors, but other anti-hormonal agents may be used for other tumors, as would be recognized by those skilled in the art.
  • the invention encompasses the use of cytotoxic compounds together with anti -hormonal compounds or the use of two or more of these compounds.
  • chemotherapeutic agent that can be used in this invention.
  • Other conventional chemotherapeutic agents that can be used with squalamine in the process of the invention include methotrexate, melphalan, thiotepa, mitoxantrone, vincristine, vinblastine, etoposide, teniposide, ifosfamide, bleomycin, procarbazine, chlorambucil, fludarabine, mitomycin C, vinorelbine, and gemcitabine.
  • 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.”
  • suitable technique such as oral, "s.q.,” “i.p.,” “i.m.,” “i.l.,” or “i.v.”
  • the terms “s.q.,” “i.p.,” “i.m.,” “i.l.,” and “i.v.” will be used to refer to subcutaneous administration of squalamine or other substances, 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 substances, respectively.
  • 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 agent.
  • carboplatin is used in combination with paclitaxel.
  • the cytotoxic chemical compound and the squalamine may be delivered simultaneously by a common pharmaceutical carrier (i.e., one injection including both squalamine and the cytotoxic chemical compound).
  • a common pharmaceutical carrier i.e., one injection including both squalamine and the cytotoxic chemical compound.
  • Other appropriate combinations of administration techniques may be used without departing from the invention. Those skilled in the art will be able to ascertain the appropriate treatment regimens, depending on the cytotoxic chemicals used, the dosages, etc., through routine experimentation.
  • the squalamine treatment procedure in accordance with the invention also may be used with radiation treatment (i.e., cobalt or X-ray treatment) as the first treatment procedure.
  • the first treatment procedure is a radiation treatment
  • the second treatment procedure is squalamine administration.
  • Radiation treatments can proceed on a schedule in combination with the squalamine treatments to provide optimum results. Such scheduling of the treatment procedures can be ascertained by the skilled artisan through routine experimentation. Any conventional radiation treatment, such as those described in Medical Oncology, supra., may be used without departing from the invention.
  • the tumor also may be treated with one or more cytotoxic chemical or anti-hormonal 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, occurs in many basic physiological processes, such as embryogenesis, ovulation, and wound healing. 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 conditions which depend on angiogenesis, such as those identified above.
  • 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
  • the cells then move, divide, and digest their way into adjacent tissue through the extracellular matrix.
  • the cells then come together to form capillaries and lay down new basement membrane.
  • This angiogenesis process is illustrated in the upper portion of Fig. 2.
  • Each of these development stages during angiogenesis is important and may be affected by antiangiogenic agents.
  • Certain compounds which are believed to be antiangiogenic compounds i.e., matrix metalloproteinase inhibitors, such as minocycline, SU101 or marimistat
  • matrix metalloproteinase inhibitors such as minocycline, SU101 or marimistat
  • downstream angiogenesis inhibitors For a discussion of matrix metalloproteinase inhibitors, please refer to Teicher, Critical Reviews in Oncology/Hematologv, Vol. 20 (1995), pp. 9-39. This document is entirely inco ⁇ orated herein by reference.
  • squalamine acts at a very early stage in the process by inhibiting the cell activation action of growth factors, i.e., it is an "upstream" angiogenesis inhibitor. As shown in Fig. 2 (toward the bottom), squalamine inhibits the sodium-proton pumps that are normally active and activated by the growth factors.
  • Inhibition of the proton pump places the cell in a quiescent state, and, in this way, capillary formation and angiogenesis is impeded. In effect, the growth factor signal is aborted in the presence of squalamine.
  • squalamine has been shown to have a capillary regression effect in newly formed capillaries.
  • a one time dose (100 ng) of squalamine was applied to capillary beds of young chick embryos that were 2-3 days old. After five minutes, this dose of squalamine appeared to have little effect on the capillary beds. In twenty minutes, however, the capillary bed appeared to be disappearing (i.e., the vessels appeared to be closed off). After forty minutes, additional capillary regression was observed.
  • the capillary bed also was observed after sixty minutes. At this time, it was noted that some of the capillary vessels were beginning to re-appear, but only the more major vessels were re-appearing. The small vessels were not re-appearing at that time.
  • squalamine-induced capillary regression is reversible, at least with respect to certain capillaries. It also was concluded that squalamine is more effective against small microcapillary blood vessels (i.e., the microvascular bed) as compared to the major blood vessels. Close histological examination of chick microvessels exposed to squalamine revealed vessel occlusion was due to shrinkage of endothelial cell volumes in cells wrapped around the vessel lumen. The applicants postulate that occlusion or regression of small blood vessels by squalamine significantly contributes to the ability of squalamine to impede the flow of nutrients and growth factors into tumors and thereby slows or blocks the rate of growth of the tumors.
  • NHE antiporter system of a cell is connected to the extracellular matrix. Activation of the NHE antiporter is necessary to induce cell growth, and interference with the NHE antiporter interrupts the matrix signal and interferes with cell growth. When endothelial cell growth is interrupted, capillary growth is impeded.
  • the NHE antiporter of cells may be activated in different ways. For example, insoluble fibronectin activates the NHE antiporter by clustering and immobilizing Integrin ⁇ v ⁇ note independent of the cell shape (the growth of anchorage-dependent cells requires both soluble mitogens and insoluble matrix molecules).
  • the attachment of stimuli to the extracellular matrix or cell attachment events involving viruses also activate the NHE antiporter.
  • the NHE antiporter When activated, the NHE antiporter induces cell growth by regulating the pH of the cell.
  • the chloride-bicarbonate exchanger and NHE are complementary pH regulators in cells.
  • 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 that the cell dies; it means only that the cell enters a quiescent state (i.e., it does not divide). If the cell returns to a normal pH, growth may resume.
  • the cell When the NHE is activated, the cell becomes more alkaline (higher pH), it pumps out protons, and growth proceeds. Interaction of various modulatory factors (i.e., serum components, secondary messengers, etc.) with one portion of the cytoplasmic region of NHE activates the antiporter, while interaction with another portion inhibits the antiporter.
  • modulatory factors i.e., serum components, secondary messengers, etc.
  • Tse, et al. "The Mammalian Na + /H + Exchanger Gene Family - Initial Structure/Function Studies," J. Am. Soc. Nephr., Vol. 4 (1993), pg. 969, et seq. This article is entirely inco ⁇ orated herein by reference.
  • 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.
  • growth factors e.g., VEGF and b-FGF
  • other stimuli such as virus attachment, addition of various mitogens, sperm attachment to an egg, etc, 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.
  • NHE1 is the antiporter found in all tissues.
  • NHE2 and NHE3 are more restrictive in their tissue distribution.
  • the effect of squalamine on NHE activity was measured to determine which isoforms of NHE were affected by squalamine.
  • NHE activity can be measured under various different cellular conditions. Acid loading a cell activates all of the antiporters and permits measurement of NHE. NHE activity also can be measured after growth factor stimulation of the cell.
  • 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 absence of bicarbonate.
  • Amilorides which are the classic inhibitors of activated NHE antiporters and which act as direct competitive inhibitors of Na + ion binding to NHE, do not turn off the antiporter activity in unstimulated cells. As illustrated in Fig. 4, amiloride and amiloride analogues specifically act against NHEl over NHE2 or NHE3. NHE3 in particular is relatively resistant to inhibition by the amilorides. In contrast to the amilorides, when NHEl activity was measured in unstimulated melanoma cells, applicants found that squalamine substantially down regulates the activity of the antiporter. The following describes the test used to determine that squalamine inhibits NHE3, but not NHEl or NHE2.
  • 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-carboxyfluorescein (BCECF).
  • BCECF pH sensitive dye 2'7'-bis(2-carboxyethyl)- 5,6-carboxyfluorescein
  • NHE activity was measured by spectrofluorometric methods using this dye and by amiloride sensitive isotopic 22 Na + cellular uptake.
  • the cells were acidified by exposure to ammonium chloride in the absence of sodium to eliminate sodium and deactivate the proton pumps.
  • the ammonium chloride was washed out by exposing the cells to tetramethyl ammonium chloride in bicarbonate free medium. The cells were consequently acidified, but in the absence of sodium, the NHE ion pumps did not activate.
  • squalamine did not act like the classic NHE inhibitor amiloride or analogues of amiloride, which are direct competitive inhibitors for sodium and, therefore, act rapidly as NHE inhibitors. Furthermore, it was observed that the NHE inhibiting effect of squalamine occurred in the absence of lactase dehydrogenase (LDH) leakage from the cell. Because LDH leakage is a non-specific marker of cytotoxicity, it was concluded that squalamine does not have a general cytotoxic effect.
  • LDH lactase dehydrogenase
  • 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 removed from the molecule, 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. Thus, 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 effect on tyrosine kinase-dependent activity, although applicants do not wish to be bound by any specific theory of operation.
  • squalamine inhibits NHE3 and not NHEl.
  • This inhibitory effect of squalamine has been found to work in a manner different from classical and known NHE3 inhibitors.
  • other inhibitors of 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 raax "), if one looks at the kinetic characteristics of the inhibition.
  • squalamine inhibits NHE with nonallosteric kinetics (i.e., nonclassical allosteric inhibition).
  • squalamine at a 1 hour pretreatment
  • This observed squalamine effect on the V max was time dependent, with a maximum effect occurring at one hour exposure. The observed effect was fully reversible within three hours after removing the cells from the medium.
  • NHE3 is important in maintaining homeostasis of the unstimulated cell.
  • prevention of cellular activation by squalamine is the mechanism through which squalamine inhibits tumor growth.
  • squalamine changes endothelial cell shape. This suggests that transport proteins which control cell volume and shape may be a squalamine target.
  • FIGs. 6a to 6c illustrate the test results where squalamine was administered subcutaneously (50 mg/kg, Fig. 6a), intraperitoneally (dose 240 ⁇ g; 10 mg/kg, Fig. 6b), and intravenously (10 mg/kg, Fig. 6c).
  • Fig. 7 illustrates the distribution of squalamine in mouse tissue two hours after i.v. administration. Some squalamine is contained in most of the tissues, with most of the squalamine concentrating in the liver and the small intestine. The test results shown in Fig. 7 indicate good squalamine distribution. Notably, however, not much squalamine is present in brain tissue, From this, applicants conclude that squalamine probably does not cross the brain/blood barrier. In treating brain tumors, it is believed that the squalamine acts on the endothelial cells in the brain, and in this way, it need not cross the brain/blood barrier
  • the mode of administration of squalamine may be selected to suit the particular therapeutic use. Modes of administration generally include, but are not limited to, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, inhalation, intralymphatic, intralesional, and oral routes.
  • the squalamine compounds may be administered by any convenient route, for example, by infusion or bolus injection, or by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and it may be administered together with other biologically active agents. Administration may be local or systemic.
  • compositions which include squalamine as an active ingredient.
  • Such compositions include a therapeutically effective amount of squalamine and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier include, but are not limited to, saline, buffered saline, dextrose, water, oil in water microemulsions such as Intralipid, glycerol, and ethanol, and combinations thereof.
  • the 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 wetting or emulsifying agents, or pH buffering agents.
  • the pharmaceutical composition may be in any suitable form, such as a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • composition also may be formulated as a suppository, with traditional binders and carriers, such as triglycerides.
  • Oral formulations may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Various delivery systems are known and may be used to administer a therapeutic compound of the invention, e.g., encapsulation in liposomes, microparticles, enteric coated systems, microcapsules, and the like.
  • 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 water or Interlipid.
  • 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 mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the pharmaceutical composition 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 for injection may be provided so that the ingredients may be mixed prior to administration.
  • 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 skilled in the art.
  • the precise dose to be employed in the formulation also will depend on the route of administration and the seriousness of the disease or disorder, and should be decided according to the judgement of the practitioner and each patient's circumstances. Effective therapeutical doses may be estimated from extrapolations of dose-response curves derived from in vitro or animal-model test systems.
  • Suitable dosages for intravenous administration are generally about 1 microgram to
  • Suitable dosage ranges for intranasal administration are generally about 0.01 mg/kg body weight to 20 mg/kg body weight.
  • Suitable dosages for oral administration are generally about 500 micrograms to
  • Suppositories generally contain, as the active ingredient, 0.5 to 10% by weight of squalamine. Oral formulations preferably contain 10% to 95%> active ingredient.
  • 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 containers filled with the pharmaceutical compositions in accordance with the invention.
  • Associated with such containers may be a notice in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • cytotoxic chemical and anti-hormonal compounds used in accordance with the invention may be present in any suitable form known to those skilled in the art. These chemical compounds also may be administered by any suitable means also known to those skilled in this art, such as orally, subcutaneously, intravenously, intraperitoneally, intralymphaticly, and intramuscularly.
  • the rabbit corneal micropocket assay is an accepted standard test.
  • an incision is made in one rabbit cornea, and a 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 growth factors to stimulate new capillary growth.
  • 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 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, one can visually assess the results qualitatively. In addition, the number of blood vessels can be counted and their length, etc., can be measured to provide a more quantitative evaluation of the results.
  • the VX2 rabbit carcinoma was implanted in 26 rabbit eyes, in the normally avascular corneal region, to act as an angiogenesis stimulus.
  • Squalamine was inco ⁇ orated into a controlled release ethylene/vinyl acetate copolymer (20% squalamine and 80% polymer by weight).
  • 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 eye of the same rabbit served as the control eye.
  • the eyes were examined weekly using a slit lamp stereomicroscope for three weeks after tumor implantment, and the Angiogenesis Index (" AI") was calculated (this calculation will be described in more detail below with reference to Fig. 8).
  • AI Angiogenesis Index
  • 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 control cornea.
  • 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 not dead), but there was essentially no vasculature associated with those tumors.
  • Fig. 8 shows a graphical representation of the results of the rabbit cornea micropocket assay test.
  • the vessel length (“L vessel ”) 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 longest vessel growth.
  • the Angiogenesis Index then is determined from these measurements by the following equation:
  • Figure 8 shows the mean Angiogenesis Index for each group of corneas (squalamine treated and untreated) in the rabbit cornea micropocket assay after 1, 2, and 3 weeks.
  • squalamine was very inhibitory to the growth of new blood vessels.
  • This data illustrates that squalamine inhibits tumor induced growth of new blood vessels or capillaries over a long time period. More specifically, squalamine exhibits high antiangiogenic activity even after three weeks.
  • squalamine may be a potent antiangiogenic agent that inhibits neovascularization. Recognizing that the exponential growth of solid tumors in the brain is dependent on neovascularization, applicants assessed the activity of squalamine in an animal model on the growth of solid tumors in the brain.
  • malignant gliomas are the most common form of cancerous tumors. These tumors are the third leading cause of death from cancer in young adults between the ages of 15 and 34. Malignant gliomas are characterized by their ability to induce the normally quiescent brain and/or CNS endothelial cells into a highly proliferative and invasive state.
  • the gliomas express vascular endothelial growth factor ("VEGF”) and other growth factors which stimulate inducible receptors on CNS endothelial cells in a paracrine manner (i.e., the VEGF originates from the tumor cell and stimulates the endothelial cells).
  • VEGF vascular endothelial growth factor
  • the CNS endothelial cells subsequently initiate angiogenic invasion and thus provide nourishment of the glioma.
  • In vitro tests were first performed to determine that squalamine acts specifically on 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.
  • tumor angiogenesis is a series of sequential and overlapping steps. First, the endothelial cells activate and proliferate. Then, proteolytic enzymes are produced and the cells migrate.
  • New basement membranes must then be generated. In this manner, new blood vessels are generated and tumor size increases.
  • the following cell lines were tested: (a) bovine 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 optimized growth media. Following attachment, the cells were exposed to solvent only or to increasing concentrations of squalamine (0, 10, 20, 30, 60, and 90 ⁇ g squalamine/ml). Cell growth was counted daily for three days using a Coulter Counter. A total of 10,000 cells per well were plated and each experimental concentration was tested in quadruplicate. The results were then averaged.
  • the 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.
  • %I Percentage endothelial cell growth inhibition
  • 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 dramatically and specifically inhibits VEGF-stimulated growth of endothelial cells in vitro.
  • squalamine is a potent inhibitor of tumor-induced angiogenesis, and this effect appears to be precipitated through specific inhibition of endothelial cell proliferation induced by VEGF.
  • 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 induced by b-FGF, PDGF bb , scatter factor (HGF or hepatocyte growth factor), conditioned tumor media, and human brain cyst fluid. Thus, as the tumor puts out a variety of different growth factors, squalamine has an inhibitory effect on several.
  • Fig. 10 further illustrates that in this animal model, the squalamine treated rats, in general, had an increased survival time.
  • BCNU which is a conventional chemotherapy agent
  • BCNU has a cumulative toxicity effect. For this reason, it is administered only one time to a patient.
  • the use of BCNU is described on pages 304 and 305 of Calabresi in Medical Oncology, supra.
  • a group of rats was given a daily squalamine dose of 20 mg/kg/day (i.p.) for more than 30 days and maintained for up to 200 days following dosing. The animals in this study remained healthy. This result indicates that squalamine has little or no toxicity.
  • squalamine is an upstream inhibitor of the angiogenesis process by inhibiting the activation of endothelial cells after growth factor interaction. Because of its angiogenesis inhibiting properties, squalamine has been demonstrated to be effective in treating solid tumors which rely on neovascularization to proliferate. Applicants tested to determine whether beneficial results could be obtained when treating tumors by combining a squalamine treatment (an upstream angiogenesis inhibitor) with a conventional cancer treatment using an alkylating agent.
  • 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 combination of squalamine and the nitrosourea BCNU (Group 4).
  • a 99.8%> reduced mean tumor size was observed when treating with both squalamine and BCNU in this group.
  • Table 5 further shows that squalamine alone (Group 3) was effective in treating the tumor.
  • the tumor size was reduced by 81.7% in Group 3, as compared to the control group.
  • 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.
  • the high dose squalamine was discontinued after five weeks of treatment because of animal weight loss and potential toxicity concerns, so these animals did not receive squalamine for the last eight weeks of the experiment.
  • the low dose squalamine treatment produced a significant (P ⁇ 0.01) inhibition in the rate of progression of the breast tumors at all times examined (Fig. 11).
  • the high dose squalamine treatment produced significant (P ⁇ 0.05) delay in progression of the breast tumors only at 30 days post-initiation (i.e., only while squalamine was still being given), but high dose squalamine also doubled the long-term cure rate in these animals compared to controls which received cyclophosphamide alone (Fig. 11).
  • Examination of the history of the long-term cure animals which received cyclophosphamide and high dose squalamine revealed that the additive effects of squalamine were manifested within two weeks after starting squalamine treatment.
  • squalamine has synergistic activity in combination with cisplatin (e.g., Fig. 12).
  • the experimental lung cancer model design involves subcutaneous injection of 5 x 10 6 tumor cells followed by a single injection of the chemotherapeutic drug on day 3 or 4.
  • Daily intraperitoneal squalamine injections with 20% Intralipid as a vehicle began the following day for some groups of mice and continued until the experiment was terminated 7-14 days later. Groups of mice receiving squalamine alone started receiving the aminosterol on the same day as aminosterol treatment in the combination chemotherapy groups.
  • Tumor volumes were then determined at termination of the experiment and compared. It was found for both the aggressively growing H460 human lung adenocarcinoma line and for the more slowly growing Calu-6 human lung adenocarcinoma line that squalamine had minimal effects on tumor growth as a mono therapeutic agent when started on day 4 or 5, but could contribute to growth inhibition if it were started on day 1. However, when used starting on day 4 or 5, in combination with cisplatin, given at or near a maximum tolerated dose, squalamine significantly and reproducibly improved tumor growth inhibition over cisplatin alone in a dose-dependent fashion for both the H460 and Calu-6 cell lines.
  • mice The murine Lewis lung adenocarcinoma was implanted subcutaneously in the hind- leg of male C57BL/6 mice and allowed to grow for one week. Groups of mice were then left untreated or treated with either squalamine (20 mg/kg/day, s.c), cyclophosphamide (125 mg/kg, i.p. on days 7, 9 and 11), cisplatin (10 mg/kg, i.p. on day 7), the combination of squalamine and cyclophosphamide, or the combination of squalamine and cisplatin. On day 20, the animals were sacrificed, and the mean number of lung metastases were determined for each group. All treatments reduced the number of metastases; however, the most effective treatments were the combination of squalamine with either of the cytotoxic agents (Fig. 13).
  • LNCaP/C4-2 human prostate cancer progression model The anti-proliferative effect of squalamine was evaluated in tissue culture by both crystal violet staining and [ 3 H] -thymidine inco ⁇ oration, on the LNCaP/C4-2 human prostate cancer progression model.
  • this lineage- derived cell line recapitulates the progression of human neoplastic prostate -disease from an androgen-dependent and minimally metastatic condition (LNCaP cells) to an androgen- independent (defined as being able to proliferate in castrate hosts) and highly aggressive state (C4-2 subline).
  • LNCaP/C4-2 progression model has been previously demonstrated to be an effective model for screening the efficacy of therapeutic agents for prostate cancer.
  • LNCaP cells admixed with MatrigelTM were implanted subcutaneously in 52 athymic mice. Those that developed measurable tumors and sufficient elevations in serum prostate specific antigen ("PSA") (measured using a standard commercially available kit such as from Abbott Diagnostics (St. Louis, MO)) underwent orchiectomy. Subsequent to castration, these animals were distributed among the four groups (1 -4) outlined below: and an additional control cohort of mice which were castrated but never treated with squalamine.
  • PSA serum prostate specific antigen
  • a third group of animals began to receive squalamine (20 mg/kg/day squalamine, five days per week with a two day respite) when their serum human PSA levels reached values of 4-10 ng/mL.
  • a fourth group of mice began squalamine treatment (20 mg/kg/day squalamine, five days per week with a two day respite)when serum human PSA values reached 20-40 ng/mL, and a fifth group began squalamine treatment when serum human PSA levels reached 100 ng/mL. It was found that the human tumor xenografts responded to castration by a slowing or cessation of tumor growth following castration for 2-4 weeks before the tumors began growing again.
  • Tissue histology was carried out on the tumor masses (or the site of tumor implantation, in the instance of the animals which underwent successful squalamine treatment) to examine the distribution of integrins in these tissues.
  • successful squalamine treatment coincided with observation of a reduction in the expression of the integrin ⁇ v ⁇ 3 and an increase in the integrin 6 ⁇ 4 .
  • the integrin ⁇ v ⁇ 3 has been associated with a greater propensity for increased angiogenesis in tumors and for a tumor to grow and metastasize (e.g., cf. B.P. Eliceiri and D.A. Cheresh, J. Clin. Invest. 103. 1227-1230 (1999)).
  • part of the mechanism by which squalamine successfully alters LNCaP tumor growth is by interfering with cellular integrin expression that is associated with tumor aggressiveness.
  • LNCaP cells were maintained as previously described (cf. J.T. Hsieh et al, Cancer Research 53. 2852-2857 (1993)). Cells were exposed to 20 ng/mL vascular endothelial growth factor (VEGF), 20 ⁇ g/mL squalamine or a combination of VEGF and squalamine. It was found that VEGF or squalamine alone had minimal effects on cell growth in media containing serum, but that the combination of VEGF and squalamine after 24 hours of exposure reduced [3H]- thymidine uptake by greater than 98%> (Table 6). As a consequence, the mechanism underlying the effectiveness of squalamine treatment of the LNCaP tumor in vivo may under certain conditions also involve direct inhibition of tumor cell growth and/or induction of tumor cell death.
  • VEGF vascular endothelial growth factor
  • mice Squalamine activity in combination with carboplatin About 5 x 10 6 cells from each of two human lung tumor lines, H460 (a rapidly proliferating large cell carcinoma cell line, ATCC HTB-177) and Calu-6 (an anaplastic carcinoma, ATCC HTB-56), were inoculated subcutaneously in the right foreleg of nude BALBc mice. Once tumors were visible (3-5 days) with a mean volume of approximately 50-80 mm 3 , mice were treated intraperitoneally with 60 mg/kg carboplatin (single dose) or 20 mg/kg/ squalamine (once daily for 5 days) or a combination of carboplatin and squalamine. There were 8 mice per group.
  • Tumor growth inhibition was scored by quantifying the size of tumors once control tumors reached a size of 1.0 gram and determining the ratio of mean treated tumor size to mean control tumor size (T/C). Tumor growth inhibition was then scored as 100%> x [1 - (T/C)].
  • mice Female Sprague Dawley nu/nu mice weighing approximately 20 grams were implanted subcutaneously by trocar with fragments of MV-522 human lung tumors harvested from subcutaneously grown tumors in nude mice hosts. When tumors reached approximately 5 x 5 mm the animals were pair-matched into treatment and control groups. Each group contained 10 mice bearing tumors, was followed individually throughout the experiment. The administration of drugs (squalamine, paclitaxel plus carboplatin, or squalamine with paclitaxel and carboplatin) or vehicle began the day the animals were pair- matched (Day 1). Drug doses and schedule were selected based upon prior measurements of maximum tolerated doses for paclitaxel plus carboplatin in mice. Intraperitoneal (i.p.) dosing of drugs was selected as an acceptable surrogate for intravenous dosing.
  • drugs squalamine, paclitaxel plus carboplatin, or squalamine with paclitaxel and carboplatin
  • TGI tumor growth inhibition
  • Some drug combinations caused shrinkage of some tumors in the MV-522 tumor xenograft model.
  • the final weight of a given tumor (Day 31) was subtracted from its own calculated weight at the start of treatment on Day 1 for the relevant mice. This difference divided by the initial tumor weight times 100% was the percent shrinkage.
  • a mean percent tumor shrinkage was then calculated from the data if more than one mouse in a group experienced a reduction in tumor size.
  • a tumor was considered to have experienced a partial regression if the reduction in tumor size was >50% at any time during the study. If the tumor completely disappeared in a mouse, this was considered a complete response or complete tumor shrinkage, which was then confirmed by histologic examination.
  • triple combination chemotherapy with squalamine, paclitaxel and carboplatin produced a mean excised tumor growth of -0.1 ⁇ 19.3 mg (p ⁇ 0.001) or a net tumor stasis for the triple therapy combination group of animals, despite no chemotherapy during the last three weeks prior to terminal sacrifice.
  • the TGI index for the squalamine/paclitaxel/carboplatin arm of this study was 96.1 %.
  • the benefit of chemotherapy on tumor size became apparent by Day 7 and differences in mean tumor sizes between the two paclitaxel and carboplatin-containing arms of this study increased thereafter. Tumors in the squalamine-treated animals scored at terminal sacrifice were significantly smaller than those seen in animals which received only paclitaxel and carboplatin.
  • mice receiving only paclitaxel and carboplatin were noted to undergo tumor shrinkage as assessed at the end of the experiment, but four mice in the triple combination chemotherapy cohort displayed tumor shrinkage over the course of the experiment, with a mean reduction in tumor size for these animals of 54.8%.
  • the transient reduction in tumor sizes was maximal on Day 14 of chemotherapy, as was seen with squalamine plus cisplatin.
  • mean excised control tumor growth was 674.3 ⁇ 75.8 mg, while the excised tumor growth in animals treated with paclitaxel and carboplatin alone was 223.4 ⁇ 82.5 mg. This corresponded to a TGI for paclitaxel plus carboplatin of 66.9%, which is similar to the 60.3%> TGI seen in the first experiment for this double drug treatment (see above).
  • mice receiving paclitaxel and carboplatin alone had tumor shrinkages, but there were at lease two mice in each squalamine-treated group whose tumors were reduced in size over the course of the experiment, with a maximum of 5 mice out of 10 displaying tumor shrinkage at Day 32 in each of the groups receiving 2 or 10 mg/kg/day squalamine.
  • mice No mice were observed with complete tumor shrinkages in any treatment group, but significant partial tumor regressions were seen in all treatment groups (including paclitaxel/carboplatin alone) which were most evident at Day 15 following initiation of chemotherapy.
  • 19 of 20 mice had a reduction in tumor burden of at least
  • Syngeneic mammary carcinoma MCA-4 tumor cells (5 x 10 5 ) were injected intramuscularly into the right legs of C3Hf Kam male mice (4 months of age). When the tumors reached 6 mm in diameter, the mice were randomly assigned to receive: (a) no treatment, (b) squalamine alone (20 mg/kg/day b.i.d. for 11-14 days), (c) 15 Gy of gamma irradiation, or (d) squalamine plus 15 Gy of gamma rays. Tumor growth was followed until tumors reached at least 12 mm in diameter. The effect of squalamine on tumor radioresponse was determined by absolute and normalized tumor growth delays (AGD and NGD).
  • AGD is defined as the time in days for tumors treated with radiation (or squalamine) to grow from 8 to 12 mm minus the time in days for untreated tumors to grow from 8-12 mm.
  • NGD is defined as the time in days for tumors treated with both squalamine and radiation to grow from 8 to 12 mm minus the time in days for tumors treated with squalamine only to grow from 8 to 12 mm.
  • An enhancement factor (EF) was determined as the ratio of NGD to AGD. The EF for squalamine with 15 Gy gamma rays calculated from the observed times for tumor growth (Table 8) is >1.5.
  • Human ovarian tumor cells were injected subcutaneously (5 x 10 7 cells per animal) in the mid-back region of female Swiss nude mice.
  • the human ovarian tumors were either the parental 2008 cell line (cf. R.J. Pietras et al, Oncogene 9, 1829-1838 (1994)) or a transfected variant overexpressing the Her-2/ «e « receptor.
  • the overexpression of the Her- 2/neu receptor is considered to make the resulting xenograft tumors more angiogenic and less responsive to chemotherapy.
  • mice were randomized to groups receiving no treatment (controls), a single dose of cisplatin (4 mg/kg), daily treatment with squalamine (2 mg/kg, days 1-10) or cisplatin plus squalamine.
  • the growth of tumors was then scored on day 28 following initiation of chemotherapy.
  • the results were very similar with the parental 2008 tumor and the transfected variant overexpressing the Her-2/ «e « receptor. Exemplary results with the parental 2008 cell line are shown in Table 9. These results can be quantified by calculating a tumor growth inhibition index for each treatment group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention se rapporte à une méthode de traitement des tumeurs qui comprend une première procédure thérapeutique mettant en oeuvre une technique classique de traitement anticancéreux et une seconde procédure thérapeutique consistant à administrer une quantité efficace de squalamine. On utilise de préférence des quantités présentant une efficacité d'un point de vue synergétique. La première procédure thérapeutique peut consister à traiter avec un ou plusieurs composés chimiques cytotoxiques classiques. Un tel composé chimique cytotoxique peut être du type nitrosourée (par exemple, BCNU), cyclophosphamide, doxorubicine, 5-fluoro-uracile, paclitaxel et ses dérivés, cisplatine ou autre agents anticancéreux contenant du platine. Cette première procédure thérapeutique peut également consister à traiter avec un ou plusieurs agents anti-hormonaux classiques. Ces agents anti-hormonaux peuvent être du type agoniste de la LHRH (lutéolibérine) ou un composé anti-androgène du type flutamide, biclutamide, nilutamide et luprolide. Ces composés anticancéreux classiques et la squalamine peuvent être administrés par toute voie adaptée. La première procédure thérapeutique peut être effectuée avant la seconde procédure thérapeutique, après celle-ci, ou simultanément à celle-ci. Cette première procédure thérapeutique peut également être un traitement classique par rayonnement. Elle peut également être une combinaison d'un traitement à base d'un ou de plusieurs composés chimiques cytotoxiques classiques et d'un traitement classique par rayonnement.
EP99952905A 1998-09-10 1999-09-10 Traitement des carcinomes a base de squalamine combinee a d'autres agents anticancereux Withdrawn EP1119361A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US15072498A 1998-09-10 1998-09-10
US150724 1998-09-10
PCT/US1999/020645 WO2000015176A2 (fr) 1998-09-10 1999-09-10 Traitement des carcinomes a base de squalamine combinee a d'autres agents anticancereux

Publications (2)

Publication Number Publication Date
EP1119361A2 true EP1119361A2 (fr) 2001-08-01
EP1119361A4 EP1119361A4 (fr) 2006-10-04

Family

ID=22535747

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99952905A Withdrawn EP1119361A4 (fr) 1998-09-10 1999-09-10 Traitement des carcinomes a base de squalamine combinee a d'autres agents anticancereux

Country Status (5)

Country Link
EP (1) EP1119361A4 (fr)
JP (1) JP2002524481A (fr)
AU (1) AU757649B2 (fr)
CA (1) CA2343133A1 (fr)
WO (1) WO2000015176A2 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0121285D0 (en) 2001-09-03 2001-10-24 Cancer Res Ventures Ltd Anti-cancer combinations

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4474813A (en) * 1980-10-24 1984-10-02 Schering Corporation Pharmaceutical preparations comprising flutamide
JPS6133121A (ja) * 1984-07-25 1986-02-17 Nissei Marine Kogyo Kk 制癌剤
WO1997028826A2 (fr) * 1996-02-12 1997-08-14 Schering Aktiengesellschaft Systemes de liberation d'agent contraceptif a effet antibacterien et/ou antiviral
WO1997040835A1 (fr) * 1996-04-26 1997-11-06 Magainin Pharmaceuticals Inc. Traitement des carcinomes par la squalamine en association avec d'autres agents anticancereux

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020520A1 (fr) * 1993-03-10 1994-09-15 Magainin Pharmaceuticals Inc. Derives steroidiens, compositions pharmaceutiques contenant ces derives steroidiens et utilisation de ces derniers comme antibiotiques ou desinfectants

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4474813A (en) * 1980-10-24 1984-10-02 Schering Corporation Pharmaceutical preparations comprising flutamide
JPS6133121A (ja) * 1984-07-25 1986-02-17 Nissei Marine Kogyo Kk 制癌剤
WO1997028826A2 (fr) * 1996-02-12 1997-08-14 Schering Aktiengesellschaft Systemes de liberation d'agent contraceptif a effet antibacterien et/ou antiviral
WO1997040835A1 (fr) * 1996-04-26 1997-11-06 Magainin Pharmaceuticals Inc. Traitement des carcinomes par la squalamine en association avec d'autres agents anticancereux

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
A. GAGLIARDI ET AL.: "Inhibition of angiogenesis by antiestrogens" CANCER RESEARCH, vol. 53, 1993, pages 533-535, XP001121715 *
BOCCON-GIBOD L. ET AL.: "Flutamide versus orchidectomy in the treatment of metastatic prostate carcinoma" EUR. UROLOGY, vol. 32, no. 4, 1997, pages 391-396, *
KAREN S. MOORE AND OTHERS: "Squalamine: An aminosterol antibiotic from the shark" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 90, February 1993 (1993-02), pages 1354-1358, XP002394985 *
KONOVALOVA ET AL: "Increasing the therapeutic efficacy of antitumor drugs" STN CHEMICAL ABSTRACTS, vol. 3, no. 111, 17 July 1989 (1989-07-17), XP002037635 *
MARSHALL J: "SHARK CARTILAGE FOR CANCER TREATMENT?" P & T, CORE MEDICAL JOURNALS, LAWRENCEVILLE, NJ, US, vol. 21, no. 3, 1 March 1996 (1996-03-01), pages 159-160, XP000602614 ISSN: 1052-1372 *
PATENT ABSTRACTS OF JAPAN vol. 010, no. 185 (C-357), 27 June 1986 (1986-06-27) & JP 61 033121 A (NISSEI MARINE KOGYO KK), 17 February 1986 (1986-02-17) *
See also references of WO0015176A2 *

Also Published As

Publication number Publication date
WO2000015176A3 (fr) 2000-07-20
CA2343133A1 (fr) 2000-03-23
AU6496299A (en) 2000-04-03
EP1119361A4 (fr) 2006-10-04
WO2000015176A2 (fr) 2000-03-23
AU757649B2 (en) 2003-02-27
JP2002524481A (ja) 2002-08-06

Similar Documents

Publication Publication Date Title
US6596712B2 (en) Treatment of carcinomas using squalamine in combination with other anti-cancer agents or modalities
AU735884B2 (en) Treatment of carcinomas using squalamine in combination with other anti-cancer agents
Shah et al. Investigational chemotherapy and novel pharmacokinetic mechanisms for the treatment of breast cancer brain metastases
US8003105B2 (en) Method of treating cancer by co-administration of anticancer agents
EP0415623A2 (fr) Torémifène ou déméthyle-torémifène ou hydroxy-torémifène pour l'inversion de la résistance "multidrug" de cellules cancéreuses contre des substances cytotoxiques
US8143226B2 (en) Tyrosine kinase receptor antagonists and methods of treatment for breast cancer
KR20160078987A (ko) 플리나불린 및 탁산의 조합에 의한 암 치료
WO2007065016A2 (fr) Methodes et compositions ameliorant l'activite et reduisant la toxicite de stents
US20220265760A1 (en) Leuprolide acetate compositions and methods of using the same to treat breast cancer
Garcia-Schürmann et al. Suramin treatment in hormone-and chemotherapy-refractory prostate cancer
WO2020236668A1 (fr) Leukadhérine-1 seule ou en association pour une utilisation dans le traitement du cancer
AU757649B2 (en) Treatment of carcinomas using squalamine in combination with other anti-cancer agents or modalities
EP3632445A1 (fr) Combinaisons avec un stéroïde c-19 pour le traitement de cancers
Mattioli et al. Long-survival in Responding Patients with Metastatic Breast Cancer Treated with Doxorubicin-Docetaxel Combination: A Multicentre Phase II Trial
JP2002524481A5 (fr)
Wolf et al. Nitroglycerin decreases medial smooth muscle cell proliferation after arterial balloon injury
Lentz Endocrine therapy of endometrial cancer
CN115920062A (zh) 联合用药物及其在预防或治疗肝癌中的应用
Baker et al. CLINICAL TRIALS SUMMARIES
Knox et al. Chemotherapy in Hormone Refractory Prostate Cancer
Rubens et al. Clinical assessment of two antiandrogen treat-ments, cyproterone acetate combined with ethi-nyl estradiol and spironolactone in hirsutism
Moore 13: Chemotherapy in Hormone Refractory Prostate Cancer
EP1843752A2 (fr) Methode de traitement du cancer

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010409

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RIC1 Information provided on ipc code assigned before grant

Ipc: A61K 31/59 20060101AFI20060818BHEP

A4 Supplementary search report drawn up and despatched

Effective date: 20060831

17Q First examination report despatched

Effective date: 20061221

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: GENAERA CORPORATION

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 35/00 20060101ALI20080627BHEP

Ipc: A61K 45/06 20060101ALI20080627BHEP

Ipc: A61K 31/4166 20060101ALI20080627BHEP

Ipc: A61K 31/277 20060101ALI20080627BHEP

Ipc: A61K 31/167 20060101ALI20080627BHEP

Ipc: A61K 31/575 20060101AFI20080627BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20081128