Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen.

PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.

Neonatal exposure to estradiol in rats influences neuroactive steroid concentrations, GABAA receptor expression, and behavioral sensitivity to anxiolytic drugs.

Gonadal steroids, in particular estradiol, exert important actions during pre- and perinatal periods in the regulation of sexual dimorphism and development of the nervous system. We have now examined the effects of neonatal estradiol administration in female rats on brain concentrations of the neuroactive steroids allopregnanolone and tetrahydrodeoxycorticosterone, expression of GABA(A) receptor subunits, and behavioral sensitivity to benzodiazepines and allopregnanolone. Administration of beta-estradiol 3-benzoate on the day of birth resulted in marked decreases in the concentrations of progesterone and allopregnanolone in the cerebral cortex at 21, 60, and 180 days after birth. The concentrations of tetrahydrodeoxycorticosterone, 17beta-estradiol, and dehydroepiandrosterone in the brain at 60 days were not affected by such treatment. Neonatal administration of beta-estradiol 3-benzoate also increased the cerebrocortical abundance of alpha(1), alpha(2), and gamma(2) subunits of the GABA(A) receptor without affecting that of alpha(3), alpha(4), alpha(5), or delta subunits. Diazepam induced a greater reduction in locomotor activity as well as a more pronounced anxiolytic-like effect in the elevated plus-maze test in rats subjected to neonatal treatment with beta-estradiol 3-benzoate than in vehicle-treated controls, while allopregnanolone induced a similar effect in both groups. These effects of estradiol suggest that it plays a major role in regulation both of GABAergic transmission and of the abundance of endogenous modulators of such transmission during development of the central nervous system.

Reduced serum level of THDOC, an anticonvulsant steroid, in women with perimenstrual catamenial epilepsy.

PURPOSE: Seizure exacerbation in catamenial epilepsy (CE) is associated with the decrease in progesterone secretion and increase in estradiol secretion during the premenstrual period. Moreover, experimental evidence suggests that tetrahydrodeoxycorticosterone (THDOC), a positive modulator of the type A receptor for gamma-aminobutyric acid (GABA), and dehydroepiandrosterone sulfate (DHEAS), a negative modulator of this receptor, might play a crucial role in modulating seizure frequency during the menstrual cycle. Following these studies it seems of interest to investigate possible variations, among other hormonal parameters, of THDOC and DHEAS in CE patients. METHODS: The serum concentrations of progesterone (P4), pregnenolone, allopregnanolone (AP), THDOC, DHEAS, cortisol, and DHEAS/cortisol ratio were measured throughout the menstrual cycle at the 7th, 11th, 15th, 19th, 23rd, and 27th day from the onset of spontaneous menstrual blood loss in young premenopausal women with CE (n = 17) and age-matched controls (n = 13). RESULTS: At each time of the study, the serum concentration of THDOC and the DHEAS/cortisol ratio were lower (p < 0.05) in women with CE than in control women. The concentrations of P4, pregnenolone, and AP did not differ between the two groups of subjects. CONCLUSIONS: The reduced serum concentration of THDOC and the reduced DHEAS/cortisol ratio detected throughout the menstrual cycle in women with CE might play a role in CE. Moreover, the peculiar pattern of CE seizure exacerbation might suggest that these neuroendocrine variations are worth investigating in other epileptic syndromes, particularly in those characterized by relevant and uncontrolled variations in seizure frequency.

Neurosteroids in nicotine and morphine dependence.

RATIONALE: Neurosteroids are implicated in various stages of drug dependence, including the acquisition phase, tolerance, and withdrawal. The neurosteroid allopregnanolone is also able to substitute for drugs with abuse potential and possesses reinforcing properties. OBJECTIVES: The effects of acute treatment with, and discontinuation of, chronic exposure to nicotine or morphine on the concentrations of allopregnanolone and its precursors, pregnenolone and progesterone, in the cerebral cortex and plasma of rats were investigated. The role of the hypothalamic-pituitary-adrenal (HPA) axis in, and the development of tolerance to, such effects were also examined. METHODS: Nicotine or morphine was administered acutely or chronically, and withdrawal syndrome was induced by spontaneous discontinuation of drug treatment or by administration of a corresponding receptor antagonist (mecamylamine and naloxone, respectively). Neurosteroids were extracted from the cerebral cortex and plasma, fractionated by high-performance liquid chromatography, and quantitated by radioimmunoassay. RESULTS: Acute intraperitoneal administration of nicotine (0.3-2 mg kg-1) or morphine (5-30 mg kg-1) induced dose- and time-dependent increases in the cerebrocortical and plasma concentrations of pregnenolone, progesterone, and allopregnanolone. The effects of both drugs were abolished by adrenalectomy-orchiectomy. Spontaneous or naloxone-precipitated morphine withdrawal and mecamylamine-precipitated (but not spontaneous) nicotine withdrawal also increased neurosteroid concentrations in the brain and plasma. A challenge dose of nicotine or morphine, administered 14 or 24 h after the last drug injection in chronic ally treated rats, failed to increase cerebrocortical neurosteroid concentrations. CONCLUSIONS: Changes in neurosteroid concentrations mediated by activation of the HPA axis may both contribute to the early acquisition phase of nicotine or morphine addiction and serve to counteract the anxiety-like behavior associated with nicotine or morphine withdrawal. However, the evidence that nicotine withdrawal did not increase neurosteroids, unless precipitated by mecamylamine, suggests that the role of these neurosteroids in spontaneous nicotine withdrawal may not be clear.

Failure of gamma-hydroxybutyric acid both to increase neuroactive steroid concentrations in adrenalectomized-orchiectomized rats and to induce tolerance to its steroidogenic effect in intact animals.

Gamma-Hydroxybutyric acid (GHB), a drug proposed in the treatment of alcohol withdrawal syndrome, increases the cerebrocortical and plasma concentrations of the neuroactive steroids allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC). In the present study, we examined the role of hypothalamic-pituitary-adrenal (HPA) axis in the effect of GHB by measuring the concentrations of these steroids in the brain and plasma of adrenalectomized-orchiectomized (Adx-Orx) rats. The acute administration of GHB (500 mg/kg, i.p.) induced in 30 min an increase in the concentrations of allopregnanolone, THDOC and their precursors pregnenolone and progesterone in different brain areas (cerebral cortex, hypothalamus and cerebellum) and plasma of sham-operated rats but had no effect on the concentrations of these compounds in Adx-Orx rats, suggesting that activation of the HPA axis mediates the effect of GHB on brain and plasma concentrations of neuroactive steroids. Moreover, we evaluated whether repeated exposure of GHB induces tolerance to its steroidogenic effects. Chronic administration of GHB (500 mg/kg, i.p., twice a day for 10 days) to intact animals failed to affect the levels of progesterone, allopregnanolone, or THDOC measured 3 or 48 h after the last drug administration, whereas a challenge injection of GHB or ethanol was still able to increase the concentrations of these steroids in brain and plasma. These results indicate that repeated exposure to GHB fails to induce tolerance or cross-tolerance to the steroidogenic action of GHB or ethanol, respectively.

Synthesis and biological evaluation of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines as new ligands of central and peripheral benzodiazepine receptors.

A large number of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines were synthesised and tested to evaluate their binding affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Relatively good PBR binding affinities were found for ligands belonging to the 3-arylmethyloxy-pyridazinoindole series, whereas only 2-aryl-indenopyridazines 7a, 8a and 10a display a weak binding affinity for CBR. To find out the main structural determinants affecting PBR affinity, a molecular modelling study based on the comparative analysis of the three-dimensional properties of four properly selected derivatives 24a, 3b, 18a and 10d, with those of highly active and selective PBR ligands, taken as reference, was performed.

Changes in GABA(A) receptor gene expression induced by withdrawal of, but not by long-term exposure to, ganaxolone in cultured rat cerebellar granule cells.

The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABA(A) receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl- currents evoked by GABA at recombinant human GABA(A) receptors (comprising alpha1beta2gamma2L or alpha2beta2gamma2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 microM ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the alpha1, alpha2, alpha3, alpha4, alpha5, gamma2L, or gamma2S subunits of the GABA(A) receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of alpha2, alpha4, and alpha5 subunit mRNAs and a decrease in the amounts of alpha1, gamma2L, and gamma2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABA(A) receptor to several positive modulators. Nevertheless, the reduction in the amounts of the alpha1 and gamma2 subunit mRNAs together with the increase in the abundance of the alpha4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.