Application of disaccharides alone and in combination,for the improvement of stability and particle properties of spray-freeze dried IgG

Purpose: Spray-freeze drying (SFD) is a recently applied method to develop pharmaceutical powders. This study aimed to analyze the competence of Trehalose, Mannitol, Lactose, and Sorbitol instability and aerosolization of Immunoglobulin G (IgG) via SFD.

Methods: Induced soluble aggregates were quantified at 0 and 3?months, and 45?°C using size-exclusion chromatography. Conformation and thermogravimetric assessments were done by Fourier transform infrared spectroscopy and differential scanning calorimetry. Laser light scattering was performed to determine the particle sizes. Aerodynamic features were characterized by twin stage impinger and scanning electron microscopy.

Results: Although sugars/polyols preferably stabilized IgG following the process, storage stabilization was achieved in Trehalose, Trehalose-Lactose, Lactose, and Trehalose-Mannitol-based powders with soluble aggregates <5%. The conformation of antibody was preserved with β sheet content from 66.28% to 76.37%. Particle sizes ranged from 5.23 to 8.12?µm. Mannitol exhibited the best aerodynamic behavior, fine particle fraction (FPF: 70%) but high degree of protein aggregation during storage.

Conclusions: SFD could favorably stabilize antibody using Trehalose and its combination with Lactose and Mannitol, and also, Lactose alone. Sorbitol disturbed IgG powder recovery. Incorporation of other types of excipient is required for efficient respiratory delivery of IgG molecules.     

Adenosine produces pulmonary vasoconstriction in sheep. Evidence for thromboxane A2/prostaglandin endoperoxide-receptor activation

Adenosine, an intermediate product in the metabolism of ATP, is thought to produce vasodilation in all vascular beds with the exception of the kidney. Due to its theoretical potential as a pulmonary vasodilator, we studied the hemodynamic effects of adenosine in the pulmonary vasculature of chronically instrumented awake sheep. Adenosine produced significant pulmonary vasoconstriction instead of the expected vasodilatation. Bolus injections of adenosine into the superior vena cava produced a dose-dependent increase in pulmonary artery pressure that was entirely due to an increase in vascular resistance, since cardiac output decreased slightly. This effect is produced via activation of specific cell surface adenosine receptors, since it was blocked by the adenosine-receptor antagonists theophylline and dipropylsulfophenylxanthine. The cell type involved in adenosine-induced pulmonary vasoconstriction appears to be located within the lung, since vasoconstriction was blunted when adenosine was infused into the left atrium, distal to the lung. However, adenosine does not directly vasoconstrict the pulmonary vasculature, because its effect could be completely abolished by cyclooxygenase inhibition with either indomethacin or ibuprofen and by a thromboxane A2/prostaglandin endoperoxide-receptor antagonist (SQ 29,548). Adenosine-induced vasoconstriction was also greatly reduced after inhibition of thromboxane synthesis. Thus, adenosine produced pulmonary vasoconstriction through generation of a thromboxane/endoperoxide product. Whether endogenous adenosine is involved in the generation of pulmonary vasoconstriction seen in pathophysiological states remains to be determined. To our knowledge, this is the first clear evidence for adenosine-induced vasoconstriction outside the kidney and for an interaction between adenosine and eicosanoid mechanisms.     

The effect of cannabis on oxidative stress and neurodegeneration induced by intrastriatal rotenone injection in rats

We investigated the effect of cannabis treatment on the development of oxidative stress and nigrostriatal cell injury induced by intrastriatal rotenone injection in rats. Rotenone was injected into the right striatum at a concentration of 5 mM (3 μl/rat). The control rats received the vehicle (DMSO). Subsequently, the effect of Cannabis sativa extract treatment on rotenone toxicity was evaluated. Starting on the second day of rotenone injection, rats were treated with C. sativa extract (5, 10, or 15 mg/kg) (expressed as Δ⁹-tetrahydrocannabinol) subcutaneously (s.c.) once daily for 30 days. Biochemical markers of oxidative stress, malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide, paraoxonase 1 (PON1) activity, catalase activity, as well as tumor necrosis factor alpha (TNF-α), were determined in different brain areas after 30 days of rotenone treatment. Histopathology and immunohistochemical expression of tyrosine hydroxylase (TH), capase 3, and inducible nitric oxide synthase (iNOS) were also performed. Results showed that intrastriatal injection of rotenone resulted in increased brain oxidative stress in the cerebral cortex, striatum, hippocampus, midbrain, and cerebellum. MDA increased by 41.4–70 %, nitric oxide increased by 48.3–77.5 %, while GSH decreased by 25.0–34.2 %. PON1 and catalase activities decreased by 43.0–60.8 % and by 14.2–36 %, respectively, in these areas. Striatal TNF-α increased by 638.9 % of control value after rotenone injection. Rotenone induced motor deficits (decreased rearing activity). Rotenone caused marked nigrostriatal neurodegeneration, decreased TH immunoreactivity, and increased both iNOS and caspase 3 immunoreactivities in the striatum. Cannabis decreased brain oxidative stress and nitric oxide release induced by intrastriatal rotenone in several brain areas. Cannabis also decreased the elevated TNF-α in the striatum. Cannabis did not protect against the immunohistochemical changes in the striatum and substantia nigra or against neuronal degeneration induced by rotenone treatment. Collectively, these results indicated that the administration of cannabis did not protect against nigrostriatal damage caused by intrastriatal rotenone.     

Inhibition of inducible nitric oxide synthase enhances anti-tumour immune responses in rats immunized with IFN-gamma-secreting glioma cells

Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, l-N(6)-(1-Iminoethyl)-l-lysine (l-NIL), and non-selective, N-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l-NIL and l-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma, and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with l-NIL, but not l-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, l-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.     

Evaluation of Intradermal Injection of Botulinum Toxin A for Facial Lifting

BACKGROUND: Intradermal botolinum toxin A (BTXA) is an advanced technique that emerged in response to the increased demand for noninvasive facial lifting and skin rejuvenation. OBJECTIVE: We sought to evaluate the safety and efficacy of intradermal injections of BTXA for facial lifting. METHODS: Twenty-five female patients with mild symmetrical facial skin laxity were enrolled in this study. All patients were treated with BTXA in one side of the face while the other side was injected with normal saline. The response to treatment was assessed by two dermatologists who evaluated global photographs using a quartile grading scale (QGS). The patient self-assessment and satisfaction questionnaires were recorded. RESULTS: A highly significant difference was found between the side injected with BTXA and the saline injected side (control) (p<0.001). Facial lifting was achieved in 58.66 percent of the sides injected with BTXA. Forty-four percent of patients were very satisfied. Older patients showed better improvement than younger patients (p=<0.001). The results persisted for 16 weeks. No adverse effects were observed. CONCLUSION: Our results suggest that intradermal injection of BTXA could be a safe and effective therapeutic option for face lifting.     

Protective Effects of Cannabis sativa on chemotherapy-induced nausea in a rat: Involvement of CB1 receptors

Cyclophosphamide is an anticancer and immunosuppressive agent used in the treatment of various malignancies but causing gastrointestinal distress. Cannabis sativa and its derivatives have been used for the treatment of human gastrointestinal disorders. A purpose of this study was to investigate the effect of C. sativa on nausea induced by cyclophosphamide in rats. The rats were divided into four groups (eight animals per group): Group 1: Normal control (saline i.p.). Group 2: Rats received cyclophosphamide (200 mg/kg i.p.) 3 consecutive days. Group 3 and 4: Rats received cyclophosphamide (200 mg/kg i.p.) across Days 1–7, and C. sativa (20 and 40 mg/kg s.c.) was administered on cyclophosphamide days 4–7. We examined intake of kaolin, normal food and changes in body weight, as an indicator of the emetic stimulus. Oxidative stress markers, antioxidant enzymes status, serotonin (5-HT), dopamine, noradrenaline and CB1R levels were evaluated in the intestinal homogenate. Moreover, histopathological study was performed. Results showed that C. sativa ameliorates cyclophosphamide-induced emesis by increasing in body weight and normal diet intake with a decrease in kaolin diet intake after 7 days. Moreover, C. sativa significantly decreases (serotonin) 5-HT, dopamine and noradrenaline, as well as decreasing oxidative stress and inflammation. Administration of C. sativa significantly increased the expression of CB1R in intestinal homogenate. Treatment with C. sativa also improved the histological feature of an intestinal tissue. These results suggested that C. sativa possess antiemetic, antioxidant and anti-inflammatory effects in chemotherapy-induced nausea in rats by activating CB1R.     

Misoprostol decreases oxidative stress and liver injury in bacterial lipopolysaccharide-induced endotoxemia in mice

The effect of misoprostol, a synthetic prostaglandin E1 analog, on the development of oxidative stress induced in mice with lipopolysaccharide (LPS) endotoxin was investigated. Misoprostol was administered by intraperitoneal route (i.p.) at doses of 10, 100, or 1,000 μg/kg at the time of LPS injection (200 μg/kg, i.p.). Mice were euthanized 4 h later. Lipid peroxidation (malondialdehyde; MDA), reduced glutathione (GSH), nitric oxide (nitrite/nitrate) levels as well as paraoxonase activity were measured in brain and liver. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities as well as DNA fragmentation were determined in the liver. The administration of LPS increased oxidative stress both in the brain and liver tissue. There were significantly increased MDA and nitrite and decreased GSH and PON1 activity in the brain and liver, respectively. In addition, LPS was associated with markedly elevated plasma ALT and AST level as well as increased liver DNA fragmentation. The administration of misoprostol at 100 or 1,000 μg/kg decreased brain MDA by 17.6 and 30 %, increased GSH by 29.8 and 33.3 %, and decreased nitric oxide by 21.74 and 42.5 %, respectively, compared with the lipopolysaccharide control group. Liver MDA decreased by 27 %, GSH increased by 47.7 %, and nitric oxide decreased by 37.2 % with misoprostol at 1,000 μg/kg. Paraoxonase activity increased in both the brain and liver by misoprostol administration. The increase in liver AST and ALT and DNA fragmentation after endotoxin administration was normalized by misoprostol. These results indicate that misoprostol can alleviate oxidative stress in the presence of a mild systemic inflammatory illness, indicating a new and potentially important therapeutic application for the drug.