Screening method for diagnosing growth hormone deficiency in pediatric patients by using maxirelin

Abstract

The present invention relates to a screening method for diagnosing growth hormone deficiency in pediatric patients by using maxirelin. The present invention further provides the substance maxirelin for use in the diagnosis of growth hormone deficiency in pediatric patients. The method includes providing at least one blood sample collected from a subject within a range of about 15 to about 100 minutes after administration of maxirelin in an amount effective to induce secretion of growth hormone, measuring a growth hormone level for each blood sample, comparing the measured growth hormone levels to a single threshold, and determining whether the measured growth hormone levels are different from the single threshold. And diagnosing whether the subject has growth hormone deficiency based on a comparison of the growth hormone level in the at least one blood sample to the single threshold. The method of the invention is a stand-alone test, does not require repetition, and does not require additional growth hormone stimulation tests for reliable diagnosis of pediatric patients.

Description

BACKGROUND OF THE INVENTION

[0002] Growth hormone (GH) is the major system-wide metabolic hormone, which regulates protein, lipid, and carbohydrate homeostasis and is essential for physical and mental growth, development, and maintenance. GH is produced in the anterior lobe of the pituitary gland under the stimulation of growth hormone-releasing hormone (GHRH) from the hypothalamic gland. GH is secreted from the pituitary in a pulsatile manner in approximately 6 - 10 random bursts over a 24-hour period.

[0003] Based on the source of GH deficiency, growth hormone deficiency (GHD) can be roughly classified into four categories: 1) pituitary or "classical" GHD, 2) hypothalamic GHD, 3) functional GHD, and 4) idiopathic GHD. GHD may become clinically apparent in children or adults. In the USA, the incidence of childhood GHD is estimated to be between 1 in 4,000 and 1 in 10,000. In the US, more than 50,000 adults have GHD, and it is reported that there are 6,000 new cases each year, including GHD children who transition to GHD adults (Human Growth Foundation www.hgfound.org).

[0004] GHD is a childhood disease characterized by reduced auxological parameters such as growth retardation and short stature. Childhood GHD can be congenital or acquired and can be isolated or combined with other pituitary hormone deficiencies. There are many well-defined etiologies of childhood GHD, but the etiology is usually unknown (idiopathic GHD). The definitions of various types of GHD and other causes of short stature are given in the International Classification of Pediatric Endocrine Diagnoses (ICPED). If untreated, childhood-onset GHD leads to permanent short stature. In children, the diagnosis of GHD relies on a detailed medical history, clinical features, auxological analysis, biochemical tests of the GH-IGF axis (GH = growth hormone; IGF = insulin-like growth factor) components, and radiological assessment of skeletal maturity and pituitary anatomy using MRI (GHRS, 2000).

[0005] Similar to the situation in adults, the diagnosis of childhood GHD relies on biochemical tests, which are based on growth hormone stimulation tests (GHSTs) that measure GH levels induced by reagents known to stimulate GH release. Currently used GHSTs, such as the insulin tolerance test (ITT) or glucagon stimulation test (GST), have not been developed and specifically approved for this purpose but are adapted from other indications and thus have limitations in performance characteristics such as specificity, sensitivity, safety, or feasibility (Molitch et al., 2011; Cook et al., 2009).

[0006] The ITT is considered the gold standard for assessing GHD. Intravenous administration of insulin is used to induce hypoglycemia, which in turn leads to GH release. However, this test is labor-intensive because intensive medical monitoring of the subject is required due to the potential risks associated with hypoglycemia (which is associated with symptoms such as tremors, lethargy, and tachycardia). Side effects are commonly reported as being dangerous. In addition, the ITT is contraindicated in subjects with seizure disorders and ischemic heart disease (Yuen 2011; Yuen 2013). Thus, the ITT is not widely used because of its inconvenience and safety issues.

[0007] The GST is an alternative that uses growth (Molitch 2011; Yuen 2011; Yuen 2013). Common side effects of the GST include nausea, vomiting, and headache. In addition, there are limitations in the test duration (3 - 4 hours), and it requires intramuscular injection. In fact, the medical need for a safe and reliable alternative test has not been truly met.

[0008] Larsen has disclosed in WO 2007 / 093820 A1 a test for diagnosing GHD based on macimorelin, an orally available peptidomimetic ghrelin receptor agonist with growth hormone secretagogue (GHS) activity.

[0009] Ghrelin effectively stimulates GH release [Kojima 1999]. The GH-releasing action of ghrelin is thought to be mediated by specific receptors present mainly at the pituitary and hypothalamic levels [Nogueiras 2006]. In membrane preparations containing GHS receptors derived from human hypothalamus and pituitary gland, macimorelin has been shown to exhibit binding potency to the human GHS receptor comparable to that of macimorelin with the natural ligand of the GHS receptor, ghrelin [Broglio 2002]. Macimorelin is readily absorbed from the gastrointestinal tract and is thought to act in the same way as ghrelin.

[0010] Macimorelin has been developed as an oral diagnostic agent for adult GH deficiency based on its ability to release GH pulses shortly after oral administration in healthy subjects.

[0011] Martinez et al. have disclosed macimorelin as a compound and its use in the treatment of GHD in WO 01 / 96300 A1.

[0012] Garcia et al. have disclosed the GHST of macimorelin in adult GHD (AGHD) (J Clin Endocrinol Metab. 2013, 2422 - 9, in J Clin Endocrinol Metab. 2018, 3083 - 3093 and in the poster entitled "Validation of Macimorelin As a Diagnostic Test for Adult Growth Hormone Deficiency (AGHD): A Phase 3 Study in Comparison with the Insulin Tolerance test (ITT)", which was presented at the 99th Annual Meeting of the Endocrine Society in 2017).

[0013] In WO 2019 / 121762 A1, a method for measuring growth hormone levels in a human or animal subject has been disclosed. The method comprises orally administering to the subject a composition containing macimorelin, collecting one, two or three post - administration samples from the subject within the range of 25 - 95 minutes after administration, and comparing the growth hormone levels in the one, two or three samples with a single threshold, wherein the single threshold is 2.8 ng / mL, and wherein the "single threshold" refers to the growth hormone level threshold used to indicate a sufficient response to macimorelin stimulation.

[0014] There is considerable debate regarding the role of GH stimulation tests in pediatric patients because low GH levels often occur during provocative tests and there are concerns about the validity and reproducibility of GHST.

[0015] Therefore, pediatric GHD diagnostic guidelines typically require the results of two GHSTs to conclude a diagnosis of GHD, unless there are typical brain defects (GHRS, 2000, GHRS 2019) where only one GHST is required.

[0016] The consensus guidelines of the GH Research Society published in 2000 (GHRS, 2000), the guidelines of the American Association of Clinical Endocrinologists published in 2003 (Gharib, 2003), and the national guidelines (Binder, 2014) recommend that in a well-standardized GHST protocol, a limited number of GHST agents should be used after an overnight fast. These include arginine (ARG), clonidine, glucagon, insulin, and levodopa.

[0017] The consensus guidelines of the GH Research Society state that the combination of GHRH and ARG as GHST is considered valuable in the diagnosis of GHD in children and adulthood, provided appropriate cut-off points are applied (GHRS, 2000). This combination has shown high sensitivity and high specificity in children and adolescents (Magnhie, 2002), late adolescents and young adults (Corneli, 2007). In the latter trial, cut-off points were established only in lean patients. Given the variation of GH secretion with body weight and obesity (Colao, 2009), there is still a need to define cut-off points suitable for overweight and obese pediatric patients (taking into account age, BMI, and waist circumference).

[0018] In children with clinical criteria for GHD, a GH peak concentration of less than 10 ng / mL has traditionally been used as the cut-off point to support the diagnosis. The sensitivity, specificity, and GH cut-off points used for different GHSTs in different studies have recently been reviewed (van Vught, 2009).

[0019] In this context, multiple GHSTs have been used in clinical practice, variably meeting the requirements of two GHSTs as part of the standard diagnostic method, either by repeating the same GHST or by performing two different GHSTs consecutively.

[0020] These two GHSTs are performed on the same day or on two consecutive days, and each test requires 4 - 6 blood samples. Especially in young children, the volume of blood collected is considered a safety issue, and attention should be paid to safety volume limits, such as those recommended by the WHO (Howie 2011). In addition to this safety topic, it should be noted that performing two tests is time-consuming and resource-intensive, and the associated burden on children and their parents as well as on pediatric endocrinologists is high.

[0021] Therefore, there is a proven need for a single test with proven safety, tolerance, ease of application, strong test characteristics in terms of sensitivity and specificity, and reliable reproducibility to replace two tests. A short press release on the present invention was published in 2020, announcing the results of a dose-finding pediatric study of macimorelin.

[0022] Brief Description of the Invention

[0023] In one aspect, the present invention provides a screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin, which comprises:

[0024] (a) providing one to five blood samples collected from a subject within a range of about 15 to about 100 minutes after administering an amount of macimorelin effective to induce growth hormone secretion;

[0025] (b) measuring the growth hormone level of each blood sample provided in step (a);

[0026] (c) comparing the measured growth hormone level obtained in step (b) with a single threshold, wherein the single threshold is 10.0 ng / mL or higher;

[0027] (d) determining that a subject whose highest growth hormone level in the blood sample obtained in step (b) is lower than the single threshold has growth hormone deficiency, and determining that a subject whose highest growth hormone level in the blood sample obtained in step (b)

[0028] is not lower than the single threshold does not have growth hormone deficiency.

[0029] In another aspect, the present invention relates to macimorelin for use in diagnosing growth hormone deficiency in pediatric patients, wherein,

[0030] (a) providing one to five blood samples collected from a subject within a range of about 15 to about 100 minutes after administering an amount of macimorelin effective to induce growth hormone secretion;

[0031] (b) measuring the growth hormone level of each blood sample provided in step (a);

[0032] (c) comparing the measured growth hormone level obtained in step (b) with a single threshold,

[0033] wherein the single threshold is 10.0 ng / mL or higher;

[0034] (d) determining that a subject whose highest growth hormone level in the blood sample obtained in step (b) is lower than the single threshold has growth hormone deficiency, and determining that a subject whose highest growth hormone level in the blood sample obtained in step (b) is not lower than the single threshold does not have growth hormone deficiency.

[0035] In a further aspect, the present invention provides a screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin, which comprises:

[0036] (a) Provide at least one blood sample that is collected from a subject within a range of about 15 to about 100 minutes after administering an effective growth hormone - secreting - inducing amount of macimorelin;

[0037] (b) Measure the growth hormone level in each blood sample;

[0038] (c) Compare each measured growth hormone level with a single threshold;

[0039] (d) Based on the comparison of the growth hormone level measured in step (b) in the at least one blood sample with the single threshold, diagnose whether the subject has growth hormone deficiency;

[0040] wherein determining whether the subject has or does not have growth hormone deficiency is based only on the growth hormone level induced by a single administration of macimorelin.

[0041] Definition

[0042] Unless otherwise indicated in the specification or claims, the terms used to interpret the methods of the present invention always have the following meanings.

[0043] As used herein, "subject" or "pediatric patient" is a human child of any gender (male or female) between the ages of about 2 years and less than 18 years. For example, the age of the subject is in the range of about 3 to less than 18, about 4 to less than 18, about 5 to less than 18, about 6 to less than 18, about 7 to less than 18, about 8 to less than 18, about 9 to less than 18, about 10 to less than 18, about 11 to less than 18, about 12 to less than 18, about 2 to less than 17, about 2 to less than 16, about 2 to less than 15, about 2 to less than 14, about 2 to less than 13, about 2 to less than 12, about 2 to less than 10, about 2 to less than 9, or about 2 to less than 8 years.

[0044] As used herein, "macimorelin" refers to a peptidomimetic compound that acts as a ghrelin receptor agonist with growth hormone secretagogue (GHS) activity. Its chemical structure and use in the treatment of GHD are disclosed in U.S. Patent 6,861,409, WO01 / 96300, and WO2007 / 093820.

[0045] As used herein, the term "effective amount" refers to the amount of a given substance that is sufficient in quantity to produce the desired effect. For example, an effective amount of macimorelin for inducing growth hormone secretion in a recipient is the amount of the compound that is capable of achieving a detectable increase in growth hormone secretion when administered to the subject.

[0046] As used in this application, the term "assay" describes the act of differentiating the presence or absence of a particular disorder based on the symptoms of a subject suspected of having the disorder. In other words, an "assay" for a disorder encompasses confirming or ruling out the disorder.

[0047] As used in this application, the term "treatment" describes the act of causing the elimination, reduction, alleviation, reversal, or prevention or delay of the onset or recurrence of any symptom of a related disorder.

[0048] As used herein, the term "blood sample" encompasses whole blood samples as well as portions of whole blood, such as serum or plasma samples. So long as two or more blood samples are used in the same methodological protocol for an assay, these blood samples are of the same type. For example, if the first sample is serum, the second sample and any subsequent samples are also serum. Further, if more than one blood sample is provided, the term "blood sample" refers to blood samples collected at different time points after administration of an amount of macimorelin effective to induce hormone secretion. Two blood samples can refer to blood samples collected after administration of an amount of macimorelin effective to induce hormone secretion, such as at about 30 ± 10 minutes and about 45 ± 10 minutes or at about 30 ± 10 minutes and about 60 ± 10 minutes. Three blood samples can refer to blood samples collected after administration of an amount of macimorelin effective to induce hormone secretion, such as at about 30 ± 10 minutes, about 45 ± 10 minutes, and about 60 ± 10 minutes or preferably at about 30 ± 10 minutes, about 45 ± 10 minutes, and about 90 ± 10 minutes or, alternatively, at about 30 ± 10 minutes, about 60 ± 10 minutes, and about 90 ± 10 minutes.

[0049] As used herein, the term "about" denotes a range of ± 10% of a reference value. For example, "about 10" defines a range of 9 to 11.

[0050] The term "single threshold" refers to indicating a sufficient response to macimorelin stimulation: instead of the threshold growth hormone level of 2.8 ng / mL typically used in adult trials, the threshold used in the method of the present invention is in a higher range of about 10.0 ng / mL or higher, such as about 10.0 - 25.0 ng / mL, about 10.0 - 20.0 ng / mL, about 10.1 - 19.5 ng / mL, about 10.2 - 19.0 ng / mL, about 10.3 - 18.5 ng / mL, about 10.4 - 18.0 ng / mL, about 10.5 - 17.5 ng / mL, about 10.6 - 17.0 ng / mL, about 11.0 - 16.5 ng / mL, about 12.0 - 16.0 ng / mL, about 13.0 - 15.5 ng / mL, about 14.0 - 15.0 ng / mL, about 15.0 - 16.0 ng / mL, about 15.5 - 18.0 ng / mL, about 16.0 - 18.0 ng / mL, about 16.5 - 18.0 ng / mL, about 17.0 - 18.0 ng / mL, about 17.5 - 20.0 ng / mL, about 18.0 - 19.0 ng / mL, about 18.5 - 19.5 ng / mL, about 19.0 - 20.0 ng / mL, about 20.0 - 21.0 ng / mL or about 25.0 - 30.0 ng / mL. The single threshold used in the method of the present invention can also be a single value, such as 10.5 ng / mL, 11.0 ng / mL, 11.5 ng / mL, 12.0 ng / mL, 12.5 ng / mL, 13.0 ng / mL, 13.5 ng / mL, 14.0 ng / mL, 14.5 ng / mL, 15.0 ng / mL, 15.5 ng / mL, 16.0 ng / mL, 16.5 ng / mL, 17.0 ng / mL, 17.5 ng / mL, 18.0 ng / mL, 18.5 ng / mL, 19.0 ng / mL, 20.0 ng / mL or 25.0 ng / mL. Additionally, the single threshold refers to ng / mL in whole blood or serum / plasma. Most preferably, the single threshold refers to ng / mL in serum. The terms "single threshold" and "cut-off point" are used interchangeably.

[0051] Weight percentage, wt%, % w / w, etc. are synonyms and refer to the concentration of a substance obtained by dividing the weight of the substance by the weight of the composition and multiplying by 100.

[0052] All patents, patent applications, and other publications cited in this application are hereby incorporated by reference in their entirety for all purposes.

[0053] Drawings

[0054] Figure 1Shows the individual mariciprilin concentrations relative to time for Group 1 (i.e., 0.25 mg / kg body weight), linear scale (Pharmacokinetics Analysis Group (PKS), number of patients (N) = 24).

[0055] Figure 2 Shows the individual mariciprilin concentrations relative to time for Group 2 (i.e., 0.5 mg / kg body weight), linear scale (PKS, N = 24).

[0056] Figure 3 Shows the individual mariciprilin concentrations relative to time for Group 3 (i.e., 1.0 mg / kg body weight), linear scale (PKS, N = 24).

[0057] Figure 4 Shows the individual GH concentrations after mariciprilin GHST relative to time for Group 1 (i.e., 0.25 mg / kg body weight), linear scale (Pharmacodynamics Analysis Group (PKS), N = 24).

[0058] Figure 5 Shows the individual GH concentrations after mariciprilin GHST relative to time for Group 2 (i.e., 0.5 mg / kg body weight), linear scale (PDS, N = 24).

[0059] Figure 6 Shows the individual GH concentrations after mariciprilin GHST relative to time for Group 3 (i.e., 1.0 mg / kg body weight), linear scale (PDS, N = 24).

[0060] Figure 7 Shows the receiver operating characteristic (ROC) analysis of mariciprilin GHST for Group 1 (i.e., 0.25 mg / kg body weight), (PDS, N = 24).

[0061] Figure 8 Shows the receiver operating characteristic (ROC) analysis of mariciprilin GHST for Group 2 (i.e., 0.5 mg / kg body weight), (PDS, N = 24).

[0062] Figure 9 Shows the receiver operating characteristic (ROC) analysis of mariciprilin GHST for Group 3 (i.e., 1.0 mg / kg body weight), (PDS, N = 24). Detailed Description of the Invention

[0064] An object of the present invention is to provide an independent method for measuring growth hormone levels in pediatric patients and for detecting GHD in pediatric patients. The object is to develop a new method that not only reduces the burden on trial administrators and trial subjects by reducing the trial duration and the number of blood draws, but also provides safe, reliable, and superior diagnostic performance.

[0065] In one aspect, the object of the present invention is surprisingly solved by providing a screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin, the method comprising:

[0066] (a) Providing one to five blood samples collected from a subject within a range of about 15 to about 100 minutes after administering an amount of macimorelin effective to induce growth hormone secretion;

[0067] (b) Measuring the growth hormone level of each blood sample provided in step (a);

[0068] (c) Comparing the measured growth hormone levels obtained in step (b) with a single threshold value, wherein the single threshold value is 10.0 ng / mL or higher;

[0069] (d) Determining that a subject in whom the highest growth hormone level in the blood sample obtained in step (b) is lower than the single threshold value has growth hormone deficiency, and determining that a subject in whom the highest growth hormone level in the blood sample obtained in step (b)

[0070] is not lower than the single threshold value does not have growth hormone deficiency.

[0071] Thus, one to five blood samples are collected from a subject not earlier than about 15 minutes and not later than about 100 minutes after administering an amount of macimorelin effective to induce growth hormone secretion.

[0072] For example, if the single growth hormone level in the blood sample is not lower than the single threshold value, it is determined that the subject does not have growth hormone deficiency. Alternatively, if the growth hormone levels in all blood samples are lower than the single threshold value, it is determined that the subject has growth hormone deficiency.

[0073] In a preferred embodiment, the screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin is an in vitro screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin.

[0074] In a preferred embodiment, the single threshold value of growth hormone is in the range of about 10.0 - about 25.0 ng / mL, preferably about 10.2 - about 20.0 ng / mL, further preferably about 12.0 - about 19.0 ng / mL, further preferably about 14.0 - about 18.0 ng / mL, more preferably about 16.0 - about 18.0 ng / mL, and most preferably about 17.0 - about 18.0 ng / mL.

[0075] In a preferred embodiment, one to four blood samples are provided in step (a), preferably one to three blood samples are provided in step (a), more preferably two or three blood samples are provided in step (a). As described above, if more than one blood sample is provided, these blood samples are collected at different time points after administration of an amount of macimorelin effective to induce hormone secretion.

[0076] In another preferred embodiment, in step (a), after administration of an amount of macimorelin effective to induce growth hormone secretion, a blood sample is collected from the subject in the range of about 20 to about 100 minutes, preferably in the range of about 25 to about 100 minutes, more preferably in the range of about 25 to about 95 minutes, and most preferably in the range of about 30 to about 90 minutes. Thus, after administration of an amount of macimorelin effective to induce growth hormone secretion, for example, not earlier than about 20 minutes but not later than about 100 minutes, preferably not earlier than about 25 minutes but not later than about 100 minutes, more preferably not earlier than about 25 minutes but not later than about 95 minutes, and most preferably not earlier than about 30 minutes but not later than about 90 minutes, a blood sample is collected.

[0077] In yet another preferred embodiment, if more than one blood sample is provided, the blood samples are collected from the subject at intervals of about 10 to about 60 minutes, preferably at intervals of about 15 to about 30 minutes. Alternatively, the blood samples can be collected at any time interval deemed appropriate by the attending physician. For example, the blood samples can be collected at intervals of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 45 or about 60 minutes.

[0078] In a further preferred embodiment, in step (a), the blood sample is a whole blood sample, a serum sample or a plasma sample. Preferably, in step (a), the blood sample is a serum sample or a plasma sample. If more than one blood sample is collected, two or more blood samples are of the same type, and thus, these blood samples are whole blood samples, serum samples or plasma samples. Most preferably, the blood sample is a serum sample.

[0079] In a further preferred embodiment, in step (a), about 0.8 mg to about 1.2 mg of masirem is administered per kg of the subject's body weight, preferably wherein in step (a), about 1.0 mg of masirem is administered per kg of the subject's body weight. Typically, the effective amount of masirem can be in the range of about 0.8 to about 0.9 mg per kg of the subject's body weight at the low-end, about 1.0, about 1.1 to about 1.2 mg per kg of the subject's body weight at the high-end, or in the range defined by any one of the low-end amounts and any one of the high-end amounts, for example, about 0.9 to about 1.1 mg / kg body weight. The effective amount of masirem can also be a single value, for example, about 0.8, about 0.9, about 1.0, about 1.1 or about 1.2 mg / kg body weight. The body weight (recorded in kg) can preferably be rounded to the nearest integer. Most preferably, in step (a), about 1.0 mg of masirem is administered per kg of the subject's body weight.

[0080] For example, a unit dose of masirem consists of 1817.2 mg of a composition for preparing an oral suspension in water. Typically, the prepared suspension contains 0.5 mg of masirem / mL of suspension. The body weight-adjusted aliquots of the reconstituted suspension are administered to pediatric subjects, yielding a pediatric dose of 1.0 mg / kg body weight. The unit dose is defined as masirem in 100% content of the free base. The mass of the free base of masirem or its free base equivalent within the unit dose is adjusted according to the content.

[0081] In a preferred embodiment, in step (a), masirem is administered in a composition comprising masirem in a suitable pharmaceutical salt, wherein preferably, the suitable pharmaceutical salt is selected from masirem acetate, masirem trifluoroacetate, or a combination thereof.

[0082] In a further preferred embodiment, in step (a), the administration of masirem is oral administration.

[0083] If the administration of masirem is oral administration, masirem can be prepared as an oral suspension. The suspension can be administered within about 90 minutes, preferably about 60 minutes, more preferably about 30 minutes after the preparation of the oral suspension. Preferably, the drinking of the oral suspension does not exceed a period of about 1 minute, preferably not exceed a period of about 30 seconds.

[0084] In a further preferred embodiment, the subject has fasted for about 10 hours, preferably about 9 hours, more preferably about 8 hours before the administration of masirem. Further preferably, in step (a), the subject fasts for about 100 minutes after the administration of masirem, which means that in step (a), the subject can fast during about 100 minutes, about 95 minutes or about 90 minutes after the administration of masirem.

[0085] In a further preferred embodiment, in step (a), a blood sample is provided which is collected from the subject about 60 ± 30 minutes after marenostrin administration. A blood sample may optionally be collected from the subject at about 30 ± 10, about 40 ± 10, about 45 ± 10, about 50 ± 10, about 60 ± 10, about 70 ± 10, about 80 ± 10, about 90 ± 10 or about 100 ± 10 minutes after marenostrin administration. A blood sample may be collected at any time point within the range of about 15 to about 100 minutes after marenostrin administration that is considered appropriate by the attending physician.

[0086] In another further preferred embodiment, in step (a), two blood samples are provided which are collected from the subject at about 30 ± 10 minutes and about 45 ± 10 minutes after marenostrin administration. Two blood samples may optionally be collected from the subject at about 20 ± 10, about 30 ± 10, about 40 ± 10, about 45 ± 10, about 50 ± 10, about 60 ± 10, about 70 ± 10, about 80 ± 10, about 90 ± 10 or about 100 ± 10 minutes after marenostrin administration. Two blood samples may be collected at any time point within the range of about 15 to about 100 minutes after marenostrin administration that is considered appropriate by the attending physician.

[0087] In another preferred embodiment, in step (a), two blood samples are provided which are collected from the subject at about 30 ± 10 minutes and about 60 ± 10 minutes after marenostrin administration.

[0088] In another preferred embodiment, in step (a), three blood samples are provided which are collected from the subject at about 30 ± 10 minutes, about 45 ± 10 minutes and about 60 ± 10 minutes after marenostrin administration, or in step (a) three blood samples are provided which are collected from the subject at about 30 ± 10 minutes, about 45 ± 10 minutes and about 90 ± 10 minutes after marenostrin administration, or in step (a) three blood samples are provided which are collected from the subject at about 30 ± 10 minutes, about 60 ± 10 minutes and about 90 ± 10 minutes after marenostrin administration. Three blood samples may optionally be collected from the subject at about 20 ± 10, about 30 ± 10, about 40 ± 10, about 45 ± 10, about 50 ± 10, about 60 ± 10, about 70 ± 10, about 80 ± 10, about 90 ± 10 or about 100 ± 10 minutes after marenostrin administration. Three blood samples may be collected at any time point within the range of about 15 to about 100 minutes after marenostrin administration that is considered appropriate by the attending physician.

[0089] If more than three blood samples are provided in step (a), these blood samples can be collected at any time point within the range of about 15 to about 100 minutes after the administration of macimorelin that is considered appropriate by the attending physician. For example, appropriate time points are about 20 ± 10, about 30 ± 10, about 40 ± 10, about 45 ± 10, about 50 ± 10, about 60 ± 10, about 70 ± 10, about 80 ± 10, about 90 ± 10 or about 100 ± 10 minutes after the administration of macimorelin.

[0090] In a particularly preferred method, in step (a), one to four, further preferably one to three, more preferably two or three blood samples are provided, which are collected from the subject at a time selected from about 30 ± 10 minutes, about 45 ± 10 minutes, about 60 ± 10 minutes and about 90 ± 10 minutes after the administration of macimorelin.

[0091] In a further preferred embodiment, in step (a), macimorelin is administered as a composition comprising macimorelin and optionally other pharmaceutically acceptable excipients such as a carrier substance. Preferably, macimorelin is administered as a composition comprising macimorelin and a sweetening agent. Suitable sweetening agents are, for example, saccharin. Advantageously, saccharin has been found to be a suitable taste masking agent for macimorelin.

[0092] In a further preferred embodiment, in step (a), macimorelin is administered as a composition comprising about 3.5% (w / w) of macimorelin (calculated as the free base), about 93.1% (w / w) of spray-dried lactose monohydrate, about 2.0% (w / w) of crospovidone type A, about 0.1% (w / w) of colloidal silicon dioxide, about 1.0% (w / w) of sodium stearyl fumarate and about 0.3% (w / w) of sodium saccharin dihydrate.

[0093] In a further preferred embodiment, the subject is a human child aged 2 to less than 18 years, preferably, the subject is a human child aged 2 to less than 17 years, more preferably, the subject is a human child aged 2 to less than 16 years.

[0094] In a further preferred embodiment, the method is an independent test, does not require repetition, and does not require additional growth hormone stimulation tests for the reliable diagnosis of growth hormone deficiency in pediatric patients.

[0095] In a further preferred embodiment, determining whether the subject has or does not have growth hormone deficiency according to step (d) is based only on the growth hormone level induced by a single administration of macimorelin.

[0096] Surprisingly, it has been found that the method of the present invention is suitable as an independent test, as no additional GHST is required for the reliable diagnosis of GHD in pediatric patients.

[0097] In another aspect, the object of the present invention is surprisingly solved by providing the substance macimorelin for diagnosing growth hormone deficiency in pediatric patients, wherein

[0098] (a) providing one to five blood samples, which are collected from the subject within the range of about 15 to about 100 minutes after administering an amount of macimorelin effective to induce growth hormone secretion;

[0099] (b) measuring the growth hormone levels of each of the blood samples provided in step (a);

[0100] (c) comparing the measured growth hormone levels obtained in step (b) with a single threshold, wherein the single threshold is 10.0 ng / mL or higher;

[0101] (d) determining that a subject whose highest growth hormone level in the blood sample obtained in step (b) is lower than the single threshold has growth hormone deficiency, and determining that a subject whose highest growth hormone level in the blood sample obtained in step (b)

[0102] is not lower than the single threshold does not have growth hormone deficiency.

[0103] The one to five blood samples are collected from the subject no earlier than about 15 minutes and no later than about 100 minutes after administering an amount of macimorelin effective to induce growth hormone secretion.

[0104] In a preferred embodiment, the substance macimorelin for diagnosing growth hormone deficiency in pediatric patients is the substance macimorelin for in vitro diagnosis of growth hormone deficiency in pediatric patients.

[0105] In a preferred embodiment, the single threshold for growth hormone is in the range of about 10.0 - about 25.0 ng / mL, preferably about 10.2 - about 20.0 ng / mL, further preferably about 12.0 - about 19.0 ng / mL, further preferably about 14.0 - about 18.0 ng / mL, more preferably about 16.0 - about 18.0 ng / mL, and most preferably about 17.0 - about 18.0 ng / mL.

[0106] In a preferred embodiment, one to four blood samples are provided in step (a), preferably one to three blood samples are provided in step (a), more preferably two or three blood samples are provided in step (a). If more than one blood sample is provided, these blood samples are collected at different time points after administration of an amount of macimorelin effective to induce hormone secretion.

[0107] In another preferred embodiment, in step (a), after administration of an amount of macimorelin effective to induce growth hormone secretion, a blood sample is collected from the subject in the range of about 20 to about 100 minutes, preferably in the range of about 25 to about 100 minutes, more preferably in the range of about 25 to about 95 minutes, and most preferably in the range of about 30 to about 90 minutes. Thus, after administration of an amount of macimorelin effective to induce growth hormone secretion, for example, not earlier than about 20 minutes but not later than about 100 minutes, preferably not earlier than about 25 minutes but not later than about 100 minutes, more preferably not earlier than about 25 minutes but not later than about 95 minutes, and most preferably not earlier than about 30 minutes but not later than about 90 minutes, a blood sample is collected.

[0108] In still another preferred embodiment, if more than one blood sample is provided, the blood samples are collected from the subject at intervals of about 10 to about 60 minutes, preferably at intervals of about 15 to about 30 minutes. Alternatively, the blood samples can be collected at any time interval deemed appropriate by the attending physician. For example, the blood samples can be collected at intervals of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 45 or about 60 minutes.

[0109] In a further preferred embodiment, in step (a), the blood sample is a whole blood sample, a serum sample or a plasma sample. Preferably, in step (a), the blood sample is a serum sample or a plasma sample. If more than one blood sample is collected, two or more blood samples are of the same type, and thus, these blood samples are whole blood samples, serum samples or plasma samples. Most preferably, the blood sample is a serum sample.

[0110] In a further preferred embodiment, in step (a), about 0.8 mg to about 1.2 mg of marenapride is administered per kg of subject body weight, preferably wherein in step (a), about 1.0 mg of marenapride is administered per kg of subject body weight. Typically, the effective amount of marenapride can be in the range of about 0.8 to about 0.9 mg per kg of subject body weight at the lower endpoint, about 1.0, about 1.1 to about 1.2 mg per kg of subject body weight at the upper endpoint, or in the range defined by any one of the lower endpoint amounts and any one of the upper endpoint amounts, for example, about 0.9 to about 1.1 mg / kg body weight. The effective amount of marenapride can also be a single value, for example, about 0.8, about 0.9, about 1.0, about 1.1 or about 1.2 mg / kg body weight. The body weight (recorded in kg) can preferably be rounded to the nearest integer.

[0111] For example, a unit dose of marenapride consists of 1817.2 mg of a composition for preparing an oral suspension in water. Typically, the prepared suspension contains 0.5 mg of marenapride / mL of suspension. Weight-adjusted aliquots of the reconstituted suspension are administered to pediatric subjects, resulting in a pediatric dose of 1.0 mg / kg body weight. The unit dose is defined as marenapride in 100% content of the free base. The mass of the free base of marenapride or its free base equivalent within the unit dose is adjusted according to the content.

[0112] In a preferred embodiment, in step (a), marenapride is administered in a composition comprising marenapride in a suitable pharmaceutically acceptable salt, wherein preferably, the suitable pharmaceutically acceptable salt is selected from marenapride acetate, marenapride trifluoroacetate or a combination thereof.

[0113] In a further preferred embodiment, in step (a), the administration of marenapride is oral administration.

[0114] If the administration of marenapride is oral administration, marenapride can be prepared as an oral suspension. The suspension can be administered within about 90 minutes, preferably about 60 minutes, more preferably about 30 minutes after the preparation of the oral suspension. Preferably, the drinking of the oral suspension does not exceed a period of about 1 minute, preferably not exceed a period of about 30 seconds.

[0115] In a further preferred embodiment, the subject has fasted for about 10 hours, preferably about 9 hours, more preferably about 8 hours before the administration of marenapride. Further preferably, in step (a), the subject fasts for about 100 minutes after the administration of marenapride, which means that in step (a), the subject can fast during about 100 minutes, about 95 minutes or about 90 minutes after the administration of marenapride.

[0116] In a further preferred embodiment, in step (a), a blood sample is provided which is collected from the subject at about 60 ± 30 minutes after administration of masirem. Optionally, a blood sample can be collected from the subject at about 30 ± 10, about 40 ± 10, about 45 ± 10, about 50 ± 10, about 60 ± 10, about 70 ± 10, about 80 ± 10, about 90 ± 10, about 100 ± 10 minutes after administration of masirem. A blood sample can be collected at any time point within the range of about 15 to about 100 minutes after administration of masirem that is considered appropriate by the attending physician.

[0117] In another further preferred embodiment, in step (a), two blood samples are provided which are collected from the subject at about 30 ± 10 minutes and about 45 ± 10 minutes after administration of masirem. Optionally, two blood samples can be collected from the subject at about 20 ± 10, about 30 ± 10, about 40 ± 10, about 45 ± 10, about 50 ± 10, about 60 ± 10, about 70 ± 10, about 80 ± 10, about 90 ± 10 or about 100 ± 10 minutes after administration of masirem. Two blood samples can be collected at any time point within the range of about 15 to about 100 minutes after administration of masirem that is considered appropriate by the attending physician.

[0118] In another preferred embodiment, in step (a), two blood samples are provided which are collected from the subject at about 30 ± 10 minutes and about 60 ± 10 minutes after administration of masirem.

[0119] In another preferred embodiment, in step (a), three blood samples are provided which are collected from the subject at about 30 ± 10 minutes, about 45 ± 10 minutes and about 60 ± 10 minutes after administration of masirem, or in step (a) three blood samples are provided which are collected from the subject at about 30 ± 10 minutes, about 45 ± 10 minutes and about 90 ± 10 minutes after administration of masirem, or in step (a) three blood samples are provided which are collected from the subject at about 30 ± 10 minutes, about 60 ± 10 minutes and about 90 ± 10 minutes after administration of masirem. Optionally, the three blood samples can be collected from the subject at about 20 ± 10, about 30 ± 10, about 40 ± 10, about 45 ± 10, about 50 ± 10, about 60 ± 10, about 70 ± 10, about 80 ± 10, about 90 ± 10 or about 100 ± 10 minutes after administration of masirem. The three blood samples can be collected at any time point within the range of about 15 to about 100 minutes after administration of masirem that is considered appropriate by the attending physician.

[0120] If more than three blood samples are provided, these blood samples can be collected at any time point within the range of about 15 to about 100 minutes after the administration of macimorelin that is considered appropriate by the attending physician. For example, suitable time points are about 20 ± 10, about 30 ± 10, about 40 ± 10, about 45 ± 10, about 50 ± 10, about 60 ± 10, about 70 ± 10, about 80 ± 10, about 90 ± 10 or about 100 ± 10 minutes after the administration of macimorelin.

[0121] In a particularly preferred embodiment, in step (a), one to four, further preferably one to three, more preferably two or three blood samples are provided, which are collected from the subject at a time selected from about 30 ± 10 minutes, about 45 ± 10 minutes, about 60 ± 10 minutes and about 90 ± 10 minutes after the administration of macimorelin.

[0122] In a further preferred embodiment, in step (a), macimorelin is administered as a composition comprising macimorelin and optionally other pharmaceutically acceptable excipients such as a carrier substance. Preferably, macimorelin is administered as a composition comprising macimorelin and a sweetening agent. Suitable sweetening agents are, for example, saccharin. Advantageously, saccharin has been found to be a suitable taste masking agent for macimorelin.

[0123] In a further preferred embodiment, in step (a), macimorelin is administered as a composition comprising about 3.5% (w / w) of macimorelin (calculated as the free base), about 93.1% (w / w) of spray-dried lactose monohydrate, about 2.0% (w / w) of crospovidone type A, about 0.1% (w / w) of colloidal silicon dioxide, about 1.0% (w / w) of sodium stearyl fumarate and about 0.3% (w / w) of sodium saccharin dihydrate.

[0124] In a further preferred embodiment, the subject is a human child aged 2 years to less than 18 years, preferably, the subject is a human child aged 2 years to less than 17 years, more preferably, the subject is a human child aged 2 years to less than 16 years.

[0125] In a further preferred embodiment, the substance is used for an independent test, without repetition, and without the need for an additional growth hormone stimulation test to reliably diagnose growth hormone deficiency in pediatric patients.

[0126] In a further preferred embodiment, it is determined whether the subject has or does not have growth hormone deficiency according to step (d) solely based on the growth hormone level induced by a single administration of macimorelin.

[0127] Surprisingly, it has been found that the method of the present invention is suitable as an independent test because no additional GHST is required to reliably diagnose GHD in pediatric patients.

[0128] In a further aspect, the object of the present invention is surprisingly achieved by providing a screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin, said method comprising:

[0129] (a) providing at least one blood sample collected from a subject within the range of about 15 to about 100 minutes after administering an amount of macimorelin effective to induce growth hormone secretion;

[0130] (b) measuring the growth hormone level of each blood sample;

[0131] (c) comparing each measured growth hormone level with a single threshold;

[0132] (d) diagnosing whether the subject has growth hormone deficiency based on comparing the growth hormone level measured in step (b) in the at least one blood sample with the single threshold;

[0133] wherein determining whether the subject has or does not have growth hormone deficiency is based only on the growth hormone level induced by administering a single dose of macimorelin.

[0134] In a preferred embodiment, the screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin is an in vitro screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin.

[0135] In a preferred method, in step (a), one to four, further preferably one to three, more preferably two or three blood samples are provided, which are collected from the subject at a time selected from about 30 ± 10 minutes, about 45 ± 10 minutes, about 60 ± 10 minutes, and about 90 ± 10 minutes after administering macimorelin.

[0136] In a preferred embodiment, in step (d), a subject whose highest growth hormone level measured in step (b) is lower than the single threshold is determined to have growth hormone deficiency, and a subject whose highest growth hormone level measured in step (b) is not lower than the single threshold is determined not to have growth hormone deficiency.

[0137] In another preferred embodiment, the single threshold for growth hormone is in the range of about 10.0 - about 25.0 ng / mL, preferably about 10.2 - about 20.0 ng / mL, further preferably about 12.0 - about 19.0 ng / mL, further preferably about 14.0 - about 18.0 ng / mL, more preferably about 16.0 - about 18.0 ng / mL, and most preferably about 17.0 - about 18.0 ng / mL.

[0138] In still further preferred embodiments, in step (a), about 0.8 mg to about 1.2 mg of macimorelin is administered per kg of subject body weight, preferably wherein in step (a), about 1.0 mg of macimorelin is administered per kg of subject body weight.

[0139] In a further preferred embodiment, the subject is a human child aged 2 to less than 18 years, preferably, the subject is a human child aged 2 to less than 17 years, more preferably, the subject is a human child aged 2 to less than 16 years.

[0140] Compared to the standard two - test format currently used by medical professionals, a main feature of the method of the present invention is the single - stimulation test format. Different from two independent tests that are performed at least one day apart and include up to 8 - 12 blood draws, the new method of the present invention only requires one test, and only requires as few as 1 - 5, preferably 2 - 4 blood draws, in order to obtain reliable diagnostic performance in terms of the accuracy, specificity, and sensitivity of detecting growth hormone deficiency, thus greatly reducing the test burden and potential harm to the tested children. Advantageously, these blood samples can be collected within a short period of time, for example, within a total of about 90 minutes after the administration of macimorelin, at intervals of about 15 to about 30 minutes.

[0141] Surprisingly, the method of the present invention has achieved a significant improvement in diagnosing growth hormone deficiency by using a higher threshold. Generally, when testing adult patients for GHD, a single threshold of about 2 - 3 ng / mL is used, while when testing pediatric patients for GHD, a single threshold of less than 10 ng / mL is used. The inventors unexpectedly found that when using a higher single threshold of 10.0 ng / mL or higher, better diagnostic performance for pediatric patients can be achieved. For example, it has been found that a single threshold of about 16.0 - 19.0 ng / mL, preferably about 17.0 - 18.0 ng / mL, very effectively indicates GHD in the independent method of the present invention.

[0142] In addition, the method of the present invention has achieved a significant improvement in diagnosing growth hormone deficiency by using a higher dose of macimorelin. Although the conventional dose of macimorelin used in current practice, especially when performing a GHD test on adult patients, is 0.5 mg / kg of patient body weight, the inventors unexpectedly found that when using a higher dose of macimorelin in the growth hormone stimulation test for pediatric patients, better diagnostic performance can be obtained. For example, it has been found that a dose of macimorelin of about 0.8 - 1.2 mg / kg body weight, preferably about 1.0 mg / kg body weight, very effectively indicates GHD in the one - test method of the present invention.

[0143] The present invention provides a method for measuring growth hormone levels in human children, including a method for assessing pituitary-related GH deficiency in a human child after a single oral administration of macimorelin to the child:

[0144] - As an independent test (only one test method and a single GH stimulation are required),

[0145] - After administration of macimorelin, for example, within a total time period of about 90 minutes, collect two to four blood samples at intervals of about 15 to about 30 minutes

[0146] - The GH cut-off point is in the range of about 10.0 ng / mL or higher, preferably about 10.0 ng / mL to about 25.0 ng / mL, more preferably about 10.2 ng / mL to about 20 ng / mL, and most preferably about 17.0 to about 18.0 ng / mL.

[0147] The following examples are provided only by way of illustration and not limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to obtain substantially the same or similar results. Examples

[0148] Childhood growth hormone deficiency (GHD) is a rare, etiologically diverse condition that causes growth retardation and short stature. Diagnosis of GHD requires an inadequate response to two different growth hormone stimulation tests (GHST). Macimorelin acetate, an effective orally administered growth hormone (GH) secretagogue, is approved by the FDA and EMA for the diagnosis of adult GHD (AGHD). This study (AEZS-130-P01) was designed to investigate macimorelin acetate as a diagnostic test for children with suspected GHD.

[0149] This was an open-label, group-comparative, dose-escalation trial to study the safety, tolerability, pharmacokinetics, and pharmacodynamics of single doses of 0.25, 0.5, and 1.0 mg / kg oral macimorelin hydrochloride in pediatric subjects with suspected GHD. Macimorelin GHST was administered between two standard GHSTs, conducted according to local clinical practice, with a recovery period of 7 - 28 days between tests. Blood samples were collected before dose administration (±15 minutes) and at 15, 30, 45, 60, 90, 120, and 360 minutes after ingestion of macimorelin acetate.

[0150] In summary, 24 pediatric subjects (8 in each of the three cohorts [C1, C2, C3]) were included in the pharmacokinetics / pharmacodynamics (PK / PD) analysis. Five males and three females were observed in C1 and C2, and seven males and one female were observed in C3. In all three cohorts, at least three subjects represented Tanner stage I or II. All 24 subjects (100%) were white, with median ages of 9.8, 9.0, and 10.5 years (range 4 - 15 years), and had a median body mass index of 16.1 kg / m 2 (12.4 - 21.4 kg / m 2 ) at screening. In summary, 88 adverse events were reported, many associated with standard GHST; none were considered related to the macimorelin trial. The maximum plasma concentration of macimorelin was observed mainly between 30 - 45 minutes. The mean C max values for C1, C2, and C3 were 3.46, 8.13, and 12.87 ng / mL, respectively. The AUC increased with dose; the mean AUC 0-6 values were 6.69, 18.02, and 30.92 h*ng / mL. The mean elimination half-lives were 1.22, 1.61, and 1.71 hours, respectively. The PK and PD curves for all three cohorts were comparable, and peak GH levels were mainly observed within 30 - 60 minutes after macimorelin ingestion.

[0151] Macimorelin acetate was safe and well tolerated in all dosing cohorts. The dose-dependent increase in C max and AUC of macimorelin in children and adolescents was closely related to data from adult subjects. A strong dose-proportional GH response was also obtained. The PD results showed that the GH responses were comparable in all dose groups, with a slight shift to earlier t max at higher macimorelin doses.

[0152] Moreover, the results of macimorelin GHST showed surprisingly high concordance with the results of two standard GHSTs and the final diagnosis as evaluated by the principal investigator. In C3, GH secretion was significantly stimulated in all eight patients. Finally, the results of macimorelin GHST as a single test showed concordance with the results of the combination of two sGHSTs and the PI evaluation in 7 out of 8 subjects.

[0153] Example 1: A macimorelin-containing composition for the diagnosis of CGHD

[0154] The macimorelin-containing composition comprises the following ingredients as listed in Table 1.

[0155] Table 1: Composition per unit dose

[0156] Composition Unit amount Percentage amount Mecasermin (content = 100%) 63.6 mg 3.5％ Lactose monohydrate, spray-dried 1691.8 mg 93.1％ Crospovidone, type A 36.3 mg 2.0％ Colloidal silicon dioxide 1.8 mg 0.1％ Sodium stearoyl fumarate 18.2 mg 1.0％ Saccharin sodium, dihydrate 5.5 mg 0.3％ Total 1817.2 mg 100.0％

[0157] One maricipril unit dose consists of a composition of 1817.2 mg for preparing an oral suspension in water. Usually, the prepared suspension contains 0.5 mg of maricipril / mL of suspension.

[0158] A weight-adjusted aliquot of the reconstituted suspension is administered to pediatric subjects to obtain a dose of 1.0 mg / kg of body weight of the child. It should be noted that in adults, a weight-adjusted aliquot of the reconstituted suspension is administered to obtain a dose of 0.5 mg / kg of body weight of the adult subject.

[0159] The unit dose is defined as maricipril in 100% content of the free base. The mass of maricipril free base or its free base equivalent in the unit dose is adjusted according to the content. Maricipril may be included in the unit dose as a suitable pharmaceutical salt. Examples of suitable pharmaceutical salts are acetate and trifluoroacetate.

[0160] The unit dose can be filled into a suitable container for easy availability in GHD testing. Examples of suitable containers are sachets or containers of suitable size made of glass or plastic.

[0161] For a 60 kg subject, the container is a sachet made of polyethylene laminated aluminum foil, and the maricipril-containing composition therein contains 63.6 mg of maricipril, 1691.8 mg of spray-dried lactose monohydrate, 36.3 mg of crospovidone type A, 1.8 mg of colloidal silica, 18.2 mg of sodium stearyl fumarate, and 5.5 mg of sodium saccharin dihydrate. When reconstituted in 120 mL of water, 2 mL provides 1.0 mg of maricipril.

[0162] Example 2 : Use of saccharin for masking off-flavors in a composition containing maricipril

[0163] In a multicenter, randomized, two-way, crossover study, 100 subjects with confirmed adult growth hormone deficiency (AGHD) received GHRH + L-Arg and maricipril as GHST to determine the diagnostic efficacy of maricipril for AGHD.

[0164] The study was conducted in two parts. In the first part, the maricipril-containing composition described in Example 1 was used at a dose of 0.5 mg / kg, but saccharin was not used, and 12 out of 52 subjects (21%) reported mild to moderate off-flavors.

[0165] During the study stoppage, saccharin was found to be a suitable taste masking agent, although the bitter taste was still not completely masked. In the second part of the study, the composition containing marzeline (containing saccharin) described in Example 1 was used, and only 1 out of 48 test subjects (2%) reported an off-taste of mild intensity.

[0166] Example 3: Pharmacokinetics, pharmacodynamics and exploratory trial characteristics of marzeline as a diagnostic test

[0167] Study AEZS-130-P01 was an open-label, group-comparative, dose-escalation trial to study the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of single oral administrations of 0.25 mg / kg, 0.5 mg / kg and 1 mg / kg of marzeline acetate in pediatric patients with suspected GHD.

[0168] The objectives were defined as follows:

[0169] Primary objectives:

[0170] · To study the safety and tolerability of marzeline acetate following escalating single oral doses of marzeline in pediatric patients with suspected GHD.

[0171] Secondary objectives:

[0172] · To study the PK of marzeline acetate in pediatric patients with suspected GHD,

[0173] · To study the PD of marzeline acetate as measured by growth hormone (GH) release in pediatric patients with suspected GHD,

[0174] · To study the PK / PD relationship following single oral dose administration of marzeline acetate in pediatric patients with suspected GHD.

[0175] Methods:

[0176] Plasma concentrations of marzeline and serum concentrations of GH were analyzed in a central laboratory.

[0177] Plasma concentration of mecasermin: In the central laboratory of Prolytic GmbH, Germany, plasma samples were analyzed for marzeline concentration using a validated liquid chromatography-mass spectrometry (LCMS / MS) method (Franz, 2005) with a limit of detection of 0.2 ng / mL.

[0178] Initial pharmacokinetics (PK) were determined by the time (t max ) of the maximum measured concentration time of marzeline plasma concentration and the maximum concentration (C max ) during the sampling period.

[0179] GH serum concentration: In the central laboratory, serum sample GH concentrations were analyzed by a validated immunochemiluminescent assay (IDS-iSYS human growth hormone (hGH), Immunodiagnostic Systems Ltd [UK]) (Manolopoulou et al., 2012). The assay was standardized to the recombinant growth hormone calibration standard WHO98 / 574 and met the recommendations for assay standardization as summarized by Clemmons (Clemmons et al., 2011).

[0180] The analytical laboratory for GH applications was: Central Laboratory Synevo Krakusa Str. 28, 93-515 Poland. Lower limit of quantification < 0.05 ng / mL.

[0181] Number of patients

[0182] In summary, macimorelin was administered as a single oral dose to 24 pediatric patients with suspected GHD, with 8 patients in each dose group. Among these 8 patients, at least 3 patients in each dose group were in pre-puberty (Tanner stage I) and puberty (Tanner stages II-IV), respectively.

[0183] Main inclusion criteria for patients: Subjects had to meet all of the following criteria to be eligible for the trial:

[0184] 1. Male or female pediatric subjects aged 1.2 to less than 18 years;

[0185] 2. Suspected GHD based on auxological and clinical criteria;

[0186] 3. Indication for performing a stimulatory growth hormone stimulation test (GHST).

[0187] Subjects who had sex steroid priming (which is part of the standard diagnostic procedure) prior to the GHST must also have sex steroid priming for the macimorelin GHST.

[0188] Investigational medicinal product (IMP) macimorelin

[0189] Single-use aluminum sachets (synonym: sachets) containing 63.6 mg of macimorelin, which when dissolved in 120 mL of water provides 0.5 mg / mL of macimorelin.

[0190] Successive groups of trial participants received increasing single oral doses of macimorelin: namely, Group 1 (C1) 0.25 mg / kg body weight, Group 2 (C2) 0.5 mg / kg body weight, and Group 3 (C3) 1 mg / kg body weight.

[0191] To determine the PK / PD of macimorelin, blood samples were collected before dosing and at 15, 30, 45, 60, 90, 120, and 360 minutes after administration of macimorelin.

[0192] As a trial, on the day of macimorelin GHST, a single oral dose of macimorelin acetate was administered to the patient.

[0193] Macimorelin GHST formulation

[0194] Macimorelin Oral suspension Prepared by the trial personnel and administered according to the following instructions (Steps #1 - Step #5), considering the dose of Group C3 (i.e., 1.0 mg / kg) as an example:

[0195] 1. Weigh the patient and determine the number of sachets / miniature sachets required (the patient requires one sachet). The body weight (recorded in kg) will be rounded to the nearest whole number;

[0196] 2. Dissolve the entire contents of the sachet in 120 mL of water in a suitable clear glass or polypropylene container (i.e., when feasible, one sachet is dissolved in 120 mL, two sachets are dissolved in 240 mL), and stir gently for at least 3 minutes (there will be a small amount of undissolved particles remaining);

[0197] 3. Based on a macimorelin dose of 1.0 mg / kg, determine the volume of suspension required corresponding to the patient's body weight, i.e., the volume of suspension required is 2 mL / kg (for example, a 30 kg patient requiring a 1.0

[0198] mg / kg macimorelin dose will require 60 mL of the prepared suspension);

[0199] 4. Measure the volume required by the patient using a graduated syringe and transfer it to a drinking glass (volumes below 20 mL should be administered using an oral syringe to minimize the risk of incomplete emptying losses; see instructions below);

[0200] 5. The suspension must be used within 30 minutes of preparation.

[0201] Fasting before GHST

[0202] Patients must fast for 8 hours before the start and throughout the sampling period of the macimorelin stimulation test.

[0203] Administration

[0204] The administration of mariciprant oral suspension is carried out under the supervision of the trial personnel. The patient is advised to drink - within a period not exceeding 30 seconds - the entire contents of the glass container prepared in step #4 of the specific dose group given above.

[0205] Blood collection

[0206] Blood samples are collected at the following time points: before dosing (sampling time window: + / - 15 minutes), then at 15, 30, 45, 60, 90, 120 minutes (+ / - 5 minute window) and 360 minutes (+ / - 10 minute window) after the administration of mariciprant. The serum concentration of GH and the plasma concentration of mariciprant are analyzed in the central laboratory.

[0207] After the first sGHST has been completed, the mariciprant GHST of the study is carried out. A recovery period of at least 1 week and at most 4 weeks is introduced between GHSTs to avoid carry-over effects or interference between subsequent GHSTs and to provide an adequate follow-up period to observe possible drug-related adverse events to the stimulants used previously.

[0208] Standard GHST used in study P01

[0209] According to local practice, two standard growth hormone stimulation tests (sGHSTs) must be carried out in the patients. The sGHST reagents are considered as "background" rather than IMPs.

[0210] For sGHSTs, the following pharmacological reagents are administered: insulin (insulin tolerance test (ITT)), arginine / growth hormone releasing hormone (GHRH), clonidine, glucagon, levodopa.

[0211] On the day of the sGHST, a single dose of the sGHST agent is administered intramuscularly (i.m.), intravenously (i.v.), subcutaneously (s.c.) or orally (depending on the formulation). The batch number is recorded on-site in the patient's medical record and in the "Standard GHST Patient Responsibility Log".

[0212] Evaluation criteria

[0213] Trial endpoints

[0214] Safety and tolerance

[0215] · Patient tolerance (including acceptability of taste and effects on sleep, appetite and digestive system symptoms), adverse events (AE);

[0216] · Determination of changes in laboratory parameters related to safety;

[0217] · Effects on vital parameters (pulse rate, blood pressure, ECG).

[0218] Pharmacokinetics

[0219] · Concentration-time curve of mariciprilin;

[0220] · Target parameters: AUC, C max , t max , T 1 / 2 .

[0221] Pharmacodynamics

[0222] · Concentration-time curve of GH;

[0223] · Target parameters: C max , t max ;

[0224] · Preliminary PK / PD: t of mariciprilin max vs. t of GH max ; C of mariciprilin max vs. C of GH max .

[0225] Others

[0226] · Establishment of the recommended dose for diagnostic purposes in pediatric patients with suspected GHD;

[0227] · Exploration of a suitable GH cut-off point for subsequent trials to establish the diagnosis of GHD in pediatric patients.

[0228] Statistical methods:

[0229] All statistical analyses were considered to be essentially exploratory. The SAS version 9.3 or higher was used to analyze the data set.

[0230] Generally, summary statistics of quantitative variables (n, arithmetic mean, standard deviation, median, minimum and maximum) and frequency tables of qualitative data were presented by dose group.

[0231] PK of mecasermin: PK parameters were analyzed for the PK analysis group (PKS), and were summarized by n (number of measurements), arithmetic mean, standard deviation and coefficient of variation (CV), median, minimum, maximum, and additionally (excluding t max ) by geometric mean, geometric standard deviation and geometric CV. For t max , frequency counts were additionally given, as well as the median, minimum and maximum.

[0232] Mecasermin PDWithout considering the availability of PK data, GH concentration data was analyzed for the PD analysis group (PDS). The GH peak concentration was correlated with the results of the clinical diagnostic method (diagnosis of GHD confirmed or not confirmed).

[0233] PK / PD analysis: The plasma concentration of macimorelin in individual patients was correlated with the corresponding GH concentration at the same time point and the results of the clinical diagnostic procedure (diagnosis of GHD confirmed or not confirmed).

[0234] Results and conclusions:

[0235] Among a total of 27 patients screened, 24 patients received the macimorelin test, with 8 patients in each of the three dosing groups (C1, C2, and C3).

[0236] Therefore, the safety population (SAF) as well as the PK analysis group (PKS), PD analysis group (PDS), and PK / PD group consisted of 24 patients.

[0237] Baseline characteristics:

[0238] In total, 17 (70.8%) patients were male, 7 (29.2%) were female, and 100% were of Caucasian origin. At screening, the median parameters for all three dosing groups were age 10.5 years (range: 4 - 15 years), height 123.35 cm (range: 46.0 - 152.5 cm), weight 25.5 kg (range: 12 - 43 kg), and body mass index (BMI) 16.1 kg / m 2 (range: 12.4 - 21.4 kg / m 2 )

[0239] The Tanner stage distribution was as follows: in C1 and C3, 4 patients showed Tanner I and 4 patients showed Tanner II; in C2, 5 patients showed Tanner I and 3 patients showed Tanner II. Application of sex steroids was initiated in two male patients in C3 by intramuscular (i.m.) administration of a testosterone depot preparation.

[0240] Regarding baseline medical history, other pituitary axis deficiencies (i.e., thyroid deficiency) were reported only for two patients in C2. As part of the standard "post - diagnosis management", IGF - 1 and IGF - BP3 values were recorded in the electronic case report form (eCRF) as collected according to local diagnostic criteria.

[0241] For 7 patients in C1 and 8 patients each in C2 and C3, IGF-1 values were given. The median in C1 was 88.00 μg / L (SD 68.72), the median in C2 was 100.00 μg / L (SD 97.90), and the median in C3 was 119.50 μg / L (SD 68.88). IGF-BP3 values were available for one C3 patient.

[0242] Bone age was shown as the median value of 102.2 months (range: 24 - 156 months). As part of the auxology parameters, height SDS was the median of -2.35 (range -3.2 - 1.7), BMI SDS -0.60 (range -2.1 - 2.0), and annual height velocity SDS -1.50 (range: -3.3 - 0.5).

[0243] SGHSTs: In summary, ITT was administered to 22 patients (i.e., 5 patients (20.8%) at the first visit (V1) and 17 patients (70.8%) at the third visit (V3)), arginine was administered to 8 patients (33.3%) at V1, and clonidine was administered to 16 patients at V1 (i.e., 11 patients (45.8%) at V1 and 5 patients (20.8%) at V3). Glucagon was administered to only one patient, and levodopa was not administered at all.

[0244] In all three groups, the treatment compliance of marzeline was 100%.

[0245] Pharmacokinetic and pharmacodynamic results:

[0246] Plasma concentration data

[0247] Generally, the plasma concentration of marzeline showed a dose-dependent increase ( Figure 1 ), with high inter-individual variability. After administration of marzeline, the plasma concentration showed a rapid increase, and the maximum level was observed between 0.25 and 2 hours. At the last sampling point 6 hours after administration, the plasma concentration decreased sharply.

[0248] Pharmacokinetics:

[0249] Generally, the plasma concentration of marzeline showed a dose-dependent increase ( Figure 1 ), with high inter-individual variability. After administration of marzeline, the plasma concentration showed a rapid increase, and the maximum level was observed between 0.25 and 2 hours. At the last sampling point 6 hours after administration, the plasma concentration decreased sharply.

[0250] The AUC and C of marzeline maxshowed a dose-dependent increase, with arithmetic mean AUC0-6 being 6.69 ng / mL in C1, 18.02 ng / mL in C2, and 30.92 ng / mL in C3, and arithmetic mean C max being 3.46 ng / mL in C1, 8.13 ng / mL in C2, and 12.87 ng / mL in C3 (Table 2).

[0251] The mean t of all three groups max was comparable, with arithmetic mean being 45.5 minutes in C1, 40.6 minutes in C2, and 31.9 minutes in C3. The mean T 1 / 2 showed a slight increase with increasing dose, i.e., 73.18 minutes in C1, 96.31 minutes in C2, and 102.85 minutes in C3.

[0252] Table 2: Summary of main pharmacokinetic parameters (PK group, N = 24)

[0253]

[0254] Pharmacodynamics:

[0255] As Figure 2 shown, after administration of macimorelin, GH concentration increased, with a tendency to have higher values with increasing dose. Large inter-patient variability was expected in the observational cohort of patients suspected of having GHD.

[0256] After administration of macimorelin, peak GH levels were observed in C1 within 0.5 - 1 hour (mean t of 52.5 minutes max (SD 11.3)), in C2 within 0.25 - 1 hour (mean t max of 37.5 minutes (SD 13.9)), and in C3 within 0.5 - 0.75 hour (mean t max of 37.5 minutes (SD 8.0)) (see Table 3).

[0257] Table 3: Summary of pharmacodynamic parameters (PK / PD group, N = 24)

[0258]

[0259] Exploratory analysis of the GH cut-off point:

[0260] Based on the GHST results and the researchers' assessments, the GH peaks diagnosed by GHD were compared. The diagnostic characteristics (i.e., sensitivity, specificity, and Youden index (unweighted, weighted)) of the GH values as cut-off point tests were listed, where the most solid expression of the diagnostic characteristics was noted at peak GH of 10.03 ng / mL for C1, at peak GH of 10.43 ng / mL for C2, and at 17.13 ng / mL for C3.

[0261] The diagnostic results of GHST are presented in Table 4. In this table, if two sGHSTs are available and both result in a GH peak ≤ 7 ng / mL, the diagnostic result of the sGHST is considered "confirmed", or if at least one peak is higher than 7 ng / mL, it is considered "unconfirmed". If two sGHST results are available and the GH peak is higher than 7 ng / ml, the result "unconfirmed" is classified as "excluded", and if the situation does not fit any of the above, it is classified as "equivocal". The researchers' assessments were based on local diagnostic standard practices. For the cut-off point test of macimorelin GHST calculated from individual peak GH values.

[0262] Table 4: Diagnostic Results of GHST (Number of Subjects = 24)

[0263]

[0264]

[0265]

[0266] Based on the above considerations, Table 4 shows the agreement between the assessments of the principal investigator (PI) and the two sGHST results: In 21 patients (87.5%), i.e., 8 confirmed and 13 unconfirmed, there was agreement between the researchers' assessments and the sGHST results. In 3 patients (12.5%), the researchers inferred GHD, while the sGHST excluded the diagnosis (in 1 patient) or was equivocal (in 2 patients).

[0267] In addition, the diagnostic results can be summarized as follows (Table 5): Among all 11 patients evaluated as "GHD" by the researchers in all three groups, only 1 patient (9.09%) in C2 had an unconfirmed macimorelin GHST for "GHD".

[0268] Out of a total of 13 patients evaluated as having "unconfirmed" GHD by the researchers, macimorelin GHST confirmed GHD in 3 patients (23.08%) in C1 and in 1 patient (7.69%) in C3.

[0269] Table 5: Summary of diagnostic results of marzirin GHST compared with sGHST and the assessments of the comparator investigators

[0270]

[0271] Considering the above data, the strongest test characteristics of the marzirin test were observed in C3; GH secretion was significantly stimulated in all 8 patients. Finally, the results of the marzirin GHST applied as a single test were shown to be consistent with the results of the combination of two sGHSTs and the PI assessments in 7 out of 8 subjects.

[0272] Receiver operating characteristic (ROC) analysis

[0273] For the GH cut-off points of all the tests, the ROC curve of C1 showed the lowest sensitivity and specificity when compared with C2 and C3 ( Figure 3 ). The relevant area under the curve (AUC) increased with increasing dose.

[0274] When comparing the characteristics of the GH cut-off points among the three groups, the 17.130 ng / mL GH cut-off point of C3 showed the strongest test characteristics, with a sensitivity of 1.0, a specificity of 0.8, a Youden index ≥ 0.80, and a ROC AUC of 0.93 (see Table 6).

[0275] Based on the test results of sGHST classified as "confirmed" vs. "unconfirmed", sensitivity analysis was performed to observe the development of the ROC AUC. Similarly, for the strongest test characteristics expressed by C3, compared with C2 (sensitivity 0.75, specificity 0.75, ROC AUC 0.563) and C1 (sensitivity 1.0, specificity 0.71, ROC AUC 0.714), it had, for example, a sensitivity of 1.00, a specificity of 0.80, and a ROC AUC of 0.933.

[0276] Table 6: Summary of ROC analysis: AUC and cut-off point characteristics by group (PDS, N = 24)

[0277]

[0278] NPV = negative predictive value, PPV = positive predictive value

[0279] PK and PD summary

[0280] In summary, the PK and PD of marzirin in C1, C2, and C3 showed comparable characteristics:

[0281] · The t max of marzirin in all three groups was comparable, with an average t maxThe value is about 0.5 to 0.75 hours;

[0282] · Mean marzeline C max showed a dose-proportional increase;

[0283] · The AUC increased with the marzeline dose;

[0284] · The maximum GH release was observed 0.25 to 2 hours after marzeline administration, with an average t max value of about 0.5 to 1 hour.

[0285] At a marzeline dose of 1.0 mg / kg body weight, the maximum values of AUC and C max were observed in C3. Additionally, the sensitivity analysis supported that the dosing in C3 had the strongest test characteristics, expressed at a cut-off point of about 17 ng / mL GH, with a specificity of 0.80, a sensitivity of 1.00, a Youden index of 0.80, and an ROC AUC of 0.933.

[0286] Safety results:

[0287] In summary, were recorded in 23 patients in SAF, 88 AEs i.e., 27 events in 8 patients in C1, 28 events in 8 patients in C2, and 33 events in 7 patients in C3.

[0288] were recorded in 21 patients in SAF patients, A total of 70 treatment-emergent adverse events (TEAEs) i.e., 22 events in 8 patients in C1 patients, 24 events in 6 patients in C2 patients, and 24 events in 7 patients in C3 patients.

[0289] None of the TEAEs were reported to be related to the mecasermin trial.

[0290] were reported during the trial No SAEs or serious TEAEs . None of the reported AEs or TEAEs caused patient withdrawal.

[0291] Most AEs were related to ITT, i.e., 62 (70.5%) events in 21 (91.3%) patients. Clonidine-related AEs (13 (14.8%)) were observed in 7 (30.4%) patients, and arginine-related AEs were reported in 1 (4.3%) patient. It should be noted that ITT was administered to 22 patients, arginine was administered to 8 patients (33.3%), clonidine was administered to 16 patients, and glucagon was administered to only 1 patient.

[0292] The AEs were reported as infectious diseases, only one case of anal fissure, and side effects of the test reagents for sGHST.

[0293] The AEs were mainly of mild to moderate intensity. ITT-related AEs included hypoglycemic symptoms (such as tremors, sweating), which were the clinical endpoints of sGHST. Related to clonidine, hypotension-related symptoms were reported, which were known side effects of this sGHST reagent.

[0294] The severe intensity of TEAE was reported in one patient in Group 1: 5 AEs (i.e., palpitations, tachycardia, hunger, hyperhidrosis, and tremors) were part of the expected hypoglycemia that occurred in patient HU0101 during ITT.

[0295] Clinical laboratories, vital signs, physical findings, ECG:

[0296] No clinically significant changes were observed in safety clinical laboratory parameters, vital signs, and physical examinations. Clinically significant abnormalities and significant changes in ECG parameters were not described.

[0297] Tolerance questionnaire:

[0298] The GHST tolerance questionnaire was completed by the patient or parent / legal guardian.

[0299] In summary, for marzeline in all three dosing groups, mostly consistency or strong consistency with the predefined statements was noted.

[0300] In C1, inconsistencies were described in one case related to "acceptable taste" and in one case related to "feeling well in the stomach the next day". In one case, strong inconsistency in "bowel movement the next day" was recorded.

[0301] For any of the marzeline trials related to the C2 questionnaire, no strong inconsistency or inconsistency was found. One case of inconsistency with "acceptable taste" was described in C3.

[0302] As handwritten notes, two patients following marzeline GHST reported "bitter taste". None of these notes were evaluated as AEs by the researchers.

[0303] In summary, after the marzeline trials, the feedback provided by the patients or parents / legal guardians demonstrated the good tolerance and safety of the trials.

[0304] Conclusion:

[0305] This trial was conducted to study the safety, tolerability, PK, and PD of macimorelin acetate following single oral doses of 0.25 mg / kg, 0.5 mg / kg, and 1.0 mg / kg in pediatric patients with suspected GHD. Additionally, it was used to identify an appropriate macimorelin dose for further testing in a test validation trial and to explore GH cut-off points for the trial.

[0306] For all three dosing groups, the C of macimorelin plasma levels was found max and t max to be within the range expected in the adult development program.

[0307] In the observed pediatric population, a dose-dependent increase in macimorelin C max (3.46 vs. 8.13 vs. 12.87 ng / mL) and mean AUC0-6 (6.69 vs. 18.02 vs. 30.92 h*ng / mL) was observed. The elimination half-life t1 / 2 was in the range of 1.25 - 1.75 hours.

[0308] Generally, the PK parameters in the pediatric population were within a similar range to those in adults.

[0309] The macimorelin dose of 0.25 mg / kg (C1) did not result in maximal stimulation of GH secretion in children, which became apparent in the evaluation of PK / PD data and comparison of the consistency between the explored GH cut-off points relative to PI assessment and sGHST results.

[0310] The dose of 0.5 mg / kg (C2) showed strong GH release, based on a high level of agreement between the principal investigator (PI) assessment of macimorelin GHST relative to sGHST and the ROC AUC of 0.80. However, the dose of 1.0 mg / kg (C3) appeared to result in more consistent and strong GH stimulation, most likely due to sufficiently high exposure to macimorelin in all subjects.

[0311] Finally, sensitivity analysis supported dosing at C3, which had the strongest test characteristics expressed at a cut-off point of approximately 17 ng / mL GH, with a specificity of 0.80, a sensitivity of 1.00, a Youden index of 0.80, and a ROC AUC of 0.93.

[0312] In addition, the results of the macimorelin GHST showed surprisingly high agreement with the results of the two standard GHSTs and the final diagnosis as evaluated by the lead investigator. In C3, GH secretion was significantly stimulated in all 8 patients. Finally, the results of the macimorelin GHST applied as a single test showed agreement with the results of the combination of the two sGHSTs and the PI assessment in 7 out of 8 subjects.

[0313] In summary, in all three dosing groups, macimorelin demonstrated good safety and tolerability, with no AEs reported in the groups. The PK and PD characteristics were within the range expected for an adult development program. In this first pediatric trial, the overall profile of macimorelin supported the selection of a macimorelin dose of 1.0 mg / kg for testing efficacy in a Phase 3 trial.

[0314] List of references

[0315]

[0316]

[0317]

[0318]

Claims

1. A method for screening blood samples from pediatric patients, comprising the following steps: (a) Measuring the growth hormone level of each of one to five blood samples, said blood samples being from a pediatric patient within the range of about 15 to about 100 minutes after administering an effective growth hormone-secreting amount of macimorelin to the pediatric patient; (b) Comparing the measured growth hormone levels obtained in step (a) with a single threshold, wherein said single threshold is 10.0 ng / mL or higher; (c) When the highest growth hormone level of the blood sample obtained in step (a) is lower than said single threshold, screening said blood sample as having growth hormone deficiency.

2. The method according to claim 1, wherein said single threshold is in the range of about 10.0 - about 25.0 ng / mL.

3. The method according to claim 2, wherein said single threshold is in the range of about 10.2 - about 20.0 ng / mL.

4. The method according to claim 2, wherein said single threshold is in the range of about 12.0 - about 19.0 ng / mL.

5. The method according to claim 2, wherein said single threshold is in the range of about 14.0 - about 18.0 ng / mL.

6. The method according to claim 2, wherein said single threshold is in the range of about 16.0 - about 18.0 ng / mL.

7. The method according to claim 2, wherein said single threshold is in the range of about 17.0 - about 18.0 ng / mL.

8. The method according to any one of claims 1 - 7, wherein one to four blood samples are provided in step (a).

9. The method according to claim 8, wherein one to three blood samples are provided in step (a).

10. The method according to claim 8, wherein two or three blood samples are provided in step (a).

11. The method according to any one of claims 1 to 10, wherein in step (a), after administering an effective growth hormone-secreting amount of macimorelin, blood samples are collected from said pediatric patient within the range of about 20 to about 100 minutes.

12. The method according to any one of claims 1 to 11, wherein if more than one blood sample is provided, the blood samples are collected from said pediatric patient at intervals of about 10 to about 60 minutes.

13. The method according to any one of claims 1 to 11, wherein in step (a), said blood sample is a serum sample or a plasma sample.

14. The method according to claim 13, wherein in step (a), about 0.8 mg to about 1.2 mg / kg of the pediatric patient's body weight of macimorelin is administered.

15. The method according to claim 13, wherein in step (a), about 1.0 mg / kg of the pediatric patient's body weight of macimorelin is administered.

16. The method according to any one of claims 1 to 15, wherein in step (a), the administration of macimorelin is oral administration.

17. The method according to any one of claims 1 to 16, wherein in step (a), one blood sample is provided, which is collected from said pediatric patient about 60 ± 30 minutes after administering macimorelin.

18. The method according to any one of claims 1 to 16, wherein in step (a), two blood samples are provided, which are collected from the pediatric patient at about 30 ± 10 minutes and about 45 ± 10 minutes after administration of marisopram.

19. The method according to any one of claims 1 to 16, wherein in step (a), two blood samples are provided, which are collected from the pediatric patient at about 30 ± 10 minutes and about 60 ± 10 minutes after administration of marisopram.

20. The method according to any one of claims 1 to 16, wherein in step (a), three blood samples are provided, which are collected from the pediatric patient at about 30 ± 10 minutes, about 45 ± 10 minutes and about 60 ± 10 minutes after administration of marisopram, or wherein in step (a), three blood samples are provided, which are collected from the pediatric patient at about 30 ± 10 minutes, about 45 ± 10 minutes and about 90 ± 10 minutes after administration of marisopram, or wherein in step (a), three blood samples are provided, which are collected from the pediatric patient at about 30 ± 10 minutes, about 60 ± 10 minutes and about 90 ± 10 minutes after administration of marisopram.

21. The method according to any one of claims 1 to 20, wherein in step (a), marisopram is administered as a composition comprising marisopram and optionally other pharmaceutically acceptable excipients.

22. The method according to any one of claims 1 to 20, wherein in step (a), marisopram is administered as a composition comprising about 3.5% (w / w) of marisopram as the free base, about 93.1% (w / w) of spray-dried lactose monohydrate, about 2.0% (w / w) of crospovidone type A, about 0.1% (w / w) of colloidal silicon dioxide, about 1.0% (w / w) of sodium stearyl fumarate and about 0.3% (w / w) of saccharin sodium dehydrate.

23. The method according to any one of claims 1 to 22, wherein the pediatric patient is a human child aged 2 years to less than 18 years.

24. The method according to any one of claims 1 to 23, wherein the substance marisopram is used in an independent test, without repetition, and without the need for additional growth hormone stimulation tests to reliably diagnose growth hormone deficiency in pediatric patients.

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