Title of Invention	A FORMULATION FROM EXTRACT OF INDIAN GREEN MUSSEL (PERNA VIRIDIS) USEFUL FOR INHIBITING OSTEOCLAST FORMATION AND BONE RESORPTION.
Abstract	Present invention provides a formulation from the Indian green mussel (Perna viridis) useful for inhibiting osteoclast formation and bone resorption obtained from fermented meat from green mussel ( Perna viridis) digested with concentrated hydrochloric acid for 15 hours at 100 ° + 2°C , wherein amount of said extract ranges from 30-100 µg unit percent supplemented with an additive, excipient, diluent or carrier such as herein described.

Title of Invention

A FORMULATION FROM EXTRACT OF INDIAN GREEN MUSSEL (PERNA VIRIDIS) USEFUL FOR INHIBITING OSTEOCLAST FORMATION AND BONE RESORPTION.

Abstract

Present invention provides a formulation from the Indian green mussel (Perna viridis) useful for inhibiting osteoclast formation and bone resorption obtained from fermented meat from green mussel ( Perna viridis) digested with concentrated hydrochloric acid for 15 hours at 100 ° + 2°C , wherein amount of said extract ranges from 30-100 µg unit percent supplemented with an additive, excipient, diluent or carrier such as herein described.

Full Text	Field of invention A formulation from the extract of Indian green mussel (Perna viridis) useful for inhibition of osteoclast formation and bone resorption . The present invention relates to an extract from green mussel that inhibits osteoclast formation and bone resorption in vitro. More particularly , the present invention relates to an extract from the Indian green mussel (Perna viridis) that inhibits the osteoclast formation and bone resorption in vitro. According to the present invention , an extract from the Indian green mussel (Perna viridis) inhibits the osteoclast differential and bone resorption activity in mouse hemopoietic osteoclast precursor cells. This extract will be useful for treatment of bone loss due to osteoporosis , rheumatoid arthritis , Paget's disease of bone , and other metabolic bone disorders. Background of invention Bone is a metabolically active and highly organized tissue . Bone contains two distinct cell types , the osteoblasts or bone forming cells and the osteoclast or bone resorbing cells. Bone remodeling is a normal process that involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. These two processes are normally integrated to maintain both the structural integrity of the skeletal system and homeostasis of bone . Both these processes are influenced by a wide variety of systemic and local factors. An imbalance of osteoblast and osteoclast functions can arise from variety of hormonal changes or perturbations of inflammatory and growth factors resulting in skeletal abnormalities characterized by increased bone mass. This may lead to excessive bone loss and eventually fracture. Osteoclasts are specialized monocyte/macrophage family members that differentiate from hemopoietic precursors. Increased osteoclast activity is seen in osteoporosis (Gross et al., 1996), rheumatoid arthritis (Seitz and Hunstein, 1985), Paget's disease (Siris, 1999), and many other diseases of clinical importance. In these disorders bone resorption exceeds bone formation resulting in decreased skeletal mass. This causes bones to become thin, fragile and susceptible to fracture. The consequences of osteoporotic bone fractures include chronic pain in bone, body deformity including height loss and muscle weakness. Osteoporosis is now a serious problem that imposes substantial limitations on the affected individuals. In human, 1 in 3 women and 1 in 12 men over 45 years are at risk of suffering painful and deforming fractures as a result of osteoporosis. More women die after hip fractures than from cancers of ovaries, cervix and uterus. Osteoporosis occurs at a relatively earlier age in Indian males and females compared to western countries (Gupta, 1996). Most bone diseases occur due to increased osteoclasts activity and increased bone resorption. Bone resorption and loss of calcium from bone are complications associated with arthritis, many cancers and with bone metastases of breast and prostate tumors. Because of lack of research into osteoporosis and related diseases, we don't know all the answers to treat these diseases. Progress in better understanding the pathogenesis and successful treatment of these diseases to date has targeted osteoclast. Drugs that inhibit the formation or activity of osteoclasts are valuable for treating these diseases. A variety of disadvantages are associated with current therapeutic agents (such as estrogen and selective estrogen receptor modulators, bisphosphonates and calcitonin) used in osteoporosis and other metabolic bone disorders (Rodan and Martin, 2000). Reference may be made to a publication wherein drugs developed for treating osteoporosis and related disorders showed adverse events and contraindications (Watts, 1999). The side effects of current therapies includes increase in the risk of breast and uterine cancers, upper gastrointestinal distress and induction of immune responses. Natural products from plants and organisms have frequently been used as a source for development of effective drugs. There is an increased interest in analysis of natural products from marine organisms. Sea animals contain metabolites which can be used for treatment of many diseases. Drugs that inhibit the formation or activity of osteoclasts and with no toxicity and harmful side effects will be valuable for treating osteoporosis, Paget's disease, and inflammation of bone associated with rheumatoid arthritis or periodontal disease. In the present invention an attempt has been made to study the effect of extract prepared from the Indian green mussel on osteoclast differentiation and bone resorption in vitro. Extract from mussels was first shown to have the anti-inflammatory activity in rats (Miller and Ormrod, 1980). In these studies mussel preparation from New Zealand green mussel (Perna canaliculus) effectively reduced the rat paw oedema but only if injected into the peritoneal cavity. Freeze-dried extract preparations from mussles have been used as anti-inflammatory treatment (Caughey et al., 1983). Reference may be made to publication wherein some preliminary account on use of mussel hydrolysate obtained from mussel meat by acid hydrolysis showing virus-inhibiting activity against influenza viruses (Bichurina et al., 1994). The extract prepared from the Indian green mussel (Perna viridis) has previously been found to be active against all influenza, herpes and hepatitis viral strains. The extract is also found to possess not only prophylactic efficacy for protection from several viral diseases but it also shows a high therapeutic property against these diseases. The process of preparation of extract was developed for the first time by the Russian scientists. A patent on the process developed by Russian scientists was also filed (Patent No. RU 2043109). The process patent on extraction of mussel hydrolysate has also been filed by National Institute of Oceanography, Goa, India (Patent No. 493/DEL/99). The main objective of the present invention is to evaluate the effect of extract prepared from the green mussel on osteoclast formation and bone resorption in vitro. Another objective of the present invention is to evaluate the effect of extract prepared from the Indian green mussel (Perna viridis) on osteoclast formation and bone resorption in vitro. Yet, another objective of the present invention is to develop an effective extract that is non-toxic to other cells. If the extract is not toxic it would be useful to prepare a drug that can be administered orally rather than parenterally. Summary of the invention Thus, the present invention relates to an extract prepared from the Indian green mussel (Perna viridis) that inhibits the osteoclast formation and bone resorption in murine hemopoietic osteoclast precursor cells. The extract is non-toxic to other cells and useful to prepare a drug that can be administered orally rather than parenterally. This extract will be useful for treatment of bone loss due to osteoporosis, rheumatoid arthritis, Paget's disease of bone, and other metabolic bone disorders. The present invention includes effect of mussel hydrolysate on osteoclastogenesis in murine hemopoietic cells, effect of mussel hydrolysate on tartrate-resistant acid phosphatase (TRAP) activity in osteoclasts, toxicity testing of mussel hydrolysate on other cell types and the effect of mussel hydrolysate on bone resorption. Description of the figures Figure 1: Depicts that mussel hydrolysate inhibits TRAP-positive red osteoclasts. Figure 2: Depicts that mussel hydrolysate dose-dependently inhibits multinuclear TRAP-positive osteoclasts. Figure 3: Depicts that mussel hydrolysate dose-dependently inhibits mononuclear TRAP-positive osteoclasts. Figure 4: Depicts that mussel hydrolysate is non-toxic to other cell types. Figure 5: Depicts that mussel hydrolysate dose-dependently inhibits bone resorption. Accordingly, the invention provides a formulation from the extract of Indian green mussel (Perna viridis) useful for inhibition of osteoclast formation and bone resorption in an animal or a human wherein the said formulation comprising a pharmaceutically acceptable amount of an extract (mussel hydrolysate) from a green mussel wherein amount of said extract ranges from 30-100 µg unit percent supplemented with an additive, excipient, diluent or carrier such as herein described. In an embodiment the invention provides a formulation as claimed in claim 1 useful for inhibition of multinuclear TRAP - positive osteoclasts. In an embodiment the invention provides a formulation as claimed in claim 1 useful for inhibition of multinuclear TRAP- positive osteoclasts. In an embodiment the invention provides a formulation as claimed in claim 1 being non-toxic to other cell types. In an embodiment the invention provides a formulation as claimed in claim 1 useful for inhibition of bone resorption. The invention further provides use of mussel hydrolysate for inhibition of osteoclast formation wherein the pharmaceutically acceptable amount of the extract is administered in an animal or a human optionally with an additive, excipient, diluent or carrier. In an embodiment the invention further provides use of mussel hydrolysate for inhibition of mononuclear TRAP-positive osteoclasts. In an embodiment the invention further provides use of mussel hyrolysate for inhibition of multinuclear TRAP-positive osteoclasts. In an embodiment the invention further provides use of mussel hydrolysate being non-toxic to other cell types. In an embodiment the invention further provides use of mussel hydrolysate for inhibition of bone resorption. The invention further provides the method for inhibition of osteoclast formation in an animal or a human wherein the pharmaceutically acceptable amount of an extract is administered in animal or human optionally with an additive, excipient, diluent or carrier. In an embodiment the invention further provides the method as claimed in claim 1 wherein the extract used for inhibition of mononuclear TRAP-positive osteoclasts. In an embodiment the invention further provides the method as claimed in claim 1 wherein the extract used for inhibition of multinuclear TRAP-positive osteoclasts. In an embodiment the invention further provides the method as claimed in claim 1 wherein the extract being used as non-toxic to other cell types. In an embodiment the invention further provides the method as claimed in claim 1 wherein the extract being used for inhibition of bone resorption. In an embodiment the invention further provides the dose for the method as claimed in 1 for inhibition of TRAP-positive multinuclear cell formation in vitro being in the range of 30 to 100 µg/ml. In an embodiment the invention further provides the dose for the method as claimed in 1 for inhibition of TRAP-positive mononuclear cell formation in vitro being in the range of 30 to 100 µg/ml. In an embodiment the invention further provides the dose for the method as claimed in 1 for inhibition of bone resorption in vitro being in the range of 30 to 100 µg/ml. Detailed description of the invention 1. Extraction of mussel hydrolysate: Samples of live bivalves, green mussel (Perna viridis) were collected directly from the sea. Bivalves were cleaned and deshelled with the help of sharp knife. Meat along with mantle fluid was removed carefully. Fermentation of meat with mantle fluid was done with enzyme protosubtiline (6 % of the weight of meat) and 6 % distilled water at a constant temperature of 40° C for two hours. Digestion of the thick paste was done with concentrated hydrochloric acid (12 % of the total meat weight) for 15 hours at 100° ± 2° C. Cooling of the resultant solution was done at room temperature. The pH of the solution was maintained by adding sodium hydroxide. Active extract was isolated by keeping the resultant solution in a separating flask for 10 days and carefully removing the middle part of the solution (Patent No. 493/DEL/99). An in vitro methods for inhibition of osteoclast formation and bone resorption using mussel hydrolysate wherein said method comprising following steps. 2. Effect of mussel hydrolysate on in vitro osteoclastogenesis Balb/c mice were killed by cervical dislocation and bone marrow cells were obtained from long bones as previously described (Wani et al., 1999). Osteoclasts were generated from stroma free population of non-adherent, macrophage-colony stimulating factor (M-CSF)-dependent osteoclast precursors from mouse bone marrow cells. These precursors were stimulated with M-CSF and receptor activator of NF-kB ligand (RANKL) to form osteoclasts. Mussel hydrolysate was tested for its effects in these stroma free systems for their ability to modulate differentiation of osteoclast progenitors of the monocyte/macrophage lineage in to osteoclasts. M-CSF-dependent, non-adherent bone marrow cells were harvested, washed twice and resuspended in αMEM containing 10 % foetal bovine serum (FBS). This suspension was added to the wells of 96-well plate containing coverslips. Cells were incubated in the presence or absence of M-CSF, RANKL and/or mussel hydrolysate, at 37° C in a humidified atmosphere of 5 % C02 in air. Cultures were fed twice a week by replacing half medium with equal quantity of fresh medium containing reagents. At varying time intervals, depending upon experimental design, cells on coverslips were prepared for tartrate-resistant acid phosphatase (TRAP) staining. 3. Tartrate-resistant acid phosphatase (TRAP) staining Osteoclast expresses Tartrate-resistant acid phosphatase (TRAP) activity. It is a marker enzyme of osteoclast. Cytochemical staining for TRAP is widely used for identifying the osteoclasts in vivo and in vitro. It is claimed to be specific for osteoclasts in bone. Staining for TRAP was carried out using naphthol AS-BI phosphate as a substrate and pararosaniline chloride as a stain for the reaction product in the presence of sodium tartrate (Fuller et al., 2000). TRAP positive osteoclast appears as red cells. 4. Bone resorption assay Osteoclast has the ability to excavate authentic resorption lacunae in vivo and in vitro. Bone resorption is the unique function of the osteoclast and is therefore the most useful means of distinguishing it from other cell types. M-CSF-dependent, non-adherent bone marrow cells were incubated for 10 days on bovine cortical bone slices in the presence of M-CSF, RANKL with or without mussel hydrolysate. Bone slices were examined for resorption pits by reflected light microscopy as previously described (Wani et al. 1999). The following examples are given by way of illustration of the present invention and therefore, should not be construed to limit the scope of the present invention. The following experiments were conducted several times with reproducible results. Assessment of inhibition of osteoclast formation and bone resorption in vitro using mussel hydrolysate In the present study, the effect of mussel hydrolysate was examined first on osteoclast differentiation from mouse bone marrow cells. Stromal cells free M-CSF-dependent osteoclast precursors were isolated from bone marrow cells. Absence of contaminating stromal cells was confirmed in cultures in which M-CSF was omitted. Osteoclasts induced by RANKL were characterized for the presence of tartrate-resistant acid phosphatase (TRAP) activity, a marker of osteoclasts. Mussel hydrolysate inhibits osteoclast formation In the presence of M-CSF (30 ng/ml) alone, only macrophages are formed (fig. 1-A). Large number of TRAP-positive osteoclasts, both mononuclear and multinuclear were formed in these cultures when both M-CSF and RANKL (30 ng/ml) were added (fig 1-B). Addition of the mussel hydrolysate dose-dependently inhibited osteoclast formation induced by RANKL (fig. 1-C & D). Mussel hydrolysate (30 and 100 ug/ml) significantly inhibited both multinuclear (with 3 or more nuclei) (fig. 2) and mononuclear (fig. 3) TRAP-positive osteoclasts. Study of mussel hydrolysate for toxicity to other cell types Effect of mussel hydrolysate was also tested for toxicity to other cell types. It was further noted that the mussel hyrdolysate was non-toxic to other cell types present in the culture and inhibited only osteoclasts. There was no toxic effect of the mussel hydrolysate on total cell number in all the cultures (fig. 4). Mussel hydrolysate inhibits bone resorption M-CSF-dependent osteoclast precursors were also incubated on bovine cortical bone slices in the presence of M-CSF and RANKL. Bone resorption was induced by RANKL. It was observed that mussel hydrolysate dose-dependently inhibited bone resorption (fig. 5). The main advantages of the present invention are: 1) That the mussel hydrolysate from the Indian green mussel (Perna viridis) inhibits osteoclast formation from hemopoietic osteoclast precursors in mice. 2) That the mussel hydrolysate from the Indian green mussel (Perna viridis) inhibits bone resorption from hemopoietic osteoclast precursors in mice. 3) That the mussel hydrolysate is non-toxic to other cells and will be useful to prepare a drug that can be administered orally rather than parenterally. 4) This invention provides a novel extract that will be useful for the treatment of bone loss in osteoporosis, rheumatoid arthritis, Paget's disease of bone and other metabolic diseases of clinical importance. References Bichurina MA, Nikitina LE, Sovetova MG, Rekhina Nl, Besedina TV, Boikov IA & Noskov FS (1994). The virus-inhibiting activity of a preparation obtained from a mussel hydrolysate. Vopr Virsol 3,134-136. Caughey DE, Grigor RR, Caughey EB, Young P Gow PJ & Stewart AW (1983). Perna canaliculus in the treatment of rheumatoid arthritis. Eur J Rheumatol Inflamm 6, 197-200. Fuller K, Lean JM, Wani MR & Chambers TJ (2000): A role for TGF in osteoclast differentiation and activation. J Cell Sci 113, 2445-2453. Gross C, Eccleshall TR & Feldman D (1996). Vitamin D receptor gene alleles and osteoporosis. In Principles of Bone Biology vol. pp. 917-934. Ed JP Bilezikian, LG Raisz, & GA Rodan. San Diego: Academic Press. Gupta A (1996). Osteoporosis in India-The nutritional hypothesis. Natl Med J India 9, 268-274. Miller TE & Ormrod D (1980). The anti-inflammtory activity of Perna canaliculus (NZ green mussel). N Z Med J 92, 187-193 Rodan GA & Martin TJ (2000). Therapeutic approaches to bone diseases. Science 289, 1508-1514 Seitz M, & Hunstein W (1985). Enhanced prostanoid release from monocytes of patients with rheumatoid arthritis and active systemic lupus erythematosus. Ann Rheum Dis 44,438-445. Siris ES (1999). Paget's disease of bone. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism vol. pp. 415-425. Ed MJ Favus. Philadelphia: Lippincott Williams and Wilkins. Wani MR, Fuller K, Kim NS, Choi Y & Chambers T (1999): Prostaglandin £2 co-operates with TRANCE in osteoclast induction from hemopoietic precursors: Synergistic activation of differentiation, cell spreading and fusion. Endocrinology 140, 1927-1935 Watts, N. B (1999). Pharmacology of agents to treat osteoporosis. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism pp. 278-283 Ed MJ Favus. Philadelphia: Lippincott Williams and Wilkins. We Claim: 1. A formulation from extract of Indian green mussel (Perna viridis) useful for inhibition of osteoclast formation and bone resorption in an animal or a human wherein the said formulation comprising a pharmaceutically acceptable amount of an extract (mussel hydrolysate) from a green mussel wherein amount of said extract ranges from 30-100 µg unit percent supplemented with an additive, excipient, diluent or carrier such as herein described. 2. A formulation from extract of Indian green mussel (Perna viridis) useful for inhibiting osteoclast formation and bone resorption substantially as herein described with reference to the examples and drawing accompanying the specification.

Full Text

Field of invention
A formulation from the extract of Indian green mussel (Perna viridis) useful for
inhibition of osteoclast formation and bone resorption .
The present invention relates to an extract from green mussel that inhibits osteoclast
formation and bone resorption in vitro.
More particularly , the present invention relates to an extract from the Indian green
mussel (Perna viridis) that inhibits the osteoclast formation and bone resorption in
vitro.
According to the present invention , an extract from the Indian green mussel (Perna

viridis) inhibits the osteoclast differential and bone resorption activity in mouse hemopoietic osteoclast precursor cells. This extract will be useful for treatment of bone loss due to osteoporosis , rheumatoid arthritis , Paget's disease of bone , and other metabolic bone disorders.
Background of invention
Bone is a metabolically active and highly organized tissue . Bone contains two distinct cell types , the osteoblasts or bone forming cells and the osteoclast or bone resorbing cells. Bone remodeling is a normal process that involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. These two processes are normally integrated to maintain both the structural integrity of the skeletal system and homeostasis of bone . Both these processes are influenced by a wide variety of systemic and local factors. An imbalance of osteoblast and osteoclast functions can arise from variety of hormonal changes or perturbations of inflammatory and growth factors resulting in skeletal abnormalities characterized by increased bone mass. This may lead to excessive bone loss and eventually fracture.
Osteoclasts are specialized monocyte/macrophage family members that differentiate from hemopoietic precursors. Increased osteoclast activity is seen in osteoporosis (Gross et al., 1996), rheumatoid arthritis (Seitz and Hunstein, 1985), Paget's disease (Siris, 1999), and many other diseases of clinical importance. In these disorders bone resorption exceeds bone formation resulting in decreased skeletal mass. This causes bones to become thin, fragile and susceptible to fracture. The consequences of osteoporotic bone fractures include chronic pain in bone, body deformity including height loss and muscle weakness.
Osteoporosis is now a serious problem that imposes substantial limitations on the affected individuals. In human, 1 in 3 women and 1 in 12 men over 45 years are at risk of suffering painful and deforming fractures as a result of osteoporosis. More women die after hip fractures than from cancers of ovaries, cervix and uterus. Osteoporosis occurs at a relatively earlier age in Indian males and females compared to western countries (Gupta, 1996).
Most bone diseases occur due to increased osteoclasts activity and increased bone resorption. Bone resorption and loss of calcium from bone are complications associated with arthritis, many cancers and with bone metastases of breast and prostate tumors. Because of lack of research into osteoporosis and related diseases, we don't know all the answers to treat these diseases. Progress in better understanding the pathogenesis and successful treatment of these diseases to date has targeted osteoclast.
Drugs that inhibit the formation or activity of osteoclasts are valuable for treating these diseases. A variety of disadvantages are associated with current therapeutic agents (such as estrogen and selective estrogen receptor modulators, bisphosphonates and calcitonin) used in osteoporosis and other metabolic bone disorders (Rodan and Martin, 2000). Reference
may be made to a publication wherein drugs developed for treating osteoporosis and related disorders showed adverse events and contraindications (Watts, 1999). The side effects of current therapies includes increase in the risk of breast and uterine cancers, upper gastrointestinal distress and induction of immune responses.
Natural products from plants and organisms have frequently been used as a source for development of effective drugs. There is an increased interest in analysis of natural products from marine organisms. Sea animals contain metabolites which can be used for treatment of many diseases.
Drugs that inhibit the formation or activity of osteoclasts and with no toxicity and harmful side effects will be valuable for treating osteoporosis, Paget's disease, and inflammation of bone associated with rheumatoid arthritis or periodontal disease.
In the present invention an attempt has been made to study the effect of extract prepared from the Indian green mussel on osteoclast differentiation and bone resorption in vitro.
Extract from mussels was first shown to have the anti-inflammatory activity in rats (Miller and Ormrod, 1980). In these studies mussel preparation from New Zealand green mussel (Perna canaliculus) effectively reduced the rat paw oedema but only if injected into the peritoneal cavity. Freeze-dried extract preparations from mussles have been used as anti-inflammatory treatment (Caughey et al., 1983). Reference may be made to publication wherein some preliminary account on use of mussel hydrolysate obtained from mussel meat by acid hydrolysis showing virus-inhibiting activity against influenza viruses (Bichurina et al., 1994).

The extract prepared from the Indian green mussel (Perna viridis) has previously been found to be active against all influenza, herpes and hepatitis viral strains. The extract is also found to possess not only prophylactic efficacy for protection from several viral diseases but it also shows a high therapeutic property against these diseases. The process of preparation of extract was developed for the first time by the Russian scientists. A patent on the process developed by Russian scientists was also filed (Patent No. RU 2043109). The process patent on extraction of mussel hydrolysate has also been filed by National Institute of Oceanography, Goa, India (Patent No. 493/DEL/99).
The main objective of the present invention is to evaluate the effect of extract prepared from the green mussel on osteoclast formation and bone resorption in vitro.
Another objective of the present invention is to evaluate the effect of extract prepared from the Indian green mussel (Perna viridis) on osteoclast formation and bone resorption in vitro.
Yet, another objective of the present invention is to develop an effective extract that is non-toxic to other cells. If the extract is not toxic it would be useful to prepare a drug that can be administered orally rather than parenterally.
Summary of the invention
Thus, the present invention relates to an extract prepared from the Indian green mussel (Perna viridis) that inhibits the osteoclast formation and bone resorption in murine hemopoietic osteoclast precursor cells. The extract is non-toxic to other cells and useful to prepare a drug that can be
administered orally rather than parenterally.
This extract will be useful for treatment of bone loss due to osteoporosis, rheumatoid arthritis, Paget's disease of bone, and other metabolic bone disorders.
The present invention includes effect of mussel hydrolysate on osteoclastogenesis in murine hemopoietic cells, effect of mussel hydrolysate on tartrate-resistant acid phosphatase (TRAP) activity in osteoclasts, toxicity testing of mussel hydrolysate on other cell types and the effect of mussel hydrolysate on bone resorption.
Description of the figures
Figure 1: Depicts that mussel hydrolysate inhibits TRAP-positive red osteoclasts.
Figure 2: Depicts that mussel hydrolysate dose-dependently inhibits multinuclear TRAP-positive osteoclasts.
Figure 3: Depicts that mussel hydrolysate dose-dependently inhibits mononuclear TRAP-positive osteoclasts.
Figure 4: Depicts that mussel hydrolysate is non-toxic to other cell types.
Figure 5: Depicts that mussel hydrolysate dose-dependently inhibits bone resorption.
Accordingly, the invention provides a formulation from the extract of Indian green mussel
(Perna viridis) useful for inhibition of osteoclast formation and bone resorption in an animal or
a human wherein the said formulation comprising a pharmaceutically acceptable amount of an
extract (mussel hydrolysate) from a green mussel wherein amount of said extract ranges from
30-100 µg unit percent supplemented with an additive, excipient, diluent or carrier such as
herein described.
In an embodiment the invention provides a formulation as claimed in claim 1 useful for
inhibition of multinuclear TRAP - positive osteoclasts.
In an embodiment the invention provides a formulation as claimed in claim 1 useful for
inhibition of multinuclear TRAP- positive osteoclasts.
In an embodiment the invention provides a formulation as claimed in claim 1 being non-toxic to
other cell types.
In an embodiment the invention provides a formulation as claimed in claim 1 useful for
inhibition of bone resorption.
The invention further provides use of mussel hydrolysate for inhibition of osteoclast formation
wherein the pharmaceutically acceptable amount of the extract is administered in an animal or
a human optionally with an additive, excipient, diluent or carrier.
In an embodiment the invention further provides use of mussel hydrolysate for inhibition of
mononuclear TRAP-positive osteoclasts.
In an embodiment the invention further provides use of mussel hyrolysate for inhibition of
multinuclear TRAP-positive osteoclasts.
In an embodiment the invention further provides use of mussel hydrolysate
being non-toxic to other cell types.
In an embodiment the invention further provides use of mussel hydrolysate for inhibition of bone resorption.
The invention further provides the method for inhibition of osteoclast formation in an animal or a human wherein the pharmaceutically acceptable amount of an extract is administered in animal or human optionally with an additive, excipient, diluent or carrier.
In an embodiment the invention further provides the method as claimed in claim 1 wherein the extract used for inhibition of mononuclear TRAP-positive osteoclasts.
In an embodiment the invention further provides the method as claimed in claim 1 wherein the extract used for inhibition of multinuclear TRAP-positive osteoclasts.
In an embodiment the invention further provides the method as claimed in claim 1 wherein the extract being used as non-toxic to other cell types.
In an embodiment the invention further provides the method as claimed in claim 1 wherein the extract being used for inhibition of bone resorption.
In an embodiment the invention further provides the dose for the method as claimed in 1 for inhibition of TRAP-positive multinuclear cell formation in vitro being in the range of 30 to 100 µg/ml.
In an embodiment the invention further provides the dose for the method as claimed in 1 for inhibition of TRAP-positive mononuclear cell formation in
vitro being in the range of 30 to 100 µg/ml.
In an embodiment the invention further provides the dose for the method as claimed in 1 for inhibition of bone resorption in vitro being in the range of 30 to 100 µg/ml.
Detailed description of the invention
1. Extraction of mussel hydrolysate:
Samples of live bivalves, green mussel (Perna viridis) were collected directly from the sea. Bivalves were cleaned and deshelled with the help of sharp knife. Meat along with mantle fluid was removed carefully. Fermentation of meat with mantle fluid was done with enzyme protosubtiline (6 % of the weight of meat) and 6 % distilled water at a constant temperature of 40° C for two hours. Digestion of the thick paste was done with concentrated hydrochloric acid (12 % of the total meat weight) for 15 hours at 100° ± 2° C. Cooling of the resultant solution was done at room temperature. The pH of the solution was maintained by adding sodium hydroxide. Active extract was isolated by keeping the resultant solution in a separating flask for 10 days and carefully removing the middle part of the solution (Patent No. 493/DEL/99).
An in vitro methods for inhibition of osteoclast formation and bone resorption using mussel hydrolysate wherein said method comprising following steps.
2. Effect of mussel hydrolysate on in vitro osteoclastogenesis
Balb/c mice were killed by cervical dislocation and bone marrow cells were obtained from long bones as previously described (Wani et al., 1999). Osteoclasts were generated from stroma free population of non-adherent, macrophage-colony stimulating factor (M-CSF)-dependent osteoclast precursors from mouse bone marrow cells. These precursors were stimulated with M-CSF and receptor activator of NF-kB ligand (RANKL) to form osteoclasts. Mussel hydrolysate was tested for its effects in these
stroma free systems for their ability to modulate differentiation of osteoclast progenitors of the monocyte/macrophage lineage in to osteoclasts.
M-CSF-dependent, non-adherent bone marrow cells were harvested, washed twice and resuspended in αMEM containing 10 % foetal bovine serum (FBS). This suspension was added to the wells of 96-well plate containing coverslips. Cells were incubated in the presence or absence of
M-CSF, RANKL and/or mussel hydrolysate, at 37° C in a humidified atmosphere of 5 % C02 in air. Cultures were fed twice a week by replacing half medium with equal quantity of fresh medium containing reagents. At varying time intervals, depending upon experimental design, cells on coverslips were prepared for tartrate-resistant acid phosphatase (TRAP) staining.
3. Tartrate-resistant acid phosphatase (TRAP) staining
Osteoclast expresses Tartrate-resistant acid phosphatase (TRAP) activity. It is a marker enzyme of osteoclast. Cytochemical staining for TRAP is widely used for identifying the osteoclasts in vivo and in vitro. It is claimed to be specific for osteoclasts in bone.
Staining for TRAP was carried out using naphthol AS-BI phosphate as a substrate and pararosaniline chloride as a stain for the reaction product in the presence of sodium tartrate (Fuller et al., 2000). TRAP positive osteoclast appears as red cells.
4. Bone resorption assay
Osteoclast has the ability to excavate authentic resorption lacunae in vivo
and in vitro. Bone resorption is the unique function of the osteoclast and is therefore the most useful means of distinguishing it from other cell types. M-CSF-dependent, non-adherent bone marrow cells were incubated for 10 days on bovine cortical bone slices in the presence of M-CSF, RANKL with or without mussel hydrolysate. Bone slices were examined for resorption pits by reflected light microscopy as previously described (Wani et al. 1999).
The following examples are given by way of illustration of the present invention and therefore, should not be construed to limit the scope of the present invention. The following experiments were conducted several times with reproducible results.
Assessment of inhibition of osteoclast formation and bone resorption in vitro using mussel hydrolysate
In the present study, the effect of mussel hydrolysate was examined first on osteoclast differentiation from mouse bone marrow cells. Stromal cells free M-CSF-dependent osteoclast precursors were isolated from bone marrow cells. Absence of contaminating stromal cells was confirmed in cultures in which M-CSF was omitted. Osteoclasts induced by RANKL were characterized for the presence of tartrate-resistant acid phosphatase (TRAP) activity, a marker of osteoclasts.
Mussel hydrolysate inhibits osteoclast formation
In the presence of M-CSF (30 ng/ml) alone, only macrophages are formed (fig. 1-A). Large number of TRAP-positive osteoclasts, both mononuclear and multinuclear were formed in these cultures when both M-CSF and RANKL (30 ng/ml) were added (fig 1-B). Addition of the mussel hydrolysate
dose-dependently inhibited osteoclast formation induced by RANKL (fig. 1-C & D). Mussel hydrolysate (30 and 100 ug/ml) significantly inhibited both multinuclear (with 3 or more nuclei) (fig. 2) and mononuclear (fig. 3) TRAP-positive osteoclasts.
Study of mussel hydrolysate for toxicity to other cell types
Effect of mussel hydrolysate was also tested for toxicity to other cell types. It was further noted that the mussel hyrdolysate was non-toxic to other cell types present in the culture and inhibited only osteoclasts. There was no toxic effect of the mussel hydrolysate on total cell number in all the cultures
(fig. 4).
Mussel hydrolysate inhibits bone resorption
M-CSF-dependent osteoclast precursors were also incubated on bovine cortical bone slices in the presence of M-CSF and RANKL. Bone resorption was induced by RANKL. It was observed that mussel hydrolysate dose-dependently inhibited bone resorption (fig. 5).
The main advantages of the present invention are:
1) That the mussel hydrolysate from the Indian green mussel (Perna viridis)
inhibits osteoclast formation from hemopoietic osteoclast precursors in
mice.
2) That the mussel hydrolysate from the Indian green mussel (Perna viridis)
inhibits bone resorption from hemopoietic osteoclast precursors in mice.
3) That the mussel hydrolysate is non-toxic to other cells and will be useful to
prepare a drug that can be administered orally rather than parenterally.
4) This invention provides a novel extract that will be useful for the treatment of bone loss in osteoporosis, rheumatoid arthritis, Paget's disease of bone and other metabolic diseases of clinical importance.
References
Bichurina MA, Nikitina LE, Sovetova MG, Rekhina Nl, Besedina TV, Boikov IA & Noskov FS (1994). The virus-inhibiting activity of a preparation obtained from a mussel hydrolysate. Vopr Virsol 3,134-136.
Caughey DE, Grigor RR, Caughey EB, Young P Gow PJ & Stewart AW (1983). Perna canaliculus in the treatment of rheumatoid arthritis. Eur J Rheumatol Inflamm 6, 197-200.
Fuller K, Lean JM, Wani MR & Chambers TJ (2000): A role for TGF in osteoclast differentiation and activation. J Cell Sci 113, 2445-2453.
Gross C, Eccleshall TR & Feldman D (1996). Vitamin D receptor gene alleles and osteoporosis. In Principles of Bone Biology vol. pp. 917-934. Ed JP Bilezikian, LG Raisz, & GA Rodan. San Diego: Academic Press.
Gupta A (1996). Osteoporosis in India-The nutritional hypothesis. Natl Med J India 9, 268-274.
Miller TE & Ormrod D (1980). The anti-inflammtory activity of Perna canaliculus (NZ green mussel). N Z Med J 92, 187-193
Rodan GA & Martin TJ (2000). Therapeutic approaches to bone diseases. Science 289, 1508-1514
Seitz M, & Hunstein W (1985). Enhanced prostanoid release from monocytes of patients with rheumatoid arthritis and active systemic lupus erythematosus. Ann Rheum Dis 44,438-445.
Siris ES (1999). Paget's disease of bone. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism vol. pp. 415-425. Ed MJ Favus. Philadelphia: Lippincott Williams and Wilkins.
Wani MR, Fuller K, Kim NS, Choi Y & Chambers T (1999): Prostaglandin £2 co-operates with TRANCE in osteoclast induction from hemopoietic precursors: Synergistic activation of differentiation, cell spreading and fusion. Endocrinology 140, 1927-1935
Watts, N. B (1999). Pharmacology of agents to treat osteoporosis. In Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism pp. 278-283 Ed MJ Favus. Philadelphia: Lippincott Williams and Wilkins.

We Claim:
1. A formulation from extract of Indian green mussel (Perna viridis) useful for
inhibition of osteoclast formation and bone resorption in an animal or a human
wherein the said formulation comprising a pharmaceutically acceptable amount of
an extract (mussel hydrolysate) from a green mussel wherein amount of said
extract ranges from 30-100 µg unit percent supplemented with an additive,
excipient, diluent or carrier such as herein described.
2. A formulation from extract of Indian green mussel (Perna viridis) useful for
inhibiting osteoclast formation and bone resorption substantially as herein
described with reference to the examples and drawing accompanying the
specification.

Documents:

761-del-2001-abstract.pdf

761-del-2001-claims.pdf

761-del-2001-correspondence-others.pdf

761-del-2001-correspondence-po.pdf

761-del-2001-description (complete).pdf

761-del-2001-drawings.pdf

761-del-2001-form-1.pdf

761-del-2001-form-18.pdf

761-del-2001-form-2.pdf

761-del-2001-form-3.pdf

761-del-2001-petition-137.pdf

« Previous Patent

Next Patent »

Patent Number

211351

Indian Patent Application Number

761/DEL/2001

PG Journal Number

45/2007

Publication Date

09-Nov-2007

Grant Date

26-Oct-2007

Date of Filing

11-Jul-2001

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG , NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	MOHAN RAMCHANDRA WANI	NATIONAL CENTER FOR CELL SCIENCES, NCCS COMPLEX, GENESHKHIND, PUNE, 411007, INDIA.
2	PRADEEP BHASKAR PARAB	NATIONAL CENTER FOR CELL SCIENCES, NCCS COMPLEX, GENESHKHIND, PUNE, 411007, INDIA.
3	ANIL CHATTERJI	NATIONAL INSTITUTE OF OCEANOGRAPHY, DONA PAULA, GOA-403004.

PCT International Classification Number

A61K 35/56

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA