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1.  Genetic Factors Influencing B-type Natriuretic Peptide-Mediated Production of Cyclic Guanosine Monophosphate and Blood Pressure Effects in Heart Failure Patients 
Natriuretic peptides (NPs) represent a critical pathway in heart failure (HF). We explored genetic determinants of pharmacodynamic effects of B-type NP (BNP) and changes in plasma cyclic guanosine monophosphate (cGMP) and blood pressure (BP). HF patients (n=135) received recombinant human BNP (nesiritide) at standard doses, and plasma cGMP levels were measured at baseline and during infusion. We tested the association of 119 single nucleotide polymorphisms (SNPs) in 4 candidate genes (NPR1, NPR2, NPR3, and membrane metallo-endopeptidase (MME)) with the change in cGMP and BP. Gene-based testing for association of genetic variation with endpoints was significant only for MME. Upon individual SNP testing, two loci in MME were associated with ΔcGMP; another (rs16824656) showed association with BP change. In summary, the pharmacodynamic effects of BNP vary substantially in HF patients and are associated with genetic variation inMME.MME genetic variation may be an important determinant of NP-mediated effects in humans.
doi:10.1007/s12265-015-9660-2
PMCID: PMC4686234  PMID: 26589601
Natriuretic peptide; Heart failure; Drug metabolism; Pharmacogenetics; Genetic polymorphisms
2.  Effect of Green Tea Catechins and Hydrolyzable Tannins on Benzo[a]pyrene-Induced DNA Adducts and Structure–Activity Relationship 
Chemical research in toxicology  2010;23(4):771-777.
Green tea catechins and hydrolyzable tannins are gaining increasing attention as chemopreventive agents. However, their mechanism of action is poorly understood. We investigated the effects of four green tea catechins and two hydrolyzable tannins on microsome-induced benzo[a]pyrene (BP)–DNA adducts and the possible structure–activity relationship. BP (1 μM) was incubated with rat liver microsomes and DNA in the presence of the test compound (1–200 μM) or vehicle. The purified DNA was analyzed by 32P-postlabeling. The inhibitory activity of the catechins was in the following descending order: epigallocatechin gallate (IC50 = 16 μM) > epicatechin gallate (24 μM) > epigallocatechin (146 μM) > epicatechin (462 μM), suggesting a correlation between the number of adjacent aromatic hydroxyl groups in the molecular structure and their potencies. Tannic acid (IC50 = 4 μM) and pentagalloglucose (IC50 = 26 μM) elicited as much DNA adduct inhibitory activity as the catechins or higher presumably due to the presence of more functional hydroxyl groups. To determine if the activity of these compounds was due to direct interaction of phenolic groups with electrophilic metabolite(s) of BP, DNA was incubated with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) (0.5 μM) in the presence of test compounds (200 μM) or vehicle. Significant inhibition of DNA adduct formation was found (tannic acid > pentagalloglucose > epigallocatechin gallate > epicatechin gallate). This notion was confirmed by analysis of the reaction products of anti-BPDE with the catechins and pentagalloglucose by electrospray ionization mass spectrometry and liquid chromatography–mass spectrometry. In conclusion, our data demonstrate that green tea catechins and the hydrolyzable tannins are highly effective in inhibiting BP–DNA adduct formation at least, in part, due to direct interaction of adjacent hydroxyl groups in their structures and that the activity is higher with an increasing number of functional hydroxyl groups.
doi:10.1021/tx900412a
PMCID: PMC5087322  PMID: 20218540
3.  Safety Evaluation of Parastar® Plus in Dogs and Assessment of Transferable Residue of Fipronil and Cyphenothrin from Dogs to Humans 
Dogs are easily infested with fleas, ticks, and other ectoparasites serving as vectors for transmitting bacterial, viral, and parasitic diseases. Therefore, the use of ectoparasiticides is inevitable and important. The present investigation was undertaken with two specific objectives: one, to evaluate the safety of fipronil and cyphenothrin in dogs after topical application of Parastar® Plus, and two, to determine the transferable residue of these insecticides from dogs to humans. Six healthy, adult dogs (medium length hair, weighing between 20.5 and 27.3 kg) received topical application of Parastar® Plus (2.68 mL; fipronil, 9.8%, and cyphenothrin, 5.2%) on the back between the shoulder blades. At predetermined intervals, dogs were given a full physical exam, and residues of fipronil and cyphenothrin were determined in dog blood and cotton glove extracts using GC/MS. Fipronil and cyphenothrin peaks eluted at 7.453 and 9.913 min, correspondingly, and the compounds were confirmed based on characteristic ions. At no time was fipronil or cyphenothrin residue detected in blood samples. In glove extracts, residues of fipronil and cyphenothrin were maximally present at 24-h posttreatment (43.84 ± 5.69 and 59.26 ± 8.97 ppm, respectively). By 48 h, the residue levels sharply declined (16.89 ± 2.82 and 17.98 ± 2.07 ppm, respectively). The insecticides’ residues were detected in insignificant amounts after 1 week (5.69 ± 2.16 and 10.00 ± 1.51 ppm, respectively), and only in trace amounts after 2 weeks. At no time did any dog show side effects, except itching at the site of Parastar® Plus application. The findings suggest that Parastar® Plus was safe for dogs, and transferable residues of fipronil and cyphenothrin were minimal, posing very little or no health concern to pet owners or veterinary personnel. Of course, veterinary personnel, who handle many dogs daily, may require proper protection to avoid cumulative exposure.
doi:10.3389/fvets.2016.00089
PMCID: PMC5043066  PMID: 27747219
parastar® plus; fipronil; cyphenothrin; pyrethroids; ectoparasiticide safety; dogs
4.  Curcumin Implants, not Curcumin Diet Inhibits Estrogen-Induced Mammary Carcinogenesis in ACI Rats 
Curcumin is widely known for its anti-oxidant, anti-inflammatory and anti-proliferative activities in cell culture studies. However, poor oral bioavailability limited its efficacy in animal and clinical studies. Recently, we developed polymeric curcumin implants that circumvents oral bioavailability issues, and tested their potential against 17β-estradiol (E2)-mediated mammary tumorigenesis. Female ACI rats were administered curcumin either via diet (1,000 ppm) or via polymeric curcumin implants (two 2-cm; 200 mg each; 20% drug load) 4 days prior to grafting a subcutaneous E2 silastic implant (1.2 cm, 9 mg E2). Implants were changed after 4½ months to provide higher curcumin dose at the appearance of palpable tumors. The animals were euthanized after 3 weeks, 3 months and after the tumor incidence reached >80% (~6 months) in control animals. The curcumin administered via implants resulted in significant reduction in both the tumor multiplicity (2±1 vs 5±3; p=0.001) and tumor volume (184±198 mm3 vs 280±141 mm3; p=0.0283); the dietary curcumin, however, was ineffective. Dietary curcumin increased hepatic CYP1A and CYP1B1 activities without any effect on CYP3A4 activity whereas curcumin implants increased both CYP1A and CYP3A4 activities but decreased CYP1B1 activity in presence of E2. Since CYP1A and 3A4 metabolize most of the E2 to its non-carcinogenic 2-OH metabolite and CYP1B1 produces potentially carcinogenic 4-OH metabolite, favorable modulation of these CYPs via systemically delivered curcumin could be one of the potential mechanisms. The analysis of plasma and liver by HPLC showed substantially higher curcumin levels via implants versus the dietary route despite substantially higher dose administered.
doi:10.1158/1940-6207.CAPR-13-0248
PMCID: PMC3992985  PMID: 24501322
Curcumin; Polymeric implants; Breast cancer inhibition; Estradiol; Hepatic cytochromes
5.  MicroRNA ‘signature’ during estrogen-mediated mammary carcinogenesis and its reversal by ellagic acid intervention 
Cancer letters  2013;339(2):175-184.
Dysregulated miRNA expression has been associated with the development and progression of cancers, including breast cancer. The role of estrogen (E2) in regulation of cell proliferation and breast carcinogenesis is well-known. Recent reports have associated several miRNAs with estrogen receptors in breast cancers. Investigation of the regulatory role of miRNAs is critical for understanding the effect of E2 in human breast cancer, as well as for developing strategies for cancer chemoprevention. In the present study we used the well-established ACI rat model that develops mammary tumors upon E2 exposure and identified a ‘signature’ of 33 significantly modulated miRNAs during the process of mammary tumorigenesis. Several of these miRNAs were altered as early as 3 weeks after initial E2 treatment and their modulation persisted throughout the mammary carcinogenesis process, suggesting that these molecular changes are early events. Furthermore, ellagic acid, which inhibited E2-induced mammary tumorigenesis in our previous study, reversed the dysregulation of miR-375, miR-206, miR-182, miR-122, miR- 127 and miR-183 detected with E2 treatment and modulated their target proteins (ERα, cyclin D1, RASD1, FoxO3a, FoxO1, cyclin G1, Bcl-w and Bcl-2). This is the first systematic study examining the changes in miRNA expression associated with E2 treatment in ACI rats as early as 3 week until tumor time point. The effect of a chemopreventive agent, ellagic acid in reversing miRNAs modulated during E2-mediated mammary tumorigenesis was also established. These observations provide mechanistic insights into the new molecular events behind the chemoprevention action of ellagic acid in and treatment of breast cancer.
doi:10.1016/j.canlet.2013.06.012
PMCID: PMC3775863  PMID: 23791885
Breast cancer; microRNAs; Ellagic acid; Estrogen; Carcinogenesis
6.  Effect of phytochemical intervention on dibenzo[a,l]pyrene-induced DNA adduct formation 
Mutation research  2015;774:25-32.
Dibenzo[a,l]pyrene (DBP) has been found to be the most potent carcinogen of the polycyclic aromatic hydrocarbons (PAHs). Primary sources for DBP in the environment are combustion of wood and coal burning, gasoline and diesel exhaust, and tires. Given the likelihood of environmental exposure to DBP and strong experimental evidence of its potency, it is likely to contribute to lung cancer development. Intervention with compounds of natural origin (“phytochemicals”) is considered an effective means to prevent cancer development and favorably modulate the underlying mechanisms, including DNA adduct formation. In this study, several agents have been identified that inhibit environmental carcinogen-induced DNA adduct formation using a cell-free microsomal system. Of the ten agents tested, resveratrol (648 ± 26 adducts/109 nucleotides), oltipraz (1007 ± 348 adducts/109 nucleotides), delphinidin (1252 ± 142 adducts/109 nucleotides), tanshinone I (1981 ± 213 adducts/109 nucleotides), tanshinone IIA (2606 ± 478 adducts/109 nucleotides) and diindoylmethane (3643 ± 469 adducts/109 nucleotides) were the most effective compared to vehicle treatment (14,062 ± 1097 adducts/109 nucleotides). DBP is metabolized by phase I metabolizing enzymes CYP1A1, CYP1A2, and CYP1B1. DBP-induced DNA adducts can be inhibited by several mechanisms. We found that all the test agents inhibited DNA adducts by inhibiting one or more of these enzymes. Oltipraz inhibited DNA adducts entirely by inhibiting the CYP450s, while resveratrol and delphinidin inhibited DNA adducts by also interacting directly with the carcinogenic metabolite, anti-dibenzo(a,l)pyrene-11,12-dihydrodiol-13,14-epoxide.
doi:10.1016/j.mrfmmm.2015.02.003
PMCID: PMC4402140  PMID: 25794985
Cytochrome P450; Dibenzo[a,l]pyrene; DNA adducts; Polycyclic aromatic hydrocarbons; 32P-postlabeling
7.  Modified silicone sling assisted temporalis muscle transfer in the management of lagophthalmos 
Indian Journal of Ophthalmology  2014;62(2):176-179.
Aim:
To evaluate the efficacy of modified temporalis muscle transfer (TMT) by silicone sling for the management of paralytic lagophthalmos.
Settings and Design:
Prospective interventional study.
Materials and Methods:
Ten patients of lagophthalmos due to facial palsy underwent modified TMT using silicone sling. The patients were followed-up for a period of 3 months. Palpebral aperture in primary gaze and during eye closure were assessed both pre- and postoperatively along with problems associated with lagophthalmos like exposure keratopathy and lacrimation.
Statistical Analysis:
Paired t-test was applied to measure the statistical outcome.
Results:
Eight patients achieved full correction of lagophthalmos with no lid gap on closing the eye. The mean (standard deviation (SD)) lid gap on eye closure was 7.7 (0.86) mm preoperatively, 0.5 (0.47) mm at 1st postoperative day, and 0.7 (0.75) mm at 3rd month. There was a reduction in mean lid gap on eye closure of 7 mm at 3 months (P < 0.0001) which is highly significant. The mean (SD) vertical interpalpebral distance during primary gaze was 12.05 (1.12) mm preoperatively, 10 (0.94) mm at 1st postoperative day, and 10.35 (1.08) mm at 3rd month. There was a reduction in mean vertical inter palpebral distance of 1.7 mm at 3 months (P = 0.001) which is significant. Exposure keratitis decreased in five out of six patients at 3 months.
Conclusion:
Modified TMT by silicone sling is a useful procedure with lesser morbidity and good outcomes for the treatment of paralytic lagophthalmos due to long standing facial palsy.
doi:10.4103/0301-4738.128629
PMCID: PMC4005234  PMID: 24618488
Lagophthalmos; silicone sling; temporalis muscle
8.  Controlled-release systemic delivery - a new concept in cancer chemoprevention 
Carcinogenesis  2012;33(8):1608-1615.
Many chemopreventive agents have encountered bioavailability issues in pre-clinical/clinical studies despite high oral doses. We report here a new concept utilizing polycaprolactone implants embedded with test compounds to obtain controlled systemic delivery, circumventing oral bioavailability issues and reducing the total administered dose. Compounds were released from the implants in vitro dose dependently and for long durations (months), which correlated with in vivo release. Polymeric implants of curcumin significantly inhibited tissue DNA adducts following the treatment of rats with benzo[a]pyrene, with the total administered dose being substantially lower than typical oral doses. A comparison of bioavailability of curcumin given by implants showed significantly higher levels of curcumin in the plasma, liver and brain 30 days after treatment compared with the dietary route. Withaferin A implants resulted in a nearly 60% inhibition of lung cancer A549 cell xenografts, but no inhibition occurred when the same total dose was administered intraperitoneally. More than 15 phytochemicals have been tested successfully by this formulation. Together, our data indicate that this novel implant-delivery system circumvents oral bioavailability issues, provides continuous delivery for long durations and lowers the total administered dose, eliciting both chemopreventive/chemotherapeutic activities. This would also allow the assessment of activity of minor constituents and synthetic metabolites, which otherwise remain uninvestigated in vivo.
doi:10.1093/carcin/bgs209
PMCID: PMC3499062  PMID: 22696595
9.  Controlled Systemic Delivery by Polymeric Implants Enhances Tissue and Plasma Curcumin Levels Compared with Oral Administration 
Curcumin possess potent anti-inflammatory and anti-proliferative activities but with poor biopharmaceutical attributes. To overcome these limitations, curcumin implants were developed and tissue (plasma, brain and liver) curcumin concentrations were measured in female ACI rats for 3 months. Biological efficacy of tissue levels achieved was analyzed by modulation of hepatic cytochromes. Curcumin implants exhibited diffusion-mediated biphasic release pattern with ~2-fold higher in vivo release as compared to in vitro. Plasma curcumin concentration from implants was ~3.3 ng/ml on day 1 which dropped to ~0.2 ng/ml after 3 months whereas only 0.2–0.3 ng/ml concentration was observed from 4–12 days with diet and was undetected subsequently. Almost 10 fold higher curcumin levels were observed in brain on day 1 from implants compared with diet (30.1±7.3 vs 2.7±0.8 ng/g) and were higher even after 90 days (7.7±3.8 vs 2.2±0.8 ng/g). Although, curcumin levels were similar in liver from both the routes (~25–30 ng/g from day 1–4 and ~10–15 ng/g at 90 days), implants were more efficacious in altering hepatic CYP1A1 levels and CYP3A4 activity at ~28 fold lower doses. Curcumin implants provided much higher plasma and tissue concentrations and are a viable alternative for delivery of curcumin to various organs like brain.
doi:10.1016/j.ejpb.2011.12.009
PMCID: PMC3345811  PMID: 22227368
Curcumin; Implants; Bioavailability; Chemoprevention; Controlled Release; Tissue curcumin levels
10.  Applying extracellular vesicles based therapeutics in clinical trials – an ISEV position paper 
Journal of Extracellular Vesicles  2015;4:10.3402/jev.v4.30087.
Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.
doi:10.3402/jev.v4.30087
PMCID: PMC4698466  PMID: 26725829
immunology; neurobiology; haematology; stem cells; tissue regeneration; tumour vaccination; regulation
11.  Potent chemopreventive/antioxidant activity detected in common spices of the Apiaceae family 
Nutrition and cancer  2015;67(7):1201-1207.
Spices are used worldwide, particularly, in the Asian and Middle-Eastern countries and considered protective against degenerative diseases, including cancer. Here, we report the efficacy of aqueous and non-aqueous extracts of eleven Apiaceae spices for free radical-scavenging activity and to inhibit cytochrome P450s in two separate reactions involving: i) 4-hydroxy-17β-estradiol (4E2), DNA and CuCl2 and ii) 17β-estradiol, rat liver microsomes, co-factors, DNA and CuCl2. Oxidative DNA adducts resulting from redox cycling of 4E2 were analyzed by 32P-postlabeling. Aqueous (5 mg/ml) and non-aqueous extracts (6 mg/ml) substantially inhibited (83% – 98%) formation of DNA adducts in the microsomal reaction. However, in non-microsomal reaction, only aqueous extracts showed the inhibitory activity (83% – 96%). Adduct inhibition was also observed at 5-fold lower concentrations of aqueous extracts of cumin (60%) and caraway (90%), and 10-fold lower concentrations of carrot seeds (76%) and ajowan (90%). These results suggests the presence of two groups of phytochemicals - polar compounds that have free radical-scavenging activity, and lipophilic compounds that selectively inhibit P450 activity associated with estrogen metabolism. Because most of these Apiaceae spices are used widely with no known toxicity, the phytochemicals from the Apiaceae spices used in foods may be potentially protective against estrogen-mediated breast cancer.
doi:10.1080/01635581.2015.1075051
PMCID: PMC4684170  PMID: 26381237
Apiaceae spices; Antioxidant activity; Free radical scavenging activity; Redox activity; Oxidative DNA adducts
12.  Chronic Electrical Stimulation of the Carotid Sinus Baroreflex Improves LV Function and Promotes Reversal of Ventricular Remodeling in Dogs with Advanced Heart Failure 
Circulation. Heart failure  2010;4(1):65-70.
Background
Autonomic abnormalities exist in heart failure (HF) and contribute to disease progression. Activation of the Carotid sinus baroreflex (CSB) has been shown to reduce sympathetic outflow and augment parasympathetic vagal tone. This study tested the hypothesis that long-term electrical activation of carotid sinus baroreflex improves left ventricular (LV) function and attenuates progressive LV remodeling in dogs with advanced chronic HF.
Methods and Results
Studies were performed in 14 dogs with coronary microembolization-induced HF (LV ejection fraction, EF ~25%). Eight dogs were chronically instrumented for bilateral CSB activation using the Rheos® System (CVRx® Inc., Minneapolis, MN) and 6 were not and served as controls. All dogs were followed for 3 months and none received other background therapy. During follow-up, treatment with CSB increased LV EF 4.0 ± 2.4 % compared to a reduction in control dogs of −2.8 ± 1.0% (p<0.05). Similarly, treatment with CSB decreased LV end-systolic volume −2.5 ± 2.7 ml compared to an increase in control dogs of 6.7 ± 2.9 ml (p<0.05). Compared to control, CSB activation significantly decreased LV end-diastolic pressure and circulating plasma norepinephrine, normalized expression of cardiac β1-adrenergic receptors, β-adrenergic receptor kinase and nitric oxide synthase and reduced interstitial fibrosis and cardiomyocyte hypertrophy.
Conclusions
In dogs with advanced HF, CSB activation improves global LV function and partially reverses LV remodeling both globally and at cellular and molecular levels.
doi:10.1161/CIRCHEARTFAILURE.110.955013
PMCID: PMC3048958  PMID: 21097604
heart failure; ventricular remodeling; gene expression; baroreflex function
13.  Promising molecular targeted therapies in breast cancer 
Indian Journal of Pharmacology  2011;43(3):236-245.
In recent years, there has been a significant improvement in the understanding of molecular events and critical pathways involved in breast cancer. This has led to the identification of novel targets and development of anticancer therapies referred to as targeted therapy. Targeted therapy has high specificity for the molecules involved in key molecular events that are responsible for cancer phenotype such as cell growth, survival, migration, invasion, metastasis, apoptosis, cell-cycle progression, and angiogenesis. Targeted agents that have been approved for breast cancer include trastuzumab and lapatinib, directed against human epidermal growth factor receptor 2 (HER2) and bevacizumab, directed against vascular endothelial growth factor (VEGF). Several other targeted agents currently under evaluation in preclinical and clinical trials include inhibitors of epidermal growth factor receptor (EGFR), dual EGFR and HER2 inhibitors, VEGF/VEGFR inhibitors, and agents that interfere with crucial signaling pathways such as PI3K/AKT/mTOR and RAS/MEK/ERK; agents against other tyrosine kinases such as Src, insulin-like growth factor (IGF)/IGF-receptor (IGFR); agents that promote apoptosis such as Poly ADP ribose polymerase inhibitors; agents that target invasion and metastasis such as matrix metalloproteinases inhibitors and others. In this review, we highlight the most promising targeted agents and their combination with mainstream chemotherapeutic drugs in clinical trials.
doi:10.4103/0253-7613.81497
PMCID: PMC3113372  PMID: 21713084
Breast cancer; chemotherapy; targeted therapy
14.  Detection of Anthocyanins/Anthocyanidins in Animal Tissues 
Dietary polyphenols may contribute to the prevention of several degenerative diseases, including cancer. Anthocyanins have been shown to possess potential anticancer activity. The aim of this study was to determine anthocyanin bioavailability in lung tissue of mice fed a blueberry diet (5% w/w) for 10 days or a bolus dose (10 mg/mouse; po) of a native mixture of bilberry anthocyanidins. All five anthocyanidins present in the blueberry were detected in the lung tissue using improved methods. The effect of various solvents on the stability of anthocyanins and their recovery from the biomatrix was analyzed. Detection of anthocyanins and their metabolites was performed by UPLC and LC-MS. Although anthocyanins were not detected, cyanidin was detected by UPLC-PDA and other anthocyanidins were detected by LC-MS, following conversion to anthocyanidins and selective extraction in isoamyl alcohol. The results show that anthocyanins can be detected in lung tissue of blueberry-fed mice and thus are bioavailable beyond the gastrointestinal tract.
doi:10.1021/jf500467b
PMCID: PMC4334289  PMID: 24650213
anthocyanins; anthocyanidins; bioavailability; lung tissue; blueberry diet
15.  POLYMERIC IMPLANTS FOR DELIVERY OF GREEN TEA POLYPHENOLS 
Journal of pharmaceutical sciences  2014;103(3):945-951.
Polymeric implants (millirods) have been tested for local delivery of chemotherapeutic agents in cancer treatment. Modeling of drug release profiles is critical as it may provide theoretical insights on rational implant design. In this study, a biodegradable poly (ε-caprolactone) (PCL) polymeric implant delivery system was tested to deliver green tea polyphenols (GTPs), both in vitro and in vivo. Factors including polymer compositions, supplements, drug loads and surface area of implants were investigated. Our data showed that GTPs were released from PCL implants continuously for long durations, and drug load was the main determining factor of GTPs release. Furthermore, the rates of in vitro release and in vivo release in the rat model followed similar kinetics for up to 16 months. A mathematical model was deduced and discussed. GTPs implants have the potential to be used locally as an alternative strategy. GTP implants have the potential to be used systemically and locally at the tumor site as an alternative strategy.
doi:10.1002/jps.23864
PMCID: PMC4009679  PMID: 24464784
16.  Sustained expression of CYPs and DNA adduct accumulation with continuous exposure to PCB126 and PCB153 through a new delivery method: Polymeric implants 
Toxicology reports  2014;1:820-833.
A new delivery method via polymeric implants was used for continuous exposure to PCBs. Female Sprague-Dawley rats received subcutaneous polymeric implants containing PCB126 (0.15% load), PCB153 (5% load), or both, for up to 45 days and release kinetics and tissue distribution were measured. PCB153 tissue levels on day 15 were readily detected in lung, liver, mammary and serum, with highest levels in the mammary tissue. PCB126 was detected only in liver and mammary tissues. However, a completely different pharmacokinetics was observed on co-exposure of PCB153 and PCB126, with a 1.8-fold higher levels of PCB153 in the liver whereas a 1.7-fold lower levels in the mammary tissue. PCB126 and PCB153 caused an increase in expression of key PCB-inducible enzymes, CYP 1A1/2 and 2B1/2, respectively. Serum and liver activities of the antioxidant enzymes, PON1 and PON3, and AhR transcription were also significantly increased by PCB126. 32P-Postlabeling for polar and lipophilic DNA-adducts showed significant quantitative differences: PCB126 increased 8-oxodG, an oxidative DNA lesion, in liver and lung tissues. Adduct levels in the liver remained upregulated up to 45 days, while some lung DNA adducts declined. This is the first demonstration that continuous low-dose exposure to PCBs via implants can produce sustained tissue levels leading to the accumulation of DNA-adducts in target tissue and induction of indicator enzymes. Collectively, these data demonstrate that this exposure model is a promising tool for long-term exposure studies.
doi:10.1016/j.toxrep.2014.09.010
PMCID: PMC4266188  PMID: 25530946
polychlorinated biphenyls (PCBs); PCB126 (3,3’,4,4’,5-pentachlorobiphenyl); PCB153 (2,2’4,4’,5,5’-hexachlorobiphenyl); Polymeric implants; DNA adducts; 32P-Postlabeling; CYPs; paraoxonase 1 (PON1)
17.  Tritium sequence analysis of oligoribonucleotides: a combination of post-labeling and thin-layer chromatographic techniques for the analysis of partial snake venom phosphodiesterase digests 
Nucleic Acids Research  1974;1(10):1329-1341.
A tritium derivative method for sequence analysis of polyribonucleotides is detailed, which is based on borotritide reduction of oligonucleotide-3′ dialdehydes generated by controlled snake venom phosphodiesterase/alkaline phosphomonoesterase digestion and periodate treatment of time point aliquots of the incubation mixture. Radioactive oligonucleotide derivatives are resolved according to chain length by PEI-cellulose1 anion-exchange TLC and their 3′-termini identified by techniques described in the preceding paper of this series2. The present tritium derivative method is compared with the one described previously2.
Images
PMCID: PMC344354  PMID: 10793693
18.  Vagus nerve stimulation improves left ventricular function in a canine model of chronic heart failure 
European Journal of Heart Failure  2013;15(12):1319-1326.
Aims
Autonomic dysfunction is a feature of chronic heart failure (HF). This study tested the hypothesis that chronic open-loop electrical vagus nerve stimulation (VNS) improves LV structure and function in canines with chronic HF.
Methods and results
Twenty-six canines with HF (EF ∼35%) produced by intracoronary microembolizations were implanted with a bipolar cuff electrode around the right cervical vagus nerve and connected to an implantable pulse generator. The canines were enrolled in Control (n = 7) vs. VNS therapy (n = 7) or a crossover study, with crossovers occurring at 3 months (C × VNS, n = 6; VNS × C, n = 6). After 6 months of VNS, LVEF and LV end-systolic volume (ESV) were significantly improved compared with Control (ΔEF Control –4.6 ± 0.9% vs. VNS 6.0 ± 1.6%, P < 0.001) and (ΔESV Control 8.3 ± 1.8 mL vs. VNS –3.0 ± 2.3 mL, P = 0.002. Plasma and tissue biomarkers were also improved. In the crossover study, VNS also resulted in a significant improvement in EF and ESV compared with Control (ΔEF Control –2.3 ± 0.65% vs. VNS 6.7 ± 1.1 mL, P < 0.001 and ΔESV Control 3.2 ± 1.2 mL vs. VNS –4.0 ± 0.9 mL, P < 0.001). Initiation of therapy in the Control group at 3 months resulted in a significant improvement in EF (Control –4.7 ± 1.4% vs. VNS 3.7 ± 0.74%, P < 0.001) and ESV (Control 1.5 ± 1.2 mL vs. NS –5.5 ± 1.6 mL, P = 0.003) by 6 months.
Conclusions
In canines with HF, long-term, open-looped low levels of VNS therapy improves LV systolic function, prevents progressive LV enlargement, and improves biomarkers of HF when compared with control animals that did not receive therapy.
doi:10.1093/eurjhf/hft118
PMCID: PMC3895958  PMID: 23883651
Vagal; Autonomic nervous system; Parasympathetic; Neurostimulation; Heart failure
19.  Chemopreventive and Therapeutic Activity of Dietary Blueberry against Estrogen-Mediated Breast Cancer 
Berries are gaining increasing importance lately for their chemopreventive and therapeutic potential against several cancers. In earlier studies, a blueberry-supplemented diet has shown protection against 17β-estradiol (E2)-mediated mammary tumorigenesis. This study tested both preventive and therapeutic activities of diet supplemented with whole blueberry powder (50:50 blend of Tifblue and Rubel). Animals received 5% blueberry diet, either 2 weeks prior to or 12 weeks after E2 treatment in preventive and therapeutic groups, respectively. Both interventions delayed the tumor latency for palpable mammary tumors by 28 and 37 days, respectively. Tumor volume and multiplicity were also reduced significantly in both modes. The effect on mammary tumorigenesis was largely due to down-regulation of CYP 1A1 and ER-α gene expression and also favorable modulation of microRNA (miR-18a and miR-34c) levels. These data suggest that the blueberry blend tested is effective in inhibiting E2-mediated mammary tumorigenesis in both preventive and therapeutic modes.
doi:10.1021/jf403734j
PMCID: PMC4334276  PMID: 24245576
blueberry; estrogen; breast cancer; chemoprevention; chemotherapy; ACI rats; miRNA modulation; blood chemistry; hematopoietic parameters
20.  Nitroxyl (HNO) a Novel Approach for the Acute Treatment of Heart Failure 
Circulation. Heart failure  2013;6(6):1250-1258.
Background
The nitroxyl (HNO) donor, Angeli’s salt (AS), exerts positive inotropic, lusitropic, and vasodilator effects in vivo that are cyclic AMP-independent. Its clinical utility is limited by chemical instability and co-generation of nitrite that itself has vascular effects. Here we report on effects of a novel, stable, pure HNO donor (CXl-1020) in isolated myoctyes, and intact hearts in experimental models and in patients with heart failure (HF).
Methods and Results
CXL-1020 converts solely to HNO and inactive CXL-1051 with a t1/2 of 2 minutes. In adult mouse ventricular-myocytes, it dose-dependently increased sarcomere shortening by 75–210% (50–500 µM), with a ~30% rise in the peak Ca2+ transient only at higher doses. Neither protein-kinase-A or soluble guanylate-cyclase inhibition altered this contractile response. Unlike isoproterenol, CXL-1020 was equally effective in myocytes from normal or failing hearts. In anesthetized dogs with coronary microembolization-induced HF, CXL-1020 reduced LV end-diastolic pressure and myocardial oxygen-consumption while increasing ejection fraction from 27 to 40% and maximal ventricular power index by 42% (both p<0.05). In conscious dogs with tachypacing-induced HF, CXL-1020 increased contractility assessed by end-systolic elastance, and provided veno-arterial dilation. Heart rate was minimally altered. In patients with systolic HF, CXL-1020 reduced both left and right heart filling pressures and systemic vascular resistance, while increasing cardiac and stroke volume index. Heart rate was unchanged, and arterial pressure declined modestly.
Conclusions
These data show the functional efficacy of a novel pure HNO donor to enhance myocardial function, and show first-in-man evidence for potential utility in heart failure.
Clinical Trial Registration
URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01096043, NCT01092325.
doi:10.1161/CIRCHEARTFAILURE.113.000632
PMCID: PMC4110196  PMID: 24107588
nitroxyl; cardiomyopathy; contractility; myocyte; pharmacology; human; canine
21.  Association of Genetic Variation with Gene Expression and Protein Abundance within the Natriuretic Peptide Pathway 
The natriuretic peptide (NP) system is a critical physiologic pathway in heart failure with wide individual variability in functioning. We investigated the genetic component by testing the association of single nucleotide polymorphisms (SNP) with RNA and protein expression. Samples of DNA, RNA, and tissue from human kidney (n=103) underwent genotyping, RT-PCR, and protein quantitation (in lysates), for four candidate genes (NP-receptor 1 [NPR1], NPR2, NPR3 and membrane metallo-endopeptidase [MME]). The association of genetic variation with expression was tested using linear regression for individual SNPs, and a principal components (PC) method for overall gene variation. Eleven SNPs in NPR2 were significantly associated with protein expression (false discovery rate ≤0.05), but not RNA quantity. RNA and protein quantity correlated poorly with each other. The PC analysis showed only NPR2 as significant. Assessment of the clinical impact of NPR2 genetic variation is needed.
doi:10.1007/s12265-013-9491-y
PMCID: PMC3795918  PMID: 23835779
Natriuretic peptide; heart failure; gene expression; pharmacogenomics; nesiritide; genetic polymorphisms
22.  Effects of Acute Intravenous Infusion of Apelin on Left Ventricular Function in Dogs with Advanced Heart Failure 
Journal of cardiac failure  2013;19(7):509-516.
Background
Apelin-13 (APLN) through apelin receptor (APJ) exerts peripheral vasodilatory and potent positive inotropic effects. We examined the effects of exogenous intravenous infusion of APLN on left ventricular (LV) systolic function in dogs with heart failure (HF, LV ejection fraction, EF~30%).
Methods and Results
Studies were performed in 7 dogs with microembolization-induced HF. Each dog received an intravenous infusion of low dose and high dose APLN followed by washout period. LV end-diastolic volume (EDV), end-systolic volume (ESV) and LV EF were measured at specified time points. APLN protein level was determined in plasma at all time points. mRNA and protein levels of APLN and APJ in LV tissue were also measured in 7 normal (NL) and 7 heart failure (HF) dogs. APLN reduced EDV only at the high dose, significantly reduced ESV and increased EF with both doses. In plasma of HF dogs, APLN levels were reduced significantly compared to NL dogs. APLN treatment in HF dogs significantly increased the plasma APLN levels at both low and high doses. Expression of APLN, but not of APJ, was reduced in LV tissue of HF dogs compared to NL.
Conclusion
Exogenous administration of APLN improved LV systolic function in dogs with advanced HF.
doi:10.1016/j.cardfail.2013.05.004
PMCID: PMC3706995  PMID: 23834927
23.  Augmentation of Left Ventricular Wall Thickness With Alginate Hydrogel Implants Improves Left Ventricular Function and Prevents Progressive Remodeling in Dogs With Chronic Heart Failure 
JACC. Heart failure  2013;1(3):252-258.
Objectives
The study tested the hypothesis that augmentation of the left ventricular (LV) wall thickness with direct intramyocardial injections of alginate hydrogel implants (AHI) reduces LV cavity size, restores LV shape, and improves LV function in dogs with heart failure (HF).
Background
Progressive LV dysfunction, enlargement, and chamber sphericity are features of HF associated with increased mortality and morbidity.
Methods
Studies were performed in 14 dogs with HF produced by intracoronary microembolizations (LV ejection fraction [EF] <30%). Dogs were randomized to AHI treatment (n = 8) or to sham-operated control (n = 6). During an open-chest procedure, dogs received either intramyocardial injections of 0.25 to 0.35 ml of alginate hydrogel (Algisyl-LVR, LoneStar Heart, Inc., Laguna Hills, California) or saline. Seven injections were made ∼1.0 to 1.5 cm apart (total volume 1.8 to 2.1 ml) along the circumference of the LV free wall halfway between the apex and base starting from the anteroseptal groove and ending at the posteroseptal groove. Hemodynamic and ventriculographic measurements were made before treatment (PRE) and repeated post-surgery for up to 17 weeks (POST).
Results
Compared to control, AHI significantly reduced LV end-diastolic and end-systolic volumes and improved LV sphericity. AHI treatment significantly increased EF (26 ± 0.4% at PRE to 31 ± 0.4% at POST; p < 0.05) compared to the decreased EF seen in control dogs (27 ± 0.3% at PRE to 24 ± 1.3% at POST; p < 0.05). AHI treatment was well tolerated and was not associated with increased LV diastolic stiffness.
Conclusions
In HF dogs, circumferential augmentation of LV wall thickness with AHI improves LV structure and function. The results support continued development of AHI for the treatment of patients with advanced HF.
doi:10.1016/j.jchf.2013.02.006
PMCID: PMC3756288  PMID: 23998003
animal models; congestive heart failure; functional mitral regurgitation; left ventricular function; pressure-volume relationship
24.  Punicalagin and Ellagic Acid Demonstrate Antimutagenic Activity and Inhibition of Benzo[a]pyrene Induced DNA Adducts 
BioMed Research International  2014;2014:467465.
Punicalagin (PC) is an ellagitannin found in the fruit peel of Punica granatum. We have demonstrated antioxidant and antigenotoxic properties of Punica granatum and showed that PC and ellagic acid (EA) are its major constituents. In this study, we demonstrate the antimutagenic potential, inhibition of BP-induced DNA damage, and antiproliferative activity of PC and EA. Incubation of BP with rat liver microsomes, appropriate cofactors, and DNA in the presence of vehicle or PC and EA showed significant inhibition of the resultant DNA adducts, with essentially complete inhibition (97%) at 40 μM by PC and 77% inhibition by EA. Antimutagenicity was tested by Ames test. PC and EA dose-dependently and markedly antagonized the effect of tested mutagens, sodium azide, methyl methanesulfonate, benzo[a]pyrene, and 2-aminoflourine, with maximum inhibition of mutagenicity up to 90 percent. Almost all the doses tested (50–500 μM) exhibited significant antimutagenicity. A profound antiproliferative effect on human lung cancer cells was also shown with PC and EA. Together, our data show that PC and EA are pomegranate bioactives responsible for inhibition of BP-induced DNA adducts and strong antimutagenic, antiproliferative activities. However, these compounds are to be evaluated in suitable animal model to assess their therapeutic efficacy against cancer.
doi:10.1155/2014/467465
PMCID: PMC4052943  PMID: 24949451
25.  Multi-layer polymeric implants for sustained release of chemopreventives 
Cancer letters  2012;326(1):33-40.
Poor oral bioavailability limits the use of many chemopreventives in the prevention and treatment of cancer. To overcome this limitation, we report an improvised implant formulation (“coated” implants) using curcumin, individual curcuminoids, withaferin A and oltipraz. This method involves the coating of blank polycaprolactone implants with 20–30 layers of 10–20% polycaprolactone solution in dichloromethane containing 0.5–2% of the test agent. The in vitro release showed that while oltipraz was released with almost zero-order kinetics over eight weeks, curcumin, individual curcuminoids and withaferin A were released with some initial burst. The in vivo release was determined by grafting implants subcutaneously in A/J mice. When delivered by coated implants, oltipraz significantly diminished lung DNA adducts in mice treated with dibenzo[a, l]pyrene compared with sham treatment (28±7 versus 54±17 adducts/109 nucleotides). Withaferin A also diminished DNA adducts, but it was insignificant. Curcumin and individual curcuminoids were ineffective. Analysis of lung, liver and brain by UPLC-fluorescence showed the presence of the three test curcuminoids indicating effectiveness of the implant delivery system. Further, based on its known antitumor activity in vivo, withaferin A given via the implants significantly inhibited human lung cancer A549 xenograft in athymic nude mice, while it was ineffective when the same total dose was administered i.p. and required over 2-fold higher dose to elicit effectiveness. Together, our data suggest that coated polymeric implants can accommodate heat-labile compounds, can furnish sustained release for long duration, and elicit DNA damage-inhibiting and anti-tumor activities.
doi:10.1016/j.canlet.2012.07.017
PMCID: PMC3462657  PMID: 22820161
Polycaprolactone; Curcumin; Withaferin A; Oltipraz; DNA adducts; Lung tumor xenograft; Bioavailability

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