WO1999026535A1

WO1999026535A1 - Reagent for tumor therapy and/or imaging

Info

Publication number: WO1999026535A1
Application number: PCT/US1998/024663
Authority: WO
Inventors: David Platt
Original assignee: David Platt
Priority date: 1997-11-20
Filing date: 1998-11-19
Publication date: 1999-06-03
Also published as: CN1299249A; IL136210A0; AU1464399A; IL136210A; EP1032304A1; CA2310841A1; JP2001523693A

Abstract

A reagent for treating or imaging a cancerous tumor in vivo comprises a carbohydrate having a paramagnetic atom bonded thereto. The carbohydrate is capable of binding or penetrating a cancerous cell. When the reagent is used for imaging, gadolinium comprises one preferred paramagnetic atom. When the reagent is used for applying thermal energy to a tumor, the paramagnetic atom preferably comprises iron. The reagent may be disposed in a carrier, and the carrier may comprise liposomes. The reagent may be used in combination with other diagnostic or therapeutic materials. One preferred material comprises gadolinium glucoside.

Description

REAGENT FOR TTIMQR THERAPY AND/OR IMAGTNG

Related Application

This patent application claims priority of provisional patent application Serial No. 60/067,081 filed November 20, 1997. Field of the Invention

This invention relates generally to materials for imaging or treating tumors and to methods for their use. More specifically, this invention relates to carbohydrate based materials which have a paramagnetic atom bonded thereto, and which are selectively taken up by tumors. Background of the Tnvention

Magnetic resonance imaging (MRI) has become an important non- invasive, medical technique over the past decade. The technique provides a mapped distribution of target nuclei.

MRI uses a strong direct current magnetic field, in conjunction with tunable gradient magnetic fields to spatially control the locations at which the net sum magnetic field reaches a preselected value. As the magnetic bias fields are varied spatially, a series of radio frequency (RF) pulses are applied. When the RF energy is at a resonance frequency of sample atoms, of a particular species and surroundings, those sample atomic nuclei absorb the RF energy and are excited to a higher spin state. The excited spin state then decays to a lower energy state of excitation, this decay often is accompanied emission of an RF pulse, this pulse is often referred to as a "spin echo." The resonance frequency of a nucleus and its resulting spin echo signal depend on a number of factors including mass, density, dipole moment, relaxation frequency, as well as the chemical bonding and electrostatic potential of its surroundings. Instrument parameters such as pulse sequence, frequency, etc. also dictate the MRI imaging signal. Due to their abundance and favorable relaxation properties protons in either aqueous or lipid environments are preferred nuclei for MRI imaging.

In order to enhance the contrast between tissues within an organism, contrast reagents are often introduced into a patient's body. Typically, contrast agents include paramagnetic metal ions such as magenese, gadolinium and iron. The large dipoles associated with paramagnetic ions, as compared to protons, perturbs the proton's relaxation time, T as a function of distance between the ion and the proton, and hence provide a strong differential signal which can be used for imaging.

Difficulties associated with the use of paramagnetic metal ion contrast agents for MRI exist. The free metal ions are generally quite toxic. In order to lessen toxicity, the metal ions have in the past typically been chelated with small organic molecules or polymers. However, chelation tends to decrease the interactive effects of the metal ions, especially at the higher RFs. Currently available chelated metal ions do not possess satisfactory cell membrane transport properties, nor biodistribution to tissues of particular interest, such as tumors. The contrast reagents of the instant invention allow for MRI imaging and monitoring of tumors that are smaller than is currently possible by computed axial tomography (CAT). Thus, tumors are detected earlier and without the introduction of radioactive tracers, thus enhancing the possibility of successful treatment.

Magnetic fields have also been shown to be a means to treat malignant tissues by inducing hyperthermia. The selective excitement of metal ions using RF radiation offers the prospect of killing cells selectively incorporating a metal ion with only minor or reversible damage to surrounding cells. Previous efforts have been hampered by difficulties in targeting the metal ion to specific cells and generating localized hyperthermia.

Brief Description of the Invention There is disclosed herein a reagent for a magnetic resonance imaging of a cancerous tumor in vivo. The reagent comprises a carbohydrate, which is capable of binding to, or penetrating, a cancerous cell. The carbohydrate has a paramagnetic atom bonded thereto, and further includes a carrier suitable for delivery of the carbohydrate to the cancerous cell. In one preferred embodiment, the paramagnetic atom comprises gadolinium. In some embodiments, the carrier may comprise liposomes, which encapsulate the carbohydrate. In other embodiments, the carbohydrate is D-glucose, and the gadolinium atom is bonded to the 2' position thereof. In other embodiments, the carbohydrate may be a glucose isomer, and the gadolinium atom may be bonded to either the 2' or 3' position. In another preferred embodiment, the reagent comprises gadolinium glucoside.

In another aspect of the present invention there is provided a method for treating a tumor cell which comprises delivering to the cell a carbohydrate capable of binding to, or penetrating, the cell. The carbohydrate has a paramagnetic atom bonded thereto. The carbohydrate material is allowed to bind or penetrate the cell, and the cell is then exposed to a magnetic field having a sufficient flux and frequency to cause resonance in the paramagnetic atom so that a Curie temperature of greater than about 60°C is generated within the tumor cell. In one embodiment, the paramagnetic atom may comprise iron, gadolinium or manganese. In accord with the present invention there is also provided a reagent for the treatment of a tumor cell. The reagent comprises a carbohydrate selected from the group consisting of monosaccharides, disaccharides and polysaccharides. The carbohydrate has an iron atom bonded thereto.

Brief Description of the Drawing Figure 1 is a structural formula for gadolinium glucoside, which is one preferred material for the practice of the present invention. Detailed Description of the Invention The present invention overcomes many of the disadvantages associated with existing contrast reagents and hyperthermic agents, including reduced toxicity and increased selectivity of tumor uptake. The present invention manipulates the ability of cells, and especially tumor cells to accumulate carbohydrates from the bloodstream. The use of carbohydrates and in particular monosaccharides as ligands to stabilize paramagnetic metal ions is the basis of the tumor therapy and imaging methodologies detailed herein.

Carbohydrates are important metabolites in cell function. As cell metabolism increases, the cellular uptake of carbohydrates similarly increases. Thus, a growing tumor mass accumulates a greater per cell percentage of available carbohydrates than will surrounding, non-replicating tissue.

In order to change the nuclear spin characteristics and electronic density of an abnormal cell mass within a body tissue, it is necessary to selectively deliver paramagnetic metal ions to the abnormal region. The delivery of the paramagnetic metal ions makes the abnormal cell mass amenable to MRI imaging and hyperthermia mediated treatments. Suitable paramagnetic metal ions operative in the instant invention include, but are not limited to, carbohydrates containing transition metals, lanthanide and actinide elements, and any of the suitable paramagnetic ions thereof. Illustrative of the ions of such elements are: Cu(II),

Cr(III), Co(II), Dy(III), Er(II), Eu(III), Fe(II), Fe(III), Gd(III), Mn(II), Ni(II), and Yb(III). Preferably, the ions are Fe(III), Gd(III), and Mn(II).

Metallated-carbohydrates that are operative in the instant invention are limited only by the requirement that they be capable of binding or penetrating a tumor cell. To this end, suitable biocompatible compounds that are operative in the instant invention illustratively include: pentose monosaccharides, such as ribose, arabinose, xylose, lyxose; hexoses, such as alose, altrose, glucose, gulose, mannose, idose, galactose, fructose, and talose; disaccharides such as sucrose, maltose, cellubiose and lactose; polysaccharides such as cellulose and agarose; the pyranoses thereof; the furanoses thereof; the glycosides thereof; the sugar alcohols thereof; and the deoxy sugars thereof. Preferably, the D enantiomers of carbohydrates are utilized, for reasons including cellular recognition and biocompatibility. More preferably, the carbohydrate is D-glucose. A dative or covalent bond between the metal ion and the carbohydrate allows for the delivery of the metal ion to either a cell surface or interior. The metal ion is alternatively bound to the carbohydrate through an alcoholic oxygen linkage, a hemiacetal, or a linkage to the carbon backbone of the carbohydrate. Preferably, the metal ion is secured to the carbohydrate via an oxygen linkage. It is appreciated that additional small ligands may be bound to the metal ion, in order to satisfy the ion valency. It is further appreciated, that the substitution of a metal ion on a carbohydrate will render the resulting metallated-carbohydrate much less likely to be metabolized by cellular enzymes. The limited degradation of the metallated-carbohydrate assures that concentrations of the free metal ion and the oxides thereof remain below toxic levels.

The metal ion is directed to various bonding sites on the carbohydrate through the use of conventional carbohydrate chemistry techniques and purifications. The use of protecting groups such as acetals and ketals to effectively protect certain of the hydroxyl groups during reactions of carbohydrates is well known in the art. Through the use of reagents such as acetone and diethoxypropane under suitable reaction conditions the metallization of carbohydrates is directed to specific sites. Owing to the importance of the C-l and C-4 binding sites in polymerization and cellular recognition, it is preferred that the carbohydrates of the instant invention be metallated at the C-2 or C-3 positions. It is recognized that positions other than C-2 and C-3 are operative positions for attachment of paramagnetic metal ions when such sites offer desired membrane transport, toxicity, or therapeutic properties. In the case of glucose, the metal ion is most preferably bonded at the C-2 position, owing to stearic considerations.

As a therapeutic reagent, the metallated carbohydrates of the instant invention are delivered to a patient in doses preferably ranging from one to five mg/kg. The metallated carbohydrates of the invention may be administered, in a suitable pharmaceutical carrier by a variety of routes, including: nasally, orally, intramuscularly, subcutaneously and intravenously. In some instances, it is desirable to administer the metallated carbohydrates of the instant invention together with an adjuvant, illustratively including mineral gels such as aluminum hydroxide, surface active substances such as lecithin, naturally occurring carbohydrates, peptides, oil emulsions and the like. After allowing for the circulation and concentration of the metallated carbohydrate in the tumor cells, the magnetic properties of the metal ion are exploited for therapeutic benefit.

The reagents of the instant invention may further include conventional formulation aids, for example stabilizers, antioxidants, osmolality adjustment agents, buffers, pH adjusting agents and the like. Where the reagent is formulated for parenteral administration, a solution in a sterile physiologically acceptable medium, for example, an isotonic or somewhat hypertonic aqueous solution is preferred. For MRI examination, the reagents of the instant invention operate by affecting the relaxation time of water protons. The time interval associated with the rate of proton energy transfer to the surrounding chemical environment, Tl, is decreased by the presence of the reagents of the instant invention, thereby increasing the local image intensity of cells wherein the reagent has concentrated. Preferably, D-glucose binding gadolinium at the C-2 position is used in MRI imaging with a conventional RF pulse sequence resonate with consistent species associated with gadolinium in an aqueous environment. A further aspect of the invention involves a method of selectively killing tumor cells through localized magnetically coupled, RF induced hyperthermia. Using the predetermined coordinates of tumorous cells which have incorporated a reagent of the instant invention, the area is exposed to a magnetic field of sufficiently low frequency that dielectric heating effects are negligible, so as to protect normal surrounding tissue. The same low frequency magnetic field induces significant magnetization, coercive forces and hysteresis losses in those cells in contact with or containing the reagent of the instant invention, thereby resulting in localized hysteresis heating. Such low frequency magnetic fields are obtained from an induction coil and a generator containing apparatus. The magnetic fields are controlled in terms of amplitude and tissue penetration depth to further minimize spurious hyperthermia. The apparatus by raising the temperature of localized areas to the range of 41-44°C preferentially destroys malignant cells, while normal tissue remains viable up to a temperature of about 48 °C. Preferably, D-glucose metallated at the C-2 position by an iron atom is utilized within the instant invention as a reagent for hyperthermia treatment of tumor cells.

Combinations of paramagnetic ion metallated-carbohydrates are within the scope of the present invention. Through the simultaneous administration of carbohydrates containing different metal ions increases in the relaxivity and contrast enhancement are obtainable. By way of example, delivery of D-glucose- Fe with D-glucose-Gd takes into account the superior contrast properties of gadolinium; relative to iron, and the superior hyperthermia properties of iron relative to gadolinium. The simultaneous administration of reagents allows for iterative MRI imaging and hyperthermal treatments.

The reagents of the instant invention are also amenable to encapsulation within an enteric coating, a vesicle, or a liposome. Encapsulation serves to protect the metallated carbohydrates of the instant invention from digestive tract metabolism or to selectively enhance uptake into particular organs, such as the reticuloendothial organs: of liver, spleen and bone marrow. Preferably, gadolinium containing carbohydrate is encapsulated within a vesicle or liposome for imaging of tissues; and an iron containing carbohydrate is used for hyperthermal treatment thereof. It is appreciated that vesicles or liposomes may further contain conventional chemotherapeutic agents such as Cis-Platin for the treatment of solid tumors and inflammatory lesions. Owing to the high metabolic rate of cancerous cells, these cells preferentially endocytosize vesicles or liposomes. The increased temperature associated with hysteresis heating of a cell containing conventional chemotherapeutic agents through the simultaneous administration with the reagents of the instant invention increases the chemical activity of the conventional chemotherapeutics through the increase in cellular temperature resulting from exposing the cells to suitable low frequency magnetic fields. In this way the reagents of the instant invention enhance the efficacy of known chemotherapeutics without directly killing the cell by means of hyperthermia.

A number of materials may be synthesized in accord with the principles of the present invention. One material having particular utility comprises gadolinium glucoside. This material has the general structure shown in Figure 1 , and comprises three glucose molecules bonded to gadolinium through the oxygens thereof via coordinate bonds.

The material of Figure 1 can be synthesized by a procedure wherein 6.9 grams (0.300 mole) of sodium are completely dissolved in a mixture of 200 ml of anhydrous isopropanol and 100 ml of tetrahydrofuran, in a first step. In a second step, anhydrous (water content less than 100 ppm) gadolinium trichloride (25.0 g, 0.0948 mole) is added in portions to the sodium solution. The resulting mixture is stirred at reflux for five hours, and then allowed to stand at room temperature for 24 hours so as to produce a colorless solution of gadolinium triisopropoxide and a sodium chloride precipitate. About 100 ml of the isopropoxide solution is evacuated in a Schlenk vessel, and the residue is dissolved in 150 ml of deoxygenated, anhydrous dimethylformamide.

A 10 ml aliquot of the resulting solution is analyzed for Gd content utilizing EDTA titration in the presence of urotropin buffer, with xylenol orange as a metallochromic indicator. 90 ml of the DMF solution having a concentration of 0.232 M/l (20.88 mole) is mixed in an inert atmosphere with 11.9 g (66.05 mole) anhydrous alpha-D-glucose in 92 ml of anhydrous DMF. The reaction mixture is heated for 30 minutes at 90-95 °C and poured into 1.2 1 of dry chloroform. This produces a precipitate which is separated on a #2 Schott glass filter and washed with 100 ml of chloroform and dried in vacuum at 100° C. This produces a yield of 11.8 g of gadolinium glucoside (81% of theoretical) having the general formula illustrated in Figure 1. Other similar materials of the present invention may be synthesized by generally similar procedures, or by other synthetic procedures which will be apparent to one of skill in the art.

Various modifications of the instant invention in addition to those shown and described herein will be apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims.

Claims

Claims 1. A reagent for magnetic resonance imaging of a cancerous tumor in vivo comprising: a carbohydrate which is capable of binding to or penetrating a cancerous cell, said carbohydrate having a paramagnetic atom bonded thereto; and a carrier suitable for delivery of said carbohydrate to the cancerous cell.

2. The reagent of claim 1 wherein said paramagnetic atom is gadolinium.

3. The reagent of claim 2, wherein said carbohydrate is D-glucose and the gadolinium atom is bonded to the 2' position thereof.

4. The reagent of claim 1, wherein said carbohydrate is a glucose isomer and the paramagnetic atom is bonded to either the 2' or 3 ' position thereof.

5. The reagent of claim 1, comprising gadolinium glucoside.

6. The reagent of claim 1, wherein said carrier comprises liposomes which encapsulate said carbohydrate.

7. A method for treating a tumor cell comprising the steps of: delivering to the cell carbohydrate capable of binding to or penetrating the cell, said carbohydrate having a paramagnetic atom bonded to a carbon atom thereof; allowing for the binding or penetrating of said carbohydrate to the cell; and exposing the cell to a magnetic field of sufficient flux and frequency to cause resonance in the paramagnetic atom of said carbohydrate, so that a Curie temperature of greater than about 60┬░ Celsius is generated within the tumor cell.

8. The method of claim 7, wherein said paramagnetic atom is selected from the group consisting of Cu, Cr, Co, Dy, Er, Eu, Fe, Gd, Mn, Ni and Yb.

9. The method of claim 7, wherein said paramagnetic atom is selected from the group consisting of Fe, Gd and Mn.

10. The method of claim 7, wherein said carbohydrate is D-glucose and the paramagnetic atom is iron and is bonded to the 2' position thereof.

11. The method of claim 7, wherein said carbohydrate is a glucose isomer and the paramagnetic atom is bonded to either the 2' or 3 ' position thereof.

12. A reagent for the treatment of a tumor cell, said reagent comprising a carbohydrate selected from the group consisting of: monosaccharides, disaccharides and polysaccharides, said carbohydrate having an iron atom bonded thereto.