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

Abstract

The present invention relates to a screening method for diagnosing growth hormone deficiency in pediatric patients using macimorelin. The present invention further provides the substance macimorelin for use in diagnosing growth hormone deficiency in pediatric patients. The method includes providing at least one blood sample taken from a subject within about 15 to about 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; measuring the growth hormone level in each blood sample; comparing the measured growth hormone level to a single threshold; and diagnosing whether the subject is suffering from growth hormone deficiency based on the comparison of the growth hormone level in the at least one blood sample to the single threshold. The method of the present invention is a stand-alone test that does not need to be repeated, and no alternative growth hormone stimulation test is required to reliably diagnose pediatric patients.

Description

[Technical Field]

[0001] [Background technology] Growth hormone (GH) is a major body-wide metabolic hormone that regulates protein, lipid, and carbohydrate homeostasis and is necessary for physical and mental growth, development, and maintenance. GH is produced in the anterior lobe of the pituitary gland upon stimulation by growth hormone-releasing hormone (GHRH) from the hypothalamus. GH is secreted from the pituitary gland in pulses of approximately 6 to 10 random bursts over a 24-hour period.

[0002] Growth hormone deficiency (GHD) can be broadly classified into four categories based on the cause of GH deficiency: 1) pituitary or "classic" GHD, 2) hypothalamic GHD, 3) functional GHD, and 4) idiopathic GHD. GHD can be clinically evident in both childhood and adulthood. In the United States, the incidence of GHD in children is estimated to be between 1 in 4,000 and 1 in 10,000. More than 50,000 adults in the United States have GHD, and 6,000 new cases are reported each year, including children with GHD who progress to GHD as adults (Human Growth Foundation www.hgfound.org).

[0003] GHD is a disorder characterized by reduced growth parameters, such as growth failure and short stature, in children. GHD in children can be congenital or acquired and can occur alone or in combination with other pituitary hormone deficiencies. There are many distinct causes of GHD in children, although the cause is often unknown (idiopathic GHD). Definitions of various types of GHD and other causes of short stature are provided in the International Classification of Pediatric Endocrine Diagnoses (ICPED). If untreated, childhood-onset GHD results in permanent short stature. In children, the diagnosis of GHD is based on a detailed medical history, clinical features, growth (growth) analysis, biochemical testing of components of the GH-IGF axis (GH = growth hormone; IGF = insulin-like growth factor), and radiological evaluation of skeletal maturation and pituitary anatomy using MRI (GHRS, 2000).

[0004] Similar to the adult situation, the diagnosis of GHD in children is by biochemical testing based on the growth hormone stimulation test (GHST), which determines GH levels that can be induced by agents known to stimulate GH release. Currently used GHSTs, such as the insulin tolerance test (ITT) or glucagon stimulation test (GST), were not specifically developed and approved for this purpose but are adapted from other indications and therefore have limitations regarding performance characteristics such as sensitivity of specificity, safety, or availability (Molitch et al., 2011; Cook et al., 2009).

[0005] ITT has been considered the standard of care for the evaluation of GHD. Intravenous administration of insulin is used to induce hypoglycemia, which then leads to GH release. However, this test is labor-intensive because it requires intensive medical monitoring of the subject due to the potential risks associated with hypoglycemia, which is associated with symptoms such as tremor, somnolence, and tachycardia. Side effects are often reported to be dangerous. Furthermore, ITT is contraindicated in subjects with seizure disorders and ischemic heart disease (Yuen 2011; Yuen 2013). Therefore, ITT is not widely used due to its inconvenience and safety issues.

[0006] GST is an increasingly available option (Molitch 2011; Yuen 2011; Yuen 2013). Common side effects of GST include nausea, vomiting, and headache. Limitations include the length of the test (3-4 hours) and the need for intramuscular injection. There remains a real unmet medical need for safe and reliable alternative tests.

[0007] A test for diagnosing GHD based on macimorelin, an orally available peptidomimetic ghrelin receptor agonist with growth hormone secretagogue (GHS) activity, was disclosed by Larsen in WO 2007 / 093820 A1.

[0008] Ghrelin potently stimulates GH release (Kojima 1999). The GH-releasing effect of ghrelin is thought to be mediated primarily by specific receptors present at the pituitary and hypothalamic levels (Nogueiras 2006). In membrane preparations containing GHS receptors derived from human hypothalamus and pituitary, macimorelin was shown to exhibit a binding potency for human GHS receptors equivalent to that of its natural ligand, ghrelin (Broglio 2002). Macimorelin is readily absorbed from the gastrointestinal tract and is presumed to exert its action in the same manner as ghrelin.

[0009] Based on the ability of macimorelin to effect GH pulse release immediately after oral administration in healthy subjects, macimorelin was developed as an oral diagnostic for GH deficiency in adults.

[0010] The compound macimorelin and its use in treating GHD was disclosed by Martinez et al. in WO 01 / 96300 A1.

[0011] Macimorelin GHST in adult GHD (AGHD) was disclosed by Garcia et al. (J Clin Endocrinol Metab. 2013-1157, J Clin Endocrinol Metab. 2018-00665, and a poster titled "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)" at the 99th Annual Meeting of the Endocrine Society in 2017).

[0012] WO 2019 / 121762 A1 discloses a method for measuring growth hormone levels in a human or animal subject. The method includes orally administering a macimorelin-containing composition to the subject, collecting one, two, or three post-administration samples from the subject within 25 to 95 minutes after administration, and comparing the growth hormone levels in the one, two, or three samples to a single threshold value, where the single threshold value is 2.8 ng / mL, the "single threshold value" referring to a threshold growth hormone level indicating an adequate response to macimorelin stimulation.

[0013] There is considerable debate about the role of GH stimulation testing in pediatric patients because low GH levels often occur during provocation testing, raising concerns about the validity and reproducibility of GHST.

[0014] Therefore, guidelines for the diagnosis of GHD in children typically require two GHST outcomes to conclude a diagnosis of GHD, unless there are typical brain defects that require the use of only one GHST (GHRS, 2000, GHRS 2019).

[0015] The GH Research Society consensus guidelines published in 2000 (GHRS, 2000) and the American Association of Clinical Endocrinologists guidelines published in 2003 (Gharib, 2003) and national guidelines (Binder, 2014) recommended that a limited number of GHST agents should be used in well-standardized GHST protocols after an overnight fast. These include arginine (ARG), clonidine, glucagon, insulin, and L-dopa.

[0016] The GH Research Society consensus guidelines state that the combination of GHRH and ARG as GHST is considered useful in diagnosing GHD in children and adults if appropriate cutoff points are applied (GHRS, 2000). This combination has been shown to have high sensitivity and specificity in children and adolescents (Maghnie, 2002) and in late adolescents and young adults (Corneli, 2007). In this latter trial, cutoff points were established only in lean patients. Given that GH secretion is a function of body weight and adiposity (Colao, 2009), appropriate cutoff points (taking into account age, BMI, and waist circumference) for overweight and obese pediatric patients have yet to be defined.

[0017] In children with clinical criteria for GHD, a peak GH concentration of less than 10 ng / mL has traditionally been used as a cutoff point to support the diagnosis. The sensitivity, specificity, and GH cutoff points used for different GHSTs in different tests have recently been reviewed (van Vught, 2009).

[0018] In this context, a variety of GHSTs are used in clinical practice, and the requirement for two GHSTs as part of a standard diagnostic procedure is variably met either by repeating the same GHST or by performing two different GHSTs sequentially.

[0019] These two GHSTs are performed either on the same day or on two consecutive days, and they require four to six blood samples per test. The amount of blood drawn, especially in young children, is considered a safety concern, and safe volume limits should be considered, as recommended, for example, by the WHO (Howie 2011). Aside from this safety issue, it should be noted that the administration of the two tests is time-consuming and resource-intensive, and the associated burden on children and their parents, as well as on pediatric endocrinologists, is significant.

[0020] Therefore, there is a demonstrated need for a single test with proven safety, tolerability, easy application, strong test characteristics with respect to sensitivity and specificity, and reliable reproducibility, instead of two tests.A brief press release about this invention was issued in 2020 announcing positive results in a dose-ranging pediatric study of macimorelin. Summary of the Invention [Means for solving the problem]

[0021] In one aspect, the present invention provides a screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin, comprising: (a) providing one to five blood samples taken from the subject within about 15 to about 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; (b) measuring the growth hormone level in each blood sample provided in step (a); (c) comparing the measured growth hormone level obtained in step (b) with a single threshold value that is equal to or greater than 10.0 ng / mL; (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 equal to or greater than the single threshold does not have growth hormone deficiency. The present invention provides a method comprising:

[0022] In another aspect, the present invention provides the substance macimorelin for use in the diagnosis of growth hormone deficiency in a pediatric patient, comprising: (a) providing one to five blood samples taken from a subject within about 15 to about 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; (b) measuring the growth hormone level in each blood sample provided in step (a); (c) comparing the measured growth hormone level obtained in step (b) with a single threshold value of 10.0 ng / mL or greater; (d) The substance macimorelin, wherein a subject whose highest growth hormone level in the blood sample obtained in step (b) is lower than a single threshold is determined to have growth hormone deficiency, and a subject whose highest growth hormone level in the blood sample obtained in step (b) is equal to or greater than the single threshold is determined not to have growth hormone deficiency.

[0023] In a further aspect, the present invention provides a screening method for diagnosing growth hormone deficiency in a pediatric patient by using macimorelin, comprising: (a) providing at least one blood sample taken from the subject within about 15 to about 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; (b) measuring the growth hormone level in each blood sample; (c) comparing each measured growth hormone level to a single threshold; (d) diagnosing whether the subject is suffering from growth hormone deficiency based on a comparison of the growth hormone level measured in step (b) in the at least one blood sample with the single threshold value; Including, The present invention relates to a method in which determining whether a subject has or does not have growth hormone deficiency is based solely on the induction of growth hormone levels by a single administration of macimorelin.

[0024] definition The terms used to describe the process of the present invention always have the following meanings, unless otherwise specified in the specification or claims.

[0025] As used herein, a "subject" or "pediatric patient" is a human child of either sex (male or female) who is between the ages of about 2 and under 18 years. For example, the subject may be between the ages of about 3 and under 18 years, about 4 and under 18 years, about 5 and under 18 years, about 6 and under 18 years, about 7 and under 18 years, about 8 and under 18 years, about 9 and under 18 years, about 10 and under 18 years, about 11 and under 18 years, about 12 and under 18 years, about 2 and under 17 years, about 2 and under 16 years, about 2 and under 15 years, about 2 and under 14 years, about 2 and under 13 years, about 2 and under 12 years, about 2 and under 10 years, about 2 and under 9 years, or about 2 and under 8 years.

[0026] 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 No. 6,861,409, WO 01 / 96300, and WO 2007 / 093820.

[0027] As used herein, the term "effective amount" refers to an amount of a given substance that is sufficient to produce a desired effect. For example, an effective amount of macimorelin for inducing growth hormone secretion in a host is an amount of the compound that is capable of achieving a detectable increase in growth hormone secretion upon its administration to a subject.

[0028] As used herein, the terms "test" and "testing" describe actions that lead to the clarification of a person suspected of having a particular condition based on the subject's symptoms and the identification of whether the condition is present or absent. In other words, "testing" for a condition encompasses the confirmation or exclusion of the condition.

[0029] The term "treat" or "treating" as used herein describes actions that lead to the eradication, reduction, alleviation, reversal, or prevention of any symptoms of the associated pathological condition, or delay in its onset or recurrence.

[0030] As used herein, the term "blood sample" encompasses whole blood samples and fractions of whole blood, such as serum or plasma samples. Whenever two or more blood samples are used in the same method scheme, these blood samples are of the same type. For example, if the first sample is serum, then the second and any subsequent samples are also serum. Furthermore, when more than one blood sample is provided, the term "blood sample" refers to blood samples taken at different time points after administration of an amount of macimorelin effective to induce hormone secretion. Two blood samples may refer to, for example, blood samples taken at about 30±10 minutes and about 45±10 minutes, or about 30±10 minutes and about 60±10 minutes, after administration of an amount of macimorelin effective to induce hormone secretion. The three blood samples may refer, for example, to blood samples taken 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 after administration of an amount of macimorelin effective to induce hormone secretion.

[0031] As used herein, the term "about" refers to a range of ±10% of a reference value. For example, "about 10" defines a range of 9 to 11.

[0032] The term "single threshold" refers to a threshold growth hormone level that indicates an adequate response to macimorelin stimulation. Instead of the 2.8 ng / mL threshold typically used in adult studies, the threshold used in the methods of the present invention may be 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, or about 12.0-16.0 ng / mL. and in a higher range of about 13.0 to 15.5 ng / mL, about 14.0 to 15.0 ng / mL, about 15.0 to 16.0 ng / mL, about 15.5 to 18.0 ng / mL, about 16.0 to 18.0 ng / mL, about 16.5 to 18.0 ng / mL, about 17.0 to 18.0 ng / mL, about 17.5 to 18.5 ng / mL, about 18.0 to 19.0 ng / mL, about 18.5 to 19.5 ng / mL, about 19.0 to 20.0 ng / mL, about 20.0 to 21.0 ng / mL, or about 25.0 to 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. Furthermore, the single threshold refers to ng per mL of whole blood or serum / plasma. Most preferably, the single threshold refers to ng per mL of serum. The terms "single threshold" and "cutoff point" are used interchangeably.

[0033] Weight percent, percent by weight, weight %, % w / w, etc. are synonyms that refer to the concentration of a substance, which is the weight of the substance divided by the weight of the composition multiplied by 100.

[0034] All patents, patent applications, and other publications cited herein are incorporated by reference in their entirety for all purposes. [Brief explanation of the drawings]

[0035] [Figure 1] Individual macimorelin concentrations are shown versus time for Cohort 1, i.e., 0.25 mg / kg body weight, linear scale (pharmacokinetic sample (PKS), number of patients (N)=24). [Figure 2] Individual macimorelin concentrations versus time for cohort 2, i.e., 0.5 mg / kg body weight, linear scale (PKS, N=24) are shown. [Figure 3] Individual macimorelin concentrations versus time for cohort 3, i.e., 1.0 mg / kg body weight, linear scale (PKS, N=24) are shown. [Figure 4] Individual GH concentrations following macimorelin GHST versus time are shown for Cohort 1, ie, 0.25 mg / kg body weight, linear scale (Pharmacodynamic Set (PDS), N=24). [Figure 5] Cohort 2, i.e., individual GH concentrations after macimorelin GHST at 0.5 mg / kg body weight, linear scale (PDS, N=24) versus time are shown. [Figure 6] Cohort 3, i.e., individual GH concentrations following macimorelin GHST at 1.0 mg / kg body weight, linear scale (PDS, N=24) versus time are shown. [Figure 7] Receiver operating characteristic (ROC) analysis of cohort 1, i.e., macimorelin GHST at 0.25 mg / kg body weight (PDS, N=24) is shown. [Figure 8] Receiver operating characteristic (ROC) analysis of cohort 2, i.e., macimorelin GHST at 0.5 mg / kg body weight (PDS, N=24) is shown. [Figure 9] Receiver operating characteristic (ROC) analysis of cohort 3, i.e., macimorelin GHST at 1.0 mg / kg body weight (PDS, N=24) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0036] It is an object of the present invention to provide a stand-alone method for measuring growth hormone levels in pediatric patients and detecting GHD in pediatric patients. The goal is to develop a new method that should be safe, reliable, and provide excellent diagnostic performance, as well as reduce the burden on test administrators and test subjects by reducing the test time duration and the number of blood draws.

[0037] The object of the present invention is surprisingly to provide a screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin, comprising: (a) providing one to five blood samples taken from the subject within about 15 to about 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; (b) measuring the growth hormone level in each blood sample provided in step (a); (c) comparing the measured growth hormone level obtained in step (b) with a single threshold value that is equal to or greater than 10.0 ng / mL; (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 equal to or greater than the single threshold does not have growth hormone deficiency. This has been solved in one aspect by providing a method comprising:

[0038] Thus, one to five blood samples are taken from the subject no earlier than about 15 minutes after administration of an amount of macimorelin effective to induce growth hormone secretion and no later than about 100 minutes after administration of an amount of macimorelin effective to induce growth hormone secretion.

[0039] For example, if the level of a single growth hormone in the blood sample is equal to or greater than a single threshold, the subject is determined not to have growth hormone deficiency. Alternatively, if the level of growth hormone in the whole blood sample is lower than a single threshold, the subject is determined to have growth hormone deficiency.

[0040] 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.

[0041] In a preferred embodiment, the single threshold value for growth hormone is within the range of about 10.0 to about 25.0 ng / mL, preferably about 10.2 to about 20.0 ng / mL, more preferably about 12.0 to about 19.0 ng / mL, even more preferably about 14.0 to about 18.0 ng / mL, more preferably about 16.0 to about 18.0 ng / mL, and most preferably about 17.0 to about 18.0 ng / mL.

[0042] 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), and more preferably two or three blood samples are provided in step (a). As explained above, when more than one blood sample is provided, these blood samples are taken at different time points after administration of an amount of macimorelin effective to induce hormone secretion.

[0043] In another preferred embodiment, in step (a), the blood sample is collected from the subject within about 20 to about 100 minutes, preferably within about 25 to about 100 minutes, more preferably within about 25 to about 95 minutes, and most preferably within about 30 to about 90 minutes, after administration of an amount of macimorelin effective to induce growth hormone secretion. Thus, for example, the blood sample is collected from about 20 minutes to about 100 minutes, preferably from about 25 minutes to about 100 minutes, more preferably from about 25 minutes to about 95 minutes, and most preferably from about 30 minutes to about 90 minutes, after administration of an amount of macimorelin effective to induce growth hormone secretion.

[0044] In yet another preferred embodiment, when 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 about 15 to about 30 minutes. Alternatively, blood samples can be collected at any time interval deemed appropriate by the attending physician. For example, 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.

[0045] 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. When more than one blood sample is collected, two or more blood samples are of the same type, and therefore, these blood samples are either a whole blood sample, a serum sample, or a plasma sample. Most preferably, the blood sample is a serum sample.

[0046] In a more preferred embodiment, about 0.8 mg to about 1.2 mg of macimorelin is administered per kg of subject body weight in step (a), preferably about 1.0 mg of macimorelin is administered per kg of subject body weight in step (a). Typically, an effective amount of macimorelin can range from about 0.8 to about 0.9 mg per kg of subject body weight at the low end and about 1.0, about 1.1 to about 1.2 mg / kg of subject body weight at the high end, or can be within a range defined by any one of the lower and upper amounts, e.g., about 0.9 to about 1.1 mg / kg of body weight. An effective amount of macimorelin can be a single value, e.g., about 0.8, about 0.9, about 1.0, about 1.1, or about 1.2 mg / kg of body weight. Body weight (recorded in kg) can preferably be rounded to the nearest integer. Most preferably, about 1.0 mg of macimorelin per kg of subject body weight is administered in step (a).

[0047] For example, one macimorelin unit dose consists of a 1817.2 mg-containing composition for preparation of an oral suspension in water. Typically, the prepared suspension contains 0.5 mg macimorelin per mL of suspension. A weight-adjusted aliquot of the reconstituted suspension is administered to pediatric subjects to provide a pediatric dose of 1.0 mg / kg body weight. The unit dose is defined as macimorelin calculated as the free base with 100% content. The mass of macimorelin free base or its free base equivalent in the unit dose is adjusted accordingly.

[0048] In a preferred embodiment, in step (a), macimorelin is administered in a composition comprising macimorelin as a suitable pharmaceutical salt, preferably the suitable pharmaceutical salt is selected from an acetate salt of macimorelin, a trifluoroacetate salt of macimorelin, or a combination thereof.

[0049] In a further preferred embodiment, in step (a), the administration of macimorelin is oral.

[0050] When macimorelin is administered orally, macimorelin can be prepared as an oral suspension. The suspension can be administered within about 90 minutes, preferably within about 60 minutes, and more preferably within about 30 minutes after preparation of the oral suspension. Preferably, the oral suspension is swallowed over a period of about 1 minute or less, preferably over a period of about 30 seconds or less.

[0051] In a further preferred embodiment, the subject fasts for about 10 hours, preferably about 9 hours, and more preferably about 8 hours prior to administration of macimorelin.More preferably, in step (a), the subject fasts for about 100 minutes after administration of macimorelin, which means that in step (a), the subject can fast for about 100 minutes, about 95 minutes, or about 90 minutes after administration of macimorelin.

[0052] In a more preferred embodiment, step (a) provides a single blood sample collected from the subject at about 60±30 minutes after administration of macimorelin. Alternatively, the single 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, or about 100±10 minutes after administration of macimorelin. The single blood sample can be collected at any time within a range of about 15 to about 100 minutes after administration of macimorelin, as deemed appropriate by the attending physician.

[0053] In another more preferred embodiment, step (a) provides two blood samples taken from the subject at about 30±10 minutes and about 45±10 minutes after administration of macimorelin. Alternatively, the two blood samples can be taken 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 macimorelin. The two blood samples can be taken at any time within a range of about 15 to about 100 minutes after administration of macimorelin, as deemed appropriate by the attending physician.

[0054] In another preferred embodiment, in step (a), two blood samples are provided taken from the subject at about 30±10 minutes and about 60±10 minutes after administration of macimorelin.

[0055] In another preferred embodiment, in step (a), three blood samples are provided that are taken from the subject at about 30±10 minutes, about 45±10 minutes, and about 60±10 minutes after administration of macimorelin; or in step (a), three blood samples are provided that are taken from the subject at about 30±10 minutes, about 45±10 minutes, and about 90±10 minutes after administration of macimorelin; or in step (a), three blood samples are provided that are taken from the subject at about 30±10 minutes, about 60±10 minutes, and about 90±10 minutes after administration of macimorelin. Alternatively, 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 macimorelin. The three blood samples can be collected at any time within the range of about 15 to about 100 minutes after administration of macimorelin, as deemed appropriate by the attending physician.

[0056] When four or more blood samples are provided in step (a), these blood samples can be taken at any time within a range of about 15 to about 100 minutes after administration of macimorelin, as deemed 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 administration of macimorelin.

[0057] In a particularly preferred method, in step (a), one to four, more preferably one to three, more preferably two or three blood samples are provided, taken from the subject at time points selected from the group consisting of about 30±10 minutes, about 45±10 minutes, about 60±10 minutes, and about 90±10 minutes after administration of macimorelin.

[0058] In a further preferred embodiment, in step (a), macimorelin is administered in a composition comprising macimorelin and, optionally, a further pharmaceutically acceptable excipient such as a carrier substance. Preferably, macimorelin is administered in a composition comprising macimorelin and a sweetener. A suitable sweetener is, for example, saccharin. Advantageously, saccharin has been found to be a suitable taste-masking agent for macimorelin.

[0059] In a further preferred embodiment, in step (a), macimorelin is administered in a composition comprising about 3.5% (w / w) macimorelin (calculated as the free base), about 93.1% (w / w) spray-dried lactose monohydrate, about 2.0% (w / w) crospovidone type A, about 0.1% (w / w) colloidal silicon dioxide, about 1.0% (w / w) sodium stearyl fumarate, and about 0.3% (w / w) sodium saccharin dihydrate.

[0060] In a further preferred embodiment, the subject is a human child aged between 2 and under 18 years old, preferably the subject is a human child aged between 2 and under 17 years old, and more preferably the subject is a human child aged between 2 and under 16 years old.

[0061] In a further preferred embodiment, the method is a stand-alone test and does not need to be repeated, and no alternative growth hormone stimulation test is required to reliably diagnose growth hormone deficiency in pediatric patients.

[0062] In a further preferred embodiment, determining whether a subject has or does not have growth hormone deficiency according to step (d) is based solely on the induction of growth hormone levels by a single administration of macimorelin.

[0063] Surprisingly, it has been found that the method of the present invention is suitable as a stand-alone test, as no additional GHST is required to reliably diagnose GHD in paediatric patients.

[0064] The object of the present invention is, surprisingly, to (a) providing one to five blood samples taken from a subject within about 15 to about 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; (b) measuring the growth hormone level in each blood sample provided in step (a); (c) comparing the measured growth hormone level obtained in step (b) with a single threshold value of 10.0 ng / mL or greater; (d) a subject whose highest growth hormone level in the blood sample obtained in step (b) is lower than the single threshold is diagnosed as having growth hormone deficiency, and a subject whose highest growth hormone level in the blood sample obtained in step (b) is equal to or higher than the single threshold is diagnosed as not having growth hormone deficiency; The solution has been reached in another aspect by providing the substance macimorelin for use in the diagnosis of growth hormone deficiency in paediatric patients.

[0065] One to five blood samples are taken from the subject no earlier than about 15 minutes after administration of the amount of macimorelin effective to induce growth hormone secretion and no later than about 100 minutes after administration of the amount of macimorelin effective to induce growth hormone secretion.

[0066] In a preferred embodiment, the substance macimorelin for use in the diagnosis of growth hormone deficiency in a pediatric patient is the substance macimorelin for use in the in vitro diagnosis of growth hormone deficiency in a pediatric patient.

[0067] In a preferred embodiment, the single threshold value for growth hormone is within the range of about 10.0 to about 25.0 ng / mL, preferably about 10.2 to about 20.0 ng / mL, more preferably about 12.0 to about 19.0 ng / mL, even more preferably about 14.0 to about 18.0 ng / mL, more preferably about 16.0 to about 18.0 ng / mL, and most preferably about 17.0 to about 18.0 ng / mL.

[0068] 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), and more preferably two or three blood samples are provided in step (a). When more than one blood sample is provided, these blood samples are taken at different time points after administration of an amount of macimorelin effective to induce hormone secretion.

[0069] In another preferred embodiment, in step (a), the blood sample is collected from the subject within about 20 to about 100 minutes, preferably within about 25 to about 100 minutes, more preferably within about 25 to about 95 minutes, and most preferably within about 30 to about 90 minutes, after administration of an amount of macimorelin effective to induce growth hormone secretion. As a result, the blood sample is collected, for example, from about 20 minutes to about 100 minutes, preferably from about 25 minutes to about 100 minutes, more preferably from about 25 minutes to about 95 minutes, and most preferably from about 30 minutes to about 90 minutes, after administration of an amount of macimorelin effective to induce growth hormone secretion.

[0070] In yet another preferred embodiment, when 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 about 15 to about 30 minutes. Alternatively, blood samples can be collected at any time interval deemed appropriate by the attending physician. For example, 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.

[0071] 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. When more than one blood sample is collected, two or more blood samples are of the same type, and therefore, these blood samples are either a whole blood sample, a serum sample, or a plasma sample. Most preferably, the blood sample is a serum sample.

[0072] In a more preferred embodiment, about 0.8 mg to about 1.2 mg of macimorelin is administered per kg of subject body weight in step (a), preferably about 1.0 mg of macimorelin is administered per kg of subject body weight in step (a). Typically, an effective amount of macimorelin can range from about 0.8 to about 0.9 mg per kg of subject body weight at the lower end and about 1.0, about 1.1, or about 1.2 mg / kg of subject body weight at the upper end, or can be within a range defined by any one of the lower and upper amounts, e.g., about 0.9 to about 1.1 mg / kg of subject body weight. An effective amount of macimorelin can also be a single value, e.g., about 0.8, about 0.9, about 1.0, about 1.1, or about 1.2 mg / kg of body weight. Body weight (recorded in kg) can preferably be rounded to the nearest integer.

[0073] For example, one macimorelin unit dose consists of a 1817.2 mg-containing composition for preparation of an oral suspension in water. Typically, the prepared suspension contains 0.5 mg macimorelin per mL of suspension. A weight-adjusted aliquot of the reconstituted suspension is administered to pediatric subjects to provide a pediatric dose of 1.0 mg / kg body weight. The unit dose is defined as macimorelin calculated as the free base with 100% content. The mass of macimorelin free base or its free base equivalent in the unit dose is adjusted accordingly.

[0074] In a preferred embodiment, in step (a), macimorelin is administered in a composition comprising macimorelin as a suitable pharmaceutical salt thereof, preferably the suitable pharmaceutical salt is selected from an acetate salt of macimorelin, a trifluoroacetate salt of macimorelin, or a combination thereof.

[0075] In a further preferred embodiment, in step (a), the administration of macimorelin is oral.

[0076] When macimorelin is administered orally, macimorelin can be prepared as an oral suspension. The suspension can be administered within about 90 minutes, preferably within about 60 minutes, and more preferably within about 30 minutes after preparation of the oral suspension. Preferably, the oral suspension is swallowed over a period of about 1 minute or less, preferably over a period of about 30 seconds or less.

[0077] In a further preferred embodiment, the subject fasts for about 10 hours, preferably about 9 hours, and more preferably about 8 hours prior to administration of macimorelin.More preferably, in step (a), the subject fasts for about 100 minutes after administration of macimorelin, which means that in step (a), the subject can fast for about 100 minutes, about 95 minutes, or about 90 minutes after administration of macimorelin.

[0078] In a more preferred embodiment, step (a) provides a single blood sample collected from the subject at about 60±30 minutes after administration of macimorelin. Alternatively, the single blood sample may 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 administration of macimorelin. The single blood sample may be collected at any time within a range of about 15 to about 100 minutes after administration of macimorelin, as deemed appropriate by the attending physician.

[0079] In another more preferred embodiment, step (a) provides two blood samples taken from the subject at about 30±10 minutes and about 45±10 minutes after administration of macimorelin. Alternatively, the two blood samples can be taken 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 macimorelin. The two blood samples can be taken at any time within a range of about 15 to about 100 minutes after administration of macimorelin, as deemed appropriate by the attending physician.

[0080] In another preferred embodiment, in step (a), two blood samples are provided taken from the subject at about 30±10 minutes and about 60±10 minutes after administration of macimorelin.

[0081] In another preferred embodiment, in step (a), three blood samples are provided that are taken from the subject at about 30±10 minutes, about 45±10 minutes, and about 60±10 minutes after administration of macimorelin; or in step (a), three blood samples are provided that are taken from the subject at about 30±10 minutes, about 45±10 minutes, and about 90±10 minutes after administration of macimorelin; or in step (a), three blood samples are provided that are taken from the subject at about 30±10 minutes, about 60±10 minutes, and about 90±10 minutes after administration of macimorelin. Alternatively, 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 macimorelin. The three blood samples can be collected at any time within the range of about 15 to about 100 minutes after administration of macimorelin, as deemed appropriate by the attending physician.

[0082] If four or more blood samples are provided, these blood samples may be taken at any time within a range of about 15 to about 100 minutes after administration of macimorelin, as deemed 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 administration of macimorelin.

[0083] In a particularly preferred embodiment, in step (a), one to four, more preferably one to three, more preferably two or three blood samples are provided that are taken from the subject at time points selected from the group consisting of about 30±10 minutes, about 45±10 minutes, about 60±10 minutes, and about 90±10 minutes after administration of macimorelin.

[0084] In a further preferred embodiment, in step (a), macimorelin is administered in a composition comprising macimorelin and, optionally, a further pharmaceutically acceptable excipient such as a carrier substance. Preferably, macimorelin is administered in a composition comprising macimorelin and a sweetener. A suitable sweetener is, for example, saccharin. Saccharin has been found to be a suitable taste-masking agent for macimorelin.

[0085] In a further preferred embodiment, in step (a), macimorelin is administered in a composition comprising about 3.5% (w / w) macimorelin (calculated as the free base), about 93.1% (w / w) spray-dried lactose monohydrate, about 2.0% (w / w) crospovidone type A, about 0.1% (w / w) colloidal silicon dioxide, about 1.0% (w / w) sodium stearyl fumarate, and about 0.3% (w / w) sodium saccharin dihydrate.

[0086] In a further preferred embodiment, the subject is a human child aged between 2 and under 18 years old, preferably the subject is a human child aged between 2 and under 17 years old, and more preferably the subject is a human child aged between 2 and under 16 years old.

[0087] In a further preferred embodiment, the substance is used in a stand-alone test and does not need to be repeated, and no alternative growth hormone stimulation test is required to reliably diagnose growth hormone deficiency in pediatric patients.

[0088] In a further preferred embodiment, determining whether a subject has or does not have growth hormone deficiency according to step (d) is based solely on the induction of growth hormone levels by a single administration of macimorelin.

[0089] Surprisingly, it has been found that the method of the present invention is suitable as a stand-alone test, since no additional GHST is required to reliably diagnose GHD in paediatric patients.

[0090] The object of the present invention is surprisingly to provide a screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin, comprising: (a) providing at least one blood sample taken from the subject within about 15 to about 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; (b) measuring the growth hormone level in each blood sample; (c) comparing each measured growth hormone level to a single threshold; (d) diagnosing whether the subject is suffering from growth hormone deficiency based on a comparison of the growth hormone level measured in step (b) in the at least one blood sample with the single threshold value; In a further aspect, the solution has been solved by a method comprising: determining whether a subject has or does not have growth hormone deficiency, wherein the method is based solely on the induction of growth hormone levels by a single administration of macimorelin.

[0091] In a preferred embodiment, the screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin is a screening method for diagnosing growth hormone deficiency in pediatric patients in vitro by using macimorelin.

[0092] In a preferred method, in step (a), one to four, more preferably one to three, more preferably two or three blood samples are provided taken from the subject at time points selected from the group consisting of about 30±10 minutes, about 45±10 minutes, about 60±10 minutes, and about 90±10 minutes after administration of macimorelin.

[0093] In a preferred embodiment, in step (d), subjects whose highest growth hormone level measured in step (b) is lower than a single threshold value are determined to have growth hormone deficiency, and subjects whose highest growth hormone level measured in step (b) is equal to or greater than the single threshold value are determined to not have growth hormone deficiency.

[0094] In another preferred embodiment, the single threshold value is within the range of about 10.0 to about 25.0 ng / mL, preferably about 10.2 to about 20.0 ng / mL, more preferably about 12.0 to about 19.0 ng / mL, even more preferably about 14.0 to about 18.0 ng / mL, more preferably about 16.0 to about 18.0 ng / mL, and most preferably about 17.0 to about 18.0 ng / mL.

[0095] In yet another preferred embodiment, about 0.8 mg to about 1.2 mg of macimorelin is administered per kg of subject body weight, preferably about 1.0 mg of macimorelin is administered per kg of subject body weight.

[0096] In a further preferred embodiment, the subject is a human child aged between 2 and under 18 years old, preferably the subject is a human child aged between 2 and under 17 years old, and more preferably the subject is a human child aged between 2 and under 16 years old.

[0097] A key feature of the method of the present invention is its single-stimulation test format, as opposed to the standard two-test format currently used by medical professionals. Rather than having two separate tests performed at least one day apart and involving as many as 8-12 blood draws, the new method of the present invention requires only one test and only 1-5, and preferably 2-4, blood draws to achieve reliable diagnostic performance in accuracy, specificity, and sensitivity for detecting growth hormone deficiency, thereby greatly reducing the testing burden and potential harm to the child being tested. Advantageously, these blood samples can be collected at intervals of about 15 to about 30 minutes within a short period of time, such as a total of about 90 minutes, after administration of macimorelin.

[0098] Surprisingly, the method of the present invention achieves a significant improvement by using a higher threshold value to diagnose growth hormone deficiency. Traditionally, a single threshold value of approximately 2-3 ng / mL is used when adult patients are tested for GHD, and a single threshold value of less than 10 ng / mL is used when pediatric patients are tested for GHD. The inventors unexpectedly discovered that better diagnostic performance can be achieved for pediatric patients when a higher single threshold value of 10.0 ng / mL or greater is used. For example, a single threshold value of approximately 16.0-19.0 ng / mL, preferably approximately 17.0-18.0 ng / mL, was found to be highly effective in indicating GHD in the stand-alone method of the present invention.

[0099] Furthermore, the method of the present invention achieves significant improvement by using higher macimorelin dosages to diagnose growth hormone deficiency. While the conventional dosage of macimorelin used in current methods is 0.5 mg / kg patient body weight, particularly when adult patients are tested for GHD, the inventors unexpectedly discovered that higher doses of macimorelin can achieve better diagnostic performance when used in growth hormone stimulation tests for pediatric patients. For example, macimorelin dosages of about 0.8 to about 1.2 mg / kg, preferably about 1.0 mg / kg body weight, have been found to be highly effective in identifying GHD in one testing method of the present invention.

[0100] The present invention includes a method for assessing pituitary-related GH deficiency in human children following a single oral administration of macimorelin to the child: - As a stand-alone test (only one test method and a single GH stimulation is required), Two to four blood samples are collected at intervals of about 15 to about 30 minutes after administration of macimorelin, for example, over a total period of about 90 minutes. -having a GH cutoff point of about 10.0 ng / mL or greater, preferably between about 10.0 ng / mL and about 25.0 ng / mL, more preferably between about 10.2 ng / mL and about 20 ng / mL, and most preferably between about 17.0 and about 18.0 ng / mL A method for measuring growth hormone levels in a human child is provided.

[0101] The following examples are offered by way of illustration only and not by way of limitation. Those of ordinary skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially the same or similar results. [Example]

[0102] Growth hormone deficiency (GHD) in children is a rare, etiologically diverse condition resulting in growth failure and short stature. Diagnosis of GHD requires an inadequate response to two different growth hormone stimulation tests (GHSTs). Macimorelin acetate, a potent, 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 in children with suspected GHD.

[0103] This was an open-label, group-controlled, dose-escalation study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of single-dose 0.25, 0.5, and 1.0 mg / kg oral macimorelin acetate in pediatric subjects with suspected GHD. Macimorelin GHST was administered between two standard GHSTs performed according to local clinical practice, with a 7- to 28-day recovery period between studies. Blood samples were collected pre-dose (±15 minutes) and at 15, 30, 45, 60, 90, 120, and 360 minutes after macimorelin acetate ingestion.

[0104] A total of 24 pediatric subjects (8 per cohort [C1, C2, C3]) were included in the pharmacokinetic / pharmacodynamic (PK / PD) analysis. Five males and three females were observed in C1 and C2, and seven males and one female in C3. At least three subjects in all three cohorts demonstrated Tanner stage I or II. All 24 subjects (100%) were Caucasian, with a median age of 9.8, 9.0, and 10.5 years (range 4-15 years) and a median body mass index of 16.1 kg / m at screening.2 (12.4-21.4 kg / m 2 Overall, 88 adverse events were reported, most of which were related to standard GHST and none were considered related to the macimorelin study. Peak plasma concentrations of macimorelin were observed primarily between 30 and 45 minutes. Mean C max The values ​​were 3.46, 8.13, and 12.87 ng / mL for C1, C2, and C3, respectively. The AUC increased with dose; the mean AUC0-6 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. PK and PD profiles were similar in all three cohorts, and peak GH levels were observed primarily within 30 to 60 minutes after macimorelin ingestion.

[0105] Macimorelin acetate was safe and well tolerated in all treatment cohorts. max The dose-dependent increases in t and AUC correlated well with data from adult subjects. A robust dose-proportional GH response was also achieved. PD results showed that GH responses were comparable across all treatment groups, with faster t max There was a slight shift to

[0106] Furthermore, the outcomes of the macimorelin GHST showed a surprisingly high agreement with the outcomes of the two standard GHSTs and with the final diagnoses assessed by the investigators. At C3, GH secretion was clearly stimulated in all eight patients. Finally, the outcomes of the macimorelin GHST administered as a single test showed agreement with the outcomes of the combined two sGHSTs and with the PI assessment in seven of the eight subjects.

[0107] Example 1: Macimorelin-containing composition for diagnosing CGHD The macimorelin-containing composition includes the following ingredients listed in Table 1.

[0108] [Table 1]

[0109] One macimorelin dose unit comprises 1817.2 mg of a composition for preparation of an oral suspension in water. Typically, the prepared suspension contains 0.5 mg of macimorelin per mL of suspension.

[0110] A weight-adjusted aliquot of the reconstituted suspension is administered to pediatric subjects to provide a dose of 1.0 mg / kg body weight in children. Note that in adults, a weight-adjusted aliquot of the reconstituted suspension is administered to provide a dose of 0.5 mg / kg body weight of the adult subject.

[0111] The unit dose is defined as macimorelin calculated as free base, with a content of 100%. The mass of macimorelin free base or its free base equivalent in the unit dose is adjusted accordingly.

[0112] Macimorelin may be included in the unit dosage as a suitable pharmaceutical salt. Examples of suitable pharmaceutical salts are acetate and trifluoroacetate.

[0113] The unit doses may be filled into suitable containers for easy access to the GHD test. Examples of suitable containers are sachets or glass of plastic containers of a suitable size.

[0114] For a 60 kg subject, the container is a polyethylene-laminated aluminum foil pouch containing a macimorelin-containing composition including 63.6 mg macimorelin, 1691.8 mg spray-dried lactose monohydrate, 36.3 mg crospovidone type A, 1.8 mg colloidal silicon dioxide, 18.2 mg sodium stearyl fumarate, and 5.5 mg sodium saccharin dihydrate. When reconstituted in 120 mL of water, 2 mL provides 1.0 mg macimorelin.

[0115] Example 2: Use of saccharin in macimorelin-containing compositions to mask unpleasant tastes In a multicenter, randomized, two-period, crossover study, 100 subjects with confirmed adult growth hormone deficiency (AGHD) were given GHRH plus L-Arg and macimorelin as GHST to determine the diagnostic efficacy of macimorelin for AGHD.

[0116] This study was conducted in two parts. In the first part, using the saccharin-free macimorelin-containing composition described in Example 1 at a dose of 0.5 mg / kg, mild to moderate unpleasant taste was reported by 12 of 52 subjects (21%).

[0117] During the study break, saccharin was found to be a suitable flavoring agent, but did not completely mask the bitter taste. In the second part of the study, using the macimorelin-containing composition (with saccharin) described in Example 1, only 1 of 48 subjects (2%) reported a mildly unpleasant taste.

[0118] Example 3: Pharmacokinetic, Pharmacodynamic, and Exploratory Properties of Macimorelin as a Diagnostic Test Study AEZS-130-P01 was an open-label, group-controlled, dose-escalation trial investigating the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of macimorelin acetate after single oral doses of 0.25 mg / kg, 0.5 mg / kg, and 1 mg / kg in pediatric patients with suspected GHD.

[0119] The objectives were defined as follows: Main: To investigate the safety and tolerability of macimorelin acetate following single escalating oral doses of macimorelin in pediatric patients with suspected GHD. Secondary: To investigate the PK of macimorelin acetate in pediatric patients with suspected GHD. To investigate the PD of macimorelin acetate, as measured by growth hormone (GH) release, in pediatric patients with suspected GHD. To explore the PK / PD relationship after a single oral dose of macimorelin acetate in pediatric patients with suspected GHD.

[0120] Methodology: Plasma concentrations of macimorelin and serum concentrations of GH were analyzed at a central laboratory.

[0121] Macimorelin plasma concentrations: Analysis of plasma samples for macimorelin concentrations was performed at a central laboratory, Prolytic GmbH, Germany, using a validated liquid chromatography-mass spectrometry (LCMS / MS) method with a detection limit of 0.2 ng / mL (Franz, 2005).

[0122] Preliminary pharmacokinetic (PK) data were obtained for macimorelin plasma concentrations over the sampling period, as well as the time to the highest measured concentration (t max ) and maximum concentration (C max ) was determined.

[0123] GH serum concentrations: Serum samples were analyzed for GH concentrations at a central laboratory using a validated immunochemiluminescence assay (IDS-iSYS Human Growth Hormone (hGH), Immunodiagnostic Systems Ltd [UK]) (Manolopoulou et al., 2012). The assay was standardized against the recombinant growth hormone calibration standard WHO 98 / 574 and conforms to the assay standardization recommendations outlined by Clemmons (Clemmons et al., 2011).

[0124] The laboratory referred for GH was Central Laboratory Synevo Lodz, Krakusa Str. 28, 93-515 Lodz, Poland. The lower limit of quantification was <0.05 ng / mL.

[0125] Number of patients Overall, macimorelin was administered as a single oral dose to 24 pediatric patients with suspected GHD, with 8 patients per treatment group. Of these 8 patients, at least 3 patients per treatment group were prepubertal (Tanner stage I) and adolescent (Tanner stages II-IV).

[0126] Key patient inclusion criteria: Subjects had to meet all of the following criteria to be eligible to participate in the trial: 1. Male or female pediatric patients aged 2 to under 18 years; 2. GHD is suspected based on growth and clinical criteria; 3. Indication of performance of the provocative growth hormone stimulation test (GHST).

[0127] Subjects who have sex steroid priming prior to the GHST as part of standard diagnostic procedure should also have sex steroid priming with the macimorelin GHST.

[0128] Investigational New Drug (IMP) macimorelin The single-use aluminum pouch (sachet) contained 63.6 mg macimorelin, which when dissolved in 120 mL of water gave 0.5 mg / mL of macimorelin.

[0129] Study participants in successive cohorts received macimorelin in ascending single oral doses: 0.25 mg / kg body weight in Cohort 1 (C1), 0.5 mg / kg body weight in Cohort 2 (C2), and 1 mg / kg body weight in Cohort 3 (C3).

[0130] To measure macimorelin PK / PD, blood samples were collected pre-dose and then at 15, 30, 45, 60, 90, 120, and 360 minutes after administration of macimorelin.

[0131] As a test, a single oral dose of macimorelin acetate was administered to patients on the day of the macimorelin GHST.

[0132] Macimorelin GHST formulation The macimorelin oral suspension was prepared by the investigator and administered according to the following instructions (Steps 1 to 5), taking the dose of Cohort C3 (i.e., 1.0 mg / kg) as an example: 1. Weigh the patient and calculate the number of pouches / sachets needed (one pouch per patient). Weight (recorded in kg) will be rounded to the nearest whole number; 2. Dissolve the entire contents of the pouch in 120 mL of water in a suitable clear glass or polypropylene container (i.e., one pouch for 120 mL, two pouches for 240 mL, as applicable) and gently stir for at least 3 minutes (a small amount of undissolved particles will remain); 3. Calculate the required volume of suspension corresponding to the patient weight based on a macimorelin dose of 1.0 mg / kg, i.e., the required volume of suspension is 2 mL / kg (e.g., a 30 kg patient requiring a macimorelin dose of 1.0 mg / kg would require 60 mL of prepared suspension); 4. Using a graduated syringe, measure the required volume for the patient and transfer it to a drinking glass (volumes less than 20 mL should be administered by oral syringe to minimize the risk of loss from incomplete swallowing; see below for instructions); 5. The suspension must be used within 30 minutes of preparation.

[0133] Fasting prior to GHST Patients were required to fast for 8 hours prior to the start of the macimorelin stimulation test and throughout the sampling period.

[0134] Administration Administration of macimorelin oral suspension was performed under investigator supervision. Patients were advised to drink the entire contents of the glass container prepared in step 4 of the cohort-specific administration instructions described above in a period of 30 seconds or less.

[0135] Blood sampling Blood samples were collected at the following time points: pre-dose (sampling time window: ±15 min), then 15, 30, 45, 60, 90, 120 min (±5 min window), and 360 min (±10 min window) after macimorelin administration. Serum concentrations of GH and plasma concentrations of macimorelin were analyzed at a central laboratory.

[0136] The investigational macimorelin GHST was performed after the first sGHST was completed. A recovery period of at least 1 week and up to 4 weeks was introduced between GHSTs to avoid carryover effects or interference with subsequent GHSTs and to allow an adequate follow-up period for observation of potentially drug-related adverse events due to the previously used inducer.

[0137] Standard GHST used in study P01 Two standard growth hormone stimulation tests (sGHST) had to be performed on patients according to local practice. The sGHST agent was considered as "background" and not as an IMP.

[0138] The following pharmacological agents are recognized for sGHST: insulin (insulin tolerance test (ITT)), arginine, arginine / growth hormone-releasing hormone (GHRH), clonidine, glucagon, and L-dopa.

[0139] A single dose of sGHST formulation was administered intramuscularly (im), intravenously (iv), subcutaneously (sc), or orally (depending on formulation) on the day of sGHST. Batch numbers were recorded on-site in the Patient Records and "standard GHST Patients Accountability Logs."

[0140] Evaluation criteria Clinical trial endpoints Safety and Tolerability Patient tolerability (including taste acceptance and effects on sleep, appetite, and gastrointestinal symptoms), adverse events (AEs); · Measuring changes in safety-related clinical laboratory parameters; ·Effects on vital parameters (pulse rate, blood pressure, electrocardiogram).

[0141] Pharmacokinetics · Macimorelin concentration-time profile; Target parameters: AUC, C max , T max , T 1 / 2 .

[0142] Pharmacodynamics ·GH concentration-time profile; Target parameters: C max , T max ; Preliminary PK / PD: T of macimorelin max vs. GH T max ;C of macimorelin max vs. GH C max .

[0143] others ·Establishment of recommended dosages for diagnostic purposes in pediatric patients with suspected GHD; · Search for a suitable GH cut-off point for subsequent testing to establish the diagnosis of GHD in pediatric patients.

[0144] Statistical methods: All statistical analyses were considered exploratory in nature. Data sets were analyzed using SAS version 9.3 or higher.

[0145] In general, summary statistics (n, arithmetic mean, standard deviation, median, minimum, and maximum) for quantitative variables and frequency tables for qualitative data were presented by treatment group.

[0146] Macimorelin PK: PK parameters were analyzed for the PK analysis set (PKS) and were analyzed by n (number of measurements), arithmetic mean, standard deviation, and coefficient of variation (CV), median, lowest, highest value, and further (T max (excluding ) are summarized by geometric mean, geometric standard deviation, and geometric CV. max Additionally, frequency counts as well as median, minimum, and maximum are also presented for each.

[0147] Macimorelin PD: GH concentration data were analyzed for the PD analysis set (PDS) regardless of availability of PK data. Peak GH concentrations were correlated with the outcome of the clinical diagnostic procedure (confirmed or unconfirmed diagnosis of GHD).

[0148] PK / PD Analysis: Individual patient plasma concentrations of macimorelin were correlated with their respective GH concentrations at the same time points, as well as the outcome of the clinical diagnostic procedure (confirmed or unconfirmed diagnosis of GHD).

[0149] Results and conclusions: Of the total 27 patients screened, 24 patients received macimorelin treatment, with 8 patients in each of the three treatment cohorts (C1, C2, and C3).

[0150] Therefore, the Safety Analysis Set (SAF) as well as the PK Analysis Set (PKS), PD Analysis Set (PDS), and PK / PD populations consisted of 24 patients.

[0151] Baseline characteristics: Overall, 17 (70.8%) patients were male and 7 (29.2%) were female, and 100% were Caucasian. At screening, the median parameters for all three treatment cohorts 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 ) was.

[0152] Tanner status was distributed as follows: in C1 and C3, 4 patients showed Tanner I and 4 patients showed Tanner II, and in C2, 5 patients showed Tanner I and 3 patients showed Tanner II. Sex steroid priming was administered in 2 male patients in C3 by im administration of a testosterone depot preparation.

[0153] Baseline medical history reported other pituitary axis deficiencies (i.e., hypothyroidism) for only two patients in C2. As part of the standard "diagnostic work-up," IGF-1 and IGF-BP3 levels were captured in the electronic case report form (eCRF) as collected according to each institution's diagnostic criteria.

[0154] IGF-1 levels were available in 7 patients in C1, 8 patients each in C2 and C3, with median values ​​of 88.00 μg / L (SD 68.72) in C1, 100.00 μg / L (SD 97.90) in C2, and 119.50 μg / L (SD 68.88) in C3. IGF-BP3 levels were available in 1 patient in C3.

[0155] The median bone age was 102.2 months (range: 24-156 months). As part of the growth parameters, the median height SDS was -2.35 (range: -3.2-1.7), BMI SDS -0.60 (range: -2.1-2.0), and annualized growth velocity SDS -1.50 (range: -3.3-0.5).

[0156] SGHST: Overall, ITT was administered to 22 patients (i.e., 5 patients (20.8%) at Visit 1 (V1) and 17 patients (70.8%) at Visit 3 (V3)), arginine to 8 patients (33.3%) at V1, and clonidine to 16 patients (i.e., 11 patients (45.8%) at V1 and 5 patients (20.8%) at V3). Glucagon was administered to only 1 patient, and L-dopa was not administered at all.

[0157] Macimorelin treatment compliance was 100% in all three cohorts.

[0158] Pharmacokinetic and pharmacodynamic results: Plasma concentration data Overall, macimorelin plasma concentrations showed a dose-dependent increase (Figure 1), with high interindividual variability. After macimorelin administration, plasma concentrations increased rapidly, with peak levels observed between 0.25 and 2 hours after administration. By the final sampling time point, 6 hours after administration, plasma concentrations had significantly decreased.

[0159] Pharmacokinetics: Overall, macimorelin plasma concentrations showed a dose-dependent increase (Figure 1), with high interindividual variability. After macimorelin administration, plasma concentrations increased rapidly, with peak levels observed between 0.25 and 2 hours after administration. By the final sampling time point, 6 hours after administration, plasma concentrations had significantly decreased.

[0160] AUC and C of macimorelin max showed a dose-dependent increase, with the arithmetic mean AUC0-6 of 6.69 h ng / mL for C1, 18.02 h ng / mL for C2, and 30.92 h ng / mL for C3. max were 3.46 ng / mL for C1, 8.13 ng / mL for C2, and 12.87 ng / mL for C3 (Table 2).

[0161] average T max was comparable among all three groups, with arithmetic means of 45.5 minutes for C1, 40.6 minutes for C2, and 31.9 minutes for C3. 1 / 2 showed a slight increase at higher doses, i.e., 73.18 min for C1, 96.31 min for C2, and 102.85 min for C3.

[0162] [Table 2]

[0163] Pharmacodynamics: As shown in Figure 2, GH concentrations increased after macimorelin administration and tended to higher values ​​with increasing doses. Large interpatient variability is expected in the observed population suspected of having GHD.

[0164] After macimorelin administration, peak GH levels occurred within 0.5–1 hour at C1 (mean T max 52.5 minutes (SD 11.3)), and within 0.25 to 1 hour for C2 (mean T max 37.5 min (SD 13.9)) and C3 within 0.5–0.75 h (mean T max 37.5 min (SD 8.0)) was observed (see Table 3).

[0165] [Table 3]

[0166] Exploratory analysis of GH cutoff points: Peak GH levels with GHD diagnosis were compared based on GHST results and investigator assessment. Diagnostic characteristics (i.e., sensitivity, specificity, and Youden index (unweighted and weighted)) of GH levels tested as cutoff points are listed, along with the most reliable representation of diagnostic characteristics to note for C1 of 10.03 ng / mL peak GH, C2 of 10.43 ng / mL peak GH, and C3 of 17.13 ng / mL peak GH.

[0167] The diagnostic outcomes of GHST are presented in Table 4. In this table, the diagnostic outcome of sGHST is considered "confirmed" if both sGHSTs are available and both result in peak GH levels of 7 ng / mL or less, or "not confirmed" if at least one of the peaks is above 7 ng / mL. The outcome "not confirmed" is classified as "ruled out" if both sGHST results are available and the GH peak is above 7 ng / mL, or as "equivocal" if the situation does not fit any of the above. Investigator assessment was based on standard diagnostic practice at each institution. Macimorelin GHST was tested against cutoff points calculated from individual peak GH values.

[0168] [Table 4] JPEG0007767387000005.jpg42170

[0169] Based on the considerations outlined above, Table 4 presents the agreement between the investigator's (PI's) assessment and both sGHST outcomes: in 21 (87.5%) patients (i.e., 8 confirmed and 13 unconfirmed), there was agreement between the investigator's assessment and the sGHST outcome. In 3 (12.5%) patients, the investigator concluded that GHD was present, but the sGHST ruled out the diagnosis (in 1 patient) or was indeterminate (in 2 patients).

[0170] Furthermore, the diagnostic results can be summarized as follows (Table 5): macimorelin GHST shows "no confirmed GHD" in only one patient (9.09%) in C2 from a total of 11 patients assessed as "GHD" by the investigator in all three cohorts.

[0171] From a total of 13 patients assessed by the investigator as "not confirmed" to have GHD, macimorelin GHST confirmed GHD in 3 (23.08%) patients at C1 and 1 (7.69%) patient at C3, respectively.

[0172] [Table 5]

[0173] Considering the data presented above, the strongest test characteristic of the macimorelin test is observed in C3: GH secretion was clearly stimulated in all eight patients. Finally, the outcomes of the macimorelin GHST applied as a single test were consistent with those of the combination of the two sGHSTs, as well as with the PI assessment, in seven of the eight subjects.

[0174] Receiver operating characteristic (ROC) analysis Of all GH cutoff points tested, the ROC curve for C1 shows the lowest sensitivity and specificity compared to C2 and C3 (Figure 3). The associated area under the curve (AUC) increases with dose escalation.

[0175] Comparing the characteristics of the GH cutoff points among the three cohorts, the cutoff point of 17.130 ng / mL GH in C3 showed the strongest test characteristics, with a sensitivity of 1.0, a specificity of 0.8, a Youden index of 0.80 or greater, and an ROC AUC of 0.93 (see Table 6).

[0176] Sensitivity analyses were performed to observe ROC AUC development based on sGHST test outcomes classified as "confirmed" versus "not confirmed." Again, the strongest test characteristics were exhibited by C3, with a sensitivity of 1.00, a specificity of 0.80, and an ROC AUC of 0.933, 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).

[0177] [Table 6]

[0178] PK and PD summary Overall, macimorelin PK and PD profiles for C1, C2, and C3 show comparable profiles: Macimorelin T max was similar in all three groups, and the mean T max The value is about 0.5-0.75 hours; Average macimorelin C max indicates a dose-proportional increase; ·AUC increases with macimorelin dose; Peak GH release was observed between 0.25 and 2 hours after macimorelin administration, with mean T max The value is approximately 0.5 to 1 hour.

[0179] The highest AUC and Cmax were observed with macimorelin at 1.0 mg / kg body weight at C3. Furthermore, sensitivity analysis supports dosing with C3, which has the strongest test characteristics, expressed at a cutoff 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 receiver operating characteristic curve (ROC) AUC of 0.933.

[0180] Safety results: Overall, 88 AEs were recorded in 23 patients with SAF: 27 events in 8 patients with C1, 28 events in 8 patients with C2, and 33 events in 7 patients with C3.

[0181] A total of 70 treatment-emergent adverse events (TEAEs) were recorded in 21 patients in SAF: 22 events in 8 patients in C1, 24 events in 6 patients in C2, and 24 events in 7 patients in C3.

[0182] None of the TEAEs were reported to be related to the macimorelin study.

[0183] No SAEs or serious TEAEs were reported during the course of this study. None of the reported AEs or TEAEs led to patient withdrawal.

[0184] The majority of AEs were ITT-related, 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. Note that ITT was administered in 22 patients, arginine in 8 (33.3%), clonidine in 16, and glucagon in only 1 patient.

[0185] AEs reported included infectious disease, one case of anal fissure, and an adverse reaction to the test agent used for sGHST.

[0186] AEs were mostly mild to moderate in intensity. ITT-related AEs included symptoms of hypoglycemia (e.g., tremor, sweating), which is the clinical endpoint of this sGHST. Hypotension-related symptoms were reported in association with clonidine, which are known side effects of this sGHST agent.

[0187] High intensity was reported in TEAEs in one patient in Cohort 1: Five AEs (i.e., palpitations, tachycardia, hunger, hyperhidrosis, and tremor) occurred in patient HU01-01 during the ITT as part of predicted hypoglycemia.

[0188] Laboratory tests, vital signs, physical examination, electrocardiogram: No clinically significant changes were observed in safety clincal laboratory parameters, vital signs, and physical examination. No clinically significant abnormalities or significant changes in electrocardiogram parameters were noted.

[0189] Tolerability Questionnaire: The GHST Tolerability Questionnaire was to be completed by the patient or parent / legal guardian.

[0190] Overall, for the most part, agreement or strong agreement with the predefined statements could be found for macimorelin in all three dose groups.

[0191] In C1, disagreements were noted in one instance regarding "acceptable taste" and in one instance related to "stomach felt fine the next day." In one instance, a strong disagreement was noted regarding "bowel movements the next day."

[0192] Neither strong nor inconsistent findings were noted on any of the macimorelin study-related questionnaires in C2.

[0193] One case of disagreement with "acceptable taste" was described in C3.

[0194] A "bitter taste" was reported by two patients after macimorelin GHST as a handwritten comment. None of these comments were assessed as an AE by the investigator.

[0195] Overall, feedback given by patients or parents / legal guardians following the macimorelin study indicates that the study was well tolerated and safe.

[0196] Conclusion: This study was conducted to investigate the safety, tolerability, PK, and PD of macimorelin acetate after 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 served to identify a suitable macimorelin dose for further testing in a validation trial and to explore a GH cutoff point for testing.

[0197] In all three treatment cohorts, macimorelin plasma levels were max and T max was found to be within the range expected from an adult development program.

[0198] In an observed pediatric population, macimorelin C max A dose-dependent increase in mean AUC0-6 (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. 1 / 2 ranged from 1.25 to 1.75 hours.

[0199] Overall, PK parameters in the pediatric population were in a similar range to those in adults.

[0200] The macimorelin dose of 0.25 mg / kg (C1) did not result in the maximal stimulation of GH secretion in children, as becomes apparent in review of the PK / PD data and comparison of the agreement between the GH cutoff points explored versus the results of PI assessment and sGHST.

[0201] The 0.5 mg / kg (C2) dose demonstrated robust GH release, a high level of agreement between investigator (PI) assessments based on macimorelin GHST versus sGHST, and a receiver operating characteristic curve (ROC) AUC of 0.80. However, the 1.0 mg / kg (C3) dose appeared to provide more consistent and robust GH stimulation in all subjects, likely due to sufficiently high macimorelin exposure.

[0202] Finally, sensitivity analysis supports dosing at C3 with the strongest test characteristics, represented by a cutoff 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 an ROC AUC of 0.93.

[0203] Furthermore, the outcomes of the macimorelin GHST showed a surprisingly high agreement with the outcomes of the two standard GHSTs and with the final diagnoses assessed by the investigators. At C3, GH secretion was clearly stimulated in all eight patients. Finally, the outcomes of the macimorelin GHST administered as a single test showed agreement with the outcomes of the combination of the two sGHSTs in seven of the eight subjects, as well as with the PI assessment.

[0204] In conclusion, in all three dosing cohorts, macimorelin demonstrated good safety and tolerability, with no reported AEs in the observed population. PK and PD profiles were within expected ranges from the adult development program. The overall characterization of macimorelin in this initial pediatric study supports the selection of the 1.0 mg / kg macimorelin dose for investigation in a Phase 3 clinical trial for study validity.

[0205] [Table 7] JPEG0007767387000009.jpg212170

Claims

1. 1. A screening method for diagnosing growth hormone deficiency in pediatric patients by using macimorelin, comprising: (a) providing one to five blood samples taken from the subject within 15 to 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; (b) measuring the growth hormone level in each blood sample provided in step (a); and (c) comparing the measured growth hormone level obtained in step (b) with the single threshold value of 16.0 to 20.0 ng / mL to determine that a subject whose highest growth hormone level in the blood sample obtained in step (b) is lower than a single threshold value has growth hormone deficiency, and to determine that a subject whose highest growth hormone level in the blood sample obtained in step (b) is equal to or greater than the single threshold value does not have growth hormone deficiency; A screening method comprising:

2. 2. The method of claim 1, wherein the single threshold value is within the range of 16.0 to 18.0 ng / mL.

3. The method according to claim 1 or 2, wherein in step (a), 1 to 4 blood samples are provided.

4. The method according to claim 1 or 2, wherein in step (a), one to three blood samples are provided.

5. 5. The method of claim 1, wherein in step (a), 0.8 mg to 1.2 mg of macimorelin is administered per kg of subject body weight.

6. The method of any one of claims 1 to 5, wherein the subject is a human child aged between 2 and under 18 years.

7. The method of any one of claims 1 to 5, wherein the subject is a human child aged between 4 and under 18 years.

8. 8. The method of any one of claims 1 to 7, wherein the method is a stand-alone test, does not need to be repeated, and no alternative growth hormone stimulation test is required to reliably diagnose growth hormone deficiency in pediatric patients.

9. 1. A composition for use in diagnosing growth hormone deficiency in a pediatric patient, comprising the substance macimorelin, wherein in diagnosing growth hormone deficiency: (a) providing one to five blood samples taken from a subject within 15 to 100 minutes after administration of macimorelin in an amount effective to induce growth hormone secretion; (b) measuring the growth hormone level in each blood sample provided in step (a); (c) comparing the measured growth hormone level obtained in step (b) with a single threshold value of 16.0 to 20.0 ng / mL; (d) a subject whose highest growth hormone level in the blood sample obtained in step (b) is lower than the single threshold is determined to have growth hormone deficiency, and a subject whose highest growth hormone level in the blood sample obtained in step (b) is equal to or higher than the single threshold is determined not to have growth hormone deficiency.

10. 10. The composition for use according to claim 9, wherein the single threshold value is within the range of 16.0 to 18.0 ng / mL.

11. The composition for use according to claim 9 or 10, wherein in step (a) 1 to 4 blood samples are provided.

12. The composition for use according to claim 9 or 10, wherein in step (a) 1 to 3 blood samples are provided.

13. The composition for use according to any one of claims 9 to 12, wherein in step (a), 0.8 mg to 1.2 mg of macimorelin is administered per kg of subject body weight.

14. The composition for use according to any one of claims 9 to 13, wherein the subject is a human child aged between 2 and under 18 years.

15. The composition for use according to any one of claims 9 to 13, wherein the subject is a human child aged between 4 and under 18 years.

16. 16. The composition for use according to any one of claims 9 to 15, wherein the substance macimorelin is used in a stand-alone test, which does not have to be repeated and no alternative growth hormone stimulation test is required to reliably diagnose growth hormone deficiency in pediatric patients.

17. 17. The composition for use according to any one of claims 9 to 16, wherein determining whether the subject has or does not have growth hormone deficiency according to step (d) is based solely on growth hormone level induction by a single administration of macimorelin.

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