madman
Super Moderator
1 | INTRODUCTION
Gonadotrophin treatment to induce puberty, spermatogenesis, and fertility in men with congenital or acquired gonadotropin deficiency is based on treatment with human chorionic gonadotrophin (hCG), aplacental heterodimeric glycoprotein hormone and natural, long‐acting analog of pituitary luteinizing hormone (LH).1,2 hCG can conveniently be administered once to three times weekly, whereas LH would require multiple injections daily.3 hCG fulfills an indispensable role in inducing spermatogenesis and fertility in gonadotrophin‐deficient infertile men as well as triggering ovulation in infertile women.4–6 Used clinically for over seven decades, hCG extracted from pregnancy urine (uhCG) had at least nine commercial brands in 1947,7 but recently only a single product (Pregnyl) remained on international markets with some products still available in Europe and India. Recombinant hCG (rhCG, choriogonadotropin alfa) was first approved by the FDA in 2000 and by the EMA in 2001 but only licensed for use in women. The patent‐based marketing monopoly for rhCG, coupled with the sponsor's failure to undertake studies in men, precluded its registration for the treatment of gonadotrophin‐deficient males, including defining appropriate dosing. So, when Pregnyl (uhCG) was withdrawn from the market abruptly in 2021, treatment of gonadotrophin‐deficient infertile men seeking fertility was severely compromised.
Single‐dose clinical pharmacology studies of uhCG and rhCG inanovulatory female infertility have shown comparable efficacy and safety, but at much higher doses than used in men.8 Only a single,non‐randomized study in healthy eugonadal men has compared uhCG with rhCG9; however, the non‐suppressed endogenous serum testosterone makes it difficult to interpret specific hCG effects instimulating serum testosterone. The present study therefore aimed to (a) determine the time course of serum testosterone, dihydrotestosterone (DHT), and estradiol responses to a single, standard, or high-dose of uhCG or rhCG doses in a randomized sequence cross‐overstudy of healthy men, (b) estimate the population pharmacokinetics (serum hCG) and pharmacodynamics (serum testosterone) of uhCG and rhCG during multi‐dose ongoing treatment of gonadotrophin‐deficient men and (c) adapt the single‐use prefilled rhCG (Ovidrel) syringe to the more frequent lower dose uses required for treatment of gonadotrophin‐deficient men.
2 | MATERIALS AND METHODS
2.1 | Single‐dose, randomized cross‐over studies
2.1.1 | Design
Studies 1 and 2 were prospective, randomized sequence, cross‐over study of uhCG and rhCG using a single standard (Study 1) or high‐dose (Study 2) hCG in healthy volunteers to investigate the time course of serum hCG, testosterone, DHT, and estradiol responses to a single‐dose hCG injection (Figure 1).
2.1.3 | Study procedures
Participants were randomized to start with uhCG or rhCG injection before subsequent cross‐over after washout. Randomization was based on a computer‐generated list prepared by someone not involved with the study of hormone administration or blood sampling. The sequence assignment was supplied in opaque envelopes marked with the participant number given sequentially as they were recruited. All injections were administered subcutaneously by the study nurse in the clinic. Treatment was open and unblinded as all pharmacological endpoints were based on objective serum hormone measures undertaken by laboratory scientists unaware of treatment assignment
Study 1 was undertaken in 2009–2010 when irregular availability of uhCG required determining a reasonable alternative using rhCG when uhCG was not available. This was despite the lack of rhCG registration studies and appropriate dosing of rhCG dosage, noting the incommensurate units of gravimetric rhCG dose and bioassay‐based uhCG units without known equivalence. We estimated that the 250‐µg single syringe dose of rhCG was equivalent to 6000 IU uhCG making the standard uhCG dose of 1500 IU equivalent to 62.5 µg rhCG. The standard 1500 IU dose of uhCG increased serum testosterone with a return to baseline by 7 days after injection.10–13 In this study, uhCG and rhCG injections were given a week apart in a random sequence with blood sampling before and on Days 1, 2, 3, 4, and 7 days after hCG injection (Figure 1).
To clarify the time course of serum testosterone after hCG injection, participants had endogenous testosterone suppressed by nandrolone decanoate (ND) injections throughout the 2‐week cross‐over study achieved by four intramuscular ND injections comprising 200 mg on Study days −3 and 7 with 100 mg on study days 1 and 11 (Figure 2) based on prior experience.3,15 Blood samples were taken before the first ND dose (Day −3) with hCG injected subcutaneously under the abdominal skin on Days 0 and 7 with venous blood sampled on Days 0, 1, 2, 3, 4, 7, 8, 9, 10, 11 and 14.
Study 2 was undertaken in 2021 primarily to administer a high hCG dose to obtain urine and serum samples for calibration of hCG assays used in anti‐doping testing. Secondarily, as this is equivalent to a single weekly therapeutic hCG dose, this study also provided blood samples to investigate the time ‐course of serum hCG, testosterone, DHT, and estradiol after a single high dose of hCG (uhCG 5000 IU or rhCG 250 µg). Subcutaneous hCG injections were administered on days 0 and 21 without suppression of endogenous testosterone and blood sampling before and at 1, 2, 3, 4, 7, 9, 11, and 16 days after each injection.
2.2 | Multidose population pharmacology study
Study 3 was an ongoing population pharmacokinetic and pharmacodynamic study conducted from 2010 onwards as an observational study of routine hCG treatment used to stimulate spermatogenesis and fertility of gonadotrophin‐deficient infertile men.16–19 The participants had gonadotrophin deficiency due to either congenital hypogonadotropic hypogonadism,6 usually presenting with failed puberty, or acquired gonadotrophin deficiency due to pituitary tumors and their surgical and/or radiotherapy treatment, all diagnosed by standard clinical criteria.20 hCG treatment was based on subcutaneous injections of 1500 IU uhCG or 62.5 µg (6 clicks) rhCG to stimulate spermatogenesis and induce fertility.16 In some men, if serum testosterone responses were suboptimal, rhCG doses were up titrated with increased to 83.3 (8 clicks) or 125 µg (12 clicks) or to higher uhCG doses (3000 or 5000 IU). Men were usually treated with uhCG unless it was unavailable due to intermittent supply shortages when rhCG was used instead. All blood sampling was conducted in the Andrology Department, CRGH with recording the time since the last hCG injection (in hours) together with anthropometric variables.
2.4 | Study drugs
Lyophilized uhCG (Pregnyl, MSD) purified from the urine of pregnant women was supplied in vials containing 1500 or 5000 IU with a 1 mL diluent. Recombinant hCG (choriogonadotrophin alfa, Ovidrel, Merck‐Serono) is supplied in a prefilled syringe containing 250 μgin 0.5 mL solution for subcutaneous injection. Nandrolone decanoate (ND; Deca‐Durabolin, MSD) is an injectable ester of 19 nor‐testosterone provided in an arachis oil vehicle at a concentration of 50 mg/mL for deep intramuscular injection.
2.5 | Hormone measurements
For Studies 1 and 2, serum testosterone, DHT, and estradiol were measured by liquid chromatography‐mass spectrometry (LCMS) in a single batch at the end of each study using a method free from cross‐reactivity of nandrolone.21,22 These steroid assays have well‐established reproducibility (all CVs <10% for at least three quality control samples per analyte spanning the working range) and lower limits of quantifiability of 25 pg/mL (testosterone), 100 pg/mL (DHT) and 2.5 pg/mL (estradiol).23The reference range for testosterone in this LCMS assay was derived from the Raine birth cohort study of 423 young men.14 In Studies 1 and 2, serum hCG, LH, FSH, and SHBG using Roche reagents by established commercial immunoassays subject to routine external and internal quality control. Additionally, for Study 3, testosterone (Roche) was measured by routine immunoassays because, over the 12 years of that study, steroid LCMS (including testosterone) was not available for routine clinical use.
2.6 | Data analysis
* For evaluating dose responses, doses of rhCG (Ovidrel) and uhCG (Pregnyl) were divided into standard (6 clicks [62.5 µg], 1500 IU), increased (8 clicks [83.3 µg], 3000 IU) and high dose (12 clicks [125 µg], 4500 or 5000 IU), respectively.
3 | RESULTS
3.1| Single dose cross‐over pharmacokinetics and pharmacodynamics
There were no differences in the pharmacokinetic variables for testosterone and hCG (Table 1 and Figure 2) and no significant sequence or cross‐over effects. The ratio (rhCG/uhCG) of AUC for testosterone was 1.27 ± 0.20 (median 1.21, interquartile range [IQR]: 0.94, 1.33;p = .23) and for hCG was 0.81 ± 0.05 (0.89, IQR: 0.77,0.90; p = .007).
Linear mixed model regression analysis indicated that the time course of serum testosterone (p = .69), DHT (p = .42), estradiol (0.38), LH (0.87), and FSH (0.85) were not significantly different for uhCG or rhCG treatment. Similarly, for each analyte, the study sequence or the interaction of hCG type and study sequence was not significant as were all covariate effects of age, BSA, or BMI (all p > .44) nor for hCG pharmacokinetics (Cmax, Tmax).
In study 2 the seven male participants were aged 37 ± 3 years with height 178 ± 2 cm, weight 84.9 ± 5.5 kg, BMI 26.7 ± 1.8 m2and BSA 2.06 ± 0.07 m2. One individual developed COVID after one hCG injection and his samples from that phase of the study were not analyzed. There were no differences in the pharmacokinetic variables for testosterone and hCG (Table 2 and Figure 3). The time course of serum DHT (p = .81) and estradiol (p = .60) did not differ according to hCG type, nor did they differ according to study sequence or their interaction (data not shown). There were no significant effects of age, BSA, or BMI on hCG pharmacokinetics (Cmax, Tmax).
Other than the incidental diagnosis of COVID-19 in one Study 2 participant, there were no emergent clinical or biochemical adverse effects reported by or detected on routine laboratory safety testing, in either Study 1 or 2.
3.2 | Study 3 multidose pharmacokinetics and pharmacodynamics
Serum samples (n = 502) were obtained from 52 gonadotrophin‐deficient men undergoing long‐term therapeutic hCG treatment with a median of 6 (IQR 3, 13) samples per person who received urinary (n = 295) or recombinant (n = 178) hCG. Linear regression of log serum hCG on time since the last hCG injection indicated a highly significant effect of time on serum hCG (pooled slope = −0.25 log (ng/mL)/day, p < 0001) creating an effective half‐life of 5.8 days (Figure 4) but the slope did not differ between types of hCG (main effect hCG type p = .15, interaction of hCG type × time p = .94). The regression of log serum hCG on time was significantly influenced, according to standardized beta coefficients, by BSA (7.3), weight (7.3), BMI (6.4)and height (3.8) but not age.
Linear regression of serum testosterone on time since the last hCG injection showed a mean serum testosterone of 20.6 ± 1.0 nmol/L with no significant slope (−0.08, p = .17) or difference between hCG types in regression on time since injection (main effect hCG type p = .56, interaction of hCG type × time p = .68). Covariates of height (0.17, p = .004), weight (−0.25, p < .0001), BSA (−0.20, p < .0001) or BMI (−0.22, p = .0001), but not age (−0.11, p = .07) had small but significant influence on the regression of serum testosterone on time.
For serum hCG and testosterone concentrations, the effects of standard doses, increased or high doses of rhCG and uhCG, showed reduced serum testosterone on higher hCG doses (Figure S1).
Serum SHBG concentrations were stable overall with a mean of 32.4 ± 1.3 nmol/L but significantly higher on uhCG (n = 296,31.5 ± 1.0 nmol/L vs. n = 180, 22.3 ± 0.7 nmol/L, p < .001) without significant change over time since last injection (p = .58) or to hCG type (p = .17). Covariates displayed negative effects on serum SHBG by BSA (−14.2, p < .001) and BMI (−0.55, p < .0001) with no effect of age (p = .49).
Consistent with the diagnosis of hypogonadotropic hypogonadism, serum LH remained very low (n = 487, 0.76 ± 0.01 IU/L) throughout the study. Similarly, at entry into the study (when participants may have already been on gonadotrophin treatment), serum FSH was low (2.2 ± 0.6 IU/L) and higher overall (as more started FSH treatment) with overall serum FSH (n = 476, 4.8 ± 0.3 IU/L) was higher and marginally correlated with serum testosterone(n = 487, p = .06).
4 | DISCUSSION
The present study is the most comprehensive study of hCG pharmacology in men, including the first controlled studies of single and multiple dosing of rhCG administration. It demonstrates that at the doses tested, uhCG and rhCG are not formally bioequivalent by regulatory pharmacokinetics standards28; however, they demonstrate such similar clinical pharmacodynamic effects on serum testosterone, their primary therapeutic objective, that the rhCG dose of 62.5 mg (6 clicks) can be considered for the first time as suitable starting dose for hCG treatment using rhCG comparable with the standard uhCG dose of 1500 IU.
The single standard dose study in healthy men with suppression of endogenous testosterone allowed a clear depiction of the time course of hCG concentrations as well as the hCG‐stimulated testicular testosterone free from being obscured by ongoing LH‐driven endogenous testosterone. The pharmacodynamic serum testosterone responses, based on Tmax, Cmax, and AUC, were statistically indistinguishable between the two SHCG products. Nevertheless, at the doses used, the ratio of AUCs was significantly lower for rhCG than for uhCG and did not meet conventional pharmacokinetic bioequivalence criteria, which required tighter confidence limits than were obtained.
While the serum testosterone profiles indicate that rhCG appears slightly more effective than uhCG at the doses used, these observational data cannot exclude differences between equal doses due to the lack of direct dose comparability of hCG doses because uhCG dosing is based on bioassay standardization whereas rhCG has a gravimetric dose and there is no reported equivalence between these metrics. The high single-dose study indicates that both hCGs have similar effects on circulating hCG as well as testicular steroid secretion at those higher doses as well noting the inability to estimate separately the unsuppressed residual endogenous testosterone in that study.
The multi‐dosing study analysis was undertaken using an observational population pharmacology approach in the therapeutic target group of gonadotrophin‐deficient men undergoing gonadotrophin (including hCG) stimulation of spermatogenesis. In this real‐world therapeutic analysis, the time course of serum hCG and testosterone did not differ significantly between the hCG products, although an expected log‐linear decline of serum hCG over time was observed. The lower serum testosterone shown by both products at high hCG doses most likely reflects the resistance of testicular testosterone production to hCG rather than desensitization to hCG. This is because these higher hCG doses were only arrived at after individual upward dose titration of hCG in treated men whose serum testosterone was not normalized on standard hCG doses.16 As a result, these two hCG products at these standard doses tested can be considered pharmacologically interchangeable for the treatment of gonadotrophin‐deficient men. This finding overcomes the difficulty that, although uhCG was registered (grandfathered) based on bioassay units for use in stimulating spermatogenesis for gonadotrophin‐deficient men, rhCG was never registered for use in men nor was any bioassay equivalence ever reported. These data also establish for the first time an evidence‐based starting dose for rhCG treatment.
Our analysis of Ovidrel pen fluid delivery demonstrates that the prefilled Ovidrel pens have an overfill loading, supplying 30 rather than 25 clicks, but with highly consistent delivery of at least five, and possibly six, sets of 5 clicks, each click being equivalent to 20 µL or 10 µg of choriogonadotrophin alfa. Thus, either six clicks delivered four times or eight clicks delivered three times, both amounting to 24 clicks, are reliable and within the capacity of the Ovidrel pens. Analysis of the used pens suggests that instruction in pen usage outside the approved single‐use mechanism is mostly effective with a majority using the pens incorrectly.
Limitations of this study include relatively small numbers in the two randomized controlled studies; however, the cross‐over design is 4–10 times more powerful/efficient than a parallel group design.29 Additionally, a cross‐over study is preferable for testing new products against older ones in humans wherein each participant acts in their own control. This reduces otherwise unexplained between‐person variance as well as accommodating testing of the period (sequence) and carry-over effects. The numbers we employed were adequate for findings of statistically significant effects. The multi‐dose study used established population pharmacology methods using real‐world data. Although a randomized controlled study design might have provided more conclusive short‐term findings, that would be at the expense of not reflecting real‐world usage which the population pharmacology method achieves. The study design limitations of the single and multiple dose studies are all traceable to the abuse of patent rights in that the monopoly patent holder for rhCG failed to conduct any clinical studies or register rhCG for use in men despite its extensive, lucrative use in IVF. Other limitations of this study are that structural differences or microheterogeneity and immunoassay cross-reactivity of the two hCG products could not be verified and required cautious interpretations of hCG immunoassay findings. Similarly, it was a limitation that a testosterone immunoassay rather than LCMS was used in the long‐term multi‐dosing study because steroid LCMS was not available for routine clinical practice during that 12‐year period.
We conclude that the urinary and recombinant forms of hCG at the standard and high doses studied produce comparable effects on serum testosterone, DHT, and estradiol although uhCG is associated with modestly higher serum hCG concentrations in regular clinical use. Despite the lack of registration of Ovidrel for men with gonadotrophin deficiency, these studies suggest it can be used with a low‐dose adaptation (62.5 µg, 6 clicks) of the click‐based delivery system of Ovidrel pens as a standard starting dose of rhCG.
Gonadotrophin treatment to induce puberty, spermatogenesis, and fertility in men with congenital or acquired gonadotropin deficiency is based on treatment with human chorionic gonadotrophin (hCG), aplacental heterodimeric glycoprotein hormone and natural, long‐acting analog of pituitary luteinizing hormone (LH).1,2 hCG can conveniently be administered once to three times weekly, whereas LH would require multiple injections daily.3 hCG fulfills an indispensable role in inducing spermatogenesis and fertility in gonadotrophin‐deficient infertile men as well as triggering ovulation in infertile women.4–6 Used clinically for over seven decades, hCG extracted from pregnancy urine (uhCG) had at least nine commercial brands in 1947,7 but recently only a single product (Pregnyl) remained on international markets with some products still available in Europe and India. Recombinant hCG (rhCG, choriogonadotropin alfa) was first approved by the FDA in 2000 and by the EMA in 2001 but only licensed for use in women. The patent‐based marketing monopoly for rhCG, coupled with the sponsor's failure to undertake studies in men, precluded its registration for the treatment of gonadotrophin‐deficient males, including defining appropriate dosing. So, when Pregnyl (uhCG) was withdrawn from the market abruptly in 2021, treatment of gonadotrophin‐deficient infertile men seeking fertility was severely compromised.
Single‐dose clinical pharmacology studies of uhCG and rhCG inanovulatory female infertility have shown comparable efficacy and safety, but at much higher doses than used in men.8 Only a single,non‐randomized study in healthy eugonadal men has compared uhCG with rhCG9; however, the non‐suppressed endogenous serum testosterone makes it difficult to interpret specific hCG effects instimulating serum testosterone. The present study therefore aimed to (a) determine the time course of serum testosterone, dihydrotestosterone (DHT), and estradiol responses to a single, standard, or high-dose of uhCG or rhCG doses in a randomized sequence cross‐overstudy of healthy men, (b) estimate the population pharmacokinetics (serum hCG) and pharmacodynamics (serum testosterone) of uhCG and rhCG during multi‐dose ongoing treatment of gonadotrophin‐deficient men and (c) adapt the single‐use prefilled rhCG (Ovidrel) syringe to the more frequent lower dose uses required for treatment of gonadotrophin‐deficient men.
2 | MATERIALS AND METHODS
2.1 | Single‐dose, randomized cross‐over studies
2.1.1 | Design
Studies 1 and 2 were prospective, randomized sequence, cross‐over study of uhCG and rhCG using a single standard (Study 1) or high‐dose (Study 2) hCG in healthy volunteers to investigate the time course of serum hCG, testosterone, DHT, and estradiol responses to a single‐dose hCG injection (Figure 1).
2.1.3 | Study procedures
Participants were randomized to start with uhCG or rhCG injection before subsequent cross‐over after washout. Randomization was based on a computer‐generated list prepared by someone not involved with the study of hormone administration or blood sampling. The sequence assignment was supplied in opaque envelopes marked with the participant number given sequentially as they were recruited. All injections were administered subcutaneously by the study nurse in the clinic. Treatment was open and unblinded as all pharmacological endpoints were based on objective serum hormone measures undertaken by laboratory scientists unaware of treatment assignment
Study 1 was undertaken in 2009–2010 when irregular availability of uhCG required determining a reasonable alternative using rhCG when uhCG was not available. This was despite the lack of rhCG registration studies and appropriate dosing of rhCG dosage, noting the incommensurate units of gravimetric rhCG dose and bioassay‐based uhCG units without known equivalence. We estimated that the 250‐µg single syringe dose of rhCG was equivalent to 6000 IU uhCG making the standard uhCG dose of 1500 IU equivalent to 62.5 µg rhCG. The standard 1500 IU dose of uhCG increased serum testosterone with a return to baseline by 7 days after injection.10–13 In this study, uhCG and rhCG injections were given a week apart in a random sequence with blood sampling before and on Days 1, 2, 3, 4, and 7 days after hCG injection (Figure 1).
To clarify the time course of serum testosterone after hCG injection, participants had endogenous testosterone suppressed by nandrolone decanoate (ND) injections throughout the 2‐week cross‐over study achieved by four intramuscular ND injections comprising 200 mg on Study days −3 and 7 with 100 mg on study days 1 and 11 (Figure 2) based on prior experience.3,15 Blood samples were taken before the first ND dose (Day −3) with hCG injected subcutaneously under the abdominal skin on Days 0 and 7 with venous blood sampled on Days 0, 1, 2, 3, 4, 7, 8, 9, 10, 11 and 14.
Study 2 was undertaken in 2021 primarily to administer a high hCG dose to obtain urine and serum samples for calibration of hCG assays used in anti‐doping testing. Secondarily, as this is equivalent to a single weekly therapeutic hCG dose, this study also provided blood samples to investigate the time ‐course of serum hCG, testosterone, DHT, and estradiol after a single high dose of hCG (uhCG 5000 IU or rhCG 250 µg). Subcutaneous hCG injections were administered on days 0 and 21 without suppression of endogenous testosterone and blood sampling before and at 1, 2, 3, 4, 7, 9, 11, and 16 days after each injection.
2.2 | Multidose population pharmacology study
Study 3 was an ongoing population pharmacokinetic and pharmacodynamic study conducted from 2010 onwards as an observational study of routine hCG treatment used to stimulate spermatogenesis and fertility of gonadotrophin‐deficient infertile men.16–19 The participants had gonadotrophin deficiency due to either congenital hypogonadotropic hypogonadism,6 usually presenting with failed puberty, or acquired gonadotrophin deficiency due to pituitary tumors and their surgical and/or radiotherapy treatment, all diagnosed by standard clinical criteria.20 hCG treatment was based on subcutaneous injections of 1500 IU uhCG or 62.5 µg (6 clicks) rhCG to stimulate spermatogenesis and induce fertility.16 In some men, if serum testosterone responses were suboptimal, rhCG doses were up titrated with increased to 83.3 (8 clicks) or 125 µg (12 clicks) or to higher uhCG doses (3000 or 5000 IU). Men were usually treated with uhCG unless it was unavailable due to intermittent supply shortages when rhCG was used instead. All blood sampling was conducted in the Andrology Department, CRGH with recording the time since the last hCG injection (in hours) together with anthropometric variables.
2.4 | Study drugs
Lyophilized uhCG (Pregnyl, MSD) purified from the urine of pregnant women was supplied in vials containing 1500 or 5000 IU with a 1 mL diluent. Recombinant hCG (choriogonadotrophin alfa, Ovidrel, Merck‐Serono) is supplied in a prefilled syringe containing 250 μgin 0.5 mL solution for subcutaneous injection. Nandrolone decanoate (ND; Deca‐Durabolin, MSD) is an injectable ester of 19 nor‐testosterone provided in an arachis oil vehicle at a concentration of 50 mg/mL for deep intramuscular injection.
2.5 | Hormone measurements
For Studies 1 and 2, serum testosterone, DHT, and estradiol were measured by liquid chromatography‐mass spectrometry (LCMS) in a single batch at the end of each study using a method free from cross‐reactivity of nandrolone.21,22 These steroid assays have well‐established reproducibility (all CVs <10% for at least three quality control samples per analyte spanning the working range) and lower limits of quantifiability of 25 pg/mL (testosterone), 100 pg/mL (DHT) and 2.5 pg/mL (estradiol).23The reference range for testosterone in this LCMS assay was derived from the Raine birth cohort study of 423 young men.14 In Studies 1 and 2, serum hCG, LH, FSH, and SHBG using Roche reagents by established commercial immunoassays subject to routine external and internal quality control. Additionally, for Study 3, testosterone (Roche) was measured by routine immunoassays because, over the 12 years of that study, steroid LCMS (including testosterone) was not available for routine clinical use.
2.6 | Data analysis
* For evaluating dose responses, doses of rhCG (Ovidrel) and uhCG (Pregnyl) were divided into standard (6 clicks [62.5 µg], 1500 IU), increased (8 clicks [83.3 µg], 3000 IU) and high dose (12 clicks [125 µg], 4500 or 5000 IU), respectively.
3 | RESULTS
3.1| Single dose cross‐over pharmacokinetics and pharmacodynamics
There were no differences in the pharmacokinetic variables for testosterone and hCG (Table 1 and Figure 2) and no significant sequence or cross‐over effects. The ratio (rhCG/uhCG) of AUC for testosterone was 1.27 ± 0.20 (median 1.21, interquartile range [IQR]: 0.94, 1.33;p = .23) and for hCG was 0.81 ± 0.05 (0.89, IQR: 0.77,0.90; p = .007).
Linear mixed model regression analysis indicated that the time course of serum testosterone (p = .69), DHT (p = .42), estradiol (0.38), LH (0.87), and FSH (0.85) were not significantly different for uhCG or rhCG treatment. Similarly, for each analyte, the study sequence or the interaction of hCG type and study sequence was not significant as were all covariate effects of age, BSA, or BMI (all p > .44) nor for hCG pharmacokinetics (Cmax, Tmax).
In study 2 the seven male participants were aged 37 ± 3 years with height 178 ± 2 cm, weight 84.9 ± 5.5 kg, BMI 26.7 ± 1.8 m2and BSA 2.06 ± 0.07 m2. One individual developed COVID after one hCG injection and his samples from that phase of the study were not analyzed. There were no differences in the pharmacokinetic variables for testosterone and hCG (Table 2 and Figure 3). The time course of serum DHT (p = .81) and estradiol (p = .60) did not differ according to hCG type, nor did they differ according to study sequence or their interaction (data not shown). There were no significant effects of age, BSA, or BMI on hCG pharmacokinetics (Cmax, Tmax).
Other than the incidental diagnosis of COVID-19 in one Study 2 participant, there were no emergent clinical or biochemical adverse effects reported by or detected on routine laboratory safety testing, in either Study 1 or 2.
3.2 | Study 3 multidose pharmacokinetics and pharmacodynamics
Serum samples (n = 502) were obtained from 52 gonadotrophin‐deficient men undergoing long‐term therapeutic hCG treatment with a median of 6 (IQR 3, 13) samples per person who received urinary (n = 295) or recombinant (n = 178) hCG. Linear regression of log serum hCG on time since the last hCG injection indicated a highly significant effect of time on serum hCG (pooled slope = −0.25 log (ng/mL)/day, p < 0001) creating an effective half‐life of 5.8 days (Figure 4) but the slope did not differ between types of hCG (main effect hCG type p = .15, interaction of hCG type × time p = .94). The regression of log serum hCG on time was significantly influenced, according to standardized beta coefficients, by BSA (7.3), weight (7.3), BMI (6.4)and height (3.8) but not age.
Linear regression of serum testosterone on time since the last hCG injection showed a mean serum testosterone of 20.6 ± 1.0 nmol/L with no significant slope (−0.08, p = .17) or difference between hCG types in regression on time since injection (main effect hCG type p = .56, interaction of hCG type × time p = .68). Covariates of height (0.17, p = .004), weight (−0.25, p < .0001), BSA (−0.20, p < .0001) or BMI (−0.22, p = .0001), but not age (−0.11, p = .07) had small but significant influence on the regression of serum testosterone on time.
For serum hCG and testosterone concentrations, the effects of standard doses, increased or high doses of rhCG and uhCG, showed reduced serum testosterone on higher hCG doses (Figure S1).
Serum SHBG concentrations were stable overall with a mean of 32.4 ± 1.3 nmol/L but significantly higher on uhCG (n = 296,31.5 ± 1.0 nmol/L vs. n = 180, 22.3 ± 0.7 nmol/L, p < .001) without significant change over time since last injection (p = .58) or to hCG type (p = .17). Covariates displayed negative effects on serum SHBG by BSA (−14.2, p < .001) and BMI (−0.55, p < .0001) with no effect of age (p = .49).
Consistent with the diagnosis of hypogonadotropic hypogonadism, serum LH remained very low (n = 487, 0.76 ± 0.01 IU/L) throughout the study. Similarly, at entry into the study (when participants may have already been on gonadotrophin treatment), serum FSH was low (2.2 ± 0.6 IU/L) and higher overall (as more started FSH treatment) with overall serum FSH (n = 476, 4.8 ± 0.3 IU/L) was higher and marginally correlated with serum testosterone(n = 487, p = .06).
4 | DISCUSSION
The present study is the most comprehensive study of hCG pharmacology in men, including the first controlled studies of single and multiple dosing of rhCG administration. It demonstrates that at the doses tested, uhCG and rhCG are not formally bioequivalent by regulatory pharmacokinetics standards28; however, they demonstrate such similar clinical pharmacodynamic effects on serum testosterone, their primary therapeutic objective, that the rhCG dose of 62.5 mg (6 clicks) can be considered for the first time as suitable starting dose for hCG treatment using rhCG comparable with the standard uhCG dose of 1500 IU.
The single standard dose study in healthy men with suppression of endogenous testosterone allowed a clear depiction of the time course of hCG concentrations as well as the hCG‐stimulated testicular testosterone free from being obscured by ongoing LH‐driven endogenous testosterone. The pharmacodynamic serum testosterone responses, based on Tmax, Cmax, and AUC, were statistically indistinguishable between the two SHCG products. Nevertheless, at the doses used, the ratio of AUCs was significantly lower for rhCG than for uhCG and did not meet conventional pharmacokinetic bioequivalence criteria, which required tighter confidence limits than were obtained.
While the serum testosterone profiles indicate that rhCG appears slightly more effective than uhCG at the doses used, these observational data cannot exclude differences between equal doses due to the lack of direct dose comparability of hCG doses because uhCG dosing is based on bioassay standardization whereas rhCG has a gravimetric dose and there is no reported equivalence between these metrics. The high single-dose study indicates that both hCGs have similar effects on circulating hCG as well as testicular steroid secretion at those higher doses as well noting the inability to estimate separately the unsuppressed residual endogenous testosterone in that study.
The multi‐dosing study analysis was undertaken using an observational population pharmacology approach in the therapeutic target group of gonadotrophin‐deficient men undergoing gonadotrophin (including hCG) stimulation of spermatogenesis. In this real‐world therapeutic analysis, the time course of serum hCG and testosterone did not differ significantly between the hCG products, although an expected log‐linear decline of serum hCG over time was observed. The lower serum testosterone shown by both products at high hCG doses most likely reflects the resistance of testicular testosterone production to hCG rather than desensitization to hCG. This is because these higher hCG doses were only arrived at after individual upward dose titration of hCG in treated men whose serum testosterone was not normalized on standard hCG doses.16 As a result, these two hCG products at these standard doses tested can be considered pharmacologically interchangeable for the treatment of gonadotrophin‐deficient men. This finding overcomes the difficulty that, although uhCG was registered (grandfathered) based on bioassay units for use in stimulating spermatogenesis for gonadotrophin‐deficient men, rhCG was never registered for use in men nor was any bioassay equivalence ever reported. These data also establish for the first time an evidence‐based starting dose for rhCG treatment.
Our analysis of Ovidrel pen fluid delivery demonstrates that the prefilled Ovidrel pens have an overfill loading, supplying 30 rather than 25 clicks, but with highly consistent delivery of at least five, and possibly six, sets of 5 clicks, each click being equivalent to 20 µL or 10 µg of choriogonadotrophin alfa. Thus, either six clicks delivered four times or eight clicks delivered three times, both amounting to 24 clicks, are reliable and within the capacity of the Ovidrel pens. Analysis of the used pens suggests that instruction in pen usage outside the approved single‐use mechanism is mostly effective with a majority using the pens incorrectly.
Limitations of this study include relatively small numbers in the two randomized controlled studies; however, the cross‐over design is 4–10 times more powerful/efficient than a parallel group design.29 Additionally, a cross‐over study is preferable for testing new products against older ones in humans wherein each participant acts in their own control. This reduces otherwise unexplained between‐person variance as well as accommodating testing of the period (sequence) and carry-over effects. The numbers we employed were adequate for findings of statistically significant effects. The multi‐dose study used established population pharmacology methods using real‐world data. Although a randomized controlled study design might have provided more conclusive short‐term findings, that would be at the expense of not reflecting real‐world usage which the population pharmacology method achieves. The study design limitations of the single and multiple dose studies are all traceable to the abuse of patent rights in that the monopoly patent holder for rhCG failed to conduct any clinical studies or register rhCG for use in men despite its extensive, lucrative use in IVF. Other limitations of this study are that structural differences or microheterogeneity and immunoassay cross-reactivity of the two hCG products could not be verified and required cautious interpretations of hCG immunoassay findings. Similarly, it was a limitation that a testosterone immunoassay rather than LCMS was used in the long‐term multi‐dosing study because steroid LCMS was not available for routine clinical practice during that 12‐year period.
We conclude that the urinary and recombinant forms of hCG at the standard and high doses studied produce comparable effects on serum testosterone, DHT, and estradiol although uhCG is associated with modestly higher serum hCG concentrations in regular clinical use. Despite the lack of registration of Ovidrel for men with gonadotrophin deficiency, these studies suggest it can be used with a low‐dose adaptation (62.5 µg, 6 clicks) of the click‐based delivery system of Ovidrel pens as a standard starting dose of rhCG.
