Control of mosquito larvae by encapsulated pathogen Bacillus thuringiensis var. israelensis

Abstract

Bacillus thuringiensis var. israelensis (B.t.i.) containing alginate microcapsules were prepared in order to maintain durable formulations which could resist several effects causing reduced efficiency during applications. B.t.i. spores were harvested through NYSM agar plates and encapsulated in Ca-alginate (0.5–2.0% w/v) gels without any significant loss of sporal or larvicidal activity. The effect of acidic pH on the larvicidal toxin was tested using Culex sp. larvae in the laboratory. The alginate microcapsules pretreated with saturated KH₂PO₄ solution gave larvicidal activity after 24–48h, by bioassay. Suspension and encapsulated forms of the pathogenic bacterium were exposed to pH variations (3.4–10.0), UV light and high temperature (50°C). Durability to Pb ^{+ +}, Cu⁺⁺, Fe^{+ +} compounds and phenol was also examined. As the alginate content increased, stability of B.t.i. drastically increased against the tested effects, but to obtain useful releasing microcapsules, 14–1.5% w/v alginate concentrations were found to be optimum.     

A Novel Method of Preparing PLGA Microcapsules Utilizing Methylethyl Ketone

Purpose. To substitute dichloromethane with a safer solvent, a solvent extraction process using methylethyl ketone (MEK) was developed to prepare poly(d,l-lactide-co-glycolide) microcapsules. Methods. The MEK dispersed phase containing PLGA and progesterone was emulsified in the MEK-saturated aqueous phase (W₁) to make a transient oil-in-water (O/W₁) emulsion. It was then transferred to a sufficient amount of water (W₂) so that MEK residing in polymeric droplets could be extracted effectively into the continuous phase. Results. This solvent extraction process provided the encapsulation efficiency for progesterone ranging from 77 to 60%. The amount of MEK predissolved in W₁ as well as the degree of progesterone payload, influenced the encapsulation efficiency. The leaching profile of MEK analyzed by GC substantiated that, upon dispersion of O/W₁ to W₂, MEK quickly diffused into the continuous phase. Such a rapid diffusion of MEK from and the ingression of water into polymeric droplets produced hollow microcapsules, as evidenced by their SEM micrographs. Conclusions. When solvent extraction/evaporation techniques are employed for preparing PLGA microcapsules, water-immiscibility of a dispersed phase is not an absolute prerequisite to the successful microencapsulation. Adjustment of an initial extraction rate of MEK and formation of a primary transient O/W₁ emulsion are found to be very crucial not only for the success of microencapsulation but also for the determination of microcapsule morphology.     

Poly(D,L-lactide-co-glycolide) encapsulated poly(vinyl alcohol) hydrogel as a drug delivery system

Purpose. The efficiency of encapsulation of water-soluble drugs in biodegradable polymer is often low and occasionally these microcapsules are associated with high burst effect. The primary objective of this study is to develop a novel microencapsulation technique with high efficiency of encapsulation and low burst effect. Method. Pentamidine was used as a model drug in this study. Pentamidine/polyvinyl alcohol (PVA) hydrogel was prepared by freeze-thaw technique. Pentamidine loaded hydrogel was later microencapsulated in poly(lactide-co-glycolide) (PLGA) using solvent evaporation technique. The microcapsules were evaluated for the efficiency of encapsulation, particle size, surface morphology, thermal characteristic, and drug release. Results. Scanning Electron Microscope (SEM) studies revealed that the microcapsules were porous. The microcapsules were uniform in size and shape with the median size of the microcapsules ranging between 27 and 94 m. The samples containing 10% PLGA showed nearly three times increase in drug loading (18-53%) by increasing the hydrogel content from 0-6%. The overall drug release from the microencapsulated hydrogel, containing 3% and 6% PVA, respectively, was significantly lower than the control batches. Conclusions. The use of a crosslinked hydrogel such as PVA can significantly increase the drug loading of highly water-soluble drugs. In addition, incorporation of the PVA hydrogel significantly reduced the burst effect and overall dissolution of pentamidine.     

Preparation and characterization of methotrexate-loaded microcapsules

Abstract

Methotrexate (MTX) has toxic effect to healthy tissues. Microencapsulation coats particles with a functional coat to optimize storage stability and to modulate release. In the present study, a new MTX encapsulated microcapsules were synthesized for controlling MTX release. Controlled drug release provides releasing of efficient dose and prevent drug side effect to tissues and also protects MTX from oxygen, pH and other interactions. MTX was encapsulated through biocompatible hyaluronic acid (HA) and sodium alginate (SA) with an encapsulation system to reduce its toxicity and for controlled release. The microcapsules prepared by vibrating nozzle were cross-linked with SA, HA and calcium chloride. Nozzle diameter and MTX concentration were changed according to loaded MTX and encapsulation efficiency were determined using HPLC. For the reliability of the data, validation studies of the HPLC method were performed. The precision of the method was demonstrated using intra- and inter-day assay relative standard deviation (RSD) values which are less than 2% in all instances. For the characterization of microcapsules, particle size, drug loading and in vitro drug release studies were performed. Diameters of MTX-loaded microcapsules were acquired approximately 160, 400 and 800?µm. Surface morphology of encapsulated microcapsules were displayed with light microscope. Eighty-nine percent MTX encapsulation efficiencies were achieved. Encapsulated MTX microcapsules showed controlled release when compared to pure MTX. While powder MTX dissolved completely in 10?min in the dissolution medium, MTX release from encapsulated MTX microcapsules became 40?h in 0.1?M PBS pH 7.4, including NaCl. MTX release from MTX-loaded microcapsules was reached to 79%. Moreover, drug efficiency was examined in vitro cell culture tests. Viability of 5RP7 cells were decreased to 88.5% for 96?h. When MTX was given directly to 5RP7 cells, viability of 5RP7 cells was decreased to 49.7% for 96?h. Flow cytometry studies also showed that, MTX microcapsules induced apoptosis. The goal of this study is to provide controlled release of MTX and to reduce the toxic effect of MTX.     

Microencapsulation of krill oil using complex coacervation

Abstract

The research work was aimed at the development of a process to yield gelatin-gum Arabic multinuclear microcapsules of krill oil (KO), via complex coacervation. On the basis of the experimental results of the screening trials, a three-level-by-three-factor Box–Behnken design was used to evaluate the effects of the ratio of the core material to the wall (RCW; x₁), the stirring speed (SP; x₂) and the pH (x₃) on the encapsulation efficiency (EE). The experimental findings indicated that x₃ has the most significant linear and quadratic effects on the EE of KO and a bilinear effect with x₁, whereas x₂ did not have any significant effect. The optimal conditions for a 92% of EE were: 1.75:1 for RCW, 3.8 for pH and 3 for SP. The microcapsules, formed by complex coacervation and without any cross-linking agent, were multinucleated, circular in shape and had sufficient stability to maintain their structure.     

Microencapsulation of insulin using a W/O/W double emulsion followed by complex coacervation to provide protection in the gastrointestinal tract

Abstract

Microcapsules containing insulin were prepared using a combination of a W/O/W double emulsion and complex coacervation between WPI (used as a hydrophilic emulsifier) and CMC or SA with further spray drying of the microcapsules in order to provide protection in the gastrointestinal tract. The microcapsules prepared exhibited high encapsulation efficiency and showed the typical structure of a double emulsion. After spray drying of these microcapsules, the integrity of the W/O/W double emulsion was maintained and the biological residual activity remained high when using the combination of 180?°C inlet air temperature and 70?°C outlet air temperature. The microcapsules exhibited low solubility at pH 2 and high solubility at pH 7 so they might protect insulin at acid pH values in the stomach and release it at intestinal pH values. The microcapsules developed in this study seem to be a promising oral delivery vehicle for insulin or other therapeutic proteins.     

Stabilization of Proteins Encapsulated in Cylindrical Poly(lactide-co-glycolide) Implants: Mechanism of Stabilization by Basic Additives

Purpose. A previous study from our group has shown that in theacidic microclimate of poly(lactide-co-glycolide) (PLGA) implants,encapsulated BSA forms insoluble noncovalent aggregates and ishydrolyzed during in vitro release. Incorporation of Mg(OH)₂ stronglyinhibits these mechanisms of instability and facilitates continuousprotein release. The purpose of this study was to determine the proteinstabilization mechanism in the presence of basic additives. Methods. BSA, as a model protein, was encapsulated in PLGAmillicylinders by a solvent extrusion method. The release of BSA fromthe PLGA millicylinders with and without basic additives (Mg(OH)₂,Ca(OH)₂, ZnCO₃ and Ca₃(PO₄)₂) in a physiological buffer was carriedout at 37°C and quantified by a modified Bradford assay. The insolubleaggregates extracted from the polymer with acetone were reconstitutedin a denaturing (6 M urea) or denaturing/reducing solvent (6 M urea/10 mM DTT) to determine the type of aggregation. Results. Aggregation of encapsulated BSA was inhibited withincreasing amount of base co-encapsulated in the polymer, irrespective of thetype of base used. The pH drop in the release medium and extent ofacid-catalyzed PLGA degradation were both inhibited in the presenceof base. The resultant effect was also reflected in an increase in wateruptake and porosity of the devices. The inhibition and mechanism ofBSA aggregation was correlated with the basicity of the additive.For Ca(OH)₂, at 3% loading, covalent BSA aggregation due tothiol-disulfide interchange was observed (indicative of ionization ofalbumin's free thiol at high pH), whereas at 3% ZnCO₃ or Ca₃(PO₄)₂, ahigher percentage of non-covalent aggregates was observed comparedto Mg(OH)₂. Decreasing the loading of BSA at constant Mg(OH)₂content caused an increase in BSA aggregation. Conclusions. The mechanism by which Mg(OH)₂ stabilizesencapsulated BSA in PLGA implants is through neutralizing the acidicmicroclimate pH in the polymer. The successful neutralization afforded by thebasic additives requires a percolating network of pores connecting bothbase and protein. The microclimate pH inside PLGA implants can becontrolled by selecting the type of basic salt, which suggests a potentialapproach to optimize the stability of encapsulated pharmaceuticals inPLGA including therapeutic proteins.     

Development of eye drops containing antihypertensive drugs: formulation of aqueous irbesartan/γCD eye drops

Abstract

Aqueous nanoparticulated eye drop formulations based on γ-cyclodextrin (γCD) complexes were developed and tested in vitro. Three antihypertensive drugs, i.e. enalapril maleate, irbesartan and verapamil HCl, that have been shown to possess IOP-lowering activity were selected for this study. All three drugs displayed B_s-type phase-solubility diagrams in aqueous γCD solutions and had relatively low affinity for γCD. Irbesartan was selected for further formulation development. The drug was relatively stable at pH 4.5 but somewhat less stable at physiologic pH. However, presence of γCD in the aqueous media enhanced the chemical stability of irbesartan. Aqueous γCD-based eye drop formulations containing 1% and 2% (w/v) irbesartan were prepared and the effect of pH on the particles size distribution and drug release investigated. Only ～2% of the drug was in solution in the pH 4.5 formulations but up to 45% in the pH 7 formulations. The pH 7 formulations, where larger fraction of the drug was in solution, displayed somewhat greater drug permeation flux but much lower drug permeation coefficients than the pH 4.5 formulations. Dynamic light scattering studies indicated the faster permeation was due to formation of smaller particles in presence tyloxapol.     

The Acidic Microclimate in Poly(lactide-co-glycolide) Microspheres Stabilizes Camptothecins

Purpose. The camptothecin (CPT) analogue, 10-hydroxycamptothecin (10-HCPT) has been shown previously to remain in its acid-stable (and active) lactone form when encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres (1). The purpose of this study was to determine the principal mechanism(s) of 10-HCPT stabilization. Methods. CPTs were encapsulated in PLGA 50:50 microspheres by standard solvent evaporation techniques. Microspheres were eroded in pH 7.4 buffer at 37°C. The ratio of encapsulated lactone to carboxylate was determined by HPLC as a function of time, initial form of drug encapsulated, fraction of co-encapsulated Mg(OH)₂, CPT lipophilicity, and drug loading. Two techniques were developed to assess the microclimate pH, including: i) measurement of H⁺ content of the dissolved microspheres in an 80:20 acetonitrile/H₂O mixture and ii) confocal microscopy of an encapsulated pH-sensitive dye, fluorescein. Results. The encapsulated carboxylate converted rapidly to the lactone after exposure to the release media, indicating the lactone is favored at equilibrium in the microspheres. Upon co-encapsulation of Mg(OH)₂, the trend was reversed, i.e., the lactone rapidly converted to the carboxylate form. Measurement of -log(hydronium ion activity) (pa*_H) of dissolved microspheres with pH-electrode and pH mapping with fluorescein revealed the presence of an acidic microclimate. From the measurements of H+ and water contents of particles hydrated for 3 days, a microclimate pH was estimated to be in the neighborhood of 1.8. The co-encapsulation of Mg(OH)₂ could both increase the pa*_H reading and neutralize pH in various regions of the microsphere interior. Varying the drug lipophilicity and loading revealed that the precipitation of the lactone could also stabilize CPT. Conclusions. PLGA microspheres prepared by the standard solvent evaporation techniques develop an acidic microclimate that stabilizes the lactone form of CPTs. This microclimate may be neutralized by co-encapsulating a base such as Mg(OH)₂, as suggested by previous work with poly(ortho esters) (2).     

Synthesis and preliminary evaluation of selected 2-aryl-5(6)-nitro- 1<Emphasis Type="Italic">H</Emphasis>-benzimidazole derivatives as potential anticancer agents

In this study we report the synthesis and preliminary evaluation of a series of six 2-aryl-5(6)-nitro-1H-benzimidazole derivatives (1–6) as potential anticancer agents. Cytotoxicity was evaluated against seven human neoplastic cell lines using the MTT assay. Compound 6 [2-(4-chloro-3-nitrophenyl)-5(6)-nitro-1H-benzimidazole] was the most active of the series, showing an IC₅₀ of 28 nM against the A549 cell line. This compound displayed a selective in vitro cytotoxic activity index (>700) in non neoplastic HACAT cells (IC₅₀ = 22.2 μM). Compounds 3 and 6 induce arrest in the S phase of the cell cycle, and compounds 1–6 induce apoptosis in the K562 cell line. Compound 6 induces poly (ADP-ribose) polymerase (PARP) inhibition activity as a potential mechanism of action. These results suggest that compound 6 could be a potent anticancer agent. Compound 3 displayed the best inhibitory activity against PARP with an IC₅₀ value of 0.05 μM, compared to the activity shown by the positive control 3-aminobenzamide (IC₅₀ = 28.5 μM).     

The effect of molecular weights of microencapsulating polymers on viability of mouse-cloned pancreatic β-cells: biomaterials,osmotic forces and potential applications in diabetes treatment

Introduction: Ideal cell-containing microcapsules should be capable of maintaining cell viability and exhibit significant structural stability to support cellular functionality. To date, such microcapsules remain unavailable; thus, this study used our well-established microencapsulating methods to examine a total of 32 different microencapsulating formulations and correlate polymers’ molecular weights (M_wt) and UDCA addition, with cell viability and microcapsules’ stability, postmicroencapsulation.

Methods: MIN6 mouse-cloned pancreatic β-cells were microencapsulated using control (n?=?16; without UDCA) and test (n?=?16; with UDCA) different polymers. Confocal microscopic imaging, cell viability, and microcapsules’ stability were assessed.

Results: Best cell viability (>50%) was obtained at average M_wt of 50,000?g/mol (poly-l-ornithine), followed by 110,000?g/mol (poly-l-lysine). There was no linear correlation between M_wt and viability. Confocal imagining showed similar microcapsules’ shape and cell distribution among all different polymers’ molecular weights, which suggests that the microencapsulating method was efficient and maintained microcapsules’ uniformity. UDCA addition resulted in enhanced osmotic stability of the microcapsules and improved cell viability, when the formulation contained 1% polylornithine, 1% polyethylene glycol, 20% Eudragit^® NM30D, 1% polytetrafluoroethylene, or 5% pentamethylcyclopentasiloxane.

Conclusions: UDCA addition improved microenvironmental conditions within the microcapsules but this effect was largely dependent on the polymer systems used.     

Polymeric Microspheres Prepared by Spraying into Compressed Carbon Dioxide

Purpose. The objective was to prepare polymeric microparticles by atomizing organic polymer solutions into a spray chamber containing compressed CO₂ (PCA-process) and to study the influence of various process parameters on their morphological characteristics. Methods. The swelling of various pharmaceutically acceptable polymers [ethyl cellulose, poly(methyl methacrylate), poly(-caprolactone), poly(dl-lactide), poly(l-lactide) and poly(dl-lactide-glycolide) copolymers] in CO₂ was investigated in order to find polymers which did not agglomerate during the spraying process. Poly(l-lactide) (L-PLA) microparticles were prepared by spraying the organic polymer solution into CO₂ in a specially designed spraying apparatus. The effect of various process (pressure and temperature of the CO₂ phase, flow rate) and formulation (polymer concentration) variables on the morphology and particle size of L-PLA-microparticles was investigated. Results. Polymers with low glass transition temperatures agglomerated even at low temperatures. The formation of microparticles was favored at moderate temperatures, low polymer concentrations, high pressures and high flow rates of CO₂. High polymer concentrations and low flow rates resulted in the formation of polymeric fibers. Colloidal L-PLA particles could also be prepared with this technique in a surfactant-free environment. Initial studies on the microencapsulation of drugs resulted in low encapsulation efficiencies. Conclusions. The PCA method is a promising technique for the preparation of drug-containing microparticles. Potential advantages of this method include the flexibility of preparing microparticles of different size and morphology, the elimination of surfactants, the minimization of residual organic solvents, low to moderate processing temperatures and the potential for scale-up.     

Influence of pH adjustment on microcapsules obtained from complex coacervation of gelatin and acacia

Abstract

The coacervation behaviour of commercial grade gelatin and acacia mixtures was studied with five different acids to adjust the coacervation pH, i.e. HCI, HNO₃, H₂SO₄, acetic acid, and citric acid. The electrical equivalence pH value (EEP) of the polymer mixture was determined by means of a streaming current detector (SCD). With all acids-except H₂SO₄-maximum coacervate yield was observed at the EEP. Using H₂SO₄ the EEP was found at a lower pH value than compared with the point of maximum coacervate yield. The quantity of coacervate at the EEP was significantly reduced in the presence of H₂SO₄ whereas with all other acids, almost no differences were found. The dependence of the coacervate volume on the added amount of acid did not change in parallel to the dry coacervate yield and there was no coincidence of the maximum coacervate volume and the EEP. The barrier properties of the capsule shells of corresponding microcapsules using indomethacin as a model drug were examined by dissolution studies. Indomethacin microcapsules showed the slowest release rate when the coacervation pH was adjusted to the EEP and not to the pH of maximum coacervate yield. As expected from the coacervation behaviour, dissolution profiles of the microcapsules were quite similar even when different acids were used for pH adjustment.     

Preparation and characterisation of multilayer films and microcapsules containing poly(N-isopropylacrylamide-co-sodium vinylsulphonate) and applicability on controlled release

The copolymers of N-isopropylacrylamide and sodium vinylsulphonate were synthesised by free radical polymerisation. The layer-by-layer self-assembly of the copolymers with poly(allylamine) hydrochloride was performed through assembling onto silicon wafer to form multilayer films and onto CaCO₃ microparticles doped with poly(styrene sulphonate) as well as deltamethrin microcrystals to form microcapsules. The multilayer films and microcapsules were characterised by atomic force microscopy, transmission electron microscopy and scanning electron microscopy. The release behaviour of deltamethrin in the microcapsules under different conditions was also investigated by high performance liquid chromatography. Results show that these deltamethrin microcapsules have good thermo-sensitive properties and deltamethrin release can be controlled via changing temperature or self-assembly layers.     

包含金纳米粒子的聚电解质微囊的制备

本文制备了含金纳米粒子的聚电解质微囊, 并进行了表征。以碳酸钙粒子为模板, 在其表面组装聚烯丙基胺盐酸盐 [poly (allyamine hydrochloride), PAH] 和金纳米粒子, 得到以碳酸钙粒子为母核, PAH/金纳米粒子为壳的核壳结构微粒。用乙二胺四乙酸二钠 (EDTA) 溶解碳酸钙, 即可得到含有金纳米粒子的聚电解质微囊。用扫描电镜表征碳酸钙粒子、含金纳米粒子的聚电解质微囊去掉母核前后的形状, 可观察到碳酸钙粒子表面包裹金纳米粒子前后的差别。用显微镜表征了微囊在溶液中的形态, 微囊在水中分散性良好。载入异硫氰酸荧光素标记的牛血清白蛋白 (FITC-bovine serum albumin, FITC-BSA) 作为模型药物, 用荧光显微镜可以观察到微囊内有一定的荧光强度, 检测得到牛血清白蛋白的包封率为 (34.31 ± 2.44) %, 聚电解质微囊载药量为 (43.75 ± 3.12) mg·g⁻¹。     

Different pH Dependency of Mitomycin C Activity in Monolayer and Three-Dimensional Cultures

Purpose. Previous studies by other investigators have shown an enhancement of mitomycin C (MMC) activity at acidic extracellular pH (pH_e) in monolayer cultures of human cells. The goal of the present study was to determine if the efficacy of intravesical MMC therapy in patients treated for superficial bladder cancer can be enhanced by using acidified dosing solutions. We evaluated (a) the effect of pH_e on MMC activity in patient bladder tumors in vitro, and (b) the pH dependency of MMC activity in 2-dimensional monolayer and 3-dimensional multilayer cultures of human bladder RT4 tumor cells. Methods. Patient bladder tumors were maintained as 3-dimensional histocultures. RT4 cells were harvested and maintained as monolayer cultures or as 3-dimensional cell pellets on a collagen gel matrix. The cell pellets were 300–450 cell layers and 4,000–5,000 µm in diameter. Tumors or cells were incubated for 2 hr with MMC-containing media at pH_eof 5, 6, and 7.4. The drug effect was measured by the inhibition of DNA precursor (thymidine) incorporation. The stability of MMC as a function of pH_e was determined. About 24% of MMC was degraded following 2 hr exposure at pH_e 5 and 2% at pH_e 6 and 7.4. Results. The drug concentrations required to inhibit thymidine incorporation by 50% (IC₅₀) were corrected for the degraded MMC at acidic pH_e. The results showed no pH-dependent MMC activity in human patient bladder tumors nor in RT4 multilayer cultures; the IC₅₀ values were about 10 µg/ml at all three pH_e. In contrast, the monolayer RT4 cultures showed a pH-dependent MMC cytotoxicity; the IC₅₀ were 0.1, 0.8 and 1.2 µg/ ml at pH_e 5,6 and 7.4, respectively (p < 0.05). Pre-incubation of multi-layered RT4 cultures in acidic pH medium for 8 hr enhanced the MMC activity; the IC₅₀ was reduced by about 5 fold at pH_e 5 and about 3 fold at pH_e 6. Similar pH-dependent MMC activity was found when multilayers were pre-treated for 1 hr with 0.5 µml nigericin, a proton ionophore known to cause the intracellular pH (pH_i) to equilibrate with pH_e. Conclusions. These data suggest that the difference in the pH dependency of MMC activity in the monolayer and multilayer systems was due to the different experimental conditions. The time lag for pH_i to equilibrate with pH_e in the multilayer systems and the instability of MMC at low pH_e imply that the efficacy of intravesical MMC therapy is unlikely to be enhanced by using acidic dosing solution.     

Constituents with α-glucosidase and advanced glycation end-product formation inhibitory activities from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> Bge.

The 75% ethanol extract from roots of Salvia miltiorrhiza Bge. (Dan shen) afforded two new compounds, 3-hydroxy-2-(2′-formyloxy-1′-methylethyl)-8-methyl-1,4-phenanthrenedione (1), (8′R)-isosalvianolic acid C methyl ester (2), and 14 known compounds. Their structures were established on the basis of spectral analysis. The ability of the compounds to inhibit α-glucosidase activity and formation of advanced glycation end-products (AGEs) was evaluated. All compounds displayed various degrees of inhibitory effects against α-glucosidase; moreover, compounds 2, 6, 11, 14, and 16 exhibited much more potent inhibition against AGEs than the positive control (aminoguanidine, AG, IC₅₀ 0.11 μM). This is the first time that compounds from this plant have been reported to have inhibitory activity against α-glucosidase.     

Diketopiperazine Formation,Hydrolysis, and Epimerization of the New Dipeptide Angiotensin-Converting Enzyme Inhibitor RS-10085

The degradation kinetics, products, and mechanisms of RS-10085(1), 2-[2-(l-ethoxycarbonyl)-3-phenylpropyl]amino-l-oxopropyl]-6,7-dimethoxy-l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(S,S,S), in aqueous solution were investigated at 40, 60, and 80°C from pH 1 to pH 13. Pseudo-first-order kinetics were observed throughout the pH range studied and the log(rate)–pH profiles reflected four kinetic processes (k _o, k_o, k_o, and k _OH) as well as the two pKa's of 1. Excellent (>98%) mass balance was obtained through products 2–5. At pH 4 or below, intramolecular cyclization leading to diketopiperazine 5 accounted for greater than 93% of the observed neutral- or water-catalyzed processes (k _o and k_o). At pH levels greater than 5, hydrolysis giving 2 predominated and was responsible for the observed neutral- or water-catalyzed (k_o) and specific base-catalyzed (k _OH) kinetic processes. Some epimerization leading to the S,S,R drug isomer (4) was also observed at pH levels greater than 7. The relative acidity of the protons at the three chiral centers of 1 was qualitatively compared and was used to explain the observed specificity in epimerization.     

A Comparison of the Bioconversion Rates and the Caco-2 Cell Permeation Characteristics of Coumarin-Based Cyclic Prodrugs and Methylester-Based Linear Prodrugs of RGD Peptidomimetics

Purpose. To compare the bioconversion rates in various biological media and the Caco-2 cell permeation characteristics of coumarin-based cyclic prodrugs (3a, 3b) and methylester-based linear prodrugs (1b, 2b) of two RGD peptidomimetics (la, 2a). Methods. Bioconversion rates of the prodrugs to the RGD peptidomimetics were determined in Hank balanced salt solution (HBSS), pH 7.4, at 37°C and in various biological media (human blood plasma, rat liver homogenate, Caco-2 cell homogenate) known to have esterase activity. Transport rates of the prodrugs and the RGD peptidomimetics were determined using Caco-2 cell monolayers, an in vitrocell culture model of the intestinal mucosa. Results. In HBSS, pH 7.4, the coumarin-based cyclic prodrugs 3a and 3b degraded slowly and quantitatively to the RGD peptidomimetics la and 2a, respectively (3a, t_1/2= 630 ± 14 min; 3b, t_1/2= 301 ± 12 min). The methylester-based linear prodrugs 1b and 2b were more stable to chemical hydrolysis (1b and 2b, t_1/2> 2000 min). Both the coumarin-based cyclic prodrugs and the methylester-based linear prodrugs degraded more rapidly in biological media containing esterase activity (e.g., 90% human blood plasma: 1b, t_1/2< 5 min; 2b, t_1/2< 5 min; 3a, t_1/2< 91 ± 1 min; 3b, t_1/2< 57 ± 2 min). When the apical (AP)-to-basolateral (BL) permeation characteristics were determined using Caco-2 cell monolayers, it was found that the methylester pro-drugs Ib and 2b underwent esterase bioconversion (>80%) to the RGD peptidomimetics 1a and 2a, respectively. In contrast, the cyclic prodrugs 3a and 3b permeated the cell monolayers intact. Considering the appearance of both the prodrug and the RGD peptidomimetic on the BL side, the methylester prodrugs 1b and 2b were approximately 12-fold more able to permeate than were the RGD peptidomimetics la and 2a. When a similar analysis of the transport data for the coumarin prodrugs 3a and 3b was performed, they were shown to be approximately 6-fold and 5-fold more able to permeate than were the RGD peptidomimetics la and 2a, respectively. Conclusions. The coumarin-based cyclic prodrugs 3a and 3b were chemically less stable, but metabolically more stable, than the methylester-based linear prodrugs. The esterase stability of the cyclic prodrugs 3a and 3b means that they are transported intact across the Caco-2 cell monolayer in contrast to the methylester prodrugs 1b and 2b, which undergo facile bioconversion during their transport to the RGD peptidomimetics. However, both prodrug systems successfully delivered more (5-12-fold) of the RGD peptidomimetic and/or the precursor (prodrug) than did the RGD peptidomimetics themselves.     

Chemical Pathways of Peptide Degradation: IX. Metal-Catalyzed Oxidation of Histidine in Model Peptides

Purpose. To elucidate the nature of the reactive oxygen species (i.e., superoxide anion radical, hydroxyl radical, and hydrogen peroxide) involved in the metal-catalyzed oxidation of histidine (His) in two model peptides. Methods. The degradation of AcAla-His-ValNH₂ (Ala-peptide) and AcCysNH₂-S-S-AcCys-His-ValNH₂ (Cys-peptide) was investigated at pH 5.3 and 7.4 in an ascorbate/cupric chloride/oxygen (ascorbate/ Cu(II)/O₂) system, both in the absence and presence of selective scavengers (i.e., catalase, superoxide dismutase, mannitol, sodium formate, isopropanol, and thiourea) of the reactive oxygen species. All reactions were monitored by HPLC. The major degradation products were characterized by electrospray mass spectrometry. Results. The Cys-peptide was more stable than the Ala-peptide at pH 5.3 and 7.4. Both peptides displayed greater stability at pH 5.3 than at 7.4. At pH 5.3, 35 ± 0.7% of the Cys-peptide and 18 ± 1% of the Ala-peptide remained after 7 hours, whereas at pH 7.4, 16 ± 3% of the Cys-peptide and 4 ± 1 % of the Ala-peptide remained. Catalase, thiourea, bicinchoninic acid, and ethylenediaminetetraacetate were effective at stabilizing both peptides toward oxidation, while superoxide dismutase, mannitol, isopropanol, and sodium formate were ineffective. The main degradation products of the Ala- and Cys-peptides at pH 7.4 appeared to be AcAla-2-oxo-His-ValNH₂ and AcCysNH₂-S-S-AcCys-2-oxo-His-ValNH₂, respectively. Conclusions. Hydrogen peroxide, Cu(I), and superoxide anion radical were deduced to be intermediates involved in the oxidation of the Ala- and Cys-peptides. Hydrogen peroxide degradation to secondary reactive oxygen species may have led to the oxidation of the peptides. The degradation of hydrogen peroxide by a Fenton-type reaction was speculated to form a complexed form of hydroxyl radical that reacts with the peptide before diffusion into the bulk solution.