Patent application title: METHOD OF TREATMENT OF DISEASES USING HOODIA EXTRACTS
Refael Aharon (Jerusalem, IL)
Yaron Ilan (Jerusalem, IL)
Yaron Ilan (Jerusalem, IL)
HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LIMITED
Desert Labs Agriculture Cooperative Association Lt
IPC8 Class: AA61K3624FI
Class name: Drug, bio-affecting and body treating compositions preparations characterized by special physical form
Publication date: 2011-09-29
Patent application number: 20110236438
A method for treating a disease comprising administering to a mammal in
need thereof an effective dosage of an extract of a plant of the genus
Hoodia, wherein the disease is selected from the group consisting of
immune-mediated disorders, immune-associated disorders, inflammatory
diseases, coronary disease, insulin resistance and liver-related
diseases. In a preferred embodiment, the Hoodia is Hoodia parviflora.
Also disclosed is a pharmaceutical composition for treating the above
diseases comprising an effective dosage of an extract of a plant of the
1. A method for treating at least one disease, comprising: administering
to a mammal in need thereof an effective dosage of an extract of a plant
of genus Hoodia, wherein the at least one disease is selected from the
group consisting of immune-mediated disorders, immune-associated
disorders, inflammatory diseases, coronary disease, insulin resistance
and liver-related diseases.
2. The method of claim 1 wherein the genus Hoodia includes at least one Hoodia species selected from the group consisting of Hoodia parviflora, Hoodia macrantha, Hoodia currorii, Hoodia lugardii, Hoodia ruschii, Hoodia tugardii and Hoodia gordonii.
3. The method of claim 2 wherein the at least one Hoodia species is Hoodia parviflora.
4. The method of claim 1 wherein the extract is administered orally.
5. The method of claim 1 wherein the liver-related diseases include at least one member selected from the group consisting of hyperlipidemia, fatty-infiltration, non-alcoholic fatty liver disease (NAFLD), cirrhosis of the liver, end stage liver disease, non alcoholic steatohepatitis (NASH), hepatitis B, hepatitis C, autoimmune hepatitis, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), inflammation of and decrease in liver function and any type of liver disease including immune mediated, viral, metabolic and drug induced.
6. The method of claim 1 wherein the immune-mediated and/or immune-associated disorders include at least one member selected from the group consisting of rheumatoid arthritis, acute and chronic graft versus host disease, systemic lupus erythmatosus, scleroderma, multiple sclerosis, non alcoholic fatty liver disease, hyperlipidemia, coronary heart disease, the metabolic syndrome or any of the diseases comprising the same, obesity, inflammatory bowel disease and immune mediated hepatitis, and any brain, lung, heart, gastrointestinal system, kidney, skin, muscle and nerve disorders that are mediated at least in part by the immune system, whether adaptive or innate.
7. The method of claim 1 wherein the coronary disease is atherosclerosis or hypertriglyceridemia.
8. The method of claim 1 wherein the disease is not type II diabetes.
9. The method of claim 1 wherein the disease is not obesity.
10. The method of claim 1 wherein the disease is not metabolic syndrome.
11. The method of claim 1 wherein the extract is a compound isolated from the Hoodia plant, and wherein said compound is not P57.
14. The method of claim 1 wherein the effective dosage of the extract of the plant of the genus Hoodia is in the form of an edible composition selected from the group consisting of a dietary supplement, a nutraceutical, a food additive, a food product, and a beverage.
15. The method of claim 14 wherein the food product is an ice-cream or a frozen-cube.
FIELD OF THE INVENTION
 This invention relates to the use of plant parts and extracts of various species of the Hoodia plant for the treatment of various diseases. In particular, the invention relates to use of the species Hoodia Parviflora.
BACKGROUND OF THE INVENTION
 Hoodia, from the Asclepiadaceae subfamily in the Apocynaceae family, is a succulent plant from Southern Africa that contains substances which suppress hunger, appetite, and thirst. The use of certain species of Hoodia as an appetite suppressant is supported by colorful folklore history and recent scientific studies. Tribesmen hunters in Africa have used Hoodia for many years to prevent hunger on long hunting trips.
 WO 98/46243 discloses a process to extract a steroidal glycoside having a specified formula from plants of the Asclepiadaceae family, and in particular from the genus Trichocaulon or Hoodia, involving treating plant material with a solvent to extract a fraction having appetite suppressant activity, separating the extraction solution from the rest of the plant material, removing the solvent from the extraction solution and recovering the extract. The solvents specifically mentioned to perform the extraction are one or more of methylene chloride (dichloromethane), water, methanol, hexane, ethyl acetate or mixtures thereof.
 U.S. Pat. No. 7,033,616 discloses pharmaceutical compositions containing an extract obtainable from a plant of the genus Trichocaulon or Hoodia having anti-diabetic activity. Also disclosed is a compound derived from the extract which may be used for the manufacture of medicaments having anti-diabetic activity.
 U.S. Patent Application No. 2005/0181077 discloses a composition for treating AIDS. The composition comprises a medicament selected from an extract of at least one of a number of plant families including Asclepiadacea, which includes Hoodia, a glyceryl ester of any of the foregoing extracts; a saponin of any of the foregoing extracts; an alkaloid of any of the foregoing extracts; a protein of any of the foregoing extracts; a fat of any of the foregoing extracts; a sugar of any of the foregoing extracts; and any mixture of any of the foregoing.
 U.S. Patent Application No. 2006/0159773 discloses herbal compositions containing Hoodia gordonii and synergistically enhancing ingredients such as green coffee bean extract. The compositions are useful in controlling obesity and supporting the treatment of various health conditions related thereto, including Syndrome X. This reference states that obesity is commonly associated with the metabolic Syndrome X, and that Hoodia gordonii has been proposed in the literature as a potentially valuable approach to the management of the metabolic Syndrome X. The main mechanism of action of Hoodia gordonii in the metabolic Syndrome X is appetite suppression, leading to control of calorie intake. This reference discloses that combinations of Hoodia gordonii and CGBE-containing chlorogenic acid or Hoodia gordonii and chlorogenic acid per se reduce insulin levels in Type II diabetes mellitus patients, patients with pre-diabetes, the metabolic Syndrome X and related conditions.
SUMMARY OF THE INVENTION
 The present invention relates to a method for treating a disease comprising administering to a mammal in need thereof an effective dosage of an extract of a plant of the genus Hoodia, wherein the disease is selected from the group consisting of immune-mediated disorders, immune-associated disorders, inflammatory diseases, coronary heart disease, insulin resistance and liver-related diseases.
 The term "Hoodia" as used herein may include any species of Hoodia, such as, but not limited to, Hoodia parviflora, Hoodia gordonii, Hoodia macrantha, Hoodia currorii, Hoodia lugardii, Hoodia ruschii, Hoodia tugardii and mixtures thereof, unless otherwise indicated. In a preferred embodiment, the Hoodia species is Hoodia parviflora.
 An extract as used herein includes, but is not limited to, liquid extracts (frozen or liquid), solid extracts or spray-dried extracts, e.g. sap and plant solids. An extract as used herein may be purified, partially purified, concentrated and/or fractionated.
 In one embodiment, the extract is an isolated compound having the biological properties of the extract, as defined below. In a preferred embodiment, the compound is not the compound named P57, an oxypregnane steroidal glycoside with the chemical name (3β,12β,14β)-3-[(O-6-Deoxy-3-O-methyl-β-D-glucopyrano- syl-(1→4)-O-2,6-dideoxy-3-O-methyl-β-D-ribo hexopyranosyl-(1→4)-2,6-dideoxy-3-O-methyl-β-D-ribo-hexopyran- osyl)oxy]-14-hydroxy-12-[[(2E)-2-methyl-1-oxo-2-butenyl]oxy]pregn-5-en-20-- one. In another embodiment, P57 contributes to the biological properties of the extract, as defined below
 It has now been discovered that the extracts used in the invention possess a biological property which is capable of strengthening the immune system. This property of the extracts also has at times an anti-inflammatory effect. It has further been found that these biological properties are not due to the compound P57, verified by activity measurements.
 In the present specification, the term biological properties or biological effect refers in general to the capability to treat a disease as defined below, in particular by strengthening the immune system, and to various biological effects, e.g. as demonstrated in the examples. In one embodiment, the above terms refer to strengthening the metabolic system.
 The diseases included within the invention include immune-mediated and immune-associated disorders, inflammatory diseases, coronary disease, insulin resistance and liver-related diseases. In a preferred embodiment, the disease is not type II diabetes. In one alternative of this embodiment, the disease is diabetes when the Hoodia is Hoodia parviflora. In another preferred embodiment, the disease is not metabolic syndrome. In another preferred embodiment, the disease is not obesity or a disease treatable by appetite suppression.
 The immune-mediated and/or immune-associated disorders may include autoimmune diseases, rheumatoid arthritis, acute and chronic graft versus host disease, systemic lupus erythmatosus, scleroderma, multiple sclerosis, hyperlipidemia, atherosclerosis, obesity, inflammatory bowel disease and immune mediated hepatitis, and any brain, lung, heart, gastrointestinal system, kidney, skin, muscle and nerve disorders that are mediated at least in part by the immune system, whether adaptive or innate. In addition any brain, lung, heart, gastrointestinal system, kidney, skin, muscle and nerves disorders that are mediated at least in part by the immune system, whether adaptive or innate are included.
 Examples of coronary related-diseases are atherosclerosis and pathological triglyceride (TG) serum levels (hypertriglyceridemia).
 The liver-related diseases may include hyperlipidemia, fatty-infiltration, non-alcoholic fatty liver disease (NAFLD), cirrhosis of the liver, hepatitis B, hepatitis C, autoimmune hepatitis, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), end stage liver disease inflammation of and decrease in liver function, and in general any type of liver disease including immune mediated, viral, metabolic and drug induced. NAFLD includes non alcoholic steatohepatitis (NASH).
 Examples of autoimmune diseases include multiple sclerosis (MS), autoimmune uveitis, autoimmune uveoretinitis, autoimmune thyroiditis, Hashimoto's disease, insulitis, Sjogren's syndrome, spontaneous abortions, experimental autoimmune myocarditis, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn's disease, lupus (SLE), psoriasis and diabetes, particularly type I.
 Additional examples of autoimmune diseases include Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Allergic asthma, Allergic rhinitis, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune thrombocytopenic purpura (ATP), Axonal & neuronal neuropathies, Bal's disease, Behnet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac sprue (nontropical), Chagas' disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, Cicatricial pemphigoid/benign mucosal pemphigoid, Cogan's syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatomyositis, Devic disease, Discoid lupus, Dressler's syndrome, Endometriosis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evan's syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Goodpasture's syndrome, Graves' disease, Guillain-Barr syndrome, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, Immunoregulatory lipoproteins, Inclusion body myositis, Insulin-dependent diabetes (type 1), Interstitial cystitis, Juvenile arthritis, Juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lyme disease, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Myasthenia gravis, Myositis, Narcolepsy, Neutropenia, Ocular cicatricial pemphigoid, Osteoarthritis, Palindromic rheumatism, Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynaud's phenomenon, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Rheumatic fever, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Autoimmune thyroid disease, Tolosa-Hunt syndrome, Transverse myelitis & necrotizing myelopathy, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Vasculitis, Vesiculobullous dermatosis, Vitiligo and Wegener's granulomatosis.
 Inflammatory diseases include sepsis, endotoxemia, pancreatitis, uveitis, hepatitis, peritonitis, keratitis, SIRS and injury-induced inflammation.
 The method of the invention may involve any of the standard means of administration such as intravenous, intramuscular, intraperitoneal, topical, transdermal, buccal, sublingual, oral (po), subcutaneous, etc. In a preferred embodiment, the extracts are administered orally.
 The present invention also relates to pharmaceutical compositions. A pharmaceutical composition as used herein means a composition comprising an extract of the invention, in admixture with acceptable auxiliaries such as, but not limited to, pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms. The auxiliaries must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof, i.e. pharmaceutically acceptable.
 "Pharmaceutically acceptable" means it is, within the scope of sound medical judgement, suitable for use in contact with the cells of humans and animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
 "Pharmaceutically acceptable dosage forms" as used herein include, but are not limited to dosage forms such as tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, lozenges, emulsions, solutions, granules and capsules, including liposome preparations. The active ingredient may also be presented as a bolus or paste. Techniques and formulations generally may be found in Remington, Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition.
 Extracts of the invention and compositions comprising such extracts may be administered under the supervision of a medical specialist, or may be self-administered.
 The exact dose and regimen of administration of an extract of the invention or a composition comprising such extract (an effective dosage or an amount effective in bringing about the biological effect) will necessarily be dependent upon the effect to be achieved (e.g. reduction in inflammation) and may vary with the route of administration, and the age and condition of the individual subject to whom the extract is to be administered.
 A dosage for humans is likely to contain from about 10 to about 10,000 mg (dry weight) per 70 kg body weight per day. The desired dose may be presented as one dose or as multiple sub-doses administered at appropriate intervals.
 The compositions may be prepared by any method well known in the art of pharmacy.
 Such methods include the step of bringing in association a Hoodia extract of the invention with any auxiliary agent.
 The invention further includes a pharmaceutical composition comprising an extract of the invention, in combination with packaging material, including instructions for the use of the composition for a use as hereinbefore described.
 Extracts of the invention may be administered in conjunction with other ingredients, including, but not limited to folic acid, vitamins, minerals, anti-oxidants, other extracts from plants or fruit, liquid flavors and so forth.
 Liquid flavors as used herein means any liquid flavor characterized by low viscosity.
 Vitamins as used herein means any vitamin such as, but not limited to, B1, B2, B3, B6, B12, Folic Acid, Vitamin C, Biotin, Pantothenic acid, K, A, D, E and so forth.
 Antioxidants as used herein are meant to encompass any antioxidant such as, but not limited to a compound that has antioxidant activity.
 Minerals as used herein means any mineral such as, but not limited to, Na, K, Cl, Ca, P, Mg, Fe, I, Cu, Zn, Mn, Fl, and so forth.
BRIEF DESCRIPTION OF THE DRAWINGS
 In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
 FIGS. 1 & 2 are bar graphs showing serum levels (u/L) of the liver enzymes aspartyl transaminase (AST) and alanine aminotransferase (ALT) in mice fed the amounts and types of Hoodia indicated;
 FIG. 3 is a bar graph showing IFN-γ levels (pg/ml) in mice as described in FIGS. 1 and 2;
 FIG. 4 is a bar graph showing relative glucose levels in different groups of ob/ob mice at various time points after glucose administration;
 FIGS. 5 & 6 are stained liver tissue sections taken from control and Hoodia fed mice, respectively;
 FIG. 7 is a bar graph showing % triglycerides per liver mass in different groups of ob/ob mice;
 FIGS. 8 & 9 are bar graphs showing the production of ATP (as expressed in luminescence units) as a function of time in a liver cell line treated with 0.1 ml (FIG. 8) and 0.3 ml (FIG. 9) of Hoodia extracts; and
 FIG. 10 is a bar graph showing the relative density of a Western Blot of phosphorylated STAT3 as compared to STAT3 for different groups of mice fed Hoodia extracts.
DETAILED DESCRIPTION OF EMBODIMENTS
Preparation of an Extract of Hoodia Parviflora
 The Hoodia parviflora extract used in the experiments described below was prepared as follows:
 Fresh Hoodia parviflora plants were washed in water and disinfectant. The washed plants were frozen and cut to size of 0.1-10 cm2, and 5-120% (v/v) water was added to the cut Hoodia plant tissue thereby obtaining suspended Hoodia parviflora. The suspended Hoodia parviflora plant tissue was further disintegrated and homogenized for 30 minutes in an ultrasonic bath filled with water at 0-10° C. Liquid Hoodia filtrate was separated from solid Hoodia sediment by centrifuging or filtering, thereby obtaining a `liquid Hoodia extract` and a `solid Hoodia extract`. All steps were carried out below 20° C.
 The administration of the plant lectin, concanavalin A (ConA) to mice induces a severe immune-mediated hepatitis within 20 hours. When injected intravenously to mice, ConA induces activation of T cells in the liver, NKT (natural killer T) cells being the most important. Together with Kupffer cells, NKT cells secrete large amounts of various hepatotoxic cytokines (IFN-γ and TNF-α) which cause severe hepatic inflammation.
 Induction of ConA Hepatitis. The ConA (MP Biomedicals, USA) was dissolved in 50 mM Tris (pH 7), 150 mM NaCl, and 4 mM CaCl2, and was intravenously injected into C57Bl/6 male mice, 10-12 weeks of age (500 μg per mouse) in a total volume of 250 μl. ConA was injected 20 h before sacrificing the mice.
 The administration of the Hoodia liquid extracts was oral (po). The exact volumes are described in FIGS. 1, 2 and 3. 5 mice per group were used. The following groups were tested in this and in the following examples (as indicated):
 ConA, DDW--control (no Hoodia);
 X2--pure Hoodia Gordonii from Africa
 P2--Hoodia Parviflora from Africa
 SAP--sap from Hoodia Parviflora (P2)
 M1--Hoodia Makarenta
 GT2,GT4--hybrid Hoodia Gordonii
 The mice were sacrificed and their liver enzymes were measured. Evaluation of serum aspartyl transaminase (AST) and alanine aminotransferase (ALT) activities were determined using an automatic analyzer. Serum IFN-γ was measured using an ELISA assay (R&D, USA). The results are summarized in FIGS. 1, 2 and 3.
 It can be seen that the groups fed with Hoodia exhibited normal or near-normal serum levels of liver enzymes and IFN-γ. Thus, the Hoodia extracts reduced liver damage in mice suffering from autoimmune hepatitis as noted by a significant decrease in ALT levels in mice. It also appears that the mice fed smaller amounts of Hoodia extract exhibited a greater reduction in liver damage. FIG. 3 shows that the anti inflammatory effect of the different extracts tested was mediated by a decrease in IFN-γ levels.
P57 Content of Different Hoodia Extracts
 The level of the compound P57 was measured in various Hoodia liquid extracts. The results are provided in the table below.
TABLE-US-00001 Hoodia species P57 (ug/ml) Hoodia Parviflora from Africa (P1) 175, 355 Hoodia Gordonii from Africa (X3) 2600, 2820
 It can be seen that the level of P57 in Hoodia Gordonii is approximately 10× higher than in Hoodia Parviflora. Despite this significant difference, the effect on autoimmune hepatitis (and on other diseases as described below) was similar with the two plant varieties. This proves that the biological properties of the extract are not due to P57.
Fatty Liver Disease
 Non-alcoholic fatty liver disease (NAFLD) is fatty inflammation of the liver when this is not due to excessive alcohol use. Non-alcoholic steatohepatitis (NASH) is the most extreme form of NAFLD, which is regarded as a major cause of cirrhosis of the liver of unknown cause.
 Work with genetically obese, insulin-resistant ob/ob mice demonstrates that hepatocytes become steatotic and die at increased rates. Thus, ob/ob mice develop NASH spontaneously. Insulin resistance, the inability of insulin to appropriately stimulate glucose uptake, is a hallmark of type 2 diabetes mellitus. Hepatic steatosis results from lipid accumulation within hepatocytes due to variable combinations of excess lipid uptake and synthesis and altered lipid secretion.
 The effect of Hoodia extracts on liver damage was assessed by the glucose tolerance test (GTT) and by determining hepatic triglyceride content.
 Leptin deficient ob/ob mice (6-8 mice per group) received daily po administrations (11 ml doses) of water, P2, SAP or GT2 for 4 weeks.
TABLE-US-00002 N = 6 N = 8 N = 8 N = 8 Control (water) P2 SAP GT2
 The mice were deprived of food for 12 hours and then given glucose. The serum glucose level was measured using Bayer Health Care strips at pre-determined times after administration of the glucose, as indicated in FIG. 4. The results for each group were normalized to 1, being the level of glucose at 0 minutes after administration. The results are summarized in FIG. 4.
 It can be seen that the Hoodia Parviflora extract improved glucose tolerance in these mice.
 The liver morphology of these mice is shown in FIGS. 5 and 6. The liver tissue sections were stained with Hematoxylin-Eosin (H&E) stain. FIG. 5 is from a control mouse while FIG. 6 is from a P2 mouse. It can be seen that administration of Hoodia Parviflora significantly decreased hepatic fat content.
 In order to further determine hepatic fat levels, liver triglycerides (TG) were measured. The mice were sacrificed and a liver lysate was prepared. TG in the lysate (Sigma) were measured. The results are shown in FIG. 7.
 It can be seen that all Hoodia extracts significantly reduced hepatic TG content.
 In summary, it may be seen that oral administration of Hoodia extracts to OB/OB mice: (1) decreases glucose levels; (2) decreases liver enzymes; (3) decreases triglyceride content in the liver; (4) decreases fat accumulation in the liver.
 P57 was isolated from Hoodia gordonii and found to have homologies to the steroidal core of cardiac glycosides. Intracerebroventricular (icv) injections of the purified P57 demonstrated that the compound has a likely central nervous system (CNS) mechanism of action. The studies demonstrated that the compound increases the content of ATP by 50-150% in hypothalamic neurons. Liver ATP content was also significantly reduced by about 60%.
 A possible mechanism for the hypothalamic Na/K-ATPase activity is that hypothalamic regulation of food intake alters intracellular concentrations of ATP. While ATP may have direct effects on K+ channel activity or Na/K-ATPase, many other phosphorylation-dependent transduction pathways may mediate the subsequent integrative response to energy sensing.
 The mechanisms that drive progression from fatty liver to steatohepatitis and cirrhosis are unknown. In animal models, obese mice with fatty livers are vulnerable to liver adenosine triphosphate (ATP) depletion and necrosis, suggesting that altered hepatic energy homeostasis may be involved. It has been shown that recovery from hepatic ATP depletion becomes progressively less efficient as body mass increases in healthy controls and is severely impaired in patients with obesity-related nonalcoholic steatohepatitis (Helena Cortez-Pinto, et al. Alterations in Liver ATP Homeostasis in Human Nonalcoholic Steatohepatitis. JAMA. 1999; 282:1659-1664).
 In order to investigate the effect of Hoodia extracts on liver metabolism, cells of a liver cell line (Hep3b) were incubated with different volumes of Hoodia extract, and ATP production was followed as a function of time by measuring luminescence production. The results are shown in FIGS. 8 (0.1 ml extract) and 9 (0.3 ml extract).
Phosphorylation of STAT3
 STAT3 (signal transducer and activator of transcription 3) has been proposed to be the main mediator of acute phase (AP) gene induction downstream of IL-6 and other gp130 cytokines. Phosphorylation of STAT3 leads to its activation and is considered important for immune modulation of liver function. Activation of STAT3 plays a role in acute phase response, protection against liver injury, promotion of liver regeneration, glucose homeostasis, and hepatic lipid metabolism. In this experiment, the effect of Hoodia extracts on the phosphorylation of STAT3 was determined.
 Psammomys obesus mice (4 mice per group) received daily po administrations of water, P2, SAP and GT2 (11 μl doses) for 4 weeks. The mice were sacrificed and the amount of phosphorylated STAT3 in the liver as compared to unphosphorylated STAT3, normalized with reference to β-actin, was measured by a Western Blot. The results (from 2 mice from each group) are shown in FIG. 10.
 It may be seen that the mice which were fed Hoodia Parviflora showed a 29% increase in phosphorylated STAT3 as compared to the control. This shows that Hoodia extract can have a positive immunomodulatory effect on the liver.
Safety and Efficacy of Oral Administration of Hoodia as a Medical Food for Patients with NAFLD
 The clinical trial was an open labeled dose escalation, safety study. The primary endpoints were based on the effect of treatment at one of the time points.
 The following protocol involved 10 men and women above 18 years with biopsy-proven NASH with a score of 4 or above, altered glucose metabolism, including diabetes (non treated, or treated with up to 2 drugs (not including insulin) without any change in medication 2 months prior to enrolment), impaired fasting glucose or impaired glucose tolerance.
 Method of Preparation:  1. Harvesting the upper part of Hoodia Parviflora plant  2. Washing the fresh Hoodia plants with vibrating washing machine  3. Freezing the washed Hoodia plants (-200 C)  4. Cutting the frozen Hoodia plants to obtain cut Hoodia plant tissue--automatic cutting machine which cuts cubes of 1 cm2  5. Blending the solids particles are less than 100 micro meter--  6. First screening ˜400 micrometer filter, solids are being removed and the liquid is now the initial basic extract which is 100% pure blended plant tissue. From 1 Kg of plant tissue we get 800 ml of initial basic extract.  7. Adding 30% (v/v) water to the initial basic extract and blend the all mix under cold conditions (refrigerated tank 40 C)  8. Crushing the suspended Hoodia plant tissue (optionally further breaking and homogenizing the suspended Hoodia plant tissue in an ultrasonic bath)  9. Squeezing the solids via mechanical filter press (filter opening size is 300 micron)--the solids are being removed  10. Clarification of the squeezed liquid by using the SWECO vibration screener (200 and 100 micrometer)  11. Fast freezing the liquid in 3 ml containers, divided into individual doses and stored at a temperature of °20-C.  12. Each ml of final clear extract contained 0.8 gr of plant tissue. Oral administration was performed based on the dose currently used in medical foods containing Hoodia in western world countries.
 Hoodia was prepared in liquid form as described below and was divided into 30 doses per patient and stored at a temperature of -20° C. All the drugs were stored at the study site.
 Hoodia was administered in a dose of 0.043 ml/kg=34 mg/Kg. The patients were treated with this dose for 30 days. During the treatment period, subjects ingested the Hoodia every day for 30 days, and were followed for clinical and laboratory effects (Table 1).
 Hoodia was ingested in the morning before breakfast at the study site. The subjects were required to refrain from food for 2 hours after taking it.
 HBA1C between 5.5 and 14% Blood was drawn for a complete blood count (CBC) and other laboratory analyses (see attached study plan in table 1). Serum was collected and archived for use in the development of surrogate markers. The subject visited the clinic on days 7, 14, 21, and 30. The Hoodia was taken every day for 30 days.
 The subjects underwent a physical examination and blood samples were collected for all the disease parameters, as well as for T cell proliferation assay and FACS analysis for CD3, CD4, CD8, NKT, CD4CD25, FoxP3, CD4CCD25LAP.
TABLE-US-00003 TABLE 1 STUDY PLAN Day Screen 1 7 14 21 30 Treatment x x x x x (daily for days 1-30) Informed X Consent Medical History X Medication History X AE Assessment X x x x x x Physical Exam X X X Vital Signs X x x x x x SMA1 X X X X X X CRP X X CBC/differential X x x x x x ESR X X Pregnancy (βHCG) X FACS X x X In vitro cytokines X x X HOMA score X X X Glucose tolerance test X X 1SMA includes: Total protein, albumin, ALT, AST, ALP, GGTP, LDH, total and LDL cholesterol, Triglycerides, uric acid, creatinine, urea (BUN), Na, K, glucose, total bilirubin
 The results regarding the effect on immune function are summarized in Table 2. It may be concluded from the results that oral administration of Hoodia Parviflora was shown to be safe. In a large proportion of treated patients it induced a significant immune modulatory effect and alleviated liver injury, improved insulin resistance and hyperlipidemia.
TABLE-US-00004 CD4 CD25 CD4 CD25 CD4 + Foxp3 CD4 + CD25 + Foxp3 CD8 CD8 + CD25 Overall mean of response (%) 10.3 -121 -100. 8. -7.8 -15.6 -5.3 No. of responders 6 6 7 6 6 10 6 Mean for responders (%) 17.2 -224. -160. -12.3 -33.3 -15.6 51. CD3 CD56 CD3 + CD56 CD3 + CD69 CD3 + CD69 + CD56 CD62 CD4 + CD62 Overall mean of response (%) -5.26 -0.9 8.9 -20.6 42.9 -10.4 -12.2 No. of responders 7 7 6 5 7 8 7 Mean for responders (%) -8.19 15.5 38.6 -74. 67.7 -13.1 -18.8 GLP-1 CD4 + CD25 + HLA-DR IVGTT AUC IL-6 (pg/ml) TNFα (pg/ml) 3 hours 0 Overall mean of response (%) -457. 1645. 8.9 5.9 27.6 27.2 No. of responders 9 7 5 5 4 1 Mean for responders (%) -508. 8.9 -37. -92. 2.08 1.67 Ratio Adiponectin/IL-6 Ratio Adiponectin/TNFα Overall mean of response (%) 1.3 1.3 No. of responders 8 7 Mean for responders (%) 17.5 25.9
Patent applications by Refael Aharon, Jerusalem IL
Patent applications by Yaron Ilan, Jerusalem IL
Patent applications by HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LIMITED
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