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An in situthermogelling, mucoadhesive formulation based on N-trimethyl chitosan chloride has been evaluated for its potential to affect the transmucosal delivery of insulin viathe nasal route. In vitrostudies at a physiologically relevant temperature (ca. 35 °C) have shown that the formulation releases most of its insulin load (ca. 70%) in a non-Fickian manner during the timescale over which the sol-to-gel transition (ca. 8 min) takes place, and also that, once gelation is complete, the release of the remainder of the therapeutic content follows first order kinetics over at least sixty minutes. Investigations on the effects of the application of the same formulation to a modelled nasal mucosa (Calu-3 cell monolayer) have indicated the capability of the formulation to induce the transient opening of tight junctions. Cytotoxic investigations have shown that the formulation exhibits negligible detrimental effects to the integrity of these monolayers. The in vivopotential of the nasal formulation to act as a once-a-day dosage form for the intranasal delivery of insulin has been demonstrated in a diabetic-rat model.

AbstractWe prove reality of the spectrum for a class of PT− symmetric, non self-adjoint quantum nonlinear oscillators of the form H=p2+ P(q) + igQ(q). Here P(q) is an even polynomial of degree 2ppositive at infinity, Q(q) an odd polynomial of degree 2r− 1, and the conditions p> 2r, |g| < for some > 0 hold.

The tumor targeting properties of a new drug carrier synthesized by bioconjugation of folic acid (FA) to β-cyclodextrins through a poly(ethylene glycol) (PEG) spacer (CD−PEG−FA) were investigated. Surface plasmon resonance demonstrated that CD−PEG−FA specifically interacts with immobilized folate binding protein (FBP) while the naked β-cyclodextrins do not display any specific interaction. In vitro studies demonstrated that CD−PEG−FA was devoid of cell toxicity. [³H]-folic acid/CD−PEG−FA competition binding investigations performed with folate receptor overexpressing human epidermal carcinoma KB cells showed that CD−PEG−FA had about 14 times lower tumor cell binding capacity than free folic acid. The carrier cell trafficking properties were investigated using rhodamine-B as fluorescent probe, which possesses 3000 and 4580 M^-1 inclusion constants for CD−PEG−FA and β-cyclodextrins, respectively. Cell-associated fluorescence measurements showed that CD−PEG−FA does not promote the rhodamine-B uptake into non-folate receptor expressing human lung carcinoma MCF7 cells while 19% higher accumulation in KB cells was found with respect to rhodamine-B loaded β-cyclodextrins. Confocal laser scanning microscopy indicated the presence of cytosolic red fluorescent spots after 2 h of incubation of KB cells with rhodamine-B included CD−PEG−FA. The fluorescent dye resided primarily in small spots, namely, endosomes and multivesicular bodies. At 1 h after pulsed incubation, wider red fluorescent cellular structures appeared as a fusion of previous structures.

Available pegylated interferon (PEG-IFN) products confer enhanced therapeutic efficacy when compared with their IFN counterparts, and the added convenience of once-weekly dosing with no novel toxicities. PEG optimises the action of the IFNs by decreasing clearance rates, thereby allowing serum concentrations to remain constant over the dosing period. The length and shape of each PEG moiety are crucial in determining the effect on pharmacokinetic and pharmacodynamic properties. In vitro activity decreases with increasing PEG size, therefore the reduction of potency at the molecular receptor should be compensated by the prolonged residence in blood, or by the higher total drug exposure. Moreover, in hepatitis C, viral suppression in blood alone may not be enough, HCV reservoirs outside the blood may play a role in HCV persistence, therefore drugs with a low volume of distribution offer much less chance for infiltrating extravascular tissue and may explain its higher relapse rate. The case of PEG-IFNs for use in the treatment of chronic hepatitis C clearly demonstrates the potential for different characteristics among PEG conjugates.

A thermodynamic analysis is developed to interpret the influence of temperature and pressure in the production of solid lipid nano- and microparticles by a high-pressure technique named particle from gas-saturated solution (PGSS). The pressure−temperature charts show three regions above the P−T solid−liquid−fluid coexistence curve, from which sub-cooled solid, solid−liquid, or liquid products can be obtained. The relation between the initial and final thermodynamic properties of the PGSS process were calculated by solving simultaneously the energy balance and a proper equation of state. The expansion of high-pressure CO2−lipid saturated solution through the micrometric nozzle was represented by a transformation at constant total enthalpy. To represent the equilibrium and thermodynamic behavior of CO2−lipid systems the perturbed-hard-sphere-chain theory (PHSCT) was used. As examples, CO2 absorption isotherms in lipids and residual properties of high-pressure tristearin−CO2 and tristearin−phosphatidylcoline−CO2 systems were calculated. Enthalpy of fusion of pure substances and formation enthalpy of microparticulate PGSS products were measured by differential scanning calorimetry (DSC). Pure lipid EOS parameters were estimated by a group contribution method, whereas the equation of state (EOS) interaction parameters were adjusted on the experimental melting point data under CO2 pressure, measured in a high-pressure windowed cell. Application of operative charts provides useful information in understanding the influence of temperature and pressure on the final properties of lipid particles produced by PGSS.

β-Cyclodextrin−poly(ethylene glycol)−folic acid conjugate (CD-PEG-FA) was synthesized according to a two-step procedure:  (1) synthesis of CD-PEG-NH2 by reaction of monotosyl-activated β-cyclodextrin with excess of 700 Da diamino-PEG; (2) synthesis of CD-PEG-FA by reaction of CD-PEG-NH2 with succinimidyl ester-activated folic acid. The CD-PEG-NH2 intermediate was purified by precipitation in acetone, and the CD-PEG-FA by gel permeation and C-18 reversed-phase chromatography. Both CD-PEG-NH2 and CD-PEG-FA were analyzed by mass spectrometry, ¹H NMR, and UV−vis spectroscopy. All analytical methods confirmed the theoretical composition of the conjugates:  the CD-PEG-NH2 intermediate was composed of CD and PEG in the molar ratio of 1:1, and the CD-PEG-FA was composed of β-cyclodextrin, PEG, and folic acid in the molar ratio of 1:1:1. The CD-PEG-FA conjugate was highly soluble in buffer (>42 mM) as compared to the unmodified β-cyclodextrin (16.3 mM). Phase solubility diagrams of β-estradiol revealed that drug solubility increases from 11 μM in buffer to 600 μM in the presence of β-cyclodextrins and 5900 μM with CD-PEG-FA. However, the affinity of β-estradiol for β-cyclodextrins decreased about 4 times with PEG and folic acid conjugation. Stability studies carried out using chlorambucil confirmed that the conjugate partially prevents drug degradation in buffer, although this effect was considerably lower than that obtained with β-cyclodextrin. Computer modeling studies showed that the folic acid linked to the β-cyclodextrins through a PEG spacer could partially interact with the cyclodextrin cavity. Finally, CD-PEG-FA displayed reduced hemolytic effect as compared to unmodified β-cyclodextrin.

Oral cavity reconstruction after removal of locally advanced tumors is particularly difficult because anatomical restoration must accurately reproduce the original structure and enable effective and fast rehabilitation of mastication, swallowing, and phonation. The authors report their 2year experience with 17 patients surgically treated for oral cavity cancer with reconstruction performed with the free anterolateral thigh flap. Thanks to its thinness and pliability, this flap has proven to be perfectly adaptable to the structural peculiarities of the resected areas and has enabled the authors to considerably reduce the cosmetic and functional complications in the donor area observed with other flaps such as the radial forearm flap. Flap grafting has always been complete and regular, and no intraoperative and postoperative complications have been observed. Swallowing recovery has always been satisfactory. On the basis of the authors' results, their current approach to oral cavity reconstruction is based on the use of flaps that enable anatomical restoration of the resected areas and reduce morbidity of the donor site. They believe that the anterolateral thigh flap can offer all of these opportunities, and the surgery can be simultaneously performed by two surgical teams.

Insulin and insulin/poly(ethylene glycol) (PEG)-loaded poly(l-lactide) (PLA) nanoparticles were produced by gas antisolvent (GAS) CO2 precipitation starting from homogeneous polymer/protein organic solvent solutions. Different amounts of PEG 6000 (0, 10, 30, 50, 100, and 200% PEG/PLA w/w) or concentration of 30% PEG/PLA with PEGs with different molecular weight (MW; 350, 750, 1900, 6000, 10,000, and 20,000) were used in the preparations. The process resulted in high product yield, extensive organic solvent elimination, and maintenance of > 80% of the insulin hypoglycemic activity. Nanospheres with smooth surface and compact internal structure were observed by scanning electron microscopy. The nanospheres presented a mean particle diameter in the range 400–600 nm and narrow distribution profiles. More than 90% of drug and PEG were trapped in the PLA nanoparticles when low MW PEGs were used in the formulation, whereas the addition of high MW PEGs significantly reduced the loading yield. In all cases, in vitro release studies showed that only a little amount of drug was released from the preparations. However, formulations containing low MW PEGs allowed for a slow but constant drug release throughout 1500 h, whereas a burst was obtained by increasing the PEG MW. In conclusion, the GAS process offers a mean to produce protein-loaded nanoparticles possessing the prerequisites for pharmaceutical applications. The PEG added to the formulation was found to play a key role in the simultaneous solute precipitation phenomena and in determining the release behavior and the chemical–physical properties of the formulation. © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1628–1636, 2001

For a comparative study of immunological properties of protein−polymer conjugates, uricase was modified with (a) poly(N-vinylpyrrolidone) 6000 Da, (b) poly(N-acriloylmorpholine) 6000 Da, (c) branched monomethoxypoly(ethylene glycol) 10000 Da, and (d) linear monomethoxypoly(ethylene glycol) 5000 Da. Spectroscopic studies performed by UV, fluorescence, and circular dichroism did not show any relevant difference in protein conformation among the native and the conjugates. Immunological studies showed that both uricase antigenicity and immunogenicity were altered by polymer conjugation to an extent that depended upon the polymer composition; in particular, monomethoxypoly(ethylene glycol) 10000 Da remarkably reduced the protein antigenicity, while unexpectedly, the poly(N-vinylpyrrolidone) derivative presented higher antigenicity than the native protein. In Balb/c mice, the native protein elicited a rapid and intense immunoresponse whereas all the conjugates induced a lower production of anti-native uricase antibodies. The rank order of immunogenicity was native uricase > uricase−poly(N-vinylpyrrolidone) ≥ uricase−poly(N-acriloylmorpholine) > uricase−monomethoxypoly(ethylene glycol) 5000 Da > uricase−monomethoxypoly(ethylene glycol) 10000 Da. The four conjugates also induced anti polymer immunoresponse. Anti poly(N-vinylpyrrolidone) and anti poly(N-acriloylmorpholine) antibodies were generated from the first immunization while low levels of anti polymer antibodies were found with both poly(ethylene glycol) conjugates only after the second immunization.

In view of developing new drug-delivery systems, microparticles of biocompatible polymer loaded with protein were produced using a supercritical antisolvent technique. As the polymer, poly(lactic acid) (PLA) of about 100 000 Da was used, whereas insulin was chosen as the protein model. To achieve microencapsulation, we started from a homogeneous solution of protein and polymer, to which the supercritical antisolvent is added; mixtures of dichloromethane and dimethyl sulfoxide were used to ensure the solubility of both the polymer and the protein. All experiments were performed in semicontinuous mode. A preliminary study with PLA alone was done in order to select the best operative condition and to control the particle dimension and their diameter distribution range, especially with mixtures of the two solvents. Then, the microspheres of PLA charged with insulin were produced; the average diameters ranged from 0.5 to 2 μm. The composition of these particles was determined analytically, and the protein bioactivity was checked.

A comparative pharmacokinetic and biodistribution investigation of polymer−protein conjugates prepared with various amphiphilic polymers was carried out using uricase as a model. Four polymer-uricase derivatives have been obtained by covalent binding of a similar number of polymer chains of (a) linear poly(ethylene glycol) (Mw 5000 Da); (b) branched poly(ethylene glycol) (Mw 10 000 Da); (c) poly(N-vinylpyrrolidone) (Mw 6000 Da); (d) poly(N-acryloilmorpholine) (Mw 6000 Da). By intravenous administration to Balb/c mice, the conjugates displayed different pharmacokinetic and organ distribution behaviors. (1) The unmodified enzyme and the poly(N-vinylpyrrolidone) conjugate were the enzyme forms with the shortest and the longest permanence in blood respectively (mean residence time 45 and 4378 min). (2) Native uricase was found to localize soon after administration significantly in heart, lungs, and liver from where it was also rapidly cleared. (3) The poly(N-acryloilmorpholine) derivative showed the highest concentration levels in liver (up to 25.5% of the dose) and considerable accumulation took also place in the other considered organs. (4) Poly(N-vinylpyrrolidone)-uricase displayed a relevant tropism for liver but low uptake indexes were found for the other organs. (5) The branched poly(ethylene glycol) derivative accumulated preferentially in liver and spleen. (6) The linear poly(ethylene glycol) conjugate was, among the various uricase forms, the species with the lowest distribution levels in all the examined organs. (7) Finally, all the enzyme forms slowly disposed in kidneys with higher levels for the poly(N-acryloilmorpholine) derivative (15% after 2880 min) and unmodified uricase (14% after 1440 min).

A biocompatible and biodegradable polyphosphazene bearing phenylalanine ethyl ester, imidazole and chlorine (10.7:1:2.5 molar ratio) as substituents of the phosphorus atoms of the polymer backbone was studied for the preparation of polymeric naproxen slow-release systems.Discs 2.5 cm in diameter and 0.5 mm (thin) or 0.65 mm (thick), loaded, respectively, with 20 and 13.5% naproxen, showed different drug release kinetics, the thin matrices releasing naproxen at a faster rate and for a shorter time. In-vivo studies in rats demonstrated the pharmacological efficacy of these two different delivery systems in the inhibition of acute or chronic inflammatory diseases. Subcutaneous implantation of the thin matrices in rats was found to reduce carrageenan oedema induced both 1 h and 7 days after implantation. Rats implanted with thick matrices showed a reduction in chronic inflammation caused by adjuvant arthritis. Approximately 78% inhibition of arthritic oedema was found 28 days after subcutaneous administration of the matrices whereas 28.7% inhibition was found after daily oral administration of naproxen. Blood levels of naproxen in arthritic rats after matrix implantation showed the presence of drug up to day 28.These positive results have encouraged us to study a controlled-release system suitable for use in man.

The changes of pharmacokinetics and distribution of ³H radio-labelled ribonuclease (RNase) and RNase adduct with monomethoxypoly(ethylene glycol) (MPEG) were studied after intravenous administration in the dose of 25 mg kg^-1 to rats. Whereas the value of total plasma clearance of RNase is close to the value of glomerular filtration rate, the value of that for MPEG-RNase is about three hundred times lower. The half-life of elimination is 79 min, 181 min and 65 h for RNase, MPEG and MPEG-RNase, respectively. The main elimination pathway of compounds under study was elimination into urine and no evident specific distribution in the examined organs (liver, kidney, muscle, lung, brain, spleen and heart) was found. The study indicates that conjugation of RNase with MPEG can improve its pharmacokinetic properties. Copyright 1993, 1999 Academic Press

We have performed a comparative study of the biodistribution in mice of native and monomethoxypolyethylene glycol-modified superoxide dismutase isolated from bovine liver after intravenous injection. Polymer modification greatly influenced the biodistribution of enzyme preparation. Monomethoxypolyethylene glycol-modified SOD (mPEG-SOD) exhibited a longer residence time and a considerably longer half-time in the blood, lungs, and heart than the native enzyme. Using a rat model of ischemia, we demonstrated that an intravenous bolus administration of mPEG-SOD reduced the size of the myocardium necrosis zone by 40% compared with a 13% reduction by native enzyme. These results suggest that mPEG-SOD is a promising agent for decreasing reperfusion injury to the cardiovascular system.

Carboxy group‐terminated synthetic polymers—branched poly‐(ethylene glycol), poly(acryloylmorpholine), and poly(vinylpyrrolidone)—were made amphiphilic by derivatization with phosphatidyl ethanolamine via the terminal carboxy group and then incorporated into lecithincholesterol liposomes prepared by the detergent dialysis method. Following the biodistribution of liposomes in mice, all three polymers were shown to be effective steric protectors for liposomes and were able to sharply increase liposome circulation times in a concentration‐dependent manner. The accumulation of liposomes in the liver decreases. The effects observed are similar to those found for liposomes modified with linear poly(ethylene glycol). At low polymer concentration, amphiphilic branched poly(ethylene glycol) seems to be the most effective liposome protector, most probably, because at the same molar content of anchoring groups, each attachment point carries two polymeric chains and doubles the quantity of liposome‐grafted polymer comparing to linear poly(ethylene glycol).

Linear and branched poly(ethylene glycol)s, with similar molecular weights, were conjugated with uricase and asparaginase, and an investigation of enzymological, immunological, and pharmacokinetic properties of the conjugates was carried out. It was found that the steric hindrance of the branched polymer has a relevant role in determining the biological properties of the conjugates. Conjugations with branched polymers inactivate the enzyme less than the linear ones. Compared to the native and the linear polymer conjugate counterparts the branched polymer derivatives: (1) are more stable to proteolysis by elastase, pronase, and trypsin, (2) stay longer in the blood with increased systemic availability after intravenous administration in mice, and (3) give rise to lower levels of antinative enzyme antibodies after immunization. These data are consistent with a greater surface area of protein covered by the branched PEG.

Monofunctional hydroxy-terminated poly(N-vinylpyrrolidone) was obtained by radical polymerization in the presence of isopropoxyethanol for protein surface modification. Superoxide dismutase was modified to various degrees by reaction with PVP-OH activated by 4-nitrophenyl chloroformate with high retention of enzymatic activity. All the polymer-enzyme conjugates showed increased body residence time after intravenous injection in rats with respect to the native enzyme. The residence time depended upon the number of bound polymer chains. PVP was found to affect the pharmacokinetic properties of the enzyme after subcutaneous, intramuscular and intraperitoneal administration. The antigenicity of superoxide dismutase was reduced to one third following modification, while its immunogenicity after intravenous, subcutaneous and intraperitoneal injection totally disappeared. The conjugates exhibited increased solubility in organic solvents and increased stability towards thermal denaturation, whereas changes in the structural properties were observed by circular dichroism measurements.

A method is described to modify urokinase by covalent binding of monomethoxypoly(ethylene glycol) (mPEG) without impairing its catalytic ac tivity towards high molecular weight substrates. The urokinase active site is protected by an inhibitor, benzamidine, bound to Sepharose during the mPEG modification in order to avoid binding mPEG chains to the active site or to the surrounding area. The mPEG modified urokinase had increased activity towards small molecular weight substrates (acetyl-Gly-methyl ester) as com pared to the unmodified enzyme, while the activity towards the high molecular weight plasminogen and the insoluble substrate fibrin clot was preserved. This did not occur when the enzyme was modified in the absence of active site pro tection. The polymer modification increased the enzyme's thermostability and the stability in plasma in vitroand prolonged in vivoretention after in travenous injection in rats.

A method to prevent the loss of enzymatic activity of proteolytic enzymes toward high molecular weight substrates that occurs upon derivatiza tion with monomethoxypoly(ethylene glycol) (mPEG) is described. It is based on the heterogenous phase enzyme modification after the enzyme is bound to an active site inhibitor immobilized on an insoluble resin. This procedure pro tects the active site and the surrounding area from mPEG linkage. Trypsin modified by mPEG in a heterogeneous phase, using benzamidine bound to Sepharose maintained a high degree of its ability to hydrolize large molecular weight substrates, such as bovine serum albumin or casein, compared to the mPEG derivatives obtained without any protection or with free benzamidine in solution.

Polyvinylpyrrolidone (PVP) oligomers with -OH terminal groups, obtained by radical polymerization using 2-mercaptoethanol as chain- transfer agent, were fractionated by gel chromatography to obtain a product with an approximate molecular weight of 1100. The hydroxyl function of such oligomer was activated with 4-nitrophenyl chloroformate to give an active car bonate derivative suitable for linking to peptide or protein amino groups in aqueous media buffered at mild alkaline pH. The linking of the PVP to the pro tein surface was accomplished without loss of enzymatic activity in the model enzyme ribonuclease. When the polymerization of PVP is carried out using 2- mercaptoacetic acid as chain-transfer agent, a much more heterogeneous prod uct was obtained. Also this, activated as succinimidyl ester, could also be bound to amine groups; however, this treatment performed on the model enzyme RNase caused inactivation.

We complete and correct some proofs of an earlier paper on distributional Borel summability and we add an application which can be useful in the discussion of semiclassical problems.

We prove that the Stark effect perturbation theory of a class of bound states uniquely determines the position and the width of the resonances by Distributional Borel Sum. In particular the small field asymptotics of the width is uniquely related to the large order asymptotics of the perturbation coefficients. Similar results apply to all the “resonances” of the anharmonic and double well oscillators.

Following 't Hooft we extend the Borel sum and the Watson-Nevanlinna criterion by allowing distributional transforms. This enables us to prove that the characteristic function of the measure of anyg-2f4 finite lattice field is the sum of a power series expansion obtained by fixing exponentially small terms in the coefficients. The same result is obtained for the trace of the double well semigroup approximated by thenth order Trotter formula.