Developments in the Management of Growth Hormone Deficiency: Clinical Utility of Somapacitan.

Growth hormone (GH) replacement therapy for growth hormone deficiency (GHD) in children and adults has for over 25 years, until recently, been administered as daily injections. This daily treatment regimen often incurs a burden to patients and caregivers, leading to high rates of non-adherence and, consequently, decreased treatment efficacy outcomes. To address this shortcoming, long-acting growth hormones (LAGHs) have been developed with the aim of reducing the burden of daily injections, thereby potentially improving treatment adherence and outcomes. Somapacitan (Sogroya®) (Novo Nordisk, Bagsværd, Denmark) is a LAGH currently approved for the treatment of adult and childhood GHD (AGHD and CGHD, respectively) in several countries. Other LAGHs, such as somatrogon (Ngenla®) (Pfizer, New York, United States) and lonapegsomatropin/TransCon GH (Skytrofa®) (Ascendis Pharma, Copenhagen, Denmark), are also currently approved and available for the treatment of CGHD in several countries. In this review, we will consider the method of protraction, pharmacokinetics (PK) and pharmacodynamics (PD), efficacy, and safety results of somapacitan in adult and pediatric trials and how these characteristics differ from those of the other aforementioned LAGHs. Additionally, the administration of somapacitan and timing of measurement of serum insulin-like growth factor-I (IGF-I) levels are summarized. Information on administration, advice on missed doses, and clinical guidelines are discussed, as well as identifying which patients are suitable for somapacitan therapy, and how to monitor and adjust dosing whilst on therapy.

Analysis of N15-rat growth hormone after incubation with rat subcutaneous tissue and immune cells using ultra-pressure chromatography-mass spectrometry.

Therapeutic proteins (TPs) are exposed to various immune cells like macrophages and neutrophils, especially after subcutaneous (SC) administration. It is well known that the immune cells can generate reactive oxygen species (ROS) and this may lead to oxidation of TPs. The oxidation can occur in the SC tissue after SC administration, during distribution to the immune organs like lymph nodes and spleen, and even in the blood circulation. The oxidation can lead to alteration of their pharmacokinetics and efficacy. Therefore, it is important to study the oxidation of TPs in the biological matrices using ultra-pressure chromatography-mass spectrometry. Rat growth hormone (rGH) was selected as a test protein due to its similarity with human growth hormone (hGH), which is widely used for treatment of growth hormone deficiency. In this manuscript, we have summarized sample processing strategy and ultra-pressure chromatography-mass spectrometry methodology to identify rGH and its degradation products after ex-vivo incubation with rat SC tissue, and in vitro incubation with rat splenocytes and canine peripheral blood mononuclear cells (cPBMCs) as a model foreign host species. We did not observe oxidation of rGH in these biological matrices. This could be due to very minor yields of oxidation products, lack of sensitivity of the mass spectrometry method, loss of protein during sample processing, rapid turnover of oxidized protein or a combination of all factors.

Short-Term Effects of Growth Hormone on Lipolysis, Glucose and Amino Acid Metabolism Assessed in Serum and Microdialysate of Healthy Young Men.

OBJECTIVE: We investigated direct effects of a therapeutic growth hormone dose on lipolysis, glucose and amino acid metabolism. METHODS: This crossover microdialysis trial involved six healthy male volunteers receiving single subcutaneous injections of both growth hormone (0.035 mg/kg) and placebo (0.9% sodium chloride). The investigation comprised three test days with standard diet. The first day served for adaptation, the second and third one for determining study data during 9 night hours with or without growth hormone. Abdominal subcutaneous microdialysate and blood were continuously collected and forwarded to a separate room next door where hourly taken samples were centrifuged and frozen until analysed. RESULTS: Growth hormone achieved the peak serum level after 3 h followed by a plateau-like course for the next 6 h. Glycerol in microdialysate started to rise 2 h following growth hormone injection achieving significance compared to placebo after 9 h (P<0.05). Serum glycerol increased 4 h after growth hormone administration achieving significance after 6 h (P<0.05). Glucose and amino acid concentrations showed neither in microdialysate nor in serum significant differences between growth hormone and placebo. Serum values of insulin and C-peptide revealed no significant difference between growth hormone and placebo. SUMMARY AND CONCLUSION: As the result of a high single subcutaneous dose of GH, persistent lipolysis can be shown in continuously collected microdialysate and blood, but no indication for gluconeogenesis or protein anabolism.

Pharmacokinetics and Pharmacodynamics of Once-Weekly Somapacitan in Children and Adults: Supporting Dosing Rationales with a Model-Based Analysis of Three Phase I Trials.

BACKGROUND: Somapacitan, a long-acting growth hormone (GH) derivative, has been well-tolerated in children with GH deficiency (GHD) and adults (healthy and adult GHD), in phase I, single- and multiple-dose trials, respectively, and has pharmacokinetic and pharmacodynamic properties supporting a once-weekly dosing regimen. OBJECTIVE: In the absence of a multiple-dose phase I trial in children with GHD, the aim was to develop a pharmacokinetic/pharmacodynamic model to predict somapacitan exposure and insulin-like growth factor-I (IGF-I) response after once-weekly multiple doses in both children and adults with GHD. METHODS: Pharmacokinetic/pharmacodynamic models were developed from pharmacokinetic and IGF-I profiles in three phase I trials of somapacitan (doses: healthy adults, 0.01-0.32 mg/kg; adult with GHD, 0.02-0.12 mg/kg; children with GHD, 0.02-0.16 mg/kg) using non-linear mixed-effects modeling. Pharmacokinetics were described using a non-linear one-compartment model with dual first- and zero-order absorption through a transit compartment, with saturable elimination. IGF-I profiles were described using an indirect response pharmacokinetic/pharmacodynamic model, with sigmoidal-effect relationship. RESULTS: The non-linear pharmacokinetic and IGF-I data were well-described in order to confidently predict pharmacokinetic/pharmacodynamic profiles after multiple doses in adults and children with GHD. Body weight was found to be a significant covariate, predictive of the differences observed in the pharmacokinetics and pharmacodynamics between children and adults. Weekly dosing of somapacitan provided elevated IGF-I levels throughout the week, despite little or no accumulation of somapacitan, in both adults and children with GHD. CONCLUSION: This analysis of somapacitan pharmacokinetic/pharmacodynamic data supports once-weekly dosing in adults and children with GHD. TRIAL REGISTRATION: ClinicalTrials.gov identifier numbers NCT01514500, NCT01706783, NCT01973244.

Somapacitan, a once-weekly reversible albumin-binding GH derivative, in children with GH deficiency: A randomized dose-escalation trial.

OBJECTIVE: To evaluate the safety, local tolerability, pharmacodynamics and pharmacokinetics of escalating single doses of once-weekly somapacitan, a reversible, albumin-binding GH derivative, vs once-daily GH in children with GH deficiency (GHD). DESIGN: Phase 1, randomized, open-label, active-controlled, dose-escalation trial (NCT01973244). PATIENTS: Thirty-two prepubertal GH-treated children with GHD were sequentially randomized 3:1 within each of four cohorts to a single dose of somapacitan (0.02, 0.04, 0.08 and 0.16 mg/kg; n=6 each), or once-daily Norditropin® SimpleXx® (0.03 mg/kg; n=2 each) for 7 days. MEASUREMENTS: Pharmacokinetic and pharmacodynamic profiles were assessed. RESULTS: Adverse events were all mild, and there were no apparent treatment-dependent patterns in type or frequency. Four mild transient injection site reactions were reported in three of 24 children treated with somapacitan. No antisomapacitan/anti-human growth hormone (hGH) antibodies were detected. Mean serum concentrations of somapacitan increased in a dose-dependent but nonlinear manner: maximum concentration ranged from 21.8 ng/mL (0.02 mg/kg dose) to 458.4 ng/mL (0.16 mg/kg dose). IGF-I and IGFBP-3, and change from baseline in IGF-I standard deviation score (SDS) and IGFBP-3 SDS, increased dose dependently; greatest changes in SDS values were seen for 0.16 mg/kg. IGF-I SDS values were between -2 and +2 SDS, except for peak IGF-I SDS with 0.08 mg/kg somapacitan. Postdosing, IGF-I SDS remained above baseline levels for at least 1 week. CONCLUSIONS: Single doses of once-weekly somapacitan (0.02-0.16 mg/kg) were well tolerated in children with GHD, with IGF-I profiles supporting a once-weekly treatment profile. No clinically significant safety/tolerability signals or immunogenicity concerns were identified.

Translational mixed-effects PKPD modelling of recombinant human growth hormone - from hypophysectomized rat to patients.

BACKGROUND AND PURPOSE: We aimed to develop a mechanistic mixed-effects pharmacokinetic (PK)-pharmacodynamic (PD) (PKPD) model for recombinant human growth hormone (rhGH) in hypophysectomized rats and to predict the human PKPD relationship. EXPERIMENTAL APPROACH: A non-linear mixed-effects model was developed from experimental PKPD studies of rhGH and effects of long-term treatment as measured by insulin-like growth factor 1 (IGF-1) and bodyweight gain in rats. Modelled parameter values were scaled to human values using the allometric approach with fixed exponents for PKs and unscaled for PDs and validated through simulations relative to patient data. KEY RESULTS: The final model described rhGH PK as a two compartmental model with parallel linear and non-linear elimination terms, parallel first-order absorption with a total s.c. bioavailability of 87% in rats. Induction of IGF-1 was described by an indirect response model with stimulation of kin and related to rhGH exposure through an Emax relationship. Increase in bodyweight was directly linked to individual concentrations of IGF-1 by a linear relation. The scaled model provided robust predictions of human systemic PK of rhGH, but exposure following s.c. administration was over predicted. After correction of the human s.c. absorption model, the induction model for IGF-1 well described the human PKPD data. CONCLUSIONS: A translational mechanistic PKPD model for rhGH was successfully developed from experimental rat data. The model links a clinically relevant biomarker, IGF-1, to a primary clinical end-point, growth/bodyweight gain. Scaling of the model parameters provided robust predictions of the human PKPD in growth hormone-deficient patients including variability.

Nanowired Delivery of Growth Hormone Attenuates Pathophysiology of Spinal Cord Injury and Enhances Insulin-Like Growth Factor-1 Concentration in the Plasma and the Spinal Cord.

Previous studies from our laboratory showed that topical application of growth hormone (GH) induced neuroprotection 5 h after spinal cord injury (SCI) in a rat model. Since nanodelivery of drugs exerts superior neuroprotective effects, a possibility exists that nanodelivery of GH will induce long-term neuroprotection after a focal SCI. SCI induces GH deficiency that is coupled with insulin-like growth factor-1 (IGF-1) reduction in the plasma. Thus, an exogenous supplement of GH in SCI may enhance the IGF-1 levels in the cord and induce neuroprotection. In the present investigation, we delivered TiO2-nanowired growth hormone (NWGH) after a longitudinal incision of the right dorsal horn at the T10-11 segments in anesthetized rats and compared the results with normal GH therapy on IGF-1 and GH contents in the plasma and in the cord in relation to blood-spinal cord barrier (BSCB) disruption, edema formation, and neuronal injuries. Our results showed a progressive decline in IGF-1 and GH contents in the plasma and the T9 and T12 segments of the cord 12 and 24 h after SCI. Marked increase in the BSCB breakdown, as revealed by extravasation of Evans blue and radioiodine, was seen at these time points after SCI in association with edema and neuronal injuries. Administration of NWGH markedly enhanced the IGF-1 levels and GH contents in plasma and cord after SCI, whereas normal GH was unable to enhance IGF-1 or GH levels 12 or 24 h after SCI. Interestingly, NWGH was also able to reduce BSCB disruption, edema formation, and neuronal injuries after trauma. On the other hand, normal GH was ineffective on these parameters at all time points examined. Taken together, our results are the first to demonstrate that NWGH is quite effective in enhancing IGF-1 and GH levels in the cord and plasma that may be crucial in reducing pathophysiology of SCI.

A novel soluble supramolecular system for sustained rh-GH delivery.

Methoxy-poly(ethylene glycol)s bearing a terminal cholanic moiety (mPEG(5kDa)-cholane, mPEG(10kDa)-cholane and mPEG(20kDa)-cholane) were physically combined with recombinant human growth hormone (rh-GH) to obtain supramolecular assemblies for sustained hormone delivery. The association constants (Ka) calculated by Scatchard analysis of size exclusion chromatography (SEC) data were in the order of 10(5)M(-1). The complete rh-GH association with mPEG(5kDa)-cholane, mPEG(10kDa)-cholane and mPEG(20kDa)-cholane was achieved with 7.5 ± 1.1, 3.9 ± 0.4 and 2.6 ± 0.4 w/w% rh-GH/mPEG-cholane, respectively. Isothermal titration calorimetry (ITC) yielded association constants similar to that calculated by SEC and showed that rh-GH has 21-25 binding sites for mPEG-cholane, regardless the polymer molecular weight. Dialysis studies showed that the mPEG-cholane association strongly delays the protein release; 80-90% of the associated rh-GH was released in 200 h. However, during the first 8h the protein formulations obtained with mPEG(10kDa)-cholane and mPEG(20kDa)-cholane showed a burst release of 8 and 28%, respectively. Circular dichroism (CD) analyses showed that the mPEG(5kDa)-cholane association does not alter the secondary structure of the protein. Furthermore, mPEG(5kDa)-cholane was found to enhance both the enzymatic and physical stability of rh-GH. In vivo pharmacokinetic and pharmacodynamic studies were performed by subcutaneous administration of rh-GH and rh-GH/mPEG(5kDa)-cholane to normal and hypophysectomised rats. The study showed that mPEG(5kDa)-cholane decreases the maximal concentration in the blood but prolongs the body exposure of the protein, which resulted in 55% bioavailability increase. Finally, rh-GH formulated with mPEG(5kDa)-cholane yielded prolonged weight increase of hypophysectomised rats as compared to rh-GH in buffer or formulated with mPEG(5kDa)-OH. After the second administration the weight of the animals treated with rh-GH formulated with mPEG(5kDa)-cholane was about 2 times higher than that obtained with equal dose of non-formulated rh-GH.

Growth hormone (GH) differentially regulates NF-kB activity in preadipocytes and macrophages: implications for GH’s role in adipose tissue homeostasis in obesity

Adipose tissue remodeling in obesity involves macrophage infiltration and chronic inflammation. NF-kB-mediated chronic inflammation of the adipose tissue is directly implicated in obesity-associated insulin resistance. We have investigated the effect of growth hormone (GH) on NF-kB activity in preadipocytes (3T3-F442A) and macrophages (J774A.1). Our studies indicate that whereas GH increases NF-kB activity in preadipocytes, it decreases NF-kB activity in macrophages. This differential response of NF-kB activity to GH correlates with the GH-dependent expression of a cadre of NF-kB-activated cytokines in these two cell types. Activation of NF-kB by GH in preadipocytes heightens inflammatory response by stimulating production of multiple cytokines including TNF-alpha, IL-6, and MCP-1, the mediators of both local and systemic insulin resistance and chemokines that recruit macrophages. Our studies also suggest differential regulation of miR132 and SIRT1 expression as a mechanism underlying the observed variance in GH-dependent NF-kB activity and altered cytokine profile in preadipocytes and macrophages. These findings further our understanding of the complex actions of GH on adipocytes and insulin sensitivity

PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins.

A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.

Clinical pharmacology considerations in biologics development.

Biologics, including monoclonal antibodies (mAbs) and other therapeutic proteins such as cytokines and growth hormones, have unique characteristics compared to small molecules. This paper starts from an overview of the pharmacokinetics (PK) of biologics from a mechanistic perspective, the determination of a starting dose for first-in-human (FIH) studies, and dosing regimen optimisation for phase II/III clinical trials. Subsequently, typical clinical pharmacology issues along the corresponding pathways for biologics development are summarised, including drug-drug interactions, QTc prolongation, immunogenicity, and studies in specific populations. The relationships between the molecular structure of biologics, their pharmacokinetic and pharmacodynamic characteristics, and the corresponding clinical pharmacology strategies are summarised and depicted in a schematic diagram.

Injectable hydrogel for sustained protein release by salt-induced association of hyaluronic acid nanogel.

A hyaluronic acid-based anionic nanogel formed by self-assembly of cholesteryl-group-bearing HA is designed for protein delivery. The HA nanogel spontaneously binds various types of proteins without denaturation, such as recombinant human growth hormone, erythropoietin, exendin-4, and lysozyme. The HA nanogel shows unique colloidal properties, in particular that an injectable hydrogel is formed by salt-induced association of the HA nanogel. A pharmacokinetic study in rats shows that an in situ gel formulation, prepared by simply mixing rhGH and HA nanogel in phosphate buffer, maintains plasma rhGH levels within a narrow range over one week. Therefore, HA nanogels offer a simple method for easy formulation of therapeutic proteins and are effective for sustained protein release systems.

Aplicaciones de la hormona del crecimiento en humanos / Indications of the use of growth hormone in humans

No disponible

Long-acting pegylated human GH in children with GH deficiency: a single-dose, dose-escalation trial investigating safety, tolerability, pharmacokinetics and pharmacodynamics.

OBJECTIVE: GH replacement therapy currently requires daily injections, which may be inconvenient and distressing for young patients. This study determined the safety, tolerability, pharmacokinetics and pharmacodynamics of escalating single doses of a pegylated GH (NNC126-0083) developed for once-weekly administration, in children with GH deficiency (GHD). DESIGN AND METHODS: Thirty children (age ≥6 and ≤12 years, weight ≥16 kg) were randomised to NNC126-0083 or daily GH treatment. The subjects discontinued their daily GH treatment 7-9 days before receiving NNC126-0083 at 0.01, 0.02, 0.04 or 0.06 mg protein/kg (n=22) or seven once-daily doses of GH at 0.035 mg protein/kg (n=8). RESULTS: NNC126-0083 was well tolerated, and no short-term safety or local tolerability issues were identified. After NNC126-0083 treatment, dose-dependent IGF1 increases were evident for maximum concentration (C(max)), but not area under the curve (AUC(0)(-)(168 h)). Mean values for IGF1 AUC(0)(-)(168 h)/168 h and C(max) were higher for GH than for NNC126-0083, although the difference was not statistically significant for cohort's 0.06 mg protein/kg. At 0.06 mg protein/kg, the resulting IGF1 response began subsiding at ∼3 days post-dose. CONCLUSION: Single doses of long-acting NNC126-0083 were safe and well tolerated in children with GHD. Increased IGF1 levels were observed in all NNC126-0083 dose groups; however, a satisfactory once-weekly IGF1 profile was not reached within the NNC126-0083 dose levels administered.

Differential effects of insufflated, subcutaneous, and intravenous growth hormone on bone growth, cognitive function, and NMDA receptor subunit expression.

The objective of this study was to characterize the effect of inhalable growth hormone (GH) delivered by an insufflator to the lungs of hypophysectomized Sprague Dawley rats. In the first cohort, the safety and efficacy of the insufflated GH were evaluated. Three experimental groups (n = 7 per group) were treated with GH for 15 d: One group received sc injection of GH daily at 200 microg/kg (SC200). Two other groups received GH by insufflation daily: 200 microg/kg (INS 200) and 600 microg/kg (INS 600). In the second set of experiments, GH was administered in three routes [SC200, INS200, intravenous (IV200)] (n=10) for 5 d, and escape latency and N-methyl D-aspartate (NMDA) receptor expression were evaluated. In the first cohort, INS200 showed similar bioactivity as SC200 in growth promotion, tibial growth, as well as escape latency on the 12th day of treatment. Insufflated GH was well tolerated without significant inflammatory responses. In the second cohort, expression of the NMDA receptor 1 and 2B in hippocampus measured after 3 or 6 d of daily treatments were significantly higher in INS200 as compared to IV200, consistent with the improvement of the escape latency. In summary, the inhalable form of GH delivered by intratracheal insufflation was safe, and its bioactivity was comparable to sc injection both in promotion of growth and acquisition of learning ability. If applied properly to human, inhalable GH would be effective for growth promotion and possibly for several disorders caused by underexpression of NMDA receptors.

Effect of recombinant porcine somatotropin (rpST) on drug disposition in swine.

Treatment of pigs with recombinant porcine somatotropin (rpST) causes a marked increase in feed utilization with increased weight-gain over untreated controls. Physiological parameters such as creatinine clearance were increased by rpST treatment. Clearance of drugs eliminated by hepatic extraction, like indocyanine green (ICG), were also increased by rpST treatment. However, clearance of intravenous (i.v.)-dosed propranolol (PPL) was not affected by rpST treatment and data from oral (p.o.)-dosing was inconclusive because of the low bioavailability, probably because of a high first-pass effect. The very low oral bioavailability indicates that intestinal metabolism of PPL is probably quite high. Analysis of urinary metabolites indicated production of the two phenolic isomers, but there was no metabolite corresponding to N-dealkylase activity; although the latter metabolite could have been eliminated in the bile with subsequent fecal elimination. PPL was an excellent in vitro substrate for measuring hepatic DME activity in vitro; two phenolic and one N-dealkylated metabolite were formed. The overall conclusions regarding this study must be that the effects of rpST on drug bioavailability and elimination were equivocal. As ICG and creatinine clearances were both increased significantly, one cannot rule out the probability that rpST would increase drug elimination in pigs as a result of increased hepatic uptake and/or renal clearance. One can only speculate that clearance of concurrently administered drugs would be increased. This would reduce residue levels, but it might also reduce efficacy.

Relative bioavailability of two drug products of somatropin obtained from either the milk of transgenic cows or bacterial culture.

BACKGROUND: Our objective was to assess the relative bioavailability of the first somatropin produced in transgenic cloned cows that carry the human growth hormone (GH) gene (Biohormon) and somatropin produced in Escherichia coli culture (HHT), the procedure most frequently used for the commercial production of the hormone. METHODS: Upon approval by an independent ethics committee and the National Regulatory Agency of Argentina, we compared the time-concentration profiles of somatropin in 24 healthy volunteers, in a randomized, 2-period, 2-sequence crossover design after inhibition of endogenous GH secretion with lanreotide, a long-acting somastostatin analogue. After the subcutaneous administration of 1.33 mg of each formulation, serum somatropin was analyzed by chemiluminescent immunoassay and IGF-I by immunoradiometric assay. Safety was assessed by clinical and laboratory parameters. Pharmacokinetic parameters were calculated with Win Nonlin 5.2 using a non-compartmental model and bioequivalence was assessed. RESULTS: The test/reference ratios of AUC, AUC(last) and C(max) were 106.4 (90% CI = 100.2-112.9), 105.3 (90% CI = 99.1-111.8) and 105.49 (90% CI = 92.6-120.1), respectively. No serious adverse events were reported and no GH antibodies were detected. CONCLUSION: This study demonstrates that a single dose of Biohormon, the first product with somatropin obtained from milk of transgenic mammals, is bioequivalent to the reference product HHT according to standard criteria.

Hipopituitarismo y neurohipófisis / No disponible

No disponible

Troubleshooting PEG-hGH detection supporting pharmacokinetic evaluation in growth hormone deficient patients.

INTRODUCTION: The ability to quantify systemic concentrations of protein therapeutics is complicated by the presence of endogenous analyte, specific binding proteins, and nonspecific matrix components in biological matrices (Lee & Ma, 2007). Further complications can be introduced following pegylation whereby polyethylene glycol (PEG) impedes epitope recognition. Due to substantial interference from high affinity binding proteins and the inability to measure systemic drug concentrations under normal conditions, acid dissociation was implemented to facilitate the pharmacokinetic evaluation of clinical samples containing pegylated human growth hormone (PEG-hGH). METHODS: A sandwich electrochemiluminescent immunosorbent assay (ECLA) was employed using an anti-PEG capture, anti-hGH detection format, thereby eliminating cross-reactivity with endogenous compound. Samples were acid treated with glycine buffered hydrochloric acid (approximately pH 2.0) to dissociate PEG-hGH from serum resident growth hormone binding protein (GHBP; 3). After neutralization with a HEPES-based neutralizing buffer, samples were diluted in a casein-based assay buffer containing 0.2% I-block, 0.1% Tween-20, 2M NaCl, and 10% normal mouse serum to eliminate nonspecific matrix effects. Meso Scale Discovery (MSD) technology was employed to achieve sensitivity requirements. RESULTS: The drug detection assay was validated in the presence and absence of acid dissociation. Validation parameters included: intra- and inter-assay accuracy and precision, selectivity (>60 lots of normal and growth hormone deficient human serum), cross-reactivity/specificity (Genotropin, hemoglobin, lipid, and bilirubin), dilutional linearity and stability (DeSilva et al., 2003; Shah et al., 1992; Smolec et al., 2005 Viswanathan et al., 2007; Food and Drug Administration, 2001). A 10-fold molar excess of GHBP was found to decrease PEG-hGH detection by >90% while acid dissociation was shown to recover >80% of the analyte. PEG-hGH clinical pharmacokinetic samples were analyzed with and without acid treatment. Only 38% of the mid-dose samples were quantifiable without acid treatment while 92% were quantifiable after acid dissociation. DISCUSSION: The implementation of acid dissociation was found to substantially increase the number of quantifiable pharmacokinetic samples over the drug exposure time course and contributed significantly to the robustness of pharmacokinetic evaluation.

Improving the pharmacokinetics/pharmacodynamics of prolactin, GH, and their antagonists by fusion to a synthetic albumin-binding peptide.

To prolong the circulation half-life of human prolactin (hPRL), human GH (hGH), and their competitive antagonists, hPRL-G129R and hGH-G120R, we examined the effects of fusing a serum albumin-binding peptide (SA20) to their amino- or carboxyl-terminus. Fusion of the SA20 peptide to the amino-terminus of the ligands was less detrimental upon their ability to induce or inhibit signal transduction and cell proliferation in vitro than fusion to the carboxyl-terminus. Pharmacokinetic (PK) studies in mice revealed that the half-life of SA20-hPRL and SA20-hGH was prolonged and their clearance was reduced in comparison with hPRL and hGH. Pharmacodynamic (PD) studies in 8-week-old female mice revealed that lobuloalveolar development in mammary glands was greater in all three groups (daily, every 2 days, or every third day over a 12-day period) of mice treated with SA20-hPRL (4 mg/kg) compared with hPRL (3.59 mg/kg). Similarly, daily administration (i.p.) of SA20-hGH (8 mg/kg) or hGH (7.15 mg/kg) to 23-day-old female mice over a 40-day period revealed the superiority of SA20-hGH over hGH as measured by weight gain, body length, and lobuloalveolar development in the mammary glands. These findings indicate that SA20 modification of hPRL, hGH, and their respective antagonists improves their PK/PD properties.