PEGylated therapeutic proteins for haemophilia treatment: a review for haemophilia caregivers.

PEGylation is the technology involving the covalent attachment of polyethylene glycol (PEG) to a protein-, peptide- or small-molecule drug to improve their pharmacokinetic, pharmacodynamic and immunological profiles, and thus, enhance the therapeutic effect. Today, PEGylation of proteins is a well-established technology and is being used in the treatment of a variety of clinical disorders. Several PEGylated coagulation proteins for haemophilia A and B are under development with the goal of prolonging the circulation half-life of factor VIII (FVIII) or factor IX. The prolongation of half-life, resulting in less frequent injections can provide significant benefits in improving the quality of life of subjects with haemophilia and improvement in adherence to treatment. A review of published literature on PEGylated therapeutic products currently approved for human use and a discussion of a PEGylated recombinant FVIII molecule (BAY 94-9027, Bayer HealthCare, Berkeley, CA, USA) currently being investigated in the pivotal clinical trial prior to registration is provided. Available safety information of PEGylated proteins containing high molecular weight PEG does not indicate any safety concerns to date, following long-term (chronic) use in animal models or patients. Chronic use of currently available PEGylated products has been shown to be safe, paving the way for chronic use of PEGylated coagulation products in persons with haemophilia.

Subacute oral toxicity of tetraethylene glycol and ethylene glycol administered to Wistar rats.

A subacute toxicity study with administration of tetraethylene glycol in dosages of 0-220-660-2000 mg/kg body weight to male and female Wistar rats via gavage was conducted in order to characterize a possible toxic action of this compound. The structurally related compound ethylene glycol is known to cause kidney toxicity. Therefore, special attention was paid to investigating possible toxic effects of tetraethylene glycol on this organ. In order to compare possible treatment-related effects of tetraethylene glycol with those known from ethylene glycol, a group of male and female rats was treated with 2000 mg ethylene glycol/kg body weight. Daily oral application of tetraethylene glycol over 4 weeks was tolerated without toxic effects up to and including 2000 mg/kg body weight. Daily oral application of ethylene glycol over 4 weeks resulted in treatment-related effects on the kidneys. A slight decrease in the urinary excretion of potassium, calcium and phosphate (males), a diminished pH-value of the urine, and a slight increase in osmolality (females) were observed. In both sexes excretion of oxalate was significantly increased and microscopic examination of urinary sediment revealed calcium oxalate crystals. Kidney weights of males and females were slightly elevated. Histopathology revealed crystals in renal tubuli, renal pelvis, and urinary bladder; tubulopathy and epithelial hyperplasia within the renal pelvis were also observed. Therefore, the study confirmed the kidney as target for ethylene glycol toxicity and gave no indications of tetraethylene glycol-induced toxic effects.

Learning and memory of rats after long-term administration of low doses of parathion.

A set of four learning and memory tests (Morris Maze I for reference memory, Morris Maze II for working memory, one-way active avoidance, and passive avoidance) were employed to address the questions whether parathion impaired cognitive functions after low, long-term exposure and could cause persistent changes in cognition. Motor activity and general behavior were investigated in a functional observational battery. Parathion was administered in rat food in low doses which caused no clinical symptoms and no or borderline brain acetylcholinesterase inhibition. Parathion doses of 0.5, 2, or 8 ppm in rat food produced the averaged uptake of 24, 100, or 400 microg/kg body weight per group per day in male rats and 36, 152, or 550 microg/kg per day in female rats in week 13. Learning tests were performed in weeks 1 to 4 and 10 to 14, as well as 30 to 34 weeks after the end of treatment, when the male and female rats were about 13 months old. Low doses of parathion given daily for 13 weeks had no cumulative or adverse effects on learning and memory, either during treatment or after the extended treatment-free period, in any of the tests. A significant improvement of learning compared to control observed in the Morris Water Maze I during the first week of treatment (males dose group 0.5 ppm) shows that parathion can improved cognitive functions in rats. Results of the study indicate that adverse effects changing learning and memory in animals may occur only at higher doses of organophosphates, at which the peripheral and brain acetylcholinesterases are inhibited to a greater extent than those in the present study.

Subchronic toxicity of 2,3,7,8-tetrabromodibenzo-p-dioxin in rats.

2,3,7,8-Tetrabromodibenzo-p-dioxin (2,3,7,8-TBDD) was administered daily to male and female rats for 91 days by gavage. Ten male and 10 female rats per group received 0.01, 0.1, 1, 3, or 10 micrograms 2,3,7,8-TBDD/kg body weight per dose per day, solubilised in arachis oil. At 1 microgram/kg per day and above, body weight gain was dose-dependently reduced by treatment. Animals in the 3 and 10 micrograms/kg dose groups showed symptoms of wasting syndrome. Fifty percent of the animals in the 3 micrograms/kg dose-group died and all animals of the highest dose (10 micrograms/kg) died or had to be killed in extremis. Hematological investigations indicated changes--mainly in the 1 and 3 micrograms/kg dose-groups--in hemoglobin content, packed cell volume and number of thrombocytes. The prothrombin-time was markedly prolonged after 3 micrograms/kg in week 13. Clinical chemistry performed at the end of treatment revealed an increase in plasma alkaline phosphatase (APh), aspartate aminotransferase, ASAT and alanine aminotransferase, ALAT (females only) in the highest surviving dose-group (3 micrograms/kg). Marginal changes of APh and ASAT were seen in rats in the 1 microgram/kg dose-group. In the same animals, total bilirubin was elevated. Triglycerides were reduced mainly at 1 and 3 micrograms/kg. Serum thyroxin was reduced, beginning with a marginal change at 0.1 micrograms/kg, triiodothyronine was elevated, starting with a dose of 1 microgram/kg. Thymus weights were reduced in rats of the 1, 3 and 10 micrograms/kg dose-groups. Histopathological analysis showed atrophy of the lymphatic tissue in thymus and spleen. Investigations of the liver indicated peliosis hepatis after treatment with 3 or 10 micrograms/kg. Activities of microsomal enzymes (ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase, UDP-glucuronyltransferase) investigated in liver, lung and kidney were dose-dependently elevated after 13 weeks of treatment. At a dose of 3.0 micrograms/kg, activities were below those of the dose 1.0 microgram/kg, probably due to liver toxicity. The induction ratio of kidney was generally higher than in liver and lung. No signs of treatment-related toxicity were observed in the 0.01 and 0.1 micrograms/kg groups after the subchronic administration of 2,3,7,8-TBDD by gavage.

Toxicity of 2,3,7,8-tetrabromodibenzo-p-dioxin in rats after single oral administration.

Five male and female rats per dose-group received 2,3,7,8-tetrabromodibenzo-p-dioxin (2,3,7,8-TBDD) once on the first day of the study. Doses of 10, 33, 100, or 300 micrograms 2,3,7,8-TBDD/kg body wt. and the vehicle control were administered by gavage. About 20% of 2,3,7,8-TBDD was excreted via feces. Severe body weight retardation was observed in the 100 and 300 micrograms/kg dose-groups. Most animals in the 300 micrograms/kg dose-group and the females receiving 100 micrograms/kg showed emaciation, rough coat and a poor health (wasting syndrome). Of the animals dosed with 300 micrograms/kg, 3 males and all females died. After 100 micrograms 2,3,7,8-TBDD/kg 3 females died. Measured 4 weeks after dosing, triiodothyronine (T3) was increased and thyroxin (T4) was reduced dose dependently in serum. A dose-dependent decrease in thymus weights was observed at necropsy and histological examinations showed that thymus and spleen were depleted of mature lymphocytes. An increase in liver-to-body weight ratio was observed in all dose-groups. The histological examination revealed hypertrophy of centrilobular hepatocytes in the liver of animals treated with 100 micrograms/kg, which was less severe at the 33 micrograms/kg dose. Hypertrophic hepatocytes were also detected in some animals at the lowest dose. Induction of enzyme activities of the mixed function oxidases ethoxycoumarin O-deethylase (ECOD), ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) in liver tissue differed for each of the three enzymes. Two days after administration, enzyme activities were increased but did not differ substantially between dose-groups. Twenty-eight days after dosing the increase in activity after 10 micrograms/kg was largest and the EROD of the 100 micrograms/kg dose-group in females was close to that of the control. This inverse dose-response relationship may be due to impaired liver cell function at higher doses.

Neurotoxicity testing during long-term studies.

Several tests and methods for the investigation of neurotoxicity were performed with female Wistar rats for up to 187 days. The methods were validated by testing 10 rats treated with beta,beta'-iminodipropionitrile (IDPN) and 10 control rats. Cage side observation of the animals revealed signs of altered behavior and motor dysfunction of the IDPN-treated rats. Results of a neuromuscular screen indicated changes in gait, righting reflex, grip strength and performance of the negative geotropism test. Investigation of the animals in activity monitors and on the accelerating rotarod showed changes of several parameters. The motor nerve conduction velocity, measured 6 months after the first treatment, was reduced by 6.7 meters per second in the IDPN group compared to controls. From the results of the tests it can be concluded that the methods chosen can be used during long-term studies but may be most useful for animals not older than 12 months.

Different properties of two voltage-dependent inward currents of the ciliate Stylonychia mytilus.

1. Membrane currents and membrane potentials of the fresh-water ciliate Stylonychia mytilus were investigated by voltage-clamp and constant-current injection techniques. 2. The Ca-dependent action potential of Stylonychia in a solution containing 0.1 mM-CaCl2 was prolonged by the addition of Mg or Na ions. 3. In a nominally Ca-free solution, containing 2 mM-MgCl2, the cells generated repetitive, spontaneous action potentials of relatively small amplitude (17 mV). The addition of 0.5 microgram concanavalin A/ml completely inhibited these action potentials in 2 mM-Mg. 4. In voltage-clamp experiments in standard solution, the inward current-voltage relationship has two maxima, confirming the existence of two different voltage-dependent Ca currents in Stylonychia: inward current I and II. In a nominally Ca-free, Mg-containing solution, the remaining inward current was inhibited by concanavalin A, a specific inhibitor of inward current I. No residual second inward current (current II) was detected in a solution containing Mg and concanavalin A. 5. Experiments, with altered ratio of Ca and Mg ions and constant concentration of divalent cations (mole-fraction experiments), showed that Mg and Ca do not inhibit each other's passage through channel I. Calculations assuming a Ca-channel model with one cation-binding site per ion channel I showed good correlation with the experimental data. 6. A similar inward current was seen after replacement of Mg by Na in nominally Ca-free solution.

Inhibition of a voltage-dependent Ca current by concanavalin A.

Incubation of the hypotrichous ciliate Stylonychia mytilus in fluorescein-labeled concanavalin A (Con A, 0.1-0.5 microgram/ml) produced a strong fluorescence of its membranelles, but comparatively weak fluorescence of the other compound cilia and of the somatic membrane. Compared to untreated cells, the frequency of spontaneous backward movements was reduced in the presence of 0.5 microgram/ml ConA. In electrophysiological experiments Con A altered the excitability of the cell membrane. The two-peak action potential lost its second component which is associated with voltage-dependent Ca channels in the membranelles. The corresponding Ca current (Ca current I) was inhibited by low concentrations of Con A (0.2-0.5 microgram/ml). A second voltage-dependent Ca current (Ca current II) was not affected. Reducing the K outward current by intracellular Cs and/or extracellular tetraethylammonium, or changing the holding potential, did not restore the Con A-sensitive Ca current I. Con A also inhibited this current when Ca was replaced by Ba. The inhibitory effect of Con A on the voltage-dependent Ca current I was prevented by 10-30 mM alpha-methyl-D-mannoside, and the lectin wheat germ agglutinin (20 micrograms/ml) did not affect the Ca currents, indicating that the Con A effect was mediated by binding to specific sugar residues on the excitable membrane. The succinylated dimeric derivative of Con A did not inhibit Ca current I up to concentrations of 5 micrograms/ml. It is concluded that the two voltage-dependent Ca currents in Stylonychia can be chemically isolated due to their different sensitivity to Con A, which appears to bind preferentially to sites near or at the Ca channel in the membranellar membrane.

Changes in voltage-dependent calcium currents during the cell cycle of the ciliate Stylonychia.

Electrophysiological properties of the hypotrichous ciliate Stylonychia mytilus were studied at two stages of its cell cycle: within 30 min after cell division and several hours thereafter. The action potential wave form, and the relative amount of two voltage-dependent calcium inward currents are significantly different in 'young' daughter cells as compared with 'adult' cells. The ratio between total inward and outward current is also larger in 'young' cells. The results provide evidence that during its cell cycle Stylonychia undergoes qualitative developmental changes with respect to its ionic channels in the membrane. These changes may explain the different cell behaviour observed up to 1 h after cell division.

Apparent delay between light-induced receptor current and receptor potential in the Limulus ventral nerve photoreceptor.

We measured receptor potential (ReP) and receptor current (ReC) under voltage-clamp conditions alternatingly evoked by successive identical 10 ms light flashes in the ventral nerve photoreceptor of Limulus. At high stimulus intensities the apparent latency of the ReP is 4-15 ms shorter than that of the ReC. At lower light intensities the difference is even larger. High amplification reveals that the light response starts with two phases corresponding to two current components. The initial first current component rises linearly, is much smaller than the second one and therefore is not detected with standard amplification. This first component, which is more pronounced at low light stimulus intensities, is caused either by a displacement or by a weak ionic current.