Peter B. Johansen

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

Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic α 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 α cell, and to a lesser extent, δ 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 α and δ cell numbers. Furthermore, the lean phenotype of Gcgr−/− mice suggests glucagon action may be involved in the regulation of whole body composition.

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia

Tissue factor pathway inhibitor (TFPI) blocks thrombin generation via the extrinsic blood coagulation pathway. Because the severe bleeding in patients with hemophilia occurs from deficiency of intrinsic blood coagulation pathway factor VIII or IX, pharmacological agents that inactivate TFPI and, therefore, restore thrombin generation via the extrinsic pathway, are being developed for treatment of hemophilia. Murine models of combined TFPI and factor VIII deficiency were used to examine the impact of TFPI deficiency on bleeding and clotting in hemophilia. In breeding studies, Factor VIII null (F8−/−) did not rescue the embryonic death of TFPI null (Tfpi−/−) mice. Tfpi+/− did not alter the bleeding phenotype of F8−/− mice. However, total inhibition of intravascular TFPI through injection of anti-TFPI antibody mitigated tail vein bleeding. Interestingly, tail blood loss progressively decreased at doses greater than needed to totally inhibit plasma TFPI, suggesting that inhibition of a sequestered pool of TFPI released at the injury site mitigates bleeding. Because TFPI is sequestered within platelets and released following their activation, the function of platelet TFPI was examined in F8−/− mice lacking hematopoietic cell TFPI that was generated by fetal liver transplantation. Blood loss after tail transection significantly decreased in Tfpi+/−;F8−/− mice with hematopoietic Tfpi−/− cells compared with Tfpi+/−;F8−/− mice with Tfpi+/+ hematopoietic cells. Additionally, following femoral vein injury, Tfpi+/−;F8−/− mice with Tfpi−/− hematopoietic cells had increased fibrin deposition compared with identical-genotype mice with Tfpi+/+ hematopoietic cells. These findings implicate platelet TFPI as a primary physiological regulator of bleeding in hemophilia.

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.

Increase of neuronal histamine in obese rats is associated with decreases in body weight and plasma triglycerides.

Objective: The purpose of the present study was to examine the metabolic effects of a specific histamine H3 receptor antagonist, the cinnamic amide NNC 0038‐0000‐1202 (NNC 38‐1202).

Prolonged effect of GlycoPEGylated rFVIIa (40k-PEG-rFVIIa) in rabbits correlates to activity in plasma

The pharmacokinetics and pharmacodynamics of 40k-PEG-rFVIIa, a GlycoPEGylated derivative of recombinant wild-type FVIIa, were compared with rFVIIa in rabbits. The procoagulant effect was determined as the weight of the clot formed in a defined segment of a facial vein. A time course study was conducted where ligation was made 10 minutes, 12 or 24 hours after i.v. injection of equimolar doses of 40k-PEG-FVIIa or rFVIIa (2 mg/kg). This dose was selected based on a dose response study and a duration of effect study with rFVIIa. The clot weight increased with increasing doses of rFVIIa, and the duration of effect correlated with the plasma FVIIa clot activity. The plasma half-life of 40k-PEG-rFVIIa measured as FVIIa clot activity was found to be 25 hours, which was 5-6 times longer than rFVIIa. The aPTT and PT were reduced, and the measured increase in thrombin-antithrombin correlated to the effect on clot formation. Thus, the effect was similar at ligation 10 minutes after administration of 40k-PEG-rFVIIa or rFVIIa. At 12 hours, the effect of rFVIIa was absent while significant effect was seen 12 and 24 hours post dosing with 40k-PEG-rFVIIa. No consumption of platelets or fibrinogen was found and no thrombi formation was seen in histological examination of various organs. In conclusion, 40k-PEG-rFVIIa has shown prolonged duration of effect that correlated to various plasma markers and FVIIa clot activity. In perspective, the data support further clinical development of 40k-PEG-rFVIIa to potentially become a long-acting recombinant treatment option for prophylaxis in haemophilia patients with inhibitors.

Abnormal joint and bone wound healing in hemophilia mice is improved by extending factor IX activity after hemarthrosis

Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX-/-) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

Growth Hormone (GH) Hypersecretion and GH Receptor Resistance in Streptozotocin Diabetic Mice in Response to a GH Secretagogue

The growth hormone (GH) and insulin-like growth factor I (IGF-I) axis were studied in streptozotocin (STZ) diabetic and nondiabetic female mice following intravenous (IV) injection of the GH secretagogue (GHS) ipamorelin or saline. On day 14, blood samples were obtained before and 10 minutes after the injection. Livers were removed and frozen for determination of the mRNA expressions of the GH receptor, GH-binding protein, and IGF-I, and hepatic IGF-I peptide. Serum samples were analyzed for GH and IGF-I. Following ipamorelin injection, the GH levels were found to be 150 ± 35 μg/L and 62 ± 11 μg/L in the diabetic compared to the nondiabetic mice (P < .05). Serum IGF-I levels were lower in diabetic than in nondiabetic animals, and rose after stimulation only in the nondiabetic animals. Furthermore, hepatic GH resistance and IGF-I mRNA levels and IGF-I peptide were increased in nondiabetic animals in response to GH stimulation, whereas the low levels per se of all these parameters in diabetic mice were unaffected. The study shows that STZ diabetic mice demonstrate a substantial part of the clinical features of type 1 diabetes in humans, including GH hypersecretion and GH resistance. Accordingly, it is proposed that STZ diabetic mice may be a better model of the perturbations of the GH/IGF-I axis in diabetes than STZ diabetic rats.

Pharmacokinetics of doxorubicin and its metabolite doxorubicinol in rabbits with induced acid and alkaline urine

SummaryThe pharmacokinetics of doxorubicin in rabbits preloaded either with ammonium chloride or sodium hydrogencarbonate have been investigated following single IV administration of 5 mg/kg.Plasma samples and urine collections were obtained over 3 h following administratio, and were assayed in duplicate for doxorubicin and its main metabolite doxorubicinol by reversed-phase high-pressure liquid chromatography.The plasma concentration of doxorubicin was fitted to an open two-compartment model.The areas under the plasma concentration-time curves (AUC) of doxorubicin in rabbits with alkaline urine were approximately half the areas in rabbits with acid urine. A pharmacokinetic analysis indicated an increase in the central volume of distribution, which is interpreted as an increase in tissue permeability in the alkaline state, due to the acid-base properties of the doxorubicin molecule.The renal excretion of doxorubicin and doxorubicinol was quantitatively similar in the two groups of rabbits. The total renal excretion of anthracyclines during the experiment was calculated to approximately 6% of the administered dose. The clearances of doxorubicin were initially three times higher than inulin clearance, but approximated this value at the end of the experiment.The renal handling of doxorubicin in the rabbits is explained by glomerular filtration followed by tubular secretion and finally by a reabsorption mechanism with limited capacity.

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 (µg/[L·7 h]), but increased to 1770 ± 269 and 1824 ± 198 (µg/[L·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.