Polyketal microparticles: a new delivery vehicle for superoxide dismutase

There is currently great interest in developing microparticles that can enhance the delivery of proteins to macrophages. In this communication, we present a new acid-sensitive polymer for drug delivery, poly(cyclohexane-1,4-diyl acetone dimethylene ketal) (PCADK). PCADK is designed to hydrolyze, after phagocytosis by macrophages, in the acidic environment of the phagosome and enhance the intracellular delivery of phagocytosed therapeutics. Other key attributes of PCADK for drug delivery are its well-characterized degradation products and straightforward synthesis. PCADK hydrolyzes into 1,4-cyclohexanedimethanol, a compound used in food packaging, and acetone, a compound on the FDA GRAS list. PCADK was synthesized using the acetal exchange reaction between 1,4-cyclohexanedimethanol and 2,2-dimethoxypropane, and could be obtained on a multigram scale in one step. The hydrolysis kinetics of the ketal linkages in PCADK were measured by 1H NMR and were determined to be pH-sensitive, having a half-life of 24.1 days at pH 4.5 and over 4 years at pH 7.4. The therapeutic enzyme superoxide dismutase (SOD), which scavenges reactive oxygen species, was encapsulated into PCADK-based microparticles using a double emulsion procedure. Cell culture experiments demonstrated that PCADK-based microparticles dramatically improved the ability of SOD to scavenge reactive oxygen species produced by macrophages. We anticipate numerous applications of PCADK in drug delivery, based on its acid sensitivity, well-characterized degradation products, and straightforward synthesis.     

Acetals as pH-sensitive linkages for drug delivery

pH-Sensitive linkages designed to undergo hydrolysis at mildly acidic pH can trigger the release of therapeutics selectively at targets such as tumor and inflammatory tissues and in the endosomes and lysosomes of cells. Acetals have the potential to be used as linkages for a range of alcohol functionalities, and, by altering their chemical structure, it is possible to tune their hydrolysis rate. The syntheses of four conjugates of model drug molecules with PEO using acetals of varying chemical structure are described herein. Primary and secondary alcohols, as well as syn-1,2-diols, were incorporated in the conjugates. The hydrolysis kinetics were investigated by HPLC, and the conjugates had half-lives ranging from less than 1 min to several days at pH 5.0, with slower hydrolysis at pH 7.4 in all cases. These acetal linkages are therefore promising for use in a variety of drug delivery applications ranging from polymer-drug conjugates to pH-sensitive micelles and nanoparticulate systems.     

Polyketal nanoparticles: a new pH-sensitive biodegradable drug delivery vehicle

In this report, we present an acid-sensitive drug delivery vehicle, termed polyketal nanoparticles, which are designed to target therapeutics to the acidic environments of tumors, inflammatory tissues, and phagosomes. The polyketal nanoparticles are formulated from poly(1,4-phenyleneacetone dimethylene ketal) (PPADK), a new hydrophobic polymer which contains ketal linkages in its backbone. The polyketal nanoparticles undergo acid-catalyzed hydrolysis into low molecular weight hydrophilic compounds and should therefore release encapsulated therapeutics at an accelerated rate in acidic environments. Importantly, the polyketal nanoparticles do not generate acidic degradation products after hydrolysis, as with polyester-based biomaterials. Dexamethasone-loaded nanoparticles, 200-600 nm in diameter, were fabricated with PPADK via an emulsion procedure using chloroform and water. The hydrolysis half-life of PPADK was measured to be 102 h at pH 7.4 and 35 h at pH 5.0. PPADK was synthesized by a new polymerization strategy based on the acetal exchange reaction. This new delivery system should find numerous applications in the field of drug delivery because of its ease of synthesis and excellent degradation properties.     

Ketal cross-linked poly(ethylene glycol)-poly(amino acid)s copolymer micelles for efficient intracellular delivery of doxorubicin

A biocompatible, robust polymer micelle bearing pH-hydrolyzable shell cross-links was developed for efficient intracellular delivery of doxorubicin (DOX). The rationally designed triblock copolymer of poly(ethylene glycol)-poly(L-aspartic acid)-poly(L-phenylalanine) (PEG-PAsp-PPhe) self-assembled to form polymer micelles with three distinct domains of the PEG outer corona, the PAsp middle shell, and the PPhe inner core. Shell cross-linking was performed by the reaction of ketal-containing cross-linkers with Asp moieties in the middle shells. The shell cross-linking did not change the micelle size and the spherical morphology. Fluorescence quenching experiments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced Stern-Volmer quenching constant (K(SV)). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study showed that the hydrolysis half-life (t(1/2)) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH, compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.     

Acid-degradable protein delivery vehicles based on metathesis chemistry

In this communication we demonstrate that acyclic diene metathesis (ADMET) polymerization is a powerful methodology for the synthesis of acid-degradable polymers based on polyketals and polyacetals. Ten new polyketals and polyacetals were synthesized, using ADMET, and a polyacetal based on anthracene aldehyde was identified, which had the physical properties needed for microparticle formulation. The antioxidant protein catalase was encapsulated into microparticles, formulated from this polyacetal, using a double emulsion procedure, and cell culture studies demonstrated that these microparticles dramatically improved the ability of catalase to scavenge hydrogen peroxide produced by macrophages. We anticipate numerous applications of ADMET for the synthesis of acid-degradable polymers based on its excellent tolerance toward functional groups and ease of synthesis.     

Application of trans and cis isomers of p-nitrophenyl-(1R, S)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate to the assay of pyrethroid-hydrolyzing esterases

A continuous-rate assay for the detection of esterases which hydrolyze synthetic pyrethroids is described. The assay is based on the release of p-nitrophenolate ion upon hydrolysis of the pyrethroid-like compound, trans- or cis-p-nitrophenyl-(1R,S)-3-(2,2-dichlorovinyl)-2, 2-dimethylcyclopropanecarboxylate, at pH 7.4 where spontaneous hydrolysis is not detected. The reagent is solubilized by 0.02% Triton X-100 in the presence of 1.0% ethanol. A simple procedure for the synthesis and separation of the isomers is described. The application of the reagent to the assay of esterases which detoxify synthetic pyrethroids in the cattle tick Boophilus microplus is reported.     

Mitochondrial 3β-hydroxysteroid dehydrogenase enzyme activity requires reversible pH-dependent conformational change at the intermembrane space

The inner mitochondrial membrane protein 3β-hydroxysteroid dehydrogenase 2 (3βHSD2) synthesizes progesterone and androstenedione through its dehydrogenase and isomerase activities. This bifunctionality requires 3βHSD2 to undergo a conformational change. Given its proximity to the proton pump, we hypothesized that pH influences 3βHSD2 conformation and thus activity. Circular dichroism (CD) showed that between pH 7.4 and 4.5, 3βHSD2 retained its primarily α-helical character with a decrease in α-helical content at lower pH values, whereas the β-sheet content remained unchanged throughout. Titrating the pH back to 7.4 restored the original conformation within 25 min. Metabolic conversion assays indicated peak 3βHSD2 activity at pH 4.5 with ~2-fold more progesterone synthesized at pH 4.5 than at pH 3.5 and 7.4. Increasing the 3βHSD2 concentration from 1 to 40 μg resulted in a 7-fold increase in progesterone at pH 4.5, but no change at pH 7.4. Incubation with guanidinum hydrochloride (GdmHCl) showed a three-step cooperative unfolding of 3βHSD2 from pH 7.4 to 4.5, possibly due to the native state unfolding to the intermediate ion core state. With further decreases in pH, increasing concentrations of GdmHCl led to rapid two-step unfolding that may represent complete loss of structure. Between pH 4 and 5, the two intermediate states appeared stable. Stopped-flow kinetics showed slower unfolding at around pH 4, where the protein is in a pseudostable state. Based on our data, we conclude that at pH 4-5, 3βHSD2 takes on a molten globule conformation that promotes the dual functionality of the enzyme.     

Discovery and optimization of 4-oxo-2-thioxo-thiazolidinones as NOD-like receptor (NLR) family,pyrin domain-containing protein 3 (NLRP3) inhibitors

Aberrant activation of NLRP3 inflammasome is present in a subset of acute and chronic inflammatory diseases. The NLRP3 inflammasome has been recognized as an attractive therapeutic target for developing novel and specific anti-inflammatory inhibitors. Cellular structure-activity relationship-guided optimization resulted in the identification of 4-oxo-2-thioxo-thiazolidinone derivative 9 as a selective and direct small-molecule inhibitor of NLRP3 with IC₅₀ of 2.4 μM, possessing favorable ex vivo and in vivo pharmacokinetic properties. Compound 9 may represent a lead for the development of anti-inflammatory therapeutics for treating NLRP3-driven diseases.     

Roles of heterogenous hepatic macrophages in the progression of liver diseases

Hepatic macrophages are key immune cells associated with the broad ranges of liver diseases including steatosis, inflammation and fibrosis. Hepatic macrophages interact with other immune cells and orchestrate hepatic immune circumstances. Recently, the heterogenous populations of hepatic macrophages have been discovered termed residential Kupffer cells and monocyte-derived macrophages, and identified their distinct population dynamics during the progression of various liver diseases. Liver injury lead to Kupffer cells activation with induction of inflammatory cytokines and chemokines, which triggers recruitment of inflammatory monocyte-derived macrophages. To understand liver pathology, the functions of different subtypes of liver macrophages should be regarded with different perspectives. In this review, we summarize recent advances in the roles of hepatic macrophages under liver damages and suggest hepatic macrophages as promising therapeutic targets for treating liver diseases.     

Fully acid-degradable biocompatible polyacetal microparticles for drug delivery

A library of polyurethanes and polyureas with different hydrophobicities containing the same acid-degradable dimethyl ketal moiety embedded in the polymer main chain have been prepared. All polymers were synthesized using an AA-BB type step-growth polymerization by reaction of bis(p-nitrophenyl carbamate/carbonate) or diisocyanate monomers with an acid-degradable, ketal-containing diamine. These polymers were designed to hydrolyze at different rates in mildly acidic conditions as a function of their hydrophobicity to afford small molecules only with no polymeric byproduct. The library of polymers was screened for the formation of microparticles using a double emulsion technique. The microparticles that were obtained degraded significantly faster at acidic pH (5.0) than at physiological pH (7.4) with degradation kinetics related to the hydrophobicity of the starting polymer. In vitro studies demonstrated the ability of the FITC-BSA loaded microparticles to be phagocytosed by macrophages resulting in a 10-fold increase in the protein uptake compared to a free protein control; in addition, the microparticles were found to be nontoxic at the concentrations tested of up to 1 mg/mL. The ease of preparation of the polymers coupled with the ability to tune their hydrophobicity and the high acid sensitivity of the microparticles identify this new class of materials as promising candidates for the delivery of bioactive materials.     

Microbial isobutyronitrile utilization under haloalkaline conditions

The utilization of isobutyronitrile (iBN) as a C and N source under haloalkaline conditions by microbial communities from soda lake sediments and soda soils was studied. In both cases, a consortium consisting of two different bacterial species capable of the complete degradation and utilization of iBN at pH 10 was selected. The soda lake sediment consortium consisted of a new actinobacterium and a gammaproteobacterium from the genus Marinospirillum. The former was capable of fast hydrolysis of aliphatic nitriles to the corresponding amides and much-slower further hydrolysis of the amides to carboxylic acids. Its partner cannot hydrolyze nitriles but grew rapidly on amides and carboxylic acids, thus acting as a scavenger of products released by the actinobacterium. The soda soil consortium consisted of two Bacillus species (RNA group 1). One of them initiated nitrile hydrolysis, and the other utilized the hydrolysis products isobutyroamide (iBA) and isobutyrate (iB). In contrast to the actinobacterium, the nitrile-hydrolyzing soil Bacillus grew rapidly with hydrolysis products, but it was dependent on vitamins most probably supplied by its product-utilizing partner. All four bacterial strains isolated were moderately salt-tolerant alkaliphiles with a pH range for growth from pH 7.0 to 8.5 up to 10.3 to 10.5. However, both their nitrile hydratase and amidase activities had a near-neutral pH optimum, indicating an intracellular localization of these enzymes. Despite this fact, the study demonstrated a possibility of whole-cell biocatalytic hydrolysis of various nitriles at haloalkaline conditions.     

Macrophage fibrinolytic activity: identification of two pathways of plasmin formation by intact cells and of a plasminogen activator inhibitor

Endotoxin-stimulated macrophages hydrolyze fibrin by a plasmin-mediated process in the absence of detectable soluble plasminogen activator (PAs). The data show that macrophages also activate plasmin by a membrane-associated plasminogen activator (PAm). In the presence of endotoxin, PAm activity increases, and plasmin is formed only by PAm. In addition, endotoxin stimulates macrophages to secrete a proteinase inhibitor that blocks PAs activity but not PAm or plasmin activity. The increased PAm activity and the PA inhibitor secretion in response to endotoxin explains the ability of intact macrophages to hydrolyze fibrin in the absence of detectable PAs. Endotoxin, 100 ng/ml, induced an intracellular PA inhibitor in cultured macrophages, and this correlated with accumulation of inhibitor in medium over the cells. The intracellular PA inhibitor was found to be 50--60 kilodaltons by gel chromatography, to be of anionic charge at pH 7.4 and to inhibit urokinase esterolytic and proteolytic activity but not preformed plasmin. These results define two pathways of plasmin formation by intact macrophages and identify the macrophage cell surface as a site of PA activity relatively protected from soluble proteinase inhibitors.     

Studies on the production and application of cellulase from Trichoderma reesei QM-9414

High yielding mutant strain, Trichoderma reesei QM-9414, was employed for the cellulase enzyme production. Enzyme production conditions (pH, inoculum age and concentration, and organic supplements) were optimized. The ability of partially purified enzyme to hydrolyze various regionally abundant lignocellulosic raw materials was studied. Enzymatic hydrolysis conditions (temperature, pH, enzyme and substrate concentrations) were optimized. Temperature 50v°C, pH 4.5, enzyme concentration 40 FPU/g substrate and substrate concentration 2.5% were found to be optimum for the maximum yields of sugars. #-glucosidase supplementation was found to increase both the sugar yield and hydrolysis rate, and shorten the reaction time significantly.     

Purified Rat Brain Microvessels Exhibit Both Acid and Neutral Sphingomyelinase Activities

Purified rat brain microvessels have been shown to hydrolyze radiolabeled sphingomyelin by means of two different enzyme systems. Enzymatic activity was detected at pH 7.4 and was strongly stimulated by magnesium or manganese and inhibited by calcium. Activity at pH 5.1 could also be found and was not dependent on any of these cations. At neutral pH and in the presence of magnesium, the rate of sphingomyelin hydrolysis did not exhibit a linear relationship with protein concentration. In contrast, increasing the protein concentration from 0.05 to 0.5 mg/ml resulted in a constant increase of sphingomyelin hydrolysis at pH 5.1. Kinetic parameters of both neutral and acid activities have been determined and were similar in magnitude to values reported previously for neural sphingomyelinases. This work demonstrates the occurrence of a neutral sphingomyelinase activity in purified rat brain microvessels, an observation raising the question of its role at the level of the blood-brain interface.     

Hydrolysis of low-density lipoprotein phospholipids in arterial smooth muscle cells

The hydrolysis of phosphatidylcholine (PC) associated with low-density lipoprotein (LDL) by homogenates of smooth muscle cells from rabbit aorta was studied. 1-Palmitoyl-2-[14C]oleoylPC associated with LDL (LDL-P[14C]OPC) or 1-linoleoyl-2-[14C]linoleoylPC associated with LDL (LDL-L[14C]LPC) was used as the substrate. The optimum pH for the formation of [14C]oleoyllysoPC from LDL-P[14C]OPC and for the formation of [14C]linoleoyllysoPC from LDL-L[14C]LPC was pH 4.5, and pH 4.5 and 7.0, respectively. These activities were designated as phospholipase A1 activities. The optimum pH values for the formation of [14C]oleate from LDL-L[14C]OPC and for the formation of [14C]linoleate from LDL-L[14C]LPC were pH 4.5 and 6.5, and pH 4.5, 6.5 and 8.5, respectively. These activities were designated as phospholipase A2 activities. Ca2+ did not affect acid phospholipase A1 activity, but decreased acid phospholipase A2 activity for the hydrolysis of LDL-L[14C]LPC. When smooth muscle cells were incubated with LDL, both phospholipase A1 and phospholipase A2 activities at pH 4.5 for the hydrolysis of LDL-L[14C]LPC increased significantly. These results indicate that phospholipases A1 and A2, which hydrolyze PC associated with LDL, exist in arterial smooth muscle cells and are involved in the metabolism of LDL incorporated into these cells.     

Kinetic properties of liver pyruvate kinase from the flounder (Platichthys flesus L.)

Pyruvate kinase, purified from flounder liver, in two forms, i.e. PK I and PK II, is characterized by sigmoid kinetics with phosphoenolpyruvate as substrate at pH 6.3, 6.7 and 7.7. K0.5 for PEP increases with increasing pH. PK I and PK II show hyperbolic kinetics with ADP, but are inhibited by ADP concentrations above 1-2 mM. K0.5 for ADP decreases with increasing pH. PK I and PK II differ in their K0.5 values for PEP with a factor of at least 2, showing the highest figures for the latter. K0.5 for ADP is about the same for the two enzyme forms. Other nucleotide diphosphates can replace ADP as the substrate. When the nucleoside diphosphates are arranged in a rank order showing decreasing effectiveness as substrate, different rank orders are obtained for PK I and PK II.     

Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman’s Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.     

Use of Trinitrophenylation for Quantification of Protease and Peptidase Activities

A sensitive and precise method for quantifying protease and peptidase activities is suggested. N-Terminal amino groups of peptides which are formed during hydrolysis of the substrates react with trinitrobenzenesulfonic acid (TNBS), and the trinitrophenyl (TNP) derivatives are determined spectrophotometrically. Spontaneous hydrolysis of TNBS is considerably diminished on trinitrophenylation at pH 7.4 rather than at pH 9-10 as is usually used. The trinitrophenylation method can be used to determine the initial rate of hydrolysis and the kinetics of reactions catalyzed by proteases and peptidases.     

Poxviral immunomodulatory proteins as new therapeutics for immunocorrection

There are numerous viral proteins known to date to modulate protective responses of their hosts. Representatives of the Poxviridae family have the greatest number of genes coding for proteins, inhibiting inflammatory responses, activities of interferons, regulating immune reactions and other protective mechanisms of macroorganisms, among viruses. This review regards poxviral immunomodulatory proteins--namely, complement-binding proteins, inhibitors of serine proteases, chemokine- and TNF-binding proteins --that were shown to be efficient therapeutics in various animal models of inflammatory and autoimmune diseases. The prospects of their usage in clinical practice for treating human inflammatory and autoimmune disorders are discussed.