JPS60252429A - Contrast agent for nmr imaging - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION 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.

It's starting to change. Current resolution is comparable to X-ray CT imaging. However, the main characteristic advantage of NMRQ is that
Different NMR relaxation times (Tl and ie contrast)
This means that it is possible to identify the This nuclear relaxation time has M
Since paramagnetic ions or stable free radicals such as n(n) and Gd(III) can have a strong influence, they are particularly useful for determining the ability of these materials to further enhance contrast. Tl of protons in water molecules in animal organs and living animals
The above materials are being explored to test whether they alter the T2 and T2 values. For example, Fillen
Donca Dias et al., The Use of Paramagnetic Contrast Agents in NMR Imaging
agnetic contrast agents in
NMRImaging) + Absts, Soc
, Mag.

Rea, Mn., 1982. 103. 104 pages;
Brad7 et al., Proton nuclear magnetic resonance imaging of focal ischemic canine hearts; paramagnetic proton signal enhancement effect (ProtonNucIear hgnetic R
e5onance 'ImhgEng of Regi
onallyIschemic Can1ne Hea
rts: effects of Paramagn
eticProton Signal Enhancement
ment), Radiolog)'+ 198L14
4. pp. 343-347; Brasch et al., Evaluation of Stable Free Radicals of Nitroxy F for Contrast Enhancement in NMR Imaging.
nof N1troxide 5table Free
Radicals for Contrast] IT
Hancement in NMRImaging
), Absts, Soc, Mag.

Res, Med, 1982. 25. 26 pages;
Brasch, Work in Progress: Contrast Enhancement Methods and Possible Applications in NMR Imaging (Work in P
logress: Methods of Contra
st Enhancement for NMRIma
ging and Potential Applica
tions), Radiology, 1983
.

Toro, p. 781-788; as well as Grossma
Gadolinium-enhanced NMR image of experimental brain membrane tumor (Ga
dol 1nium Enhanced NMRImag
es of experimental BrainA
l) scess), J, Co+nput, A35t,
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 (Me
ndonca Dias et al., The Use of Paramagnetic Contrast Agents for NMR Imaging
genetic contrast agents in
NMRImaging) *Ab+sts, Sa
c, Mag, Res, Med, 1982゜1
．． 05, page 106). The present invention, conceived under these circumstances, reduces the toxicity of NMR contrast agents and enhances their usefulness by using paramagnetic materials and micellar particles together, which have properties that meet the unique demands of NMR imaging. The purpose is to increase.

(1, Margin) Another important issue to consider is that the maximum volume that micelles, such as vesicles, can occupy within a tissue generally does not exceed about 0.1 volume factor. This means that it is necessary to produce a contrast effect with micelles of extremely small volume. However, in this regard, paramagnetic NMR contrast agents are used in other imaging modalities where the signal or attenuation is simply proportional to the number per unit volume, regardless of whether the contrast agent is chemically bound or trapped. It is fundamentally different from contrast agents such as X-ray absorbers and gamma-ray emitters. In NMR,
Contrast agents (ions or stable free radicals) act to increase the relaxation rate of protons in the bulk water surrounding the free child 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 the paramagnetic material TI is encapsulated in phospholipid vesicles as in a preferred embodiment of the invention, the paramagnetic material typically binds less than 0.1 volume fraction of the total trapped water. is expected. Under these conditions, the presence of contrast agent encapsulated in the t1 vesicles (therefore, no detectable change in the NMR image will occur). Andtasko et al., “Di/g
NMR examination of fast water diffusion in the lipid bilayer in lumitoyl lecithin vesicles (NM, R5 study of Rapid
water DiffusionAcross Li
pid Bylayers in Dipalymit
oylLecithin Vesicles),
Biochem, Biophys, Res.

See Corrym, 1974.60813-819. The present invention solves 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.
(ormulation).

It is known that phospholipid vesicles are concentrated in certain tissues and therefore may have an additional enhancing effect from tissue-specific iK. For example, phospholipid vesicles were observed to accumulate in transplanted tumors in mice. (Proffi)
tt) et al., “Liposomal inhibition of the reticuloendothelial lineage: improved tumor imaging with unilamellar vesicles (Liposomal
Blockade of the Reticulo
en-dothereal SS75te: Imp
roved Tumor Imaging withS
rnall Unilamella Vesicles
J, Scie-nce, 1
983.220.502-505 and Proffitt et al., Tumor imaging potential of liposomes containing In-I-NTA; biodistribution in mice (Tu.
mor -Imaging Potential of
Liposomes Loaded wj th In
-11-NTA: Biodist-rfbutio
n in Mice) J, Journal of Nuclear Medicine (Journal of Nuc)
learMedians), 1983, 24
+ See pages 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. Conventionally, it has been reported that the use of a manganese-labeled anti-myosin monoclonal antibody selectively shortens the T1 relaxation time of the pumped heart. Brad3
) et al., ``Selective shortening of infrared myocardial relaxation time by using manganese-labeled monoclonal antibodies (5electi
ve Decrease in theRalaxat
ion Times of Infrayid Myo
cardium with the use of a
Manganese-Labeled Monoc
lonalAntibody), Sac-Mhgn
, Res, Med, , Works in Progress, Second Annual φ Meeting (5econd Ann
ua1Me@ting), 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 attached contrast agent carrier, the vesicle, amplifies the contrast-enhancing effect of NMR compared to when ions are bound to antibodies alone.

Ru. Typically, paramagnetic compounds are encapsulated in vesicles.

The surface of the vesicles may optionally be attached with antibodies such as anti-mision, anti-fibrin, or with another surface modification (5urfa) corresponding to a specific cell receptor in a particular tissue.
ce modtficatlons) may be optionally given.

Components of vesicles include phospholipids such as distearoyl phosphatidylcholine (DSPC), di/9 lumitoyl phosphatidylcholine (DPPC'), and simyristoylphosphatidylcholine (DMPC). Examples of paramagnetic substances include transition metals, and
The lanthanide and actinide series of the periodic table, such as Gd (
IN)5Mn(II), Cu(II), Cr(Ml
)4NoFe(II), F'e(11), Co(Il), E
r(lI), nickel (■).

And, these ions and diethylenetriaminepentaacetic acid (
I) TPA), ethylenecyaminetetraacetic acid (EDTA)
) or complexes with other ligands. Another paramagnetic compound is formed into lipid vesicles in an aqueous medium containing the paramagnetic material using stable free means such as organic nitroxides, e.g. sonication, homogenization, chelate dialysis, etc., and then External substances may be removed from the vesicles by methods such as ultrafiltration and gel filtration. Additionally, it is easy to modify the internal solution of paramagnetic material by combining it with a charged polymeric material, such as poIJ-L-IJ, for example, to maximize the rate of relaxation per unit dose.

Details g! 1. Clear Definitions and Abbreviations When used in Kikiri #11, "micellar particles" and 6 mic/I/'' refer to particles generated by the aggregation of amphiphilic molecules. Mediatic molecules are biological lipids.

By "vesicles" we mean micelles, usually spherical, often derived from lipids that form bilayer membranes and called 'lifosomes'. The methods for forming these vesicles are no longer known in the art. It is a very well known place.

Depending on the technical method used, the envelope of the vesicle is transformed into a single spherical bilayer shell (5hell) (unilamellar vesicle (e uni
lamellar vesicles) or containing multiple layers within the vesicle envelope (lamellar vesicles)
cles ) ) is also possible.

DSPC=distearoylphosphatidylcholine ch
- Cholesterol DPPC = Dino Q Lumitoyl Phosphatidylcholine DM
I) C=simyristoylphosphatidylcholine DTPA
= diethylenetriaminepentaacetic acid ED'l"A - ethylenediaminetetraacetic acid SUV - small unilamellar vesicles (
small unilamellarveslcle
) Micelle materials and preparation method Deionized water or 4.0 mM Na2HPO4,0,9
The paramagnetic compound forceps were prepared in weight-NaC, pH 7.4 buffer (PBS).

10.0 μmol of GdC 3+ 6H*0(9
9,999%, made by Aldrich
) was dissolved in K 100 mol of trisodium citrate (analytical reagent, manufactured by Mallinckrodt).
ical reagent + Mallinakrod
t)) to bring the pH of this solution to neutral! l1u
A stock solution of 10.0 mM citric acid (to mM GdU) was prepared by adjusting the volume to 10.0 m in a weighing flask.

Manganese (Mn)(II)-citrate 9 ml of water contains 10.0 μmol of M nck·4 HzO (Baker analyzed) and 1
00 μmol of sodium citrate was added to adjust the solution to neutrality, and the volume was adjusted to 10.0 − in a weighing flask to prepare a stock solution of 1.0 rnM Mr+ (II) and 10.0 mM citric acid.

Gallinium (Gd) (lit) -DTPA] Om
2.10 mmol of DTP in a weighing flask
A was dissolved in a minimum amount of 6N NaOH and 2.0 m m
ol GdCj! Add s ・6H20 and add I)H to 6N
Combine 7.4 with NaOH and then transfer the solution in the flask for 10. Increase to 200 mM G
A stock solution of d(l[)-210mM DTPA was made.

Lantern (La XI) -DTPA LaC,j! 200 m VI La (II
) - A stock solution of 210 mM DTPA was made.

This stock solution was made and prepared in a similar manner to Gd(I)-DTPA.

The above substances with average molecular weights of 25,000 and 4,000 were purchased from Sigma Chemical Corporation (Sigma Ch.
Retrieved from e-mlcal Co.).

98% were obtained from Mallinckrodt.

DSPC is Cal-Bioche
m) synthetic material was used.

16~ DSPC and 4~ Cholesterol were dissolved in 2~HC~3. 0.16mM cholesterol Cu in CHCk for the purpose of quantifying lipids in the final preparation.
-4-4='E) (56,5mci/mmole
) 10μ of @ solution was added. The lipid solution was evaporated to dryness in a vacuum desiccator and stored therein if not used immediately.

Small vehicle-lamellar vesicles (SUVs) were created as follows by adding 200 mM Gd, fl) - DTPA preserved ion row assembly solution 2.0 - to the dry fried lipid tubes described above. This mixture was bound to the dry lipid tube to form a complex.

This is an ultrasonic with a microchip installed.
Inc. (Ultra 5onics I)
nc, ) sonicated at a power level of 56 W using a probe. The tube 1 was cooled by being partially immersed in a water bath. Also, during the ultrasonic wave, a sufficient amount of N gas was flowed onto the sample. Sonicate for a total of 15 minutes or more until the sample solution becomes slightly opalescent.

3. Place Sephadex G-50 swollen in PBS in advance on a plastic cylinder, apply this solution to a column prepared by centrifuging it, and collect the eluate from the column with vesicles. The column was centrifuged along with the installed glass tube. The vesicle concentration in the final preparation was determined using the reagents present in the volcano in a PBS John counter. The laser particle counter model 200 was manufactured by Nicomp Instruments.
The average vesicle diameter was measured in the 2-year-old menses). Vesicle diameter was 600±100X in all tests.

Measurement of NMR Relaxation Time Unless otherwise specified, TI and T2 were measured at 20 MHz using a Norse NMR spectrophotometer connected via an interface to a microcomputer (IBMPC). T1 is the inversion-recovery method (tnversion-recovery method).
covery method) (Fuzzy Tea, Sea-1 (Fa, rrar, T, C,) r
Pesocar E, Dee.

Press (Academic Press) = Knee York), T2 is measured with Meiboom (Meiboom).
) and modified by Gill (G11l) (Mayboon.

xy,, (MeibOOm, s, ), Gil D, (G11l, D,), Instra A, (R
ev, Sci, In5trun, ), 1958, vol. 29, p 688-691) Carr-purcell sequence:,ys(Carr-purcell 5eq)
uence) (car h.

qi, (CRrr+ 1-1.,'Y,),ノ(-cell E,H.

Experiments), Huiz, Rev, (Ph
ys, Rev, ), 1. fit (b, est
ftt) was performed automatically on a computer. The reported TI and T2 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%. TI 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. 1゛1 value (Praxis)
II using a NMR spectrophotometer with a probe temperature of 25°C and 1
Measured at 0 MHW.

Animal Experiments EMT6 tumor tissue was implanted subcutaneously into the flank of male Pulp/c (Ba1b/c) mice and allowed to grow for 10 days. Once every 10 days, mice were injected intravenously with 200 μl of vesicle solution or control buffer. At intervals, mice were sacrificed and tumor sections were collected. Sections of the liver and pancreas were also taken in some experiments. Tissues were washed with PBS, blotted, weighed, and emptied with N within % hours after section collection.
MR relaxation was measured.

A portion of the stock electrolyte was added to PBS buffer.900-
Measured at 10 MHz T Tt f3: using the −90° method.

Probe temperature was 25°C. Concentrations of Er-EDTA are given in mM and concentrations of Mn-citrate are given in μM.

Figure 2 shows 1/T by Gd ions added in various forms.
1 change. Water (Gd/citric acid) or PBS (Gd
/DTPA and Qd/DTPA in vesicles) shows the influence of magnetic ion complex concentration. Gd in inserted PBS
- DSPC/cholesterol vesicles were prepared with increasing concentrations of DTPA. All lipids (vesicles>m degrees) were adjusted using PBS so that the final total lipid concentration was 8.3 degrees.

Figure 4 shows the relaxation rate of mouse tissues and tumors. 200 mM in DSPC/cholesterol vesicles
G4-DTPA (10mg/m!=lipid) (GdV
E5), 200mM in DSPC/covsterol vesicles
La-DTPA (at La V), 2.0mM in PES
Gd-DTPA (Ga But; ) or PBS
(BuF)k were each injected into 200111 Bulb/C mice. Mice were sacrificed 16 hours later and tissue sections were collected. Relaxation times are the average of at least 3 animals.

Figure 5 shows the point I for the relaxation rate of Gd-DTPA solution.
The effect of addition of J-L-IJ lysine is shown.

2.0 mM Gd-DTPA in H2O 2. Om
An aliquot of poly-luridine, measured by dry weight, was dissolved in 1 ml of poly-luridine. TI and T2 were measured as described in the specification. Figure 6 shows the time course of 1/T1 in mouse tumors. 200 μJ-i of 10 mp/m lipid vesicle preparation containing 200 mM Gd-DTPA inside, EMT6 tumor-bearing Pal 7”/10 days after implantation.
c Injected intravenously into the tail of mice. The mouse was coated with gold at intervals, and the TI of the tumor was measured immediately after sectioning.

Not injected (○) or 2.0mM G in PBS
d-DTPA200μ! (b) was used as a control. This graph is a collection of three separate experiments.

For circles and mouths, each point represents one tumor sword. Regarding Δ, T1 values were sometimes 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 shows Er-EDTA
and the relaxation rate of Mn citrate solution. 10
17T1 values at MHz, 25°C are plotted as a function of ion concentration. The average value of 1/TL of mouse soft tissue is shown in the graph. The concentration of Er-EDTA is mM, Mn-
Citrate concentrations are given in μM. 1 in PBS solution
．． Addition of 8 mM Er-EDTA complex increases 1/T1 of mouse tissue to 2,4 s-'. The same relaxation rate is obtained with the Mn-citrate complex at only 0.1.7 mM. The weak complex of Mn is strongly complexed with Er-E.
Enhances relaxation by 100 times compared to DTA. This is M
i to n water: 8. , , reflects the inherently strong relaxivity of the M-n(It) ion, as well as its accessibility.

Figure 2 shows the relaxation effect of Gd (III). 2
Adding Gd citrate to H40,1 at 0 MHz has a relaxing effect. This decrease results in an increase in the complex. In solutions containing Gd-DTPA complex gold encapsulated in DSPC-cholesterol vesicles, 1/TI is still Ml; at 1 mM Gd-DTPA it increases to a value of 2.5 s (unit ion equivalent) than free Gd-DTPA. Although not effective, the vesicles still have a substantial effect on water relaxation 211.

Figure 3 shows the internal paramagnetic ion complex concentration of Gd-D, T
Figure 3 shows the effect on the relaxation rate of PA-encapsulated vesicles. 1/Ts and 1/T1 of vesicle solution internal Gd-DT
The PA concentration increases linearly up to 150mM.

According to the results shown in Figure 3, Gd-DTP was added at a concentration of 15
It has been shown that up to 0 mM, intravesicular MTl has a longer exchange lifetime than τb.

4th cell encapsulated Gd-DTPA has a mitigating effect on tumor and tumor in mice, diamagnetic lanthanide ion complex intravesicular La-DTPA (pancreatic) or PBS buffer and PBS buffer plus 2.0mM Gd/
DTPA (C tumors) resulted in a significant decrease in T, in pancreatic and EMT6 tumors compared to controls. Qd/
In the case of DTPA-vesicle-treated mice, the non-drug-injected control was 1-44 days after the change in paramagnetic species of the vesicle and macromolecule complexes, such as the rotational correlation time. A combination of interrelated factors determines the minimum concentration of T1 protonated conventional material. The amount of paramagnetic material to be encapsulated can vary, but typically the paramagnetic material will be at most about 50 mM in the vesicle, depending on the specific material used and the factors listed above. . The maximum amount will be determined by cost, toxicity and vesicle formation considerations, but will usually not exceed the encapsulation concentration vM.

Figure 5 shows the relaxation rate increasing with the addition of polymer. The relaxation effect of Gd-DTPA is shown in Table 1. The relaxation effect of Gd-DTPA is shown in Table 1. A 40% increase in the relaxation rate 1/T+ is obtained, and at 1/T2 30 this v-I is 1! We show that the increase in relaxation rate is not due to an increase in viscosity, and that in addition of PO')-LyS in all concentration ranges, -L-Lys is
7d -DTPA has a small effect due to positive charge voltage IJ -L
The negative binding inversely to ys is effectively 1;
ysb or some similar positively charged macromolecule,
The Gd single unit ion can be used to measure the increase in oxidation effect and therefore the decrease in the compatibility of the preparation. FM over time,
The alleviating effect on T6 lung tumors is shown in FIG. The maximum effect of Gd-DTPA encapsulated in vesicles is achieved 3-4 hours after injection of the drug. The average effect over the 4-hour period was approximately the same over the 16-hour period post-injection, suggesting that the effect was 16 hours post-injection.

EM implanted subcutaneously in Ba1b/c mice
Three different ribbon booms were tested at higher doses than those used for the data in Figure 6 for palliative effects on T6 tumors. The drug was injected into the mice, and then sacrificed after a certain period of time. Tumor and liver Tl values were measured within % hours after animal sacrifice. The results regarding tumors are shown in Table 1, and the results regarding liver are shown in Table 2. Animals receiving buffer only received 960±41
Mean tumor T1 value of ms (n=25) and 392±3
They had a mean liver MI'r+ value of 1 m5 (n=24). 1:1 pspc/cH 24 hours after injection
oL# vs. coma, tumor relaxation time was 665 ± 28 ms
(n=4), and the liver of those animals was 37
It had an average Tl value of 0±13 m8 (n=4). 44% of the changes in TI in tumors were in the liver (6
%) Yoshi is also physically thick.

With the various commonly used lipome formulations, the majority of the vesicular dose is deposited in the liver (and lungs). The specific formulations of the invention are much more specific to tumors, at least in terms of their effect on NMR relaxation times. Therefore, the vesicle-encapsulated paramagnetic complexes of the present invention fulfill the requirements of contrast agents for NMR imaging, i.e., T
Lowers the l value. In this case, the originally long T1 (average 960 m5) of the tumor before the contrast agent was introduced would leave the tumor dark in the NMR image, but when the contrast agent was injected, the tumor appeared brighter in the scan.

Table 1. Tumor relaxation rate valve (Balb)/c Mouse flank OEMT6 oval tumor (10 days after tumor growth) Vesicle encapsulation Contrast medium K is Tt (ms) Standard deviation n = Number of mice PBS control 962 Seven meters 974± 50 920±1
9 926 Average value n=9 n=1i n=4 g60
±41G・D2G・8G.

Lipid injection cap = 250 - 300 m Typical lipid concentration 20 / Table ■, Liver relaxation rate Tumor-bearing pulp/C mouse After injection of contrast agent, vesicle-encapsulated NMR contrast agent value is T5Cms) Standard deviation n = Number of mice 1-2 2-5 5-8 24 Gd/DTPA 200mM n-2n = 3n-4 ti Injection volume = 250-300 Typical lipid concentration 20 jng/ml 24+ where contrast medium for NMR imaging is extremely useful To be honest, the maximum toxicity is 1/T with τ<J- limit! 11;-tM must have specificity for the tissue type. The present invention can provide these features. The polymer aggregate is 1 of Gd-DTPA solution.
As shown by the addition of P'JLys to /Tl and 1/T2 (see Figure 5), the relaxation effect per unit ion can be increased. Paramagnetic ions, which are normally toxic, can be combined with strong chelates in polymeric assemblies that block their circulation (e.g., facilitated by preparing vesicles). The heel of (
It is obtained by the complex nature of the micelle aggregate that traps the Frog 5 and Buddha distribution by the 1 Po relaxation effect. In the case of phospholipid vesicles, this reflects a differential effect on tissue relaxation rates (see Figure 4, Table I and This is evidenced by the specific effect of Gd-DTPA on tumor mitigation (see Figures 4 and 6).

To obtain tissue specificity, antibodies can be attached to vesicles of L.
iposomes to Cells), Biochemistry, 1981
20, pp. 4229-4238, the disclosure of which is incorporated herein by reference). Antimyosin is NM of myocardial infarction
Enables R imaging. More recently, science.

1983, pages 222.1129-1131 Antibodies to a 5ynthetic
Fibrtn-LikePeptide T(lnd
to Human Fibrin but not
Fibrinogen) was reported to produce antifibrin. This antibody is expected to be concentrated in blood clots where ficillin has formed. There are other surface modifications for cell identification that are known to alter the biodistribution of small vesicles bound to antifibrin. For example, carbohydrate receptor analogs bound to the vesicle surface have been found to target vesicles. (Mouse of Lipid Ves
icles: 5 specificity of Ca
rboh)'drateReceptor Analo
guests for Leukoc)'tes in M
tce).

Proc, Nat'l Acad, Sci.
USA 77, 4430-4434 (1980) Mauck et al. Vesicular Targeting;
: Timed, Re1ease for Leu
koc)'tea in Mice by 5ubcut
aneous 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 drawings]

Figure 1 shows the longitudinal relaxation rate as a function of paramagnetic ion concentration, and Figure 2 shows the Gd doped in various forms.
The vertical axis shows the relaxation rate d as a function of the ion, and Figure 3 shows 1/T1 and 1/T2 on the vertical axis as a function of the capsule blue magnetic ion complex concentration.
Figure 4 shows the relaxation time of mouse organs after rare earth/DTPA complex injection, and Figure 5 shows the longitudinal relaxation rate as a function of the amount of poly-lysine added. Figure 6 shows the time course of 1/T1 in BMTa tumors. Applicant Tsu 1 Work Mata 9-・Wasa+・I〉Two Positive Reite'
Engraving of the drawing F' (no change in content) I2+sul fIX "l/I 20MHz HhL 38°C 1 伎 (mMl Menin G-2° within -1? [Gd/DTPA] (mM) Me:) θ- 3
゜(S11111 N-high-R spout 1-litre roasted, shogu lilyshi'ngaro 2.
07已6. f/shiTF'A (38'C) 0 2 4 6 8 10 [Tsume Lee LYS] ) k-1 (mgs/2.0.ml
l Metsu λ9,5 Hocho 6 Time I=Hei'I Oval Tsukuda Female Sword W Hei Ito Existence Change A7
θ・5 Procedural Amendment June 1985 Manabu Shiga, Commissioner of the K-Japan Patent Office2, Title of the invention Contrast agent for NMR imaging 3, Relationship to the person making the amendment Case Patent applicant name Name Vestar 94 Norch Inc. Horated 4, Sokujin Yamada Building 6, 1-1-14 Shinjuku, Shinjuku-ku, Tokyo, Number of inventions increasing due to amendment ■ Official drawings will be supplemented as shown in the attached sheet. (No change in content)
(3) Supplement the power of attorney and its translation as shown in the attached sheet. On the same date, United States of America, April 27, 1984.
['', we have submitted a priority claim certificate regarding No. 6011.721.

Claims (1)

[Scope of Claims] (1) A nuclear magnetic resonance contrast agent comprising micellar particles and a paramagnetic substance encapsulated therein. (2) The contrast agent according to claim 1, wherein the micelle particles are vesicles. (3) A contrast agent according to claim 2, wherein the vesicles are prepared with a promoter that promotes water exchange and vesicle stability across the vesicle bilayer. (4) The contrast agent according to claim 3, wherein the vesicles are prepared together with cholesterol. (5) Any one of claims 1 to 4, characterized in that the micelle particles are bound to antibodies, carbohydrates, or other cell-specific targeting agents to specifically recognize the target. Contrast agent as described in . (6) The contrast agent according to claim 5, wherein the targeting agent is an anti-myosin antibody. (7) The contrast agent according to claim 5, wherein the targeting agent is an anti-fibrin antibody. (8) Imaging according to any one of claims 1 to 3 and 5, wherein the paramagnetic substance is a transition metal or a salt of the lanthanide or actinide series of the periodic table. agent. (9) The paramagnetic substance is a salt of paramagnetic ions selected from manganese, copper, gadolinium, erbium, chromium, iron, cobalt and nickel. Contrast agent. αG Claim 1, wherein the paramagnetic substance is a paramagnetic compound containing stable free radicals.
The contrast agent according to any one of Items 1 to 3 and 5. Ql) The contrast agent according to any one of claims 1 to 3 and 5, wherein the paramagnetic substance is a paramagnetic compound of a paramagnetic ion and a chelate. (1) The contrast agent according to any one of claims 1 to 3 and 5, characterized in that the paramagnetic substance is present at a concentration of at least about 50 mM. Claims 1-3, wherein the substance is present in a concentration of about 50 mM to about IM
The contrast agent according to any one of Items 1 and 5. 11. The contrast agent according to claim 10, wherein a charged polymer is added to the paramagnetic material to increase the efficiency of the material. 05) The contrast agent according to claim 11, wherein the charged polymer is poly-L-lysine. θQ Nuclear magnetism consisting of phospholipid vesicles prepared with promoters that promote water exchange across the vesicle bilayer and vesicle stability, and a paramagnetic material that is a paramagnetic ion encapsulated in the vesicle and bound to a chelate. Resonance contrast agent. (17) The contrast agent according to claim 16, wherein the vesicles are prepared together with cholesterol. 18. The contrast agent according to claim 17, wherein the paramagnetic substance is Gd-DTPA. 182. The contrast agent of claim 181, wherein the paramagnetic material is present at a concentration of at least about 50 mM. (2G) The contrast agent according to claim 19, wherein the paramagnetic substance is present at a concentration of about 50 rnM to about IM. (2) A micellar particle and a paramagnetic substance associated therewith. (221) The contrast agent according to claim 21, wherein the paramagnetic substance is associated with a chelating agent fixed to the surface of the micelle particle.