Topical Semisolid Drug Product Critical Quality Attributes with Relevance to Cutaneous Bioavailability and Pharmacokinetics: Part I-Bioequivalence of Acyclovir Topical Creams.

PURPOSE: To develop a toolkit of test methods for characterizing potentially critical quality attributes (CQAs) of topical semisolid products and to evaluate how CQAs influence the rate and extent of active ingredient bioavailability (BA) by monitoring cutaneous pharmacokinetics (PK) using an In Vitro Permeation Test (IVPT). METHODS: Product attributes representing the physicochemical and structural (Q3) arrangement of matter, such as attributes of particles and globules, were assessed for a set of test acyclovir creams (Aciclostad® and Acyclovir 1A Pharma) and compared to a set of reference acyclovir creams (Zovirax® US, Zovirax® UK and Zovirax® Australia). IVPT studies were performed with all these creams using heat-separated human epidermis, evaluated with both, static Franz-type diffusion cells and a flow through diffusion cell system. RESULTS: A toolkit developed to characterize quality and performance attributes of these acyclovir topical cream products identified certain differences in the Q3 attributes and the cutaneous PK of acyclovir between the test and reference sets of products. The cutaneous BA of acyclovir from the set of reference creams was substantially higher than from the set of test creams. CONCLUSIONS: This research elucidates how differences in the composition or manufacturing of product formulations can alter Q3 attributes that modulate myriad aspects of topical product performance. The results demonstrate the importance of understanding the Q3 attributes of topical semisolid drug products, and of developing appropriate product characterization tests. The toolkit developed here can be utilized to guide topical product development, and to mitigate the risk of differences in product performance, thereby supporting a demonstration of bioequivalence (BE) for prospective topical generic products and reducing the reliance on comparative clinical endpoint BE studies.

Deformable liposomes as enhancer of caffeine penetration through human skin in a Franz diffusion cell test.

OBJECTIVE: The permeation of hydrophilic molecules through the skin is still a challenge due to the barrier posed by stratum corneum, the outermost layer of the skin. Liposomes have frequently been used as carriers for different types of drugs and may also function as permeation enhancers. Propylene glycol has also been used as an edge activator in liposomes to increase the permeation. The aim of this work was to prepare liposomes containing an edge activator and loaded with caffeine to evaluate the potential of caffeine reaching the deeper layers in the skin. METHODS: The formulations were prepared by a top-down process using high-pressure homogenization at 200 00 psi for 10 min. They were characterized by size, polydispersity index (PI), zeta potential (ZP), pH, caffeine content and encapsulation efficiency (EE%) on preparation (time zero) and after 30 days. Cytotoxicity of blank and loaded liposomes was assessed by MTT proliferation assay with a normal keratinocyte cell line (HaCaT). In vitro permeation tests were performed with human skin in Franz cells over 24 h, and caffeine concentration was determined in the skin surface, stratum corneum, dermo-epidermal fraction and receptor medium by HPLC. RESULTS: The caffeine liposomes with (DL-Caf) or without propylene glycol (CL-Caf) showed, respectively, mean size 94.5 and 95.4 nm, PI 0.48 and 0.42, ZP + 1.3 and + 18.1 mV and caffeine content of 78.57 and 80.13%. IC50 values of caffeine in DL-Caf (3.59 v/v %) and CL-Caf (3.65 v/v %) were not significantly different from conventional blank liposome (3.27 v/v %). The DL-Caf formulation presented the best capability to enhance the caffeine permeation through the skin, resulting 1.94-folds higher than caffeine solution. Furthermore, the caffeine flux from DL-Caf was 1.56- and 3.05-folds higher than caffeine solution and CL-Caf, respectively. On the other hand, CL-Caf showed the lowest caffeine penetration revealing the importance of edge activator to aid hydrophilic drug penetration to all skin layers. CONCLUSION: The DL-Caf formulation tested was able to improve the permeation of caffeine through the stratum corneum and dermo-epidermal layers, suggesting that this delivery system may be effective for deep skin delivery of hydrophilic drugs.

OBJECTIF: La perm´eation de mol´ecules hydrophiles `a travers lapeau reste un d´efi en raison de la barri`ere oppos´ee par la couchecorn´ee, la couche la plus externe de la peau. Les liposomes ontfr´equemment ´et´e utilis´es comme supports pour diff´erents types dem´edicaments et peuvent ´egalement fonctionner comme des amplificateursde perm´eation. Le propyl`ene glycol a ´egalement ´et´e utilis´ecomme activateur dans les liposomes pour augmenter la perm´eation.Le but de ce travail ´etait de pr´eparer des liposomes contenantun activateur et charg´es de caf´eine pour ´evaluer le potentiel de lacaf´eine atteignant les couches les plus profondes de la peau. MÉTHODES: Les formulations sont pr´epar´ees par homog´en´eisationhaute pression `a 200 00 psi pendant 10 min. Elles sontcaract´eris´es par la taille des liposomes, l'indice de polydispersit´e(PI), le potentiel zˆeta (ZP), le pH, la teneur en caf´eine et l'efficacit´ed'encapsulation (EE%) `a la pr´eparation (temps z´ero) et apr`es 30jours. La cytotoxicit´e des liposomes `a blanc et charg´es est ´evalu´eepar un test de prolif´eration MTT avec une lign´ee cellulaire de k´eratinocytesnormale (HaCaT). Des tests de perm´eation in vitro sontr´ealis´es avec de la peau humaine dans des cellules de Franz pendant24 h, et la concentration de caf´eine est d´etermin´ee `a la surfacede la peau, dans la couche corn´ee, la fraction dermo-´epidermique et le milieu r´ecepteur par HPLC. RÉSULTATS: Les liposomes contenant de la caf´eine avec (DL-Caf)ou sans propyl`ene glycol (CL-Caf) pr´esentent respectivement unetaille moyenne de 94,5 et 95,4 nm, PI 0,48 et 0,42, ZP + 1,3 et +18,1 mV et une teneur en caf´eine de 78,57 et 80,13%. Les valeursIC50 de la caf´eine dans DL - Caf (3,59 %v/v) et CL - Caf (3,65 %v/v) ne sont pas significativement diff´erentes de celles du liposome `ablanc conventionnel (3,27 %v/v). La formulation DL-Caf est cellequi permet la meilleure perm´eation de la caf´eine, avec une quantit´ede caf´eine dans la peau 1,94 fois plus ´elev´ee que la solution decaf´eine. De plus, le flux de caf´eine de DL-Caf est 1,56 et 3,05 foisplus ´elev´e que la solution de caf´eine et CL-Caf, respectivement.D'autre part, CL-Caf montre la plus faible p´en´etration de caf´eine,r´ev´elant l'importance de l'activateur pour aider `a la p´en´etration dela mol´ecule hydrophile dans toutes les couches de la peau. CONCLUSION: La formulation DL-Caf test´ee am´eliore la perm´eationde la caf´eine `a travers la couche corn´ee et les couches dermo-´epidermiques, ce qui sugg`ere que ce syst`eme d'administration peutˆetre efficace pour l'administration cutan´ee profonde de mol´eculeshydrophiles.

Compartmental modeling of skin transport.

The primary objective of this study is to introduce a simple and flexible mathematical approach which models transport processes in skin using compartments. The main feature of the presented approach is that the rate constants for exchange between compartments are derived from physiologically relevant diffusional transport parameters. This allows for better physical interpretation of the rate constants, and limits the number of parameters for the compartmental model. The resulting compartmental solution is in good agreement with previously published solutions for the diffusion model of skin when ten or more compartments are used. It was found that the new compartmental model with three compartments provided a better fit of the previously publish water penetration data than the diffusion model. Two special cases for which it is difficult to implement the diffusion model were considered using our compartmental approach. In both cases the compartmental model predictions agreed well with the diffusion model.

Topical and cutaneous delivery using nanosystems.

The goal of topical and cutaneous delivery is to deliver therapeutic and other substances to a desired target site in the skin at appropriate doses to achieve a safe and efficacious outcome. Normally, however, when the stratum corneum is intact and the skin barrier is uncompromised, this is limited to molecules that are relatively lipophilic, small and uncharged, thereby excluding many potentially useful therapeutic peptides, proteins, vaccines, gene fragments or drug-carrying particles. In this review we will describe how nanosystems are being increasingly exploited for topical and cutaneous delivery, particularly for these previously difficult substances. This is also being driven by the development of novel technologies, which include minimally invasive delivery systems and more precise fabrication techniques. While there is a vast array of nanosystems under development and many undergoing advanced clinical trials, relatively few have achieved full translation to clinical practice. This slow uptake may be due, in part, to the need for a rigorous demonstration of safety in these new nanotechnologies. Some of the safety aspects associated with nanosystems will be considered in this review.

Human skin penetration and local effects of topical nano zinc oxide after occlusion and barrier impairment.

Public health concerns continue to exist over the safety of zinc oxide nanoparticles that are commonly used in sunscreen formulations. In this work, we assessed the effects of two conditions which may be encountered in everyday sunscreen use, occlusion and a compromised skin barrier, on the penetration and local toxicity of two topically applied zinc oxide nanoparticle products. Caprylic/capric triglyceride (CCT) suspensions of commercially used zinc oxide nanoparticles, either uncoated or with a silane coating, were applied to intact and barrier impaired skin of volunteers, without and with occlusion for a period of six hours. The exposure time was chosen to simulate normal in-use conditions. Multiphoton tomography with fluorescence lifetime imaging was used to noninvasively assess zinc oxide penetration and cellular metabolic changes that could be indicative of toxicity. We found that zinc oxide nanoparticles did not penetrate into the viable epidermis of intact or barrier impaired skin of volunteers, without or with occlusion. We also observed no apparent toxicity in the viable epidermis below the application sites. These findings were validated by ex vivo human skin studies in which zinc penetration was assessed by multiphoton tomography with fluorescence lifetime imaging as well as Zinpyr-1 staining and toxicity was assessed by MTS assays in zinc oxide treated skin cryosections. In conclusion, applications of zinc oxide nanoparticles under occlusive in-use conditions to volunteers are not associated with any measurable zinc oxide penetration into, or local toxicity in the viable epidermis below the application site.

Measurement of compartment elasticity using pressure related ultrasound: a method to identify patients with potential compartment syndrome.

PURPOSE OF THE STUDY Decision-making in treatment of an acute compartment syndrome is based on clinical assessment, supported by invasive monitoring. Thus, evolving compartment syndrome may require repeated pressure measurements. In suspected cases of potential compartment syndromes clinical assessment alone seems to be unreliable. The objective of this study was to investigate the feasibility of a non-invasive application estimating whole compartmental elasticity by ultrasound, which may improve accuracy of diagnostics. MATERIAL AND METHODS In an in-vitro model, using an artificial container simulating dimensions of the human anterior tibial compartment, intracompartmental pressures (p) were raised subsequently up to 80 mm Hg by infusion of saline solution. The compartmental depth (mm) in the cross-section view was measured before and after manual probe compression (100 mm Hg) upon the surface resulting in a linear compartmental displacement (Δd). This was repeated at rising compartmental pressures. The resulting displacements were related to the corresponding intra-compartmental pressures simulated in our model. A hypothesized relationship between pressures related compartmental displacement and the elasticity at elevated compartment pressures was investigated. RESULTS With rising compartmental pressures, a non-linear, reciprocal proportional relation between the displacement (mm) and the intra-compartmental pressure (mm Hg) occurred. The Pearson's coefficient showed a high correlation (r2 = -0.960). The intraobserver reliability value kappa resulted in a statistically high reliability (κ = 0.840). The inter-observer value indicated a fair reliability (κ = 0.640). CONCLUSIONS Our model reveals that a strong correlation between compartmental strain displacements assessed by ultrasound and the intra-compartmental pressure changes occurs. Further studies are required to prove whether this assessment is transferable to human muscle tissue. Determining the complete compartmental elasticity by ultrasound enhancement, this application may improve detection of early signs of potential compartment syndrome. Key words: compartment syndrome, intra-compartmental pressure, non-invasive diagnostic, elasticity measurement, elastography.

Iontophoresis-mediated transdermal permeation of peptide dendrimers across human epidermis.

PURPOSE OF THE STUDY: The overall aim of the present work was to elucidate the effects of iontophoresis on assisting permeation/deposition of peptide dendrimers across/within human skin. PROCEDURES: A series of peptide dendrimers containing arginine and histidine as terminal acids were synthesized and characterized. These dendrimers were subjected to passive and iontophoretic permeation studies across human epidermis. RESULTS: The synthesized peptide dendrimers were found to be stable in epidermal, dermal and skin extracts up to 6 h. Passive diffusion studies revealed that none of the synthesized peptide dendrimers permeated human epidermis up to 6 h, although minute concentrations of low molecular weight dendrimers were detected in receptor medium at the end of 24 h. Application of iontophoresis significantly increased the permeation of all the tested peptide dendrimers across human skin in a molecular weight-dependent manner compared to simple passive diffusion. Electromigration was found to be the dominant mechanism behind the iontophoretic permeation of peptide dendrimers across human skin. CONCLUSIONS: The present study demonstrates that iontophoresis is an effective technique in enhancing the transdermal permeation of peptide dendrimers. MESSAGE OF THE PAPER: This study foresees the possibility of applying peptide dendrimers in iontophoretic delivery of drugs and macromolecules across/within the skin.

Mortality following hip fracture: trends and geographical variations over the last 40 years.

Hip fractures are an ever increasing cause of morbidity and mortality. Treatment of this condition requires an all-encompassing approach from prevention to post-operative care. It is important in such a situation to gather data on the incidence and trends of hip fractures to aid in the future treatment planning of this important condition. A review of all articles published on the outcome after hip fracture over a four decade period (1959-1998) was undertaken to determine any changes that had occurred in the demographics of patients and mortality over this time period. The mean age of patients sustaining hip fractures was found to be steadily increasing over the study period at a rate of 1 year of age for every 5-year time period. The mean age in the 1960s was 73 years to a mean of 79 years in the 1990s. No notable differences were seen in the proportion of male patients over the years but a definite downward trend was noticed with regard to intracapsular fractures. The mortality at 6 and 12 months after injury remained essentially unchanged over the four decades reviewed. Mortality after a hip fracture remains significant, being 11-23% at 6 months and 22-29% at 1 year from injury. Geographical variations exist in the mortality after hip fracture. More detailed international comparisons are required to determine if these differences in outcome are accounted for by the variations in the demographics of patients or due to diversities in treatment methods.

Association between chronic fatigue syndrome and the corticosteroid-binding globulin gene ALA SER224 polymorphism.

Chronic fatigue syndrome (CFS) is characterized by idiopathic fatigue of greater than 6 months' duration with postexertional exacerbation and many other symptoms. A trend toward relative hypocortisolism is described in CFS. Twin and family studies indicate a substantial genetic etiologic component to CFS. Recently, severe corticosteroid-binding globulin (CBG) gene mutations have been associated with CFS in isolated kindreds. Human leukocyte elastase, an enzyme important in CBG catabolism at inflammatory sites, is reported to be elevated in CFS. We hypothesized that CBG gene polymorphisms may act as a genetic risk factor for CFS. A total of 248 patients with CFS defined by Centers for Disease Control criteria, and 248 controls were recruited. Sequencing and restriction enzyme testing of the CBG gene coding region allowed detection of severe CBG gene mutations and a common exon 3 polymorphism (c.825G-->T, Ala-Ser224). Plasma CBG levels were measured in 125 CFS patients and 198 controls by radioimmunoassay. Total and free (calculated and measured) cortisol levels were ascertained in single samples between 8-10 a.m. The age of onset (mid 30s) and gender ratio (2.2:1, female:male) of the patients were similar to those reported in U.S. epidemiologic studies. A trend toward a preponderance of serine224 homozygosity among the CFS patients was noted, compared with controls (chi2 = 5.31, P = 0.07). Immunoreactive-CBG (IR-CBG) levels were higher in Serine/Alanine (Ser/Ala) than Ala/Ala subjects and higher again in Ser/Ser subjects, this effect was strongest in controls; Ser/Ser: 46.1+/-1.8 (n = 31, P = 0.03) vs. Ser/Ala: 42.4+/-1.0 (n = 56, P = 0.05) vs. Ala/Ala: 40.8+/-1.7 microg/mL (n = 21). Despite higher CBG levels, there was a nonsignificant trend toward lower total and free plasma cortisol in serine allele positive patients, total cortisol: Ser/Ser: 13.3+/-1.4 (n = 34) vs. Ser/Ala: 14.0+/-0.7 (n = 66) vs. Ala/Ala: 15.4+/-1.0 (n = 23). Homozygosity for the serine allele of the CBG gene may predispose to CFS, perhaps due to an effect on hypothalamic-pituitary-adrenal axis function related to altered CBG-cortisol transport function or immune-cortisol interactions.

Early rise in blood pressure following administration of adrenocorticotropic hormone-[1-24] in humans.

1. An elevation in blood pressure has been consistently observed 24 h after adrenocorticotropic hormone (ACTH) administration and is caused by increased ACTH-stimulated cortisol secretion, in association with increased cardiac output. The aim of the present study was to investigate the previously undefined time of onset of this increase in blood pressure in normal humans. 2. Ten normal healthy volunteers received 250 mg ACTH-[1-24], in 500 mL normal saline, infused at a constant rate over 8 h. Six subjects also received a placebo infusion (normal saline only). Blood pressure, heart rate and cortisol levels were determined hourly. Adrenocorticotropic hormone (ACTH-[1-24] plus native ACTH) was measured at 0, 1, 7 and 8 h. 3. Infusion of ACTH-[1-24] produced maximal secretion rates of cortisol, resulting in a mean peak plasma level of 985 +/- 46 nmol/L at 8 h. In response, blood pressure and heart rate rose significantly by 2 h and remained generally elevated for the duration of the infusion. 4. The early onset of haemodynamic responses is consistent with classical steroid receptor-mediated genomic mechanisms, but could be due non-genomic mechanisms. 5. The cardiovascular consequences of therapeutic use of ACTH are well recognized. This results of the present study suggest that even diagnostic administration of ACTH, delivered over a few hours, may raise blood pressure.

Familial corticosteroid-binding globulin deficiency due to a novel null mutation: association with fatigue and relative hypotension.

Corticosteroid-binding globulin is a 383-amino acid glycoprotein that serves a hormone transport role and may have functions related to the stress response and inflammation. We describe a 39-member Italian-Australian family with a novel complete loss of function (null) mutation of the corticosteroid-binding globulin gene. A second, previously described, mutation (Lyon) segregated independently in the same kindred. The novel exon 2 mutation led to a premature termination codon corresponding to residue -12 of the procorticosteroid-binding globulin molecule (c.121G-->A). Among 32 family members there were 3 null homozygotes, 19 null heterozygotes, 2 compound heterozygotes, 3 Lyon heterozygotes, and 5 individuals without corticosteroid-binding globulin mutations. Plasma immunoreactive corticosteroid-binding globulin was undetectable in null homozygotes, and mean corticosteroid-binding globulin levels were reduced by approximately 50% at 18.7 +/- 1.3 microg/ml (reference range, 30-52 microg/ml) in null heterozygotes. Morning total plasma cortisol levels were less than 1.8 microg/dl in homozygotes and were positively correlated to the plasma corticosteroid-binding globulin level in heterozygotes. Homozygotes and heterozygote null mutation subjects had a high prevalence of hypotension and fatigue. Among 19 adults with the null mutation, the systolic blood pressure z-score was 12.1 +/- 3.5; 11 of 19 subjects (54%) had a systolic blood pressure below the third percentile. The mean diastolic blood pressure z-score was 18.1 +/- 3.4; 8 of 19 subjects (42%) had a diastolic blood pressure z-score below 10. Idiopathic chronic fatigue was present in 12 of 14 adult null heterozygote subjects (86%) and in 2 of 3 null homozygotes. Five cases met the Centers for Disease Control criteria for chronic fatigue syndrome. Fatigue questionnaires revealed scores of 25.1 +/- 2.5 in 18 adults with the mutation vs. 4.2 +/- 1.5 in 23 healthy controls (P < 0.0001). Compound heterozygosity for both mutations resulted in plasma cortisol levels comparable to those in null homozygotes. Abnormal corticosteroid-binding globulin concentrations or binding affinity may lead to the misdiagnosis of isolated ACTH deficiency. The mechanism of the association between fatigue and relative hypotension is not established by these studies. As idiopathic fatigue disorders are associated with relatively low plasma cortisol, abnormalities of corticosteroid-binding globulin may be pathogenic.

The insulin hypoglycemia test: hypoglycemic criteria and reproducibility.

The insulin hypoglycemia test (IHT) is widely regarded as the "gold standard" for dynamic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. This study aimed to investigate the temporal relationship between a rapid decrease in plasma glucose and the corresponding rise in plasma adenocorticotropic hormone (ACTH), and to assess the reproducibility of hormone responses to hypoglycemia in normal humans. Ten normal subjects underwent IHTs, using an insulin dose of 0.15 U/kg. Of these, eight had a second IHT (IHT2) and three went on to a third test (IHT3). Plasma ACTH and cortisol were measured at 15-min intervals and, additionally, in four IHT2s and the three IHT3s, ACTH was measured at 2.5- or 5-min intervals. Mean glucose nadirs and mean ACTH and cortisol responses were not significantly different between IHT1, IHT2 and IHT3. Combined data from all 21 tests showed the magnitude of the cortisol responses, but not the ACTH responses, correlated significantly with the depth and duration of hypoglycemia. All subjects achieved glucose concentrations of of < or = 1.6 mmol/l before any detectable rise in ACTH occurred. In the seven tests performed with frequent sampling, an ACTH rise never preceded the glucose nadir, but occurred at the nadir, or up to 15 min after. On repeat testing, peak ACTH levels varied markedly within individuals, whereas peak cortisol levels were more reproducible (mean coefficient of variation 7%). In conclusion, hypoglycemia of < or = 1.6 mmol/l was sufficient to cause stimulation of the HPA axis in all 21 IHTs conducted in normal subjects. Nonetheless, our data cannot reveal whether higher glucose nadirs would stimulate increased HPA axis activity in all subjects. Overall, the cortisol response to hypoglycemia is more reproducible than the ACTH response but, in an individual subject, the difference in peak cortisol between two IHTs may exceed 100 nmol/l.

Adrenocorticotropin stimulation tests in patients with hypothalamic-pituitary disease: low dose, standard high dose and 8-h infusion tests.

OBJECTIVES: Low doses of ACTH [1-24] (0.1, 0.5 and 1.0 microg per 1.73 m2) may provide a more physiological level of adrenal stimulation than the standard 250 microg test, but not all studies have concluded that the 1.0 microg is a more sensitive screening test for central hypoadrenalism. Eight-hour infusions of high dose ACTH [1-24] have also been suggested as a means of assessing the adrenals' capacity for sustained cortisol secretion. In this study, we compared the diagnostic accuracy of three low dose ACTH tests (LDTs) and the 8-h infusion with the standard 250 microg test (HDT) and the insulin hypoglycaemia test (IHT) in patients with hypothalamic-pituitary disease. SUBJECTS AND DESIGN: Three groups of subjects were studied. A healthy control group (group 1, n = 9) and 33 patients with known hypothalamic or pituitary disease who were divided into group 2 (n = 12, underwent IHT) and group 3 (n = 21, IHT contraindicated). Six different tests were performed: a standard IHT (0.15 U/kg soluble insulin); a 60-minute 250 microg HDT; three different LDTs using 0.1 microg, 0.5 microg and 1.0 microg (all per 1.73 m2); and an 8-h infusion test (250 microg ACTH [1-24] at a constant rate over 8 h). RESULTS: Nine out of the 12 patients in group 2 failed the IHT. Three out of 12 patients from group 2 who clearly passed the IHT, also passed all the ACTH [1-24] stimulation tests. Seven of the 9 patients who failed the IHT, failed by a clear margin (peak cortisol < 85% of the lowest normal). Two of the 7 also failed all the ACTH [1-24] tests. Five of the 7 patients had discordant results, four passed the 0.1 LDT, one (out of four) passed the 0.5 LDT, none (out of three) passed the 1.0 LDT, two passed the HDT and three passed the 8-h test. Two patients were regarded as borderline fails in the IHT. Both passed the ACTH [1-24] tests, although one was a borderline pass in the 8-h test. Only five out of the 21 patients in group 3 showed discordance between the HDT and the LDTs. One patient passed the HDT and failed the 0.1 LDT, four patients failed the HDT but passed some of the different LDTS. CONCLUSIONS: We conclude that in the diagnosis of central hypoadrenalism, ACTH [1-24] stimulation tests may give misleading results compared to the IHT. The use of low bolus doses of ACTH [1-24] (1.0, 0.5 or 0.1 microg) or a high dose prolonged infusion does not greatly improve the sensitivity of ACTH [1-24] testing. Dynamic tests that provide a central stimulus remain preferable in the assessment of patients with suspected ACTH deficiency.

Interactions between the stimulated hypothalamic-pituitary-adrenal axis and leptin in humans.

Leptin, produced by adipocytes, has homeostatic effects on body fat mass through inhibition of appetite and stimulation of the sympathetic nervous system. Several studies have reported that high-dose exogenous glucocorticoids increase circulating leptin concentrations in humans. Conversely, leptin has inhibitory effects on the hypothalamic-pituitary-adrenal (HPA) axis, both at the hypothalamic and adrenal levels. We hypothesized that acute hypercortisolism, in the physiological range, may not alter leptin secretion. Four stimuli of the HPA axis were administered to eight healthy male volunteers in a placebo-controlled study. On separate afternoons, in a randomised order, fasting subjects received i.v. injections of saline, naloxone (125 microg/kg); vasopressin (0.0143 IU/kg); naloxone and vasopressin in combination; or insulin (0.15 U/kg; a dose sufficient to induce hypoglycaemia). Plasma concentrations of adrenocorticotrophic hormone (ACTH), cortisol and leptin were measured before and for 120 min after the injection. The cortisol secretory response was greatest after insulin-hypoglycaemia, this response was significantly greater than that following naloxone, naloxone/vasopressin, or vasopressin alone. Despite the cortisol release, leptin concentrations were not increased after any stimulus. Insulin-hypoglycaemia was associated with a decrease in leptin concentration at 60 and 90 min, while naloxone did not alter leptin concentrations. However, basal leptin concentrations were positively correlated with integrated ACTH and cortisol responses to naloxone, but did not correlate with ACTH or cortisol responses to the other stimuli. Thus acute elevations of plasma cortisol, in the physiological range, do not appear to influence plasma leptin concentrations. The fall in plasma leptin concentration after insulin-induced hypoglycaemia may reflect catecholamine secretion after this stimulus.

Comparison of adrenocorticotropin (ACTH) stimulation tests and insulin hypoglycemia in normal humans: low dose, standard high dose, and 8-hour ACTH-(1-24) infusion tests.

The efficacy of the standard high dose ACTH stimulation test (HDT), using a pharmacological 250-microg dose of synthetic ACTH-(1-24), in the diagnosis of central hypoadrenalism is controversial. The insulin hypoglycemia test is widely regarded as the gold standard dynamic stimulation test of the hypothalamo-pituitary-adrenal (HPA) axis that provides the most reliable assessment of HPA axis integrity and reserve. Alternatively, a prolonged infusion of ACTH causes a continuing rise in plasma cortisol levels that may predict the adrenals' capacity to respond to severe ongoing stress. In nine normal subjects, we compared plasma ACTH and cortisol levels produced by three i.v. bolus low doses of ACTH-(1-24) (0.1, 0.5, and 1.0 microg/1.73 m2; LDTs) with those stimulated by hypoglycemia (0.15 U/kg insulin) and with the cortisol response to a standard 250-microg dose of ACTH-(1-24). The normal cortisol response to an 8-h ACTH-(1-24) infusion (250 microg at a constant rate over 8 h) was determined using three modern cortisol assays: a high pressure liquid chromatography method (HPLC), a fluorescence polarization immunoassay (FPIA), and a standard RIA. In the LDTs, stepwise increases in mean peak plasma ACTH were observed (12.4 +/- 2.0, 48.2 +/- 7.2, 120.2 +/- 15.5 pmol/L for the 0.1-, 0.5-, and 1.0-microg LDTs, respectively; P values all <0.0022 when comparing peak values between tests). The peak plasma ACTH level after insulin-induced hypoglycemia was significantly lower than that produced in the 1.0-microg LDT (69.6 +/- 9.3 vs. 120.2 +/- 15.5 pmol/L; P < 0.0002), but was higher than that obtained during the 0.5-microg LDT (69.6 +/- 9.3 vs. 48.2 +/- 7.2 pmol/L; P < 0.02). In the LDTs, statistically different, dose-dependent increases in peak cortisol concentration occurred (355 +/- 16, 432 +/- 13, and 482 +/- 23 nmol/L; greatest P value is 0.0283 for comparisons between all tests). The peak cortisol levels achieved during the LDTs were very different from those during the HDT (mean peak cortisol, 580 +/- 27 nmol/L; all P values <0.00009. However, the mean 30 min response in the 1.0-microg LDT did not differ from that in the HDT (471 +/- 22 vs. 492 +/- 22 nmol/L; P = 0.2). In the 8-h ACTH infusion test, plasma cortisol concentrations progressively increased, reaching peak levels much higher than those in the HDT [995 +/- 50 vs. 580 +/- 27 nmol/L (HPLC) and 1326 +/- 100 vs 759 +/- 31 nmol/L (FPIA)]. Significant differences in the basal, 1 h, and peak cortisol levels as determined by the three different assay methods (HPLC, FPIA, and RIA) were observed in the 8-h infusion tests. Similarly, in the HDTs there were significant differences in the mean 30 and 60 min cortisol levels as measured by HPLC compared with those determined by FPIA. We conclude that up to 30 min postinjection, 1.0 microg/1.73 m2 ACTH-(1-24) stimulates maximal adrenocortical secretion. Similar lower normal limits at 30 min may be applied in the 1.0-microg LDT and the HDT, but not when lower doses of ACTH-(1-24) are administered. The peak plasma ACTH level produced in the 1.0-microg LDT is higher than in the insulin hypoglycemia test, but is of the same order of magnitude. The peak cortisol concentration obtained during an 8-h synthetic ACTH-(1-24) infusion is considerably higher than that stimulated by a standard bolus 250-microg dose, potentially providing a means of evaluating the adrenocortical capacity to maintain maximal cortisol secretion. Appropriate interpretation of any of these tests of HPA axis function relies on the accurate determination of normal response ranges, which may vary significantly depending on the cortisol assay used.

Pituitary-adrenal responses to combined oral D-fenfluramine and intravenous naloxone in humans.

1. 1. Fenfluramine is an optically active 5-hydroxytryptamine (5-HT) releaser and re-uptake inhibitor. Increased brain 5-HT mediates appetite suppression, the D enantiomer being more active than L- or DL-fenfluramine. Fenfluramine also stimulates the hypothalamic-pituitary-adrenal (HPA) axis, leading to suggestions that this could act as a marker for its biological actions. However, the D enantiomer appears less active than a comparable DL racemate dose in animals, while effects of D-fenfluramine on the human HPA axis remain unproven. The aim of the present study was to clarify this. 2. Seven healthy human volunteers (three male, four female; 18-58 years) received 30 mg oral D-fenfluramine or placebo, followed by 125 micrograms/kg, i.v. naloxone or placebo, in randomized, double-blinded, placebo-controlled afternoon studies. We measured plasma adrenocorticotropic hormone (ACTH) and cortisol levels in samples taken at intervals throughout the study period. 3. In contrast to previous results with DL-fenfluramine, we found no dynamic responses to D-fenfluramine alone and no augmentation of responses to naloxone. 4. Central pathways to HPA axis activation are apparently not stimulated by D-fenfluramine at this dose in humans, in contrast with DL-fenfluramine, where the L enantiomer may be more selective for proposed corticotropin-releasing hormone-mediated, post-synaptic 5-HT2 or noradrenergic mechanisms. As previously reported, D-fenfluramine significantly blunted the circadian fall in basal plasma cortisol, providing in vivo evidence for serotonergic involvement in circadian regulation.

Inhibition of naloxone-stimulated adrenocorticotropin release by alprazolam in myotonic dystrophy patients.

Myotonic dystrophy (DM) is an autosomal dominant disorder causing myotonia, progressive muscle weakness, and endocrine abnormalities including hypothalamic-pituitary-adrenal (HPA) axis hyperresponsiveness to CRH-mediated stimuli. This ACTH hyperresponsiveness appears directly related to the underlying genetic abnormality. Naloxone (Nal)-mediated CRH release causes ACTH release in normal humans and an ACTH hyperresponse in DM. Alprazolam (APZ) attenuates the ACTH release in response to Nal in normal individuals, probably by inhibiting CRH release. This study investigates the effects of APZ on Nal-induced HPA axis stimulation in DM. The ACTH response to Nal in DM subjects was significantly reduced by APZ. Despite this DM patients have a relative resistance to APZ inhibition of Nal-induced ACTH/cortisol release. APZ caused a smaller percentage reduction in AUC for ACTH in DM compared with controls. These findings provide further insight into the mechanism(s) of the HPA axis abnormalities in DM. In DM, there may be an increase in tonic opioid inhibition to CRH release with compensatory increases in stimulatory pathways. Alternatively, these patients may have a basal increase in pituitary vasopressin levels or an enhanced AVP/CRH synergistic mechanism at the level of the corticotroph.

Diurnal effects of fluoxetine and naloxone on the human hypothalamic-pituitary-adrenal axis.

1. Central serotonergic pathways are hypothesized to be involved in the stimulation of hypothalamic adrenocorticotropic hormone (ACTH) secretagogue release by both circadian- and stress-induced mechanisms. We aimed to investigate this hypothesis by measuring the effect of the highly specific serotonin re-uptake inhibitor fluoxetine (FX) on ACTH and cortisol release in the morning and in the afternoon in humans, both by itself and in combination with the opioid antagonist naloxone (Nal). Naloxone causes ACTH release in humans by removing an endogenous inhibitory opioid tone on central noradrenergic pathways stimulatory to hypothalamic corticotropin-releasing hormone (CRH) secretion. Serotonergic agents may act directly or indirectly through these central noradrenergic pathways and, if so, would be expected to be additive to or synergistic with Nal in causing ACTH and cortisol release. 2. Oral FX (40 mg) was given at approximately 07.00 or 11.00 h, either alone or with intravenous Nal 3 h later, to normal human volunteers. Plasma ACTH and cortisol levels were measured for 5 h after FX dosing. 3. Fluoxetine produced a small but non-significant increase in Nal-stimulated ACTH and cortisol release in both morning and afternoon studies. Naloxone alone did not cause different ACTH and cortisol responses in the morning and afternoon. 4. These results suggest that serotonergic pathways are not major regulators of the hypothalamic-pituitary-adrenal axis in humans or that FX has counteracting acute inhibitory effects on the axis, such as inhibition of hypothalamic arginine vasopressin secretion, which has been demonstrated in chronic animal studies.

Aspirin inhibits vasopressin-induced hypothalamic-pituitary-adrenal activity in normal humans.

PGs influence ACTH secretion. However, their specific role in modulating the activity of the human hypothalamic-pituitary-adrenal (HPA) axis remains unclear. Acetylsalicylic acid (aspirin) inhibits the synthesis of PGs from arachidonic acid by blocking the cyclooxygenase pathway. In this study we administered a single, clinically relevant dose of aspirin before HPA axis stimulation by a bolus dose of iv arginine vasopressin (AVP) to seven normal males using a randomized, placebo-controlled, single blinded design. Aspirin significantly reduced the cortisol response to AVP [mean peak increase from basal, 221.1 +/- 20.1 vs. 165.4 +/- 22.5 nmol/L (P = 0.0456); mean integrated response, 11,199.3 +/- 1,560.0 vs. 6,162.3 +/- 1,398.6 nmol.min/L (P = 0.0116) for placebo aspirin/AVP and aspirin/ AVP, respectively]. The ACTH response was reduced, but did not reach statistical significance [mean peak increase from basal, 7.5 +/- 2.2 vs. 4.3 +/- 0.3 pmol/L (P = 0.0563); mean integrated response, 142.6 +/- 36.0 vs. 96.2 +/- 8.7 pmol.min/L (P = 0.12) for placebo aspirin/ AVP and aspirin/AVP, respectively]. PGs may influence ACTH secretion by being stimulatory or inhibitory to the HPA axis at different levels, such as hypothalamic or pituitary. Which effect predominates in vivo during dynamic activation of the axis may depend on the level at which the secretory stimulus acts. We showed that when normal male volunteers were treated with the PG synthesis inhibitor, aspirin, they had a blunted HPA axis response to the pituitary corticotroph stimulator, AVP.