Inhibition of ribonuclease A by nucleoside–dibasic acid conjugates

We report the inhibition of the ribonucleolytic activity of ribonuclease A (RNase A) by nucleoside–dibasic acid conjugates for the first time. Agarose gel and precipitation assays show that the spacer length and the pK_a of the carboxylic group have an important role in the inhibitory capacity. Kinetic experiments indicate a competitive mode of inhibition with inhibition constant (K_i) value of 132 ± 3 μM for Oxa-aT. Docking studies revealed that the carboxylic group of the most active compounds is within hydrogen bonding distance of His-12, Lys-41 and His-119.     

Preparation and <Emphasis Type="Italic">In Vitro</Emphasis>/<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Microparticle Formulations Containing Meloxicam

In this study, we have formulated chitosan-coated sodium alginate microparticles containing meloxicam (MLX) and aimed to investigate the correlation between in vitro release and in vivo absorbed percentages of meloxicam. The microparticle formulations were prepared by orifice ionic gelation method with two different sodium alginate concentrations, as 1% and 2% (w/v), in order to provide different release rates. Additionally, an oral solution containing 15 mg of meloxicam was administered as the reference solution for evaluation of in vitro/in vivo correlation (ivivc). Following in vitro characterization, plasma levels of MLX and pharmacokinetic parameters [elimination half-life (t _1/2), maximum plasma concentration (C _max), time for C _max (t _max)] after oral administration to New Zealand rabbits were determined. Area under plasma concentration–time curve (AUC_0–∞) was calculated by using trapezoidal method. A linear regression was investigated between released% (in vitro) and absorbed% (in vivo) with a model-independent deconvolution approach. As a result, increase in sodium alginate content lengthened in vitro release time and in vivo t _max value. In addition, for ivivc, linear regression equations with r ² values of 0.8563 and 0.9402 were obtained for microparticles containing 1% and 2% (w/v) sodium alginate, respectively. Lower prediction error for 2% sodium alginate formulations (7.419 ± 4.068) compared to 1% sodium alginate formulations (9.458 ± 5.106) indicated a more precise ivivc for 2% sodium alginate formulation.     

Extracellular production of <Emphasis Type="Italic">Streptomyces exfoliatus</Emphasis> poly(3-hydroxybutyrate) depolymerase in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. T104: determination of optimal biocatalyst conditions

The phaZ _Sex gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T _m) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine–histidine–carboxylic acid in the active site. High-performance liquid chromatography (HPLC)–mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.     

New pemetrexed‐peptide conjugates: synthesis,characterization and in vitro cytostatic effect on non‐small cell lung carcinoma (NCI‐H358) and human leukemia (HL‐60) cells

Pemetrexed (Pem) is a novel antimetabolite type of anticancer drug that demonstrated promising clinical activity in a wide variety of solid tumors, including non‐small cell lung carcinoma and malignant pleural mesothelioma. It inhibits enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary. The heteroaromatic ring system of Pem has a modifiable amino group, which opens a possibility to apply a new strategy to conjugate Pem to carrier molecules. Considering this as well as the necessity of untouched carboxylic groups of Pem in the new conjugates, we developed a new synthesis strategy. Here, we describe the synthesis and the characterization of new Pem‐peptide conjugates in which cell‐penetrating octaarginine or/and lung‐targeting H‐Ile‐Glu‐Leu‐Leu‐Gln‐Ala‐Arg‐NH₂ peptide is attached to the drug by thioether bond. The conjugates characterized by RP‐HPLC and MS exhibited cytostatic effect in vitro on non‐small cell lung carcinoma as well as on human leukemia cell lines. The IC₅₀ values of the conjugates were similar, but the conjugates with H‐Ile‐Glu‐Leu‐Leu‐Gln‐Ala‐Arg‐NH₂ sequence were slightly more effective. Our data show that the in vitro cytostatic effect of the free Pem was essentially maintained after conjugation with cell‐penetrating or cell‐targeting peptides. Thus, the conjugation strategy reported could lead to the development of a new generation of active Pem conjugates. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Improved Bioavailability of Poorly Water-Soluble Drug Curcumin in Cellulose Acetate Solid Dispersion

Curcumin (Cur), one of the most widely used natural active constituents with a great variety of beneficial biological and pharmacological activities, is a practically water-insoluble substance with a short biologic half-life. The aim of this study was to develop a sustained-release solid dispersion by employing water-insoluble carrier cellulose acetate for solubility enhancement, release control, and oral bioavailability improvement of Cur. Solid dispersions were characterized by solubility, in vitro drug release, Fourier transform infrared spectroscopy, X-ray diffractometry, and differential scanning calorimetry studies. The in vivo performance was assessed by a pharmacokinetic study. Solid-state characterization techniques revealed the amorphous nature of Cur in solid dispersions. Solubility/dissolution of Cur was enhanced in the formulations in comparison with pure drug. Sustained-release profiles of Cur from the solid dispersions were ideally controlled in vitro up to 12 h. The optimized formulation provided an improved pharmacokinetic parameter (C _max = 187.03 ng/ml, t _max = 1.95 h) in rats as compared with pure drug (C _max = 87.06 ng/ml, t _max = 0.66 h). The information from this study suggests that the developed solid dispersions successfully enhanced the solubility and sustained release of poorly water-soluble drug Cur, thus improving its oral bioavailability effectively.     

Interaction of salicylate and ibuprofen with the carboxylic acid: CoA ligases from bovine liver mitochondria

Neither salicylate nor ibuprofen was a substrate or inhibitor of the long-chain fatty acid: CoA ligase. In contrast, all three xenobiotic-metabolizing medium-chain fatty acid:CoA ligases (XL-I, XL-II, and XL-III) had activity toward salicylate. The K_m value for salicylate was similar for all three forms (2 to 3 μM), but XL-II and XL-III had higher activity at V_max. For ibuprofen, only XL-III catalyzed its activation, and it had a K_m for ibuprofen of 36 μM. Studies of salicylate inhibition of XL-I, XL-II, and XL-III revealed that it inhibited the benzoate activity of all three forms with K₁ values of ca. 2 μM, which is in agreement with the K_m values obtained with salicylate as substrate. Kinetic analysis revealed that salicylate conjugation by all three forms is characterized by substrate inhibition when salicylate exceeds ca. 20 μM. Substrate inhibition was more extensive with XL-I and XL-III. Previous work on the ligases employed assay concentrations of salicylate in the range of 0.1 to 1.0 mM, which are clearly inhibitory, particularly toward XL-I and XL-III. Thus, activity was not properly measured in previous studies, which accounts for the fact that salicylate conjugation was only found with one form, which is most likely XL-II since it has the highest V_max activity and shows the least amount of substrate inhibition. Studies with ibuprofen indicated that it inhibited XL-I, XL-II, and XL-III, with K₁ values being in the range of 75–125 μM. The short-chain ligase was inhibited by both salicylate and ibuprofen with K₁ values of 93 and 84 μM, respectively. It was concluded that pharmacological doses of salicylate, but not ibuprofen, will affect the metabolism of medium-chain fatty acids and carboxylic acid xenobiotics and that the previously described mitochondrial ibuprofen:CoA ligase activity is attributable to XL-III. © 1996 John Wiley & Sons, Inc.     

Combined Use of Crystalline Sodium Salt and Polymeric Precipitation Inhibitors to Improve Pharmacokinetic Profile of Ibuprofen through Supersaturation

To maximize the pharmacological effect of a pain reliever such as ibuprofen, early onset of action is critical. Unfortunately, the acidic nature of ibuprofen minimizes the amount of drug that can be solubilized under gastric conditions and would be available for immediate absorption upon entry into the intestine. Although the sodium salt of ibuprofen has higher solubility, rapid conversion from the salt to the poorly soluble free acid phase occurs under gastric conditions. Therefore, the combination of the highly soluble sodium salt form of ibuprofen with polymers was evaluated as an approach to prolong supersaturation of ibuprofen during the disproportionation of the salt. Binary combinations of ibuprofen sodium with polymers resulted in the identification of several formulations that demonstrated high degrees and extended durations of supersaturation during in vitro dissolution experiments. These formulations included HPMC, polyvinyl pyrrolidone-vinyl acetate copolymer (PVP-VA64), methylcellulose (MC), and hydroxypropyl cellulose (HPC). The in vitro supersaturation observed with these ibuprofen-polymer formulations translated to an increase in C_max and an earlier T_max for the PVP-VA64, MC, and HPC formulations relative to ibuprofen only controls when administered orally to rats under fasted conditions. Based on these observations, combining ibuprofen sodium with polymers such as PVP-VA64, MC, or HPC is a viable formulation approach to prolong supersaturation in the stomach and enable an optimized pharmacokinetic profile in vivo where rapid onset of action is desired.     

Synthesis of the amino acid conjugates of <Emphasis Type="Italic">epi</Emphasis>-jasmonic acid

The TES ether of the C6-hydroxy derivative of naturally occurring epi-jasmonic acid (epi-JA) was designed as epimerization-free equivalent of epi-JA. The TES ether was synthesized from (1R,4S)-4-hydroxycyclopent-2-enyl acetate in 13 steps. The acid part of the ether was activated with ClCO₂Bu ⁱ and subjected to condensation with l-amino acid at room temperature for 48 h. The TES group in the condensation product was removed in HCO₂H (0°C, 30 min) and the resulting hydroxyl group was oxidized with Jones reagent (acetone, 0°C, 30 min) to furnish the amino acid conjugate of epi-JA. The amino acids examined are l-isoleucine, l-leucine, l-alanine, l-valine, and d-allo-isoleucine, which afforded the conjugates in 48–68% yields with 89–96% diastereomeric purity over the trans isomers. Similarly, the possible three stereoisomers of epi-JA were condensed with l-isoleucine successfully, producing the corresponding stereoisomers in good yields.     

Energy-dependent export of the 13-oxooctadecadienoic acid–glutathione conjugate from HT-29 cells and plasma membrane vesicles

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, β,γ-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K_m of 325.5 μM and a V_max of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K_m of 169 μM and a V_max of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.     

Heterologous expression and characterization of a proxidomal ascorbate peroxidase from <Emphasis Type="Italic">Populus tomentosa</Emphasis>

The present study reported for the first time, cloning, expression and characteristics of a Proxidomal APX gene (PpAPX) from Populus tomentosa. The PpAPX gene encodes a protein of 287 amino acid residues with a calculated molecular mass of 31.58 kDa. The over-expressed recombinant PpAPX protein showed high activity towards the substrates ascorbate aicd (ASA) and H₂O₂. At fixed ASA concentrations, the K _m and V _max values were 0.12 ± 0.01 mM and 23.4 ± 4.2 mmol/min mg for H₂O₂. And at fixed H₂O₂ concentrations, the K _m and V _max values were 0.53 ± 0.04 mM and 20.0 ± 2.3 mmol/min mg for ASA.     

Chemical Characterization,Crossfeeding and Uptake Studies on Hydroxamate Siderophore of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

We report the production of two types of siderophores namely catecholate and hydroxamate in modified succinic acid medium (SM) from Alcaligenes faecalis. Two fractions of siderophores were purified on amberlite XAD, major fraction was hydroxamate type having a λ_max at 224 nm and minor fraction appeared as catecholate with a λ_max of 264 nm. The recovery yield obtained from major and minor fractions was 297 and 50 mg ml⁻¹ respectively. The IEF pattern of XAD-4 purified siderophore suggested the pI value of 6.5. Cross feeding studies revealed that A. faecalis accepts heterologous as well as self (hydroxamate) siderophore in both free and iron complexed forms however; the rate of siderophore uptake was more in case of siderophores complexed to iron. Siderophore iron uptake studies indicated the differences between hydroxamate siderophore of A. faecalis and Alc E, a siderophore of Alcaligenes eutrophus.     

Modulation of Glutamate and Glycine Transporters by Niflumic,Flufenamic and Mefenamic Acids

Three fenamates—niflumic, flufenamic and mefenamic acids—were tested for effects on substrate-induced currents of glutamate and glycine transporters (EAAT1, EAAT2, GLYT1b and GLYT2a) expressed in Xenopus laevis oocytes. All fenamates inhibited EAAT1 currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 53 ± 4% (P < 0.001). EAAT2 currents were less sensitive, but 100 μM flufenamic acid inhibited the I _max by 34 ± 5% (P = 0.006). All fenamates inhibited GLYT1b currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 61 ± 1% (P < 0.001). At 100 μM, effects on the GLYT2a I _max were mixed: 13 ± 2% inhibition by flufenamic acid (P = 0.002), 30 ± 6% enhancement by niflumic acid (P = 0.002), and no effect by mefenamic acid. Minor effects on substrate affinity suggested non-competitive mechanisms. These data could contribute to the development of selective transport modulators.     

Chloride channel blockers activate an endogenous cationic current in oocytes of<Emphasis Type="Italic"> Bufo arenarum</Emphasis>

A two-electrode, voltage-clamp technique was used to measure the effect of the Cl^– channel blockers, 9-anthracene carboxylic acid and niflumic acid, upon the ionic currents of oocytes of the South American toad Bufo arenarum. The main results were: (1) both blockers produced a reversible increase of the outward currents on a dose-dependent manner; (2) the activated outward current was voltage dependent; (3) the 9-anthracene carboxylic acid-sensitive current was blocked with barium; and (4) the effect of 9-anthracene carboxylic acid was more pronounced in a zero-K⁺ solution than in standard (2 mmol l^–1) or high (20 mmol l^–1) K⁺ solutions, indicating that a K⁺ conductance is activated. The effect of the Cl^– channel blockers could be due to a direct interaction with endogenous cationic channels. Another possible explanation is that Cl^– that enter the cell during depolarizing steps in control solution inhibit this cationic conductance; thus, the blockade of Cl^– channels by 9-anthracene carboxylic acid and niflumic acid would remove this inhibition, allowing the cationic current to flow freely.Abbreviations 9-AC 9-anthracene carboxylic acid - E_r reversal potential - NA niflumic acid - NSC non-selective cation channel     

Characterization of long-chain fatty acid uptake in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Studies evaluating the uptake of long-chain fatty acids in Caulobacter crescentus are consistent with a protein-mediated process. Using oleic acid (C18:1) as a substrate, fatty acid uptake was linear for up to 15 min. This process was saturable giving apparent V_max and K_m values of 374 pmol oleate transported/min/mg total protein and 61 μM oleate, respectively, consistent with the notion that one or more proteins are likely involved. The rates of fatty acid uptake in C. crescentus were comparable to those defined in Escherichia coli. Uncoupling the electron transport chain inhibited oleic acid uptake, indicating that like the long-chain fatty acid uptake systems defined in other gram-negative bacteria, this process is energy-dependent in C. crescentus. Long-chain acyl CoA synthetase activities were also evaluated to address whether vectorial acylation represented a likely mechanism driving fatty acid uptake in C. crescentus. These gram-negative bacteria have considerable long-chain acyl CoA synthetase activity (940 pmol oleoyl CoA formed/min/mg total protein), consistent with the notion that the formation of acyl CoA is coincident with uptake. These results suggest that long-chain fatty acid uptake in C. crescentus proceeds through a mechanism that is likely to involve one or more proteins.     

Molecular characterization of phenylalanine ammonia lyase gene from <Emphasis Type="Italic">Cistanche deserticola</Emphasis>

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis–Menten kinetics with a K _m of 0.1013 mM, V _max of 4.858 μmol min⁻¹, K _cat of 3.36 S⁻¹, and K _cat/K _m is 33,168 M⁻¹ S⁻¹. The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol⁻¹ when l-Phenylalanine was used as a substrate; l-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg²⁺, Pb²⁺, and Zn²⁺) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a K _i of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with K _i = 0.056 μM.     

Injectability Evaluation: An Open Issue

The current work aimed to propose a system of scoring to rationalize and support the selection of the optimal diameter and length of needles. Four formulations at different viscosity and needles ranging from 21 to 26 G and length ranging from 16 to 40 mm were used. Plunger-stopper breakloose force, maximum force (F _max), and dynamic glide force were measured by a texture analyzer at the crosshead speed of 1 mm/s. Testing was carried out into air or human subcutaneous tissue. The manual injectability of the highest viscosity product was assessed by ten evaluators. The comparison of the panel test score and the quantitative measurements of the forces permitted to score a given needle-syringe-formulation system keeping also in consideration the pressure created in the subcutaneous space and muscles at the injection site. In particular, the following relationship was drawn: at the F _max up to 250 mPa, the injection was practically impossible; at F _max ranging from 160 to 250 mPa, the injection was very difficult; at F _max in the 125–160 mPa range, the injection was feasible, though with some difficulty; when the values of F _max were lower 125 mPa, the injection went smoothly. On the basis of these preliminary data, a system of scoring the needle-syringe-formulation system is proposed to rationalize and support the selection of the optimal diameter and length of needles, keeping also in consideration the pressure created in the subcutaneous space and muscles at the injection site.     

Hybrid fluorescent conjugates of COX-2 inhibitors: Search for a COX-2 isozyme imaging cancer biomarker

The observation that the cyclooxygenase-2 (COX-2) isozyme is over-expressed in multiple types of cancer, relative to that in adjacent non-cancerous tissue, prompted this investigation to prepare a group of hybrid fluorescent conjugates wherein the COX inhibitors ibuprofen, (S)-naproxen, acetyl salicylic acid, a chlororofecoxib analog and celecoxib were coupled via a linker group to an acridone, dansyl or rhodamine B fluorophore. Within this group of compounds, the ibuprofen-acridone conjugate (10) showed potent and selective COX-2 inhibition (COX-2 IC₅₀ = 0.67 μM; SI = 110.6), but its fluorescence emission (λ_em = 417, 440 nm) was not suitable for fluorescent imaging of cancer cells that over-express the COX-2 isozyme. In comparison, the celecoxib-dansyl conjugate (25) showed a slightly lower COX-2 potency and selectivity (COX-2 IC₅₀ = 1.1 μM; SI > 90) than the conjugate 10, and it possesses a better fluorescence emission (λ_em = 500 nm). Ultimately, a celecoxib-rhodamine B conjugate (28) that exhibited moderate COX-2 potency and selectivity (COX-2 IC₅₀ = 3.9 μM; SI > 25) having the best fluorescence emission (λ_em = 580 nm) emerged as the most promising biomarker for fluorescence imaging using a colon cancer cell line that over-expresses the COX-2 isozyme.     

Physiological responses to nitrogen and sulphur addition and raised temperature in <Emphasis Type="Italic">Sphagnum balticum</Emphasis>

Sphagnum, the main genus which forms boreal peat, is strongly affected by N and S deposition and raised temperature, but the physiological mechanisms behind the responses are largely unknown. We measured maximum photosynthetic rate (NP_max), maximum efficiency of photosystem II [variable fluorescence (F _v)/maximum fluorescence yield (F _m)] and concentrations of N, C, chlorophyll and carotenoids as responses to N and S addition and increased temperature in Sphagnum balticum (a widespread species in the northern peatlands) in a 12-year factorial experiment. NP_max did not differ between control (0.2 g N m⁻² year⁻¹) and high N (3.0 g N m⁻² year⁻¹), but was higher in the mid N treatment (1.5 g N m⁻² year⁻¹). N, C, carotenoids and chlorophyll concentration increased in shoot apices after N addition. F _v/F _m did not differ between N treatments. Increased temperature (+3.6°C) had a small negative effect on N concentration, but had no significant effect on NP_max or F _v/F _m. Addition of 2 g S m⁻² year⁻¹ showed a weak negative effect on NP_max and F _v/F _m. Our results suggest a unimodal response of NP_max to N addition and tissue N concentration in S. balticum, with an optimum N concentration for photosynthetic rate of ~13 mg N g⁻¹. In conclusion, high S deposition may reduce photosynthetic capacity in Sphagnum, but the negative effects may be relaxed under high N availability. We suggest that previously reported negative effects on Sphagnum productivity under high N deposition are not related to negative effects on the photosynthetic apparatus, but differences in optimum N concentration among Sphagnum species may affect their competitive ability under different N deposition regimes.     

Anhydride Prodrug of Ibuprofen and Acrylic Polymers

The objective of this study was to synthesize anhydride prodrugs for carboxylic-acid-bearing agents such as non-steroidal anti-inflammatory drugs, shield the carboxylic acid group from irritative effects, and obtain sustained release patterns. Ibuprofen was used as a representative drug for anhydride derivatization. Conjugates of ibuprofen with carboxylic acid moieties of different acrylic polymers were prepared by dehydration reaction using acetic anhydride. Products were characterized by infrared spectroscopy, nuclear magnetic resonance, and scanning electron microscopy followed by preparation of microspheres with different sizes from the conjugate Eudragit® L-100-ibuprofen. The drug release was monitored by high-performance liquid chromatography. Ibuprofen was bound to the polymers via an anhydride bond in high reaction yields (75–95%) with drug loading of up to 30% (w/w). These anhydride derivatives hydrolyzed and release the drug at different periods ranging from 1 to 5 days, depending on the hydrophobicity and the cross-linking of the conjugates. The release of drug from the microspheres was correlated to their size and ranged from 2 to almost 8 days. This study demonstrates the promise of anhydride prodrug for extending drug action while shielding the carboxylic acid group.     

Growth and physiological activity in <Emphasis Type="Italic">Larix kaempferi</Emphasis> seedlings inoculated with ectomycorrhizae as affected by soil acidification

To evaluate the effect of ectomycorrhizal colonization on growth and physiological activity of Larix kaempferi seedlings grown under soil acidification, we grew L. kaempferi seedlings with three types of ectomycorrhizae for 180 days in acidified brown forest soil derived from granite. The soil had been treated with an acid solution (0 (control), 10, 30, 60, and 90 mmol H⁺ kg⁻¹). The water-soluble concentrations of Ca, Mg, K, Al, and Mn increased with increasing amounts of H⁺ added to the soil. Ectomycorrhizal development significantly increased in soil treated with 10 and 30 mmol H⁺ kg⁻¹ but was significantly reduced in soil treated with 60 and 90 mmol H⁺ kg⁻¹. The concentrations of Al and Mn in needles or roots increased with increasing H⁺ added to the soil. The total N in seedlings significantly increased with increasing H⁺ in soil and colonization with ectomycorrhiza. The maximum net photosynthetic rate at light and CO₂ saturation (P _max) was greater in soil treated with 10 mmol H⁺ kg⁻¹ than in controls, and was less is soils treated with greater than with 30 mmol H⁺ kg⁻¹, especially with 60 and 90 mmol H⁺ kg⁻¹. However, colonization with ectomycorrhiza significantly reduced the concentration of Al and Mn in needles or roots and increased the values of P _max and total dry mass (TDM). The relative TDM of L. kaempferi seedlings was approximately 40% at a (BC, base cation)/Al ratio of 1.0. However, ectomycorrhizal seedlings had a 100–120% greater TDM at a BC/Al ratio of 1.0 than non-ectomycorrhizal seedlings, even though the acid treatment reduced their overall growth.