Introduction: Studies of gonadotropin-releasing hormone analogues (intramuscular [IM] leuprolide acetate [LA] and triptorelin) for treatment monitoring of central precocious puberty (CPP) demonstrate this approach is effective for confirming pubertal hormone suppression. Herein, we provide new data using subcutaneous LA (SC LA), suggesting similar efficacy for treatment monitoring. Methods: PubMed, Embase, and CINAHL were searched for studies of GnRHa used to monitor treatment of CPP. The titles and the abstracts were reviewed; 5 studies were selected. Additionally, new unpublished data for SC LA from the original phase 3 trial (primary data published by Klein et al.) were evaluated. Serum luteinizing hormone (LH) and leuprolide levels at screening, 1, 4, and 6 h after the first dose SC LA were analyzed and plotted. Results: Data from 162 children (155 girls) were evaluated. SC and IM LA produced overlapping median LH concentration curves and peak LH concentrations after the first dose. For IM LA, subsequent doses yielded suppressed peak LH levels (2.7 IU/L [mean]). For SC LA, subsequent doses also resulted in significant suppressed peak LH levels (0.2 ± 0.02 IU/L) and achieved sex-steroid hormone suppression of >98%. Conclusions: Compared to IM LA and triptorelin, long-acting SC LA shows similar burst kinetics and rapid LH rise after the first dose, followed by similar suppression of LH and sex steroids after subsequent doses. Since IM LA and triptorelin have demonstrated usefulness that is comparable to that of traditional GnRH stimulation testing for monitoring CPP, we presume that SC LA may be similarly employed.

The gonadotropin-releasing hormone analogue (GnRHa) stimulation test has historically been used for diagnosing and monitoring treatment efficacy in children with central precocious puberty (CPP). However, this test is not always practical due to costs related to lengthy hospital or clinical testing, and additional injections that may negatively impact the child’s therapeutic experience. Therefore, alternative methods of assessing therapeutic efficacy would be valuable. This manuscript presents an overview of the studies supporting the suitability of therapeutic dosing of CPP medications (intramuscular [IM] leuprolide acetate [LA], IM triptorelin pamoate, and subcutaneous [SC] LA) for treatment monitoring. Four studies of injectable CPP medications have demonstrated that these therapies achieved similar stimulated luteinizing hormone (LH) levels compared to a traditional GnRHa stimulation test. However, the effect of SC LA (approved by the US FDA for treatment in May 2020) on LH levels has not been directly compared to those measured as part of a traditional stimulation test. As several studies have shown that SC LA has similar efficacy on the physical characteristics of puberty as IM LA, we postulate that therapeutic SC LA may also be used to monitor the response to CPP treatment, in place of a traditional GnRHa stimulation test.

The gonadotropin-releasing hormone (GnRH) stimulation test has been the historic gold standard for diagnosing central precocious puberty (CPP) [1, 2]. The procedure for this test consisted of a subcutaneous (SC) or intravenous injection of native GnRH followed by measurement of stimulated luteinizing hormone (LH), follicle-stimulating hormone (FSH), and sex-steroid concentrations [3]. However, the lack of availability of native GnRH (Factrel®; gonadorelin) in the USA [2, 4] led to the use of GnRH analogues (GnRHa) for stimulation-testing, generally performed with daily SC leuprolide acetate (LA). This entails a SC injection of GnRHa, followed by measurement of stimulated LH and FSH levels 1–2 h post-injection and sex-hormone levels at similar times and 18–24 h later. Although also used for monitoring therapeutic efficacy, GnRHa stimulation tests are not always practical; the tests are expensive and may not be covered by insurance [2, 5], and the additional injections may negatively impact the child’s therapeutic experience. As CPP treatment frequently continues for several years, the extra time spent in the clinic or in hospital infusion centers with a formal 2–3 h GnRHa stimulation test (followed by an additional venipuncture 18–24 h later) may be bothersome for children and their families. Due to these limitations of the GnRHa stimulation test, other monitoring options, such as random measurement of ultrasensitive LH and/or sex-steroid concentrations or hormone levels following the therapeutic injections themselves, are often used instead. Some clinicians may rely solely on clinical indices of hypothalamic-pituitary-gonadal axis suppression without routinely conducting biochemical monitoring to confirm treatment efficacy.

As therapies for CPP may also function as stimulation tests for treatment monitoring post-injection, such an approach could eliminate the need for separate GnRHa stimulation tests and the aforementioned associated issues in many patients. A number of studies have assessed the suitability of intramuscular (IM) LA and triptorelin pamoate (TP) for treatment monitoring [6‒9]. The initial study assessing the pattern of gonadotropin and estradiol (E2) secretion in girls with exaggerated thelarche used 20-μg/kg SC LA as a stimulation test as opposed to native GnRH, with the rationale that this GnRHa “induces follicle-stimulating hormone and LH release, which in turn stimulates secretion of ovarian E2 in girls with an activated hypothalamic-pituitary-ovarian axis.” [10] It is postulated that the agonist has a greater effect on LH and FSH protein synthesis, and not merely on their release. Moreover, in the recent phase 3 study of 45-mg 6-month IM LA, Klein et al. [11] noted that, in 2 treatment-naïve girls who missed their prescribed GnRHa stimulation test in week 24 due to COVID-19, LH concentrations were <4 IU/L in 30 min and 1 h after the second study drug injection at week 24 [11]. Their standard stimulation tests at weeks 12, 20, 44, and 48 confirmed suppression. Thus, long-acting (6-month) GnRHa’s appear to release sufficient amounts of “free” agonist to allow for a quasi-GnRHa-stimulation test during therapeutic dosing [6‒11].

This manuscript presents an overview of the relevant literature and data supporting the suitability of utilizing therapeutic GnRHa’s for treatment monitoring, with a focus on 45-mg 6-month SC LA [12], and how these results demonstrate that this approach is an effective method for monitoring pubertal suppression (Table 1). Comparisons across studies should be taken with caution due to differences in the times that laboratory values were measured in relation to the GnRHa injection, variability in the laboratory methods used among studies, and other possible methodologic differences.

All new unpublished data for SC LA are from the same phase 3 trial in which primary data were published by Klein et al. [12] GnRHa stimulation tests for diagnosis were performed at screening, and repeated for treatment monitoring at 12, 24, 36, and 48 weeks to assess gonadotropin and sex-steroid suppression during treatment [12]. Blood samples for random LH, FSH, and E2 or T were also collected at 4, 20, and 44 weeks [12]. To evaluate the pharmacokinetics (PK) of the study drug, blood samples for analysis of leuprolide concentrations were collected at screening, 1, 4, and 6 h after the first dose of 45-mg 6-month SC LA, and also at 4, 12, 20, 24, 36, 44, and 48 weeks [12]. The cutoff for adequate LH suppression to prepubertal levels was defined as LH <4 IU/L [13‒15]. Mean serum LH and mean serum leuprolide levels at screening, 1, 4, and 6 h after the first dose of the study drug were analyzed and plotted.

PubMed, Embase, and CINAHL were searched. Reference lists of relevant studies were examined manually to identify additional relevant studies. The following eligibility criteria guided the literature search and selection: English language, relevant literature, and data supporting the suitability of utilizing therapeutic GnRHa’s for treatment monitoring with a focus on 45-mg 6-month SC LA [12]. The titles and the abstracts were reviewed; 5 studies were selected (Table 1).

Evidence that GnRHa Injections Can Be Used as Stimulation Tests

Evidence from First-Dose LH Levels

Studies of 7.5-mg and 3.75-mg IM LA (Bhatia and Brito, respectively) [6, 7] and 7.5-mg and 60-μg/kg IM TP (Strich and Salerno, respectively) [8, 9] have shown that peak LH levels achieved following the first therapeutic dose treating CPP are at least as high as those seen after a stimulation test using native GnRH (28 vs. 22–27 IU/L [median], 23 vs. 18 IU/L [mean], 19 vs. 15 IU/L [mean], and 70 vs. 31 IU/L [mean] for Bhatia, Brito, Strich, and Salerno, respectively) [6‒9].

The first dose of 45-mg 6-month SC LA induced mean peak LH levels (shown in Fig. 1a) higher than those seen during a traditional GnRHa stimulation test at screening (43.4 [previously unpublished data] vs. 23.5 IU/L [12]), and higher than those seen in the reports of Bhatia (27.5 IU/L [median]) [6], Brito (22.6 IU/L [mean]) [7], and Strich (19.1 IU/L [mean]) [8].

Evidence from Subsequent-Dose LH Suppression

Three studies have compared peak LH levels after subsequent doses of GnRHa to those after a traditional stimulation test. Two studies of 7.5-mg and 3.75-mg IM LA demonstrated that peak LH levels were suppressed to similar levels as seen in a traditional GnRH stimulation test (0.8 vs. 0.5–0.6 IU/L [median] and 2.7 vs. 1.4 IU/L [mean] for Bhatia and Brito, respectively) [6, 7]. Use of 60-μg/kg IM TP resulted in similarly suppressed peak LH levels (Salerno, 2.0 IU/L [mean]) compared to a GnRH stimulation test (2.1 IU/L) [9].

In addition to these 3 studies, 1 additional study reported peak LH levels after subsequent doses of a GnRHa. Strich (7.5-mg IM TP) found a mean peak LH concentration of 1.1 IU/L (range 0.2–2.1 IU/L) in suppressed children and suggested that the maximum peak LH response reflecting suppression should be 2.1 IU/L [8]. Strich also noted that this suggested cut-off is slightly lower than that proposed by Bhatia (3 IU/L) [6, 8]. Although Strich did not compare LH levels after subsequent doses of GnRHa to LH concentrations after a traditional stimulation test, there were similar peak LH levels in response to 7.5-mg IM LA on subsequent dosing (Strich, 1.1 IU/L [mean] vs. Bhatia, 0.8 IU/L [median]) [6, 8], which are also lower than those previously reported for 60-μg/kg IM TP (Salerno, 2.0 IU/L [mean) [9].

Two of these studies (Strich and Salerno) also reported that LH concentrations after 7.5-mg and 60-μg/kg IM TP injections were discriminative between children who were and were not adequately suppressed clinically [8, 9]. In both studies, clinicians were able to subsequently achieve suppression by increasing the TP dose or injection frequency [8, 9]. Salerno also reported clinical parameters, which were consistent with laboratory measurements indicating lack of suppression [9].

Bhatia (IM LA) and Salerno (IM TP) Demonstrate Similar First-Dose LH Stimulation and Subsequent-Dose LH Suppression to a GnRH Stimulation Test

Bhatia and Salerno suggest 7.5-mg IM LA and 60-μg/kg IM TP, respectively, can be used for stimulation testing because they both induce peak LH levels similar to those seen during traditional stimulation tests after the first and subsequent (if the patient is suppressed) doses [6, 9].

Brito (IM LA), Strich (IM TP), and Klein (SC LA) Demonstrate Similar Peak LH Levels as Bhatia (IM LA) and Salerno (IM TP)

Brito (3.75-mg IM LA) showed similar peak LH levels (22.6 IU/L [mean]) in response to the first dose as Bhatia (7.5-mg IM LA, 27.5 IU/L [median]) and similarly suppressed peak LH levels (2.7 IU/L [mean]) after subsequent doses as both Bhatia (IM LA, 0.8 IU/L [median]) and Salerno (60-μg/kg IM TP, 2.0 IU/L [mean]) [6, 7, 9]. Strich (7.5-mg IM TP) showed similar peak LH levels after the first dose (21.6 IU/L [mean]) to Bhatia (27.5 IU/L [median]) and lower peak LH levels after subsequent doses (1.1 IU/L [mean]) than Salerno (2.0 IU/L [mean]) [6, 8, 9].

Use of 45-mg 6-month SC LA showed a rapid increase in mean peak LH levels, 27.6 IU/L at 1 h and 43.4 IU/L at 4 h after the first dose (shown in Fig. 1a) (previously unpublished data), and significant suppression of peak LH levels to 0.2 ± 0.02 IU/L after subsequent doses (shown in Fig. 1b) (data previously published but reformatted here for clarity [12]). Therefore, data from SC LA are similar to both IM LA and IM TP, which implies utility for stimulation testing.

SC LA Demonstrates Similar PK to IM LA

SC LA has similar PK compared to IM LA. In a head-to-head study in adult men, mean serum leuprolide concentrations showed rapid increases in the burst phase followed by continuous decreases in both groups [16]. A phase 3 study evaluating the efficacy, PK, and safety of 45-mg 6-month SC LA in children with CPP (60 girls, 2 boys) showed an initial burst release of leuprolide within 1–6 h [12]. Subsequently, serum leuprolide concentrations decreased and remained stable during the plateau phase [12].

SC LA Demonstrates a Similar LH Burst to IM LA

As expected, the similar burst kinetics of leuprolide between SC and IM LA results in overlapping median LH concentration curves and median peak LH concentrations after the first dose [16].

SC LA Demonstrates Subsequent Dose Suppression of Sex Steroids

In the phase 3 study of 45-mg 6-month SC LA in children with CPP, most girls achieved prepubertal levels of E2 (attaining the target of <20 pg/mL) after the second dose (98% at week 36 and 48) [12]. Both boys achieved prepubertal testosterone concentrations (<28.4 ng/dL) at week 36 [12].

Healthcare providers may consider utilizing various injectable long-acting CPP therapies as substitutes for GnRHa stimulation tests for treatment monitoring, along with sequential assessments of clinical pubertal status, growth velocity, and bone age. Additionally, using a long-acting CPP therapy to both replace the GnRHa stimulation test and simultaneously treat CPP would eliminate the extra injections necessary for GnRHa stimulation tests while incurring no additional costs for caregivers and/or the healthcare system. Our proposed approach would be simple to implement as it would entail removing the stimulation tests for treatment monitoring and would not require offices to acquire any additional supplies or provide extra training. As serum LH levels increase 1–6 h after a dose of SC LA (and other GnRH agonists), LH samples taken in this time window would be considered stimulated. While some clinicians may not perform routine biochemical monitoring to assess treatment efficacy if clinical indices of hypothalamic-pituitary-gonadal axis suppression are not indicative of the need, having the option of a long-acting CPP therapy to both treat and monitor CPP allows for a convenient biochemical evaluation that would provide a more complete picture of a child’s treatment response.

While addressing discordances between clinical and laboratory parameters may be occasionally challenging, study data indicate that using CPP therapy to both replace the GnRHa stimulation test and treat CPP allows clinicians to identify and manage children with otherwise equivocal laboratory control. There is evidence that peak LH concentrations after therapeutic GnRHa injections were sufficient to identify children who did not achieve LH suppression [8, 9]. Clinicians responded quickly by increasing the dose or injection frequency, thus demonstrating 1 strategy for managing children with inadequate pubertal hormonal suppression [8, 9]. Importantly, LH levels of <4 IU/L were achieved by ≥85% of children who were administered 45-mg 6-month SC LA, and children who did not achieve LH levels of <4 IU/L were well suppressed by clinical measures [12].

One limitation of our conclusions is that the PK/PD data presented from the head-to-head comparison of SC and IM LA were not from children [16]. However, as the observed PK/PD effects after the first injection of 45-mg 6-month SC LA in children with CPP were similar to those in the head-to-head study [12], the data may allow for the interpretation that we have put forward. Additionally, as most patients with CPP are females, no newer data on boys have been available for analysis.

Ultimately, using long-acting CPP therapies as substitutes for GnRHa stimulation tests could improve the child’s treatment experience, which is particularly important as CPP treatment may continue for several years. There are two scenarios that would be encountered when using long-acting CPP therapies as GnRHa stimulation tests. The first, and most likely, is that post-dose LH and/or sex-steroid levels are well-suppressed. In this case, using the CPP therapy to monitor treatment is extremely valuable as it eliminated the traditional GnRHa stimulation test, confirmed suppression, and no alternative action is required. The second scenario is that post-dose LH and/or sex-steroid levels indicate lack of suppression. If this occurs, this is also extremely valuable information as the clinician would be quickly alerted as to the need for consideration of a change in therapy, potentially increasing the dose or injection frequency to achieve suppression.

Compared to IM LA, SC LA yields a similar burst release of leuprolide and induces a similar rapid rise in stimulated LH levels in response to the first dose [12, 16], followed by sex-steroid suppression after subsequent doses [12]. Given that IM LA has demonstrated usefulness as a stimulation test to monitor CPP, 45-mg 6-month SC LA may be similarly used, as inferred from the studies of other GnRHa’s as well as from preliminary data of peak LH levels after the first therapeutic injection. The assessment of long-acting GnRHa-induced suppression is an important aspect of care in children with CPP. For assessment or confirmation of adequate biochemical pubertal hormone suppression in those already on GnRHa treatment, using a therapeutic 45-mg 6-month dose of SC LA as a means to monitor treatment efficacy may provide greater flexibility, utility, and interpretability compared to random ultrasensitive LH levels, random sex-hormone measurements, and/or traditional GnRHa stimulation tests. It may also prove more cost-effective and easier than GnRHa testing for the patient and family.

Editorial support was provided by Xelay Acumen Group, Inc. All the authors have authorized the submission of their manuscript via Xelay Acumen Group, Inc. and have approved all statements and declarations, including conflicting interests and funding.

All study documents were approved by an Institutional Review Board or Independent Ethics Committee for each site prior to initiation of the study. All aspects of the study were conducted in accordance with International Council on Harmonisation Good Clinical Practice principles. Caregivers gave written informed consent and children gave assent, when applicable. The central study protocol was reviewed and approved by the New England Independent Review Board, IRB #15-164. The study protocol was reviewed and approved by ethics committees at each of the participating sites [12].

Dr. Silverman is a consultant for ENDO Pharma, Tolmar, Inc., Enteris BioPharma, OPKO Biologics, Pfizer, Novo Nordisk, Ascendis, and Myovant Sciences, and serves on Speakers Bureaus for AbbVie and Pfizer. Dr. Geffner receives research support from Ascendis, Diurnal, Neurocrine Biosciences, Novo Nordisk, Pfizer, and Spruce Biosciences; serves on advisory boards or as a consultant for Adrenas Therapeutics, Ascendis, Eton Pharmaceuticals, Neurocrine Biosciences, Novo Nordisk, Pfizer, and Spruce Biosciences; serves as an adjudication committee member for ICON Clinical Research, LLC/Aeterna Zentaris; and receives royalties from McGraw-Hill and UpToDate. Dr. Benson is a consultant for Arbor Pharmaceuticals; receives salary and stock options as a board member of Kihealth; and receives research support from Beta Bionics, Diurnal, Novo Nordisk, and Sanofi/Provention Bio.

The study was funded by Tolmar, Inc.

Drs. Silverman, Geffner, and Benson had access to the full data and approved the paper in its current form, and contributed to the study analysis and reporting. Drs. Silverman and Geffner were on the Tolmar Data and Safety Monitoring Board. Dr. Benson was an investigator for the phase 3 trial of 45-mg 6-month SC LA.

The data that support the findings of this study are available from the corresponding author upon reasonable request. Restrictions apply to the availability of data generated or analyzed during this study to preserve patient confidentiality or because they were used under license. The corresponding author L.S. [Lawrence.silverman@atlantichealth.org] will on request detail the restrictions and any conditions under which access to some data may be provided.

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