FORMULATION OF PURE DIMETHOXY CURCUMIN-HUMAN SERUM ALBUMIN AND A PROCESS FOR THE PREPARATION THEREOF

Abstract

A formulation of pure dimethoxy curcumin-human serum albumin (DMCHSA) comprising a pure di-methoxy curcumin (DMC) bound to human serum albumin (HSA), wherein molar ratio of DMC to HSA is in the range of 3.0-6.0. Further, there is provided a highly soluble and safe intravenous formulation of pure dimethoxy curcumin-human serum albumin retaining proven biological activities and a process for the preparation thereof. Even further, there is provided a process for preparing formulation of pure dimethoxy curcumin-human serum albumin (DMCHSA) by preferential binding of DMC to HSA, which excludes other curcuminoids such as Demethoxy curcumin (DeMC) and Bidemethoxy curcumin (BiDeMC) present in the added chemical, increasing the purity of 80% DMC in starting raw material to >99% in the product DMCHSA.

Claims

1. A formulation of pure dimethoxy curcumin-human serum albumin (DMCHSA) comprising a pure di-methoxy curcumin (DMC) bound to human serum albumin (HSA), wherein molar ratio of DMC to HSA is in the range of 3.0-6.0.

2. The formulation as claimed in claim 1, wherein the molar ratio of DMC to HSA is in the range of 3.0-4.0.

3. The formulation as claimed in claim 1, wherein the DMCHSA is non-toxic in a dose range of 1 mg to 15 mg curcumin per kg body weight administered intravenously in mice and the DMCHSA upon entering cell cytoplasm is nontoxic at low dose range and cytotoxic at higher dose to both cancer cell lines and primary human cells.

4. A process for preparing formulation of pure dimethoxy curcumin-human serum albumin (DMCHSA) wherein said process comprises the steps of: (i) stirring a pharmacopoeia grade commercially obtained solution containing 1 g to 2 g human serum albumin (HSA) per 5 m1 to 10 m1 saline; (ii) adding 10-12 mg of curcumin mixture/ curcuminoids comprising 80 to 95 % of di-methoxy curcumin dissolved in 100 .Math.1 dimethyl sulfoxide (DMSO) to the solution of step (i) slowly in the amount 5ul at time under gentle mixing to obtain a mixture; (iii) incubating the mixture of step (ii) for 1 hour to 2 hour at 20° C. to 24° C. to obtain a conjugate; and (iv) eluting the conjugate using molecular sieving process to obtain a DMCHSA.

5. The process as claimed in claim 4, wherein the curcumin mixture/curcuminoids is added in amount of 12 mg or 10 mg per 1 g HSA in 5 m1 and the 10 mg reduced waste of DMC in the unbound form.

6. The process as claimed in claim 4, wherein the curcumin mixture/ curcuminoids comprises di-methoxy curcumin (DMC), demethoxycurcumin (DeMC) and Bidemethoxycurcumin (BiDeMC).

7. The process as claimed in claim 4, wherein the process comprises the steps of: (i) stirring the pharmacopoeia grade commercially obtained solution containing HSA in the saline; (ii) adding a 0.4 M - 0.6 M solution of mixed curcuminoid containing di-methoxy curcumin (DMC), demethoxycurcumin (DeMC) and Bidemethoxycurcumin (BiDeMC) and dissolved in DMSO, to the HSA solution; (iii) adding curcuminoid containing dimethoxy curcumin solution in an aliquot of 0.0001 to 0.0002 vol of HSA at a time; (iv) making up the final curcumin to HSA proportion of 12:1000 in mg to obtain a solution; (v) mixing the solution continuously for 1-2 hour at a temperature of solution kept between 20° C. to 24° C.; (vi) centrifuging the mixture of step (v) at 1000 g to 2000 g for 5-10 min. to settle the particulate (insoluble) of excess undissolved curcumin, comprising DMC and other contaminating curcuminoids; (vii) decanting the supernatant to a fresh tube removing precipitated curcuminoids and injecting the clear solution to a column packed with Sephadex G25; (ix) separating DMCHSA from free curcuminoids by size exclusion principle and pooling eluted fractions of DMCHSA with high absorbance at 280 nm and 420 nm.

8. The process as claimed in claim 7, wherein the DMCHSA of step (ix): (i) pass through porous membranes of 0.22 .Math.m porosity for sterilization and estimating the actual concentration of the curcumin per unit volume of solution using spectrophotometry; (ii) dispensing into small vials achieving defined DMC content in each vial and Freeze-drying the product in the vial into dry powder form and vacuum sealing and storing between 2-8° C.; and (iii) dissolving the dry powder in water to obtain soluble DMC to a maximum of 2.0 mg m1.sup.-1.

9. The process as claimed in claim 4, wherein preferential binding of DMC to HSA excludes other curcuminoids such as Demethoxy curcumin (DeMC) and Bidemethoxy curcumin (BiDeMC) present in the added raw chemical/ curcumin mixture/ curcuminoids, increasing the purity of 80% DMC in starting raw material to >99% in the product DMCHSA.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0079] The disclosure may be more fully understood by reading the following detailed description of the embodiment, with reference made to the accompanying drawings as follows:

[0080] FIG. 1 shows a summary of the process of conjugate preparation, with FIG. 1A showing stirring/mixing of raw materials-HSA and 80% pure curcuminoid obtained from Sigma Chemicals, USA; FIG. 1B showing centrifuged settled Curcumin; FIG. 1C shows decanted fluid; FIG. 1D shows column chromatography; FIG. 1E shows aliquots; and FIG. 1F shows lyophilized DMCHSA;

[0081] FIG. 2 depicts data showing selective binding of and elimination of unbound curcumin or its derivatives, with FIG. 2A showing FTIR of DMCHSA Raw material; FIG. 2B demonstrating FTIR of HSA-bound curcumin eluted from column; FIG. 2C showing unbound curcumin eluted from column; and FIG. 2D showing unbound curcumin derivatives with different chemical characteristics washed out from column using 30% ethanol;

[0082] FIG. 3 illustrates stability of DMCHSA as compared to fast degrading free curcumin, with

[0083] FIG. 3A illustrating UV-Vis Spectral Properties of degrading free curcumin in 2h; FIG. 3B. illustrating overlaid UV-Vis spectra showing stable nature of DMCHSA 0h-48h period; FIG. 3C showing FTIR spectrum of Fresh DMCHSA; FIG. 3D showing FTIR Spectrum of 24h stored DMCHSA; FIG. 3E showing FTIR Spectrum of 48h stored DMCHSA; and FIG. 3F showing FTIR spectrum of 72h stored DMCHSA;

[0084] FIG. 4 illustrates increasing stability of DMCHSA upon storing lyophilized samples, with FIG. 4A showing FTIR spectrum of 6 m stored DMCHSA; FIG. 4B, Functional stability of 9 m stored DMCHSA showing dose dependent cytotoxic effect on cancer cell line A549; FIG. 4C, HPLC elution profile of raw curcumin; FIG. 4D, HPLC elution profile of curcumin extracted from fresh DMCHSA; and FIG. 4E, HPLC elution profile of DMC eluted from DMCHSA stored for 9 months.

[0085] FIGS. 5A-5C illustrate raw material independent conjugation of curcumin to has, with FIG. 5A showing overlaid NMR spectra comparing 3 products of DMCHSA prepared using 3 different curcumin; FIG. 5B showing overlaid FTIR spectra comparing 3 products of DMCHSA prepared using 3 different curcumin; and FIG. 5C depicting a graph comparing 3 products of DMCHSA prepared using 3 different curcumin for dose dependent cytotoxic effect on A549.

[0086] FIGS. 6A-6C illustrate cell specific transport of DMCHSA into cytoplasm across cell membrane, with FIG. 6A showing endocytosis into A549; FIG. 6B showing endocytosis into endothelial cells; and FIG. 6C showing endocytosis into chrondrocytes.

[0087] FIGS. 7A-7D illustrate cell-specific cytotoxic effect, with FIG. 7A showing a graph illustrating dose dependent cytotoxic effect of DMCHSA on A549; FIG. 7B showing a graph illustrating dose dependent cytotoxic effect of DMCHSA on MCF-7; FIG. 7C showing a graph illustrating dose dependent cytotoxic effect on EC; and FIG. 7D showing a graph illustrating dose dependent cytotoxic effect on chondrocytes.

[0088] FIG. 8 depicts a demonstration of functional stability of DMCHSA as compared to free DMC in fibroblasts (L929 mouse myeloma cell lines), with the data showing significant increase in the numbers of viable fibroblasts in 24 h-48 h when treated with free curcumin (FIG. 8B) as compared to when treated with DMCHSA (FIG. 8A) indicating that free curcumin is less cytotoxic and permit cell proliferation increasing the numbers of viable cells in 24 h to 48 h.

[0089] FIG. 9 illustrates anti-inflammatory effect of DMCHSA, with FIG. 9A showing a graph illustrating down regulation of inflammatory markers upon treating TNF-α activated EC with DMCHSA; and FIG. 9B showing a graph illustrating down-regulation of inflammatory markers upon treating TNF-αactivated chondrocytes with DMCHSA;

[0090] FIGS. 10A-10B show blood compatibility and non haemolytic nature of DMCHSA, with FIG. 10A showing control blood smear stained showing normal RBC morphology; and FIG. 10B showing DMCHSA treated blood smear stained showing normal RBC morphology;

[0091] FIG. 11 depicts pharmacokinetics showing absorption, distribution, metabolism and elimination (ADME) of intravenous administered ADME in mice model, with FIG. 11A showing fluorescent images of DMCHSA administered to mice in 1 h to 48 h period showing whole body distribution; FIG. 11B showing accumulation of DMCHSA in liver in 24 h; FIG. 11C showing accumulation of DMCHSA in liver after 48 h; and FIG. 11D showing fluorescent images of urine sample demonstrating elimination in urine in 1 h to 96 h.

DETAILED DESCRIPTION

[0092] According to this disclosure, there is provided a formulation of pure dimethoxy curcumin-human serum albumin (DMCHSA) and a process for the preparation thereof.

[0093] In accordance with one or more embodiments, the formulation of pure dimethoxy curcumin-human serum albumin (DMCHSA) is a highly soluble and safe intravenous formulation of pure dimethoxy curcumin-human serum albumin retaining proven biological activities.

[0094] The problem was approached in the following manner: [0095] 1. Exploit higher hydrophobicity of DMC for its selective binding to HSA. [0096] 2. Apply molecular sieving principle to remove unbound curcuminoids from the product [0097] 3. Use HPLC analysis to prove homogeneity of the bound molecule in the product [0098] 4. Use fluorescent conjugated albumin to prove transport of DMCHSA into cell cytoplasm [0099] 5. Use MTT assay to identify cytotoxic response to different cell lines and primary cells [0100] 6. Use cultures of cancer cell lines MCF-7 and A549 to determine effect of DMCHSA for inducing cancer cell death [0101] 7. Demonstrate stability and higher cytotoxic activity of DMCHSA as compared to free DMC [0102] 8. Use cytokine stimulation to generate inflammatory ECs and chondrocytes and test anti-inflammatory effect of DMCHSA. [0103] 9. Employ standard ISO10993-part4 for establishing hemocompatibility and intravenous safety of DCMHSA [0104] 10. Employ ISO10993 to evaluate systemic toxicity level of DCMHSA upon intravenous administration in mice model. [0105] 11. Use mice model to conduct pharmacokinetics (PK) studies determining the absorption, distribution, metabolism and elimination (ADME) of DMCHSA [0106] 12. Demonstrate scale up production of DMCHSA using pharmacopoeia grade HSA and curcuminoid containing 80% DMC as raw materials to obtain homogenous product.

[0107] In the present embodiment, the ability of HSA to preferentially bind more hydrophobic molecules has been exploited. One or more embodiments demonstrate a process to increase dimethoxy curcumin (DMC) purity through selective conjugation to albumin. The less hydrophobic curcuminoids such as demethoxy curcumin (DeMC) and bi-demethoxy curcumin (BiDeMC) present in an impure mixture of commercially available curcuminoid raw material and excess unbound DMC were removed from the reaction mixture by molecular sieving principle. Human serum albumin (HSA) in pure form as specified in pharmacopoeia suitable for clinical intravenous administration was another major raw material. The stability of the pure product of DMCHSA was increased by freeze drying into a powder form enabling long term storage. Upon dissolving in aqueous medium, high concentrations of DMCHSA for producing different dose range for biological use. Said conjugate has a molar ratio of DMC to HSA in the range of 3.0-6.0, which theoretically permits receptor mediated entry into cells.

[0108] According to embodiments, there is provided a highly soluble albumin conjugated form of DMC for prospective application in curing inflammatory diseases and cancer. Said albumin is selected from different commercially available, pharmacopoeia grade human serum albumin (HSA).

[0109] In an embodiment, there is provided an albumin DMC conjugate wherein said conjugate has a molar ratio of DMC to albumin is in the range of 3.0 -6.0.

[0110] In another embodiment the albumin is pharmacopoeia grade human serum albumin (HSA) safe for intravenous administration.

[0111] In yet another embodiment, the molar ratio of DMC to HSA ranges from 3-4 upon selecting 80% curcumin as raw material for conjugation.

[0112] In one embodiment, there is provided a process of preparing the albumin conjugate wherein said process comprises the steps of: [0113] Gently stirring a pharmacopoeia grade solution containing HSA in physiological saline (with stabilizers),using a magnetic stirrer [0114] Adding curcumin dissolved in dimethyl sulfoxide (DMSO) to the above solution slowly at time under gentle mixing; [0115] Incubating the aforesaid mixture for about 1 hour at 20° C. -24° C.; and [0116] Eluting the conjugate using molecular sieving principle.

[0117] In one embodiment, there is provided a process of preparing the albumin conjugate wherein said process comprises the steps of: [0118] Gently stirring a pharmacopoeia grade solution containing 1000 mg (1 g) HSA per 5 ml physiological saline (with stabilizers),using a magnetic stirrer; [0119] Adding curcumin (10-12 mg) dissolved in 100 .Math.1 dimethyl sulfoxide (DMSO) to the above solution slowly- 5ul at time under gentle mixing; [0120] Incubating the aforesaid mixture for ~1 hour at 20° C. -24° C.; and [0121] Eluting the conjugate using molecular sieving principle.

[0122] In accordance with this embodiment, different concentrations of curcuminoid mixture was added such as 12 mg or 10 mg per 1 g HSA in 5 ml and the binding was comparable; but 10 mg reduced waste of DMC in the unbound form.

[0123] While the free DMC metabolizes to other inactive products at physiological pH, the DMCHSA showed several months stability in the lyophilized form. Increased transportation of the DMCHSA formulation across cell membrane, into cytoplasm of cancer cell (A549) as compared to endothelial cells (EC) and chondrocyte is demonstrated and could be receptor-mediated action. Also, aspects of this disclosure demonstrate intake of bound curcumin by cancer cells and inflammatory cells inducing cytotoxicity and down regulation of inflammatory response, respectively.

[0124] The dissolved DMCHSA was experimentally demonstrated to cross membrane of various cell types including cancer cell lines, inflammatory primary cells such as endothelial cells and chondrocytes. Low dose range of DMCHSA was found to be non-toxic and at high dose the molecule induced cytotoxicity and produced non-viable cells in culture. The higher cytotoxic effect of free DMCHSA on fibroblast cell line as compared to free DMC indicates that free molecule undergo degradation and is less active. Fibroblasts continued to multiply producing more viable cells in culture in the presence of free DMC, whereas the similar dose of DMCHSA caused cytotoxic effect reducing the numbers of fibroblasts in 24h-48h of culture. Also, at low non-cytotoxic dose, the DMCHSA prevented cytokine induced inflammatory response in endothelial cells and chondrocytes, indicating its potential use for treating inflammation, and thus to prevent atherosclerotic and arthritic diseases. At high dose range DMCHSA turned both breast and lung cancer cells into nonviable phenotype in respective cultures, indicating its potential to treat cancer.

[0125] In the in vitro experiments, absence of DMCHSA induced change to RBC morphology (12.5 .Math.g/ml bound curcumin/ml blood) or hemolysis was eliminated upon treating high concentration (0.2 mg bound curcumin/ml blood); thus ensuring intravenous safety.

[0126] In vivo animal studies showed the essential distribution of the bound molecule throughout the body, accumulation in liver for further metabolism and elimination through renal route. Being a cytotoxic anti-cancer drug, the pharmacokinetics study carried out suggests adequate distribution and clearance for preventing other normal tissues from excessive cytotoxic damage.

[0127] Albumin solution and dissolved curcumin are taken and mixed at high concentration but in low volume of DMSO. The mixture is centrifuged to remove precipitated (undissolved) curcuminoids from the mixture and subjected to molecular sieving to remove un-reacted curcuminoids and unreacted albumin.

[0128] The conjugated fractions were identified with the help of diode array spectrophotometer. The spectral peak ratio of albumin (280 nm) and curcumin (420 nm) was used to eliminate fractions of unbound DMC, other curcuminoids and albumin.

[0129] The primary objective was to standardize conditions for preparation of DMCHSA complex and to establish purity of the conjugate, yield, stability and activity. Parameters tested were the effect of different raw materials on the binding efficiency and purity. Other aims were to establish conjugation of DMC to HSA by spectroscopic methods, prove stability and solubility of the bound DMC and demonstrate biological activity of the complex on cancer cell lines and primary human inflammatory cells in vitro. Also, scaled up production of DMCHSA was demonstrated indicating commercial value of the product.

Preparation of Conjugate

[0130] Pharmacopoeia grade HSA from three different sources were used to establish reproducibility. All the three sources used are pharmacopoeia grade but produced and marketed under different trade names and all products are standard preparations of 20 g of albumin in 100 ml (200 mg/ml) containing 123.5-136.5 mM NaCl, 16 mM Acetyltryptophan and 16.0 mM Sodium Caprylate. The HSA solution was withdrawn from the original container using a syringe and needle. The solution was transferred to a small beaker and kept stirring on a magnetic stirrer. From a 0.5 M stock in DMSO, small aliquots of curcumin were added to HSA, with continuous stirring for 1 h, maintaining the temperature between 20° C.-24° C. The solution was then centrifuged for 10 min at 1000 g, to settle the particulate (insoluble) of excess undissolved DMC, and other contaminating curcuminoids. The clear but deep yellow supernatant was then decanted and injected to gel filtration column packed with Sephadex G-25 beads and pre-equilibrated with physiological saline. The peak that eluted the conjugate was identified by absorbance measurement in the first elution peak. The fractions which showed minimum A280/A420 ratio were pooled and used for further evaluations. All fractions with <2 ratio was pooled, sterile filtered using 0.22 .Math.m Millipore syringe filter and dispensed. The column was regenerated for next run by washing with 3 bed volumes 30% ethanol and re-equilibrating with saline.

Conjugate Characterization

[0131] Characterization of conjugated product was carried out with UV-visible absorption spectroscopy (Diode array spectrophotometer, Hewlett Packard 8453), infrared (Jasco 6300 FT-IR spectrometer) /Raman spectroscopy (Bruker RFS 100/s FT-Raman spectroscope).

Detection of Curcumin Concentration in 1 Ml Aliquot

[0132] Purified, pooled conjugate was dispensed into 1 ml fractions and lyophilized. To extract the bound curcumin, 0.1:9 mixture of water DMSO was added, vortex mixed, centrifuged to remove the protein debris and the absorbance of the supernatant was measured at 420 nm (Max absorption of curcumin) in a diode array spectrophotometer. For quantification of the extracted curcumin, a standard curve was prepared using serially diluted curcumin in DMSO. Molar concentration of curcumin in 1 ml was estimated based on the MW of 368.

Detection of Albumin in 1 Ml Aliquot

[0133] To 1 vial of lyophilized DMCHSA, 1 ml of water was added. After complete dissolution the protein concentration was estimated using Lowry’s method. Molar concentration of Albumin in 1 ml conjugate was estimated based on the MW of 66000.

[0134] Binding Ratio: The ratio of curcumin to albumin was estimated based on the molar concentration of each constituent in the conjugate.

Raw Material Validation

[0135] Commercially available curcumin (80% or 99% or USP grade Sigma-Aldrich, USA), identified by different catalogue numbers and pharmacopoeia grade human serum albumin (Intas Pharmaceutical Ltd India) were used. Objective was to demonstrate that irrespective of the purity of the curcumin, bound form attained homogenous chemical form of curcumin i.e. dimethoxy curcumin, and all products showed similar FTIR spectra and NMR spectra. In the unbound form which eluted in the later fractions of the column washing contained other chemical forms of curcumin or degradation products are detected. The conjugate chemical characteristics is found to be independent of the raw material (curcumin) used for reaction. Both overlaid NMR spectra and overlaid FTIR spectra of the products prepared using different grades of commercially available curcumin is demonstrated.

Identification of Excluded Impurities

[0136] In order to prove other impurities in the raw material curcuminoid are excluded from DMCHSA, upon binding followed by size exclusion chromatography, the following experiment was done.

[0137] The reaction mixture for preparing DMCHSA contained 200 mg HSA and 2.88 mg C7727. The mixture was subjected to molecular sieving chromatography on 25 ml Sephadex G-25 column. Five ml fractions were collected and were analysed by both A280 and A420 absorbance. Fractions with highest HSA content, medium HSA content and very low HSA content but with high DMC absorbance were pooled and designated as P1, P2, P3 respectively. After completing elution with 2 bed volume of saline, another 100 ml was collected as Pool4 (P4). Once the column wash volume completed 150 ml, (i.e. 6× bed volumes), still the column shows yellow colour. Therefore, the column was washed with 50 ml of 30% ethanol and the eluted fractions were collected as pool5 (P5).

[0138] Both Pools 4 and 5 were lyophilized and FTIR spectrum was recorded and compared with raw material as the drug001 as sample for comparison. The FTIR spectrum of DMCHSA in P2 with 280 nm and 420 nm absorbance also was also recorded.

[0139] The different chemical characteristic of the unbound curcumin was established by FTIR spectroscopy analysis of the pooled fractions of later eluted unbound chemical. Comparison of the original raw material, conjugated DMCHSA and unbound chemicals are demonstrated.

Storage Stability of DMCHSA Solution

[0140] The liquid stability of the product was analysed at 24 h interval for 48 h or 72 h. The UV spectra recorded at different storage period are shown

[0141] FTIR spectra are shown to substantiate the chemical degradation after 48 h of storage of DMCHSA solution.

[0142] From both analysis it is confirmed that DMCHSA solution showing minor degradation after 48 h.

[0143] All liquid stored samples were analysed for functional efficacy in terms of cytotoxicity to lung cancer cells A549.

[0144] Sample Preparation: [0145] Freshly prepared DMCHSA-80.01 [0146] 24h liquid stored DMCHSA-80.01 [0147] 48h liquid stored DMCHSA-80.01

[0148] A549 cells (2.5×10.sup.3) were seeded into each well of 96 well plates. The cells were allowed to grow till it appeared nearly confluent. Standard MTT assay was carried out to determine the cytotoxic effect of DMCHSA-80.01. Functional stability up to 48 h of liquid storage is confirmed.

[0149] Therefore, lyophilisation immediately after preparation of DMCHSA as normal practice is found essential.

Storage Stability of Lyophilized DMCHSA

[0150] The lyophilized samples were used for respective periods as prepared for liquid stability, were lyophilized and recorded FTIR spectrum. The spectral properties are compared Spectral characteristics showed noticeable difference in the liquid sample stored for 72 h.

[0151] Thus, liquid stability of DMCHSA stored for 48 h is evident.

[0152] The lyophilized sample stored in 2-8° C. for 6 m to 8 m was verified for the chemical and functional stability and the results are demonstrated.

Transport of DMCHSA Across Cell Membrane

[0153] The cytotoxic effect could vary depending on the number of molecules entering the cells and the final concentration achieved in the cell cytoplasm. Therefore, transport of DMCHSA across cell membrane (endocytosis) was measured in different cells such as cancer cells (A549), endothelial cells (EC) and chondrocytes (CC) and compared. As expected, receptor mediated endocytosis of DMCHSA could take place in all cells.

[0154] To track endocytosis, two aliquot of DMCHSA-80 (total 0.4 mg Curcumin bound to 50 mg albumin) was dissolved in carbonate buffer pH 9.0 and tagged with FITC (50 mM) using standard method. Unreacted FITC was removed by gel-filtration on Sephadex G-25. Fractions with >2.0 A495:A280 ratios were pooled, sterile filtered (0.22 .Math.m) and lyophilised as 0.2 ml aliquots for tracking experiments. The curcumin from aliquots of FITC-tagged DMCHSA were extracted into 9:1 DMSO-water mixture and curcumin was quantified based on standard curve.

[0155] For analysis of DMCHSA endocytosis, A549, EC and CC cultures (>70% confluent) were treated with 30 uM concentration of FITC-DMCHSA. The cultures were allowed to grow under standard conditions. The cells were analysed using fluorescence microscope (Leica system) and the fluorescent images were captured using LAS camera and software. The cells were harvested by trypsinization and was analysed for fluorescence quantification using FITC channel. The mean fluorescence intensity of 4 replicate culture samples were computed to get average MFI in each case (FIGS. 9A-9D)

[0156] The results indicated that while > 90% cells endocytosed DMCHSA in 8 h, the numbers of molecule entering the cell is different depending on the cell type, in turn increasing the mean fluorescence intensity (MFI) upon flow cytometry analysis. The MFI in A459 is double that of CC and ~10 fold higher than that in primary human EC. Accordingly, cytotoxic response of primary EC was also lower as compared to chondrocytes or to the cancer cells- A549.

[0157] Thus the cytotoxicity to A549>Chondrocytes>Endothelial cell has been established

Cytotoxic Effect of DMCHSA in Cell Lines and Primary Cells

[0158] For cytotoxicity assay, both lung cancer cell line A549 and breast cancer cell line MCF-7 were grown under standard culture conditions. Once the cells achieved 70-80% confluence, cell viability assay was carried out in A549, MCF-7, ECs and CCs using graded concentration of conjugate for a period of 24 h.

[0159] Cytotoxicity was assayed by employing the standard MTT assay. This is a colorimetric assay that measures the percentage of metabolically active cells. The test is based on the principle that MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. Reduction of yellow MTT in the reagent by mitochondrial succinate dehydrogenase is the first step. This formazan production is directly proportional to the viable cell numbers and inversely proportional to the degree of cytotoxicity. The formazan are then solubilized with an organic solvent (e.g. DMSO) and the released, solubilised formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viable cells. Thus the metabolic activity of each culture treated with different dose of DMCHSA was determined by the standard MTT assay and compared to those of untreated cells.

[0160] Cancer cell lines (A549,MCF-7), Fibroblast cell line (L929), and primary cells (ECs and CCs) were cultured in 96-well plates containing 100 .Math.l medium, kept this plate in CO.sub.2 incubator (5% CO.sub.2) at 37° C. for 24 h. DMCHSA solution was freshly prepared by dissolving the lyophilized conjugate in DMEM F12 medium and added (0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.4 mg/ml) in 96 well plate for 24 h. The cytotoxicity of DMCHSA was compared with equivalent concentrations of free DMC by adding similar molar concentrations of the solutions into the fibroblast culture medium. In all cultures standard MTT assay quantified viable cells after treating with DMCHSA/free DMC. Briefly, commercial MTT (0.5 mg/ml) reagent was dissolved in medium. This solution was filtered through a 0.22 .Math.m filter and stored at 2 - 8° C. After removal of 100 .Math.l medium, MTT dye solution was added and the plates were incubated at 37° C. for 4 h in a humidified 5% CO.sub.2 incubator, followed by 100 .Math.l of DMSO added to each well, and mixed thoroughly to dissolve the dye crystals.

[0161] The absorbance was measured using an ELISA plate reader at 570 nm. High optical density readings corresponded to a high intensity of dye colour. The fractional absorbance was calculated by the formula: % Cell survival/PR = Mean absorbance in test × 100 Mean absorbance in control

[0162] Cytotoxicity of DMCHSA on cells was calculated as cell growth inhibition rate (IR),IR = 100-PR. Compiled results are presented in graphs. The effect on MCF-7>A549>CC>EC in dose dependent manner is demonstrated.

Anti-Inflammatory Response of DMCHSA

[0163] The endothelial cells (ECs) are an important structural and functional component of blood vessels and help in maintaining the integrity of vessel walls. When these cells are exposed to harmful and toxic components they begin to show the after effects by secreting certain cytokine molecules or by expressing cell adhesion molecules on their surface. This sudden change in the cell behaviour is referred to as the endothelial dysfunction and it occurs when the cells are under stress. These stress conditions contribute to the development of atherosclerosis which is marked by the deposition of cholesterol molecules beneath the wall of blood vessels. Slowly as the disease progresses, the blood vessel narrows and hardens resulting in obstructed blood flow making the target tissue which was being supplied with this same vessel devoid of oxygen and nutrients. As time passes the tissue begins to deteriorate and integrity gets destabilized. So, all these events involve immense role played by various cells of the immune system as well as inflammatory mediators, which are either produced by the ECs itself or act on ECs to initiate inflammation.

[0164] Another major disease is arthritis which is marked by inflammation of the cartilaginous tissue present at the joints. It is a major causative of movement impairment and also sometimes may lead to mental depression also. The initial events involve cartilage injury whose causes vary, followed by the action of inflammatory cytokines which worsen the situation. Chondrocytes help in the secretion of extracellular matrix (EDMC) that provides the required strength for cartilage. These cells are embedded in the matrix itself in specialized pouches called lacunae. But once there is an insult to the tissue, matrix undergoes biochemical and pathophysiological changes that disrupts the integrity of cartilage.

[0165] To evaluate the effect of DMCHSA, inflammatory response in ECs/chondrocytes was established using inflammatory inducer-Tumour necrosis factor-α (TNF-α). Near confluent stage of EC monolayers or CC were incubated with graded concentrations of cytokines: TNF-α (10 ng/ml) for 24 h; the expression of inflammatory markers: Nuclear factor-kB (NF-kB), monocyte chemo-attractant protein-1 (MCP-1), endothelin-1 (ET-1), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule (VCAM-1) were determined to be up-regulated by Quantitative Real Time polymerase chain reaction (qRT-PCR). The expression of the following inflammatory markers: Nuclear factor-kB (NF-kB), cyclooxygenase-2 (COX-2), interleukin-8 (IL-8), Matrix metalloproteinases -13 (MMP-13) and Tissue inhibitor of MMPs (TIMP-1) were determined by qRT-PCR in chondrocytes. The cytokine-induced up regulation of inflammatory markers were calculated against normal ECs/CCs and using Glyceraldehyde 3-phophate dehydrogenase (GAPDH) as house-keeping gene. For analysing the effect of DMCHSA on such activated ECs, initially 24 h exposure of >70% confluent ECs to TNFα (10 ng /ml) was done. Further, the activated ECs/CCs were treated with different dose of DMCHSA (5 to 60 .Math.M) for an additional 24 h. qRT- PCR was carried out as described above to estimate the effects of DMCHSA on the marker expressions.

[0166] The cell culture experiments thus have proven the anti-inflammatory activity of DMCHSA. A biphasic effect is seen which is due to increased cytotoxicity at higher dose. This is because cytotoxic effect can also up regulate inflammatory genes. Therefore, to achieve anti-inflammatory effect, the dose of DMCHSA should be selected carefully (FIGS. 8A-8B).

Intravenous Safety

[0167] Safety of DMCHSA for intravenous infusion is another aspect. As part of primary qualification of DMCHSA for intravenous applications, the hemocompatibility is an important parameter to be studied.

[0168] The test is done as per ISO10993-part4 which is recommended for blood contacting materials and biomedical devices. Human blood is collected using 3.8% citrate as anticoagulant. An aliquot of blood is centrifuged and plasma haemoglobin is measured as part of ISO17025 quality system to qualify the blood sample for testing the effect of material on hemolysis and RBC morphology. Whole blood is subjected to total blood count using Sysmex 4500 automated haematology analyzer. Blood smears are prepared for analyzing RBC morphology (control).

[0169] One millilitre of anticoagulated human blood was treated with 6.25 .Math.g to 200 .Math.g of DMCHSA for 30 min. Using the whole blood, smears were prepared and stained (Lieshman’s) for observing RBC morphology (Test) under microscope to compare control RBCs with test RBCs.

[0170] Blood was centrifuged at 1000 g for 10 min. Platelet poor plasma is aspirated and subjected to spectrophotometric detection of plasma hemoglobin. Based on the plasma hemoglobin, the % hemolysis was calculated. % hemolysis= (plasma hemoglobin/total hemoglobin in whole blood) ×100

[0171] No morphological change to RBC was observed upon treating blood with 12.5 .Math.g, some cells with slight crenations were seen without any lysis were seen above 25 .Math.g/ml blood No other abnormal change in morphology were noted in all concentration studied. The % haemolysis was <0.1% at a concentration of 200 .Math.g /ml blood, which is similar to normal blood. The concentration (0.2 mg ml.sup.-1) is which is much higher than expected therapeutic dose for inducing apoptosis as per the in vitro data.

Pharmacokinetics in In Vivo Model

[0172] Further, the absorption, distribution, metabolism and elimination (ADME) upon intravenous administration of DMCHSA determines the safety and predicts the resident time available for the molecule to localize in the cancer/inflammatory tissues and cells.

[0173] For labelling of Vivotag-750 S to DMCHSA, required amount was dialyzed in carbonate buffer (pH 8.5) for 24 h with three exchange of dialyzing fluid. Vivotag-750 was dissolved in DMSO. The required quantity of fluorochrome (300 .Math.g for 1 mg protein) was added slowly as 5 .Math.L aliquots and incubated at 20-25° C. for 2 h. The resultant mixture was purified using Sephadex G-25 columns to remove unreacted Vivotag-750 S and 1ml fractions were collected, analyzed, spectrophotometrically at A280, A420 and A750. Fractions found maximum binding with Vivotag-750 S to conjugate were selected, pooled, aliquots of 0.5ml were stored at 4° C.

[0174] All the protocols used for the studies were approved by Institutional animal ethical committee (IAEC-SCT/IAEC-263/February/2018/95-06.04.2018). Briefly, hair of Swiss albino mice was completely removed using approved methods to eliminate auto-fluorescence. All animals were housed in metabolic cages prior to experiment for acclimatization. DMCHSA80.002 labelled with Vivotag-750 S was administered at a dose equivalent (Albumin 0.5 mg/kg and of 8.7 .Math.g/kg) through tail vein injected in normal saline and images were taken at 24 h, 48 h, 72 h and 96 h by Xenogen IVIS Spectrum (Caliper Life Science). A set of animals were euthanized at the same intervals, organs like brain, heart, lungs, liver, spleen, gastrointestinal system and kidney were isolated, washed in saline, blotted on a paper and images were taken. Urine and faecal samples were collected at the same time intervals and imaged for determining the route of elimination.

[0175] From the whole animal images, it is evident that, DMCHSA distribution in blood is attained at 2 h administration with significant clearance in 24 h and seen localized in liver at 48 h. Maximum elimination is in urine at 24 h and 48 h, which sustain till 72 h. Trace is seen in faecal matter at 48 h and 72 h.

[0176] Therefore, DMCHSA is safe for intravenous transfusion and it is eliminated through the renal route after reasonable resident time in the body for cellular uptake and action.

Example 1

[0177] For setting up reaction; 3 g liquid stable albumin (15 ml of 20 g dL.sup.-1 solution) was aspirated aseptically from therapeutic preparation. From 0.5 M stock of curcumin in DMSO 200 ul was added to albumin i.e. (~36.8 mg of curcumin) slowly with gentle continuous stirring. After 1 h, the mixture was gel filtered using Sephadex G-25 packed to get bed volume of 25 ml and equilibrated with sterile filtered normal saline. The chromatography system Akta Prime Plus (GE Health Science USA) with UV detector (280 nm) and associated software was used to run the purification protocol in automatic mode. Five 5ml mixture was injected for each run and 3 such runs were made to purify 15 ml reaction mixture. The eluted protein peak was collected as two ml fractions based on A280. Bound molecule in the elute was identified by measuring absorbance at 280 nm (A280) for protein and 420 nm (A450) for bound curcumin (1). Selected fractions with maximum curcumin-specific and albumin-specific peaks were pooled, filtered (0.22 .Math.m), dispensed into 1ml aliquots and lyophilized.

[0178] A calibration curve was created using graded concentrations of curcuminoid solution in DMSO; A425 was measured using quartz cuvette and HP8453 Diode Array Spectrophotometer by selecting software in the “quantification’ mode. The calibration curve was stored for later measurement of concentration of curcumin in DMCHSA.

[0179] For measurement of DMC in DMCHSA one lyophilized aliquot was dissolved in 1 ml water, 10 ul was added to 990 ul of DMSO and measured the concentration using the calibration curve and total content in 1 ml was calculated based on which the yield in the pool was calculated.

[0180] Albumin in the DMCHSA was measured using a calibration curve stored in the HP8453 system by Lowry’s protein assay method. Standard procedure of dilution is applied if concentration is higher than the range in the calibration curve. Based on the measured protein per ml, yield in the pool is calculated.

[0181] Finally, the molar binding ratio was calculated to determine how many molecules of curcumin is bound to 1 molecule of albumin in different conditions of reaction.

TABLE-US-00001 Raw material Concentr ation of reactants Reactant volumes Concentration /ml After purification Volume of pooled fractions Total yield Recovery (%) Drug 001 36.8 mg 200 ul 0.4 mg 66 ml 26.4 mg 71.7 % Albumin (Intas) 3 g 15 ml 24 mg 1584 mg 52.8 % Molarity of drug in the final product: 0.0048 M Molarity of Albumin in the final product: 0.0016 M Binding ratio: 3.0

Example 2

DMCHSA-10g Batch

[0182] Albumin from Kedrion (pharmacopoeia grade) was used and drug from Sigma Chemicals was used. Fifty ml containing 10 g albumin was withdrawn from the sterile vial and added into a 250 ml beaker. The solution was kept under laminar flow placed in Class 10000 room.

[0183] Drug (C7277) dissolved in 1.5 ml DMSO was added in small aliquots into the albumin under continuous stirring, over a period of 1h. Temperature of the reaction mixture was 20° C. -24° C.After adding the entire drug, solution was kept under stirring for another 1h. The reaction mixture was transferred into 2 numbers of 50 ml, sterile centrifuge tubes and were centrifuged at 10000 g for 10 min. The unreacted drug settled at the bottom was discarded after decanting the reaction mixture into a fresh 50 ml tube.

[0184] The supernatant was further filtered using 0.45um syringe filter. For the 10 g batch, larger column was packed with Sephadex G-25, bed volume 350 ml.

[0185] Program Method was Flow rate 5 ml per min; Injection at 10 ml (Break point 1); Injection volume 50 ml; Fraction collection started at 120 ml (break point 2); Fraction size-10 ml; Total fractions collected 40

[0186] The column was run using normal saline in which the beads were equilibrated by passing 1 L of saline 0.50 ml injection loop purchased from GE Health, USA was used for loading DMCHSA into the column.

[0187] After sample, elution of pure DMCHSA started after ~ 120 ml of normal saline was passed. The eluted conjugate was collected in 10 ml fractions. All fractions having high protein content (280 nm) and drug content (420 nm) were pooled. Total fractions pooled were 25.The concentration of drug in the conjugate and albumin in the conjugate were determined using the calibration curves. Pooled sample was sterile filtered using 0.22um membrane filtered. The conjugate was dispensed into different vials to contain 1 mg /vial, 3 mg/vial and 5 mg/vial. All vials were subjected to lyophilisation and were closed with rubber stopper under vacuum. The rubber caps were closed tightly by crimping aluminium caps.

TABLE-US-00002 Raw material Concentr ation of reactants Reactant volumes Concentration/ ml After purification Volume of pooled fractions Total yield Recovery (%) Drug 001 120 mg 1.5 ml 0.3 mg 250 ml 75 mg 62.5 % Albumin (Kedrion) 10 g 50 ml 16.29 mg 4072.5 mg 40.7 % Molarity of drug in the final product: 0.05 M Molarity of albumin in the final product: 0.015 M Binding Ratio = 3.3

[0188] Inference: Irrespective of the batch size, only 60% to 70% drug was bound to albumin. The binding ratio was consistently between 3.0 to 3.5 when 80% pure drug was used. The unreacted drug was removed from the product efficiently by simple molecular sieving chromatography.