Patent Application
20160303264
Liposomes have proved a valuable tool for delivering various pharmacologically active molecules, such as anti-neoplastic agents, to cells, organs, or tumors. Liposome delivery has been shown to improve the pharmacokinetic profile and widen the therapeutic index of certain anticancer drugs, especially the anthracycline class. Improved efficacy is in part a result of passive targeting to tumor sites based on the enhanced permeability and retention (EPR) effect. To fully exploit this process, drug carriers should be engineered to retain drug while circulating, thereby preventing premature drug release before accumulating in the tumor but still allowing for release of drug once in the vicinity of the tumor. Antibody-targeted nanoparticles, such as immunoliposomes comprising external antibodies or antibody fragments that immunospecifically bind, for example, HER2 or epidermal growth factor receptor, represent another strategy for more efficient drug delivery to tumor cells.
It has been found, however, that deposition of liposomal drugs into tumors varies and tumors that exhibit higher liposomal drug deposition will have improved clinical outcomes. Liposomal drugs have been shown to accumulate in tumors via a mechanism termed the enhanced permeability and retention (EPR) effect whereby liposomes preferentially escape from the bloodstream into the tumor interstitium via leaky tumor vasculature and then become trapped in the tumor by virtue of their large size and the reduced levels of functional lymphatics in the tumor. However, the degree to which liposomal particles can deposit into tumors has been shown to be highly variable in both preclinical tumor models and in clinical studies.
Compositions and non-invasive methods allowing the determination of whether or not a liposomally-delivered therapeutic agent is suitable for use in a patient (e.g., to predict clinical outcomes of targeted and untargeted liposomal cancer therapeutics) are therefore needed.
Provided herein are liposomal imaging agents that can be used to predict low or high deposition of liposomal drugs in lesions (e.g., localized pathology such as cancers, malignant or benign tumors, and sites of inflammation or infection) in a patient, and ultimately which patients will benefit from a particular liposomal drug, as well as methods for their use. Also disclosed herein are methods for non-invasive imaging, and more particularly, for non-invasive imaging for use in predicting the utility of liposomal therapeutics. Such methods are useful in imaging cancer or another disease (e.g., a localized infectious or inflammatory disease), and/or for drug delivery to a target site, e.g., tumor tissue. In some embodiments, the method further comprises treating a patient, e.g., a patient having an infection, a localized inflammatory condition, or a cancerous tumor. For example, a preparation of liposomes may contain a chemotherapeutic agent, such as a taxane, a topoisomerase inhibitor (e.g., irinotecan or topotecan), or an anthracycline (e.g., doxorubicin), in the liposomal interior space and liposomes comprised by the preparation may be loaded with a radiolabel suitable for PET imaging, such as 64Cu, thus allowing for imaging and treatment to result from the same administration of the liposomal preparation.
In a first aspect, disclosed herein is a method of preparing a patient for PET imaging of a lesion in the patient, the method comprising administering to the patient an injection comprising a dose of a preparation of 64Cu-loaded liposomal doxorubicin, the liposomes comprised by the preparation having an average diameter of 75-110 nm, wherein the dose comprises 3-5 mg/m2 doxorubicin and is formulated to deliver 10.8 (+/−15%, optionally +/−10%) millicuries (mCi) of 64Cu when administered to the patient. In one embodiment, the lesion is a benign tumor or a malignant tumor, optionally a brain tumor. In another embodiment, the 64Cu-loaded liposomes are immunoliposomes. In one embodiment, the immunoliposomes are HER2-targeted immunoliposomes. In another embodiment, the immunoliposomes are EphA2-targeted immunoliposomes. In some embodiments the liposomes comprise a gradient-loadable chelator. In one embodiment, the chelator is 4-DEAP-ATSC.
In a second aspect, disclosed herein is a method of imaging a lesion in a patient, the method comprising administering to the patient an injection comprising a preparation of 64Cu-loaded liposomal doxorubicin, the liposomes comprised by the preparation having an average diameter of 75-110 nm, at a dose of 3-5 mg/m2 doxorubicin; then within 48 hours following the injection, obtaining a PET scan of a region of the patient, the region comprising the location of the lesion. In one embodiment, the lesion is a site of inflammation, a site of infection, a benign tumor or a malignant tumor, optionally a malignant brain tumor. In another embodiment, the dose of liposomal doxorubicin is formulated to deliver to the patient, when administered, 10.8 (+/−15%, optionally +/−10%) mCi of 64Cu. In another embodiment, the PET scan is obtained within 24 hours, within 12 hours, within six hours, within 3 hours, within 2 hours, or within 1 hour following the injection.
In a third aspect, disclosed herein is a method of imaging a lesion in a patient, the method comprising: (a) administering to the patient an injection comprising a preparation of 64Cu-loaded liposomes, the injection administered at a dose of 10.8 mCi of 64Cu (+/−15%); and (b) within 48 hours following the injection, obtaining a PET scan of a region of the patient, the region comprising the location of the lesion. In one embodiment, the preparation comprises liposomes with an average diameter of 75-110 nm. In another embodiment, the PET scan is obtained within 3 hours following the injection. In one embodiment, the within 3 hours is within 2 hours or within 1 hour. In one embodiment, the 64Cu-loaded liposomes are immunoliposomes.
In one embodiment, the immunoliposomes are HER2-targeted immunoliposomes. In another embodiment, the immunoliposomes are EphA2-targeted immunoliposomes. In some embodiments the liposomes comprise a gradient-loadable chelator. In one embodiment, the chelator is 4-DEAP-ATSC. In one embodiment, the liposomes further comprise a chemotherapeutic agent. In some embodiments the chemotherapeutic agent is doxorubicin or irinotecan or a taxane. In one embodiment, the lesion is a brain tumor.
In a fourth aspect, disclosed herein is a method of treating and imaging a patient, the method comprising: (a) administering to the patient a first injection comprising immunoliposomal doxorubicin that does not comprise detectable levels of 64Cu, the injection administered at a dose of at least 25, at least 30, at least 35, at least 40, or at least 45, or 50 mg/m2 of doxorubicin; (b) at between one and 6 hours following the first injection, administering to the patient a second injection comprising 64Cu-loaded immunoliposomal doxorubicin, the doxorubicin comprised by the second injection consisting of a dose of at least 3, at least 4, at least 5, at least 6, or 7 mg/m2 of doxorubicin, said dose comprising at 10.8 mCi of 64Cu+/−15%; and (c) obtaining at least two PET/CT scans of a region of pathology in the patient, wherein each scan is obtained at a different time point, and wherein time elapsed from the injection of (a) until a final scan of the at least two scans is obtained is no more than three days. In one embodiment, the immunoliposomal doxorubicin is HER2-targeted. In another embodiment, the immunoliposomal doxorubicin is EphA2-targeted.
In a fifth aspect, disclosed herein are compositions comprising 64Cu-loaded liposomes containing doxorubicin, such compositions being useful in practicing the methods disclosed herein. In one embodiment, the liposomes are HER2-targeted liposomes. In another embodiment, the liposomes are EphA2-targeted immunoliposomes. In some embodiments, the composition is adapted for administration to a human patient at a dose of at least 0.028, at least 3, at least 4, at least 5, at least 6, or 7 mg/m2 of doxorubicin. In one embodiment, the liposomes comprise a gradient-loadable chelator. In one embodiment, the chelator is 4-DEAP-ATSC. In another embodiment, the composition comprises about 5, about 7, about 10, about 10.8, about 12, or about 15 mCi of 64Cu. In one embodiment, the liposomes comprise hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and poly(ethylene glycol) (PEG)-derivatized distearoylphosphatidylethanolamine (PEG-DSPE) at a 3:1:0.05 molar ratio. In one embodiment, the poly(ethylene glycol) of the PEG-DSPE has a molecular weight of about 2000.
Other features and advantages will be apparent from the detailed description, and from the claims.
The present invention provides compositions and methods for non-invasive imaging, and more particularly, non-invasive imaging for liposomal therapeutics, as well as methods of treating patients comprising the use of such methods for non-invasive imaging prior to administration of liposomal therapeutics.
The invention is based, at least in part, on the discovery that diacetyl 4,4′bis(3-(N,N-diethylamino)propyl)thiosemicarbazone (4-DEAP-ATSC) is useful as a non-invasive imaging reagent for determining whether a subject is a candidate for treatment with a liposomal therapeutic, as well as for monitoring treatment of a subject with a liposomal therapeutic.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.
By “Liposome A” is meant a 64Cu-loaded liposome that does not contain any drug.
By “Liposome B” is meant 64Cu-loaded, HER2-targeted liposomal doxorubicin. Exemplary methods of preparation, dosage and administration of Liposome B may be found, e.g., in co-pending Patent Publication No. WO/2012/078695.
By “Liposome C” is meant 64Cu-loaded irinotecan sucrosofate liposome injection. Liposome C can be prepared in accordance with U.S. Pat. No. 8,147,867.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the term “subject” or “patient” is a human patient.
By “mGy” is meant milligray, which is a measure of an absorbed dose of ionizing radiation. A Gy is defined as the absorption of one joule of radiation energy by one kilogram of matter.
By “mBq” is meant megabecquerel, which is a measure of radioactivity. One Bq is defined as the activity of a quantity of radioactive material in which one nucleus decays per second.
By “doxorubicin equivalent” is meant, in the case of liposomal doxorubicin, the total mass of doxorubicin in each dose. That is, the dosage of liposomal doxorubicin is determined based on the amount of doxorubicin in a particular volume of liposome preparation.
A substance “loaded liposomal” drug or preparation (e.g., 64Cu-loaded liposomal doxorubicin), or substance “loaded liposomes” refer to a liposomal preparation in which the substance is entrapped within liposomes comprised by the preparation or to liposomes comprising the substance.
By “lesion,” as used herein, is meant a region in an organ or tissue that has suffered damage through injury or disease, such as a tumor (benign or malignant) or localized sites of inflammation or infection.
By “EphA2” is meant ephrin type-A receptor 2. Eph receptors are a unique family of receptor tyrosine kinases that play critical roles in embryonic patterning, neuronal targeting, and vascular development during normal embryogenesis. Eph receptor tyrosine kinases and their ligands, the ephrins, are also frequently overexpressed in a variety of cancers and tumor cell lines. EphA2 is overexpressed in, e.g., breast, prostate, lung, and colon cancers.
Disclosed herein are liposomal imaging and drug delivery agents having at least two components: (1) A liposome, which will be suspended or solubilized in a liquid medium (such as a buffer or other pharmaceutically acceptable carrier); (2) a chelator moiety capable of chelating a metal ion; and optionally (3) a metal ion suitable for imaging or otherwise assessing the in vitro or in vivo uptake of the liposomal imaging agent into cells, organs, or tumors. In some embodiments, the metal ion has a valency of 2 or 3 or 4. In exemplary embodiments, the metal ion has a valency of 2. Exemplary liposomal imaging agents are described in PCT/US13/37033.
The liposomes of the liposomal imaging agents disclosed herein can be any liposome known or later discovered in the art. In certain embodiments, the liposome comprises hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and poly(ethylene glycol) (PEG) (Mol. weight 2000)-derivatized distearoylphosphatidylethanolamine (PEG-DSPE) (3:1:0.05 molar ratio).
In other embodiments, the liposome comprises poly(ethylene glycol)-derivatized phosphatidylethanolamines such as 1,2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine-N-[methoxy(poly(ethylene glycol))]; 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl ethanolamine-N-[methoxy(poly(ethylene glycol))]; 1,2-dimyristoyl-sn-glycero-3-phosphatidyl ethanolamine-N-[methoxy(poly(ethylene glycol))]; or 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine-N-[methoxy(poly(ethylene glycol))]. In certain embodiments, the molecular weight of PEG is 750, 1000, 1500, 2000, 3000, 3500, or 5000.
In certain embodiments the liposome comprises poly(ethylene glycol)-derivatized diacyl glycerols such as such as 1,2-distearoyl-glyceryl-[methoxy(poly(ethylene glycol))]; 1,2-dimyristoyl-glyceryl-[methoxy(poly(ethylene glycol))]; 1,2-dipalmitoyl-glyceryl-[methoxy(poly(ethylene glycol))]; or 1,2-dioleoyl-glyceryl-[methoxy(poly(ethylene glycol))]. In certain embodiments, the molecular weight of PEG is 750, 1000, 1500, 2000, 3000, 3500, or 5000.
In other embodiments the liposome comprises 1,2-dioctadecyl glycero-N-[methoxy(poly(ethylene glycol))]; dihexadecyl glycero-N-[methoxy(poly(ethylene glycol))]; or ditetradecyl glycero-N-[methoxy(poly(ethylene glycol))]. In certain embodiments, the molecular weight of PEG is 750, 1000, 1500, 2000, 3000, 3500, or 5000.
In various embodiments the liposome comprises PEG-ceramides, such as N-octdecanoyl-sphingosine-1-{succinoyl[methoxy(poly(ethylene glycol))]}; N-tetradecanoyl-sphingosine-1-{succinoyl[methoxy(poly(ethylene glycol))]}; N-hexadecanoyl-sphingosine-1-{succinoyl[methoxy(poly(ethylene glycol))]}; N-octdecanoyl-sphingosine-1-[methoxy(poly(ethylene glycol))]; N-tetradecanoyl-sphingosine-1-[methoxy(poly(ethylene glycol))]; or N-hexadecanoyl-sphingosine-1-[methoxy(poly(ethylene glycol))]. In certain embodiments the molecular weight of PEG is 750, 1000, 1500, 2000, 3000, 3500, or 5000.
Additional examples of suitable nanoparticle or liposome forming lipids that may be used in the compositions or methods include, but are not limited to, the following: phosphatidylcholines such as diacyl-phosphatidylcholine, dialkylphosphatidylcholine, 1,2-dioleoyl-phosphatidylcholine, 1,2-dipalmitoyl-phosphatidylcholine, 1,2-dimyristoyl-phosphatidylcholine, 1,2-distearoyl-phosphatidylcholine, 1-oleoyl-2-palmitoyl-phosphatidylcholine, 1-oleoyl-2-stearoyl-phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholine and 1-stearoyl-2-oleoyl-phosphatidylcholine; phosphatidylethanolamines such as 1,2-dioleoyl-phosphatidylethanolamine, 1,2-dipalmitoyl-phosphatidylethanolamine, 1,2-dimyristoyl-phosphatidylethanolamine, 1,2-distearoyl-phosphatidylethanolamine, 1-oleoyl-2-palmitoyl-phosphatidylethanolamine, 1-oleoyl-2-stearoyl-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylethanolamine and N-succinyl-dioleoyl-phosphatidylethanolamine; phosphatidylserines such as 1,2-dioleoyl-phosphatidylserine, 1,2-dipalmitoyl-phosphatidylserine, 1,2-dimyristoyl-phosphatidylserine, 1,2-distearoyl-phosphatidylserine, 1-oleoyl-2-palmitoyl-phosphatidylserine, 1-oleoyl-2-stearoyl-phosphatidylserine, 1-palmitoyl-2-oleoyl-phosphatidylserine and 1-stearoyl-2-oleoyl-phosphatidylserine; phosphatidylglycerols such as 1,2-dioleoyl-phosphatidylglycerol, 1,2-dipalmitoyl-phosphatidylglycerol, 1,2-dimyristoyl-phosphatidylglycerol, 1,2-distearoyl-phosphatidylglycerol, 1-oleoyl-2-palmitoyl-phosphatidylglycerol, 1-oleoyl-2-stearoyl-phosphatidylglycerol, 1-palmitoyl-2-oleoyl-phosphatidylglycerol and 1-stearoyl-2-oleoyl-phosphatidylglycerol; pegylated lipids (lipids comprising polyethylene glycol); pegylated phospoholipids such as phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-1000], phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000], phophatidylethanolamine-N-[methoxy(polyethylene glycol)-3000], phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-5000]; lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, lyso-phosphatidylglycerols, lyso-phosphatidylserines, ceramides, sphingolipids, e.g., sphingomyelin; phospholipids; glycolipids such as ganglioside GMI; glucolipids; sulphatides; phosphatidic acid, such as di-palmitoyl-glycerophosphatidic acid; palmitic fatty acids; stearic fatty acids; arachidonic fatty acids; lauric fatty acids; myristic fatty acids; lauroleic fatty acids; physeteric fatty acids; myristoleic fatty acids; palmitoleic fatty acids; petroselinic fatty acids; oleic fatty acids; isolauric fatty acids; isomyristic fatty acids; isostearic fatty acids; sterol and sterol derivatives such as cholesterol, cholesterol hemisuccinate, cholesterol sulphate, and cholesteryl-(4-trimethylammonio)-butanoate, ergosterol, lanosterol; poly-oxyethylene fatty acids esters and polyoxyethylene fatty acids alcohols; poly-oxyethylene fatty acids alcohol ethers; polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol oxy-stearate; glycerol polyethylene glycol ricinoleate; ethoxylated soybean sterols; ethoxylated castor oil; polyoxyethylene polyoxypropyl-ene fatty acid polymers; polyoxyethylene fatty acid stearates; di-oleoyl-sn-glycerol; dipalmitoyl-succinyl glycerol; 1,3-dipalmitoyl-2-succinylglycerol; 1-alkyl-2-acyl-phosphatidylcholines such as i-hexadecyl-2-palmitoyl-phosphatidylcholine; 1-alkyl-2-acyl-phosphatidylethanolamines such as 1-hexadecyl-2-palmitoyl-phosphatidylethanolamine; 1-alkyl-2-acyl-phosphatidylserines such as 1-hexadecyl-2-palmitoyl-phosphatidylserine; 1-alkyl-2-acyl-phosphatidylglycerols such as 1-hexadecyl-2-palmitoyl-phosphatidylglycerol; 1-alkyl-2-alkyl-phosphatidylcholines such as 1-hexadecyl-2-hexadecyl-phosphatidylcholine; 1-alkyl-2-alkyl-phosphatidylethanolamines such as 1-hexadecyl-2-hexadecyl-phosphatidylethanolamine; 1-alkyl-2-alkyl-phosphatidylserines such as 1-hexadecyl-2-hexadecyl-phosphatidylserine; 1-alkyl-2-alkyl-phosphatidylglycerols such as 1-hexadecyl̂-hexadecyl-phosphatidylglycerol; N-Succinyl-dioctadecylamine; palmitoylhomocysteine; lauryltrimethylammonium bromide; cetyltrimethyl-ammonium bromide; myristyltrimethylammonium bromide; N-[1,2,3-dioleoyloxy)-propyl]-N,N,Ntrimethylammoniumchloride (DOTMA); 1,2-dioleoyloxy-3 (trimethyl-ammonium)propane (DOTAP); and 1,2-dioleoyl-c-(4′-trimethylammonium)-butanoyl-sn-glycerol (DOTB).
The liposomes contained in the liposomal imaging agents disclosed herein can be untargeted liposomes or targeted liposomes, e.g., liposomes containing one or more targeting moieties or biodistribution modifiers on the surface of the liposomes. A targeting moiety can be any agent that is capable of specifically binding or interacting with a desired target. In one embodiment, a targeting moiety is a ligand. The ligand may preferentially bind to and/or internalize into, a cell in which the liposome-entrapped entity exerts its desired effect (a target cell). A ligand is usually a member of a binding pair where the second member is present on, or in, a target cell(s) or in a tissue comprising the target cell. Examples of suitable ligands include: folic acid, protein, e.g., transferrin, a growth factor, an enzyme, a peptide, a receptor. A targeted liposome wherein a targeting moiety is an antibody or a target antigen-binding fragment thereof (generally an immunoglobulin) is called an “immunoliposome”.
In certain embodiments, the liposomes of the liposomal imaging agents exhibit a transmembrane gradient formed by a gradient-forming agent such as a substituted ammonium compound. Alternate loading modalities are described, e.g., in U.S. Pat. No. 8,147,867. Preferably, the higher concentration of the gradient forming agent is in the interior (inner) space of the liposomes. In addition, a liposome composition disclosed herein can include one or more trans-membrane gradients in addition to the gradient created by the substituted ammonium and/or polyanion disclosed herein. For example, liposomes contained in liposome compositions disclosed herein can additionally or alternately include a transmembrane pH gradient, ion gradient, electro-chemical potential gradient, and/or solubility gradient.
It will be appreciated that when a trapping agent is used, excess gradient forming agent can be removed from the liposomes (e.g., by diafiltration) after the loaded component has been entrapped within the liposome.
The metal chelating moiety of the liposomal imaging agent can be any agent capable of stably chelating a divalent metal cation and being retained in the interior of the liposome. Examples of such metal chelating moieties include the compound 4-DEAP-ATSC:
Additional examples of suitable chelators include compounds represented by Formula (IV):
in which
Q is H, substituted or unsubstituted C1-C6alkyl or —(CH2)n—NR3R4; R1, R2, R3 and R4 are each independently selected from H, substituted or unsubstituted C1-C6alkyl, or substituted or unsubstituted aryl or wherein either or both of (1) R1 and R2 and (2) R3 and R4 are joined to form a heterocyclic ring;
and
n is independently, for each occurrence, an integer from 1 to 5.
In some embodiments the metal ion chelated by the chelator is a divalent metal cation. The metal cation for use in the liposomal imaging agents disclosed herein can be any suitable divalent metal cation, e.g., of the alkaline earth, transition metal, lanthanide, or actinide series. A divalent metal cation can be selected according to the intended use of the liposomal imaging agent.
For example, for use in positron emission computed tomography (PET/CT scanning), a positron-emitting radioisotope (such as a divalent ion of 44Sc2+, 64Cu2+, 110In2+ or 128Cs2+) can be employed. In certain embodiments, the divalent metal cation is 64Cu2+. In some embodiments, an x-ray computerized tomography (x-ray CT) scan is performed concomitantly with a PET/CT scan and the images aligned and overlaid upon each other (a PET/x-ray overlay).
Gradient-based drug loading technologies, in which, e.g., electrochemical gradients drive the accumulation of drugs in the liposome interior, can be used to prepare liposomes. Thus, a liposome having, e.g., an electrochemical gradient between the interior and the exterior of the lipid bilayer can be loaded with cationic chelation complexes of divalent metals by addition of the cationic chelator complex to the liposome preparation.
In general, liposomes can be prepared according to any method known in the art. Other methods for producing nanoparticles/liposomes are disclosed, e.g., in U.S. Patent Application Nos. 20030118636; 20080318325; and 20090186074 and U.S. Pat. Nos. 4,192,869; 4,397,846; 4,394,448; 4,394,149; 4,241,046; 4,598,051; 4,429,008; 4,755,388; 4,911,928; 6,426,086; 6,803,053; 7,871,620; 8,147,867 and 8,329,213.
Alternatively, a liposome can be loaded with an un-complexed chelator moiety (i.e., without a metal cation complexed to the chelator moiety), followed by addition of the divalent metal cation to the liposomal preparation. In one embodiment, the intraliposomal pH is adjusted so that 64Cu penetrates the lipid bilayer and forms a complex with the chelator inside the liposome.
The Liposomes disclosed herein may be used for patient stratification or determination of the suitability of a patient for a candidate liposome-based therapy. An exemplary method of determining whether a patient is a candidate for therapy with a liposomal therapeutic agent is as follows:
In another aspect, the invention provides a method of monitoring treatment of a location within the patient by a liposomal therapeutic agent, the method comprising:
In general, the liposomal imaging agents disclosed herein may be used to image a variety of neoplasias including, but not limited to, glioma, astrocytoma, chordoma, craniopharyngioma, acoustic neuroma, medulloblastoma, meningioma, metastatic brain tumors, pituitary tumors, oligodendroglioma, schwannoma, CNS lymphoma, ependymoma, pineal tumors, brain stem glioma, rhabdoid tumors, juvenile pilocytic astrocytoma, primitive neuroectodermal tumors, optic nerve glioma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric carcinoma, gastro-esophageal junction cancer, esophageal cancer, colon carcinoma, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, lymphoma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
In another embodiment, liposomal imaging agents may be used to image vascular damage caused by a variety of infectious agents including, but not limited to, bacteria, fungi, and viruses. Likewise, the liposomal imaging agents may be used to monitor a patient during treatment for vascular disorders such as hand-foot syndrome (also known as palmar-plantar erythrodysesthesia (PPE), plantar palmar toxicity, palmoplantar keratoderma, and cutaneous toxicity), which is a side effect of some chemotherapy drugs. Hand-foot syndrome results when a small amount of an anti-neoplastic agent leaks out of the smallest blood vessels in the palms of the hands and soles of the feet. The amount of drug in the capillaries of the hands and feet increases due to the friction and subsequent heat that is generated in those extremities. As a result, more drug may leak out of capillaries in these areas. Once out of the blood vessels, the chemotherapy drug damages surrounding tissues. Liposomal imaging agents may be used to image such damage and treatment of the patient can be adjusted accordingly, either by adjusting the dose of drug or by increasing adjunctive therapies such as administration of anti-inflammatory therapeutics. Liposomal imaging agents may also be used to predict those patients who are most likely to experience such side effects and prophylactic adjunctive therapies may be employed.
The quantity of liposome composition necessary to image a target cell or tissue can be determined by routine in vitro and in vivo methods. Safety testing of such compositions will be analogous to those methods common in the art of drug testing. Typically the dosages for a liposome composition disclosed herein ranges between about 0.0007 and about 10 mg of the liposomes per kilogram of body weight. In an exemplary embodiment, the dosage is about 0.0007 mg of the liposomes per kilogram of body weight.
Typically, the liposome pharmaceutical composition disclosed herein is prepared as a topical or an injectable, either as a liquid solution or suspension. However, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
The liposome composition disclosed herein can be administered in any way which is medically acceptable which may depend on the neoplasia being imaged. Possible administration routes include injections, by parenteral routes such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intraepidural, intrathecal, or others, as well as oral, nasal, ophthalmic, rectal, vaginal, topical, or pulmonary, e.g., by inhalation. The compositions may also be directly applied to tissue surfaces.
Although 64Cu-Liposome B has not been tested in humans, 60Cu-ATSM and 64Cu-ATSM have been evaluated in human trials as potential imaging agents. The 64Cu with 4-DEAP-ATSC used label Liposome B is derived from 64Cu-ATSM. In human studies there were no clinically significant changes in vital signs or laboratory test results after injection of 60Cu-ATSM and 64Cu-ATSM. No adverse events or clinically detectable pharmacologic effects related to either 60Cu-ATSM or 64Cu-ATSM were observed.
A Parent Study is enrolled that is a Phase 1, multi-center, open-label, dose-escalation, safety, and pharmacokinetic clinical study of intravenously administered Liposome B monotherapy and combination therapy for patients with advanced HER2 positive breast cancer. Disclosed herein are methods and procedures for a Companion Study that will also be enrolled; the Companion Study is an open label, multicenter, single-dose, radiation dosimetry, and biodistribution study of 64Cu-Liposome B in patients with advanced cancers. Ten to 45 evaluable patients will be enrolled. Patients will be screened and eligibility confirmed to participate in the Parent Study. A minimum of 6-10 patients is anticipated to obtain sufficient radiation dosimetry assessments. The number of patients may be extended depending on human biodistribution and acquired image quality. After the radiation dosimetry has been evaluated, dosing may be adjusted.
Each patient receives one dose of study treatment (unlabeled Liposome B+64Cu-Liposome B), h unlabeled Liposome B, according to the schedule set forth in the Parent Protocol. Participation on this companion protocol will last until all required assessments are completed, approximately 48 hours post-dose. All subsequent study visits and treatment administration will be conducted according to the parent protocol. Dose levels are described below in Table 1.
64Cu-Liposome B
aThe dose range indicated is an approximate dose; the actual dose will depend on the time of administration of 64Cu-Liposome B and the patient's body surface area (BSA).
bThis dose will only be used if patients are to enroll in a parent protocol cohort using this dose.
cAdditional doses may be examined as deemed appropriate by the Investigators, Sponsor, and Medical Monitor.
A variation of plus or minus (+/−) 15% in the dose in millicuries (mCi) of 64Cu administered in accordance with this disclosure is provided for in the methods disclosed herein. This is needed because dosage of 64Cu (e.g., in mCi/mL) is measured at the radiopharmacy and variability will occur due to alterations such as changes in the timing of the delivery of 64Cu preparations from the radiopharmacy to the clinic and in the timing of administration to the patient following delivery, which can significantly alter the dose administered due to the short half-life (about 12.7 hours) of 64Cu. In one embodiment, the about 10.8 mCi is between about 9.72 and about 11.88 mCi. In another embodiment, the about 10.8 mCi is between about 9.18 and about 12.42 mCi.
In some embodiments it is desirable to limit the amount of radioactivity that is administered to a patient. Thus, in some embodiments, a patient may be given a reduced dose of 64Cu-Liposome B. As a non-limiting example, a reduced dose maybe used for patients of small stature, for patients who have already recently been exposed to radiation in another capacity, or for patients who are scheduled for an extended imaging time period. Such a reduced dose may comprise, for example, a total of 5 mCi, 7 mCi, or 10 mCi.
By the same token, in cases where a shortened imaging time is necessary, or in cases where increased signal-to-background ratio of the image is desired, the amount of 64Cu may be increased up to about 15 mCi, or higher if the radiation dosimetry profile is deemed tolerable. For example, an increased dose might be used if a patient is suspected of having a number of smaller metastatic lesions with low 64Cu-Liposome B uptake, or high background tissue signal, making the increased resolution desirable. An increased dose might also be used in the case of late stage cancer patients who are permitted a higher dose of radiation.
The total dose of Liposome B is administered in two stages: (1) unlabeled Liposome B (non-radioactive) followed by (2)64Cu-Liposome B (radioactive) up to six hours later. Following administration of 64Cu-Liposome B, a transmission scan is acquired using a low-dose CT scan. Patients are imaged in a supine position on a PET/CT scanner in high-sensitivity three-dimensional (3D) mode. Each patient undergoes 2-3 scan sessions at different times, as assigned upon enrollment. After the radiation dosimetry has been evaluated, the number of scans and time points is adjusted if necessary. Subsequent cycles of unlabeled Liposome B will be administered under the parent Protocol. The study schema is outlined below.
Safety data, including AEs and SAEs, is monitored on an ongoing basis by the study Investigators, the Medical Monitor, and a Sponsor representative as part of routine investigator meetings. Patients are enrolled and dosed according to protocol unless it has been determined that dose-limiting toxicities have occurred in any of the first 6-10 patients. Once 6-10 patients have enrolled, the dosimetry data is reviewed to determine if the dose of 64Cu-Liposome B should be adjusted. Any decision or recommendations made during the Investigator meetings is documented in the meeting minutes.
The plasma pharmacokinetic (PK) analyses are performed at the specified times described in Table 2 and Table 3. Blood plasma samples (˜5 mL) is collected and analyzed for unlabeled Liposome B (Table 2). Blood samples taken after 64Cu-Liposome B administration are analyzed for radioactivity using a gamma counter (Table 3). The actual time of blood collection must be documented in the respective electronic case report form, and any deviations outside of the time limits must be commented upon. The scheduled blood sampling times are used for the PK analysis; however, any deviations outside the limits (real times) are relevant and the data sets are then adjusted for the PK evaluations and the real times are used.
64Cu-Liposome B dosing
64Cu-Liposome B (within 1 hr)
64Cu-Liposome B dosing
On the day of treatment with 64Cu-Liposome B, transmission scans are acquired using a low-dose CT scan. After administration of the 64Cu-Liposome B, patients are imaged in a supine position on a PET/CT scanner in high-sensitivity mode. Patients are assigned in an alternating fashion to either early or late scan groups at the time of enrollment, to ensure data are gathered across various time points. Each patient undergoes 2-3 scan sessions at different times as described in Table 4 below. Vital signs are measured and recorded prior to and at the end of each PET/CT scanning procedure.
64Cu-Liposome B
64Cu-Liposome B
64Cu-Liposome B
64Liposome B
64Cu-Liposome B
aEach patient is assigned to a scan group at the time of enrollment.
bThe 2nd scan is optional for Scan Group 1 and the 3rd scan is optional for Scan Group 2. Once the dosimetry has been determined, the scan time points are adjusted
The labeling of liposomes with 64Cu is performed using a novel, gradient-loadable chelator named 4-DEAP-ATSC. 4-DEAP-ATSC was derived from ATSM, a copper (Cu) chelator. 4-DEAP-ATSC tightly binds Cu and, by virtue of its amphipathic nature, is able to carry the Cu across liposomal membranes. The manufacturing of, e.g., 64Cu-loaded HER2-targeted liposomal doxorubicin (Liposome B) involves the generation of a trans-liposomal membrane pH gradient that is used to load doxorubicin into the acidic interior of the liposomes. Following manufacturing, there is a residual gradient remaining that can be used to load 4-DEAP-ATSC (and its complex with Cu) into HER2-targeted liposomal doxorubicin. Once inside the liposomes, 4-DEAP-ATSC is believed to become protonated, which then restricts its ability to cross the liposomal membrane, resulting in entrapment of 64Cu in the interior of the liposome. Loading of copper into liposomes is described in detail in, e.g, co-pending patent application PCT/US13/37033.
64Cu:4-DEAP-ATSC has been successfully loaded into liposomal formulations that contain chemotherapeutic agents via the residual chemical gradient. Examples of such liposomal formulations include the HER2-targeted doxorubicin-loaded Liposome B, the irinotecan-loaded Liposome C, as well as the commercially available doxorubicin-loaded Doxil®. 64Cu:4-DEAP-ATSC with chelation efficiency >90% was mixed with varying amounts of Liposome B, Liposome C, or Doxil®. The mixture was then incubated in a water bath at 65° C. for 10 minutes and the loading procedure was subsequently quenched in an ice water bath. Using size exclusion chromatography, it was determined that more than 90% of 64Cu:4-DEAP-ATSC can be loaded into Liposome B (Table 5), Liposome C (Table 6), and Doxil® (Table 7) below.
64Cu-loaded Liposome B
64Cu
64Cu-loaded Liposome C
64Cu
64Cu-loaded Doxil ®
64Cu
64Cu was shown to be effectively retained in the liposome after incubation of 64Cu:4-DEAP-ATSC-loaded liposome (Liposome A) in human plasma for 48 hours (
The stability of the 64Cu-Liposome B was evaluated in vitro by incubation of 64Cu-Liposome B in human plasma at 37° C. for up to 48 hours. Size exclusion chromatography was then performed to separate liposomal 64Cu from free 64Cu, and radioactivity was quantified by gamma counter, shown in
Naïve CD-1 mice were injected with 64Cu-Liposome B, free 64Cu or 64Cu:4-DEAP-ATSC complex. Plasma samples were collected via saphenous vein puncture at designated time points. The 64Cu and doxorubicin contents in the plasma were analyzed via gamma-counting or HPLC, respectively. All data are decay-corrected to the injection time. B, the ratio of 64Cu to doxorubicin was calculated from the 64Cu-Liposome B data in A.
The pharmacokinetics of 64Cu-Liposome B was evaluated in non-tumor bearing CD-1 mice, and was assessed by measuring both 64Cu and doxorubicin in plasma samples, as shown in
A biodistribution study was performed in BT-474-M3 xenograft tumor-bearing mice to determine the correlation between 64Cu levels and doxorubicin levels in the tumor and other tissues following dosing with 64Cu-Liposome B. Mice (n=4) were dosed with 3 mg/kg of 64Cu-Liposome B by tail vein injection. Twenty-four hours post-injection, mice were perfused with 20 mL phosphate-buffered saline and tissues harvested. 64Cu content was measured by gamma-counter and doxorubicin content measured by HPLC, correcting for extraction efficiency. 64Cu data are decay-corrected to the time of injection. *p<0.01. Similar values of 64Cu and doxorubicin were measured in the tumor, as shown in
Preclinical studies have examined the effect of liposome targeting on total tumor deposition. These studies have shown that the targeting of PEGylated liposomes to the HER2 receptor on tumors did not affect its pharmacokinetics or overall tumor deposition compared to an untargeted liposome. Kirpotin et al labeled liposomes with 67Ga and showed similar tumor deposition % injected dose per gram (% i.d./g) for a HER2-targeted liposome and a corresponding untargeted liposome (Cancer Research (66)6732 (2006). Similar results were obtained by comparing tumor deposition by HER2-targeted Liposome B and untargeted liposomes (disclosed in co-pending Patent Application Serial No. PCT/US2011/064496) in an NCI-N87 (ATCC® #CRL-5822™) gastric carcinoma mouse xenograft model, as well as in BT474-M3 breast carcinoma mouse xenograft model in which the two liposome formulations only result in difference in tumor cell uptake (
PET/CT imaging was performed in BT-474-M3 tumor bearing mice injected intravenously with 64Cu-Liposome B. 64Cu-Liposome B accumulated mainly in the liver and spleen, as well as in circulation as a result of the long-circulating characteristics of the disclosed liposomes (
The dosimetry of 64Cu-Liposome B at the organ level was studied in the mouse using standard methods and predicted human radiation absorbed doses to the kidneys, liver and spleen of 0.083 mGy/MBq (0.307 (rad/mCi)), 0.069 (0.256) and 0.06 (0.220), respectively. At the whole organ level, it is predicted that the kidney will be the dose-limiting organ.
The proposed starting radiation dose of 64Cu-Liposome B for humans is 400 MBq (with a range of 320-440 MBq). The radiation dose may be adjusted after obtaining improved estimates of dosimetry in humans. Based on preclinical dosimetry estimates in mice, the predicted radiation absorbed doses to the kidneys, liver and spleen are 33.2, 27.6 and 24 mGy, respectively. These values are consistent with radiation absorbed doses observed in other clinical studies with 64Cu-labeled agents and with radiolabeled liposomes.
PET/CT imaging was performed on human cancer patients after administration of 64Cu-Liposome B at a dose of approximately 400 MBq. Radiation dosimetry from 11 patients was estimated to result in radiation absorbed doses to the kidneys, liver, and spleen at 8.0, 46.4, and 59.6 mGy. 400 MBq of administered 64Cu-Liposome B was able to provide adequate PET image quality for quantification assessment from <3 h to at least 48 h post-injection. As can be seen from the images in
Endnotes
While the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations including such departures from the present disclosure that come within known or customary practice within the art. Any combination or combinations of each of the embodiments disclosed in the dependent claims is contemplated within the scope of this disclosure. The disclosure of each and every U.S., international, or other patent or patent application or publication referred to herein is hereby incorporated herein by reference in its entirety for all purposes.
This application is a 35 U.S.C. §371(c) United States national phase application of PCT/US2014/062007, filed Oct. 23, 2014, which claims the benefit of and priority to U.S. Provisional Patent Application No. 61/894,854, filed Oct. 23, 2013. The entire contents of the foregoing applications are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/62007 | 10/23/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61894854 | Oct 2013 | US |
