HK1092701B - Heterocarpin, a plant-derived protein with anti-cancer properties - Google Patents

Description

Protein of plant origin having anti-cancer properties, hatecavir

The present invention relates to a plant derived human GHRH (human growth hormone releasing hormone) binding protein with anti-cancer properties.

Growth hormone ("GH") is a 191 amino acid protein that stimulates the production of many growth factors, such as insulin-like growth factor I (IGF-1), and initiates the growth of many tissues, bone, connective tissue, muscle, and internal organs. GH also has physiological activity, increasing nucleic acid, protein synthesis and lipolysis reducing urine secretion (Frohman L.A. & Kineman, r.d. handbook of physiology, hormone control of growth, Kostyo, j.l & Goodman, h.m. eds (oxford university press, new york, 1999), page 189-.

GH synthesis is regulated by positive or negative acting factors secreted by the hypothalamus. The major factor controlling GH production is "growth hormone releasing hormone" (GHRH), a 44 amino acid peptide in humans.

GH and GHRH are associated with a number of diseases. Among these, the following diseases should be mentioned in particular: cancer (especially prostate and lung cancer), acromegaly, diabetic retinopathy and nephropathy; for these pathologies, treatment with GHRH antagonists is indicated. The industry continues to investigate GHRH antagonists because of the many diseases that are potentially involved.

The applicant has therefore just isolated a new protein of plant origin having the property of binding to human GHRH.

A subject of the present invention is therefore an isolated protein obtainable by extraction from the plant Pilocarpus isophyllus (Pilocarpus isophyllus), characterized in that it has a molecular weight of about 90.9kDa and comprises fragments of the peptide sequences seq.d. No.1, seq.d. No.2 and seq.d. No.3, said protein being capable of existing in glycosylated or non-glycosylated form. For the sake of simplicity of the following disclosure, this protein is hereinafter referred to as "hecodine".

The sequences of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 are as follows:

SEQ.ID.NO.1：KLIGARYFDK

SEQ.ID.NO.2：YGEDIIVGVIDSGV

SEQ.ID.NO.3 PESESY

the nomenclature used above to define the peptide, as in the remainder of the present application, is defined by the "IUPAC-IUB Commission on Biochemical nomenclature", in which the N-terminal amino acid (amino group) appears to the left and the C-terminal amino acid (carboxyl group) appears to the right, according to the standard. The term "natural amino acid" denotes the natural L-amino acid found in natural proteins: gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp and His.

A protein is said to be "isolated" if it is removed from its original environment. Specifically, a native protein is "isolated" if it is separated from the biological material with which it coexists in the native system.

The present invention preferably relates to the non-glycosylated form of htecavir.

According to a preferred variant of the invention, the ditocarpine is obtained from a cell extract of the plant Pilocarpus isophyllus cultivated in vitro.

Furthermore, a subject of the invention is also a monoclonal antibody or an antigen-binding fragment of a monoclonal antibody that specifically binds to htemite.

Ottecan has the property of binding to human GHRH. In vitro, ottocarpine binds human GHRH and thereby inhibits cyclic AMP synthesis induced when human GHRH binds to its receptor. In vivo, in rats, the htradine/human GHRH complex is formed in the blood compartment and inhibits in a dose-dependent manner a mole-to-mole ratio of 10 μ g of human GHRH-induced GH synthesis. Ottecan has the property of binding to human GHRH.

These properties make the compounds of the invention suitable for pharmaceutical use. Thus, another subject of the invention is the use of glycosylated or non-glycosylated htecavir as a medicament. The invention also relates to a pharmaceutical composition comprising as active ingredient a glycosylated or non-glycosylated htecavir, said composition further comprising one or more pharmaceutically acceptable excipients. Another subject is the use of a glycosylated or non-glycosylated form of htecavir for the preparation of a medicament antagonistic to the effect of GHRH for the treatment of a proliferative disease, in particular cancer, for the treatment of acromegaly or for the treatment of diabetic retinopathy or nephropathy. With respect to cancer, hattachin is particularly suitable for the preparation of a medicament for the treatment of benign tumors and pancreatic tumors, hypothalamic-pituitary ganglionic cell tumors, bronchial, intestinal and hepatic tumors, sympathoadrenergic tumors, pheochromocytoma, pituitary adenoma and thyroid tumors. Heterocarpin is particularly suitable for the preparation of a medicament for the treatment of cancers that depend on the growth factor GHRH, in particular for the treatment of cancers selected from small cell lung cancer and breast cancer, especially small cell lung cancer.

One subject of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to htemite as a medicament. Also relates to a pharmaceutical composition comprising as active ingredient a monoclonal antibody or antigen-binding fragment thereof that specifically binds to htemite, said composition further comprising one or more pharmaceutically acceptable excipients. Also relates to the use of a monoclonal antibody or antigen-binding fragment thereof that specifically binds to htecavir for the preparation of a medicament that antagonizes the effects of GHRH for the treatment of proliferative diseases, especially cancer, for the treatment of acromegaly or for the treatment of diabetic retinopathy and nephropathy. For cancer, the monoclonal antibody or antigen-binding fragment thereof is particularly suitable for the preparation of a medicament for the treatment of benign tumors and tumors of the pancreas, hypothalamic-pituitary ganglion cells, bronchial, intestinal and hepatic tumors, sympathoadrenergic tumors, pheochromocytoma, pituitary adenoma and thyroid.

The invention also relates to the use of htecapine as an excipient in a pharmaceutical composition for the sustained release of GHRH. Also relates to a pharmaceutical composition comprising GHRH, ditocapi and one or more pharmaceutically acceptable excipients.

Finally, a further subject of the invention is a process which makes it possible to extract and isolate haltecavir from cells of the plant Pilocarpus isophyllus, preferably from in vitro cultures. These processes essentially comprise a step of extracting the cells from the plant isopychium pilocarpa with water at a temperature of 0 to 50 ℃, preferably 4 to 25 ℃, followed by a filtration step to separate a filtrate enriched in castocarpine from the isopychium cells, and one or more steps of separating castcarpine from the other components extracted from the plant isopychium pilocarpa.

According to a first variant, these extraction and separation methods essentially comprise the following successive steps:

a) a step of extracting the cells from the plant Pilocarpus isophyllus with water at a temperature of 0 to 50 ℃, preferably 4 to 25 ℃, said extraction step being followed by a filtration step of separating a pittecavir-rich filtrate from the Pilocarpus isophyllus cells;

b) a step of precipitating the extracted proteins, for example by adding ammonium sulphate, followed by a step of separating the precipitate (by filtration or, preferably, by centrifugation);

c) dissolving the precipitate recovered in step b) in water; and

d) a step of gel filtration chromatography in order to separate the aptocarpine from the other components of the solution.

According to another variant, these extraction and separation methods essentially comprise the following successive steps:

a) a step of extracting the cells from the plant Pilocarpus isophyllus with water at a temperature of 0 to 50 ℃, preferably 4 to 25 ℃, said extraction step being followed by a filtration step of separating a pittecavir-rich filtrate from the Pilocarpus isophyllus cells;

b) a step of degreasing (delipidation) the solution obtained in a), acidified by addition of a non-oxidizing acid (for example, hydrochloric acid, sulfuric acid or phosphoric acid) at a pH preferably ranging from 2 to 4, using liquid-liquid extraction (preferably by means of an organic solvent such as dichloromethane, heptane, hexane or cyclohexane);

c) by contacting the defatted solution obtained in b) with polyvinylpyrrolidone (or nylon 66) and then on a macroporous resin (preferably a polystyrene-based resin such as a resin)HP-20) to remove tannins by filtration;

d) adjusting the filtrate obtained after step c) to a basic pH (preferably a pH between 9 and 11) by adding a base such as ammonium hydroxide, sodium hydroxide or potassium hydroxide;

e) one or more steps of filtration on an anion-exchange resin, the eluent of the one or more filtration steps preferably being a buffer at pH9 to 11 and optionally containing a salt (e.g. sodium chloride or ammonium sulphate) concentration gradient, the purpose of the one or more steps being to separate the ditocarpine from the other components of the solution;

f) a desalting step comprising passing the solution obtained in step e) through a resin (e.g. a resin) that separates the components of the mixture based on their molecular weightG25 or200HR) and eluting the mixture from the resin with water.

Pharmaceutical compositions containing the compounds of the invention may be in solid form such as, for example, powders, pills, granules, tablets, liposomes, gelatin capsules or suppositories. The pill, tablet or gelatin capsule may be coated with a substance that protects the composition from the gastric acid or enzymes in the stomach of the subject for a sufficient time to allow the composition to pass through the small intestine of the subject without being digested. The compound may be administered locally, for example, to the actual site of a tumor. The compounds may also be used according to a sustained release method (e.g., by using a sustained release composition or infusion pump). Suitable solid carriers may be, for example, calcium phosphate, magnesium stearate, magnesium carbonate, talc, sugar, lactose, dextrose, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone and waxes.

Pharmaceutical compositions containing the compounds of the present invention may also be presented in liquid form such as solutions, emulsions, suspensions or sustained release formulations. Suitable liquid carriers may be, for example, water, organic solvents (e.g., glycerol) or glycols (e.g., polyethylene glycol), and mixtures thereof in water in various proportions.

Administration of the medicament of the present invention can be carried out by topical, oral, parenteral routes, by intramuscular injection, and the like.

The dosage of the compounds of the present invention provided for the treatment of the above-mentioned diseases or disorders varies depending on the method of administration, the age and weight and condition of the subject to be treated, and will ultimately be at the discretion of the attendant physician or veterinarian. Such an amount as determined by an attending physician or veterinarian is referred to herein as a "therapeutically effective amount".

According to the present invention, ditocarpine can be prepared by the method described hereinafter.

Preparation of htecavir

According to a preferred variant of the invention, the in vitro culture of calli or cell suspensions derived from different organs of the plant is carried out. These tissues cultured in semi-solid or liquid media are capable of biosynthesizing compounds having biological properties.

"callus" in this application refers to a macroscopic population of undifferentiated cells cultured on a semi-solid nutrient medium. The term "undifferentiated cells" in the present application denotes cells which are capable of proliferating under certain conditions in the form of callus or cell suspension without any morphogenesis. Finally, "cell suspension" refers to a microscopic population of undifferentiated cells that can form in a culture of liquid nutrient media.

The selection of nutrient media, hormones, culture conditions and the extraction of these in vitro cultures and analysis of the extracts form an integral part of the invention.

For example, cells from the Pilocarpus heterophyllus seed can be cultured in suspension according to the procedure thereafter.

These organs were sterilized according to conventional methods prior to culture. The embryo organ in vitro also serves as a callus formation (callogensis) starting material without prior sterilization. The preferred basal nutrient medium is one of the media commonly used for in vitro culture: it is Gamborg medium (in Gamborg et al, nutritional requirements for suspension cultures of soybean root cells, exp. cell Res. (1968), 50(1), 151-. The carbon source is sucrose but glucose can also be used in a concentration of 1 to 120g/l, preferably about 30 g/l. The macro-ingredient content can also be halved. The addition of auxin or auxin and cytokinin to the culture medium preferably combines these two hormones, usually 2, 4-dichlorophenoxyacetic acid and kinetin, but alpha-naphthylacetic acid (NAA), beta-indoleacetic acid (IAA), beta-indolebutyric acid (IBA) or picloram may also be combined with kinetin or Benzyl Aminopurine (BAP). The concentration of auxin may be 0.1 to 10mg/l (for example 1mg/ml may be selected) and the concentration of cytokinin may be 0.01 to 2mg/l (for example 0.06mg/ml may be selected). Vitamins are those vitamins associated with different basal media. The culture is carried out in light or dark. The temperature is from 10 ℃ to 33 ℃, but is preferably about 23 ℃. The pH of the medium is 4 to 6.5 and is preferably adjusted to 5.8 before sterilization. In addition, agar may or may not be added to the medium.

Primary callus appears after several days of culture and can be isolated from the initial implant, taken out and subcultured after about 1 month, and then cultured on agar semi-solid medium (in test tubes or petri dishes) with intervals of 4 to 8 weeks, preferably 6 weeks, so that callus can be preserved for several years by serial subculture on new medium. The callus may also be subcultured in stirred liquid medium (in Erlenmeyer flasks or bioreactors) for 2 to 6 weeks, preferably 3 weeks.

Strains are distinguished by their genetic origin, culture conditions, appearance and lack of morphogenesis.

The freeze-dried isopychnum cells are extracted with water at a temperature of 0 to 50 c, preferably 4 to 25 c. The resulting extract is freeze-dried and then redissolved at a suitable concentration (e.g., about 30% dry matter). The precipitated protein is dissolved in a minimum amount of water by adding a concentrated ammonium sulfate solution (e.g., at a concentration representing 70 to 90% of the saturation concentration) and the insoluble material is separated by centrifugation. The protein is then separated by column chromatography (the eluent is preferably water) and the ditocarpine can be recovered (identifiable by its molecular weight of about 90.9 kDa).

Preparation of antibody specifically binding to htemite

The invention provides binding agents, such as antibodies, that specifically bind to htecavir. Such a binding agent is said to "specifically bind" if it reacts at a detectable level (e.g., by an ELISA assay) with the protein and does not detectably react with other proteins. "binding" refers to the non-covalent association between two different molecules to form a complex. Binding capacity can be assessed, for example, by determining the binding constant for complex formation. The binding constant is the value of the complex concentration value divided by the product of the concentration values of the non-complex components. The two products are said to be "bound" when the binding constant reaches 103 l/mol. Binding constants can be determined using methods well known to those skilled in the art.

Any agent that can meet the above criteria can be considered a binding agent.

In the present invention, the binding agent is preferably an antibody or a fragment thereof. Antibodies can be prepared by any technique available to those skilled in the art (see Harlow and Lane, antibodies: A laboratory Manual, Cold spring harbor laboratory, 1988). Generally, antibodies can be produced by cell culture techniques, including those that produce monoclonal antibodies, or by gene transfection of antibodies into host cells from bacteria or mammals to produce recombinant antibodies.

Among other techniques, those described later are preferable. An immunogen containing hattachin is injected into a group of mammals (e.g., mice, rats, rabbits, sheep, or goats). At this step, htalcaine can be used as the unmodified immunogen. Alternatively, if the ottecan is associated with a transporter protein such as bovine serum albumin orA greater immune response can then be induced by the combination of hemocyanines. Preferably, the immunogen is injected into the host animal according to a predetermined protocol and the animal is periodically bled. Polyclonal antibodies specific for haltecavir can be purified from these antisera, for example, by affinity chromatography using haltecavir conjugated to a suitable solid support.

Pharmaceutical composition for releasing GHRH

The compositions can be prepared from Hitachi and GHRH in particular according to one of the methods described in the publications De Wolf and Brett, pharmacological reviews (2000), 52, 207-.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Similarly, all publications, patent applications, all patents, and all other references mentioned herein are incorporated by reference.

The examples given below serve to illustrate the above process and should in no way be considered as limiting the scope of the invention.

Obtaining the product of Hitachi

Example 1:

in vitro culture of cells:

the seeds of Pilocarpus jaborandi are germinated and the stems resulting from the germination are removed. The stems were cultured in Gamborg medium (Gamborg et al, nutritional requirements for suspension culture of soybean root cells, exp. CellRes. (1968), 50(1), 151-158) to which had been added 30g/l sucrose, 1mg/l 2, 4-dichlorophenoxyacetic acid and 0.06mg/l kinetin. The culture was carried out in a test tube at a temperature of 23 ℃ in the dark. Subculture was performed every 6 weeks under conventional conditions. These lines were grainy in appearance with a light brown pigment.

A study of the growth kinetics of the strains was carried out for a period of 8 weeks, the kinetics being based on the increase in the mass of fresh and dry matter from the biomass. Calli from both tubes were combined and composed as a twice weekly harvest, the first harvest was performed at time 0. The callus and galactan were then harvested and freeze dried. Growth was observed to be exponential up to 6 weeks before a stationary growth phase occurred.

Extraction of cell cultures:

25g of freeze-dried cells of Pilocarpus jaborandi were extracted twice by immersing the cells in 375ml of 4 ℃ water, standing overnight at 4 ℃, then in 250ml of 4 ℃ water for 4 hours and finally washed with 125ml of 4 ℃ water. Each of the aqueous solutions thus obtained was filtered through a glass filter containing diatomaceous earth (celite) in vacuo to separate the cell residue from the aqueous solution. The resulting aqueous solutions were combined and lyophilized to give 9.4g of dry matter. The lyophilized dry extract was then dissolved in 31ml of water at 20 ℃ to obtain a solution containing 30% dry extract. Ammonium sulfate, 17.4g, was added in small portions under stable magnetic stirring to precipitate a protein fraction. The protein precipitate was then separated from the ammonium sulfate solution by centrifugation at 3000 rpm for 20 minutes. The ammonium sulfate solution was decanted and the precipitated protein was dissolved in 22ml of water, centrifuged again and filtered to remove insoluble particles.

The filtrate was then subjected to gel filtration chromatography. It was injected into a Superdex-loaded container prepared according to the manufacturer's recommendations^TM20(Amersham Pharmacia Biotech, reference No. 17-1043-01; mean diameter of microparticles 13 μm) (Buchi N.cndot. 19678, L: 230 mm; inner diameter: 26mm) using ultrapure Water (Water's Milli-Q) as an eluent at a flow rate of 5 ml/min. Multiple 40ml fractions were collected and active protein was found in fractions 3 and 4. These fractions were freeze-dried to give about 14.2mg of active product.

The purity of the product obtained is demonstrated by a single band appearing on an electrophoretic gel containing sodium dodecyl sulfate (SDS PAGE). The product corresponding to this band is designated herein after as a hetercar.

Example 2:

the cells were cultured in vitro according to the same method as described in example 1, and the cultured cells were extracted according to the method described hereinafter.

100g of freeze-dried isophyllum rutaecarpa cells were extracted with 2 l of deionized water at 20 ℃ and the mixture was kept under stirring overnight. By covering with a bed of diatomaceous earth (previously washed with acid; 1 to 2cm thick)) The cells and the extract were filtered under suction on a filter plate (pore 3, diameter 20 cm). The recovered cells were washed with 400ml of deionized water and then removed. The aqueous filtrate was then acidified by addition of about 10ml of 18% hydrochloric acid. The acidic solution was defatted by liquid-liquid extraction using 400ml dichloromethane. The dichloromethane phase was decanted and purged. The degreased solution was subjected to rotary evaporation to remove residual dichloromethane. About 30g of polyvinylpyrrolidone was then added to the defatted solution (pH about 3.0) and the mixture was stirred for about 30 minutes to remove tannins. The mixture was filtered through a filter plate (porosity 3, diameter 10cm) covered with a bed of a mixture containing 25g of diatomaceous earth (previously washed with acid) and 25g of polyvinylpyrrolidone by suction through the bed. The filtrate was then passed through 400ml preactivated according to the manufacturer's instructionsHP-20(Mitsubishi Chemical Company). The resulting filtrate was made basic (pH10) by adding about 60ml of 20% ammonium hydroxide solution. After 30 minutes of standing, a little precipitate appeared. 1g of diatomaceous earth (previously washed with acid) was added to the basic solution, which was then filtered through a membrane filter (0.22 μm) by suction. About 2 liters of filtrate was then passed through the filter cartridgeOn the purifierQ XL 16/10 column, which has been pre-equilibrated with piperazine/HCl 0.1M buffer, pH 10.2, at a flow rate of 0.5ml per minute (Column andpurifiers are all products of amersham biosciences). The column was connected with 6 column volumes of starting buffer pH 10.2, 5 column volumes of the same buffer containing 0.2M NaCl, and 10 column volumes of the same buffer containing 1M NaClAnd (5) continuously washing. Most of the htecans were recovered in the first 3 column volumes of buffer containing 1M NaCl. The active fraction is passed throughA G25 column (bed volume: 260ml) was desalted using deionized water as eluent. The active fraction was found in the first column volume corresponding to the retention volume and was freeze-dried to yield 170mg of ditocarpine. The resultant htecavir was essentially a band on the SDSPAGE gel.

Characterization of Hitachi

Analysis and microsequencing

Samples were loaded onto 10% polyacrylamide gels. After migration, the gel was fixed and stained with coomassie brilliant blue.

The gel lanes depicted in FIG. 3 corresponding to lanes 1, 2, 3, 4 and 5 are the molecular weight marker (Amarsham), the 0.5, 1 and 2 μ g content of the final ditocarpine fraction as obtained in example 1 and the molecular weight marker (Amersham), respectively. Determination of molecular weight by standard molecular weight mapping using standard computational tools well known to those skilled in the art (e.g., Viber Lourmat's Bio-Profile biolD software) allows for the indication that the molecular weight of Hitacalcin is 90.9 kilodaltons (+ -1.6 kilodaltons).

For the microsequencing analysis of the protein, the polyacrylamide bands containing the protein were excised and digested in the presence of 0.4. mu.g of endolysine-C (Sigma) at 35 ℃ for 18 hours in 300. mu.l of digestion buffer containing 50mM Tris (pH 8.6), 0.03% sodium dodecyl sulfate. The resulting peptides were separated by HPLC on a DEAE-C18 embedded column with a diameter of 1 mm. The separation gradient was based on a mixture of acetonitrile (from 2 to 70%) and 0.1% trifluoroacetic acid (TFA). Sequencing was then performed on a proximity sequencer (applied biosystems). Three peaks have been sequenced by this method, making it possible to characterize the htecans in a unique way. The corresponding sequences are identified in the present invention as seq.id.no.1, seq.id.no.2 and seq.id.no. 3.

Analysis of the glycoproteins was performed by detecting the glycated structures of the glycoproteins separated on SDS-PAGE gels. The detection system is an improvement on the "Periodic Acid-Schiff" method and results in the appearance of a magenta band, which is proved to be glycoprotein (Sigma). For the hetekins as obtained in example 1, the results reproduced in fig. 4 were obtained.

Pharmacological Properties of Heterocarpine

Stable transfection of the human GHRH receptor (hGHRH-R):

human embryonic kidney cells HEK-293 (cell line developed by doctor Stuart Sealfon (mountain Medical School, N.Y.) which express the human GHRH receptor in a stable manner) were obtained from doctor Kelly Mayo (Northwestern University, Chicago, IL).

Cell culture and membrane preparation:

the above HEK-293 cells stably transfected with human GHRH receptor were cultured in DMEM (Dulbecco's modified Eagle Medium, high glucose content, supplied by Life technologies) supplemented with 0.4mg/ml G418(Life technologies) in the presence of 10% fetal bovine serum and 4mM L-glutamine (Life technologies). The cells were incubated in a medium containing 50mM HEPES (pH 7.4), 5mM magnesium chloride (MgCl)₂) 2mM ethylene glycol-bis (2-amino-ethyl) -N, N, N ', N' -tetraacetic acid (EGTA) and 50. mu.g/ml bacitracin in buffer A and sonicated in the same buffer A. The homogenized cells were centrifuged at 39,000g for 10 min at 4 ℃, suspended in buffer A and centrifuged again at 40,000g for 10 min at 4 ℃. Total membrane protein was quantified by Bradford's technique. The precipitated membrane was then stored at-80 ℃ until use.

Competitive binding assay for hGHRH-R

Membranes of HEK-293 cells stably transfected with human GHRH receptors were treated with a solution containing 50mM HEPES (pH 7.4), 5mM MgCl₂2mM EGTA, 50. mu.g/ml bacitracin and 0.5% Bovine Serum Albumin (BSA) to a concentration of 100. mu.g/ml. Contacting the membrane with 0.05nM in the presence of increasing concentrations of htabalin¹²⁵I]GHRH (1-44 amide) was incubated at 23 ℃ for 2 hours at a final volume of 200. mu.l.The reaction was stopped by rapid filtration over 96-well GF/C filters pre-loaded with 0.1% polyethylenimine. The filter was then washed 3 times at 4 ℃ using a Packard 96-well filtration station (filtration station) with a wash buffer containing 50mM Tris (pH 7.4). The so dried filters were immersed in 20. mu.l scintillation cocktail (Microscint O, Packard) and Topcout counts (Packard) were performed. Nonspecific activity was determined in the presence of 100nM hGHRH. The resulting hGHRH (0.001nM to 100nM) dose-response curves and results are included in FIG. 1.

Competitive formation of cyclic AMP:

HEK-293 cells stably transfected with human GHRH receptor were distributed in 48-well culture plates and cultured for 3 days. The medium was removed and replaced with medium B containing 250. mu.l DMEM (Dulbecco's modified Eagle Medium, high glucose content; supplied by Life technologies) in the presence of 0.5% BSA, 0.5mM 3-isobutyl-1-methylxanthine (IBMX) and preincubated for 5 minutes at 37 ℃. At the end of the pre-incubation period, the htecavir was detected for an additional 20 minutes. The observed concentrations are reported in figure 2. Incubation was stopped by adding 100 μ l 0.1M HCl and aliquots were analyzed for cyclic AMP content using the FlashPlate kit (new england Nuclear).

Analysis of GH in rats:

the level of GH in rats (male Sprague Dawley) was measured in blood samples by an enzyme-immunological assay developed by Spi-Bio (Spi-Bio, France). Rats were treated by intravenous injection of increasing doses of htag (vehicle only, with 1.3 and 10nmol of htag) followed 10 minutes later by intravenous injection of 10 μ g (3nmol) of hGHRH. The growth hormone levels in the blood samples were measured 10 minutes after the injection of hGHRH as described above. The results obtained are shown in FIG. 5.

Measurement of antitumor activity:

subcutaneous injection of human tumor cells, specifically H-69 small cell lung carcinoma cells, into athymic mice resulted in approximately 80mm approximately 10 days after the first transplantation³The human tumor xenograft of (1). Increment passage every two days (vector only, 2)5mg/kg, 5mg/kg and 10mg/kg) of htecamin. Tumor volumes were measured every 4 days throughout the treatment.

Brief description of the drawings:

figure 1 is a graph representing the inhibition of binding of human GHRH to human GHRH receptors with increasing concentrations of htocarpine.

Figure 2 is a graph representing inhibition of cyclic AMP production in cells stably transfected with the human GHRH receptor in the presence of 10nM human GHRH as the concentration of ditocarpine increases.

FIG. 3 is a reproduction of the SDS-PAGE protein gel plate showing the presence of Hitacalcin with a molecular weight of 90.9 kDa.

FIG. 4 is a reproduction of the SDS-PAGE protein gel plate showing that ditocarpine is a glycoprotein (FIG. 4B).

FIG. 5 presents in bar graph the inhibition of GH synthesis in rats in the presence of 10 μ g human GHRH with increasing concentrations of ditocarpine.

Sequence listing

<110> scientific research and application consulting company

<120> plant-derived proteins with anti-cancer properties: heterocarpin

<130>RS 329 FR

<140>FR 02/15560

<141>2002-08-26

<160>3

<170> PatentIn version 2.1

<210>1

<211>10

<212>PRT

<213> Pilocarpus Heterophyllus (Pilocarpus Heterophyllus)

<400>1

Lys Leu Ile Gly Ala Arg Tyr Phe Asp Lys

1 5 10

<210>2

<211>14

<212>PRT

<213> Pilocarpus jaborandi

<400>2

Tyr Gly Glu Asp Ile Ile Val Gly Val Ile Asp Ser Gly Val

1 5 10

<210>3

<211>6

<212>PRT

<213> Pilocarpus jaborandi

<400>3

Pro Glu Ser Glu Ser Tyr

1 5

Claims (5)

1. Use of a human GHRH binding protein obtained by extraction from the plant Pilocarpus heterophyllus for the preparation of a medicament for the treatment of cancer growing dependent on the growth factor GHRH, wherein said protein is characterized by a molecular weight of about 90.9kDa and comprises fragments of the peptide sequences seq.ID.no.1, seq.ID.no.2 and seq.ID.no. 3; furthermore, the protein can be present in glycosylated or non-glycosylated form.

2. Use according to claim 1, characterized in that the protein has been obtained from an extract of the plant Pilocarpus heterophyllus cells cultured in vitro.

3. Use according to claim 1 or 2, characterized in that the cancer that grows dependent on the growth factor GHRH is selected from the group consisting of small cell lung cancer and breast cancer.

4. Use according to claim 3, characterized in that the cancer that grows dependent on the growth factor GHRH is small cell lung cancer.

5. Use according to claim 3, characterized in that the cancer that grows dependent on the growth factor GHRH is breast cancer.