Patent Description:
Any references in the description to diagnostic methods practised on the human or animal body refer to the compounds and pharmaceutical compositions of the present invention for use in said methods.

The adrenal glands are above the kidneys and are part of the body's endocrine system. They produce a number of different hormones; those involved in metabolism (cortisol), salt and water balance (aldosterone) and sex steroids (oestrogen and testosterone). Cortisol release is regulated by interaction and feedback with the hormones of the hypothalamus and the anterior pituitary gland. Corticotropin releasing hormone (CRH) produced by the hypothalamus stimulates adrenocorticotropin (ACTH) production in the pituitary gland which in turn stimulates the production of cortisol in the adrenal cortex of the adrenal gland. Cortisol has itself a negative feedback on the hypothalamus and the pituitary gland inhibiting the production of CRH and ACTH.

Cortisol is referred to as a stress hormone and has several other physiological functions such as maintaining blood pressure, regulating protein, carbohydrate and fat metabolism, regulating the effects of insulin and influencing the immune system's inflammatory responses. Adrenal insufficiency is a rare endocrine disorder and can affect people of all ages and sex and is characterised by insufficient production of cortisol (and in some cases aldosterone) causing symptoms such as muscle weakness, loss of weight, low mood, cramps and exhaustion, and has pronounced effects on mental and physical development in children. Adrenal insufficiency (Al) is caused by an impaired function of either i) the adrenal glands (primary adrenal insufficiency), ii) the pituitary gland (secondary adrenal insufficiency) or iii) the hypothalamus (tertiary adrenal insufficiency) and can present with the slow but progressive loss of cortisol (and in some cases aldosterone) resulting in steadily worsening symptoms which, if undiagnosed and not treated with adequate hormone replacement therapy, can ultimately result in adrenal crises, a potential life threatening condition requiring immediate emergency treatment with hydrocortisone.

The most common cause in adults from the developed world is autoimmune destruction of the adrenal gland but worldwide it is tuberculosis. The commonest paediatric cause is the prescription of steroid medication. Adrenal insufficiency is usually permanent but may be transient, especially in children, and therefore periodic diagnostic testing is required in some patients (for example weekly, monthly or <NUM>-monthly).

A diagnosis of adrenal insufficiency can be confirmed by the short synacthen test (SST) using the synthetic analogue of ACTH, tetracosactide, to monitor cortisol production. The test is typically conducted at a hospital where a blood sample is obtained from the patient to determine the amount of cortisol present in the blood. Then a dose of tetracosactide is injected to stimulate the adrenal glands and after a short period of time, such as after <NUM>, <NUM> and/or <NUM>, cortisol levels in the blood are measured again to monitor the response of the adrenal glands to the stimulant. A failure to produce cortisol in response to the stimulant can be indicative of adrenal insufficiency and may provide information of the interaction of hypothalamus, pituitary and adrenal glands.

In recent years requests for SSTs have risen in line with increased paediatric steroid usage and heightened awareness of the adrenal insufficiency steroids can cause. However, although the SST is a common diagnostic method for adrenal insufficiency, cannulation and blood sampling are required making it invasive, time-consuming and resource-intensive, which, when considering children in particular, is not desirable.

The disclosure relates to compositions comprising tetracosactide and chitosan suitable for nasal administration and to a diagnostic test for detecting adrenal insufficiency comprising the delivery of said tetracosactide composition to the nasal cavity which allows measurement of the cortisol response of a subject in the saliva in a non-invasive manner.

We disclose but do not claim a liquid pharmaceutical composition adapted for nasal administration comprising adrenocorticotropic hormone (ACTH) or a synthetic ACTH analogue, a bioadhesive excipient and including one or more other pharmaceutical excipients.

According to an aspect of the invention there is provided a liquid pharmaceutical composition adapted for nasal administration comprising an effective dose of adrenocorticotropic hormone (ACTH) or a synthetic ACTH analogue selected from: tetracosactide, tetracosactide acetate or SEQ ID NO <NUM>, a bioadhesive excipient and including one or more other pharmaceutical excipients for use in a method of diagnosis of adrenal insufficiency in a paediatric subject, wherein the bioadhesive excipient is chitosan and wherein the method comprises application of the composition to the nasal cavity.

ACTH is used in treatment and as a diagnostic agent. Synthetic forms of ACTH are also available, most notably in the form of tetracosactide which is also known as tetracisactrin, cosyntropin and the acetate ester tetracosactide acetate or tetracisactrin acetate.

Synthetic analogues consist typically of the first <NUM> amino acids of ACTH and retain full function of the native peptide hormone. Other known analogues consist of the first <NUM> or <NUM> amino acids of SEQ ID NO <NUM> and retain the full function of the native peptide hormone.

In a preferred embodiment of the invention said analogue is tetracosactide, preferably tetracosactide acetate.

Suitably tetracosactide has the molecular formula C<NUM>H<NUM>N<NUM>O<NUM>S and a molecular weight of <NUM>/mol.

Suitably, said tetracosactide acetate comprises between <NUM>-<NUM> mole of acetic acid per mole of peptide.

Tetracosactide stimulates the release of corticosteroids such as cortisol from the adrenal glands, and is used for the ACTH stimulation test to assess adrenal gland function.

Bioadhesives or mucoadhesives may prolong the residence time of a drug dosage form at the site of absorption, for example the nasal mucosa, and in turn may be able to enhance the absorption and subsequently the efficacy of the drug.

According to the invention said bioadhesive excipient is chitosan. Chitosan, is a polysaccharide derived from chitin by partial deacetylation of poly-N-acetyl-D-glucosamine comprising β-(<NUM>→<NUM>)-linked <NUM>-acetamido-<NUM>-dexoy-D-glucopyranose and β-(<NUM>→<NUM>)-linked N-acetyl-D-glucosamine units. Chitosan may facilitate paracellular transport of large polar compounds.

Suitably, said chitosan is deacetylated between <NUM>-<NUM> %, more suitably between <NUM>-<NUM>%, even more suitably between <NUM>-<NUM>%. Suitably said chitosan is deacetylated between <NUM>-<NUM>% or <NUM>-<NUM>% more suitable chitosan is deacetylated to <NUM>% or more suitably to <NUM>%.

Pharmaceutically acceptable salt forms of chitosan are selected from the group consisting of hydrochloride, lactate, glutamate, maleate, acetate, formate, propionate, malate, malonate, adipate, succinate and nitrate.

In a preferred embodiment said chitosan is chitosan glutamate.

The chitosan glutamate preferably has a molecular weight of between <NUM>-<NUM>/mol. The molecular weight may be more conveniently expressed in terms of the viscosity of a <NUM>% solution of chitosan (or its salt) in <NUM>% acetic acid in water. The preferred molecular weight according to the present invention results in a viscosity ranging from <NUM> to <NUM> mPa. s, especially from <NUM> to <NUM> mPa. s, more particularly from <NUM> mPa. s to <NUM> mPa. When using chitosan with a molecular weight resulting in a viscosity of a <NUM>% solution in <NUM>% acetic acid in water lower than <NUM> mPa. s the absorption enhancing effect of chitosan is considered to be strongly reduced. When using chitosan with a molecular weight resulting in a viscosity of a <NUM>% solution in <NUM>% acetic acid in water higher than <NUM> mPa. s the resulting final composition may be too viscous to conveniently administer.

In a further preferred embodiment of the invention said chitosan is unmodified.

In an alternative embodiment of the invention said chitosan is modified.

In a further embodiment of the invention said modified chitosan is a quaternary chitosan or thiolated chitosan.

The term "chitosan" is used in this application to encompass chitosan in unmodified, modified and salt forms such as for example chitosan glutamate.

The pH range in which chitosan or its salts is soluble depends upon the deacetylation grade of the chitosan. The lower the deacetylation of the chitosan the higher the pH can be at which the chitosan remains soluble.

The pH of the composition according to the present invention may range from <NUM> to <NUM>. The pH of the composition comprising the preferred form of chitosan may range from <NUM> to <NUM> or from <NUM> to <NUM>. Preferably the pH range of the composition is from <NUM> to <NUM>. The preferred range of pH is between <NUM> and <NUM>.

In a preferred embodiment said composition comprises chitosan at a concentration of between <NUM>- <NUM> % (w/v), more suitably between <NUM>-<NUM> % (w/v) or <NUM>-<NUM>% (w/v).

Suitably, said composition comprises chitosan at a concentration of between <NUM> and <NUM> % (w/v).

Suitably said composition comprises chitosan at a concentration of <NUM> % (w/v).

In a preferred embodiment of the invention said composition further comprises tonicity adjusting agents, preservatives and acids and buffers to adjust and/ or maintain pH.

In a preferred embodiment of the invention said tonicity adjusting agents are selected from the group consisting of sodium chloride, glucose, dextrose, mannitol, sorbitol or lactose.

In a further preferred embodiment of the invention said tonicity adjusting agents is sodium chloride.

The tonicity of the composition should approximately be equal to the tonicity of plasma.

In a preferred embodiment of the invention said composition comprises preservatives selected from the group of quaternary ammonium salts such as lauralkonium chloride, benzalkonium chloride, benzododecinium chloride, cetyl pyridium chloride, cetrimide, domiphen bromide; alcohols such as benzyl alcohol, chlorobutanol, o-cresol, phenyl ethyl alcohol; organic acids or salts thereof such as benzoic acid, sodium benzoate, potassium sorbate, parabens; or complex forming agents such as EDTA.

In a preferred embodiment of the invention said preservative is benzalkonium chloride.

In a further preferred embodiment of the invention said buffer comprises sodium acetate.

In a further preferred embodiment of the invention said acid is selected from the group consisting of hydrochloric acid, lactic acid, glutamic acid, maleic acid, acetic acid, formic acid, propionic acid, malic acid, malonic acid, adipic acid, succinic acid or nitric acid.

In a further preferred embodiment of the invention said acid is acetic acid.

In a preferred embodiment of the invention said composition comprises of the following components:.

In a preferred embodiment of the invention said composition comprises between <NUM>-<NUM>/ml tetracosactide acetate.

In a preferred embodiment said composition is administered at an effective dose of between <NUM>-<NUM>µg tetracosactide acetate.

In a preferred embodiment said composition is administered at a dose between <NUM>-<NUM>µg tetracosactide acetate.

In a preferred embodiment said composition is administered at an effective dose selected form the group consisting of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>µg tetracosactide acetate.

In a preferred embodiment said composition is administered at an effective dose of <NUM>µg tetracosactide acetate.

We disclose but do not claim that said effective dose is administered once in <NUM>.

The intranasal composition according to the invention can form an aerosol or is applied to the nasal cavity as a liquid. The property to form an aerosol mainly depends upon the viscosity of the composition. When the composition is too viscous, the composition will not allow the formation of a spray. It can be sprayed and still the droplets reside long enough in the nasal cavity to allow for a good bioavailability. The viscosity of the solution may range between <NUM>-<NUM> mPa. The compositions preferably adhere to the mucosa, at least to some extent, and this facilitates retention of the composition of the mucosa and/or enhances the absorption of the active ingredient.

In a preferred embodiment of the invention said adrenal insufficiency is caused by a condition selected from the group consisting of: primary or secondary or tertiary adrenal failure examples of which include congenital adrenal hyperplasia, late-onset congenital adrenal hyperplasia, glucocorticoid-remediable aldosteronism (GRA), Addison's disease or tuberculosis.

In a preferred embodiment of the invention said paediatric subject includes neonates (<NUM>-<NUM> days old), infants (<NUM> - <NUM> months old), young children (<NUM> months - <NUM> years old), prepubescent [<NUM>-<NUM> years old] or adolescents [<NUM>-<NUM>].

In a preferred embodiment of the invention said composition is for use in a method of diagnosis of adrenal insufficiency in a paediatric subject comprising the following steps:.

In a further preferred method of the invention said paediatric subject is selected from the group consisting of neonates, infants, children or adolescents.

In a preferred method of the invention step i) is repeated <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and/or <NUM> after intranasal administration of said composition according to the invention.

In a preferred method of the invention step i) is repeated <NUM>, <NUM>, <NUM> or <NUM> times after intranasal administration of said composition according to the invention.

In a further preferred method of the invention step i) is repeated at frequent intervals such as <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>-minute intervals after intranasal administration of said composition according to the invention. Step i) can also be repeated at <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>-minute intervals after intranasal administration of said composition according to the invention which are non-linear e.g. the second sample is taken after <NUM>, the third sample taken after <NUM> minutes and the forth sample taken after <NUM> from the time point when the first sample is taken.

In an alternative further preferred method of the invention step i) is repeated at frequent intervals such as <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>-minute intervals after intranasal administration of said composition according to the invention.

We disclose but do not claim a device comprising the pharmaceutical composition according to the invention wherein said device is adapted to deliver said composition as an aerosol or in liquid form.

Preferably, said composition is delivered into the nasal cavity.

Preferably said device is selected from the group consisting of pipettes, syringes, nasal spray pumps, dropper bottles, squeeze bottles, metered-dose spray pumps, single-dose spray devices, nasal pressurized metered-dose inhalers, mucosal atomiser devices or nebulisers.

We disclose but do not claim a kit comprising a container for collecting saliva, a composition according to the invention comprising ACTH or an ACTH synthetic analogue.

"AUC(<NUM>-t)" is the area under the concentration time curve from time zero until the last quantifiable time point i.e. <NUM> minutes.

"AUC(<NUM>-∞)" area under the concentration time curve from time zero until infinity.

"Bioavailability (F)" is concerned with both the amount of drug present in the systemic circulation and the rate of systemic absorption. Bioavailability is often measured by comparing administration via a non-intravenous route with the i. Absolute bioavailability is dependent on the dose and the area under the concentration-time profiles (AUC) following administration via the route of interest and i. v route (<FIG>. Two different formulations of the same drug may have the same bioavailability as measured by the AUC but one formulation may release drug more quickly compared to the other, resulting in a higher initial concentration (Cmax) at an earlier time (Tmax), therefore the formulations would not be bioequivalent. In this study proving bioequivalence is not necessary but enough of the intranasal dose must be absorbed and rapidly enough to produce an equivalent cortisol response to the <NUM> mcg i.

"Consisting essentially" means having the essential integers but including integers which do not materially affect the function of the essential integers.

An embodiment of the invention will now be described by example only and with reference to the following figures:.

All used healthy adult, male volunteers.

The materials and methods below are from NeSST (<NUM>st study). I have indicated when differences between the studies are relevant.

These were four pharmacokinetic (PK) studies as described above (studies <NUM>-<NUM> & <NUM>). All were open-label. A crossover design was used, such that the same individual received the IV comparator and IN formulations. STUDY <NUM> was a repeatability PK study and therefore no IV comparator was used. The crossover design, where between occasion variables are minimized (e.g. fasting or fed conditions, time of day, concomitant medication), is the recommended methodology for generating bioequivalence data (European Medicines Agency <NUM>). The study design was based on that recommended for bioequivalence studies, although this was a bioavailability study. It was considered unnecessary to demonstrate that the two formulations (i. n) have the same (bioequivalence) but instead show that an adequate amount of Synacthen is absorbed to produce equivalence in the resultant cortisol response (Table <NUM> and <NUM>).

In keeping with pharmacokinetic trials of this kind the subjects were neither randomized nor blinded and did not receive a placebo. Studies <NUM>,<NUM>,<NUM> + <NUM> were conducted from Sheffield Children's NHS Foundation Trust (SCH), Sheffield, UK and studies <NUM>+<NUM> from Sheffield Teaching Hospitals NHS Trust, Sheffield, UK.

The participants for studies <NUM>-<NUM> were healthy, male volunteers between the ages <NUM>-<NUM>. The healthy children in study <NUM> were aged between <NUM>-<NUM> years. European Medicines Agency (EMA) guidelines were followed and only adult, male volunteers were enrolled in the initial studies due to the possible adverse effects of the drug on a fetus or child and to reduce variability not attributable to the difference between routes of administration. EMA guidance states that "this model, in vivo healthy volunteers, is regarded as adequate in most instances to detect formulation differences and to allow extrapolation of the results to populations for which the reference medicinal product is approved (the elderly, children, patients with renal or liver impairment, etc.)".

European Medicines Agency guidance was followed and <NUM> subjects were selected as the minimum number for a bioavailability study.

Basic demographic data was collected on all volunteers at visit one. This included age and ethnic group. Volunteers had their height and weight measured. This allowed calculation of body mass index (BMI) and body surface area (BSA) to enable volume of distribution calculations for Synacthen.

At each visit subjects were asked to report coryzal symptoms beginning within <NUM> hours of the test (which may impact on nasal absorption due to changes in the mucosa) and this was additionally checked on all subsequent visits.

Paired samples of blood and saliva were taken throughout each visit. These were analysed for plasma Synacthen and serum cortisol and salivary cortisol and cortisone levels.

Following recruitment, subjects were asked to attend the Children's Clinical Research Facility (CCRF) at SCH or RHH for their visits, each separated by a minimum of a week (fortnight in study <NUM>). This allows for a sufficient washout period between administrations, at least <NUM>-half lives of the drug.

Prior to each visit volunteers were asked to abstain from alcohol and recreational drugs for <NUM> hours and to take <NUM> of dexamethasone on retiring the night before the visit and a second <NUM> dose after breakfast on the morning of the visit (<NUM> if child younger than <NUM> years). There are no commercially available Synacthen assays, or any for research purposes, and therefore it was necessary to temporarily suppress the volunteers' endogenous production of ACTH to enable use of an ACTH assay, with the inference that anything detected was Synacthen.

Each visit commenced between <NUM> and <NUM>. Volunteers were not required to fast. v cannula was sited and the volunteer was asked to rest, lying down, for <NUM> minutes to recover from the physiological stress of cannulation. Ten-fifteen minutes before the first samples were taken the subject was asked to rinse their mouth thoroughly with water to minimise contamination that may compromise salivary cortisol assay performance.

Additionally it was requested that they refrain from eating or drinking, other than water, during the visit. The volunteers were asked to remain supine for the duration of the test.

Nasal Synacthen was administered by atomiser syringe (Mucosal atomizer device™, Wolfe Tory Medical Inc. Utah, USA). The Mucosal atomizer device™ (MAD) atomises liquids to <NUM>-<NUM> microns allowing rapid absorption into the bloodstream.

Between <NUM>-<NUM> mls were given up each nostril per administration:.

The pharmacokinetic, metabolic, toxicological and clinical properties of tetracosactide following i. v injection are well known and are described in the Summary of Product Characteristics. Tetracosactide nasal solution (TNS) has the same active pharmaceutical ingredient but with the addition of an excipient, chitosan, to aid absorption when administered nasally.

Pharmacokinetic extrapolation using NeSST Study (study <NUM>) AUC data estimated that <NUM> mcg of intranasal Synacthen would be required to give an equivalent response to the <NUM> mcg i. TNS is an aqueous solution containing either <NUM>/ml (providing <NUM> mcg tetracosactide) or <NUM>/ml (containing <NUM> mcg of tetracosactide) in the form of the acetate salt. In order to deliver <NUM> mcg and <NUM> mcg one spray (of <NUM>) per nostril (<NUM> in total) was required. Two TNS solutions also contained chitosan glutamate (a cationic biopolymer which acts as a mucoadhesive/bioadhesive agent. In addition, the three TNS solutions contained sodium chloride for tonicity adjustment, benzalkonium chloride as a preservative, and acetic acid and sodium acetate as a buffer to adjust/maintain pH (Table <NUM>).

Tetracosactide is used as the acetate salt form in the existing injection product. This salt form is reported to be sparingly soluble in water (approximately <NUM>-<NUM>/ml), however this solubility is sufficient to provide a solution suitable for intranasal administration. Two of the three TNS formulations contained chitosan (in the glutamate salt form). A chitosan glutamate concentration of <NUM>/ml was selected for the TNS formulations.

Chitosan glutamate has been tested for safety and toxicity in a number of animal species using different routes of administration. The preclinical data augment the considerable clinical data on intranasal chitosan in both human volunteers and patients. Nasal formulations containing chitosan, both in the form of solutions containing <NUM>/ml chitosan as well as powders, have been administered to over <NUM> people in clinical trials and in excess of <NUM> doses have been administered. Collectively no safety or tolerability issues were identified in any study and these data do not identify any specific hazard that would preclude the use of chitosan in further clinical trials (personal communication, Peter Watts, Phormulate Consulting Ltd).

Paired blood and saliva samples were taken at:.

Saliva was obtained by passive drool technique, with <NUM>, dribbled down a straw into a salicap container.

Abbott Architect chemiluminescent microparticle immunoassay method. Abbott Diagnostics quoted functional sensitivity (with applied <NUM>% confidence interval) as <NUM> nmol/L, a linear range of <NUM>-<NUM> nmol/L, and <<NUM>% total CV for serum samples in the ≥<NUM> to ≤<NUM> nmol/l range. Quoted limit of detection (LoD) is <NUM> nmol/L; typical daily Quality Control precision (CV) at the Sheffield Children's Hospital was <NUM>-<NUM>% at <NUM>, <NUM> and <NUM> nmol/l levels. Quoted cross-reactivity with dexamethasone was <NUM>%.

CORTISOL: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. Derived assay characteristics showed the assay to be linear up to <NUM> nmol/L, with lower limits of quantitation of <NUM> nmol/L and intra- and inter assay imprecision of <<NUM>% over three levels of internal QC, with recovery and accuracy within acceptable limits. Additionally interference studies demonstrated high specificity. The cortisol assay was unaffected by the presence of dexamethasone.

CORTISONE: LC-MS/MS Derived assay characteristics showed the assay to be linear up to <NUM> nmol/L, with lower limits of quantitation of <NUM> nmol/L and intra- and inter assay imprecision of <<NUM>% over three levels of internal QC, with recovery and accuracy within acceptable limits. The cortisone assay was unaffected by the presence of dexamethasone.

The aim of the study was to determine the bioavailability of intranasal Synacthen and ascertain if equivalence (for the purposes of an adrenal suppression test) is possible via the nasal route.

The analysis was performed using WinNonLin <NUM> (Pharsight, Missouri, USA). This is the industry standard for PK, pharmacodynamic (PD) and non-compartmental analysis.

Prior to analysis the concentration-time data for i. v and nasal Synacthen were visually inspected by plotting the individual profiles in Microsoft® Excel. Any baselines effects (from endogenous substances i.e. ACTH or an interfering substrate detected in the assay) should be removed from the PK profile prior to analysis and thus the -<NUM> minute Synacthen values were subtracted from all concentration time points. If necessary the values were fixed after the concentration fell to zero, as PK analysis cannot compute negative numbers. All plasma concentrations reported as missing or below the lower limit of quantification were excluded from the analysis. Data was then arranged (subject, time, concentration, dose and route) for import into the pharmacokinetic (PK) software Phoenix WinNonLin <NUM>.

The standard PK parameters for bioavailability, time to maximum plasma concentration (Tmax), maximum plasma concentration (Cmax), area under the concentration time curve from time zero until the last quantifiable time point (AUC<NUM>-t), area under the concentration time curve from time zero until infinity (AUC<NUM>-∞) and terminal half-life (terminal t½) were calculated for each individual using standard methods within the Phoenix WinNonLin <NUM> non compartmental analysis software. For calculating the terminal t½ a minimum of three of the last data points were used.

Bioavailability of the i. n formulation to the i. v formulation were assessed on the basis of Cmax and AUC<NUM>-t (AUC<NUM>-∞ was not used as the Synacthen was eliminated very rapidly with plasma concentrations being virtually zero by time t).

The concentration-response data in terms of the Cmax for the Synacthen and cortisol data was collated manually in Microsoft® Excel. The concentration-response data were analysed using a number of known models (Emax, sigmoidal Emax, linear and power function), models were fitted using non-linear regression. Weighted residuals were calculated to determine the difference between the observed and model predicted cortisol values at each Synacthen concentration, these were weighted based on the concentration of Synacthen to allow for potential analytical errors. The sum of the squares of the weighted residual values was calculated and the solver function in Excel used to fit model parameters to minimise this value. The best-fit model was determined using the Akaike information criteria.

In addition to the pharmacokinetic modelling mean cortisol and Synacthen at the various time points were compared by paired t-tests with a Bonferroni correction applied. The timing of the peak cortisol response, the dose-response relationship between nasal Synacthen and cortisol production and the inter-individual variability of nasal Synacthen have all been deduced.

The mean plasma Synacthen responses to the four different doses of Synacthen are heightened both by the increase in dose and the addition of chitosan (<FIG>, <FIG>, <FIG>). The resultant cortisol response was similarly affected by both dose and the addition of chitosan (<FIG>, <FIG>, <FIG>). The peak in plasma Synacthen following administration with <NUM> mcg i. v Synacthen is much lower compared with the nasal formulations however the cortisol peak is similar to those seen following <NUM> mcg with chitosan and <NUM> mcg nasal Synacthen and much greater with <NUM> mcg with chitosan (<FIG>+<NUM>).

The two <NUM> mcg i. v tests produced broadly similar plasma Synacthen responses, both with a suppressed cortisol response. Considerable variability in plasma Synacthen levels with all formulations and serum cortisol response following nasal administration are demonstrated by the large ranges and SDs and can be seen by the large error bars (<FIG>, <FIG>). The calculation of CVs gives a measure of variability and is considerably smaller for the i. v dose than the nasal formulations. When chitosan is added to <NUM> mcg tetracosactide it reduces the variability of peak plasma Synacthen values (CV falls from <NUM>% to <NUM>%) however with the <NUM> mcg tetracosactide dose the addition of chitosan appears to worsen variability (<NUM>% to <NUM>%). The addition of chitosan reduced the variability of peak serum cortisol at both doses (<NUM> mcg <NUM>% to <NUM>% and <NUM> mcg from <NUM>% to <NUM>%). The timing of the cortisol peak is seen to vary more after nasal Synacthen than following i. v administration (table <NUM>).

In study <NUM> six volunteers received the same dose of nasal Synacthen on two occasions, in addition to the administration with the same dose/formulation in study <NUM> (although this had been with an unprimed mucosal atomiser device so a lower and potentially more variable dose). There was no difference between all three visits for Synacthen Cmax, plasma cortisol Cmax and AUC<NUM>-t, salivary cortisone Cmax and AUC<NUM>-t. There was a difference between visits for Synacthen AUC<NUM>-t and AUC<NUM>-inf. The difference was due to visit <NUM>, where a slightly lower dose was given due to MAD lack of priming - there was no statistical difference between visits <NUM> and <NUM>. (<FIG>, <FIG>, <FIG> + <NUM> and <NUM>-<NUM>).

The cortisol response to the higher dose of nasal Synacthen given in the first NeSST Study (study <NUM>) (<NUM> mcg), the combined mean of the two <NUM> mcg i. v tests from both studies and the three nasal doses from the NeSST2 Study are included for a visual comparison of the effect of increase dose and addition of a nasal drug enhancer (<FIG>).

The median absolute bioavailability of the <NUM> mcg with chitosan intranasal dose was approximately <NUM>% (range <NUM> to <NUM>%) of the <NUM> mcg i. The bioavailability with <NUM> mcg tetracosactide showed the greatest variability, <NUM>% (<NUM>-<NUM>%) and the highest bioavailability was achieved with the <NUM> mcg tetracosactide and chitosan formulation, <NUM>% (<NUM>-<NUM>%). The time to maximum concentration (Tmax) was longer for intranasal formulations, <NUM>-<NUM> minutes, compared with five minutes following i. v administration and was longest with the <NUM> mcg tetracosactide and chitosan preparation (<NUM> compared to <NUM> minutes). The maximum concentration (Cmax) was approximately <NUM>% following <NUM> mcg with chitosan, <NUM>% after <NUM> mcg tetracosactide and <NUM>% following <NUM> mcg tetracosactide with chitosan, much greater than those seen with the nasal doses given in the NeSST Study (study <NUM>). Table <NUM>.

The bioavailability of the <NUM> mcg Synacthen intranasal dose increased by <NUM>-fold following formulation with chitosan, although the populations were not the same as the results are combined from the NeSST and NeSST2 studies. The bioavailability of the 500mcg intranasal dose was higher than anticipated without the addition of chitosan and the addition of the nasal enhancer only increased the bioavailability by <NUM>-fold. Table <NUM>.

Mean residence time (MRT) measures the time that molecules of Synacthen remain in the body after injection/nasal ingestion to elimination and therefore, as one would expect, the MRT was longer for the nasal formulations than for the i. v preparation. At both <NUM> and <NUM> mcg the MRT was reduced following the addition of chitosan, but dose escalation appeared not to influence it. Similarly the elimination (or terminal) half-life of plasma Synacthen was shorter following the addition of chitosan to both the <NUM> and <NUM> mcg doses. It was almost the same for the i. v and <NUM> mcg tetracosactide with chitosan formulations. Despite a shorter half-life, the cortisol response appeared more prolonged with the higher nasal doses. Table <NUM>.

Following both the <NUM> mcg tetracosactide with chitosan and the <NUM> mcg tetracosactide doses individuals showed a similar cortisol response to the <NUM> mcg i. v test but consistency was lacking. As expected there was more variability in the data following intranasal administration compared with i. Table <NUM>.

Plasma cortisol rose <NUM> minutes after Synacthen administration (figure 8a). Plasma cortisol Tmax was at <NUM> minutes for all participants. The IV 250mcg SST was the least variable, with standard deviations ranging from ±<NUM>-<NUM> nmol/I for time-points. Mean plasma cortisol concentration:.

Cortisol levels increased <NUM> minutes after intranasal Synacthen administration, later than in the IV test (figure 8c). Mean peak cortisol was lower in the lower dose IN test than the IV test, at <NUM>. 3nmol/l (±<NUM>).

Plasma cortisol results for IN 500mcg were more variable than the IV test, as shown by higher standard deviations, except at <NUM> minutes post Synacthen. Results were nearly three times as spread at <NUM> and <NUM> minutes, although the lower mean concentration and varying Cmax and Tmax of the IN 500mcg test should be taken into account. Mean plasma cortisol concentration:.

Participants showed a rise from baseline after <NUM> minutes (figure 8b). Unfortunately, two participants had a cannula failure midway through the test and so blood results were missed from samples beyond <NUM> and <NUM> minutes. Standard deviation, and thus variability, was higher in the IN <NUM> test than the IV test and similar to the IN 500mcg test.

The IV 250mcg test showed the highest mean Cmax, at <NUM> (±<NUM>) nmol/l, ranging from <NUM>-<NUM> nmol/l.

Unpaired t-tests compared the mean Cmax of each nasal test against the IV test. There was a statistically significant difference between the mean Cmax in the IN 500mcg test (-<NUM>. 9nmol/l (p=<NUM>, Cl -<NUM> to -<NUM>.

In the IN 500mcg test, Tmax was more variable: the majority reached Cmax at <NUM> or <NUM> minutes, but one participant was as early as <NUM> minutes. An unpaired t-test compared Cmax results against early Tmax (≤<NUM> minutes (N=<NUM>)) and late Tmax (≥ <NUM> minutes (N=<NUM>)). There was a statistically significant difference in Cmax for the early Tmaxs (-<NUM>. 6nmol/l (p=<NUM>, C! -<NUM> to -<NUM>. 5nmol/l)) showing that those who peak early get lower peak concentrations.

The mean Cmax of the IN <NUM> test was in between the two other tests at <NUM>. 9nmol/l (±<NUM>), the difference between this test and the IV was not statistically significant (-<NUM>. 3nmol/l (p=<NUM>, Cl <NUM> to -89nmol/l)). Tmax was at <NUM> minutes by the majority of participants (N=<NUM>) and ≤<NUM> minutes by the rest (N=<NUM>). The mean difference was statistically significant (early: -152nmol/l (p=<NUM>, C! -<NUM> to -<NUM>) (Table <NUM>).

Relationship between serum cortisol, salivary cortisol and salivary cortisone.

The relational analysis of the NeSST Study (study <NUM>) salivary samples, both to each other and the paired serum sample revealed a correlation, but not as tight as had been anticipated. The data from the NeSST2 Study (study <NUM>) (<FIG>, <FIG>) and study <NUM> (NeSST2 1b) (<FIG>, <FIG>) were analysed in the same way. The NeSST Study and NeSST2 Study data were combined to give approximately <NUM> paired samples. Tighter relationships than previously were seen between serum and salivary cortisol, salivary cortisol and cortisone and serum cortisol and salivary cortisone. A clear exponential, or biphasic response is seen between salivary and serum cortisol. A tight linear relationship between salivary cortisol and cortisone is observed and between serum cortisol and salivary cortisone.

The relationship between the timing of the serum cortisol peak and the peaks in salivary cortisol and cortisone following administration with <NUM> mcg tetracosactide and chitosan were examined in a number of ways to determine recommended sampling times (Table <NUM>).

When viewing the graph of mean cortisol response to <NUM> mcg tetracosactide and chitosan the peak times for peak salivary cortisol and cortisone appear to occur slightly later, at <NUM> minutes, compared to the <NUM> minute peak seen when measuring the response in serum. However the modal peak time occurs at <NUM> minutes when measuring serum cortisol and earlier at <NUM> minutes for the salivary markers, but at <NUM> minutes for all markers when the median is used; (<FIG>).

Assessment of correlation between salivary cortisol and cortisone identified a strong positive correlation (Pearson's r= <NUM>, p<<NUM>; (<FIG>), R<NUM> <NUM> (<FIG>)).

Assessment of correlation between serum and salivary cortisol identified a strong positive correlation (Pearson's r= <NUM>, p<<NUM>); (<FIG>).

Pearson's correlation coefficient identified a strong positive association between plasma cortisol and salivary cortisone (r=<NUM>, p<<NUM>), which was stronger than the correlation found between plasma cortisol and salivary cortisol (<FIG>).

As shown in table <NUM> and <NUM> the bioavailability of 500mcg IN is approximately <NUM>-fold higher that 1000mcg based on AUC0-inf. <NUM>-fold higher is based on AUC0-Last.

Claim 1:
A liquid pharmaceutical composition adapted for nasal administration comprising an effective dose of adrenocorticotropic hormone (ACTH) or a synthetic ACTH analogue selected from: tetracosactide, tetracosactide acetate or SEQ ID NO <NUM>; a bioadhesive excipient and including one or more other pharmaceutical excipients for use in a method of diagnosis of adrenal insufficiency in a paediatric subject, wherein the bioadhesive excipient is chitosan and wherein the method comprises application of the composition to the nasal cavity.