EP0433312A4

EP0433312A4 - Carrier system and method for enhancement of magnetic resonance imaging

Info

Publication number: EP0433312A4
Application number: EP19890909661
Authority: EP
Inventors: George Y. Wu; Catherine H. Wu
Original assignee: University of Connecticut
Current assignee: University of Connecticut
Priority date: 1988-08-24
Filing date: 1989-08-17
Publication date: 1991-12-04
Also published as: JPH04501560A; EP0433312A1; WO1990001900A1

Abstract

Enhanced magnetic resonance imaging contrast is provided by targeting paramagnetic material specifically to selected mammalian cell types. A carrier system for the paramagnetic material utilizes receptor mediated endocytosis to endow cell specificity and is formed by complexing the paramagnetic material to a glycoprotein-chelator conjugate. The conjugate is formed by bonding receptor-specific ligands to chelating agents through covalent bonding. The carrier not only exhibits a high degree of cell spcificity but also provides significant accumulation of the paramagnetic material within the cell to contribute to the improved enhancement of the magnetic resonance contrast.

Description

CARRIER SYSTEM AND METHOD FOR ENHANCEMENT OF MAGNETIC RESONANCE IMAGING

Background and Summary of the Invention

The present invention relates generally to contrast enhancement of magnetic resonance imaging. More particularly, it relates to a new and improved carrier system and method for the targeted delivery of paramagnetic substances of specific types of cells. Magnetic resonance imaging, MRI, relies on the response of electrons, atoms, or nuclei to certain discreet radiation frequencies as a result of spaced quantization in a magnetic field. Hydrogen nuclei or protons, when stimulated by electromagnetic radiation in the presence of a magnetic field, emit signals in the radio frequency range. The intensity of those signals is proportional to the concentration of the protons present.

In the human body, water is quantitatively the most important source of protons. Therefore, variations in the intensity of the proton signals can be used to form images that delineate differing types of body tissue. Advantageously, MRI provides a non-invasive technique for studying soft body tissue and detecting both the presence and location of tumors and the like. This technique utilizes and relies upon the specific differences in the radio frequency signals emitted by tumors and other space-occupying lesions relative to the surrounding normal tissue. Unfortunately, the signal intensity for normal liver tissue is very similar to that of tumor tissue resulting in poor contrast and, therefore, difficult detectabi1ity.

SUBSTITUTE SHEE Paramagnetic contrast agents have been used heretofore to enhance the differences between certain tissues with primary emphasis focusing on paramagnetic complexes, such as complexes of gadolinium ions with appropriate chelating agents, such as diethylenetriamine pentaacetic acid, DTPA. The effective paramagnetic substances typically form hydrates with the surrounding water molecules and result in an altered signal emission by these protons. However, the complex or chelate must possess a low dissociation constant and must be stable over long periods of time since free gadolinium ions are known to be toxic. Although the gadolinium/DTPA complex has found wide usage as an experimental tool, other complexing materials such as the acids of macrocyclic polyamines discussed by Knop et al in the Journal of Computer Assisted Tomography, Volume 2, No. 1, Jan.-Feb., 1987, pages 35-42 have been employed. Other investigators have studied the behavior of chelate complexes as conjugates of specific biological materials by covalent bonding to those materials to the chelating agent. The utilization of a conjugate employing proteins such as human serum albumin has been discussed by Schmiedl et al in Radiology, Volume 162, No. 1, Jan., 1987, pages 205-210, while conjugate with monoclonal antibodies have been investigated by Manabe et al in Biochimica et Biophysica Acta, Volume 883 (1986), pages 460-467. However, utilization of these conjugates is believed to result primarily in a surface treatment of the tissue being studied and frequently is incapable of providing a sufficient concentration of contrast agent to result in a markedly discernible differentiation between closely related specific cell types. Most, if not all, mammalian cells possess cell-surface binding sites or receptors that recognize, bind, and internalize specific biological molecules, i.e., ligands. These molecules, once recognized and bound by the receptors, can be internalized by the target cells via receptor-mediated endocytosis. Normal liver cells, hepatocytes, possess unique surface receptors that can recognize asialoglycoproteins, glycoproteins possessing exposed terminal galactose residues. Following recognition by these receptors, internalization and intracellular degradation of the asialoglycoproteins occurs. However, primary hepatocellular carcinomas, as well as other metastatic tumors in the liver lack this glycoprotein receptor.

Accordingly, it is an object of the present invention to provide a new and improved carrier system for paramagnetic contrast agents used in magnetic resonance imaging, particularly a system that can reliably deliver paramagnetic substances in a cell-specific manner to mammalian cells. Included in this object is a provision for a carrier in the form of a conjugate that exhibits a high degree of cell-specificity coupled with the ability to form stable complexes with the paramagnetic material.

Another feature of the present invention is the use of receptor-mediated endocytosis to endow cell specificity to the contrast agent carrier system. This involves the use of cell-surface receptors as naturally existing entry mechanisms for the nontoxic, cell-specific delivery of the agent. Included in this feature is the provision for a unique image enhancing complex capable of targeting the contrast agent to specific cells possessing particular receptors that recognize the carrier. These features and advantages of the present invention all directly contribute to the ef iciency and targetability to specific cell types, particularly hepatocytes, and form the basis not only for delivery in an efficient and effective manner while avoiding the disadvantages of prior contrast agent, but also provide enhanced accumulation of the contrast agent within the targeted cell and therefore a more discernible differentiation between specific cell types.

Other features and advantages will be in part obvious and in part pointed out more in detail hereinafter.

These and related advantages are achieved in accordance with the present invention by providing a new and improved delivery system and method for targeting paramagnetic contrast agents to specific cell types to achieve enhanced concentration of the contrast agent within the specific cells. The system and method utilize a conjugate of a receptor-specific ligand that is recognized by the targeted cell and a complexing agent for the paramagnetic contrast agent. The conjugate, loaded with contrast agent, is delivered to the cell where it is recognized and internalized via a receptor-mediated endocytotic mechanism.

A better understanding of the features and advantages of the present invention will be obtained from the following detailed description and from the illustrated applications of the invention. This includes the several components of the invention and the relation of one or more of such components with respect to each of the others and the process including the several steps thereof and their relationship with each of the others as well as the features, characteristics, compositions, properties, and relation of elements described and exemplified herein.

Description of a Preferred Embodiment

The enhanced results of the present invention are achieved by providing a paramagnetic contrast agent carrier that is targeted to a specific cell type based on the presence of specific receptors on the target cells. For ease of understanding and clarity of description, the invention will be described hereinafter primarily in connection with liver cells or hepatocytes. Normal hepatocytes comprise a vast majority of the cell volume of the liver and possess unique cell-surface receptors that can recognize, bind and internalize specific types of glycoproteins, namely galactose terminal (asialo-) glycoproteins. Abnormal hepatocytes, such as primary hepatocellular carcinomas, as well as other metastatic tumors in the liver lack these specific receptors. Accordingly, by targeting contrast agents to the normal hepatocytes, these cells will produce the most profound contrast enhancement effects while the tumors will remain substantially unaffected. Additionally, contrary to the cell surface coating effect achieved by other techniques, recognition of the carrier by the normal hepatocyte receptors will result in internalization and intracellular degradation thereof. This, in turn, has been found to lead to unexpectedly greater accumulation of the stable contrast agent complex within the cell. Such accumulation may account for the significantly enhanced contrast noted for these images. The targetable MRI carrier system of the present invention consists essentially of three principal components: (1) a ligand, such as a glycoprotein possessing exposed terminal galactose residues, capable of being recognized by unique receptors present on the surface of specific cells or types of cells; (2) an image enhancing paramagnetic substance, such as gadolinium, that has the ability to alter magnetically-induced proton resonance signal emission, and (3) a complexing a chelating agent such as diethylenetriamine pentaacetic acid, DPTA, capable of binding the paramagnetic material with a high degree of affinity in a complex that is relatively stable within the cell. Additionally, the complexing agent must be capable of being chemically coupled to the ligand by covalent bonding to form a conjugate that retains its receptor recognizability while binding the paramagnetic material in a stable manner.

As mentioned, this specific targeted delivery of MRI contrast agent to individual mammalian cells using a soluble carrier system is based on the fact that most if not all mammalian cells possess certain surface binding sites that recognize and internalize specific ligands. Typically these ligands are proteins having functional groups that are exposed sufficiently to be recognized by the cell receptors. The particular proteins used will vary with the particular target cell. Typically glycoproteins having certain exposed terminal carbohydrate groups are used. For specific targeting to hepatocytes, galactose-terminated or asialo-ligands formed by chemical or enzymatic desialylation are employed. These can be prepared by treating glycoproteins that possess terminal sialic acid to expose the terminal galactose residues. Although the naturally occurring serum glycoproteins are preferred, other galactose terminal forms of proteins can be used. For example, hepatocyte-targetable asialoglycoprotein ligands containing more numerous amino groups may be prepared by coupling lactose to nongalactose-bearing proteins by reductive lactosamination or by coupling of thioglycosides.

Because a variety of different receptors exist on the surfaces of various cells, cell-specific targeting to other non-hepatic cells may be possible based on the use of other ligand components i.e., mannose-6-phosphate glycoproteins for fibroblasts, intrinsic factor-Vit B₁₂ f°^r enterocytes, insulin for fat cells, and the like.

The paramagnetic material preferably consists of any of those materials currently utilized to enhance MRI. Gadolinium is currently the most effective material for this purpose. However, the paramagnetic substances may be any one of the various forms of ferric iron, manganese and other rare earth metals such as europium or dysprosium known to be effective for improving contrast of the images. It is also possible that if appropriate contrast agents for CAT scanning could be coupled to a glycoprotein, a similar enhancement in image contrast could be obtained.

The third component of the system is a chelating agent that complexes with the paramagnetic material and, at the same time, can be coupled with the receptor-specific glycoprotein. A variety of chelating agents may be employed although, as mentioned, diethylenetriamine pentaacetic acid, DTPA, is the preferred material. Other polyamine polycarboxylic acid chelating agents that have been used to complex gadolinium include 1,4,7,10-tetraazacyclododecane-N, N', N", N¹" tetraacetic acid (DOTA) ; 1,4,8,11- tetrazacyclotetradecane-N, N', N", N"'; tetraacetic acid (TETA) and 1,4,7-triazacyclononane-N, N', N", N^H (NOTA) .

In the preferred embodiment, a conjugate that has a high affinity for the paramagnetic material is prepared by chemically coupling the chelating agent directly to the asialo-ligand. Chelation of the metal ion by the conjugate rends the cytotoxic character of the gadolinium ion nontoxic and readily excretable since the resultant chelate is very stable. The amount of contrast agent bound to the carrier conjugate will vary from a molar ratio of about 5:1 to 15:1. However, larger quantities of chelating agent can be coupled to the asialoglycoprotein if, as mentioned, the glycoprotein is first covalently joined to a polycationic linker to increase the number of free a ino groups on the protein. As a result, many more molecules of the chelator and, therefore, of gadolinium, are bound than is the case without the polycation. When treated in this manner, the molar ratio of gadolinium to conjugate increased to approximately 90:1.

Preparation Of The Carrier With Complexed Contrast Acrent

To determine whether the paramagnetic material could be bound in a stable form to the conjugate, the human serum glycoprotein, orosomucoid, was desialylated to expose terminal galactose residues thereby forming asialoorosomucoid (AsOR) . In order to couple gadolinium to this protein, a chelating agent, such as DTPA, was covalently coupled to the AsOR. The conjugate was separated from excess chelator by dialysis followed by molecular sieve chromatography. Radioactive gadolinium, ¹⁵³Gd, was incubated with the conjugate in acetate buffer at pH 6 and subsequently chromatographed on a gel filtration column. A radioactive peak containing protein was assessed by gel electrophoresis followed by staining with Coomassie Blue. The gel was subsequently dried and photographic film exposed to it. A single radioactive band was obtained corresponding in mobility to asialoorosomucoid, AsOR. The gadolinium loaded conjugate was stable to heat at 80° C and to denaturation with SDS. The molar ratio of gadolinium to conjugate and to AsOR was calculated to fall in the range of 5:1 to 15:1, the molar ratio of gadolinium to DTPA being essentially 1:1.

As a control, AsOR without DTPA was mixed with ¹⁵³Gd. In the control, no radioactivity was associated with the protein after column chromatography.

Endocytosis Of Paramagnetic Complex

To determine whether the contrast agent complexed with the conjugate can be targeted to cells that possess specific receptors, the radiolabeled ¹⁵³Gd conjugate was incubated with the hepatocyte-derived cell line HepG2, receptor (+) cells, and with Morris 7777, receptor (-) cells, at 37°C in a medium containing a saturating concentration of radiolabeled conjugate. At regular time intervals, the cells were removed, washed free of the medium containing the conjugate, counted for gamma radioactivity and the amount of cell protein present was determined by Bio-Rad assay. Receptor (+) cells took up the radiolabeled conjugate at a linear rate at 37°C, reaching a high of 600 ng/10⁶ cells. Uptake of the conjugate was blocked by a competing excess of AsOR indicating that the uptake was directed by the asialoglycoprotein component of the conjugate. Uptake of radioactivity by the receptor (-) cells was barely detectable and not significantly different from background confirming that recognition of the conjugate is not non-specific; it is limited exclusively to receptor (+) cells.

Accumulation Of Paramagnetic Material

Radioactive iodine-labeled proteins reach a maximum accumulation in receptor (+) cells in approximately three hours, with an accumulation within the cell that is ten times the amount bound to the surface. For the Gd-conjugate of the present invention, uptake by the cell increased linearly with time. Maximum accumulation required twenty-five hours of incubation and was almost 100 times that bound to the surface. This indicates that the chelated Gd targeted to the receptor (+) cells appears to exit such cells far more slowly and, therefore, accumulates within those cells. The accumulation of Gd-chelate within the targeted cells offers the possibility of enhancing magnetic resonance imaging since the contrast is proportional to the concentration of the gadolinium present. No toxicity was noted in these cells even after three days of incubation. Effect of Increased Free Amino Groups

A high molecular weight polylysine was modified to form a polypeptide with exposed galactose residues and used to make an asia-polylysine-chelator conjugate recognizable by asialoglycoprotein receptors. In this case, lactose was chemically coupled to the residual amino groups of the polylysine to form the desired (asialo-) polypeptide. This resultant conjugate was shown to be capable of binding approximately 90 molecules of gadolinium per molecule of polylysine, and was effectively taken up by receptor (+) cells when administered in culture medium.

Targeted Delivery of an MRI Contrast Agent to Liver in Vivo

To evaluate delivery of Gd to hepatocytes in vivo, rats were injected with a targetable conjugate containing ¹⁵³Gd. Measurement of residual radioactivity in the blood at regular intervals revealed that the half-life of the conjugate was approximately 3 minutes. After 15 minutes the organ distribution of the injected counts was determined to be: liver, 88%; spleen, 0.1%; kidney, 1%; lungs, 0.5%. The intrahepatic Gd concentration was calculated to be .15 mM at 15 minutes. Co-injection of a 1000-fold excess of asialoorosomucoid to compete for uptake by asialoglycoprotein receptors in vivo resulted in: blood retention of 64% of the injected counts, the liver had only 4.7% after 15 minutes. These data indicate that an MRI contrast agent can be targeted specifically to liver and reach concentrations necessary for MRI contrast in vivo.

BSTITUTESHEET As will be appreciated by those skilled in the art, various modifications, adaptations, and variations of the foregoing specific disclosure can be made without departing from the teaching of the present invention.

Claims

WE CLAIM:

1. A carrier system for the targeted delivery of MRI contrast enhancement agents to specific cell types comprising a paramagnetic material and a conjugate suited for forming a complex with said paramagnetic material, said conjugate comprising a receptor-specific ligand for said cell type and a complexing agent chemically bonded to said ligand.

2. The system of claim 1 wherein said complexing agent is a chelating agent.

3. The system of claim 1 wherein the complexing agent is a polyamine polycarboxylic acid.

4. The system of claim 1 wherein the complexing agent for the paramagnetic material is selected from the group considering of diethylenetriamine pentaacetic acid; 1,4,7,10-tetraazacyclododecane-N, N', N", N¹" tetracetic acid; 1,4,8,11-tetraazacyclotetradecane - N, N', N", N"' tetraacetic acid and 1,4,7- triazacyclononane-N, N', N", N"' tetraacetic acid.

5. The system of claim 1 wherein the receptor-specific ligand is a glycopolyamino acid having an exposed terminal carbohydrate recognizable by a receptor of said cell type.

6. The system of claim 5 wherein the receptor-specific glycopolyamino acid is selected from the group consisting of glycoproteins, glycopolycations and conjugates of glycoproteins and polycations.

7. The system of claim 1 wherein the receptor-specific ligand has exposed terminal galactose residues.

8. The system of claim 1 wherein the receptor-specific ligand is an asialoglycoprotein.

UBSTITUTE SHEET 9. The system of claim 8 wherein the complexing agent comprises diethylenetriamine pentaacetic acid.

10. The system of claim 1 wherein the paramagnetic material contains paramagnetic substances selected from the group consisting of manganese, ferric iron and rare earth metals.

11. The system of claim 1 wherein the paramagnetic material is gadolinium.

12. A method of enhancing contrast in MRI radioimages comprising the steps of forming a receptor-specific carrier for a paramagnetic material, said carrier being suitable for the targeted delivery of said material to a specific cell type, loading the paramagnetic material onto said carrier whereby said material is bound thereto in a stable manner, and delivering said carrier and bound paramagnetic material to said cell type for recognition of said carrier by surface receptors of said cell.

13. The method of claim 12 wherein said carrier is a conjugate formed by bonding a receptor-specific ligand to a complexing agent for the paramagnetic material.

14. The method of claim 13 wherein said complexing agent is a chelating agent.

15. The method of claim 14 wherein the chelating agent is diethylenetriamine pentaacetic acid.

16. The method of claim 12 wherein said carrier is a conjugate of a receptor-specific glycoprotein having exposed receptor responsive residues.

17. The method of claim 16 including the step of modifying the glycoprotein to increase the number of free amino groups thereon.

18. The method of claim 12 wherein the paramagnetic material is a form of gadolinium.