DAB-4 linked nitroxide citrate, useful as MRI contrast agent

Abstract

A method of imaging a joint of a subject comprises administering to the subject an amount of a salt of DAB-4 linked nitroxide citrate effective to enhance an MRI image of the joint and taking an MRI image of said joint.

Claims

1. A method of imaging cartilage in a joint of a subject comprising: administering to the subject a citrate salt of diaminobutyl poly(propyleneimine) (DAB)-4 linked nitroxide and taking an magnetic resonance imaging (MRI) image of cartilage in the joint of the subject.

2. The method of claim 1 wherein the citrate salt is a 1:1 molar ratio citric acid:DAB-4 linked nitroxide.

3. The method of claim 1 wherein the image is taken 30-45 minutes after the DAB-4 linked nitroxide citrate is administered.

4. The method of claim 1 further comprising evaluating cartilage status of said joint.

5. A method of imaging a joint of a subject comprising: administering to the subject an amount of a citrate salt of DAB-4 linked nitroxide effective to enhance an MRI image of said joint and taking an MRI image of said joint.

6. The method of claim 5 wherein the citrate salt is a 1:1 molar ratio citric acid:DAB-4 linked nitroxide.

7. The method of claim 5 wherein the image is taken 30-45 minutes after the DAB-4 linked nitroxide citrate is administered.

8. The method of claim 5 further comprising evaluating cartilage status of said joint.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A: MRI T1W (weighted) image for DAB-8 linked nitroxide citrate was obtained at 27 minutes after immersion of normal cartilage in the contrast agent is shown.

(2) FIG. 1B: MRI T1W (weighted) image for DAB-4 linked nitroxide citrate was obtained at 29 minutes after immersion of normal cartilage in the contrast agent is shown.

(3) FIG. 1C: MRI T1 value map image for DAB-8 linked nitroxide citrate was obtained at 27 minutes after immersion of normal cartilage in the contrast agent is shown.

(4) FIG. 1D: MRI T1 value map image for DAB-4 linked nitroxide citrate was obtained at 29 minutes after immersion of normal cartilage in the contrast agent is shown.

(5) FIG. 1E: A pre-contrast T1 value map for normal cartilage is shown.

(6) FIG. 2A: MRI Ti1 value map for DAB-4 linked nitroxide citrate in cartilage, indicating high levels of contrast media and more glycosaminoglycans (GAG) in the cartilage is shown.

(7) FIG. 2B: Histology of the same specimen stain with Saframin-O/fast Green is shown. Comparison of FIG. 2a) with FIG. 2b) shows high GAG concentration in the same areas as indicated by DAB-4 linked nitroxide on the T1 value map and the stain on the histology map.

(8) FIG. 3: Results for the MTT assay using the citrate, maleate and tartrate salts of DAB-4 linked nitroxide, DAB-8 linked nitroxide and Magnevist are shown.

(9) FIG. 4: Results for the Picogreen assay using the citrate, maleate and tartrate salts of DAB-4 linked nitroxide, DAB-8 linked nitroxide and Magnevist are shown.

(10) FIG. 5: Results for the MTT data normalized to gDNA/ml using the citrate, maleate and tartrate salts of DAB-4 linked nitroxide, DAB-8 linked nitroxide and Magnevist are shown.

(11) FIG. 6: Data from the Modified Mankin scores for join degeneration using DAB-4 linked nitroxide citrate, Magnevist and PBS, the latter two as controls is shown.

(12) FIG. 7: Data from Synovial Inflammation scores for join degeneration using DAB-4 linked nitroxide citrate, Magnevist and PBS, the latter two as controls is shown.

(13) FIG. 8: T1-weighted MRI image of a control knee joint is shown.

(14) FIG. 9: T1-weighted MR image of a knee joint 10 minutes after intraarticular injection of DAB-linked nitroxide citrate is shown.

EXAMPLE 1

Synthesis of DAB-4 Linked Nitroxide (Scheme 1)

(15) In a solution of DMF (10 mL) containing DAB-4 (MW=316.5, 316 mg, 1 mmoles) was added N,N-diisopropylethylamine (DIEA, MW=129, d=0.78, 5.0 mmoles, 0.65 gm=0.83 mL) and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MW=186, 5.0 mmoles, 0.93 gm) and the reaction was stirred at room temperature for hr. Then, benzotriazole-1-yl-tris-(dimethylamino)phosphonium hexafluorophosphate, (BOP, MW=442, 5.0 mmoles, 2.21 gm) was added. This reaction was stirred overnight, about 16 hr, at room temperature. At that point, a sodium chloride solution (50 mL), sodium bicarbonate saturated (5 mL) and methylene chloride (100 mL) were added. The organic layer was separated, dried over anhydrous sodium sulfate and evaporated to dryness. The remaining residue was dried under high vacuum to remove remaining DMF. The resultant oil was chromatographed using silica gel (230-400 mesh, EMD Chemicals, distributed by VWR International, Bridgeport, N.J.). Elution with chloroform/acetone (40 mL/10 mL) brought down a small amount of a yellow compound, which was discarded. Changing to the following mixture: chloroform (25 mL)/methanol (25 mL) plus triethylamine (0.3 mL) afforded product one peak by TLC (silica gel, chloroform/methanol or alumina plates, chloroform/methanol). Mass spec (MW=989) confirms compound.

(16) ##STR00001##

EXAMPLE 2

Synthesis of DAB-4 Linked Nitroxide (Scheme 1a)

(17) In a solution of DMF (10 mL) containing DAB-4 (MW=316.5, 316 mg, 1 mmoles) was added 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MW=186, 5.0 mmoles, 0.93 gm) and O-(berizotrizol-yl)-N,N,NN-tetramethyluronim hexafluorophosphate (HBTU, MW=379, 1.9 gm, 5 mmoles). The reaction was stirred at room temperature for hr. Then, N,N-diisopropylethylamine (DIEA, MW=129, d=0.78, 5.0 mmoles, 0.65 gm=0.83 mL) was added. This reaction was stirred overnight, about 16 hr, at room temperature. At that point, a sodium chloride solution (50 mL), sodium bicarbonate saturated (5 mL) and methylene chloride (100 mL) were added. The organic layer was separated, dried over anhydrous sodium sulfate and evaporated to dryness. The remaining residue was dried under high vacuum to remove remaining DMF. The resultant oil was chromatographed using silica gel (230-400 mesh, EMD Chemicals, distributed by VWR international, Bridgeport, N.J.). Elution with chloroform/acetone (40 mL/10 mL) brought down a small amount of a yellow compound, which was discarded. Changing to the following mixture: chloroform (25 mL)/methanol (25 mL) plus triethylamine (0.3 mL) afforded product one peak by TLC (silica gel, chloroform/methanol or alumina plates, chloroform/methanol). Mass spec (MW=989) confirms compound.

(18) ##STR00002##

EXAMPLE 3

Synthesis of Citric Acid Salt of DAB-4 Linked Nitroxide (Schemes 1 and 1a)

(19) Citric acid salt is prepared as follows. DAB-4-linked nitroxide (MW=989, 0.74 gm, 0.7 mmoles) prepared in Example 1 or 2, dissolved in dry CHCl.sub.3, and citric acid anhydrous (MW=192, 0.14 gm, 0.7 mmoles dissolved in absolute ethanol are mixed together and evaporated to dryness. MW of citrate salt is 1,181.

EXAMPLE 4

Superiority of DAB-4 Linked Nitroxide Citrate for MRI Contrast of Cartilage

(20) Comparison of DAB-4 linked nitroxide citrate and DAB-8 linked nitroxide citrate for enhancement of normal cartilage. Cylindrical immature bovine cartilage plugs were imaged by MR at 7 Tesla initially in saline and then in DAB-4 linked nitroxide citrate or DAB-8 linked nitroxide citrate. T1-weighted (T1W) images (TR=750 ms, TE=7 ms) and T1-value maps were performed. Images and T1 maps obtained 30 minutes after immersion in the contrast agents are shown in FIG. 1. Diffusion of the contrast agent was restricted to occur only from the periphery and not from the top nor the bottom. The figures show: a) DAB-8 linked nitroxide citrate T1W image 27 min after immersion b) DAB-4 linked nitroxide citrate T1W image 29 min after immersion c) DAB-8 linked nitroxide citrate T1 value map 27 min after immersion d) DAB-4 linked nistroxide citrate T1 value map 29 min after immersion e) For comparison, a pre-contrast T1 value map for the cartilage

(21) The DAB-8 linked nitroxide citrate shows much poorer penetration into the cartilage. The enhancement is difficult to detect on the T1W images. The T1 value maps show the T1 values drop (contrast penetration) for only a very thin rim of cartilage, only about the distance for the DAB-4 linked nitroxide citrate.

EXAMPLE 5

Effectiveness of DAB-4 Linked Nitroxide Citrate to Enhance Contrast for Articular Cartilage Assessment as Measured by GAG Targeting

(22) Cartilage-bone plug excised from a human tibial plateau that was retrieved from total knee replacement surgery was equilibrated in DAB-4 linked nitroxide citrate. T1 value map (as acquired for FIG. 1) is compared to histology in FIG. 2: a) T1 value map: orange color is lower T1 value indicating more contrast agent and more glycosaminoglycans (GAG) in the cartilage. b) Histology of the same specimen stained with Safranin-O/fast green.
Comparison of FIG. 2a) with FIG. 2b) shows high GAG concentration in the same areas indicated by DAB-4 linked nitroxide on the T1 value map and the stain on the histology map.

EXAMPLE 6

Summary of Cytotoxicity Studies Comparing Salts of Dendrimer-Linked Nitroxides Based on DAB-4 and DAB-8 Dendrimer Cores with Magnevist and Positive and Negative Controls

(23) Assays: Modified MTT assay for metabolic activity (absorbance).

(24) Quantitative picogreen for cell proliferation (gDNA/ml).

(25) Assay data analysis: Expressed as % of the positive controls for MTT and picogreen. MTT absorbance measures then normalized to the gDNA/ml generated by the picogreen assay.

(26) Cell cultures: RES-LTC chondrosarcoma cell line. Expresses hyaline-like chondrocyte phenotype and reliably produces large, homogeneous cell populations

(27) Compounds Tested:

(28) Positive controlculture medium only

(29) Negative control5 M Staurosporine

(30) Magnevist1 mM, 5 mM, 7.5 mM, 10 mM

(31) DAB-4 linked nitroxide Citrate1 mM, 5 mM, 7.5 mM, 10 mM

(32) DAB-4 linked nitroxide Maleate1 mM, 5 mM, 7.5 mM, 10 mM

(33) DAB-4 linked nitroxide Tartrate1 mM, 5 mM, 7.5 mM, 10 mM

(34) DAB-8 linked nitroxide Citrate1 mM, 5 mM, 7.5 mM, 10 mM

(35) DAB-8 linked nitroxide Maleate1 mM, 5 mM, 7.5 mM, 10 mM

(36) DAB-8 linked nitroxide Tartrate1 mM, 5 mM, 7.5 mM, 10 mM

(37) Methods:

(38) Cell cultures in 96 well plates. Each compound, at a fixed concentration, was added in triplicate for each assay. MTT and picogreen assays in separate plates (assays interfere with each other). All plates treated together. Assays run at 48 hour timepoint. Microscopy performed to confirm findings.

(39) Results for the MTT assay are shown in FIG. 3. At concentrations above 1 mM, DAB-8 linked nitroxide shows less metabolic activity than positive controls (untreated), or DAB-4 linked nitroxide and Magnevist. DAB-4 linked nitroxide showed less metabolic activity than positive controls but greater than for DAB-8 linked nitroxide. The citrate salt of DAB-4 linked nitroxide showed more metabolic activity than the maleate or tartrate salts.

(40) Results for the Picogreen assay are shown in FIG. 4. DAB-8 linked nitroxide citrate shows lower cell proliferation than positive control, (untreated), DAB-4 linked nitroxide maleate and Magnevist at all concentrations. All DAB-4 linked nitroxide salts show a dose-dependent effect. The 7.5 mM DAB-4 linked nitroxide tartrate appears to be an outlier. Magnevist at 10 mM shows low cell proliferation. It is not clear if the lower cell proliferation is due to cell death or an initial delay in proliferation.

(41) Results for MTT data normalized to gDNA/ml are shown in FIG. 5. This is a measure of the metabolic activity of the cells normalized to the concentration of cells (gDNA/ml), i.e. the health of the cells. DAB-4 linked nitroxide salts perform similar to positive controls. DAB-8 linked nitroxide salts show effects above 1 mM. Magnevist appears abnormally high at 10 mM.

EXAMPLE 7

Toxicity Studies of DAB-4 Linked Nitroxide Citrate in Rabbit Joints

(42) The potential inflammatory and structural effects of PBS, Magnevist, and DAB-4 linked nitroxide citrate were assessed using a single intra-articular injection model (data were taken from studies conducted by Arthroteq testing laboratory). Twelve female New Zealand White rabbits (2.7-3.3 kg) were randomly assigned to one of two study groups that received bilateral intra-articular injections with Magnevist (n=4) in the right stifle and sterile PBS in the left stifle, or DAB-4 linked nitroxide citrate (n=8, 0.3 mL of 10 mM) in the right stifle and sterile PBS in the left stifle (n=8, 0.3 mL).

(43) Rabbits were euthanized two weeks post-injection. Both left and right stifles were harvested and immediately fixed in 10% neutral buffered formalin, then set for histological processing. Five-micron thick slides from each knee were stained with Hematoxylin & Eosin and Safranin-O.

(44) No animals presented clinical signs of inflammation or lameness during the study. Modified Mankin scoring did not uncover any signs of structural damage and/or degenerative response due to Magnevist, DAB-4 linked nitroxide citrate, or PBS injections (FIG. 6). Custom synovial inflammation scoring reflected the absent of an inflammatory response in all stifles and no difference between Magnevist or DAB-4 linked nitroxide and the PBS-injected stifles were observed (FIG. 7).

(45) FIG. 6: No differences were detected in the Modified Mankin subscores or total score between any treatment groups as assessed by ANOVA. All tests were performed at a significance level of p<0.05. Mean cartilage degenerative changes assessed by Modified Mankin Scoring (MeanS.D.) for all study groups. A higher score (maximum of 19) indicates greater joint degeneration. Mean subscores for a nave group of age-matched rabbits normally fall in a range between 0 and 1.0. Total Mankin Score for a nave group of age-matched rabbits normally falls in a range between 0 and 3.0.

(46) FIG. 7: No differences were detected in the Synovial Inflammation subscores or total score between any treatment groups as assessed by ANOVA. All tests were performed at a significance level of p<0.05. Average scores (MeanS.D.) for Synovial Inflammation for all study groups. A higher score indicates greater inflammation. Only two of the seven sub-categories presented non-zero scores. A mean score between 0 and 1.0 is typical for nave age-matched rabbits.

EXAMPLE 8

MRI Images of a Knee Joint Using DAB-4 Linked Nitroxide Citrate

(47) Coronal T1-weighted MR images of a human knee were taken in a human cadaver. The cartilage surfaces are poorly differentiated as shown in FIG. 8. Coronal T1-weighted MR contrast images were also taken ex vivo in a human cadaver 10 minutes after intraarticular injection of 25 ml of a 5 mM solution of DAB-4 citrate diluted in PBS. As shown in FIG. 9, the joint fluid (asterisk) is now bright and outlines the cartilage surfaces. A partial thickness cartilage defect (arrow) is easily identified. All images were performed on a Siemens Skyra 3 Tesla magnet using a 15 channel transmit/receive QED knee coil. (14 cm FOV (field of view); 3 mm thick slice; 384384 matrix; TR 800 ms; TE 8.6 ms; Turbo factor of 2; receiver bandwidth 350 Hz/pixel.)

(48) From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.