MRI CONTRAST AGENT FOR USE IN THE DIAGNOSIS OF EARLY CHANGES IN THE ENDOTHELIUM OF BLOOD VESSELS

Abstract

MRI contrast agent for use in the diagnosis of early changes in the endothelium of blood vessels

Claims

1. An MRI contrast agent for use in the diagnosis of early changes in blood vessel endothelium, in the form of an aqueous suspension containing as a hydrophilic contrast agent Gd-diethylenetriaminepentaacetate-bis (methylamide) encapsulated within single-layer liposomes from 50 nm to 200 nm in size, wherein the lipid membrane of liposomes is made of a mixture containing: phosphatidylcholine, cholesterol and polyethylene glycol covalently bounded to a phosphatidylethanolamine molecule (PEG-PE).

2. The contrast agent according to claim 1, characterized in that the molar ratio of contrast agent to lipid components is from 1:50 to 2:1.

3. The contrast agent according to claim 1, characterized in that the molar ratio of lipids to cholesterol in the lipid membrane is from 10:1 to 5:1.

4. The contrast agent according to claim 1, characterized in that the monolayer liposomes contain PEG2000-PE in an amount of up to 10 mol % of all amphiphilic components.

5. The contrast agent according to claim 1, characterized in that it contains at least one of the components selected from the group consisting of caldiamide, sodium hydroxide, osmotically active substance, antioxidant, and/or a mixture thereof.

6. The contrast agent according to claim 5, characterized in that the osmotically active substance contains a substance selected from the group consisting of saline, mono- or disaccharide solution and/or a mixture thereof.

7. The contrast agent according to claim 1, characterized in that the concentration of the hydrophilic contrast agent in the aqueous phase of the suspension is from 1 to 200 g/L.

Description

EXAMPLE 1

[0019] Gd-diethylenetriaminepentaacetate-bis(methylamide), also known as gadodiamide, dissolved in an aqueous solution of sodium caldiamide at pH 7.45 was added to purified phosphatidylcholine (Lipoid GmbH, Germany) dissolved in propylene glycol. The whole mixture was stirred at 60° C. until a homogeneous, milky suspension was formed. Then the suspension thus obtained was extruded through polycarbonate filters with a pore diameter of 100 nm to obtain a homogenous liposome gel characterized by uniform size of liposomes encapsulating the contrast agent.

TABLE-US-00001 TABLE 1 Contrast agent composition. % by weight CONTRAST AGENT: Gd-diethylenetriaminepentaacetate-bis(methylamide) 14.8 ADDITIONAL SUBSTANCES: Purified phosphatidylcholine 30 Propylene glycol 20 Caldiamide sodium 0.62 Sodium hydroxide 0.01 Purified water 34.57 TOTAL 100

[0020] The molar ratio of Gd-diethylenetriaminepentaacetate-bis(methylamide) to amphiphilic substances is about 1:2. The average size of liposomes with an encapsulated contrast agent in an aqueous solution at pH 7.4 is 124 nm with PDI=0.12. The encapsulation efficiency was 77%±5%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methylamide) was determined by ultrafiltration.

[0021] FIG. 1 shows the homogeneity of the liposome suspension. The left panel shows the distribution of liposome sizes with an encapsulated contrast agent in a pH 7.4 aqueous solution and the right panel shows the quality of matching the correlation function to experimental data. The encapsulation efficiency was 77%±5%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methylamide) was determined by ultrafiltration.

EXAMPLE 2

[0022] Gd-diethylenetriaminepentaacetate-bis(methylamide) dissolved in an aqueous solution of sodium caldiamide at pH 7.45 was added to purified phosphatidylcholine (Lipoid) with the addition of cholesterol dissolved in propylene glycol. The whole mixture was stirred at 60° C. until a homogeneous, milky suspension was formed. Then the suspension thus obtained was extruded through polycarbonate filters with a pore diameter of 100 nm to obtain a homogenous liposome gel characterized by uniform size of liposomes encapsulating the contrast agent.

TABLE-US-00002 TABLE 2 Contrast agent composition. % by weight CONTRAST AGENT: Gd-diethylenetriaminepentaacetate-bis(methylamide) 14.8 ADDITIONAL SUBSTANCES: Purified phosphatidylcholine 27 Cholesterol 3 Propylene glycol 20 Caldiamide sodium 0.62 Sodium hydroxide 0.01 Purified water 34.57 TOTAL 100

[0023] The molar ratio of Gd-diethylenetriaminepentaacetate-bis(methylamide) to amphiphilic substances is about 1:2. The average size of liposomes with an encapsulated contrast agent in an aqueous solution at pH 7.4 is 132 nm with PDI=0.17. The encapsulation efficiency was 87%±6%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methyl amide) was determined by ultrafiltration.

[0024] FIG. 2 shows the homogeneity of the liposome suspension. The left panel shows the distribution of liposome sizes with an encapsulated contrast agent in a pH 7.4 aqueous solution and the right panel shows the quality of matching the correlation function to experimental data. The encapsulation efficiency was 87%±6%. The encapsulation efficiency of gadolinium was determined using ultrafiltration. Example 2 shows that the lipid composition of the liposome can change the encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methyl amide).

EXAMPLE 3

[0025] Gd-diethylenetriaminepentaacetate-bis(methylamide) dissolved in an aqueous solution of sodium caldiamide at pH 7.45 was added to purified phosphatidylcholine (Lipoid) with the addition of cholesterol and PEG-PE (polyethylene glycol with a molecular weight of 2000 Da covalently bound to a phosphatidylethanolamine molecule) dissolved in propylene glycol. The whole mixture was stirred at 60° C. until a homogeneous, milky suspension was formed. Then the suspension thus obtained was extruded through polycarbonate filters with a pore diameter of 100 nm to obtain a homogenous liposome gel characterized by uniform size of liposomes encapsulating the contrast agent.

TABLE-US-00003 TABLE 3 Contrast agent composition. % by weight CONTRAST AGENT: Gd-diethylenetriaminepentaacetate-bis(methylamide) 14.8 ADDITIONAL SUBSTANCES: Purified phosphatidylcholine 22 Cholesterol 6 PEG-PE 2 Propylene glycol 20 Caldiamide sodium 0.62 Sodium hydroxide 0.01 Purified water 34.57 TOTAL 100

[0026] The molar ratio of Gd-diethylenetriaminepentaacetate-bis(methylamide) to amphiphilic substances is about 1:2. The average size of liposomes with an encapsulated contrast agent in an aqueous solution at pH 7.4 is 120 nm with PDI=0.2. The encapsulation efficiency was 74%±8%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methylamide) was determined by ultrafiltration.

EXAMPLE 4

[0027] Gd-diethylenetriaminepentaacetate-bis(methylamide) dissolved in an aqueous solution of sodium caldiamide at pH 7.45 was added to purified phosphatidylcholine (Lipoid) dissolved in propylene glycol. The whole mixture was stirred at 60° C. until a homogeneous, milky suspension was formed. Then the suspension thus obtained was extruded through polycarbonate filters with a pore diameter of 100 nm to obtain a homogenous liposome gel characterized by uniform size of liposomes encapsulating the contrast agent.

TABLE-US-00004 TABLE 4 Contrast agent composition. % by weight CONTRAST AGENT: Gd-diethylenetriaminepentaacetate-bis(methylamide) 20 ADDITIONAL SUBSTANCES: Purified phosphatidylcholine 30 Propylene glycol 20 Caldiamide sodium 0.84 Sodium hydroxide 0.01 Purified water 29.15 TOTAL 100

[0028] The molar ratio of Gd-diethylenetriaminepentaacetate-bis(methylamide) to amphiphilic substances is about 2:3. The average size of liposomes with an encapsulated contrast agent in an aqueous solution at pH 7.4 is 105 nm with PDI=0.15. The encapsulation efficiency was 82%±5%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methyl amide) was determined by ultrafiltration.

EXAMPLE 5

[0029] Gd-diethylenetriaminepentaacetate-bis(methylamide) dissolved in an aqueous solution of sodium caldiamide at pH 7.45 was added to purified phosphatidylcholine (Lipoid) dissolved in propylene glycol. The whole mixture was stirred at 60° C. until a homogeneous, milky suspension was formed. Then the suspension thus obtained was extruded through polycarbonate filters with a pore diameter of 100 nm to obtain a homogenous liposome gel characterized by uniform size of liposomes encapsulating the contrast agent.

TABLE-US-00005 TABLE 5 Contrast agent composition. % by weight CONTRAST AGENT: Gd-diethylenetriaminepentaacetate-bis(methylamide) 0.1 ADDITIONAL SUBSTANCES: Purified phosphatidylcholine 30 Propylene glycol 20 Caldiamide sodium 0.01 Sodium hydroxide 0.01 Purified water 49.88 TOTAL 100

[0030] The molar ratio of Gd-diethylenetriaminepentaacetate-bis(methylamide) to amphiphilic substances is about 1:300. The average size of liposomes with an encapsulated contrast agent in an aqueous solution at pH 7.35 is 110 nm with PDI=0.1. The encapsulation efficiency was 84%±6%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methyl amide) was determined by ultrafiltration.

EXAMPLE 6

[0031] Gd-diethylenetriaminepentaacetate-bis(methylamide) dissolved in an aqueous solution of sodium caldiamide at pH 7.45 was added to purified phosphatidylcholine (Lipoid) dissolved in glycerol. The whole mixture was stirred at 60° C. until a homogeneous, milky suspension was formed. Then the suspension thus obtained was extruded through polycarbonate filters with a pore diameter of 100 nm to obtain a homogenous liposome gel characterized by uniform size of liposomes encapsulating the contrast agent.

TABLE-US-00006 TABLE 6 Contrast agent composition. % by weight CONTRAST AGENT: Gd-diethylenetriaminepentaacetate-bis(methylamide) 1 ADDITIONAL SUBSTANCES: Purified phosphatidylcholine 30 Glycerol 20 Caldiamide sodium 0.04 Sodium hydroxide 0.01 Purified water 48.95 TOTAL 100

[0032] The molar ratio of Gd-diethylenetriaminepentaacetate-bis(methylamide) to amphiphilic substances is about 1:30. The average size of liposomes with an encapsulated contrast agent in an aqueous solution at pH 7.4 is 106 nm with PDI=0.12. The encapsulation efficiency was 86%±7%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methyl amide) was determined by ultrafiltration.

EXAMPLE 7

[0033] Gd-diethylenetriaminepentaacetate-bis(methylamide) dissolved in saline at pH 7.45 was added to purified phosphatidylcholine (Lipoid) and vitamin E dissolved in propylene glycol. The whole mixture was stirred at 60° C. until a homogeneous, milky suspension was formed. Then the suspension thus obtained was extruded through polycarbonate filters with a pore diameter of 100 nm to obtain a homogenous liposome gel characterized by uniform size of liposomes encapsulating the contrast agent.

TABLE-US-00007 TABLE 7 Contrast agent composition. % by weight CONTRAST AGENT: Gd-diethylenetriaminepentaacetate-bis(methylamide) 1 ADDITIONAL SUBSTANCES: Purified phosphatidylcholine 20 Propylene glycol 30 Sodium hydroxide 0.01 Vitamin E 1 Saline 47.99 TOTAL 100

[0034] The molar ratio of Gd-diethylenetriaminepentaacetate-bis(methylamide) to amphiphilic substances is about 1:20. The average size of liposomes with an encapsulated contrast agent in an aqueous solution at pH 7.4 is 112 nm with PDI=0.21. The encapsulation efficiency was 88%±6%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methyl amide) was determined by ultrafiltration.

EXAMPLE 8

[0035] Gd-diethylenetriaminepentaacetate-bis(methylamide) dissolved in an aqueous solution of sodium caldiamide at pH 7.45 was added to purified phosphatidylcholine (Lipoid) dissolved in propylene glycol. The whole mixture was stirred at 60° C. until a homogeneous, milky suspension was formed. Then the suspension thus obtained was extruded through polycarbonate filters with a pore diameter of 100 nm to obtain a homogenous liposome gel characterized by uniform size of liposomes encapsulating the contrast agent.

TABLE-US-00008 TABLE 8 Contrast agent composition. % by weight CONTRAST AGENT: Gd-diethylenetriaminepentaacetate-bis(methylamide) 14.8 ADDITIONAL SUBSTANCES: Purified phosphatidylcholine 20 Propylene glycol 20 Caldiamide sodium 0.62 Sodium hydroxide 0.01 Purified water 44.57 TOTAL 100

[0036] The molar ratio of Gd-diethylenetriaminepentaacetate-bis(methylamide) to amphiphilic substances is about 1:20. The average size of liposomes with an encapsulated contrast agent in an aqueous solution at pH 7.4 is 118 nm with PDI=0. 1. The encapsulation efficiency was 82%±6%. The encapsulation efficiency of Gd-diethylenetriaminepentaacetate-bis(methylamide) was determined by ultrafiltration.

The Effectiveness of MRI Contrast According to the Invention was Tested in an Animal Model of Endothelial Dysfunction.

Animal Model of Endothelial Dysfunction

[0037] The experiments were performed using female ApoE/LDLR.sub.−/− mice of different ages: 4-, 6-, 8-, 12- and 28-week-old.sub.(19) in comparison to control young (8-week-old) mice (C57BL/6) without endothelial dysfunction. All mice (body weight 20-30 g) were housed under standard pathogen-free conditions (LD: 12/12, humidity: 60%, temperature: 23° C.).

Magnetic Resonance Imaging (MRI)

[0038] MRI experiments were carried out using a 9.4 T scanner (BioSpec 94/20 USR, Bruker, Germany). During the experiment, mice were anesthetized using isoflurane (Aerrane, Baxter Sp. z o.o., Poland, 1.5% by volume) in an oxygen and air mixture (1:2). Cardiac activity, respiration rate and body temperature (maintained at 37° C., using circulating warm water) were monitored using Monitoring and Gating System (SA Inc., Stony Brook, N.Y., USA). Mice were imaged in the supine position to test endothelial function and permeability in various blood vessels.

In Vivo MRI Protocol for the Assessment of Endothelial Permeability

[0039] Endothelial permeability measurements were performed using two gadolinium contrast agents (CA); reference gadolinium albumin binding contrast agent (Galbumin, BioPAL, Worcester, Mass., USA—25 mg/mL, 4.5 mL/kg, iv) and new preparation according to the invention—Gd-diethylenetriaminepentaacetate-bis(methylamide) placed inside liposomes. Relaxation time (T.sub.1) before and 30 min after intravenous CA administration was measured to assess BCA permeability using the VFA technique.sub.(20), by sampling the signal using varying values of flip angles (FA), and then fitting the result to the expected T.sub.1-dependent signal model.sub.(21). 3D images of the aortic arch were acquired using the 3D IG-FLASH sequence, to obtain high Bi field profile uniformity within measured subslices. Imaging parameters included the following: T.sub.R 10 ms, T.sub.E 1.1 ms, FOV 30×30×4 mm.sub.3, matrix size 192×160×8, number of repetitions 12. Eight FAs were used: 2°, 4°, 6°, 8°, 14°, 20°, 30° and 50°. FA values were set by changing the RF pulse length with constant amplifier power. The total scanning time for all angles was 16 min.

[0040] Data analysis: The obtained images were used to calculate T.sub.1 maps before and after CA administration. The signal model was fitted pixel by pixel using Matlab software. Two T.sub.1 maps (before and after CA administration) were compared to estimate the number of pixels for which T.sub.1 had changed significantly (by more than 50%) after CA administration (Npx50, marked in red in FIG. 3C). The threshold (50%) was determined experimentally.

[0041] FIG. 3 shows an illustration of the methodology for MRI-based in vivo assessment of endothelium-dependent response and endothelial permeability. Endothelium-dependent response to acetylcholine (Ach), expressed as changes in vessel volume, was assessed in the brachiocephalic artery (BCA) visible on the 3D image of the aortic arch (A) and in the abdominal aorta (AA) positioned on the sagittal view of the mouse (B). Assessment of vessel response to flow-mediated dilation (FMD), also expressed as changes in vessel volume, was performed in the femoral artery (FA), visible on the 3D image of the left hind limb (D). Pixels for which T.sub.1 had changed more than 50% after CA (Npx50) were marked in red on a representative cross-sectional image of blood vessels arising from the aortic arch (C).

[0042] The obtained data are presented as the mean and standard deviation. Statistical tests were done using STATISTICA 10 (Stat Soft Inc., USA) using a parametric test (one-way analysis of variance (ANOVA) with a Tukey test). A p value of 0.05 was considered statistically significant.

Demonstration that the Subject of the Invention in the Form of a Hydrophilic Gadolinium Encapsulated in Liposomes is an MRI Contrast Agent for the Detection of Early Changes in the Endothelium of Blood Vessels.

[0043] Prepared liposomes, in which Gd-diethylenetriaminepentaacetate-bis(methylamide) (GD liposome) was encapsulated were used as a magnetic resonance imaging (MRI) contrast agents for in vivo detection of endothelial dysfunction based on changes in endothelial permeability in ApoE/LDLR.sub.−/− mice. For comparison, assessment of the endothelial permeability was performed using albumin-binding gadolinium contrast agent (GD albumin).

The Development of Increased Endothelial Permeability in Brachiocephalic Artery (BCA) in ApoE/LDLR.SUB.−/− Mice

[0044] In the presented experiment, the contrast agent having the composition of Example 8 was used. As shown in FIG. 4B, the preparation of Gd-diethylenetriaminepentaacetate-bis (methylamide) encapsulated in the liposome allowed the detection of early changes in endothelial permeability, observed as an increase in Npx50, which was already observed in 4-week-old ApoE/LDLR.sub.−/− mice (Npx50: increase by 100% in comparison to control mice, C57BL/6). Changes in endothelial permeability were exacerbated by atherosclerosis progression in ApoE/LDLR.sub.−/− mice, and a 4-fold increase in Npx50 was observed in 28-week-old ApoE/LDLR.sub.−/− mice. In contrast to the administration of the liposomal contrast agent, intravenous injection of GD albumin caused significant changes in the Npx50 parameter (increase by about 100%) only in 28-week-old ApoE/LDLR.sub.−/− mice, Furthermore, in 4-12-week-old ApoE/LDLR.sub.−/− mice did not show a significant increase in vascular wall permeability assessed using GD albumin (increase in Npx50 between 3% and 30%, in comparison to control mice; FIG. 4A).

[0045] FIG. 4 shows an increase of the endothelial permeability in brachiocephalic artery in ApoE/LDLR.sub.−/− mice. Number of pixels around BCA lumen for which T.sub.1 has changed by more than 50% (Npx50) after using albumin-binding gadolinium (A: GD-albumin) contrast agent or Gd-diethylenetriaminepentaacetate-bis(methylamide) encapsulated in liposomes (B: GD-liposome) are expressed as a percentage of control in ApoE/LDLR.sub.−/− mice (white columns) of different ages: 4-week-old ApoE/LDLR.sub.−/− mice (ApoE_4 W, n=4), 6-week-old ApoE/LDLR.sub.−/− mice (ApoE_6 W, n=4), 8-week-old ApoE/LDLR.sub.−/− mice (ApoE_8 W, n=6), 12-week-old ApoE/LDLR.sub.−/− mice (ApoE_12 W, n=5) and 28-week-old ApoE/LDLR.sub.−/− mice ((ApoE_28 W, n=6). Control is 8-week-old C57BL/6 mice (C57_8W, n=6, black columns).

Progression of an Impairment of the Endothelium-Dependent Response to Acetylcholine Administration in the BCA and the Abdominal Aorta (AA) in ApoE/LDLR.SUB.−/− Mice

[0046] To assess the sensitivity of endothelial dysfunction detection using the new contrast agent, being an invention, impaired endothelial function in ApoE/LDLR−/− mice was also assessed by other MRI-based techniques; detection of endothelium-dependent response to acetylcholine (FIG. 5) and measurement of increase in flow after short-term vessel occlusion (FMD, FIG. 6). As described in our recent publication.sub.(22), endothelium-dependent response to acetylcholine (Ach, FIG. 5B) in abdominal aorta (AA) is the most sensitive in vivo method to detect changes in endothelial phenotype. Intraperitoneal administration of Ach resulted in the detection of significantly impaired AA functional response in 4-week-old ApoE/LDLR.sub.−/− mice (AA volume change: about 23% in comparison to about 34% in control mice). Further progression of the impairment of the AA response to Ach was observed in 6-8-week-old ApoE/LDLR.sub.−/− mice and as the paradoxical vasoconstriction in 12- and 28-week-old ApoE/LDLR.sub.−/− mice (change in AA volume: about −8% and −23%, respectively). Changes in the BCA endothelial function, described as impaired endothelium-dependent vasodilatation in response to Ach, were only observed in 12-week-old mice (volume changes: about 2%, FIG. 5A). The BCA response to Ach in 4 to 8-week-old ApoE/LDLR.sub.−/− mice remained unchanged (changes in BCA volume: approximately 10%) and was comparable to the normal vasodilatatory response observed in the 8-week-old control mice (volume change in the BCA: 11.26%). Moreover, impairment of endothelium-dependent response to increase in flow after short-term vessel occlusion (FMD) was observed not earlier than in 12-week-old ApoE/LDLR.sub.−/− mice (volume change in the FA: about 16% in comparison to about 34% in control mice, FIG. 6). FMD response in younger, 8-week-old ApoE/LDLR.sub.−/− mice remained unchanged, as compared to control mice (FA volume change: about 31%).

[0047] These experiments clearly show that the assessment of changes in endothelial permeability using the liposomal contrast agent being an invention, allows the detection of functional changes in vascular endothelium at a similar early stage of endothelial dysfunction development as the assessment of endothelial-dependent response to Ach administration in in abdominal aorta, which was the most sensitive method for detection of changes in endothelial phenotype, in our previous studies.sub.(22). The detection of functional changes in BCA as well as in the FMD response, the latter is the gold standard in the assessment of endothelial function in clinical conditions.sub.(12,13), was possible at a later stage of endothelial dysfunction development, in comparison to the assessment using a liposomal contrast agent being a subject of an invention.

[0048] FIG. 5 shows the progression of the impaired endothelium-dependent response to acetylcholine administration in the brachiocephalic artery and the abdominal aorta in ApoE/LDLR.sub.−/− mice. Changes in the end-diastolic volume of brachiocephalic artery (A: BCA-ACH) and abdominal aorta (B: AA-ACH) 25 minutes after administration of acetylcholine in ApoE/LDLR.sub.−/− mice (white columns) of different ages: 4-week-old ApoE/LDLR.sub.−/− mice, 6-week-old ApoE/LDLR.sub.−/− mice, 8-week-old ApoE/LDLR.sub.−/− mice, 12-week-old ApoE/LDLR.sub.−/− mice, 28-week-old ApoE/LDLR.sub.−/− mice as compared to 8-week-old control C57BL/6 mice, (C57_8W, n=4, black columns). Statistics: one-way ANOVA followed by Tukey's post hoc test (normality was assessed using the Shapiro-Wilk test); *p<0.05, **p<0.01, ***p<0.001.

[0049] FIG. 6 shows the progression of the impaired endothelium-dependent response to an increase in flow after short term vessel occlusion (FMD) in the femoral artery (FA) in ApoE/LDLR.sub.−/− mice of different ages: 8-week-old ApoE/LDLR.sub.−/− mice (ApoE_8 W, n=6), 23-week-old ApoE/LDLR.sub.−/− mice (ApoE_12 W, n=6) and 28-week-old ApoE/LDLR.sub.−/− mice (ApoE_28 W, n=5), as compared to the 8-week-old control C57BL/6 mice (C57_8W, n=5, black columns). Statistics: one-way ANOVA followed by Tukey's post hoc test (normality was assessed using the Shapiro-Wilk test); *p<0.05, **p<0.01, ***p<0.001.

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