Liposomal composition of gadobutrol and process for preparation thereof

Abstract

The present invention relates to a liposomal composition of Gadobutrol. The present invention also relates to a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients. The present invention also relates to a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients, wherein Gadobutrol encapsulated into a liposome carrier of size less than 200 nm and unbound free Gadobutrol limit of less than 2%. The present invention also relates to a process for the preparation of liposomal composition of Gadobutrol.

Description

[0001] LIPOSOMAL COMPOSITION OF GADOBUTROL AND PROCESS FOR PREPARATION THEREOF FIELD OF INVENTION

[0002] The present invention relates to a liposomal composition of Gadobutrol.

[0003] The present invention also relates to a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients.

[0004] The present invention also relates to a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients, wherein Gadobutrol encapsulated into a liposome carrier having size less than 200 nm and unbound free Gadobutrol limit of less than 2%.

[0005] The present invention also relates to a process for the preparation of liposomal composition of Gadobutrol.

[0006] BACKGROUND OF INVENTION

[0007] Gadobutrol is a paramagnetic contrast agent for magnetic resonance imaging, which consists of a neutral complex consisting of gadolinium (III) and the macrocyclic dihydroxy -hydroxymethylpropyl- tetraazacyclododecane- triacetic acid (butrol).

[0008] Gadobutrol shortens the T1 and T2 relaxation times in a magnetic field environment. When T1 -weighted magnetic resonance sequences are used, the T1 shortening effect is most sensitively detected at the prescribed dosage. When gadolinium is injected at high concentrations (during bolus injection), it induces a local magnetic field in-homogeneities, which results in a decrease in signal in T2-weighted sequences. Gadobutrol is chemically known as 4,7,10-tris(carboxymethyl)-l-[(2S,3R)-1 ,3,4-trihydroxybutan-2-yl]- 1 ,4,7, 10-tetraaza- 13-gadolinatetracyclo[5.5.1.0A{4,13}.0A{ 10,13 }]tridecane l,4,7,10-tetraium-13-uide. The molecular formula is Ci8H31GdN4O9 and the molecular weight is 604.7 g / mol. The structural formula is

[0009]

[0010] Gadobutrol solution for injection (GADOVIST® 7.5 ml, 10 ml, 15 ml, 30 ml, 65 ml) marketed by Bayer (PTY) LTD. However, the currently used gadolinium (Gd) chelates restrict the time window for image acquisition due to their rapid elimination from blood and their rapid diffusion into the extravascular space, which prevents their use in steady-state imaging, particularly for MR angiography (MRA).

[0011] US 20220211880 Al discloses liposome compositions of contrast agent for targeted diagnostics, enhancing the detection of molecular and cellular changes with great precision.

[0012] CN 115364246 A discloses a method for preparing a targeted MRI contrast agent using gadolinium and this contrast agent effectively targets specific lymphatic vessels for MRI imaging and is safe and reliable for biological use.

[0013] JPH 10513466 A discloses a liposome formulation for intravascular space imaging, specifically designed for diagnostic imaging. The liposome preparation includes: 40-90% phospholipid or amphiphile, 10-50% sterol, 0-25% charge carrier. The liposomes have an average diameter of 100-400 nm, and the active ingredient is an X-ray, MRI contrast agent, or radiodiagnostic agent.

[0014] CN 106620726 A discloses a gadobutrol injection and a preparation method which includes following components : Gadobutrol, calcobutrol sodium, pH adjusting agent and water, the pH value of the Gadobutrol parenteral solution is 6.7 ~ 8.0, Characterized in that, the pH adjusting agent is phosphate.

[0015] The goal of present invention is to prepare long-circulating polyethylene glycol-bearing ((PEG)ylated) liposomes encapsulating Gadobutrol chelate, and characterize and demonstrate their utility for MRI. The long -circulating liposomal formulations allow for high spatial resolution imaging without the confounding effects of clearance and extravascular diffusion of the agent complicating the data and image analysis.

[0016] Thus to overcome drawbacks associated with currently available formulations, the inventors of the present invention have developed Liposomal composition of gadobutrol contrast agent for enhanced magnetic resonance imaging which provides enhanced permeation and retention effect in cancer imaging with improved stability of the liposomal Gadobutrol.

[0017] OBJECTIVE OF INVENTION

[0018] The objective of the present invention is to provide a liposomal composition of Gadobutrol.

[0019] Another objective of the present invention is to provide a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients. Another objective of the present invention is to provide a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients, wherein Gadobutrol encapsulated into a liposome carrier having size less than 200 nm and unbound free Gadobutrol limit of less than 2%.

[0020] Another objective of the present invention is to provide a process for the preparation of liposomal composition of Gadobutrol.

[0021] SUMMARY OF INVENTION

[0022] Accordingly, the present invention provides a liposomal composition of Gadobutrol.

[0023] One embodiment of the present invention provides a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients.

[0024] Another embodiment of the present invention provides a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients, wherein Gadobutrol encapsulated into a liposome carrier having size less than 200 nm and unbound free Gadobutrol limit of less than 2%.

[0025] Another embodiment of the present invention provides a liposomal composition of Gadobutrol comprising one or more excipients selected from phospholipids, hydrophilic lipids, sterol, tonicity modifiers, buffers and vehicles.

[0026] Another embodiment, the present invention relates to encapsulation of Gadobutrol into liposome comprising a mixture of at least one phospholipid and sterol such as cholesterol.

[0027] Another embodiment of the present invention provides a liposomal composition comprising:

[0028] a) Gadobutrol, b) phospholipid mixture,

[0029] c) hydrophilic lipid,

[0030] d) tonicity modifiers,

[0031] e) buffers, and

[0032] f) vehicles.

[0033] Another embodiment of the present invention provides a liposomal composition comprising:

[0034] a) Gadobutrol,

[0035] b) Calcobutrol sodium,

[0036] c) Tromethamine,

[0037] d) Hydrogenated Soybean Phosphatidylcholine (HSPC),

[0038] e) Cholesterol,

[0039] f) methoxy-poly(ethyleneglycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE),

[0040] g) Sucrose,

[0041] h) Sodium Chloride,

[0042] i) Trometamol and

[0043] j) Sodium Hydroxide.

[0044] Another embodiment of the present invention provides a liposomal composition comprising:

[0045] a) Gadobutrol in the weight range of 100 mg / mL to 1000 mg / mL,

[0046] b) Calcobutrol sodium in the weight range of 0.1 mg / mL to 5 mg / mL, c) Tromethamine in the weight range of 0.1 mg / mL to 10 mg / mL,

[0047] d) Hydrogenated Soybean Phosphatidylcholine (HSPC) in the weight range of 1 mg / mL to 20 mg / mL,

[0048] e) Cholesterol in the weight range of 1 mg / mL to 10 mg / mL,

[0049] f) methoxy-poly(ethyleneglycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) in the weight range of 1 mg / mL to 10 mg / mL, g) Sucrose in the weight range of 50 mg / mL to 200 mg / mL,

[0050] h) Sodium Chloride in the weight range of 10 mg / mL to 100 mg / mL, i) Trometamol in the weight range of 0.1 mg / mL to 5 mg / mL, and

[0051] j) Sodium hydroxide in the weight range of 0.001 mg / mL to 2 mg / mL,

[0052] Another embodiment of the present invention provides a process for the preparation of liposomal composition of Gadobutrol.

[0053] Another embodiment of the present invention provides a process for the preparation of liposomal composition comprising:

[0054] a) preparing Gadobutrol hydration solution and

[0055] b) preparing Gadobutrol loaded liposomes.

[0056] Another embodiment of the present invention provides a process for the preparation of liposomal composition comprising:

[0057] a) dissolving Calcobutrol sodium in water for injection and stirring until clear solution is obtained,

[0058] b) dissolving sodium hydroxide and stirring until a clear solution is obtained, c) dissolving Tromethamine and stirring until a clear solution is obtained, d) adding Gadobutrol and stirring until a clear solution is obtained,

[0059] e) adjusting the pH to 6.6 - 8 using IN HC1, making up the volume to the required batch size and filtering the solution through a 0.22 pm filter, f) co-dissolving Hydrogenated Soybean Phosphatidylcholine (HSPC), methoxy-poly(ethyleneglycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) and Cholesterol in ethanol at about 74°±4°C, g) hydrating the lipids of step (f) with solution obtained in step (e),

[0060] h) extruding the coarse lipid dispersion under pressure through three stacked polycarbonate membrane of size 200nm and 80nm (0.2 / 0.08 / 0.08p), i) removing the unentrapped Gadobutrol by dialysis or tangential flow filtration (TFF) against buffer solution containing 645 mM sodium chloride, 292 mM sucrose & 10 mM Tro metamol and

[0061] j) storing the liposomes obtained in above step.

[0062] BRIEF DESCRIPTION OF DRAWINGS

[0063] Fig.l: In-Vitro cell magnetic resonance (MR) images of non-liposomal Gadobutrol Vs Gadobutrol liposomes of present application

[0064] Fig.2: In-Vivo Biodistribution at 0.5hr of non-liposomal Gadobutrol Vs Gadobutrol liposomes of present application

[0065] Fig.3: In-Vivo Biodistribution at Ihr of non-liposomal Gadobutrol Vs Gadobutrol liposomes of present application

[0066] Fig.4: In-Vivo Biodistribution at 2hr of non-liposomal Gadobutrol Vs Gadobutrol liposomes of present application

[0067] Fig.5: In-Vivo Biodistribution at 4hr of non-liposomal Gadobutrol Vs Gadobutrol liposomes of present application

[0068] DETAILED DESCRIPTION OF THE INVENTION

[0069] The term "comprising", which is synonymous with "including", "containing", or "characterized by" here is defined as being inclusive or open-ended, and does not exclude additional, unrecited elements or method steps, unless the context clearly requires otherwise.

[0070] The present invention provides a liposomal composition of Gadobutrol.

[0071] One embodiment of the present invention provides a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients. Another embodiment of the present invention provides a liposomal composition comprising Gadobutrol and one or more pharmaceutically acceptable excipients, wherein Gadobutrol encapsulated into a liposome carrier having size less than 200 nm and unbound free Gadobutrol limit of less than 2%.

[0072] The Gadobutrol used in the present invention is Gadobutrol Monohydrate.

[0073] The concentration of Gadobutrol in the present invention is 100 mg / mL to 1000 mg / mL. The concentration of Gadobutrol expressed in moles is between 165.365 mM to 1653 mM in the hydration layer; more preferably 1 M (eq to 623 mg of Gadobutrol monohydrate); with the encapsulation efficiency for gadobutrol inside the liposome by passive loading is approximately 5%.

[0074] Gadobutrol hydration solution contains Gadobutrol Monohydrate, Free Gadolinium chelating agent and buffering agents.

[0075] The Free Gadolinium chelating agent used in the Gadobutrol hydration solution is Calcobutrol Sodium present in an amount of 0.1 mg / mL to 5 mg / mL (0.195 mM to 9.792 mM).

[0076] The buffering agents used in the Gadobutrol hydration solution are Tromethamine and sodium hydroxide.

[0077] The concentration of Tromethamine is 0.1 mg / mL to 10 mg / mL (0.825 mM to 82.549 mM) and Sodium hydroxide is 0.001 mg / mL to 2 mg / mL (0.025 mM to 50.125 mM).

[0078] The pH of the Gadobutrol hydration solution is adjusted to 6.6 - 8 using IN HC1. Another embodiment of the present invention provides a liposomal composition of Gadobutrol comprising excipients selected from phospholipids, sterols, hydrophilic lipids, tonicity modifiers, buffers and vehicles.

[0079] Another embodiment, the present invention relates to encapsulation of Gadobutrol into liposome comprising a mixture of at least one phospholipids, sterols and hydrophilic lipids.

[0080] The liposome forming phospholipids used in the present invention may include but not limited to, hydrogenated soy phosphatidyl choline (HSPC), distearoyl phosphatidyl choline (DSPC), phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine.

[0081] The concentration of hydrogenated soy phosphatidyl choline (HSPC)is from 1 mg / mL to 20 mg / mL (1.265 mM to 25.311 mM)

[0082] The sterol used in the present invention is cholesterol. The concentration cholesterol from 1 mg / mL to 10 mg / mL (2.586 mM to 25.862 mM).

[0083] The hydrophilic lipid includes, methoxy polyethylene glycol (mPEG), with a molecular weight of 2000 Daltons, terminated distearoyl phosphatidyl ethanolamine (DSPE).

[0084] The concentration of MPEG-2000-DSPE is from 1 mg / mL to 10 mg / mL (0.367 mM to 3.673 mM).

[0085] Phospholipid mixture in liposomes contains DSPC / HSPC, Cholesterol, and MPEG 2000-DSPE at a molar ratio of 56:38:5 and the total lipid concentration is between 20 mM to 23 mM, more preferably 21.5 mM. The tonicity modifiers used in the present invention are sucrose and sodium chloride.

[0086] The concentration sucrose is from 50 mg / mL to 200 mg / mL (146.113 mM to 584.453 mM).

[0087] The concentration sodium chloride is from 10 mg / mL to 100 mg / mL (171.11 mM to 1711 mM).

[0088] The buffers used in liposomes of present invention are Trometamol and sodium hydroxide.

[0089] The concentration Trometamol is from 0.1 mg / mL to 5 mg / mL (0.825 mM to 41.274 mM).

[0090] The vehicle used in the present invention is water for injection in quantity sufficient.

[0091] In another embodiment lipids are hydrated and passively loaded with gadobutrol using an aqueous solution of gadobutrol monohydrate, calcobutrol sodium, sodium hydroxide and tromethamine; having a pH of 6.6 to 8.

[0092] In another embodiment, the liposomes were suspended in a Sucrose-Sodium chloride & Trometamol having pH 6.6-8. The resulting stable liposomal formulation can be stored at refrigerated storage condition for 24 months.

[0093] The liposomal composition, containing gadobutrol or its pharmaceutically acceptable salt, a gadolinium based macrocyclic paramagnetic contrast agent, encapsulated into the liposome for enhanced magnetic resonance imaging. The liposomal composition contains unbound free MRI contrast agent limit of less than 2%.

[0094] The liposomal composition having the mean particle diameter of liposome is less than 200nm.

[0095] The liposomal composition is stored in refrigerated condition and is stable at this condition for 24 months.

[0096] The following examples describe the nature of the invention and are given only for the purpose of illustrating the present invention in more detail and are not limitative.

[0097] EXAMPLES

[0098] Example 1: Preparation of Gadobutrol hydration solution (non-liposomal Gadobutrol solution)

[0099] Category QUANTITY Sl.No Ingredients

[0100] Formula (mg / mL) 1 Gadobutrol Active ingredient

[0101] 623 Monohydrate

[0102] 2 Free Gadolinium

[0103] Calcobutrol Sodium 0.513 chelating agent

[0104] 3 Tromethamine Buffering agent 1.211

[0105] 4 Sodium Hydroxide Buffering agent 0.023

[0106]

[0107] Collect approximately the required amount of WFI (Water for Injection) [temperature: 70°C to 80°C]. Purge nitrogen for over 1 hour, then cool the WFI to 20- 25 °C. Take the required amount of WFI and begin stirring while purging with nitrogen. Dissolve the required quantity of Calcobutrol sodium until a clear solution is obtained. Then dissolve the required quantity of sodium hydroxide and stir with continuous nitrogen purging until a clear solution is obtained. Next, dissolve the required quantity of Tromethamine and stir with continuous nitrogen purging until a clear solution is obtained. Then add the required quantity of Gadobutrol and stir with continuous nitrogen purging until a clear solution is formed. Heat the solution to 80°C for no more than 5 minutes under continuous stirring and nitrogen purging. Then, cool the solution to 20-25°C and adjust the pH 6.6 - 8 using IN HC1. Finally, make up the volume to the required batch size. Filter the solution through a 0.22 pm filter

[0108] Example 2: Preparation of Gadobutrol loaded liposome

[0109] Category QUANTITY Sl.No Ingredients

[0110] Formula (mg / mL) 1 HSPC phospholipid mixture 9.58

[0111] 2 Cholesterol Sterol 3.19

[0112] 3 MPEG-2000-DSPE hydrophilic lipid 3.19

[0113] 4 Sucrose tonicity modifiers 100

[0114] 5 Sodium Chloride tonicity modifiers 37.72

[0115] 6 Trometamol Buffering agent 1.211

[0116]

[0117] The liposomes were prepared by ethanol-inj ection method. Lipids including HSPC, MPEG-2000-DSPE and Cholesterol at a molar ratio of 56:38:5 were codissolved in ethanol at about 74°±4°C. Lipids are hydrated using aquous solution prepared by using formula in example 1. The bulk were homogenized at NLT 10,000 RPM and the temperature was maintained upto 74°±4°C for 30mins to obtain the coarse liposomal dispersion. Then coarse lipid dispersion were extruded under pressure through three stacked polycarbonate membrane of size 200nm and 80nm (0.2 / 0.08 / 0.08p), extrusion was continued up to 8 to 10 cycles to obtain liposomes with size less than 200nm.The unentrapped Gadobutrol was removed by dialysis or tangential flow filtration (TFF) against buffer solution containing 645 mM sodium chloride, 292 mM sucrose & 10 mM Trometamol. The prepared liposome can be stored in refrigerated condition.

[0118] Biological Data

[0119] In- Vitro Cell magnetic resonance (MR) data

[0120] A 3 T human magnetic resonance scanner was used to obtain MR images and the resulting signal intensity ratio of HepG2 cell pellets in order to examine the potential of liposomes to improve T1 -weighted contrast of cell populations. Gadobutrol liposomes, non-liposomal Gadobutrol, and SNT buffer were used to incubate HepG2 cells, respectively. Cells incubated with non-liposomal Gadobutrol showed slight improvement in contrast over cells incubated with SNT buffer. In the contrast, there was a noticeable increase in the cells that were exposed to Gadobutrol liposomes. In addition, a quantitative analysis of the signal intensity was conducted to determine the variation in MR images. The non-liposomal Gadobutrol -treated cells showed a slight improvement in signal intensity to 105 ± 2%, while the cells incubated with Liposomal Gadobutrol showed an increase to 140 + 3%. The SNT buffer group served as a baseline.

[0121] Table 1

[0122] IN VITRO Cell MR

[0123] non-liposomal LIPOSOMAL Gadobutrol 1.0 GADOBUTROL Relative Intensity

[0124] (SNT buffer % as 105% 140%

[0125] base line)

[0126]

[0127] Gd / f ®ram o;

[0128] ki t<np _ _

[0129] *SNT - Sucrose , NaCl, Trometamol buffer

[0130]

[0131] Biodistribution data:

[0132] Applicant investigated the distribution of liposomal gadobutrol in HepG2 tumorbearing nude mice in order to ascertain whether it can be delivered into a tumour in vivo. ICP-AES was used to measure the amounts of gadolinium in the tissues after the animals were killed at various intervals following injections of liposomal gadobutrol and non-liposomal Gadobutrol, respectively (Dose-O.IM Gd / Kg). According to the data, liposomal gadobutrol consistently showed increased gadolinium levels in every organ but the kidney. The clearance of liposomal gadobutrol was comparatively slow. The distribution of liposomal Gadobutrol in HepG2 tumor-bearing nude mice was studied to see if it could be delivered into a tumour in vivo. Once liposomal Gadobutrol and non-liposomal Gadobutrol were injected, the animals were sacrificed at various intervals. Tumour uptake of liposomal Gadobutrol peaked at 2% ID / g after one hour. In vivo, since non-liposomal Gadobutrol is basically a small molecule, which led to extremely low organ uptake and quick blood clearance. Kidney uptake levels of non-liposomal Gadobutrol peaked at 1.41% ID / g at 1 hour. At all time points, there was greater tumour uptake of liposomal Gadobutrol than that of non-liposomal Gadobutrol. The tumor-targeting of liposomal Gadobutrol was higher than that of non- liposomal Gadobutrol, according to these Ex-vivo biodistribution data. In addition to accomplishing our goal, this study showed the higher tumour uptake and residence time in tumor when compared to the control.

[0133] Table 2

[0134] In Vivo Biodistribution 0.5hr

[0135] Live Lun

[0136] Heart Spleen Kidney Tumor Muscle r g

[0137] non-liposomal 0.3 0.28 0.29 0.22 2.28 0.24 0.26

[0138]

[0139] %ID Gd / fi t %ID Gd / fi tgram osseugram osseu

[0140] Gadobutrol

[0141] LIPOSOMAL GADOBUTRO 2.2 2.04 5 0.94 0.2 1.4 0.6 L

[0142]

[0143] Table 3

[0144] In Vivo Biodistribution Ihr

[0145] Live Lun

[0146] Heart Spleen Kidney Tumor Muscle r g

[0147] non-liposomal

[0148] 0.2 0.25 0.22 0.48 1.41 0.54 0.55 Gadobutrol

[0149] LIPOSOMAL GADOBUTRO 0.3 1.51 2.32 1.25 0.87 2 0.46 L

[0150]

[0151] Table 4

[0152] In Vivo Biodistribution 2hr

[0153] Live Lun Kidne

[0154] Heart Spleen Tumor Muscle r g y

[0155] non-liposomal

[0156] 0.19 0.33 0.24 0.13 1.21 0.27 0.13 Gadobutrol

[0157] LIPOSOMAL GADOBUTRO 0.38 1.42 1.7 0.22 0.86 0.53 0.45 L

[0158]

[0159] %ID Gd / fi tgram osseu

[0160] Table 5

[0161] In Vivo Biodistribution 4hr

[0162] Live Lun Kidne

[0163] Heart Spleen Tumor Muscle r g y

[0164] non-liposomal

[0165] 0.18 0.18 0.37 0.24 0.45 0 0.12 Gadobutrol

[0166] LIPOSOMAL GADOBUTRO 0.19 1.37 1.72 0.41 0.64 0.22 0.31 L

[0167]

Claims

I / WE CLAIM1. A liposomal composition comprising Gadobutrol and pharmaceutically acceptable excipients.

2. The composition as claimed in claim 1, wherein pharmaceutically acceptable excipients are selected from Gadolinium chelating agent, phospholipids, hydrophilic lipids, sterol, tonicity modifiers, buffers and vehicles.

3. The composition as claimed in claim 2, wherein Free Gadolinium chelating is Calcobutrol Sodium present in an amount of 0.1 mg / mL to 5 mg / mL.

4. The composition as claimed in claim 2, wherein phospholipids selected from hydrogenated soy phosphatidyl choline (HSPC), distearoyl phosphatidyl choline (DSPC), phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, the concentration of phospholipids and cholesterol is from 1 mg / mL to 20 mg / mL.

5. The composition as claimed in claim 2, wherein sterol is cholesterol, concentration cholesterol from 1 mg / mL to 10 mg / mL.

6. The composition as claimed in claim 2, wherein hydrophilic lipid is methoxy polyethylene glycol (mPEG), with a molecular weight of 2000 Daltons, terminated distearoyl phosphatidyl ethanolamine (DSPE), concentration of hydrophilic lipid is from 1 mg / mL to 10 mg / mL.

7. The composition as claimed in claim 2, wherein tonicity modifiers are sucrose and sodium chloride, buffers are Trometamol and sodium hydroxide, vehicle is water for injection.

8. The composition as claimed in claim 1, wherein phospholipid mixture in liposomes contains HSPC, Cholesterol, and MPEG 2000-DSPE at a molar ratio of 56:38:5.

9. The process for preparation of composition as claimed in claim 1, wherein said process comprising:a) preparing Gadobutrol hydration solution (non-liposomal Gadobutrol solution) andb) preparing Gadobutrol loaded liposomes10. The process for preparation of composition as claimed in claim 1, wherein said process comprising:a) dissolving Calcobutrol sodium in water for injection and stirring until clear solution is obtained,b) dissolving sodium hydroxide and stirring until a clear solution is obtained, c) dissolving Tromethamine and stirring until a clear solution is obtained, d) adding Gadobutrol and stirring until a clear solution is obtained,e) adjusting the pH to 6.6 - 8 using IN HC1, making up the volume to the required batch size and filtering the solution through a 0.22 pm filter, f) co-dissolving Hydrogenated Soybean Phosphatidylcholine (HSPC), methoxy-poly(ethyleneglycol)-distearoylphosphatidylethanolamine (MPEG-2000-DSPE) and Cholesterol in ethanol at about 74°±4°C, g) hydrating the lipids of step (f) with solution obtained in step (e),h) extruding the coarse lipid dispersion under pressure through three stacked polycarbonate membrane of size 200nm and 80nm (0.2 / 0.08 / 0.08p), i) removing the unentrapped Gadobutrol by dialysis or tangential flow filtration (TFF) against buffer solution containing 645 mM sodium chloride, 292 mM sucrose & 10 mM Tro metamol andj) storing the liposomes obtained in above step in refrigerated condition.

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