Development of Injectable Fiducial Markers for Image Guided Radiotherapy with Dual MRI and CT Visibility

Abstract

Radiation therapy or radiotherapy (RT) is a powerful treatment where precision and accuracy is crucial. Image Guided Radiotherapy (IGRT) facilitates more accurate position verification, correcting for anatomic changes related to internal organ movement. IGRT thereby helps reduce toxicity of radiotherapy and increases relapse-free survival. An inter-correlation point with a fixed position and volume (a marker) can be applied to indicate the point of treatment clearly in both imaging modalities and to localize and track tumors in real time. In this study, we present the development of a marker based on lactose octaacetate:octapropionate 1:1 containing 3 mM PLA-DTPA(Gd), 40% triglyceride, 5% propylene carbonate and 10% XSAIB (sucrose based CT-contrast agent). The injectable marker had high CT contrast (>1000 HU) and displayed clearly visible, stable T.sub.1 contrast enhancement (T.sub.10.900 ms) in the rim over at least 3 weeks with clinically observable resolution.

Claims

1. A composition for at least MR imaging, comprising non-water soluble carbohydrates, wherein at least 50% of the non-water soluble carbohydrates are carbohydrates selected from derivatives of lactose, maltose, trehalose, raffinose, glucosamine, galactosamine, lactosamine, sucrose or derivatives of sucrose, or mixed saccharides, or derivatives of disaccharides with at least two pyranose saccharide units, trisaccharides, tetrasaccharides, or mixtures thereof, and wherein the composition is a liquid before administration into the human or animal body and increases in viscosity by more than 1,000 centipoise (cP) after administration, wherein the composition contains at least one imaging contrast agent, and wherein the composition provides a phase separation which provides a clear contrast distinction in MR imaging, wherein the clear contrast distinction is bright vs dark in two different phases of the composition after administration into the human or animal body, and wherein either the bright or dark phase is provided by said non-water soluble carbohydrates.

2. (canceled)

3. The composition for at least MR imaging according to claim 1, wherein the composition is intended for combined MR and CT imaging, and wherein the composition is arranged to provide a clear distinction between CT contrast and MRI contrast in a marker.

4. The composition for at least MR imaging according to claim 1, wherein the composition is a liquid before administration into the human or animal body that increases in viscosity by more than 10,000 centipoise (cP) after administration into the human or animal body.

5. The composition for at least MR imaging according to claim 1, wherein the composition is a liquid before administration and has the ability to transform into a gel-like material after administration.

6. The composition for at least MR imaging according to claim 1, wherein the composition becomes a solid material after administration, such as a crystalline or amorphous solid.

7. The composition for at least MR imaging according to claim 1, wherein said at least one imaging contrast agent is more concentrated on the surface of the administered material after administration than inside the administered material for at least 1 hour to 3 months after administration, such as for at least 4 hours to 1 month, such as for at least 1 day to 2 months, such as for at least 2 days to 3 months after administration.

8. The composition for at least MR imaging according to claim 1, wherein said at least one imaging contrast agent is more concentrated less than 1 cm away from the administered material after administration than inside the administered material for at least 1 hour to 3 months after administration, such as for at least 4 hours to 1 month, such as for at least 1 day to 2 months, such as for at least 2 days to 3 months after administration.

9. The composition for at least MR imaging according to claim 1, wherein an increase in viscosity after administration into the human or animal body is due to diffusion of a solvent-like molecule out of the administered material and into surrounding tissue.

10. The composition for at least MR imaging according to claim 1, wherein the non-water soluble carbohydrates are disaccharides with structures selected from: ##STR00011## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.8, R.sub.6, R.sub.7, and R.sub.8 in formulae I, II and III are selected collectively from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are independently selected from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl; or wherein all groups of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are selected collectively from the group consisting of acetyl, isobutyryl or propionyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are independently selected from the group consisting acetyl, isobutyryl or propionyl; and wherein both pure anomers and mixtures of - and -anomers of the above mentioned structural variations are claimed.

11. The composition for at least MR imaging according to claim 1, wherein the non-water soluble carbohydrates are trisaccharides with structures selected from: ##STR00012## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 in formulae IV are selected collectively from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are independently selected from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl; or wherein all groups of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are selected collectively from the group consisting of acetyl, isobutyryl or propionyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are independently selected from the group consisting acetyl, isobutyryl or propionyl; and wherein both pure anomers and mixtures of - and -anomers of the above mentioned structural variations are claimed.

12. The composition for at least MR imaging according to claim 1, wherein at least 50% of the non-water soluble carbohydrates are mono- or oligosaccharides containing at least one amino sugar unit.

13. The composition for at least MR imaging according to claim 12, wherein the amino sugar has the structure: ##STR00013## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 in formulae V are selected collectively from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently selected from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl, and mono-, di-, tri- or tetra-saccharide derivatives; or wherein all groups of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are selected collectively from the group consisting of acetyl, isobutyryl or propionyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently selected from the group consisting acetyl, isobutyryl or propionyl; and wherein both pure anomers and mixtures of anomers such as - and -anomer centres of the above mentioned structural variations are claimed.

14. The composition according to claim 1, wherein the non-water soluble carbohydrates comprises poly(ethylene glycol-b-caprolactone) (PEG-PCI), sucrose acetate isobutyrate (SAIB), poly(D,L-lactic acid), or poly(lactic-co-glycolic acid) (PGLA), or a combination thereof.

15. The composition for at least MR imaging according to claim 1, wherein the non-water soluble carbohydrates comprise mixed saccharides of furanose and pyranose.

16. The composition for at least MR imaging according to claim 1, wherein said at least one imaging contrast agent makes the composition visible by PET imaging, SPECT imaging, Ultrasound imaging, CT imaging, MR imaging, x-ray imaging, fluoroscopy imaging, fluorescence imaging, or OCT imaging.

17. The composition for at least MR imaging according to claim 1, wherein the composition contains two imaging contrast agents that are visible by at least two imaging methods, such as visible by at least two imaging methods chosen from PET imaging, SPECT imaging, Ultrasound imaging, CT imaging, MR imaging, x-ray imaging, fluoroscopy imaging, fluorescence imaging, or OCT imaging.

18. The composition for at least MR imaging according to claim 1, wherein the composition contains an imaging contrast agent for CT imaging and an imaging contrast agent for MR imaging.

19. The composition for at least MR imaging according to claim 1, wherein the composition contains an MR imaging contrast agent containing Gadolinium associated with a chelate.

20. The composition for at least MR imaging according to claim 1, wherein the composition contains an MR imaging contrast agent containing Gadolinium associated with a chelate that is covalent linked to a polymer.

21. The composition for at least MR imaging according to claim 1, wherein the composition contains an MR imaging contrast agent containing Gadolinium associated with a chelate that is covalent linked to poly lactic acid (PLA).

22. The composition for at least MR imaging according to claim 1, wherein the composition contains an MR imaging contrast agent containing Gadolinium associated with a chelate chosen from PLA-DTPA or PLA-DOTA polymer chelates.

23. The composition for at least MR imaging according to claim 1, wherein the composition contains a CT contrast agent and an MR imaging contrast agent containing Gadolinium associated with a chelate, and wherein the CT contrast agent is hydrophobic.

24. The composition for at least MR imaging according to claim 1, wherein the composition comprises a pharmacologically active compound that is released into the surrounding tissue, such as chemotherapy that enhances the effect of radiotherapy.

25. The composition for at least MR imaging according to claim 1, arranged to be administered to the human or animal body through a syringe, an endoscope or a bronchoscope to the target tissue preferably wherein the composition after insertion into the human or animal body constitutes a medical or surgical implant for tissue or surgical adhesion which preferably is wound dressing, a hemostat, enhances tissue regeneration, is a void filler.

26. A medical or surgical implant comprising a composition for at least MR imaging according to claim 1, wherein the composition is part of a sprayable composition.

Description

SPECIFIC EMBODIMENTS OF THE PRESENT INVENTION

[0055] Below some specific embodiments are listed.

[0056] According to one embodiment of the present invention, the clear contrast distinction is bright vs dark in two different phases of the composition after administration into the human or animal body. This is further seen in the figures.

[0057] According to yet another embodiment of the present invention the composition is intended for combined MR and CT imaging, and where the composition is arranged to provide a clear distinction between CT contrast and MRI contrast in a marker. As one example, CT may be only be seen in the dark MR area.

[0058] Furthermore, according to one embodiment of the present invention, the composition is a liquid before administration into the human or animal body that increases in viscosity by more than 10,000 centipoise (cP) after administration into the human or animal body. Moreover, the composition may be arranged to be a liquid before administration and with the ability to transform into a gel-like material after administration. Furthermore, the composition may be provided to become a solid material after administration, such as a crystalline or amorphous solid.

[0059] Moreover, according to yet another specific embodiment of the present invention, said at least one imaging contrast agent is more concentrated on the surface of the administered material after administration than inside the administered material for at least 1 hour to 3 months after administration, such as for at least 4 hours to 1 month, such as for at least 1 day to 2 months, such as for at least 2 days to 3 months after administration. In another embodiment, said at least one imaging contrast agent is more concentrated less than 1 cm away from the administered material after administration than inside the administered material for at least 1 hour to 3 months after administration, such as for at least 4 hours to 1 month, such as for at least 1 day to 2 months, such as for at least 2 days to 3 months after administration.

[0060] Furthermore, according to one embodiment of the present invention, an increase in viscosity after administration into the human or animal body is due to diffusion of a solvent-like molecule out of the administered material and into surrounding tissue.

[0061] According to one specific embodiment of the present invention, the non-water soluble carbohydrates are disaccharides with structures selected from:

##STR00001##

[0062] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 in formulae I, II and Ill are selected collectively from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are independently selected from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl;

[0063] or wherein all groups of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are selected collectively from the group consisting of acetyl, isobutyryl or propionyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are independently selected from the group consisting acetyl, isobutyryl or propionyl;

[0064] and wherein both pure anomers and mixtures of - and -anomers of the above mentioned structural variations are claimed.

[0065] According to yet another embodiment, the non-water soluble carbohydrates are trisaccharides with structures selected from:

##STR00002##

[0066] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 in formulae IV are selected collectively from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are independently selected from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl;

[0067] or wherein all groups of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are selected collectively from the group consisting of acetyl, isobutyryl or propionyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10 and R.sub.11 are independently selected from the group consisting acetyl, isobutyryl or propionyl;

[0068] and wherein both pure anomers and mixtures of - and -anomers of the above mentioned structural variations are claimed.

[0069] At least 50% of the non-water soluble carbohydrates may be mono- or oligosaccharides containing at least one amino sugar unit. Moreover, according to another embodiment, the amino sugar has the structure:

##STR00003##

[0070] wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 in formulae V are selected collectively from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently selected from the group consisting of hydrogen, alkanoyl, hydroxyl-substituted alkanoyl, and acyloxy-substituted alkanoyl, alkanyl, hydroxysubstituted alkanyl and acyloxy substituted alkanyl, and mono-, di-, tri- or tetra-saccharide derivatives;

[0071] or wherein all groups of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are selected collectively from the group consisting of acetyl, isobutyryl or propionyl; or wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently selected from the group consisting acetyl, isobutyryl or propionyl;

[0072] and wherein both pure anomers and mixtures of anomers such as - and -anomer centres of the above mentioned structural variations are claimed.

[0073] According to one specific embodiment, the non-water soluble carbohydrates comprises poly(ethylene glycol-b-caprolactone) (PEG-PCI), sucrose acetate isobutyrate (SAIB), poly(D,L-lactic acid), or poly(lactic-co-glycolic acid) (PGLA), or a combination thereof. According to another embodiment, the non-water soluble carbohydrates comprise mixed saccharides of furanose and pyranose.

[0074] Moreover, according to one specific embodiment of the present invention, said at least one imaging contrast agent makes the composition visible by PET imaging, SPECT imaging, Ultrasound imaging, CT imaging, MR imaging, x-ray imaging, fluoroscopy imaging, fluorescence imaging, or OCT imaging.

[0075] Moreover, according to yet another embodiment, the composition contains two imaging contrast agents that are visible by at least two imaging methods, such as visible by at least two imaging methods chosen from PET imaging, SPECT imaging, Ultrasound imaging, CT imaging, MR imaging, x-ray imaging, fluoroscopy imaging, fluorescence imaging, or OCT imaging.

[0076] Furthermore, according to one embodiment of the present invention the composition contains an imaging contrast agent for CT imaging and an imaging contrast agent for MR imaging.

[0077] According to one specific embodiment of the present invention, the composition contains an MR imaging contrast agent containing Gadolinium associated with a chelate. The composition may contain an MR imaging contrast agent containing Gadolinium associated with a chelate that is covalent linked to a polymer. Moreover, the composition may contain an MR imaging contrast agent containing Gadolinium associated with a chelate that is covalent linked to poly lactic acid (PLA). Moreover, the composition may contain an MR imaging contrast agent containing Gadolinium associated with a chelate chosen from PLA-DTPA or PLA-DOTA polymer chelates. Furthermore, the composition may contain a CT contrast agent and an MR imaging contrast agent containing Gadolinium associated with a chelate, and wherein the CT contrast agent is hydrophobic. As mentioned before, the hydrophobic CT contrast agent may be relatively uniformly distributed throughout the whole formulation, and the amphiphilic Gd-chelating constructs are capable of diffusing to the rim.

[0078] According to one embodiment, the composition comprises a pharmacologically active compound that is released into the surrounding tissue, such as chemotherapy that enhances the effect of radiotherapy.

[0079] Furthermore, according to yet another specific embodiment the composition is intended to be administered to the human or animal body through a syringe, an endoscope or a bronchoscope to the target tissue preferably wherein the composition after insertion into the human or animal body constitutes a medical or surgical implant for tissue or surgical adhesion which preferably is wound dressing, a hemostat, enhances tissue regeneration, is a void filler.

[0080] The present invention also embodies a medical or surgical implant comprising a composition according to the present invention, wherein the composition is part of a sprayable composition.

[0081] Moreover, the present invention provides a composition system which may be used as a tissue marker for guided surgery and/or imaging by one or multiple imaging modalities. The composition system may allow for detection of the tissue marker by an external imaging modality if administered or implanted into a mammalian body. Exemplary external imaging modalities include, but are not limited to, X-ray imaging, such as CT imaging, MRI, PET imaging, single photon emission computed tomography (SPECT) imaging, nuclear scintigraphy imaging, ultrasonography imaging, ultrasonic imaging, near-infrared imaging and/or fluorescence imaging.

EXAMPLES

Example 1: Synthesis of Carbohydrate Esters

[0082] General experimental conditions: All reactions were carried out under inert atmosphere (N.sub.2). Water sensitive liquids and solutions were transferred via syringe. Water used for washing of the isolated products was in all cases MilliQ water. Organic solutions were concentrated by rotary evaporation at 30-60 C. at 200-0 mbar. Thin layer chromatography (TLC) was carried out using aluminum sheets pre-coated with silica 60F (Merck 5554). The TLC plates were inspected under UV light or developed using a cerium ammonium sulphate solution (1% cerium (IV) sulphate and 2.5% hexa-ammonium molybdate in a 10% sulfuric acid solution).

[0083] Reagents: DOTA-NHS was purchased from Macrocyclics. All other chemicals were purchased from Sigma Aldrich and were used as received. Dry pyridine was obtained by drying over molecular sieves (4 ) for 2-3 days prior to use.

[0084] Instrumentation: Nuclear Magnetic Resonance (NMR) was conducted on a Bruker Ascend 400 MHzoperating at 401.3 MHz for .sup.1H and 100.62 MHz for .sup.13Cwith a 5 mm HBroadband Dual Channel z-gradient Prodigy cryoprobe at 298 K using the residual solvent as internal standard. All coupling constants (J) are expressed in Hz. The FID files were processed in Mnova Suite. In .sup.1H-NMR spectra of , anomeric mixtures, the integral of H-1 of the most abundant anomer was always set to 1.0, and the percentage of each anomeric species was calculated from the integral ratio of H-1 and H-1 . MALDI-TOF MS was conducted on a Bruker Autoflex Speed mass spectrometer. The matrix used for MALDI-TOF was a mixture of 2,5 dihydroxy benzoic acid (DHB) spiked with sodium trifluoroacetate in ethanol (60 mg/mL).

[0085] General Experimental Procedure for Synthesis of Carbohydrate Esters

[0086] Lactose (typically 10-100 g) was suspended in dry pyridine under inert atmosphere (N.sub.2). Hereafter, acetic, propionic or isobutyric anhydride (2.2 eq. pr. hydroxyl group) was carefully added. Then, a catalytic amount of DMAP (0.1 eq.) was added. The reactions were heated to 48 C. overnight, and then continued for 24 hours at room temperature, until TLC and MALDI-TOF showed complete acylation of the starting material. The reactions were concentrated under reduced pressure and co-evaporated with toluene. The concentrates were dissolved in CHCl.sub.3 and washed with NaHCO.sub.3(aq.) (5), brine (1) and water (1). The organic phases were dried with MgSO.sub.4 (s), filtered, concentrated under reduced pressure and dried in vacuo. Yields and reported spectra of individual sugar esters can be found below.

[0087] , Lactose Octaacetate

##STR00004##

[0088] Yield: 93.7% yield (mixture of anomers: 30% and 70% 3). .sup.1H-NMR: (400 MHz, Chloroform-d) 6.24 (d, J=3.7 Hz, 1H, H-1 ), 5.66 (d, J=8.3 Hz, 1H, H-1 ), 5.44 (dd, 10.28, 9.53 Hz, 0.4H), 5.37-5.31 (m, 2H), 5.23 (t, J=9.1 Hz, 1H), 5.15-5.00 (m, 3H), 4.99-4.91 (m, 2H), 4.50-4.41 (m, 3H), 4.17-4.05 (m, 4H), 3.99 (ddd, J=10.2, 4.3, 2.1 Hz, 0.4H, H5 ), 3.91-3.78 (m, 3H), 3.75 (ddd, J=9.9, 4.8, 2.0 Hz, 1H, H5 ), 2.19-1.93 (singlets, 32H, CH.sub.3 acetyls). MALDI TOF-MS: Calc [M+Na].sup.+: 701.59. Found: 701.51.

[0089] , Lactose Octapropionate

##STR00005##

[0090] Yield: 84% (mixture of anomers: 30% and 70% ). .sup.1H-NMR (400 MHz, Chloroform-d) 6.26 (d, J=3.7 Hz, 1H, H1-), 5.68 (d, J=8.3 Hz, 1H, H-1 ), 5.47 (dd, 10.3, 9.2 Hz, 0.4H), 5.38-5.33 (m, 2H), 5.26 (t, J=9.2 Hz, 1H), 5.15-5.00 (m, 3H), 5.02-4.91 (m, 2H), 4.49-4.41 (m, 3H), 4.15-4.03 (m, 4H), 3.98 (ddd, J=10.1, 3.9, 1.8 Hz, 0.4H, H5 ), 3.91-3.77 (m, 3H), 3.73 (ddd, J=9.9, 4.6, 2.0 Hz, 1H, H5 ), 2.47-2.15 (m, 23H), 1.19-0.99 (m, 34H). MALDI TOF-MS: Calc [M+Na].sup.+: 813.80. Found: 813.42.

[0091] , Lactose Octaisobutyrate

##STR00006##

[0092] Yield: 89.5% (mixture of anomers: 30% and 70% ). .sup.1H NMR (400 MHz, Chloroform-d) 6.26 (d, J=3.8 Hz, 1H, H-1), 5.68 (d, J=8.3 Hz, 1H, H-113), 5.48 (dd, J=10.3, 9.3 Hz, 0.4H), 5.40-5.34 (m, 2H), 5.27 (t, J=9.5 Hz, 1H), 5.18-5.00 (m, 3H), 5.03-4.91 (m, 2H), 4.50-4.41 (m, 3H), 4.24-4.02 (m, 4H), 3.95 (ddd, J=10.1, 3.8, 1.7 Hz, 0.4H, H5 ), 3.91-3.80 (m, 3H), 3.70 (ddd, J=9.9, 4.5, 2.0 Hz, 1H, H5 ), 2.70-2.32 (m, 11H), 1.26-1.01 (m, 68H). MALDI TOF-MS: Calc [M+Na].sup.+: 926.02. Found: 925.70.

Example 2: Synthesis of Fluorescently Labeled PLA

[0093] PLA-RhB

##STR00007##

[0094] PLA-NH.sub.2 (Mn2500) (260 mg, 0.1 mmol) was suspended in dry DCM (5 mL). Then, a pre-mixed mixture of Rhodamine-B (105, 0.2 mmol), EDC-HCl (80 mg, 0.4 mmol) and DMAP (106 mg, 0.9 mmol) dissolved in 5 mL dry DCM was added, and the reaction was continued at r.t. for 2 days, where Kaiser test was negative, indicating full functionalization. The solvents were removed in vacuo, and the crude mixture was dissolved in DMSO and purified by dialysis (Mw cutoff: 1000 da) against MQ water for 14 days. Yield: 299 mg (97%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.31 (d (br), J=7.7 Hz, 1H), 7.95 (br. t, J=7.4 Hz, 1H), 7.87 (br. t, J=7.6 Hz, 1H), 7.57-7.44 (m, 2H), 7.05-7.00 (m, 1H), 6.39-6.24 (m, 5H), 5.20 (q, J=7.1 Hz, 33H), 3.65 (dd (br), J=13.8, 6.9 Hz, 4H), 3.31 (br. q, J=7.1 Hz, 8H), 3.12-2.94 (m, 2H), 1.47 (d, J=7.1 Hz, 99H), 1.08 (t, J=6.9 Hz, 12H).

Example 3: Synthesis of Gd-Chelators

[0095] DOPE-DOTA

##STR00008##

[0096] Diacylphosphatidylethanolamine (DOPE) (12 mg, 0.016 mmol) was suspended in dry dichloromethane (3 mL), followed by addition of DOTA-NHS (18 mg, 0.036 mmol) and triethyl amine (50 L). The reaction was continued for 2.5 days at r.t., where after kaisertest (negative) and MALDI-TOF of the reaction mixture indicated completion. The solvent was removed in vacuo, and the crude mixture was dissolved in MeOH:H.sub.2O 40:60 and purified by preparative HPLC (Xterra C8 column, MeCN/H.sub.2O/TFA system. Gradient: 50->100% MeCN in 10 minutes). Yield: 13.5 mg, 74%. .sup.1H NMR (400 MHz, Chloroform-d) 7.93 (s, 1H), 5.39-5.28 (m, 5H), 5.21 (dddd (br), J=5.6, 3.0 Hz, 1H), 4.32 (dd, J=12.0, 3.1 Hz, 1H), 4.09 (dd, J=12.1, 6.8 Hz, 1H), 3.95-3.78 (m, 4H), 3.49 (dd, J=14.6, 7.3 Hz, 8H) 3.09 (dd, J=14.6, 7.3 Hz, 15H), 2.63 (s, 4H), 2.25 (dt, J=10.0, 7.5 Hz, 4H), 1.97 (q, J=6.4 Hz, 8H), 1.40-1.13 (m, 40H), 0.84 (t, 6H). MALDI-TOF MS: Calc [M+H].sup.+: Calc 1131.45, Found: 1131.5.

[0097] PLA-DTPA

##STR00009##

[0098] PLA-NH.sub.2 (Mn2500) (260 mg, 0.1 mmol) was suspended in dry pyridine (10 mL) followed by addition of DTPA-dianhydride (57.5 mg, 0.16 mmol) and a catalytic amount of DMAP (1.3 mg, 0.01 mmol). The reaction was continued for 1.5 day, where Kaiser test (negative) indicated the reaction was completed. 5 mL MQ water was added and stirred for 2 h to hydrolyze any residual anhydride. The solvents were removed in vacuo, and the crude mixture was dissolved in DMSO and purified by dialysis (Mw cutoff: 1000 da) against MQ water for 10 days. Yield: 285 mg (96%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.09 (t, J=5.08 Hz, NH, 1H), 5.20 (q, J=7.0 Hz, 33H), 5.47 (s, 1H, OH), 4.21 (q, J=7.0 Hz, 2H), 4.17-4.01 (m, 2H)), 3.69-3.21 (m, 10H), 3.14 (q, J=7.0 Hz, 2H), 3.06-2.81 (m, 6H), 1.74 (p, J=7.0 Hz, 2H), 1.47 (d, J=7.0 Hz, 99H).

[0099] PLA-DOTA

##STR00010##

[0100] PLA-NH.sub.2 (Mn2500) (80 mg, 0.032 mmol) was suspended in dry dichloromethane (3 mL), followed by addition of DOTA-NHS (35 mg, 0.0704 mmol) and triethyl amine (40 L). The reaction was continued for 2.5 days at r.t., where Kaiser test (negative) indicated completion. The solvent was removed in vacuo, and the crude mixture was dissolved in DMSO and purified by dialysis (Mw cutoff: 1000 da) against MQ water for 8-10 days. Yield: 89.6 mg (97%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.23 (s, NH, 1H), 5.21 (q, J=7.0 Hz, 33H), 4.21 (q, J=7.0 Hz, 2H), 4.16-3.99 (m, 2H), 3.49-3.34 (m, 6H), 3.24-3.03 (m, 4H), 3.03-2.87 (m, 9H), 2.70-2.61 (m, 4H), 1.79 (p, J=7.0 Hz, 2H), 1.63 (d, J=7.1 Hz, 1H), 1.47 (d, J=7.0 Hz, 99H).

Example 4: In-Vitro Confocal Microscopy of Markers Containing Fluorescent Amphiphiles

[0101] Laser scanning confocal microscopy was conducted using a Leica TCS SP5 Scanning Laser Confocal Microscope (61 wet objective, 561 nm excitation DPPS Laser) on 5-10 uL volumes of different marker compositions (see Table 1) in PBS buffer in order to investigate distribution of fluorescently labeled amphiphiles in the markers. A predominant distribution to the marker rim indicates the possibility of primary contrast enhancement at the marker rim when formulating the corresponding Gd chelators. The markers were prepared in 8 well microscope slides containing PBS buffer and imaged the same day. Multiple images were acquired for each sample with a 0.5 um spacing between z-stack frames. Image processing was performed using FIJI. All formulations listed in Table 1 show predominant accumulation of fluorophore at the marker-water interface. Representative results are shown in FIG. 9.

TABLE-US-00001 TABLE 1 Specifications for preparation of fluorophore containing marker formulations. Concentration Composition (w/w %) (g/L) Formu- Sugar Tri- Fluoresc. lation# ester glyceride Solvent Amphiphile 1 LAP 1:1 GTH PC EtOH PLA-RhB 65 40 5 0.006 2 LAP 1:1 GTH PC EtOH PLA-RhB 50 40 10 0.006 3 LOIB GTO PC EtOH PLA-RhB 60 40 0.006 4 LAP 1:1 GTH PC EtOH PLA-RhB 75 20 5 0.006 5 LAP 1:1 GTH PC EtOH DOPE-RhB 65 40 5 0.001 6 LAP 1:1 GTH PC EtOH DOPE-RhB 50 40 10 0.001 7 LOIB GTO PC EtOH DOPE-RhB 60 40 0.001 8 LAP 1:1 GTH PC EtOH DOPE-CF 65 40 5 0.04 9 LAP 1:1 GTH PC EtOH DOPE-CF 50 40 10 0.04 10 LOIB GTO PC EtOH DOPE-CF 60 40 0.04 LAP 1:1: Lactose octaacetate:octapropionate 1:1. LOIB: Lactose octaisobutyrate. GTH: Glycerol trihexanoate. GTO: Glycerol trioctanoate. PC: Propylene carbonate. EtOH: Ethanol. PLA: Poly-(L-lactide). DOPE: Diacylphosphatidylethanolamine, RhB: Rhodamine B. CF: Carboxy fluorescein.

Example 5: In-Vitro MRI Imaging of Markers

[0102] Single markers (50 L) containing 3 mM concentrations of different Gd-chelators (see Table 2) were injected into 2 mL glass vials containing PBS buffer and investigated using a PharmaScan 7T micro MRI scanner by T1 RARE imaging (Flip angle: 90. TR: 1000 ms. TE: 6.8 ms. Echo spacing: 6.8 ms, averages: 7, repetitions: 7. Rare factor: 2. Slice thicknes s: 0.7 mm, slice package of 8. FOV: 2020 mm.sup.2. Resolution: 256256 voxels) in order to visualize T1 enhancement at the marker-water interface. The markers were MRI scanned while still in the PBS vials within 1 week after injection. All scans were conducted using a 3D mouse volume coil (Bruker RF volume coil with 3 cm inner diameter). Image data processing was performed on ParaVision software version 6.0.1. All markers displayed T.sub.1 contrast enhancement at the marker-water interface. The results are shown in FIG. 10.

TABLE-US-00002 TABLE 2 Specifications for preparation of Gd-containing marker formulations for in-vitro MRI scan. Composition (w/w %) Concentration Formu- Sugar Tri- CT (mM) lation# ester glyceride Solvent contrast Gd-chelator 1 LAP 1:1 GTH PC x-SAIB BSA-DTPA(Gd) 45 40 5 10 3 2 LAP 1:1 GTH PC x-SAIB DOPE-DOTA(Gd) 45 40 5 10 3 3 LAP 1:1 GTH PC x-SAIB PLA-DTPA(Gd) 45 40 5 10 3 4 LAP 1:1 GTH PC x-SAIB PLA-DOTA(Gd) 45 40 5 10 3 LAP 1:1: Lactose octaacetate:octapropionate 1:1. GTH: Glycerol trihexanoate. PC: Propylene carbonate. PLA: Poly-(L-lactide). DOPE: Diacylphosphatidylethanolamine, BSA: Bis(stearylamide). DOTA: 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid. DTPA: diethylenetriaminepentaacetic acid. x-SAIB: 6,6-di-triidobenzene-isobuturic-sucrose.

TABLE-US-00003 TABLE 3 In-vivo T.sub.1 relaxation table of material rims formulated with the above listed species in the stated concentrations. Base formulation was in all cases the LAP 1:1, 40% GTH, 5% PC matrix. In-vivo T.sub.1 values T1 rim T1 rim PLA- DOPE- mM PLA- DTPA(Gd) mM DOPE- DOTA(Gd) DTPA(Gd) (msec) DOTA(Gd) (msec) 3 mM 876.93 3 mM 409.95 1.5 mM 917.79 1.5 mM 779.40 0.75 mM 1089.45 0.75 mM 942.46 0.3 mM 1145.45 0.3 mM 1212.26 No Gd 1421.35 No Gd 1677.27 T1 rim BSA-DPTA(Gd), T1 rim PLA-DOTA(Gd), 3 mM (msec) 3 mM (msec) 1188.27 949.28

Example 6: In-Vivo CT and MRI Imaging of Subcutaneous Markers

[0103] 6.1:

[0104] The formulations from Example 5 were injected in 50 L volumes subcutaneously in the hind leg of NMRI Nude mice (n=1 for each formulation). The mice were MRI and CT scanned 1 day and 1 week post injection, utilizing the same scanners, software and coil as in Example 5. Both CT scan (Inveon small animal CT scanner (Siemens Medical Systems), processsing performed on Inveon software), T1 RARE imaging (Flip angle: 90. TR: 1500 ms, TE: 8 ms, Echo spacing: 8 ms, averages: 2, repetitions: 1, Rare factor: 4. FOV: 3535 mm.sup.2. Resolution: 256256 voxels. Slice thickness: 0.7 mm, slice packages of 9-16 depending on marker size) and T1 RARE mapping (Flip angle: 90. TR's: 5500, 4000, 3000, 1500, 800, 400 and 200 ms. TE: 7.5 ms, Echo spacing: 7.5 ms. Rare factor: 2. Averages: 2, repetitions: 1. FOV: 3535 mm.sup.2, collected into a matrix of 192192 voxels, slice packages of 9-16 depending on marker size) was performed, in order visualize the markers as well as measure the T.sub.1 relaxation time at the marker rim. Data processing was performed on ParaVision software version 6.0.1. While the Gd-chelating lipids seemed to leak out of the marker over time, the Gd-chelating PLA analogues did not result in visible leakage but seemed to stay predominantly at the marker-water interface, causing a bright T.sub.1 enhancement of the marker rims. The results from day 7 are shown in FIG. 11.

[0105] 6.2:

[0106] The best performing formulation from FIG. 11, with the clearest contrast enhancement (FIG. 11 a-b. Formulation 3, Table 2), was, injected subcutaneously along with an additional formulation containing 20% triglyceride (see Table 4) in the hind leg of NMRI Nude mice (50 L volumes, n=4). The mice were then CT and MRI scanned at specific timepoints post injection (MRI: 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks and 6 weeks. CT: week 1, 3 and 6) in order to assess marker volumes, as well as CT and MRI (T.sub.1) contrast enhancement over time. The same scan methods, software and equipment as described in 6.1 was employed. All markers displayed T.sub.1 enhancement and high CT contrast enhancement over the entire study period. After ended study, the mice were euthanized and the markers removed surgically, no visible irritation or inflammation was present around the markers. The results are shown in FIGS. 12.1 and 12.2.

TABLE-US-00004 TABLE 4 Specifications for preparation of Gd-containing marker formulations for 6-week study in-vivo of subcutaneous markers. Composition (w/w %) Concentration Formu- Sugar Tri- CT (mM) lation# ester glyceride Solvent contrast Gd-chelator 1 LAP 1:1 GTH PC x-SAIB PLA-DTPA(Gd) 45 40 5 10 3 2 LAP 1:1 GTH PC x-SAIB PLA-DTPA(Gd) 65 20 5 10 3 LAP 1:1: Lactose octaacetate:octapropionate 1:1. GTH: Glycerol trihexanoate. PC: Propylene carbonate. PLA: Poly-(L-lactide). DTPA: diethylenetriaminepentaacetic acid. x-SAIB: 6,6-di-triidobenzene-isobuturic-sucrose.

Example 7: In-Vivo CT and MRI Imaging of Intramuscular Marker

[0107] Formulation 1 from Table 4, Example 6.2 (25 L) was injected into the thigh muscle of an NMRI Nude mouse to monitor marker stability in frequently moving tissue. The mouse did not show any signs of pain or difficulty moving around, the weight of the mouse also remained stable throughout the experiment. MRI scan was conducted 7 days post injection using the same scanner, T1 RARE scan method and coil as listed in Example 6.1. Image processing was performed on ParaVision software version 6.0.1. At 7 days post injection, CT scan of the marker was also performed on the same equipment as listed in Example 5-6. The MRI images showed T.sub.1 enhancement at the marker rim. The results are shown in FIG. 13.

CONCLUSION

[0108] The lactose acetate:propionate 1:1 formulation containing 3 mM PLA-DTPA(Gd), 40% GTH and 5% PC performed well as dual MRI and CT marker over the observation period of 3 weeks. The stable T.sub.1 contrast enhancement displayed sufficient strength and sufficient resolution making it amenable to be observable in patients when using standard clinical MRI facilities and setup. These agents have the potential to result in a novel, commercially applicable, injectable marker for treatment planning and monitoring during IGRT. Future development includes adjusting the triglyceride level and addition of polyfunctional Gd-chelating polymers to achieve both enhanced T.sub.1 contrast as well as a stable 3D structure optimal for tracking in-vivo.

Abbreviations

BSA: Bis(Stearylamide)

CF: Carboxyfluorescein

CT: Computed Tomography

DOPE: Diacylphosphatidylethanolamine

[0109] DOTA: 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid.
DTPA: diethylenetriaminepentaacetic acid

EBUS: Endobronchial Ultrasound

[0110] EDC: 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide.

EtOH: Ethanol

EUS: Endoscopic Ultrasound

[0111] G: Gauge (needle size)

GTH: Glycerol Trihexanoate

GTO: Glycerol Trioctanoate

IGRT: Image Guided Radiotherapy

[0112] LAP: Lactose octaacetate:octapropionate 1:1
LI: Lactose octaisobutyrate
MALDI-TOF: Matrix assisted laser desorption/ionization time-of-flight mass spectrometry.

MRI: Magnetic Resonance Imaging

PBS: Phosphate Buffered Saline

PC: Propylene Carbonate

PLA: Polylactic Acid

[0113] RARE: Rapid Acquisition with Relaxation Enhancement

RhB: Rhodamine B

RT: Radio Therapy

SAIB: Sucrose Acetate Isobutyrate

S.C.: Subcutaneous

T: Tesla

[0114] T.sub.1: Longitudinal relaxation
T.sub.2: Transverse relaxation
TE: Echo time

TLC: Thin Layer Chromatography

TR: Repetition Time

Wt %: Weight %

[0115] XSAIB: 6,6-di-triidobenzene-isobuturic-sucrose or IodoSAIB

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