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Artemisinin vs Cancer








Narendra SINGH, et al. : Artemisinin/Iron vs Cancer

http://www.australiannationalreview.com/wormwood-iron-annihilate-98-cent-cancer-cells-16-hours/
Dec 1, 2015

Wormwood and iron annihilate 98 per cent of cancer cells in 16 hours

Doctors and pharmaceutical companies reject the idea that diet and healthy lifestyle choices can lead to a cancer-free life and can prevent or delay the evolution of this deadly disease.

According to a study published by Life Sciences, artemisinin, a derivate of the wormwood plant can kill 98 per cent of breast cancer cells in just 16 hours when paired with iron. On its own, the herb managed to reduce the breast cancer cells by 28 per cent, but when it joined forces with iron, normal cells were not affected by the treatment and had a better outcome.

The problem with mainstream cancer treatment is that it attacks not only cancer cells, but also the healthy ones, making the road to recovery heavier and in some cases even impossible because an acidic system with a stagnant lymphatic system that stems from poor dietary and lifestyle choices allows the disease to thrive. However, the body can be quickly alkalized and flooded with nutrients when people consume a predominantly plant-based and unprocessed diet.

Artemisinin has been used in the past as a tool against malaria, but new studies show it can fight against cancer too. In the study published by Life Sciences last year, when subjects were given an iron supplement, the artemisinin was able to target only “bad” cells and left the “good” ones untouched. Researchers’ results demonstrated that it “mediates the cell cycle arrest of human breast cancer cells and represents a critical transcriptional pathway by which artemisinin controls human reproductive cancer cell growth.”

Scientists Narendra Singh and Henry Lai from the University of Washington made the initial discovery. Lai synthesized to compound to target only the diseased cells, which picked it up without knowing that there is a “bomb” (wormwood derivate) hidden inside. The compound is being licensed by the University of Washington to Artemisia Biomedical Inc., a company founded by Singh and Lai in Newcastle, Washington for development and commercialisation.

According to the study’s abstract, “artemisinin reacts with iron to form free radicals that kill cells.” Previous studies have shown that the compound is “more toxic to cancer cells than to normal cells.”

Tomikazu Sasaki, chemistry professor at University of Washington and senior author of the study which focuses on artemisinin’s anti-cancer benefits judged that “side effects are a major limitation to current chemotherapies.” Although the compound kills about 100 cancer cells for every healthy cell, the researchers added a small chemical tag to artemisinin which sticks to the “iron needed here” protein signal. The cancer cell waited for the protein machinery to deliver iron molecules and engulfed everything. As Lai described it, “the compound is like a little bomb-carrying monkey riding on the back of a Trojan horse.”

During ancient times, wormwood was used to treat stomach disorders. Now, the herb is being used to bring down high temperature due to fever and can be applied directly to the skin to treat minor wounds, burns or insect bites.



http://depts.washington.edu/bioe/resources/artemisinin-cancer/

The Department of Bioengineering and University of Washington do not advocate the use of artemisinin to treat cancer.

The US Food and Drug Administration does not currently approve the use of artemisinin for the treatment of any disease.

Research on artemisinin and cancer is still in very early stages. Human use of artemisinin should be considered experimental and taking artemisinin or any other drug should be approached with extreme caution and responsibility. Always consult with your physician before beginning any new therapy or treatment.

Please contact UW Bioengineering Research Professor Narendra Singh for further information.


http://depts.washington.edu/bioe/wp-content/uploads/2013/11/Artemisinin-and-Cancer1.pdf

Publications Relating to Effects of Artemisinin & Analogs on Cancer ( 10 Decemeber 2015 )








US5578637
Methods of inhibition or killing cancer cells using an endoperoxide

Inventor(s): LAI HENRY C [US]; SINGH NARENDRA
Methods of inhibiting or killing cancer cells are disclosed wherein compounds having an endoperoxide moiety that is reactive with heme are administered under conditions which enhance intracellular iron concentrations. Representative endoperoxide compounds include endoperoxide bearing sesquiterpene compounds such as artemisinin and its analogs, arteflene and its analogs, 1,2,4-trioxanes and 1,2,4,5-tetraoxanes. Intracellular iron concentrations may be enhanced by the administration of iron salts or complexes.

FIELD OF THE INVENTION

The present invention relates to the inhibition or killing of cancer cells. More particularly, the present invention relates to the systemic and topical treatments of cancer cells with sesquiterpene compounds.

BACKGROUND OF THE INVENTION

Artemisinin (Qinghaosu) and its analogs are the treatments of choice for cerebral or chloroquine resistant malaria or for patients with chloroquine allergy. Artemisinin is a naturally occurring substance, obtained by purification from sweet wormwood, Artemisia annua L. Artemisinin and its analogs are sesquiterpene lactones with a peroxide bridge, and are characterized by very low toxicity and poor water solubility. Artemisinin is known as a humoral immunosuppressive agent which is less active than cyclophosphamide, the latter being one of the major chemotherapeutic agents for carcinomas. Artemisinin stimulates cell-mediated immunity, and yet decreases abnormally elevated levels of polyamine regulatory proteins. It also markedly inhibits nucleic acid and protein syntheses. Further, it affects cellular membrane functions and decreases hepatic cytochrome oxidase enzyme system activity. Still further, it is virustatic against influenza and cidal against three groups of pathogenic parasites.

Known analogs of artemisinin which have higher solubility in water are dihydroartemisinin, artemether, artesunate, arteether, propylcarbonate dihydroartemisinin and artelinic acid. Dihydroartemisinin has an antimalarial potency which is 60% higher than that of artemisinin. Artemether and artesunate have antimalarial potencies which are 6 times and 5.2 times, respectively, that of artemisinin. In terms of their ability to inhibit nucleic acid synthesis, dihydroartemisinin, artemether, artesunate, arteether, and propylcarbonate dihydroartemisinin all have 100 times the activity of artemisinin, and protein synthesis is stimulated to an even greater extent by these compounds. Artesunate stimulates the immune system at low doses and inhibits it at high doses. Artelinic acid is the most water-soluble and the most stable of the group. Two of the compounds in this group have been demonstrated to display synergistic activity with doxorubicin (a chemotherapeutic agent) and miconazole (an antifungal agent) in the in vitro killing of Plasmodium falciparum, the etiologic agent of malaria.

The very low toxicity of these compounds to humans is a major benefit. Artesunate, for example, is twice as safe as artemether and only one-fiftieth as toxic as chloroquinine, the most common antimalarial. The first manifestation of toxicity of these compounds is generally a decreased reticulocyte count. Other manifestations include transient fever, decreased appetite and elevated blood transaminase levels, the latter an indication of hepatotoxicity.

U.S. Pat. No. 4,978,676 discloses the use of artemisinin and artemisinin analogs in the treatment of skin conditions such as psoriasis, blistering skin diseases, viral warts, and hemorrhoids.

U.S. Pat. No. 4,978,676 discloses the use of combinations artemisinin and artemisinin analogs with monocarboxylic acids, esters or amides in the treatment of papulosquamous skin diseases, including psoriasis, an eczematous skin diseases, including seborrheic and atopic dermatitis.

U.S. Pat. No. 5,219,880 discloses the use of artemisinin and artemisinin analogs in the treatment of warts, molluscum contagiosum and hemorrhoids.

U.S. Pat. No. 5,225,427 discloses certain 10-substituted ether derivatives of dihydroartemisinin alleged to exhibit antimalarial and antiprotozoal activity.

Artemisinin alone has been shown to be toxic to cancer cells in vitro at 20 to 180 .mu.M range (Sun et al., "Antitumor Activities of 4 Derivatives of Artemisic Acid and Artemisinin B in vitro," Chung-Kuo-Yao-Li-Hsueh-Pao 13:541-543 (1992)). The effect was found to be more effective for hepatoma and embryonic lung cells than against human gastric cancer cells. In another study (Woerdenbag et al., "Cytotoxicity of Artimisinin-related Endoperoxides to Erlich Ascites Tumor Cells," J. Nat. Prod. 56(6):849-856 (1993)), artemisinin was shown to have an IC50 value of 29.8 .mu.M on Ehrlich ascites tumor cells. Several derivatives of dihydroartemisinin (artemether, arteether, sodium artesunate, artelinic acid, and sodium artelinate) had IC50 values ranged from 12.2 to 19.9 .mu.M. A ether dimer of dihydroartemisinin was found to have an IC50 of 1.4 .mu.M. However, the toxicity of the dimer to normal cells was not tested. The authors of the latter paper concluded that, "The artemisinin-related endoperoxides showed cytotoxicity to Ehrlich ascites tumor cells at higher concentrations than those needed for in vitro antimalarial activity, as reported in the literature." However, serum concentrations at the levels reported by the two papers cannot be reached in vivo.

Artemisinin is a relatively safe drug with little side-effects even at high doses. Oral dose of 70 mg/kg/day for 6 days has been used in humans for malaria treatment. Furthermore, more potent analogs of this and similar compounds are also available. Higher efficacy of artemisinin action also can be achieved by other means. For example, artemisinin is more reactive with heme than free iron (Hong, et al. "The Interaction of Artemisinin with Malarial Hemozoin," Mol. Biochem. Parasit. 63:121-128 (1974)). Heme can be introduced into cells using transferrin (Stout, D. L., "The Role of Transferrin in Heme Transport," Biochim. Biophy. Res. Comm. 189:765-770 (1992)) or the heme-carrying compound hemoplexin (Smith et al., "Expression of Haemopexin-Transport System in Cultured Mouse Hepatoma Cells," Biochem. J. 256:941-950 (1988); Smith et al., "Hemopexin Joins Transferrin as Representative Members of a Distinct Class of Receptor-Mediated Endocytic Transport System," Europ. J. Cell Biol. 53:234-245 (1990)). The effectiveness of artemisinin also can be enhanced by increasing oxygen tension, decreasing intake of antioxidants, and blockade of peroxidase and catalase by drugs such as miconazole (Meshnick et al., "Activated Oxygen Mediates the Antimalarial Activity of Qinghaosu," Prog. Clin. Biol. Res. 313:95-104 (1989); Krungkrai et al., "The Antimalarial Action on Plasmodium falciparum of Qinghaosu and Artesunate in Combination with Agents Which Modulate Oxidant Stress," Tran. Roy. Soc. Trop. Med. Hyg. 81:710-714 (1989); Levander et al., "Qinghaosu, Dietary Vitamin E, Selenium, and Cod Liver Oil: Effect on the Susceptibility of Mice to the Malarial Parasite Plasmodium yoelii," Am. J. Clin. Ntr. 50:346-352 (1989)).

The endoperoxide moiety of artemisinin and its analogs has been found to be necessary for antimalarial activity, and analogs lacking this group have been found to be inactive. In the presence of heme, the endoperoxide bridge undergoes reductive decomposition to form a free radical and electrophilic intermediates. Accordingly, endoperoxide bearing compounds other than artemisinin and its analogs have been found to have antimalarial activity. For example, arteflene (Ro. 42-1611; Biirgen et al., "Ro. 42-1611, A New Effective Antimalarial: Chemical Structure and Biological Activity," Sixth International Congress for Infectious Diseases, Abst. 427, p. 152, April 1994, Prague), and the 1,2,4-trioxanes, such as the fenozans (Peters et al., "The Chemotherapy of Rodent Malaria. XLIX. The Activities of Some Synthetic 1,2,4-Trioxanes Against Chloroquinine-Sensitive and Chloroquinine-Resistant Parasites. Part 2: Structure-Activity Studies on cis-fused Cyclopenteno-1,2,4-Trioxane (Fenozans) Against Drug-Sensitive and Drug-Resistant Lines of Plasmodium berghei and P. yoelii spp. NS In Vivo," Annals of Tropical Medicine and Parasitology, 87(1):9-16 (1993)), and the 1,2,4,5-tetraoxanes (Vennerstrom et al., "Dispiro-1,2,4,5-tetraoxanes: A New Class of Antimalarial Peroxides," J. of Medicinal Chemistry, 35( 16):3023-3027 (1992)).

SUMMARY OF THE INVENTION

It has been discovered that the anticancer activity of compounds having an endoperoxide moiety that is reactive with heme and iron, of which artemisinin and its analogs, arteflene and its analogs, 1,2,4-trioxanes and 1,2,4,5-tetraoxanes are representative examples, is substantially enhanced both in vitro and in vivo when administered under conditions which enhance intracellular iron concentrations.

In one embodiment of the invention, the endoperoxide bearing compounds have a sesquiterpene structure, particularly an oxygenated tricyclic sesquiterpene structure with an endoperoxide group, and preferably those which are sesquiterpene lactones or alcohols, carbonates, esters, ethers and sulfonates thereof. Examples of such compounds include artemisinin; dihydroartemisinin; carbonate, sulfonate, ester and ether derivatives of dihydroartemisinin, notably artemether, arteether, artesunate and artesunate salts, and dihydroartemisinin propyl carbonate; and the bis-ether artelinic acid.

Iron agents useful for enhancing intracellular iron concentrations in connection with the practice of the invention include pharmaceutically acceptable iron salts and iron complexes.

In addition to the sesquiterpene compounds and the iron enhancing agents, compositions of the present invention may further comprise the administration of conventionally used antineoplastic agents, such as androgen inhibitors, antiestrogens, cytotoxic agents, hormones, nitrogen mustard derivatives, steroids and the like, as a means of further enhancing clinical efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a time response curve of molt-4 human leukemia cells incubated in holotransferrin (12 .mu.M) and dihydroartemisinin (200 .mu.M), as described in Example 1. Dihydroartemisinin was added at time zero, one hour after the addition of holotransferrin. Each response curve is the average from four experiments.

FIG. 2 is a time response curve of normal human lymphocytes incubated in holotransferrin (12.mu.M) and dihydroartemisinin (200 .mu.M), as described in Example 1. Dihydroartemisinin was added at time zero, one hour after the addition of holotransferrin. Each response curve is the average from four experiments.

FIG. 3 is a dose-response relationship of molt-4 cells (FIG. 3A) and lymphocytes (FIG. 3B) exposed to holotransferrin and dihydroartemisinin. Treatment 1 is control samples with no drug added. Samples in treatments 2-6 contained 0, 1, 10, 50, and 200 .mu.M of dihydroartemisinin, respectively, plus 12 .mu.M of holotransferrin. Holotransferrin was added at 1 hr before the addition of dihydroartemisinin. Cell counts were done at 8 hours after addition of dihydroartemisinin. Each bar represents the average from four experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, it has been discovered that the ability to kill cancer cells of compounds having an endoperoxide moiety that is reactive with heme and iron, particularly artemisinin and artemisinin analogs, arteflene and its analogs, 1,2,4-trioxanes and 1,2,4,5-tetraoxanes, can be significantly enhanced by increasing the iron concentration in the body of a patient, such as by the administration of iron salts or complexes, followed by administration of the endoperoxide compound. Analogs of artemisinin, in general molecules containing sesquiterpene lactone with a peroxide, arteflene and its analogs, 1,2,4-trioxanes and 1,2,4,5-tetraoxanes are particularly preferred for this purpose. Both iron salts or complexes and the endoperoxide compound can be administered orally. It is presently particularly preferred that iron salt or complex be administered before the endoperoxide compounds such that cancer cells will be preloaded with iron to induce free radical formation with the endoperoxide compound.

Transferrin is an endogenous protein which transports iron and heme from the circulation into cells. Transferrin binds to transferrin receptors on cell surface, and via endocytosis is taken inside the cell and iron is then released. Most cancer cells express higher cell surface concentration of transferrin receptors than normal cells and have high rates of iron influx via transferrin receptors, because iron is needed in cell growth and division. For example, human hepatoma cells can express 800,000 transferrin receptors per cell on the cell surface, whereas normal lymphocytes generally express no transferrin receptors. The entire population of transferrin receptors on a mouse teratocarcinoma stem cell can be internalized within 6 min.

An artemisinin molecule, as a representative endoperoxide compound of the invention, is a sesquiterpene lactone containing an endoperoxide bridge that can be catalyzed by iron to form free radicals. Its antimalarial action is due to its reaction with-the iron in free heme molecules in malaria parasite with the generation of free radicals leading to cellular destruction. The present invention takes advantage of this property of artemisinin and targets it towards cancer cells. This selectivity in action is because cancer cells have higher concentration of transferrin receptors on their cell membrane and pick up iron at a higher rate than normal cells. In the presence of artemisinin, increase in iron concentration inside cancer cells will lead to free radical formation intracellularly and cell death.

In accordance with the present invention, a human or animal patient is treated by enhancing the iron concentration in extracellular fluids in the patient and administering to the patient a compound comprising an endoperoxide group that is reactive with heme or iron.

In connection with the present invention, compounds may be employed, in general, that possess an endoperoxide group that reacts in the presence of heme to form free radicals. Representative, presently preferred endoperoxide compounds are set forth herein, although it will be apparent that other endoperoxide compounds will be useful for this purpose.

Preferred endoperoxide bearing sesquiterpene compounds of the present comprise compounds of the formula: ##STR1## wherein R is ##STR2## where R1 is hydrogen, hydroxyl, alkyl, or has the formula: ##STR3## wherein R2 is alkyl or aryl and n is 1 to 6, and the pharmaceutically acceptable salts thereof. As used herein, the term "alkyl" means lower alkyl having from 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. Alkyl groups of the invention may be straight-chain or branched-chain groups, with straight-chain groups preferred. The term "aryl" preferably refers to phenyl and benzyl, with phenyl the most preferred. Pharmaceutically acceptable salts include the alkali or alkaline earth metal salts, preferably sodium or potassium, with sodium the most preferred.

The presently particularly preferred sesquiterpene compounds of the invention include artemisinin, where R is ##STR4## dihydroartemisinin (R1 .dbd.--OH), artesunic acid (R1 .dbd.--OCO(CH2)2 CO2 H), and artesunate, artemether (R1 .dbd.--OCH3) and arteether (R1 .dbd.--OC2 H5). The presently most particularly preferred sesquiterpene compound of the invention is dihydroartemisinin.

Other representative endoperoxide compounds of the invention include arteflene (Ro. 42-1611) and its analogs (Biirgen et al., supra), 1,2,4-trioxanes (Peters et al., supra) and 1,2,4,5-tetraoxanes (Vennerstrom et al., supra).

Preferred agents for enhancing intracellular iron levels for use in combination with the sesquiterpenes of the present invention include pharmaceutically acceptable iron salts and iron complexes. Iron salts useful in the practice of the invention include ferrous fumarate, ferrous sulfate, ferrous carbonate, ferrous citrate, ferrous gluconate and ferrous lactate. Iron complexes useful in the practice of the invention generally include pharmaceutically acceptable complexes comprising iron, such as, for example, ferrocholinate, ferroglycine sulfate, dextran iron complex, peptonized iron, iron sorbitex, ferric oxide and saccharated iron, as well as iron complexed with iron binding proteins and glycoproteins, such as the holoferritins and holotransferrins.

The concentrations of the endoperoxide compounds in the formulations to be applied in the practice of the present invention will generally range up to the maximally tolerated dosage, but the concentrations are not critical and may vary widely. For artemisinin and its analogs, however, best results will be obtained using formulations containing the compounds at levels of from about 0.1 to about 100 mg per kilogram of body weight per day, preferably from about 1 to about 90 mg per kilogram of body weight per day, and most preferably from about 1 to about 75 mg per kilogram of body weight per day. The precise amounts employed by the attending physician will vary, of course, depending on the compound, route of administration, physical condition of the patent and other factors. The daily dosage may be administered as a single dosage or may be divided into multiple doses for administration. The amount of the compound actually administered for treatment will be a therapeutically effective amount, which term is used herein to denote the amount needed to produce a substantial clinical improvement. Optimal amounts will vary with the method of administration, and will generally be in accordance with the amounts of conventional medicaments administered in the same or a similar form. Topical or oral administration, for instance, may typically be done from once to three times a day.

The concentrations of agents for enhancing intracellular iron concentrations in the practice of the present invention will generally range up to the maximally tolerated dose for a particular subject and agent, which will vary depending on the agent, subject, disease condition and other factors. Dosages ranging from about 1 to about 100 mg of iron per kilogram of subject body weight per day will generally be useful for this purpose.

This procedure will be most effective for the treatment of aggressive cancer, in which large number of transferrin receptors are expressed on the cell surface. However, the procedure may not be effective in the treatment of certain types of cancer. For example, some adult T-cell leukemia have defective internalization of transferrin receptors and may not be susceptible to this treatment. (Vidal et al., "Human T Lymphocyte Virus I Infection Deregulates Surface Expression of the Transferrin Receptors," J. Immunol. 141:984-988 (1988)) Furthermore, this procedure can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other traditional cancer therapy.

More potent and water soluble analogs of artemisinin and similar compounds, e.g., dihydroartemisinin, artesunate, artether, and artemether, etc., are available. Higher efficacy of artemisinin action also can be achieved by other means. For example, artemisinin is more reactive with heme than free iron. Heme can be introduced into cells using transferrin or the heme-carrying compound hemoplexin. The effectiveness of artemisinin also can be enhanced by increasing oxygen tension, decreasing intake of antioxidants, and blockade of peroxidase and catalase by drugs such as miconazole.

The topical formulations may further include one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface active agents. Keratolytic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur.

The amounts of each of these various types of additives will be readily apparent to those skilled in the art, optimal amounts being the same as in other, known formulations designed for the same type of administration. Stratum corneum penetration enhancers, for example, will typically be included at levels within the range of about 0.1% to about 15%.

The optimal systemic formulation of the basic combination of the present invention, i.e., the combination of sesquiterpene lactone with an intracellular iron enhancing agent, may vary from one such combination to the next.

The formulations of the present invention may further include as optional ingredients one or more agents already known for their use in the inhibition of cancer cells, for added clinical efficacy. Such combinations will in some cases provide added benefit. These agents include, for example, androgen inhibitors, such as flutamide and luprolide, antiestrogens, such as tomoxifen, antimetabolites and cytotoxic agents, such as daunorubicin, fluorouracil, floxuridine, interferon alpha, methotrexate, plicamycin, mecaptopurine, thioguanine, adriamycin, carmustine, lomustine, cytarabine, cyclophosphamide, doxorubicin, estramustine, altretamine, hydroxyurea, ifosfamide, procarbazine, mutamycin, busulfan, mitoxantrone, carboplatin, cisplatin, streptozocin, bleomycin, dactinomycin and idamycin, hormones, such as medroxyprogesterone, estramustine, ethinyl estradiol, estradiol, leuprolide, megestrol, octreotide, diethylstilbestrol, chlorotrianisene, etoposide, podophyllotoxin and goserelin, nitrogen mustard derivatives, such as melphalan, chlorambucil, methlorethamine and thiotepa, steroids, such as betamethasone, and other antineoplastic agents, such as live Mycobacterium bovis, dicarbazine, asparaginase, leucovorin, mitotane, vincristine, vinblastine and taxotere. Appropriate amounts in each case will vary with the particular agent, and will be either readily known to those skilled in the art or readily determinable by routine experimentation.

The endoperoxide compounds and iron agents of the invention may be employed in vitro, in vivo or ex vivo for killing of target cancer cells. For in vivo applications, compositions of the endoperoxide compounds of the invention generally comprise an amount of the endoperoxide compounds effective, when administered to a human or other animal subject, to localize a sufficient amount of the endoperoxide compounds at target tissue sites to facilitate target cell killing, together with a pharmaceutically acceptable carrier. Any pharmaceutically acceptable carrier may be generally used for this purpose, provided that the carrier does not significantly interfere with the stability or bioavailability of the sesquiterpene compounds of the invention.

The compositions of the invention can be administered in any effective pharmaceutically acceptable form to warm blooded animals, including human and other animal subjects, e.g., in topical, lavage, oral, suppository, parenteral, or infusable dosage forms, as a topical, buccal, or nasal spray or in any other manner effective to deliver the agents to a site of target cells. The route of administration will preferably be designed to optimize delivery and localization of the agents to the target cells.

For topical applications, the pharmaceutically acceptable carrier may take the form of liquids, creams, lotions, or gels, and may additionally comprise organic solvents, emulsifiers, gelling agents, moisturizers, stabilizers, surfactants, wetting agents, preservatives, time release agents, and minor amounts of humectants, sequestering agents, dyes, perfumes, and other components commonly employed in pharmaceutical compositions for topical administration.

Compositions designed for injection may comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, suspensions or emulsions. Examples of suitable nonaqueous carriers, diluents, solvents, or vehicles include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters such as ethyl oleate. Such compositions may also comprise adjuvants such as preserving, wetting, emulsifying, and dispensing agents. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents into the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved or suspended in sterile water, saline, or other injectable medium prior to administration.

Solid dosage forms for oral or topical administration include capsules, tablets, pills, suppositories, powders, and granules. In solid dosage forms, the compositions may be admixed with at least one inert diluent such as sucrose, lactose, or starch, and may additionally comprise lubricating agents, buffering agents, enteric coatings, and other components well known to those skilled in the art.

Actual dosage levels of the compositions of the invention may be varied so as to obtain amounts of the sesquiterpene compound and iron at the site of target cells, especially tumor cells, effective to obtain the desired therapeutic or prophylactic or diagnostic response. Accordingly, the selected dosage level will depend on the nature and site of the target cells, the desired quantity of sesquiterpene compound and iron required at the target cells for target cell inhibition or killing purposes, the nature of the sesquiterpene compound and iron agent employed, the route of administration, and other factors. Generally, for oral delivery routes of administration, effective administration doses will include from about 1 to about 100 mg/kg of iron containing agent, more preferably from about 10 to about 90 mg/kg of iron containing agent per kilogram of body weight of the subject per day, and from about 0.1 to about 100 mg/kg of an endoperoxide compound of the invention, more preferably from about 1 to about 90 mg/kg of the endoperoxide compound per kilogram of body weight of the subject per day. The foregoing doses may be administered as a single dose or may be divided into multiple doses for administration, e.g., up to three times per day.

EXAMPLES

EXAMPLE 1

In Vitro Cancer Cell Inhibition

Molt-4-lymphoblastoid cells and human lymphocytes were used in the experiment. Molt-4-lymph cells were purchased from the American Type Culture Collection (Rockville, Md.). They are acute lympoblastic leukemia cells from human peripheral blood. Cultures were maintained in RPMI-1640 (Gibco, Long Island, N.Y.) supplemented with 10% fetal bovine serum (Hyclone, New Haven, Conn.). Cells were cultured at 37 DEG C. in 5% CO2 /95% air and 100% humidity, and were split 1:2 at a concentration of approximately 1.times.10@6 /ml. Approximate cell number before experiment were between 150.times.10@3 to 300.times.10@3 per ml. Human lymphocytes were isolated from fresh blood obtained from a healthy donor and isolated using a modification of the Ficoll-hypaque centrifugation method of Boyum, A., "Isolation of Mononuclear Cells and Granulocytes from Human Blood," Scand. Clin. Lab. Invest. 21:77-89 (1968). In this method, 20-100 .mu.l of whole blood obtained from a finger prick was mixed with 0.5 ml of ice-cold RPMI-1640 without phenol red (GIBCO, N.Y.) in a 1.5 ml heparinized microfuge tube (Kew Scientific Inc., Columbus, Ohio). Using a Pipetman, 100 .mu.l of cold lymphocyte separation medium (LSM) was layered at the bottom of the tube. The samples were centrifuged at 3500 rpm for 2 min in a microfuge (Sorvall, Microspin model 245) at room temperature. The lymphocytes in the upper portion of the Ficoll layer were pipetted out. Cells were washed twice in 0.5 ml RPMI-1640 by centrifugation for 2 min at 3500 rpm in the microfuge. The final pellet consisting of approximately 0.4-2.0.times.10@5 lymphocytes was resuspended in RPMI-1640. Cell viability was determined before experiments using trypan blue exclusion and found to be more than 95%.

Cells (Molt-4 and lymphocytes) were aliquoted in 0.1 ml volumes into microfuge tubes. Human holotransferrin (Sigma Chemicals, St. Louis, Mo.) was added to samples of the cells. Different concentrations of freshly prepared dihydroartemisinin dissolved in complete medium were added 1 hr later to the tubes. The final concentration of holotransferrin was 12 .mu.M and dihydroartemisinin was either 1, 10, 50, or 200 .mu.M. Equal volume of medium was added to control samples (i.e., samples without holotransferrin nor dihydroartemisinin). Cells were kept in an incubator at 37 DEG C. under 5% CO2 and 95% air during the experiment. At 1, 2, 4, and 8 hrs after the addition of dihydroartemisinin, the cell number was counted from a 10 .mu.l aliquot from the samples using a hemocytometer. The cells were thoroughly mixed by repeated pipeting before an aliquot was taken for counting. In the case of Molt-4 cells, cell viability was not determined because it is not correlated with cell loss as rapid cell disintegration was observed.

Data are expressed as percentage of cell count at a certain time point compared to cell count at the time when dihydroartemisinin was added (time zero in FIGS. 1 and 2). Time-response curves were compared by the method of Krauth, J., "Nonparametric Analysis of Response Curves," J. Neurosci. Method 2:239-252 (1980). The level of the curves, i.e., ao of the orthogonal polynomial coefficient, were compared with the median test. .chi.@2 was calculated with Yates's correction for continuity. The difference between two data points was also compared by the median test. A difference at p<0.05 was considered statistically significant. The Probit analysis was used to determine LD50 s, i.e., the concentration of dihydroartemisinin that causes a decrease in cell count by 50% in 8 hrs, from the dose-response data.

The percent cell count at different times after the addition of 200 .mu.M of dihydroartemisinin of Molt-4 cells and lymphocytes are shown in FIGS. 1 and 2, respectively. In both Molt-4 and lymphocytes, no significant difference in cell counts was observed between control samples and samples exposed to holotransferrin (12 .mu.M) alone (.chi.@2 =0.5, df=1, non-significant) during the 8-hr incubation period. Compared to controls, a significant decrease in cell count (FIG. 1) was observed in Molt-4 cells exposed to dihydroartemisinin alone (.chi.@2 =4.5, df=1, p<0.035 compared to control), and a combination of dihydroartemisinin and holotransferrin (.chi.@2 =4.5, df=1, p<0.035 compared to control). In addition, percent cell counts of the combined drug treatment were significantly less than those treated with dihydroartemisinin alone (.chi.@2 =4.5, df=1, p<0.035 compared to control).

In the case of lymphocytes, exposure to dihydroartemisinin alone or dihydroartemisinin plus holotransferrin, cell counts were significantly less than those of the controls (.chi.@2 =4.5, df=1, p<0.035 compared to control). However, the addition of holotransferrin did not significantly further enhance the effect of dihydroartemisinin alone (.chi.@2 =0.5, df=1, no significant difference between the dihydroartemisinin alone and holotransferrin+dihydroartemisinin response curves).

FIGS. 3A and 3B show the dose-response relationship of dihydroartemisinin for Molt-4 and lymphocytes, respectively, incubated in 12 .mu.M of holotransferrin. The percent cell counts from samples at 8 hrs after addition of various concentrations of dihydrotransferrin is presented. Dose-dependent decreases in cell counts were observed. For Molt-4 cells, a significant difference (from control samples, .chi.@2 =4.5, df=1, p<0.035) was observed at 1 .mu.M of dihydroartemisinin and higher. Smaller decreases in cell counts were observed with lymphocytes under similar treatment conditions. A significant difference from control samples was observed only at a concentration of dihydroartemisinin of 50 .mu.M and higher. Probit analysis of the data showed that the LD50 s for Molt-4 cells and lymphocytes were 2.59 and 230 .mu.M, respectively.

The foregoing results demonstrate that combined incubation in holotransferrin and dihydroartemisinin can selectively destroy human cancer cells, whereas the effect is significantly less on normal lymphocytes. Artemisinin alone has been shown to be toxic to cancer cells in vitro at 20 to 180 .mu.M range (Sun et al., supra). The effect was found to be more effective for hepatoma and embryonic lung cells than against human gastric cancer cells. However, serum concentrations at these levels cannot be reached in vivo. Addition of holotransferrin increases the potency and selectivity of the drug and may decrease the time of cell killing. In the combined treatment, considerable cell death was observed at a concentration of dihydroartemisinin of 1 .mu.M after 8 hrs of incubation. Furthermore, there is reason to believe that artemisinin can work at lower concentrations in vivo than in vitro. Culture medium may contain 19-30 .mu.M of free iron and could cause destruction of dihydroartemisinin molecules before they can gain entry into cells.

EXAMPLE 2

In Vivo Dog Study 1

A 7 year old male canine basset hound was diagnosed with lymphosarcoma of the lymph nodes. Artemisinin (10 mg per day, i.v., approx. 0.3 mg/kg) and ferrous sulfate (10 mg per day, p.o.) was given in three settings of five days each, with an interval between each treatment period of three to five days. The ferrous sulfate was administered six hours before artemisinin administration. The diameter size of inguinal and submandibular, right and left lymph nodes was reduced to half within five days of treatment. A reduction in size was observed in prescapular and popliteal lymph nodes. The diameter of all lymph nodes increased within two weeks of cessation of therapy. The dog survived for five months after treatment and then was euthanised.

EXAMPLE 3

In Vivo Dog Study 2

A female canine retriever was operated on for hemangiopericytoma of the right thigh. One week after the operation, artemisinin (10 mg per day, p.o.) plus ferrous sulfate (10 mg per day, p.o.) therapy was initiated and continued for 23 consecutive days. As in Example 2, iron sulfate was given six hours before artemisinin each day. No signs of tumor recurrence were seen during or at three months after therapy period.

EXAMPLE 4

In Vivo Dog Study 3

A 12 years old, female canine was diagnosed with a malignant mast cell tumor, grade 11, on the right thoracic wall. The dog underwent surgery, and was treated with ferrous sulfate (10 mg per day, p.o.) and artemisinin (10 mg per day, p.o. ) for seven days and then on artemisinin alone (10 mg per day, p.o.) for the next ten days. Examination after four weeks of therapy, showed no signs of tumor recurrence. The canine owner reported no sign of recurrence at four months after treatment.



http://www.sciencedaily.com/releases/2014/11/141118091609.htm

Cheap malaria drug could treat colorectal cancer effectively too, say experts

Medical experts say a common malaria drug could have a significant impact on colorectal cancer providing a cheap adjunct to current expensive chemotherapy.

A pilot study by researchers at St George's, University of London, has found the drug artesunate, which is a widely used anti-malaria medicine, had a promising effect on reducing the multiplication of tumour cells in colorectal cancer patients who were already going to have their cancer surgically removed.

Colorectal cancer (CRC) makes up about 10 percent of the annual 746,000 global cancer cases in men and 614,000 cases in women.

In the UK, 110 new cases are diagnosed daily, with older patients particularly at risk of death. Prognosis even with the best available treatments does not increase disease free or overall survival beyond 60 percent, five years after diagnosis.

Professor Sanjeev Krishna, an infectious disease expert at St George's who jointly-led the study, said: "There is therefore a continuing and urgent need to develop new, cheap, orally effective and safe colorectal cancer treatments.

"Our approach in this study was to take a close look at an existing drug that already had some anticancer properties in experimental settings, and to assess its safety and efficacy in patients.

"The results have been more than encouraging and can offer hopes of finding effective treatment options that are cheaper in the future."

"Larger clinical studies with artesunate that aim to provide well tolerated and convenient anticancer regimens should be implemented with urgency, and may provide an intervention where none is currently available, as well as synergistic benefits with current treatment regimens," added Professor Devinder Kumar, a leading expert in colorectal cancer at St George's and joint-lead of this study.

For most patients globally, access to advanced treatments is difficult as they are too expensive to be widely available, or associated with significant morbidity thereby further compromising their survival.

"In the St George's study, patients were examined and then were given either the anti-malaria drug artesunate or a placebo. After 42 months following surgery, there were six recurrences of cancer in the placebo group (of 12 patients) and one recurrence in an artesunate recipient (of 10 patients).The survival beyond two years in the artesunate group was estimated at 91% whilst surviving the first recurrence of cancer in the placebo group was only 57%.

This is the first randomized, double blind study to test the anti-CRC properties of oral artesunate. The anticancer properties of artemisinins have been seen in the laboratory previously but this is the first time their effect has been seen in patients in a rigorously designed study.



http://www.ebiomedicine.com/article/S2352-3964%2814%2900034-6/abstract
EBioMedicine, 2014;
DOI: 10.1016/j.ebiom.2014.11.010
 
A Randomised, Double Blind, Placebo-Controlled Pilot Study of Oral Artesunate Therapy for Colorectal Cancer

Sanjeev Krishna, et al.


Abstract
Background

Artesunate is an antimalarial agent with broad anti-cancer activity in in vitro and animal experiments and case reports. Artesunate has not been studied in rigorous clinical trials for anticancer effects.

Aim

To determine the anticancer effect and tolerability of oral artesunate in colorectal cancer (CRC).

Methods

This was a single centre, randomised, double-blind, placebo-controlled trial. Patients planned for curative resection of biopsy confirmed single primary site CRC were randomised (n = 23) by computer-generated code supplied in opaque envelopes to receive preoperatively either 14 daily doses of oral artesunate (200 mg; n = 12) or placebo (n = 11). The primary outcome measure was the proportion of tumour cells undergoing apoptosis (significant if >7% showed Tunel staining). Secondary immunohistochemical outcomes assessed these tumour markers: VEGF, EGFR, c-MYC, CD31, Ki67 and p53, and clinical responses.

Findings

20 patients (artesunate = 9, placebo = 11) completed the trial per protocol. Randomization groups were comparable clinically and for tumour characteristics. Apoptosis in >7% of cells was seen in 67% and 55% of patients in artesunate and placebo groups, respectively. Using Bayesian analysis, the probabilities of an artesunate treatment effect reducing Ki67 and increasing CD31 expression were 0.89 and 0.79, respectively. During a median follow up of 42 months 1 patient in the artesunate and 6 patients in the placebo group developed recurrent CRC.
Interpretation

Artesunate has anti-proliferative properties in CRC and is generally well tolerated.



http://www.wikipedia.org

Artemisinin

Systematic (IUPAC) name: (3R,5aS,6R,8aS,9R,12S,12aR)-Octahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one
Identifiers
CAS Number     63968-64-9
ATC code     P01BE01
PubChem     CID: 68827
ChemSpider     62060 Yes
UNII     9RMU91N5K2 Yes
KEGG     D02481 Yes
ChEBI     CHEBI:223316 Yes
ChEMBL     CHEMBL77
Synonyms     Artemisinine, qinghaosu
Chemical data
Formula     C15H22O5
Molecular mass     282.332 g/mol
Physical data
Density     1.24 ± 0.1 g/cm3
Melting point     152 to 157 °C (306 to 315 °F)
Boiling point     decomposes

Artemisinin, also known as qinghaosu (Chinese: 青蒿素), and its semi-synthetic derivatives are a group of drugs that possess the most rapid action of all current drugs against Plasmodium falciparum malaria.[1] It was discovered by Tu Youyou, a Chinese scientist, who was awarded half of the 2015 Nobel Prize in Medicine for her discovery.[2] Treatments containing an artemisinin derivative (artemisinin-combination therapies, ACTs) are now standard treatment worldwide for P. falciparum malaria. Artemisinin is isolated from the plant Artemisia annua, sweet wormwood, an herb employed in Chinese traditional medicine. A precursor compound can be produced using genetically engineered yeast.

Chemically, artemisinin is a sesquiterpene lactone containing an unusual peroxide bridge. This peroxide is believed to be responsible for the drug's mechanism of action. Few other natural compounds with such a peroxide bridge are known.[3]

Artemisinin and its endoperoxides derivatives have been used for the treatment of P. falciparum related infections but low bioavaibility, poor pharmacokinetic properties and high cost of the drugs are a major drawback of their use.[4] Use of the drug by itself as a monotherapy is explicitly discouraged by the World Health Organization,[5] as there have been signs that malarial parasites are developing resistance to the drug. Therapies that combine artemisinin or its derivatives with some other antimalarial drug are the preferred treatment for malaria and are both effective and well tolerated in patients. The drug is also increasingly being used in Plasmodium vivax malaria,[6] as well as being a topic of research in cancer treatment.

Medical use
Uncomplicated malaria

Artemisinins can be used alone, but this leads to a high rate of recrudescence (return of parasites) and other drugs are required to clear the body of all parasites and prevent recurrence. The World Health Organization (WHO) is pressuring manufacturers to stop making the uncompounded drug available to the medical community at large, aware of the catastrophe that would result if the malaria parasite developed resistance to artemisinins.[7]

The WHO has recommended artemisinin combination therapies (ACT) be the first-line therapy for P. falciparum malaria worldwide.[8] Combinations are effective because the artemisinin component kills the majority of parasites at the start of the treatment, while the more slowly eliminated partner drug clears the remaining parasites.[9]

Several fixed-dose ACTs are now available containing an artemisinin component and a partner drug which has a long half-life, such as mefloquine (ASMQ[10]), lumefantrine (Coartem), amodiaquine (ASAQ), piperaquine (Duo-Cotecxin), and pyronaridine (Pyramax). Increasingly, these combinations are being made to GMP standard. A separate issue concerns the quality of some artemisinin-containing products being sold in Africa and Southeast Asia.[11][12]

Artemisinins are not used for malaria prophylaxis (prevention) because of the extremely short activity (half-life) of the drug. To be effective, it would have to be administered multiple times each day.

Severe malaria

Artesunate administered by intravenous or intramuscular injection has proven superior to quinine in large, randomised controlled trials in both adults [13] and children.[14] Combining all trials comparing these two drugs, artesunate is associated with a mortality rate that is approximately 30% lower than that of quinine.[14] Reasons for this difference include reduced incidence of hypoglycaemia, easier administration and more rapid action against circulating and sequestered parasites. Artesunate is now recommended by the WHO for treatment of all cases of severe malaria. Effective treatment with ACT (Artemisinin Combination Therapy) has proven to lower the morbidity and mortality from malaria within two years by around 70%.[15]

Cancer treatment

Artemisinin is undergoing early research and testing for the treatment of cancer.[16] Artemisinin has anticancer effects in experimental models of hepatocellular carcinoma.[17] Artemisinin has a peroxide lactone group in its structure, and it is thought that when the peroxide comes into contact with high iron concentrations (common in cancerous cells), the molecule becomes unstable and releases reactive oxygen species. It has been shown to reduce angiogenesis and the expression of vascular endothelial growth factor in some tissue cultures. Recent pharmacological evidence demonstrates the artemisinin derivative dihydroartemisinin targets human metastatic melanoma cells in vitro with induction of phorbol-12-myristate-13-acetate-induced protein 1 dependent mitochondrial apoptosis that occurs downstream of iron-dependent generation of cytotoxic oxidative stress.[18] A pilot study on the use of the artemisinin derivative artesunate yielded promising results for the treatment of colorectal cancer [19]

Helminth parasites

A serendipitous discovery was made in China while searching for novel anthelmintics for schistosomiasis that artemisinin was effective against schistosomes, the human blood flukes, which are the second-most prevalent parasitic infections, after malaria. Artemisinin and its derivatives are all potent anthelmintics.[20] Artemisinins were later found to possess a broad spectrum of activity against a wide range of trematodes, including Schistosoma japonicum, S. mansoni, S. haematobium, Clonorchis sinensis, Fasciola hepatica, and Opisthorchis viverrini. Clinical trials were also successfully conducted in Africa among patients with schistosomiasis.[21] A randomized, double-blind, placebo-controlled trial also revealed the efficacy against schistosome infection in Côte d'Ivoire[22] and China.[23]

Adverse effects

Artemisinins are generally well tolerated at the doses used to treat malaria.[24] The side effects from the artemisinin class of medications are similar to the symptoms of malaria: nausea, vomiting, anorexia, and dizziness. Mild blood abnormalities have also been noted. A rare but serious adverse effect is allergic reaction.[24][25] One case of significant liver inflammation has been reported in association with prolonged use of a relatively high-dose of artemisinin for an unclear reason (the patient did not have malaria).[26] The drugs used in combination therapies can contribute to the adverse effects experienced by those undergoing treatment. Adverse effects in patients with acute P. falciparum malaria treated with artemisinin derivatives tend to be higher.[27]

Resistance

Clinical evidence for artemisinin resistance in southeast Asia was first reported in 2008,[28] and was subsequently confirmed by a detailed study from western Cambodia.[29][30] Resistance in neighbouring Thailand was reported in 2012,[31] and in Northern Cambodia, Vietnam and Eastern Myanmar in 2014.[32][33] Emerging resistance was reported in Southern Laos, central Myanmar and North-Eastern Cambodia in 2014.[32][33] The parasite's kelch gene on chromosome 13 appears to be a reliable molecular marker for clinical resistance in southeast Asia.[34]

In April 2011, the WHO stated that resistance to the most effective antimalarial drug, artemisinin, could unravel national (India) malaria control programs, which have achieved significant progress in the last decade. WHO advocates the rational use of antimalarial drugs and acknowledges the crucial role of community health workers in reducing malaria in the region.[35]

Mechanism of action

Most artemisinins used today are prodrugs of the biologically active metabolite dihydroartemisinin, which is active during the stage when the parasite is located inside red blood cells. Although there is no consensus regarding the mechanism of action through which artemisinin derivatives kill the parasites,[36][37] several lines of evidence indicate that artemisinins exert their antimalarial action by radical formation that depends on their endoperoxide bridge. When the parasite that causes malaria infects a red blood cell, it consumes hemoglobin within its digestive vacuole, a process that generates oxidative stress.[38] In the primary theory of the mechanism of action, the iron of the heme directly reduces the peroxide bond in artemisinin, generating high-valent iron-oxo species and resulting in a cascade of reactions that produce reactive oxygen radicals which damage the parasite and lead to its death.[39] However, this mechanism has been debated and other hypotheses have been described.[40] One alternative is that artemisinins disrupt cellular redox cycling.[41] Artesunate has been shown to potently inhibit the essential Plasmodium falciparum exported protein 1 (EXP1), a membrane glutathione S-transferase.[42] Recently Shandilya et al. suggested a free radical mechanism in which artemisinin gets activated by iron present in the food vacuole which in turn inhibits PfATP6 by closing the phosphorylation, nucleotide binding, and actuator domains leading to loss of function of PfATP6 of the parasite and its death.[43]

Numerous studies have investigated the type of damage oxygen radicals may induce. For example, Pandey et al. have observed inhibition of digestive vacuole cysteine protease activity of malarial parasites by artemisinin.[44] These observations were supported by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin and inhibition of hemozoin formation by malaria parasites, although this inhibition was not seen in an in vitro B-hematin inhibition assay.[45] Electron microscopic evidence linking artemisinin action to the parasite's digestive vacuole has been obtained showing that the digestive vacuole membrane suffers damage soon after parasites are exposed to artemisinin.[46] This would also be consistent with data showing that the digestive vacuole is already established by the mid-ring stage of the parasite's blood cycle,[47] a stage that is sensitive to artemisinins but not other antimalarials. However, fluorescently tagged artemisinin was seen in the Golgi, ER and mitochondria, rather than the digestive vacuole, suggesting that the vacuolar damage may be a downstream effect, and also that tiny ring stages (containing minimal digested material) are highly susceptible to artemisinins.[48][49][50]

Other suggested targets include the mitochondrial electron transport chain [51] and the parasite's SERCA pump (PfATP6/PfSERCA).[52] Evidence to support the PfATP6 hypothesis includes studies reporting specific interactions between SERCAs and artemisinins,[52][53] undertaken in a Xenopus oocyte system that generated valuable results despite the challenges of working with low amounts of heterologous expressed material.[54] These findings have subsequently been confirmed in an independent series of experiments that use yeast expressing this calcium ATPase. Artemisinins selectively and reproducibly inhibit the yeast growth by their actions on PfATP6.[55] This methodology also provided information on the effects of mutations in PfATP6 on drug sensitivity.[53] In French Guiana and Senegal reduced sensitivity to artemisinins have been associated with single nucleotide polymorphisms in PfATP6.[56] In Tanzania, a significant increase in a predicted but previously undiscovered polymorphism in PfATP6 has been found after a period of artemisinin combination therapy.[57] The mammalian calcium ATPase SERCA1a was also inhibited by artemisinins in coupled enzyme assays,[58] and mutations in either SERCA1a or PfATP6 modulate their sensitivity to artemisinin.[53] Anomalously, if either PfATP6 or SERCA1a are detergent solubilised, they do not show sensitivity to artemisinins.[59][60][61]

Dosing

Artemisinin derivatives have half-lives on the order of an hour. Therefore, they require at least daily dosing over several days. For example, the WHO-approved adult dose of co-artemether (artemether-lumefantrine) is four tablets at 0, 8, 24, 36, 48, and 60 hours (six doses).[62][63]

Artemisinin is not soluble in water, therefore Artemisia annua tea is postulated not to contain pharmacologically significant amounts of artemesinin.[64] Artemisia tea is therefore not recommended as a substitute for the ACTs; however, clinical studies have been suggested.[65]

Production and price

China and Vietnam provide 70% and East Africa 20% of the raw plant material. Seedlings are grown in nurseries and then transplanted into fields. It takes about 8 months for them to reach full size. The plants are harvested, the leaves are dried and sent to facilities where the artemisinin is extracted using solvent, typically hexane. Alternative extraction methods have been proposed.[66] The market price for artemisinin has fluctuated widely, between $120 and $1200 per kilogram from 2005 to 2008.[67]

The Chinese company Artepharm created a combination artimisinin and piperaquine drug marketed as Artequick.[68] In addition to clinical studies performed in China and southeast Asia, Artequick was used in large scale malaria eradication efforts in the Comoros Islands. Those efforts, conducted in 2007, 2012, and 2013–2014, produced a 95–97% reduction in the number of malaria cases in the Comoros Islands.[69]

After negotiation with the WHO, Novartis and Sanofi-Aventis provide ACT drugs at cost on a nonprofit basis; however, these drugs are still more expensive than other malaria treatments.[70] Artesunate injection for severe malaria treatment is made by the Guilin Factory in China where production has received WHO prequalification,[71] an indicator of drug quality.

High-yield varieties of Artemisia are being produced by the Centre for Novel Agricultural Products at the University of York using molecular breeding techniques.[67]

Using seed supplied by Action for Natural Medicine (ANAMED), the World Agroforestry Centre (ICRAF) has developed a hybrid, dubbed A3, which can grow to a height of 3 m and produce 20 times more artemisinin than wild varieties. In northwestern Mozambique, ICRAF is working together with a medical organisation, Médecins sans frontières, ANAMED and the Ministry of Agriculture and Rural Development to train farmers on how to grow the shrub from cuttings, and to harvest and dry the leaves to make artemisia tea.

In April 2013, Sanofi announced the launch[72] of a production facility in Garessio, Italy, to manufacture the anti-plasmodial drug on a large scale. The partnership to create a new pharmaceutical manufacturing process was led by PATH’s Drug Development program (through an affiliation with OneWorld Health), with funding from the Bill & Melinda Gates Foundation and based on a modified biosynthetic process for artemisinic acid, initially designed by Jay Keasling at the University of California, Berkeley and optimized by Amyris. The reaction is followed by a photochemical process creating singlet oxygen to obtain the end product. Sanofi expects to produce 25 tons of artemisinin in 2013, ramping up the production to 55–60 tons in 2014. The price per kg will be $350–400, roughly the same as the botanical source.[73] Despite concerns that this equivalent source would lead to the demise of companies, which produce this substance conventionally through extraction of A. annua biomass, an increased supply of this drug will likely produce lower prices and therefore increase the availability for ACTs treatment. In August 2014, Sanofi announced the release of the first batch of semisynthetic artemisinin. 1.7 million doses of Sanofi's ArteSunate AmodiaQuine Winthrop (ASAQ Winthrop), a fixed-dose artemisinin-based combination therapy will be shipped to half a dozen African countries over the next few months.[74]...



Related Patents for Artemisinin vs Cancer


KR20150087715
COMPOSITION CONTAINING ARTEMISIA ANNUA EXTRACT, AND ARTEMISININ OR DIHYDROARTEMISININ FROM ARTEMISIA ANNUA EXTRACT FOR PREVENTING OR TREATING LIVER CANCER
Inventor(s):     KIM JONG SANG, et al.
The present invention relates to a composition which contains a mugwort (Artemisia annua) extract as an active ingredient and used for the prevention and treatment of the liver cancer. More specifically, the composition includes artemisinin or dihydroartemisinin in the mugwort extract. The mugwort extract, artemisinin, or dihydroartemisinin can significantly alleviate the liver cancer to be utilized for the prevention and treatment of the liver cancer.

US8940787
Use of Artemisinin for Treating Tumors Induced by Oncogenic Viruses and for Treating Viral Infections
In certain aspects, the invention relates to methods of treating proliferative cervical disorders (such as cervical cancer and cervical dysplasia) and treating virus infection by administering artemisinin-related compounds. In certain aspects, the invention relates to methods of treating a tumor induced by an oncogenic virus, methods of killing or inhibiting a squamous cell carcinoma, and methods of inhibiting the replication of a virus, by administering artemisinin-related compounds.


US2014275240
Suppression and prevention of tumors and treatment of viruses
Combinations of betaine and vitamin C are used to suppress or prevent malignant tumors or to treat viruses, e.g., by combining the two ingredients in a product consumed by a human, dog, or cat, such as an aqueous liquid such as grape juice, the ingredients being provided in containers with instructions for use, or in finished products, especially with support of tests demonstrating the effectiveness of the treatment for, e.g., preventing tumors in populations known to be at risk of developing tumors, or, treating existing cancers in combination with other cancer drugs such as anastrozole and/or fulvestrant and/or artemisinin either concurrently or sequentially to prevent the cancer from growing when the cancer drug is not being used, or in the treatment of viruses.


CN102485237
Sweet wormwood herb brewed liquid, brewing technology thereof, and preparation method of starter thereof
Inventor(s):     ZHIFEN BEI, ET AL.
The invention relates to sweet wormwood herb brewed liquid, which is a pure brewed liquid brewed with a modified ancient traditional fermentation technology and with sweet wormwood herb and glutinous rice as raw materials. A main effective component of sweet wormwood herb, artemisinin, exists in plant cell glands, and is not soluble in water. Currently, both organic solvent extraction and chemical synthesis are disadvantaged in complicated technology, high cost and a problem of toxicity. According to the invention, a multi-strain starter which has both functions of saccharification and fermentation, a rice feeding method, and a composite fermentation technology are adopted. The technology is characterized in that: the temperature during the whole brewing process is controlled below 35 DEG C; a fermentation period is long (above 90 days); and prepared sweet wormwood herb medicinal brewed liquid contains various nutrients and complete effective components in sweet wormwood herb. As a result of cytotoxic tests upon breast cancer cells and lung cancer cells, the cells can be completely killed within 20h. The brewed liquid is taken by more than 20 people. It is found that the brewed liquid has certain ideal effects against some stubborn diseases, and no toxic or side effect is found.


US2010279976
USE OF ARTEMISININ AND ITS DERIVATIVES IN CANCER THERAPY
Inventor(s):     WANG HUI, et al.
A method for treating cancer in a mammal includes administering to the mammal in need thereof a therapeutically effective amount of artemisinin (ART) or its derivative, such as dihydroartemisinin (DHA), artemether (ARM), or artesunate (ARS) alone or in combination with a chemotherapeutic agent, such as gemcitabine and carboplatin. A method for inhibiting tumor cell proliferation includes contacting a tumor cell with ART or its derivative, such as DHA, ARM, and ARS, in an amount effective to inhibit tumor cell proliferation or in combination with a chemotherapeutic agent, such as gemcitabine and carboplati


KR20100026030
AN EXTRACT OF EFFECTIVE COMPONENT FOR PROTECTION AGAINST OTHER KINDS OF CANCER, METHOD FOR EXTRACTING THE SAME AND AN ANTI-CANCER MEDICINE COMPRISING IT
Inventor(s):     KWEON OH KWAN, et al.
PURPOSE: A method for isolating a compound isolated from Artemisa annua extract is provided to easily extract artemisinin, arteannuin B1, and artemisinic acid and ensure anti-cancer effect. CONSTITUTION: An Artemisa annua extract isolated using dichloromethane contains a compound of chemical formula 1, 2 or 3 as an active ingredient. A method for isolating from Artemisa annua extract comprises: a step of extracting Artemisa annua using methylene chloride through reflux to obtain Artemisa annua methylene chloride extract; a step of performing silica column chromatography and adding organic solvent to obtain eluted liquid; a step of fractioning eluted liquid through silica column chromatography to obtain first fraction; and a step of performing HPLC to obtain second fraction.


JP2009051803
DISCOVERY OF NEW METHOD FOR EXTRACTION
Inventor(s):     NAGAURA YOSHIAKI, et al.
PROBLEM TO BE SOLVED: To provide a new method for extraction, that is, a prophylactic means and a therapeutic means for patients suffering from viral diseases such as malaria mediated by Anopheles or HIV or HCV or HPV or highly virulent H5N1 type influenza virus or cancer. ;SOLUTION: Bark of Quina or an annual herb Artemisia annua is finely powdered (hereinafter abbreviated to the Artemisia annua). A tea bag containing the resultant fine powder of the Artemisia annua introduced together with sodium hydrogencarbonate or sodium carbonate into the interior of the tea bag is formed. An aqueous solution or an alcohol aqueous solution in the interior of a tea cup in which the pH concentration of the aqueous solution in the interior of the tea cup is regulated to >=8.0 is used to carry out alkali extraction of artemisinin or an artemisinin derivative which is an active ingredient contained in the fine powder of the Artemisia annua. The resultant aqueous solution or alcohol aqueous solution is drunk or internally used as the therapeutic means for the patients suffering from the viral diseases such as malaria which is the name of disease or HIV or the cancer.




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