GB2273046A - Synergistic combination to improve secretion of endogenous growth hormone - Google Patents

Abstract

The combination of growth hormone releasing factor (GRF) and the synthetic molecule (I) L-692,429, a non-peptidal growth hormone secretagogue, is seen to synergistically increase the endogenous production of growth hormone by the pituitary gland. Compositions containing GRF and L-692,429 are also disclosed. <IMAGE>

Description

TITLE OF THE INVENTION SYNERGISTIC COMBINATION TO IMPROVE THE SECRETION OF ENDOGENOUS GROWTH HORMONE BACKGROUND OF THE INVENTION Growth hormone releasing factor (GRF and also referred to as somatoliberin) is a 44 amino acid polypeptide produced by the hypothalamus which stimulates the production of growth hormone by the pituitary gland. Growth hormone is required by the body for many metabolic effects including to increase the rate of protein synthesis; decrease the rate of carbohydrate utilization in cells; and increase the mobilization of free fatty acids and the use of fatty acids for energy. A deficiency of natural growth hormone secretions can result in various medical disorders such as dwarfism.

L-692,429 is a non-peptide substituted benzolactam which has the effect of stimulating the natural production of growth hormone by the pituitary.

The compound is known and is described in European Patent Publication 92,302,143.0.

SUMMARY OF THE INVENTION The instant invention is concerned with the synergistic combination of GRF and L-692,429 to stimulate the pituitary to secrete more growth hormone than would be expected from the administration of each drug alone. Thus, it is an object of the instant invention to describe the effect of each of GRF and L-692,429 individually and in synergistic combination.

It is a further object to describe the joint administration of these two drugs as well as the administration of L-692,429 in the presence of GRF to stimulate the release of endogenous growth hormone. A still further object is to d cribe the composition containing GRF and L-692,429 for the administration to animals, including humans, to stimulate the release of growth hormone. Further ob: cts will become apparent from a reading of the following description.

DESCRIPTION OF THE INVENTION The non-peptide growth hormone secretagogue L-692,429 is disclosed in European Patent Publication 92,302,143.0 and has the following structure:

This compound is shown to promote the secretion of endogenous growth hormone by the pituitary when administered to a human or animal patient.

Growth hormone releasing factor (GRF) is normally secreted by the hypothalamus and also stimulates the release of endogenous growth hormone.

It has been unexpectedly discovered that when L-692,429 is administered in the presence of GRF, the rate of growth hormone secretion is increased to an extent that is beyond what would be expected from either- drug alone and also what could be predicted from the properties of the individual compounds. That is, L-692,429 and GRF synergistically combine to provide much higher levels of growth hormone secretions.

While it often occurs that those in need of increased levels of growth hormone have depressed levels of GRF, in those cases where GRF secretions are still present, the synergistic effects of the combination of L-692,429 and GRF will be noted with the administration of L-692,429 alone. The synergism will promote the secretions of growth hormone beyond that of the natural secretions occuring due to the presence of endogenous GRF and also beyond what would be expected from the administration of L-692,429.

When the body is not producing significant quantities of endogenous GRF, the synergistic effects can be achieved by the co-adminstration of L-692,429 and GRF.

The effects of the synergism of L-692,429 and GRF can be observed or measured either invivo or in vitro, however, it is somewhat easier and faster to measure the effect in isolated pituitary cells in vitro. In this manner, the absolute levels of both L-692,429 and GRF can be accurately controlled, and the precise effects of the two compounds, singularly and together, can be accurately measured. In these in vitro experiment, carried out in isolated rat pituitary cells the effects of L-692,429 alone was to increase the level of growth hormone secretions by 500% and GRF alone was seen to increase the level of growth hormone secretions by 400%. When administered together the isolated rat pituitary cells increased growth hormone secretions in excess of 1800%.

The increases in growth hormone secretions can also be measured in vivo by determining the basal levels of endogenous GRF and adminstering both compounds, jointly and singularly to the test animal and measuring the levels of growth hormone secretions.

DESCRIPTION OF THE DRAWING The attached figure is a plot of the levels of L-692,429 administered against the levels of growth hormone secretions of isolated rat pituitary cells.

The two curves show the levels of growth hormone secretions resulting from L-692,429 alone (open circles) and in the presence of 10 nM of GRF (solid circles). The Y axis shows the basal levels of growth hormone with no GRF and no L-692,429 to be about 30 ng per well. With 10 nM of GRF the growth hormone secretion is about 150 ng per well. It is noted that the level of growth hormone secretions for L-692,429 rise to a constant value of about 150 ng per well which is about the same as the level of growth hormone secretion induced by 10 nM of GRF.

With both L-692,429 and a constant level of GRF of 10 nM, the growth hormone secretions increase to between 400 and 500 ng per well which is considerably beyond what would be expected from a combination of the two compounds.

DESCRIPTION OF THE UTILITY OF THE INVENTION The synergistic combination of the growth hormone releasing compounds L-692,429 and GRF are useful in vitro as unique tools for understanding how growth hormone secretion is regulated at the pituitary level. This includes use in the evaluation of many factors thought or known to influence growth hormone secretion such as age, sex, nutritional factors, glucose, amino acids, fatty acids, as well as fasting and non-fasting states. In addition, the synergistic combination of compounds of this invention can be used in the evaluation of how other hormones modify growth hormone releasing activity. For example, it has already been established that somatostatin inhibits growth hormone release. Other hormones that are important and in need of study as to their effect on growth hormone release include the gonadal hormones, e.g., testosterone, estradiol, and progesterone; the adrenal hormones, e.g., cortisol and other corticoids, epinephrine and norepinephrine; the pancreatic and gastrointestinal hormones, e.g., insulin, glucagon, gastrin, secretin; the vasoactive intestinal peptides, e.g., bombesin; and the thyroid hormones, e.g., thyroxine and triiodothyronine. The compounds of Formula I can also be employed to investigate the possible negative or positive feedback effects of some of the pituitary hormones, e.g., growth hormone and endorphin peptides, on the pituitary to modify growth hormone release. Of particular scientific importance is the use of these compounds to elucidate the subcellular mechanisms mediating the release of growth hormone.

The synergistic combination of L-692,429 and GRF can be administered to animals, including man, to release growth hormone ia vivo. For example, the compounds can be administered to commercially important animals such as swine, cattle, sheep and the like to accelerate and increase their rate and extent of growth, and to increase milk production in such animals. In addition, these compounds can be administered to humans in vivo as a diagnostic tool to directly determine whether the pituitary is capable of releasing growth hormone. For example, the compounds of Formula I can be administered in vivo to children.

Serum samples taken before and after such administration can be assayed for growth hormone.

Comparison of the amounts of growth hormone in each of these samples would be a means for directly determining the ability of the patient's pituitary to release growth hormone.

Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of Formula I in association with a pharmaceutical carrier or diluent. Optionally, the synergistic combination of active ingredients of the pharmaceutical compositions can comprise a growth promoting agent in addition to at least one of the compounds of Formula I or another composition which exhibits a different activity, e.g., an antibiotic or other pharmaceutically active material.

Growth promoting agents include, but are not limited to, TRH, diethylstilbesterol, theophylline, enkephalins, E series prostaglandins, compounds disclosed in U.S. Patent No. 3,239,345, e.g., zeranol, and compounds disclosed in U.S. Patent No. 4,036,979, e.g., sulbenox or peptides disclosed in U.S. Patent No.

4,411,890.

A still further use of the disclosed novel combination of L-692,429 and GRF is in combination with other growth hormone secretagogues such as GHRP-6, GHRP-1 as described in U.S. Patent Nos. 4,411,890; and publications WO 89/07110 and WO 89/07111 and B-HT920.

As is well known to those skilled in the art, the known and potential uses of growth hormone are varied and multitudinous. Thus, the administration of the synergistic combination of the compounds of this invention for purposes of stimulating the release of endogenous growth hormone can have the same effects or uses as growth hormone itself.These varied uses of growth hormone may be summarized as follows: stimulating growth hormone release in elderly humans; Prevention of catabolic side effects of glucocorticoids, treatment of osteoporosis, stimulation of the immune system, treatment of retardation, acceleration of wound healing, accelerating bone fracture repair, treatment of growth retardation, treating renal failure or insufficiency resulting in growth retardation, treatment of physiological short stature, including growth hormone deficient children, treating short stature associated with chronic illness, treatment of obesity and growth retardation associated with obesity, treating growth retardation associated with Prader-Willi syndrome and Turner's syndrome; Accelerating the recovery and reducing hospitalization of burn patients;Treatment of intrauterine growth retardation, skeletal dysplasia, hypercortisolism and Cushings syndrome; Induction of pulsatile growth hormone release; Replacement of growth hormone in stressed patients; Treatment of osteochondrodysplasias, Noonans syndrome, schizophrenia, depression, Alzheimer's disease, delayed wound healing, and psychosocial deprivation; treatment of pulmonary dysfunction and ventilator dependency; Attenuation of protein catabolic response after a major operation; reducing cachexia and protein loss due to chronic illness such as cancer or AIDS.Treatment of hyperinsulinemia including nesidioblastosis; Adjuvant treatment for ovulation induction; To stimulate thymic development and prevent the age-related decline of thymic function; Treatment of immunosuppressed patients; Improvement in muscle strength, mobility, maintenance of skin thickness, metabolic homeostasis, renal hemeostasis in the frail elderly; Stimulation of osteoblasts, bone remodelling, and cartilage growth; Stimulation of the immune system in companion animals and treatment of disorders of aging in companion animals; Growth promotant in livestock; and stimulation of wool growth in sheep.

The compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual, or topical routes of administration and can be formulated in dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.

Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.

Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or a suppository wax.

Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.

The dosage of active ingredients in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredients be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Generally, for L-692,429 dosage levels of between 0.001 to 100 mg/kg., preferably between 0.02 and 100 mg/kg. of body weight daily are administered to patients and animals, e.g., mammals, to obtain effective release of growth hormone in those cases where normal levels of GRF are suppressed. In those cases where normal levels of GRF are present, or when GRF is being administered along with L-692,429, somewhat lower levels of L-692,429 may be used, as low as 0.0005 mg/kg.The amount of GRF administered in combination with L-692,429 can vary from the basal level of GRF present in the animal or, where the natural levels of GRF are suppressed, the amount of GRF administered can vary from 0.0001 to 0.1 mg/kg, preferably from 0.001 to 0.1 mg/kg.

The following examples are provided for the purpose of further illustration only and are not intended to be limitations on the disclosed invention.

EXAMPLE 1 MATERIALS AND METHODS Materials Dulbecco's Modified Eagle Medium (DMEM), horse and fetal bovine sera, glutamine (100-fold concentrated), non-essential amino acids (100-fold concentrated), nystatin (10,000 U/ml), gentamycin (50 mg/ml), Hank's Balanced Salt Solution and collagenese were purchased from Gibco (Grand Island, NY). Human GRF-(1-40)-OH, was from Peninsula Laboratories (San Carlos, CA). Hyaluronidase was purchased from Sigma (St. Louis, MO). Rat GH RIA kits were obtained from Dr. A. F. Parlow (Harbor-UCLA Medical Center, Torrance, CA). L-692,429 and was synthesized as described in EP 92,302,143.0. and dissolved in DMSO before use.

Methods Pituitary glands were aseptically removed from 150-200 g Wistar male rats and placed in Hank's Balanced Salt Solution (HBSS) in a 50-ml polypropylene centrifuge tube. The collection tube was centrifuged for 5 minutes at 250 x g, and HBSS was removed by aspiration. Pituitary glands were transferred to a disposable petri plate and minced with a scalpel. The minced tissue was then transferred to a 50-ml disposable centrifuge tube by suspending the tissue fragments in three successive 10-ml aliquots of HBSS.

The tissue suspension was then centrifuged at 1200 x g for 10 minutes, and HBSS was removed by aspiration.

The pellet was resuspended in 30 ml 0.2% collagenase and 0.2% hyaluronidase in HBSS. Cell dispersion was carried out in a 37"C water bath, with vortexing at 15 and 30 minutes. The undigested pituitary fragments were allowed to settle for 3-5 minutes and transferred with a pipette to a second centrifuge tube, and 20 ml of fresh digestion mixture was added. After 20 minutes in a water bath, the contents of the tube were vortexed for 30 seconds, and the two digests were pooled and passed through a 102in Nytex filter (Frankel Co., Philadelphia, PA) to remove undigested material. The ensuing cell suspension was centrifuged at 1200 x g for 10 minutes. The supernatant fluid was discarded, and the pellet was resuspended in 15 ml culture medium and counted.For culture, the cell suspension was adjusted to approximately 1.5 x 105 cells/ml, and 1.0 ml of this suspension was placed in each well of a 24-well tray (Costar, Cambridge, MA). Incubation was carried out in a humidified 5% C02-95% air atmosphere at 37"C. The culture medium consisted of Dulbecco's Modified Eagle Medium containing 0.37% NaHC03, 10% horse serum, 2.5% fetal bovine serum, 1% nonessential amino acids, 1% glutamine, 1% nystatin and 0.1% gentamycin.

After 3-4 days in culture, cells were washed twice with the above culture medium containing 25 mM HEPES, adjusted to pH 7.4 with 2N NaOH, and incubated at 37"C for 15 minutes in 1 ml of the same medium with test agents in quadruplicate. After incubation, the medium was removed and centrifuged at 2000 x g for 15 minutes to remove any cellular material. The supernatant fluid was removed and growth hormone content in the culture medium was measured by a double antibody RIA procedure.

Results The results of this experiment are shown in the figure which shows the effects of increasing levels of L-692,429 on growth hormone secretions in the presence or absence of GRF. The incubation of the rat pituitary cells was carried out at 37"C for 15 minutes whereupon the levels of growth hormone were measured.

Each point represents the mean SEM of quadruplicate incubations. If no SEM is shown in the figure, it is smaller than the symbol representing the mean.

Claims (10)

WHAT IS CLAIMED IS:

1. A method for synergistically increasing the secretions of growth hormone in an animal which comprises administering to such animal an effective amount of L-692,429 in the presence of an effective amount of GRF.

2. The method of Claim 1 where the amount of GRF synergizing with L-692,429 is the basal level of GRF present in the animal.

3. The method of Claim 1 where the GRF synergizing with L-692,429 is co-administered with L-692,429.

4. The method of Claim 3 where the amount of GRF administered is from 0.0001 to 0.1 mg/kg and the amount of L-692,429 administered is from 0.001 to 100 mg/kg.

5. The method of Claim 4 where the amount of GRF administered is from 0.001 to 0.1 mg/kg and the amount of L-692,429 administered is from 0.02 to 100 mg/kg.

6. A synergistic combination of L-692,429 and GRF.

7. The synergistic combination of L-692,429 and GRF of Claim 6 in which the GRF is endogenously present in an animal and the L-692,429 is administered to such animal.

8. The synergistic combination of Claim 6 in which both the L-692,429 and GRF are administered to an animal.

9. The synergistic combination of Claim 8 which contains from 0.001 to 100 mg/kg of L-692,429 and from 0.001 to 0.1 mg/kg of GRF.

10. The synergistic combination of Claim 9 which contains from 0.02 to 100 mg/kg of L-692,429 and from 0.001 to 0.1 mg/kg of GRF.