Factor VIII with a 237 amino acid B-domain has an extended half-life in F8-knockout mice.

Essentials Factor (F)VIII with an intermediate-length B-domain showed higher levels in murine gene therapy. FVIII with different B-domain lengths were analysed. FVIII variants with B-domains between 186 and 240 amino acids (aa) have extended half-life in mice. Reduced cell binding of FVIII with a 237aa B-domain may explain the extended half-life. SUMMARY: Background Factor VIII consists of the A1-domain, A2-domain, B-domain, A3-domain, C1-domain, and C2-domain. FVIII with an intermediate-length B-domain of 226 amino acids (aa) has previously been evaluated in murine gene therapy studies. Objective To characterize FVIII with intermediate-length B-domains in vitro and in vivo in F8-knockout (KO) mice. Methods and results FVIII molecules with B-domains of 186-240aa had longer half-lives in F8-KO mice than FVIII molecules with shorter or longer B-domains. FVIII with a B-domain containing the 225 N-terminal aa fused to the 12 C-terminal aa of the wild-type B-domain (FVIII-237) had a 1.6-fold extended half-life in F8-KO mice as compared with FVIII with a 21aa B-domain (FVIII-21). The in vitro and in vivo activity of FVIII-237 were comparable to those of FVIII-21, as was binding to von Willebrand factor. Cell binding to LDL receptor-related protein 1 (LRP-1)-expressing cells was markedly reduced for FVIII-237 as compared with FVIII-21, whereas the affinity for LRP-1 was not reduced in surface plasmon resonance (SPR) studies. FVIII-21 cell binding and internalization could be inhibited by a fragment consisting of the 226 N-terminal aa of the FVIII B-domain, and SPR analysis suggested that this B-domain fragment might bind with weak affinity to FVIII-21. Conclusion Reduced cell binding of FVIII-237 might explain the observed extended half-life in F8-KO mice. This may contribute to the increased FVIII levels measured in murine gene therapy studies using FVIII constructs with similar B-domain lengths.

Preclinical pharmacokinetics and biodistribution of subcutaneously administered glycoPEGylated recombinant factor VIII (N8-GP) and development of a human pharmacokinetic prediction model.

Essentials N8-GP is an extended half-life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous (SC) FVIII dosing might reduce the treatment burden of prophylaxis. SC N8-GP has a favorable PK profile in animal models and disappears from skin injection sites. Combined animal (SC) and clinical (IV) data suggest that daily SC dosing may provide prophylaxis. SUMMARY: Background N8-GP is an extended half-life recombinant factor VIII (FVIII) for the treatment of hemophilia A. Subcutaneous administration of FVIII may reduce the treatment burden of prophylaxis; however, standard FVIII products have low bioavailability after subcutaneous dosing in animals. Objective To evaluate the pharmacokinetics, effectiveness and local distribution of subcutaneously administered N8-GP in preclinical models and predict the human pharmacokinetic (PK) profile. Methods The pharmacokinetics of subcutaneously administered N8-GP were evaluated in FVIII knockout (F8-KO) mice and cynomolgus monkeys; a human PK prediction model in hemophilia A patients was developed. The hemostatic effect was evaluated in a tail vein bleeding model in F8-KO mice. The injection-site distribution and absorption of subcutaneously administered N8-GP were assessed in F8-KO mice by the use of temporal fluorescence imaging and immunohistochemistry. Results Subcutaneously administered N8-GP had a bioavailability, a first-order absorption rate and a half-life, respectively, of 24%, 0.094 h-1 and 14 h in F8-KO mice, and 26%, 0.33 h-1 and 15 h in cynomolgus monkeys. A dose-dependent effect of subcutaneously administered N8-GP on blood loss was observed in mice. A minimal amount of N8-GP was detected at the injection site 48-72 h after single or multiple dose(s) in F8-KO mice. Subcutaneously administered N8-GP was localized to the skin around the injection site, with time-dependent disappearance from the depot. PK modeling predicted that subcutaneously administered N8-GP at a daily dose of 12.5 IU kg-1 will provide FVIII trough levels of 2.5-10% in 95% of patients with severe hemophilia A. Conclusions Subcutaneously administered N8-GP may provide effective hemophilia A prophylaxis. A phase I clinical trial is underway to investigate this possibility.

Danish, national cross-sectional observational study on the prevalence of prior major osteoporotic fractures in adults presenting with hip fracture-limitations and scope for fracture liaison services in prevention of hip fracture.

Fracture liaison services prevent hip fractures by identifying other osteoporotic fractures that generally debut at a younger age. However, this study showed that a minority of hip fracture patients are already known to the health services through having had prior osteoporotic fractures. Identification of vertebral fractures in particular is lacking. INTRODUCTION: The purpose of this study was to examine the prevalence of prior major osteoporotic fractures (MOF) in the prior 10 years preceding hip fracture in order to provide information about the potential for prevention of hip fractures by fracture liaison services (FLS). METHODS: We included all patients aged 50+ with surgically treated hip fracture in one calendar year (N = 8158) in the Danish Hospital Discharge Register. Prior fractures were identified using the same data source. A prior hip fracture was only included as a prior fracture if occurring more than 6 months before the present fracture. RESULTS: A total of 28% of hip fracture patients (32% of women and 19% of men) had at least one recognized MOF in the preceding 10 years. Forearm and humerus fractures constituted > 70% of prior MOF. In both genders, vertebral fractures only represented a small percentage (2.6%) of previously recognized MOF. Men were less likely than women to have experienced a prior MOF, chiefly due to fewer forearm and humerus fractures. CONCLUSION: The majority of hip fractures-and in particular hip fractures in men-occur without a previously treated MOF that could have resulted in early detection and treatment of osteoporosis. With current treatment modalities, a maximum of one in six hip fractures in Denmark can be prevented through FLS initiatives. Identification of patients with vertebral fractures for assessment and treatment is therefore critical for successful prevention of hip fractures using this strategy.

Development of a tail vein transection bleeding model in fully anaesthetized haemophilia A mice - characterization of two novel FVIII molecules.

INTRODUCTION: The tail tip bleeding model and the tail vein transection survival model in mice are important tools for assessment of in vivo effect in haemostasis research. While the tail vein transection model exhibits the best sensitivity to pharmacological intervention it uses death or near-death as endpoint which is fully avoided in the tail tip bleeding model. AIM: The aim of this study was to develop a new tail bleeding model maintaining the sensitivity of the previous survival model but avoiding death/near-death as endpoint. METHODS: Combining the two existing tail bleeding models we developed an optimized version of the survival model with full anaesthetic coverage and short duration of experiments. Using this model, we characterized the effect of turoctocog alfa, a B-domain truncated FVIII molecule (NovoEight(®) ), as well as the prolonged half-life version of the same molecule (turoctocog alfa pegol, N8-GP). RESULTS: Data showed that the model was sensitive to clinically relevant doses of both turoctocog alfa as well as N8-GP when dosed for 'on demand' treatment. The model also correctly identified a longer duration of effect for N8-GP compared with turoctocog alfa. Moreover, the model allowed the use of mice of both genders and was reproducible over time. CONCLUSION: The optimized tail vein transection bleeding model is sensitive to standard as well as half-life prolonged FVIII molecules and should be a valuable alternative to both the tail tip bleeding model, enhancing sensitivity to pharmacological intervention, as well as to the previously used tail vein transection survival model, avoiding death or near-death as endpoint.

Recombinant human factor VIIa and a factor VIIa-analogue reduces heparin and low molecular weight heparin (LMWH)-induced bleeding in rats.

BACKGROUND: Heparin and low molecular weight heparin (LMWH) are widely used for prevention and treatment of thromboemobolic events, but may occasionally cause uncontrollable bleeding. Heparin can readily be antagonized with protamine, but this is less effective against LMWH. OBJECTIVES: To test the effects of rFVIIa or an analogue of rFVIIa, NN1731, on heparin- and LMWH-induced bleeding in rats. METHODS: Initially the doses of heparin and tinzaparin (a LMWH) were determined by dose-titration. Following pretreatment with heparin or tinzaparin in rats, tail-transection was performed, and the effect of rFVIIa and NN1731 on the bleeding was observed. RESULTS: rFVIIa (5, 10 and 20 mg kg(-1)) reduced bleeding time and blood loss caused by heparin- and tinzaparin-induced bleeding, using doses of 200 IU kg(-1) (n = 8) and 500 IU kg(-1) (n = 9), respectively. Similarly, 10 mg kg(-1) NN1731 significantly reduced both heparin- and tinzaparin-induced bleeding to the normal level. Following severe anticoagulation with 1800 IU kg(-1) tinzaparin, 10 mg kg(-1) NN1731 reduced and normalized the bleeding, while the effect of 20 mg kg(-1) rFVIIa failed to reach statistical significance. These data are consistent with the hypothesis that rFVIIa/NN1731 are capable of generating sufficient thrombin locally on the surface of activated platelets to induce hemostasis in the presence of heparin/LMWH. CONCLUSIONS: This study suggests that rFVIIa and NN1731 may have the potential to control bleedings caused by heparin or LMWH.

Influence of a selective histamine H3 receptor antagonist on hypothalamic neural activity, food intake and body weight.

OBJECTIVE: This study was conducted to elucidate whether antagonistic targeting of the histamine H3 receptor increases hypothalamic histamine levels, in parallel with decreases in food intake and body weight. METHODS: The competitive antagonist potency of a recently synthesized histamine H3 receptor antagonist, NNC 38-1049, was studied in intact HEK293 cells expressing human or rat histamine H3 receptor, in which NNC 38-1049 was allowed to antagonize the effect of the H3 receptor agonist R-alpha-methylhistamine on isoprenaline-induced accumulation of cAMP. The affinity of NNC 38-1049 for a number of variants of the histamine receptor was also determined. Following single dosing of normal rats with NNC 38-1049, hypothalamic histamine levels were assessed by means of microdialysis. Plasma and brain levels of NNC 38-1049 and acute effects on food intake and energy expenditure were followed after oral doses of 3-60 mg/kg. Potential side effects were examined with rat models of behaviour satiety sequence (BSS), pica behaviour and conditioned taste aversion (CTA). Intakes of food and water together with body weight were recorded for 15 days during daily dosing of dietary obese rats. RESULTS: NNC 38-1049 was found to be a highly specific and competitive antagonist towards both human and rat histamine H3 receptors, and measurable amounts of NNC 38-1049 were found in the plasma of rats following single oral doses of 3-60 mg/kg and in the brain after 15-60 mg/kg. Following single intraperitoneal injections of NNC 38-1049 (20 mg/kg), significant increases in extracellular histamine concentrations were observed. The same dose did not change BSS or pica behaviour acutely, nor did it induce CTA following repeated administration for 7 days. Reductions in food intake were seen very soon after administration, and occurred in a dose-dependent fashion. Energy expenditure was unchanged, but the respiratory quotient (RQ) tended to decrease at higher doses, indicating an increase in lipid oxidation. Twice daily administration of 20 mg/kg of NNC 38-1049 in old and dietary obese rats resulted in sustained reduction of food intake throughout a 2-week study, and was associated with a highly significant (P<0.01) decrease in body weight compared with controls (-18.4+/-3.4 vs +0.4+/-2.7 g). The same dose of NNC 38-1049 produced an acute decrease of water intake, but 24 h intakes were not significantly changed. CONCLUSIONS: The results of this study strongly support the idea that an increase in the hypothalamic concentration of histamine produces a specific reduction of food intake and that this effect can be translated into a decrease in body weight.

Adipogenic and orexigenic effects of the ghrelin-receptor ligand tabimorelin are diminished in leptin-signalling-deficient ZDF rats.

OBJECTIVE: The aim was to investigate the possible interactions of the two peripheral hormones, leptin and ghrelin, that regulate the energy balance in opposite directions. METHODS: Leptin-receptor mutated Zucker diabetic fatty (ZDF) and lean control rats were treated with the ghrelin-receptor ligand, tabimorelin (50 mg/kg p.o.) for 18 days, and the effects on body weight, food intake and body composition were investigated. The level of expression of anabolic and catabolic neuropeptides and their receptors in the hypothalamic area were analysed by in situ hybridization. RESULTS: Tabimorelin treatment induced hyperphagia and adiposity (increased total fat mass and gain in body weight) in lean control rats, while these parameters were not increased in ZDF rats. Treatment with tabimorelin of lean control rats increased hypothalamic mRNA expression of the anabolic neuropeptide Y (NPY) mRNA and decreased hypothalamic expression of the catabolic peptide pro-opiomelanocortin (POMC) mRNA. In ZDF rats, the expression of POMC mRNA was not affected by treatment with tabimorelin, whereas NPY mRNA expression was increased in the hypothalamic arcuate nucleus. CONCLUSION: This shows that tabimorelin-induced adiposity and hyperphagia in lean control rats are correlated with increased hypothalamic NPY mRNA and decreased POMC mRNA expression. The elimination of tabimorelin-induced adiposity and hyperphagia in ZDF rats may be due to lack of POMC mRNA downregulation. In conclusion, we suggest that ghrelin-receptor ligands exert their adipogenic and orexigenic effects via hypothalamic mechanisms that are dependent on intact leptin-receptor signalling.

Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice.

Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic alpha cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr(-/-)). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr(-/-) mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to alpha cell, and to a lesser extent, delta cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr(-/-) mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and alpha and delta cell numbers. Furthermore, the lean phenotype of Gcgr(-/-) mice suggests glucagon action may be involved in the regulation of whole body composition.

The growth hormone secretagogue ipamorelin counteracts glucocorticoid-induced decrease in bone formation of adult rats.

The ability of the growth hormone secretagogue (GHS) Ipamorelin to counteract the catabolic effects of glucocorticoid (GC) on skeletal muscles and bone was investigated in vivo in an adult rat model. Groups of 8-month-old female rats were injected subcutaneously for 3 months with GC (methylprednisolone) 9 mg/kg/day or GHS (Ipamorelin) 100 microg/kg three times daily, or both GC and GHS in combination. The maximum tetanic tension of the calf muscles was determined in vivo in a materials testing machine. The maximum tetanic tension was increased significantly, and the periosteal bone formation rate increased four-fold in animals injected with GC and GHS in combination, compared with the group injected with GC alone. In conclusion, the decrease in muscle strength and bone formation found in GC-injected rats was counteracted by simultaneous administration of the growth hormone secretagogue.

Daily low-dose administration of growth hormone secretagogue stimulates pulsatile growth hormone secretion and elevates plasma insulin-like growth factor-1 levels in pigs.

Repeated administration of growth hormone secretagogues (GHSs) has proven to be a delicate matter owing to development of tolerance. The aim of the present study was to define conditions during which the responsiveness to the orally active NN703 was maintained over several days. Growing pigs were fitted with stomach and vascular catheters, permitting unstressed intragastric administrations and blood sampling. NN703 or vehicle was administered once daily. When NN703 was given at a dose of 18 mg/kg, there was a massive acute increase in plasma growth hormone (GH) levels, but this was only seen on the first day of administration. A dose of 1.8 mg/kg did not cause a significant acute increase in plasma GH concentrations, whereas stimulation of pulsatile GH release was sustained over a 4-d period. During the first 7 h following injection of vehicle, the area under the curve of plasma GH was 1211+/-144 (microg/[L x 7 h]), but increased to 1770+/-269 and 1824+/-198 (microg/[L x 7 h]) on the first and fourth day of NN703 administration, respectively. Deconvolution analysis of the 7-h profiles revealed that the GH mass per burst as well as the GH burst amplitude were significantly (p < 0.001) increased during treatment with NN703, which led to an increase in pulsatile GH secretion rate (p < 0.001). Insulin-like growth factor-1 plasma concentrations increased steadily during NN703 administration (p < 0.01) and decreased after termination of treatment. The sustained increase in GH pulsatility observed with low-dose NN703 treatment suggests that development of tolerance to this GHS may be obviated by minimization of dose.

Comparison of methods of analysis of body composition in hypophysectomized rats treated with rat growth hormone.

The present study compared estimates of body composition derived from dual-emission X-ray absorptiometry (DEXA) and from chemical analyses. The primary aim was to compare the two methods because growth hormone (GH) may cause fluid retention, and DEXA does not distinguish water from lean mass. Hypophysectomized rats were fed ad libitum and were treated with continuous infusions of rat GH in doses of 0, 10, 30, and 100 microg/day for 14 days. By chemical analysis, a decrease in percentage fat from 12.9% in the control group to 11.3%, 11.0%, and 10.2% in the low, medium, and high dose groups was observed (P < 0.0001). The fat percentages were about 3-4% higher by DEXA, but showed the same decline (P < 0.03). Lean mass increased from 74.4% in the control group to 75.8%, 78.0%, and 78.6% in the treatment groups (P < 0.001). A significant increase in the wet weight of the quadriceps muscle, but no difference in dry weight was observed in all four treatment groups, indicating that the increase in muscle weight was exclusively caused by water. This accumulation of water was reflected in the total water content of the carcasses, which increased from 62.0% in the control group to 64.9%, 66.1%, and 66.8% in the GH groups (P < 0.0001). The protein content decreased from 19.8% in the control group to 19.4%, 19.1%, and 18.9% in the GH groups (P < 0.001). Regardless of the decrease in protein, the GH treated groups contained more water in relation to protein as the g water/g protein ratio was increased by 13% from 3.14 in the control group to 3.55 in the group treated with the highest GH dose (P < 0.0001). Also, a close relationship between feed intake and body weight were found, together with increases in epiphyseal growth plate width, insulin-like growth factor I (IGF-I), and insulin-like growth factor binding protein 3 (IGFBP-3). In conclusion, the study shows that estimation of lean mass by DEXA should be carefully evaluated when used in connection with treatment of drugs that cause water retention.

Pharmacological characterisation of a new oral GH secretagogue, NN703.

NN703 is a novel orally active GH secretagogue (GHS) derived from ipamorelin. NN703 stimulates GH release from rat pituitary cells in a dose-dependent manner with a potency and efficacy similar to that of GHRP-6. The effect is inhibited by known GHS antagonists, but not by a GH-releasing hormone antagonist. Binding of (35)S-MK677 to the human type 1A GHS receptor (GHS-R 1A) stably expressed on BHK cells was inhibited by GHRP-6 and MK677 as expected. NN703 was also able to inhibit the binding of (35)S-MK677. However, the observed K(i) value was lower than expected, as based on the observed potencies regarding GH release from rat pituitary cells. Similarly, the effect of NN703 on the GHS-R 1A-induced inositol phosphate turnover in these cells showed a lower potency, when compared with GHRP-6 and MK677, than that observed in rat pituitary cells. The effect of i.v. administration of NN703 on GH and cortisol release was studied in swine. The potency and efficacy of NN703 on GH release were determined to be 155+/-23 nmol/kg and 91+/-7 ng GH/ml plasma respectively. A 50% increase of cortisol, compared with basal levels, was observed for all the tested doses of NN703, but no dose-dependency was shown. The effect of NN703 on GH release after i. v. and oral dosing in beagle dogs was studied. NN703 dose-dependently increased the GH release after oral administration. At the highest dose (20 micromol/kg), a 35-fold increase in peak GH concentration was observed (49.5+/-17.8 ng/ml, mean+/-s.e.m.). After a single i.v. dose of 1 micromol/kg the peak GH plasma concentration was elevated to 38.5+/-19.6 ng/ml (mean+/-s.e.m.) approximately 30 min after dosing and returned to basal level after 360 min. The oral bioavailability was 30%. The plasma half-life of NN703 was 4.1+/-0.4 h. A long-term biological effect of NN703 was demonstrated in a rat study, where the body weight gain was measured during a 14-day once daily oral challenge with 100 micromol/kg. The body weight gain was significantly increased after 14 days as compared with a vehicle-treated group. In summary, we here describe an orally active and GH specific secretagogue, NN703. This compound acts through a similar mechanism as GHRP-6, but has a different receptor pharmacology. NN703 induced GH release in both swine and dogs after i.v. and/or p.o. administration, had a high degree of GH specificity in swine and significantly increased the body weight gain in rats.

Ipamorelin, a new growth-hormone-releasing peptide, induces longitudinal bone growth in rats.

Ipamorelin is a new and potent synthetic pentapeptide which has distinct and specific growth hormone (GH)-releasing properties. With the objective of investigating the effects on longitudinal bone growth rate (LGR), body weight (BW), and GH release, ipamorelin in different doses (0, 18, 90 and 450 microg/day) was injected s.c. three times daily for 15 days to adult female rats. After intravital tetracycline labelling on days 0, 6, and 13, LGR was determined by measuring the distance between the respective fluorescent bands in the proximal tibia metaphysis. Ipamorelin dose-dependently increased LGR from 42 microm/day in the vehicle group to 44, 50, and 52 microm/day in the treatment groups (P<0.0001). There was also a pronounced and dose-dependent effect on BW gain. The treatment did not affect total IGF-I levels, IGFBPs, or serum markers of bone formation and resorption. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the metaphysis of the tibia did not change significantly with treatment. The responsiveness of the pituitary to a provocative i.v. dose of ipamorelin or GHRH showed that the plasma GH response was marginally reduced (P<0.03) after ipamorelin, but unchanged after GHRH. The pituitary GH content was unchanged by ipamorelin treatment. Whether ipamorelin or other GH secretagogues may have a place in the treatment of children with growth retardation requires demonstration in future clinical studies.

Methylprednisolone does not inhibit the release of growth hormone after intravenous injection of a novel growth hormone secretagogue in rats.

The present study was undertaken to study the growth hormone-releasing properties and growth-promoting effect of a GH secretagogue ipamorelin (IPA) in rats given the synthetic glucocorticoid methylprednisolone (MP). In a first experiment, rats received either saline or MP (5.0 mg/kg) for 8 days. Treatment with MP significantly (P< 0.001) decreased body weight gain, but the acute response to either IPA or growth hormone releasing hormone (GHRH) in terms of plasma GH was not changed. In a second experiment, venous catheters were surgically implanted. On the next day, rats were randomly allocated to receive saline alone, MP alone (5.0 mg/kg) or MP plus IPA in doses of 0.4 or 1.6 mg/kg/day for 10 days. IPA was administered intravenously four times a day.MP treatment significantly (P< 0.05) retarded recovery from surgery in terms of body weight. Thus, saline treated animals lost 4.0 +/- 3.5 g over the entire experimental period, whereas animals receiving MP lost 13. 6 +/- 2.9 g. When IPA was given together with MP, losses in body weight were significantly (P< 0.05) reduced to 2.3 +/- 2.0 and 1.6 +/- 2.0 g in animals given the high and low dose of IPA, respectively. In parallel with this IGF-I levels increased. In conclusion, this work shows that MP does not disrupt the response of the GH-IGF-I axis to an exogenous stimulus like IPA, and repeated stimulation leads to increases in IGF-I and of body weight gain.

Ototoxicity due to hydroxychloroquine: report of two cases.

We report two cases of irreversible sensineuronal hearing loss due to hydroxychloroquine treatment. The first patient was a 44-year-old woman with long-standing systemic lupus erythematosus and the second case was a 44-year-old man with clinical manifestations consistent with subacute cutaneous lupus erythematosus. They both developed irreversible hearing loss after several years of hydroxychloroquine treatment. Sensineuronal deafness has previously been reported in connection with chloroquine treatment, but this is the first report of ototoxicity associated with hydroxychloroquine.

Pharmacokinetic evaluation of ipamorelin and other peptidyl growth hormone secretagogues with emphasis on nasal absorption.

1. The pharmacokinetics of three new peptidyl growth hormone secretagogues, ipamorelin (NNC 26-0161), NNC 26-0194 and NNC 26-0235, were compared with two well-known hexapeptides, GHRP-2 and GHRP-6, in the male rat following different routes of administration. 2. Following i.v. bolus injection, plasma concentrations of the peptides declined biexponentially. Ipamorelin differed markedly from the other peptides investigated, demonstrating a systemic plasma clearance 5-fold lower than that of GHRP-6. Ipamorelin was mainly excreted in the urine, whereas GHRP-6 was predominantly excreted in the bile. NNC 26-0194 and NNC 26-0235 also showed high biliary excretions. Ipamorelin and the two NNC peptides were moderately resistant towards metabolism as 60-80% of the administered dose could be recovered from bile and urine as intact peptide. 3. After intranasal application, the bioavailability of ipamorelin was estimated at approximately 20%. Higher bioavailabilities of approximately 50% were determined for NNC 26-0235, NNC 26-0194 and GHRP-2, whereas the nasal absorption of GHRP-6 was somewhat lower. Thus, the peptides could be easily transported across the nasal epithelium suggesting that the nasal route seems promising for systemic delivery of this family of peptidyl growth hormone secretagogues.

Absorption and metabolism of the absorption enhancer didecanoylphosphatidylcholine in rabbit nasal epithelium in vivo.

The absorption enhancer, didecanoylphosphatidylcholine (DDPC), improves the nasal absorption of human growth hormone in rabbits. We elucidated the uptake and the metabolism of 1,2-di[1-14C]decanoyl-L-3-phosphatidylcholine and 1,2-didecanoyl-L-3-phosphatidyl[N-methyl-3H]choline in rabbit nasal mucosa in vivo. One minute after nasal application of DDPC, 4.4-7.5% of the applied dose was found absorbed into the mucosa. The retained radioactivity left the tissue in <2 hr. The lipophilic tissue extract revealed that, at t = 1 min, only 1.1-1.4% of the applied dose was found as intact DDPC in the nasal mucosa. The other labeled compounds were decanoic acid, DDPC reacylated with endogenous fatty acids, a neutral lipid, and very small amounts of lyso-DDPC and phosphatidylethanolamine. The water-soluble metabolites revealed formation of phosphorylcholine, glycerophosphorylcholine, cytidinediphosphatecholine, and a slight amount of choline. The detection of these metabolites suggests that DDPC was rapidly cleared from the nasal mucosa, partly by degradation by phospholipases. In addition, data illustrated reutilization of DDPC metabolites in the formation of cytidinediphosphatecholine and phosphatidylethanolamine, together with DDPC being reacylated with endogenous fatty acids. The rapid formation of decanoic acid raises the possibility that this acid may contribute significantly to the drug-enhancing properties of DDPC.

Effects of heparin and aminoguanidine on glomerular basement membrane thickening in diabetic rats.

The effects of heparin and aminoguanidine on glomerular basement membrane thickening were studied in streptozotocin diabetic Sprague-Dawley rats. A placebo-treated group and a non-diabetic group served as controls. All diabetic rats remained severely hyperglycaemic (23 mmol/l) throughout the 8-month study period. At the end of this time relative kidney weight was significantly increased in diabetic control rats (4.9 +/- 0.5 g/kg b.w.) compared with non-diabetic rats (3.3 +/- 0.3 g/kg). This increase was not affected by the intervention treatments. Glomerular basement membrane thickness increased 32% in diabetic control rats (240 +/- 24 nm) compared with non-diabetic rats (182 +/- 20 nm). This increase was prevented by s.c. treatment with both unfractionated and low molecular weight heparins, while basement membrane thickness was the same in animals treated with oral heparins and aminoguanidine and untreated diabetic rats. Macroscopic malignant kidney tumours were seen in three aminoguanidine-treated rats. In conclusion, subcutaneously administered heparin prevents diabetes-induced glomerular basement membrane thickening.

Epithelial transport and bioavailability of intranasally administered human growth hormone formulated with the absorption enhancers didecanoyl-L-alpha-phosphatidylcholine and alpha-cyclodextrin in rabbits.

The transepithelial transport of biosynthetic human growth hormone (hGH) formulated with the absorption enhancers didecanoyl-L-alpha-phosphatidylcholine (DDPC) and alpha-cyclodextrin (alpha-CD) was studied after intranasal administration to rabbits. Plasma concentrations of the hormone were determined until 240 min post administration by ELISA, and the absolute bioavailability was estimated to be in the vicinity of 20%. The localization of hGH was studied 15 min after application of the powder formulation in the initial absorptive phase. To visualize the hormone, a two-step indirect immuno-gold technique was used on semithin and ultrathin cryosections and Epon sections. Polyclonal rabbit anti-hGH was used as primary antibody and gold-conjugated goat anti-rabbit IgG as secondary antibody, succeeded by silver enhancement. Growth hormone was mainly found in the cytoplasm and nuclei of ciliated cells, showing distinct morphological signs of early necrosis, and in lamina propria, including the venules. Minute amounts of hGH were found in endocytotic vesicles in morphologically normal epithelial cells and in the intercellular compartment. We conclude that the major transport route of hGH formulated with absorption enhancers DDPC and alpha-CD was transcellular through lethally damaged ciliated cells.

Antibiotic activity in serum following single and repeated oral administration of sodium fusidate in volunteers.

The pharmacokinetics of antibiotic activity were investigated in 10 healthy, female volunteers receiving a single oral dose of sodium fusidate (500 mg) followed after 48 h by repeated oral dosing of 250 mg b.i.d. for 5 consecutive days. By use of turbidimetry, drug-related antibiotic activity in serum was determined and expressed as fusidic acid equivalents. After a single dose and repeated dosing, the peak concentrations were (mean +/- SE): 30 +/- 3 micrograms/ml and 27 +/- 3 micrograms/ml, respectively (NS), and the trough concentration at steady state was 8.4 +/- 1.8 micrograms/ml. The experimental and predicted accumulation ratios were 2.1 +/- 0.1 versus 1.6 +/- 0.2, respectively (P < 0.16). By use of a model independent method, the terminal elimination half-lives were estimated to be 11 +/- 1 h and 13 +/- 2 h after a single dose and repeated doses, respectively (NS). The total clearances of antibiotic activity were 2.0 +/- 0.4 l/h after a single dose and 1.6 +/- 0.2 l/h after repeated doses (P < 0.11). Model dependent pharmacokinetic parameters were also obtained by fitting a two-compartment open model to the median serum concentrations which, with respect to half-life and clearance, gave values close to those observed by use of the model independent approach. Safety-wise, biochemical parameters were within the normal range. However, a statistically significant increase in ASAT and a decrease in leucocytes were observed. The tolerability of the drug was good and only minor adverse events were reported.