IgG1 Aggregation and Particle Formation Induced by Silicone–water Interfaces on Siliconized Borosilicate Glass Beads: A Model for Siliconized Primary Containers

Understanding and mitigating particle formation in prefilled syringes are critical for ensuring stability of therapeutic proteins. In the current study, siliconized beads were used as a model for the silicone–water interface to evaluate subvisible particle formation and aggregation of a monoclonal antibody (IgG1). Agitation with siliconized beads greatly accelerated the formation of protein aggregates and particles, an effect that was enhanced at pH 7.4 relative to pH 5 and in the presence of 0.5 M sucrose or 150 mM NaCl. Aggregation and particle formation were minimal in samples agitated without siliconized beads or in quiescent samples with siliconized beads. At pH 5, 0.01% (w/v) polysorbate 20 substantially inhibited aggregation during agitation with siliconized beads, but had minimal protective effect at pH 7.4. Transient exposure of IgG₁ formulations to the silicone–water interface by flowing formulations through a column packed with siliconized beads led to the formation of subvisible particles, with increased levels observed at pH 7.4 compared to pH 5. Agitation of protein formulations in the presence of siliconized glass beads provides a model for baked-on silicone oil–water interface in prefilled syringes and a means by which to evaluate particle formation and aggregation during formulation screening. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:852–865, 2013     

The Effects of pH and PEG 400–Water Cosolvents on Oxytetracycline–Magnesium Complex Formation and Stability

The effects of pH and PEG 400 on the stoichiometry, conformation, and stability of the magnesium–oxytetracycline (Mg⁺²–OTC) complex were evaluated. Circular dichroism (CD) and HPLC were used to investigate Mg⁺²–OTC complex formation and determine the stability of the complexes formed. The stoichiometry of the complex was determined to be a 1:1 molar ratio of Mg⁺² to OTC regardless of changes in pH, in the range 7–10, and regardless of the percentage of polyethylene glycol (PEG) 400 in solution. CD showed that the conformation assumed by Mg⁺²–OTC complex is sensitive to changes in pH, however, little to no effect was found when the PEG 400 concentration was varied. PEG 400 was found to effect the magnitude of complexation as evident by the dependence of CD peak intensity on the cosolvent concentration in solution. The Job's method confirmed that the formation of this complex increased with increasing PEG 400 concentration and was most favored at pH 8. HPLC analyses of OTC solutions at pH 9 revealed the formation of multiple degradation products after storage at 50°C. The incidence and magnitude of OTC degradation products were reduced in the presence of Mg⁺² and PEG 400. Despite the HPLC results of maintained OTC stability in magnesium-complexed solutions over time, visual inspection showed these solutions to have darkened, indicating that an oxidative process is responsible for initial degradation of OTC. Therefore, the need for additional measures (i.e., antioxidants) was established to ensure the long-term stability of OTC in solution.     

Quantification of the Effects of Ionic Strength,Viscosity, and Hydrophobicity on Protein–Ligand Binding Affinity

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Effects of saffron and its constituents, crocin-1, crocin-2, and crocetin on α-synuclein fibrils

Journal of Natural Medicines - Saffron, the stigma of Crocus sativus Linné (Iridaceae family), has been known to inhibit aggregation of β-amyloid, a nerve tissue protein. α-Synuclein...     

Effects of intrathecal 6-hydroxydopamine, α1 and α2 adrenergic receptor antagonists on antinociception of propofol in mice

AIM: To investigate the relationship between spinal cord norepinephrine, alpha1 and alpha2 adrenergic receptors and antinociception of propofol in mice. METHODS: Kunming mice were used. Antinociceptive tests were investigated with the tail-immersion test and the acetic acid-induced writhing test. The effects of subcutaneous (sc), intrathecal (ith) and intracerebroventricular (icv) injection propofol on pain threshold were observed. The influences of pretreatment with ith 6-hydroxydopamine, alpha1R antagonist prazosin, or alpha2R antagonist yohimbine on the antinociception of propofol were studied. RESULTS: Significant antinociception was produced by propofol (25, 50 mg/kg, sc) and propofol (20, 40 microg, ith) in tail-immersion test and acetic the acid-induced writhing test (P<0.05 or P<0.01). Icv propofol (10, 20, and 40 microg) did not produce any effect on pain threshold in mice (P>0.05). The 6-hydroxydopamine (5 and 10 microg), prazosin (5 and 10 microg), or yohimbine (5 and 10 microg) ith alone did not affect basal tail-flick latency (TFL) in conscious mice, but significantly reduced the TFL as measured by tail-immersion test in propofol (50 mg/kg, sc)-treated mice, compared with basal TFL and vehicle groups (P<0.05 or P<0.01). CONCLUSION: The spinal cord is a target of propofol antinociception. In mice propofol antinociception is partly mediated by spinal norepinephrine, alpha1R and alpha2R.     

Multi-variate approach to global protein aggregation behavior and kinetics: Effects of pH,NaCl, and temperature for α-chymotrypsinogen A

A global characterization of nonnative aggregation is presented for α-chymotrypsinogen A (aCgn) as a function of temperature (T), pH, and [NaCl]. Changes in unfolding free energy, native-state second osmotic virial coefficient (B₂₂), and aggregation pathways and kinetics were qualitatively and quantitatively determined using a combination of size-exclusion chromatography, multi-angle laser light scattering, and circular dichroism and fluorescence spectroscopy. Results were analyzed quantitatively using multi-variate statistical models and a recently developed mechanistic model that naturally accounts for changes in aggregation pathway due to competition between unfolding, nucleation, chain polymerization, aggregate condensation, and phase separation. State diagrams are presented that show the natural progression between different aggregation behaviors or pathways. Together, the results show that pH and [NaCl] determine both the rates of aggregation and what aggregation behavior or pathway holds. In contrast, T affects primarily only aggregation rates, in large part due to changes in unfolding free energy. Finally, it is shown that B₂₂ correlates strongly with which type of aggregation pathway is followed, suggesting a potentially useful approach for predicting and controlling physical properties of the resulting aggregates. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:645–662, 2010     

Effects of isoprenaline,adrenaline and selective α1- and α2- adrenoceptor stimulation on hypoxic pulmonary vasoconstriction in rat isolated perfused lungs

In the rat pulmonary vasculature perfused with blood in situ vasoconstriction induced by hypoxia was reversed by isoprenaline (doses > 1 ng) and adrenaline (doses > 30 ng) and exacerbated by phenylephrine but not UK 14304. Doses of adrenaline < 30 ng had no effect, except in the presence of propranolol (1 μM) or phentolamine (3 μM) when they caused vasoconstriction and vasodilation respectively, showing that, at dose levels < 30 ng, adrenaline's β- adrenoceptor vasodilator properties were balanced by its α- adrenoceptor vasoconstrictor properties. The pressor effects of adrenaline, in the presence of propranolol, were antagonised by prazosin (0.1 μM) but not by equi-molar concentrations of rauwolscine. These results suggest that the α- adrenoceptor agonist property of adrenaline is of benefit to its use as an inhaled bronchodilator because unopposed β- adrenoceptor stimulation can reverse hypoxic pulmonary vasoconstriction in poorly ventilated regions of the lung, promote further ventilation/ perfusion mismatching and lower PaO₂. They further suggest that adrenaline affects pulmonary vascular tone in the rat via α₁- adrenoceptors, stimulation of α₂- adrenoceptors having no effect.     

Effects of α,β-unsaturated sulphones and phosphonium salts on ecto-ATPase activity and contractile responses mediated via P2x-purinoceptors

• 1.1. In the guinea-pig urinary bladder and vas deferens, several α,β-unsaturated sulphones and phosphonium salts that were tested inhibited ecto-ATPase activity. The sulphones were more active in the bladder but the phosphonium salts were more effective in the vas deferens.
• 2.2. These compounds either potentiated or inhibited purinergic contractile responses in the guinea-pig urinary bladder and vas deferens.
• 3.3. α,β-Unsaturated sulphones and phosphonium salts represent a new promising class of compounds, capable of modulating purinergic neurotransmission.

     

Effects of intracerebroventricular administration of prostaglandin D2 on behaviour,blood pressure and body temperature as compared to prostaglandins E2 and F2α

The present work examined some central nervous actions of prostaglandin D₂ (PGD₂), which is the most prevalent prostaglandin in rodentorain. The effects of PGD₂ were compared with those of PGE₂ and PGF₂. The prostaglandins were administered intracerebroventricularly (ICV) to conscious rats using the method of Herman (1970). All three prostaglandins studied produced depressive behavioral effects, causing obvious sedation at doses of 2.0 g and 20.0 g ICV. PGD₂ and PGE₂ significantly reduced spontaneous motor activity at doses of 2.0 g and 20.0 g ICV. PGF₂ was less effective; only 20.0 g significantly inhibited motor activity. At a dose of 20.0 g ICV all three compounds were shown to block convulsions induced by pentylenetetrazol. PGD₂, the most effective prostaglandin in this respect, was still slightly anticonvulsive at a dose of 2.0 g ICV. PGF₂ hat the weakest anticonvulsive potency. PGE₂ and PGF₂ (2.0 g and 20.0 g ICV) caused a marked hypertensive effect, whereas PGD₂ at the same dose levels only produced a small increase in blood pressure. PGE₂ and PGF₂ (2.0 g and 20.0 g) also exerted marked pyrogenic actions. The effects of PGD₂ on body temperature were variable. When given at a dose of 20.0 g ICV, it caused slight hyperthermia whereas a lower dose (2.0 g ICV) induced a moderate fall in body temperature. These findings suggest a relationship between the actions of the different prostaglandins on blood pressure and body temperature.A preliminary report was given at the Spring Meeting of the Deutsche Pharmakologische Gesellschaft, March 1983 (Förstermann and Heldt, 1983)     

Effects of antidepressant drugs and electroconvulsive shock on pre- and postsynaptic α2-adrenoceptor function in the brain: rapid down-regulation by sibutramine hydrochloride

Clonidine (0.1 mg/kg IP)-induced hypoactivity and mydriasis responses were respectively used as functional indices of pre- and postsynaptic ₂-adrenoceptors in mouse brain. A single injection of various antidepressant drugs had no effect on either response when measured 24 h later. However, 14 days' treatment with sibutramine HCl (3 mg/kg IP), dothiepin (50 mg/kg IP), amitriptyline (10 mg/kg IP), desipramine (10 mg/kg IP) or tranylcypromine (10 mg/kg IP) markedly attenuated both clonidine-induced hypoactivity and mydriasis. Repeated administration of zimeldine (10 mg/kg IP), mianserin (10 mg/kg IP) or clenbuterol (5 mg/kg IP) had no effect on either response. Subchronic treatment with sibutramine HCl (3 mg/kg IP; 3 days) also attenuated pre- and postsynaptic ₂-adrenoceptor function. Five ECS (200 V, 2 s) spread over 10 days, but not a single shock, reduced the hypoactivity and mydriasis responses to clonidine. Together, the results indicate that pre- and postsynaptic ₂-adrenoceptor function is attenuated by repeated treatment with those antidepressants which acutely increase synaptic levels of noradrenaline. These adrenergic receptor populations are also desensitized by ECS, although this effect is probably mediated via a different mechanism. Finally, the rapid down-regulation observed with sibutramine HCl is not confined to-adrenoceptors alone, because pre- and postsynaptic ₂-adrenoceptor function is also attenuated by 3 days of treatment with this novel antidepressant drug.     

Effects of the specific α4β2 nAChR antagonist, 2-fluoro-3-(4-nitrophenyl) deschloroepibatidine, on nicotine reward-related behaviors in rats and mice

Rationale

Alleviating addiction to tobacco products could prevent millions of deaths. Investigating novel compounds selectively targeting α4β2 nAChRs hypothesized to have a key role in the rewarding effects of nicotine may be a useful approach for future treatment.

Objectives

The present study was designed to evaluate 2-fluoro-3-(4-nitrophenyl) deschloroepibatidine (4-nitro-PFEB), a potent competitive antagonist of neuronal α4β2 nAChRs, in several animal models related to nicotine reward: drug discrimination, intracranial self-stimulation (ICSS), conditioned place preference, and limited access to self-administration.

Methods

Long Evans rats were trained in a two-lever discrimination procedure to discriminate 0.4 mg/kg nicotine (s.c.) from saline. Male Sprague–Dawley rats were stereotaxically implanted with electrodes and trained to respond for direct electrical stimulation of the medial forebrain bundle. ICR mice were evaluated using an unbiased place preference paradigm, and finally, male Wistar rats were implanted with intrajugular catheters and tested for nicotine self-administration under limited access (1 h/day).

Results

4-Nitro-PFEB attenuated the discriminative stimulus effects of nicotine, but alone did not produce nicotine-like discriminative stimulus effects. Nicotine-induced facilitation of ICSS reward thresholds was reversed by 4-nitro-PFEB, which alone had no effect on thresholds. 4-Nitro-PFEB also blocked the conditioned place preference produced by nicotine, but alone had no effect on conditioned place preference. Finally, 4-nitro-PFEB dose-dependently decreased nicotine self-administration.

Conclusions

These results support the hypothesis that neuronal α4β2 nAChRs play a key role in mediating the rewarding effects of nicotine and further suggest that targeting α4β2 nAChRs may yield a potential candidate for the treatment of nicotine dependence.     

Sazetidine-A,a selective α4β2 nicotinic acetylcholine receptor ligand: effects on dizocilpine and scopolamine-induced attentional impairments in female Sprague–Dawley rats

Background  

Neuronal nicotinic receptor systems have been shown to play key roles in cognition. Nicotine and nicotinic analogs improve attention and nicotinic antagonists impair it. This study was conducted to investigate the role of α4β2 nicotinic receptors in sustained attention using a novel selective α4β2 nicotinic receptor ligand, sazetidine-A.     

Effects of glutathione, N-acetyl-cysteine, α-lipoic acid and dihydrolipoic acid on the cytotoxicity of a 2-pyridylcarboxamidrazone antimycobacterial agent in human mononuclear leucocytes in vitro

A series of antioxidants was used to explore the cytotoxicity of one particularly toxic antimycobacterial 2-pyridylcarboxamidrazone anti-tuberculosis agent against human mononuclear leucocytes (MNL), in comparison with isoniazid (INH) to aid future compound design. INH caused a significant reduction of nearly 40% in cell recovery compared with control (P < 0.0001), although the co-incubation with either glutathione (GSH, 1 mM) or (NAC, 1 mM) showed abolition of INH toxicity. In contrast, the addition of GSH or NAC 1 h after INH failed to protect the cells from INH toxicity (P < 0.0001). The 2-pyridyl-carboxamidrazone ‘Compound 1’ caused a 50% reduction in cell recovery compared with control (P < 0.001), although this was abolished by the presence of either GSH or NAC. A 1 h post incubation with either NAC or GSH after Compound 1 addition failed to protect the cells from toxicity (P < 0.001). Co-administration of lipoic acid (LA) abolished Compound 1-mediated toxicity, although again, this effect did not occur after LA addition 1 h post incubation with Compound 1 (P < 0.001). However, co-administration of dihydrolipoic acid (DHLA) prevented Compound 1-mediated cell death when incubated with the compound and also after 1 h of Compound 1 alone. Pre-treatment with GSH, then removal of the antioxidant resulted in abolition of Compound 1 toxicity (vehicle control, 63.6 ± 16.7 versus Compound 1 alone 26.1 ± 13.6% versus GSH pre-treatment, 65.7 ± 7.3%). In a cell-free incubation, NMR analysis revealed that GSH does not react with Compound 1, indicating that this agent is not likely to directly deplete membrane thiols. Compound 1’s MNL toxicity is more likely to be linked with changes in cell membrane conformation, which may induce consequent thiol depletion that is reversible by exogenous thiols.     

Effects of carbamazepine and metabolites on IL-2, IL-5, IL-6, IL-10 and IFN-γ secretion in epileptic patients: the influence of co-medication

Carbamazepine is a widely used anticonvulsive agent. Its metabolic pathway leads not only to the major active metabolite, carbamazepine-10,11-epoxide, but also to minor terminal metabolites such as iminostilbene and acridine. Carbamazepine is usually well-tolerated, but it may lead to rare, but serious, hypersensitive reactions associated with hypereosinophilia. The mechanisms of hypersensitivity reactions to carbamazepine are still largely unknown, and the implications of the cell-mediated immune response (Th1 pathway) or the humoral immune response (Th2 pathway) are still not understood in these reactions. It is also unclear whether the parent drug or its subsequent metabolites are the primary trigger agent. In our study, we performed ex vivo experiments to evaluate the stimulation of cytokine secretion by carbamazepine, carbamazepine-10,11-epoxide, iminostilbene and acridine. IL-5, IL-6 and IL-10 were quantified as markers of the Th2 pathway, and IL-2 and IFN-γ were used as markers of the Th1 pathway. Blood samples (n=24) were obtained from epileptic patients routinely treated with carbamazepine alone or co-treated with lamotrigine or valproate. The concentrations of cytokines in the plasma were determined before and after 3 h stimulation with drugs. We found a significantly positive effect of co-treatment with valproate on the basal level of IL-5 (p<0.01) and IL-10 (p<0.05). IL-5 production increased only in blood stimulated with a high level of acridine (33 μM), whereas IL-6 production was less specifically stimulated (p<0.05). Because IL-5 is the most potent stimulating factor of the eosinophils, we suggest that the potential helper effect of valproate and acridine can lead to hypersensitive reactions to carbamazepine in the context of the humoral immune response.     

Effects of Piper nigrum extracts: Restorative perspectives of high-fat diet-induced changes on lipid profile,body composition,and hormones in Sprague–Dawley rats

Abstract

Context: Piper nigrum Linn (Piperaceae) (PnL) is used in traditional medicine to treat gastric ailments, dyslipidemia, diabetes, and hypertension.

Objective: The present study explores the possible protective effects of P. nigrum extracts on high-fat diet-induced obesity in rats.

Materials and methods: High-fat diet-induced obese rats were treated orally with 200?mg/kg bw of different extracts (hexane, ethylacetate, ethanol, and aqueous extracts) of PnL for 42?d. The effects of PnL extracts on body composition, insulin resistance, biochemical parameters, leptin, adiponectin, lipid profile, liver marker enzymes, and antioxidants were studied.

Results and discussion: The HFD control group rats showed a substantial raise in body weight (472.8?±?9.3?g), fat% (20.8?±?0.6%), and fat-free mass (165.9?±?2.4?g) when compared with normal control rats whose body weight, fat%, and fat-free mass were 314.3?±?4.4?g, 6.4?±?1.4%, and 133.8?±?2.2?g, respectively. Oral administration of ethyl acetate or aqueous extracts of PnL markedly reduced the body weight, fat%, and fat-free mass of HFD-fed rats. In contrast to the normal control group, a profound increase in plasma glucose, insulin resistance, lipid profile, leptin, thiobarbituric acid reactive substance (TBARS), and the activities of lipase and liver marker enzymes, and a decrease in adiponectin and antioxidant enzymes were noted in HFD control rats. Administration of PnL extracts to HFD-induced obese rats significantly (p?<?0.05) restored the above profiles.

Conclusion: PnL extracts significantly reduced the body weight, fat%, and ameliorated HFD-induced hyperlipidemia and its constituents.     

Development of Infrared Imaging to Measure Thermogenesis in Cell Culture: Thermogenic Effects of Uncoupling Protein-2, Troglitazone,and β-Adrenoceptor Agonists

Purpose. Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours. In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography. Methods. Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone. Adipocytes were exposed to rotenone, FCCP, cycloheximide, troglitazone, or CL316243. Thermogenesis was measured using infrared thermography. Results. Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2. Further, thermogenesis in adipocytes was stimulated by CL316243, a ₃-adrenoceptor agonist being developed to treat obesity. The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis. In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes. Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes. Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes. Conclusions. The results, taken together with the high resolution (0.002°C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.     

The effects of nicotine, varenicline, and cytisine on schedule-controlled responding in mice: differences in α4β2 nicotinic receptor activation

Nicotine, varenicline, and cytisine are pharmacotherapies for tobacco dependence; the extent to which their in vivo effects vary as a function of differences in nicotinic acetylcholine receptor agonism is not clear. Male C57BL/6 J mice responding under a fixed ratio 30 schedule of food delivery were used to establish the potency and time course of nicotine, varenicline, and cytisine; antagonism was examined with the non-competitive, non-selective antagonist mecamylamine and the competitive, α4β2 nicotinic receptor antagonist dihydro-β-erythroidine (DHβE). Intraperitoneal nicotine, varenicline, and cytisine dose-dependently decreased responding; nicotine was more potent (ED₅₀ value = 0.83 mg/kg) than varenicline (ED₅₀ value = 2.51 mg/kg) and cytisine (ED₅₀ value = 2.97 mg/kg). The agonists had a similar time course including a rapid onset (5 min or less) and relatively short duration of action (30 min). Mecamylamine dose-dependently attenuated the rate-decreasing effects of a fixed dose of nicotine (1.78 mg/kg), varenicline (5.6 mg/kg), and cytisine (5.6 mg/kg). Mecamylamine (1 mg/kg) produced parallel rightward shifts in the dose–response curves for nicotine (3.3-fold), varenicline (3.1-fold), and cytisine (2.3-fold). In contrast, DHβE (3.2 mg/kg) produced 2-fold antagonism of nicotine and did not antagonize varenicline or cytisine. The data strongly suggest that nicotinic acetylcholine receptors mediate the effects of the agonists to decrease operant responding in mice. However, α4β2 receptor agonism appears to contribute partially to the rate-decreasing effects of nicotine but not to the rate-decreasing effects of varenicline and cytisine. Differential activation of α4β2 receptors in vivo might contribute to differences in the effectiveness of these smoking cessation aids.     

Novel 4-substituted-2(1H)-phthalazinone derivatives: synthesis, molecular modeling study and their effects on α-receptors

Novel 4-(4-bromophenyl)phthalazine derivatives connected via an alkyl spacer to amine or N-substituted piperazine were designed and synthesized as promising α-adrenoceptor antagonists. The structures of the phthalazine derivatives were established using elemental and spectral analyses. Twelve of the tested compounds displayed significant α-blocking activity. Molecular modeling studies were performed to rationalize the biological results. Among the tested compounds, 7j displayed the best-fitting score and the highest in vitro activity.