WO2004110497A2 - Mitomycin conjugates cleavable by thiols - Google Patents
Mitomycin conjugates cleavable by thiols Download PDFInfo
- Publication number
- WO2004110497A2 WO2004110497A2 PCT/US2004/013820 US2004013820W WO2004110497A2 WO 2004110497 A2 WO2004110497 A2 WO 2004110497A2 US 2004013820 W US2004013820 W US 2004013820W WO 2004110497 A2 WO2004110497 A2 WO 2004110497A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mitomycin
- lipid
- drug
- conjugate
- liposomes
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
-
- 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
- A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
- A61K31/105—Persulfides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- 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
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to a method for reducing the cytotoxicity of mitomycin C, and to a method of administering mitomycin C to a multi-drug resistant cell.
- Mitomycin C is provided in the form of a prodrug conjugate comprised of a hydrophobic moiety linked to the drug via a cleavable linkage. More particularly, the prodrug conjugate is comprised of a lipid linked to the drug via a cleavable linkage, io the lipid being incorporated into a liposomal formulation.
- the prodrug conjugate is cleavable under mild thiolytic conditions in vivo for release of mitomycin C in an unmodified state.
- Mitomycin is an established chemotherapeutic agent given for several different types of cancer, including breast, stomach, gullet and bladder cancer.
- the agent acts by cross-linking DNA so the cancer cells are unable to proliferate.
- common side effects due to the toxicity include fever, nausea, vomiting, bone marrow depression, and others (HARRISON'S PRINCIPLES OF
- Drug toxicity is not the only problem associated with chemotherapy. Another problem is drug resistance. Some tumor types, e.g., non-small cell lung cancer and colon cancer, exhibit primary resistance, i.e., absence of response on the first exposure to currently available, conventional chemotherapeutic agents. Other tumor types exhibit 25 acquired resistance, which develops in a number of drug-sensitive tumor types. Drug resistant cancer cells demonstrate two types of acquired drug resistance; cells exhibiting single agent resistance or resistance to single class of anti-cancer drugs with the same mechanism of action. The second type involves cells broadly resistant to several or many chemically diverse anti-cancer drugs with different mechanisms of action. This
- 3 o second type of acquired resistance is known as multi-drug resistance.
- Multi-drug resistance is also found in some tumor cells, such as renal and colon tumors, exhibiting primary resistance. That is, in contrast to an acquired multidrug resistance, certain tumor types are non-responsive to initial treatment with many chemotherapeutic agents.
- Multidrug-resistance is often associated with increased expression of a normal gene, the MDR1 gene, for a cell surface glycoprotein, P-glycoprotein, involved in drug efflux. P-glycoprotein expression correlates with a decrease in intracellular drug accumulation; that is, the P-glycoprotein acts as an energy-dependent pump or transport 5 molecule that removes drugs from the cell, preventing the drug from accumulating in the cell.
- P-glycoprotein is normally primarily expressed at epithelial and endothelial surfaces and seems to play a role in absorption and/or secretion. It is an active transporter that pumps hydrophobic drugs out of cells, reducing their cytoplasmic io concentration and therefore toxicity. In normal cells, P-glycoprotein functions to eliminate toxic metabolites or xenobiotic compounds from the body (Endicott, J. and Ling, V., Annu. Rev. Biochem., 58:137-171 , (1989)).
- Cancers which express P-glycoprotein include cancers derived from tissues which normally express the MDR1 gene, namely cancers of the liver, colon, kidney, is pancreas and adrenal. Expression of the gene is also seen during the course of chemotherapy with multidrug-resistant drugs in leukemias, lymphomas, breast and ovarian cancer, and many other cancers. These cancers initially respond to chemotherapy , but when the cancer relapses, the cancer cells frequently express more P-glycoprotein. There are cancers derived from tissues which do not normally express the MDR1 gene, namely cancers of the liver, colon, kidney, is pancreas and adrenal. Expression of the gene is also seen during the course of chemotherapy with multidrug-resistant drugs in leukemias, lymphomas, breast and ovarian cancer, and many other cancers. These cancers initially respond to chemotherapy , but when the cancer relapses, the cancer cells frequently express more P-glycoprotein. There are cancers derived from tissues which do not normally express
- P-glycoprotein but in which P-glycoprotein expression increases during the development of the cancer.
- One example is chronic myelogenous leukemia, which when it goes into blast crisis, expresses more P-glycoprotein irrespective of the previous treatment history (Gottesman, M.M. Cancer Research, 53:747-754 (1993)).
- the MDR1-enco ⁇ ed P-glycoprotein pump recognizes and transports many 25 different substances, including most natural product anti-cancer drugs such as doxorubicin, daunorubicin, vinblastine, vincristine, actinomycin D, paclitaxel, teniposide and etoposide (Gottesman, M. et al., Current Opinion in Genetics and Development, 6:610-617 (1996)). More generally, the drugs often involved in multidrug-resistance are alkaloids or antibiotics of plant or fungal origin, and they include the vinca alkaloids,
- Liposomes are closed lipid vesicles used for a variety of therapeutic purposes, and in particular, for carrying therapeutic agents to a target region or cell by systemic administration of liposomes.
- Liposomes having a surface grafted with chains of water-soluble, biocompatible polymer, in particular polyethylene glycol, -have 5 become important drug carries. These liposomes offer an extended blood circulation lifetime over liposomes lacking the polymer coating. The grafted polymer chains shield or mask the liposome, thus minimizing nonspecific interaction by plasma proteins. This in turn slows the rate at which the liposomes are cleared or eliminated in vivo since the liposome circulate unrecognized by macrophages and other cells of io the reticuloendothelial system. Furthermore, due to the enhanced permeability and retention effect (Maeda H. et al., J.
- the liposomes tend to accumulate in sites of damaged or expanded vasculature, e.g., tumors, sites of inflammation.
- An extended blood circulation time is often desired to allow systemically is administered liposomes to reach a target region, cell or site.
- a blood circulation lifetime of greater than about 12 hours is preferred for liposomal-therapy to a tumor region, as the liposomes must systemically distribute and then extravasate into the tumor region.
- mitomycin C It would be desirable to provide a formulation of mitomycin C that can be
- a liposome composition having a long blood circulation lifetime and capable of retaining an entrapped drug for a desired time, yet able to release the drug on demand. It would also be desirable to provide a formulation of mitomycin C that is as efficacious as the drug in free form, yet has a reduced systemic toxicity. Furthermore, it would 25 be desirable to release the cytotoxic mitomycin C in response to the endogenous conditions in the tumor.
- the invention includes a method for reducing the in vivo cytotoxicity of mitomycin C, comprising providing mitomycin C in the form of a liposome composition comprised of a vesicle-forming lipid and of between about 1 to about 30 mole percent of a conjugate having the general form:
- L is a hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer
- R 1 is mitomycin C covalently attached to the dithiobenzyl moiety
- orientation of the CH 2 R 1 group is selected from the ortho position and the para position.
- mitomycin C is covalently attached by a urethane
- L is selected from the group consisting of cholesterol, a diacylglycerol, and a phospholipid.
- mitomycin C is covalently linked to the dithiobenzyl moiety to form a conjugate having the structure:
- R 4 represents a residue of mitomycin C, where the secondary amine in the aziridine moiety of mitomycin C forms a urethane linkage between the dithiobenzyl and mitomycin C.
- the invention includes a method for administering mitomycin C to a multi-drug resistant cell, comprising providing mitomycin C in the form of a liposome composition comprised of a vesicle-forming lipid and of between about 1 to about 30 mole percent of a conjugate having the general form:
- L is a hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer
- R 1 is mitomycin C covalently attached to the dithiobenzyl moiety
- orientation of the CH 2 R 1 group is selected from the ortho position and the para position.
- Fig. 1 shows a synthetic reaction scheme for preparation of para- diacyldiglycerol-dithiobenzylalcohol for further reaction with amine-, hydroxy- or carboxyl-containing drugs;
- Fig. 2A shows a general reaction scheme for attachment of an amino- containing drug to a reactive diacyldiglycerol-dithiobenzylcarbonate
- Fig. 2B shows the products after thiolytic cleavage of the conjugate in Fig.
- Fig. 3A shows a synthetic reaction scheme for preparation of a diacyldiglycerol-dithiobenzyl-mitomycin-C conjugate
- Fig. 3B shows the products after thiolytic cleavage of the conjugate in Fig. 3A;
- Fig. 4 shows a synthetic reaction scheme for preparation of a cholesterol- dithiobenzyl-mitomycin-C conjugate;
- Fig. 5 shows another synthetic reaction scheme for preparation of a cholesterol-dithiobenzyl-mitomycin-C conjugate
- Figs. 6A-6C show the structures of three lipid-dithiobenzyl-mitomycin-C conjugates, para-distearoyl-DTB-mitomycin-C (Fig. 6A), para-dipalmitoyl-DTB- mitomycin-C (Fig. 6B) and o/t/70-dipalmitoyl-DTB- mitomycin-C (Fig. 6C);
- Figs. 7A-7B are HPLC chromatograms for liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 7A) and HSPC/cholesterol/mPEG- DSPE/lipid-DTB-mitomycin C (Fig. 7B), where each figure shows a series of chromatograms as a function of time of incubation of the liposomes in the presence of cysteine;
- Fig. 8 is a plot showing the percent of mitomycin C released from liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed diamonds) and HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (closed circles) as a function of time of incubation in the presence of cysteine;
- Figs. 9A-9B are plots showing the percent of mitomycin C released from liposomes comprised, of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 9A) and HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (Fig. 9B) as a function of time of incubation in the presence of cysteine at concentrations of 150 ⁇ M (closed 5 symbols) and at 1.5 mM (open symbols);
- Fig. 10 is a plot of growth rate of M109 cells, expressed as a percentage based on growth of M109 cells in the absence of drug and cysteine, as a function of mitomycin C amount, in nM, for free mitomycin c (open triangles), liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed squares), and io liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB-mitomycin C (open circles);
- Fig. 11 A is a plot of growth rate of M109 cells, expressed as a percentage based on growth of M109 cells in the absence of drug or cysteine, as a function of mitomycin C concentration in nM. Shown are cells treated mitomycin C in free form is (open triangles) and with mitomycin C in free form plus 1000 ⁇ M cystein (closed triangles). Also shown are cells treated with the liposome formulation comprised of HSPC/PEG-DSPE/lipid-DTB-mitomycin C (open circles) and with the liposome formulation with additional cysteine added at concentrations of 150 ⁇ M (open diamonds), 500 ⁇ M (closed circles) and 1000 ⁇ M (open squares);
- Fig. 11 B is a plot of growth rate of M109 cells, expressed as a percentage based on growth of M109 cells in the absence of drug or cysteine, as a function of mitomycin C concentration in nM. Shown are cells treated mitomycin C in free form (open triangles) and with mitomycin C in free form plus 1000 ⁇ M cysteine (closed triangles).
- Fig. 12 is a plot showing the percent increase in cytotoxicity (as determined by (IC50 nO cysteine /IC50 CyStei n e )x100)) of free mitomycin C (closed squares), mitomycin C
- liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB- mitomycin C (closed circles), and liposomes comprised of HSPC/mPEG-DSPE/lipid-
- DTB-mitomycin C (open triangles) to M109 cells in vitro at various concentrations of cysteine
- Fig. 13A is a plot showing the concentration of mitomycin C in the blood of rats as a function of time in hours following intravenous injection of free mitomycin C (open squares), liposomes comprised of HSPC/cholesterol/mPEG-DSPE/lipid-DTB- mitomycin C (closed diamonds), and liposomes comprised of HSPC/mPEG- DSPE/lipid-DTB-mitomycin C (closed circles);
- Fig. 13B is a plot showing the percent of injected dose remaining in the blood of rats as a function of time in hours following intravenous injection of free mitomycin C (open squares), liposomes comprised of HSPC/cholesterol/mPEG- DSPE/lipid-DTB-mitomycin C (closed diamonds), and liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed circles); io [0035] Fig.
- FIG. 14 is a plot showing the mean body weight, in grams, as a function of time, in days, after injection of free mitomycin C (open squares) or of mitomycin C in the form of a liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed circles); [0036] Fig.
- FIG. 15A is a plot showing median footpad size, in mm, as a function of is days after inoculation with M109 tumor cells in the paw of mice, where the mice were left untreated (control mice; (open squares)) or were treated with free mitomycin C (open triangles) or with liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB- mitomycin C (closed circles); [0037]
- Fig. 15B is a plot showing median footpad size, in mm, as a function of
- mice 20 days after inoculation with M109 tumor cells in the paw of mice, where the mice were left untreated (control mice; (open squares)) or were treated with free mitomycin C (open triangles) at 2 mg/kg (dashed line) or 4 mg/kg (solid line), or with liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed circles) at 2 mg/kg (dashed line) or 4 mg/kg (solid line); 25 [0038] Fig.
- 16A is a plot showing median footpad size, in mm, as a function of days after inoculation with M109 tumor cells in the paw of mice, where the mice were left untreated (control mice; (open squares)) or were treated with free mitomycin C (open triangles) at 6 mg/kg or with three doses given on days 5, 12, and 19 of liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C at 6 mg/kg
- Fig. 16B is a plot showing the percent of mice alive with a footpad tumor size of less than 4 mm, as a function of days after tumor inoculation, for the mice treated as set forth in Fig. 16A;
- Fig. 17 is a plot of percent survival as a function of time after inoculation with C26 tumor cells in mice left untreated (squares), treated with free mitomycin C (triangles) at 6 mg/kg, or treated with liposomes comprised of HSPC/mPEG- 5 DSPE/lipid-DTB-mitomycin C at a single dose of 6 mg/kg (circles) or two doses of 6 mg/kg and cysteine (diamonds);
- Fig. 18 is a plot of median footpad size, in mm, as a function of time after inoculation with M109-R tumor cells in mice left untreated (open squares), treated with free mitomycin C (open triangles) at 8 mg/kg, treated with one dose (closed io circles, solid line) or two doses (closed circles, dashed line) of liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C at 8 mg/kg; [0042] Fig.
- FIG. 19A is a plot of median weight, in grams, as a function of days after tumor inoculation, for mice left untreated (open squares), treated with two 10 mg/kg doses of doxorubicin entrapped in liposomes having a coating of polyethylene glycol is chains (Stealth ® , open triangles), treated with two doses of liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C at 10 mg/kg (closed circles) without cysteine (closed circles, solid line) or with 5 mg/kg cysteine (closed circles, dashed line); [0043] Fig. 19B is a plot of median footpad thickness, in mm, as a function of days
- mice left untreated (open squares), treated with two 10 mg/kg doses of doxorubicin entrapped in liposomes having a coating of polyethylene glycol chains (Stealth ® , open triangles), treated with two doses of liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C at 10 mg/kg (closed circles) without cysteine (solid line) or with 5 mg/kg cysteine (dashed line); and 25 [0044] Fig.
- 19C is a plot of the percentage of mice alive with a footpad tumor of less than 5 mm as a function of days after tumor inoculation of M109R cells, for mice left untreated (open squares), treated with two 10 mg/kg doses of doxorubicin entrapped in liposomes having a coating of polyethylene glycol chains (Stealth ® , open triangles), treated with two doses of liposomes comprised of HSPC/mPEG-
- hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer intends any material comprising a hydrophobic portion capable of being 5 integrated with the hydrophobic bilayer region of a liposomal lipid bilayer.
- Such hydrophobic moieties are typically lipids, including amphipathic lipids having a hydrophobic lipid tail and a hydrophilic polar head, such as phospholipids and diacylglycerols.
- Triglycerides, sterols, derivatives of phospholipids, diacylglyerols, sterols and triglycerides and other lipids derived from a natural source or synthetically io prepared are also contemplated.
- Polypeptide refers to a polymer of amino acids and does not refer to a specific length of a polymer of amino acids.
- peptide oligopeptide, protein, and enzyme are included within the definition of polypeptide. This term also includes post-expression modifications of the
- polypeptide for example, glycosylates, acetylations, phosphorylations, and the like.
- PEG poly(ethylene glycol); mPEG, methoxy-PEG; DTB, dithiobenzyl; DSPE, distearoyl phosphatidylethanolamine; HSPC, hydrogenated soy phosphatidylcholine; MMC, mitomycin C.
- the invention includes a conjugate of the form:
- L is a hydrophobic moiety suitable for incorporation into a liposomal lipid bilayer
- R 1 represents a therapeutic drug residue covalently attached to the dithiobenzyl moiety
- orientation of the CH 2 R 1 group is selected from the ortho position and the para position.
- the hydrophobic moiety, L is typically a lipid such as a diacylglycerol, a sterol, a phospholipid, derivatives of these lipids, other naturally-occurring lipids and their synthetic analogs.
- a therapeutic drug is attached to the dithiobenzyl moiety by a covalent linkage, thereby forming a drug residue, represented by R 1 in the 5 structure.
- the linkage will vary according to the drug and the reaction chemistry, as will be appreciated by those of skill in the art.
- the therapeutic drug is covalently attached to the diithiobenzyl moiety by a linkage selected from the group consisting of urethane, amine, amide, carbonate, thio- carbonate, ether and ester.
- R 4 represents the therapeutic drug residue.
- a drug containing a primary or secondary amine such as mitomycin C, mitomycin A, bleomycin and therapeutic polypeptides to name a few, is reacted to from a urethane linkage with the amine moiety in the drug.
- R 4 represents the drug residue and the linkage derives from a moiety in the drug.
- 20 carbonate linkage include fluorodeoxyuridine, iododeoxyuridine, etoposide, AZT, acyclovir, vidarabine, arabinosyl cytosine, pentostatin, quinidine, mitoxantrone and atropine.
- the linkage derives from reaction with a carboxylic acid moiety in the 25 therapeutic drug, and an example of a conjugate having an ester linkage between chlorambucil and dithiobenzyl is described below.
- Methotrexate is another example of a drug capable of forming an ester linkage with the dithiobenzyl moiety of the conjugate.
- R 4 represents a residue of the therapeutic drug.
- the conjugate includes an ether linkage, which takes the form of O-R 4 , where R 4 represents the therapeutic drug residue.
- the 5 linkage typically derives from reaction with an alcohol functionality on the drug.
- a conjugate with the drug 5-fluorouracil where an amine linkage is formed is one example, set forth in U.S. Patent No. 6,342,244.
- An amide linkage io can also be formed with a peptide as the therapeutic agent, where the free carboxyl of an amino acid residue, such as an aspartic acid or glutamic acid, is condensed with dithiobenzylamine.
- FIG. 1 shows a synthetic reaction scheme for preparation of exemplary conjugates in accord with the invention.
- synthesis of an intermediate compound, para-diacyldiglyceroldithiobenzalcohol (Compound IV) is prepared for further reaction with a selected therapeutic drug.
- Compound IV is prepared, as described in Example 1 , by reacting 3-mercapto-1 ,2-propanediol
- R can be a fatty acid having from about 8 to about 24 carbon atoms.
- Example 1 details the reaction procedure where R is stearic acid.
- R is a fatty acid having from about 12 to about 22 carbon atoms.
- Compound V is also readily reacted with a drug containing a reactive amine moiety (R'-NH 2 ) to yield a lipid-DTB-drug conjugate where the drug is joined to the DTB by a urethane linkage (Compound Vl).
- Compound IV is also readily reacted with a drug containing a reactive hydroxyl moiety (R 1 OH) to form a lipid-DTB-drug conjugate where the drug is joined to the DTB by a carbonate linkage (Compound VII).
- drugs are contemplated for use in the conjugate of the invention.
- the invention contemplates drugs having an amine (NH or NH 2 ), carboxyl, sulfhydryl or hydroxyl moiety suitable for reaction.
- "suitable for reaction” implies that the drug has one of the recited moieties capable of reacting with the dithiobenzyl moiety, in the form of, for example, dithiobenzyl alcohol.
- Exemplary drugs include 5-fluorouracil, which has an NH group suitable for reaction, chlorambucil, which has a reactive carboxyl and mitomycin C, which has a reactive amine (aziridine group).
- Example 1 also details the reaction conditions for preparation of ortho- diacyldiglyceroldithiobenzalcohol, which can serve as a intermediary compound to form the conjugate.
- Figs. 2A-2B show preparation of a lipid-DTB-drug conjugate (Fig. 2A), and thiolytic cleavage of the conjugate in the presence of a reducing agent (Fig. 2B). As shown in Fig. 2A, Compound VII of Fig.
- hydrophobic moiety R is derived from a fatty acid R"(CO)OH, such as stearic acid (CH 3 (CH 2 ) I eCO 2 H), is reacted with an amine-containing drug, H 2 N-drug, in the presence of phosgene (COCI 2 ).
- This reaction yields the lipid-DTB-drug conjugate illustrated in Fig. 2A.
- the conjugate upon exposure to reducing conditions, i.e., a reducing agent such as cysteine or glutathione, decomposes to yield the products shown in Fig. 2B.
- thiolytic cleavage of the conjugate results in regeneration of the drug in an unmodified, natural state.
- Fig. 3A shows the synthesis of the mitomycin C prodrug conjugate. In the reaction scheme shown, mitomycin C (Compound XVII, Fig.
- Fig. 3B shows the thiolytic decomposition of a diacyldiglycerol-DTB- mitomycin-C conjugate. In the presence of a reducing agent, the conjugate decomposes to regenerate mitomycin C (Compound XVII) and the other products io shown.
- hydrophobic moiety in the conjugate can be selected from any number of hydrophobic moieties, e.g., lipids.
- a diacyldiglycerol lipid can be used to form conjugates having the structure:
- R 2 and R 3 are hydrocarbons having between about 8 to about 24 carbon atoms.
- lipids are contemplated.
- Fig. 4 shows another embodiment where cholesterol is used as the hydrophobic moiety in the conjugate. Cholesterol (Compound XIV) is reacted with
- Fig. 6B shows a para-dipalmitoyl-DTB-mitomycin C conjugate. It will also be 5 appreciated that the conjugate can also have an isomeric linkage. This is evident by the orffto-dipalmitoyl-DTB-mitomycin C conjugate as shown in Fig. 6C.
- the mitomycin C prodrug conjugate is io provided in the form of a liposome composition comprised of a vesicle-forming lipid and the mitomycin C prodrug conjugate.
- Liposomes are closed lipid vesicles used for a variety of therapeutic purposes, and in particular, for carrying therapeutic agents to a target region or cell by systemic administration of liposomes.
- liposomes having a surface coating of hydrophilic polymer chains, such as polyethylene glycol is (PEG), are desirable as drug carries as these liposomes offer an extended blood circulation lifetime over liposomes lacking the polymer coating.
- the polymer acts as a barrier to blood proteins thereby preventing binding of the protein and recognition of the liposomes for uptake and removal by macrophages and other cells of the reticuloendothelial system.
- Liposomes include a conjugate in combination with a lipid, which in one embodiment is a vesicle-forming lipid, and, optionally, other bilayer components.
- a lipid which in one embodiment is a vesicle-forming lipid, and, optionally, other bilayer components.
- lipid-forming lipids are lipids that spontaneously form bilayer vesicles in water.
- the vesicle-forming lipids preferably have two hydrocarbon chains, typically acyl chains, and a polar head group.
- Examples include the phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (Pl), and sphingomyelin (SM).
- PC phosphatidylcholine
- PE phosphatidylethanolamine
- PA phosphatidic acid
- Pl phosphatidylinositol
- SM sphingomyelin
- lipid for use in the present invention is hydrogenated soy phosphatidylcholine (HSPC).
- HSPC hydrogenated soy phosphatidylcholine
- diacylglycerols are diacylglycerols.
- lipids can be obtained commercially or prepared according to published methods.
- the vesicle-forming lipid may be selected to achieve a degree of fluidity or rigidity, to control the stability of the liposome in serum, and to control the rate of release of an entrapped agent in the liposome.
- Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer can be prepared by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g., up to 5 about 8O 0 C.
- Rigid lipids i.e., saturated, contribute to greater membrane rigidity in the lipid bilayer.
- Other lipid components, such as cholesterol are also known to contribute to membrane rigidity in lipid bilayer structures.
- Lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase io transition temperature, e.g., at or below room temperature (about 20-25 0 C).
- a relatively fluid lipid typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase io transition temperature, e.g., at or below room temperature (about 20-25 0 C).
- the liposome can also include other components that can be incorporated into lipid bilayers, such as sterols. These other components typically have a hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and a polar head group moiety oriented toward the exterior, polar surface is of the membrane.
- Another lipid component in the liposomes of the present invention is a vesicle-forming lipid derivatized with a hydrophilic polymer.
- a derivatized lipid results in formation of a surface coating of hydrophilic polymer chains on both the inner and outer lipid bilayer surfaces.
- Hydrophilic polymers suitable for derivatization with a vesicle-forming lipid include polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate,
- polystyrene resin 25 polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol, and polyaspartamide.
- the polymers may be employed as homopolymers or as block or random copolymers.
- a preferred hydrophilic polymer chain is polyethyleneglycol (PEG), preferably as a PEG chain having a molecular weight between about 500 to about
- Liposomes of the present invention include typically between about 1 and about 30 mole percent of the lipid-DTB-drug conjugate, preferably between about 5 and about 30 mole percent, more preferably between about 5 and about 20 mole percent.
- liposomes comprised of the vesicle-forming lipid hydrogenated soy phosphatidylcholine (HSPC), distearoyl 5 phosphatidylethanolamine derivatized with methoxy-polyethylene glycol (mPEG- DSPE) and the conjugate shown in Fig. 6A, para-distearoyl-DTB-mitomycin C (Compound XVIII) were prepared as described in Examples 4A-4B.
- HSPC vesicle-forming lipid hydrogenated soy phosphatidylcholine
- mPEG- DSPE distearoyl 5 phosphatidylethanolamine derivatized with methoxy-polyethylene glycol
- Fig. 6A para-distearoyl-DTB-mitomycin C
- One of the liposome formulations included cholesterol (Example 4A), with the lipids HSCP/cholesterol/mPEG-DSPE/para-distearoyl-DTB-mitomycin C (Compound XVIII) io present at a molar ratio of 60/30/5/5.
- the lipids HSCP/mPEG-DSPE/para-distearoyl-DTB-mitomycin C (Compound XVII) were present at a molar ratio of 90/5/5.
- Liposomes were prepared as described in Examples 4A-4B and were characterized in vitro to determine the rate of release of mitomycin C following exposure to reducing agent. For the in vitro studies, reducing conditions were induced by addition
- cysteine typically at a concentration of about 150 ⁇ M, to the test medium.
- endogenous reducing conditions may be sufficient to effect thiolytic decomposition of the lipid-DTB-drug conjugate for release of the drug.
- reducing conditions in vivo can be artificially induced by administration of a suitable reducing agent, such as cysteine or glutathione.
- the liposome formulations e.g., HSPC/cholesterol/mPEG-DSPE/conjugate Compound XVIII (hereinafter the "cholesterol-containing formulation”) and HSPC/mPEG-DSPE/conjugate Compound XVIII (hereinafter the "cholesterol-free liposome formulation”) were incubated at 37 0 C in the presence of 150 ⁇ M cysteine for 24 hours. Samples were withdrawn at selected time points and analyzed by high
- Figs. 7A-7B show HPLC chromatograms for two liposome formulations. In Fig. 7A, the results for the cholesterol-free liposome formulation are shown. At time zero, there is no detectable free mitomycin C and all measurable drug is in the form of a lipid-DTB-drug conjugate that is liposome bound. As the incubation time increases, the amount of mitomycin C released from the liposomes and detectable in free form increases, with a corresponding decrease in the presence of conjugate-bound mitomycin C. 5 [0081] Fig.
- FIG. 7B shows the results for the liposome formulation containing cholesterol.
- the first sample taken at time zero there was no detectable free mitomycin C.
- a small amount of free drug was detected, indicating decomposition of the liposome-bound lipid-DTB- mitomycin conjugate.
- liposomes containing cholesterol io yield a slower conjugate decomposition rate and accordingly slower release of the drug.
- Fig. 8 is a plot showing the percent of mitomycin C. released from the two liposome formulations, as determined from the chromatograms in Figs. 7A-7B.
- the cholesterol-free liposomes (closed diamonds) had a higher rate of release than the is liposomes containing cholesterol (closed circles). More than 50% of the mitomycin C was released from the liposome-bound conjugate after 2 hours for the cholesterol- free formulation. For both formulations, greater than 80% of the drug was released at the end of the 24 hour incubation period.
- the two liposome formulations were incubated in 1.5 mM
- Figs. 9A-9B show the percent of mitomycin C released from the lipid-DTB- drug conjugate incorporated into the cholesterol-free liposomes (HSPC/PEG- DSPE/lipid-DTB-mitomycin C). The percent release during incubation with 150 ⁇ M are also shown (closed diamonds) for comparison. As seen, incubation at a higher 25 concentration of reducing agent (1.5 mM, open diamonds) causes an increase in the rate of conjugate decomposition and rate of drug release. [0084] Fig. 9B shows the results for the liposome formulation containing cholesterol. Liposomes incubated in 1.5 mM (open circles) have a significantly higher decomposition rate than the same liposomes incubated in 150 ⁇ M cysteine (closed
- the percent growth rate of M109 mouse carcinoma cells determined from the cytotoxicity studies is shown in Fig. 10.
- the percent growth rate is expressed as a percentage based on growth rate of M109 cells in the absence of mitomycin C and of cysteine and is shown as a function of mitomycin C concentration, in nM.
- the growth rate of cells was determined as described in Example 6. As seen, the percent of cell growth rate decreases as the cysteine concentration is increased for both the liposomes containing cholesterol (open circles) and the cholesterol-free liposome formulation (closed squares). It can also be seen that cysteine has no effect on the activity of free mitomycin c and that mitomycin C is released from the conjugate to effectively inhibit cell growth.
- Figs. 11 A-11 B The in vitro growth rate of M109 mouse carcinoma cells treated with mitomycin C in free form or with mitomycin C in the form a liposome-bound lipid-DTB- drug conjugate is shown in Figs. 11 A-11 B. In Fig. 11 A the results for the liposome formulation containing no cholesterol are shown. In the plot, the growth rate of M109 cells is expressed as a percentage based on growth of M109 cells in the absence of drug and cysteine and is shown as a function of mitomycin C concentration in nM. The cells treated with mitomycin C in free form (open triangles) and with mitomycin C in free form plus 1000 ⁇ M cysteine (closed triangles) exhibit a decrease in growth rate due the toxicity of the drug in free form.
- Fig. 11 B is a similar plot for the liposome formulation containing cholesterol.
- the same pattern was observed for cells treated with the liposome composition containing cholesterol plus additional cysteine at concentrations of 150 ⁇ M (open diamonds), 500 ⁇ M (closed circles) and 1000 ⁇ m (open squares). That is, as the concentration of cysteine increased, the cell growth rate decreased. This indicates a cysteine-induced release of mitomycin C in direct correlation with cysteine concentration.
- the in vitro growth rate of cells treated with mitomycin C in free form was the same as the growth rate of cells treated with mitomycin C in free form plus 1000 ⁇ M cysteine (closed triangles).
- Fig. 12 shows the percent increase in cytotoxicity as a function of cysteine concentration, in ⁇ M, of free mitomycin C and of the liposome formulations. Increase in cytotoxicity was determined by the percent drop in IC50, e.g., IC50 in the presence of cysteine relative to IC50 in the absence of cysteine time 100 ((IC50 no cy s t e i ne/IC50 cyst e i ⁇ e)x100)). As seen, the percent of cytotoxicity increases significantly as the cysteine concentration is increased for both the liposomes containing cholesterol (open triangles) and the cholesterol-free liposome formulation (closed circles).
- Cytotoxicity of free mitomycin C (closed squares) is not effected by the presence of cysteine.
- the cytotoxicity data shows that the cholesterol-free liposome formulation is more affected by cysteine.
- the IC50 of the cholesterol-free liposome formulation at certain cysteine concentrations is only 2-fold lower than that of the free drug alone.
- the liposome formulation containing cholesterol is less cytotoxic than the cholesterol- free liposome formulation.
- cysteine has no cytotoxic effect of the tumor cells and no effect on the cytotoxicity of free mitomycin C. It is also apparent from the data that cysteine increases in a dose-dependent fashion the cytotoxcity of liposome-bound mitomycin C.
- cysteine-mediated release of 5 mitomycin C from the lipid-DTB-drug conjugate is mostly accounted for by cysteine-mediated release of 5 mitomycin C from the lipid-DTB-drug conjugate.
- Figs. 13A-13B The in vivo pharmacokinetics of the liposomes containing cholesterol and the cholesterol-free liposome formulation was determined in rats. As described in io Example 7, the animals were treated with a single bolus intravenous injection of approximately 0.1 mg/mL mitomycin C in free form or incorporated into liposomes in the form of the lipid-DTB-mitomycin C conjugate in accord with the invention. After injection, blood samples were taken and analyzed for amount of mitomycin C. The results are shown in Figs. 13A-13B. is [0093] Fig. 13A shows the concentration ( ⁇ g/mL) of mitomycin C in the blood of rats as a function of time in hours following intravenous injection.
- Mitomycin C As seen, free mitomycin C (open squares) administered intravenously in free form is rapidly cleared from the blood. Mitomycin C in the form of a liposome-bound lipid-DTB-drug conjugate remains in circulation for a substantially longer period of time. Mitomycin C
- Fig. 13B shows the percent of injected dose remaining in the blood as a function of time in hours following intravenous injection of the test formulations. 25 Virtually none of the dose of free mitomycin C (open squares) remains in the blood at time points greater than about 5 minutes. However, at 20 hours after injection of the liposome formulations, about 15-18 percent of the dose of mitomycin C remains in circulation. This indicates the mitomycin C-DTB-lipid conjugate remains stable in the liposome while in circulation and that minimal thiolytic cleavage occurs in plasma.
- Step ® liposomes which have an extended blood circulation lifetime and enhanced accumulation in tumors.
- Fig. 14 The reduction in toxicity of mitomycin C when the drug is incorporated into liposomes in the form of a drug-DTB-lipid prodrug conjugate is illustrated in Fig. 14.
- the liposomes were comprised of HSPC, mPEG-DSPE and para-distearoyl-DTB- mitomycin C in a molar ratio of 90/5/5 (the cholesterol-free formulation described above).
- Three 10 mg/kg doses of liposomes were injected into female Balb/c mice at a dose of 10 mg drug/kg. Control animals received free mitomycin C, at a dose of 10 5 mg/kg. The weight of the animals was taken 3, 7, and 11 days after administration of the test substance, as shown in Fig. 14.
- liposomes prepared as described in Example 4 were tested in two mouse carcinoma models: an M109 footpad inoculation modes with tumor size as the endpoint, and a C26 intraperitoneal tumor model with survival as the endpoint. Test mice were inoculated with tumor cells (Example 8) and subsequently treated with free mitomycin C or mitomycin C in the form of a prodrug is conjugate incorporated into liposomes.
- mice were treated with a test compound intravenously, at a dose of 2 mg/kg.
- a second intravenous dose was given 13 days after tumor inoculation.
- the footpad size was measured a regular intervals. The results are
- Fig. 15A 20 shown in Fig. 15A for control mice left untreated (open squares) and for animals treated with free mitomycin C (open triangles) or with the liposomal formulation (HSPC/mPEG-DSPE/lipid-DTB-mitomycin C; closed circles).
- the tumor size of the untreated control animals increased continuously over the test period.
- Animals treated with mitomycin C experienced slower tumor growth, with the liposomal 25 formulation providing higher efficacy relative to mitomycin C in free form, as evidenced by a smaller footpad size for animals treated with mitomycin C in the form of a prodrug conjugate incorporated into liposomes.
- Fig. 15B shows the results from a similar study but with mitomycin C doses of 2 mg/kg and 4 mg/kg.
- mice 30 function of days after inoculation with M109 tumor cells in the paw of mice.
- Figs. 16A-16B show the median footpad size, in mm, as a function of days after inoculation with M109 tumor is cells in the paw of mice.
- mice treated with liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C at 6 mg/kg on days 5, 12, and 19 (closed circles, closed diamonds) had a slower tumor growth rate than mice treated with free mitomycin C (open triangles). Cysteine was administered subcutaneously on days 6-
- Fig. 16B shows the percent of mice alive with a footpad tumor size of less 25 than 4 mm, as a function of days after tumor inoculation, for the mice treated as set forth in Fig. 16A.
- This plot records as descending steps two types of events: deaths (toxic deaths) and tumor measures greater than 4 mm. All of the mice left untreated (open squares) had tumors greater than 4 mm after about test day 23. Mice treated with the liposomal formulation (closed circles, closed diamonds) had tumors less than
- mice were inoculated intraperitoneally with 10 6 C26 tumor cells. Five days after inoculation, the mice were treated with 6 mg/kg intravenously in free form or as a drug-DTB-lipid conjugated incorporated into liposomes. The results are shown in Fig. 17, where the percent survival as a function of time after inoculation with C26 tumor cells in mice is plotted. Mice left untreated (squares) failed to survive past test day 23. At test day 40, only 10% of the mice treated with 6 mg/kg free mitomycin C (triangles) were living.
- mice treated with 6 mg/kg mitomycin C in the form of a prodrug in a liposome were living. It is noteworthy that the mice treated with the liposomal formulation could tolerate a substantially higher dose, e.g, about 2- fold and in some cases 3-fold higher, of mitomycin C than when the drug in free form.
- M109R cells a subline of M109 cells selected for multi-drug resistance
- Fig. 18 shows the median footpad size, in mm, as a function of time after is inoculation with M109R tumor cells. Mice left untreated (open squares) had a continual increase in tumor size. Mice treated with 8 mg/kg liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C (closed circles, solid line) had a smaller footpad size than mice treated with a similar dose free mitomycin C (open triangles), until about day 130. Mice treated with two 8 mg/kg doses of liposomes comprised of
- Figs. 19A-19B show the results of similar test mice but the mitomycin C dose was 10 mg/kg and cysteine was administered to one of the test groups.
- Fig. 19A shows the median weight of the test mice, in grams, as a function of days after
- mice left untreated (open squares), treated with two 10 mg/kg doses of doxorubicin entrapped in liposomes having a coating of polyethylene glycol chains (Stealth ® , open triangles), treated with two doses of liposomes comprised of HSPC/mPEG-DSPE/lipid-DTB-mitomycin C at 10 mg/kg (closed circles) without cysteine (closed circles solid line) or with 5 mg/mouse cysteine (closed circles,
- mice treated with liposomal-doxorubicin had a loss of weight, indicating that this was indeed the maximal tolerated dose that they could tolerate. In contrast, no weight loss was observed with liposomal MMC prodrug with or without cysteine.
- Fig. 19B shows the median footpad thickness for the test animals.
- Fig.19C The data from this study is also presented in Fig.19C as the percentage of mice alive with a footpad thickness of less than 5 mm as a function of days after tumor inoculation.
- Figs. 18-19 The data shown in Figs. 18-19 indicates that mitomycin C administered in the form of drug-lipid conjugate incorporated into liposomes is able to be taken up by multi-drug resistant cells, and accumulate in the cells to an amount sufficient for cytotoxicity.
- the M109R cells were unresponsive to liposome-entrapped doxorubicin (Fig. 19B), as expected for this drug-resistant carcinoma model. is [0107] From the foregoing, various aspects and features of the invention are apparent.
- mitomycin C can be administered at 2-fold or 3-fold the dose of the drug in free form.
- the studies herein also show that multi-drug resistant cells are able to take up the mitomycin C when administered in the form of the lipid-DTB-drug conjugate.
- the research literature indicated that various primary tumors have an increased level of thioredoxin, a disulfide reducing enzyme, relative to healthy tissue (Powis et al., Free 25 Radical Biology & Med., 29:312 (2000); Engman, L., et al., Bioorganic and Medicinal Chemistry 11 :5091 , (2000)).
- the increased level of thioredoxin in tumor cells offers a unique synergy with the mitomycin C conjugate described here, since a natural source of a reducing enzyme is concentrated in the target tissue.
- the warm lipid solution was rapidly added to the warm (63-67°C) hydration medium, with mixing, to form a suspension of liposomes having heterogeneous sizes.
- the suspension was mixed for one hour at 63-67°C.
- the liposomes were sized to the desired mean particle diameter by controlled extrusion through polycarbonate filter cartridges housed in Teflon-lined stainless steel vessels.
- the liposome suspension was maintained at 63-65 0 C throughout the extrusion process, a period of 6-8 hours.
- Ethanol was removed from the liposome suspension by diafiltration.
- a histidine/sodium chloride solution was prepared by dissolving histidine (10 mM) and sodium chloride (150 mM) in sterile water. The pH of the solution was adjusted to approximately 7. The solution was filtered through a 0.22 ⁇ m Durapore filter. The liposome suspension was diluted in approximately a 1 :1 (v/v) ratio with the histidine/sodium chloride solution and diafiltered through a polysulfone hollow-fiber ultrafilter. Eight volume exchanges were performed against the histidine/sodium chloride solution to remove the ethanol. The process fluid temperature was maintained at about 20-30 0 C. Total diafiltration time was approximately 4.5 hours.
- Liposome particle size was measured by dynamic light scattering and the amount of "free", unbound mitomycin C in the external suspension medium was measured by HPLC.
- Liposomes were prepared as described above with a lipid composition of HSPC, mPEG-DSPE and para-distearoyl-DTB-mitomycin C in a molar ratio of 90/5/5. Specifically, 88.5 mg HPSC, 17.9 mg mPEG-DSPE (PEG MW 2000 Daltons) and 7.3 mg of the conjugate were dissolved in 1 mL ethanol. Liposome size, lipid and drug concentration and free mitomycin C concentration in the external suspension medium were determined after each processing step.
- Liposomes prepared as described in Examples 4A-4B were diluted in 0.6 M octaylglucopyranoside. The liposomes were incubated in the presence of 150 mM cysteine at 37 0 C. Samples with withdrawn at time zero, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours. A 20 ⁇ L volume was analyzed by HPLC using a Water Symmetry C8 3.5 x 5 cm column. The flow rate was 1 mL/min and the mobile phase gradient as follows:
- Liposomes prepared as described in Example 4A-4B, were composed of 5 HSPC/mPEG-DSPE/distearoyl-DTB-mitomycin C (90/5/5) or
- HSPC/cholesterol/mPEG-DSPE/distearoyl-DTB-mitomycin C (90/45/5/5).
- the liposome preparations were sterile filtered through 0.45 ⁇ m cellulose membranes and were not downsized via extrusion. After liposome formation, mitomycin C concentration was determined by absorbance at 360 nm in liposomes solubilized by io 10-20 fold dilution in isopropanol and the phospholipid concentration was determined by inorganic phosphate assay.
- the liposomes containing cholesterol had an average diameter of 275 ⁇ 90 nm.
- the cholesterol-free liposomes had an average diameter of 150 ⁇ 50 nm.
- the phospholipid concentration in both liposome formulations was 10 ⁇ M/mL and the is concentration of mitomycin C in both formulations was 120 ⁇ g/mL.
- the cultures were fixed by the addition of 50 ⁇ l 2.5% glutaraldehyde to each well for 10 minutes.
- the plates were washed three times with deionized water, once with 0.1 M borate buffer (pH 8.5) and then 35 stained for 60 minutes with 100 ⁇ l methylene blue (1 % in 0.1 M buffer borate, pH 8.5) at room temperature (20-25°C).
- the plates were rinsed in five baths of deionized water to remove non-cell bound dye and then dried.
- the dye was extracted with 200 ⁇ L 0.1 N HCI for 60 minutes at 37 0 C and the optical density was determined using a microplate spectrophotometer.
- the percent growth is inhibition or percent of control growth rate was obtained by dividing the growth rate of drug-treated cells by the growth rate of the untreated, control cells.
- the drug concentration which caused a 50% inhibition of the control growth rate (IC 50 ) was calculated by interpolation of the two closest values of the growth inhibition curve.
- Mitomycin C was assayed in the range 10 "8 -10 '5 M. The liposomal
- Liposomes containing cholesterol and cholesterol-free liposomes were prepared as described in Example 5A and 5B.
- a solution of mitomycin C in free form was prepared by dissolving 11.9 mg of mitomycin C in 119 ⁇ L ethanol. After dissolution, approximately 11.8 ⁇ L of a solution of 10 mM histidine/150 mM saline was added. Prior to use, the mitomycn C solution was diluted to 100 ⁇ g/mL with the histidine/saline solution and filtered.
- a single intravenous injection of the test formulation was administered as a bolus dose.
- Blood samples were taken from each animal at the following times after injection: 30 seconds, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours and 96 hours.
- the quantity of mitomycin C in the blood samples was determined by the HPLC procedure given below.
- a 200 mM io iodoacetamine solution was prepared by placing 199.3 mg of iodoacetamide in 5.1 mL of 7.5% EDTA. 15 ⁇ L of the 200 mM iodoacetamide solution was placed in each 1 ⁇ L of blood sample.
- the io concentration ranges were 0.05-5.0 ⁇ g/mL and 0.1-5 ⁇ g/mL for mitomycin C and mitomycin C conjugate, respectively.
- the final volume was adjusted to 1 mL with methanol.
- a similar procedure was followed to prepare quality control samples.
- the concentrations of quality control samples was 0.1 , 0.5 and 5 ⁇ g/mL for mitomycin C and 0.1 , 1 and 5 ⁇ g/mL for mitomycin C conjugate in rat plasma.
- the samples were is spun down at 3,000 rpm for 10 minutes at room temperature. 300 ⁇ L of supernatant was transferred to HPLC vials containing 300 ⁇ L insert for injection.
- ⁇ 100 ⁇ L of plasma sample was denatured with 900 ⁇ L of methanol followed 20 by centrifugation for 10 minutes at 3,000 rpm. An aliquot of 300 ⁇ L supernatant was transferred to an HPLC vial containing a 300 ⁇ L insert for injection.
- a Supelco® C-8, 5 ⁇ , 4.6mm x 5 cm column was used.
- the mobile phase 25 A was 10 mM ammonium phosphate, pH 7.
- Mobil phase B was methanol.
- the flow rate was 1 mL/min and detection was by UV at 360 nm.
- the injection volume was 40 ⁇ L and the typical run time was 15 minutes.
- the gradient program was as follows:
- EXAMPLE 8 In Vivo Studies is [0151] Female 10-week-old BALB/c mice were maintained in a specific Spathogen-free facility. M109 cells or M109R cells were grown in in vitro suspension. The mice were injected into the right hind footpad with 50 ⁇ l _ (10 6 cells). The footpad thickness was measured with calipers until completion of the study, when the mice were sacrificed, the final number of tumors recorded, and
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES04751275T ES2380620T3 (en) | 2003-04-30 | 2004-04-29 | Treatment of multi-drug resistant tumors using a mitomycin C conjugate |
EP04751275A EP1617873B1 (en) | 2003-04-30 | 2004-04-29 | Treatment of multi-drug resistant tumours by a conjugate of mitomycin C |
AU2004247004A AU2004247004B2 (en) | 2003-04-30 | 2004-04-29 | Mitomycin conjugates cleavable by thiols |
AT04751275T ATE541589T1 (en) | 2003-04-30 | 2004-04-29 | TREATMENT OF MULTI-RESISTANT TUMORS WITH A CONJUGATE OF MITOMYCIN C |
JP2006532557A JP5009621B2 (en) | 2003-04-30 | 2004-04-29 | Mitomycin conjugates capable of thiol cleavage |
CA2524179A CA2524179C (en) | 2003-04-30 | 2004-04-29 | A composition and its use for adminstration of mitomycin c in vivo for treating a multi-drug resistant tumor |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US46707003P | 2003-04-30 | 2003-04-30 | |
US60/467,070 | 2003-04-30 | ||
US10/714,085 | 2003-11-14 | ||
US10/714,085 US7303760B2 (en) | 1999-04-23 | 2003-11-14 | Method for treating multi-drug resistant tumors |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2004110497A2 true WO2004110497A2 (en) | 2004-12-23 |
WO2004110497A3 WO2004110497A3 (en) | 2005-03-24 |
Family
ID=33555221
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2004/013820 WO2004110497A2 (en) | 2003-04-30 | 2004-04-29 | Mitomycin conjugates cleavable by thiols |
Country Status (9)
Country | Link |
---|---|
US (1) | US7303760B2 (en) |
EP (1) | EP1617873B1 (en) |
JP (1) | JP5009621B2 (en) |
KR (1) | KR20060033711A (en) |
AT (1) | ATE541589T1 (en) |
AU (1) | AU2004247004B2 (en) |
CA (1) | CA2524179C (en) |
ES (1) | ES2380620T3 (en) |
WO (1) | WO2004110497A2 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005053749A2 (en) * | 2003-11-26 | 2005-06-16 | Alza Corporation | Thiol-cleavable linkage between polymer and ligand |
US7276248B2 (en) | 1999-04-23 | 2007-10-02 | Alza Corporation | Conjugate having a cleavable linkage for use in a liposome |
US7303760B2 (en) | 1999-04-23 | 2007-12-04 | Alza Corporation | Method for treating multi-drug resistant tumors |
EP2491945A1 (en) | 2006-09-28 | 2012-08-29 | Pierre Fabre Medicament | Method for generating active antibodies against a resistance antigen, antibodies obtained by said method and their uses |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9937261B2 (en) * | 2014-06-09 | 2018-04-10 | Lipomedix Pharmaceuticals Ltd. | Combination therapy comprising a liposomal prodrug of mitomycin C and radiotherapy |
AU2016233143B2 (en) * | 2015-03-17 | 2021-03-25 | Alberto Gabizon | Methods for the treatment of bladder cancer |
WO2018089481A1 (en) * | 2016-11-08 | 2018-05-17 | Mallinckrodt Llc | Mitomycin c prodrug liposome formulations and uses thereof |
EP3908261A1 (en) | 2019-01-11 | 2021-11-17 | Lipomedix Pharmaceuticals Ltd. | Liposome composition comprising liposomal prodrug of mitomycin c and method of manufacture |
JPWO2022050369A1 (en) * | 2020-09-04 | 2022-03-10 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2254336A1 (en) | 1973-12-17 | 1975-07-11 | Kyowa Hakko Kogyo Kk | |
EP0317957A2 (en) | 1987-11-23 | 1989-05-31 | Bristol-Myers Squibb Company | Drug-monoclonal antibody conjugates |
EP0317956A2 (en) | 1987-11-23 | 1989-05-31 | Bristol-Myers Squibb Company | Anti-tumor prodrugs |
EP0510197A1 (en) | 1990-01-11 | 1992-10-28 | Nippon Shinyaku Company, Limited | Fat emulsion |
WO1997036904A1 (en) | 1996-04-02 | 1997-10-09 | Sagami Chemical Research Center | Mitomycin c derivative and non-receptor tyrosine kinase inhibitor |
WO1999029302A1 (en) | 1997-12-05 | 1999-06-17 | Katarina Edwards | Drug delivery system with two-step targeting |
WO2000064484A2 (en) | 1999-04-23 | 2000-11-02 | Alza Corporation | Conjugate having a cleavable linkage for use in a liposome |
Family Cites Families (21)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4179337A (en) * | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
GB8430252D0 (en) * | 1984-11-30 | 1985-01-09 | Beecham Group Plc | Compounds |
US4766106A (en) * | 1985-06-26 | 1988-08-23 | Cetus Corporation | Solubilization of proteins for pharmaceutical compositions using polymer conjugation |
US4917888A (en) * | 1985-06-26 | 1990-04-17 | Cetus Corporation | Solubilization of immunotoxins for pharmaceutical compositions using polymer conjugation |
JPH01113391A (en) | 1987-10-24 | 1989-05-02 | Kyowa Hakko Kogyo Co Ltd | Mitomycin derivative |
US5103556A (en) * | 1988-05-05 | 1992-04-14 | Circon Corporation | Method of manufacturing an electrohydraulic probe |
US4902502A (en) * | 1989-01-23 | 1990-02-20 | Cetus Corporation | Preparation of a polymer/interleukin-2 conjugate |
DE69329073T2 (en) * | 1992-03-23 | 2001-01-18 | Georgetown University Washingt | TAXOL ENCLOSED IN LIPOSOMES AND METHOD OF USE |
WO1994005259A1 (en) * | 1992-09-02 | 1994-03-17 | Georgetown University | Method of encapsulating anthracycline glycosides in liposomes |
US5395619A (en) * | 1993-03-03 | 1995-03-07 | Liposome Technology, Inc. | Lipid-polymer conjugates and liposomes |
TW520297B (en) * | 1996-10-11 | 2003-02-11 | Sequus Pharm Inc | Fusogenic liposome composition and method |
EP0932390A1 (en) | 1996-10-11 | 1999-08-04 | Sequus Pharmaceuticals, Inc. | Therapeutic liposome composition and method |
JPH1160499A (en) | 1997-08-22 | 1999-03-02 | Hiroshi Maeda | Antitumor agent |
US6180095B1 (en) * | 1997-12-17 | 2001-01-30 | Enzon, Inc. | Polymeric prodrugs of amino- and hydroxyl-containing bioactive agents |
PL196533B1 (en) * | 1998-04-28 | 2008-01-31 | Applied Research Systems | Method for the stepwise attachment of polyethylene glycol (peg) in series to polypeptide |
US7303760B2 (en) | 1999-04-23 | 2007-12-04 | Alza Corporation | Method for treating multi-drug resistant tumors |
WO2000064483A2 (en) | 1999-04-23 | 2000-11-02 | Alza Corporation | Releasable linkage and compositions containing same |
US7112337B2 (en) | 1999-04-23 | 2006-09-26 | Alza Corporation | Liposome composition for delivery of nucleic acid |
WO2001026625A2 (en) | 1999-10-08 | 2001-04-19 | Alza Corp | Neutral-cationic lipid for nucleic acid and drug delivery |
US7052686B2 (en) | 2000-09-29 | 2006-05-30 | Schering Corporation | Pegylated interleukin-10 |
US7260330B2 (en) * | 2002-11-04 | 2007-08-21 | The Boeing Company | Optical communication system using correlation receiver |
-
2003
- 2003-11-14 US US10/714,085 patent/US7303760B2/en not_active Expired - Lifetime
-
2004
- 2004-04-29 JP JP2006532557A patent/JP5009621B2/en not_active Expired - Fee Related
- 2004-04-29 ES ES04751275T patent/ES2380620T3/en active Active
- 2004-04-29 AT AT04751275T patent/ATE541589T1/en active
- 2004-04-29 WO PCT/US2004/013820 patent/WO2004110497A2/en active Application Filing
- 2004-04-29 AU AU2004247004A patent/AU2004247004B2/en not_active Ceased
- 2004-04-29 KR KR1020057020690A patent/KR20060033711A/en not_active Application Discontinuation
- 2004-04-29 EP EP04751275A patent/EP1617873B1/en active Active
- 2004-04-29 CA CA2524179A patent/CA2524179C/en active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2254336A1 (en) | 1973-12-17 | 1975-07-11 | Kyowa Hakko Kogyo Kk | |
EP0317957A2 (en) | 1987-11-23 | 1989-05-31 | Bristol-Myers Squibb Company | Drug-monoclonal antibody conjugates |
EP0317956A2 (en) | 1987-11-23 | 1989-05-31 | Bristol-Myers Squibb Company | Anti-tumor prodrugs |
EP0510197A1 (en) | 1990-01-11 | 1992-10-28 | Nippon Shinyaku Company, Limited | Fat emulsion |
WO1997036904A1 (en) | 1996-04-02 | 1997-10-09 | Sagami Chemical Research Center | Mitomycin c derivative and non-receptor tyrosine kinase inhibitor |
WO1999029302A1 (en) | 1997-12-05 | 1999-06-17 | Katarina Edwards | Drug delivery system with two-step targeting |
WO2000064484A2 (en) | 1999-04-23 | 2000-11-02 | Alza Corporation | Conjugate having a cleavable linkage for use in a liposome |
Non-Patent Citations (4)
Title |
---|
DIAZ, C ET AL.: "Synthesis of disulfide-containing phospholipid analogs for the preparation of head group-specific lipid antigens: generation of phosphatidylserine antibodies", BIOCONJUGATE CHEMISTRY, vol. 9, no. 2, March 1998 (1998-03-01), pages 250 - 254, XP002125967, DOI: doi:10.1021/bc970156x |
MAEDA H. ET AL., J. CONTROLLED RELEASE, vol. 65, no. 1-2, 2000, pages 271 |
VAAGE J ET AL.: "Therapy of Primary and Metastatic Mouse Mammary Carcinomas with Doxorubicin Encapsulated in Long Circulating Liposomes", INTERNATIONAL JOURNAL OF CANCER, vol. 51, no. 6, 1992, pages 942 - 948, XP000979548, DOI: doi:10.1002/ijc.2910510618 |
ZALIPSKY, S ET AL.: "New liposomal prodrug of mitomycin C", PROCEEDINGS - 28TH INTERNATIONAL SYMPOSIUM ON CONTROLLED RELEASE OF BIOACTIVE MATERIALS AND 4TH CONSUMER & DIVERSIFIED PRODUCTS CONFERENCE, vol. 1, 23 June 2001 (2001-06-23), pages 437 - 438, XP008039801 |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7238368B2 (en) | 1999-04-23 | 2007-07-03 | Alza Corporation | Releasable linkage and compositions containing same |
US7276248B2 (en) | 1999-04-23 | 2007-10-02 | Alza Corporation | Conjugate having a cleavable linkage for use in a liposome |
US7285622B2 (en) | 1999-04-23 | 2007-10-23 | Alza Corporation | Releasable linkage and compositions containing same |
US7303760B2 (en) | 1999-04-23 | 2007-12-04 | Alza Corporation | Method for treating multi-drug resistant tumors |
US7592307B2 (en) | 1999-04-23 | 2009-09-22 | Alza Corporation | Releasable linkage and compositions containing same |
US7608687B2 (en) | 1999-04-23 | 2009-10-27 | Alza Corporation | Releasable linkage and compositions containing same |
WO2005053749A2 (en) * | 2003-11-26 | 2005-06-16 | Alza Corporation | Thiol-cleavable linkage between polymer and ligand |
WO2005053749A3 (en) * | 2003-11-26 | 2006-11-09 | Alza Corp | Thiol-cleavable linkage between polymer and ligand |
EP2491945A1 (en) | 2006-09-28 | 2012-08-29 | Pierre Fabre Medicament | Method for generating active antibodies against a resistance antigen, antibodies obtained by said method and their uses |
EP2494984A1 (en) | 2006-09-28 | 2012-09-05 | Pierre Fabre Medicament | Method for generating active antibodies against a resistance antigen, antibodies obtained by said method and their uses |
EP2497491A1 (en) | 2006-09-28 | 2012-09-12 | Pierre Fabre Medicament | Method for generating active antibodies against a resistance antigen, antibodies obtained by said method and their uses |
Also Published As
Publication number | Publication date |
---|---|
AU2004247004A1 (en) | 2004-12-23 |
JP2007502323A (en) | 2007-02-08 |
CA2524179C (en) | 2012-06-19 |
US7303760B2 (en) | 2007-12-04 |
JP5009621B2 (en) | 2012-08-22 |
AU2004247004B2 (en) | 2010-10-14 |
WO2004110497A3 (en) | 2005-03-24 |
US20040161455A1 (en) | 2004-08-19 |
ES2380620T3 (en) | 2012-05-16 |
ATE541589T1 (en) | 2012-02-15 |
CA2524179A1 (en) | 2004-12-23 |
EP1617873A2 (en) | 2006-01-25 |
EP1617873B1 (en) | 2012-01-18 |
KR20060033711A (en) | 2006-04-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6365179B1 (en) | Conjugate having a cleavable linkage for use in a liposome | |
US7238368B2 (en) | Releasable linkage and compositions containing same | |
CA2059649A1 (en) | Cell internalizable conjugates and complexes including intracellularly cleavable moieties | |
JPS63258423A (en) | Liposome/anthraquinone drug composition and therapy therewith | |
US20040022842A1 (en) | Liposome preparations containing oxaliplatin | |
US7303760B2 (en) | Method for treating multi-drug resistant tumors | |
JP2002541088A (en) | Compositions and methods for the treatment of lymphoma | |
EP1341497A2 (en) | Receptor antagonist-lipid conjugates and delivery vehicles containing same | |
ES2329374T3 (en) | PHARMACEUTICAL FORMULATIONS THAT USE SHORT CHAIN SPHYNOLIPIDS AND USES OF THE SAME. | |
MXPA05001222A (en) | Conjugates of porphyrin compounds with chemotherapeutic agents. | |
US20220087975A1 (en) | Liposome composition comprising liposomal prodrug of mitomycin c and method of manufacture |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2004247004 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 543291 Country of ref document: NZ Ref document number: 2524179 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2004751275 Country of ref document: EP Ref document number: 1020057020690 Country of ref document: KR Ref document number: 2006532557 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2225/KOLNP/2005 Country of ref document: IN |
|
ENP | Entry into the national phase |
Ref document number: 2004247004 Country of ref document: AU Date of ref document: 20040429 Kind code of ref document: A |
|
WWP | Wipo information: published in national office |
Ref document number: 2004247004 Country of ref document: AU |
|
WWP | Wipo information: published in national office |
Ref document number: 2004751275 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1020057020690 Country of ref document: KR |