CA1245553A - Contrast agents for nmr imaging - Google Patents
Contrast agents for nmr imagingInfo
- Publication number
- CA1245553A CA1245553A CA000480146A CA480146A CA1245553A CA 1245553 A CA1245553 A CA 1245553A CA 000480146 A CA000480146 A CA 000480146A CA 480146 A CA480146 A CA 480146A CA 1245553 A CA1245553 A CA 1245553A
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- Prior art keywords
- vesicles
- contrast agents
- paramagnetic
- para
- process according
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
- G01R33/00—Arrangements or instruments for measuring magnetic variables
- G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
- G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
- G01R33/48—NMR imaging systems
- G01R33/54—Signal processing systems, e.g. using pulse sequences ; Generation or control of pulse sequences; Operator console
- G01R33/56—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution
- G01R33/5601—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution involving use of a contrast agent for contrast manipulation, e.g. a paramagnetic, super-paramagnetic, ferromagnetic or hyperpolarised contrast agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1806—Suspensions, emulsions, colloids, dispersions
- A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
Abstract
ABSTRACT OF THE DISCLOSURE
An agent for contrast enhancement in nuclear magnetic resonance imaging is disclosed. Vesicles such as phospholipid vesicles are associated with or enclose a paramagnetic material.
The vesicles may be formulated with an agent, such as cholesterol, to promote vesicle stability and water exchange across the vesicle bilayer. The vesicles may or may not have antibodies or other cell recognition targeting agents attached to the surface to provide specific targeting. The vesicles provide enhanced target specificity, reduced burden of toxic contrast material and amplified contrast enhancement.
An agent for contrast enhancement in nuclear magnetic resonance imaging is disclosed. Vesicles such as phospholipid vesicles are associated with or enclose a paramagnetic material.
The vesicles may be formulated with an agent, such as cholesterol, to promote vesicle stability and water exchange across the vesicle bilayer. The vesicles may or may not have antibodies or other cell recognition targeting agents attached to the surface to provide specific targeting. The vesicles provide enhanced target specificity, reduced burden of toxic contrast material and amplified contrast enhancement.
Description
~L2455S3 The invention described herein relates to enhanced contrast in nuclear magnetic resonance (~MR) imaging through ves (c /es , the use of a paramagnetic material in association with ~r~h~-,~"J
lar pa~-ti~l-~ such as phospholipid vesicles.
NMR imaging of humans is fast becoming a major diag-nostic tool. Resolution is now on a par with X-ray CT imaging, but the key advantage of ~MR is its ability to discriminate between tissue types (contrast) on the basis of differing ~MR
relaxation times, Tl and T2. Because nuclear relaxation times can be strongly affected by paramagnetic ions such as Mn(II) and Gd(III) or stable free radicals, these materials have been explored to determine their ability to ~24~3 172/~0 provide further contrast, specifically to test whether they alter water proton Tl and T2 valuas in excised animal organs and in live animals; see, for example, Mendonca Dias et al, The Use of Paramagnetic Contrast Agents in NMR Imaginq, Absts. Soc. Mag.
Res. Med., 1982, pages 103, 104; Brady et al, Proton Nuclear Magnetic Resonance Imaging of Regionally Ischemic Canine Hearts:
effects of Paramagnetic Proton Signal_Enhancement, Radiology, 1982, 144, pages 343-347; and ~rasch et al, Evaluation of Nitroxide Stable Free Radicals for Contrast Enhancement in NMR
Imagin~, Absts. Soc. Mag. Res. Med., 1982, pages 25, 26; Brasch, Work in Progress: Methods of Contrast 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, J. Comput. Asst. Tomogr., 1984, 8, p. 204-207. Results reported show that contrast is enhanced by a variety of paramagnetic agents.
However, useful compounds, due to the nature of the candidate paramagnetic materials, may be toxic at the concen-trations required for optimal effect, and findin~ contrast agents for which the toxicity is low enough to make possible their eventual use in medical diagnosis is regarded as the most serious and difficult problem in the field, Mendonca Dias et al, The Use of Paramagnetic Contrast Agents in NMR Imaging, Absts. Soc. Mag.
Res. Med., 1982, pages 105, 106. The invention described herein is thus designed to reduce toxicity and increase the utility of NMR contrast agents by associating a paramagnetic material with a cle~
~uu~L~ eieY~- having properties tailored to the unique demands of NMR imaging Another significant problem which must be addressed is that the maximum tissue volume occupied by micelles such as J~Z~sS3 vesicles generally does not exceed about 0.1~, which means that the micelle must be capable of affecting an image with a very small volume percentage. In this regard, however, paramagnetic NMR contrast agents differ fundamentally from contrast agents used as X-ray absorbers, gamma ray emitters or the like in other imaging modalities in which the signal or attenuation is simply proportional to the number per unit volume, no matter how they are chemically bound or entrapped. In NMR, the agent (ion or stable ree radical) acts to increase the relaxation rate of bulk .. . . . . . . .... . ...
water protons surrounding the free electron spin. The phenomenon depends on rapid exchange of water on and off an ion or rapid diffusion of water past an organic free radical. In such case, . .
the net relaxation rate is a weighted average for free and bound water.
Encapsulation of the paramagnetic material within a phospholipid vesicle, as in one preferred form of this invention, would seem to deny access of the para~agnetic agent to all but the entrapped water, typically less than 0.1~ of the total volume. Under--such conditions, the NMR image would not be altered-detectably by the presence of vesicle-encapsulated contrast agent. Only if water exchanges sufficiently rapidly across the bilayer is the relaxation rate of the bulk water enhanced, Andrasko et al, NMR Study of Rapid Water Diffusion . .
Across Lipid Bylayers in DipalymitoYl Lecithin Vesicles, Biochem. Biophys. Res. Comm., 1974, 60, p. 813-819. The present invention addresses this problem by providing a formulation of micelle and paramagnetic material that simultaneously maximizes micelle stability while permitting adequate rates of water exchange across the membrane.
~24S5~3 72/40 Phospholipid vesicles are known to concentrate in certain tissues, so additional enhancement will come from tissue specificity. For example, phospholipid vesicles have been observed to accumulate in implanted tumors of mice, Proffitt et al, Liposomal Blockade of the Reticuloendothelial System:
Improved Tu_or Imaging with Small Unilamella Vesicles, Science, 1983 220 p. 502-505, Proffitt et al, Tumor-Imaging Potential of Li~osomes ~oaded with In~ NTA: Biodistribution in Mice, Journal of Nuclear Medicine, 1983, 24, p. 45-51.
~e~/c/es The invention also extends the use of ~f~L~d~
L6l4~_as contrast agent carriers to applications where the micelles are attached to antibodies. While it has been reported that a selective decrease in Tl relaxation times of excised heart may be obtained using manganese-labeled monoclonal antimyosin antibody, Brady,-et al, Selective Decrease in the Relaxation ~-Times of Infr rèd Myocardium with the Use of a Manganese-Labelled Monoclonal Antibody, Soc. Magn. Res. Med., Works in Progress, Second Annual Meeting, 1983,-p. lO, heretofore, due in large méasure to considerations such as toxicity referred to above, the practical use of such antibodies has been significantly restricted. With the present invention, however, increased sensitivity is obtained and specificity is maintained by attach-~ ~e~l~le ment of antibody to the surface of mio~lular particlcc. The antibodies provide high specificity for cell or tissue types, while the attached vesicle agent carriers amplify the NMR
contrast enhancement over what can be achieved with ions bound to antibody alone.
.
lZ45~5~
The invention described herein is directed to pre-parations of vesicles such as small unilamellar vesicles, with which a paramagnetic material is associated, typically para-magnetic compounds enclosed within the vesicles. The vesicles may or may not have antibodies, such as antimyosin, or anti-fibrin, attached to the surface or have other surface modifica-t-ions for which there are specific cell receptors in certain tissue.
Examples of vesicle constituents are phospholipids such as distearoylphosphatidylcholine (DSPC), dipalmitoylphos-phatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC). Examples of paramagnetic materials are salts of trans-ition metals and the lanthanide and actinide series of the periodic table such, as Gd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II~, Er(III), nickel(II) and complexes of such ions with diethylenetriaminepentaacetic acid (DTPA), ethylenediami-~etetraacetic acid (EDTA) and other ligands. Other paramagnetic compounds include stable free radicals such as organic nitroxides.
Vesicle-encapsulated contrast agents may be prepared by forming the lipid vesicles in an aqueous medium containing the paramagnetic agent by any suitable means such as sonication, homogenization, cholate dialysis and the like, and then freeing the vesicles of external agent by ultrafiltration, gel filtrat-ion or similar method. Moreover, the internal solution of the paramagnetic material may be altered readily -to maximize the relaxation rate per unit of agent as for example, by formulation with a charged polymeric material such as poly-L-Lysine.
According to the present invention there are provided magnetic resonance imaging contrast agents comprising vesicles with paramagnetic material encapsulated therein, said vesicles formulated with an agent -to promote vesicle stability and water excnange across the vesicle bilayer for a time sufficient to ~Z4555;~
enable scanning to be effected.
According to another aspect of the present invention there is provided a process for preparing magnetic resonance imaging contrast agents comprising vesicles with paramagnetic material encapsulated therein ar.d formulated with an agent to promote vesicle stability and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effec-ted which comprises encapsulating paramagnetic material in vesicles and formulating said vesicles with an agent to promote vesicle stabi~ity and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effected.
As used herein, "vesicle" and "micelles" refer to particles which result from aggregations of amphiphilic mole-cules. In this invention, preferred amphiphiles are biological lipids.
"Vesicle" refers to a micelle which is in a generally spherical form, often obtained from a lipid which forms a bi-layered membrane and is referred to as a "liposome". Methods for forming these vesicles are, by now, very well known in the art. Typically, they are prepared from a phospholipid, for example, distearoyl phosphatidylcholine or lecithin, and may in-clude other materials such as neutral lipids, and also surface modifiers such as positively or negatively charged compounds.
Depending on the techniques for their preparation, the envelope may be a simple bilayered spherical shell (a unilamellar vesicle) or may have multiple layers within the envelope (multi-lamellar vesicles). DSPC = distearoyl phosphatidylcholine Ch = cholesterol DPPC = dipalmitoylphosphatidylcholine DMPC = dimyristoylphosphatidylcholine DTPA = diethylenetriaminepentaacetic acid EDTA = ethylenediaminetetracetic acid SUV = small unilamellar vesicles ~' iZ4S5S3 Materials and Preparation of Micelles Complexes of paramagnetic compounds were prepared in deionized water or in a buffer of 4.0 mM Na2HPO4, 0.9% (by weight) NaCl, pH 7.4 (PBS).
Gd(III)-citrate. A stock solu-tion of 1.0 mM Gd(III) 10.0 mM citrate was made by dissolving 10.0 ~moles GdC13.6H2O
(99.999%, Aldrich) in 9 ml deionized water and adding 100 llm~lesNa3citrate (analytical reagen-t, Mallinckrodt). The pH
was adjus-ted to neutrality and the volume brought to 10.0 mL in a volumetric flask.
Mn(II)-citrate. A stock solu-tion of 1.0 mM Mn (II) 10.0 mM citrate was made by adding 10.0 ~moles MnC12.4H20 (Baker analyzed) and 100 ~moles sodium citrate to 9 ml water.
The solution was neutralized and made up to 10.0 ml in a volumetric flask.
Gd(III)-DTPA. A stock solution of 200 mM Gd(III)-210 mM DTPA was made by dissolving 2.10 m~oles DTPA in minimum 6 N NaOH in a 10 ml volumetric flask. 2.0 mmoles GdC13.6H20 were added and the pH adjusted to 7.4 with 6 N NaOH, after which the sample was made up to 10.0 ml in the flask.
La(III)-DTPA. A stock solution of 200 mM La(III)-210 mM DTPA was made up in a manner analogous to the Gd(III) DTPA stock using LaC13.7H2O (99.999%, Aldrich).
Er(III)-EDTA. Stock solutions were prepared in a manner analogous to Gd(III)-DTPA.
Poly-L-lysine hydrobromide of approximate average molecular weights 25,000 and 4,000 were obtained from Sigma Chemical Co.
Cholesterol (98%) was from Mallinckrodt. DSPC was synthetic material from Cal-Biochem.
DSPC/cholesterol vesicle-encapsulated NMR contrast agent. 16 mg DSPC and 4 mg cholesterol were dissolved in 2 ml .. . .
, S5S~
CHC13. 10~1 of a solution of 0.16 mM cholesterol [u-14C] (56.5 mCi/mmole) in CHC13 were added for purposes of quantitating lipid concentra-tions in the final preparations. The lipid sol-ution was evaporated to dryness in a vacuum dessicator and stored in the same, if not used immediately.
Small unilamellar vesicles (SUV) were formed in a solution of 200 mM Gd(III)-DTPA by adding 2.0 ml of the stock ion complex to the dried lipid tube. The mixture was sonicated using an Ultrasonics, Inc. probe with a microtip at a power level of 56 W. The tube was cooled by partial immersion in a water bath, and N2 was flowed over the sample during sonication.
Total time of sonication was 15 min. or more until the solution was slightly opalescent.
Paramagnetic agent outside the vesicles was separated from the SUVs by passage through columns of Sephadex G-50 swollen in PBS, that had been loaded into 3 ml plastic syringe bodies and precentrifuged. The vesicle solution was placed at the top of the syringe and centrifuged with a glass tube pos-itioned to collect the eluate. 300 ~1 PBS was used to elute the vesicles from the columns. The procedure was repeated a total of 3 times to reduce the outside concentration of free agent and to exchange it for PsS.
Vesicle concentration in the final preparation was measured by counting an ali~uo-t of the solution in the scintil-lation counter, using a standard cocktail. Average vesicle size was measured in a laser particle sizer Model 2~0 (Nicomp Instruments). The vesicle size was measured to be 600, + 100, A in all experiments.
NMR Relaxation time measuremen-ts Unless otherwise indicated, measurements of Tl and T2 were made at 20 MHz with a pulsed ~MR spectrometer (IBM PC/20) *Trademar~
1~9~553 60724-1587 interfaced to a microcomputer (IBM PC). Tl was measured by the inversion-recovery method (Farrar, T.C., Becker, E.D., Pulse and Fourier Transform NMR, 1971, Academic Press, New York.) and T2 by the Carr-Purcell sequence (Carr, H.Y., Purcell, E.H., Effects of Diffusion on Free Precession in NMR Experiments, Phys. Rev., 1954, 94, p. 630-633.), as modified by Meiboom and Gill (Meiboom S., Gill D., Modified Spin-Echo Method Eor Measuriny Nuclear Relaxation Times, Rev. Sci. Instrum., 1958, 29, p. 688-691.) Least-squares best fits of the data to single exponential recoveries were done automatically by the computer.
Values of Tl and T2 reported are for a p~robe temperature of 38C. Tl values are estimated to have an experimental uncer-tainty of + 10% and a reproducibility of + 5%. T2 values are generally accurate to within + 20% and reproducible to + 5%.
This precision is sufficient clearly to demonstrate the effects claimed. Some values of Tl were measured with a Praxis II NMR
spectrometer operating at 10 MHZ and a probe temperature of 25 C.
Animal Studies EMT6 tumor tissue was transplanted subcutaneously into the flank of female Balb/c mice and allowed to grow for 10 days.
On the 10th day, mice were injec-ted i.v. with 200ul of vesicle solution or control buffer. Mice were sacrificed a-t intervals, and the tumors were dissected. In some experiments liver and spleen were also dissected. The tissue was rinsed in PBS, lightly blotted, weighed, and wrapped in air tight plastic bags.
NMR relaxation measurements were made within 2 hr of dissection to limit water loss and consequent changes in Tl and T2.
Figure l is a plot of longitudinal relaxation rate as a function of added paramagnetic ion concentration. Aliquo-ts of the stock solutions were added to PBS buffer. Tl measuremen-ts were made at 10 MHz using the 90- -90 method. Probe tempera-~ ' ~2~5S53 -10- 6072~-1587 ture was 25. Concentration scale for Er-EDTA is in mM units while for Mn-citrate the units are ~M.
In Figure 2, the dependence of l/Tl on added Gd ion in various forms is illustrated. Aliquots of stock solutions were added to water (Gd/citrate) or PBS (Gd/DTPA and Gd/DTPA
in vesicles) to give the to-tal concentration of ion indicated.
Figure 3 illustrates internal paramagnetic ion complex concentration effects on l/Tl and l/T2. DSPC/choleste-rol vesicles were prepared with increasing concentrations of Gd-DTPA in PBS encapsulated inside. The lipid (vesicle) con-centrations were all adjusted with PBS to be equal at 8.3 mg/
ml total lipid final concentration.
Figure 4 illustrates relaxation rates of mouse tissue and tumors. Balb/c mice were injected with 200~1 of 200 mM
Gd-DTPA in DSPC/cholesterol vesicles (10 mg/ml lipid) (Gd Ves), 200 mM La-DTPA in DSPC/cholesterol vesicles (La Ves), 2.0 mM
Gd-DTPA in PBS (Gd Buf) or PBS(Buf). After 16 hrs, the mice were sacrificed and the tissues dissected. Relaxation times ; are the average for at least 3 animals.
Figure 5 shows the effect of added poly-L-lysine on relaxation rates of Gd-DTPA solutions. Dry weighed aliquots of poly-L-lysine were dissolved in 2.0 ml of 2.0 mM Gd-DTPA in H2O. Tl and T2 were measured as described in the text.
Figure 6 illustrates the time course of l/Tl for mouse tumors. Preparations of 10 mg/ml lipid vesicles con-taining 200 mM Gd-DTPA inside were injected (200~1) into the tail vein of Balb/C mice having 10 day old EMT6 tumors from previous implants. The mice were sacrificed at intervals and Tl of the tumors measured i~ediately after dissection.
Controls were either no injection (0) or 200~1 of 2.0 mM Gd-DTPA in PBS (~). Three separate experiments are collected in this graph. E'or the O and L7 data, the points each represent .,, ~4 5S;~
Tl for a single -tumor. For the ~ data, 2 or 3 Tl values were occasionally measured for a single tumor.
Referring now in more detail to the figures of drawing, the improved results of the present invention will be discussed.
In Figure 1, relaxation rates of Er-EDTA and Mn citrate solutions are shown. Values of l/Tl are plotted as a function of ion con-centration at 10 MHz and 25C. The average value of l/Tl for mouse soft tissue is indicated on the graph. The concentration scale for Er-EDTA is millimolar while that for Mn-citrate is micromolar. Addition of 18 ~ Er-EDTA complex to a P~S solution increases l/Tl to the mouse tissue value of 2.4 s-l. The same relaxation rate is achieved with only 0.17 mM Mn-citrate com-plex. The weak complex of Mn is 100 times more efficient for relaxation enhancement than the strongly complexed Er-EDTA, re-flecting the intrinsically stronger relaxation power of the Mn (II) ion as well as the greater accessibility of the Mn to water.
In Figure 2, the relaxation effects of Gd(III) are shown. At 20 MHz the addition of Gd-citrate to H2O increases l/Tl to a value of 8.1 s at 1.0 mM. When complexed to DTPA, :~Z~5553 the ion has one-half the relaxation effect. This reduction occurs because of displacement of water binding sites by the DTPA functional groups, partly balanced by an increased rota-tional correlation time of the complex. With the Gd-DTPA com-plex encapsulated in DCPC-cholesterol vesicles, the solution 1/T1 is still increased to a value of 2.5 s-l for 1.0 mM total Gd-DTPA. While less efficient than free Gd-DTPA per unit ion, the vesicles stili have a substantial effect on water relaxa-tion.
The efEect of internal paramagnetic ion complex con-centration on relaxation rates for vesicle-encapsulated Gd-DTPA
is shown by Figure 3. 1/Tl and 1/T2 for vesicle solutions increase linearly up to 150 mM internal Gd-DTPA concentration.
Using the equation, 1 Pb 1 , wherein b T1 obsd = (Irlb+~b) T1a is for inside the vesicle and a is outside, ~ b is the life-time of water protons inside, T1b is the net relaxation time of water inside (made small by the paramagnetic agent), and Pb is the fraction of water inside the vesicle, predicts a linear dependence of 1/T1 on paramagnetic ion concentration until the value f T1 becomes on the order of or less than ~ b. The results shown by Figure 3 suggest that up to 150 m~ ~d-DTPA
concentration, T1 inside the vesicles is greater than the ex-change lifetime, ~ b.
Relaxation effects on mouse tissue and tumors are illustrated in Figure ~. The T1 values of BALB/C mouse liver, spleen, kidney and EMT6 tumor tissue are compared 16 hours after injection of paramagnetic agents or controls. Vesicle encapsulated Gd-DTPA promotes a significant reduction in T1 for spleen and for E~T6 tumors compared to the control of the dia-magnetic lanthanide ion complex of LA-DTPA in vesicles (spleen) or PBS buffer and PBS buffer plus 2.00 m.m. Gd/DTP~.
i~ . , .
~2~lS55;:~
(tumor). In the case of Gd/DTPA-vesicle treated mice, the Tl values averaged 17% less than controls wi.thout injected agent.
The foregoing data allow an estimate of the mini~um Gd/DTPA or other paramagnetic species concentrations inside vesicles which provide contrast enhancement. There is a complex set of interrelatin~ factors, such as proton exchange rate across membranes of the tumor cells, wash out rate of free Gd/DTPA from lipid vesicles, and altered rotational correlation time of the complex in a macromolecular environment, which contributes to the Tl proton relaxation rate and subsequent contrast enhancement.
The amount of accumulated vesicles in a particular tissue to be imaged dictates the minimum concentration of encapsulated para-magnetic material. For this Murine tumor model, it has been inferred that approximately 0.1~ of the tumor volume is occupied by intact vesicles.
While the quantity of paramagnetic material to be encap-su~ated will vary, depending upon the specific material used as well as the factors mentioned above, in general, the paramagnetic matexial will be at least approximately 50~M in the vesicles.
The maximum quantity will be dictated by considerations of cost, toxicity and vesicle formulation, but ordinarily will not be above about lM encapsulated concentration.
Figure 5 illustrates the enhanced relaxation rates through addition of a polymer. The relaxation effect of Gd-DTPA
can be enhanced by the addition of the positively charged polymer, poly-L-lysine. Figure 5 shows the result of adding poly-L-Lys of average M~ 25,000 to a solution of 2.0 mM Gd-DTPA
in H20. A 40~ increase in relaxation rate l/Tl is obtained and an 30~ increase in 1/T2. The effect of added poly-L-lysine plateaus above 3 mg/ml showing a "weak binding" situation. This ~2455S3 172/~0 leveling off also shows that the increased relaxation rate is ~.ot due to an increase in viscosity, since the effect there would be linear in added poly-Lys over the whole concentration range.
Smaller molecular weight poly-L-Lys is less effective on a weight basis. Gd-DTPA is a negatively charged complex which binds reversibly to the positive charge of the poly-Lys. The large size and consequent slow tumbling of the macromolecule made relaxatio~ of the paramagnetic ion more efficient. This effect can be used by co-encapsulating Gd-DTPA and poly-Lys or some like positively charged macromolecule to increase the effect per unit ion of Gd and thus decrease net toxicity of the preparation.
Time course of relaxation effect on ~MT6 tumors is shown in Figure 6. The maximal effect of vesicle-encapsulated Gd-DTPA
is achieved 3-4 hrs after injection of the agent. The average effect at 4 hrs is approximately equal to that at 16 hrs post-injection, suggesting that a steady-state condition obtains where the rate of uptake by tumor is matched by loss of agent to the circulàtion.~
Three different liposome formulations were tested at doses higher than used for data of ~igure 6 for their relaxation effects on EMT6 tumors subcutaneously implanted in Balb/c mice.
The mice were injected intravenously w1th the agent and then sacrificed at intervals. Tumor and liver Tl values were measured within l/2 hour of sacrifice of the animal. The results are set forth in Table I for tumors and in Table II for liver. Animals receiving only buffer had an average tumor T1 value of 960+41 ms (n= 25) and an average liver Tl value of 392+31 ms (n=24). The tumor relaxation time decreased to 665+2~ ms (n=4) at 24 hours post injection for the 1:1 DSPC/CHOL formulation, while the livers of these animals had average Tl values of 370+13 ms 55S~
(n=4). The Tl change of 44~ for the tumors is substantially larger than that for the liver (6%)l With many liposome formulations in common use, liver (and spleen) accumulate the largest fraction of the vesicle dose. The particular formulation of the present invention is thus far more specific for the tumor, at least in its effect on NMR relaxation times. The vesicle-encapsulated paramagnetic complex of the present invention accordingly fulfills the requirement of an NMR imaging contrast agent; that is, it Leads to reduced values of Tl in selected tissues. In this case, the ori~inal long Tl of the tumor before contrast agent (average 960 ms) will leave the tumor dark in an NMR image, while, after injection of agent, the tumor would appear brighter in the scan.
12~5SS3 Table I. TUMOR RELAXATION RATE
EMT6 Tumor in Flank of 3alb/c .~ouse (10 day tumor growth) Vesicle-encapsulated NMR Contrast Agent Values are Tl (in ms) + standard deviation n = number of mic:e . Formulation * Post-injection Time (hr) Notes .
_ PBS Control962 + 24974 + 50920 i 19 926 Global Average n = 9 n = 11 n = 4 960 + 41 n = 25 DPPC/CHOL 2:1869 + 28840 + 30845 + 40 Gd/DTPA 200 mN
n = 14 n = 13 n = 13 DSPC/CHOL 2:1812 + 45768 + 30769 + 28 GD/DTPA 200 mMl n = 8 n = 8 n = 4 DSPC/CHOL 1:1 710 + 19720 + 21 665 + 28 Gd/DTPA 200 mM
n = 2 n = 3 n = 4 * Injection volume = 250-300 ml Lipid concentration generally 20 mg/ml ~2~5553 Table II. LIVER RELAXATION RATE
Tumor bearing Balb/c Mouse Post Contrast Agent Injection Vesicle-encapsulated NMR Contrast Agent Values are Tl (in ms) + standard deviation n = number of mice Formulation * Post-injection Time (hr) Motes 1-2 2-5 5-~ 24 PBS Control400 + 39380 + 26411 + 6 412 Global Average n = 8 n = 12 n = 3 n = 1 392 + 31 n = 24 DPPC/CHOL 2:1379 + 35377 + 29375 + 34 Gd/DTPA 200 mN
n = 11 n = 11 n = 11 ,. . ... . .. . ..
DSPC/CHOL 2:1349 + 17345 + 13379 + 29 GD/DTPA 200 mMl n = 11 n = 11 n = 7 DSPC/CHOL 1:1 330 + 32 342 + 11 370 + 13 Gd/DTPA 200 mM
n = 2 n = 3 n - 4 * Injection volume = 250-300 ml Lipid concentratiorl generally 20 mg/ml ~LZ9~5553 For an NMR imaging contrast agent to be most useful, it must yield the maximum increase of l/Tl possible with minimum toxicity, and have specificity for tissue type. The invention provides these features. A macromo:Lecular assembly can increase the relaxation effect per unit ion, as demons-trated by the effect of added poly-Lys on l/Tl and l/T2 of Gd-DTPA solutions (Figure 5). Low toxicity is gained by associating the normally toxic paramagnetic ion with a strong chelate in a macromolecular assembly (e.g. encapsulation in a vesicle) which keeps the ion out of circulation. NMR relaxation is enhanced by formulating the vesicle to maximizeaccess of H20 protons to the ion. This was accomplished as shown by the strong relaxation effect of encapsulated Gd-DTPA (Figure 2). Tissue specificity is provided by the complex nature of the micellularassembly for ~hich biological recognitionprocesses causethe macromolecule to distribute to certain sites. This is demonstrated for phospholipid vesicles by the differential influence on tissue relaxation rates (Figure 4,tables I and II) and by the specific effect on tumor relaxation of Gd-DTPA encapsulated in vesicles versus approximately the same total concentration of Gd-DTPA free in solution (Figures 4and 6).
It has been described herein that antibodies can be bound to vesicles to obtain tissue specificity, Martin et al, Immunospecific Targeting of Liposomes to Cells, Biochemistry, 1981, 20, p. 4229~4238. Antimyosin has potential for NMR
imaging ofinfarcted heart muscle. Moreover preparation of antifibrin has recently been reported; Hui et al, Monoclonal Antibodies to a Synthetic Fibrin-Like Peptide Bind to Human Fibrin but not Fibrinogen, Science, 1983, 222 p. 1129-1131.
This ~Z~555~
antibody would be expected to concentrate at the sites of blood clots, where ibrin has been formed. Vesicle agent carriers attached to antifibrin could provide NMR contrast for imaging clots and thrombin in blood vessels. There are, ho~ever, other surface modiications which provide for cell recognition that are known to alter the biodistribution of the vesicles. For example, carbohydrate receptor analogues bound to the vesicle surface have been shown to target vesicles. (Mauk, et al., Targeting of Lipid Vesicles: Specificity of Carbohydrate Receptor Analogues for Leukocytes in Mice, Proc. Nat'l. Acad. Sci. USA 77, 4430-4434 (1980); Mauk, et al., Vesicle Targeting: Timed Release for Leukocytes in Mice by Subcutaneous Injection, Science 207, 309-311 (1980).) Such targeting by surface modifications are directly applicable for altering the biodistribution of para-magnetic ion.
lar pa~-ti~l-~ such as phospholipid vesicles.
NMR imaging of humans is fast becoming a major diag-nostic tool. Resolution is now on a par with X-ray CT imaging, but the key advantage of ~MR is its ability to discriminate between tissue types (contrast) on the basis of differing ~MR
relaxation times, Tl and T2. Because nuclear relaxation times can be strongly affected by paramagnetic ions such as Mn(II) and Gd(III) or stable free radicals, these materials have been explored to determine their ability to ~24~3 172/~0 provide further contrast, specifically to test whether they alter water proton Tl and T2 valuas in excised animal organs and in live animals; see, for example, Mendonca Dias et al, The Use of Paramagnetic Contrast Agents in NMR Imaginq, Absts. Soc. Mag.
Res. Med., 1982, pages 103, 104; Brady et al, Proton Nuclear Magnetic Resonance Imaging of Regionally Ischemic Canine Hearts:
effects of Paramagnetic Proton Signal_Enhancement, Radiology, 1982, 144, pages 343-347; and ~rasch et al, Evaluation of Nitroxide Stable Free Radicals for Contrast Enhancement in NMR
Imagin~, Absts. Soc. Mag. Res. Med., 1982, pages 25, 26; Brasch, Work in Progress: Methods of Contrast 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, J. Comput. Asst. Tomogr., 1984, 8, p. 204-207. Results reported show that contrast is enhanced by a variety of paramagnetic agents.
However, useful compounds, due to the nature of the candidate paramagnetic materials, may be toxic at the concen-trations required for optimal effect, and findin~ contrast agents for which the toxicity is low enough to make possible their eventual use in medical diagnosis is regarded as the most serious and difficult problem in the field, Mendonca Dias et al, The Use of Paramagnetic Contrast Agents in NMR Imaging, Absts. Soc. Mag.
Res. Med., 1982, pages 105, 106. The invention described herein is thus designed to reduce toxicity and increase the utility of NMR contrast agents by associating a paramagnetic material with a cle~
~uu~L~ eieY~- having properties tailored to the unique demands of NMR imaging Another significant problem which must be addressed is that the maximum tissue volume occupied by micelles such as J~Z~sS3 vesicles generally does not exceed about 0.1~, which means that the micelle must be capable of affecting an image with a very small volume percentage. In this regard, however, paramagnetic NMR contrast agents differ fundamentally from contrast agents used as X-ray absorbers, gamma ray emitters or the like in other imaging modalities in which the signal or attenuation is simply proportional to the number per unit volume, no matter how they are chemically bound or entrapped. In NMR, the agent (ion or stable ree radical) acts to increase the relaxation rate of bulk .. . . . . . . .... . ...
water protons surrounding the free electron spin. The phenomenon depends on rapid exchange of water on and off an ion or rapid diffusion of water past an organic free radical. In such case, . .
the net relaxation rate is a weighted average for free and bound water.
Encapsulation of the paramagnetic material within a phospholipid vesicle, as in one preferred form of this invention, would seem to deny access of the para~agnetic agent to all but the entrapped water, typically less than 0.1~ of the total volume. Under--such conditions, the NMR image would not be altered-detectably by the presence of vesicle-encapsulated contrast agent. Only if water exchanges sufficiently rapidly across the bilayer is the relaxation rate of the bulk water enhanced, Andrasko et al, NMR Study of Rapid Water Diffusion . .
Across Lipid Bylayers in DipalymitoYl Lecithin Vesicles, Biochem. Biophys. Res. Comm., 1974, 60, p. 813-819. The present invention addresses this problem by providing a formulation of micelle and paramagnetic material that simultaneously maximizes micelle stability while permitting adequate rates of water exchange across the membrane.
~24S5~3 72/40 Phospholipid vesicles are known to concentrate in certain tissues, so additional enhancement will come from tissue specificity. For example, phospholipid vesicles have been observed to accumulate in implanted tumors of mice, Proffitt et al, Liposomal Blockade of the Reticuloendothelial System:
Improved Tu_or Imaging with Small Unilamella Vesicles, Science, 1983 220 p. 502-505, Proffitt et al, Tumor-Imaging Potential of Li~osomes ~oaded with In~ NTA: Biodistribution in Mice, Journal of Nuclear Medicine, 1983, 24, p. 45-51.
~e~/c/es The invention also extends the use of ~f~L~d~
L6l4~_as contrast agent carriers to applications where the micelles are attached to antibodies. While it has been reported that a selective decrease in Tl relaxation times of excised heart may be obtained using manganese-labeled monoclonal antimyosin antibody, Brady,-et al, Selective Decrease in the Relaxation ~-Times of Infr rèd Myocardium with the Use of a Manganese-Labelled Monoclonal Antibody, Soc. Magn. Res. Med., Works in Progress, Second Annual Meeting, 1983,-p. lO, heretofore, due in large méasure to considerations such as toxicity referred to above, the practical use of such antibodies has been significantly restricted. With the present invention, however, increased sensitivity is obtained and specificity is maintained by attach-~ ~e~l~le ment of antibody to the surface of mio~lular particlcc. The antibodies provide high specificity for cell or tissue types, while the attached vesicle agent carriers amplify the NMR
contrast enhancement over what can be achieved with ions bound to antibody alone.
.
lZ45~5~
The invention described herein is directed to pre-parations of vesicles such as small unilamellar vesicles, with which a paramagnetic material is associated, typically para-magnetic compounds enclosed within the vesicles. The vesicles may or may not have antibodies, such as antimyosin, or anti-fibrin, attached to the surface or have other surface modifica-t-ions for which there are specific cell receptors in certain tissue.
Examples of vesicle constituents are phospholipids such as distearoylphosphatidylcholine (DSPC), dipalmitoylphos-phatidylcholine (DPPC), and dimyristoylphosphatidylcholine (DMPC). Examples of paramagnetic materials are salts of trans-ition metals and the lanthanide and actinide series of the periodic table such, as Gd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II~, Er(III), nickel(II) and complexes of such ions with diethylenetriaminepentaacetic acid (DTPA), ethylenediami-~etetraacetic acid (EDTA) and other ligands. Other paramagnetic compounds include stable free radicals such as organic nitroxides.
Vesicle-encapsulated contrast agents may be prepared by forming the lipid vesicles in an aqueous medium containing the paramagnetic agent by any suitable means such as sonication, homogenization, cholate dialysis and the like, and then freeing the vesicles of external agent by ultrafiltration, gel filtrat-ion or similar method. Moreover, the internal solution of the paramagnetic material may be altered readily -to maximize the relaxation rate per unit of agent as for example, by formulation with a charged polymeric material such as poly-L-Lysine.
According to the present invention there are provided magnetic resonance imaging contrast agents comprising vesicles with paramagnetic material encapsulated therein, said vesicles formulated with an agent -to promote vesicle stability and water excnange across the vesicle bilayer for a time sufficient to ~Z4555;~
enable scanning to be effected.
According to another aspect of the present invention there is provided a process for preparing magnetic resonance imaging contrast agents comprising vesicles with paramagnetic material encapsulated therein ar.d formulated with an agent to promote vesicle stability and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effec-ted which comprises encapsulating paramagnetic material in vesicles and formulating said vesicles with an agent to promote vesicle stabi~ity and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effected.
As used herein, "vesicle" and "micelles" refer to particles which result from aggregations of amphiphilic mole-cules. In this invention, preferred amphiphiles are biological lipids.
"Vesicle" refers to a micelle which is in a generally spherical form, often obtained from a lipid which forms a bi-layered membrane and is referred to as a "liposome". Methods for forming these vesicles are, by now, very well known in the art. Typically, they are prepared from a phospholipid, for example, distearoyl phosphatidylcholine or lecithin, and may in-clude other materials such as neutral lipids, and also surface modifiers such as positively or negatively charged compounds.
Depending on the techniques for their preparation, the envelope may be a simple bilayered spherical shell (a unilamellar vesicle) or may have multiple layers within the envelope (multi-lamellar vesicles). DSPC = distearoyl phosphatidylcholine Ch = cholesterol DPPC = dipalmitoylphosphatidylcholine DMPC = dimyristoylphosphatidylcholine DTPA = diethylenetriaminepentaacetic acid EDTA = ethylenediaminetetracetic acid SUV = small unilamellar vesicles ~' iZ4S5S3 Materials and Preparation of Micelles Complexes of paramagnetic compounds were prepared in deionized water or in a buffer of 4.0 mM Na2HPO4, 0.9% (by weight) NaCl, pH 7.4 (PBS).
Gd(III)-citrate. A stock solu-tion of 1.0 mM Gd(III) 10.0 mM citrate was made by dissolving 10.0 ~moles GdC13.6H2O
(99.999%, Aldrich) in 9 ml deionized water and adding 100 llm~lesNa3citrate (analytical reagen-t, Mallinckrodt). The pH
was adjus-ted to neutrality and the volume brought to 10.0 mL in a volumetric flask.
Mn(II)-citrate. A stock solu-tion of 1.0 mM Mn (II) 10.0 mM citrate was made by adding 10.0 ~moles MnC12.4H20 (Baker analyzed) and 100 ~moles sodium citrate to 9 ml water.
The solution was neutralized and made up to 10.0 ml in a volumetric flask.
Gd(III)-DTPA. A stock solution of 200 mM Gd(III)-210 mM DTPA was made by dissolving 2.10 m~oles DTPA in minimum 6 N NaOH in a 10 ml volumetric flask. 2.0 mmoles GdC13.6H20 were added and the pH adjusted to 7.4 with 6 N NaOH, after which the sample was made up to 10.0 ml in the flask.
La(III)-DTPA. A stock solution of 200 mM La(III)-210 mM DTPA was made up in a manner analogous to the Gd(III) DTPA stock using LaC13.7H2O (99.999%, Aldrich).
Er(III)-EDTA. Stock solutions were prepared in a manner analogous to Gd(III)-DTPA.
Poly-L-lysine hydrobromide of approximate average molecular weights 25,000 and 4,000 were obtained from Sigma Chemical Co.
Cholesterol (98%) was from Mallinckrodt. DSPC was synthetic material from Cal-Biochem.
DSPC/cholesterol vesicle-encapsulated NMR contrast agent. 16 mg DSPC and 4 mg cholesterol were dissolved in 2 ml .. . .
, S5S~
CHC13. 10~1 of a solution of 0.16 mM cholesterol [u-14C] (56.5 mCi/mmole) in CHC13 were added for purposes of quantitating lipid concentra-tions in the final preparations. The lipid sol-ution was evaporated to dryness in a vacuum dessicator and stored in the same, if not used immediately.
Small unilamellar vesicles (SUV) were formed in a solution of 200 mM Gd(III)-DTPA by adding 2.0 ml of the stock ion complex to the dried lipid tube. The mixture was sonicated using an Ultrasonics, Inc. probe with a microtip at a power level of 56 W. The tube was cooled by partial immersion in a water bath, and N2 was flowed over the sample during sonication.
Total time of sonication was 15 min. or more until the solution was slightly opalescent.
Paramagnetic agent outside the vesicles was separated from the SUVs by passage through columns of Sephadex G-50 swollen in PBS, that had been loaded into 3 ml plastic syringe bodies and precentrifuged. The vesicle solution was placed at the top of the syringe and centrifuged with a glass tube pos-itioned to collect the eluate. 300 ~1 PBS was used to elute the vesicles from the columns. The procedure was repeated a total of 3 times to reduce the outside concentration of free agent and to exchange it for PsS.
Vesicle concentration in the final preparation was measured by counting an ali~uo-t of the solution in the scintil-lation counter, using a standard cocktail. Average vesicle size was measured in a laser particle sizer Model 2~0 (Nicomp Instruments). The vesicle size was measured to be 600, + 100, A in all experiments.
NMR Relaxation time measuremen-ts Unless otherwise indicated, measurements of Tl and T2 were made at 20 MHz with a pulsed ~MR spectrometer (IBM PC/20) *Trademar~
1~9~553 60724-1587 interfaced to a microcomputer (IBM PC). Tl was measured by the inversion-recovery method (Farrar, T.C., Becker, E.D., Pulse and Fourier Transform NMR, 1971, Academic Press, New York.) and T2 by the Carr-Purcell sequence (Carr, H.Y., Purcell, E.H., Effects of Diffusion on Free Precession in NMR Experiments, Phys. Rev., 1954, 94, p. 630-633.), as modified by Meiboom and Gill (Meiboom S., Gill D., Modified Spin-Echo Method Eor Measuriny Nuclear Relaxation Times, Rev. Sci. Instrum., 1958, 29, p. 688-691.) Least-squares best fits of the data to single exponential recoveries were done automatically by the computer.
Values of Tl and T2 reported are for a p~robe temperature of 38C. Tl values are estimated to have an experimental uncer-tainty of + 10% and a reproducibility of + 5%. T2 values are generally accurate to within + 20% and reproducible to + 5%.
This precision is sufficient clearly to demonstrate the effects claimed. Some values of Tl were measured with a Praxis II NMR
spectrometer operating at 10 MHZ and a probe temperature of 25 C.
Animal Studies EMT6 tumor tissue was transplanted subcutaneously into the flank of female Balb/c mice and allowed to grow for 10 days.
On the 10th day, mice were injec-ted i.v. with 200ul of vesicle solution or control buffer. Mice were sacrificed a-t intervals, and the tumors were dissected. In some experiments liver and spleen were also dissected. The tissue was rinsed in PBS, lightly blotted, weighed, and wrapped in air tight plastic bags.
NMR relaxation measurements were made within 2 hr of dissection to limit water loss and consequent changes in Tl and T2.
Figure l is a plot of longitudinal relaxation rate as a function of added paramagnetic ion concentration. Aliquo-ts of the stock solutions were added to PBS buffer. Tl measuremen-ts were made at 10 MHz using the 90- -90 method. Probe tempera-~ ' ~2~5S53 -10- 6072~-1587 ture was 25. Concentration scale for Er-EDTA is in mM units while for Mn-citrate the units are ~M.
In Figure 2, the dependence of l/Tl on added Gd ion in various forms is illustrated. Aliquots of stock solutions were added to water (Gd/citrate) or PBS (Gd/DTPA and Gd/DTPA
in vesicles) to give the to-tal concentration of ion indicated.
Figure 3 illustrates internal paramagnetic ion complex concentration effects on l/Tl and l/T2. DSPC/choleste-rol vesicles were prepared with increasing concentrations of Gd-DTPA in PBS encapsulated inside. The lipid (vesicle) con-centrations were all adjusted with PBS to be equal at 8.3 mg/
ml total lipid final concentration.
Figure 4 illustrates relaxation rates of mouse tissue and tumors. Balb/c mice were injected with 200~1 of 200 mM
Gd-DTPA in DSPC/cholesterol vesicles (10 mg/ml lipid) (Gd Ves), 200 mM La-DTPA in DSPC/cholesterol vesicles (La Ves), 2.0 mM
Gd-DTPA in PBS (Gd Buf) or PBS(Buf). After 16 hrs, the mice were sacrificed and the tissues dissected. Relaxation times ; are the average for at least 3 animals.
Figure 5 shows the effect of added poly-L-lysine on relaxation rates of Gd-DTPA solutions. Dry weighed aliquots of poly-L-lysine were dissolved in 2.0 ml of 2.0 mM Gd-DTPA in H2O. Tl and T2 were measured as described in the text.
Figure 6 illustrates the time course of l/Tl for mouse tumors. Preparations of 10 mg/ml lipid vesicles con-taining 200 mM Gd-DTPA inside were injected (200~1) into the tail vein of Balb/C mice having 10 day old EMT6 tumors from previous implants. The mice were sacrificed at intervals and Tl of the tumors measured i~ediately after dissection.
Controls were either no injection (0) or 200~1 of 2.0 mM Gd-DTPA in PBS (~). Three separate experiments are collected in this graph. E'or the O and L7 data, the points each represent .,, ~4 5S;~
Tl for a single -tumor. For the ~ data, 2 or 3 Tl values were occasionally measured for a single tumor.
Referring now in more detail to the figures of drawing, the improved results of the present invention will be discussed.
In Figure 1, relaxation rates of Er-EDTA and Mn citrate solutions are shown. Values of l/Tl are plotted as a function of ion con-centration at 10 MHz and 25C. The average value of l/Tl for mouse soft tissue is indicated on the graph. The concentration scale for Er-EDTA is millimolar while that for Mn-citrate is micromolar. Addition of 18 ~ Er-EDTA complex to a P~S solution increases l/Tl to the mouse tissue value of 2.4 s-l. The same relaxation rate is achieved with only 0.17 mM Mn-citrate com-plex. The weak complex of Mn is 100 times more efficient for relaxation enhancement than the strongly complexed Er-EDTA, re-flecting the intrinsically stronger relaxation power of the Mn (II) ion as well as the greater accessibility of the Mn to water.
In Figure 2, the relaxation effects of Gd(III) are shown. At 20 MHz the addition of Gd-citrate to H2O increases l/Tl to a value of 8.1 s at 1.0 mM. When complexed to DTPA, :~Z~5553 the ion has one-half the relaxation effect. This reduction occurs because of displacement of water binding sites by the DTPA functional groups, partly balanced by an increased rota-tional correlation time of the complex. With the Gd-DTPA com-plex encapsulated in DCPC-cholesterol vesicles, the solution 1/T1 is still increased to a value of 2.5 s-l for 1.0 mM total Gd-DTPA. While less efficient than free Gd-DTPA per unit ion, the vesicles stili have a substantial effect on water relaxa-tion.
The efEect of internal paramagnetic ion complex con-centration on relaxation rates for vesicle-encapsulated Gd-DTPA
is shown by Figure 3. 1/Tl and 1/T2 for vesicle solutions increase linearly up to 150 mM internal Gd-DTPA concentration.
Using the equation, 1 Pb 1 , wherein b T1 obsd = (Irlb+~b) T1a is for inside the vesicle and a is outside, ~ b is the life-time of water protons inside, T1b is the net relaxation time of water inside (made small by the paramagnetic agent), and Pb is the fraction of water inside the vesicle, predicts a linear dependence of 1/T1 on paramagnetic ion concentration until the value f T1 becomes on the order of or less than ~ b. The results shown by Figure 3 suggest that up to 150 m~ ~d-DTPA
concentration, T1 inside the vesicles is greater than the ex-change lifetime, ~ b.
Relaxation effects on mouse tissue and tumors are illustrated in Figure ~. The T1 values of BALB/C mouse liver, spleen, kidney and EMT6 tumor tissue are compared 16 hours after injection of paramagnetic agents or controls. Vesicle encapsulated Gd-DTPA promotes a significant reduction in T1 for spleen and for E~T6 tumors compared to the control of the dia-magnetic lanthanide ion complex of LA-DTPA in vesicles (spleen) or PBS buffer and PBS buffer plus 2.00 m.m. Gd/DTP~.
i~ . , .
~2~lS55;:~
(tumor). In the case of Gd/DTPA-vesicle treated mice, the Tl values averaged 17% less than controls wi.thout injected agent.
The foregoing data allow an estimate of the mini~um Gd/DTPA or other paramagnetic species concentrations inside vesicles which provide contrast enhancement. There is a complex set of interrelatin~ factors, such as proton exchange rate across membranes of the tumor cells, wash out rate of free Gd/DTPA from lipid vesicles, and altered rotational correlation time of the complex in a macromolecular environment, which contributes to the Tl proton relaxation rate and subsequent contrast enhancement.
The amount of accumulated vesicles in a particular tissue to be imaged dictates the minimum concentration of encapsulated para-magnetic material. For this Murine tumor model, it has been inferred that approximately 0.1~ of the tumor volume is occupied by intact vesicles.
While the quantity of paramagnetic material to be encap-su~ated will vary, depending upon the specific material used as well as the factors mentioned above, in general, the paramagnetic matexial will be at least approximately 50~M in the vesicles.
The maximum quantity will be dictated by considerations of cost, toxicity and vesicle formulation, but ordinarily will not be above about lM encapsulated concentration.
Figure 5 illustrates the enhanced relaxation rates through addition of a polymer. The relaxation effect of Gd-DTPA
can be enhanced by the addition of the positively charged polymer, poly-L-lysine. Figure 5 shows the result of adding poly-L-Lys of average M~ 25,000 to a solution of 2.0 mM Gd-DTPA
in H20. A 40~ increase in relaxation rate l/Tl is obtained and an 30~ increase in 1/T2. The effect of added poly-L-lysine plateaus above 3 mg/ml showing a "weak binding" situation. This ~2455S3 172/~0 leveling off also shows that the increased relaxation rate is ~.ot due to an increase in viscosity, since the effect there would be linear in added poly-Lys over the whole concentration range.
Smaller molecular weight poly-L-Lys is less effective on a weight basis. Gd-DTPA is a negatively charged complex which binds reversibly to the positive charge of the poly-Lys. The large size and consequent slow tumbling of the macromolecule made relaxatio~ of the paramagnetic ion more efficient. This effect can be used by co-encapsulating Gd-DTPA and poly-Lys or some like positively charged macromolecule to increase the effect per unit ion of Gd and thus decrease net toxicity of the preparation.
Time course of relaxation effect on ~MT6 tumors is shown in Figure 6. The maximal effect of vesicle-encapsulated Gd-DTPA
is achieved 3-4 hrs after injection of the agent. The average effect at 4 hrs is approximately equal to that at 16 hrs post-injection, suggesting that a steady-state condition obtains where the rate of uptake by tumor is matched by loss of agent to the circulàtion.~
Three different liposome formulations were tested at doses higher than used for data of ~igure 6 for their relaxation effects on EMT6 tumors subcutaneously implanted in Balb/c mice.
The mice were injected intravenously w1th the agent and then sacrificed at intervals. Tumor and liver Tl values were measured within l/2 hour of sacrifice of the animal. The results are set forth in Table I for tumors and in Table II for liver. Animals receiving only buffer had an average tumor T1 value of 960+41 ms (n= 25) and an average liver Tl value of 392+31 ms (n=24). The tumor relaxation time decreased to 665+2~ ms (n=4) at 24 hours post injection for the 1:1 DSPC/CHOL formulation, while the livers of these animals had average Tl values of 370+13 ms 55S~
(n=4). The Tl change of 44~ for the tumors is substantially larger than that for the liver (6%)l With many liposome formulations in common use, liver (and spleen) accumulate the largest fraction of the vesicle dose. The particular formulation of the present invention is thus far more specific for the tumor, at least in its effect on NMR relaxation times. The vesicle-encapsulated paramagnetic complex of the present invention accordingly fulfills the requirement of an NMR imaging contrast agent; that is, it Leads to reduced values of Tl in selected tissues. In this case, the ori~inal long Tl of the tumor before contrast agent (average 960 ms) will leave the tumor dark in an NMR image, while, after injection of agent, the tumor would appear brighter in the scan.
12~5SS3 Table I. TUMOR RELAXATION RATE
EMT6 Tumor in Flank of 3alb/c .~ouse (10 day tumor growth) Vesicle-encapsulated NMR Contrast Agent Values are Tl (in ms) + standard deviation n = number of mic:e . Formulation * Post-injection Time (hr) Notes .
_ PBS Control962 + 24974 + 50920 i 19 926 Global Average n = 9 n = 11 n = 4 960 + 41 n = 25 DPPC/CHOL 2:1869 + 28840 + 30845 + 40 Gd/DTPA 200 mN
n = 14 n = 13 n = 13 DSPC/CHOL 2:1812 + 45768 + 30769 + 28 GD/DTPA 200 mMl n = 8 n = 8 n = 4 DSPC/CHOL 1:1 710 + 19720 + 21 665 + 28 Gd/DTPA 200 mM
n = 2 n = 3 n = 4 * Injection volume = 250-300 ml Lipid concentration generally 20 mg/ml ~2~5553 Table II. LIVER RELAXATION RATE
Tumor bearing Balb/c Mouse Post Contrast Agent Injection Vesicle-encapsulated NMR Contrast Agent Values are Tl (in ms) + standard deviation n = number of mice Formulation * Post-injection Time (hr) Motes 1-2 2-5 5-~ 24 PBS Control400 + 39380 + 26411 + 6 412 Global Average n = 8 n = 12 n = 3 n = 1 392 + 31 n = 24 DPPC/CHOL 2:1379 + 35377 + 29375 + 34 Gd/DTPA 200 mN
n = 11 n = 11 n = 11 ,. . ... . .. . ..
DSPC/CHOL 2:1349 + 17345 + 13379 + 29 GD/DTPA 200 mMl n = 11 n = 11 n = 7 DSPC/CHOL 1:1 330 + 32 342 + 11 370 + 13 Gd/DTPA 200 mM
n = 2 n = 3 n - 4 * Injection volume = 250-300 ml Lipid concentratiorl generally 20 mg/ml ~LZ9~5553 For an NMR imaging contrast agent to be most useful, it must yield the maximum increase of l/Tl possible with minimum toxicity, and have specificity for tissue type. The invention provides these features. A macromo:Lecular assembly can increase the relaxation effect per unit ion, as demons-trated by the effect of added poly-Lys on l/Tl and l/T2 of Gd-DTPA solutions (Figure 5). Low toxicity is gained by associating the normally toxic paramagnetic ion with a strong chelate in a macromolecular assembly (e.g. encapsulation in a vesicle) which keeps the ion out of circulation. NMR relaxation is enhanced by formulating the vesicle to maximizeaccess of H20 protons to the ion. This was accomplished as shown by the strong relaxation effect of encapsulated Gd-DTPA (Figure 2). Tissue specificity is provided by the complex nature of the micellularassembly for ~hich biological recognitionprocesses causethe macromolecule to distribute to certain sites. This is demonstrated for phospholipid vesicles by the differential influence on tissue relaxation rates (Figure 4,tables I and II) and by the specific effect on tumor relaxation of Gd-DTPA encapsulated in vesicles versus approximately the same total concentration of Gd-DTPA free in solution (Figures 4and 6).
It has been described herein that antibodies can be bound to vesicles to obtain tissue specificity, Martin et al, Immunospecific Targeting of Liposomes to Cells, Biochemistry, 1981, 20, p. 4229~4238. Antimyosin has potential for NMR
imaging ofinfarcted heart muscle. Moreover preparation of antifibrin has recently been reported; Hui et al, Monoclonal Antibodies to a Synthetic Fibrin-Like Peptide Bind to Human Fibrin but not Fibrinogen, Science, 1983, 222 p. 1129-1131.
This ~Z~555~
antibody would be expected to concentrate at the sites of blood clots, where ibrin has been formed. Vesicle agent carriers attached to antifibrin could provide NMR contrast for imaging clots and thrombin in blood vessels. There are, ho~ever, other surface modiications which provide for cell recognition that are known to alter the biodistribution of the vesicles. For example, carbohydrate receptor analogues bound to the vesicle surface have been shown to target vesicles. (Mauk, et al., Targeting of Lipid Vesicles: Specificity of Carbohydrate Receptor Analogues for Leukocytes in Mice, Proc. Nat'l. Acad. Sci. USA 77, 4430-4434 (1980); Mauk, et al., Vesicle Targeting: Timed Release for Leukocytes in Mice by Subcutaneous Injection, Science 207, 309-311 (1980).) Such targeting by surface modifications are directly applicable for altering the biodistribution of para-magnetic ion.
Claims (41)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Magnetic resonance imaging contrast agents comprising vesicles with paramagnetic material encapsulated therein, said vesicles formulated with an agent to promote vesicle stability and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effected.
2. The contrast agents of claim 1 in which said vesicles are formulated with cholesterol.
3. The contrast agents of claim 1 in which antibodies, carbohydrates, a cell recognition targeting agent is attached to said vesicles, to provide specific targeting.
4. The contrast agents of claim 3 wherein the cell recognition targeting agent is an antibody or a carbohydrate.
5. The contrast agents of claim 3 or 4 in which said targeting agent is the antibody antimyosin.
6. The contrast agents of claim 3 in which said targeting agent is the antibody antifibrin.
7. The contrast agents of claim 1 or 3 in which said paramagnetic material is a salt of a transition metal or the lanthanide or actinide series of the Periodic Table.
8. The contrast agents of claim 6 in which said paramag-netic material is a salt of a paramagnetic ion selected from manganese, copper, gadolinum, erbium, chromium, iron, cobalt and nickel.
9. The contrast agents of claim 1 in which said paramag-netic material is a paramagnetic compound including a stable free radical.
10. The contrast agents of claim 1 or 3 in which said paramagnetic material is a paramagnetic compound of a paramag-netic ion and a chelate.
11. The contrast agents of claim 1 or 3 in which said paramagnetic material is present in a concentration of at least approximately 50 mM.
12. The contrast agents of claim 1 in which said para-magnetic material is present in a concentration of from about 50 mM to approximately 1 M.
13. The contrast agents of claim 9 in which the effi-ciency of said paramagnetic material is enhanced by the addi-tion of a charged polymer thereto.
14. The contrast agents of claim 9 in which said charged polymer is poly-L-lysine.
15. Magnetic resonance imaging contrast agents comprising phospholipid vesicles formulated with an agent to promote vesicle stability and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effected with paramagnetic material encapsulated therein, said paramagnetic material being a paramagnetic ion associated with a chelate.
16. The contrast agents of claim 15 in which said vesicle are formulated with cholesterol.
17. The contrast agents of claim 16 in which said para-magnetic material is Gd-DTPA.
18. The contrast agents of claim 17 in which said para-magnetic material is present in a concentration of at least approximately 50 mM.
19. The contrast agents of claim 18 in which said para-magnetic material is present in a concentration of from about 50mM to approximately 1 M.
20. Magnetic resonance imaging contrast agents comprising vesicles having a paramagnetic material associated therewith, said vesicles formulated with an agent to promote vesicle stability and water exchange across the bilayer for a time sufficient to enable scanning to be effected.
21. The contrast agents of claim 20 in which said para-magnetic material is associated with a chelating agent bound to the surface of said vesicles.
22. A process for preparing magnetic resonance imaging contrast agents comprising vesicles with paramagnetic material encapsulated therein and formulated with an agent to promote vesicle stability and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effected which comprises encapsulating paramagnetic material in vesicles and formulating said vesicles with an agent to promote vesicle stability and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effected.
23. A process according to claim 22 wherein said vesicles are formulated with cholesterol.
24. A process according to claim 22 wherein antibodies, carbohydrates, or other cell recognition targeting agents are attached to said vesicles, to provide specific targeting.
25. A process according to claim 24 wherein said targeting agent is the antibody antimyosin.
26. A process according to claim 24 wherein said targeting agent is the antibody antifibrin.
27. A process according to claim 22 wherein said para-magnetic material is a salt of a transition metal or the lanthanide or actinide series of the Periodic Table.
28. A process according to claim 22 wherein said para-magnetic material is a salt of a paramagnetic ion selected from manganese, copper, gadolinum, erbium, chromium, iron, cobalt, and nickel.
29. A process according to claim 22 wherein said para-magnetic material is a paramagnetic compound including a stable free radical.
30. A process according to claim 22 wherein said para-magnetic material is a paramagnetic compound of a paramagnetic ion and a chelate.
31. A process according to claim 22 wherein said para-magnetic material is present in a concentration of a least approximately 50 mM.
32. A process according to claim 22 wherein said para-magnetic material is present in a concentration of from about 50 mM to approximately 1M.
33. A process according to claim 22 wherein the effi-ciency of said paramagnetic material is enhanced by the addition of a charged polymer thereto.
34. A process according to claim 33 wherein said charged polymer is poly-L-lysine.
35. A process for preparing magnetic resonance imaging contrast agents comprising phospholipid vesicles formulated with an agent to promote vesicle stability and water exchange across the vesicle bilayer fox a time sufficient to enable scanning to be effected with paramagnetic material encapsulated therein, said paramagnetic material being a paramagnetic ion associated with a chelate, which comprises encapsulating para-magnetic material in phospholipid vesicles and formulating said vesicles with an agent to promote vesicle stability and water exchange across the bilayer for a time sufficient to enable scanning to be effected.
36. A process according to claim 35 wherein said vesicles are formulated with cholesterol.
37. A process according to claim 35 wherein said para-magnetic material is Gd-DTPA.
38. A process according to claim 37 wherein said para-magnetic material is present in a concentration of at least approximately 50 mM.
39. A process according to claim 38 wherein said para-magnetic material is present in a concentration of from about 50 mM to approximately 1M.
40. A process for preparing magnetic resonance imaging contrast agents comprising vesicles having a paramagnetic material associated therewith which comprises associating paramagnetic material with vesicles and formulating said vesicles with an agent to promote vesicle stability and water exchange across the vesicle bilayer for a time sufficient to enable scanning to be effected.
41. A process according to claim 40 wherein said para-magnetic material is associated with a chelating agent bound to the surface of said vesicles.
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| US06/720,954 US4728575A (en) | 1984-04-27 | 1985-04-10 | Contrast agents for NMR imaging |
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| CA1245553A true CA1245553A (en) | 1988-11-29 |
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| EP (1) | EP0160552B1 (en) |
| KR (1) | KR920010391B1 (en) |
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| HU (1) | HU194741B (en) |
| IE (1) | IE58354B1 (en) |
| IL (1) | IL74985A (en) |
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| ATE61117T1 (en) | 1991-03-15 |
| EP0160552B1 (en) | 1991-02-27 |
| IL74985A0 (en) | 1985-08-30 |
| DE3581830D1 (en) | 1991-04-04 |
| IL74985A (en) | 1989-03-31 |
| DK188785A (en) | 1985-10-28 |
| DK188785D0 (en) | 1985-04-26 |
| ES542604A0 (en) | 1987-01-16 |
| HU194741B (en) | 1988-03-28 |
| US4728575A (en) | 1988-03-01 |
| EP0160552A2 (en) | 1985-11-06 |
| AU4170385A (en) | 1985-10-31 |
| KR920010391B1 (en) | 1992-11-27 |
| EP0160552A3 (en) | 1986-08-20 |
| ES8703019A1 (en) | 1987-01-16 |
| AU585506B2 (en) | 1989-06-22 |
| DK168853B1 (en) | 1994-06-27 |
| KR850007562A (en) | 1985-12-07 |
| IE851035L (en) | 1985-10-27 |
| IE58354B1 (en) | 1993-09-08 |
| HUT37569A (en) | 1986-01-23 |
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