FORM2
THE PATENTS ACT, 1970
(39 of 1970)
& Patent Rules 2003
Provisional SPECIFICATION
(See section 10; rule 13)

1. Title of the invention. -

"Novel agents for treatment ailments and dysfunctions "

2. Applicant

(a) Regain Biotechnology Pvt Ltd

b) "Rose"Premier Park,
Bunglow No. l,Domnic Lane no.2 Orlem,Malad (W) Mumbai - 400 064, India
(c) an Indian Company.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed

Field of the Invention
The present invention is related to the use of novel agents effective for differential killing of abnormal cells such as cancer cells without damaging or being toxic to normal cells. Further these agents may be used for treating a host of ailments including various types of cancers, skin diseases, prevention and reversal of ageing process, prevention of inflammatory reactions, cure of bacterial infections, cure of fungal infections, etc.
Background of the invention
A variety of approaches have been made to control and cure cancer involving proto oncogenes the antioncogenes and the suicide genes (Portsmouth D. Hlavaty J. and Renner M. 2007, Mol.Aspects.Med 28:4-41) The general approach has been to detect cancer at an early stage, and destroy the cancer cells. The second has been to interfere in the replication of DNA, the third has been to prevent supply of blood to the newly formed tumours and the recent approaches have been to strengthen the immune system to fight against the transformed cells.
In the case of Antiproliferative Therapy, drugs that act on rapidly dividing cells are most effective during the S phase of cell cycle generally causing DNA damage that may initiate apoptotic cell death but not without associated side effects thereby becoming toxic to bone marrow cells, impairing wound healing, damaging hair follicles and gastro-intestinal epithelium or the drugs themselves being carcinogenic [Hans-Peter Lipp, 1999.].
Conventional therapy for cancer has not been highly successful for a variety of reasons. Generally, cancer chemotherapy is painful and debilitating. Often it is ineffective, or its effect on prolonging survival is only short. Further they are expensive. Despite major advances in the basic understanding of carcinogenesis, metastasis, and angiogenesis, most of the discoveries in these fields have not been applicable in regular therapeutic practice.
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In an article in J. Cell Biochem 58, 175 (1995) King and Cidlowski have used acridone derivatives to interfere in different phases of the cell cycle, which has resulted in the induction of apoptosis. However such studies have met with limited success as factors that could induce the apoptotic pathways in the cancer cells could also turn them on in normal cells resulting in low differential killing thereby leading to extensive tissue degeneration in the cancer patients. Also, when tested in vivo, it was found that the hormones of the animal could rescue the cancer cells from drug induced apoptotic killing( Thimmaiah.P and DSouza C, 2003 PhD thesis)
Skin diseases account for about 13% of all disorders and a quarter of all occupational diseases. Dermatitis is the second most common cause of occupational disease. Conditions such as contact dermatitis, folliculitis, acne pigmentary disorders and neoplasms are generally treated by topical applications of therapeutic molecules such as salicylic acid, zinc oxide, variety of antibiotics with antibacterial or antifungal functionalities, steroids generally in appropriate delivery systems [Ostrenga.J. Steinmetz,C. and Poulsen.B. J.Pharmaceutical Sci.60:1175-1179,2006]. However it is well established that chronic skin diseases generally do not respond to conventional treatments. While antibiotics are capable of controlling bacterial or fungal infections, development of drug resistance in the long term create limitations for the treatment of chronic skin diseases. Also clinicians are concerned about the unknown risks of using many of the topical medications( McNeill A.M. and Koo J Y. Int. J.Dermatol. 46: 656-658, 2007)
Ageing is a complex physiological process involving a variety of metabolic reactions. There are no specific diagnostic markers of ageing or prevention of aging at a molecular level, although collagen synthesis used as a marker and antioxidants have been applied for the prevention of aging process. The results from such attempts continue to be inconclusive.
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In the case of treatment of inflammatory processes, steroidal anti-inflammatory molecules and nonsteroidal anti-inflammatory molecules have been used. While steroids prevent inflammatory pathways, because of the side effects it is not a preferred therapeutic molecule. Among the non-steroidal anti-inflammatory molecules aspirin is predominant. The aspirin and cyclooxygenease inhibitors act downstream to the production of arachidonic acid. However, to date no efficient anti-inflammatory molecule, has been found that acts at the level of inhibiting cellular pholipase A2. Efficient PLA2 inhibitors are not yet found. Efficient PLA2 inhibitors would act as an efficient anti-inflammatory molecule. (Rainsford K.D. Sub-cell. Biochem.42:3-27, 2007)
It has been a long standing need to provide effective agents to tackle diverse ailments including various types of cancers, skin diseases, prevention and reversal of ageing process, prevention of inflammatory reactions, cure of bacterial infections, cure of fungal infections, etc.
Summary of the invention
The main object of the invention is to provide effective agents for differential killing of abnormal cells such as cancer cells without damaging or being toxic to normal cells so that they can be used for treating several ailments including various types of cancers, skin diseases, prevention and reversal of ageing process, prevention of inflammatory reactions, cure of bacterial infections, cure of fungal infections, etc.
Another object of the inventions is to provide effective agents for differential killing of abnormal cells without being significantly toxic to normal cells even at 1000 times the concentration that was required to kill the cancer cells.
Another object of the invention is to provide effective antidermatitis agents that would partition into the membranes of the skin cells in a facile manner so that they can be used to treat chronic skin diseases.
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Yet another object of the invention is to provide agents that reduce the thickness of the epidermis, which is a desirable trait in maintenance of skin quality.
Yet another object of the invention is to provide agents to function as controlled PLA2 inhibitors recognizing the fact that basal generation of eicosanodis is essential for normal cellular function.
It is yet another object of the invention to provide agents with anti bacterial and antifungal activities within minimal risk of the bacteria or fungi developing resistance to the said agents.
Yet another object of the invention is to explore natural sources for such agents for the applications mentioned above.
Yet another object of the invention is to develop processes for the extraction of the effective agents from diverse natural sources.
Thus in accordance with this invention, extracts are prepared from promising natural sources, characterized and then tested for diverse functionalities as potential candidates for the treatment of various diseases and / or abnormal physiological conditions.
Detailed description of the Invention
1. Preparation of the agents
Various natural sources such as sunflower oil, safflower oil, ground nut oil, soybean oil, sesame oil and other unsaturated oils may be selected for the preparation of the agents of this invention.
In a specific embodiment of this invention Sesame oil was selected as the preferred natural source for the preparation of the novel agents. Sesame oil was aged in a vessel optionally layered with fine layer of carbon particles, optionally in the presence of activated clay. The sesame oil undergoes auto oxidation during the process of ageing to transform to a red viscous liquid which when further
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aged forms a solid white material. The oxidative process may be catalysed by addition of metal oxides such as ferric oxide, lead oxide, mercury oxide, wherein lead oxide is the preferred choice. The ageing process may also be done by bubbling oxygen or air in the vessel. Further the process may be accelerated by the addition of the red viscous oil and or the said white material formed in the sesame oil during the process of ageing.
The red viscous liquid and the white material obtained during the ageing process function as agents with distinctive effects on cell cultures and for the diverse treatments mentioned above.
The red viscous liquid and the white material may further be subjected to saponification followed by acidification to obtain another set of active agents. These active agents may also be further derivatized to form phospholipids, which may then be used to form liposomes as effective delivery agents.
In addition to the saponification product the red viscous oil and the white material contain Glycerol, and fatty acids in ratios of 16:0,18:0, 18:1, and 18:2 measured by gas chromatography. The other fatty acids with higher saturation and longer chains may be present in traces.
The saponified product can be converted to stearic acid by treatment with dry hydriodic acid followed by zinc-acetic acid reduction. The saponified product had a characteristic fluorescence excitation at 375 nm and emission at 440 nm. Some of the characteristics of the prepared agents by the above process are given in tables 1-3.
2. Demonstrating the efficacy of the prepared agents
2.1 Differential Toxicity
In order to demonstrate the differential cytotoxic effects of the prepared agents, invitro experiments were performed using cultured cells. Both primary culture and cell lines were used in the study.
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To demonstrate differential cytotocity, normal cells like HDCS, and VERO and transformed cells like HeLa, KB and MSF-8 were used.
Since transformed cells divide rapidly and do not have contact inhibition, a model of normal cells dividing rapidly was used in the study. This is a primary cell culture prepared from chick embryo.
In order to test the functionality of the prepared agents to distinguish between normal and transformed cells, a mixed culture was prepared consisting of HeLa and HDCS. When the cells were confluent, they were treated with the prepared agent. The left over cells were tested for their ability to be agglutinated by phytohemoagglutinin.
The cytotoxicity of the prepared agents on cancer cells in vivo was tested using a mouse model. Yoshida sarcoma was grown in the peritoneal cavity of swiss mice. Third day after the inoculation of the tumour cells, the prepared agent was injected into the peritomal cavity of the mice in one group and intradermal in another group. The differential action on the growth of tumour was measured in comparison with untreated tumour control.
Normal cells in culture were transformed using viruses and ability of the prepared agent to kill the transformed cells was tested.
2.1.1 Differential Cytotoxic Effect with potential for cancer treatment:
The prepared agent was cytototxic to transformed cells like HeLa, KB and MFS-8 at 0.1 mg/ml whereas it did not show any cytotoxic effect on HDCS and VERO at 100 mg/ml concentration (Fig. 1 -2).
The prepared agent was also not cytotoxic to chick embryo culture at 100 mg/ml (Fig. 2) the biological activity for the crude product as well as prepared agent prepared by hydrolyzing the aged oil indicating that the prepared agent was the active principle of the aged oil. There was no difference in the action of aged oil or the prepared agent on cytotoxicity either to transformed cells or normal cells
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when tested under identical conditions. However the maximum cytotoxic effect with the prepared agent was 0.1 um/ml whereas the auto oxidised oil was 10 mg/ml. The original starting oil was not cytotoxic.
The effect of prepared agent on Yoshida sarcoma in mice is shown in Fig. 3.
When the tumor was treated with either the prepared agent or completely aged oil the effect was equally only when injected into the site of the tumor. However when injected subcutaneously it showed no effect.
2.2 Effect of the prepared agent on Skin Diseases:
The prepared agent and a placebo controlled double blind study were carried out.
The subjects were chosen from among the chronic dermatitis patients who had the disease for 5 years and longer. The subjects were divided into two groups of which one group received the placebo made from the untreated sesame oil in a carrier (Lanoline) and the second group received the prepared agent made by mixing 10% of the crude product in the carrier (Lanoline). Chronic dermatitis patients were asked to apply the ointment three times a day.
In order to unravel the mechanism of action on skin, in vitro and in vivo treatment were done on rat skin.
Rats were depilated on the abdomen one set of rats was used as control and the other set was treated with the aged oil for 10 days once daily. The rats were subjected to gross physical examination for sign of irritation, scratch marks, inflammation or any other reaction. After sacrificing the rats, the blood was examined for specific signs of toxicity. Skin from control and treated portion of the skin was fixed in Bouins fixative, sectioned and stained to measure the thickness of the epidermis.
Newborn mouse ear was cut and placed in organ culture. It was painted on one side with either the crude product or the prepared agent. The culture medium
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was changed on every third day. The skin was then fixed in Bouins fixative and sectioned. The thickness of the epidermis was measured.
The skin constituents of treated and control skin were determined. The skin was also used to measure conversion of 3H progesterone to hydrocortisone.
Incorporation of the 14C acetate into lipids of control and treated skin was determined.
In the placebo controlled double blind study 23 patients reported relief from itching within 3 days. 15 patients reported that itching subsided within one week. After one month the skin was normal in these cases (total of 38 patients) 4 patients had relief from itching within one week and skin was normal after one month but had occasional relapse. 4 patients had symptomatic relief as long as the ointment was applied. Even after two months the skin had not returned to normal and 2 patients had no relief. All the patients were available for follow up. 27 patients had relief. The patients whose skin returned to normal were not available for follow up. 25 patients who had received placebo did not find any relief. They discontinued the use of the ointment after one week. Out of the 100 patients in this study 75 received the active ointment. Out of these only 6 patients did not show any cure. A typical case is shown in Fig 4
The gross examination of rat skin treated with the aged oil did not show any scratch marks or any other marks of inflammation or toxicity. The blood picture also did not show any signs of toxicity. The mucopolysacchardies and glycoproteins in the skin on treatment decreased, D4 3 - oxo steroids in the skin increased. Conversion of 3H progesterone to hydrocortisone also increased. Incorporation of 14C acetate into phospholipids of treated skin increased compared with the control (Fig 5) the thickness of the control skin was 30.7 ± 2.2 urn whereas that of treated skin was 17.5 + 1.4 urn (Fig. 6).
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2.3 Testing the effectiveness on ageing
Rat skin was treated in vitro and in vivo with white material as well as prepared agent.
The skin of rats was depilated, and treated with the aged oil once daily for 10 days. The skin was then removed and one protion was fixed in Bouin's fixative. The other portion was tested for muropoly saceharides, glycoprotein and hormonal steroids.
The mucopolysaccharides, glycoprotein and A4, 3-oxo steroids in the control and treated skin is shown in Fig.7. The mucopolysacchardies and glycoproteins in treated skin had decreased and the A4, 3- oxo steroids increased.
The thickness of the control epidermis was 30.7 ± 2.2 urn and that of treated epidermis was 17.5 ±1.4 urn. Similar results were obtained from skin treated in vitro in organ culture (Fig. 8).
2.4 Effect of the prepared agent on Inflammatory Reactions
Purified synovial fluid (inflammatory) PLA2 was used as the target enzyme. The substrate was Ecoli membrane having radio labeled fatty acid. The enzyme activity in the presence and absence of prepared agent was measured.
The prepared agent was also used as an inhibitor in the lipoxygenase assay.
The prepared agent inhibited PLA2 by 45%. It also inhibited lipoxygenase by over 90% (Fig. 9,10).
Bacterial culture was prepared and grown on a Petridish. A central well was made and 10 pi of prepared agent was added. Norfloxacine disc was added on the dish for comparison. The prepared agent was mixed with Vaseline and applied on acne for two individuals and one individual with atopic eczema.
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The zone of inhibition by prepared agent was larger that of norfloxacine (Fig. 11). It also cured the acne of two individuals. The atopic eczema was cured and had no relapse for 3 years (Fig. 12).
2.6 Effect of the prepared agent on Antifungal Activity
Fungal culture was grown on agar plate and the prepared agent was added on the plate and zone of inhibition was measured.
The prepared agent was mixed with Vaseline and treated on athletes' foot and other fungal infections.
The prepared agent inhibited the growth of aspergellus (Fig 13). It also cured 8 cases of fungal infection including 5 athletes foot infection.

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