WO2014087413A1

WO2014087413A1 - Nanoparticles comprising sorafenib

Info

Publication number: WO2014087413A1
Application number: PCT/IN2012/000787
Authority: WO
Inventors: Vishwa Vidya Peetham University Amrita
Original assignee: Vishwa Vidya Peetham University Amrita
Priority date: 2012-12-03
Filing date: 2012-12-03
Publication date: 2014-06-12

Abstract

This invention relates to nanomedicine formulation comprised of undoped or metal cluster doped protein nanoparticles loaded with a tyrosine kinase inhibitor, sorafenib. The loading of sorafenib in the protein nanoparticles greatly improves its solubility in bio-fluid whereby improving its bioavailability and therapeutic efficacy. Additionally, the protein nanoparticles can be targeted specifically to the disease site by conjugating active biomolecules such as antibodies, peptides, small sugar molecules, vitamins, etc. The proteins selected for the nanoparticle preparations are derived from natural sources and are highly biocompatible. The protein nanoparticles are preferably doped with nanosized atomic clusters of gold, copper, iron, platinum or silver for imparting improved properties such as optical contrast, magnetic contrast, modulation of zeta potential and desired pharmacokinetics. The metal nanocluster doped protein nanoparticles loaded/embedded with sorafenib possess altered surface charge enabling enhanced renal clearance, whereas, the undoped protein nanoparticles carrying sorafenib adopt a hepatic route of clearance.

Description

ANOPAR ICLES COMPRISING SORAFENIB

FIELD OF INVENTION

The invention is related to a nanomedicine formulation for the treatment of cancer and its associated manifestations and the methods for the preparation of the same. More specifically, the invention is related to protein-sorafenib nanoparticles, composed of proteins embedded with sorafenib molecules or its salts. Sorafenib is a multi-kinase inhibitor having high therapeutic efficacy in the treatment of cancer and related diseases. However, since the free molecule is highly hydrophobic and less aqueous soluble, the dissolution of conventional microcrystalline sorafenib tablets is poor in biological fluids. The poor aqueous dissolution leads to poor absorption of the drug in to the systemic circulation. Thus, sorafenib has limited bioavailability, which limits its therapeutic outcome. Hence, it is advisable to improve the aqueous solubility of sorafenib. The nanoparticle formulations comprising of protein and sorafenib are intended to have better dissolution properties, better absorption characteristics, cell specific targeting and uptake, enhanced bioavailability with less damage to normal cells, in vitro as well as in vivo self- tracking capability, charge tunability etc. The size of the individual nanoparticles comprising of protein and sorafenib is less than l OOOnm.

BACKGROUND

Background regarding nanomedicine formulations for the treatment of cancer

Cancer is a cellular proliferative disorder involving dynamic changes in the genome. Conventional treatment regime is mainly based on cytotoxic chemo drugs, which hinder the cell division by inducing damage to DNA. Thus, they are harmful not only to cancer cells but also to the normal cells. In the case of hematological malignancies such as leukemia, cytotoxic drugs can affect all the types of blood cells including the immune cells, ultimately making the patient highly susceptible to infections. This necessitates the administration of wide-spectrum antibiotics to the patient during the course of treatment to which the patients may present drug tolerance problems. In addition to this, cancer cells possess extensive DNA repair mechanisms, which are sufficient to reduce the effects of cytotoxic drugs. Moreover, the cost involved in the cancer treatment is extremely high with regard to chemo-drugs as well as antibiotics. Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr.

Shantikumar Nair In contrast to this conventional regime, molecular targeted therapy using small molecule inhibitors is more advantageous since it exploits the differences between normal cells and the cancer cells with respect to the cell-signaling cascade so that they can be selectively destroyed.

Protein kinases play crucial role in cancer development, its progression, metastasis and drug resistance. Inhibition of critical signal transduction pathways involving protein tyrosine kinases in cancer cells offers a survival disadvantage to them. Targeting the protein kinome using smal l molecule inhibitors is beneficial over conventional drugs in terms of cancer specific targeting since they target specifically the deregulated pathways manifested in cancer cells, which are not present in normal cells. For e.g. chronic myeloid leukemia (CML) is manifested by a reciprocal translocation between chromosomes 9 and 22 resulting in the formation of a bcr-abl fusion protein exhibiting constitutive tyrosine kinase activity. Imatinib, a small molecule inhibitor targeted to the bcr-abl active site offers survival disadvantages to bcr-abl^+vc myeloid cells. The normal cells both hemetopoietic and non-hematopoietic cells, which are bcr-abl^'ve, are not affected by imatinib.

Though the application of tyrosine kinase inhibitors for the treatment of cancer and its associated manifestations appears really promising, the bioavailability of most of the small molecule inhibitors in the physiological environment is extremely poor owing to their hydrophobic nature. This limits its dissolution potential in biological fluids and hence the active molecules fail to reach the target organs in the required concentration. To evoke a desired response in the target cells, high concentration of drug may be required which increases the toxic side effects. Poor bioavailability of small molecule inhibitors is a major problem for therapeutic formulations intended for oral as wel l as parenteral administration. In the case of parenteral administration such as intravenous administration, the hydrophobic drugs may be sequestered by serum proteins, which bind the drug in a non-speci fic manner and prevent its uptake by the target cells. Oral ly administered drugs having solubility less than about l Omg/ml; tend to-be eliminated from the gastro-intestinal tract before being absorbed in to circulation. Ultimately, very few molecules reach the target organ, thus delaying the therapeutic outcome. Hence improving the aqueous solubility of small molecule inhibitors has immense implications for cancer treatment.

X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Shantikumar Nair Nanoparticle formulations of tyrosine kinase inhibitors can greatly improve the patient compl iance by both increasing the bioavailability, aqueous dissolution and also making the cancer cell specific targeting of molecular targeted inhibitors. Most of the proteins are amphiphilic having a deeply buried hydrophobic core and hydrophilic side chains. Protein nanoparticles with hydrophobic cavity can be used for incorporating the poorly soluble drugs, whereas the hydrophilic side chains of the protein are exposed to the surrounding medium, which will improve the dissolution properties. The protein nanoparticles carrying the drug molecule will prevent the non-specific adsorption of serum proteins and opsonin molecules, which are otherwise involved in the reduced cellular uptake of drug molecules. Moreover the surface chemistry of protein nanoparticles involving different types of functional groups enable the conjugation of a wide variety of cancer targeting ligands and monoclonal antibodies. The nano- drug formulations can be made multifunctional in terms of optical and magnetic contrast by doping them with metallic nanocluster based contrast agents. Suitably designed nanocarrier loaded with tyrosine kinase inhibitors can deliver the drug in a targeted fashion to the cells. This wi l l enhance the therapeutic outcome of the molecularly targeted drug active molecule. Doping the protein nanocarrier with NIR emitting optical contrast agents and/or magnetic contrast agents can do the in vitro or in vivo tracking of the drug molecule.

Background regarding Sorafenib tosylate

Sorafenib tosylate (also known as BAY 43-9006) is a small molecular inhibitor targeting multiple kinase pathways in cancer. It has a molecular weight of 637.03 with a molecular formula of Qi

It is a white to yellowish or brownish solid substance practically insoluble in water, slightly soluble in alcohols and soluble in DMSO and DMF. Sorafenib tosylate chemical name is 4- {4-[3-(4-Chloro-3-trifluoromethyl phenyl) ureido] phenoxy} pyridine-2-carboxylic acid methyl amide 4-methylbenzenesuIfonate. Sorafenib is marketed as Nexavar by Bayer. Each film coated tablets contains 274 mg of sorafenib tosylate, which corresponds to 200 mg of sorafenib, as active substance. '

X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Sliantikumar Nair Other ingredients are microcrystalline cellulose, croscarmellose, hypromellose, sodium laurilsulfate, ma nesium stearate, titanium dioxide and red ferric oxide (as colorants).

The active component has a bioavailability of 29-49% with a protein binding of 99.5% and a half- life of 25-48h. The metabolism is through CYP3A4-mediated hepatic oxidation and glucuronidation and excreted through feces (77%) and urine ( 19%). It blocks the enzyme RAF kinase, a critical component of the RAF/MEK/ERK signaling pathway that controls cell division and proliferation. It has also shown to inhibit CRAF, BRAF, V600E, KIT, FLT-3 and RET. It also inhibits the VEGFR-2/PDGFR-P signaling cascade (including VEGFR-2/3, PDGFR-β and RET), thereby blocking tumor angiogenesis. Thus it acts on both serjne/threonine kinases (RAF kinases) and also receptor tyrosine kinases (KIT, FLT-3 , VEGEFR-2/3 and PDGFR-β). The above said kinases have implications in several types of cancers, the mutations of which have prognostic significance. For e.g. mutations of BRAF have been associated with melanomas, mutations of KIT have been associated with gastrointestinal stromal tumors and mutations of FLT-3 have been associated with acute myelogenous leukemia. Moreover in highly drug resistant forms of chronic myelogenous leukemia, it can induce apoptosis by STAT5 inhibition and anti-apoptotic Mcl- 1 down-regulation.

Sorafenib tosylate is practically insoluble in water. Hence the dissolution rate and bioavailability of conventional sorafenib tosylate formulations are likely poor in physiological environments. In order to have maximum effect, patients are advised to take the tablets one or two hours before having food, thus increasing the likel ihood of patient compliance problems. Moreover the distribution of sorafenib in non-target organs can cause the undesirable side effects, which can be overcome if the drug active component is suitably targeted. These problems limit the therapeutic outcome for all treatments requiring sorafenib.

Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr.

Shandk mar Nair The present invention fulfils all such needs required for improving the therapeutic efficacy, improved dissolution, cancer specific targeting as well as charge turiability for desired biodistribution and clearance combined with optical/magnetic contrast enabling trackability of the formulation in the in vivo environment.

Prior Art related to sorafenib nanoformulation: Process for the preparation of sorafenib and its salts are discussed in the US patent No. US 2009/0253913A 1 dated Oct.8, 2009. Treatment of cancer and its related manifestations using sorafenib is discussed in the US patent No. US 2009/02 15835A 1 dated Aug.27, 2009. Preparation of albumin protein nanopartieles for the delivery of therapeutically active components is discussed in the US patent No. US2003/0068362A 1 dated Apr.10, 2003; -The application of protein ^■nanopartieles- for the administration of anticancer drug pac!ttaxei, is discussed in the US patent, US 6, 537, 579 B l dated Mar.25, 2009. The formulation is FDA approved and marketed as Abraxane for the treatment of metastatic breast cancer. Nanoparticulate formulations of sorafenib have been discussed in the US patent No. US 2008/0213374A 1 dated Sep.4, 2008. This is related to the application of various proteins and polymers as surface stabilizing agents for sorafenib drug active molecule and its salts and the methods for the preparation of the same. In contrast to this earl ier invention, we have used new proteins and metallic nanocluster doped proteins for making nanopartieles of sorafenib. The ne proteins are not covered under the prior art but are important class of proteins for sorafenib delivery. In our method, these protein molecules are eross ink-ed for better encapsulation and stabilization of sorafenib drug active molecule. The cross-linking methods are selected in such a way that the chemical stability of -sorafenib remains unchanged. The doping of metallic nanoclusters in the protein matrix enables the proteins to acquire a cationic nature which are otherwise negatively charged in the physiological pH owing to their p a < 7.0. The incorporation of metal nanoclusters also improves the interactions' between sorafenib and the protein at the molecular level compared to undoped protein. The metal nanocluster doping in protein-sorafenib nanopartieles also impart characteristic features like optical and magnetic contrast, which enables the tracking of the system in vitro as well as in vivo.

Amrita Vishwavidyapeetham represented by its Director, Centre of Nandsciences, Dr.

Shantikumar Nair Summary of the invention

In the current invention, a poorly aqueous soluble multi-kinase inhibitor sorafenib and its salts are incorporated in to a multifunctional protein nanocarrier. Pure molecules of sorafenib as well its salts are completely soluble only in organic solvents such as DMSO and DMF, which cannot be used for human applications. The incorporation of sorafenib, a multi-kinase inhibitor, in protein nanoparticles can effectively kill caricer cells compared to free sorafenib. The protein molecules are cross-linked for effective encapsulation of the drug. The current invention improves the therapeutic efficacy of poorly aqueous-soluble sorafenib and its salts by increasing the dissolution rate, enhanced uptake as well as enables the cancer cell specific targeting. The nanoparticles are made multifunctional in terms of optical imaging and magnetic imaging. Doping the nanocarrier with red-NIR emitting fluorescent gold provides optical contrast. Magnetic contrast is provided by paramagnetic platinum nanoc!usters. The doping of metallic nanoclusters in -protein nanoparticles imparts a positive charge to the protein nanocarrier, which has profound implications in terms of enhanced cellular uptake and renal clearance apart from providing optical/magnetic contrast. The targeting efficacy of the nanoforrnulation can be achieved by conjugating with a wide array of cancer targeting ligands and monoclonal antibodies against cancer cell-specific surface antigens; the examples of which include folic acid, transferrin, and monoclonal antibodies against CD33, EGFR, and CD 123 etc.

Amrita Visit wavidyapeeth am represented by its Director, Centr of Nanosclences, Dr.

Shantikumar Nair

Description of the drawings:

Fig. l

a) Scanning Electron Microscopic (SEM) image of albumin-sorafenib nanoparticles (Alb-Sora- NPs) showing spherical particles, b) dynamic light scattering (DLS) showing size distribution of - Alb-Sora-NPs with mean particle size is 84+24.7nm, c) molecular modeling of interactions between sorafenib and albumin using 'Auto dock' molecular docking tool, (i) sorafcnib molecule in bal l model, (ii) bovine serum albumin active site in ribbon model, (iii) sorafenib interacting with albumin active site, (iv) amino acid residues in the active site (ball and stick model) interacting with sorafenib. Sorafenib (20 A) fits in to the active site of bovine serum albumin (¾ A), interacting with the amino acid residues in the active site, d) schematic of albumin nanoparticles carrying sorafenib molecules. The surface of albumin is surrounded by hydrophilic carboxyl and amine side chains enabling aqueous dissolution and -the hydrophobic core of albumin carries sorafenib molecules, e) photograph of albumin-sorafenib nanomedicirie formulation, f) photoluminescence spectra of free sorafenib in DMSO and albumin-sorafenib nanoparticles in aqueous medium. The corresponding shift in the excitation and emission characteristics of sorafenib and albumi -sofaferiib nanoparticles is due to^" the interaction of sorafenib and albumin at the molecular level.

Fig.2 ' ^{■ " ,!" : ' ;}

a) Transmission electron microscopic image (TEM) of metallic nanoclusters of gold (Au₂s) doped in albumin represented as Au-BSA showing ~ 1 nm size Au clusters in BSA, b) atomic force microscopic image (AFM) of Au-BSA recorded from a film formed over atomically flat mica substrate, c) UV-VIS absorption spectrum of BSA and Au-BSA, d) photograph of Au-BSA under visi ble l ight, e) photograph of Au-BSA excited using UV handheld lamp @ 365nm, schematic representation of Au-BSA, g) zefa potential analysis of BSA nanoparticles and Au-BSA nanoparticles showing negative charge for BSA nanoparticles and positive charge for Au cluster doped BSA nanoparticles, h) fluorescence spectra of Au-BSA showing an excitation peak maximum @ 5 I Onm and emission @ 656nm with the emission tail extending to the NIR region, i) photostabi l ity analysis of Au-BSA under 530nm excitation for 2 h with intermittent recording of/ intensity of emission during 1 5min interval in Kodak in vivo imaging station.

Ainrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Shantikumar Nair Fig.3

a) Graphical representation of inflammatory cytokine expression by albumin nanoparticles in human peripheral blood mononuclear cells (PBMCs for 24 h, cytokine induction by positive control was set as 100% and that of negative control was set as 0%. The cytokines analyzed were IL8, ILi p, IL6, IL 10, TNF and IL12 P70, b) scatter plot of -cytokine induction: (i) untreated cells, (ii) ^g/ml bacterial lipopolysaccharide treated cells i.e. positive control, (iii) 10μ /ηι1 albumin nanoparticle treated cells, (iv) 50(ig7mT of albumin nanoparticle treated cells, c) hemolysis analysis of free sorafenib and albumiri-sorafenib nanoparticles, negative control was untreated cells and positive control was 1 % triton X 100 treated cells; inset shows the SE image of red blood cells treated with 20μΜ albumin-sorafenib nanoparticles, d) cytotoxicity analysis of free sorafenib and albumiri-sorafenib nanoparticles on human PBMCs.

Fig.4

a) Reactive oxygen species (ROS) generation assay using 500 g/ml Au-BSA; negative control was untreated cells, whereas positive control was hydrogen peroxide (H₂O₂) treated cells, b) cytotoxicity analysis of Au-BSA on PBMCs and human umbilical vein endothelial cells (HUVEC), c) Propidium iodide (PI) staining of cells treated with 500μg/ml of Au-BSA for 24 h using flow cytometry to assess the membrane integrity of cells.

Fig.5

Schematic representation of bioconjugation of CD33 mAb with Au-NCs using EDC-NHS chemistry. The carboxyl group of mAb is activated by EDC and stabilized by sulfo-NHS, and the resulting semi-stable amine reactive CD33 mAb-NHS ester reacts with the protein stabilized Au- NCs leading to the formation of-Au-NC-GD33 throagh-a strong-amide -bond. :~r :. ·:-:::--^■■. -

X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr.

Shantikumar Nair Fig.6

Targeted uptake of Au-BSA nanoparticles conjugated to cancer cell targeting ligands and monoclonal antibodies: a_ra₂) fluorescent microscopic image showing human lung adenocarcinoma A549 cells with low expression of folate receptors (FR^low) treated with l mg/ml Au-BSA conjugated to folic acid (Au-BSA-FA) for 24 h, b b₂) human oral carcinoma KB cells (FR^h'^8h) treated with 1 mg/ml Au-BSA-FA for 24 -h, C†) confocal microscopic image shewing PBMCs (CD33^l0W) treated with 4.5 g/ml AU-NC-CD33 for 1 h, C₂)-PBMCs tfeated With^" 9.0ug/rhT AU-NC-CD33 for 2 h, d,) KG l a (CD33^h,gh) cells treated with 4^g/ml Au-NC-CD33 for 1 h, d₂) KG 1 a cells treated with 9.(^g/ml Au-NC-CD33 for 2 h.

Fig.7 ■ - - - — =-^■· >^{■ ■} - - ^^.. ^- ^

Biodistribution of BSA nanoparticles and Au-BSA nanoparticles in mice at different times intervals recorded using Kodak in vivo imaging system: aj-aio) BSA nanoparticle biodistribution in mice b|-b_I0) Au-BSA nanoparticle bio listribution in mice.

Fig.8 ^' _ a,) Ex-vivo images of vital-organs and urine from BSA nanoparticle injected mice after 48 h, a₂) ex-vivo images of vital organs and urine from Au-BSA nanoparticle injected mice after 48 h, b) graphical representation of relative accumulation of BSA nanoparticle and Au-BSA nanoparticle in the vital organs, of mice, c) graphical representation of accumulation of Au-BSA nanoparticle in the vital organs of mice at different intervals, d_rd₂) fluorescence intensity in kidneys and urine after 48 h of BSA nanoparticle administration, d₃-d₄) fluorescence intensity in kidneys and urine after 48 h of Au-BSA nanoparticle administration, e) grapTiical representation oFrel^ intensity in kidney and urine at 24 h and 48h in mice injected with Au-BSA nanoparticles. ' ^: - ^; - ^■-

X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Shantikumar Nair Fig.9

Characterization of drug resistant K562 CML cells: a) Fluorescence in situ hybridization (FISH) analysis of imatinib-dasatinib resistant K.562 cells showing multiple bcr-abl fusions (encircled regions), b) gel picture showing bcr-abl amplieon (304bp), lane 1 is water-control; lane-2 is DN A negative control; lane 3 is bcr-abl -ampUeen-irorn imatinib-dasatinib- resistant K562-; lane-4 is ber- abl amplieon from chronic phase CML patient, c) MTT cell viability assay on imatinib-dasatinib resistant K562.

Fig.10

Cytotoxicity analysis of free sorafenib and albumin-sorafenib nanoparticles on imatinib- dasatinib resistant K562: a) MTT cell viability assay for a duration of 24 h, b) MTT cell viability assay for a duration of 48 -h,-c) apoptosis assay using annexin V-PI staining for a duration of 24 h, d) apoptosis assay using annexin V-PI staining for a duration of 48 h. Ι ΟμΜ concentration of free sorafenib and albumin-sorafenib nanoparticles was used for the apoptosis assay. _: - „, ,_..: : _..

Fig.l l

Microscopic images of imatinib-dasatinib resistant K.562 CML cells treated with 5μΜ of free sorafenib and albumin sorafenib nanoparticles for a duration of 48h.

a,-a₂) untreated control cells, b_rb₂) 5μΜ free sorafenib treated cells b₃) enlarged image of cells treated with 5μΜ free sorafenib, c_rc₂) 5uM aibumin-sorafenib nanoparticles treated cells, c₃) enlarged image of cells treated with 5μΜ albumin-sorafenib nanoparticles.

AnnexinV-PI staining of drug treted cells: a,) untreated cells, a₂) 5μΜ free sorafenib treated cells, a₃) 5μΜ albumin-sorafenib treated cells.

X Amrita Vishwavidyapeetham represented by Us Director, Centre of Nanosciences, Dr. Shantikumar Nair Detailed description of the specific embodiments

The invention provides compositions comprising sorafenrtr or its salt and at^' least ^"oner protein; ^" which can act as a nanocarrier for the drug active molecule. The sorafenib molecules interact with the protein nanocarrier non-covalently and hence the active molecule remains chemically unchanged throughout the process. The protein nanocarrier is selected from different types of proteins derived from natural sources and are highly biocompatible. The amphiphilic nature of the proteins enables them to deliver both hydrophilic as well as hydrophobic drugs. The drug active molecule can also be a salt in the crystalline phase, semi-crystalline phase, amorphous phase or a combination. The formulation can be . administered as a parenteral injection (intravenous, intramuscular or subcutaneous), oral administration as liquid, solid or aerosol; The proteins can be used individually or in combination for preparing nanoformulations of sorafenib. The proteins modified or unmodified for preparing nanoformulations include but are not limited to transferrin, albumin, casein, soy protein, protamine etc. We have taken a representative protein bovine serum albumin (BSA) for the drawings described. This is applicable to all other proteins and proteins modified with metallic cluster doping. The -mult kinase inhibitor sorafenib. is erobedded in the- hydrophobic pocket of BSA nanoparticles. The albumin-sorafenib nanopartie!es (Alb-Sora-NPs) acquire a spherical morphology as- shown in the scanning electron micrograph (fig. la): The nanoparticles preferably have a hydrodynamic diameter < 200nm as shown in fig.lb. Sorafenib molecules interact with the amino-acid residues in the active site of BSA_S as-shewn in the in silieo modeled images in fig.lc. The schematic representation of sorafenib _. embedded BSA nanoparticles is shown in fig.ld, where the inset (fig.le) shows the photograph of albuminr sorafenib nanoformulations The molecular level interactions of sorafenib. and BSA as; shown: in the molecular modeling also reflect in the photoluminescence characteristics of albumin-sorafenib nanoparticles as shown in fig. If.

IX Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Sliantikumar Nair The protein is either unmodified or modified with metal nanoclusters providing optical contrast and/or magnetic contrast as well as improved cationic nature for the formulation enabling enhanced cellular uptake of the drug active molecule. The metallic nanoclusters of gold, silver and platinum are used for doping. The doping of metallic nanoclusters in to the protein matrix imparts magnetic/optical contrast. Metallic nanocluster doping can change the properties of the host protein to a great extent. This is very well represented by the .doping of gold nanoclusters comprising of as few as 25 atoms of gold in to BSA. The gold nanoclusters have size <l nm as shown in fig.2a and b. UV-VIS absorption spectrum shows the absorption characteristics of BSA and Au-BSA (fig.2c). Au-BSA iias a characteristic golden brown colour, which when excited under UV produces bright red luminescence as shown in fig.2d and e. the schematic representation of Au-BSA is shown in fig.lf. The doping of metal lic nanoclusters such as gold nanoclusters can change the zeta potential of protein, thus making it cationic. This is well represented in fig.2g. This has immense significance in the biodistribution and clearance of protein drug delivery systems. Metallic nanocluster doped protein nanocarriers can undergo renal clearance whereas the unmodified proteins cannot pass through the glomerualr filter membrane of kidney. The photoluminescence excitation and emission spectrum of Au-BSA is shown in fig.2h. The emission of Au-BSA extends to the near infrared region, which enables the in vivo tracking of the protein nanocarrier. Unlike other organic dye based probes for molecular imaging, Au- BSA possesses extremely high photostability even after continuous irradiation for 2 h as shown in fig.2i.

The metall ic cluster doped/undoped protein nanocarriers, exhibit biocompatibility in terms of non-inflammatory nature (fig.3a and b). The system didn't cause hemolysis of red blood cells (RBCs) derived from healthy donors (fig.3c). The morphology of RJBCs was as good as the control cells, which were not treated with albumin-sorafenib nanoparticles (inset of fig.3c). The toxicity of albumin-sorafenib nanoformulation to human peripheral blood mononuclear cells (PBMCs) was assessed along with free sorafenib, which shows that the formulation did not induce much toxicity compared to free sorafenib (fig.3d).

X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Shantikumar Nair Cellular stress in terms of reactive oxygen species (ROS) production was assessed after treating the cells with Au-BSA, which showed that Au-BSA did not induce ROS generation, which indicates the cytocompatability of Au-BSA (fig.4a). Similarly Au-BSA did not show toxicity to PBMCs as inferred from the MTT cell viability assay as shown in fig.4b. The membrane integrity of the cells treated with Au-BSA were stained with propidium iodide (PI) also indicates the nontoxic nature of Au-BSA (fig.4c). Au-BSA can be conjugated to cancer targeting ligands and monoclonal antibodies to enable the cancer specific uptake of the carrier comprising of drug. EDC-sulfo NHS cross-linking chemistry can be employed for the conjugation of cancer targeting ligands. The schematic of bioc^'onjuagtion of Au-BSA to CD33 monoclonal antibody to target the primitive population of acute leukemia (AMU) cells over expressing the CD33 myeloid antigen is shown in fig. 5. The examples for targeted uptake of Au-BSA are shown in the following drawings. Fig.6b shows the^'targeVed^' uptai e of Au-SSA^' conjugated to folic acid in folate receptor over-expressing (FR^{+ e}) oral carcinoma cells whereas fig.6a shows the corresponding negative control, which are FR^'ve lung cancer cells. Similarly fig.6d shows the targeted uptake of Au-BSA conjugated to CD33 monoclonal antibody in CD33 over expressing primitive AML cells whereas fig.6c shows the negative control i.e. CD33^'ve'^1ow PBMCs. ^"

Tlte doping of metallic narioclusters of gold ί n BSA alters the charge of the protein and makes it cationic. Unmodi fied proteins because of their negative charge cannot pass through the glomerular fi lter membrane of kidney. In contrast to this Au-BSA adopts a different biodistribution and clearance profile compared to BSA nanoparticles. This is indicated in fig.Ta and b. it is evident from the drawing that BSA and Au-BSA underwent complete clearance from the animal body after 48 h. the vital organs of the mice were removed and analyzed separately for the presence of the particles (fig.Sai ^"and a₂ .^" The ^"enhanced uptake and clearance of Au-BSA compared to BSA nanoparticl^'es is evident from the plot Au-BSA ^'and^'

BSA nanoparticles from various organs (fig.8b) and with respect to different time points (fig.8c). The accumulation of Au-BSA in kidney and the corresponding elimination through urine compared to BSA nanoparticles is shown in fig.8d. The corresponding graphical plot is represented in the fig.8e.

I X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Shantikumar Nair Drug resistant forms of leukemia have shown in vitro sensitivity to sorafenib at micro molar concentrations. Sorafenib ^* down-regulates STAT5 and the "anti -apoptotic protein Mct- 1 downstream of STAT5. Y t, the poor aqueous -solubility of sorafenib signvf»€antly reduces -its therapeutic efficacy in the clinical scenario. The efficacy of albumin-sorafenib nanoparticles was tested in multi-drug resistant chronic myeloid leukemia (CML) cells. CML harbors a bcr-abl fusion gene encoding a 2 l Ot Da protehrhavmg constitutive tyrosine kmaseactivity. The clinically used drugs against CML suctra^ima^

to over expression of bet;*ablj

alternative signaling pathways such as STAT5. Since sorafenib employs a bcr-abl independent- mechanism to kill the CML cellsv they ^"are acliye against drug resistant CML. The drug resistant CML cell line, which we developed, carried bcr-abl · over expression as determined . h . fluorescence in situ hybridization FISH (fig.9a). Eig.9b shows tJae 3il4bp icx-abl amplicoa iiom polymerase chain reaction (PCR), The- multi-drug resistance of the cells^' is represented by the MTT cell viability assay using imatinib and dasatinib as shown in fig.9c. The sensitivity of the drug resistant CML cells to sorafenib and albumin sorafenib nanoparticles for- 24 h and 48 h -is shown in fig.10a and b respectively which represents the enhanced cytotoxic nature of albumin- sorafenib nanoparticles compared to free sorafenib. Apoptosis assay done using FITC conjugated annexin-V and Pf also showed that albumin-sorafenib nanoparticles have greater apoptotic potential than free sorafenib (fig. LOc and d).

Albumin-sorafenib nanoparticles resulted in evident morphological changes and loss of membrane integrity compared to free sorafenib as shown by the microscopic images in fig.l la, b and c. The cells treated with free sorafenib and albumin-sorafenib nanoparticles were stained with FITC conjugated annexin-V and ^rPi and the flnorescent-microscopic images recorded shows · the apoptotic cells after free sorafenib and.alburain_^sorafenib nanoparticles treatmen (fig.12).

Preferred characteristics^; of^uItifanctiOnal rotern-sorafenib nairoformulatrons

The advantages of protein-sorafenib nanoformu!ations are the following but are not limited to:

tgsfsJX Atnrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr.

Shantikumar Nair Targeting of the drug active molecule: The' Incorporation of sorafenib and -its salts irrthe protein nanoparticle also enable the targeting. of...the-.-img..active-4W)lecu|.e--to.-th^^an^--(½Hs--wkheut- affecting the normal cells. The targeting can be done using the protein itself which -possess a spontaneous cancer targeting capability or another protein, which can be either a cancer specific ligand or a monoclonal anti-body, conjugated to the protein. The targeting of the drug active molecule specifically to the cancer cells, combined with the enhanced dissolution rate and bioavailabilty can significantly, in^rove te

targeting of protein-sorafenib nanoparticles can be performed in solid tumors as . ell- as hematological malignancies. For e.g. gold nanocluster doped BSA rianbconjugtes can be conjugated to folic acid and targeted to folate receptor over-expressing cancers such as oral- cancer and breast cancer. Similarly they. can also ..be.. conjugated to..CD3imonocIonal antibody for targeting the CD33 myeloid surface autigeh over-expressed in primitive population of acute myeloid leukemia (AML) cells. This mode of active targeting of drug is extremely important in eradicating not only the mature -cells in cancer but also the cancer stem -cell population responsible for the cancer drug resistance.

Charge modulation and renal clearance of nanocluster doped protein-sorafenib nanoformulation: Sorafenib i s a multi -kinase^' inhibitor appro ved Tot cehatxell. carcinoma.. The non-specific binding/adsorption of protein and other molecules prevent its accumulation in the kidneys and a few non-bound molecules reach kidney. This can be of two major reasons; (i) nonspecific protein adsorption impart a negative charge to the drug, which will prevent it from filtered through the negatively charged glomerular filter membrane of kidney, (ii) the size of protein adsorbed sorafenib- is larger- compared to- free sorafenib- molecules and" -cannot pass through the glomerular filter membrane.. Since mosi.of the proteins, have pKa.less than 7.0 and hence are negatively chargeddn thei ^

renal targeting. Metallic nanoclusters of gold doped in to protein nanoparticles carrying sorafenib alter its charge and make the system positively charged and enable them to accumulate- in the kidney and slowly undergo clearance. This has profound implication for improving the therapeutic efficacy of sorafenib for the treatment of renal cell carcinoma (RCC). X Atnrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr.

Shantikumar Nair In vivo and in vitro tracking of sorafenib and its salts: The modification of the protein carrier with metallic nanoclusters having -in vivo optical contrast and mapfctiif ^'tonfrast- ^'-eha&T^s the tracking of sorafenib actiwagenWrc vrrro as wett^'SSin VfVd ^'

Pharmacokinetic profile of protein- sorafenib nanoformtdatmn: The improved in vivo dissolution characteristics, bioavailability and cell specific targeting greatly improve the pharmacokinetic profile of sorafenib and its salts. The entrapment of sorafenib and its salts in a protein matrix, enable the slow release of the drug from the- nanofoimulation with the action of proteases or acidic pH on the<proiem nanoparticles. ^1■■ - " ' * ^{■ ^} " -

Examples

The following example

a protein having inherent cancer cell targeting capability.

Example 1

Transferrin-sorafenib nanoparticles (Tf-Sora NPs): In a typical synthesis procedure, sorafenib tosylate dissolved in DMSO is slowly added drop-wise to 5ml of l Omg/ml holo- transferrin dissolved in double distilled water kept under continuous stirring at 37°C. The final concentration of the sorafenib is ad usted-to 5ee M;- he-s Tlurrjn s^" ke t for stirring at 37°C for 2 h and then kept for overnight stirring at 4°C. This enables the effective interaction of the protein with the drug. The individual protein molecules are cross-linked using a zero length cross-linker 1 -ethyl -3- (3-dimethylaminopropyl)-carbodiimide (EDC). The concentration of EDC is optimized in such a way as to maintain the particle size within l OOnm. The cross-linking reaction is interrupted using an EDC quencher Glycine. The Tf-Sora nanofoimulation is dialyzed to remove the unreacted components andiyophilized to^'m^'ake^{" "}a it ^'poWdef.^' 5irrin y7 5r0tein-sorafenib nanoformulations are prepared with other proteins also.

The following example discusses the preparation of protein-sorafenib nanoformulation, using a dietary protein.

X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Shantikumar Nair Example 2

Soy protein isolate (SPI) is obtained from soybean by removing soybean oil and soy carbohydrates. SPI contains more : than 90% projein^^^

water consist of sphere-like protein particles. Soyprotein nanoparticles are prepare _.bx lJkaHne_ denaturation of SPI at 3 C- using 1 M NaOH. Sorfanib tosylate dissolved in DMSO is added drop-wise in to the SPI solution to make up the final concentration of sorafenib to 500μΜ. The encapsulation of sorafenib i , the soy pratein nanoparticles is indicated »by..th©-enhaneed- turbidky- of the formulation. Similarl other proteins-can . be ..used - for- the- prer^ration - nanopartic;les_rfor _. carrying sorafenib active molecule - ·_; . ; : „: : ,,_..,_..,.„ . _v, , _:,

The following example discusses the preparatw^ef_: protein^

a hydrophilic protein doped with metallic clusters.

Example 3 ; .

Au-Albumin-sorafenib nanoparticles (Au Alb-Sora NPs): Au-Alb-Sora NPs): In a typical synthesis procedure, 5ml of 1 OmM HAuCl₄.,is adde.d dropwise to. 5ml ,oL20mg ml human serum albumin dissolved in double distilled water kept- under -continuous stirring at 37°C. 1 5.7mM sorafenib tosylate dissolved in DMSO is slowly added drop-wise to the solution so that the final concentration of the drug -active component is 500uM 1 M NaOH is added drop-wise in to the solution to enable the reduction of HAuC . The reduction process leads to the formation of gold nanoclusters comprising magic numbers of Au atoms. The solution mixture is stirred continuously overnight at 4°C for effective mixing of the protein with the .drug, The individual protein molecules are cross-linked using a zero lengt cross-lirrker EDC. The concentration of EDC is optimized in such a way as to maintain the particle size within l OOnm. The cross-linking reaction is interrupted using an ED€ quencher- Glycine, The nartoformulation · is dialyzed to remove the unreacted components and yophilized' to ma e^" ί fine: ^' powder. The- lyophilized - powder is resuspended in milHQ water or PBS. These nanoparticles exhibit a rcd- IR emission having a peak maximum at 650nm and the emission extending up to 800nm. Similarly, metallic clusters of platinum and sil ver can also be used for thepreparatton; The preparation is iot limited to HSA alone but to other proteins also.

X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr. Shantikumar Nair The following example discusses the preparation of protein-sorafenib nanoformulation^ using a hydrophobic protein doped -wkh metallic clusters; -

Example 4 -- - —

Au-Casein-sorafenib nanoparticles (Au-Cas-Sora NPs): Sorafenib tosyate powder is dissolved in DMSO to prepare a stock -soratran of†5-.7m l Omg of β-casein from bovineTnilk is dissolved in 5ml of phosphate buffered saline (PBS), pH- 7,4,- PBS contained 80mM aCl₍.5.7mM- Na₂HPQ4,. , and 3mM NaH₂P04.

solution under stirring at 37°C. The solution is kept stirring for 30min for adequate interaction of HAuCl₄ ions with β-casein. After 30ffiin, add 1 M NaOH drop-wise in to the solution till the solution turns transparent yellow. The solution mixture is kept under stirring for 12 h at 37°C. The body colour of the solution turns to goltfe^br wrr

metallic clusters of platinum a^

The following example discusses the preparation of protein-sorafenib nanoformulation, using a cationic protein having cell penetrating capability. .-■—

Example 5

Protamine is a highly cationic peptide composed of a few amino acids. Owing to the small size as well as its cationic nature, it enhances the¹ uptake of anionic agents such as negatively charged drugs as well as nucleic acids. Being aqueous soluble it can greatly enhance the solubility of poorly water-soluble drugs, rmg/mf protamine is dissolved in double distilled water; Sorfanib tosylate dissolved in DMSO is added drop-wise in to the protamine solution to make up the final concentration of sorafenib to 500μΜ. The solution is stirred continuously for .3 Jx. at... .?G.far_. effective comp!exation BeWe ffie^{^}pYofamm^ and sorafenib^' tosylate.^' - - .·■- . ^{' '■}-·■- -· ; .

The following example discusses the preparation of protein-sorafenib nanoformulation using co-acervation method _? r^X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr.

Shantikumar Nair Example 6

Bovine serum albumin (BSA) is dissolved in distilled water at a concentration of 5mg/ml. Sorafenib tosylate is added to BSA drop-wise at a final concentration of ΙΟΟμΜ and stirred continuously for effective complexation at room temperature. To this mixture 100% ethanol is added at BSA:ethanol volume ratio 1 : 1. This co-acervatioh process induces the^' deriatufatioh of BSA and initiates the formation of nanoparticles in the size range bTT0O 2O0hm^". Tlie dehatured protein molecules are cr6ss-lin e ^"ulmg^"ED^"C. ^"The solution is continuously stirred at room temperature for 3h. Lyophilizing the sample enables the removal of ethanol. The fine powder obtained can be resuspendedTh^~waterl5FPBl ^{~~ "" " " "} · ^{~ ~}

The following example discusses the preparation of protein-sorafenib nanoparticles conjugated to a monoclonal antibody targeting a myeloid antigen in leukemia cells

Example 7

In a typical synthesis procedure, sorafenib tosylate dissolved in DMSO is slowly added drop-wise to 5ml of l Omg ml

continuous stirring at 37°C. The final concentration of the sorafenib is adjusted to 500μΜ. The solution is kept under stirring at 37°C for 2 h and then kept for overnight stirring at 4°C. This enables the effective interaction of the protein with the drug. The individual protein molecules are cross-linked using a zero length cross-linker l -ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC). The concentration of EDC is optimized in such a way as to maintain the" particle size within l OOnm. The cross-linking reaction is interrupted using an EDC quencher Glycine. The albumin-sorafenib nanoformu!ation is dialyzed to remove the unreacted components and lyophilized to make a fine powder. The Iyophilized powder is resuspended in PBS (pH 7.4) before antibody conjugation. O^g of CD33 monoclonal antibodyTs^''activated^''lising^"¾fn¾^'"EDC^' and 8mg of sulfo-NHS for ~ 15 min. The activated antibody is added to albumin-sorafenib nanoparticles and reaction is continued for ~ 2 h at room temperature in the dark. The unreacted components are removed by centrifugation at l OOOOrpm for 15minutes.

Shantikumar Nair

Claims

Claims:

1- We claim . a ._.;nanGrnedie>ne;;produ9t consisting. Qf;stab.le -.ptoiein-bQUDCfc ..

sorafenib

a) Nanoparticles of a protein in its native or denatured form having an average size < I OGOnm b) Sorafenib or its salts in monomeric or aggregated -form- loaded bound within the above protein nanoparticles ; - _. · ^■: ...·.... . :

2. The composition o lairri I , wherein the protein nanoparticle Is formed from at least one protein from the group of: total human serum protein, protamine, transferrin, mucin, soy protein, apoferritin, ferritin, lectin, lactoferrin, gluten, whey protein, prolamines^' such ^{" '}as^' gliadih; KoMein, ecafin,' ^' zefri', aveni^'n, or combinations thereof

3. The composition of claim 1 , wherein the sorafenib used may be bound to all part of the protein or embedded as aggregates within the protein nanoparticles.

4.

bound protein nanoparticles will have enhanced cancer uptake as these proteins are needed by the rapid ly prol i ferating cafi^'cer cells.

5. The comppsition iri claim. 1 , wherein the sprafenib-bqund protein . nanoparticle is preferably conjugated with cancer targeting ligand, at least any one from the group of: folic acid^mannose, hyaluronic. acid,..ant&

factor receptor, vascular growth factor receptor, prominine -1 (CD 133), CD44, GD I 23, CD24, CD 1 1 7, CD33, C-kit receptor (Cd 1 17), transferrin receptor, integrins, Her2 receptor, somatostatin. receptor, estrogen receptor, progesteron receptor, prostate specific antigen receptor, mucine protein, p-glycoprotein, oligosaccharides, etc

6. Claim 1 -5 wherein the nanomedicine product is doped with metal clusters.

7. A process for claims 1 - 6 above involving an acqueous method wherein the sorafenib is reacted with the protein in aqueous medium or a mixture of aqueous and organic medium, preferably at room temperature. ^f ^X Amrita Vishwavidyapeetham represented by its Director, Centre of Nanosciences, Dr.

Shantikumar Nair