DESC:FIELD OF INVENTION
The present invention pertains to the field of pharmaceutical compositions. Particularly, the invention
relates to pharmaceutical compositions comprising myosin inhibitor and TGF beta (TGFß). More
particularly, the invention relates to pharmaceutical compositions comprising blebbistatin and TGF
beta-1, kits comprising blebbistatin and TGF beta-1 (TGFß1), systems for delivering the composition
and uses of TGF beta-1 and blebbistatin in preventing and treating cartilage diseases.
BACKGROUND OF THE INVENTION
Cartilage has a very limited regeneration potential. Hence, cartilage diseases and disorders are very
difficult to treat.
The current practices in use such as “Debridement” is complicated and not a regenerative method as it
involves the smoothening of the rough edge of cartilage using instruments and mechanical shaver.
Another technique “Marrow stimulation” makes tiny holes below the damaged cartilage to trigger
cartilage formation, however, suffers from repairment if multiple surgeries and multiple attempts.
Further, mosaicplasty a technique in which the healthy cartilage is taken from one area and moved to
the damaged site. This cannot be done if there is large area of defect and limited to small area of damage.
Another widely accepted technique is Autologous chondrocyte implantation where chondrocytes are
removed from a small piece of cartilage of the patient followed by cell isolation. Few months later, the
laboratory grown cells are implanted into the knee. This technique also required multiple surgeries.
Injection of autologous chondrocyte suspension into the site injury or implantation of tissue engineered
porous scaffolds are the current strategies for regeneration of cartilage in tissue engineering.
Both strategies suffer from limitations. In case of injection of autologous chondrocyte suspension into
the site injury, cells may leak from the site of the graft in the case of load bearing defects.
In the case of implantation of tissue engineered porous scaffolds, architectural mismatch between the
preformed 3D scaffold and the defect site often leads to a lack of proper integration between the neoengineered
construct and the surrounding host cartilage.
There is a long-felt need for alternative approaches for replacing damaged cartilage tissue. From this
point of view, 3D bioprinting has the potential to offer a paradigm shift, as this will deposit cells in
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micrometer precision, in order to develop relatively large cartilage grafts in arbitrary shapes suitable for
the particular defect and in a patient-specific manner.
Other treatments such as administration of oral non-steroidal anti-inflammatory drugs (NSAIDs), intraarticular
injections of corticosteroids, or hyaluronic acid provides only temporary inflammation and
pain relief. Further, the bioactive factors reside in the joint for less than 2 months, necessitating frequent
injections. Lacking available options for long-term repair and/or regeneration, these patients are often
neglected and almost certainly destined for total joint replacement.
Thus, the present invention thus contemplates to overcome the problems of the prior art to solve a
long-standing problem of providing a pharmaceutical composition with improved efficacious effects
for treatment of cartilage disorders. Further, the approach used for development of this invention
would make the treatment of cartilage disorders more accessible and affordable.
OBJECTS OF THE INVENTION
An important object of the invention is to provide a pharmaceutical composition comprising myosin
inhibitor and TGF beta.
Another important object of the invention is to provide a system for delivering pharmaceutical
composition at the site of action.
Yet another important object of the invention is to provide a kit comprising blebbistatin and TGF beta-
1 for treatment of cartilage diseases.
Another important objection of the invention is to provide a pharmaceutical composition comprising
myosin inhibitor and TGF beta along with or without cells such as chondrocytes or mesenchymal stem
cells.
SUMMARY OF THE INVENTION
Technical Problem
The technical problem to be solved in this invention is to provide a pharmaceutical composition and kits
comprising pharmaceutical compositions which provides treatment for cartilage diseases including the
degeneration of cartilage and cartilage injury.
Solution to the problem
The problem has been solved by the development of compositions and kits comprising myosin
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inhibitors and TGF beta. In particular, blebbistatin and TGF beta-1 have been used for preparing the
pharmaceutical composition for the treatment of cartilage diseases. Further, compositions comprising
blebbistatin and TGF beta-1 have been developed for treatment of cartilage diseases. The invention also
provides kits comprising blebbistatin and TGF beta-1 for treatment of cartilage diseases.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts hematoxylin and eosin staining of cartilages after administration of only TGF Beta,
and TGF Beta along with Blebbistatin.
Figure 2 depicts safranin-O staining of cartilage after administration of only TGF Beta, and TGF Beta
along with Blebbistatin.
Figure 3 depicts the gene expression profile of different genes expressed by chondrocytes in the
cartilage.
Figure 4 depicts prepared UCMSC pellets for experiments.
Figure 5 depicts Real-Time Polymerase Chain Reaction (RT-PCR) study to analyse the key genes in
chondrogenesis.
Figure 6 depicts the Alcian Blue, a cationic dye-bind to sulphated glycosaminoglycans (sGAGs) and
glycoproteins (GP), a major extracellular matrix of cartilage and stain with intense blue colour.
Figure 7 depicts the immunofluorescent staining of Collagen II, another major extracellular matrix protein
present in cartilage.
Figure 8 depicts the effect of the pharmaceutical composition in in-vivo model.
DETAILED DESCRIPTION OF THE INVENTION
At the very outset, it may be understood that the ensuing description only illustrates a particular form of
this invention. However, such a particular form is only an exemplary embodiment, without intending to
imply any limitation on the scope of this invention. Accordingly, the description and examples are to be
understood as exemplary embodiments for teaching the invention and not intended to be taken
restrictively.
The details of one or more embodiments of the invention are set forth in the accompanying description
below including specific details of the best mode contemplated by the inventors for carrying out the
invention, by way of example. It will be apparent to one skilled in the art that the present invention may
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be practiced without limitation to these specific details.
Abbreviations
ACAN: Aggrecan
BLB: Blebbistatin
BSA: Bovine Serum Albumin
cDNA: complementary DNA
CIM: Chondrogenic induction medium
LAP: Lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate
PBS: Phosphate Buffered Saline
PEGDA: Polyethylene Glycol Diacrylate
ROCK inhibitor: Rho-kinase inhibitors
RT-PCR: Real-Time Polymerase Chain Reaction
RT-qPCR: quantitative reverse transcription Real-Time Polymerase Chain Reaction
SOX9: SRY-Box Transcription Factor 9
sGAGs: sulphated Glycosaminoglycans
TGF ß1: Transforming Growth Factor-1
UMSC: Umbilical Mesenchymal Stem Cells
Definitions:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which the methods belong. Although any
compositions and methods similar or equivalent to those described herein can also be used in the
practice or testing of the methods and compositions, representative illustrative methods and
compositions are now described.
Where a range of values is provided, it is understood that each intervening value between the upper and
lower limit of that range and any other stated or intervening value in that stated range, is encompassed
within by the methods and compositions. The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also encompassed within by the methods and
compositions, subject to any specifically excluded limit in the stated range. Where the stated range
includes one or both of the limits, ranges excluding either or both of those included limits are also
included in the methods and compositions.
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It is appreciated that certain features of the methods, which are, for clarity, described in the context of
separate embodiments, may also be provided in combination in a single embodiment. Conversely,
various features of the methods and compositions, which are, for brevity, described in the context of a
single embodiment, may also be provided separately or in any suitable sub-combination. It is noted that,
as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude
any optional element. As such, this statement is intended to serve as antecedent basis for use of such
exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements
or use of a "negative" limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual
embodiments described and illustrated herein has discrete components and features which may be
readily separated from or combined with the features of any of the other embodiments without departing
from the scope or spirit of the present methods. Any recited method can be carried out in the order of
events recited or in any other order that is logically possible.
The term “pharmaceutical composition” as used herein refers to a pharmaceutical concoction or
formulation comprising a myosin inhibitor and TGF beta. Preferably, the myosin inhibitor is Blebbistatin
and TGF beta is TGF beta-1.
Blebbistatin also known with a chemical name 1-Phenyl-1,2,3,4-tetrahydro--hydroxpyrrolo[2,3-b]-7-
methylquinolin-4-one, is cell permeable, specific inhibitor of non-muscle myosin II (ROCK inhibitor).
ROCK inhibitors regulate cell shape and migration by promoting cellular contraction and also ROCK
increases the activity of the motor protein MYOSIN II by two different mechanisms, Firstly,
phosphorylation of the myosin light chain (MLC) increases the myosin II ATPase activity. Thus, several
bundled and active myosins, which are asynchronously active on several actin filaments, move actin
filaments against each other. Secondly, ROCK inactivates MLC phosphatase, leading to increased levels
of phosphorylated MLC. Thus, in both cases, ROCK activation by Rho induces the formation of
actin stress fibers, actin filament bundles of opposing polarity, containing myosin II, tropomyosin,
caldesmon and MLC-kinase, and consequently of focal contacts, which are integrin-based adhesion
points with the extracellular substrate.
The TGF ß -1 is a polypeptide member of the transforming growth factor beta superfamily of cytokines.
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It tightly regulates the rate of chondrocyte maturation through the interactions of Smad-mediated
signaling, the Wnt signaling pathway and plays essential roles in nearly every aspect of cartilage
formation and maintenance including extracellular matrix formation.
The term “delivery system” refers to a system capable of delivering compositions to a treatment site in
an individual. Preferably, the compositions can be delivered using an injectable syringe or deposited over
the cartilage.
The term “subject” or “patient” refers to an animal which can be treated by kits and compositions of the
invention. The “subject” may have, be at risk for, or be believed to have or be at risk for a disease or
condition that can be treated by compositions, kits and/or methods of the present invention. Subjects that
can be treated in accordance with the invention include vertebrates, with mammals such as bovine, canine,
equine, feline, ovine, porcine, and primate (including humans and non-human primates) animals being
particularly preferred examples.
The term “pharmaceutically acceptable carrier” refers to refers to adjuvant, excipient, sweetening agent,
diluent, preservative, dye/colorant, flavor enhancer, wetting agent, dispersing agent, suspending agent,
stabilizer, isotonic agent, solvent, or emulsifier excipients, disintegrants, precipitation inhibitors,
surfactants, glidants, binders, lubricants, and other additives and vehicles with which the compound is
administered.
The term “pharmaceutically acceptable carrier” as used herein refers to the acceptance or use of the carrier
in the pharmaceutical industry. Preferably the carrier is approved by the Federal Drug Administration
(FDA) for use in humans. Exemplary carriers include physiological solutions including but not limited
to liposomes, polymeric micelles, microspheres, nanoparticles, anhydrous lactose, lactose monohydrate,
glucose, dextrose, normal saline, phosphate buffered saline (PBS), bovine serum albumin (BSA), sorbitol,
xylitol, mannitol etc.
The present invention discloses compositions comprising TGF beta and a myosin inhibitor. The present
disclosure also provides kits comprising pharmaceutical composition, and the delivery system along with
an instruction manual for generating matrix of chondrocytes. The present disclosure provides
composition, method of producing the composition, methods of treatment and using the composition to
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treat cartilage diseases.
The inventors have found that compositions comprising Blebbistatin alone or along with TGF beta have
a synergistic effect on the therapeutic efficacy in treatment of a diseases or disorder of the cartilage.
Further, the inventors have contemplated a unique approach in preparation of kits comprising
Blebbistatin along with TGF beta which can be administered together for treatment of cartilage
disorders.
The compositions, kits and methods of the present invention are highly efficacious and represents
improved therapeutic approaches as compared to the prior art.
Before the compositions and methods of the present disclosure are described in greater detail, it is to be
understood that the invention is not limited to particular embodiments and may vary. It is also to be
understood that the terminology used herein is for the purpose of describing particular embodiments only,
and is not intended to be limiting, since the scope of the compositions and methods will be limited only
by the appended claims.
Pharmaceutical composition and method of preparing pharmaceutical compositions
The present disclosure provides methods of preparing pharmaceutical compositions. The methods
involve preparing a composition comprising TGF beta and a myosin inhibitor. Preferably, the myosin
inhibitor is Blebbistatin and TGF beta-1 from the TGF superfamily.
In one embodiment, the composition is free of any contaminants.
Systems for delivery of cell compositions
The present disclosure provides a system for delivering the pharmaceutical compositions to a site
of action in an individual.
A subject system comprises a delivery system that includes an injectable material. The injectable material
comprises composition comprising TGF beta-1 and a myosin inhibitor, such as Blebbistatin. The
injectable material may further comprise one or more pharmaceutically acceptable carriers or excipients.
A suitable delivery system can include two syringes, each holding a composition to be admixed. The
subject system for delivering the composition to a treatment site in an individual comprises:
a. a first delivery system comprising a myosin inhibitor such as Blebbistatin; and
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b. a second delivery system comprising TGF beta-1 in combination
A suitable delivery system can include a syringe, a syringe and a needle, a syringe and a flexible tubing,
capillary cell delivery system, cannula cell delivery systems and the like. A syringe can include a single
chamber, or two or more chambers. Preferably, the delivery system includes two or more syringes or a
delivery system with two chambers.
Treatment of cartilage disorders and diseases
In one embodiment, the invention provides a method of treating a disease or disorder of cartilage,
comprising the step of administering the compositions to a treatment site in an individual.
In another embodiment, the disease or disorder of the cartilage is treated by the use of blebbistatin
and other myosin inhibitors with or without TGF beta-1 to treat cartilage related diseases either in
injectable form or any other form of medication.
In another embodiment, the method of treating a disease or disorder using the composition is
supplemented with tissue engineering or any other form of tissue replacement.
In an embodiment, the present invention provides a pharmaceutical composition comprising, blebbistatin,
a growth factor TGF beta-1, along with one or more pharmaceutically acceptable carriers or excipients.
In an embodiment, the present invention provides a pharmaceutical composition wherein the
concentration of blebbistatin is in the range of 1-30000 ng/ ml and of TGF beta-1 is in the range of 1-20
ng /ml.
In another embodiment, the present invention provides a pharmaceutical composition, wherein the
pharmaceutically acceptable carriers and excipients are selected from but not limited to liposomes,
polymeric micelles, microspheres, nanoparticles, normal saline, anhydrous lactose, lactose monohydrate,
sorbitol, xylitol, mannitol, phosphate buffered saline, bovine serum albumin.
In another embodiment, the present invention provides a pharmaceutical composition, wherein the said
composition induces matrix production by chondrocytes and mesenchymal stem cells.
In another embodiment, the present invention provides a method, wherein the matrix production involves
the steps of:
a. Culturing cells selected from mesenchymal stem cells, chondrocytes in chondrogenic
induction media;
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b. Exposing the cultured cells obtained in step (a) to the composition as claimed in claim 1; and
c. Obtaining a matrix of chondrocytes.
In another embodiment, the present invention provides a pharmaceutical composition, as and when used
in the preparation of a medicament for the treatment of cartilage diseases including the degeneration of
cartilage and cartilage injury.
In another embodiment, the present invention provides a pharmaceutical composition, wherein the mode
of delivery of the said composition is selected from but not limited to epidural, intra-articular, intrathecal
route.
In another embodiment, the present invention provides a system, wherein the delivery system is selected
from a syringe, a syringe and a needle, a syringe and a flexible tubing, capillary cell delivery system,
cannula cell delivery systems, a patch and the like.
In an embodiment, the present invention provides a kit comprising the pharmaceutical composition and
the delivery system, along with an instruction manual for treatment of cartilage diseases including the
degeneration of cartilage and cartilage injury.
The following examples particularly describe the manner in which the invention is to be performed. But
the embodiments disclosed herein do not limit the scope of the invention in any manner.
EXAMPLES
Example 1: Preparation of pharmaceutical composition
The first component of the pharmaceutical composition is blebbistatin, a myosin inhibitor. The stock is
prepared where 1 mg of blebbistatin is dissolved in 500 µL of DMSO (2 mg/ml) and is stored in -20°C.
The obtained solution is further aliquoted into working volumes to avoid repeated freeze-thaw cycles. The
final concentration (working concentration) used for the study was 20000ng/ml (0.02 mg/ml).
The second component of the pharmaceutical composition is TGF beta-1. The stock is prepared where 20
µg of TGB beta-1 is dissolved in 1 mL of sterile distilled water (20 µg /ml). The final concentration
(working concentration) used for the study was 10 ng/ml.
Further, the first component blebbistatin, a myosin inhibitor, the second component growth factor TGF
beta-1, along with one or more active ingredients or excipients, wherein blebbistatin and TGF beta-1 are
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added one after another (no sequence need to be followed) to the cell culture medium at a concentration
given above, i.e., 20000 ng/ml of blebbistatin and 10 ng/ ml of TGF beta-1.
Example 2: Culturing UCMS in the formulated pharmaceutical composition
1.1 Umbilical cord Mesenchymal Stem Cell (UMSC) pellet preparation:
Around 0.03×106 UMSCs were centrifuged in 1.5 ml microfuge tube at 1200 RPM for 10 min
resulting in pellet formation.
1.2 Chondrogenic induction medium (CIM) composition:
A final concentration of 50 µg/mL L-Ascorbic-2-phosphate, 6.25 µg mL of Insulin Transferrin
Selenium-E was taken for each. The concentration of Transforming Growth Factor-1 (TGF ß1) was
10 ng/ mL and the concentration of Blebbistatin taken was 0.02 mg/ml.
1.3 Inducing stem cells for matrix production
The experiment was divided into four groups as illustrated in Figure 4:
? Group 1: Control sample, which consisted only of umbilical cord-derived mesenchymal stem cells.
? Group 2: Comprised of chondrogenic induction media to provide the in vivo cartilage environment
along with TGF beta-1, a commonly used growth factor to regenerate cartilage.
? Group 3: Comprised of chondrogenic induction media with Blebbistatin, a myosin inhibitor (smallmolecule
inhibitor of myosin II).
? Group 4: Comprised of chondrogenic induction media with TGF beta-1 and Blebbistatin, myosin
inhibitors (small-molecule inhibitor of myosin II).
Example 3: Efficacy of pharmaceutical compositions
The inventors observed that blebbistatin in combination with Transforming growth factor -1 (TGF
beta-1) can induce matrix production by UCMS.
The application potential of the pharmaceutical composition to treat any cartilage defect or degenerative
diseases was proved by analytical studies.
3.1 Alcian Blue Staining:
UMSC pellet was subjected to cryosectioning up to 5-micron thickness using a cryotome. The sections
were collected onto poly-l-lysine precoated slides. Alcian blue (Himedia) staining was performed on the
sections. Alcian blue staining was done by incubating the sections in 1% Alcian blue solution for 30
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minutes. After that, the stain was removed by rinsing with tap water and then dehydrated. The stained
sections were observed under light microscopy (EVOS M 7000 ThermoFisher Scientific, USA).
As illustrated in Figure 6, Group 1 i.e., the control sample, stained less with Alcian blue indicating the
reduced or no synthesis of sGAGs and GP whereas Group 2 showed the increased synthesis of sGAGs
and GP compared to Group 3 but less than Group 4. The Group 4 combination observed to be stained
intensively indicated the role of the combination in the synthesis of more cartilage-specific extracellular
matrices.
3.2 Immunofluorescence analysis:
The sections were washed with 1× PBS and fixed with 4% paraformaldehyde at 4°C for 10 min. Then,
the sections were permeated with 0.1%TritonX-100 for 15 min. To prevent non-specific staining, the
sections were incubated with 0.1% BSA for 1 hour. Afterwards, sections were incubated with primary
antibody anti-collagen type II (Santa Cruz, USA) at 4°C overnight in a humid chamber. In the next step,
the primary antibody was removed from the sections and washed with 1× PBS. Then the sections were
incubated with Alexa Flour 594 conjugated goat anti-mouse (Elabscience, USA) for 1 hour. Finally, the
sections were stained with DAPI for 5 min and were observed and imaged using (EVOS M 7000
ThermoFisher Scientific, USA) fluorescent microscope.
As illustrated in Figure 7, Group 1 i.e., the Control sample, on day 7 did not synthesis Collagen II as
indicated by the red colour stain, however, on day 14 it was maximum among the time points. However,
Group 2, on day 14 stained and maintained the expression until day 21, whereas Group 3 showed an early
(as early as on day 7) and increased synthesis of collagen II and maintained throughout the experiment
i.e., day 21. Interestingly, in Group 4, the combinations of CIM+TGF+BLB were observed to be the best
among the groups as it synthesised more collagen II at all time points indicating the ability of the combined
formulation to support the differentiation of UCMSCs to chondrocytes, the cartilage cells and thereby
cartilage regeneration.
3.3 Gene expression analysis:
The quantitative reverse transcription (RT-qPCR) analysis was used to examine the expression profile of
standard chondrogenic differentiation markers. For this purpose, the total RNA was extracted from the
UMSC pellets using TRIzol (Ambion, Life Technologies, USA) according to the manufacturer’s
instructions. The complementary DNA (cDNA) was synthesized using the QuantiTect Reverse
Transcription Kit (Qiagen) by employing the pure form of total RNA (1µg) through reverse transcriptase
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reaction. Chondrogenic markers like aggrecan (ACAN) and SRY-Box Transcription Factor 9 (SOX9)
expression were studied using Real-Time PCR Detection System (Bio-Rad Laboratories, USA). GAPDH
was used as a reference gene to normalize the targeted gene expression. The relative gene expression
quantification was determined using 2-?CT and 2-??CT methods and reported as a fold difference
between the test group gene expression and the reference. The list of primers used in this study and their
sequence are listed in Table 1.
Table 1. List of primers for RT-qPCR analysis:
Primer Forward primer Reverse primer
GAPDH 5'-CTCTCTGCTCCTCCTGTTCG-3' 5'-TTTCTCTCCGCCCGTCTTC-3'
SOX9 5'-GAACAAGCCGCACGTCAAG-3' 5'-CTCTCGCTTCAGGTCAGCC-3'
ACAN 5'-CCCAAGACTACCAGTGGATCG-3' 5'-CGTTTGTAGGTGGTGGCTGTG-3'
As illustrated in Figure 5, Group 1 and Group 2, expresses less SOX9, the transcription factor expresses
during the chondrocyte differentiation and cartilage development, whereas Group 3 comprising of
chondrogenic induction media with Blebbistatin, a myosin inhibitor small-molecule inhibitor of myosin
II shows higher gene expression compared to only TGF beta1 but less than Group 4. The RT-PCR results
conclude that the combination of CIM+TGF+BLB provides a better differentiation of UCMSCs towards
chondrocytes.
The inventors observed that a pharmaceutical composition comprising blebbistatin, a nontoxic,
nonhazardous chemical compound and other myosin inhibitors, alone and along with growth factor TGF
beta-1 to treat cartilage related diseases either injectable form or supplemented with tissue engineering
or any other form of tissue replacement.
Example 4: Culturing Chondrocytes in the formulated pharmaceutical composition
4.1 Chondrocyte isolation and culturing:
The knee joints of the goats aged 10-12 months were procured from a local slaughterhouse in Kandi,
Hyderabad. Chondrocytes were isolated from the articular cartilage of goats by enzymatic digestion.
Cartilage was dissected from the knee joint and washed with PBS.
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Further, the cartilage tissue was digested using 0.1% collagenase type II for 24 hour and the resultant
solution passed through a 70 µm filter. The single-cell suspension obtained was plated at a seeding density
of 6000 cells/cm2 and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal
bovine serum, 100 µg/ml penicillin–streptomycin, 50 µg/ml gentamycin, 100 µg/ml amphotericin-B, 5
ng/ml FGF-2and 2 ng/ml TGFß1. After subculture the cells from passage 3 were used for the experiments.
4.2 Inducing chondrocytes/stem cells for matrix production:
The experiment was divided into three groups as illustrated in Figure 1:
? Group 1: Control sample, which consisted only of sub-cultured chondrocytes from passage 3.
? Group 2: Comprised of sub-cultured chondrocytes along with TGF beta-1, a commonly used growth
factor to regenerate cartilage.
? Group 3: Comprised of sub-cultured chondrocytes along with TGF beta-1 and Blebbistatin, myosin
inhibitors (small-molecule inhibitor of myosin II).
4.3 Efficacy studies of pharmaceutical composition:
The inventors observed that blebbistatin in combination with Transforming growth factor -1 (TGF
beta-1) can induce matrix production in chondrocytes. The application potential of the pharmaceutical
composition to treat any cartilage defect or degenerative diseases was proved by histology haematoxylin
and eosin staining (Figure 1), safranin O staining (Figure 2) and gene expression profile by RT PCR
(Figure 3).
4.3.1 Haematoxyllin and Eosin Staining:
As illustrated in Figure 1, Group 2 i.e., the culture of chondrocytes in presence of TGF beta-1 alone
produced more extracellular matrix compared to Group 1 i.e., the Control sample (“only cells”) Group
3 where both TGF beta-1 and Blebbistatin were added showed the highest matrix production among all
the conditions. The blue colour indicates the cells, and the pink/purple indicates the matrix synthesized
by the cells. It can be concluded that the addition of Blebbistatin in TGF beta-1 enhanced the
extracellular matrix in articular chondrocytes.
4.3.2 Safranin-O Staining:
As illustrated in Figure 2, Group 2 i.e., the culture of chondrocytes in presence of TGF beta-1 alone
produced more acidic proteoglycan and glycosaminoglycan, one of the components of cartilage tissue
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extracellular matrix than in condition “only cells” i.e., Group 1, without TGF beta. Further, the
proteoglycan and glycosaminoglycan synthesis in the culture condition where both TGF beta-1 and
Blebbistatin was added, i.e., Group 3 demonstrated the highest among all the conditions as indicated by
the intensity of purple or pink colour.
4.3.3 Gene expression profile of different genes expressed by chondrocytes in the cartilage:
As illustrated in Figure 3, it was observed that among all the conditions, the chondrogenic specific genes
were upregulated in the conditions where TGF beta-1 and blebbistatin were added together, i.e., Group
3. The genes related to extracellular matrix such as Aggrecan and Collagen II were upregutaed in the
TGF beta-1 and blebbistatin added together. The SOX-9, the transcription factor for chondrogenesis is
also expressed higher in the TGF beta-1 and blebbistatin combination. Whereas collagen-1, the over
synthesis of which leads to an inferior cartilage was found to be less less in Group 3 i.e., when TGF
beta-1 and blebbistatin are together. The gene COMP-1 is found to be increased in TGF beta-1 and
blebbistatin combination compared to any other condition facilitates the collagen–collagen interactions
and microfibril formation in the produced matrix by chondrocytes.
Example 5: Effect of the composition in in-vivo model
Rabbits were chosen for further conducting animal studies to determine the composition efficacy. The
rabbits were procured/obtained from Jeeva Life Sciences, Plot No 98, Shanthi Nagar, Uppal,
Hyderabad-500039.
For the experimental set up, four groups were made with three rabbits in each group. Gender was not a
criterion of the chosen rabbits for this study. In each group, the weight of the rabbits was in the range
of 1.8 – 2.5 kg. The hindlimb cartilage of the rabbits was surgically exposed for both the legs. A 3 mm
punch/defect with an approximate depth of 500 micrometer was made on the legs articular cartilage
with 3 mm biopsy punch and thereafter the animals were grouped.
1. In the first group, the defect was filled with 0.5 x 106 MSC cells in 2-3 microliter of 20%
Polyethylene Glycol Diacrylate (PEGDA) with lithium phenyl (2,4,6-trimethylbenzoyl)
phosphinate (LAP)-0.05%, a free radical photo-initiator used to initiate chain polymerization upon
visible blue light (405 nm) exposure for a minute for photo-crosslinking. Later 200 ng of TGF beta-
1 with 20000 ng of Blebbistatin in 20 microlite of 0.1 % BSA (carrier protein) and 200 microliter
of normal saline was injected intra-articularly in the knee once in a week for total of 4 weeks.
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2. In the second group, the defect was filed with 3 microliters of PEGDA with LAP 0.05% along with
200 ng of TGF beta-1, with 20000 ng of Blebbistatin and 1 minute visible blue light exposure. 200
ng of TGF beta-1, with 20000 ng of Blebbistatin in 20 microlite of 0.1 % BSA (Carrier protein) and
200 microliter of normal saline (Vehicle) was injected intra-articularly in the knee once in a week
for total of 4 weeks.
3. In the third group, the defect was created and left without filling the PEGDA or LAP and without
application of any formulation. Later 200 ng of TGF beta-1, with 20000 ng of Blebbistatin in 20
microlite of 0.1 % BSA (carrier protein) and 200 microliter of normal saline was injected intraarticularly
in the knee once in a week for 4 weeks.
4. In the fourth group, which is the positive control group the defect was left untreated without
injection, and the formulation, PEGDA or MSCs.
Results:
On day 30, following observations were made for all the four groups, which is further illustrated in
Figure 8:
1. In the first group, the defect size was reduced by cartilage regeneration. Only moderate hyaline
cartilage formation was observed, and tissue regeneration was not complete.
2. In Group-2, it was observed that mixed hyaline to fibrocartilage was present in the defect area with
a complete defect filling.
3. In Group-3 the repaired tissue was mostly of fibrocartilage along with some hyaline cartilage.
4. In Group-4, from the gross appearance itself it was evident that the defect was not healed and also
the histology shows only the partial filling of the defect. The cell arrangement in the repaired tissues
was irregular and disorganized in all the groups. The tidemark was not visible in any of the groups.
However, the regenerated tissue in groups 2 and 3 did not show any abnormal calcification or blood
vessel formation.
Conclusion:
In the result, the inventors have demonstrated the differentiation of umbilical cord-derived mesenchymal
stem cells that are not differentiated to any cell types are also getting differentiated towards cartilagespecific
cells in the presence of Blebbistatin and TGF beta-1 combination in a short period as early as 7
17
days. Additionally, the inventors have demonstrated increased expression of cartilage-specific
transcription factor SOX 9 and Aggrecan the gene that regulates the synthesis of glycosaminoglycans.
The major cartilage matrix protein collagen II was also synthesized at an increased level in the presence
of Blebbistatin and TGF beta-1 combined formulation.
Further, the goat articular cartilage chondrocytes that are terminally differentiated demonstrated the
increased extracellular matrix production and the upregulation of the cartilage-specific genes in the
presents of Blebbistatin and TGF beta-1 together.
Advantages of the Invention
? Unlike the conventionally available techniques this pharmaceutical composition/formulation can be
injected or can be applied directly at the site with minimal invasive modes such as in one surgical
incision. ,CLAIMS:We claim:
1. A pharmaceutical composition comprising, blebbistatin, a growth factor TGF beta-1,
along with one or more pharmaceutically acceptable carriers or excipients.
2. The pharmaceutical composition as claimed in claim 1, wherein the concentration of
blebbistatin is in the range of 1-30000 ng/ ml and of TGF beta-1 is in the range of 1-
20 ng /ml.
3. The pharmaceutical composition as claimed in claim 1, wherein the pharmaceutically
acceptable carriers and excipients are selected from but not limited to liposomes,
polymeric micelles, microspheres, nanoparticles, normal saline, anhydrous lactose,
lactose monohydrate, sorbitol, xylitol, mannitol, phosphate buffered saline, bovine
serum albumin.
4. The composition as claimed in claim 1, wherein the said composition induces matrix
production by chondrocytes and mesenchymal stem cells.
5. The composition as claimed in claim 1 to 4, wherein the matrix production involves
the steps of:
a. Culturing cells selected from mesenchymal stem cells, chondrocytes in
chondrogenic induction media;
b. Exposing the cultured cells obtained in step (a) to the composition as claimed in
claim 1; and
c. Obtaining a matrix of chondrocytes.
6. The composition as claimed in claim 1, as and when used in the preparation of a
medicament for the treatment of cartilage diseases including the degeneration of
cartilage and cartilage injury.
7. The composition as claimed in claim 1, wherein the mode of delivery of the said
composition is selected from but not limited to epidural, intra-articular, intrathecal
route.
8. A system for delivery of composition as claimed in claim 1, wherein the delivery
system is selected from a syringe, a syringe and a needle, a syringe and a flexible
tubing, capillary cell delivery system, cannula cell delivery systems, a patch and the
like.
9. A kit comprising the composition as claimed in claim 1 and the delivery system as
claimed in claim 8, along with an instruction manual for treatment of cartilage
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diseases including the degeneration of cartilage and cartilage injury.