GB2193095A

GB2193095A - Contrast agent for NMR imaging

Info

Publication number: GB2193095A
Application number: GB08717854A
Authority: GB
Inventors: Gianni Valensin; Gil Navon
Original assignee: Ramot at Tel Aviv University Ltd
Current assignee: Ramot at Tel Aviv University Ltd
Priority date: 1986-07-29
Filing date: 1987-07-28
Publication date: 1988-02-03
Also published as: IL79559A0; GB8717854D0; DE3724985A1; FR2602145A1; FR2602145B3

Description

GB2193095A 1

SPECIFICATION

Contrast agent for NMR imaging The invention relates to novel contrast media for NMR-Medical Imaging. Amongst others the 5 novel contrast media have an improved stability compared with preparations of similar proper ties; they result in enhanced water proton relaxation rate. The novel contrast media are provided in the form of liposomes containing paramagnetic ions bound to physiologically acceptable macro-molecules.

NMR imaging (MRI) is a comparatively new technique which provides a 3dimensional picture 10 of the human body or of certain organs thereof in a non-invasive manner. The diagonistic value of 1H MRI is greatly enhanced when the proton density information is superimposed on proton relaxation time information. It is established that the proton relaxation times of tissue water reflect not only the composition, and the structural complexity of the tissue, but also is physio- logical or pathologic state MR[ contrast agents are very useful for improving the delineation of 15 structures of organs, for characterizing physiological functions and for the further differentiation of tissues.

- For this purpose there are generally used paramagnetic ions or stable free radicals which dramatically shorten water relaxation times at relatively low concentrations. The use of such materials as contrast enhancing agents has two quite serious problems, namely the toxicity of 20 the agents and the problem of delivery to the desired target tissues. Some of the most effective paramagnetic relaxation probes, such as Mn2+ and Gd3+ or stable nitroxides are quite toxic, even at low dosages. Furthermore the metabolic routes of these have not been fully established. The toxicity problem can be overcome to a certain extent by the complexing of such ions with a strong complexing agent, such as DTPA, EDTA, but this limits the use of the complexed agent 25 to the blood stream and to blood vessels. Recently the use of the Mn2± DTPA entrapped in multilamillar liposomes was investigated by Caride et al., Mag.Resonance Imaging 2, 107(1984).

It was found that the entrapment in liposomes alters the biodistribution of the metal chelate and that 54Mn accumulation did very markedly increase in the spleen and in the liver, with some reduction in the heart and kidneys relative to free Mn-DTPA. The accumulation in the liver seems 30 to indicate leakage of the complex from the liposomes and their subsequent dissociation, There are provided contrast agents for NMR imaging in medicine. The MRI contrast enhancers of the present invention comprise paramagnetic ions bound to physiologically acceptable macro molecules which are entrapped within liposomes. The binding of the paramagnetic ions to macromolecules enhances the water proton relaxation rate and thus smaller quantities of such 35 ions can be used. This is of importance in view of the substantial toxicity of such ions. The macromolecule-bound ions tend to leak to a much lesser degree from the liposomes, thus resulting in an extended useful lifetime inside the body. The contrast agents of the invention, due to the use of specific liposomes, make possible an improved targeting to specific organs as well as to- normal or tumorous tissues. Liposome types developed for targeting drugs to certain 40 organs of the humarl body can be used for this effect, see for example, Weinstein, UCLA Symp.Mol.Cell Biol. 4, 441 (1983). The paramagnetic ions may be bound to suitqble macromo lecules. Macromolecules of choice are certain proteins, and especially human serum proteins so as to reduce immune reaction problems. The binding properties of the proteins can be used for the bonding of the ions: BSA is known to bind manganese and gandoliniurn with proton 45 relaxation enhancement: Biochem 2, 910 (1963) and Biochern 10 (1971), 2834. Experiments carried out by us have shown that there can be advantageously used human serum albumin as well as betaand garnma-globulins. The experiments have demonstrated that a 10% (w/w) solution of such protein dialyzed against 1 mM' Mn2+' the fraction of bound Mn2+ was 68%, 53% and 14% respectively for the above defined three types of serum proteins, respectively. 50 According to a further embodiment of the invention, the pramagnetic ions are complexed by means of a strong complexing agent such as DTPA or EDTA. Ions of choice are Mn2+ and Gd3+, but the same system can be used with other suitable metal ions. The thus obtained complexes give a significant relaxation enchancement, and the entrapment of such complex inside the liposomes does not reduce the relaxation effect which seems to be due to the fast diffusion of 55 water molecules across the liposome membrane system, thus producing a fast exchange on the NMR time'scale and thus a weighed average of relaxation times.

The preparation of liposomes entrapping proteins is well known in the art and need not be described here in detail. See, for example, textbooks such as Liposome Technology, Vol. 1 to 3, Boca Baton, Florida, CRC Press, 1984. 60 In the following Example the vesicles were prepared as set out on Biochemistry 20 833 (1981).

- The following Examples are provided in order to illustrate the present invention and they are to be construed in a non-fimitative manner. It is clear that a variety of different ions, proteins, chelating agents and mode of preparation of complexes and liposomes can be resorted to 65 2 GB2193095A 2 without departing from the scope and spirit of the invention.

EXAMPLES

EXAMPLE 1:

The starting material was 0.3 mi egg lecitine (phosphatidyl choline, Sigma) in dioxane. The 5 dioxane was removed by evaporation in a stream of nitrogen. 0.5 mi of CI- IC13 was added, then evaporated and Iyophilized. 0,06 gr. n-octyl-fl-D-glucopyranoside was added with 0.5 mi CHC13.

The mixture was shaken, evaporated and Iyophilized. 1 mi of 10% human serum albumin solution with 2 mM, MnCI,, Hepes 20 mM, NaC] 130 mM, was added and the solution was dialyzed against two changes of 250 mi of the same solution without the protein's first dialysis 10 for 24 h., and the second one-for 48 h. The content of the dialysis bag was washed by repeated (3 times) ultracentrifugation at WC, each time for 1 h. The final precipitate consists of washed vesicles, which contain Mn-HSA. 11 -Q EXAMPLE 2: 15

A run was carried out as in Example 1, except that 10% fl-Globulin was used instead of HSA.

Vesicles were obtained in a similar manner.

EXAMPLE 3:

A run was carried out as in Example 1, except that 10% a-Globulin was used instead of HSA. 20 Similar veiscles were obtained.

EXAMPLE 4:

A run was carried out as in Examples 1-3, but with 1 mM WC12 instead of 2 mM. Vesicles containing a corresponding concentration of Mn 2+ were obtained. 25 EXAMPLE 5:

Runs were carried out as in Examples 1 and 4, but with I9G-EDTA conjugate. Vesicles containing this conjugate with the Mn2+ were obtained.

30 EXAMPLE 6:

Runs were carried out as in Examples 1 and 4, but with HSA-EDTA conjugate. Vesicles containing the conjugate with W+ were obtained.

EXAMPLE 7, 35

A number of runs were carried out as in Examples 1-6, but with Gd Cl, replacing MnC12.

Vesicles containing the bound Gd31 cations were obtained.

EXAMPLE 8:

Runs were carried out as in Examples 1, 4 and 7, except that IgG-DTPA conjugate replaced 40 the HSA. Corresponding vesicles were obtained.

EXAMPLE 9:

Runs were carried out as in Examples 1, 4 and 7, except that HSA-DTPA conjpgate replaced the HSA. Corresponding vesicles were obtained. 45 Results of Manganese Binding and Proton Relaxation Rates for Liposomes contaiping Mn 2+ and Serum Proteins In the following there is presented a series of examples of the effects observed:

There were measured by atomic absorption manganese ion concentrations in the buffers 50 (blank) and in the suspensions of the liposomes, which contained 10% (w/w) of proteins from human serum. The volume, occupied by the liposomes, was about 20% of the suspension. The excess manganese concentration in the suspension over that of the buffer indicates binding of manganese to the proteins in the vesicles. It is seen from the Table that the largest binding was obtained for the serum albumine. 55 The measurements were made in two typical frequencies: 21 MHz and 42 MHz, which are used in NMR imaging.

The results of the T1 relaxation time show a dramatic (up to 33-fold) decrease of T1 over that of the blank, which contained manganese in equlibrium with the liposomes. Even when we normalise the results to manganese concentration, a relaxation enhancement of up to factor of 60 16 is obtained. The best results were obtained for albumin as it binds more Mn 2+ and it gives also large relaxation enhancement.

Corresponding results were obtained with the liposomes containing Gd3+.

The results for Mn 2+ and Gd3+ bound to protein conjugated with EDTA and DTPA give less relaxation per metal ion, but more metal ions bound per protein. Therefore, the choice between 65 3 GB2193095A 3 the different systems depends on the particular application and the clinical results.

The T1 for suspensions of liposomes containing human serum albumin and W+ ions at 21 and 42 MHz are given in Table 2. The concentrations of free manganese ions were kept constant throughout the preparation of the liposomes, including during the process of removal of external proteins. Thus, the additional Mn2+ Concentrations in the liposome suspensions are due 5 to W+ binding to the proteins inside the liposomes.

For control experiments we measured T1 relaxation times containing -empty vesicles- i.e. vesicles containing buffer without W+, as well as vesicles containing Mn2+ at the same concentration as the outside solutions. Although there was some shortening of T1 in these samples compared. with the blank solutions, the effect of vesicles containing HSA on T1 relaxa10 tion rates is much larger. A comparison to solutions of serum albumin as described in Table 1 should take into consideration the small amount of albumin and bound Mn2+ in the suspension of the liposomes (Table 2). In fact, the normalized effect of the bound W+ , T1,-1/A1Vin2+ is similar in the two experiments. In an additional experiment which is not described in Table 2 we washed vesicles loaded with 10% HSA and 3mM Mn2+ with buffer solution without W+. 15 The results for the total Mn2+ concentration in the suspension as measured by atomic absorption were [Mn2+1=0.31 mM and Tl=46.3 ms at a frequency of 42 MHz. The molar relaxivity, T-,l/[1Vin2+]=69.7 is comparable to the previous experiments. Thus, the fact that the bound manganese was enclosed in liposomes did not affect its relaxation enhancing properties.

It can be concluded that the relaxation obtained in the sytems of the invention is greater by a 20 large factor for the same amount of the toxic, paramagnetic metal ions.

Furthermore, toxicity is reduced significantly since the metal ions are entrapped in the liposomes.

T A B L E 1 SamD] e WC +.,mM T ms T -1 - 1. -1 2+ Tl ms T - 1 -1.1 2+ 1 1]P's T, / Mn lp 'S TIO / AMn Blank 300C 3000 A] bumi n 98C, 1020 s-Globul in 102C, 1180 r-Glubul in - 1210 1720 Blank 0,75 156 6.1 8.1 176 5.4 7.1 A] bumi n 2,34 j 11.7 206. 130. 6.1 158. 99.

A -G] obul in 1 1.62 13.6 67. 77. 16.0 57. 65.

^-Globulin n Q7 19 13 7 Blank 1.31 M. 11.6 8.8 90. 10.3 8.2 AI bumi n 3.12 4.2 226. 125. 5.0 189 104.

o -G] obul i n 2.65 7.4 123 92. 8.6]OS 78.

--GI obul i n 1 M 1 15--- 97 - -1 _.1 -1 A -M- 22 2 ill - Blank. 2.64 44. 22.4 3.5 52. 18.9 7.2 AI bumi n 5.93 3.5 263. so. 4.2 219. 6.

a -G] obul i n 4.99 4.7 190. 81. 5.1 179.76.

-Globulin 3.'07 102. 19.5 32. 74.

--- --- 15. j==44. G) a) T 1 p = T 1 - - T, (0)-1 W 0 where Tl(O) Is the value of 171 of an identical solution without a paramagnetic ion.

TABLE 2

Water Proton Spin-Lattice Relaxation Tinics' in Suspensions of Vesicles with and without 1 luman Scrum Albumin and W' b Vesicles containing Blank E.111Ply Vesicles C free M n 2+ d Vesicles containing FISA and Mn 2+ [NI 1,24 1 T, IMn 21 1 7,1 INIn 2+ 1 T1 1M11 2+] [11SA] Tif 7-1p'l A h 1 n 2+ g (111,5f) (ms) (111A1) (Ills) (niA]) (ms) (M AI) (MAI) (ms) (S-' MAr-1) 0.455 244 0.545 168 -0.455 120 0.758 0.222 36 64.2 C)l 123 1.09 85 0.93 75 1.213 0.195 25 97.8 UR6 67 2.1C 46 1.86' 44 2.52 0.248 Is 66.6 At Ni NI R frcquency ol'.1 2 M 117.

All olutions contained 130 inAl NaG, 20 m.Al llepes buRr p11 7.0.

Vesielcs contained Bullcr as in footnote 1). Mn 24 wis added to the outside solution.

Vesteles preliwed lly dialysis against solutions identical to those given as Blank.

Vcsieles 1)rcl),xtcd as desenbed in the experimental solution. They were washed with the solutions given as 1 1,1 relaxation tiniesof thesanic sol utions at a frcqtictic of21 Mlizwcrc 33. 25.5. and 14 ms, rcsi..cctix.ely. ir.1.1 ' is the difficience txtx,..ecn T71 ortlicsuspcnsions of 5- csielcs with 1 ISA and N1n2' ind those conta; r4111g 1 N1112f. only AMn 2e is the (lin'crcnce of N4,12+ concentration in the same two suspensions. i M Dianieter or veRicles slandard deviation: 340 74 rim.

Ohimeter of Y(.ieles,1,ttioard deviation: 402 111) rim, 6 GB2193095A 6

Claims

1. An MRI contrast enhancer comprising a liposome containing macromolecule-bound paramagnetic ions.

2. An MRI contrast enhancer according to claim 1 where the paramagnetic ions are selected from Mn2+ and Gd3+. 5 3. An MRI contrast enhancer wherein the macromolecules are physiologically acceptable proteins.

4. An MRI contrast enhancer according to claims 3, wherein the protein is selected from serum protein.

5. An MRI contrast enhancer according to claim 4, where the serum protein is selected from 10 serum albumin, beta-globulin and gamma globulin.

6. An MRI contrast enhancer according to any of claims 1 to 5, wherein the ions are bound to the protein by absorption forces of the protein. P, 7. An MRI contrast enhancer according to claims 1 to 5, wherein the paramagnetic ions are ic complexed with a strong complexing agent. 15 8. An MRI contrast enhancer according to claim 7, where the complexing agent is EDTA or DTPA.

9. An MRI contrast enhancer according to claims 1 to 8, where the liposome (vesicle) is a phospholipid liposome.

10. An MR[ contrast enhancer according to claims 1 to 8, wherein there is used a synthetic 20 polymer liposome.

11. MRI contrast enhancer systems for use as NIVIR medical imaging agents, substantially as hereinbefore described and -with reference to any of the Examples.

12. An MRI contrast enhancer according to any of claims 1 to 11 in injectable unit dosage 25 form.

Published 1988 at The Patent Office, State House, 66/71 High Holborn, London WC1R 4TP. Further copies may be obtained from The Patent Office, Sales Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed by Burgess & Son (Abingdon) Ltd. Con. 1/87.

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