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Context Children with congenital adrenal hyperplasia (CAH) and adrenal insufficiency (AI) require daily hydrocortisone replacement with accurate dosing. Objective Prospective study of efficacy and safety of hydrocortisone granules in children with AI and CAH monitored by 17-OHP (17-hydroxyprogesterone) saliva profiles. Methods Seventeen children with CAH (9 male) and 1 with hypopituitarism (male), aged from birth to 6 years, had their hydrocortisone medication changed from pharmacy compounded capsules to hydrocortisone granules. Patients were followed prospectively for 2 years. In children with CAH, the therapy was adjusted by 17-OHP salivary profiles every 3 months. The following parameters were recorded: hydrocortisone dose, height, weight, pubertal status, adverse events, and incidence of adrenal crisis. Results The study medication was given thrice daily, and the median duration of treatment (range) was 795 (1–872) days, with 150 follow-up visits. Hydrocortisone doses were changed on 40/150 visits, with 32 based on salivary measurements and 8 on serum 17-OHP levels. The median daily mg/m2 hydrocortisone dose (range) at study entry for the different age groups 2–8 years, 1 month to 2 years, Interpretation This first prospective study of glucocorticoid treatment in children with AI and CAH demonstrates that accurate dosing and monitoring from birth results in hydrocortisone doses at the lower end of the recommended dose range and normal growth, without occurrence of adrenal crises.

Context Measurement of salivary glucocorticoids is an accepted method for testing adrenal function but there are few data on stability during home collection. Current salivary collection techniques require active participation or present a choking hazard and are unsuitable for young children. Objective We sought to compare different salivary collection methods; assess the stability of salivary glucocorticoids under conditions replicating home collection; and assess patient tolerability and caregiver acceptability of a salivary collection device for young children, a swab encased in an infant pacifier (SaliPac). Methods Six healthy adults collected salivary samples using a Salivette Cortisol, passive drool, and SalivaBio at night, waking, and 3 Pm for five days. Time to collect 1-mL saliva using the SalivaBio and SaliPac and caregiver acceptability were assessed in 30 children younger than 6 years. Saliva was stored at 4 °C, room temperature (RT), and 50 °C for 24, 48, 72 hours and 1 week to replicate potential postage conditions. Salivary cortisol and cortisone concentrations were measured by mass spectrometry. Results There was no difference in salivary glucocorticoid concentrations using the 3 collection methods. Salivary cortisol and cortisone were stable for 72 hours at RT and 4 °C, and repeated freeze-thaw cycles did not cause significant degradation. In children younger than 6 years the SalivaBio and SaliPac were well tolerated and collected sufficient saliva for salivary steroid analysis in less than 4 minutes. Conclusion Salivette, passive drool, and SalivaBio collect samples with comparable salivary cortisol and cortisone concentrations, which are stable under conditions replicating home collection. SaliPac is an acceptable device for salivary sampling in young children.

Congenital adrenal hyperplasia (CAH) is the most common form of adrenal insufficiency in childhood; it requires cortisol replacement therapy with hydrocortisone (HC, synthetic cortisol) from birth and therapy monitoring for successful treatment. In children, the less invasive dried blood spot (DBS) sampling with whole blood including red blood cells (RBCs) provides an advantageous alternative to plasma sampling. Potential differences in binding/association processes between plasma and DBS however need to be considered to correctly interpret DBS measurements for therapy monitoring. While capillary DBS samples would be used in clinical practice, venous cortisol DBS samples from children with adrenal insufficiency were analyzed due to data availability and to directly compare and thus understand potential differences between venous DBS and plasma. A previously published HC plasma pharmacokinetic (PK) model was extended by leveraging these DBS concentrations. In addition to previously characterized binding of cortisol to albumin (linear process) and corticosteroid-binding globulin (CBG; saturable process), DBS data enabled the characterization of a linear cortisol association with RBCs, and thereby providing a quantitative link between DBS and plasma cortisol concentrations. The ratio between the observed cortisol plasma and DBS concentrations varies highly from 2 to 8. Deterministic simulations of the different cortisol binding/association fractions demonstrated that with higher blood cortisol concentrations, saturation of cortisol binding to CBG was observed, leading to an increase in all other cortisol binding fractions. In conclusion, a mathematical PK model was developed which links DBS measurements to plasma exposure and thus allows for quantitative interpretation of measurements of DBS samples.

Context There is no licensed oral native testosterone (NT) because of challenges in the formulation. Licensed oral formulations of the ester, testosterone undecanoate (TU), require a meal for absorption and generate supraphysiological dihydrotestosterone (DHT) levels. Objective To develop an oral NT formulation. Design and methods A lipid-based formulation of native testosterone filled into soft-gelatin capsules at 40 mg per capsule was designed with 2 years of stability at ambient temperature. Pharmacokinetic comparison studies of this oral lipidic NT formulation to oral TU were conducted in dogs and hypogonadal men. Results In dogs, 40 mg NT was well absorbed under fasted conditions whereas 40 mg TU required a high-fat meal: for NT, the mean fed/fasted AUC ratio was 1.63 and for TU 7.05. In hypogonadal men, fed and fasted NT had similar pharmacokinetics: Cmax mean 26.5 vs 30.4 nmol/L (769 vs 882 ng/dL), AUC0–10 h 87 vs 88.6 h nmol/L. NT (fed state) showed a testosterone AUC increase of 45% between 120 and 200 mg, and NT 200 mg gave a similar mean AUC0–10 h to TU 80 mg: 87 vs 64.8 h nmol/L. Serum TU levels were variable and on a molar basis were ~ten-fold higher than serum testosterone levels after TU 80 mg fed. The DHT: testosterone AUC0–10 h ratio was more physiological for NT than TU being 0.19 vs 0.36. There were no emerging safety concerns with NT. Conclusion This novel oral lipidic native testosterone formulation has potential advantages over oral TU of dosing independently of food and a lower risk of supraphysiological DHT levels. Significance statement There is no licensed oral testosterone because of challenges in formulation, and the oral formulations of the ester, testosterone undecanoate, require a fatty meal for absorption and generate supraphysiological dihydrotestosterone levels. We have overcome the design challenges and formulated an oral native testosterone that can be taken with or without food and provides physiological levels of testosterone and dihydrotestosterone in hypogonadal men. This formulation, DITEST, has the potential advantage of being oral for patients who do not tolerate injections and less risk of adverse events that might theoretically be associated with elevated dihydrotestosterone levels. Future studies will need to define the dosing regimen for replacement in hypogonadal men.

Purpose The objective of the present work was to screen whether a novel pediatric hydrocortisone granule formulation can be co-administered with common food matrices and liquids. Methods Pediatric hydrocortisone granules were studied using a biopredictive in vitro approach. Experiments included an in situ chemical compatibility study of active ingredient and drug product with liquid dosing vehicles and soft foods commonly ingested by infants, pre-school- and school children. Drug solubility and stability experiments in the different vehicle types and, drug release/dissolution experiments mimicking age-related pediatric gastric conditions after administering the hydrocortisone granules together with the dosing vehicles and after different exposure/mixing times were performed. Results In the simulated dosing scenarios applied in dissolution experiments, in vitro dissolution in gastric conditions was rapid and complete. Results of the chemical compatibility/stability studies indicated that mixing with the different dosing vehicles studied should not be an issue regarding drug degradation products. Conclusions A novel in vitro approach ensuring a proper risk assessment of the use of dosing vehicles in the administration of pediatric dosage forms was established and applied to a novel pediatric hydrocortisone drug product. The studied dosing vehicles were shown to not alter performance of the drug product and are thus considered suitable for administration with hydrocortisone granules.

Context Population studies frequently measure cortisol as a marker of stress, and excess cortisol is associated with increased mortality. Cortisol has a circadian rhythm, and frequent blood sampling is impractical to assess cortisol exposure. We investigated measuring salivary cortisone and examined the sampling frequency required to determine cortisol exposure. Methods Serum and saliva with cortisol and cortisone were measured by liquid chromatography–tandem mass spectrometry in independent cohorts. The relationship between serum cortisol and salivary cortisone was analyzed in cohort 1 using a linear mixed effects model. The resulting fixed effects component was applied to cohort 2. Saliva cannot easily be collected when a patient is sleeping, so we determined the minimum sampling required to estimate cortisol exposure [estimated area under the curve (eAUC)] using 24-hour cortisol profiles (AUC24) and calculated the relative error (RE) for eAUC. Results More than 90% of variability in salivary cortisone could be accounted for by change in serum cortisol. A single serum cortisol measurement was a poor estimate of AUC24, especially in the morning or last thing at night (RE >68%); however, three equally spaced samples gave a median RE of 0% (interquartile range, −15.6% to 15.1%). In patients with adrenal incidentalomas, eAUC based on three serum cortisol samples showed a difference between those with autonomous cortisol secretion and those without (P = 0.03). Interpretation Accepting that most people sleep 7 to 8 hours, ∼8-hourly salivary cortisone measurements provide a noninvasive method of estimating 24-hour cortisol exposure for population studies., Accepting that most people sleep 7 to 8 hours, ∼8-hourly salivary cortisone measurements provide a noninvasive method of estimating 24-hour cortisol exposure in population studies.

The goal of hormone replacement is to mirror physiology. Hydrocortisone granules and modified release formulations are being developed to optimise cortisol replacement in the rare disease of adrenal insufficiency. To facilitate clinical development, we built and verified a physiologically based pharmacokinetic (PBPK) model for the endogenous hormone cortisol (hydrocortisone) in healthy adults, and children and adults with adrenal insufficiency. The model predicted immediate-release hydrocortisone pharmacokinetics in adults across the dose range 0.5 to 20 mg, with predicted/observed AUCs within 0.8 to 1.25-fold. The model also tightly predicted pharmacokinetic parameters for modified-release formulations, with AUCs within 0.8 to 1.25-fold after single and multiple dosing. Predicted modified-release formulation pharmacokinetics (PK) in 12 to 18-year olds showed PK to be similar to adults. This hydrocortisone PBPK model is a useful tool to predict adult and paediatric pharmacokinetics of both immediate- and modified-release hydrocortisone formulations, and develop clinical dosing regimens.

Steroid 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates endogenous glucocorticoids and catalyses an important step in bile acid synthesis. Endogenous and synthetic glucocorticoids are potent regulators of metabolic phenotype and play a crucial role in hepatic glucose metabolism. However, the potential of synthetic glucocorticoids to be metabolised by AKR1D1 as well as to regulate its expression and activity has not been investigated. The impact of glucocorticoids on AKR1D1 activity was assessed in human liver HepG2 and Huh7 cells; AKR1D1 expression was assessed by qPCR and Western blotting. Genetic manipulation of AKR1D1 expression was conducted in HepG2 and Huh7 cells and metabolic assessments were made using qPCR. Urinary steroid metabolite profiling in healthy volunteers was performed pre- and post-dexamethasone treatment, using gas chromatography-mass spectrometry. AKR1D1 metabolised endogenous cortisol, but cleared prednisolone and dexamethasone less efficiently. In vitro and in vivo, dexamethasone decreased AKR1D1 expression and activity, further limiting glucocorticoid clearance and augmenting action. Dexamethasone enhanced gluconeogenic and glycogen synthesis gene expression in liver cell models and these changes were mirrored by genetic knockdown of AKR1D1 expression. The effects of AKR1D1 knockdown were mediated through multiple nuclear hormone receptors, including the glucocorticoid, pregnane X and farnesoid X receptors. Glucocorticoids down-regulate AKR1D1 expression and activity and thereby reduce glucocorticoid clearance. In addition, AKR1D1 down-regulation alters the activation of multiple nuclear hormone receptors to drive changes in gluconeogenic and glycogen synthesis gene expression profiles, which may exacerbate the adverse impact of exogenous glucocorticoids.

Objectives There is no licensed, dose-appropriate formulation of hydrocortisone for children with adrenal insufficiency (AI) and patients rely on compounded adult medication. The aim of this study was to evaluate the absorption, palatability and safety of Infacort®, an immediate-release, granule formulation of hydrocortisone with taste masking. Study design Single site with satellites attended by a “flying” doctor from investigator site. Open-label, single-dose study in three consecutive child cohorts (n=24) with AI; Cohort 1, children aged 2 to Au: the age ranges quoted are slightly different in each of the abstract, main text, Table 1 and Supplemental table. These have now been unified as shown here. . Methods Fasted children were given a single dose of Infacort® as dry granules administered directly from a capsule or spoon followed by a drink. The primary endpoint was the maximum serum cortisol concentration up to 240 minutes after Infacort® administration. Secondary endpoints were palatability and adverse events (AEs). Results All children showed an increase in cortisol above baseline after Infacort® (p

Context Optimization of hydrocortisone replacement therapy is important to prevent under- and over dosing. Hydrocortisone pharmacokinetics is complex as circulating cortisol is protein bound mainly to corticosteroid-binding globulin (CBG) that has a circadian rhythm. Objective A detailed analysis of the CBG circadian rhythm and its impact on cortisol exposure after hydrocortisone administration. Design and methods CBG was measured over 24 hours in 14 healthy individuals and, employing a modelling and simulation approach using a semi-mechanistic hydrocortisone pharmacokinetic model, we evaluated the impact on cortisol exposure (area under concentration-time curve and maximum concentration of total cortisol) of hydrocortisone administration at different clock times and of the changing CBG concentrations. Results The circadian rhythm of CBG was well described with two cosine terms added to the baseline of CBG: baseline CBG was 21.8 µg/mL and interindividual variability 11.9%; the amplitude for the 24 and 12 hours cosine functions were relatively small (24 hours: 5.53%, 12 hours: 2.87%) and highest and lowest CBG were measured at 18:00 and 02:00, respectively. In simulations, the lowest cortisol exposure was observed after administration of hydrocortisone at 23:00-02:00, whereas the highest was observed at 15:00-18:00. The differences between the highest and lowest exposure were minor (≤12.2%), also regarding the free cortisol concentration and free fraction (≤11.7%). Conclusions Corticosteroid-binding globulin has a circadian rhythm but the difference in cortisol exposure is ≤12.2% between times of highest and lowest CBG concentrations; therefore, hydrocortisone dose adjustment based on time of dosing to adjust for the CBG concentrations is unlikely to be of clinical benefit.

Aim Patients with classic congenital adrenal hyperplasia (CAH)have poor health outcomes. In the absence of a comprehensive observational study, this manuscript provides a model to esti-mate the lifetime disease burden of adults with classic CAH. Methods The model, built in Excel, comprises subdomains addressing the health consequences of CAH and synthesises evidence from clinical and epidemiological studies on health out-comes.Results The model estimates that adults with classic CAH will implement ‘sick day rules’ (doubling or tripling glucocorticoid and/or use of parenteral therapy) 171 times over their lifetime and attend hospital for adrenal crisis on 11 occasions. In a population of 1000, over 200 will die of a condition complicated by adrenal crisis resulting, on average, in a loss of 7 years of life.Patients with CAH may also suffer from excess CVD events.Treatment with glucocorticoids almost doubles the risk of bone fractures in patients with CAH compared to the general popula-tion, leading on average to an additional 08 fractures perpatient with CAH over their lifetime.Conclusions The disease burden model highlights gaps in evidence, particularly regarding intensity of care and adrenal crisis,and the relationship between control of CAH and risks of CVD, osteoporosis, diabetes and infertility. The model can be used for research on the impact of new clinical pathways and therapeutic interventions in terms of clinical events and cost.

Context: The assessment absolute bioavailability of oral hydrocortisone is complicated by its saturable binding to cortisol binding globulin (CBG). Previous assessment of bioavailability used a cortisol radioimmunoassay which has cross reactivity with other steroids. Salivary cortisone is a measure of free cortisol and LC-MS/MS is the gold standard method for measuring steroids. We here report the absolute bioavailability of hydrocortisone calculated using serum cortisol and salivary cortisone measured by LC-MS/MS. Methods: 14 healthy male dexamethasone suppressed volunteers were administered 20 mg hydrocortisone either intravenously or orally by tablet. Samples of serum and saliva were taken and measured for cortisol and cortisone by LC-MS/MS. Serum cortisol was corrected for saturable binding using published data and pharmacokinetic parameters derived using the program WinNonlin. Results: The mean (95% CI) bioavailability of oral hydrocortisone calculated from serum cortisol, unbound serum cortisol and salivary cortisone was 1.00 (0.89-1.14); 0.88 (0.75-1.05); and 0.93 (0.83-1.05), respectively. Conclusion: The data confirm that, after oral administration, hydrocortisone is completely absorbed. The data derived from serum cortisol corrected for protein binding, and that from salivary cortisone, are similar supporting the concept that salivary cortisone reflects serum free cortisol levels and that salivary cortisone can be used as a non-invasive method for measuring the pharmacokinetics of hydrocortisone.

Summary Objective Hydrocortisone via nasogastric (NG) tube is used in sick children with adrenal insufficiency; however, there is no licensed formulation for NG administration. Methods We investigated hydrocortisone recovery after passage through NG tubes in vitro for three formulations: liquid suspension, crushed tablets mixed with water, and hydrocortisone granules designed for oral administration to children. Cortisol was measured by LC‐MS/MS. Results Hydrocortisone content was variable and recovery low after preparation in syringe and prior to passage through NG tubes. For doses, 0.5 and 2.0 mg mean percentage recovery was as follows: liquid suspension 57% and 58%; crushed tablets 46% and 30%; and hydrocortisone granules 78% and 71%. Flushing the administering syringe increased recovery. Hydrocortisone recovery after passage with flushing through 6‐12Fr gauge NG tubes was variable: liquid suspension 61%‐92%, crushed tablets 40%‐174%, hydrocortisone granules 61%‐92%. Administration of hydrocortisone granules occluded 6 and 8Fr NG tubes; however, administration using a sampling needle to prevent granules being administered gave a recovery of 74%‐98%. Conclusions The administration of hydrocortisone through NG tubes is possible; however, current methods deliver a variable dose of hydrocortisone, generally less than that prescribed. Attention should be placed on the technique used to optimize drug delivery such as flushing of the administering syringe. Hydrocortisone granules block small NG tubes but behaved as well as the commonly used liquid suspension when prepared with a filtering needle that filters out granules.

Context:The classic androgen synthesis pathway proceeds via DHEA, androstenedione and testosterone to 5α-dihydrotestosterone (DHT). However, DHT synthesis can also be achieved by an alternative pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens and, in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid therapy is unknown.Objective:We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation and their response to conventional glucocorticoid (GC) therapy and modified release hydrocortisone.Methods:We employed urinary steroid metabolome profiling by gas chromatography-mass spectrometry for 24-h steroid excretion analysis, studying the impact of conventional GC...

Research into adrenal insufficiency (AI) and congenital adrenal hyperplasia (CAH) in children has focused largely on clinical consequences for patients; and until recently, the wider experience of the condition from the perspective of other family members has been neglected. In a mixed methods study, we captured the experiences of parents of young children affected by AI/CAH, including their views on the psychosocial impact of living with and managing the condition. Semi-structured interviews were carried out in the UK and an online survey was developed, translated and disseminated through support groups (UK and the Netherlands) and outpatient endocrinology clinics (Germany). Challenges associated with diagnosis, treatment, support and the future were identified. For UK parents, the diagnosis period was characterised by a lack of awareness amongst healthcare professionals and occurrences of adrenal crisis. Parents reported burden, anxiety and disruption associated with the intensive treatment regimen. Parents adjusted and gained confidence over time yet found delegating responsibility for medication difficult and worried about the future for their child. Access to psychological support and contact with other families was reported as highly beneficial. The findings of the study provide critical context for future studies and for informing how parents and families can be better supported. Prenatal genetic counselling for parents who already have an affected child will include an explanation of recurrence risk but should also focus on providing information and reassurance about diagnostic testing and care for their newborn.

Alkindi® is a novel oral multi-particulate taste-masked formulation of hydrocortisone for use in children with the rare disease adrenal insufficiency. The objective of the present work was to study the biorelevant dissolution and compatibility properties of Alkindi® granules following exposure to administration fluids including breast milk, artificial milk, whole milk and water. To provide in vitro data for a representative patient collective, dosing conditions in neonates, infants and pre-school children were assessed. Experiments included the physicochemical characterisation of the different administration fluids, hydrocortisone solubility experiments and, dissolution experiments in which initial gastric conditions after the administration of a 0.5 or 5 mg dose with 50–200 mL fluid were simulated. The total duration of the dissolution experiments was 240 min to screen both dissolution and compatibility with the different fluids. Dissolution of the 0.5 mg dose was fast and complete in all scenarios, i.e. ≥80% of the dose was released in the neonate and the infant scenario and ≥75% in the pre-school children setup within 30 min. Results for the 5 mg dose were ∼5–10% lower in all simulated patient scenarios. The results obtained in the present study confirm the compatibility and in-use chemical stability of Alkindi® with all studied dosing matrices and that in vivo dissolution and bioavailability of the product will not be affected by the composition of the co-administered fluids studied.

Treatment of congenital adrenal hyperplasia (CAH) is suboptimal. Inadequate suppression of androgens and glucocorticoid excess are common and current glucocorticoid formulations cannot replace the cortisol circadian rhythm.The primary objective was to characterize the pharmacokinetic profile of Chronocort, a modified-release hydrocortisone formulation, in adults with CAH. Secondary objectives included examining disease control following 6 months of Chronocort with dose titration.Sixteen adults (eight females) with classic CAH participated in an open-label, nonrandomized, Phase 2 study at the National Institutes of Health Clinical Center. Twenty-four-hour blood sampling was performed on conventional glucocorticoids and following 6 months of Chronocort. Chronocort was initiated at 10 mg (0700 h) and 20 mg (2300 h). Dose titration was performed based on androstenedione and 17-hydroxyprogresterone (17-OHP) levels and clinical symptomatology.The primary outcome was cortisol pharmacokinetics of Chronocort and secondary outcomes included biomarkers of CAH control (androstenedione and 17-OHP).In patients with CAH, Chronocort cortisol profiles were similar to physiologic cortisol secretion. Compared with conventional therapy, 6 months of Chronocort resulted in a decrease in hydrocortisone dose equivalent (28 ± 11.8 vs 25.9 ± 7.1 mg/d), with lower 24-hour (P = .004), morning (0700-1500 h; P = .002), and afternoon (1500-2300 h; P = .011) androstenedione area under the curve (AUC) and lower 24-hour (P = .023) and morning (0700-1500 h; P = .02) 17-OHP AUC.Twice-daily Chronocort approximates physiologic cortisol secretion, and was well tolerated and effective in controlling androgen excess in adults with CAH. This novel hydrocortisone formulation represents a new treatment approach for patients with CAH.

Objectives To evaluate the risks of depression and all-cause mortality, healthcare utilisation costs and treatment adherence in congenital adrenal hyperplasia (CAH) in the United Kingdom. Design and methods A retrospective, matched-cohort study using UK primary-care data from the Clinical Practice Research Datalink linked to hospital and death certification data. Patients diagnosed with CAH and having ≥1 corticosteroid prescription were matched 1:10 to reference subjects. Risk of death and lifetime prevalence of depression were compared using Cox regression models. Direct financial costs were estimated for healthcare contacts. Treatment adherence was measured by medical possession ratio (MPR). Results 605 patients with CAH were identified; 562 were matched. 270 CAH patients (2700 controls) were linkable to death-certificate data, with adjusted hazard ratio for all-cause mortality 5.17 (95% CI 2.81–9.50). Mean (s.d.) age at death in CAH patients was 54.8 (23.9) vs 72.8 (18.0) years in control patients. The prevalence ratio of depression in CAH vs control patients was 1.28 (95% CI 1.13–1.45). Mean (s.d.) annual healthcare costs were higher in CAH than controls: at age 0–6 years, £7038 (£14 846) vs £2879 (£13 972, P P P = 0.007) and ≥41 years, £4204 (£4863) vs £1651 (£2303, P Conclusions This first analysis of CAH in routine UK healthcare suggests that patients with CAH have increased mortality, depression and healthcare utilisation and low treatment adherence.

Optimisation of hydrocortisone replacement therapy in children is challenging as there is currently no licensed formulation and dose in Europe for children under 6 years of age. In addition, hydrocortisone has non-linear pharmacokinetics caused by saturable plasma protein binding. A paediatric hydrocortisone formulation, Infacort® oral hydrocortisone granules with taste masking, has therefore been developed. The objective of this study was to establish a population pharmacokinetic model based on studies in healthy adult volunteers to predict hydrocortisone exposure in paediatric patients with adrenal insufficiency. Cortisol and binding protein concentrations were evaluated in the absence and presence of dexamethasone in healthy volunteers (n = 30). Dexamethasone was used to suppress endogenous cortisol concentrations prior to and after single doses of 0.5, 2, 5 and 10 mg of Infacort® or 20 mg of Infacort®/hydrocortisone tablet/hydrocortisone intravenously. A plasma protein binding model was established using unbound and total cortisol concentrations, and sequentially integrated into the pharmacokinetic model. Both specific (non-linear) and non-specific (linear) protein binding were included in the cortisol binding model. A two-compartment disposition model with saturable absorption and constant endogenous cortisol baseline (Baseline cort,15.5 nmol/L) described the data accurately. The predicted cortisol exposure for a given dose varied considerably within a small body weight range in individuals weighing

Objectives Due to the lack of paediatric-licensed formulations, children are often treated with individualized pharmacy-compounded adult medication. An international web-based survey about the types of medication in children with adrenal insufficiency (AI) revealed that the majority of paediatric physicians are using pharmacy-compounded medication to treat children with AI. Observations of loss of therapy control in children with congenital adrenal hyperplasia with compounded hydrocortisone capsules and regained control after prescribing a new hydrocortisone batch led to this ‘real world’ evaluation of pharmacy-compounded paediatric hydrocortisone capsules. Methods Capsule samples were collected randomly from volunteering parents of treated children suffering from congenital adrenal hyperplasia from all over Germany. Analysis of net mass and hydrocortisone content by high-performance liquid chromatography with ultraviolet (HPLC-UV) detection method was performed based on the European Pharmacopeia. Results In a total of 61 batches that were sent, 5 batches could not be analysed because of missing dose information, insufficient number of capsules or were not possible to be evaluated. Fifty-six batches containing 1125 capsules were evaluated. 21.4% of the batches revealed insufficiency in uniformity of net mass or drug content and additional 3.6% failed because they did not contain the labelled drug. Conclusions Compounded medication is a possible cause of variation of steroid doses in children with adrenal insufficiency or congenital adrenal hyperplasia, putting these vulnerable patients at risk of poor disease control and adrenal crisis. These data may apply to other individualized compounded oral medication as well, emphasizing the need for development of licensed paediatric formulations approved by regulatory authorities.

BackgroundPatients with congenital adrenal hyperplasia (CAH) have low to no biosynthesis of cortisol and require lifelong cortisol replacement. Optimisation of hydrocortisone (HC, synthetic cortisol) therapy in this population is important, since too low or high cortisol concentrations increase the risk of adrenal crisis or Cushing’s syndrome1. HC has nonlinear pharmacokinetics (PK) caused by saturable binding to corticosteroid binding globulin (CBG)2. The objective of this analysis was to extend an established paediatric HC PK-model3 with dried blood spot (DBS) data in order to further characterise the binding behaviour of HC in children.MethodsA semi-mechanistic adult PK model for a novel HC formulation4 has previously been reduced to a paediatric model using sparse plasma samples from a phase III study in 24 patients with adrenal insufficiency5. Plasma and DBS concentrations of cortisol were collected and additional DBS HC concentrations were obtained from a follow-up study. The relation between plasma and DBS samples was characterised by a graphical evaluation, after which nonlinear mixed-effects modelling was applied using NONMEM 7.4.ResultsPlasma concentrations of cortisol were substantially higher than the corresponding DBS concentrations. The plasma/DBS ratio ranged between 2 to 8 within and between children, while the relation between the cortisol DBS concentrations and cortisol plasma concentrations showed nonlinear behaviour mirroring the nonlinear binding kinetics to CBG.ConclusionsOur graphical analysis identified substantial differences and high inter- and intraindividual variabilities between plasma and DBS samples. A nonlinear mixed-effects model is being set up to quantify these findings and allow for further prediction of HC exposure. Afterwards, effect biomarkers can be included in order to evaluate cortisol replacement therapy and to optimise the HC treatment in paediatric patients.ReferencesSpeiser PW, et al. J Clin Endocrinol Metab 2010;95(9):4133–4160.Lentjes EG, WM, et al:J Clin Endocrinol Metab. 1999;84(2):682–687.Melin J:Dissertation 2017.Melin J, et al:Clin Pharmacokinet. 2018;57(4):515–527.Neumann U. et al:Clin Endocrinol. 2018;88(1):21–29.Disclosure(s)Nothing to disclose

SummaryObjective It is not possible with current hydrocortisone replacement to mimic the diurnal cortisol profile in patients with adrenal insufficiency. Previous attempts with modified-release technology were unsuccessful. Our objective was to develop hydrocortisone formulations that recreate the diurnal cortisol profile using multiparticulate technology. Design and Measurements Screening by in vitro dissolution profiles, pharmacokinetic (PK) testing in dexamethasone-suppressed dogs and humans, and comparison with a reference population. Setting Field laboratories and clinical research facility. Results Formulations were generated using an enteric (delayed release) design configuration with an extended (sustained release) dissolution profile. In vitro dissolution confirmed delayed and sustained hydrocortisone release. However, in dogs and humans, sustained release resulted in reduced bioavailability. A formulation, DIURF-006, was developed that maintained delayed release but omitted the sustained-release functionality. PK characterization of DIURF-006 showed that, despite absence of a sustained-release component, absorption was sufficiently sustained to deliver extended hydrocortisone absorption. In dexamethasone-suppressed volunteers (n = 16) receiving a twice-daily ‘toothbrush’ regimen (20 mg at 23:00 h and 10 mg at 07:00 h), DIURF-006 gave a similar cortisol profile to physiological cortisol levels: DIURF-006 vs physiological, Geomean AUC 5610 vs 4706 h * nmol/l, Geomean Cmax 665 vs 594 nmol/l and Median Tmax 8·5 h vs clock time 08:12 h for peak cortisol. The relative bioavailability of DIURF-006 vs hydrocortisone was 89%, and cortisol levels increased linearly with doses between 5 and 30 mg. Conclusion A multiparticulate oral hydrocortisone formulation with only an enteric coat provides delayed and sustained absorption and when given in a ‘toothbrush’ regimen provides physiological cortisol exposure.

Background We developed a modified-release hydrocortisone, Chronocort, to replace the cortisol rhythm in patients with congenital adrenal hyperplasia. Food, alcohol and pH affect drug absorption, and it is important to assess their impact when replicating a physiological rhythm. Subjects and methods In vitro dissolution to study impact of alcohol and pH on Chronocort. A phase 1, three-period, cross over study in 18 volunteers to assess the impact of food on Chronocort and to compare bioavailability to immediate-release hydrocortisone. Results In vitro dissolution of Chronocort was not affected by gastrointestinal pH up to 6.0 nor by an alcohol content up to 20% v/v. Food delayed and reduced the rate of absorption of Chronocort as reflected by a longer Tmax (fed vs fasted: 6.75 h vs 4.5 h, P = 0005) and lower Cmax (549.49 nmol/L vs 708.46 nmol/L, ratio 77% with CI 71–85). Cortisol exposure was similar in fed and fasted state: Geo LSmean ratio (CI) AUC0t for fed/fasted was 108.33% (102.30–114.72%). Cortisol exposure was higher for Chronocort compared to immediate-release hydrocortisone: Geo LSmean ratios (CI) 118.83% (111.58–126.54%); however, derived free cortisol showed cortisol exposure CIs were within 80.0–125.0%: Geo LSmean ratio (CI) for AUC0t 112.73% (105.33–120.65%). Conclusions Gastric pH ≤6.0 and alcohol do not affect hydrocortisone release from Chronocort. Food delays Chronocort absorption, but cortisol exposure is similar in the fasted and fed state and exposure as assessed by free cortisol is similar between Chronocort and immediate-release hydrocortisone.

Context: The classic androgen synthesis pathway proceeds via dehydroepiandrosterone, androstenedione, and testosterone to 5α-dihydrotestosterone. However, 5α-dihydrotestosterone synthesis can also be achieved by an alternative pathway originating from 17α-hydroxyprogesterone (17OHP), which accumulates in congenital adrenal hyperplasia (CAH). Similarly, recent work has highlighted androstenedione-derived 11-oxygenated 19-carbon steroids as active androgens, and in CAH, androstenedione is generated directly from 17OHP. The exact contribution of alternative pathway activity to androgen excess in CAH and its response to glucocorticoid (GC) therapy is unknown. Objective: We sought to quantify classic and alternative pathway-mediated androgen synthesis in CAH, their diurnal variation, and their response to conventional GC therapy and modified-release hydrocortisone. Methods: We used urinary steroid metabolome profiling by gas chromatography–mass spectrometry for 24-hour steroid excretion analysis, studying the impact of conventional GCs (hydrocortisone, prednisolone, and dexamethasone) in 55 adults with CAH and 60 controls. We studied diurnal variation in steroid excretion by comparing 8-hourly collections (23:00–7:00, 7:00–15:00, and 15:00–23:00) in 16 patients with CAH taking conventional GCs and during 6 months of treatment with modified-release hydrocortisone, Chronocort. Results: Patients with CAH taking conventional GCs showed low excretion of classic pathway androgen metabolites but excess excretion of the alternative pathway signature metabolites 3α,5α-17-hydroxypregnanolone and 11β-hydroxyandrosterone. Chronocort reduced 17OHP and alternative pathway metabolite excretion to near-normal levels more consistently than other GC preparations. Conclusions: Alternative pathway-mediated androgen synthesis significantly contributes to androgen excess in CAH. Chronocort therapy appears superior to conventional GC therapy in controlling androgen synthesis via alternative pathways through attenuation of their major substrate, 17OHP., We studied diurnal urinary steroid excretion in glucocorticoid-treated patients with congenital adrenal hyperplasia and found increased alternative pathway androgen synthesis that was ameliorated by modified-release hydrocortisone.

Supercritical CO(2) has the potential to be an excellent environment within which controlled release polymers and dry composites may be formed. The low temperature and dry conditions within the fluid offer obvious advantages in the processing of water, solvent or heat labile molecules. The low viscosity and high diffusivity of scCO(2) offer the possibility of novel processing routes for polymer drug composites, but there are still technical challenges to overcome. Moreover, the low solubility of most drug molecules in scCO(2) presents both challenges and advantages. This review explores the current methods that use high pressure and scCO(2) for the production of drug delivery systems and the more specialized application of the fluid in the formation of highly porous tissue engineering scaffolds.

Context: Measuring serum cortisol to evaluate stress, adrenal disease, and monitor hydrocortisone replacement requires venepuncture. Conversely, salivary measurements are noninvasive. Objective: This study aimed to investigate measurement of salivary cortisol and cortisone as alternatives to serum cortisol. Design and Setting: This was a prospective cross-over study in a clinical research facility. Patients and Methods: Over three periods (Period 1, 24-h physiological cortisol rhythm; Periods 2 and 3, after 20 mg oral and iv hydrocortisone) 14 male volunteers had serum and saliva cortisol and cortisone, serum albumin, cortisol-binding globulin, and free cortisol measured. Data were analyzed for rhythm parameters and correlations. Linear mixed-effects modelling was performed to determine the relationship between serum cortisol and salivary cortisone. Results: Serum cortisol and cortisone showed similar circadian rhythms with large peak:trough ratios (cortisol median ratio, 11). Albumin and cortisol-binding globulin showed minor peak:trough ratios Conclusion: Salivary cortisol is frequently undetectable and contaminated by oral hydrocortisone. In contrast, salivary cortisone reflects serum cortisol and provides a noninvasive alternative to measuring serum cortisol levels.

Microcellular foams were produced from polycaprolactone (PCL) using a method based on the rapid release of supercritical CO2. The average pore size in the structure was found to vary with the selected vent time and the final microstructure was a combination of open and closed cells. Thermal analysis showed that the degree of crystallinity of all foams was in the region of 70%. This was found to be consistent with the observation of lamellar textures on the inner surface of the pores. There was also a pronounced decrease in compressive modulus with increasing vent time and average pore size.

In the field of drug delivery, the ability to control the size, morphology, and release of drug particles is fundamental to good targeting, but is often hampered by harsh processing conditions or inadequate methods; likewise for the processing of polymeric controlled-release systems. However, the use of supercritical fluids such as supercritical CO 2 (scCO 2 ) has provided a ‘clean’ and effective alternative to traditional methods of drug and polymer processing. In particular, scCO 2 has a number of unique properties that make it possible to process both bioactive molecules and amorphous polymers without using toxic organic solvents or elevated temperatures. Here, we review the positive impact that supercritical fluids have had on the micronization, encapsulation, and impregnation of molecules of interest to both the pharmaceutical and biotechnology industries.

A new method of surface-selective laser sintering (SSLS) leads to the fabrication of three-dimensional (3D) composite scaffolds (spatial resolution ∼200 μm) that are both bioactive and biodegradable. Moreover, the scaffolds can have very precise dimensions and intricate structure. Conventionally, in selective laser sintering (SLS), the polymer absorbs infrared (λ=10.6 μm) radiation and this leads to a volumetric absorption by the whole polymer particle. In other words, each particle of polymer is completely melted and fuses to the next in order to form the desired morphology. In our experiments we have used near-infrared (λ=0.97 μm) laser radiation, which polymer particles do not absorb at all. To initiate the sintering process a small quantity (< 0.1 wt.-%) of carbon microparticles were homogeneously distributed on the surfaces of the polymer particles. Thus, the melting process was limited to only the surfaces of each particle. The carbon microparticles are strong absorbers of laser radiation, and this opens up the technique to a range of polymers that up till now could not be processed by laser sintering. More importantly, since the laser melts only the surfaces of the particles, delicate bioactive species trapped within each particle retain their activity throughout the processing. We have demonstrated the application of this technique by the incorporation of the enzyme ribonuclease A into particles of poly(d,l-lactic) acid (PLA) and the assembly of 3D matrices at three different laser intensities, using a 0.97 μm wavelength continuous wave (CW) diode laser.

The ability to deliver, over time, biologically active osteogenic growth factors by means of designed scaffolds to sites of tissue regeneration offers tremendous therapeutic opportunities in a variety of musculoskeletal diseases. The aims of this study were to generate porous biodegradable scaffolds encapsulating an osteogenic protein, bone morphogenetic protein 2 (BMP-2), and to examine the ability of the scaffolds to promote human osteoprogenitor differentiation and bone formation in vitro and in vivo. BMP-2-encapsulated poly(DL-lactic acid) (PLA) scaffolds were generated by an innovative supercritical fluid process developed for solvent-sensitive and thermolabile growth factors. BMP-2 released from encapsulated constructs promoted adhesion, migration, expansion, and differentiation of human osteoprogenitor cells on three-dimensional scaffolds. Enhanced matrix synthesis and cell differentiation on growth factor-encapsulated scaffolds was observed after culture in an ex vivo model of bone formation developed on the basis of the chick chorioallantoic membrane model. BMP-2-encapsulated polymer scaffolds showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation and within diffusion chambers implanted into athymic mice as assessed by X-ray analysis and immunocytochemistry. The generation of three-dimensional biomimetic structures incorporating osteoinductive factors such as BMP-2 indicates their potential for de novo bone formation that exploits cell-matrix interactions and, significantly, realistic delivery protocols for growth factors in musculoskeletal tissue engineering.

Exposure of poly(DL-lactic acid) (PDLLA), and related polymers, to supercritical CO2 (scCO2) at or below, physiological temperatures leads to very effective plasticization and liquefying of the polymers. The phenomenon arises from the high solubility and interaction of the scCO2 in the polymer. Under these unique conditions, temperature and solvent labile molecules can be mixed efficiently into the liquefied polymer. This liquefied polymer/drug/CO2 mixture can then be sprayed into a collecting chamber, and during this process particles of drug-loaded polymer are formed. This process is very different from rapid expansion and antisolvent based techniques that have been previously reported. In this article, we describe a method of controlling particle size during the spray process by introducing a backpressure of N2 in the collecting chamber. This backpressure dynamically regulates the loss of CO2 from the issuing polymer/CO2 mixture, leading to control over sprayed particle size. In situ observation of the viscosity of the plasticized polymer indicates that a backpressure of 68 bar or greater is necessary to ensure the production of fine particles. The influences of backpressure and saturation temperature on particle size for the sprayed products are discussed in terms of observed PDLLA/CO2 mixture viscosities.

Treatment of neonates and infants with adrenal insufficiency is unsatisfactory because unlicensed hydrocortisone formulations are used.The objectives were to survey current hydrocortisone prescribing practice and develop a novel hydrocortisone formulation, Infacort.The use of hydrocortisone by European pediatric endocrinologists was surveyed. Based on this, an oral hydrocortisone granule formulation, Infacort, with taste masking was developed and evaluated in vitro and then in vivo in a phase I pharmacokinetic study.The survey showed that pediatricians use a variety of unlicensed compounded adult medications at doses of between 0.5 and 5 mg. Infacort was formulated with a taste-masking layer stable for at least 5 minutes in aqueous media and was produced in unit doses of 0.5, 1, 2, and 5 mg. Infacort 10 mg is the bioequivalent of a 10-mg hydrocortisone tablet (mean area under the curve from zero to infinity [AUC(0-inf)] ratio, 101%; 90% confidence interval, 96-107%). Mean cortisol maximum concentration (C(max)) and AUC(0-inf) values after administration of Infacort were linear with dose and dose proportional when adjusted for saturable plasma protein binding. Subjects rated Infacort as "not good or bad" for smell (86%), feel in the mouth (71%), and taste (79%). No serious adverse events were reported.This phase 1 study demonstrates that Infacort is safe, well tolerated, of neutral taste, bioequivalent to hydrocortisone licensed for adults, and shows dose proportionality with respect to cortisol exposure. Infacort is expected to facilitate optimization of hydrocortisone dosing in neonates and children with adrenal insufficiency; however, clinical studies will be required to demonstrate efficacy in this patient age group.

Current treatment in Europe for adrenal insufficiency in neonates and infants is unsatisfactory as unlicensed adult dosage formulations are used. These are difficult to administer and may give rise to inconsistencies in dose as the content uniformity of the dosage form cannot be verified. As there is no licensed hydrocortisone formulation for children < 6 years hydrocortisone is often compounded by pharmacies using adult hydrocortisone tablets. In a recent study of compounded hydrocortisone up to 20% of the batches did not meet European Pharmacopeial accuracy and precision criteria (ECE 2014 Abstract #1278). This medication safety study investigated hydrocortisone individually and extemporaneously compounded for paediatric use in adrenally insufficient patients, meaning the current therapy is inadequate in up to every 5th child treated. Thus, there is a need for specifically designed and licensed hydrocortisone formulations for this vulnerable paediatric patient group especially neonates and infants.

Synthetic scaffold materials are used in tissue engineering for a variety of applications, including physical supports for the creation of functional tissues, protective gels to aid in wound healing and to encapsulate cells for localized hormone-delivery therapies. In order to encourage successful tissue growth, these scaffold materials must incorporate vital growth factors that are released to control their development. A major challenge lies in the requirement for these growth factor delivery mechanisms to mimic the in-vivo release profiles of factors produced during natural tissue morphogenesis or repair. This review highlights some of the major strategies for creating scaffold constructs reported thus far, along with the approaches taken to incorporate growth factors within the materials and the benefits of combining tissue engineering and drug delivery expertise.

We demonstrate that mammalian cells can survive for 5 min within high-pressure CO 2 . Cell survival was investigated by exposing a range of mammalian cell types to supercritical CO 2 (scCO 2 ) (35°C, 74 bar; 1 bar = 100 kPa) for increasing exposure and depressurization times. The myoblastic C2C12 cell line, 3T3 fibroblasts, chondrocytes, and hepatocytes all displayed appreciable but varying metabolic activity with exposure times up to 1 min. With depressurization times of 4 min, cell population metabolic activity was ≥70% of the control population. Based on survival data, we developed a single-step scCO 2 technique for the rapid production of biodegradable poly( dl -lactic acid) scaffolds containing mammalian cells. By using optimum cell-survival conditions, scCO 2 was used to process poly( dl -lactic acid) containing a cell suspension, and, upon pressure release, a polymer sponge containing viable mammalian cells was formed. Cell functionality was demonstrated by retention of an osteogenic response to bone morphogenetic protein-2 in C2C12 cells. A gene microarray analysis showed no statistically significant changes in gene expression across 4,418 genes by a single-class t test. A significance analysis of microarrays revealed only eight genes that were down-regulated based on a δ value of 1.0125 and a false detection rate of 0.

In this study, we use supercritical carbon dioxide as a processing medium for the fabrication of poly(DL-lactic acid) P(DLLA) microparticles that encapsulate a protein material. We have previously demonstrated that this polymer and a dry powder of a protein can be mixed under supercritical carbon dioxide conditions (above 31.1 degrees C and 73.8 bar) and that the protein component retains its biological activity. In this paper, we progress the work to demonstrate that the plasticized polymer and dry powder protein mixture can be sprayed to form solid polymer particles that encapsulate the protein. Particle size range is between 10 and 300 microm after spraying. Ribonuclease A and lysozyme were encapsulated in the polymer without significant loss of enzymatic activity. Biological assays of insulin and calcitonin confirm retention of activity after fabrication of the microparticles and release of the peptides/proteins.

Liquefying a polymer with supercritical carbon dioxide (scCO2) and then spraying the polymer/CO2 melt through a nozzle is a promising way to generate polymeric particles. This technique is of particular interest since the processing can be performed at lowered temperatures and in the absence of organic solvents. We use poly(ethylene glycol) (PEG) as the processing polymer to explore the general control principles for particle size and morphology for crystalline polymers. The morphology for sprayed particles can be controlled by manipulating the processing conditions. For example, decreasing processing pressure and increasing temperature favour the production of spherical particles. Furthermore, decreasing the bore diameter of the nozzle can increase the percentage of spherical particles in the product. Compared to particle morphology, the effect on particle size is less dramatic when either temperature, pressure or the bore diameter of the nozzle are altered. The spherical particles produced are mostly hollow in structure with a singe internal void and a thin outer crust.

This paper presents the results of work on the development and modification of laser methods of rapid prototyping and fabrication of individual implants and matrices for tissue engineering. A technology has been developed for shaping mineral-polymer bioactive structures, based on laser photopolymerization of a liquid mixture of polyfunctional acrylic oligomers with microparticles of hydroxyapatite. A method is proposed and developed for surface-selective laser sintering, making it possible to sinter polymeric microparticles without melting them completely, but only submelting their surface. This made it possible to obtain bioactive polymeric matrices based on bioresorbable aliphatic polyesters.