US20070111936A1

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

Info

Publication number: US20070111936A1
Application number: US11/274,906
Authority: US
Inventors: Vladimir Pak; Stephane Gagne
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-11-15
Filing date: 2005-11-15
Publication date: 2007-05-17
Also published as: CN101437531A; WO2007056852A1; JP2009515909A; RU2438695C2; UA94924C2; US20080318840A1; KR20080067376A; EP1959978B1; CA2669549A1; RU2008123804A; EP1959978A4; US8071547B2; EP1959978A1

Abstract

The invention relates to a composition comprising exogenous alpha-fetoprotein, a first compound reversibly bound to exogenous alpha-fetoprotein in vitro, and an unbound second compound wherein the first and second compound are anticancer drugs or combinations of anticancer drugs and wherein the second compound reversibly binds to recycled exogenous alpha-fetoprotein in vivo. A process for the butanol extraction of alpha-fetoprotein obtained from porcine 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 invention also relates to a method of using these compositions to prevent, treat or inhibit a malignant neoplasm expressing an alpha-fetoprotein receptor.

Description

FIELD OF INVENTION

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.

BACKGROUND OF THE INVENTION

During ontogeny in mammals and other vertebrates, some proteins are produced at various stages of embryonic fetal and neoplastic growth. These proteins are termed oncofetal antigens as they can be found in fetal and tumor-associated growth. They may appear, disappear or vanish to small amounts depending on the specific phase of development. 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). 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. There are also genetic variants of HAFP, including phase-specific expression of HAFP MRNA.
Mizejewski G. J. et al., Tumour Biol. 7(1): 19-36, (1986) describes the cyclic physiology of AFP as the “developmental clock” where the structure and function of AFP changes throughout the course of development. In humans, AFP functions as a tumor marker and it also functions as a fetal defect marker during embryogenesis.
Various chemical preparations, such as alkylating agents, antimetabolites, alkaloids, antibiotics, hormones and immunomodulators, have been used in the prior art to treat cancer. However, these preparations lack specificity in that they do not specifically target tumor cells.
HAFP has been successfully used as a carrier or transporter for the specific delivery of anticancer drug conjugates to tumor cells. The overexpression of HAFP receptors (HAFPR) on the surface of malignant cells versus the negligible incidence on normal cells has promoted the use of HAFP as a carrier or transporter for delivering anticancer drugs (Severin, S. E. et al., 37(2):385-92,1995. and Severin, S. E. et al., Dokl Akad Nauk. 366(4): 561-4, 1999.) to cancer cells. 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. doxorubicin, daunomycin, calichemicin, carboxyphospharnide, cleomycetin, chlorbutin, cis-platinum and mothotrexate (Moskaleva et al., Cell Biol Int. 21(12):793-91997, 1997). However, the binding procedure of HAFP to these anticancer drugs requires a prior step of conjugation with HAFP. This additional conjugation step not only complicates the preparation of such anticancer conjugates (i.e. HAFP-doxorubicin) but forces the covalent binding of HAFP and anticancer drug by chemical modification of HAFP. In addition, since 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. Moreover, 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. This loss of anticancer drugs requires a higher dosage which leads not only to an increase in the costs of production but also increases the possibility of potential side effects to the treatment. This step must occur before the conjugation step and contributes to the high cost of purified HAFP. HAFP may also be purified by use of HAFP-specific monoclonal antibodies which are very expensive.
Since many tumor cells demonstrate multi-drug resistance (MDR), the use of only one anticancer drug in the prior art methods (i.e. HAFP-estrone-doxorubicin conjugate) is a limiting factor in the treatment of malignant neoplasms. Finally, the prior art anticancer drugs are alkylating agents and antibiotics which target the DNA and therefore are not fully effective as it is known that 50% of cancers have damaged p53 protein (Bykov, V J et al., Nat. Med. 8(3):282-8, 2002).
There is therefore a need for a simple, inexpensive, non-invasive and efficient method of specifically killing cancer cells.
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.

SUMMARY OF THE INVENTION

Briefly stated, the invention provides compositions and methods for preventing, treating or inhibiting a malignant neoplasm expressing an alpha-fetoprotein receptor (AFPR). Such compositions 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.
Within certain specific embodiments, 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.
In a preferred embodiment the first and second compounds are anticancer drugs which induce apoptosis. In a most preferred embodiment 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.
Within certain specific embodiments, the compositions as described above are comprised of first and second compounds which have separate dosage forms.
Within other aspects of the invention, the 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.
Within other aspects, 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.
Within other aspects, 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.
Typically the anticancer drugs trigger cancer cell apoptosis by targeting the mitochondria. Preferably 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.
In one embodiment the first compound may comprise more than one anticancer drug (i.e. betulinic acid and CD 437).
In another embodiment the second compound may comprise more than one anticancer drug (i.e. betulinic acid and CD 437). In yet another embodiment 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. These and other embodiments, features and advantages of the present invention will become apparent with reference to the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

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.
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.

DETAILED DESCRIPTION OF THE INVENTION

The term reversible as used herein 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.
The term anticancer drug as used herein means a substance that when administered to a patient induces apoptosis of the cancer cells expressing AFPR. Examples of 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.
The term a therapeutically effective amount as used herein 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.
The term 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.
The term exogenous as used herein 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.
This is possible since there are different binding domains on AFP (Mizejewski, Gerald J., Exp. Biol. Med. 226(5): 377-408 at 383, 2001.) for binding different anticancer drugs. 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. 92(13):1042-53., (2000) reviews the mechanism of inducing apoptosis through the mitochondrion destruction by acting on the mitochondrial membranes and/or on the permeability transition pore complex (PTPC) as well as listing the drugs that may induce apoptosis through such pathway.
Depending on the characteristics of the person to be treated and the aggressiveness of the disease, 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. For mice, the amount is between 3.7×10⁻⁴mg and 6.3×10⁻³mg. (Natural Compounds in Cancer Therapy, 2001). In addition, in preparing the AFP for use in the current invention, a non-aqueous solvent is used to de-bind the AFP from its existing binding elements such as ligands.
Typically 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. As stated above, 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). Typically 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.
Typically, the second compound is present in an amount that is at least 10-fold lower than the amount used in the prior art methods. For example, in Pezzuto et al. the recommended dosage of betulinic acid is 3000 μg, in the current invention betulinic acid (second compound) can be used at 150 μg.
It was noticed that PAFP is unstable and can aggregate, precipitate or become inactivated during the manipulations. Therefore, 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. Once bounded to PAFP, an anticancer drug will not de-bind until the anticancer drug is delivered to the tumor cell expressing AFPR. Typically the first compound is one type of anticancer drug and is present in a daily concentration of no more than 150 μg. Typical anticancer drugs include, but are not limited to thapsigargin, atractyloside, betulinic acid, CD437, arsenic trioxide and lonidamine. As for example, betulinic acid is derived from Betulin, a substance found in abundance in the outer bark of white birch trees (Betula alba). Other examples of 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.
The 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.
The 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.
The inventive compositions may be mixed with appropriate fillers in relation to the method of administration to be chosen. For example, 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.

EXAMPLE 1

PAFP Isolation and Purification
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^thand 14^thweek 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^thweek 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. Next 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. As shown in 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.

EXAMPLE 2

PAFP is Bioequivalent to HAFP
It was not known if PAFP exhibits the same biological properties as HAFP. Therefore, we tested our preparation of PAFP with 2 immuno enzyme kits for quantitative determination of alpha-fetoprotein in human serum and amniotic fluid. The first one is a membrane EIA Alpha-fetoprotein test (cat. #410-1 produced by IND Diagnostic Inc., Vancouver, Canada) which uses a monoclonal antibody to HAFP. The results were negative confirming a difference of HAFP and PAFP in their chemical structure. The second kit that was used contains a polyclonal antibody to HAFP (T-8456-T-8456-AFP-EIA-BEST-Strip“, manufactured by Vector-BEST, Novosibirsk, Russia) which detected the presence of PAFP. It was not known if PAFP would provoke the same biological response as HAFP (i.e. apoptosis) as the amino-acid ratio for the porcine source is different from the human source. To determine if PAFP is bio-equivalent to the human AFP (HAFP) we:

- a. checked if PAFP would bind to HAFPR; and
- b. checked if PAFP would be able to bind to anticancer drugs.
  With the mice (see examples 4 and 6 below) and human trials (see example 5 below) using the PAFP and the anti-cancer drugs (atractyloside, thapsigargin and betulinic acid), we were able to confirm the bio-equivalence of PAFP.

EXAMPLE 3a

In Vitro Binding of PAFP and the First Compound
The upper phase containing unbound PAFP and the first compound (at least one type of anticancer drug) 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 use the diafiltration process to remove small molecules and other non-desired elements (i.e. impurities) such as the salt which is used as a biological buffer during the collection of the raw material (embryo's fluids). 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.
Between 3 and 4 “wash” (3-4 volumes of washing solution (i.e. water) for one volume of the retentate) are performed to further concentrate and eliminate any remaining impurities such as ballast proteins. 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.

EXAMPLE 3b

Atractyloside

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.

EXAMPLE 3c

Thapsigargin

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.

EXAMPLE 3d

Betulinic Acid

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.

EXAMPLE 4

In vivo data—leukemia: PAFP+first compound kills tumor cells in mice after 24 days of tumor cells inoculation.

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
The mice were transplanted with 20,000 P-388 cells subcutaneous in side of the body. 100% survived the transplant.

Each group of 10 mice received daily 0.2 ml of the related substance the next day after they have been inoculated with the P-388 cells.

TABLE 1


Groups of 10 mice	Survival	Tumor appearance

1	Died 1, but because	Tumors very small
	of accident due to
	not right tumor cells
	transplantation
2	Died 1	Tumors are midsize
3	Died 1	Tumors are midsize
4	Died 4	Tumors are slightly
		over midsize
5	Died 5	Tumors are larger
		then in group 4
control, oil	half animals died (5)	Tumors are
		extremely large
control, water	All animals died (10)	Tumors were
		mesurable

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.
Results:

1. The growth of the tumors in preparation A and T groups is suppressed by 85% and 79% correspondently as compared to controls groups (feed with 0.2 ml of water or 0.2 ml of oil) up to the 24-th day after the tumor was injected. At that day, all the mice in controls were dead.
2. The survival rate was measured when 50% of the mice were alive. In the group taking the “Preparation A”, the mice increased their survival by 1.5 times compared to the control (water). For the group taking the “Preparation T”, the mice survived 1.35 times longer than the control group taking only water. At the 38th day of the experiment, 3 mice (out of 10) in the group taking the “Preparation A” were still alive and one mice was still alive in the group taking the “Preparation T” (FIG. 4).
3. In one of three mice that survived (group of “Preparation A”), we observed a regression of the growth of the tumor.
Conclusions:
1. “Preparation A” group and “Preparation T” groups, at the demonstrated concentrations, can efficiently inhibit the growth of tumor.
2. The intake of anticancer drugs atractyloside and thapsigargin when bound with PAFP can enlarge the longevity of life of the cancer suffering animals.

EXAMPLE 5

In Vivo Data—Tumor: PAFP-atractyloside Product Kills Tumor Cells in Humans
Eight patients (classified stage IV in their cancer progression by their respective doctors) ingested PAFP-atractyloside product for one month and have been followed by their doctor for an additional period of four months. The patients have reported several benefits such as a reduction in the growth of the main tumor, a reduction in size of the main tumor in some cases, a reduction in the spread of metastasis and an overall better quality of life. The conclusions of that human study were the following:

1. The oral intake of PAFP-atractyloside product (in starch and oil) in daily doses of 2-6 capsules by oral intake was found to be safe and had no side effect during the course of the treatment.
2. The decrease in tumor and metastasis size as well as the occasional pain at the location of metastasis confirms the specific anti-cancer action of PAFP-atractyloside product.
3. The improvement of self feeling and physical activity can be induced by PAFP-atractyloside product alone or in combination with other drugs.
4. It seems that there is a dose dependant response of the PAFP-atractyloside product observed by a reduction in tumor mass and metastasis and sometime accompanied by an acute immune reaction (increase in body temperature, local pain at the site of tumor/mestastasis) as the dose was increased.
5. The PAFP-atractyloside product is orally bioavailable from the confirmation of its anti-cancer properties.

EXAMPLE 6

In vivo data—leukemia: a composition comprising exogenous PAFP reversibly bound to a first compound in vitro and a second compound kills tumor cells in mice

#1—Control solution is water
#2—Control solution is oil
#3—PAFP reversibly bound to a first compound (betulinic acid) in vitro (PAFP-betulinic acid product)
#4—A composition comprising exogenous PAFP reversibly bound to a first compound (betulinic acid) in vitro (PAFP-betulinic acid product) and an additional second compound (betulinic acid)

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. By comparing the group #3 where there is no additional amount of betulinic acid (i.e. PAFP-betulinic acid product. alone), with the group #4 where there is an additional amount of betulinic acid, we clearly see the advantage of using a second compound which can be delivered to cancer cells by reversibly binding to the recycled exogenous PAFP. Surprisingly, there is a clear additional benefit (synergistic effect) of using this method (group #4) which results are superior over the use of PAFP-betulinic acid product alone (group #3). These results suggest the use of any anticancer drug—having binding capabilities to AFP to treat malignant neoplasms expressing the receptors of AFP by first bringing the bound anticancer drug or chemotherapeutic agent to the tumor cells and then, through the delivery mechanism of action, bringing an additional, second anticancer drug or chemotherapeutic agent which will bind in vivo and improve the therapy and health benefits.
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 μg within 0.2 ml of oil acting as the carrier. In a weight ratio, Betulinic acid is present at a daily dose of 19.6 nanograms.

EXAMPLE 7

In vivo data-tumor: a composition comprising exogenous PAFP reversibly bound to a first compound in vitro and a second compound used to kill solid tumour type of cancers in human.
Preliminary results in 5 patients (4 women and one man) with solid type of tumours.
Patient #1: woman, 57 years old, localized breast cancer
Patient #2: woman, 63 years old, breast cancer metastasized to the bones
Patient #3: woman, 44 years old, with ovarian breast cancer.
Patient #4: woman, 60 years old, breast cancer with metastasis to the lymph nodes
Patient #5: man, 58 years old, testicular cancer.

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.
The patients took a daily dosage of two softgels.
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 μg of PAFP and 6 μg of betulinic acid per softgel. The excess or additional betulinic acid is present in an amount of 150 μg per softgel.
Each patient took 2 softgels per day (on an empty stomach, one in the morning and one before bedtime).
The preliminary results have shown that 4 out of 5 patients have shown a slow down of their cancer progression and an overall better quality of life (reported by the patients by having less pain, felling more energetic and a having better appetite). The patients did not report any side effects related to the treatment with the softgels.
The foregoing are specific examples of certain aspects of the present invention. Many other embodiments, including modifications and variations thereof, are also possible and will become apparent to those skilled in the art upon a review of the invention as described herein. Accordingly, all suitable modifications, variations and equivalents may be resorted to, and such modifications, variations and equivalents are intended to fall within the scope of the invention as described herein and within the scope of the appended claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide exemplary, procedural or other details supplementary to those set forth herein.

REFERENCES

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Costantini et al., mitochondrion as a novel target of anticancer chemotherapy. J. Natl. Cancer Inst. 92(13):1042-53., (2000).
Fulda, S. et al., activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J. Biol. Chem. 18; 273 (51): 33942-8, 1998b.
Mizejewski, G. J. et al., studies of the intrinsic antiuterotropic activity of murine alpha-fetoprotein. Tumour Biol. 7(1): 19-36, (1986).
Mizejewski, Gerald J., alpha-fetoprotein structure and function relevance to isoforms, epitopes and conformational variants. Exp. Biol. Med. 226(5): 377-408, 2001.
Moskaleva et al., alpha-fetoprotein-mediated targeting—a new strategy to overcome multidrug resistance of tumour cells in vitro. Cell Biol Int. 21(12):793-91997, 1997.
Pak, V. N. et al., US Patent Application Publication No. 2003/0186945) Method of Treatment of Malignant Neoplasms and Complex Preparation having Antineoplastic Activity for Use in Such Treatment.
Pezzuto et al., US Patent Application Publication (Patent Application no. 2003/0186945 A1), method of preparing and use of prodrugs or betulinic actid derivatives, October 2003.
Severin, S. E. et al., Alpha-fetoprotein-mediated targeting of anti-cancer drugs to tumor cells in vitro. Biochem Mol Biol Int. 37(2):385-92, 1995.
Severin, S. E. et al., Antitumor activity of a covalent conjugate of the endiene antibiotic esperamicin A1 with human alpha-fetoprotein. Dokl Akad Nauk. 366(4): 561-4, (1999).

Claims

1. A composition comprising exogenous alpha-fetoprotein (AFP), a first compound reversibly bound to exogenous AFP 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 AFP in vivo.

2. The composition of claim 1 wherein the first compound and the second compound are the same anticancer drug.

3. The composition of claim 1 wherein the first compound and the second compound are different anticancer drugs.

4. The composition of claim 1 wherein the first compound comprises at least two anticancer drugs.

5. The composition of claim 1 wherein the second compound comprises at least two anticancer drugs.

6. The composition of claim 1 wherein the anticancer drugs induce apoptosis.

7. The composition of claim 6, where the anticancer drugs are selected from the group consisting of atractyloside, thapsigargin, betulinic acid, CD 437, arsenic trioxide and lonidamine.

8. The composition of any one of claims 1 to 7 wherein the first compound and the second compound comprise separate dosage forms.

9. A method of preventing, treating or inhibiting a malignant neoplasm, expressing an alpha-fetoprotein receptor (AFPR), the method comprising administering an effective amount of a composition comprising exogenous AFP, a first compound reversibly bound to exogenous AFP in vitro, and a second compound to a in need thereof, wherein the first compound and the second compound are anticancer drugs, and wherein the second compound reversibly binds to recycled, exogenous AFP in vivo.

10. The method of claim 9 wherein the first compound and the second compound are the same anticancer drug.

11. The method of claim 9 wherein the first compound and the second compound are different anticancer drugs.

12. The method of claim 9 wherein the first compound comprises at least two anticancer drugs.

13. The method of claim 9 wherein the second compound comprises at least two anticancer drugs.

14. The method of claim 9 wherein the anticancer drugs induce apoptosis.

15. The method of claim 14 wherein the anticancer drug are selected from the group consisting of atractyloside, thapsigargin, betulinic acid, CD 437, arsenic trioxide and lonidamine.

16. The method of any one of claims 9 to 15 wherein the first compound and the second compound are administered in separate dosage forms.

17. A process for the butanol extraction of AFP comprising the following sequential steps:

collecting porcine blood and amniotic fluid during early embryogenesis;

separating the blood and the amniotic fluid into a supernatant and a precipitate;

collecting the supernatant resulting from step (b);

concentrating the supernatant resulting from step (c) to form a concentrated solution;

adding butanol to the concentrated solution resulting from step (d) to produce a 5-10% butanol solution;

stirring the butanol solution resulting from step (e);

separating the butanol solution resulting from step (f) into an upper non-aqueous phase and a lower aqueous phase; and

collecting the non-aqueous phase resulting from step (f) to produce a final solution having unbound AFP.

18. A process for the in vitro binding AFP and a first compound comprising the following sequential steps:

mixing unbound AFP and a first compound;

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;

filtering the AFP-first compound mixture resulting from step (b) to form a filtrate having impurities and a retentate;

washing the retentate resulting from step (c) to form a washed retentate;

filtering the washed retentate resulting from step (d) to form a final solution; and

drying the final solution resulting from step (e) to form a dried, AFP-first compound product.