WO2016145256A1 - Use of components of cobra venom for treatment of chronic kidney disease - Google Patents

Abstract

A composition of sterile cobra venom and a method for its oral administration to provide significant analgesic effects to a human and/or animal are disclosed. Such cobra venom compositions comprise a sterilized solution preserved by the addition of one or more suitable food-grade preservatives. The venom composition may be conveniently administered orally by means of a metered spray device. Further disclosed is a method of treating chronic kidney disease in a subject comprising administering to the subject an effective amount of a composition comprising a cobra venom cytotoxin. In certain aspects, the cobra venom cytotoxin is cardiotoxin. In yet further aspects of the invention, the cardiotoxin is a heat modified cardiotoxin

Description

USE OF COMPONENTS OF COBRA VENOM FOR TREATMENT OF CHRONIC

KIDNEY DISEASE

[0001] FIELD OF THE INVENTION.

[0002] This invention relates generally to the field of pharmaceutical and healthcare products for the treatment of pain and chronic kidney disease, and more particularly to formulations of sterile cobra venom suitable for oral administration, and products comprising these formulations in liquid and spray forms.

[0003] BACKGROUND OF THE INVENTION.

[0004] Millions of people around the world suffer from untreated pain related to a variety of illnesses and ailments, as well as from unidentified causes. Humans have searched for effective painkillers for many, many years. Natural pain-killing compositions have been discovered from various sources as varied as willow bark, opium poppies (a source of morphine, codeine, and thebaine), and snake venoms. Opium, for example, was used as a narcotic by Hippocrates, introduced to Persia and India by Alexander the Great, and used as a painkiller by Paracelsus during the Renaissance.

[0005] Despite their effectiveness as analgesics, opiate drugs such as morphine and codeine are classified as narcotics and their use is subject to complex legal and medical regulations in most countries. Furthermore, opiate drugs have a high potential for addiction and abuse.

[0006] Clinical investigations from the 1930's through the 1950's revealed that cobra venom is a potent pain killer with activity superior to morphine, but without the known adverse effects of opiates. In the United States, cobra venom for medicinal use is only available in the form of homeopathic products that contain extremely low concentrations of the active product. The use of medicinal products in homeopathy centers on the logarithmic dilution of the active product to a specific point where it is then deemed ready for use. These dilutions are so high that they may result in the absence of the original product in the formulation administered to the patient. In homeopathy, the preferred dilution of cobra venom is a 1:10,000 dilution, although the volume actually administered has been quite variable over the years. In 1870's Europe, the preferred dose was 10-4 (1: 10,000 dilution). Present guidelines, as provided in the Homeopathic Pharmacopoeia of the United States (USHP), list the recommended dilutions in the range of 10-6 to 10-8.

[0007] In Chinese medicine, the venom is prepared on demand and small quantities, usually sufficient for one week, are given to the patient. Alternatively, the dried venom is mixed into lactose (triturated) and provided as small pills. The venom solution is then mixed with tea or water. The dosage to be used is left to the discretion of the treating physician.

[0008] Unfortunately, at these low dilutions, the direct ingestion of cobra venom left subjects with unpleasant side effects that included irritated and sore throat, headache, nausea, vomiting, abdominal cramps and pain, sudden bowel movements and diarrhea. Given the existence of such problematic side effects, not surprisingly the utilization of oral cobra venom as a pain remedy declined, and was ultimately abandoned in Western medicine.

[0009] The use of cobra venom as a treatment for pain enjoyed a short resurgence in the 1930' s following research and clinical studies that revealed that cobra venom had very potent analgesic activity. However, during this period cobra venom was administered only by injection, requiring that the venom solution be rendered sterile prior to use. This was accomplished by prolonged exposure of the venom to heat at approximately 60° Celsius. While clinically successful and safe, it required frequent injections of cobra venom by physicians and this method of administration also fell out of favor by the 1970' s.

[0010] Considering the favorable outcomes related to pain relief reported for injectable cobra venom in trials conducted during the 1930' s to the 1970' s, although associated with side- effects, and considering the problems associated with opiate-based medications, what are needed are compositions and methods for providing analgesic levels of cobra venom in an orally- administered form suitable for self-administration by a patient in need of the substantial pain relief that cobra venom may provide and free of the side-effects which have previously limited its use.

[0011] Additionally, there are an estimated 26 million American adults with chronic kidney disease (CKD) and, with the increasing levels of obesity, millions of others are at increased risk. The major causes of CKD are diabetes, high blood pressure and other inflammatory processes. High blood pressure is almost always present during all stages of chronic kidney disease. Post-infectious conditions and lupus are among the many causes of glomerulonephritis. Glomerulonephritis is the inflammation and damage of the filtration system of the kidneys, which can cause kidney failure. Other causes of chronic kidney disease include HIV infection, sickle cell disease, heroin abuse, amyloidosis, kidney stones, chronic kidney infections, and certain cancers. There is no cure for chronic kidney disease. Untreated, it usually progresses to end-stage renal disease. Lifelong treatment may control the symptoms of chronic kidney disease though it must be managed in close consultation with a health care practitioner.

[0012] Current medications to treat CKD include drugs to lower blood pressure, diuretics and Vitamin K. Common adverse drug reactions include hypotension, cough, high potassium, headache, dizziness, fatigue, nausea, skin rash and a metallic taste. In some patients, the medication may cause a further decline in kidney function. Use of analgesics such as acetaminophen (Tylenol) and ibuprofen (Motrin, Advil) regularly over long durations of time can cause analgesic nephropathy, another cause of kidney disease. Certain other medications can also damage the kidneys such as chemotherapy. There is a need in the art for improved compositions and methods for the treatment of CKD.

[0013] SUMMARY OF THE INVENTION.

[0014] The present invention relates to a novel oral formulation of cobra venom, and methods for the oral administration of cobra venom. More particularly, the invention provides formulations of sterile solutions of cobra venom containing a preservative that are suitable for oral administration in several forms, including as beverages and oral sprays that can be used with an oral delivery device to permit convenient, metered administration of the venom. The resulting solutions and delivery systems are safe for the storage and administration of cobra venom over extended periods of time.

[0015] In one aspect, the invention provides a composition of cobra venom suitable for oral administration. Such a composition comprises a sterile cobra venom solution admixed with a food-grade preservative. In various aspects, the preservative may be chosen from among the group consisting of methyl paraben, sodium benzoate, potassium sorbate, and combinations thereof.

[0016] In various aspects of the invention, a sterile cobra venom solution may be formulated at a final homeopathic concentration of 3X, 4X, and/or 5X. Such homeopathic concentrations generally may contain from about 0.035 to about 0.35 mg/ml, from about 0.0035 to about 0.035 mg/ml, and from about 0.00035 to about 0.0035 mg/ml of venom protein, respectively.

[0017] In another aspect, the invention provides a method for the oral administration of a composition comprising a sterile cobra venom solution and a food-grade preservative, the method comprising administering the composition as a spray or jet. Some aspects of the invention provide a healthcare product comprising a solution of sterile cobra venom admixed with a food-grade preservative, the venom in the solution having a homeopathic formulation of from about 3X to about 5X, and a metered pump configured to deliver a volume of the solution in the range of from about 0.05 to about 1 ml.

[0018] In various aspects of the invention, a composition is provided as a beverage. Compositions may also be provided for release from edible films, for example, which may be placed on the mucosa within the mouth.

[0019] In another aspect, disclosed is a method of treating chronic kidney disease in a subject comprising administering to the subject an effective amount of a composition comprising a cobra venom cytotoxin. In further aspects, the cobra venom cytotoxin is cardiotoxin. In yet further aspects, the composition further comprises cobrotoxin and cobra venom factor. According to certain aspects, the cardiotoxin is a modified cardiotoxin. In further aspects, the cardiotoxin is heated at from about 50°C to about 100°C for a period of from about 1 minute to about 300 minutes. In yet further aspects, the cardiotoxin is modified through oxidation. In still further aspects, the cardiotoxin is administered at a dose of from about 1 to about 3000 μg/kg daily. In yet further aspects, the cardiotoxin is administered at a dose of from about 1 to about 1000 μg/kg daily.

[0020] DETAILED DESCRIPTION OF THE INVENTION.

[0021] As used herein, the term "pharmaceutically acceptable carrier" refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly (anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0022] As used herein, the term "subject" refers to the target of administration, e.g., an animal. Thus the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term "patient" includes human and veterinary subjects. In some aspects of the disclosed methods, the subject has been diagnosed with a need for treatment of one or more cancer disorders prior to the administering step.

[0023] As used herein, the term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is a human. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).

[0024] As used herein, the terms "administering" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0025] As used herein, the terms "effective amount" and "amount effective" refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a "therapeutically effective amount" refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0026] Methods for Preparation of Oral Formulations of Cobra Venom and/or Its Components

[0027] The present invention relates to an oral formulation of cobra venom, and methods for the oral administration of cobra venom and components of cobra venom. More particularly, the invention provides formulations of sterile solutions of cobra venom and/or its components admixed with one or more food- grade preservative(s), the combination of venom and preservative(s) being suitable for oral administration in several forms, including, for example, as beverages, oral sprays, lozenges, and edible films that can be used with an oral delivery device to permit convenient, metered administration of the venom. The resulting solutions and delivery systems provide safe storage and administration of cobra venom and/or its components over extended periods of time.

[0028] Cobra venom is comprised of 1-2% of cobra venom factor, 40-50% of cardiotoxin, 10% of neurotoxin and other peptides. Homeopathic cobra venom preparations may be made from the venom of the Asian cobra (e.g., Naja tripudians) and related species according to methods provided in the United States and European Homeopathic Pharmacopoeias. Historically, cobra venoms were selected by homeopaths based upon their neurotoxic activity for treatment of disorders of the nervous system. Without intending to be bound by theory, it was therefore believed that the principal active components in the venoms are most likely neurotoxins.

[0029] Clinical investigations from the 1930's into the 1950's revealed that cobra venom was a potent pain killer, with activity superior to morphine, yet without the known adverse effects of opiates. In contrast to the opioid drugs which bind to opioid receptors (G-protein coupled receptors acting by GABAnergic neurotransmission), the venom neurotoxins are known to primarily target the cholinergic system by blocking the activity of acetylcholine, although it is possible that other receptors or targets may be involved in the analgesic effect.

[0030] In those studies, sterile cobra venom solution was administered by subcutaneous injection where dry mouth, dizziness and nausea were the principle side effects. Occasionally allergic reactions were reported. However, during this period, cobra venom was administered only by injection, necessitating that the venom solution be rendered sterile prior to use. This was accomplished by prolonged exposure of the venom to heat at approximately 60° Celsius. While clinically successful and safe, the prolonged onset of action and necessity for frequent injections by physicians was inconvenient and problematic, and this method of administration also fell out of favor by the 1970' s.

[0031] Preparing an injectable form of a cobra venom solution is now straightforward. However, developing a convenient and effective oral venom formulation that avoids the known problems associated with oral administration presents several challenges. While cobra venom toxicity can be reduced by chemical oxidation or high heat, this process also reduces its anti- analgesic activity because the disulfide bonds of the peptides are irreversibly opened by oxidation. Consequently higher doses of the resulting product are required for pharmacodynamics effects. Toxicology studies in mice were conducted from which it was determined that mice could drink a 1 mg/ml solution of cobra venom for 28 days with a daily intake of 350 mg/kg. By contrast, injection of merely 10-12 micrograms was a fatal dose. The mice in this study gained weight, were quite active, and were apparently unaffected by the ingestion of cobra venom at this concentration via their drinking water. This perplexing and unexpected finding led to question why the side effects of oral administration of cobra venom as described in the prior art from the last century, being so to detrimental in humans, were seemingly absent in current studies with mice.

[0032] Without intending to be bound by theory, it may be that the clinical tests on humans described in the homeopathic and scientific literature, which were conducted with orally administered cobra venom, were flawed because the venom product may have been contaminated with bacteria. Furthermore, the dosing of the material employed a concentration that was too high. Today, it is possible to remove such bacterial contamination by sterile filtration without compromising the quality of the venom. When cobra venom is prepared under sterile conditions, no abdominal problems are experienced. Sterile filtration may be accomplished, for example, using a filter having a pore size of 0.45 μιη or smaller. Sterilization may also be accomplished by prolonged low-heat treatment or by pasteurization.

[0033] Providing a sterile composition for oral administration is facilitated by the addition of a food-grade preservative to the venom (i.e., at least one food-grade preservative). It has been determined that the use of such a preservative has no adverse effect on the efficacy of the venom composition for achieving analgesia after oral administration. Suitable food-grade preservatives include, for example, methyl paraben, sodium benzoate, and potassium sorbate, with the use of other suitable preservatives being known to those of skill in the art and within the scope of the present invention, given the present disclosure.

[0034] Throat irritation had previously been consistently reported when cobra venom was taken by mouth. While homeopathic rules guide the dilution strength of the product from the "mother tincture" (in this case raw venom), the dose that is applied has been left to the discretion of the homeopath. In the 1873 copy of Guy's Hospital Report, Taylor reported that he received 4.2 grains (273 mg) of dried cobra venom and, allowing for the loss of water through desiccation, it represented half a drachm (1.78 g) or 15% of the venom. Clinically, cobra venom was used at dilutions as low as 1:1000 (10-3). The volumes or quantities administered were quite small, often consisting of a pill or a drop of the diluted tincture. However, side effects associated with this form of administration caused the characteristic irritated and sore throat associated with cobra venom administration.

[0035] In conventional Western medicine, a defined quantity of a drug is administered to a patient in need thereof. For example, 1 mg of a drug can be given in a volume of 1 ml or 10 ml. In homeopathic medicine, however, the same volume of material is administered, regardless of the dilution factor. Furthermore, the dilution factors that used are quite large, tenfold at a minimum, and more commonly 100-fold or 1000-fold in order to reach the exceedingly high dilutions that are routinely used by homeopathic doctors.

[0036] In the 1800's, it would not have been feasible to accurately weigh out the minute quantities of drugs that ended up in the final dilutions given to patients. The only way to obtain such small amounts of the active ingredient was to make progressive ten-fold dilutions, starting with a stock solution of known (high) concentration, and repeatedly diluting it. For purposes of comparing the dosages of venom in formulations described in the homeopathic literature of the 1800's with venom solutions of today (which are described in terms of protein concentrations (e.g. in mg/ml or μg/ml), the protein content of raw cobra toxin as it is derived from the snake was first measured and determined to be about 350 mg/ml. With this information in hand, it was possible to estimate the approximate protein concentration that would be present in a homeopathic remedy prepared from raw venom, in which the starting solution (i.e., "mother tincture") has a protein concentration of approximately 350 mg/ml (Table 1). Table 1.

Approximate protein concentration in dilutions of cobra venom used in homeopathic formulations.

[0037] Serial dilutions such as these did not provide for dilution of cobra venom by a factor of 2X or 5X. It occurred that intermediate dilutions, i.e., greater than undiluted (referred to as "mother tincture" in the homeopathic literature) but less than ten-fold, might be very useful, if they provided the advantage of arriving at a dose that is therapeutically effective, safe, and lacking in the unpleasant side effects discussed above.

[0038] With the goal of achieving a standard dose of the active ingredient, a therapeutically effective (3X) homeopathic dose (e.g., 0.35 mg in 1 ml, if starting with a mother tincture having a concentration of 350 mg/ml) was produced that could be administered in dosages such as a single dose in a 10-fold greater volume, or as 10 4X doses, or as 100 5X doses. Use of these dilutions might reduce the possibility of experiencing the adverse effects that caused this medication to be abandoned as a potent orally- administered analgesic. Tests were therefore conducted as described in the Examples below, using several liquid formulations based on homeopathic doses ranging from 2X to 5X. Results of clinical testing with human subjects with back pain showed effective reduction of back pain using dilutions of cobra venom stock solutions corresponding to each of these homeopathic doses. Positive results were achieved with a minimum of side effects reported previously using other dosage ranges.

[0039] From the standpoint of modern drug manufacturing, formulations in the 2X to 5X range were shown to provide several advantages over prior art formulations. For example, preparing the venom as a diluted liquid solution facilitates the preparation of a sterile product, and easier handling by automated systems. [0040] The addition of a suitable edible preservative permits the sterile solution to be dispensed into containers for long-term storage and prevents the solution from becoming adulterated during the period of use. A metered spray or jet permits the venom to be administered as a controlled dose that allows frequent administration with limited esophageal irritation. The formulation and metered dose permits the venom solution to be administered over periods of days to weeks. These formulations may be useful for pain relief in both humans and/or animals.

[0041] A certain degree of effectiveness appears to accompany administering the venom to the mucous membranes of the mouth, such as would be achieved by an oral spray. This may also be accomplished, along with the additional benefit of potentially providing modified release compositions, using edible films such as those known to those of skill in the art. Such films and dissolvable strips have been made, for example, using pullulan, whey proteins, and other carbohydrates and proteins known to those of skill in the art of pharmaceutical formulation and administration.

[0042] The invention may be further described by means of the following non-limiting examples.

EXAMPLES OF FORMULATIONS FOR ORAL ADMINISTRATION OF COBRA VENOM

Example 1. Activity of Native Cobra Venoms in Animal Models of Pain

[0043] A study set out to establish if analgesia or anti-inflammatory inflammation could be exerted by the parenteral and oral administration of cobra venom and identify if there are significant interspecies differences in the activities of the venoms and characterize the likely active agents. Employing the commonly used mouse models of pain: formalin, hot-plate and acetic acid writhing tests, this study sought to compare the safety and efficacy of orally and parenterally administered cobra venom. The pharmacodynamic activity of venoms from several species of cobra was examined. In the acetic acid writhing assay, the activity of the venoms was compared to the opiate standard, morphine. The results revealed that the intraperitoneal and intragastric administration of venoms from either N. siamensis, N. naja, N. atra, N. kaouthia, and O. hannah, had analgesic effects in mice with an excellent therapeutic window. Attempts to estimate the circulating levels of neurotoxins utilizing a receptor binding assay failed. However, interpretation of the toxicity data in addition to the highly variable levels of alpha neurotoxins between cobra venom species suggested that the active component were not neurotoxins but another venom component. Surprisingly, oral cobra venoms at 100 meg/Kg suppressed writhing 6 hours into the study comparably to that of oral morphine at 5000 meg/Kg at 1 hour into the study.

Example 2. Activity of Pasteurized Venom in Animal Models of Pain

[0044] Employing the commonly used mouse models of pain: formalin, hot-plate and acetic acid writhing tests, a study sought to compare the safety and efficacy of orally and parenterally administered cobra venom that was pasteurized. The pharmacodynamic activity of venoms from several species of cobra was examined. In the acetic acid writhing assay, the activity of the venoms was compared to the opiate standard, morphine. The results revealed that, contrary to published reports, the oral administration of pasteurized N. kaouthia venom (Nyloxin) had anti-inflammatory and analgesic effects. Oral pasteurized venom at 100 meg/Kg suppressed writhing 6 hours into the study comparably to that of oral morphine at 5000 meg/Kg at 1 hour into the study.

Example 3. Oral Administration of a 5 mg/ml ("Homeopathic IX") Sterile Cobra Venom Liquid in Subject With Chronic Back Pain

[0045] From a stock solution of 400mg/ml of sterile filtered cobra venom, a dilution was prepared by suspending 0.125 ml (50 mg) of the stock solution in 10 ml of saline, to reach a final venom concentration of 5 mg/ml. No secussion was required, because it was a final dilution.

[0046] This dilution is designated herein as a "homeopathic IX." However, it is to be noted that the terminology used in this context is not precise with respect to concentration, but rather covers a range of concentrations, because of the historical absence of dilutions of less than tenfold. For example, in classic homeopathy, starting with a mother tincture having a 350 mg/ml protein concentration (a modern concept not known or incorporated into homeopathic calculations), there is no intermediate dilution between IX (corresponding to 35 mg/ml) and 2X (corresponding to 0.035 mg/ml). As used herein, any solution having a protein concentration in the range between 35 mg/ml and 0.35 mg/ml would be described in homeopathic terminology as a "IX" homeopathic formulation because fractions (e.g., 1.1X, 1.2X, etc.) are not used in homeopathic designations. However, as noted throughout the Examples, actual protein concentrations present in the samples are also provided.

[0047] A subject with chronic back pain was administered the IX product prepared as described above, suspended in 10 ml of saline, by mouth. The reported taste was very unpleasant, provoking lacrimation and coughing. The unpleasant aftertaste persisted for some time, accompanied by a slight feeling of nausea, which may have been due to drinking the saline, rather than being attributable to the venom. The subject noted that his throat was tender and had a scratchy feeling similar to that of a sore throat treated with a numbing agent. The subject reported that stiffness in the back was noticeable, but not back pain. The subject also noted eyelids feeling heavy. A slight headache was noted 90 minutes after ingestion of the IX solution that persisted for 8 hours. Throat symptoms were reported to be back to normal after 4 hours. No intestinal disturbances were reported.

Example 4. Oral Administration of a 0.333 mg/ml ("Homeopathic 3X") Sterile Cobra Venom Liquid to a Subject

[0048] From a stock solution of 350 mg/ml of sterile filtered cobra venom, an aliquot of 0.143 ml (50 mg) was suspended in 10 ml of purified water (final concentration 5mg/ml) with 1 minute of secussion. This solution was mixed with 140 ml of pure orange juice giving a final concentration of 0.333 mg/ml (equivalent to a homeopathic 3X). The formulation as described was administered to a subject with chronic back pain.

[0049] Upon ingestion, the subject reported no adverse effects save for a minor taste sensation in the mouth. None of the side effects traditionally associated with oral venom ingestion— such as esophageal irritation, lacrimation, coryza (acute inflammation of the mucous membrane of the nasal cavities; head cold), or intestinal disturbances were reported. Importantly, the same amount of venom drug was delivered (i.e., 50 mg) as in Example 1 above, however the volume of liquid was much greater (150 ml vs. 10 ml.) This result indicated that the same dosage (50 mg venom protein) that previously caused unacceptable side effects was completely tolerable when delivered in a much higher volume of liquid.

Example 5. Oral Administration of a 1 mg/ml ("Homeopathic 2X") Sterile Cobra Venom Liquid in a Subject With Chronic Back Pain

[0050] The subject with chronic back pain of Example 1 was administered 0.0125 ml (5 mg) of a 400 mg/ml mother tincture of sterile filtered cobra venom solution, taken orally in 5 ml water (final concentration 1 mg/ml). No secussion was required because it was a final dilution.

[0051] In this example, a IX solution would contain 40 mg/ml protein, a 2X solution would contain 4 mg/ml protein, and a 3X solution would contain 0.4 mg/ml protein. The final solution given to the patient, having an intermediate concentration of 1 mg/ml, which is less than 2X (4 mg/ml) but greater than 3X (0.4 mg/ml) is designated as being equivalent to a homeopathic 2X or 1C).

[0052] At the time of administration, the subject's back pain was estimated to be 4-5 on a scale of 1-10. The patient reported that the taste of the diluted solution (as compared with a IX solution described in Example 1) was not nearly as harsh as before, even with the solution being rinsed around in the mouth before swallowing. The subject reported, however, that the taste worsened over time. Ninety minutes after administration, the patient reported a pain level of 0.5-1, with no adverse effects. Second and third administrations of a 5 mg dose at 8 and 24 hours after the first administration resulted in no adverse responses.

Example 6. Oral Administration of a 0.4 mg/ml ("Homeopathic 3X") Sterile Cobra Venom Liquid in a Subject With Chronic Back Pain

[0053] subject, while experiencing back pain at a level of 3-4 on a scale of 10, was administered an oral cobra venom product prepared as described in Examples 1 and 2 above, but using a 3X formulation (0.4mg/ml) in water. More specifically, the oral formulation was prepared by using 0.02 ml from a mother tincture of 400 mg/ml (aliquot thus containing 8 mg venom). The aliquot was diluted into 20 ml of purified water, yielding a 3X formulation with a final venom concentration 0.4 mg/ml.

[0054] The subject noted taste deterioration, and throat sensations as described above. A dull headache was noted 90 minutes later. Back pain was reduced to 1 to 1.5. Twelve hours later, the subject reported that the headache persisted, but the backache was reduced to a pain level of 0.5 on a scale of 1-10. Second and third doses of the 3X formulation were taken on the second and third days, respectively. No adverse effects were noted, including absence of the characteristic throat irritation associated with previous homeopathic formulations of cobra toxin. Back pain levels were reported to be less than 0.5. No gastrointestinal upset was experienced.

Example 7. Oral Administration of a 0.07 mg/ml ("Homeopathic 4X") Sterile Cobra Venom Beverage in a Subject With Chronic Back Pain

[0055] The subject in this Example was experiencing back pain at a level reaching 7- 8 upon standing, and settling in around 5. A beverage of cobra venom was prepared by adding 0.01 ml (3.5 mg) of a 350 mg/ml sterile cobra venom "mother tincture" solution to purified water containing 5% pure lime juice, and 0.2% citric acid, made up to a final volume of 50 ml. Final concentration of the venom in the beverage solution was 0.07 mg/ml. Having a final concentration greater than 5X (0.0035 mg/ml), but less than 4X (0.035 mg/ml), this formulation would be designated as within the range of a homeopathic 4X solution.

[0056] The subject reported minor irritation to the throat, although it was deemed to possibly have been attributable to either the venom or the lime juice. Within 1 hour, the subject's pain level was reduced to 3-4, and the subject could stand up and sit down easily. The subject was also able to touch his toes easily, which was usually not the case, suggesting some relaxation of muscles. Seven hours post administration, the pain level was further reduced to a level of 2- 3. The subject also reported a significant improvement in sleep quality. Notably, the characteristic headache, typically experienced with the liquid formulations as described in Examples 1-3, failed to appear after ingestion of the beverage formulation. No gastrointestinal upset was experienced.

Example 8. Pain Relief Product Comprising Flavored 0.035 mg/ml ("Homeopathic 4X") Sterile Cobra Venom Solution in an Oral Spray Dispenser

[0057] A formulation of sterile cobra venom at "4X" with citric acid, flavoring and methyl paraben was prepared. To do so, a 2X dilution was made from a mother tincture solution of 350 mg/ml (2X = 3.5 mg/ml). To produce a 4X solution, the 2X solution was diluted 1:100, to produce a solution with a final venom concentration of 0.035 mg/ml. The formulation was filled into bottles to a volume of 20 ml (final concentration of venom 0.035 mg/ml). In this case, the final protein concentration (0.035 mg/ml) corresponded precisely to that calculated according to Table 1 for a homeopathic 4X formulation. Bottles were fitted with a pump dispenser configured to deliver 0.1 ml of solution per actuation.

[0058] Over the course of two weeks, several such dispensers comprising the flavored sterile cobra venom composition were used to deliver two sprays, four times per day. Each spray dose of 0.1 ml volume contained 0.0035 mg of venom protein. Accordingly, a two- spray dose would deliver 0.007 mg venom per application. Use of the spray as directed (two sprays, four times per day), thus delivers a total dose of 0.028 mg of venom per day.

[0059] At two weeks, a sterility test was conducted on the contents by spreading a 0.5 ml volume of the product onto bacterial agar plates. The results showed no growth was observed following three days of incubation. Example 9. Oral Administration of 0.035 mg/ml ("Homeopathic 4X") Sterile Cobra Venom by Oral Spray in Subjects With Chronic Back Pain

[0060] A formulation of sterile cobra venom at 4X homeopathic concentration as described in Example 5 was prepared and packaged in pump dispensers. The product was provided to six subjects with various types of chronic pain, with instructions to administer two sprays every 3- 4 hours daily.

[0061] In general, a satisfactory reduction in pain was achieved in over 70% of the subjects. No gastrointestinal upset was reported, although a minor esophageal irritation was experienced, described as a dryness which diminished with continued use. In no case was the esophageal irritation sufficiently uncomfortable to discourage continued use of the pain relief product.

Example 10. Oral Administration of a 0.07 mg/ml ("4X") Sterile Cobra Venom by Oral Spray in Subjects With Chronic Back Pain

[0062] A formulation of cobra venom at 4X with citric acid, flavoring and methyl paraben was prepared in the manner described in Example 5 and packaged in pump dispensers. In this case, however, the final concentration of venom in the spray formulation was 0.07 mg/ml. If fractions were permitted, this formulation could be described as equivalent to a "3.8X" homeopathic formulation, as compared with the 4X formulation of Example 7.

[0063] This product was provided to 20 subjects with various types of chronic pain, who were instructed to take two sprays every 3-4 hours daily. In this group as well, a satisfactory reduction in pain was achieved in over 70% of the subjects. No gastrointestinal upset was reported, although minor esophageal irritation was experienced, as noted by subjects described in Example 6.

Example 11. Oral Administration of 0.175 mg/ml ("4X") Sterile Cobra Venom by Oral Spray in Subjects With Chronic Back Pain

[0064] A formulation of sterile cobra venom was prepared as described above, this time at the protein concentration of a homeopathic "3.5X" as defined in Example 8. Final concentration of venom in this formulation was 0.175 mg/ml. The spray formulation included citric acid, flavoring and methyl paraben, and was packaged in pump dispensers.

[0065] The product was provided to eight subjects with various types of chronic pain. Patients were instructed to take two sprays every 3-4 hours daily. In this instance, a satisfactory reduction in pain was achieved in over 90% of the subjects. Improved pain response was noted over the previous 4X formulation. No gastrointestinal upset was reported, although minor esophageal irritation as described above was observed. Nevertheless, this irritation was not severe enough to cause the subjects to discontinue use of the product.

Example 12. Stability of liquid formulations of cobra venom

[0066] Formulations of cobra venom in water, with strengths of 0.035 mg/mL, 0.14 mg/mL and 0.28 mg/mL, in addition to 5% xylitol, 1% flavoring, 0.2% sodium benzoate, 0.1% potassium hydrogen phosphate with a pH ranging from 4.0-7.0 were prepared and tested for stability at 25 and 30 degrees Celsius. These formulations were found to be stable and to surprisingly retain their biological activity for at least 2 years.

Example 13. Stability of liquid formulations of cobra venom under accelerated conditions

[0067] Formulations of cobra venom in water, with strengths of 0.035 mg/mL, 0.14 mg/mL and 0.28 mg/mL, in addition to 5% xylitol, 1% flavoring, 0.2% sodium benzoate, 0.1% potassium hydrogen phosphate with a pH ranging from 4.0-7.0 were prepared and tested for stability at 49 degrees Celsius. These formulations were found to be stable and to surprisingly retain their biological activity for at least 3 months, equivalent to 3 years as determined from the Arrhenius equation.

Use of Components of Cobra Venom for Treatment of Chronic Kidney Disease

[0068] Formulations of the product as described and claimed herein have been determined to be effective for the reduction of chronic pain symptoms, such as chronic back pain, in human subjects. These analgesic benefits are delivered without significant adverse effects and without potential for addiction, representing a significant advance over opioid- based analgesics. Other formulations of the product as described and claimed herein have been determined to be effective for treatment of chronic kidney disease (CKD) in human subjects.

[0069] Additionally, cardiotoxin-like peptide has been reported to have analgesic activity when administered by injection. Cardiotoxin has demonstrated efficacy in several animal models of rheumatoid arthritis, cancer pain and metastasis, neuropathic and joint pain. Disclosed herein is the finding that cardiotoxin is effective in acute renal failure induced by glycerol. It has been found that intragastric administered of heat-modified cardiotoxin reduces proteinuria and can improve biological parameters in the Adriamycin-induced kidney disease model. [0070] The production of cardiotoxin from venom sources is published and relatively straight forward. The peptide has also been cloned and expressed in a bacterial host though it is abundant and easily obtained from natural sources. According to the invention, physical heating of cobra venom reduces cardiotoxin toxicity using a reversible denaturation process. Cardiotoxin is dissolved in D.I. water, and heated at 80-100 °C for 1-10 minutes and then allowed to cool slowly. Heat-modified cardiotoxin has a stronger effect on nephropathy than raw cardiotoxin.

[0071] The compositions and methods disclosed herein exhibit the following characteristics: 1) modified cardiotoxin has low toxicity and high pharmacological activity; 2) cardiotoxin reduces proteinurea in acute and chronic neuropathy; 3) cardiotoxin reduces serum creatinine, blood urea nitrogen, which are symptoms of renal failure; 4) cardiotoxin reduces hyperlipidemia in chronic neuropathy induced by adriamycin; 5) cardiotoxin can improve low serum protein symptom in chronic neuropathy induced by adriamycin; 6) cardiotoxin improves electrolyte disorder in chronic neuropathy induced by adriamycin; 7) cardiotoxin increases the antioxidant ability of subjects with chronic neuropathy induced by adriamycin, and reduce injury caused by lipid peroxidation; 8) cardiotoxin reduces nephritic pathology injury caused by acute and chronic nephritic syndrome; 9) cardiotoxin shows efficacy at low dose ranging from 1-3000 μg/kg to treat acute and chronic nephritic syndrome; For human being, the effective dose range is 1-1000 μg/kg; and 10) cardiotoxin can be delivered by many ways such as oral administration, dermal delivery, oral or nasal mucosa spray and injection.

[0072] According to certain embodiments, a heat modification process can also be used to create a crude purified cardiotoxin preparation from raw cobra venom by precipitating out the larger molecular weight proteins than tend to be more labile and in this way create a cost effective oral formulation. In another embodiment, a crude purified cardiotoxin preparation from raw cobra venom can be prepared using dialysis or any other suitable size exclusion technology. The crude purified cardiotoxin preparations can thereafter be modified by dissolution in D.I. water followed by heating at 80-100 °C for 1-10 minutes and then slow cooling. From the modified cardiotoxin solution, pharmaceutical formulations can be prepared such as orally disintegrating tablets (films), oral solution, capsules, tablets, spray and transdermal delivery. The modified cardiotoxin solution can then be highly purified by methods such as by ion exchange chromatography or isoelectric-focusing.

[0073] In certain embodiments, an injectable formulation is prepared. For injectable formula, the modified cardiotoxin would preferably be highly purified as by chromatography. It can be formulated in saline at 0.1 to 2 mg/ml using 0.007-0.01% benzalkonium chloride as a preservative where it will remain stable for 2 years when refrigerated. This formula would be especially useful for infusion at dialysis centers for the treatment of advanced cases of CKD. The efficacy of the preparations can be assessed using a simple red cell lysis assay.

[0074] In certain aspects, disclosed herein is a method of treating chronic kidney disease in a subject comprising administering to the subject an effective amount of a composition comprising a cobra venom cytotoxin. In further aspects, the cobra venom cytotoxin is cardiotoxin. In yet further aspects, the composition further comprises cobrotoxin and cobra venom factor. According to certain aspects, the cardiotoxin is a modified cardiotoxin. In certain aspects, the modified cardiotoxin is oxidized by heating at from about 50°C to about 100°C for a period of from about 1 minute to about 300 minutes.

[0075] In further aspects, the active ingredient in the composition consists essentially of modified cardiotoxin. In still further aspects, the cardiotoxin is administered at a dose of from about 1 to about 3000 μg/kg daily. In yet further aspects, the cardiotoxin is administered at a dose of from about 1 to about 1000 μg/kg daily.

[0076] According to certain embodiments, the composition further comprises a pharmaceutically acceptable carrier thereof. In further embodiments, the composition further comprises an excipient.

[0077] According to certain aspects, the cardiotoxin is diluted in saline at 0.1 to 2 mg/ml and 0.007-0.01% benzalkonium chloride. In further aspects, composition is administered by way of infusion. According to still further aspects, the composition is administered parenterally, transdermally or orally. In yet further aspects, the composition is administered as an orally disintegrating tablet, oral solution, capsule, spray, injection or skin transfer paste.

[0078] According to certain aspects, the chronic kidney disease is caused by diabetes, hypertension, chemical poisoning, inflammation, or autoimmune disorders. Instill further aspects, the method further comprises administering the composition in conjunction with at least one other treatment or therapy.

[0079] Disclosed herein is a method of preparing a composition for the treatment of chronic kidney disease comprising: dissolving cobra venom in D.I. water; and heating the cobra venom at from about 50°C to about 100°C for a period of from about 1 minute to about 300 minutes. In certain aspects, the cobra venom is from the species Naja naja, kaouthia, atra or siamensis. In yet further aspects, the cobra venom comprises raw venom. In still further aspects, the cobra venom comprises cardiotoxin, cobrotoxin, and cobra venom factor.

[0080] The invention may be further described by means of the following non-limiting examples.

EXAMPLES OF FORMULATIONS OF COMPONENTS OF COBRA VENOM FOR TREATMENT OF CHRONIC KIDNEY DISEASE

Example 14. Effects of cardiotoxin in the rat model of nephritic syndrome induced by glycerol

[0081] After rats were fasted for 24 hours, and grouped into 5 groups as negative control, positive model, modified cardiotoxin low dose (20μg/kg), modified cardiotoxin median dose (40μg/kg), modified cardiotoxin high dose (80μg/kg). The animals except control group were subjected to single intramuscular injection of hypertonic glycerol (50% v/v, 8 ml/kg). The animals in treatment groups were administrated intragastrically modified cardiotoxin at doses of l-3000μg/kg daily. Three days after disease induction by glycerol, the animals were anesthetized with 5% chloral hydrate. All animals were assessed for plasma creatinine, blood urea nitrogen, and renal function assays along with renal histopathological examination.

[0082] The experimental results showed the following: compared with the negative control, 4 rats in positive model group had enlarged renal coefficients, increased urea, serum creatinine, urea and cholesterol. However, the animals treated with modified cardiotoxin had lower renal coefficient and decreased serum markers when comparing with the model.

[0083] Compared with the negative control group, pathology diagrams of 4 rats in the model group illustrated swollen epithelial cells in the glomeruli, fatty degeneration, dead epithelial cells, coagulation necrosis in part of proximal and distal convoluted tubules, erythrocyte casts in most of the renal tubules. The glomeruli were pressed and deformed by swollen renal tubules, some of which exfoliated. However, the renal pathological diagrams of rats treated by modified cardiotoxin indicated a significant reduction in these pathological indicators.

Example 15. Rat experiment with nephritic syndrome induced by Adriamycin

[0084] Adriamycin-induced nephropathy (AN) was used as an animal model of chronic progressive glomerular disease. AN is characterized by prodocyte injury followed by glomerulosclerosis, tubulointerstitial inflammation and fibrosis. The type of structural and functional injury is very similar to that of chronic proteinuric renal disease in humans.

[0085] The Wistar rats were grouped into 5 groups as negative control, model, modified cardiotoxin low dose (20μg/kg), modified cardiotoxin median dose ( ^g/kg), modified cardiotoxin high dose (80μg/kg). The animals except control group were subjected to single tail intravenous administration of 6mg/kg adriamycin. The animals in therapy groups were intragastrically administrated modified cardiotoxin at doses of l-3000μg/kg daily. At one week prior to induction of adriamycin, first week, second week, third week, fourth week post induction of adriamycin, all animals were collected 24 hours urea in metabolic cages, and tested protein in urea by Bradford method. At the fourth week post induction, the animals were anesthetized with 5% chloral hydrate, collected blood for albumin, globulin, triglyceride, cholesterol, serum creatinine, blood urea, serum cystatin C, serum superoxide dismutase (SOD), and serum electrolyte measurement. Then, all animals were sacrificed to obtain the kidneys, paraffin and thinnest slices were made.

[0086] The rats in the model group had increased urine protein excretion, serum cystatin C, serum creatinine, urea, sodium, chloride and phosphate consistent with the nephrotic syndrome. The animals in model group also had dramatically increased total cholesterol and triglycerides, decreased serum albumin, increased serum globulin, and decreased the ratio of serum albumin to serum globulin. The animals treated with modified cardiotoxin, especially at dose of 40 μg/kg, significantly reversed these disease markers.

[0087] In Kidney tissues stained by HE method: the slit diaphragm in saline control did not showed any mesangial proliferation, glomerular nodule lesions, and glomerular sclerosis, and their renal corpuscle had a normal clear structure. Furthermore, the animals induced by adriamycin had glomerulus damaged with subsequent tubulointerstitial injury, as evidence of renal glomerulus deformed by pressing, renal glomerular capillary adhered to glomerular basement membranes, renal granular capillary containing granules that are easily stained by eosin, vacuolar degeneration and necrosis of epithelial cells, as well as inflammation cells infiltrating interstitium and renal capillary. Kidney tissues stained by PAS method

[0088] The animals treated with modified cardiotoxin had good improvement from glomerular injury, reversal of obstruction was confirmed by reduction of obvious protein cast in renal tubules and interstitial inflammatory cell infiltration when comparing with control group.

[0089] Kidney tissues stained by PAS and Masson methods showed adriamycin treatment caused the development of thicker renal glomerular basement membranes, renal capillary damage and atrophy, and renal tubular marked protein. The animals treated with modified cardiotoxin showed less thickness of renal glomerular basement membrane, less of renal tubular marked protein, less pathological change, when comparing with model group.

[0090] However, by comparing with model group, the animals treated with modified cardiotoxin at dose of 20μg/kg did not have podocyte cell foot processes fused, increased collagen, and the animals treated at doses of 40 μg/kg and 80 μg/kg did not exhibit shrivelled glomerular basement membranes and less fusion of podocyte cell foot processes.

[0091] Although the present invention has been described with reference to preferred embodiments, persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.

Claims

CLAIMS What is claimed is:

1. A method of treating chronic kidney disease in a subject comprising administering to the subject an effective amount of a composition comprising a cobra venom cytotoxin, wherein the active ingredient of the composition consists essentially of cardiotoxin.

2. The method of claim 1 wherein the cardiotoxin comprises a modified cardiotoxin.

3. The method of claim 2 wherein the cardiotoxin is modified by heating at from about 50°C to about 100°C for a period of from about 1 minute to about 300 minutes.

4. The method of claim 3, wherein the cardiotoxin is modified by heating at about 100°C for about 10 minutes.

5. The method of claim 1 wherein the cardiotoxin is administered at a dose of from about 1 to about 3000 μg/kg daily.

6. The method of claim 5 wherein the cardiotoxin is administered at a dose of from about 10 to about 1000 μg/kg daily.

7. The method of claim 5, wherein the cardiotoxin is diluted in saline at 0.1 to 2 mg/ml and 0.007-0.01% benzalkonium chloride

8. The method of claim 5, wherein the composition is administered by way of infusion.

9. The method of claim 1, the composition further comprising a pharmaceutically acceptable carrier thereof.

10. The method of claim 9, further comprising an excipient.

11. The method of claim 1, wherein the composition is administered parenterally, transdermally or orally.

12. The method of claim 1, wherein the composition is administered as an orally disintegrating tablet, oral solution, capsule, spray, injection or skin transfer paste.

13. The method of claim 1, wherein the chronic kidney disease is caused by diabetes, hypertension, chemical poisoning, inflammation, or autoimmune disorders.

14. The method of claim 1 further comprising administering the composition in conjunction with at least one other treatment or therapy.

15. A method of preparing a composition for the treatment of chronic kidney disease comprising: a) fractionating cobra venom to obtain cardiotoxin; b) dissolving the cardiotoxin in D.I. water at a strength of no more than lOmg cardiotoxin per ml of water; c) thereafter modifying the cardiotoxin by heating from about 50°C to about 100°C for a period of from about 1 minute to about 300 minutes; and d) thereafter preparing a composition comprising the modified cardiotoxin, wherein the active ingredient of the composition consists essentially of the modified cardiotoxin.

16. The method of claim 15, wherein the cobra venom is from the genus Naja.

17. A composition for treatment of chronic kidney disease in a subject, comprising a composition prepared according to claim 15.