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
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
  • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
• • 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
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to methods for treating cells resistant to neoplastic agents.
• cytotoxic agents that help to produce a positive outcome in cancer patient therapy
• many cancer cells develop resistance or are resistant to the neoplastic agents currently of choice for chemotherapeutic treatment.
• the development of drug resistance substantially compromises the efficacy of cancer therapy.
• Multidrug resistance cells are one example of cells that are resistant to antineoplastic agents. In this case, the cells are resistant to more than one antineoplastic agent. Multidrug resistance is a well-defined phenomenon. Often cancer cells that become resistant to one class of anticancer drugs (i.e., Vinca alkaloids, anthracyclines, taxanes, including paclitaxel, epipodophyllotoxins, and the like) also demonstrate resistance to other anticancer drugs. Development of multidrug resistance creates a significant impediment in the generation of positive outcomes for many cancer patients. Multidrug resistant agents have a number of general features in common; they are generally lipophili, weakly basic molecules of greater than about 300 daltons or larger molecular weight.
• Multidrug resistant cells tend to accumulate anticancer drugs at a level lower than cells that are not multidrug resistant (Beck, WT, Adv. Enzym. Regul 1984, 22:207). Accumulation of drug at lower levels has been shown in some models to be associated with an increase in activity or in the amount of a family of transmembrane channel proteins.
• transmembrane channel proteins that are capable of decreasing the intercellular concentration of anti-cancer drugs are the integral membrane proteins P- glycoproteins (Pgp, Endicott JA and Ling, V. Annu. Rev. Biochem. 1989, 58:137).
• the proteins appear to bind to antineoplastic agents and release the agents into the extracellular milieu.
• Expression of the MDR1 cDNA, the DNA encoding Pgp is sufficient to produce a multidrug resistance phenotype (Gros et al, Nature 1986, 323:728). These proteins are present in rodents and in man.
• MRP multidrug resistance-associated protein
• Another protein associated with resistance to antineoplastic agents is the multidrug resistance-associated protein (MRP) (Grant CE et al, Cancer Res.
• MRP multidrug resistance-associated protein
• MRP has been shown to confer multidrug resistance to doxorubicin, vincristine, etoposide and colchicine.
• MRP has been shown to confer multidrug resistance to doxorubicin, vincristine, etoposide and colchicine.
• Elevated levels of Pgp have been observed in a variety of cancers including, but not limited to, Acute Myelogenous Leukemia, Non-Hodgkin's Lymphoma, multiple myeloma as well as in a variety of solid tumors including, but not limited to, cancers of the adrenal, colon, kidney, lung and breast (see Beck and Dalton, supra).
• cancers having an origin in a variety of tissues and cells can develop multidrug resistance. Therefore, there is a need to identify and to use compounds that remain toxic to otherwise multidrug resistant cells.
• antineoplastic agents In addition to multidrug resistance, there are other types of resistance to antineoplastic agents that have been observed. These include, for example, resistance to one or more antineoplastic agents as a result of a mutated protein.
• resistance to antineoplastic agents results from mutations in microtubules or in mutations in tubulin dimers.
• Cellular resistance to taxanes such as paclitaxel, can be multifactorial. For example, cellular resistance to the taxane family has been associated in some instances with a mutation in the ⁇ - tubulin subunit. Again, as in the case of multidrug resistant cells, there is a need for neoplastic agents that remain toxic to taxane-resistant cells.
• the present invention relates to methods for treating cells with discodermolide.
• the invention relates to methods for treating cells with discodermolide in vivo.
• the invention in another aspect of this invention, relates to a method for inhibiting the growth of multidrug resistant cells comprising the step of contacting at least one multidrug resistant cell with a growth-inhibiting amount of discodermolide.
• the multidrug resistant cell is resistant to taxanes, for example paclitaxel.
• the multidrug resistant cells are growth inhibited in vivo or in culture.
• the cell is from a mammal and more preferably from a human.
• the invention relates to a method for inhibiting the growth of a cancer cell comprising the steps of: contacting at least one cancer cell with a growth inhibiting amount of discodermolide wherein the cancer cell is resistant to at least one antineoplastic agent.
• the cancer cell is selected from the group consisting of a leukemia cell, a lymphoma cell and a solid tumor cell.
• the cancer cell is a multidrug resistant cell.
• the cell comprises a mutation in ⁇ -tubulin and in another embodiment, the cell over-produces glutathione.
• the cell is in a mammal.
• the invention also relates to a method for promoting apoptosis in a multidrug resistant cell comprising the steps of contacting a multidrug resistant cell with discodermolide; and inducing apoptosis in the cell.
• the multidrug resistant cell is resistant to paclitaxel.
• the cell can be a cell in culture or in vivo.
• the cell is from a mammal and more preferably from a human.
• the invention further relates to a method for inhibiting the growth of cancer cells having a ⁇ -tubulin mutation comprising the steps of; contacting at least one cancer cell with a growth inhibiting amount of discodermolide wherein the cell comprises a mutation in the protein ⁇ - tubulin; and inhibiting cell division in the cell.
• the cell is resistant to paclitaxel or to another antineoplastic agent.
• growth inhibition occurs in vivo and more preferably growth inhibition occurs in a mammal, preferably a human.
• the invention also relates to a method for inhibiting growth of a tumor resistant to at least one antineoplastic agent comprising the step of: contacting a tumor with discodermolide wherein the tumor comprises cells resistant to at least one antineoplastic agent.
• the cells have a mutation in a ⁇ -tubulin protein and in another embodiment the cells overproduce glutathione.
• the cells are multidrug resistant.
• the at least one neoplastic agent is paclitaxel.
• the cells comprise raf-1 and wherein raf-1 is phosphorylated in the presence of discodermolide.
• the tumor is selected from the group of tumors consisting of lung, prostate, colon, breast, ovarian, kidney, brain, pancreatic esophageal, head and neck, gastric, and liver tumors.
• Figure 1 is a table illustrating the antiproliferative activity of discodermolide in various cancer cell lines. Various cancer cell lines were incubated with increasing concentrations of discodermolide or paclitaxel and the IC 50 for cell proliferation is determined by methylene blue staining.
• Figure 2(a) provides the mean tumor volumes and Figure 2(b) provides mean body weights in a study to assess paclitaxel-resistant (Pgp-l/MRP)-overexpressing human colon tumor xenograft (HCT 15 cells) sensitivity to discodermolide.
• Pgp-l/MRP paclitaxel-resistant
• HCT 15 cells human colon tumor xenograft
• D— refers to control solution of 16.7% Crm.- 8.3%EtOH/D5 W, iv lx (d.14); --•-- refers to mice receiving discodermolide, iv, 15 mg/kg, lx (d.l4); ⁇ ⁇ refer to mice receiving discodermolide, iv, 7.5 mg/kg, lx (d.14); --0 -refers to mice receiving discodermolide, iv, 2.5 mg/kg, lx (d 14); --o— refers to mice receiving 12.5% Crm-12.5%EtOH/D5W, iv, lx/day (d. 14-16); and ⁇ x — refers to mice receiving paclitaxel, iv, 15 mg/kg, lx/day (d. 14-18).
• Figure 3(a) provides the mean tumor volumes and Figure 3(b) provides mean body weights in a study to assess paclitaxel-resistant (Pgp-l)-overexpressing human colon tumor xenograft (MIP 101 cells) sensitivity to discodermolide.
• Pgp-l paclitaxel-resistant
• MIP 101 cells human colon tumor xenograft
• -- ⁇ -- refers to control solution of 16.7% Crm.-8.3%EtOH/D5W, iv lx (d.14); — •-- refers to mice receiving discodermolide, iv, 15 mg/kg, lx (d.l4); ⁇ 4 ⁇ refers to mice receiving discodermolide, iv, 7.5 mg/kg, lx (d.14); -- -- refers to mice receiving discodermolide, iv, 2.5 mg/kg, lx (d 14); — o— refers to mice receiving 12.5% Crm-12.5%EtOH/D5W, iv, lx/day (d. 14-16); and-x-refers to mice receiving paclitaxel, iv, 15 mg/kg, lx/day (d. 14-18).
• Figure 4 assesses paclitaxel-resistant 1 A9PTX22 ( ⁇ -tubulin mutation) cell sensitivity to discodermolide.
• --•-- refers to 1A9 cells receiving paclitaxel;
• — o— refers to 1A9PTX22 cells receiving paclitaxel;
• -- ⁇ — refers to 1A9 cells receiving discodermolide;
• ⁇ ⁇ 7 — refers to 1 A9PTX22 cells receiving discodermolide.
• Figure 5 illustrates results of experiments demonstrating that Paclitaxel-resistant 1 A9
• PTX22 cells are sensitive to discodermolide in nude mice.
• Figure 5(a) refers to treatments starting 24 hours after animals were implanted subcutaneously (sc) with hollow fibers (3 fibers/animal, one fiber/each cell line, six animals/compound). Paclitaxel was administered IV, once daily for 5 days at 15 mg/kg. Vehicle control was administered according to the paclitaxel schedule.
• Figure 5(b) refers to the identical regimen as 5(a) but here discodermolide rather than paclitaxel was administered iv, as a single 15 mg/kg injection. Vehicle control was administered according to the discodermolide schedule.
• discodermolide is a metabolite of the marine sponge Discodermia dissoluta (See Gunasekera, et al., J Org. Chem. 55:4912, 1990. Correction: J Org. Chem. 56:1346, 1991).
• discodermolide is believed to function much the same way as paclitaxel, the active substance in the drug TAXOL. Like paclitaxel, discodermolide acts to inhibit cold-induced depolymerization of purified tubulin, and interferes with microtubule dynamics in cells (ter Haar E, et al., Biochemistry 1996; 35:243-50). Proliferating cells treated with the compound are arrested during mitosis, and subsequently undergo apoptosis (Balachandran R, et al., Anti-Cancer Drugs 1998; 9:67-76.
• the present invention provides data to demonstrate that discodermolide is effective for inhibiting the growth of cancer cells in vivo.
• the present invention demonstrates the efficacy of discodermolide in vivo in cells that have demonstrated resistance to at least one antineoplastic agent.
• the studies described below demonstrate that discodermolide is useful for treating cells both in vivo and in culture and in treating cancer cells where the cancer cells are resistant to at least one antineoplastic agent because, for example, the cancer cells are multidrug resistant; the cancer cells over produce glutathione or because the cancer cells have a mutation in one or more proteins rendering the cells resistant to the antineoplastic agent..
• resistant to at least one neoplastic agent is used herein to refer to cells, for example, that are multidrug resistant; cells that are resistant to platinum or to other alkylating agents because they tend to over produce glutathione and to cells that have a mutation in one or more cells that render the cells resistant to a particular chemotherapeutic agent.
• cells resistant to taxanes include a mutation in ⁇ -tubulin protein
• present studies support the use of discodermolide in cases where one or more antineoplastic agents have failed to adequately inhibit growth of the cancer cells.
• the term “resistance” is used herein to refer to cells that are able to survive in the presence of at least one neoplastic agent where the normal cell counterpart (i.e., a growth regulated cell of the same origin) would either show signs of cell toxicity, cell death or cell quiescence (i.e., would not divide).
• the term “inhibit the growth of as used in herein refers to the ability of a particular antineoplastic agent to limit or reduce the growth potential of a cell, preferably a cancer cell.
• a particular antineoplastic agent can inhibit the growth of a cell by reducing the rate at which a particular cell divides, it can cause the cells to remain in a quiescent (i.e., non- dividing state) or it can induce cell cytotoxicity and/or cell death, including apoptosis.
• cancer cells from cancers resistant to at least one antineoplastic agent that can benefit from discodermolide therapy include leukemias and lymphomas, as well as solid tumors such as tumors of the colon, spleen, prostate, liver, lung and breast.
• Figure 1 includes a Table that illustrates that discodermolide is effective in inhibiting the growth of a number of different types of cancer cells.
• this invention relates to the use of discodermolide to inhibit the growth of multidrug resistant cells.
• Multidrug resistance is a term known in the art that refers to cells which are resistant (i.e., the cells survive) to more than one antineoplastic agent.
• the term "antineoplastic agent" is used herein to refer to molecules that are able to inhibit growth of a cancer cell and are used in therapies to treat cancer in mammals.
• Cells can be multidrug resistant through a genetic mutation even though the cells have not been exposed to one or more antineoplastic agents. More commonly, multidrug resistance results from exposure to one antineoplastic agent which then selects for cells that are resistant to more than one other antineoplastic agent.
• Multidrug resistance is a major challenge in cancer chemotherapy because the resistance severely impairs the effectiveness of a number of clinically important drugs.
• Drugs that are known to induce multidrug resistance include, for example, Actinomycin D, anthracyclines such as daunorubicin, doxorubicin, etoposide, mitoxantrone, taxanes, such as paclitaxel, topoisomerase inhibitors such as etoposides, and Vinca alkaloids such as vinblastine and vincristine, vinorelbine and colchicine,.
• Multidrug resistance occurs both in culture and in vivo. In general, cell lines that display the multidrug resistance phenotype are resistant to natural products, but retain their sensitivity to alkylating agents and antimetabolites.
• a multidrug resistance gene family has been identified and appears to be part of the ABC (ATP-binding cassette) superfamily (reviewed by Bellamy in Annu. Rev. Pharm. Toxicol. 1996, 36:161-83). The more common member of this family is the protein, Pgp which is described vide supra.
• a second protein associated with multidrug resistance is MRP. MRP also confers resistance to numerous natural products. Like Pgp, MRP can be elevated in patients with acute and chronic leukemia and solid tumors.
• the cells which are resistant to at least one antineoplastic agent are preferably contacted with discodermolide in vivo, preferably in a mammal. Although the cells can also be contacted with discodermolide in culture.
• Multidrug resistance can be monitored in culture or in vivo. Methods for monitoring and assessing multidrug resistance are well known and for that reason will not be described in detail here. In culture, cells which are able to grow or to survive in the presence of more than one antineoplastic agent as compared with matched, growth controlled cultures of cells including those obtained from normal, differentiated tissue are said to be multidrug resistant. It is also possible to assess multidrug resistance in vivo and to assess multidrug resistance over time for or during a particular treatment regime. For example, immunocytochemical assays are known in the art that assess levels of Pgp protein or other proteins belonging to the multidrug resistance family of proteins.
• RNA assays or immunoblots have been described in the literature to monitor multidrug resistance as have in situ hybridization studies and flow cytometric assays.
• HCT-15 and MIP 101 two different human tumor xenografts are separately implanted subcutaneously in athymic nude mice (see Example 2 below).
• NVP XAA296-NX results in statistically significant (p ⁇ 0.01) and reproducible inhibition of tumor growth in both tumor models.
• the HCT-15 model is completely refractory to paclitaxel treatment, while the MIP 101 model is resistant to paclitaxel when administered at 15 mg/kg, once daily for the first five days.
• Discodermolide administered as a single injection produces dose-dependent, statistically significant (p ⁇ 0.01) inhibition of tumor growth for all tested doses in both xenograft models.
• Toxicity appears to be tumor-dependent since an independent experiment demonstrates that na ⁇ ve (non-tumor bearing) athymic nude mice dosed with single injections of discodermolide lost no more than 4% of body weight one week after dosing and fully recovered to the control levels in 3 weeks after dosing.
• These experiments demonstrate the antitumor efficacy of discodermolide in two tumor models that were resistant to paclitaxel. In both cases discodermolide was able to induce apoptosis.
• the invention relates to methods for inhibiting the growth of cancer cells having mutation in a cellular protein that renders the cells resistant to at least one antineoplastic agent.
• An example of this mechanism of resistance are cells having a ⁇ - tubulin mutation that renders the cells resistant to taxanes, such as paclitaxel.
• the invention involves contacting at least one cell with a growth-inhibiting amount of discodermolide.
• cell growth inhibition can be monitored in blood borne tumors by assessing tumor load over time. Similarly, the size of the tumor in situ can be monitored as can the progression or lack thereof of metastases. All of these methods are well known to those of ordinary skill in the art of oncology drug testing.
• Example 3 details experiments assessing discodermolide sensitivity using a paclitaxel-resistant ovarian carcinoma cell line, 1 A9PTX22 and its parental cell line, 1 A9, which is sensitive to paclitaxel. In these studies cells remain sensitive to discodermolide irrespective of the presence of a ⁇ -tubulin mutation.
• amino acid sequences for the two isotypes of native human ⁇ -tubulin are provided below as SEQ ID NO:l and SEQ ID NO:2:
• the invention relates to the use of Discodermolide to treat cells resistant to platinating agents such as cisplatin and its analogues.
• the ovarian cell lines 2008 and C13 are tested for sensitivity to discodermolide and to paclitaxel.
• C13 is resistant to paclitaxel and demonstrates an overproduction of glutathione.
• 2008 cells have IC 50 s of 0.06 and 0.8 for discodermolide and paclitaxel respectively while the cisplatin resistant cells C13 have IC 50 S of 0.03 and 12.
• contacting is used in this invention to refer to any suitable delivery method for bringing discodermolide in contact with the cancer cells that are resistant to at least one antineoplastic agent.
• a suitable delivery method for bringing discodermolide in contact with the cancer cells that are resistant to at least one antineoplastic agent For culture applications, merely adding solutions of discodermolide in a pharmaceutically acceptable buffer of cell culture medium is sufficient.
• discodermolide can be delivered to the cancer cells resistant to at least one antineoplastic agent using any suitable method known to those of ordinary skill in the art of drug delivery. Intravenous delivery and peritoneal delivery is preferred and those skilled in the art of drug delivery are familiar with the apparati designed for drug delivery via this route of administration.
• the pharmaceutically acceptable formulations comprising pharmacologically active discodermolide alone, or in combination with one or more pharmaceutically acceptable carriers, preferably suitable for parenteral application will be readily discernible to those of ordinary skill in the art.
• Such formulations may include suitable excipients.
• Preferred delivery formulations are provided in the examples below. These formulations include combinations of discodermolide with cremaphor, propylene glycol, propylene glycol with D5W, ethanol D5W or with saline.
• effective doses used are typically those at the IC 50 concentration (see Figure 1).
• In vivo acute toxic doses are determined in clinical trial by treating at fractions of the IC 50 and assess toxicity.
• Effective doses of discodermolide for the mouse are about (+/- 5 mg/kg) 15 mg/kg given as one treatment every three weeks; for the rat; about (+/- 0.5) 3 mg/kg administered as one treatment every three weeks; and for marmoset; about (+/- 0.5) 1 mg/kg given as one treatment every three weeks.
• Preferred dosages and dosing regimes for man will of course be perfected following clinical trials using methods well known to those of ordinary skill in the art of clinical trials and will be optimized for particular types of cancer; however expected dosages are preferably from about 10 mg/kg to about 300 mg/kg in humans and more preferably from about 50 mg/kg to about 150 mg/kg of discodermolide.
• a stock solution of discodermolide (natural product) at 10 mg/ml in 95 % v/v ethanol is prepared and stored at -20 °C. Aliquots are diluted directly either in cell culture media (for in vitro assays) or in phosphate buffered saline (PBS; for all in vivo experiments). Cells and cell culture conditions
• the following cell lines are obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA): human colon carcinomas HCT-15 (CCL 225) and HCT-116 (CCL 247), human lung adenocarcinoma A549 (CCL 185), human large cell carcinoma NCI- H460 (HTB 177), estrogen-independent breast carcinoma MDA-MB-231 (HTB 177), prostate cancer cell line Du 145 (HTB 81).
• the human KB-31 (drug-sensitive) and B-8511 (multidrag- resistant, Pgp 170 overexpressing) epidermoid carcinoma cells are obtained from Dr. R. M.
• cells are seeded at 1.5 x 103/well into 96-well microtiter plates and incubated overnight. Compounds are added in serial dilutions on day 1. The plates are than incubated for additional 5 days. This allowed the control cultures to undergo at least 3 cell divisions. After incubation the cells are fixed with 3.3 % v/v glutaraldehyde, washed with water and stained with 0.05% w/v methylene blue. After washing, the dye is eluted with 3 % v/v HC1 and the optical density measured at 665 nm with a SpectraMax 340 (Bucherer, Basel, Switzerland).
• IC 50 values are determined by a computerized system (SoftPro, Bucherer, Basel, Switzerland) using the formula (OD test - OD start) / (OD control - OD start) x 100. IC 50 is defined as the drug concentration which leads to 50% of cells per well compared to control cultures (100%) at the end of the incubation period.
• Material Natural discodermolide (sample 1) is obtained from Harbor Branch Oceanographic
• Synthetic discodermolide is prepared using any number of methods described in the art including, for example, the methods of Smith AB, PCT Publication Number WO 00/04865, the contents of which is incorporated by reference herein.
• Paclitaxel is obtained from Calbiochem (La Jolla, CA, USA).
• Cell culture materials are from Integra BioSciences (Wallisellen, Switzerland).
• solvents are HPLC Gradient grade from Merck (Darmstadt, Germany).
• Liquid media, fetal bovine serum (FBS) and media additives are from Gibco/BRL (Basel, Switzerland). Results Antiproliferative activity The antiproliferative profile of discodermolide is determined against a panel of human tumor lines.
• the compound showed potent antiproliferative activity in vitro with the IC 50 values in the low nanomolar range (- 2 - 24 nM) for drug-susceptible cell lines.
• Paclitaxel is a potent cytotoxic agent but is much less active than discodermolide against HCT-15 colon cells ( ⁇ 120 vs. ⁇ 8 nM IC 50 respectively).
• the loss of activity of paclitaxel was several fold higher than that shown by discodermolide. Discussion
• Discodermolide is more potent than paclitaxel against MCF-7/ADR cells, which is multidrug resistant due to overexpression of Pgp 170, protein kinase-C, and glutathione S- transferase.
• the HCT-15 human colon tumor cell line is purchased from the American Type Culture Collection, Rockville, MD, Accession Number ATCC CCL 225.
• the MIP 101 human colon tumor cell line is obtained from Dr. R. Kramer (Bristol Meyers Squibb) and was previously described (Niles RM, et al. Cancer Invest. 1987;5(6):545-52). These cells are Pgp-1 (human Pgp) overexpressors, making the cells resistant to paclitaxel.
• mice bearing tumors are sorted into groups of eight for the study. The sorting process produced groups balanced with respect to mean and range of tumor size.
• Discodermolide is isolated from the sponge Discodermia dissoluta using the methods of (Gunasekera SP, et al. supra). Multiple batches of compound of similar purity (all > 95% pure, as determined by mass spectrometry, and nuclear magnetic resonance analysis) are used throughout the various studies. Solid discodermolide is dissolved in pure ethanol to create a stock solution which is diluted just before dosing with Cremophor EL (Crm) and D5W to a final concentration of 16.7% Cremophor EL, 8.3% ethanol and 75% D5W. The compound is administered intravenously (iv).
• discodermolide is tested against both tumors at five different dosing schedules: (i.) 30 mg/kg dosed as two 15 mg/kg injections on day 1 of the experiment, (ii.) 30 mg/kg dosed as three 10 mg/kg injections on days 1, 2, and 3, (iii.) 15 mg/kg dosed as a single injection on day 1, (iv.) 20 mg/kg dosed as a 15 mg/kg injection on day 1, followed by a 5 mg/kg injection on day 11, and (v.) 10 mg/kg dosed as a 7.5 mg/kg injection on day 1 followed by a 2.5 mg/kg injection on day 11.
• discodermolide is dosed as one injection on the first day of the experiment, at 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg, or 15 mg/kg.
• 7.5 mg/kg injection on day 1 followed by a 2.5 mg/kg injection on day 11 is repeated from the first study.
• Positive control animals receive clinical formulations of paclitaxel (TAXOL) diluted 4-fold with D5W and administered iv once daily for five consecutive days. Vehicle control for paclitaxel is administered according to paclitaxel's schedule.
• Tumors are measured, and individual animal body weights are recorded once weekly. Standard experiments are conducted for 3 full weeks from the initial dosing.
• mice To assess toxicity of discodermolide on non-tumor bearing animals, four groups of 8 naive nude mice are dosed with the compound, iv, once (5mg/kg, 7.5 mg/kg, 10 mg/kg, or 15 mg/kg). The control group is dosed with the vehicle alone (16.7% Cremophor EL, 8.3% ethanol and 75% D5W). Body weights are recorded once weekly. Calculations of results
• Antitumor activity is expressed as % T/C (comparing ⁇ tumor volumes for treatment group to vehicle control group). Regressions are calculated using the formula: (1-T/T 0 ) x 100%, where T is the tumor volume for the treatment group at the end of the experiment, and To is the tumor volume at the beginning of the experiment.
• Results are determined for the first experiment in the HCT-15 colon tumors with discodermolide administered at: (i.) 30 mg/kg dosed as two 15 mg/kg injections on day 1 of the experiment, (ii.) 30 mg/kg dosed as three 10 mg/kg injections on days 1, 2, and 3, (iii.) 15 mg/kg dosed as a single injection on day 1, (iv.) 20 mg/kg dosed as a 15 mg/kg injection on day 1, followed by a 5 mg/kg injection on day 11, and (v.) 10 mg/kg dosed as a 7.5 mg/kg injection on day 1 followed by a 2.5 mg/kg injection on day 11..
• Corresponding body weight changes are 3.5% (gain), -1.9%, -7.9%, -8.5%, -12.9%, and -11.0%.
• mice administered (i.) 30 mg/kg dosed as two 15 mg/kg injections on day 1 of the experiment, (ii.) 30 mg/kg dosed as three 10 mg/kg injections on days 1, 2, and 3, (iii.) 15 mg/kg dosed as a single injection on day 1, (iv.) 20 mg/kg dosed as a 15 mg/kg injection on day 1, followed by a 5 mg/kg injection on day 11, and (v.) 10 mg/kg dosed as a 7.5 mg/kg injection on day 1 followed by a 2.5 mg/kg injection on day 11.
• Results are presented graphically in Figure 2.
• Discodermolide dosed at 10 mg/kg, administered as a 7.5 mg/kg injection on day 1 followed by a 2.5 mg/kg injection on day 11 gives 35% T/C with 11.4% body weight loss.
• One animal in that group dies from apparent drug toxicity.
• Single injections of 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg, or 15 mg/kg discodermolide on day 1 of the experiment produces 59% T/C, 57% T/C, 46% T/C, 37% T/C, 22% T/C, and 18% T/C, respectively.
• Corresponding body weight losses are 7.9%, 10.2%, 13.2%, 18.7%, 15.5%, and 11.7%. All antitumor efficacy results are statistically significant (p ⁇ 0.01).
• paclitaxel showed statistically significant inhibition of tumor growth (54% T/C, p ⁇ 0.01).
• Results are summarized in Figure 3.
• HCT-15 and MIP 101, discodermolide, administered as single injection, demonstrated a dose-dependent, statistically significant inhibition of tumor growth at all doses between 2.5 mg/kg and 15 mg/kg.
• the HCT-15 model is totally refractory to treatment with paclitaxel, while the MIP 101 model shows no response in the first study (82% T/C), although in the second study paclitaxel produces a modest, but statistically significant inhibition of tumor growth (54% T/C, P ⁇ 0.01 ).
• Discodermolide at a total dose of 10 mg/kg administered as two injections, a 7.5 mg/kg on day 1 followed by a 2.5 mg/kg on day 11 produces 38% T/C in the first study, and 35% T/C in the retest.
• the dose response study is repeated in the third experiment.
• Antitumor efficacy of the discodermolide demonstrates good reproducibility for all doses between 2.5 mg/kg, and 15 mg/kg (62% T/C and 54% T/C for 2.5 mg/kg, 44% T/C and 43% T/C for 5 mg/kg, 43% T/C and 23% T/C for 7.5 mg/kg, 40% T/C and 27% T/C for 10 mg/kg, 28% T/C and 25% T/C for 12.5 mg/kg, 27% T/C and 19% T/C for 15 mg/kg). In the repeat of the dose response study body weight losses are higher, reaching 23%, compared to 13% in the original study. In conclusion, discodermolide shows dose-dependent antitumor efficacy in two known multidrug resistant tumor lines grown as xenografts in nude mice.
• Example 3 In vitro and In vivo antitumor effect of discodermolide to paclitaxel resistant cells having ⁇ - tubulin mutation. Cell lines and tissue culture
• the LS174T human colon tumor cell line is purchased from the American Type Culture Collection, Rockville, MD.
• the 1 A9 and the 1 A9PTX22 ovarian tumor cell lines are obtained from Dr. T. Fojo, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
• the 1 A9 is a clone of the ovarian carcinoma cell line, A2780 (Eva A, et al Nature 1982, 295:116-119.).
• the 1 A9PTX22 subline is isolated as an individual clone from the 1A9 cell line in a single step selection by exposure to 5 ng/mL paclitaxel in the presence of 5 ⁇ g/mL verapamil.
• the 1 A9PTX22 cell line is found to be 24-fold more resistant to paclitaxel than the parental 1 A9 (Giannakakou P, et al., J. Biol. Chem. 1997, 272(4): 17118-17125). Resistance to paclitaxel is maintained following 2 years of culturing in a drug-free media, and was attributed to the Ala 364 ⁇ Thr mutation in ⁇ -tubulin that is found in the 1 A9PTX22 cell line.
• All cell lines are propagated and expanded in RPMI 1640 medium containing 10% heat-inactivated FBS (Life Technologies, Grand Island, NY) in a tissue culture incubator (37 °C, controlled, humidified atmosphere containing 5% CO 2 ). Cell expansions are performed in T75 tissue culture flasks (COSTAR, Corning, NY). For hollow fiber preparations, cells are harvested at 70-90% confluency using 0.25% Trypsin-EDTA (Life Technologies, Grand Island, NY).
• 1 A9 and 1 A9PTX22 cells are plated in 96-well plates at 5x10 4 cells/well, placed in a tissue culture incubator and allowed to attach overnight. The next morning, the number of viable cells in the "time 0" plate (3 wells for each cell line) is determined using an MTT assay (Alley MC, et al, Cancer Res. 1988, 48:589-601). At the same time drugs are added in serial, 10-fold dilutions, to the experimental plates. Corresponding vehicles are added to control plates. Experimental and control plates are then incubated in the tissue culture incubator for 72 hours. After the incubation the number of viable cells is determined in each plate using the MTT assay.
• IC50S defined as a concentration of a given compound causing 50% inhibition of cell growth
• PVDF hollow fibers (Spectrum, Gardena, CA) are soaked in 70% EtOH for 72 hours before use. After this step, all handling of fibers are done under a biological laminar flow hood using aseptic procedures. Individual fibers are flushed with 3mL of the ice-cold tissue culture media using a syringe equipped with a 20-gauge needle. Next, each fiber is filled with an appropriate cell suspension (lxlO 6 cells/mL for the 1 A9 and 1 A9PTX22 cells, and 0.5xl0 6 cells/mL for the LS 174T cells), and both ends of the fiber are sealed with a hot flat needle holder.
• an appropriate cell suspension lxlO 6 cells/mL for the 1 A9 and 1 A9PTX22 cells, and 0.5xl0 6 cells/mL for the LS 174T cells
• hollow fibers 1.5 cm microcapsules (further called “hollow fibers”), each containing approximately 15 uL of the appropriate cell suspension. After separation, individual hollow fibers are placed in 6 well plates (6 fibers in 5 mL media per well), and are incubated overnight at 37 °C in the tissue culture incubator. Implantation of hollow fibers Outbred athymic n /nu) female mice ("Chris: Athymic Nude-nu", Charles River
• Ketamine/Xylazine 150 mg/kg, and 12 mg/kg body weight, respectively.
• an 11 -gauge trocar containing one or two hollow fibers is inserted into an incision made with scissors at the nape of the neck of an animal, and fibers are released by retracting the trocar while depressing the plunger. This procedure is repeated until all three hollow fibers are implanted.
• One wound clip is used to close the skin incision After the surgery each animal receives a single, subcutaneous injection of 0.4 mg/kg butorphenol to relieve any potential pain. Animals recover from the anesthesia on a heating pad, before returning to their cages.
• Group 1 Discodermolide, 15 mg/kg, iv, once.
• Group 2 Vehicle for discodermolide (16.7% Crem. EL, 8.3% Ethanol, 75% D5W), iv, once.
• Group 3 Paclitaxel, 15 mg/kg, iv, daily for 5 days.
• Group 4 Vehicle for paclitaxel (12.5% Cremophor EL, 12.5% Ethanol, 75% D5W), iv, daily for
• Discodermolide is isolated from the sponge Discodermia dissoluta using the method of Gunasekera SP (supra). Multiple batches of compound of similar purity (all > 95% pure, as determined by mass spectrometry, and nuclear magnetic resonance analysis) are used throughout the various studies. For the in vitro cytotoxicity assays all compounds are dissolved in DMSO and are added to the plates with cells to obtain desired concentrations. The amount of DMSO in cell cultures did not exceed 0.1% v/v. Paclitaxel is purchased from Sigma/Aldrich, (St. Louis, MO).
• solid discodermolide is dissolved in pure ethanol to create a stock solution which is diluted just before dosing with Cremophor EL and D5W to a final concentration of 16.7% Cremophor EL, 8.3% ethanol and 75% D5W.
• the compound is atlministered to mice as a single, 15 mg/kg iv injection.
• Positive control animals receive clinical formulations of paclitaxel (TAXOL) diluted 4-fold with D5W (12.5% Cremophor EL, 12.5% ethanol and 75% D5W final concentrations) and administered iv, at 15 mg/kg, once daily for five consecutive days. Vehicle controls are administered according to the corresponding drug schedules.
• results of the first experiment are summarized in Figure 5, however, all experiments were performed in duplicate.
• the IC 50 s for paclitaxel, and discodermolide are 0.8 ng/ml, and 6 ng/ml, respectively.
• the corresponding IC 50 S are: 15 ng/ml, and 3 ng/ml.
• IC 50 s for, paclitaxel, and discodermolide are 0.4 ng/ml, and 3 ng/ml, respectively.
• the paclitaxel-resistant 1 A9PTX22 cell line the corresponding IC 50 s are 9 ng/ml, and 3 ng/ml.
• Paclitaxel dosed iv, at 15 mg/kg, once daily, for 5 days produced T/C of 8%, 2%, and 90%, in LS 174T, 1A9, and 1A9PTX22 cell lines, respectively.
• Discodermolide is administered as a single, 15 mg/kg, iv injection, gave 13%, 14%, and 13% T/C in the respective cell lines.
• animals dosed with paclitaxel lost 5% of their body weights
• animals dosed with discodermolide lost 10% of their body weights. Discussion
• the 1A9PTX22 cell line was derived from a 1A9 clone of an ovarian carcinoma cell line A2780 (Eva A, et al. Nature 1982, 295:116-119) by exposure to paclitaxel (Giannakakou P, et al. J. Biol. Chem. 1997, 272(4): 17118-17125).
• the 1A9PTX22 cell line shows 24-fold resistance to paclitaxel in vitro, compared to the parental 1A9. This level of resistance is maintained after the cell line is cultured for 2 years in the absence of paclitaxel.
• the 1 A9PTX22 cell line contains an Ala 364 - ⁇ Thr mutation in ⁇ -tubulin, and that paclitaxel does not induce polymerization of the mutated tubulin prepared from the 1 A9PTX22 cells (Giannakakou P., supra). Taken together these data suggest that mutations in ⁇ - tubulin are likely responsible for the resistance to paclitaxel.
• the 1 A9PTX22 cell line is 20-fold more resistant to paclitaxel then the parental 1 A9 cells (for 1 A9 cells IC 50 s were 0.8 ng/mL, and 0.4 ng/mL, and for 1A9PTX22 cells IC 50 s are 15 ng/mL, and 9 ng/mL).
• Doxorubicin used as a mechanistically unrelated cytotoxic control, produced the IC50S of 3 ng/mL for 1 A9 cells, and 3-5 ng/ml for 1 A9PTX22 cells, showing that the latter is only slightly (2 fold) less sensitive to this compound than the parental 1 A9 cell line.
• discodermolide and paclitaxel are dosed iv, at optimal concentrations and dosing schedules as described in Example 2. Discodermolide is administered once, at 15 mg/kg, and paclitaxel is administered once daily for 5 days at 15 mg/kg.
• the 1 A9PTX22 cell line is sensitive to treatment with discodermolide (13% regression in the first study, and 13% T/C in the retest, both p ⁇ 0.01), but is completely refractory to paclitaxel (79% T/C in the first study, and 90% T/C in the retest, both p > 0.05).
• the LS 174T cell line is equally sensitive to both compounds.
• A549, a human non-small cell lung carcinoma and MDA-MB-435, a human breast carcinoma, used in this study are obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).
• A549 cells are maintained in RPMI 1640 containing 10% FBS.
• 1A9, a single-cell clone of the human ovarian carcinoma cell line A2780 and PTX22, the paclitaxel-resistant subline, used in this study are obtained from M. Wartman (Novartis Pharmaceuticals).
• A9 cells and PTX22 cells are maintained in RPMI 1640 supplemented with 10% FBS.
• PTX22 maintenance media also contained 15ng/mL paclitaxel and 5 ⁇ g/mL verapamil. Drug is removed from the media for 5-7 days before use in an experiment. All maintenance media contained 100 units/mL penicillin and 100 ⁇ g/mL streptomycin.
• Antiproliferative assays :
• Cell lines are trypsinized and counted using a Coulter counter. Cells were plated in 96 well plates (190 ⁇ L/well) at the following densities: 1,000 cells/well for A549 and 3,000 cells/well for MDA-MB-435. The number of cells plated results in cell densities of 75-90% confluence by the time of harvest. Plates are seeded on day 0. On day 1 test compounds are added to triplicate wells in a final volume of 10 ⁇ L media. Initial cell density for each cell line is measured on day 1 by adding 10 ⁇ L MTS mixture (see below), incubating for 4 h and recording absorbance at 490 nm (A490).
• MTS mixture is prepared fresh on day of addition to cell plates at a ratio of 10 ⁇ L of a 0.92 mg/mL solution of phenazine methosulfate (PMS) to 190 ⁇ L of a 2 mg/mL solution of MTS (3- (4,5-dimemylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt).
• PMS phenazine methosulfate
• Test compounds are prepared as stock solutions in DMSO. Test compound dilutions are made in 2% DMSO/cell maintenance media and diluted into assay plates to give 0.1% DMSO final in all wells.
• Cells are plated at a density of 1.5 x 10 6 cells per 100 mm plate. The next day cells are treated with vehicle control (0.1% DMSO) or test compound for 24 h. Cells are harvested by washing monolayers twice with PBS, then lysing with 300 ul of lysis buffer[20 mM Tris (pH 8.0), 2 nM EDTA, 100 mM NaCl, 0.5% NP40, 0.0125% DOC, 2.5% glycerol, 1 mM vanadate, 25 mM sodium fluoride and protease inhibitor cocktail (1 :500 dilution, Sigma)]. Lysates were spun at 12,000 x g and the supernatant transferred to a new tube.
• Protein concentration of the lysates is determined using BCA protein assay Reagent (Pierce). Samples (75 ⁇ g) are resolved by SDS-PAGE on a 7.5% or 14% tris-glycine gel for Raf-1 and Bcl-x L , respectively, and transferred to nitrocellulose.
• a Storm 860 (Molecular Dynamics) is used to detect fluorescent product according to the manufacturer's instructions.
• results The effects on cell proliferation following a 72 h exposure to discodermolide was measured by MTS assays.
• the IC 50 values are determined in two independent experiments to be 45 and 60 nM for A549 cells, 3 and 15 nM for MDA-MB-435 cells, 10 and 39 nM for 1A9 cells and 23 and 37 nM for PTX-22 cells.
• Cells are treated with 50, 100 and 180 nM discodermolide and MDA-MB-435 cells are treated with 2, 20, 50 and 90 nM discodermolide to determine the effects of discodermolide treatment on Raf-1 and BC1-XL phosphorylation.
• the paclitaxel concentration used as a positive control in the study was the same as used in Example 2.
• Raf-1 and BC1-X are phosphorylated following 24 hour treatment of A549 and MDA-MB-435 cells with paclitaxel or discodermolide. Phosphorylation of Raf-1 is observed by the appearance of additional bands migrating more slowly and phosphorylation of BC1-X i is observed by the broadening of a single band. Raf-1 phosphorylation is concentration-dependent, since in A549 and MDA-MB-435 cells the doublet is only observed at the higher concentrations tested. These results are consistent with previous studies in which the effects of paclitaxel on Raf-1 phosphorylation are also shown to be concentration dependent (Torres K and Horwitz SB. Cancer Res. 1998:58:3620-3626).
• Raf-1 phosphorylation is not required for cell death since low concentrations of paclitaxel led to apoptosis without Raf-1 phosphorylation perhaps through p21 and or p53 mediated apoptotic pathways.
• the minimum paclitaxel concentration required for Raf-1 phosphorylation coincides with the induction of the G2/M block suggesting that Raf-1 activation may be a component of the signal cascade activated during the mitotic checkpoint. Since the discodermolide concentration required to induce Raf-1 phosphorylation was greater than the ICso value, it is likely that similar concentration specific discodermolide activities also exist.
• the paclitaxel 72 hour IC 50 value shifts from 6 nM on 1 A9 cells to 80 nM on PTX-22 cells (13-fold less sensitive).
• discodermolide shows no cross-resistance as measured by IC 50 , which are 25 and 3011M on 1 A9 and PTX-22 cells, respectively.
• Raf-1 is phosphorylated in 1 A9 parental cells treated with both paclitaxel and discodermolide and in the PTX-22 cells treated with discodermolide., Raf-1 phosphorylation is not observed in the PTX-22 cells following paclitaxel treatment.

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