US20070111936A1 - Complex of alpha-fetoprotein and inducers of apoptosis for the treatment of cancer - Google Patents

Complex of alpha-fetoprotein and inducers of apoptosis for the treatment of cancer Download PDF

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US20070111936A1
US20070111936A1 US11/274,906 US27490605A US2007111936A1 US 20070111936 A1 US20070111936 A1 US 20070111936A1 US 27490605 A US27490605 A US 27490605A US 2007111936 A1 US2007111936 A1 US 2007111936A1
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compound
afp
anticancer drugs
composition
pafp
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Vladimir Pak
Stephane Gagne
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Priority to US11/274,906 priority Critical patent/US20070111936A1/en
Priority to CA002669549A priority patent/CA2669549A1/en
Priority to KR1020087013966A priority patent/KR20080067376A/ko
Priority to JP2008540415A priority patent/JP2009515909A/ja
Priority to CNA2006800510671A priority patent/CN101437531A/zh
Priority to PCT/CA2006/001867 priority patent/WO2007056852A1/en
Priority to EP06804732.3A priority patent/EP1959978B1/de
Priority to UAA200808027A priority patent/UA94924C2/uk
Priority to RU2008123804/15A priority patent/RU2438695C2/ru
Priority to US12/093,842 priority patent/US8071547B2/en
Publication of US20070111936A1 publication Critical patent/US20070111936A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/36—Arsenic; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the field of medicine, oncology in particular, and can be used for the treatment of cancer in humans and animals.
  • AFP Alpha-fetoprotein
  • AFP is an example of one such oncofetal antigen and is a member of the albuminoid gene superfamily. The molecular weight of AFP can vary from 64,000 to 72,000 daltons depending on the animal and origin and the method used for its purification. AFP is a glycoprotein containing 3-5% carbohydrate which percentage also varies depending on the animal and origin.
  • AFP appears to be present in two basic molecular forms: 1) an unbound form, and 2) a bound form in which AFP is complexed to various ligands (e.g. fatty acids, estrogens, phytosteroids) wherein there exists different conformations (holoforms) of AFP depending on the nature and concentration of the bound ligand(s).
  • ligands e.g. fatty acids, estrogens, phytosteroids
  • HAFP Human AFP
  • HAFP Molecular variants of Human AFP
  • HAFP also exist and are attributed to carbohydrate microheterogeneity (i.e. different carbohydrate moieties binding at various glycosylation sites on HAFP) and alternations in isoelectric points.
  • HAFP phase-specific expression of HAFP MRNA.
  • AFP functions as a tumor marker and it also functions as a fetal defect marker during embryogenesis.
  • HAFP has been successfully used as a carrier or transporter for the specific delivery of anticancer drug conjugates to tumor cells.
  • HAFP receptors HAFP receptors
  • the high specificity of HAFP for cancer cells contributes to a safer and more effective drug profile. Conjugation of HAFP with numerous anticancer drugs has been reported (e.g.
  • HAFP exists in so many different forms, the amount of unbound, properly glycosylated HAFP available for conjugation with an anticancer drug is present in such a minute amount that the HAFP:anticancer drug conjugation molar ratio is in the range from 1:3 to 1:5, requiring a higher quantity of the formed conjugate to obtain the desired clinical outcome.
  • these prior art methods have all focused on invasive methods of administration (e.g. injection) using human AFP which is an expensive and a limited source of AFP.
  • the method of purification of HAFP in these prior art preparations requires the use of affinity chromatography to remove albumin. The removal of albumin is required to prevent the loss of the anticancer drugs also binding to albumin.
  • HAFP may also be purified by use of HAFP-specific monoclonal antibodies which are very expensive.
  • the present invention may provide one or more of the foregoing advantages or other advantages which will become apparent to persons skilled in the art after review of the present application.
  • compositions and methods for preventing, treating or inhibiting a malignant neoplasm expressing an alpha-fetoprotein receptor comprise an exogenous alpha-fetoprotein, a first compound reversibly bound to exogenous alpha-fetoprotein in vitro, and a second compound wherein the first compound and the second compound are anticancer drugs and wherein the second compound reversibly binds to recycled, exogenous alpha-fetoprotein in vivo.
  • the invention also provides for a process for butanol extraction of alpha-fetoprotein obtained from blood and amniotic fluid during early embryogenesis and a process for the in vitro binding of alpha-fetoprotein and a first compound are also described.
  • the first and second compounds within the compositions described above are the same anticancer drug. In other specific embodiments, the first and second compounds are different anticancer drugs. In some specific embodiments, the composition includes a first compound comprising at least two anticancer drugs. In other specific embodiments, the composition includes a second compound comprising at least two anticancer drugs. The at least two anticancer drugs may be the same anticancer drugs or they may be different anticancer drugs.
  • the first and second compounds are anticancer drugs which induce apoptosis.
  • the first and second compounds are anticancer drugs which induce apoptosis and are selected from the group consisting of, but not limited to atractyloside, thapsigargin, betulinic acid, CD 437, arsenic trioxide and lonidamine.
  • compositions as described above are comprised of first and second compounds which have separate dosage forms.
  • compositions described above are provided in therapeutically effective amounts to a patient, as a method of preventing, treating or inhibiting a cancerous cell expressing an alpha-fetoprotein receptor.
  • the invention is a process for the butanol extraction of the alpha-fetoprotein which typically comprises the following steps: a) collecting porcine blood and amniotic fluid during early embryogenesis, b) separating the blood and the amniotic fluid into a supernatant and a precipitate, c) collecting the supernatant resulting from step (b), (d) concentrating the supernatant resulting from step (c) to form a concentrated solution, (e) adding butanol to the concentrated solution resulting from step (d) to produce a 5-10% butanol solution, (f) stirring the butanol solution resulting from step (e); (g) separating the butanol solution resulting from step (f) into an upper non-aqueous phase and a lower aqueous phase; and (h) collecting the non-aqueous phase resulting from step (f) to produce a final solution having unbound AFP.
  • the invention is a process for the in vitro binding of exogenous AFP and a first compound which typically comprises the following sequential steps: (a) mixing unbound AFP and a first compound, (b) incubating the unbound AFP and the first compound for about 10 minutes wherein the first compound reversibly binds to the unbound AFP to form an AFP-first compound mixture, (c) filtering the AFP-first compound mixture resulting from step (b) to form a filtrate having impurities and a retentate, (d) washing the retentate resulting from step (c) to form a washed retentate, (e) filtering the washed retentate resulting from step (d) to form a final solution, and, (f) drying the final solution resulting from step (e) to form a dried, AFP-first compound product.
  • the anticancer drugs trigger cancer cell apoptosis by targeting the mitochondria.
  • the AFP is from a mammalian source, such as human or rodent. More preferably the AP the AFP is from a porcine source (PAFP).
  • the AFP may be isolated from nature or it may be a recombinant form of AFP.
  • the first compound may comprise more than one anticancer drug (i.e. betulinic acid and CD 437).
  • the second compound may comprise more than one anticancer drug (i.e. betulinic acid and CD 437).
  • the first compound and the second compound may both comprise more than one anticancer drug.
  • the anticancer drugs used in this invention have been selected for their specificity in a) binding AFP, and b) for triggering cancer cell apoptosis through direct mitochondria destruction.
  • FIG. 1 Schematic of a pathway leading to apoptosis of cancer cells.
  • FIG. 2 Gel Electrophoresis of AFP concentrate alone (lane “C”), AFP concentrate bound to Atractyloside (lane “A”) and standard molecular weight markers. Lane “B” is discarded for another experiment.
  • FIG. 3 Tumor volume growth over time (in days) after innoculation of tumor cells P-388 in mice.
  • 1-preparation A PAFP-atractyloside product
  • 2-preparation T PAFP-thapsigargin product
  • 3-preparation S Spleen extract
  • 4-preparation A+S 5-preparation T+S
  • control oil control water.
  • FIG. 4 Survival rate of mice over time (in days) after innoculation of tumor cells P-388 in mice; 1-preparation A (PAFP-atractyloside product); 2-preparation T (PAFP-thapsigargin product); 3-preparation S (Spleen extract); 4-preparation A+S; 5-preparation T+S; control oil; control water.
  • 1-preparation A PAFP-atractyloside product
  • 2-preparation T PAFP-thapsigargin product
  • 3-preparation S Stpleen extract
  • 4-preparation A+S 5-preparation T+S
  • control oil control water.
  • FIG. 5 Tumor volume growth over time (in days) after innoculation of tumor cells P-388 in mice; 1 control (water); 2 control (oil); 3 PAFP-betulinic acid product; 4 PAFP-betulinic acid product and an additional amount of betulinic acid.
  • reversible means capable of being returned to the original (‘unbound’) condition, wherein the exogenous AFP, after delivering a compound to a tumor cell (i.e. atractyloside, thapsigargin, betulinic acid, CD 437, arsenic trioxide and lonidamine) is recycled back to the extracellular medium in an unbound form where it binds to another compound (i.e. atractyloside, thapsigargin, betulinic acid, CD 437, arsenic trioxide and, lonidamine).
  • the recycling of the exogenous AFP may occur more than two times.
  • anticancer drug means a substance that when administered to a patient induces apoptosis of the cancer cells expressing AFPR.
  • drugs suitable for use with the current invention include, but are not limited to, dexamethasone, oligomicin B, rotenone, hydroxychloroquine phosphate, quercetin, vitamin A, vitamin D2 and D3, curcumin, germander, raw soya flour, green tea, flax oil, adrenal cortex powder, olive leaf extract, garlic, paprika, DHEA, and heavy metals such as zinc, lead, copper, nickel, cadmium, and chemotherapeutic agents.
  • a therapeutically effective amount means an amount of a composition of the present invention that when administered to a patient ameliorates or alleviates a symptom of the cancer (solid or non-solid) herein described.
  • the specific dose of a composition administered according to this invention will, of course, be determined by the particular circumstances surrounding the case including, for example, the composition administered, the route of administration, the state of being of the patient, and the type of cancer being treated. Cancers suitable for treatment with the current invention are those cancers in which the cancerous cells express AFPR.
  • Some examples include, but are not limited to; bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney cancer (renal cell), leukemia, liver, lung cancer, melanoma, non-hodgkin's lymphoma, ovarian, pancreatic cancer, prostate cancer, skin cancer (non-melanoma), testis and thyroid cancer.
  • patient means all mammals including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep, pigs, and rabbits.
  • the present invention involves the use of exogenous alpha-fetoprotein (AFP) acting as a carrier or transporter for delivering anticancer compounds to cancer cells resulting in the apoptosis of the cancer cells.
  • AFP exogenous alpha-fetoprotein
  • exogenous means originating from outside the patient or organism.
  • the invention relates to a composition of exogenous alpha-fetoprotein (AFP) and a first compound reversibly bound to exogenous AFP in vitro to form an AFP-first compound product, and a second compound wherein the first compound and the second compound are anticancer drugs (i.e. atractyloside, thapsigargin, betulinic acid, CD 437, arsenic trioxide and lonidamine), and wherein the second compound reversibly binds to recycled, exogenous AFP in vivo.
  • the first compound and the second compound may be the same anticancer drug (i.e. betulinic acid) or they may be different anticancer drugs (i.e. betulinic acid and CD 437). More than one type of anticancer drug may bind to AFP in vitro.
  • binding domains on AFP Mizejewski, Gerald J., Exp. Biol. Med. 226(5): 377-408 at 383, 2001.
  • Some binding domains interact with hydrophobic drugs, while other binding domains interact with hydrophilic or amphiphilic drugs.
  • These anticancer drugs have been described in previous work or experiments to target mitochondria and induce cell apoptosis (Fulda, S. et al., J. Biol. Chem. 18; 273 (51): 33942-8, 1998., Pezzuto et al. (Patent Application #20030186945 of October 2003). Costanini et al., J. Natl. Cancer Inst.
  • the person will take a daily dosage of the inventive composition where the total daily intake of AFP will be between 0.07 mg and 1.2 mg.
  • the amount is between 3.7 ⁇ 10 ⁇ 4 mg and 6.3 ⁇ 10 ⁇ 3 mg.
  • a non-aqueous solvent is used to de-bind the AFP from its existing binding elements such as ligands.
  • the solvent is butanol, although other alcohols, such as isobutanol and pentanol may also be employed.
  • the amount of butanol to be employed is important as too small a volume does not allow for an optimal collection of the AFP fraction and, a too high volume of butanol does not allow for the collection of AFP as it gets dissolved in the total volume instead of being concentrated on the top layer of the supernatant from the concentrate.
  • butanol allows for the “de-binding” of the elements that might already be binding to the AFP and provides for a higher yield of the unbound form of AFP.
  • the composition of the current invention may contain varying molar ratios of the first and second compound, such as from an equimolar ratio to an overabundance of the first compound in relation to AFP and from an equimolar ratio to an overabundance of the second compound in relation to AFP.
  • the in vitro binding conditions of the first compound and AFP will depend on the properties of the first compound (i.e. hydrophilic or hydrophobic).
  • AFP is mixed with the first compound where the molar ratio of the AFP and the first compound ranges from 1:1 to 1:3. More typically AFP is mixed with the first compound where the molar ratio of the AFP and the first compound ranges from 1:1 to less than 1:3. This low ratio requires less quantity of the first compound to obtain a desired biological response compared to other previous methods.
  • the first compound may be composed of one or more anticancer drugs. However, these drugs must bind to different binding domains on AFP as mentioned above.
  • the second compound is present in an amount that is at least 10-fold lower than the amount used in the prior art methods.
  • the recommended dosage of betulinic acid is 3000 ⁇ g
  • betulinic acid (second compound) can be used at 150 ⁇ g.
  • the time for in vitro binding of PAFP and the first compound is 18-25° C. for 10 minutes which is considerably lower than the prior art where the standing time at 18-25° C. was for 10-12 hours (U.S. Pat. No. 6,878,688).
  • This shorter time of the in vitro binding of PAFP and the first compound may be attributed to (1) a high concentration of unbound PAFP in the concentrate obtained by the ultrafiltration and butanol extraction, and (2) the microhomogenicity of PAFP compared to the HAFP which, as stated above is microheterogenous.
  • an anticancer drug will not de-bind until the anticancer drug is delivered to the tumor cell expressing AFPR.
  • the first compound is one type of anticancer drug and is present in a daily concentration of no more than 150 ⁇ g.
  • Typical anticancer drugs include, but are not limited to thapsigargin, atractyloside, betulinic acid, CD437, arsenic trioxide and lonidamine.
  • betulinic acid is derived from Betulin, a substance found in abundance in the outer bark of white birch trees (Betula alba).
  • anticancer drugs suitable for use with the current invention include, but are not limited to, dexamethasone, oligomicin B, rotenone, hydroxychloroquine phosphate, quercetin, vitamin A, vitamin D2 and D3, curcumin, germander, raw soya flour, green tea, flax oil, adrenal cortex powder, olive leaf extract, garlic, paprika, DHEA, and heavy metals such as zinc, lead, copper, nickel, cadmium, and chemotherapeutic agents.
  • compositions of the current invention may further comprise a pharmaceutical agent to form pharmaceutical compositions.
  • Pharmaceutical compositions of the current invention can be prepared by procedures known in the art using well known and readily available ingredients.
  • compositions may be administered in any currently known method of administration including but not limited to oral dosage forms (i.e. capsules, softgels, and tablets), and suppositories, injectables and topical formulations.
  • oral dosage forms i.e. capsules, softgels, and tablets
  • suppositories injectables and topical formulations.
  • the inventive compositions may be mixed with appropriate fillers in relation to the method of administration to be chosen.
  • omega-3 fatty acids is a good choice of vehicle as it serves as limiting bacteria growth and improves the ability for the inventive compositions to be taken up by the cancer cell which needs more fatty acids than normal cells due to their need in building their cell membrane.
  • Fatty acids also protect the AFP compositions from digestion by blood proteases.
  • PAFP is collected from the liver and blood of porcine embryos, the amniotic fluid, and the placenta.
  • the time of collection of the fluids is crucial during the pregnancy since it affects the properties of PAFP influencing the parameters and success of the pre-binding step. If collected too early or too late, the glycosylation of PAFP is different and the subsequent binding properties of PAFP are changed.
  • the best time to collect the raw material containing PAFP is between the 6 th and 14 th week of embryogenesis.
  • the period of collection of the fluids from the embryo is important as the glycosylation of PAFP varies during embryogenesis and the yield of fluids diminishes significantly after the 14 th week of gestation.
  • the blood and amniotic fluid is kept at 4-10° C. for 12 to 24 hours for natural sedimentation.
  • the supernatant is collected and transferred to a different container.
  • the supernatant is concentrated 3-5 times by ultrafiltration using a 50 kDa membrane. During the ultrafiltration process, the temperature does not exceed 15° C. This concentration step results in a high yield of PAFP.
  • butanol is added to a final concentration of between 5%-10%. Typically the final concentration of butanol is about 8%. After stirring the raw material and butanol for 2 minutes, we let the mixture incubate for one minute so that the solution separates into an upper non-aqueous phase and a lower aqueous phase. We then collect the upper layer.
  • the upper layer contains unbound PAFP which will be used after diafiltration (to remove Butanol) for the in vitro binding of PAFP and anti-cancer drugs.
  • This extraction step increases the yield of PAFP in the unbound form.
  • the higher the concentration of PAFP in this layer the higher the quantity of anticancer drugs that can bound to the PAFP thus providing for a high output of PAFP-anticancer drug product.
  • FIG. 2 we can see, in lane A and lane C, two major protein bands.
  • the upper band (70 kDA) is represented as the PAFP and the lower band (66 kDA) which correspond to standard molecular weight of albumin.
  • HAFP human AFP
  • the upper phase containing unbound PAFP and the first compound are mixed for one minute and will incubate for an additional 10 minutes at 10-15° C. to allow for the binding of PAFP and the first compound to form a PAFP-first compound mixture.
  • the incubation may occur for a longer time however we find that there is no significant benefit to increasing the time of incubation.
  • the PAFP-first compound mixture then undergoes ultrafiltration using a 50 kDa membrane.
  • We also use the diafiltration step to get rid of any unbound first compound.
  • the non-desired elements from the diafiltration are discarded as filtrate and the retentate, containing the PAFP-first compound mixture is used in the next step.
  • Albumin is not removed from the PAFP-first compound mixture as albumin does not interfere with the efficacy of the current inventive compositions.
  • washing solution i.e. water
  • a final filtration step is performed on a 0.22 micron membrane to obtain a final sterile solution.
  • Albumin is not removed from the final product as it does not interfere with the efficacy of the inventive composition.
  • the final solution is flash-frozen at ⁇ 45° C. until it is completely frozen.
  • the PAFP-first compound product is then dried using a freeze dryer until the layer is completely dried.
  • the dried PAFP-first compound product is then grinded to fine particles making sure that the temperature of the powder doesn't exceed 35° C.
  • a powder of 80 mesh or smaller will facilitate the incorporation in various preparations or delivery systems.
  • AFP retentate 1 liter of AFP retentate with a final protein concentration of 35 mg/ml (approximately 21 grams of AFP per liter) is combined with 50 ml of water with dissolved 250 mg of atractyloside (1:1 molar ratio AFP:actractyloside). After 10 minutes at 4-15° C. exposure, the mixture was ultrafiltrated and diafiltrated with water (2-3 volumes) and 1 liter of the final solution was collected for freeze-dry process to produce the AFP-atractyloside product.
  • AFP retentate 0.5 liter of AFP retentate with a summary protein concentration of 8 mg/ml (approximately 1.7 grams of AFP per liter) was combined with 10 mg of Thapsigargin dissolved in 10 ml of alcohol (1:1 molar ratio AFP:thapsigargin). After 30 minutes at 15-25° C. exposure, the mixture was ultrafiltrated and diafiltrated with water (2-5 volumes) and 0.2 liter of the final solution was collected for freeze-dry process to produce the AFP-thapsigargin product.
  • AFP retentate 2 liters of AFP retentate with a summary protein concentration of 20 mg/ml (approximately 28 grams of AFP for 2 liters) was combined with 500 mg of betulinic acid dissolved in 100 ml of DMSO which was added drop wise (1:1 molar ratio AFP:betulinic acid). After 10 minutes at 25-37° C. exposure and diafiltration, 1 liter of the final solution was collected for freeze-dry process to produce the AFP-betulinic acid product.
  • mice received daily 0.2 ml of the related substance the next day after they have been inoculated with the P-388 cells.
  • mice The growth of the P-388 lymphocytic leukemia in mice during daily oral intake of 0.2 ml of control and preparations 1-5 (in vehicle of oil), starting one day after the inoculation of 20,000 tumor cells per mouse is shown in FIG. 3 .
  • leukemia a composition comprising exogenous PAFP reversibly bound to a first compound in vitro and a second compound kills tumor cells in mice
  • FIG. 5 shows the growth of leukosis P-388 cells in mice during a daily oral intake of placebo groups (water #1 and oil #2) and preparations 3 and 4 suspended in 0.2 ml of oil.
  • the feeding started the day after the inoculation of 20,000 tumor cells per mouse (10 mice in each group).
  • FIG. 5 demonstrates the advantage of using a second compound in the inventive composition.
  • the second compound (betulinic acid) is dissolved in DMSO (0.5 mg/ml) and 2 microlitres of this solution is added to 0.2 ml of oil to be given daily to the treated mice of group #4.
  • Betulinic acid was meant to reversibly bind to recycled, exogenous PAFP in vivo and to be subsequently delivered to tumor cells.
  • PAFP is mixed with the first compound in a molar ratio of 1:2 where the composition is present in the amount of 1.5 ⁇ g within 0.2 ml of oil acting as the carrier.
  • Betulinic acid is present at a daily dose of 19.6 nanograms.
  • the aim of the study was to see how patients with solid type of cancers being either inoperable, refractory to existing treatments or had recurrence of cancer after operation were able to improve after receiving the product.
  • Each softgel comprises PAFP-betulinic acid product (i.e.betulinic acid reversibly bound to exogenous PAFP in vitro) and excess betulinic acid (i.e. additional betulinic acid not bound to PAFP).
  • the PAFP-betulinic acid product is in a dosage of 300 ⁇ g of PAFP and 6 ⁇ g of betulinic acid per softgel.
  • the excess or additional betulinic acid is present in an amount of 150 ⁇ g per softgel.

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US11/274,906 US20070111936A1 (en) 2005-11-15 2005-11-15 Complex of alpha-fetoprotein and inducers of apoptosis for the treatment of cancer
CA002669549A CA2669549A1 (en) 2005-11-15 2006-11-15 Compositions of alpha-fetoprotein and inducers of apoptosis for the treatment of cancer
KR1020087013966A KR20080067376A (ko) 2005-11-15 2006-11-15 알파 태아 단백질의 조성물 및 암 치료를 위한세포자멸사의 유도인자
JP2008540415A JP2009515909A (ja) 2005-11-15 2006-11-15 癌治療のためのアルファフェトプロテインおよびアポトーシス誘導剤の組成物
CNA2006800510671A CN101437531A (zh) 2005-11-15 2006-11-15 甲胎蛋白的组合物和用于治疗癌症的细胞程序死亡的诱导剂
PCT/CA2006/001867 WO2007056852A1 (en) 2005-11-15 2006-11-15 Compositions of alpha-fetoprotein and inducers of apoptosis for the treatment of cancer
EP06804732.3A EP1959978B1 (de) 2005-11-15 2006-11-15 Zusammensetzungen von alpha-fetoprotein und apoptose-induktoren zur behandlung von krebs
UAA200808027A UA94924C2 (uk) 2005-11-15 2006-11-15 Композиція глікозилованого альфа-фетопротеїну та індукторів апоптозу для лікування раку
RU2008123804/15A RU2438695C2 (ru) 2005-11-15 2006-11-15 Композиция альфа-фетопротеина и индукторов апоптоза для лечения рака
US12/093,842 US8071547B2 (en) 2005-11-15 2006-11-15 Compositions of alpha-fetoprotein and inducers of apoptosis for the treatment of cancer

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WO2010003232A1 (en) * 2008-07-07 2010-01-14 Constab Pharmaceutical Inc. Treating cancer using atractyloside
CN102666576A (zh) * 2009-10-22 2012-09-12 里卡多·J·莫罗 结合甲胎蛋白 (afp) 受体的肽和其用途
IN2014MN01944A (de) * 2012-03-06 2015-07-10 Univ Illinois
MX359210B (es) * 2012-03-06 2018-09-19 Univ Illinois Composición de combinación de pac-1 y tamoxifeno.
JP6280129B2 (ja) * 2012-11-13 2018-02-14 ナント ホールディングス アイピー，エルエルシー カルシウムフラックスアゴニスト及びその方法
FI20130341L (fi) 2013-11-19 2015-05-20 Safemed Ltd Oy Huonosti vesiliukoisten lääkeaineiden kuljetus metalli-ioneilla tasapainotetun alfafetoproteiinin mukana
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GB201418710D0 (en) 2014-10-21 2014-12-03 Iron Therapeutics Holdings Ag Dosage regimen
CA2974152A1 (en) * 2015-01-28 2016-08-04 University Health Network Drug complexes comprising alpha-fetoprotein
RU2642957C2 (ru) * 2016-03-11 2018-01-29 Федеральное государственное бюджетное учреждение науки Уральский научно-практический центр радиационной медицины ФМБА России (ФГБУН УНПЦ РМ ФМБА России) Липосома, фармацевтическая композиция и лекарственное средство для лечения местных радиационных поражений кожи, применение липосом и способ для лечения местных радиационных поражений кожи
CN106581021B (zh) * 2016-11-17 2020-04-21 浙江省医学科学院 苍术苷和5-氟尿嘧啶联合在制备预防和治疗直肠癌药物中的应用
IL274663B2 (en) 2017-11-17 2024-01-01 Univ Illinois Cancer therapy through MEK dual signaling disruption
RU2665922C1 (ru) * 2018-04-24 2018-09-05 Федеральное государственное бюджетное учреждение науки "Федеральный исследовательский центр "Казанский научный центр Российской академии наук" Фосфониевые соли на основе бетулиновой кислоты, обладающие цитотоксической активностью в отношении аденокарциномы предстательной железы

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US6534479B1 (en) 1995-01-24 2003-03-18 Martinex R & D Inc. Recombinant alpha-fetoprotein hybrid cytotoxins for treating and diagnosing cancers
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RU2179452C1 (ru) * 2000-06-22 2002-02-20 Пак Владимир Николаевич Способ лечения злокачественных новообразований и комплексный препарат, обладающий противоопухолевым действием, для осуществления способа
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EP1959978A1 (de) 2008-08-27
UA94924C2 (uk) 2011-06-25
CN101437531A (zh) 2009-05-20
CA2669549A1 (en) 2007-05-24
JP2009515909A (ja) 2009-04-16
KR20080067376A (ko) 2008-07-18
WO2007056852A1 (en) 2007-05-24
US20080318840A1 (en) 2008-12-25
US8071547B2 (en) 2011-12-06
RU2438695C2 (ru) 2012-01-10
EP1959978A4 (de) 2009-12-09
RU2008123804A (ru) 2009-12-27

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