CH649783A5 - PROCESS FOR THE PRODUCTION OF HUMAN PROLACTIN. - Google Patents

Description

The present invention relates to a process for the production of human prolactin which consists in multiplying human cells.

Human prolactin is a hormone secreted by the cells of the acidophilic adenomas of the anterior pituitary gland, which stimulates the development of the mammary gland, prostate and seminal vesicles. No process for the mass production of human prolactin has been developed to date.

The incumbent studied methods for the mass production of inexpensive human prolactin. His efforts led to the unexpected discovery that human cells, obtained by multiplication of human cells capable of producing human prolactin, with the use of a warm-blooded animal, have a production capacity of human prolactin much greater than that tissue cultures in vitro: a production of human prolactin per cell 2 to 50 times higher.

The process mentioned at the start is therefore characterized according to the invention by the characterizing part of claim 1.

The method of the invention allows an increased production of human prolactin, requires no less or much less nutrient medium containing an expensive serum for cell multiplication, and makes maintenance of the culture medium during cell multiplication much easier than in the case of tissue culture in vitro. In particular, all human cells capable of producing human prolactin can be easily multiplied, using the nutritive liquid of the organism of a warm-blooded animal, by transplantation of these cells into the organism of the animal or suspending these cells in a diffusion chamber designed to receive the nutritive liquid from the organism, the animal being fed in the ordinary way. The process is also characterized by more stable and abundant cell multiplication, as well as by an increase in the production of human prolactin per cell.

As regards the cells which can be used in the invention, any cell which produces human prolactin and which multiplies easily in the organism of a warm-blooded animal can be used. For example, it is possible to use according to the invention human cells naturally producing human prolactin, such as the acidophilic cells of the anterior pituitary gland of man, the cells transformed by the EB virus or by irradiation with X-rays. , and that of an acidophilic adenoma of the pituitary gland of a human subject; lung carcinoma cells, producing ectopic human prolactin, and established cell lines of the above cells. Also the use of established, easy-to-maintain human lymphoblastoid lines, into which genes controlling the production of human prolactin have been introduced, according to genetic recombination techniques using enzymes such as DNA ligase, nuclease and DNA polymerase or by cell fusion with agents such as polyethylene glycol or Sendai virus, causes remarkably greater cell multiplication when these cells are transplanted into the body of a warm-blooded animal; the production of human prolactin, per cell, is about 2 to 10 times or more higher. In addition, as the transplantation of the aforementioned human lymphoblastoid lines causes the formation of massive tumors, and these tumors are hardly contaminated by the cells of the host animal and easily disintegrate, the multiplied living human lymphoblast cells can be easily harvested.

As regards the animals which can be used in the invention, any animal in which the cells multiply can be used. For example, it is advantageous to use poultry such as chick or pigeon, or mammals such as dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse or naked mouse. As such a cell transplant provokes an undesirable immune reaction, the use of a newborn or very young animal or at the earliest possible stage, for example in the form of an egg, embryo or fetus, is desirable. . To reduce the immune response, the animal may be treated with x-rays or y-rays at approximately 200 to 600 rems or by injection of an antiserum or agent before cell transplantation. immunosuppressant prepared according to a conventional method. Since naked mice used as warm-blooded animals exhibit a weaker immune response, even when they are adults, they can conveniently be transplanted with established human cells, so that they multiply rapidly in their body without any such pretreatment is necessary.

Stabilized cell multiplication can be achieved and human prolactin production increased by repeated transplantation with one or more combinations of different warm-blooded animals; for example, we can first implant human cells in the hamster, so that they multiply, then re-implant them in the naked mouse. In addition, repeat transplantation can be performed with animals belonging to the same class or division as well as the same species or genus.

Human cells can be implanted at any site in the animal, as long as they multiply there: for example, implantation can be carried out in the allantoic cavity, or intravenously, intraperitoneally or subcutaneously.

In addition to the direct transplantation of cells into the organism of an animal, in order to multiply conventional established human cell lines capable of producing human prolactin, it is possible to use the nutritive liquid of the organism of an animal, by introducing into the body of this animal, for example intraperitoneally, a conventional diffusion chamber of various shape and size, provided with a porous filter membrane, an ultrafilter or hollow fibers having pores of '' about 10 "7 to 10 ~ 5 m in diameter, preventing contamination of the diffusion chamber

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by the cells of the host animal and allowing the nutritive liquid of the organism of the animal to supply the cells. In addition, the diffusion chamber can be designed, if necessary, to be able to be placed for example on the host animal and allow the circulation of the liquid of the animal's organism in the diffusion chamber, as well as the observation cell suspension in the chamber by one or more transparent side windows placed on one or more walls of the chamber; the room is replaceable and exchangeable with a new room; this increases the multiplication of cells during the life of the animal and also the production of cells per animal without having to sacrifice the latter. In addition, when such a diffusion chamber is used, as it is easy to harvest the multiplied human cells and there is no immune reaction, due to the absence of direct contact between the human cells and the cells. of the host animal, it is possible to use, according to the invention, any warm-blooded animal, without any prior treatment to reduce the immune reaction.

The animal in which human cells are implanted can easily be fed in the conventional manner, even after the transplantation of the cells, and no particular care is necessary.

Maximum cell multiplication is obtained approximately 1 to 20 weeks after cell transplantation. When the established human cell line which is implanted in the animal is a human, tumor or human lymphoblastoid cell line, the maximum multiplication of cells is achieved 1 to 5 weeks after transplantation due to their speed extremely high multiplication.

The number of human cells obtained per host can be between approximately 10 7 and 10 12 or more. In other words, the number of human cells transplanted into the animal's body increases by a factor of about 102 to 107 or more, which is about 101 to 106 or more times greater than numbers obtained by the tissue culture method in vitro with a nutrient medium; the cells can therefore be used appropriately for the production of human prolactin.

As for the method of inducing human prolactin, any method can be used, provided that the human cells obtained by the method indicated above release human prolactin. For example, the human cells obtained by multiplication suspended in the ascites liquid and harvested in this liquid, or by extraction of the massive tumor formed under the skin and harvested after disintegration of this tumor, are suspended at a concentration of '' approximately 104 to 108 cells / ml, in a nutritive medium maintained at a temperature of approximately 20 to 40 ° C, then subjected to the action of a prolactin inducer at this temperature, for approximately 1 to 50 h to induce production of human prolactin.

The preferred prolactin inducers are organic compounds such as reserpine, phenothiazine or chlorproma-zine; hormones such as TRH (thyrotropin releasing hormone) and estrogen; amino acids such as lysine, arginine and cysteine; and mineral salts such as sodium, potassium or calcium chloride, or magnesium sulfate. In this case, other hormones, such as human somatotropic hormone (hGH), can be produced simultaneously according to the invention.

Human prolactin thus obtained can easily be collected according to purification and separation techniques using conventional operations, in particular salting out, dialysis, filtration, cen-trifugation, concentration or / and lyophilization. If a more purified human prolactin preparation is desired, it can be obtained in the highest purity state by combining the above techniques with other conventional operations, such as adsorption and desorption with ion exchange, filtration. on gel, affinity chromatography, isoelectric point fractionation and electrophoresis.

The human prolactin preparation thus obtained is identical from the immunological point of view to the NIH standard human prolactin preparation; it is therefore advantageously possible to use the preparation alone or in combination with one or more agents for administration by injection, by external or internal route, for the preparation and treatment of human diseases such as hypogalactia and for a diagnostic purpose.

In the present description, the production of human prolactin is determined by a radioimmunological method, described by P. Hang et al., "Proc. Nat. Acad. Sci. USA ”, vol. 68, pp. 1902-1906 (1971) and it is expressed by weight per milliliter of cell suspension, relative to the preparation of NIH standard human prolactin. Simultaneous production of hGH is determined by the radioimmunological method, as described by S.M. Glick et al., "Nature", vol. 199, p. 784 (1963), and is expressed by weight relative to the NIH standard human hormone'somatotropic preparation.

Several nonlimiting embodiments of the invention are described below.

Example 1:

Naked adult mice are implanted subcutaneously, which are then fed in the usual manner for three weeks, with human cells of disaggregated acidophilic adenoma, obtained by chopping, after extraction from a patient suffering from acidophilic adenoma. of the pituitary gland. The massive tumors obtained, formed under the skin and each weighing approximately 10 g, are extracted and broken up by chopping and suspended in a physiological saline solution containing trypsin. After washing the cells with Earle's medium 199 without glucose (pH 7.2), supplemented with 10% by volume of fetal calf serum, the cells are resuspended, to obtain a concentration of approximately 105 cells / ml , in a fresh preparation of the same medium having a concentration of 30 mmol in L-arginine as a prolactin inducer, then incubation at 37 ° C for 6 h to induce the production of human prolactin. Next, the cells are treated with ultrasound and the human prolactin in the supernatant is determined. Human prolactin production is approximately 600 ng / ml cell suspension. The simultaneous production of hGH is approximately 500 ng / ml of cell suspension. The control cells, obtained by m vitro culture of human acidophilic adenoma cells, are treated as above with the prolactin inducer, in Earle's medium 199 (pH 7.2) supplemented with 10% by volume of fetal serum. calf and incubation at 37 ° C. The production of human prolactin is only about 100 ng / ml of cell suspension.

Example 2:

Human cells of disaggregated acidophilic adenoma, obtained by hashing, are suspended together, after extraction of a patient suffering from acidophilic adenoma of the pituitary gland, with human leukemic lymphoblastoid cells of Namalwa line,

in a container with a saline solution of concentration 140 mmol in NaCl, 1 mmol in KCl, 1 mmol in NaH2PO4 and 2 mmol in CaCl2, to obtain concentrations of each cell type of approximately 103 cells / ml. The frozen cell suspension is mixed with a preparation of the same saline solution, containing Sendai virus previously inactivated by ultraviolet irradiation; transferred to an oven at 37 ° C about 5 min after mixing, and stirred in the oven for about 30 min, to obtain cell fusion, with introduction of the human prolactin production capacity of human cells acidophilic adenoma in the human leukemic lymphoblastoid cell line. After cloning, in the conventional manner, of the hybridoma cell strain capable of producing human prolactin, this strain is implanted intraperitoneally into adult naked mice, which are fed in the usual way for 5 weeks. The massive tumors produced, weighing approximately 15 g each, are extracted and treated as in Example 1, to induce the production of human prolactin, but the L-arginine 30 mmol is replaced by approximately 10 ng of TRH and that incubation is carried out at 37 ° C. The production of human prolactin is approximately 3300 ng / ml of cell suspension.

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A control experiment is carried out, as in Example 1, by in vitro culture of the hybrid of the human leukemic lymphoblastoid line Namalwa and exposure of the multiplied cells to the action of the prolactin inducer. Human prolactin production is only about 200 ng / ml cell suspension.

Example 3:

After injecting an antiserum prepared with a rabbit, according to a conventional method, into newborn hamsters to reduce their possible immune reaction to cell transplantation, the animals are implanted, subcutaneously, the human leukemic lymphoblastoid line JBL , which has been given the ability to produce human prolactin from human acidophilic adenoma cells, as in Example 2; the hamsters are then fed in the usual way, for 3 weeks. The massive subcutaneous tumors obtained, weighing approximately 10 g each, are extracted and treated as in Example 1, to induce the production of human prolactin; this production is approximately 1800 ng / ml of cell suspension. The simultaneous production of hGH is approximately 2000 ng / ml of cell suspension.

A control experiment, as in Example 1, is carried out by in vitro culture of the hybrid of the human leukemic lymphoblastoid line JBL, and exposure of the multiplied cells to the action of the prolactin inducer. Human prolactin production is only about 200 ng / ml suspension.

Example 4:

Intravenously, newborn rats are implanted with a human leukemic lymphoblastoid line Namalwa, to which the ability to produce human cellular prolactin from human acidophilic adenoma cells has been conferred, as in Example 2; then the rats are fed in the usual way for 4 weeks. The massive tumors produced, weighing about 40 g each, are extracted and treated as in Example 2 to induce the production of human prolactin; this production is approximately 2700 ng / ml of cell suspension.

A control experiment, as in Example 1, is carried out by in vitro culture of the hybrid of the human leukemic lymphoblastoid line Namalwa, and exposure of the multiplied cells to the action of the prolactin inducer. The production of human prolactin is then only about 100 ng / ml of cell suspension.

Example 5:

Adult mice are irradiated with approximately 400 rems of X-rays to reduce their immune reaction, and they are implanted, subcutaneously, human cells of acidophilic adenoma obtained as in Example 1; then the mice are fed in the usual way for 3 weeks. We extract massive tumors under

skin products, weighing approximately 15 g each, and treated as in Example 2, to induce the production of human prolactin; this production is approximately 800 ng / ml of cell suspension.

A control experiment, as in Example 1, is carried out by in vitro culture of human cells with acidophilic adenoma and exposure of the multiplied cells to the action of the prolactin inducer. The production of human prolactin is then only about 200 ng / ml of cell suspension.

Example 6:

Cells of the human leukemic lymphoblastoid line JBL, which have been given the capacity to produce human prolactin, are cells suspended in physiological saline solution from human cells of acidophilic adenoma, as in Example 3; the cells are then transferred into a cylindrical diffusion chamber, of plastic material, with an internal volume of approximately 10 ml, provided with a filtering membrane having pores of approximately 0.5 µm in diameter; the chamber is inserted into the peritoneum of an adult rat. After feeding the rat for 4 weeks in the usual way, the chamber is removed. The density of human cells in the chamber is about 2 x 109 cells / ml, which is about 103 times or more higher than what is obtained by in vitro culture with a carbon dioxide incubator. The cells thus obtained are treated as in Example 1, to induce the production of human prolactin: this is approximately 1900 ng / ml of cell suspension. The simultaneous production of hGH is approximately 2200 ng / ml of cell suspension.

A control experiment, as in Example 1, is carried out by in vitro culture of hybrid cells of the human leukemic lymphoblastoid line JBL, then exposure of the multiplied cells to the action of a prolactin inducer. Human prolactin production is only about 200 ng / ml cell suspension.

Example 7:

In the allantoic cavity of embryonated eggs, which have been preincubated at 37 ° C. for 5 d, cells of the human leukemic lymphoblastoid line JBL are implanted, to which the ability to produce human prolactin from human cells has been conferred. The acidophilic adenoma, as in Example 3. After incubating the eggs at this temperature for another week, the multiplied human cells are collected. The cells are treated as in Example 2, to induce the production of human prolactin; this production is approximately 1700 ng / ml of cell suspension.

In a control experiment, as in Example 1, by in vitro culture of hybrid cells of the human leukemic lymphoblastoid line JBL, and exposure of the multiplied cells to the action of the prolactin inducer, only about 200 ng of human prolactin per milliliter of cell suspension.

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Claims (5)

649783 1. A method for producing human prolactin, which consists in multiplying human cells capable of producing human prolactin and exposing the multiplied cells to the action of a prolactin inducer to induce the production of human prolactin, characterized in that the multiplication takes place by transplantation in the body of a warm-blooded animal or in a device enabling these cells to be supplied with nutritive liquid from the body of a warm-blooded animal. 2. Method according to claim 1, characterized in that the human cells are acidophilic, intact cells of the anterior pituitary gland, such cells transformed by the EB virus or by x-ray irradiation, human adenoma cells acidophile from the pituitary gland, lung carcinoma cells or established cell lines of such cells. 2 3. Method according to claim 1, characterized in that the human cells are hybrid cells, obtained by cell fusion of a human lymphoblastoid line, and of one of the human cells described in claim 2. 4. Method according to one of claims 1 to 3, characterized in that the prolactin inducer is reserpine, phenothiazine, chlorpromazine, a hormone of the thyrotropin releasing hormone type, TRH, estrogens, an amino acid, in particular lysine , arginine or / and cysteine, a mineral salt, in particular sodium, potassium or calcium chlorides, magnesium sulfate, or more of such substances. 5. Method according to one of claims 1 to 4, characterized in that the warm-blooded animal is a poultry, in particular chick or pigeon, or a mammal, in particular dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse or naked mouse.