A supramolecular hydrogel for spatial-temporal release of auxin to promote plant root growth.

Short peptide-based hydrogels have attracted extensive research interests in drug delivery because of their responsive properties. So far, most drug molecules have been conjugated with short peptides via an amide bond, restricting the release of the native drug molecules. In this study, we demonstrated the effectiveness of an auxin-based hydrogelator linked by a hydrolysable ester bond. Hydrogel I, formed by the gelator (NAA-G'FFY) linked with an ester bond, was able to release 1-naphthaleneacetic acid (NAA), whereas hydrogel II, formed by the gelator without an ester bond (NAA-GFFY), was not. By mixing NAA-G'FFY with Fmoc-GFFY to form a two-component hydrogel, the spatial and temporal release of NAA was achieved, promoting on-site auxin responses including primary root elongation and lateral root formation in the model plant Arabidopsis thaliana. The strategy of using a hydrolysable ester bond to connect drug molecules and self-assembling peptides could lead to the development of supramolecular hydrogels with more controllable drug release profiles.

Drug-fortified liposomes as carriers for sustained release of NSAIDs: The concept and its validation in the animal model for the treatment of arthritis.

Drug-fortified cationic liposomes of 6­methoxy­2­naphthylacetic acid (6­MNA) were prepared and characterized by various techniques. The residence time of drug-fortified liposomes in joint cavity was evaluated by intra-articular (IA) administration of the radio-labeled (99mTc) liposomal formulation in the inflamed joints in rats. The cationic liposomal formulation composed of 6­MNA (3) as an active agent, its double salt (4) with the lipid 1,2­distearoyl­sn­glycero­3­phosphoethanolamine (DSPE), and pharmaceutically acceptable excipients such as hydrogenated soyabean phospatidylcholine (HSPC) and 1,2­dioleyloxy­3­trimethylammoniumpropane chloride (DOTAP) were developed using thin film hydration technique. The cryo-TEM analysis confirmed that the prepared optimized liposomal formulation (DFL-2) was a mixture of small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In addition, the TEM analysis confirmed that the prepared liposomes were of spherical in shape having liposome size in the range of 500-900 nm and zeta potential of about +30 mV. The developed cationic liposomes exhibited sustained release profile of payload of 6­MNA for over >12 h and about five times higher retention in the inflamed animal joints after 24 h (by scintigraphy of the joints) as compared to the plain 6­MNA solution when administered by IA route. The anti-inflammatory activity of prepared liposomal composition is evaluated by Freund's adjuvant induced arthritic model in rats. The liposomal formulation was well tolerated by all animals indicating good biocompatibility. Further, the cationic liposomal formulation treated group showed decreased erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) level in comparison to the control and the standard groups in the in vivo study. The improved efficacy of the drug-fortified liposomal formulation was due to the coupled effect of longer retention and sustained release of the active drug 6­MNA in the joints. From the obtained results it could be concluded that the combined effect of the cationic charge on the drug-fortified liposomes and the inherent affinity of the active agent towards the synovial joint tissues, coupled with slow release of the active drug due to double salt approach at the site of administration could potentially decrease the frequency of IA drug administration. Hence such a formulation could prove to be a therapeutic boon for the management of late stage arthritis.

Cell dedifferentiation and multiplication of Burdock (Arctium Lappa) as a medicinal plant.

Arctium lappa L. (Burdock) is an important plant with various pharmacological effects. According to the importance of this plant, optimization of its tissue culture will lead to more investigation and application of it. The aim of this study was to develop protocols for callus induction and shoot regeneration of A.  lappa. In order to optimize of tissue culture in A. lappa, callus induction, indirect regeneration and direct regeneration were carried out in factorial experiment based on Completely Randomized Designs (CRDs). Hypocotyl and cotyledon were cultured on the Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BAP) for callus induction. In indirect regeneration experiment various levels of BAP and α-Naphthaleneacetic acid (NAA) and two types of explants (calli derived from cotyledon and hypocotyl) were investigated. In direct regeneration section, various levels of BAP plus 2 mg/l NAA and different explants (cotyledon, hypocotyl and bud) were compared. In both cotyledon and hypocotyl, the maximum callus induction was observed on a media containing 2 mg/l 2,4-D plus 1 mg/l BAP (100% and 76.19% respectively). The highest percentage of indirect regeneration (65%) was observed at 1 mg/l BAP plus 0.5 mg/l NAA on calli from hypocotyl. The highest percentage of direct regeneration (90.33) was observed in hypocotyl with a lateral bud explant on MS medium supplemented with 0.5 mg/l BAP plus 2 mg/l NAA. In this study, optimization of tissue culture protocol for A. lappa was carried out as a research technique, as well as technique for further exploitation of this plant.

Auxin-cytokinin interaction and variations in their metabolic products in the regulation of organogenesis in two Eucomis species.

In the current study, we evaluated the effect of α-naphthaleneacetic acid (NAA) individually or in combination with different cytokinins (CKs) including benzyladenine (BA), meta-topolin (mT) and isopentenyladenine (iP) on organogenesis, auxin and CK content in Eucomis autumnalis subspecies autumnalis (EA) and Eucomis zambesiaca (EZ). These species were used as model plants due to their ornamental and medicinal properties. Three leaf explants were inoculated in screw-cap jars containing 30mL Murashige and Skoog (MS) media supplemented with 5µM NAA alone or in combination with 5µM CK (BA, mT or iP). After 10 weeks (EA) or 15 weeks (EZ), parameters including shoot and root growth as well as plant fresh weight were recorded. For analysis of auxin and CK content, whole plantlets were harvested, pooled and freeze-dried for the different treatments. In both species, shoot and root proliferation as well as plant biomass were generally higher when NAA was combined with the individual CK than in NAA or CK treatment. The highest concentration of indole-3-acetic acid (IAA, 619pmolg-1 DW) and 2-oxindole-3-acetic acid (OxIAA, 2381pmolg-1 DW) were observed in EA-treated with NAA alone while mT treatment (without NAA) had the most abundant indole-3-acetyl-l-aspartic acid (IAAsp, 904 and 582pmolg-1 DW for EA and EZ, respectively) in both species. A significant concentration of total endogenous CK accumulated in both Eucomis regenerants from mT and mT+NAA when compared to the other treatments. The majority of the detected CKs were of the aromatic CK-type, mainly free bases. The potential physiological roles of these quantified phytohormones in relation to the observed morphological responses are discussed.

Potential of Piperazinylalkylester Prodrugs of 6-Methoxy-2-Naphthylacetic Acid (6-MNA) for Percutaneous Drug Delivery.

Piperazinylalkyl ester prodrugs (4a-5d) of 6-methoxy-2-naphthylacetic acid (6-MNA) (1) were synthesized and evaluated in vitro for the purpose of percutaneous drug delivery. These ionizable prodrugs exhibited varying aqueous solubilities and lipophilicities depending on the pH of the medium. The prodrugs (4a-5c) showed higher aqueous solubility and similar lipophilicity at pH 5.0 and lower aqueous solubility and higher lipophilicity at pH 7.4 in comparison to 6-MNA. The chemical and enzymatic hydrolyses of the prodrugs was investigated in aqueous buffer solutions (pH 5.0 and 7.4) and in 80% human serum (pH 7.4) at 37°C. The prodrugs showed moderate chemical stability (t 1/2 = 6-60 h) but got readily hydrolyzed enzymatically to 6-MNA with half-life ranging from 10-60 min. In the in vitro permeation study using rat skin, the flux of 6-MNA and the prodrugs was determined in aqueous buffers of pH 5.0 and 7.4. The prodrug (5b) showed 7.9- and 11.2-fold enhancement in skin permeation compared to 6-MNA (1) at pH 5.0 and 7.4, respectively. It was concluded that the parent NSAIDs having favorable pharmacokinetic and pharmacodynamic properties coupled with increased skin permeability of their prodrugs could give better options for the treatment of rheumatic diseases.

Enhanced skin permeation of 6-methoxy-2-naphthylacetic acid by salt formation.

The aim of this work was to prepare salts of 6-methoxy-2-naphthylacetic acid (6-MNA) to improve its physicochemical properties for percutaneous application. 6-MNA, an active metabolite of non-steroidal anti-inflammatory drug nabumetone has long half life and has the tendency to penetrate well into synovial fluid. The physicochemical properties of 6-MNA salts were investigated by solubility measurements, differential scanning calorimetry (DSC) and infrared (IR). The DSC thermograms and Fourier transform infrared (FT-IR) spectra indicated that 6-MNA formed salts with organic and alkali metal bases. Among the series, salts formed with amine bases (ethanolamine, diethanolamine, triethanolamine and diethylamine) had lower melting points while alkali metal salt (sodium) had higher melting point than 6-MNA. The salts had higher solubilities than 6-MNA as determined in phosphate buffer at pH 5.0 and 7.4. There is no significant difference in partition coefficient (log P) values between salts and 6-MNA at pH 5.0 but, at pH 7.4, the log P values for the salts increased by 4-10 times as compared to 6-MNA. In vitro permeation studies showed that all the salts increased the flux in comparision to 6-MNA, and the ethanolamine salt (1b) was found to be having 7.7 and 9.4 times higher permeability as compared to 6-MNA at pH 5.0 and 7.4, respectively.

Preparation and characterization of 1-naphthylacetic acid-silica conjugated nanospheres for enhancement of controlled-release performance.

Chemical pesticides have been widely used to increase the yield and quality of agricultural products as they are efficient, effective, and easy to apply. However, the rapid degradation and low utilization ratio of conventional pesticides has led to environmental pollution and resource waste. Nano-sized controlled-release formulations (CRFs) can provide better penetration through the plant cuticle and deliver the active ingredients efficiently to the targeted tissue. In this paper we reported novel conjugated nanospheres derived from 1-naphthylacetic acid (NNA), 3-aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate and their application as a controlled-release plant growth regulator. The NNA and APTES conjugate was prepared through a covalent cross-linking reaction and subsequent hydrolyzation and polycondensation to synthesize NNA-silica nanospheres. The release data indicated that the release of NNA was by non-Fickian transport and increased as particle size decreased. It was also found that the acidity-alkalinity was enhanced and as the temperature increased, the release of the active ingredient was faster. The nanoconjugate displayed a better efficacy in promoting root formation than NNA technical. The present study provides a novel synthesis route for CRFs comprising a pesticide, with long-duration sustained-release performance and good environmental compatibility. This method may be extended to other pesticides that possess a carboxyl group.

Serum protein binding displacement: theoretical analysis using a hypothetical radiopharmaceutical and experimental analysis with 123I-N-isopropyl-p-iodoamphetamine.

INTRODUCTION: The binding of radiopharmaceutical to serum proteins is thought to be an important factor that restricts its excretion and accumulation in tissue. We calculated the effect of inhibitors of serum protein binding using a hypothetical radiopharmaceutical. In vitro experiments and protein binding inhibitor-loaded monkey scintigraphy were then conducted using (123)I-N-isopropyl-p-iodoamphetamine (IMP) as the radiopharmaceutical. METHODS: Free fraction ratios of radiopharmaceutical were calculated with one radiopharmaceutical, two serum proteins and two specific inhibitors in the steady state at various serum protein concentrations. In vitro protein binding inhibition studies using human, rat and monkey sera were performed with site-selective displacers of specific binding sites: 400 microM 6-methoxy-2-naphthylacetic acid (6MNA; a major nabumeton metabolite) as a serum albumin Site II inhibitor and 400 microM erythromycin (ETC) as an alpha(1)-acid glycoprotein (AGP) site inhibitor. Scintigraphy with or without 6MNA loading of monkeys was performed. RESULTS: The theoretical findings roughly corresponded to the experimental results. Approximately 75% of IMP bound to serum albumin Site II and AGP in the species examined. The free fraction of IMP (25.0+/-0.6% for human, 22.8+/-0.4% for monkey, 23.7+/-0.3% for rat) increased with loading of specific protein binding inhibitors (6MNA: 28.0+/-0.3% for human, 24.5+/-0.7% for monkey, 24.3+/-0.2% for rat; ETC: 26.3+/-0.4% for human, 29.5+/-1.1% for monkey, 26.0+/-0.7% for rat) and was serum protein concentration dependant based on the results of calculations. Simultaneous administration of 6MNA and ETC produced a higher free fraction ratio of IMP (31.9+/-1.0% for human, 34.6+/-0.4% for monkey, 27.0+/-0.3% for rat) than summation of the single administrations of 6MNA and ETC (domino effect) in human, rat and monkey sera. Rapid cerebral accumulation was observed with 6MNA loading in monkey scintigraphy. CONCLUSIONS: 6MNA appears to change the pharmacokinetics and brain accumulation of IMP in monkeys. Further studies in human are required.

In vitro plant regeneration from leaf-derived callus of Cimicifuga racemosa.

Cimicifuga racemosa (L.) Nutt., also known as Black Cohosh, is among the top 10 selling medicinal herbs in the United States. The rhizomes have been used to relieve menopausal discomfort. This plant is wild crafted and conservationists have expressed concerns with the sustainability of C. racemosa. Excised tissues from young leaves of C. racemosa were cultured on Murashige and Skoog's medium (MS) supplemented with various concentrations of NAA and TDZ for production of callus. The optimum callus growth and maintenance was in 1.0 microM NAA plus 0.5 microM TDZ. Two-month-old calli were sub-cultured on different concentrations of cytokinins (BA, kinetin, 2ip, TDZ) or in combination with GA(3) for shoot induction. The rate of shoot induction and proliferation was higher in MS media supplemented with 2.0 or 4.0 microM of TDZ. Concentrations of TDZ greater than 4.0 microM suppressed shoot growth. Adding 3.5 microM of GA(3) into media containing BA increased shoot growth. The presence of GA(3) with kinetin or TDZ did not affect shoot production. For rooting, shoots were transferred to MS medium with activated charcoal supplemented with various auxins (IAA, IBA and NAA), roots were noticed 20 days after transference. Activated charcoal was an essential component for vigorous rooting formation. Our results suggest that conservation of C. racemosa is possible through in vitro multiplication of leaf-derived callus.

Efficacy of the selective prostaglandin synthase type 2 inhibitor nimesulide in blocking basal prostaglandin production and delaying glucocorticoid-induced premature labor in sheep.

OBJECTIVE: The purpose of the study was to determine the effects of selective prostaglandin synthase type 2 inhibitors on basal prostaglandin concentrations in the fetal and maternal circulations and on the labor-associated increase in prostaglandin production in sheep. STUDY DESIGN: The effects of maternal nimesulide (0.01, 0.1, and 1 mg/kg) and 6-methoxy-2-naphthylacetic acid (1, 5, and 10 mg/kg) administration were examined (n = 5) at 134 +/- 1 days' gestation. At 138 days' gestation premature labor was induced by fetal dexamethasone infusion (1 mg/d). Ewes were treated with either vehicle or nimesulide infusion (20 mg. d-1. kg-1, n = 5 per group). RESULTS: Nimesulide and 6-methoxy-2-naphthylacetic acid decreased basal prostaglandin production in a concentration-dependent manner. Delivery of nimesulide-treated ewes was delayed by >/=17 hours with respect to that of control ewes (53.9 +/- 2.6 hours). In 2 nimesulide-treated ewes labor did not progress to delivery despite membrane rupture. The increase in prostaglandin concentrations usually seen during dexamethasone-induced labor was abolished in nimesulide-treated ewes and also in their fetuses. CONCLUSIONS: Highly selective inhibitors of prostaglandin endoperoxidase H synthase 2 may be required to spare fetal prostaglandin production and limit potential side effects during the suppression of preterm labor.

Disposition and excretion of 6-methoxy-2-naphthylacetic acid, the active metabolite of nabumetone in horses.

OBJECTIVE: To examine, in horses, the disposition and excretion of the active metabolite 6-methoxy-2-naphthylacetic acid (6MNA) of the nonsteroidal anti-inflammatory prodrug nabumetone. DESIGN: Pharmacokinetic analysis of 6MNA after oral administration of nabumetone and IV administration of 6MNA. PROCEDURE: Using a crossover design, 5 horses were orally administered 3.7 mg of nabumetone/kg of body weight. After a 3-week washout period, 4 horses were administered 2.5 mg of 6MNA/kg, IV. RESULTS: Absorption of nabumetone from the gastrointestinal tract and its metabolism to 6MNA had a median appearance half-life of 0.88 hour. The elimination half-life was 11 hours. Area under the plasma concentration time curve for 6MNA after oral administration of nabumetone was 120.6 mg/h/L. A dose of 2.5 mg/kg of 6MNA administered IV resulted in plasma concentration nearly equivalent to that induced by the orally administered dose. Disposition of 6MNA was modeled as a one-compartment, first-order elimination. The area under the plasma concentration time curve for IV administration of 6MNA was 117.0 mg/h/L, and the specific volume of distribution was 0.247 L/kg. The distribution half-life and the elimination half-life were 0.56 and 7.90 hours, respectively. Percentage of total dose recovered in urine for the 36-hour collection period after the oral and IV administrations was 7.4 and 5.3%, respectively. CONCLUSIONS: Metabolism of nabumetone to 6MNA, as reported in other species, also occurs in horses. There were a number of additional metabolites of nabumetone in urine that could not be fully identified and characterized.

Antiinflammatory and gastrointestinal effects of nabumetone or its active metabolite, 6-methoxy-2-naphthylacetic acid (6MNA). Comparative studies with indomethacin.

6MNA, the active metabolite of the nonacidic antiinflammatory drug nabumetone, was investigated using intravenous administration for effects on (1) carrageenan paw edema and gastric irritancy compared with either oral nabumetone or both oral and intravenous indomethacin when given acutely and (2) gastrointestinal irritancy when given in repeat dosing studies. Oral doses of nabumetone or intravenous 6MNA produced effective antiinflammatory activity together with significant inhibition of paw exudate PGE2. Antiinflammatory oral doses of nabumetone or intravenous 6MNA produced minimal effects on gastric 6-keto PGF1 alpha production with an absence of gastric damage, in contrast with indomethacin. In repeat dose studies, 6MNA failed to induce gastrointestinal damage even at doses where general toxicity was evident. These results show that in the rat, 6MNA is an effective antiinflammatory drug but even in very high intravenous doses does not have the propensity to induce gastrointestinal damage.

Anti-inflammatory and gastrointestinal effects of nabumetone or its active metabolite, 6MNA (6-methoxy-2-naphthylacetic acid): comparison with indomethacin.

6MNA, the active metabolite of the non-acidic anti-inflammatory drug nabumetone, was investigated using intravenous administration for effects on (a) carrageenan paw oedema and gastric irritancy compared to either oral nabumetone or both oral and intravenous indomethacin when given acutely and (b) gastrointestinal irritancy when given in repeat dosing studies. An oral dose of nabumetone or intravenous 6MNA produced effective anti-inflammatory activity together with significant inhibition of paw exudate PGE2. An anti-inflammatory oral dose of nabumetone or intravenous 6MNA produced minimal effects on gastric 6-keto-PGF1 alpha production, with an absence of gastric damage, in contrast to indomethacin. In repeat dose studies, 6MNA failed to induce gastrointestinal damage even at doses where general toxicity was evident. These results show that in the rat 6MNA, the active metabolite of nabumetone, is an effective anti-inflammatory drug but, even in very high intravenous doses, does not have the propensity to induce gastrointestinal damage.

Pharmacokinetics and metabolism of 14C isobytylnaphtyl acetic acid in rat.

After oral administration to rats, absorption of INAA was slow but complete. Plasma level curves reached a plateau for INAA as well as for the two metabolites, which were rapidly formed (MI and MII). The plateau concentration led to an increase of the apparent elimination half-life, which was short after i.v. administration due to the small volume of distribution and to the high rate of metabolism. In any case the half-life was independent of the dose and the pharmacokinetics of INAA remained linear from 1.5 to 15 mg/kg. The two rapidly formed plasma metabolites were eliminated more slowly than INAA. INAA and its metabolites were distributed only sparsely in all tissues under investigation, probably due to the high protein binding. Both routes of administration resulted in elimination of the radioactivity mainly by the urine. Besides the two main metabolites with known structures (MI and MII) small amounts of INAA and two additional metabolites were detected.

Studies on the metabolism of arylacetic acids. 7. The influence of varying dose size upon the conjugation pattern of 2-naphthylacetic acid in the guinea pig, mouse and hamster.

1. The influence of dose size upon the metabolic conjugation of 2-naphthylacetic acid with various amino acids and glucuronic acid has been studied in the guinea pig, mouse and hamster. 2. Guinea pigs conjugated 2-naphthylacetic acid with glycine and glucuronic acid. 3. Mice conjugated 2-naphthylacetic acid with glycine, taurine and glucuronic acid. Taurine conjugation had the highest capacity, and both this and the glycine mechanism were saturated at doses above 100 mg/kg. 4. Hamsters utilized glutamine, glycine, taurine and glucuronic acid for the conjugation of 2-naphthylacetic acid. No conjugation pathway was saturated by doses up to 200 mg/kg. 5. The thus-far unique ability of 2-naphthylacetic acid to evoke multiple amino acid conjugations, using the taurine and glutamine mechanisms hitherto unknown in these species, appears to be due to its affinity for previously unrecognized enzyme systems, rather than to saturation of 'normal' pathways revealing novel routes at high doses.

Cell-mediated T lymphocyte responses against syngeneic cells modified with amino-reactive hapten (AED-NH2): H-2Dk serves as an element for cell-mediated lympholysis to amino-reactive hapten (AED-NH2)-modified self.

Spleen cells from C3H/He mice immunized to the newly synthesized amino-reactive hapten, 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide (AED-NH2), were stimulated in vitro with AED-NH2 modified syngeneic cells. After 5 days of culture, effector cells were assayed for their cytotoxic activity against AED-NH2-modified target blast cells. In contrast to other amino-reactive haptens reported so far, a strong cytotoxic activity against AED-NH2-modified syngeneic cells was found in H-2b mice as well as in H-2k mice. Furthermore, Dk-restricted anti-AED-NH2 CTL recognition was observed in H-2k mice as shown by cold target inhibition. Previous studies have demonstrated the predominant influence of K over D region self determinants, and of the chemical reactivity of the haptenic reagent in Ir gene control of CTL response to hapten-self. The present report illustrates the importance of the hapten itself in genetic regulation of these CTL responses.

A new simple controlled release delivery system.

The chemical pulping of wood without subsequent drying affords low-cost hollow fibers with microporous multilamellar cell walls saturated with water. Simple impregnation of these never-dried pulp fibers with solutions of biologically active chemicals followed by drying yields collapsed cellulose ribbons containing entrapped impregnant. The release characteristics of exemplary composites containing an analgesic (acetylsalicylic acid), a growth stimulant (alpha-naphthylacetic acid), or a herbicide (2,4-dichlorophenoxyacetic acid) are reported both for in vitro and in vivo evaluations.

Naproxen in rheumatoid arthritis. Extended trial.

121 patients with active rheumatoid arthritis, 91 of whom had proved intolerant of other nonsteroidal anti-inflammatory agents, were treated for a mean of 10 months with naproxen. A dosage of 250 mg twice daily produced sustained improvement in most of the standard clinical measurements. 28 patients complained of side effects, with a lower than expected incidence of gastrointestinal complaints and no drug-induced rash being recorded. 19 patients withdrew from the trial because of side effects, while a further 22 withdrew because the drug was ineffective. Naproxen is a useful drug for long-term use in patients with rheumatoid arthritis, including those who have proved intolerant of or experienced inadequate symptomatic relief from other nonsteriodal anti-inflammatory agents.

Naproxen absorption in children.

A study was carried out in 9 children (mean age 10.8 years) suffering from rheumatic disorders to determine peak plasma levels and plasma curve of naproxen following the administration of a single oral dose of a suspension formulation. The results suggest that there is no significant difference in naproxen peak blood levels and half-life between adults and children and a twice-daily dosage regime, as in adults, should be suitable, therefore, for therapeutic use in children. The formulation was well-tolerated and no untoward effects were noted.