WO2001072203A2 - Therapy of proliferative disorders by direct irradiation of cell nuclei with tritiated nuclear targeting agents - Google Patents
Therapy of proliferative disorders by direct irradiation of cell nuclei with tritiated nuclear targeting agents Download PDFInfo
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- WO2001072203A2 WO2001072203A2 PCT/US2001/008446 US0108446W WO0172203A2 WO 2001072203 A2 WO2001072203 A2 WO 2001072203A2 US 0108446 W US0108446 W US 0108446W WO 0172203 A2 WO0172203 A2 WO 0172203A2
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- nuclear targeting
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0491—Sugars, nucleosides, nucleotides, oligonucleotides, nucleic acids, e.g. DNA, RNA, nucleic acid aptamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/0493—Steroids, e.g. cholesterol, testosterone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
- A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
- A61K51/1234—Liposomes
Definitions
- This invention relates to the field of proliferative disease therapy with low- energy radionuclides, in particular the direct irradiation of tumor cell nuclei with tritiated nucleic acid precursors.
- Proliferative disorders are characterized by the uncontrolled growth of cells of certain tissue type or types, and can be classified as cancerous or non-cancerous.
- Cancerous proliferative disorders are characterized by the uncontrolled growth of cells with a malignant phenotype, meaning that the cells evade both the normal controls on cell growth and position.
- the cells not only form cancerous lesions ⁇ e.g., tumors or neoplasms) but can invade underlying tissue or migrate to other areas of the body and establish cancerous lesions there.
- metastasis The process of malignant cell migration in cancerous proliferative disorders.
- Non-cancerous proliferative disorders are characterized by the uncontrolled growth of cells with a benign phenotype, meaning that the cells evade only the normal controls on growth, but cannot metastasize.
- CAD coronary artery disease
- coronary angioplasty is a non-surgical procedure designed to restore the patency of blocked or partially blocked coronary arteries. Typically, this procedure is carried out with a balloon catheter. Coronary angioplasty results in successful revascularization in more than 90% of coronary artery disease patients. More than 300,000 coronary angioplasty procedures were performed in the United States in 1990. However, the major limitation of coronary angioplasty is a 30-40% restenosis rate which occurs in the first six months following the procedure.
- vascular smooth muscle cell (VSMC) proliferation has been identified as playing an important role in the development of atherosclerosis and restenosis following coronary angioplasty.
- the presence of VSMCs has been confirmed in both types of lesions, and is due primarily to a change from a contractile to a synthetic phenotype in VSMCs. This phenotypic change is associated with VSMC proliferation, migration from media to intima, and the synthesis of extracellular matrix, all of which results in neointimal formation (narrowing of the artery).
- vascular restenosis represents an acute response to balloon injury culminating in a significant renarrowing by neointimal formation of an initially patent vessel in the course of a few months.
- brachytherapeutic stents For example, Albiero et al. reported high (43% to 50%) restenosis rates and high need for target vessel revascularization at four to six months in patients with radioactive stents.
- Proliferative disorders may be treated with ionizing radiation.
- Ionizing radiation is cytotoxic because it disrupts DNA either by direct impact on a component of the molecule or by generating free-radical intermediates which cause chemical damage to the DNA (Hall EJ, Radiobiology for the Radiologist (4 th ed.), J.B. Lippincott Co., Philadelphia 1994, pp. 39-40).
- When sufficient radiation- induced DNA damage accumulates in a cell the cell dies (Carrano AV (1973) "Chromosome Aberrations and Radiation-Induced Cell Death," Mutat. Res 17: 355-366).
- the effect of ionizing radiation on other cell components is negligible in terms of inducing cell death.
- the cytoreductive effects of radiation therapy for proliferative disorders is dependent on how much of the radiation reaches the proliferating cell's nucleus.
- the volume of a tissue consists primarily of extracellular space and cytoplasm. Therefore, radiation applied externally as a therapy for proliferative disorders is largely ineffective because the incident particles deposit their energy in structures outside the nucleus of the proliferative cells. To ensure that a sufficient amount of radiation reaches the proliferative cell nuclei, clinicians must use high doses of externally applied radiation. These high radiation doses can cause damage to surrounding normal tissue.
- brachytherapy The direct application of a therapeutic radiation source is called "brachytherapy.”
- Brachytherapy maximizes the dose absorbed by the proliferative tissue and reduces the radiation damage of the surrounding normal tissue.
- brachytherapy techniques include the implantation of radionuclide-containing sources, e.g., I25 I or 103 Pd "seeds" for treatment of prostate cancer.
- Brachytherapy is also used in the treatment of restenosis after the removal of vascular occlusions.
- Current treatment of restenoses include the temporary or permanent placement of a device containing a radioisotope source at the site of restenosis.
- U.S. Pat. No. 5,059,166 to Fischell et al. discloses a stent where the radioisotope source is contained either in the surface coating of the stent or in the metal alloy that forms the stent.
- U.S. Pat. No. 5,199,929 to Dake et al. discloses a catheter with a radioisotope source permanently attached to the distal end.
- US Pat. No. 5,899,882 to Waksman discloses a closed-end lumen catheter that contains strontium-90.
- brachytherapy techniques still allow the majority of the radiation dose to be absorbed by non-nuclear structures in the proliferative cells.
- brachytherapy techniques still allow the majority of the radiation dose to be absorbed by non-nuclear structures in the proliferative cells.
- brachytherapy techniques still allow the majority of the radiation dose to be absorbed by non-nuclear structures in the proliferative cells.
- brachytherapy techniques still
- Leclerc et al in US patent 5,821 ,354, discloses the delivery of high-energy radionuclides to tumor cell nuclei with short stretches of radiolabeled complementary DNA.
- particles emitted by the high-energy radionuclides generally have a penetration distance in tissue of several millimeters to several centimeters.
- a cell typically has a cytoplasmic diameter of about 20-40 microns, and nuclear diameter of 1-2 microns.
- Most of the emitted particles in the method described by Leclerc et al will escape the cell nucleus to expend their energy in the cytoplasm or extracellular space.
- High energy radionuclides delivered to cell nuclei for example in tissue characteristic of proliferative disorders, will therefore expend their energy largely outside the nucleus.
- a therapy system for proliferative disorders utilizing a radionuclide of lower energy, so that the emitted particle has limited penetration within the cell.
- the particles emitted by the radionuclide would have an average energy low enough that virtually none would escape the cell nucleus, but still possess sufficient energy to cause cell death.
- Tritium is a hydrogen isotope having one proton and two neutrons (atomic weight: approx. 3) that emits a low-energy beta particle.
- the average energy of the emitted beta particle is approximately 0.0055 MeV (Gregory DP and Landsman DA. (1958) "Average Decay Energy of Tritium.” Phys. Rev. 109: 2091-2097), which corresponds to an average penetration in tissue of less than one micron (Caro LG (1962) "High Resolution Autoradiography. II. The Problem of Resolution.” J. Cell. Biol. 15: 189-199).
- tritium has a physical half-life of approximately 12.3 years, it has an effective biological half-life of approximately 12 days (Caro, supra).
- beta particles emitted from tritium are considered too weak to cause significant DNA damage (Straus et al. "The Uptake, Excretion, and Radiation Hazards of Tritiated Thymidine in Humans," Cancer Res. 37: 610-618).
- Tritium has been used as a molecular tag in a wide range of in-vivo studies. Tritium has also been used to measure cell proliferation (Meyer JS et al. (1976) "Tritiated Thymidine Labeling Index of Benign and Malignant Human Breast Epithelium.” J. Surg. Oncol. 8: 165-181 ; Denekamp et al. (1973) "In Vitro and In Vivo Labeling of Animal Tumors With Tritiated Thymidine.” Cell. Tissue Kinet. 6: 217-227) or patterns of cell division in normal and tumor tissue (Post et al. (1977) "The Proliferative Patterns of Human Breast Cancer Cells In Vivo.” Cancer 39: 1500-1507; Young et al.
- tritium delivered directly to the cell nuclei of tissues associated with proliferative disorders by the compounds and methods of the present invention is effective in killing cells associated with proliferative disorders.
- the present invention thus provides methods of treating proliferative disorders utilizing the low-energy radionuclide tritium, which is delivered directly to proliferative tissue cell nuclei.
- target tissue tissue associated with a proliferative disorder
- target cells cells of tissue associated with a proliferative disorder
- target tissue and target cells may also be described with the name of the particular disorder or tissue; i.e. "tumor cells” or "restenotic tissue.”
- tritiated nuclear targeting agent Delivery of tritium to target cell nuclei is accomplished by associating tritium with an agent that specifically targets the nucleus of a target cell.
- Agents that specifically target a target cell nucleus are called “nuclear targeting agents.”
- Nuclear targeting agents associated with tritium radionuclides are called “tritiated nuclear targeting agents.”
- tritiated nuclear targeting agent and “agent” are used interchangeably.
- a method of treating a proliferative disorder comprising the steps of:
- tissue associated with a proliferative disorder wherein said tissue comprises target cells having DNA-containing nuclei; b) administering an effective amount of a tritiated nuclear targeting agent to said subject so that the target cells are exposed to the tritiated nuclear targeting agent;
- the tritiated nuclear targeting agent causes target cell death.
- the tritiated nuclear targeting agent is a steroid hormone.
- the agent is an oligonucleobase.
- the agent is a DNA precursor molecule.
- the agent is administered by direct application to the target tissue.
- Such direct application includes the delivery of the agent by a medical device such as a catheter.
- the agent is administered by direct injection into the target tissue.
- the agent is administered systemically to the subject.
- the agent is administered by repeated systemic injections, repeated direct applications to the target tissue, or repeated injections into the target tissue.
- the agent is administered in a sustained dose, for example by sustained systemic or subcutaneous infusion over a prolonged period of time.
- the tritiated nuclear targeting agent is associated with a structure that serves to protect the agent from degradation or clearance by the body.
- the structure can also serve to direct the agent to the target cell.
- the structure is a liposome.
- the liposome is modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems.
- the liposome carries targeting groups that direct the liposome to the target cells. Further embodiments include modified and unmodified micelles and microcapsules associated with a tritiated nuclear targeting agent.
- a pharmaceutical formulation for treating proliferative disorders comprising a tritiated nuclear targeting agent, wherein the pharmaceutical formulation is at least sterile and pyrogen free.
- Fig. 1 shows the in vitro cell survival in percent for three different cancer cell lines (4047 rat colon cancer, and BT-20 human breast cancer and MCF-7 human breast cancer) incubated with tritiated water and tritiated thymidine.
- cancer cell lines 4047 rat colon cancer, and BT-20 human breast cancer and MCF-7 human breast cancer
- Fig. 2 shows the results of the direct injection of 0.4 ⁇ Ci of 3 H-thymidine into human BT-20 breast cancer tumors grown on nude mice.
- R the ratio of initial tumor size to final rumor size
- Twenty animals are represented, comprising 10 animals in each of two groups. Animals are paired together roughly by tumor size at time of treatment.
- the present invention provides methods of treating proliferative disorders in a subject by direct delivery of tritium to the nuclei of target cells.
- a subject can be any animal suffering from a proliferative disorder, including birds, fish, and mammals.
- the expression "animal” includes human being. It is preferred that the subject is a mammal, for example a rodent ⁇ e.g. ; mouse, rat, rabbit, guinea pig, etc.) or a human being. Most preferably, the subject is a human being.
- a proliferative disorder is characterized by the uncontrolled growth of cells of certain tissue type or types.
- Such disorders can be classified as cancerous or non-cancerous, depending largely on whether cells associated with the proliferative disorder can evade the normal controls on cell growth and position (cancerous), or whether the cells have evaded only the controls on growth (non-cancerous). Both forms of proliferative disorders may be treated according to the present invention.
- Cancerous proliferative disorders are often associated with the growth of tumors.
- Tumors associated with cancerous proliferative disorders include, but are not limited to: breast, prostate, ovarian, lung, colorectal, brain (i.e, glioma) and renal tumors.
- Cancerous proliferative disorders may also cause the uncontrolled growth of diffuse malignant cell populations, such as in the leukemias.
- Non-cancerous proliferative disorders include, but are not limited to, the following: hemangiomatosis in the newborn, secondary progressive multiple sclerosis, chronic progressive myelodegenerative disease, neurofibromatosis, ganglioneuromatosis, keloid formation, Pagets Disease of the bone, uterine and breast fibrocystic disease, Peronies and Duputren's fibrosis, cirrhosis, atherosclerosis and vascular restenosis.
- CAD coronary artery disease
- VSMCs vascular smooth muscle cells
- the present methods, compounds and formulations are well suited to controlling non-cancerous proliferative diseases such as VSMC proliferation by direct delivery of tritium to the nuclei of VSMCs.
- the present methods, compounds and formulations are also well suited to controlling the proliferation of cancerous proliferative diseases, e.g., by direct delivery of tritium to the nuclei of tumor cells.
- tritium is a hydrogen isotope having one proton and two neutrons that emits a low-energy beta particle. It has been found that tritium located in the nuclei of target cells emits beta particles that do not escape the cell nucleus, but possess sufficient energy to cause cell death.
- Direct delivery of the tritium to a target cell nucleus is accomplished by associated atoms of that isotope with a nuclear targeting agent.
- the nuclear targeting agent associates with or is incorporated into the target cell DNA.
- nuclear targeting agents include, for example, steroid hormones, oligonucleobases and nucleic acid precursors.
- the steroid hormones are small, hydrophobic molecules derived from cholesterol. If administered to the bloodstream, steroid hormones are transported to target cells by binding reversibly to specific carrier proteins in the blood. Steroid hormones are released from the carrier proteins near a target cell, and diffuse through the target cell membrane to bind reversibly to steroid hormone receptor proteins in the target cell cytosol.
- the cytosolic hormone/receptor complex has an affinity for DNA that causes these complexes to accumulate in the cell nucleus.
- Steroid hormones can also be applied directly to target cells, e.g., by administration via catheter or other placement device, direct injection or subcutaneous infusion near the target tissue site.
- Steroid hormones applied directly to target cells are taken up by the tumor cells and transported to the target cell nucleus as described above.
- Steroid hormones useful in the present invention include, for example, cortisol, estradiol, testosterone, progesterone, tamoxifen and their analogs and derivatives. It is understood that, as used herein, "steroid hormone” includes both agonists and antagonists of naturally occurring steroid hormones.
- Preferred steroid hormones include those that bind to estrogen receptors expressed in target cells (for example deriving from breast or uterine tissue), and those that bind to testosterone receptors expressed in target cells (for example deriving from prostate or testicular tissue).
- oligonucleobase is a polymer of nucleobases that can hybridize to complementary sequences of target cell DNA. By hybridization, it is meant that complementary oligonucleobases join with the target cell DNA by Watson-Crick base-pairing, i.e., by forming a duplex. It is preferred that oligonucleobases are administered directly to target cells, e.g., by direct injection or subcutaneous infusion near the target tissue. It is particularly preferred that oligonucleobases be associated with a structure such as a liposome, to protect the oligonucleobase from degradation in the body.
- nucleobases comprising an oligonucleobase comprise purine or pyrimidine bases (or a derivative or analog thereof) which are covalently linked to a sugar moiety.
- Nucleobases include, for example, nucleotides, nucleosides, and nucleotoids.
- Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2'-deoxyriboside, and have a linkage to other nucleobases that do not contain a phosphorous atom.
- Nucleotoids are pentosefuranosyl-containing nucleobases having linkages that contain a phosphorous atom; e.g., phosphorothioates, phosphoroamidates and methylphosphonates.
- Nucleotides are pentosefuranosyl-containing nucleobases that are linked by phosphodiester groups.
- Nucleobases are either of the ribo- or deoxyribo-type. Ribo-type nucleobases contain pentosefuranosyl moieties wherein the 2' carbon is substituted with a hydroxy, alkyl, or halogen.
- Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases, and include nucleobases which do not contain a pentosefuranosyl moiety.
- a preferred oligonucleobase comprises ribonucleotides joined by phosphodiester bonds (RNA).
- RNA phosphodiester bonds
- a particularly preferred oligonucleobase comprises deoxyribonucleotides joined by phosphodiester bonds (DNA). Oligonucleobases may be nuclease resistant.
- oligonucleobases of the invention may contain modified internucleobase linkages, such as phosphorothioate linkages.
- oligonucleobase includes unmodified oligomers of oligonucleobases as well as oligomers wherein one or more purine or pyrimidine moieties, sugar moieties or internucleobase linkages is chemically modified.
- nuclease resistance is conferred on oligonucleobases of the invention by providing nucl ease-resistant internucleobase linkages. Many such linkages are known in the art, e.g., phosphorothioate: Zon and Geiser, Anti-Cancer Drug Design.
- Additional nucl ease-resistant linkages include phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, alkylphos- photriester such as methyl- and ethylphosphotriester, carbonate such as carboxymethyl ester, carbamate, morpholino carbamate, 3'-thioformacetal, silyl such as dialkyl(C,-C 6 )- or diphenylsilyl, sulfamate ester, and the like.
- ribo-type nucleobases of the present oligonucleobases can be nuclease-resistant.
- Suitable nuclease resistant ribo-type nucleobases can be selected from the group consisting of 2AX-nucleosides, 2'AX-nucleotoids and 2'AR-nucleotides, where: A is oxygen or a halogen (preferably fluorine, chlorine or bromine);
- X is hydrogen or C, .6 alkyl; R is C
- Preferred nuclease resistant ribo-type nucleobases are 2'-O methyl ribo-type nucleobases, and particularly preferred are 2'-O methyl ribonucleotides.
- Oligonucleobases can be any size polymer which is directed to the cell nucleus. Preferably, any oligonucleobase of at least 2 nucleobases to about 5000 nucleobases may be used in the present invention. More preferably, the oligonucleobase is about 10 to about 40 nucleobases in length.
- oligonucleobases are known to those skilled in the art.
- the nucleobase sequence of the oligonucleobase targeting agent is not important therapeutically; the sequence is only used to direct the oligonucleobase to the target cell DNA.
- the oligonucleobase is complementary to the coding strand of a specific target cell DNA sequence. Oligonucleobases complementary to the coding strand of the specific target cell DNA sequence will not hybridize with extranuclear RNA derived from that same sequence.
- preferred oligonucleobase sequences are those of proto- oncogenes and oncogenes or fragments thereof.
- Proto-oncogenes are normal cellular genes, the alteration of which engenders a transforming allele or "oncogene.” Damage to one or more proto- oncogenes has been found in a variety of human malignancies. A large number and variety of human tumors contain consistent point mutations in proto- oncogenes. Chromosomal translocations also contribute to tumorigenesis by activating proto-oncogenes to oncogenes, e.g. , the translocation of c-abl to the BCR locus to form the hybrid oncogene bcr-abl which has been correlated with the occurrence of Philadelphia chromosome-positive leukemias.
- proto-oncogenes carry abnormally amplified domains of DNA that can include proto-oncogenes and magnifytheir expression (Alitalo& Schwab, Adv. Cancer Res.47, 235-282, 1986).
- the potential of proto-oncogenes to participate in tumorigenesis arises from the fact that their protein products are relays in the biochemical circuitry that governs the phenotype of vertebrate cells (Bishop, Cell 64, 235-248, 1991).
- useful proto-oncogene sequences include, but are in no way limited to, sequences derived from the proto-oncogenes c-myb and c-myc.
- One such sequence is the c-myc sequence CAC GTT GAG GGG CAT (SEQ ID NO: 1 ).
- Other c-myc sequences useful in the present invention included SEQ ID NOS: 2 - 5, 13 and 14 from WO 94/15646 of Thomas Jefferson University, the entire disclosure of which is incorporated by reference in its entirety.
- WO 99/34814 of Temple University discloses sequences of the interferon responsive transcript (IRT-1) gene, which are also useful in the present invention.
- the IRT-1 gene is active in proliferating vascular smooth muscles cells.
- the disclosure of WO 99/34814 is herein incorporated by reference in its entirety.
- Nucleic aid precursors are substances which are incorporated into DNA or
- Nucleic acid precursors include any of the above-mentioned nucleobases which can be incorporated into DNA or RNA by the cell, especially the nucleotide bases.
- Preferred nucleotide bases include adenosine, cytidine, guanosine, thymidine, and uridine, and analogs and derivatives thereof.
- Thymidine is preferred because this nucleotide base is only incorporated into DNA. Thus, virtually all tritiated thymidine administered to a subject would deliver the tritium radionuclide to the tumor cell nucleus.
- Nucleic acid precursors also include the component molecules of nucleobases, and include, for example, purines or pyrimidines.
- Purines and pyrimidines include, for example, adenine, guanine, cytosine, thymine and uracil, and their derivatives or analogs.
- Nucleic acid precursors are actively taken up by proliferating cells, such as tumor cells, and transported to the cell nucleus for incorporation into DNA or RNA. It is preferred that nucleic acid precursors of the present invention be incorporated into the DNA.
- a nuclear targeting agent can be directly labeled with tritium by the substitution of tritium for a hydrogen on the nuclear targeting agent.
- a nuclear targeting agent can be synthesized in the presence of tritium so that the tritium is incorporated into the atomic structure of the nuclear targeting agent.
- any substance associated with at least one tritium nucleus is called "tritiated.”
- a tritiated substance is also designated in this specification by use of the prefix " 3 H-", for example as in “ 3 H- thymidine.”
- tritiated nuclear targeting agents useful in the present invention are commercially available.
- tritiated nucleic acid precursors are available from Amersham Pharmacia Biotech, Inc., 800 Centennial Ave., P.O. Box 1327, Piscataway, NJ 08855 USA, such as 3 H-adenosine, 3 H-guanosine, 3 H-cytidine, 3 H- thymidine and 3 H-uridine.
- Tritiated steroid hormones are also available from Amersham Pharmacia Biotech, including 3 H-testosterone, 3 H-oestradiol, 3 H- progesterone, 3 H-corticosterone, 3 H-dexamethasone and 3 H-tamoxifen.
- a list of available tritiated steroid hormones is given in a table entitled "Selection Guide - Steroid Receptors" on page 84 of the 1999 Amersham Pharmacia Biotech catalog, which table is herein incorporated by reference.
- tritiated nucleobases and/or tritiated nucleic acid precursors can be administered directly to subjects as a nuclear targeting agent, or can be used to synthesize oligonucleobases which are then administered to subjects as a nuclear targeting agents. Techniques for synthesizing oligonucleobases from nucleobases or nucleic acid precursors are well-known to those skilled in the art.
- the tritiated nuclear targeting agents of the invention can be administered by any method designed to expose target cells to the agent so that the agent is taken up by the target cells and transported to the cell nucleus. Parenteral administration is preferred.
- suitable parenteral administration methods include intravascular administration (e.g.
- intravenous bolus injection intravenous infusion
- intra-arterial bolus injection intra-arterial infusion and catheter instillation into the vasculature
- peri- and intra-target tissue injection ⁇ e.g., peri-tumoral and intra- tumoral injection
- direct application to the target tissue, for example by a catheter or other placement device.
- Suitable parenteral methods also include subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps). It is preferred that subcutaneous injections or infusions be given in the area near the target tissue, particularly if the target tissue is on or near the skin.
- the administration of the agent is by injection or direct application, the injection can be in a single dose or in multiple doses.
- the infusion can be a single sustained dose over a prolonged period of time or multiple infusions. Injection of the agent into the target tissue is preferred. Multiple injections of the agent into the target tissue are particularly preferred.
- Nucleic acid precursors are generally taken up only by actively growing cells. Thus, to ensure that substantially all target cells take up and incorporate tritiated nucleic acid precursors, it is preferred that the target cells be chronically exposed to this particular type of nuclear targeting agent. Chronic exposure is defined as exposure for a period of time during which the majority of target cells enter the S-phase of the cell cycle (i.e., are actively growing). For agents that do not depend on the cell cycle for uptake, i.e. the steroid hormones and oligonucleobases, it is not critical that tumor cells pass through S-phase. It is particularly preferred that target tissue from cancerous proliferative disorders, for example tumors, be chronically exposed to tritiated nucleic acid precursors.
- the period of time during which the majority of target cells enter the S- phase of the cell cycle can be readily determined by one of ordinary skill in the art ⁇ see e.g., Tubiana M and Malaise E (1976), Cancer Treatment Reports 60: 1887- 1895).
- majority of target cells means about 80%, preferably about 90%, and more preferably about 95% or greater of a target cell population has entered the S-phase.
- a population of target cells includes discreet groupings ⁇ e.g., tumors, specific restenotic areas) or the entire number of target cells in a subject.
- One way to ensure chronic exposure of target cells to 3 H-nucleic acid precursors is by peri- or intra-target tissue injection of a high dose of the agent.
- a high dose of 3 H-thymidine can be injected into a tumor (intra-tumoral injection) or around or near the tumor site (peri-tumoral injection). Injection of a high dose in this manner ensures that effective levels of agent persist in the target tissue for several days.
- An alternative strategy for chronic exposure involves multiple injections or infusions of agent over time to maintain a sufficient concentration for days or weeks, so that actively cycling target cells will incorporate sufficient agent to cause target cell death.
- Another strategy for chronic exposure involves the sustained infusion of agent for a period of time during which the majority of target cells enter the S-phase of the cell cycle.
- the sustained infusion can be, for example, intravascular or subcutaneous.
- Tritiated nuclear targeting agents can also be associated with a structure, for example a liposome, micelle or microcapsule, to facilitate the direction of the agent to target cells.
- the structure can also serve to protect the agent from degradation or clearance by the body.
- Tritiated nuclear targeting agents associated with structures are administered as described above. It is preferred that the amount of agent administered to a subject suffering from a proliferative disorder is expressed in terms of the tritium activity. Tritium activity is typically given in terms of microCuries ( ⁇ Ci), but of course can be expressed in any units of radioactivity relevant to disintegration of tritium nuclei. Those skilled in the art are familiar with techniques for measuring the activity of tritium in tritium-labeled compounds.
- Tritiated nuclear targeting agents are administered to a subject suffering from a proliferative disorder in any amount effective to cause target cell death.
- an amount of tritiated nuclear targeting agent effective to cause target cell death is any amount which causes a measurable decrease in viable target cells in the subject.
- An effective amount of tritiated nuclear targeting agent is referred to herein as a "dose.”
- Techniques to determine the number of viable target cells in a subject include biochemical and histological techniques for detecting cell death or necrotic tissue. Such biochemical and histological techniques are familiar to those skilled in the art.
- Target cell death can also be inferred from a reduction in target tissue size upon treatment with tritiated nuclear targeting agents.
- Reduction in target tissue size can be ascertained visually or by diagnostic imaging methods including, for example, X-ray, magnetic resonance imaging, ultrasound, and scintigriphy. Diagnostic imaging methods used to ascertain reduction in target tissue size can be employed with or without contrast agents. Such diagnostic imaging methods (both with and without contrast agents) are well known to those of skill in the art.
- Reduction in target tissue size can also be ascertained by physical means.
- physical means include, for example, palpation of the target tissue mass, or measurement of the target tissue mass at different times during treatment with a measuring instrument such as a caliper.
- a dose of tritiated nuclear targeting agent can be based on the approximate or estimated mass of the target tissue to be treated.
- Techniques for approximating or estimating target tissue mass are well known in the art.
- target tissue ⁇ e.g., tumor can be estimated by calculating the approximate tissue volume and considering one gram of mass equivalent to one cubic centimeter of tissue volume.
- a dose of tritiated nuclear targeting agent based on target tissue mass can be at least about 1 ⁇ Ci/gram of tumor, and is preferably between about 1- 1000 ⁇ Ci/gram of tumor. More preferably, the dose is at least about 60 ⁇ Ci/gram of target tissue. Particularly preferably, the dose is at least about 100 ⁇ Ci/gram of target tissue. It is preferred that doses of tritiated nuclear targeting agent based on target tissue mass be injected directly into the target tissue. However, doses based on target tissue mass can also be administered systemically (e.g., intravascularly, subcutaneously, intramuscularly or intraperitoneally) or by direct application to the target tissue.
- systemically e.g., intravascularly, subcutaneously, intramuscularly or intraperitoneally
- a dose of tritiated nuclear targeting agent can also be based on the approximate or estimated body weight of the subject to be treated.
- body weight doses are used for systemic administrations including, for example, intravascular injections and infusions, subcutaneous depositions or infusions, and intramuscular or intraperitoneal administrations.
- single injection intravascular doses in humans can range from about 5 - 3000 ⁇ Ci/kg of body weight, and are preferably between about 700 - 1000 ⁇ Ci/kg of body weight. Such doses are more preferably greater than about 1000 ⁇ Ci/kg of body weight.
- the same doses for single intravascular injections can also be used for multiple intravascular injections, although for multiple injections the dose may also be lower.
- multiple intravascular injection doses in humans are preferably greater than about 250 ⁇ Ci/kg body weight, and particularly preferably greater than about 500 ⁇ Ci/kg body weight.
- Single sustained infusion intravascular doses can be the same as those used for single and multiple intravascular injections, but may also be lower.
- doses for single sustained infusions are preferably greater than about 90 ⁇ Ci/kg body weight, and more preferably are greater than about 100 ⁇ Ci/kg.
- Multiple sustained infusion intravascular doses can be the same as those used for single and multiple injection and single sustained intravascular doses, but may also be lower.
- doses for multiple sustained intravascular infusions are preferably greater than about 35 ⁇ Ci/kg, and more preferably are greater than about 50 ⁇ Ci/kg.
- Subcutaneous, intramuscular and intraperitoneal doses can be the same as for the intravascular doses but are preferably between about 200 and 1000 ⁇ Ci/kg body weight.
- a dose of tritiated nuclear targeting agent can further be based on the approximate or estimated surface area of the subject to be treated. Surface area doses for a given subject are typically expressed in terms of ⁇ Ci tritiated nuclear targeting agent square meter of surface area (m 2 ). It is preferred to base doses of a tritiated nuclear targeting agent on the surface area of a subject, because better inter-species dose comparisons can be made. Also, doses based on surface area allow doses to be determined for human adults and children without further adjustment.
- Table 1 provides approximate surface area to weight ratios for various species.
- the surface area to weight ratio can be used to convert body weight doses expressed in terms of ⁇ Ci/kg to surface area doses.
- the surface area to weight ratio is also used to calculate the conversion factors found in Table 2.
- the conversion factors in Table 2 can be used to convert doses expressed in terms of ⁇ Ci/kg from one species to another.
- doses based on surface area be administered systemically, as describe above for doses based on body weight.
- surface area doses can be administered by peri- or intra-target tissue injection or by direct application to the target tissue.
- the present invention also provides pharmaceutical formulations comprising the tritiated nuclear targeting reagents.
- Pharmaceutical formulations of the present invention are characterized as being at least sterile and pyrogen-free.
- pharmaceutical formulations include formulations for human and veterinary use.
- compositions include agents mixed with a physiologically acceptable carrier medium to form solutions, suspensions or dispersions.
- Preferred physiologically acceptable carrier media are water or normal saline.
- Pharmaceutical formulations can also include conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include, for example, stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
- Suitable additives include, for example, physiologically biocompatible buffers (e.g., fromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTP A-bisamide), or, optionally, additions ⁇ e.g., 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
- physiologically biocompatible buffers e.g., fromethamine hydrochloride
- additions e.g., 0.01 to 10 mole percent
- chelants such as, for example, DTPA or DTPA-bisamide
- calcium chelate complexes as for example calcium DTPA, CaNaDTP A-bisamide
- additions ⁇ e.g., 1 to 50 mole percent for example, calcium
- the tritiated nuclear targeting agent is carried by a structure.
- structures useful for carrying an agent include liposomes, micelles, and microcapsules.
- the structure can serve to more effectively direct the agent to target cells.
- the structure can also serve to protect the agent from degradation or clearance by the body.
- Structures useful in the present invention can be modified to affect their biodistribution, for example by having opsonization inhibition moieties or targeting groups bound to the surface of the structure.
- a structure has both opsonization inhibition moieties and targeting groups bound to its surface.
- the structure is a liposome.
- liposome refers to a generally spherical entity formulated from amphiphilic compounds which is characterized by the presence of at least one internal void.
- Preferred amphiphilic compounds are lipids.
- the amphiphilic compounds may be in the form of a one or more monolayers or bilayers. Where a liposome comprises more than one mono- or bilayer, the mono- or bilayers are generally concentric.
- the liposomes described herein include such entities commonly referred to as liposomes, bubbles, microbubbles, microspheres, vesicles and the like.
- the amphiphilic compounds may be used to form a unilamellar liposome (comprised of one monolayer or bilayer), an oligolamellar liposome (comprised of about two or about three monolayers or bilayers) or a multilamellar liposome (comprised of more than about three monolayers or bilayers).
- liposome also refers to multivesicular liposomes, which are liposomes comprising multiple non-concentric voids. For examples of multivesicular liposomes and methods of their preparation, see US 5,993,850 of Sankaram et al.
- Multivesicular liposomes are especially useful for sustained or timed release of the tritiated nuclear targeting agents.
- the internal voids of the liposomes may be filled with a liquid, including, for example, an aqueous liquid, a gas, a gaseous precursor, and/or a solid or solute material.
- tritiated nuclear targeting agents are carried by liposomes.
- carrier by liposomes means the agent is embedded within the wall of the liposome, encapsulated in the liposome or attached to the liposome.
- attachment to means that the agent is associated in some manner to the inside and/or the outside wall of the liposome, such as through a covalent or ionic bond, or other means of chemical or electrochemical linkage or interaction.
- encapsulated in means that the agent is located in the internal liposome void.
- embedded within means the agent is within the liposome wall.
- the agent can be positioned variably, such as, for example, entrapped within the internal void of the liposome, incorporated onto the internal/external surfaces of the liposome and/or enmeshed within the liposome structure itself.
- liposomes carrying tritiated nuclear targeting agents are within the skill of those in the art.
- the agent is lipophilic or amphiphilic
- efficient embedding within the liposome wall can be achieved by preparing a mixture of liposome-forming material and the agent, e.g. , in a dried film, and hydrating the mixture. This procedure will form liposomes with lipophilic or amphiphilic agent embedded predominantly in the wall of the liposomes.
- Useful techniques for incorporating amphiphilic compounds into liposome membranes are disclosed, e.g. , in Grant et al., Magn. Res. Med., 11 :236-243 (1989); Kabalka et al., Magn. Res. Med., 8:89-95 (1988); and Hnatowich et al., J. Nucl. Med., 22:810-816 (1981), the disclosures of which are herein incorporated by reference in their entirety.
- the amphiphilic material used in forming the liposomes can be conjugated with the agent prior to liposome formation.
- Liposomes formed with amphiphilic material conjugated with the agent will carry the agent attached to both the inner and outer liposome surfaces.
- the agent can also be conjugated to the liposome after it has been formed, which will result in the agent being carried only on the outside surface of the liposome.
- Passive loading may also be employed for preparing liposomes with encapsulated hydrophilic trititated nuclear targeting agents.
- the hydrophilic agent is usually dissolved in the aqueous medium used to hydrate a film of liposome-forming material.
- the aqueous medium containing the film is sonicated to form liposomes encapsulating the agent dissolved in the aqueous solution.
- encapsulation efficiencies of between about 5-20% are typically obtained, with the remainder of the agent being in the bulk aqueous phase.
- An additional processing step for removing non-encapsulated agent is therefore usually required.
- water-soluble materials are encapsulated in liposomes; see e.g., Bangham et al. J. Mol. Biol., 13:238-252 (1965); D. Papahadjopoulos and N. Miller. Biochim. Biophys.
- This loading method typically involves an agent having an ionizable amine group which is loaded by adding it to a suspension of liposomes prepared to have a lower inside to higher outside ion gradient, for example a pH gradient.
- the liposomes of the present invention deliver the carried agent at or near the target cells.
- the liposomes can fuse with a target cell, be taken up by a target cell, or release their contents outside a target cell. Regardless, the agent will be delivered to the target cell nucleus.
- Liposomes are particularly effective at delivering agents to tumor cells. Suitable doses of tritiated nuclear targeting agents carried by liposomes are as disclosed above.
- the liposomes of the present invention can be any size.
- liposomes useful in the present invention can have a diameter of about 0.1 to about 2,000 microns.
- Preferred liposomes are those having a diameter of about 100 to 1 ,000 microns, others having a diameter of about 10 to 100 microns, and still others having a diameter of about 1 to 100 microns.
- a preparation of liposomes typically has a distribution of sizes.
- a liposome preparation in a range of about 0.1 to 10 microns with a size distribution within less than about a 20% standard deviation of the average diameter is preferred.
- a liposome preparation in a range of about 0.1 to 10 microns with a size distribution within about 10% of the average diameter is still more preferred.
- Delivery of an agent may be enhanced by the external application of energy to the delivery site, for example by the application of ultrasound to the tumor site.
- the ultrasound energy will either enhance the fusion of the liposomes with the target cell, or cause the liposomes to break, thus exposing target cells to the trititated nuclear targeting agent.
- Modified liposomes are "modified liposomes.” Modified liposomes carry components on their outer surface that affect biodistribution, for example opsonization inhibiting moieties or targeting groups with a specific affinity for a target cell. A modified liposome can comprise opsonization inhibiting moieties and targeting groups.
- Opsonization-inhibiting moieties are typically large hydrophilic polymers that are bound to the liposome membrane.
- an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
- These opsonization inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by themacrophage-monocyte system (MMS) and reticuloendothelial system (RES), e.g., as described in U.S. Pat. No.
- Liposomes modified with opsonization inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
- Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature.
- target tissue characterized by such microvasculature defects for example solid tumors, will efficiently accumulate these liposomes; see Gabizon, et al, P.N.A.S., USA, 18:6949-53 (1988).
- the reduced uptake by the RES lowers the toxicity of stealth liposomes carrying tritiated nuclear targeting agent by preventing significant accumulation in the liver and spleen.
- liposomes that are modified with opsonization inhibition moieties deliver the agent to tumor cells, whereupon the agent will be transported to the target cell nucleus.
- Opsonization inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons.
- Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives, e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GM,.
- PEG polyethylene glycol
- PPG polypropylene glycol
- synthetic polymers such as polyacrylamide or poly N-vinyl
- Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
- the opsonization inhibiting polymer maybe a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
- the opsonization inhibiting polymers may also be natural polysaccharides containing amino acids or carboxylic acids, e.g.
- the opsonization inhibiting polymer can be bound to the liposome membrane by any one of numerous well-known techniques.
- an N- hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane.
- a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH 3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60 °C.
- a targeting group is bound to the outer surface of the liposome.
- a targeting group is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
- the carbohydrate portion of the liposome membrane is oxidized, e.g., by exposure to sodium metaperiodate to yield aldehyde groups, which are highly reactive and will bind the target group to the membrane.
- the target group can be linked to a lipid-soluble anchor, and the anchor is then intercalated into the liposome membrane.
- Suitable targeting groups include compounds selected or designed to target a target cell; for example polyclonal or monoclonal antibodies, fragments of antibodies, chimeric antibodies, an enzyme or enzyme substrate, a lectin, a saccharide ligand of a lectin, and small molecule ligands.
- a preferred small molecule ligand is the E. coli heat stable enterotoxin ST, which specifically targets cells of colorectal origin, such as metastasized colorectal cancer cells.
- the E. coli heat stable enterotoxin ST is described in Waldman, US patent 5,518,888, the disclosure of which is herein incorporated by reference in its entirety.
- Preferred antibodies include antibodies to tumor-associated antigens. Specific examples include, for example, B72.3 antibodies (described in U.S. Pat. Nos.
- ING-1 and other genetically engineered antibodies which are described in WO-A-90/02569, B174 antibodies (developed at Biomira, Inc. of Edmonton, Canada), which recognize squamous cell carcinomas, B43 antibodies which are reactive with certain lymphomas and leukemias, and other antibodies which may be of particular interest. All references cited in this paragraph are herein incorporated by reference in their entirety.
- Suitable targeting groups include antibodies directed to cell surface antigens of neovascular endothelium ⁇ e.g., anti-CD 105 antibodies) or ligands with affinity for cell surface receptors of neovascular endothelium.
- vascular smooth muscle cells are a major component of restenotic lesions seen in patients undergoing treatment for coronary artery disease.
- Other useful antibodies include those directed to the product of the IRT-1 gene, as described in WO 99/34814 supra.
- compositions of liposomes carrying tritiated nuclear targeting agents may be formulated as described above, but may contain additional emulsifiers and/or viscosity modifiers designed to keep the liposomes in suspension.
- Suitable viscosity modifiers include, for example, carrageenan, cellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, magnesium aluminum silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, propylene glycol alginate, silicon dioxide, sodium alginate, tragacanth, and xanthan gum.
- Suitable emulsifiers include, for example, poloxamers and their derivatives, polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitan mono-laurate, sorbitan mono-oleate, sorbitan mono-palmitate, sorbitan monostearate, stearic acid, and emulsifying wax.
- Pharmaceutical formulations according to the present invention comprising liposomes can be prepared in a manner fully within the skill of the art.
- the structure is a micelle.
- a micelle is formed by the spontaneous organization of amphiphilic materials in solution into particles with a hydrophobic core and a hydrophilic corona.
- a micelle has little or no internal void. Therefore, micelles generally only carry tritiated nuclear targeting agents as associated with the surface of the micelle or as part of the micellar structure.
- a micelle can also carry agent entrapped within the micellar structure.
- Tritiated nuclear targeting agents carried by micelles are preferably amphiphilic or are linked to a lipophilic anchor such that they are incorporated into the micellar structure.
- hydrophobic agents can be carried within a micelle's hydrophilic core.
- a hydrophilic agent can be carried within a micelle's hydrophilic corona.
- Micelles can also be modified to affect their biodistribution, for example by association with opsonization inhibitors or targeting groups.
- a surface modifier can be associated with a micelle by attachment ⁇ e.g., covalent or ionic bond, or other means of chemical or electrochemical linkage or interaction) to the micellar surface, or by incorporation of the surface modifier into the micellar structure.
- a long-chain hydrophilic polymer or a targeting group can be conjugated to a lipophilic anchor and assembled into the micellar structure along with the other amphiphilic material. Examples of opsonization inhibiting moieties and targeting groups useful for modifying micelles are as described for liposomes above.
- Micelles are particularly useful in delivering the agents to tumors residing in the lymphatic system, especially when administered by subcutaneous injection or infusion.
- Micelles modified with opsonization inhibiting moieties are useful in delivering agent to solid tumors when administered intravascularly, as they will accumulate in tissue fed by porous or leaky microvasculature ⁇ see Gabizon et al, supra).
- a preparation of micelles typically has a distribution of sizes.
- a micelle preparation a size distribution within less than about a 20% standard deviation of the average diameter is preferred.
- a micelle preparation in a with a size distribution within about 10% of the average diameter is still more preferred.
- the micelles of the invention are between about 5 nanometers and about 50 nanometers in diameter.
- Pharmaceutical formulations of micelles can be prepared as described above for liposomes, using techniques well known to those of skill in the art.
- the structure is a microcapsule.
- Microcapsules are fine dispersions of solids or droplets of liquid onto which a thin film coating has been applied.
- the average diameter of microcapsules may vary from one micron to several hundred microns depending on the materials used and their method of production.
- the coating of microcapsules comprises an non-amphiphilic organic polymer, including for example amines (e.g., mono-, di-, tri-, terra-, and higher amines, mixtures thereof, and mixtures thereof with monoamines), alginic acid, arabic acid, cellulose sulfate, carboxymethyl cellulose, carrageenans, chondroitin sulfate, heparin, polyacrylic acid, polyoxyethylene cross-linked polyacrylic acid, polyphosphazine, glycolic acid esters of polyphosphazine, lactic acid esters of polyphosphazine, hyaluronic acid, polygalacturonic acid, polyphenylene sulfonic acid, and polyvinylcarboxylic acid, polymerizable aldehydes, derivatives thereof and mixtures thereof.
- amines e.g., mono-, di-, tri-, terra-, and higher amines, mixtures thereof, and mixtures thereof with monoamines
- microcapsules suitable for use in the present invention are found in US 5,686,1 13 of Speaker et al, US 5,501,863 of Rossling et al. and US 5,993,374 of Kick, the disclosures of which are incorporated herein by reference in their entirety.
- Microcapsules can optionally be modified to affect their biodistribution, for example to alter the microcapsule biodistribution by addition of targeting groups or opsonization-inhibiting moieties.
- Microcapsules modified in this way have similar characteristics and advantages as modified liposomes and micelles.
- Opsonization inhibiting moieties and targeting groups useful in surface- modifying microcapsules are as described for liposomes and micelles above. Techniques for preparing and modifying microcapsules are well known in the art (see, for example, US 5,686,1 13, US 5,501,863, and US 5,993,374, supra).
- microcapsules of the invention can be any size. Particularly preferred microcapsules are those which are small enough to pass through the pulmonary capillary bed; i.e. those with an diameter of approximately 8 microns. However, microcapsules useful in the present invention can have a diameter of about 0.1 to about 2,000 microns. Preferred microcapsules are those having a diameter of about 100 to 1,000 microns, others having a diameter of about 10 to 100 microns, and still others having a diameter of about 1 to 100 microns.
- a preparation of microcapsules typically has a distribution of sizes. A microcapsule preparation in a range of about 0.1 to 10 microns with a size distribution within less than about a 20% standard deviation of the average diameter is preferred. A microcapsule preparation in a range of about 0.1 to 10 microns with a size distribution within about 10% of the average diameter is still more preferred.
- compositions of microcapsules can be prepared as described above for liposomes and micelles, using techniques well known to those of skill in the art (see, for example, US 5,501,863, supra).
- the tritiated nuclear targeting agents are used to treat rumors.
- the agents are used to treat restenotic lesions occurring in patients being treated for coronary artery disease.
- Clinicians have used mechanical devices (e.g., stent, atherectomy, laser, rotablator, etc.) to physically remove restenotic plaques, including the proliferating vascular smooth muscle cells (VSMCs), which occur after coronary angioplasty.
- VSMCs vascular smooth muscle cells
- intravascularly administered agents are carried in a structure modified with targeting groups which direct the structure to VSMCs.
- the agent can be carried in a liposome, micelle or microcapsule which is optionally modified ⁇ e.g., with an opsonization inhibition moiety and/or with a targeting group comprising an anti- VSMC antibody). Determination of dosage amounts and dosage regimens is as described above.
- Direct application of agent to the restenotic lesion can be accomplished by any device capable of reaching the lesion, such as a catheter designed to deliver a therapeutic drug solution.
- a catheter designed to deliver a therapeutic drug solution for example, U.S. Pat. No. 5,087,244 to Wolinsky et al. discloses a catheter having a perforated inflatable balloon for expressing drug to the vascular wall.
- U.S. Pat. No. 5,021,044 to Sharkawy discloses an infusion catheter having a plurality of effluent flow ports along its outer wall, each having a successively larger diameter in the distal direction.
- each effluent flow port through the wall of the catheter is placed in fluid communication with a fluid source by a unique flow passageway extending throughout the length of and within the wall of the catheter body.
- US Pat. No. 6,027,487 to Crocker discloses a low profile infusion catheter for delivering thrombolytic drugs to a preselected site, with improved flexibility characteristics and relatively uniform delivery over a preselected axial length.
- Other useful catheters include the perforated or porous balloon catheters as described in Wolinsky et al. (1990) J. Am. Coll. Cardiol. 15: 475-481 and US Pat. No. 5,993,374 to Kick. The disclosures of all references cited in this paragraph are herein incorporated by reference in their entirety. Use of such catheters to deliver the present agent to restenotic lesions is well known to those of skill in the art.
- Example 1 Killing of tumor cells in vitro with 3 H-thymidine.
- FBS FBS The cells were divided into 4 groups as follows:
- Group 1 control Group 2: incubated with 3 H-water (New England Nuclear Life Sciences
- Group 3 incubated in H-thymidine (specific activity 89.9 Ci/mmol; New England Nuclear Life Sciences Products, Boston, MA) at final concentrations of 0.1 , 0.3, 0.5, 0.7, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0 ⁇ Ci/ml media
- Group 4 incubated in unlabelled thymidine at concentrations 1, 2, 3, and 4 mM (so that concentration of thymidine was identical to that of the labeled thymidine in Group 3).
- Tumor cells 5xl0 6
- the mice were weighed and their physical condition was visually monitored every other day.
- Approximate tumor volumes were measured every other day using calipers to measure the height (H), width (W), and length (L) of each tumor.
- One group received a dose of 0.4 ⁇ Ci of 3 H-thymidine (specific activity 89.9 Ci/mmol; New England Nuclear Life Sciences Products, Boston, MA) in 20 ⁇ l of saline injected directly into the tumor.
- the other group received 20 ⁇ l of saline injected directly into the tumor. Tumor growth was monitored and recorded daily for a period of 3 weeks.
- mice Over the course of the experiment, no systemic ill effects were observed in either the experimental or control groups of mice. All mice continued to gain weight and there were no apparent behavioral aberrations. Tumor measurements were normalized for variations in initial volume by calculating the results as:
- R is the ratio of initial tumor volume (SI) to tumor volume three weeks after treatment (S2).
- SI initial tumor volume
- S2 tumor volume three weeks after treatment
- BT-20 breast cancer tumors are grown in the flanks of nude mice as in Example 2 except that the tumors are grown to an approximate volume of 500 mm 3 .
- the mice are randomly divided into two groups.
- the first group receives an intravenous injection of 10 ⁇ Ci of 3 H-thymidine diluted in 20 ⁇ l of normal saline for an approximate dose of 0.5 ⁇ Ci per gram of body mass.
- the second group receives 10 ⁇ Ci of 3 H-thymidine diluted in 20 ⁇ l of normal saline injected into the center of the tumor.
- Ten animals from each group are sacrificed at 1 hour, 24 hours, 48 hours, 96 hours, and 120 hours following administration of 3 H-thymidine.
- Levels of 3 H-thymidine are determined for each animal in blood, bone marrow, liver, spleen, kidney and small intestines since these represent the major sites of 3 H-thymidine incorporation following systemic administration.
- each rumor is divided into 8 sections by bisecting the lesion three times. The level of 3 H-thymidine is determined separately for each section.
- Each sample of normal or tumor tissue is weighed and the level of tritium determined using methodology described by Larson et al. (1981) J. Nucl. Med. 22(10) 869-874, the entire disclosure of which is herein incorporated by reference.
- Each sample is oxidized using an automatic sample oxidizer. Tracer content is determined using a liquid scintillation counter.
- the Larson et al. method may overestimate the amount of 3 H- thymidine present in the cell nuclei since some of the tritium presumably detaches from the thymidine and some of the tritiated thymidine may be metabolized.
- additional analyses are performed on the blood and tumor samples from two of the animals in each group. These samples are lysed and fractionated using HPLC. The presence of tritium in each fraction is determined using a liquid scintillation counter. Any sample that has tritium present is further analyzed with reverse phase HPLC and compared to the retention times for water, thymidine, and thymine (the major thymidine metabolite) standards.
- 3 H-thymidine dose will be retained in the tumor at one hour.
- the 3 H-thymidine concentration will be reduced by about 50% by approximately 120 hours. Only about 10% of the administered dose is expected to be found in the bone marrow and intestine. Significant concentrations of 3 H-thymidine are expected to be found in the blood and liver at 1 hour and 24 hours.
- This experiment is designed to measure tumor growth, systemic toxicity, concentration and distribution of intratumorally injected 3 H-thymidine in tumor, liver, stomach, and blood in the nude mouse after 4 weeks total exposure to 3 H- thymidine.
- the relative amounts of 3 H-thymidine, thymidine, and water in blood, liver, stomach, and tumor tissue of the mice at the end of the treatment regimen are also measured.
- a total of thirty 4-6 week old Nu/Nu female nude mice (Charles River Laboratory, Wilmington, MA) are used: 10 mice as untreated controls and 20 mice subjected to the 3 H-thymidine treatment.
- 10 x 10 6 BT-20 human breast cancer cells are inoculated subcutaneously into the flanks of the mice. Animal weights are recorded daily and animals are closely monitored for signs of illness.
- a set of 6 measurements per day is made on a single tumor in the control group on one day of each week to determine the standard deviation versus volume in the volume measurements.
- the 20 mice of the treatment group are given intratumoral injections of 2 ⁇ Ci 3 H-thymidine in 20 ⁇ l of saline weekly for two weeks. Tumor growth is monitored for a total of 4 weeks, whereupon the mice are sacrificed and the tumors and other tissue are harvested. Lines are drawn along the anterior-posterior length of the harvested tumors. In addition, the top, left and right of the harvested tumors is marked. The tumors are quartered, and the tip from each quarter that was in the tumor center is removed and combined into one sample. Thus there are 5 tumor samples - one from each quarter and 1 from the tumor center. The issue samples collected from the stomach, liver and blood are also processed.
- tissue solubilizer Soluene-50, Packard Instruments.
- a portion of the sample in is placed in Hionic-Fluor LSC cocktail and the radioactivity counted in a scintillation counter.
- TLC Thin Layer Chromatography
- the concentration of 3 H-thymidine, 3 H-thymine, and 3 H- water in all tissue samples is determined by correcting the count rates for quenching and converting counts to concentration of 3 H-thymidine from a standard.
- 3 H-thymidine concentration is considered separately for each of the 5 pieces and also as two different groups; mean of the center sample versus the mean of the outer (4 quarters), and mean of the of all 5 samples for each tumor.
- the data is blocked by replicate to minimize inherent variability in the measurements deriving from the scintillation counting technique and the heterogeneity of the tumors. The same scintillation procedure is followed for determining amounts of
- Tumor growth is also analyzed by calculating the percent reduction in tumor volume over the course of the experiment.
- the error in this measurement is determined from the measured standard deviation and the standard formula for calculating the error for a percent difference.
- Systemic toxicity is not directly measured, but any signs of illness are recorded. Weight is also monitored and plotted daily. Slight variations in the above described experimental design are not expected to significantly change the anticipated results.
- Intratumoral injection is expected to significantly increase the achievable concentration of 3 H-thymidine in tumors and minimize the systemic 3 H- thymidine concentration. This is because most of the 3 H-thymidine will likely be retained in the tumor and the slow "leaching" of 3 H-thymidine into the blood would allow the agent to be metabolized into 3 H-thymine which is not incorporated into DNA, and thus has little or no cytotoxicity.
- Example 5 Dose Response Characteristics for Intratumoral and Intravenous Injections of 3 H-Thymidine.
- BT-20 human breast cancer rumors are grown in the flanks of six experimental groups of twenty nude mice as in Example 3.
- the tumors are grown to volume of approximately 500 mm 3 .
- One group of ten mice serves as untreated controls.
- the average weight of the experimental and control mice is 20 grams. In each of the six experimental groups, ten mice receive a given
- the doses are represented in Table 3 as total amount of 3 H-thymidine given ( ⁇ Ci/20 ⁇ l saline), the equivalent dose based on approximate tumor mass ( ⁇ Ci/gram of tumor) and the equivalent dose based on approximate body weight ( ⁇ Ci/kg body wt.).
- Equivalent doses for a 60 kg human calculated from Table 2 above are given in parentheses in the "dose ⁇ Ci/kg body wt.” column.
- Table 3 Doses for administration to nude mice carrying human xenograft tumors.
- mice are monitored daily, and the tumors measured every two days for a minimum of two weeks or until tumor regrowth begins.
- the data are expressed as ratios of the tumor volume at any given time point to its pre- treatment volume. Tumor measurements are normalized for variations in initial volume by calculating the results as:
- r is the ratio of tumor volume at a given time point (S,) to initial tumor volume (S,).
- r is the inverse of ratio "R” used in Example 2 above (R is equivalent to S, / S,).
- R is equivalent to S, / S,).
- Table 4 Anticipated approximate regression of human xenograft tumors in nude mice injected systemically with the indicated dose of 3 H-thymidine.
- the dose from intratumorally injected 3 H- thymidine to the tumor and other bodily systems is dependent on biological processes.
- a pharmacokinetic model describing the transport of 3 H-thymidine in the single dose case is utilized.
- the two-compartment first-order linear pharmacokinetic model chosen describe the 3 H-thymidine transport from a single intratumoral dose is taken from Talarida, Manual of Pharmacologic Calculations with Computer Programs, Springer- Verlag, New York, NY, 1987, and is represented schematically below:
- the two compartments are the tumor and the blood plasma, respectively.
- the model includes three key parameters T 0 , k, and k 2 , where
- T 0 is the initial amount of 3 H-thymidine found in the tumor nuclei; and k, and k 2 are diffusion constants which describe diffusion of 3 H- thymidine out of the tumor into the plasma, and then out of the plasma, respectively.
- Equation 1 The model can be described using Equations 1 and 2:
- Equations 1 and 2 k, and k 2 are as defined above;
- T is time in a particular compartment (tumor or plasma);
- P is the amount of 3 H-thymidine in the plasma
- T is the amount of 3 H-thymidine in the tumor
- dT/dt is the change in the amount of 3 H-thymidine in the rumor over time
- dP/dt is the change in the amount of 3 H-thymidine in the plasma over time.
- BT-20 tumor cells (10 million cells in 0.2 ml PBS) are inoculated into the right flank of each mouse as in Example 4 above. The tumors are grown to volumes of 300-500 mm 3 over the course of 3-4 weeks.
- each group of 60 is divided into ten subgroups of six animals each. As the rumors have variable growth rates, the subgroups will be chosen such that the mean tumor volume and standard deviations are similar.
- 3 H-thymidine, specific activity 89.9 Ci/mmol, (New England Nuclear Life Sciences Products, Boston, MA), in 20 ⁇ l of saline is injected into the tumors on each mouse at a dose of 4 ⁇ Ci/g of tumor (group I), 16 ⁇ Ci/g of tumor (group II), and 50 ⁇ Ci/g tumor (group III).
- One subgroup of six mice from each group is sacrificed by asphyxiation at the following time points: 24 hours post-injection, and then every three days for the next 28 days (a total of 10 time points).
- the time points have been chosen according to the known bi-exponential decay governing systemic 3 H-thymidine excretion (Straus MJ et al (1977), supra).
- the first point i.e. 24 hours
- the last data point (-28 days) is roughly 3 times the long (10.8 day) half-life reported by Straus.
- the other time points are taken at an equal distribution in between.
- Levels of 3 H-thymidine are determined by liquid scintillation counting for each mouse in tumor, blood, liver, stomach, and small intestine samples. To measure the distribution and retention of 3 H-thymidine in the tumors, each rumor is divided into 8 sections by bisecting the lesion three times. The tip of each section corresponding to the tumor center is sliced off and these pieces are pooled. The level of 3 H-thymidine is determined separately for each of the 9, (i.e. 8 + center) sections for each tumor as in Example 3 above.
- the half-life for retention of 3 H-thymidine in the tumors is measured by fitting the concentration of 3 H-thymidine in the tumor samples at each time point to a theoretical exponential curve predicted by the pharmacokinetic model outlined above.
- Half-life is determined separately for three different divisions of tumor tissue: whole tumors, tumor outer pieces, and tumor centers, to check for effects that may be caused by the vascular distribution. The error for each point is the standard deviation in the measurement.
- the half-life for the three groups of six animals per subgroup and ten time periods is analyzed using two-way ANOVA.
- Reduction in tumor volume is estimated as in Example 4 for each treatment subgroup over the course of the experiment.
- the data is analyzed by calculating the percentage of rumor reduction as in Example 4.
- the error in this measurement is determined from the measured standard deviation and the standard formula for calculating the error for a ratio given above in Equation 1. Systemic toxicity is not directly measured, but is monitored as described above in Example 4.
- Example 8 Determination of a Preliminary In Vivo Dose-Response Curve and Determination of the Tumor Toxicity of Single Intratumoral Injections of Escalating Amounts of 3 H-Thymidine.
- mice Six experimental groups of 18 4-6 week old Nu/Nu female nude mice (Charles River Laboratory, Wilmington, MA) are used. In each of the six groups an additional group of 6 mice serves as an uninoculated control. Thus the total number of experimental mice is 108 and total number of uninoculated control mice is 36.
- Human BT-20 breast cancer tumors are grown in the flanks of all experimental mice as in Example 4 above. Tumors are grown to an approximate volume of 300-500 mm 3 over the course of 3-4 weeks. Each experimental group is then divided into two subgroups: one treatment subgroup of 12 mice and one untreated (but inoculated) control subgroup of six mice. This experimental setup provides 80% power assuming the same standard deviation in the groups as seen in the previous examples. Because the tumors have variable growth rates, subgroups are chosen such that the mean rumor volume and standard deviation are similar for each subgroup.
- 3 H-thymidine in normal saline is injected into the tumors of each treatment subgroup of each group as follows: amount 3 H-thymidine dose
- mice are monitored daily and the tumors measured daily by microcaliper for a minimum of 3 weeks or until tumor re-growth begins.
- Tumor volumes are calculated as in Example 2.
- Three daily tumor volumes are measured for the first group so that the error in the volume measurement can be determined as in Example 4.
- the mean volumes and standard deviations are calculated and growth rate curves are monitored during the experiment. The data are expressed as percent difference of the tumor volume at any given time point to its pre-injection volume.
- Tumor volume data is analyzed as described above in the in Example 4 by calculating percent tumor reduction and initial tumor volume. Systemic toxicity is not directly measured, but is monitored as described above in Example 4. Concentrations and distribution of 3 H-thymidine in the tumors are determined at the end of treatment as described in Example 4.
- the dose response curve which plots the mean percent tumor reduction versus dose, is calculated from the tumor volume measurements. From this curve the ED 90 , which is the minimal effective dose for eradicating tumors in 90% of the treated mice, is found.
- the above example gives a theoretical ED 90 of 16 ⁇ Ci/g tumor, which is used as the dose for the multiple administration regimen of Example 9 below.
- An intratumoral multiple dose regimen is determined using the theoretical half-life of 3 H-thymidine retention in tumors from Example 7 and the theoretical ED 90 from Example 8.
- the superposition method of Sharget and Yu, supra is used to extend the two compartment pharmacokinetic model presented in Example 6 from the single dose to the multidose case. This assumes that previous doses do not affect the pharmacokinetics of subsequent doses.
- the maximum and minimum of the multi-dose regimen can be averaged together to give an estimate of the mean concentration in the tumor during multiple injections (Sharget and Yu, supra).
- an effective multiple dose regimen is one intratumoral injection of 16 ⁇ Ci/g rumor per week, over three weeks.
- This dose regimen is tested experimentally as follows. 60 4-6 weeks old female nude mice in 2 groups of 30 mice are used.
- Each group of 30 mice is further divided into two subgroups (10 control mice and 20 treatment mice). As before, the treatment mice will be divided into subgroups that consist of similar distribution of tumor volumes.
- 10 x 10 6 BT20 human breast cancer cells are inoculated subcutaneously in flanks of the mice as in Example 4. Animal weights are recorded daily and animals are closely monitored for signs of illness.
- tumor volumes are measured with a caliper and recorded every other day, and sets of 6 measurements per day are made on a single tumor in the control group on one day of each week to determine the standard deviation versus volume in the volume measurements. Tumors are grown to 300-500 mm 3 in volume.
- mice from one treatment subgroup receive an intratumoral injection of 16 ⁇ Ci/20 ⁇ l normal saline (a dose of 32 ⁇ Ci/gram of tumor). The dose is repeated every 7 days for a total of 3 doses.
- the mice from the other treatment subgroup receive 16 ⁇ Ci 3 H- thymidine by i.v. injection (a dose of -800 ⁇ Ci/kg body wt.) once a week for three weeks. Slight variations in the above described experimental design are not expected to significantly change the anticipated results.
- mice receiving i.v. injection it is expected that the tumors will show moderate regression (r of approximately 0.8) after three doses. Mice receiving intratumoral injection will show complete and prolonged tumor regression (r of approximately zero).
- liposomes are prepared encapsulating an aqueous solution of 100 ⁇ Ci/ml 3 H-thymidine in normal saline.
- the liposomes are composed of lactosyl cerebroside, phosphatidylglycerol, phosphatidylcholine and cholesterol in molar ratios of 1 :1 :4:5.
- the liposomes so prepared are passed through a 0.4 polycarbonate membrane and suspended in saline, and are separated from non- encapsulated material by column chromatography in 135 mM sodium chloride, 10 mM sodium phosphate pH 7.4.
- the amount of encapsulated 3 H-thymidine is measured by scintillation counting.
- the liposomes thus prepared are used without further modification, or are modified as described below.
- a quantity of the liposomes prepared in (A) above are charged to an appropriate reaction vessel to which is added a stirring a solution of 20 mM sodium metaperiodate, 135 mM sodium chloride and 10 mM sodium phosphate (pH 7.4).
- the resulting mixture is allowed to stand in darkness for 90 minutes at a temperature of about 20 °C.
- Excess periodate is removed by dialysis of the reaction mixture against 250 ml of buffered saline (135 mM sodium chloride, 10 mM sodium phosphate, pH 7.4) for 2 hours.
- the product is a liposome having a surface modified by oxidation of carbohydrate hydroxyl groups to aldehyde groups.
- Various targeting groups or opsonization inhibiting moieties are conjugated to the liposome surface via these aldehyde groups.
- the cmyc oligonucleotide is synthesized with [methyl, l',2'- 3 H]thymidine 5'-triphosphate (Amersham Pharmacia Biotech, Inc., cat. # TRK576).
- DMPE dimyristoylphosphatidylethanolamine
- 5 ml of anhydrous methanol containing 2 equivalents of triethylamine and 50 mg of m-maleimidobenzoyl N-hydroxysuccinimide ester Kitagawa and Aikawa, J. Biochem. 79,233-236, 1976.
- the resulting reaction is allowed to proceed under a nitrogen gas atmosphere, at room temperature, overnight.
- the resulting reaction mixture is subjected to thin layer chromatography on Silica gel H in chloroform/methanol/water (65/25/4), which reveals quantitative conversion of the DMPE to a faster migrating product.
- Methanol is removed under reduced pressure and the products redissolved in chloroform.
- the chloroform phase is extracted twice with 1 % sodium chloride and the maleimidobenzoyl-phosphatidylethanolamine (MBPE) purified by silicic acid chromatography with chloroform/methanol (4/1) as the solvent. Following purification, thin-layer chromatography indicates a single phosphate containing spot that is ninhydrin negative.
- the MBPE is an activated phospholipid for coupling sulfhydryl containing compounds, including proteins, to the liposomes.
- the vesicles are separated from the unencapsulated tritiated nuclear targeting reagent by column chromatography in 100 mM sodium chloride-2 mM sodium phosphate (pH 6.0).
- the fraction containing the vesicles is mixed with 2 mg of antibody and 0.2 ml of a freshly prepared solution of sodium cyanoborohydride and are reacted at room temperature overnight.
- the vesicles containing the antibody on their surface are separated from non-attached antibody.
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AU2001255179A AU2001255179A1 (en) | 2000-03-24 | 2001-03-16 | Therapy of proliferative disorders by direct irradiation of cell nuclei with tritiated nuclear targeting agents |
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WO1998023299A2 (en) * | 1996-11-26 | 1998-06-04 | Angiogene Inc. | Radiolabeled dna oligonucleotide, method of preparation and therapeutic uses thereof |
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US5847104A (en) * | 1995-05-22 | 1998-12-08 | Hybridon, Inc. | Method of tritium labeling oligonucleotide |
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WO1998023299A2 (en) * | 1996-11-26 | 1998-06-04 | Angiogene Inc. | Radiolabeled dna oligonucleotide, method of preparation and therapeutic uses thereof |
US5821354A (en) * | 1996-11-26 | 1998-10-13 | Angiogene Inc. | Radiolabeled DNA oligonucleotide and method of preparation |
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