A THERMOSENSITIVE HYDROGEL AND A PROCESS OF PREPARATION THEREOF

Abstract

A thermosensitive hydrogel comprising a) polyvinyl alcohol, b) poloxamer 407; c) bovine serum albumin (BSA); d) doxorubicin; and e) Proflavine hemisulfate (PfHS), wherein the thermosensitive hydrogel is a nano-transformable gel. The present disclosure also relates to the method of preparing the same.

Specification

Description:FIELD OF THE INVENTION:
[0001] The present disclosure relates to the field of pharmaceuticals. More particularly, the present disclosure relates to a thermosensitive hydrogel comprising Doxorubicin and Proflavine hemisulfate. The present disclosure also relates to a method of preparing the said thermosensitive hydrogel.

BACKGROUND OF THE INVENTION
[0002] Cancer refers to a large group of diseases characterized by the growth of abnormal cells which disregards the normal rules of cell division. Cancer is a leading cause of fatality which is characterized by the ability of the abnormal cells to metastasize across the body, spreading tumors at multiple sites, consequently leading to death.
[0003] Conventional treatment modalities for cancers include surgery, chemotherapy, radiation therapy, immunotherapy, etc. These treatment modalities are inefficient and have major drawbacks due to the complexity and heterogeneity of the tumor microenvironment.
[0004] Hydrogels are hydrophilic three-dimensional polymeric networks that have high water sorption ability. Injectable hydrogels have been used for various biomedical applications due to their shear thinning ability, controlled drug release, minimal invasiveness, low toxicity and good swelling behaviour. Most often these hydrogels are physically or chemically crosslinked and are combined with other therapeutic modalities such as photothermal, photodynamic, chemodynamic, sonodynamic therapies etc., for the treatment. However, the main drawbacks of these hydrogels are poor stability, faster degradation, and lesser mechanical strength.
[0005] Therefore, there is a need to provide thermosensitive hydrogels that can be used for combining therapeutic modalities that can be used for the treatment and management of cancer.

OBJECTS OF THE INVENTION
[0006] Some of the objectives of the present disclosure, with at least one embodiment herein satisfied, are listed herein below:
[0007] It is the primary objective of the present disclosure to provide a thermosensitive hydrogel that addresses the above-mentioned problems.
[0008] It is the primary objective of the present disclosure to provide a thermosensitive hydrogel comprising Doxorubicin and Proflavine hemisulfate.
[0009] It is yet another objective of the present disclosure to provide a thermosensitive hydrogel for increased therapeutic efficacy.
[0010] It is another objective of the present disclosure to provide the combination therapy with lesser toxicity.
[0011] . It is another objective of the present disclosure to provide the thermosensitive hydrogel that provides controlled drug delivery.
[0012] It is yet another objective of the present disclosure to provide a simple and cost-effective method for the preparation of thermosensitive hydrogel.

SUMMARY OF INVENTION
[0013] The present disclosure relates to a thermosensitive hydrogel comprising
a) polyvinyl alcohol,
b) poloxamer 407;
c) bovine serum albumin (BSA);
d) doxorubicin; and
e) Proflavine hemisulfate (PfHS),
wherein the thermosensitve hydrogel is a nano-transformable gel; and
wherein the ratio of doxorubicin and PfHS is in range of 5:1 to 25: 1.
[0014] The present disclosure also relates to a method for preparing the thermosensitive hydrogel comprising doxorubicin and Proflavine hemisulfate, the method comprising the steps of:
a. preparing poloxamer 407 solution, PVA solution and BSA solution separately in a mili Q water;
b. adding the PVA solution to the poloxamer 407 solution under constant stirring in an ice cold bath condition for at least a minute;
c. adding DOX and PfHS with continuous stirring; and
b. adding bovine serum albumin to the reaction mixture obtained in step (a) under vigorous stirring for 5 to 10 minutes to obtain a thermosensitive hydrogel.
BRIEF DESCRIPTION OF DRAWINGS
[0015] The present disclosure contains the following drawings that simply illustrates certain selected embodiments of the nanocarrier composition and processes that are consistent with the subject matter as claimed herein, wherein:
[0016] Figure 1 depicts the thermosensitive gel system with a CT contrast agent and synergistic two drug combination
[0017] Figure 2 depicts fixation of the percentage of addition of iohexol by checking the stability of the gel under irreversible conditions
[0018] Figure 3 depicts the Inversion test which shows the thermos-responsive behaviour of the hydrogel
[0019] Figure 4 depicts the micro CT image showing the loaded iohexol and fluorescent nature of the loaded drugs under UV light.
[0020] Figure 5 depicts the cytotoxicity on 4T1 cell line and doxorubicin
[0021] Figure 6 depicts the combination index of the drugs
[0022] Figure 7 depicts the % cell viability of the drugs alone and in combination.

DESCRIPTION OF THE INVENTION:
[0023] A detailed description of various exemplary embodiments of the disclosure is described herein. It should be noted that the embodiments are described herein in such detail as to communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.
[0024] The terminology used herein is to describe particular embodiments only and is not intended to be limiting to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising", or "includes" and/or "including" or "has" and/or "having" when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
[0025] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0026] The term "photothermal therapy" refers to irradiation of laser light within NIR range (650 to 1100 nm) on light-absorbing material that converts light energy into heat energy causing hyperthermia. The materials responsive to this therapy could be NIR dyes or metallic nanoparticle. Photothermal therapy in the context of the present invention is effectuated with the use of one or more "photothermal agent."
[0027] The term "photodynamic therapy" refers to the irradiation of light within the NIR range on light-absorbing material that produces reactive oxidative species.
[0028] The term "chemotherapy" refers to the use of chemotherapeutic drugs (chemicals) for destroying the cancer cells. The term "chemo-photothermal therapy" refers to a combination of both chemotherapy and photothermal therapy.
[0029] The term "thermosensitive" as used herein refers to the property of the hydrogels to exhibit a phase a reversible transition from sol to gel at physiological temperature (37° C.) and irreversible transition at elevated temperature (55° C.).
[0030] The present disclosure relates to a thermosensitive hydrogel comprising
a) polyvinyl alcohol (PVA),
b) Poloxamer 407;
c) bovine serum albumin (BSA);
d) doxorubicin; and
e) Proflavine hemisulfate (PfHS),
wherein the thermosensitve hydrogel is a nano-transformable gel, and
wherein the ratio of doxorubicin and PfHS is in range of 5:1 -25:1.
[0031] In an embodiment of the present disclosure, the PVA is a water-soluble polymer represented by the general formula

[0032] In another embodiment of the present disclosure, Poloxamer 407 (tradename: Pluronic-F127) is a triblock copolymer consisting of a central hydrophobic block of polypropylene glycol flanked by two hydrophilic blocks of polyethylene glycol (PEG). It is represented by the general formula:

[0033] In another embodiment of the present disclosure, a concentration of the polyvinyl alcohol is in a range from 2-15% v/v.
[0034] In another embodiment of the present disclosure, a concentration of doxorubicin is in a range of 5 to 10 µg
[0035] In another embodiment of the present disclosure, a concentration of PfHS is in a range of 0.25 to 1 µg
[0036] In yet another embodiment of the present disclosure, a concentration of the pluronic (PF)-127; is in a range from 20-50% wt/v.
[0037] In another embodiment of the present disclosure, a concentration of the bovine serum albumin, a protein is in a range from 20-40% wt/v.
[0038] In an embodiment of the present disclosure, the protein-polymer based hydrogel is characterized by thermosensitivity and ability to deliver DOX and PfHS in a safe and efficacious manner. In an embodiment, the irreversibility of the thermosensitive hydrogel is due to the denaturation of BSA.
[0039] In an embodiment of the present disclosure, the hydrogel contains one or more additional component selected from a group comprising photothermal agents, photosensitizers, and contrast agent.
[0040] In an embodiment, the contrast agent is selected from the group consisting of Iodine-based contrast agents or Iohexol.
[0041] In yet another embodiment of the present disclosure, a concentration of the contrast agent is in a range from 10-30%
[0042] In another embodiment of the present disclosure, the hydrogels may further comprise a pharmaceutically acceptable carrier or excipient. The carriers include, but are not limited to sterile aqueous media, solid diluents or fillers, excipients, and various non-toxic organic solvents.
[0043] The present disclosure relates to a method for preparing the thermosensitive hydrogel comprising DOX and PfHS , comprising the steps of:
a. preparing poloxamer 407 solution, PVA solution and BSA solution separately in a mili Q water;
b. adding the PVA solution to the poloxamer 407 solution under constant stirring in an ice cold bath condition for at least a minute;
c. adding DOX and PfHS with continuous stirring; and
d. adding bovine serum albumin to the reaction mixture obtained in step (a) under vigorous stirring for 5 to 10 minutes to obtain a thermosensitive hydrogel with drugs.
[0044] The present disclosure also relates to a method for preparing the thermosensitive hydrogel comprising DOX, PfHS and contrast agent, comprising the steps of:
a. preparing poloxamer 407 solution, PVA solution and BSA solution separately in a mili Q water;
b. adding the PVA solution to the poloxamer 407 solution, followed by contrast agent under constant stirring in an ice-cold bath condition for at least a minute;
c. adding DOX and PfHS with continuous stirring; and
d. adding bovine serum albumin to the reaction mixture obtained in step (a) under vigorous stirring for 5 to 10 minutes to obtain a thermosensitive hydrogel with drugs.
[0045] In another embodiment, the present disclosure provides a method of treating cancer, wherein the thermosensitive hydrogel can target primary and metastasized secondary tumors.
[0046] In another embodiment, the present disclosure provides a method of treating cancer, wherein the thermosensitive hydrogel can be used for treatment of primary and metastasized secondary tumors.
[0047] In another embodiment, the present disclosure provides the use of hydrogel for targeted drug delivery to primary and metastasized secondary tumors and for bio-imaging.

ADVANTAGES OF THE PRESENT INVENTION
[0048] In accordance with the present disclosure, the thermosensitive gel has the following advantages:
(a) Affordable: The hydrogels developed are considerably inexpensive and has the potential to act as a cancer therapeutic.
(b) Thermosensitive: The hydrogels when injected in the tumor can exhibit a phase a reversible transition from sol to gel at physiological temperature (37° C.) and irreversible transition at elevated temperature (55° C.) thus forming a depot
(c) Sustained Release: These hydrogels have the potential to be used as a depot for sustained release of nanoparticle for photothermal therapy and/or for micellar delivery of drug/dyes for therapeutics and bio imaging
(d) Dual modality treatment: The hydrogels are extremely effective for dual modality treatment, such as combining photothermal therapy with chemotherapy (chemo-photothermal therapy) or photodynamic therapy (photothermal-photodynamic therapy).
(e) Targeted delivery: The hydrogels can be used for targeted drug delivery to primary & metastasized secondary tumors and for bio-imaging
(f) Absence of undesirable effects: The hydrogels are highly efficacious in treatment of cancer without having any visible side effects
(g) Therapeutic efficacy: The hydrogels combined with Dox and PfHS exhibit synergistic therapeutic efficacy when compared to Dox and PfHS individually.
[0049] The present disclosure will be explained using the following examples:

EXAMPLE
Material:
[0050] Pluronic (PF)-127, PVA (low molecular weight-10,000 kDa), BSA, Milli-Q water, Doxorubicin, Proflavine hemisulfate (PfHS), and Iohexol were obtained from sigma Aldrich.

Example 1:
Preparation of thermosensitive gel comprising Doxorubicin and Proflavine hemisulfate (PfHS)
[0051] Precursor solutions were prepared separately. 40% PF-127 was prepared by adding 20g of PF-127 in 50ml milli-Q water. 10%PVA was prepared by dissolving 1g in 10ml and 35% BSA is prepared by adding 3.5g in 10ml. All the precursor solutions were prepared and stored in 4°C until further use for the formation of Nano-transformable gel, 3ml of 40%PF-127 was taken and stirred at 1000 rpm in ice-cold bath condition for at least a minute. Then 600µL of 10% PVA was added and stirred for a minute, addition of drugs was carried out and 300 µL of BSA was added to obtain the thermosensitive gel comprising Doxorubicin and Proflavine hemisulfate .

Example 2:
Preparation of thermosensitive gel comprising Doxorubicin and Proflavine hemisulfate with contrast agent Iohexol,
[0052] Precursor solutions were prepared separately. 40% PF-127 is prepared by adding 20g of PF-127 in 50ml milli-Q water. 10%PVA was prepared by dissolving 1g in 10ml and 35% BSA was prepared by adding 3.5g in 10ml. All the precursor solutions were prepared and stored in 4°C until further use. For the formation of Nano-transformable gel, 3ml of 40%PF-127 was taken and stirred at 1000 rpm in ice-cold bath condition for at least a minute. Then 600µL of 10% PVA was added and stirred for a minute, followed by which 300ul of Iohexol was also added and stirring was allowed for 2 minutes. Further, the drugs were added and finally, 300 µL of BSA solution was added and stirring was kept for 5 minutes.

Example 3: Stability analysis of Thermosensitive gel:
[0053] Fixation of the percentage of addition of iohexol by checking the stability of the gel under irreversible conditions (Group-1 is kept at 60°C for 5 minutes and Group-2 is kept at 60°C for 10 minutes). The drug loaded gel(0.5ml) was taken and kept in refrigerator (for achieving 4°C) and in water bath (for reaching 37°C and 60°C).The gel was stable and stayed in place and is not influenced by gravity which showed that shear force is requied for its derangement. (figure 2)
[0054] Nanotransformable gel containing nile red(1mg/mL)- 9% Iohexol shows the reversibility at 4°C and irreversibility at 60°C.

Example 4: Inversion Test of thermosensitive gel:
[0055] Inversion tests were performed to analyse the thermoresponsive nature of thermosensitive gel. The coloring agent Nile red is added to visualise the sol gel nature of the thermosensitive gel.

[0056] At low temperature (at 4°C), the thermosensitive gel shows viscous form and at high temperatures (at 60°C), the thermosensitive gel shows sol form as shown in figure 3.

Example 5: Loading of drugs
IC-50 was determined for doxorubicin and PfHs in 4T-1 breast cancer cell line, It was 3µg for PfHs and 30µg for doxorubicin as shown in figure 5. Further to obtain the synergistic concentrations, different combinations were tried lesser than the IC-50 values and also at their IC-50 values.
Combination 1- 3µg of Dox and 0.25 µg of PfHS
Combination 2- 5µg of Dox and 0.25 µg of PfHS
Combination 3- 9µg of Dox and 0.5 µg of PfHS
Combination 4- 30µg of Dox and 3µg of PfHS
Combination 2 and 3 showed good synergism and Combination 4 showed additive effect according to the Compusyn report (figure 6).
Combination 3 also showed effective killing with 40% viability which was equal to result of Combination 4.
Thus, we can achieve good cytotoxicity at Combination 3 (9µg of Dox and 0.5µg of PfHS) which was much lesser than the IC-50 values.

Example 6: Drug Loading in hydrogel:
The drugs are loaded during the synthesis of gel before the addition of BSA. The drug loaded gels are stored in 4°C until further use. The drug concentration of doxorubicin is 9µg/mL and for PfHS it is 0.5µg/mL. Micro-CT image showing the loaded iohexol and fluorescent nature of the loaded drugs under UV light (figure 4).

Example 7: Synergistic effect of Combination:
The concentration of Dox and PfHS was taken as 9 & 0.5 µg/mL and loaded in the gel system. The combination (Dox+PfHS) showed cell viability(%) around 24.4±0.6 µg/mL while for Dox and PfHS it was 30.1±0.6 µg/mL and 43.6±0.3 µg/mL respectively in MTT assay suggesting that Dox and PfHS combined exhibits synergistic activity in 4T1 cancer cell line (figure 7). Lesser cell death was observed for the drug loaded gels (i.e.,Dox alone and PfHS alone) than the gel with combination(Dox+PfHS) confirming the synergistic activity.
, Claims:WE CLAIM
1. A thermosensitive hydrogel comprising:
a) polyvinyl alcohol,
b) poloxamer 407;
c) bovine serum albumin (BSA);
d) doxorubicin; and
e) Proflavine hemisulfate (PfHS),
wherein the thermosensitve hydrogel is a nano-transformable gel; and
wherein the ratio of doxorubicin and PfHS is in range of 5:1 to 25:1.

2. The hydrogel as claimed in claim 1, further comprises a contrast agent, wherein the contrast agent is selected from the group consisting of Iodine-based contrast agents and Iohexol.
3. The hydrogel as claimed in claim 1, wherein a concentration of the polyvinyl alcohol is in a range from 2-15% v/v.
4. The hydrogel as claimed in claim 1, wherein a concentration of doxorubicin is in a range of 5 to 10 µg
5. The hydrogel as claimed in claim 1, wherein a concentration PfHS is in a range of 0.25 to 1 µg
6. The hydrogel as claimed in claim 1, wherein a concentration of the poloxamer 407
is in a range from 20-50% wt/v.
7. The hydrogel as claimed in claim 1, wherein a concentration of the bovine serum albumin is in a range from 20-40% wt/v.
8. The hydrogel as claimed in claim 2, wherein a concentration of the contrast agent is in a range from 10 to 30% wt/v.
9. A method for preparing the thermosensitive hydrogel as claimed in claim 1, comprising the steps of:
a. preparing poloxamer 407 solution, PVA solution and BSA solution separately in a mili Q water;
b. adding the PVA solution to the poloxamer 407 solution under constant stirring in an ice cold bath condition for at least a minute;
c. adding DOX and PfHS with continuous stirring; and
d. adding bovine serum albumin to the reaction mixture obtained in step (c) under vigorous
stirring for 5 to 10 minutes to obtain a thermosensitive hydrogel with drugs.

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