Differential diagnosis and treatment of the states of growth hormone insufficiency (or deficiency) with an alpha-2-adrenergic agonist and a growth hormone releasing peptide
This is a continuation of application Ser. No. 07/637,362 filed Jan. 4, 1991, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the differential diagnosis of the various states of growth hormone insufficiency or deficiency. The invention also relates, in those cases in which a positive response could be expected, to the treatment with an alpha-2-adrenergic agonist and a growth hormone-releasing peptide to induce growth hormone secretion in children, particularly those children growing between the tenth (P 10 ) and third (P 3 ) percentile, and most particularly children with stature below the third percentile (P 3 ). 
     BACKGROUND OF THE INVENTION 
     From a clinical point of view, it is clear that it would be helpful to use a biochemical test to better understand and differentiate the various states of growth hormone (GH) deficiency. The availability of such a test would permit the identification of the defect leading to the GH insufficiency and its degree; as well as to better decide when a GH treatment is needed and when it must be started. All conventional dynamic tests (i.e. clonidine, levodopa, arginine, insulin hypoglycemia) (Gil-Ad, Lancet 2:278, 1979;  Plotnik, J. Clin. Endocrinol. Metab., 48:811, 1979; Frasier, Pediatrics, 53:929, 1974; Underwood, &#34;Normal and Aberrant Growth,&#34; Williams Textbook of Endocrinology (eds. Wilson and Foster) p. 155, 1985, Saunders, Philadelphia) are unable to differentiate between GH-deficiency due to pituitary defect and that due to hypothalamic dysfunction involving the mechanisms engaged in GH neuroregulation. Moreover, these tests do not distinguish between hypothalamic defects leading to transiently or chronically impaired GH secretion; or particularly between Constitutional Growth Delay and Familial Short Stature, especially when, as is frequently observed, the former condition coexists with the genetic cause. 
     It was expected that the availability of growth hormone-releasing hormone (GHRH) would improve the ability to define GH-deficient states; however, the majority of growth hormone deficient subjects have a hypothalamic dysfunction and the majority of subjects tested have GH secretion in response to GHRH regardless of the etiology of their GH deficiency (Chatelain, Pediatric Research, 19:610, 1985). Otherwise, non-responder subjects could have been tested in a refractory hypothalamic somatotroph rhythm (HSR) phase (Devesa, Clinical Endocrinology (Oxf), 30:367, 1989), occurring either physiologically, during a normal trough period, or pathologically, as a consequence of a chronically increased somatostatinergic tone. Therefore, the use of GHRH alone has now been discarded for that purpose. 
     It is known that administering an agent to a normal subject that interferes with the hypothalamic release of somatostatin will enhance growth hormone release. This effect has been shown for clonidine, an alpha-2-adrenergic agonist, and pyridostigmine, a cholinergic agonist, although the mechanism of action is different for each drug (Ghigo, J. Endocrinol. Invest., 12:99, 1989). Likewise, galanin has been shown to potentlate GHRH-induced GH secretion in normal subjects (Davis, J. Clin. Endocrinol. Metab., 65:1248, 1987) via the cholinergic pathways (Chatterjee, J. Endocrinol., 116:R1-R2, 1988). It has also been recently postulated that the pyridostigmine plus GHRH test would allow the differentiation of GH deficiency due to pituitary secretory inability from that due to a hypothalamic defect, without distinguishing between the different states of GH neurosecretory dysfunctions (i.e., Congenital Delay of Growth, GH Neurosecretory Dysfunction) and Familial Short Stature (Ghigo, &#34;Effects of the Enhancement of the Cholinergic Activity on Growth Hormone Secretion in Children: Clinical Implications,&#34; Recent Advances in Basic and Clinical Neuroendocrinology (eds. Casanueva and Dieguez), pp. 241-250, 1989, Excerpta Medica, Amsterdam). However, the fact that alpha-2-adrenoceptor blockade blunted the enhanced GH response to pyridostigmine plus GHRH (Devesa, [Devesa Mugica, the inventor herein] Abstracts Book, XII Panamerican Congress of Endocrinology, held November 2-8, 1990, Recife, Brazil) indicates that false negative responses (i.e. false pituitary defects) may also occur when utilizing this test. Moreover, pyridostigmine does not represent a useful treatment of short stature (Ghigo, &#34;Effects of the Enhancement of the Cholinergic Activity on Growth Hormone Secretion in Children: Clinical Implications,&#34;  Recent Advances in Basic and Clinical Neuroendocrinlogy (eds. Casanueva and Dieguez), pp. 241-250, 1989, Excerpta Medica, Amsterdam). While it has been shown that pretreatment with clonidine enhances the GH response to GHRH in short, normal children and adults (Reiter, J. Pediatr. Endocrinol., 3:21, 1988; Devesa [Devesa Mugica, the inventor herein] et al. J. Clin. Endocrinol. Metab., 71:1581-1588, 1990, it was not known whether such treatment could be extended to the differential diagnosis and treatment of the various states of GH-insufficiency in children. 
     SUMMARY OF THE INVENTION 
     It has now been discovered that the various GH-deficient states may be effectively differentiated by cojointly administering an alpha-2-adrenergic agonist, such as clonidine, and a growth hormone releasing peptide, such as GHRH. This test both explores the functionality of the main hypothalamic structures involved in GH control, and directly assesses the secretory integrity of somatotropes. Therefore, no false negative responses are expected to occur. Moreover, it minimizes, at high GH levels, the variability in GH responses to GHRH, thus allowing its use in the treatment of short children having a hypothalamic dysfunction responsible for their diminished linear growth. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Until now, all of the known provocative tests for GH secretion were unable to characterize the various GH-deficient states, and the unique effective treatment for hypothalamic GH deficiency was the administration of exogenous GH. A unique and effective test for differential diagnosis and treatment of such patients has now been found. This test involves administering cojointly to children of short stature an effective amount of an alpha-2-adrenergic agonist and a growth hormone releasing peptide. 
     The alpha-2-adrenergic agonist may be any of those which produce a post-synaptic stimulation of the alpha-2-adrenergic agonist pathway so as to inhibit somatostatin release by the hypothalamus into the hypothalamic pituitary portal system. Alpha-2-adrenergic agonists which may be utilized include clonidine (2-(2,6-dichloroanilino)-2-imidazoline), guanfacine, guanabenz, guanclofine, guanoxabenz (U.S. Pat. No. 4,910,215) and medatomidine (U.S. Pat. No. 4,910,214). Clonidine is preferred. The dosage is adjusted in accordance with the needs of the patient and the result desired. Typically, clonidine is administered orally at a dosage of about 100 to about 300 ug/m 2 , preferably about 150 ug/m 2 . It is preferably administered from 0 to about 120 minutes, most preferably about 60 minutes, prior to administering the growth hormone releasing peptide. 
     The growth hormone releasing peptide which may be utilized includes those peptides which stimulate a GH response at the GHRH level, i.e. stimulate the pituitary somatotropes. Such peptides include GHRH itself in its various known active forms such as growth hormone-release factors (GRF) GRF 1-44, GRF 1-40, GRF 1-37 and GRF 1-29. GHRH is typically administered by injection intravenously (i.v) or subcutaneously (s.c.), and may be advantageously delivered in a pulsatile manner (e.g. by infusion pump) or continuously (e.g. long-term preparations). The dosage is typically about 1 ug/kg if administered by i.v. bolus, or about 10 ug/kg s.c., for acute tests, or about 3 ug/kg/h for chronic treatment. Obviously, the dosage can be adjusted to meet the needs of the particular patient under diagnosis or treatment and the desired objective. 
     Other growth hormone-releasing peptides which can be effectively utilized are those short chain peptides (4-11 amino acids, preferably 5-7 amino acids) which have been recently found to stimulate a GH response similar to GHRH. These peptides include, but are not limited to those active peptides which are disclosed in U.S. Pat. No. 4,223,019, U.S. Pat. No. 4,223,020, U.S. Pat. No. 4,223,021, U.S. Pat. No. 4,224,316, U.S. Pat. No. 4,226,857, U.S. Pat. No. 4,228,155, U.S. Pat. No. 4,228,156, U.S. Pat. No. 4,228,157, U.S. Pat. No. 4,228,158, U.S. Pat. No. 4,410,512, U.S. Pat. No. 4,410,513, U.S. Pat. No. 4,411,890, U.S. Pat. No. 4,839,344, U.S. Pat. No. 4,880,777, U.S. Pat. No. 4,880,778, WO 89/07110, WO 89/07111 and WO 89/10933. Of the peptides disclosed in the above-identified patents, especially preferred are peptides of the formula: 
     AA 1  -His-AA 3  -Ala-TrP-D-Phe-Lys-NH 2  wherein AA 1  is H or Ala and AA 3  is preferably D-Trp, as well as analogs and derivatives thereof with similar activity. 
    
    
     EXAMPLE 
     Twenty-four children (17 boys and 7 girls) with stature below the third percentile (P 3 ), participated in the study with informed consent (parent and child). Ages of the children ranged from 7 to 13.9 years. In all but seven of the children, weight was more than or equal to P 3 . Three of these children showed evidence of malnourishment. Two children had received prophylactic cranial irradiation due to brain tumors, five and three years before the study. Three children had been treated with GH for 9 months; their growth velocity increased during this therapy from 3 to 8 cm/yr. All children had been previously studied for their GH insufficiency. Studies included growth velocity, bone age, classical provocative tests for GH secretion (i.e. clonidine, insulin hypoglycemia and propranolol plus exercise), plasma somatomedin C (Sm C) (Insulin Growth Factor-I (IGF-I)) analysis, and analysis of the spontaneous pattern of nocturnal GH secretion. On the basis of a low GH response (&lt;10 μg/L) to provocative tests, 50% of the children appeared to have a GH-deficiency after challenge with clonidine; this also occurred in 42% of the cases when tested with propranolol plus exercise, and in five of the six cases in which an insulinic hypoglycemia was achieved. Despite these results, in all but six children, indices of total and pulsatile nocturnal GH release appeared to be normal; plasma Sm C values were below normal limited for age in 18 of them. Mean (±SEM) plasma Sm C was 488±376 U/L. Mean (±SEM) growth velocity was 3.7±0.9 cm/yr. 
     For comparison purposes, twenty age-matched normal children (14 boys and 6 girls) were studied. Studies in this group included evaluation of growth velocity and bone age at the time of performing the combined test with clonidine plus GHRH. 
     Studies commenced at 09.00 h after an overnight fast and bed rest and thirty minutes after the insertion of a nonthrombogenic catheter for blood withdrawal in a forearm vein. The study consisted of administering clonidine (Catapresan, Boehringer Ingelheim, Spain) orally at 150 micrograms/m 2  at time 0, followed by administering GHRH (GRF 1-29, Serono, Spain) as an intravenous bolus at 1 microgram/kg at time 60 minutes. An additional control for short children consisted of administering a placebo at time 0, followed by administering GHRH at time 60 minutes. Blood samples for GH analysis (RIA, BioMerieux, France) were taken at 0 and 60 minutes and at 15 minute intervals for one hour following GHRH administration. The mean intra-assay coefficient of variation was 5.7, 3.2 and 4.3% at mean GH concentrations of 1.5, 10 and 22 micrograms/L respectively. To avoid interassay variations, all samples from a subject were run in the same assay. 
     Pretreatment with clonidine led to a clear GH response to GHRH in normal children, with a peak value of 41.6±3.5 μg/L. A multiple regression analysis showed that there was a significant positive correlation between the amplitude of GH response and both chronological and bone age (R=0.832; F=37.6; p=0.001; y=a+b×BA+c×CA, where y=GH peak, BA=bone age, CA=chronological age). This was also observed between GH peaks and the bone age/growth velocity ratio in this group of normal children. 
     Baseline plasma GH levels in short children were not different during the 2 study days. GHRH-induced GH response after pretreatment with placebo was deficient in 9 patients (GH peak &lt;10 μg/L), but no relationship was observed between this low response to GHRH and low responses in the other provocative tests, plasma Sm C, bone age or growth velocity. Pretreatment with clonidine led to a significantly higher GH response to GHRH in all but four children, but the range of these positive responses oscillated between 23.5 and 80.7 μg/L. A multivariate regression analysis established the existence of a close relationship (R=0.71; p=0.01) between the amplitude of clonidine plus GHRH elicited GH peaks and growth velocity, bone age and plasma Sm C levels, in those children in which a positive GH response to this test was found. Partial F table for this analysis showed that the strongest probability corresponded to growth velocity (F=8.92; p=0.009). 
     To confirm the uniqueness of such combined clonidine plus GHRH test in the differentiation of GH-deficient states, the theoretical GH responses to this test in short children were calculated on the basis of the equation obtained from the multiple regression analysis relating chronological and bone age with the amplitude of GH peaks in normal children. The percentage of the deviation between theoretical values thus obtained and real values after the clonidine plus GHRH test ranged between -435 and +41%. Responses between the -20 to +20% range were considered to be normals, as they fitted between the standard deviation from the mean in the group of normal children. Plotting those responses against the standard deviation of the bone age to growth velocity ratio, five well defined populations were showed to exist: 1) Low responders with low growth velocity for bone age; 2) Low responders with normal growth velocity for bone age; 3) Normal responders with low growth velocity for bone age; 4) Normal responders with normal growth velocity for bone age; and 5) High responders with low growth velocity for bone age. 
     The results of these experiments are summarized in the following Table I, Table II and Table III: 
     
                                           TABLE I                                 
__________________________________________________________________________
Patient Group                                                             
(Number of                                                                
Short Children)                                                           
        % DT-R  GY    Sm C  MCGH CA    BA                                 
__________________________________________________________________________
1(6)    -211 ± 163                                                     
                 2.8 ± 0.06                                            
                      318 ± 62                                         
                            4.1 ± 2.1                                  
                                 10.9 ± 2.1                            
                                       8.3 ± 2                         
2(5)    -69 ± 36                                                       
                4.5 ± 0.4                                              
                      394 ± 13                                         
                            5.2 ± 0.9                                  
                                  11 ± 1.3                             
                                       8.1 ± 1.7                       
3(5)      9 ± 6.2                                                      
                3.6 ± 0.4                                              
                      434 ± 73                                         
                            4.9 ± 2                                    
                                 8.4 ± 1.2                             
                                       5.9 ± 1.7                       
4(5)    4 ± 9                                                          
                4.4 ± 1                                                
                       976 ± 345                                       
                            5.9 ± 1                                    
                                  11 ± 2.2                             
                                       8.4 ± 3                         
5(3)    36 ± 5                                                         
                3.3 ± 0.3                                              
                      263 ± 55                                         
                            7.8 ± 1.8                                  
                                 8.3 ± 1.2                             
                                       4.6 ± 1.5                       
__________________________________________________________________________
 % DTR = Deviation between theoretically calculated and real GH peak      
 response to clonidine plus GHRH. GY = Growth velocity (cm/yr.). Sm C     
 (U/L).                                                                   
 MCGH = mean plasma GH concentration (μg/L) during a nocturnal period  
 sampling. CA, BA = Chronological and bone age (years), respectively.     
 Values are the mean ± SE.                                             
 
    
     
                       TABLE II                                                    
______________________________________                                    
                         Mean GH peak                                     
        Mean GH peak response                                             
                         response to clonidine                            
Group   to GHRH          plus GHRH                                        
______________________________________                                    
Normal  --               41.6 ± 3.5                                    
                                    (μg/L)                             
children                                                                  
Short   19.5 ± 15.7   42.2 ± 19                                     
children                                                                  
______________________________________                                    
 
    
     
                       TABLE III                                                   
______________________________________                                    
Number of positive (+, &gt;10 μg/L) or negative (-, &lt;10 μg/L)          
GH responses to the different provocative tests.                          
Patient                                                                   
Group                                                                     
(Short                                                                    
Children)                                                                 
        P + E   Clo     IH    GHRH  Clo + GHRH                            
______________________________________                                    
1       +3,-3   +0,-6   -6    +2,-4 +4,-2                                 
2       +3,-2   +4,-1   --    +3,-2 +5                                    
3       +3,-1   +2,-2   --    +3,-1 +4                                    
4       +4,-1   +3,-2   --    +3,-2 +5                                    
5       +1,-1   +1,-2   +1       +3 .sup. +3.sup.1                        
______________________________________                                    
 .sup.1 Two children in this group did not increase GH peak after clonidin
 plus GHRH as compared to that observed after GHRH alone. P + E =         
 propranolol plus exercise. Clo =  clonidine. IH = insulinic hypoglycemia.
 
    
     This data is evidence of the uniqueness of the clonidine plus GHRH test to allow better differentiation among the diverse GH-deficient states, and therefore optimize the decision about which children must be treated and when the treatment must start. While classical GH tests in this and other studies, including pyridostigmine plus GHRH (Ghigo, &#34;Effects of the Enhancement of the Cholinergic Activity on Growth Hormone Secretion in Children: Clinical Implications,&#34; Recent Advances in Basic and Clinical Neuroendocrinology (eds. Casanueva and Dieguez), pp. 241-250, 1989, Excerpta Medica, Amsterdam), do not differentiate between pituitary or hypothalamic defect, or among theoretically different hypothalamic dysfunction, the combined administration of clonidine and GHRH allowed these categorizations to be made well beyond any provocative test previously studied. 
     According to the results of this test, children from Group 1 are GH-deficients, from pituitary origin in two of them, and hypothalamic (neurosecretory dysfunction) in the other four. While the first two need to be treated with GH, the other four would benefit from a clonidine and GHRH treatment. Group 2 is composed of children with Constitutional Growth Delay; therefore no GH therapy is needed in this group, while a clonidine plus GHRH treatment would improve growth velocity in these children. Group 3 and Group 4 were composed of children having Familial Short Stature with or without Constitutional Growth Delay. The main difference between the two groups was due to the beginning of puberty in most of the children of Group 4, while all of the children in Group 3 were prepubertal. A more complete analysis of these children revealed the existence of differences in the GH response to clonidine plus GHRH between those cases in which Familial Short Stature was associated with Constitutional Growth Delay and those cases in which sexual maturation was normal. Children from Group 5 were malnourished, hence pretreatment with clonidine did not significantly enhance the GH response to GHRH. 
     In summary, the combined test with clonidine and GHRH allows the differentiation among diverse GH-deficient states. This single test places the subjects in a similar basal state thus eliminating interferences due to the hypothalamic-somatotroph rhythm (Devesa, [Devesa Mugrcal] Clinical Endocrinology (Oxf), 30:367, 1989). GH responses would therefore be a real expression of the pituitary reserve in hormone, indicating the functionality of the hypothalamus-somatotrope axis. Hence, this test does not give false negative responses and facilitates the differential diagnosis of GH-deficiency without the need for other provocative tests or the analysis of spontaneous GH secretion. Also, some children growing below the third percentile and showing a positive response to this test, independently of the degree of deviation from theoretically calculated values, would benefit from such a treatment. The superior effect of the pretreatment with alpha-2-adrenergic agonists such as clonidine is believed to result from the direct inhibition of somatostatin, unlike the indirect inhibition that occurs with the cholinergic agonists. 
     Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. It is preferred, therefore, that the present invention be limited not by the specific disclosure herein, but only by the appended claims.