JPH0586380B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to contrast enhancement in nuclear magnetic resonance (NMR) imaging by the use of paramagnetic materials in conjunction with micellar particles such as phospholipid vesicles. Prior Art Human NMR imaging is becoming a major diagnostic tool. Current resolution is comparable to X-ray CT imaging. However, the main characteristic advantage of NMR is that the different NMR relaxation times (T ₁ and
This means that differences (i.e., contrasts) between tissue types can be identified based on T ₂ ). Since this nuclear relaxation time can be strongly influenced by paramagnetic ions such as Mn () and Gd () or stable free radicals, in order to determine the ability of these materials to further enhance contrast, we These are the _T1 values of protons in water molecules in excised animal organs and living animals.
The above substances are being searched to test whether they alter T ₂ values. for example,
Mendonca Dias et al., The Use of Paramagnetic Contrast Agents in NMR Imaging
Paramagnetic Contrast Agents in NMR
Imaging), Absts.Soc.Mag.Res.Med., 1982,
pp. 103, 104; Brady et al., Proton nuclear magnetic resonance imaging of canine hearts with focal ischemia: paramagnetic proton signal enhancement effect (Proton Nuclear
Magnetic Resonance Imaging of Regionally
Ischemic Canine Hearts: effects of
Paramagnetic Proton Signal Enhancement),
Radiology, 1982, 144, pp. 343-347; Brasch
Evaluation of Nitroxide Stable Free Radicals for Contrast Enhancement in NMR Imaging
Radicals for Contrast Enhancement in NMR
Imaging), Absts.Soc.Mag.Res.Med., 1982,
pp. 25, 26; Brasch, Work in Progress: Methods of Contrast for NMR Imaging and Possible Applications
Enhancement for NMR Imaging and
Potential Applications), Radiology, 1983,
147, p. 781-788; and Grossman et al., Gadolinium Enhanced NMR Images of Experimental Brain Abscess.
experimental Brain Abscess), J.Comput.Asst.
Tomogr., 1984, 8, p.204-207. As shown in these papers, contrast is enhanced by various paramagnetic materials. However, potentially useful compounds, due to their paramagnetic properties, can be toxic at the concentrations required for optimal efficacy. And the discovery of contrast agents that are sufficiently low in toxicity to be successfully used in medical diagnosis is considered the most critical and difficult problem in the field (Mendonca Dias
et al., The Use of Paramagnetic Contrast Agents for NMR Imaging
in NMR Imaging), Absts.Soc.Mag.Res.Med.,
1982, pp. 105, 106). The present invention, conceived under these circumstances, aims to reduce the toxicity of NMR contrast agents and improve their usefulness by using paramagnetic substances and micellar particles together, which have properties that meet the unique requirements of NMR imaging. The purpose is to enhance sexuality. Another important issue to consider is that the maximum volume that micelles, such as vesicles, occupy within a tissue generally does not exceed about 0.1% by volume. This means that it is necessary to produce the contrast effect with a very small volume % of micelles. However, in this respect,
Paramagnetic NMR contrast agents are contrast agents used in other imaging modalities, e.g. They are fundamentally different from bodies, gamma ray emitters, etc.
In NMR, contrast agents (ions or stable free radicals) act to increase the relaxation rate of protons in the bulk water surrounding the free electron spins. This phenomenon depends on the rate of exchange of water desorbing ions or the rate of diffusion of water in organic free radicals. In such cases, the net relaxation rate is a weighted average of free and bound water. When paramagnetic materials are encapsulated in phospholipid vesicles, as in a preferred embodiment of the invention, it is expected that the paramagnetic materials will typically bind less than 0.1% by volume of the total trapped water. be done. Under these conditions, the presence of contrast agent encapsulated in the vesicles
No detectable changes in the NMR image will occur.
The relaxation rate of bulk water is accelerated only if water is exchanged quickly enough through the double membrane. Andrasko et al., “NMR of fast water diffusion in lipid bilayer membranes in dipalmitoyl lecithin vesicles.
Test (NMR Study of Rapid water Diffusion)
Across Lipid Bylayers in Dipalymitoyl
Lecithin Vesicles), Biochem.Biophys.Res.
See Comm. 1974, 60 pp. 813-819. The present invention seeks to solve this problem by creating a formulation of micelles and paramagnetic substances that simultaneously achieves maximum micelle stability and an adequate rate of water exchange through the membrane.
formulation). concentration of phospholipid vesicles in certain tissues;
It is therefore known that tissue specificity can provide additional enhancement effects. For example, phospholipid vesicles were observed to accumulate in transplanted tumors in mice. Proffitt et al., “Liposomal Blockade of the Reticuloendothelial Lineage: Improved Tumor Imaging with Unilamellar Vesicles”
Reticuloendothelial System：Improved
Tumor Imaging with Small Unilamella
"Vesicles", Science, 1983, 220 ,
pp. 502-505, and Prof. et al., In.
Tumor imaging potential of liposomes containing NTA: biodistribution in mice (Tumor
−Imaging Potential of Liposomes Loaded
with In−−NTA: Biodistribution in
Mice,” Journal of Nuclear Medicine,
See 1983, 24 , pp. 45-51. The present invention further encompasses the use of the micellar particles as a contrast agent carrier in applications such as those in which the micelles are used in conjunction with antibodies. Previously, it has been reported that the use of a manganese-labeled anti-myosin monoclonal antibody selectively shortens the T ₁ relaxation time of isolated hearts. Brady et al., “Selective Decrease in Infrared Myocardial Relaxation Time by Using Manganese-Labeled Monoclonal Antibodies.”
the Relaxation Times of Infrared
Myocardium with the Use of a Manganese
-Labeled Monoclonal Antibody), Soc.
Magn.Res.Med., Works in Proqress, Second Annual
Meeting (Second Annual Meeting),
See 1983, p. 10. However, there are considerable restrictions on the practical use of such antibodies, mainly considering the toxicity as described above. In the present invention, improved sensitivity and maintenance of specificity were achieved by attaching antibodies to the surface of micelle particles. Antibodies confer a high degree of specificity to cell or tissue types, and the contrast agent carrier, the attached vesicle, amplifies the contrast-enhancing effect of NMR compared to when ions are bound to antibodies alone. SUMMARY OF THE INVENTION It is an object of the present invention to provide a formulation of micellar particles, such as small unilamellar vesicles, in combination with a paramagnetic material. Typically, paramagnetic compounds are encapsulated in vesicles. The surface of the vesicles may optionally be attached with antibodies such as anti-misions, anti-fibrin, or other surface modifications that correspond to specific cell receptors in particular tissues. You may give. Components of vesicles include phospholipids such as distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC). Examples of paramagnetic substances include transition metals and the lanthanide and actinide series of the periodic table, such as Gd(), Mn(), Cu
(), Cr(), Fe(), Fe(), Co(), Er
(), nickel (), and complexes of these ions with diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), or other ligands. Other paramagnetic compounds contain stable free radicals such as organic nitroxides. To produce the contrast agent in the form of vesicles, suitable means such as ultrasound treatment, homogenization treatment,
Lipid vesicles may be formed in an aqueous medium containing a paramagnetic substance using chelate dialysis or the like, and then external substances may be removed from the vesicles by ultrafiltration, gel filtration, or the like. Additionally, it is easy to modify the internal solution of paramagnetic materials by incorporating them with charged polymeric materials, such as poly-L-lysine, for example, to maximize the rate of relaxation per unit dose. Detailed Description Definitions and Abbreviations As used herein, “micellar particles”
and "micelle" means particles resulting from the aggregation of amphiphilic molecules. In the present invention, preferred amphiphilic molecules are biological lipids. "Vesicles" refers to micelles, usually spherical, often derived from lipids that form bilayer membranes and called "liposomes." The methods for forming these vesicles are now very well known in the art. Typically, these vesicles are prepared from phospholipids, such as distearoylphosphatidylcholine or lecithin, but may also contain other substances such as neutral lipids, or positively or negatively charged compounds. Surface modifiers may also be included. Depending on the technical method used to prepare these vesicles, the envelope of the vesicles forms a single spherical bilayer shell (a unilamellar vesicle).
vesicle) or can contain multiple layers within the vesicle envelope (multi-lamellar vesicle)
vesicles)). DSPC = distearoyl phosphatidylcholine Ch = cholesterol DPPC = dipalmitoyl phosphatidylcholine DMPC = dimyristoyl phosphatidylcholine DTPA = diethylenetriaminepentaacetic acid EDTA = ethylenediaminetetraacetic acid SUV small unilamellar
vesicle) Micelle materials and preparation method Deionized water or 4.0mM Na ₂ HPO ₄ , 0.9% by weight
Complexes of paramagnetic compounds were prepared in NaC, pH 7.4 buffer (PBS). Gadolinium (Gd) ()-citrate 10.0 μmol GdC ₃ in 9 ml deionized water. 6H ₂
O (99.999%, Aldrich) was dissolved and added with 100 mol of sodium citrate (analytical reagent, Mallinckrodt).
Add reagent, Mallinckrodt)) to this solution.
Adjust the pH to neutral and bring the volume to 10.0 using a weighing flask.
A stock solution of 1.0mM Gd() and 10.0mM citric acid was prepared according to the volume of the sample. Manganese (Mn) ()-citrate Add 10.0 μmol of MnC ₂ 4H ₂ O (Baker analyzed) and 100 μmol of sodium citrate to 9 ml of water, adjust the solution to neutrality, and weigh. Adjust the volume to 10.0ml in a flask.
A stock solution of 1.0mM Mn() and 10.0mM citric acid was prepared. Gadolinium (Gd) () - DTPA Dissolve 2.10 mmol DTPA in the minimum amount of 6NNaOH in a 10 ml weighing flask and add 2.0 mmol GdC ₃ .
Add 6H ₂ O and adjust the pH to 7.4 with 6NNaOH, then
The solution was increased to 10.0 ml in the flask.
A stock solution of 200mMGd()-210mMDTPA was prepared. Lanthanum (La) () - DTPA LaC ₃・7H ₂ O (99.999%, manufactured by Aldrich)
A stock solution of 200mMLa()-210mMDTPA was prepared using a method similar to the Gd()DTPA stock solution. This stock solution was made in a similar manner to Erbium (Er)()-EDTA Gd()-DTPA. Poly-L-lysine-hydrogen bromide The above substances with average molecular weights of 25,000 and 4,000 were purchased from Sigma Chemical Corporation (Sigma
Chemical Co.). The one containing 98% cholesterol was obtained from Mallinckrodt.
DSPC was a synthetic material from Cal-Biochem. DSPC/NMR encapsulated in cholesterol vesicles
Contrast agent: 2 ml of 16 mg DSPC and 4 mg cholesterol
of _CHC3 . A 0.16mM cholesterol [u- ^14C ] (56.5mCi/mmole) solution in _CHC3 was used to quantify lipids in the final preparation.
Added 10μ. The lipid solution was evaporated to dryness in a vacuum desiccator and stored therein if not used immediately. 200mMGd()-DTPA preserved ion complex solution
Small unilamellar vesicles (SUVs) were created as follows by adding 2.0 ml to the above dry lipid tube. This mixture was bound to the dry lipid tube to form a complex. Ultrasonics Inc., which has a microchip attached to this
Inc.) probe at a power level of 56 W. The tube was cooled by partially immersing it in a water bath. Also, during the ultrasound, a sufficient amount of N ₂ gas was flowed over the sample. Sonication was applied for a total of 15 minutes or more until the sample solution appeared slightly opalescent. Pre-pour into a 3ml plastic cylinder.
This solution was applied to a column prepared by mounting Cephadex G-50 swollen with PBS and centrifuging it to separate paramagnetic compounds outside the vesicles from the SUV. Briefly, the vesicle solution was placed on top of the cylindrical column and the column was centrifuged with a glass tube placed to collect the eluate from the column. Vesicles were then eluted from the column using 300μ PBS. This operation was repeated a total of three times to reduce the reagent freely present outside the vesicles and replace it with PBS. The vesicle concentration in the final preparation was measured in a scintillation counter by aliquoting the solution using a standard cocktail. Laser Particle Counter Model 200 (Nicomp Instruments)
The average vesicle diameter was measured in a 100-μm-sized vesicle (Instruments). In all experiments, the vesicle diameter was 600±100 Å. Measurement of NMR Relaxation Times Unless otherwise specified, T ₁ and T ₂ were measured at 20 MHz using a pulsed NMR spectrophotometer connected via an interface to a microcomputer (IBMPC). T ₁ is an inversion-recovery method (Farrar, TC), Becker, ED, pulse and Fourier transform NMR (Pulse
and Fourier Transform NMR), 1971, Academic Press, New York), T ₂ was measured at Meiboom.
and Gill (Meiboom, S.), Gill D.
(Gill, D.), Modified Spin-Echo Method for nuclear relaxation time measurements.
Measuring Nuclear Relaxation Times), Rev. Sai. Instrument. (Rev.Sci.
Instrum.), 1958, Vol. 29, p688-691) Carr-Purcell Sequence
sequence) (Carr HY),
Purcell E. H. (Purcell, E.H.)
Effects of Diffusion on Free Precession in NMR Experiments
Precession in NMR Experiments), Fiz.
Rev. (Phys.Rev.), 1954, vol. 94, p.630−633)
It was measured with Single exponential recovery
A least squares best fit of the data to cxponential recovery was automatically performed on the computer. The reported T ₁ and T ₂ values are for a probe temperature of 38°C. The experimental uncertainty of the _T1 value is estimated to be ±10% and the reproducibility to be ±5%. _T2 values are generally accurate to within ±20%, and their reproducibility is up to ±5%. This accuracy is clearly sufficient to demonstrate the effectiveness of the present invention. Some of the T ₁ values are Praxis
Probe temperature 25℃ using NMR spectrophotometer.
Measured at 10MHz. Animal Experiments EMT6 tumor tissue was implanted subcutaneously into the flank of male Balb/c mice and allowed to grow for 10 days. On day 10, mice were injected intravenously with 200μ of vesicle solution or control buffer. At intervals, mice were sacrificed and tumor sections were collected. Liver and spleen sections were also taken in some experiments. Tissues were washed with PBS, blotted dry, weighed, and wrapped in airtight plastic bags. NMR relaxation was measured within 1/2 hour after sectioning to minimize water loss and consequential changes in T ₁ and T ₂ . FIG. 1 is a plot of longitudinal relaxation rate (1/T ₁ ) as a function of added paramagnetic ion concentration. A portion of the stock solution was added to the PBS buffer. T ₁ was measured at 10 MHz using the 90°−−90° method. Probe temperature was 25°C. Er−EDTA
The concentrations of Mn-citrate are given in μM and the concentrations of Mn-citrate are given in μM. FIG. 2 shows the change in 1/T ₁ due to Gd ions added in various forms. water (Gd/citric acid) to obtain the indicated total ion concentration.
or PBS (Gd/DTPA and Gd/DTPA in vesicles)
An aliquot of the stock solution was added to. Figure 3 shows the influence of the paramagnetic ion complex concentration inside the capsule (vesicle) on 1/T ₁ and 1/T ₂ . DSPC/cholesterol vesicles were prepared by increasing the concentration of Gd-DTPA in encapsulated PBS. The final total lipid concentration was 8.3 mg/ml.
All lipid (vesicle) concentrations were adjusted using PBS. Figure 4 shows the relaxation rate of mouse tissues and tumors. DSPC/cholesterol in vesicles
200mM Gd-DTPA (10mg/ml lipid) (GdVES),
200mM La− in DSPC/cholesterol vesicles
DTPA (La VES), 2.0mM Gd−DTPA in PBS
(Gd BUF) or PBS (BUF) were each injected into 200μ Bulb/C mice. Mice were sacrificed 16 hours later and tissue sections were collected. Relaxation times are the average of at least 3 animals. FIG. 5 shows the effect of poly-L-lysine addition on the relaxation rate of the Gd-DTPA solution. An aliquot of poly-L-lysine measured by dry weight was dissolved in 2.0 ml of 2.0 mM Gd-DTPA in _H2O . T ₁ and T ₂ were measured as described in the specification. FIG. 6 shows the time course of 1/T ₁ in mouse tumors. 200mM Gd− inside
200μ of 10mg/ml lipid vesicle formulation containing DTPA
was intravenously injected into the tail of EMT6 tumor-bearing Valve/c mice 10 days after transplantation. Mice were sacrificed at intervals and tumor _T1 was measured immediately after sectioning. Not injected (○) or in PBS
200μ (□) of 2.0mM Gd-DTPA was used as a control. This graph is a collection of three separate experiments. For ○ and □, each point represents T ₁ of one tumor. For △, the T ₁ value was measured two or three times for one tumor. In the following, the improved results of the invention will be described with reference to a more detailed description of the drawings. Figure 1 is
The 1/T ₁ values at 10 MHz and 25° C. are plotted as a function of ion concentration, showing the relaxation rates of Er-EDTA and Mn citrate solutions. The average value of 1/T ₁ for mouse soft tissue is shown in the graph. Concentrations of Er-EDTA are given in mM and concentrations of Mn-citrate are given in μM. When 18mM Er-EDTA complex is added to PBS solution, 1/ _T1 of mouse tissue increases to 2.4s ^-1 . In the Mn-citrate complex, only a small amount
The same relaxation rate is obtained at 0.17mM. Weak complexes of Mn enhance relaxation by a factor of 100 compared to strongly complexed Er-EDTA. This reflects the inherently stronger relaxivity of Mn() ions as well as the highest accessibility of Mn to water. Figure 2 shows the relaxation effect of Gd().
When Gd citrate is added to _H2O at 20MHz, 1/ _T1 increases to a value of 8.1s ^-1 at 1.0mM. When complexed with DTPA, the ion has a relaxing effect of 1/2. This decrease is due to the replacement of water binding sites by DTPA functional groups, which is partially equilibrated by the increased rotational correlation time of the complex. DSPC-Gd-DTPA encapsulated in cholesterol vesicles
In solutions containing the complex, 1/T ₁ is still the total Gd−
Increases to a value of 2.5s ^-1 at 1mM DTPA.
The vesicles still have a substantial effect on water relaxation, even if less effective than free Gd-DTPA per unit ion. Figure 3 shows the internal paramagnetic ion complex concentration, Gd
- shows the effect on the relaxation rate of DTPA encapsulated vesicles. 1/T ₁ and 1/T ₂ of vesicle solution
increases linearly up to an internal Gd-DTPA concentration of 150 mM. Formula 1/T ₁ obsd=Pb/(T ₁ b+τb) +1/T ₁ a [where b is inside the vesicle and a is outside,
τb is the lifetime of the internal water proton, T ₁ b is the net relaxation time of the internal water (reduced by the paramagnetic agent), and Pb is the fraction of the vesicle internal water.
T ₁ is shown to be linearly dependent on the paramagnetic ion concentration until the value of T ₁ is equal to or less than τb. According to the results shown in Figure 3,
It has been shown that the vesicle internal T ₁ is larger than the exchange lifetime τ up to a Gd-DTPA concentration of 150 mM. The fourth study showed the alleviating effect on tissues and tumors in mice.
As shown in the figure. The T ₁ values of liver, spleen, kidney and EMT6 tumor tissues of Balb/c mice are compared 16 hours after injection of paramagnetic agent or control. Vesicle-encapsulated Gd-DTPA is a paramagnetic lanthanide ion complex containing intravesicular La-
DTPA (spleen) or PBS buffer and
PBS buffer plus 2.0mM Gd/DTPA (tumor) resulted in a significant decrease in _T1 of spleen and EMT6 tumors compared to control. Gd/DTPA
- _T1 values are on average 17% lower in vesicle-treated mice compared to controls without drug injection. With the above data, it is possible to determine the minimum concentration of Gd/DTPA or other paramagnetic species inside the vesicle that will increase contrast. There is a complex of interrelated factors such as the rate of proton exchange across the tumor cell membrane, the rate of free Gd/DTPA washout from lipid vesicles, and the changing rotational correlation time of the complex in the macromolecular environment.
T ₁ helps the proton relaxation rate and thus increases the contrast. The amount of accumulated vesicles in the parapod organ to be imaged determines the minimum concentration of encapsulated paramagnetic material. For the present murine tumor model, it was estimated that approximately 0.1% of the tumor volume was accounted for by the vesicles themselves. The amount of paramagnetic material encapsulated can vary, but depending on the specific material used and the factors listed above, generally the paramagnetic material is present in the vesicle at least about
It would be 50mM. The maximum amount will be determined by considerations of cost, toxicity, and vesicle formation, but will usually not exceed an encapsulation concentration of about 1M. Figure 5 shows the rate of relaxation increasing with the addition of polymer. The relaxation effect of Gd-DTPA can be increased by the addition of a positively charged polymer, namely poly-L-lysine. Figure 5 shows average molecular weight (MW)
25000 poly-L-lysine in _2.0mM Gd-
The results are shown when added to DTPA solution. A 40% increase in relaxation rate 1/T ₁ was obtained, and a 30% increase in 1/T ₂ . An equilibrium state (plateau) of the effect of adding poly-L-lysine of 3 mg/ml or more indicates a "weak binding" state. This equilibration also shows that the increase in relaxation rate is not due to an increase in viscosity, since the effect is linear for poly-Lys addition over the entire concentration range. Poly-L-Lys having a molecular weight smaller than this is less effective on a weight basis. Gd-DTPA is a negatively charged complex that binds inversely to positively charged poly-Lys. The large size, and therefore the slow tumbling of the macromolecules, made the relaxation of paramagnetic ions more effective. This result suggests that simultaneous encapsulation of Gd-DTPA and poly-Lys, or any similar positively charged macromolecule, increases the effectiveness per Gd unit ion and thus reduces the net toxicity of the preparation. Can be used for over time
The alleviating effect on EMT6 tumors is shown in Figure 6. The maximum effect of Gd-DTPA encapsulated in vesicles is achieved 3-4 hours after injection of the drug. The average effect at 4 hours was approximately equal to 16 hours post-injection, suggesting that a steady state is achieved in which the rate of uptake by the tumor matches the loss through circulation. Three different liposome formations were tested at higher doses than those used for the data in Figure 6 for palliative effects on subcutaneously implanted EMT6 tumors in Balb/c mice. Mice were injected intravenously with the drug and then sacrificed after a certain period of time. Tumor and liver T ₁ values were measured within 1/2 hour after animal sacrifice. The results regarding tumors are shown in Table 1, and the results regarding liver are shown in Table 1. Animals receiving buffer alone had a mean tumor _T1 value of 960±41ms (n=25) and a mean liver T1 value of 392±31ms (n=24).
It had a T ₁ value. 24 hours after injection,
For the 1:1 DSPC/CHOL preparation, tumor relaxation time was reduced to 665 ± 28 ms (n = 4), and the livers of these animals had a mean T ₁ value of 370 ± 13 ms (n = 4). Ta. The 44% change in _T1 in the tumor is substantially greater than that in the liver (6%). With the various commonly used liposome formulations, the majority of the vesicle dose is accumulated in the liver (and spleen). The specific formulation of the present invention has extremely high specificity for tumors, at least in terms of the effect on NMR relaxation time! Therefore, the vesicle-encapsulated paramagnetic complex of the present invention satisfies the requirements for a contrast agent for NMR imaging, i.e., it is selected as a contrast agent for NMR imaging. decreases the T ₁ value in the affected tissue. In this case, the tumor's inherently long T ₁ (average 960 ms) before contrast agent injection leaves the tumor black in the NMR image, but when the contrast agent is injected, the tumor appears brighter in the scan. [Table] [Table] For a contrast agent for NMR imaging to be extremely useful, it must have minimal toxicity and maximum 1/2
It must provide an increase in T ₁ and be specific to tissue type. The present invention can provide these features. The polymer aggregate is Gd−
As shown by the effect of added polyLys on 1/T ₁ and 1/T ₂ of the DTPA solution (see Figure 5), the relaxation effect per unit ion can be increased.
Normally toxic paramagnetic ions are rendered less toxic when associated with strong chelates (eg, encapsulated in vesicles) in polymeric assemblies that prevent circulation of the ions. NMR relaxation is facilitated by preparing the vesicles to maximize access of H ₂ O protons to the ions. This was achieved due to the strong relaxation effect of the encapsulated Gd-DTPA (see Figure 2). Tissue specificity is achieved through the complex nature of the micelle assembly, which allows macromolecules to be distributed to specific locations through biological recognition processes. In the case of phospholipid vesicles, this is due to the differential impact on tissue relaxation rates (see Figure 4, Table and Reference) and the approximately equivalent total concentration of free Gd-DTPA in solution compared to vesicle-encapsulated Gd-DTPA. (See Figures 4 and 6). He stated that antibodies can be attached to vesicles to obtain tissue specificity (Immunospecific targeting of liposomes to cells).
of Liposomes to Cells, Biochemstry, 1981, 20, pages 4229-4238 (this disclosure is incorporated herein by reference). Antimyosin enables NMR imaging of myocardial infarction. More recently, Science, 1983
Hui, 222, pages 1129-1131
monoclonal antibodies to a synthetic fibrin-like peptide bond that binds to human fibrin but not to fibrinogen.
Fibrin−Like Peptide Bind to Human Fibrin
but not Fibrinogen), production of antifibrin was reported. This antibody is expected to be concentrated in blood clots where fibrin is formed. Vesicular agent carriers bound to antifibrin can provide NMR contrast for imaging clots and thrombin within blood vessels. However, there are other surface modifications for cell identification that are known to alter the biodistribution of vesicles. For example, carbohydrate receptor analogs bound to the vesicle surface have been found to target vesicles. (Mauk)
et al., Lipid vesicle targeting: specificity of carbohydrate receptor analogs for murine leukocytes.
Lipid Vesicles: Specificity of Carbohydrate
Receptor Analogues for Leukocytes in
Mice), Proc.Nat′l.Acad.Sci., USA 77, 4430
-4434 (1980) Vesicle Targeting: Timed Release of Mouse Leukocytes by Subcutaneous Injection (Vesicle
Targeting: Timed Release for Leukocytes in
Mice by Subcutaneous Injection, Science
207, 309–311 (1980)). Targeting through such surface modifications can be directly used to alter the biodistribution of paramagnetic ions.

[Brief explanation of the drawing]

Figure 1 shows the longitudinal relaxation rate as a function of paramagnetic ion concentration, Figure 2 shows the longitudinal relaxation rate as a function of Gd ions added in various forms, and Figure 3 shows the longitudinal relaxation rate as a function of the paramagnetic ion concentration. 1/T ₁ and 1/T ₂ on the vertical axis as a function of the encapsulated paramagnetic ion complex concentration.
Figure 4 shows rare earth/DTPA
Figure 5 shows the relaxation time of mouse organs after injection of the complex; Figure 5 shows the longitudinal relaxation rate as a function of the amount of poly-lysine added; Figure 6 shows the
This figure shows the time course of 1/T ₁ in EMT ₆ tumors.

Claims (1)

[Scope of Claims] 1. A nuclear magnetic resonance contrast agent for tissue scanning, comprising a vesicle and a paramagnetic substance contained therein, in order for the vesicle to provide contrast for tissue scanning, The vesicles are prepared with promoters to allow for adequate water proton exchange across the vesicle bilayer and to promote vesicle stability for a sufficient period of time for the vesicles to be distributed in vivo for tissue scanning. The contrast agent characterized in that: 2. The contrast agent according to claim 1, characterized in that the vesicles are prepared together with cholesterol. 3. Claim 1, wherein an antibody, carbohydrate, or other cell recognition targeting agent is bound to the micelle particles, and specifically recognizes a target.
2. The contrast agent according to any one of paragraphs 1 and 2. 4. Claim 1, wherein said paramagnetic substance is present in a concentration of at least about 50mM.
The contrast agent according to any one of Items 1 to 3. 5. A contrast agent according to any of claims 1 to 4, wherein the paramagnetic substance is present at a concentration of about 50 mM to about 1M. 6. The contrast agent according to any one of claims 1 to 5, wherein the paramagnetic substance is a transition metal or a salt of the lanthanide or actinide series of the periodic table. 7. The contrast agent according to claim 6, wherein the paramagnetic substance is a salt of paramagnetic ions selected from manganese, copper, gadolinium, erbium, chromium, iron, cobalt and nickel. . 8. The contrast agent according to claim 6 or 7, wherein the paramagnetic substance is a paramagnetic compound of a paramagnetic ion and a chelate. 9. The contrast agent according to any one of claims 1 to 5, wherein the paramagnetic substance is a paramagnetic compound containing stable free radicals. 10. A contrast agent according to any one of claims 1 to 9, characterized in that a charged polymer is added to the paramagnetic material to increase the efficiency of this material. 11. The contrast agent according to claim 10, wherein the charged polymer is poly-L-lysine. 12. The contrast agent according to any one of claims 1 to 5, wherein the paramagnetic substance is a paramagnetic ion associated with a chelate. 13. The contrast agent according to claim 10, wherein the paramagnetic substance is Gd-DTPA. 14. The contrast agent according to any one of claims 1 to 13, wherein the tissue to be scanned is a tumor tissue. 15. The contrast agent according to any one of claims 1 to 14, wherein the paramagnetic substance is associated with a chelating agent bound to the surface of the vesicle.