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The potential of poly (2-oxazoline) or POZ to be a versatile and broad based platform for drug delivery with wide utility in multiple therapeutic areas has long been recognized by experts in the field. This feature article provides a case study which describes the chemistry and preclinical studies underlying the Investigational New Drug Application for SER-214, a POZ conjugate of rotigotine, for the treatment of Parkinson’s disease. We report the chemistry, preclinical safety and pharmacology, and the early clinical safety, tolerability and pharmacokinetic data from the Phase I study in patients. SER-214 utilizes a POZ polymer and proprietary custom linker technology to deliver a sustained dose of rotigotine over a period of seven days following a single subcutaneous administration – a result not observed by any other polymer approach that we are aware of. As such, this candidate drug has the promise to be a major advancement in the treatment of Parkinson’s disease. Furthermore, this feature article also highlights the versatility of the POZ polymer platform (POZ™) to deliver cancer drugs by actively targeting cancer cells. Preclinical data reported in this feature showcase the polymer’s attributes in facilitating targeted delivery with folic acid and antibody targeting agents (ADCs). The ability of POZ to reliably delivery large payloads of anticancer drugs is of particular importance when pursuing low-receptor-density targets on the cancer cell. The data presented in this feature, much of it for the first time, establish the broad utility of the POZ polymer platform in drug development. Together with its ease of manufacture, ability to attach drugs to the polymer, and ability to administer an appropriate dose to patients, the results underscore the need to further explore and expand the untapped potential of POZ for the development of new therapeutics for unmet medical needs.

We formed viscous, mucoadhesive gels by complexing PEGylated proteins with poly(acrylic acid) (PAA) at pH 3. These complexes dissociate at pH 7.4, and under this condition the PEGylated protein is released from the gel. We propose this system as a means for prolonged mucosal delivery of a PEGylated protein to nasal, ophthalmic or vaginal sites. In this paper we have studied the effect of the molecular weight of the PEG used in the PEGylated protein (5 kD, 20 kD and 40 kD PEG) and the effect of added free PEG (18.5 kD) on the in vitro release rate of the PEGylated protein from complexes with PAA. The PEGylated proteins we studied included papain, MW = 23.7 kD and pI 8.75, and soybean trypsin inhibitor (STI), MW = 20.1 kD and pI 4.6.

A series of three nonionic surfactants which undergo acid-catalyzed hydrolysis, O -[(2,2-dialkyl-1,3-dioxolan-4-yl) methoxy] – O ′-methoxy poly(ethylene glycol), or cyclic ketal (CK), were synthesized. The surfactants shared similar HLB values (9.2–9.8) but differed in the relative length of their two alkyl tails. Water/CK/isooctane microemulsion systems formed by each of the three surfactants also shared similar phase behavior, physical properties, and surfactant hydrolysis kinetics. The apparent critical microemulsion concentration for three CK surfactants, 0.060–0.075 g g −1 (in both water and isooctane), and the efficiency, the minimum amount of surfactant required to totally mix water and oil into single microemulsion phase, 0.35–0.40 g g −1 , are significantly higher than values possessed by regular, linear, alkyl ethoxylate surfactants. The underlying causes for the high values were investigated. Due to the broad distribution of its ethoxylate head group size, CK surfactants were distributed among all phases within two- and three-phases systems, with CKs possessing shorter ethoxylate groups partitioning to the oil phase and CKs with larger ethoxylate groups to the aqueous phase. Small-angle neutron scattering experiments suggest that CK exists as monomers or very small pre-micellar aggregates in isooctane at 12% and as ellipsoidal aggregates in water at 2.5–25%. Surface pressure versus surface area measurements of CK surfactant monolayers at the water-air interface demonstrate two differences from linear alkyl ethoxylate monolayers: the occurrence of a collapse at moderately low surface pressure (30 mN m −1 ) and high surface area per molecule (0.59 nm 2 ) and the inability to undergo dissolution when subjected to a constant, moderately high surface pressure (25 mN m −1 ). The results suggest that CK molecules are not able to pack as efficiently as linear alkyl ethoxylates at interfaces, and are strongly anchored to monolayers in aqueous media.

Solvolysis/dehydrohalogenation rates of 2-chloro-2-methyladamantane (CMA) in 15 hydrogen-bond acidic and/or basic solvents are studied. The rates of reaction in these solvents have been correlated with the solvation equation developed by Kamlet, Abraham, and Taft. The linear solvation energy relationship (LSER) derived from this study is given by the following equation: log k = -5.409 + 2.219 + 2.505alpha(1) - 1.823beta(1) where , alpha(1), and beta(1) are the solvation parameters that measure the solvent dipolarity/polarizability, hydrogen-bond acidity (electrophilicity), and hydrogen-bond basicity (nucleophilicity). A high correlation coefficient (r = 0.996, SD = 0.191) was achieved. The cavity term, which includes the Hildebrand parameter for solvent cohesive energy density, delta(H), was not found to be statistically significant for this reaction substrate. The resulting equation allows calculated rates of reaction in other solvents and provides insight into the reaction pathway. In a previously reported correlation for another tertiary chloride, tert-butyl chloride (TBC), the coefficients for alpha(1) and are significantly larger and the coefficient for is statistically significant. In addition, the coefficient for beta(1) in the TBC correlation is positive, rather than negative, indicating that the transition states for TBC and CMA are significantly different. These results demonstrate why the uses of simple solvolytic correlation methods may be invalid even for comparisons of similar type substrates, e.g., tertiary chlorides. Also, these results provide confidence in the use of multiple linear regression analysis for predicting solvolytic rates in additional solvents.

An oil-soluble pH-degradable nonionic surfactant with poly(ethylene glycol) monomethyl ether as the hydrophile and a cyclic ketal as the hydrophobe was synthesized for use in microemulsion-based protein extraction. The surfactant solubilized water in isooctane. Dynamic light-scattering measurements showed formation of fairly monodisperse water-in-oil microemulsions of radii 4−6 nm, with very strong intermicellar attractive interactions. The ternary phase diagram for the system surfactant/water/isooctane at 23 °C consists of one-, two-, and three-phase regions as well as gel-like phases. The well-known “fish” pattern occurred for the phase diagram of temperature vs surfactant concentration at a fixed ratio of water-to-oil (1/1 g/g). The surfactant remained stable at neutral pH for several days but degraded rapidly when a mildly acidic phosphate buffer (pH = 5) was encapsulated in the water-in-oil microemulsion solution. Degradation occurred more rapidly when the microemulsion solution was brought in contac...

Poly(ethyleneglycol) or PEG has proven to be of great value for a range of biomedical applications. A review the properties of PEG that lead to these applications is reported. Emphasis is placed on pharmaceutical uses of PEG–proteins, with specific discussion of the attributes of PEGylated α-interferon for treatment of hepatitis C. In this latter case the choice of PEG reagent is critical to the properties of the drug, and therefore a brief presentation of PEG reagents for protein PEGylation will be given. PEGylation chemistries can be divided into first- and second-generation approaches. The first-generation chemistries are generally restricted to low-molecular-weight methoxy-PEGs because of the problem of diol contamination and resulting difunctional reagents. Problems with weak linkages and side reactions are also encountered. Second-generation PEGylation reagents avoid weak linkages and side reactions. Also they can be purified to remove diol contaminants, and as a consequence, high-molecular-weight PEGs can be used. These relatively simple chemical advances have given new vigor to PEGylation as a technology. The benefits of using high-molecular-weight, second-generation PEG reagents are demonstrated by using PEG–α-interferon as an example. In this case it is observed that a greatly improved drug is provided for treatment of hepatitis C.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Organophosphorous hydrolase (OPH) was physically and covalently immobilized within photosensitive polyethylene glycol (PEG)-based hydrogels. The hydroxyl ends of branched polyethylene glycol (b-PEG, four arms, MW = 20,000) were modified with cinnamylidene acetate groups to give water-soluble, photosensitive PEG macromers (b-PEG-CA). The b-PEG-CA macromers underwent photocrosslinking reaction and formed gels upon UV irradiation (>300 nm) in the presence of erythrosin B. Native OPH was pegylated with cinnamylidene-terminated PEG chains (MW = 3400) to be covalently linked with the b-PEG-CA macromers during photogelation. The effect of pegylation on the stability of the enzyme was determined. Furthermore, the effect of enzyme concentration, wavelength of irradiation, and photosensitizer on the stability of the entrapped enzyme was also investigated. The pegylated OPH was more stable than the native enzyme, and the OPH-containing gels exhibited superior stability than the soluble enzyme preparations.

Novel water-soluble unsaturated poly(ether amide)s (PEAs) were synthesized by low-temperature polycondensation of fumaryl chloride and amine-terminated poly(ethylene glycol) (Jeffamine®). The unsaturated copolymers were further chemically modified with thiols to provide reactive pendant functional groups. Hydrogels based on these copolymers were prepared by copolymerization of the PEA with N-vinyl pyrrolidone exposure to ultraviolet (UV) irradiation. The resulting hydrogels exhibited a high swelling ratio, and the magnitude of swelling depended on the molecular weight of Jeffamine®. The swelling ratio and equilibrium water content tended to increase with increasing chain length of the Jeffamine® used in copolymer synthesis.

We have investigated the reaction chemistry of methoxypoly(oxyethylene)propionitrile, I, to synthesize methoxypoly(oxyethylene)propionic acid, II. We have found that II can be prepared by converting I first to the corresponding amide. Subsequent hydrolysis of the amide then yields II.

Capillary electrophoresis (CE) was investigated for characterizing poly(ethylene glycol) (PEG) attachment ("PEGylation") and PEG removal ("dePEGylation") of proteins. Lysozyme was used as a model protein because it is one of the best understood enzymes, has a high ionic strength (high pI value; thus making it suitable for CE), and has a tertiary structure that is known with high resolution. Several PEG derivatives, both hydrolytically degradable and nondegradable and with varying reactivities toward amino groups, were used to couple to amino groups (six epsilon-amino and one alpha-amino) on the surface of the protein. Capillary electrophoresis was found to be useful in following both the PEGylation and dePEGylation of lysozyme. Capillary electrophoresis separation is based on the size of the conjugate, which is determined by the number and molecular weight of the PEG that is attached. Baseline resolution was obtained between the peaks for each PEG chain attached per protein molecule ("PEGmers") for PEG molecular weights5000, although individual PEGmers could be recognized at lower molecular weights without baseline separation. Highly modified lysozyme showed complete inactivation, but when released from the degradable PEG, regained60% of the original in vitro activity. The sites of PEGylation were determined using a tryptic map of the modified and unmodified protein. Typically, peptide fragments are separated by reversed-phase HPLC, but we show that CE can provide a complementary separation technique for determining sites of PEGylation. Capillary electrophoresis has advantages of high efficiency separations, rapid analysis, and ease of use.

Electrokinetic surface characterization is sensitive to both charge groups (pK and surface density) and neutral polymer coatings (molecular weight, surface density, grafting chemistry). It allows nondestructive, semiquantitative evaluation of coating stability, as regards alteration of the above parameters in relation to various environments. Characterization of surface electroosmosis versus pH (2 to 11) was used to evaluate poly(ethylene glycol) (PEG) coatings of biotechnical significance following a 3-week 22 °C exposure to 0.5 M sodium phosphate solutions of pH 4−11. The coatings involved epoxide-functionalized PEGs of Mr 3400 (E-PEG 3400) covalently grafted to aminosilane-modified fused quartz capillaries. Coatings made from PEGs with an epoxide group at one end (mE-PEG 5000) were stable at pH 4−11 whereas those made from PEGs with epoxide groups at both ends (diE-PEG 3400) were only stable at pH 4−9. During storage at pH 11 the latter exhibited time-dependent generation of surface negative charge gro...

This paper reviews recent studies by the authors on the surface characterization of biomedically significant materials through electroosmosis determination. The surfaces studied include transparent and nontransparent materials such as quartz, ceramics, paper, and cast polymer capillaries, slides, and particles, in both native and surface modified form. The method is nondestructive, relatively fast, mechanistically simple, automatable to varying degrees, and can be used to analyze samples under physiologically compatible conditions. New experimental and mathematical modeling approaches allow estimates to be obtained with regard to the surface density and pK of various chemical groups, as well as the thickness of polymer or other surface coatings. Surface modifications which may be characterized include, covalent alteration via radiofrequency plasma discharge or organosilane grafting, noncovalent alteration via polymer adsorption, and covalent grafting of neutral polymers, such as poly(ethylene glycol) or dextran. Results complement those from other surface analysis techniques, and correlate with physiologically significant phenomena such as protein adsorption.

Graft copolymers of poly(ethylene glycol) (PEG) on a chitosan backbone (PEG-g-chitosan) have been synthesized and their aqueous solution properties were investigated. At pH 6.5 the graft copolymers are 100% soluble, while chitosan phase separates from solution at those conditions. These interesting graft copolymers may be especially suitable as carriers for delivery of anionic drugs, such as proteins, glycosaminoglycans, and DNA plasmids or oligonucleotides.

Intelligent polymers exhibit sharp, reversible phase changes in response to small changes in environmental conditions. For example, a small temperature change can cause a sharp precipitation or gelation of a smart polymer solution. Conjugation of these unusual polymers to biomolecules such as enzymes, ligands, lipids, and drugs can lead to many new and exciting applications in medicine and biotechnology. (1-4) This presentation reviews the principles, methodolgies and applications of these smart polymer-biomolecule systems, with special focus on temperature-sensitive polymer-protein conjugates.

A series of noncyclic acetal-linked cleavable surfactants were simply prepared by condensation of aldehydes with poly(ethylene glycol) monomethyl ethers. All of the products were characterized by1H nuclear magnetic resonance. Their hydrophile-lipophile balance, surface tension, cloud point, critical micelle concentration, and foam height were determined. Hydrolysis kinetic studies, followed by gas chromatography, showed that they had higher hydrolytic reactivity in acidic solution than cyclic acetal-linked cleavable surfactants.

Poly(ethylene glycol) coatings are known to control a variety of surface phenomena including nonspecific protein adsorption, wetting, and electrokinetic effects. These and other properties make such coatings useful for biocompatible materials and in improving the efficiency of analytical and preparative separations where analyte-surface interactions play a role. In the present study terminally activated poly(ethylene glycol) derivatives were grafted onto quartz functionally activated with (3-aminopropyl)triethoxysilane, (3-mercaptopropyl)trimethoxysilane, or poly(ethylenimine). Grafts and activated surfaces were electro-kinetically characterized by measuring the pH dependence of induced electroosmotic fluid flow. A site-dissociation model of electroosmotic fluid flow based on the Smoluchowski equation, the Gouy-Chapman theory of the electrical double layer, and exact solutions to the Poisson-Boltzmann equation is presented in order to relate electroosmosis to the chemistry of the surface. Results include indications of relative coating stability and estimates of grafting density as well as effective coating thickness.

Two enzymes (subtilisin and thermolysin) have been modified with a polyethylene glycol (PEG, MW 3400) having an acrylate group at one terminus and an active ester at the other terminus and then incorporated into polyacrylates during free-radical initiated polymerization in a variety of organic solvents. Despite the high activity of the radical intermediates, both enzymes remain active and stable after incorporation. The activity and stability of the resulting biopolymer produced by subtilisin have been compared, in both aqueous and organic media, to those for the native, PEG-modified, and traditionally-immobilized subtilisins. In aqueous solution, the biopolymer had a reduced Km and k,, relative to the native subtilisin, but the specificity constant (kcatlKm) was only reduced to oneninth by incorporation into the polymer. In a flow-cell reactor, the biopolymer achieved the longest half-life (> 100 days) relative to the other forms of the enzyme. The enzyme’s tolerance toward both heat and a miscible organic solvent was also enhanced by incorporation into the polymer.

Poly(acrylonitrile-co-vinyl chloride) (PAN/VC) anisotropic membranes were chemically modified with poly(ethylene oxide) (PEO) (5000 and 20,000 g/mol) by one of two aqueous reactions: (a) acid hydrolysis of the nitrile group to a carboxylic acid with which amine-terminated PEO (PEO-NH(2)) reacted or (b) base reduction of the nitrile group to an amine with which PEO-succinimide (PEO-SC) reacted. Approximately 1.3% of the bulk material was modified with PEO-NH(2) whereas 1.8 to 3.5% was modified with PEO-SC as determined by proton nuclear magnetic resonance ((1)H NMR) and attenuated total reflectance Fourier transform infrared (ATR FTIR) spectra. Approximately 50 to 75% less bovine serum albumin (BSA) adsorbed to PEO-grafted single skin fibers than to unmodified PAN/VC. Transport properties of modified and unmodified fibers were compared by passive diffusion, convective nominal molecular weight cutoff, and hydraulic permeability. Neither hydraulic permeability nor nominal molecular weight cutoff of BSA changed appreciably after surface modification with PEO indicating that pore structure was not adversely affected by the chemistry involved in grafting poly(ethylene oxide). However, in the absence of any membrane conditioning, the apparent diffusion of alpha-chymotrypsinogen (24,000 g/mol) was enhanced in PEO-grafted PAN/VC fibers possibly as a result of reduced sorption of the permeating protein. In vivo biocompatibility in the brain tissue of rats was judged by histological assessment of the host's cellular response to fibers implanted for 30 days; biocompatibility of both PAN/VC and PAN/VC-g-PEO was satisfactory but improved slightly with PEO grafting. (c) 1994 John Wiley & Sons, Inc.

Trifluoroacetolysis rates for 3-methyl-2-butyl tosylate (4) and knetic isotope effects at C(1) (k H /k D =1.083 per H atom), C(2) (k H /k D =1.10), and C(3) (k H /k D =1.82) were determined. The products are 2-methyl-2-butyl trifluoroacetate (5, 98-5%) and 3-methyl-2-butyl trifluoroacetate (6, 1.5%). GC-MS analysis of products from labeled tosylates 4-1-d 3 and 4-2-d showed that 42% of the apparently unrearranged 6 had a methyl group shifted from the original C(3) to the original C(2), whereas 3.6% methyl shift occurred in 5

Microparticle capillary electrophoresis was used to characterize the surface of quartz capillaries grafted with the glycidyl ether of poly(ethylene glycol) (E-PEG). Site dissociation modeling of capillary electrokinetic behavior provided estimates of surface group pK and density, plus the distance (d) from the surface to the hydrodynamic plane of shear. Native quartz appeared to possess silanol groups of pK 3.6 and 6.9 whose surface densities varied with quartz treatment. Aminopropylsilane derivatization of quartz silanol groups in toluene yielded a coating which was stable (6 h) at pH 10.3 and 60 °C. Aqueous grafting of E-PEG to this surface was relatively independent of pH (7.3-10.3) and reaction time (6-24 h) but was significantly influenced by reaction temperature (25-95 °C) and salt composition. PEG-grafted capillaries exhibited greatly reduced electroosmosis from pH 2 to 11. Significant grafting could be obtained under mild conditions (6 h, 35 °C, 0.4 M K(2)SO(4), pH 6.9). These results suggest that PEG chains increasingly extend normal to a surface as their grafting density increases, and that PEG conformation influences grafting density. The methods described should aid the use of PEG-coated surfaces in a variety of applications.

Polyoxazoline polymers with methyl (PMOZ), ethyl (PEOZ), and propyl (PPOZ) side chains were prepared by the living cationic polymerization method and purified by ion-exchange chromatography. The following properties of polyoxazoline (POZ) were measured: apparent hydrodynamic radius by aqueous size-exclusion chromatography, relative lipophilicity by reverse-phase chromatography, and viscosity by cone-plate viscometry. The PEOZ polymers of different molecular weights were first functionalized and then conjugated to model biomolecules such as bovine serum albumin, catalase, ribonuclease, uricase, and insulin. The conjugates of catalase, uricase, and ribonuclease were tested for in vitro activity using substrate-specific reaction methods. The conjugates of insulin were tested for glucose lowering activity by injection to naïve Sprague-Dawley rats. The conjugates of BSA were injected into New Zealand white rabbits and serum samples were collected periodically and tested for antibodies to BSA. The safety of POZ was also determined by acute and chronic dosing to rats. The results showed that linear polymers of POZ with molecular weights of 1 to 40 kDa can easily be made with polydispersity values below 1.10. Chromatography results showed that PMOZ and PEOZ have a hydrodynamic volume slightly lower than PEG; PEOZ is more lipophilic than PMOZ and PEG; and PEOZ is significantly less viscous than PEG especially at the higher molecular weights. When PEOZ was attached to the enzymes catalase, ribonuclease, and uricase, the in vitro activity of the resultant bioconjugates depended on the extent of protein modification. POZ conjugates of insulin lowered blood glucose levels for a period of 8 h when compared to 2 h for insulin alone. PEOZ, like PEG, was also able to successfully attenuate the immunogenic properties of BSA. The POZ polymers (10 and 20 kDa) are safe when administered intravenously to rats, and the maximum tolerated dose (MTD) was greater than 2 g/kg. Blood counts, serum chemistry, organ weights, and the histopathology of key organs were normal. These results conclude that POZ has the desired drug delivery properties for a new biopolymer.

We have achieved the synthesis and isolation, without substantiel label scrambling, of 2-(phenylthio)-ethyl-2,2-d 2 chloride (2-Cl), a compound useful in modeling reactions of mustard and other highly reactive β-thioethyl chlorides. 2-Cl undergoes slow label scrambling upon dissolution in hot, dry acetonitrile or acetone. Rates of scrambling of the deuterium label in 2-Cl have been measured by following proton NMR changes. Our present work clearly demonstrates that cyclic sulfonium ions are involved in these scrambling reactions. In anhydrous or aqueous acetone, thiourea traps the sulfonium ion intermediate to give stable, label-scrambled thiouronium salts. In anhydrous acetone, in competition with formation of the thiouronium salts, chloride return occurs giving 2-Cl and its label-scrambled isomer, 2-(phenylthio)ethyl-1,1-d 2 chloride (1-Cl). However, no chloride return is observed in 60% aqueous acetone. Mechanistic implications of these results are presented

There has been much recent interest in the use of poly(ethylene glycol)s (PEGs) for a variety of biotechnical applications. In the present work we have immobilized several cellulose derivatives and dextran on polystyrene surfaces and have measured the extent of fibrinogen adsorption onto the coated surfaces. Immobilization was achieved by adsorption onto clean polystyrene and by covalent linkage of oxidized polysaccharides to polyethylenimine which was ionically bound to polystyrene. Covalently bound polysaccharides, and adsorbed polysaccharides that are strongly held, compare well with poly(ethylene glycol) in preventing fibrinogen adsorption. The same polymers were coupled to polystyrene latex particles to permit examination by analytical microparticle electrophoresis. This investigation suggests that adsorbed polysaccharides form thicker layers than do covalently bound polysaccharides. Despite the polysaccharides being bound at many points along the polymer chain while PEG is bound only at the polymer terminus, the functional equivalence of polysaccharide and PEG coatings is of significance in interpreting the protein-rejecting ability of polymer-modified surfaces.

Analytical microparticle electrophoresis was used to characterize various polymer coatings known to control protein adsorption and related phenomena of biotechnical significance. The electrophoretic mobility of polystyrene latex microspheres and the electro-osmosis associated with quartz capillaries were characterized over the pH range 2–11. Such characterization provides information related to surface modification. Aminopropylsilane and mercaptopropylsilane were shown to be effective sublayers for covalent attachment of hydrophilic polymers to quartz glass surfaces. Poly(ethylenimine) was similarly verified as an effective sublayer for polystyrene latex. Polymer coatings based on poly(ethylene glycol) and three polysaccharides, dextran, ethyl(hydroxyethyl)cellulose, and hydroxypropylcellulose, were found to reduce capillary electro-osmosis and microsphere electrophoretic mobility significantly over a broad pH range. This reduction corresponds to the ability of these coatings to reduce protein adsorption and control surface wetting by aqueous polymer two-phase systems.

The effects of relative humidity on polymer sorption properties as revealed by changes in hydrogen-bonding acidity and E T (30) values are studied. Thin films of four polymers containing Reichardt's dye [2,6-diphenyl-4-(2,4,6-triphenyl-N-pyridinio)phenolate] were subjects to varying levels of relative humidity, and the UV-visible spectra of the polymer films were obtained. The effect of humidity on the solvatochromic parameters was found to vary greatly, dependending on the polymer, indicating that polymer sorption properties (such as solute solubility and membrane permeability) are a function of the humidity to which the polymer is exposed

The wetting behavior of phase-separated aqueous solutions of dextran and poly (ethylene glycol) on quartz substrates coated with covalently bound dextran fractions was examined as a function of coating molecular weight. The effect of dissolved polymer size was also investigated by using two-phase test systems containing high and low molecular weight dextrans. Sharply cut dextran fractions of Mw from 27,900 to 205,000 were attached to quartz activated with 3-aminopropyldimethylethoxysilane through reductive amination of the dextran's reducing end group in the presence of sodium cyanoborohydride. X-ray photoelectron spectroscopy and contact angle measurements in the two-phase polymer solutions were used to assess the quantity and quality of the surface coatings. Our results demonstrate that wetting by the dextran-rich phase improves, as evidenced by a declining contact angle, with increasing molecular weight of the bound dextran, decreasing molecular weight of the free dextran in solution and increasing time of exposure to the polymer phases. The usual relationship of increasing contact angle with increasing interfacial tension is not observed in these phase-separated polymer mixtures.

The solvatochromic method of Kamlet, Abraham, and Taft (KAT) has been used extensively for characterizing solvents, solutes, and solvent-solute interactions. Here we extend this method to characterize solid polymers as solvent phases by preparing thin films of the polymers with dissolved dyes and measuring the UV-visible absorption spectra of the films. The resulting solvatochromic measures of dipolarity/ polarizability, hydrogen-bond acidity and hydrogen-bond basicity show great potential for determining solubility and sorption properties of polymers with gaseous, liquid, and solid solutes, as well as with bulk liquid solvents. Additionally, the solvatochromic parameters can be used to predict optical properties of molecules dissolved in polymers (e.g., for nonlinear optical applications). This particular application is illustrated by predicting the absorption maximum of an important nonlinear optical dye dispersed in various polymers. The E~(30) solvatochromic scale of solvent polarity developed by Reichardt is also applied to the polymers, and the results are compared to those obtained by the KAT method.

Utilisation de la RMN pour la determination du degre de substitution dans un tosylate de PEG, de la quantite de PEG present dans des monomethoxy ethers de PEG commerciaux et de la masse moleculaire de PEG etalon a masse moleculaire faible

A new electrophilic polymer, the picryl ehter of methoxypolyethylene glycol (M-PEG-OPi), was prepared by reaction of the lithium alkoxide of methoxypolyethylene glycol with picryl chloride. The resultant M-PEG-OPi was characterized by 200 MHz 1H and 13C-NMR, microanalysis, and hydrolytic behavior. The rate constant for hydrolysis in pH 8.0 buffer is consistent with that expected for SNAr hydrolysis of a polyethylene glycol picrate. Reaction of n-propylamine with M-PEG-OPi in (CD3)2SO was followed by 1H-NMR and UV-visible spectroscopy. Evidence is presented for formation of a C-1 Meisenheimer complex; no C-3 adduct was observed in these experiments despite the steric bulk of the polymeric “group” on the picryl ring. Addition of a large excess of amine results in formation of a 2:1 dianionic complex, as shown by the UV-visible spectrum. The possibility is discussed of using the reaction of M-PEG-OPi with protein amino groups as an analytical tool for measuring the number of available amino groups on protein chains.

alpha-Chymotrypsin and lysozyme were solubilized in a water/O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl)methoxy]-O'-methoxy poly(ethylene glycol) (CK-2,13 surfactant)/isooctane water-in-oil microemulsion solution at 1.5-2 and 10 g l(-1) for 0.15 and 1.2 M: CK-2,13, respectively. Upon contact with an equal volume of 0.1 M: NaH(2)PO(4)/Na(2)HPO(4) buffer, pH 5, a three-phase system (Winsor-III system) was formed, consisting of a surfactant-rich middle phase and aqueous and isooctane-rich "excess" phases. Both enzymes were rapidly released into the aqueous excess phase, with 70% recovery of each in 30 and 60 min for microemulsion solutions containing 0.15 and 1.2 M: surfactant, respectively. The recovered enzymes retained90% of their original specific activity.

Protein and peptide drugs hold great promise as therapeutic agents. However, many are degraded by proteolytic enzymes, can be rapidly cleared by the kidneys, generate neutralizing antibodies and have a short circulating half-life. Pegylation, the process by which polyethylene glycol chains are attached to protein and peptide drugs, can overcome these and other shortcomings. By increasing the molecular mass of proteins and peptides and shielding them from proteolytic enzymes, pegylation improves pharmacokinetics. This article will review how PEGylation can result in drugs that are often more effective and safer, and which show improved patient convenience and compliance.

The chemical attachment of poly(ethylene glycol) [PEG] to therapeutic proteins produces several benefits, including enhanced plasma half-life, lower toxicity, and increased drug stability and solubility. In certain instances, pegylation of a protein can increase its therapeutic efficacy by reducing the ability of the immune system to detect and mount an attack on the compound. A PEG-protein conjugate is formed by first activating the PEG moiety so that it will react with, and couple to, the protein. PEG moieties vary considerably in molecular weight and conformation, with the early moieties (monofunctional PEGs; mPEGs) being linear with molecular weights of 12kD or less, and later moieties being of increased molecular weights. PEG2, a recent innovation in PEG technology, involves the coupling of a 30kD (or less) mPEG to lysine that is further reacted to form a branched structure that behaves like a linear mPEG of much larger molecular weight. These compounds are pH and temperature stable, and this factor along with the large molecular weight may account for the restricted volume of distribution seen with drugs utilising these reagents. Three PEG-protein conjugates are currently approved for clinical use in the US, with more under clinical development. Pegademase is used in the treatment of severe combined immunodeficiency disease, pegaspargase for the treatment of various leukaemias, and pegylated interferon-alpha for chronic hepatitis C virus infections. As illustrated in the case of the 2 pegylated interferon-alphas, all pegylated proteins are not equal. The choice of PEG reagent and coupling chemistry is critical to the properties of the PEG-protein conjugate, with the molecular weight of the moiety affecting its rate and route of clearance from the body, and coupling chemistry affecting the strength of the covalent attachment of PEG to therapeutic protein.

A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35degreesC at 5% phosphate, and increased linearly to 63degreesC at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 66: 211-218, 1999. (Less)

Covalent linkage of poly(ethylene glycol) (PEG) to drug molecules results in water-soluble conjugates with altered bioavail-ability, pharmacokinetics, immunogenic properties, and biological activities. For drugs bearing one or more amino groups, reductive amination is a potentially useful method for conjugation to PEG. PEG acetaldehyde has been used for this purpose, but its ease of polymerization under certain conditions and its susceptibility to air oxidation have caused some problems in its application. A simple and reliable method for preparation and use in reductive amination of PEG acetaldehyde hydrate generated in situ by hydrolysis of PEG acetaldehyde diethylacetal is demonstrated. PEG acetaldehyde diethylacetal is prepared in high yield and purity by reaction of PEG with chlorodiethylacetal in dioxane in the presence of finely powdered sodium hydroxide under heterogeneous conditions. PEG acetaldehyde hydrate is generated in solution by hydrolysis in aqueous acids. Solutions of the hydrate may be used directly, in conjunction with sodium cyanoborohydride, to effect reductive amination. We demonstrate application of these methods in PEGylation of lysozyme and chitosan to form water-soluble methoxy poly(ethylene glycol) (mPEG) derivatives and PEG-chitosan hydrogels.

Hydrogels have become increasingly important in the biomedical field. This paper describes synthesis and characterization of two types of novel degradable poly(ethylene glycol) (PEG) hydrogels with potential utility as delivery carriers for bioactive drugs. The simplest gel is prepared by one-step polycondensation of difunctional PEG acid and branched PEG “polyol”. The second type of the novel degradable PEG hydrogel was prepared in a two-step process, in which an ester-containing, amine-reactive PEG derivative was synthesized and then reacted with a branched PEG amine to form the gel. The two-step gels are formed in very mild conditions, and therefore fragile drugs such as proteins can be loaded during gel formation. Because most proteins have free amino groups in the sequence, these proteins can be covalently linked to the hydrogel network. This covalent attachment provides a new way to achieve long-term controlled release of proteins. These hydrogels have a wide range of degradation rates. Upon hydrolysis, these PEG hydrogels will degrade into low molecular weight PEG derivatives, which can be easily cleared by the body.

This volume provides an interdisciplinary analysis of current biological applications of poly(ethylene glycol) (PEG). It includes a wide array of topics useful to materials scientists, organic chemists, biochemists, and bioengineers interested in drug delivery systems, pharmaceuticals and other biomaterials. The applications discussed include PEG-modified proteins, liposomes, drugs, surfaces of materials, and hydrogels. The volume also includes a review of PEG-oligonucleotides and a concise summary of the toxicology of PEG and its derivatives.

There is a group of polymers which phase separate in water solution when the temperature is increased. These polymers have a lower critical solution temperature (LCST), which is also called the cloud point of the system. Above the critical temperature the polymers are not soluble in water. Examples of thermo-separating polymers are ethylene oxide (EO)/propylene oxide (PO) random copolymers and hydrophobically modified cellulose derivatives.1,2 A water phase and a liquid, concentrated polymer phase are formed at temperatures above the cloud point of the EO/PO copolymer. Figure 1 shows the cloud point diagram for the EO/PO random copolymer Ucon 50-HB-5100. Factors determining the cloud point are EO/PO ratio, molecular weight and salt concentration. Non-ionic surfactants, such as Triton X-114, also have a LCST in water, and this property has been used for isolation of membrane proteins.3

The chapters of this book describe the synthesis and use of a variety of active PEG derivatives designed to couple PEG to other materials. Despite the availability of these derivatives, there remains a need for new derivatives with presently unavailable properties and work continues in this area. Desirable properties include selectivity, stability, and ease of preparation. For example, it would be desirable to have derivatives that react with nucleophilic groups on proteins, but which do not react with water. Derivatives of a wide range of reactivities are always in demand. Similarly, there would be advantages to having derivatives that react with groups other than the commonly used amino groups. And, of course, the need for derivatives that can be prepared cheaply and easily in large quantity is critical for commercialization of the many biomedical and biotechnical applications of PEG chemistry.

At first glance, the polymer known as poly(ethylene glycol) or PEG appears to be a simple molecule. It is a linear or branched, neutral polyether, available in a variety of MWs, and soluble in water and most organic solvents. Despite its apparent simplicity, $${HO - {{\left( {C{H_2}C{H_2}O} \right)}_n} - C{H_2}C{H_2}OH} \hfill \\ {poly(ethylene\,glycol)}$$ this molecule is the focus of much interest in the biotechnical and biomedical communities. Primarily this is because PEG is unusually effective at excluding other polymers from its presence when in an aqueous environment. This property translates into protein rejection, formation of two-phase systems with other polymers, nonimmunogenicity, and nonantigenicity. In addition, the polymer is nontoxic and does not harm active proteins or cells although it interacts with cell membranes. It is subject to ready chemical modification and attachment to other molecules and surfaces, and when attached to other molecules it has little effect on their chemistry but controls their solubility and increases their size. These properties, which are described in more detail below, have led to a variety of important biotechnical and biomedical applications, a summary of which is also presented below.