Plasma proteomic changes in response to surgical trauma and a novel transdermal analgesic treatment in dogs.

Assessment of pain responses and inflammation during animal surgery is difficult because traditional methods, such as visual analogue scores, are not applicable while under anaesthesia. Acute phase proteins (APPs), such as C-reactive protein and haptoglobin, that are typically monitored in veterinary research, do not show a significant change until at least 2 h post-surgery and therefore, immediate pathophysiological changes are uncertain. The current study used sequential window acquisition of all theoretical mass spectra (SWATH-MS) to investigate plasma proteome changes that occur immediately following surgery in dogs and also to assess the efficacy of a novel transdermal ketoprofen (TK) formulation. Castration was chosen as surgical model in this study. The procedure was performed on twelve dogs (n = 6 in two groups) and blood samples were collected at 0 h, 1 and 2 h after surgery for proteomic analysis. Following surgery, there was a general downregulation of proteins, including complement C- 3, complement factor B, complement factor D, transthyretin, and proteins associated with lipid, cholesterol, and glucose metabolisms, reflecting the systemic response to surgical trauma. Many of these changes were diminished in the transdermal group (TD) since ketoprofen, a non-steroidal anti-inflammatory drug (NSAID), inhibits prostanoids and the associated chemotactic neutrophil migration to site of tissue injury. SIGNIFICANCE: SWATH-MS Proteomic analysis revealed significant changes in plasma proteins, predominantly involved in early acute phase and inflammatory response at 1 & 2 h after surgery in castrated dogs. Pre-operative application of transdermal ketoprofen formulation had reduced the systemic immune response, which was confirmed by negligible alteration of proteins in transdermal treated group. A key outcome of this experiment was studying the efficacy of a novel transdermal NSAID formulation in dogs.

Pharmacokinetic Evaluation of a Novel Transdermal Ketoprofen Formulation in Healthy Dogs.

Dogs undergo various surgical procedures such as castration, ovariohysterectomy, and other orthopedic procedures, which are known to cause inflammation and pain. Non-steroidal anti-inflammatory drugs (NSAIDs) are very effective analgesics for alleviating postoperative pain in veterinary medicine. Ketoprofen is currently approved in Australia and the United States for treating different painful conditions in dogs. This study evaluated the pharmacokinetic parameters of ketoprofen after intravenous (IV) and transdermal (TD) administration in healthy dogs. A novel transdermal ketoprofen (TDK) formulation containing 20% ketoprofen, dissolved in a combination of 45:45% isopropanol and Transcutol, along with 10% eucalyptus oil, was developed and evaluated for in vitro dermal permeation using Franz diffusion cells. A crossover study was then conducted to determine the pharmacokinetic parameters of the formulation in six dogs following IV ketoprofen (1 mg/kg) and TDK (10 mg/kg) administration. A liquid chromatography-mass spectrometry (LC-M/MS) method was used to measure plasma concentrations of ketoprofen over time, and a non-compartmental analysis determined the pharmacokinetic parameters. The mean terminal elimination half-life (T½ h), AUC0-t (µg·h/mL), and mean residence time (MRT, h) between IV and TDK groups were 4.69 ± 1.33 and 25.77 ± 22.15 h, 15.75 ± 7.72 and 8.13 ± 4.28 µg·h/mL, and 4.86 ± 1.81 and 41.63 ± 32.33 h, respectively. The calculated bioavailability (F%) was ~7%, with a lag time of 30 min to achieve effective plasma concentrations after the application of TDK.

Data-Independent Acquisition Enables Robust Quantification of 400 Proteins in Non-Depleted Canine Plasma.

Mass spectrometry-based plasma proteomics offers a major advance for biomarker discovery in the veterinary field, which has traditionally been limited to quantification of a small number of proteins using biochemical assays. The development of foundational data and tools related to sequential window acquisition of all theoretical mass spectra (SWATH)-mass spectrometry has allowed for quantitative profiling of a significant number of plasma proteins in humans and several animal species. Enabling SWATH in dogs enhances human biomedical research as a model species, and significantly improves diagnostic and disease monitoring capability. In this study, a comprehensive peptide spectral library specific to canine plasma proteome was developed and evaluated using SWATH for protein quantification in non-depleted dog plasma. Specifically, plasma samples were subjected to various orthogonal fractionation and digestion techniques, and peptide fragmentation data corresponding to over 420 proteins was collected. Subsequently, a SWATH-based assay was introduced that leveraged the developed resource and that enabled reproducible quantification of 400 proteins in non-depleted plasma samples corresponding to various disease conditions. The ability to profile the abundance of such a significant number of plasma proteins using a single method in dogs has the potential to accelerate biomarker discovery studies in this species.

Bombesin conjugated solid lipid nanoparticles for improved delivery of epigallocatechin gallate for breast cancer treatment.

Epigallocatechin-gallate (EGCG) is a potent anti-cancer therapeutic which effectively controls the growth of cancerous cells through a variety of different pathways. However, its molecular structure is susceptible to modifications due to cellular enzymes affecting its stability, bioavailability and hence, overall efficiency. In this study, we have initially encapsulated EGCG in the matrix of solid lipid nanoparticles to provide a stable drug carrier. To confer additional specificity towards gastrin releasing peptide receptors (GRPR) overexpressed in breast cancer, EGCG loaded nanoparticles were conjugated with a GRPR-specific peptide. In-vitro cytotoxicity studies showed that the peptide-conjugated formulations possessed greater cytotoxicity to cancer cell lines compared to the non-conjugated formulations. Further, in-vivo studies performed on C57/BL6 mice showed greater survivability and reduction in tumour volume in mice treated with peptide-conjugated formulation as compared to the mice treated with non-conjugated formulation or with plain EGCG. These results warrant the potential of the system designed in this study as a novel and effective drug delivery system in breast cancer therapy.

Supplementation of oat (Avena sativa L.) extract abates alcohol-induced acute liver injury in a mouse model.

Dietary supplementation of oats has been associated with reduced risk of cardiovascular disease, diabetes, and gastrointestinal disorders. The role of oat extract as prophylactic in treating acute liver injury is not thoroughly established. We, therefore, hypothesized that oat extract would exert protective effect against alcohol-induced acute liver injury in a mouse model. To test this hypothesis, male C57BL/6 mice were pretreated with phenolic-enriched ethyl acetate (EA) fraction of oats (prepared by fractionating aqueous ethanolic extract with solvents of increasing polarity) at dosages of 125 and 250 mg kg-1 d-1 for 12 consecutive days. Acute liver injury was induced by administering 5 doses of 50% ethanol intragastrically (10 g/kg body weight) to mice at an interval of 12 hours. The alcohol-induced liver injury was evaluated by measuring serum levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, antioxidant parameters, mitochondrial function, and histology of liver tissue. Our results demonstrated that pretreatment with EA fraction at 250 mg kg-1 d-1 significantly (P < .001 for aspartate aminotransferase, alanine aminotransferase, and thiobarbituric acid-reactive species and P < .01 for lactate dehydrogenase and nitrites) reduced the levels of liver injury markers and significantly (P < .001 for glutathione reductase and glutathione S-transferase; P < .01 for catalase, superoxide dismustase, and vitamin C; P < .05 for reduced glutathione and NAD(P)H quinone dehydrogenase 1) increased the levels of antioxidant defenses. Furthermore, EA-pretreated mice showed mechanistic inhibition of nuclear factor κB signaling pathway through decreased phosphorylation and degradation of IκBα. We conclude that phenolic-enriched EA fraction of oats has immense potential to serve as dietary intervention against alcohol-induced liver damage.

Fabrication of surfactant-stabilized nanosuspension of naringenin to surpass its poor physiochemical properties and low oral bioavailability.

BACKGROUND: Nanosuspension is a biphasic system consisting of native drug particles dispersed in an aqueous surfactant or polymeric solution with a particle size between 10 to 1000 nm. In contrast to other drug delivery systems, nanosuspension offer the unique advantage of increasing solubility of the native drug resulting into faster drug absorption and hence achieving faster maximum plasma concentration. HYPOTHESIS/PURPOSE: The present study aims to evaluate surfactants/polymer stabilized nanosuspensions of naringenin (NN), a phytomedicine, to surpass its poor physiochemical properties and low oral bioavailability. STUDY DESIGN: Optimization and characterization (DLS, SEM, PXRD and DSC) of nanosuspensions followed by in-vitro drug dissolution studies and pharmacokinetic study in male Sprague-Dawley rats were performed. METHODS: Nanosuspensions were prepared by precipitation-ultrasonication method with varying concentrations of different surfactants and polymer such as sodium cholate (SC), sodium lauryl sulphate (SLS), poly ethylene glycol 4000 (PEG), polysorbate 80 (Tween® 80), poloxomer-188 and D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS or Vitamin E-TPGS). RESULTS: Nanosuspension prepared with 0.5% w/v d-α-Tocopherol polyethylene glycol 1000 succinate (TPNS) and 7.5 mg NN, showed the smallest size of 118.1 ±â€¯2.7 nm. TPNS showed increase in drug dissolution in simulated gastric fluid pH 1.2 (SGF) and phosphate buffer pH 6.8 (PB). TPNS demonstrated an improved pharmacokinetic profile compared to pure NN resulting 2.14 and 3.76 folds increase in Cmax and AUC, respectively. In addition, TPNS were stable over a period of six months. CONCLUSION: The developed formulation strategy of nanosuspension could be exploited to improve the solubility and bio-availability of poorly soluble NN and other phytomedicines.

Ferulic acid protects lipopolysaccharide-induced acute kidney injury by suppressing inflammatory events and upregulating antioxidant defenses in Balb/c mice.

Sepsis-induced acute kidney injury (AKI) is responsible for 70-80% mortality in intensive care patients due to elevated levels of endotoxin, Lipopolysaccharide (LPS) caused by gram-negative infections. Ferulic acid (FA), a phenolic phytochemical is known for its renal protection on various induced models of nephrotoxicity. However, the curative effect of FA in LPS-induced AKI is not well studied. This study aimed to investigate the effect of FA on LPS-induced AKI in mice model and to understand the protective mechanisms involved, to provide evidence for FA in the treatment of AKI. Balb/c mice were treated with FA at 50 mg/kg and 100 mg/kg dosages after LPS stimulation (10 mg/kg). At the end of the intervention, we determined the concentrations of serum creatinine and blood urea nitrogen, inflammatory cytokines and histopathological changes in animals. Also, the relative protein expression level of TLR4 mediated NF-κB signaling pathway were studied in kidney tissues. FA treated animals showed upregulation of antioxidant defenses and suppression of inflammatory events by inhibiting TLR-4 mediated NFκB activation. However, LPS alone administered group, resulted in rapid renal damage with increased levels of blood urea nitrogen and modest increase in creatinine; decreased antioxidant defenses and release of inflammatory cytokines. The histopathological analysis also revealed the protective action of the FA against sepsis induced fibrosis and renal damage. Our findings demonstrated that FA exhibits marked protective effects on LPS-induced AKI in mice suggesting its chemopotential role for treating AKI in humans.

Capsaicin, the pungent principle of peppers, ameliorates alcohol-induced acute liver injury in mice via modulation of matrix metalloproteinases.

Alcohol, the most common cause for hepatic injury, may further deteriorate the hepatic tissue when left unattended. Capsaicin, the pungent principle of chilli peppers, possesses antioxidant and anti-inflammatory properties and is a proven dietary antioxidant in various ailments. However, its role in alcohol-induced hepatic injury is unclear. In this study, we investigated the effects of capsaicin on the hepatic tissue of mice treated with alcohol. Acute liver injury was induced in mice by oral gavage of 5 doses of 10 mL/kg of 50% ethyl alcohol at an interval of 12 h. The tissue antioxidant levels along with the mitochondrial functional parameters and matrix metalloproteinase levels were evaluated in the hepatic tissues of mice following alcohol challenge. The results showed that alcohol intake significantly attenuated the hepatic antioxidant levels and mitochondrial function. These changes were accompanied by enhanced serum hepatic injury markers and matrix metalloproteinases. However, capsaicin treatment (10 and 20 mg/kg, oral) throughout the experimental period caused a drastic improvement in the hepatic tissue of the alcohol-treated mice, reflected by the normalization of hepatic enzyme and protein levels along with restored histological alterations. These results indicate that capsaicin, as a dietary intervention, may prevent alcohol-induced acute liver injury.

Large Amino Acid Transporter 1 Selective Liposomes of l-DOPA Functionalized Amphiphile for Combating Glioblastoma.

Despite significant progress in neurosurgery and radiation therapy during the past decade, overall survivability (OS) of glioblastoma patients continues to be less than 2 years. The scope of systemic chemotherapy is greatly limited by poor drug transport across the blood brain barrier (BBB) and, thereby, suboptimal drug accumulation in glioma tissue. To this end, use of large amino acid transporter-1 (LAT1) overexpressed both on brain capillary endothelial cells (BCECs) and glioma cells has begun. Prior reports on the use of LAT1 mediated delivery of model drugs showed their brain accumulations. However, in depth in vivo glioblastoma regression studies aimed at examining the therapeutic potential of LAT1 mediated delivery of potent chemotherapeutics to brain tumor tissues have not yet been undertaken. Herein, we report on the development of a nanometric (100-135 nm) promising LAT1 selective liposomal drug carrier prepared from a novel l-3,4-dihydroxyphenylalanine (l-DOPA) functionalized amphiphile (Amphi-DOPA). In vitro studies using Rh-PE labeled liposomes of Amphi-DOPA both in untreated glioma (GL261) cells and in GL261cells preincubated with LAT1 antibody revealed LAT1 mediated cellular uptake. Intravenously administered NIR-dye labeled liposomes of Amphi-DOPA in glioblastoma-bearing mice showed preferential accumulation of the dye in brain tissue. Notably iv administration of WP1066-loaded liposomes of Amphi-DOPA enhanced the overall survivability of C57BL/6J mice bearing orthotopically established mouse glioblastoma by ∼60% compared to that for the untreated mouse group. Furthermore, we show that the OS of established glioblastoma-bearing mice can be significantly enhanced (by >300% compared to that for the untreated mouse group) when the presently described LAT1 mediated targeted chemotherapy with WP1066-loaded liposomes of Amphi-DOPA is combined with in vivo DC-targeted DNA vaccination using a survivin (a glioblastoma antigen) encoded DNA vaccine. The present findings open a new door for LAT1 mediated systemic chemotherapy of glioblastoma.

Polydatin alleviates alcohol-induced acute liver injury in mice: Relevance of matrix metalloproteinases (MMPs) and hepatic antioxidants.

BACKGROUND: Alcohol, a most commonly consumed beverage, is the foremost cause of liver injury throughout the world. Polydatin, a stilbenoid glucoside, was known to possess antioxidant and anti-inflammatory properties and is being investigated for use in various disorders. PURPOSE: The present study was intended at investigating the hepatoprotective efficacy of polydatin against acute-alcohol induced liver injury model in mice. STUDY DESIGN: C57BL/6 mice were fed with five doses of 50% ethyl alcohol (10ml/kg body weight) to induce acute liver injury. Effect of polydatin against alcohol induced hepatic injury was investigated by giving 50 or 100mg/kg polydatin, orally, for 8 days. METHODS: Serum markers of liver injury, morphology, histology and fibrosis of liver tissue, levels of enzymatic and non-enzymatic antioxidants and the mitochondrial respiratory enzyme activities in liver tissue were investigated. The activities and the protein expression of matrix metalloproteinases (MMP-2 and -9), the expression of NF-κB in the liver tissue were also studied. RESULTS: Polydatin pre-treatment significantly alleviated the alcohol induced hepatic injury by reducing the serum liver injury markers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), attenuating oxidative stress and restoring antioxidant balance in the hepatic tissue. Simultaneously, polydatin pre-treatment also prevented alcohol induced mitochondrial damage and refurbished the matrix metalloproteinases levels of the hepatic tissue. CONCLUSION: The findings of the present study suggest that polydatin may have a potential benefit in preventing alcohol-induced acute hepatic injury.