Heterocarpin, a plant-derived protein with anti-cancer properties

Translation of description of equivalent WO 2004017987 A2

Heterocarpine, a plant-derived protein with anti-cancer properties

The present invention relates to a plant protein with anti-cancer properties which binds human GHRH (human Growth Hormone releasing hormone releasing hormone or human growth hormone).

Growth hormone ( "GH") is a protein of 191 amino acids which stimulates the production of numerous growth factors, such as Y Insulin-Like Growth Factor I (IGF-1) and triggers the growth of a large number of tissues (skeleton, connective tissue, muscle and viscera). GH also has physiological activities, increasing the synthesis of nucleic acids, proteins and lipolysis whilst reducing urinary secretions (Frohman LA & Kineman, RD, Handbook of Physiology, Hormonal Control of Growth, edited by Kostyo, JL & Goodman HM (Oxford Univ. Press, New York, 1999), p. 189-221).

GH synthesis is regulated by factors with positive or negative action secreted by the hypothalamus. The main factor controlling the production of GH is the "Growth Hormone Releasing Hormone" (GHRH), peptide 44 amino acids in humans.

GH and GHRH are involved in many diseases. Among them, it should be mentioned including cancer (particularly prostate or lung), Pacromégalie, retinopathy and diabetic nephropathy; to these pathologies, treatment with GHRH antagonists is indicated. Because of the number of diseases potentially concerned, the industry continues to research GHRH antagonists.

The plaintiff therefore just isolated a new protein of vegetable, which property is to set human GHRH.

The invention therefore firstly relates to an isolated protein obtainable by extraction from the plant Pilocarpus heterophyllus, which is characterized in that it has a molecular mass of approximately 90.9 kDa and comprises fragments of peptide sequences SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3, wherein said protein is likely to be in a glycosylated or non-glycosylated form. To simplify the following discussion, this protein will be designated hereinafter as "heterocarpine". Said sequences 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 YGEDEVGVIDSGN SEQ. ID. NO. 3 PESESY

The nomenclature used above (as in the rest of this application) to define the peptides is that specified by the "--UPAC-RJB Commissioner is Biochemical Nomenclature" in which, in agreement with the conventional representation, the amino acid at N-terminal (amino group) appears to the left and the amino acid at the C-terminal (carboxyl group) appears to the right. The term "natural amino acid" means one of natural L-amino acids found in naturally occurring 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 called "isolated" if it is taken out of its original environment. In particular, a natural protein is isolated if it is separated from the biological material with which it coexists in the natural system.

The invention preferably relates to heterocarpine in its unglycosylated form.

In a preferred embodiment of the invention, heterocarpine is obtained from an extract of the plant cells cultured in vitro Pilocarpus Heterophyllus.

The invention moreover also relates to a monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds Phétérocarpine.

Heterocarpine has the property of binding human GHRH. In vitro, heterocarpine binds human GHRH and thus inhibits the synthesis cyclic dAMP induced during the fixing of human GHRH on its receptor. In vivo, in rats, the complex heterocarpine / human GHRH is formed in the blood compartment and inhibited in a dose dependent manner GH synthesis induced by 10 micrograms of human GHRH in a mole to mole ratio. Heterocarpine has the property of binding human GHRH.

These properties make the compounds of the invention suitable for pharmaceutical use. The invention also relates, as medicaments, heterocarpine in glycosylated and non-glycosylated form. It also relates to pharmaceutical compositions containing, as active principle, heterocarpine in a glycosylated or unglycosylated form, said composition also comprising one or more pharmaceutically acceptable excipients. It further relates to the use of heterocarpine in glycosylated and non-glycosylated form for preparing medicaments intended to antagonize the effects of GHRH, to treat proliferative diseases (including cancer), to treat acromegaly or to treat retinopathy and diabetic nephropathy. Regarding cancer, heterocarpine be particularly suitable for preparing a drug intended to treat carcinoid and pancreatic tumors, gangliocytomas hypothalamic pituitary, bronchial carcinoma, intestinal and liver, sympathoadrenergic tumors, pheochromocytoma, pituitary adenomas and thyroid carcinomas. Heterocarpine will be particularly suitable for preparing a medicament for treating cancers whose growth is dependent on the GHRH growth factor, in particular for preparing a medicament for treating a cancer selected from lung cancer and small cell cancer breast (particularly lung small cell carcinoma).

The invention also relates to, as a medicament, a monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds heterocarpine. It also relates to a pharmaceutical composition comprising, as active principle, a monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds heterocarpine, said composition also comprising one or more pharmaceutically acceptable excipients . It further relates to the use of a monoclonal antibody, or an antigen-binding fragment thereof, which specifically binds heterocarpine, for preparing medicaments intended to antagonize the effects of GHRH, to treat proliferative diseases (including cancer), to treat acromegaly or to treat retinopathy and diabetic nephropathy. As regards cancer, said monoclonal antibody or said antigen binding fragment thereof will be particularly suitable for preparing a medicament intended to treat carcinoid and pancreatic tumors, hypothalamo-pituitary gangliocytomas, bronchial carcinomas, intestinal and liver, sympathoadrenergic tumors, pheochromocytoma, pituitary adenomas and thyroid carcinomas.

The invention further relates to the use of heterocarpine as an excipient in a pharmaceutical composition for the prolonged release of GHRH. It also relates to a pharmaceutical composition comprising GHRH, heterocarpine and one or more pharmaceutically acceptable excipients.

On the other objects of the invention are finally the processes for extracting and isolating the heterocarpine from cells of the plant Pilocarpus Heterophyllus, said cells preferably originating from in vitro cultures. These methods include essentially an extraction step of the cells of the plant Pilocarpus Heterophyllus with water at a temperature of 0 to 50 ° C, and preferably from 4 to 25 ° C, said extraction stage being followed by a step of filtration to separate the rich heterocarpine filtrate cells Pilocarpus Heterophyllus and one or more separation steps heterocarpine other components extracted from the plant Pilocarpus Heterophyllus.

In a first variant, these extraction and isolation processes essentially include the following successive stages: a) a step of extraction of the cells of the plant Pilocarpus Heterophyllus with water at a temperature of 0 to 50 ° C, and preferably from 4 to 25 ° C, said extraction stage being followed by a filtration stage to separate the heterocarpine-rich filtrate from the Pilocarpus Heterophyllus cells; b) a precipitation step of the extracted protein, 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 chromatographic step by gel filtration to separate the heterocarpine from the other components of the solution.

According to another variant, these extraction and isolation processes essentially include the following successive stages: a) a step of extraction of the cells of the plant Pilocarpus Heterophyllus with water at a temperature of 0 to 50 ° C, and preferably from 4 to 25 ° C, said extraction stage being followed by a filtration stage to separate the heterocarpine-rich filtrate from the Pilocarpus Heterophyllus cells; b) a degreasing step of the solution obtained in a), acidified by addition of a non-oxidizing acid (for example hydrochloric acid, sulfuric or phosphoric acid) at a pH of preferably between 2 and 4, using a liquid-liquid extraction (preferably using an organic solvent such as dichloromethane, heptane, hexane or cyclohexane); c) a step of removing the tannins by contacting the delipidated solution obtained in c) with polyvinylpyrrolidone (or nylon 66) followed by filtration on large-pore resin (preferably a resin based polystyrenes such as Diaion resin^® HP-20); d) at alkaline pH passage (preferably between pH 9 and 11) the filtrate obtained after step c) by adding a base such as ammonium hydroxide, sodium hydroxide or h ydroxyde of potassium; e) one or more stages of filtration through an ion exchange resin, the eluent for this or these filtration steps being preferably a buffer solution having a pH between 9 and 11 and optionally containing a salt concentration gradient (such as for example sodium chloride or ammonium sulfate) in order to separate the heterocarpine from the other components of the solution; and f) a desalting step consisting of passing the solution obtained in step e) on a resin separating the constituents of a mixture according to their molecular weight (such as Sephadex resin^® Or Superdex G25^® 200 HR) and elution of said mixture on said resin with water.

The pharmaceutical compositions containing a compound of the invention may be in solid form such as, for example, powders, pills, granules, tablets, liposomes, gelatin capsules or suppositories. Pills, tablets or capsules may be coated with a substance capable of protecting the composition from the action of gastric acid or enzymes in the subject's stomach for a period of time sufficient to allow the composition of pass undigested into the small intestine of the latter. The compound can also be administered locally, for example at the same location of a tumor. The compound can also be administered according to a sustained release process (for example using a sustained release composition or an infusion pump). Suitable solid supports can be, for example, calcium phosphate, magnesium stearate, magnesium carbonate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, cellulose methyl, sodium carboxymethyl cellulose, polyvinylpyrrolidine and wax.

The pharmaceutical compositions containing a compound of the invention may also be in liquid form such as, for example, solutions, emulsions, suspensions or a sustained release formulation. Appropriate liquid supports can be, for example, water, organic solvents such as glycerol or glycols such as polyethylene glycol, similarly their mixtures, in varying proportions, in water.

Administration of a drug of the invention may be made by topical, oral, parenteral, intramuscular injection, etc. The dose of a compound of the present invention to provide for the treatment of diseases or disorders mentioned above, varies according to the mode of administration, age and body weight of the subject to be treated and the condition of the latter, and will be finally decided by the attending physician or veterinarian. Such a quantity determined by the doctor or vet is here called "therapeutically effective amount".

According to the invention, can be prepared heterocarpine by the method described below.

Preparing heterocarpine

According to a preferred variant of the invention, in vitro cultures of callus or cell suspensions from different organs of the plant were carried out. These tissues cultured on semi-solid or liquid medium are capable of bio-synthesizing compounds with biological properties.

By "callus" is meant in this application a macroscopic cluster of undifferentiated plant cells cultured on semi-solid nutrient medium. By "undifferentiated cells" are designated herein cells that have the ability under certain conditions to multiply in the form of a callus or cell suspension without any morphogenesis phenomenon. Finally, "cell suspension" refers to undifferentiated cells which can form microscopic clusters cultured in a liquid nutrient medium.

The choice of the nutrient medium, hormones, culture conditions are an integral part of the invention as well as the extraction and analysis of the extract from these in vitro cultures.

Pilocarpus Heterophyllus seed cells can be cultured in suspension for example according to the procedure below.

The organs are decontaminated according to the usual methods prior to culture. in vitro plantlets organs were also used as starting material to callogenesis without requiring prior disinfection. The preferred nutrient base medium is one of the commonly used for in vitro culture media: it is the middle of Gamborg (described in Gamborg et al, Nutrient requirements of suspension cultures of Soybean root cells, Exp Cell Res.. . (1968), 50 (1), 151-158). The carbon source is sucrose but glucose may also be employed at a concentration of 1 to 120 g / 1, preferably 30 g / 1 about. The content can also be reduced by macronutrients by a factor 2. The media is supplemented with auxin or auxin and cytokinin with a preference for the association of two hormones, generally 2,4-dichlorophenoxyacetic acid and kinetin, but the α-naphthaleneacetic acid (NAA), the β-indoleacetic acid (IAA), the β-indolbutanoïque acid (IBA) or picloram can also be combined with kinetin or benzylaminopurine (BAP). The concentration can vary from 0.1 to 10 mg / 1 for auxin (one can choose e.g.

1 mg / 1), and 0.01 to 2 mg / 1 cytokinin (can be chosen for example 0.06 mg / 1). The vitamins are those associated with different base areas. Cultures are made in light or darkness. The temperature may vary from 10 ° C to 33 ° C, but will preferably be about 23 ° C. The pH of the medium is between 4 and 6.5 and preferably adjusted to 5.8 before sterilization. The medium may also be supplemented or not supplemented agar.

The primary calluses appear after a few days of culture and can be separated from the original implant, removed and transplanted after about 1 month then cultured on semisolid agar medium (tube or Petri dish) with passages 4 to 8 weeks, preferably 6 weeks, we can thus maintain a cal for years by successive subcultures on new media. One can also dub the callus in an agitated liquid culture medium (Erlenmeyer flask or bioreactor) with subcultures

2-6 weeks, preferably 3 weeks.

The strains obtained are distinguished by their genetic origin, culture conditions, appearance and absence of morphogenesis.

Cells lyophilized Pilocarpus Heterophyllus are extracted with water at a temperature of 0 to 50 ° C, and preferably from 4 to 25 ° C. The extract thus obtained is lyophilized before being redissolved to a suitable concentration (e.g., about 30% of dry matter). The proteins precipitated by addition of a concentrated solution of ammonium sulphate (for example at a concentration representing 70 to 90% of the saturation concentration) is dissolved in a minimum of water and the insolubles were recovered by centrifugation. The proteins are then separated by column chromatography (the eluent being preferably water) and heterocarpine (identifiable by its molecular mass of approximately 90.9 kDa) can then be recovered.

Preparation of antibodies binding specifically heterocarpine

The present invention provides binding agents, such as antibodies which specifically bind heterocarpine. Such an agent is said to be "specifically binding" a protein if it reacts at a detectable level (e.g. by ELISA) with said protein and do not detectably react with other proteins. "The binding" refers to a noncovalent association between two separate molecules such that a complex is formed. The binding ability can be assessed, for example, by determining the binding constant for complex formation. The binding constant is the value obtained when the value of the concentration of the complex is divided by the product of the values ​​of concentration of uncomplexed components. 2 products are called "bound" when fixing the constant reaches 103 1 / mol. The binding constant may be determined using methods well known to those skilled in the art.

Any agent capable of meeting the above criteria can be regarded as a binding agent.

In the present invention, a fixing agent is preferably an antibody or a fragment thereof. The antibodies may be prepared by any technique available to those skilled in the art (cf. Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies or via transfection of antibody genes into host cells of bacteria or mammals in order to produce recombinant antibodies.

Among other techniques, it is preferable to use those described below. An immunogen containing heterocarpine is injected into a group of mammals (eg, mice, rats, rabbits, sheep or goats). In this step, heterocarpine may serve as the immunogen without modification. Alternatively, a superior immune response may be induced if the heterocarpine is joined to a carrier protein such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule, and the animals are bled periodically. Polyclonal antibodies specific for the heterocarpine can thus be purified from such antisera, for example, by affinity chromatography using the heterocarpine coupled to a suitable solid support.

pharmaceutical compositions for the release of GHRH

These compositions can in particular be prepared from heterocarpine and GHRH according to the methods described in the review of De Wolf and Brett, Pharmacological Reviews (2000), 52, 207-236 and references cited therein. Unless they are defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by an ordinary specialist in the field to which this invention belongs. Similarly, all publications, patent applications, all patents and all other references mentioned here are incorporated by reference.

The following examples are presented to illustrate the above procedures and should in no case be considered as limiting the scope of the invention.

OBTAINING HETEROCARPINE

Example 1:

in vitro cell culture:

A Pilocarpus Heterophyllus seed is germinated and the stem resulting from this germination is removed. Said rod is cultured in a medium of Gamborg (Gamborg et al., Nutrient requirements of suspension cultures of Soybean root cells, Exp. Cell Res. (1968), 50 (1), 151-158) supplemented with 30 g / 1 sucrose, 1 mg / 1 2,4-dichlorophenoxyacetic acid and 0.06 mg / 1 kinetin. Cultivation is carried out in the tubes at a temperature of 23 ° C and in the dark. Subcultures are carried out every 6 weeks under normal conditions. The strains of granular appearance, have a beige pigmentation. A stem growth kinetics, based on the mass increase of fresh and dry matter of the biomass, was carried out over 8 weeks. The calluses from 2 tubes together and form a bi-weekly harvest, the first harvest taking place at time 0. Calluses and gelose are then harvested and freeze dried. It is observed that the growth is exponential up to 6 weeks of culture before the onset of a stationary growth phase.

Extracting cell cultures:

25 g of lyophilized Pilocarpus Heterophyllus cells are extracted 2 times by immersion in 375 ml of water at 4 ° C and left overnight at 4 ° C and then in 250 ml of water at 4 ° C for 4 hours and finally washed with 125 ml of water at 4 ° C. Each resulting aqueous solution is vacuum filtered through a glass filter topped celite to remove cell debris from the aqueous solution. The aqueous solutions are combined and then lyophilized to obtain 9.4 g of dry matter. The lyophilized dry extract is then dissolved in 31 ml of water at 20 ° C to obtain a solution containing 30% solids. 17.4 g of ammonium sulfate are added in small portions with constant magnetic stirring to precipitate the protein fraction. The protein precipitate is then separated from the ammonium sulphate solution by centrifugation at 3000 rpm for 20 minutes. The ammonium sulfate solution was decanted and the precipitated proteins are dissolved in 22 ml of water, re-centrifuged and filtered to remove insoluble particles. The resulting filtrate was then chromatographed by gel filtration. It is injected into a column (Buchi No. 19678, L = 230 mm; inner diameter = 26 mm) filled with Superdex ™ 200 (Amersham Pharmacia Biotech, Catalog No. 17-1043-01; average particle diameter of 13 microns) prepared as recommended by the manufacturer using ultrapure water (Milli-Q water's) as eluent at a flow rate of 5 ml per minute. 40 ml fractions are collected and thus the active protein is found in the third and the fourth fraction. These fractions are lyophilized to yield about 14.2 mg of active product.

The purity of the product is demonstrated by the appearance of a single band on electrophoresis gel containing sodium dodecyl sulfate (SDS PAGE). The product corresponding to this band is designated in the following as heterocarpine.

Example 2:

Cells grown in vitro according to the same procedure as described in Example 1 above are extracted according to the method described below.

100 g lyophilized Pilocarpus Heterophyllus cells are extracted using 2 liters of demineralized water at 20 ° C, the mixture is kept stirred overnight.

Cells and extract are filtered by suction on frit (porosity 3, diameter 20 cm) covered by a bed of celite (previously washed with acid, 1 to 2 cm thick).

The recovered cells were washed with 400 ml of demineralized water before being eliminated. The aqueous filtrate is then acidified to pH 3.0 by addition of about 10 ml of 18% hydrochloric acid. The acidified solution is then delipidated by liquid-liquid extraction using 400 ml of dichloromethane. The dichloromethane phase is decanted then eliminated. The delipidated solution is subjected to rotary evaporation to remove residual dichloromethane. About 30 g of polyvinylpyrrolidone are then added to the defatted solution (pH about 3.0) and the mixture is stirred for about 30 minutes to remove tannins. The mixture was filtered on a bed by suction frit (porosity 3, diameter 10 cm) covered with a mixed bed consisting of 25 g of Celite

(Previously washed with acid) and 25 g polyvinylpyrrolidone. The filtrate is then passed through a bed of 400 ml of Diaion^® HP-20 (Mitsubishi Chemical Company) pre-activated according to the manufacturer's instructions. The resulting filtrate is then made alkaline (pH 10) by adding about 60 ml of ammonium hydroxide solution 20%. A slight precipitation appears after 30 minutes of rest. 1 g of celite (previously washed with acid) is added to the alkaline solution which is then filtered by suction through a membrane filter (0.22 .mu.m). Approximately 2 liters of filtrate are then passed through a column HiPrep^® Q XL 16/10, mounted on an Akta purifier^® and pre-equilibrated to pH 10.2 with a piperazine / HCl buffer 0.1, with a flow rate of 0.5 ml per minute (HiPrep column^® and the Akta purifier^® Both are products of the company Amersham Biosciences). The column was then successively washed with 6 column volumes of pH 10.2 buffer starting, 5 column volumes of the same buffer containing 0.2 NaCl concentration; and 10 column volumes of the same buffer containing a concentration of the NaCl. The majority of heterocarpine is recovered in the first three 3 column volumes buffer containing concentration \ M NaCl. The active fractions were desalted by passage through a Sephadex^® G25 (bed volume: 260 ml) using demineralized water as eluent. The active fractions, found in the first column volume corresponding to the dead volume, are then lyophilized to obtain 170 mg of heterocarpine. Heterocarpine thus obtained is practically single-band on SDS-PAGE gel.

Characterisation of HETEROCARPINE

Analysis and microsequencing:

The samples are loaded onto a polyacrylamide gel at 10%. After migration, the gels are fixed and stained with Coomassie blue.

The gel tracks represented in Figure 3 corresponding to the tracks 1, 2, 3, 4 and 5 are the molecular weight marker respectively (Amersham), 0.5, 1 and 2 ug of the content of the final fraction of heterocarpine such obtained in example 1 and the molecular weight marker (Amersham). The determination of molecular weight using a standard curve of the molecular weight marker using standard IT tools and well known to those skilled in the art (eg, Bio-Profil BiolD software Viber Lourmat) can show heterocarpine that has a molecular weight of 90.9 kilodaltons (± 1.6 kiloDaltons). For analysis by microsequencing of protein, band of polyacrylamide containing the protein is cut out and digested in 300 .mu.l of digestion buffer containing 50 mM Tris (pH 8.6), 0.03% sodium dodecyl sulfate at 35 ° C for 18 hours in the presence of 0.4 mcg endolysin-C (Sigma). The peptides obtained are separated by HPLC on-line column of DEAE-C18 1 mm in diameter. The separation gradient is based on a mixture of acetonitrile (from 2 to 70%) and 0.1% trifluoroacetic acid (TFA). The sequencing is then performed on a Procise sequencer (Applied Biosystem). Three peaks have been sequenced, to characterize uniquely heterocarpine. The corresponding sequences are identified herein as SEQ. ID. NO. 1, SEQ. ID. NO. 2 and SEQ. ID. NO. 3.

Analysis of the glycoproteins is performed by detecting sweet structures of glycoproteins separated by SDS-PAGE gel. This detection system is a modification of the "Periodic acid-Schiff" and led to the appearance of magenta bands showing glycoproteins (Sigma). Is obtained for the heterocarpine as obtained in Example 1, the result reproduced in Figure 4.

PHARMACOLOGICAL PROPERTIES OF HETEROCARPINE

Stable Transfections of the Human GHRH Receptor (hGHRH-R):

Embryonic kidney human cells, HEK-293 (a cell line developed by Dr. Stuart Sealfon, Mount Sinai Medical School, New York, New York) stably expressing the human GHRH receptor were obtained from Dr. Kelly Mayo (Northwestern University, Chicago, IL).

Cell Culture and Membrane Preparation:

HEK-293 cells stably transfected with the human GHRH receptor are described above cultured in DMEM (Eagle medium modified by Dulbecco's high glucose content; supplied by Life technologies) supplemented with 0.4 mg / ml G418 (Life technologies) in the presence of 10% fetal calf serum and 4 mM L-glutamine (Life technologies). Cells are homogenized in buffer containing 50 mM HEPES (pH 7.4), 5 mM magnesium chloride (MgCl₂), 2 mM ethylene glycol bis (2-aminoethyl) -N, N, N ', N'-tetraacetic acid (EGTA), and 50 ug / ml bacitracin then are subjected to sonication in the same buffer A. cells Homogenates are centrifuged at 4 ° C at 39,000 g for 10 minutes, suspended in buffer A and re-centrifuged at 4 ° C at 40 000 g for 10 minutes. The total membrane proteins are quantified by the Bradford technique. The pelleted membranes are thus stored at -80 ° C for later use.

competitive binding assay on hGHRH-R:

The membranes of HEK-293 cells stably transfected with the human GHRH receptor are diluted to a concentration of 100 .mu.g / ml in the reaction buffer containing 50 mM HEPES (pH 7.4), 5 mM MgCl₂, 2 mM EGTA,

50 mcg / ml bacitracin and 0.5% bovine serum albumin (BSA). The membranes are incubated with 0.05 nM [¹²⁵I] GHRH (l-44 amide) (Amersham) in a final volume of 200 .mu.l in the presence of increasing concentrations of heterocarpine for 2 hours at 23 ° C. The reaction is stopped by rapid filtration on 96-well

GF / C pre-loaded in 0.1% polyethylenimine. The filters are then washed three times with

4 ° C with washing buffer containing 50 mM Tris (pH 7.4) using a filtration station Packard 96 well. The filters thus dried are submerged in 20 .mu.l of scintillation cocktail (Microscint O, Packard) and are subject to a count on the Topcount (Packard). The non-specific activity is determined in the presence of 100 nM of hGHRH. A dose-response curve is generated for hGHRH (0.001 nM-100 nM) and the results obtained are given in Figure 1.

Competitive Formation of Cyclic AMP:

HEK-293 cells stably transfected with the human GHRH receptor are distributed in 48 well culture plates and cultured for 3 days. The culture medium is then removed and replaced with medium B containing 250 .mu.l of DMEM (Eagle medium modified by Dulbecco's high glucose content; supplied by Life technologies) in the presence of 0.5% BSA, 0.5 mM 3-isobutyl-l-methylxanthine (-OBMX) and pre-incubated for 5 minutes at 37 ° C. at the end of the pre-incubation, heterocarpine is tested for an additional 20 minutes. Concentrations observed are reported in Figure 2. The incubation is stopped by adding 100 .mu.l of HC1 0.1 and the aliquots are analyzed for their cyclic AMP content using the FlashPlate kit (New England Nuclear).

GH dosing in rats:

GH levels in rats (male, Sprague Dawley) were measured in blood samples by enzyme-immunological test developed by Spi-Bio (Spi Bio, France). The rats are treated by intravenous injection of heterocarpine in increasing doses (vehicle alone, 1, 3 and 10 nmol), and then, 10 minutes later by intravenous injection of 10 mcg (3 nmol) of hGHRH. Ten minutes after injection of hGHRH, levels of growth hormone are measured in blood samples as described above. The results obtained are shown in Figure 5.

Measurement of antitumor activity:

Human tumor cells, particularly lung cancer cells with small cell H-69 are injected under the skin of nude mice to produce a human tumor xenograft of about 80 mm³ ten days after the first transplant. Mice are treated every other day by intravenous injection of heterocarpine in increasing doses (vehicle alone, 2.5 mg / kg, 5 mg / kg and 10 mg / kg). Tumor volume is then measured every 4 days throughout the duration of treatment.

Brief description of the figures:

Figure 1 is a curve representing human GHRH fixing inhibition on the human GHRH receptor as a function of increasing concentrations of heterocarpine.

Figure 2 is a curve showing the inhibition of cyclic AMP production in cells transfected in a stable manner with the human GHRH receptor in the presence of 10 nM of human GHRH as a function of increasing concentrations of heterocarpine.

Figure 3 is a reproduction of a protein gel SDS-PAGE slab showing the presence of heterocarpine having a molecular weight of 90.9 kDa.

Figure 4 is a reproduction of a protein gel SDS-PAGE slab showing that heterocarpine is a glycoprotein (Panel B).

Figure 5 is a representation in the form of bar graphs depicting the GH synthesis inhibition in rats in the presence of 10 micrograms of human GHRH as a function of increasing concentrations of heterocarpine.