One-Step Precise Characterization of Drug Delivery Systems by PULCON Magnetic Resonance Spectroscopy.

Polymers are extensively used for the realization of drug delivery systems across multiple scales, from nanomedicines to microparticles and macroscopic implantable devices, for their favorable biodegradation profiles and tunable physicochemical features. The accurate quantification of the polymer content is key to finely controlling drug loading and release and ensuring reproducibility, yet it continues to be a major challenge in the design and development of delivery systems. In this study, we introduce a novel protocol based on the PULCON technique to quantify, with a routine NMR spectroscopy analysis, the precise concentration of polymers in various delivery systems. Specifically, the PULCON protocol is applied to characterize the physicochemical and pharmaceutical properties of nanoparticles, microparticles, and implantable devices realized by combining three extensively used polymers, namely, poly(lactic-co-glycolic acid) (PLGA), poly(vinyl alcohol) (PVA), and poly(ethylene glycol) (PEG). Without using internal calibration procedures, in a single step, the PULCON protocol precisely quantifies the concentration of each polymer and the drug content. This approach can be readily implemented on standard NMR spectrometers, enabling accurate characterization of drug delivery systems and facilitating their effective development.

Control of targeting ligand display by pH-responsive polymers on gold nanoparticles mediates selective entry into cancer cells.

Selective targeting of cells for intracellular delivery of therapeutics represents a major challenge for pharmaceutical intervention in disease. Here we show pH-triggered receptor-mediated endocytosis of nanoparticles via surface ligand exposure. Gold nanoparticles were decorated with two polymers: a 2 kDa PEG with a terminal folate targeting ligand, and a di-block copolymer including a pH-responsive and a hydrophilic block. At the normal serum pH of 7.4, the pH-responsive block (apparent pKa of 7.1) displayed a hydrophilic extended conformation, shielding the PEG-folate ligands, which inhibited cellular uptake of the nanoparticles. Under pH conditions resembling those of the extracellular matrix around solid tumours (pH 6.5), protonation of the pH-responsive polymer triggered a coil-to-globule polymer chain contraction, exposing folate residues on the PEG chains. In line with this, endocytosis of folate-decorated polymer-coated gold nanoparticles in cancer cells overexpressing folate receptor was significantly increased at pH 6.5, compared with pH 7.4. Thus, the tumour acidic environment and high folate receptor expression were effectively exploited to activate cell binding and endocytosis of these nanoparticles. These data provide proof-of-concept for strategies enabling extracellular pH stimuli to selectively enhance cellular uptake of drug delivery vectors and their associated therapeutic cargo.

Sub-unit vaccine against S. aureus-mediated infections: set-up of nano-sized polymeric adjuvant.

The aim of this work was the design of a novel adjuvanted system for vaccination against S. aureus-mediated infections: in particular, poly-lactide-co-glycolide (PLGA) nanoparticles were developed in order to efficiently load and boost a sub-unit model vaccine, namely a purified recombinant collagen binding bacterial adhesin fragment (CNA19). At first, the assessment of the actual immunogenicity of free CNA19 via subcutaneous administration was evaluated, in order to consider it as subunit antigen model. Secondly, for the development of CNA19 loaded PLGA nanoparticles, a preliminary study was focused on the production of well-formed nanoparticles by w/o/w double emulsion method exploiting ultrasonication cycles under mild conditions, then the optimization of the freeze-drying conditions and different CNA19 loading methods were considered (encapsulation, adsorption of on blank or CNA19 encapsulated nanoparticles). The set-up preparation method (process yield of about 83%) permitted to obtain CNA19 loaded nanoparticles with spherical shape, narrow size distribution (187.41 ± 51.2 nm), a slightly negative zeta-potential (-2.91 ± 0.64 mV) and to elicit satisfactory protein encapsulation efficiency (75.91 ± 4.22%) and loading capacity (8.59 ± 0.33 µg CNA19/nanoparticles mg). Then, CNA19 loaded PLGA nanoparticles were characterized by (i) an in vitro release test performed at different temperatures, namely 4°C, 25°C and 37°C, testing the antigen integrity (SDS-PAGE) and activity (ELISA); (ii) an in vitro stability study in terms of dimension and surface charge performed in a 21 days period of time. At 37°C there was evidence of a sustained release of the antigen, in active form, for almost 240 h with a burst release of about 20% in the first 2h. At 4°C stability tests and activity assays allowed to identify storage conditions useful to maintain CNA19 activity and easily NP re-suspendability with intact physical characteristics. Furthermore the evaluation of CNA19 loaded nanoparticles cytotoxicity (up to 10.652 mg PLGA/ml) by MTT assay and the study of cellular up-take assessed on human fibroblasts confirmed the feasibility to formulate a dosage form useful for vaccination against S. aureus-mediated infections.

A once-a-day dosage form for the delivery of insulin through the nasal route: in vitro assessment and in vivo evaluation.

An in situ thermogelling, mucoadhesive formulation based on N-trimethyl chitosan chloride has been evaluated for its potential to affect the transmucosal delivery of insulin via the nasal route. In vitro studies 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 vivo potential 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.

Synthesis, physico-chemical and biological characterization of a paclitaxel macromolecular prodrug.

Paclitaxel was attached to poly(hydroxyethylaspartamide) via a succinic spacer arm by a two-step protocol: (1) synthesis of 2'-O-succinyl-paclitaxel; (2) synthesis of PHEA-2'-O-succinyl-paclitaxel. The 2'-O-succinyl-paclitaxel derivative and the macromolecular conjugate were characterized by UV, IR, NMR and mass spectrometry analysis. The reaction yields were over 95% and the purity of products over 98%. Paclitaxel release and degradation from 2'-O-succinyl-paclitaxel occurred at a faster rate at pH 5.5 than 7.4. After 30 h of incubation at pH 5.5 and 7.4 the released free paclitaxel was about 40 and 20%, respectively. In plasma both drug release and degradation were found to occur at a higher rate than in buffer at pH 7.4 suggesting that an enzymatic mechanism could be involved. The paclitaxel release and degradation from PHEA-2'-O-succinyl-paclitaxel were negligible at pH 5.5 and 7.4 and very slow in plasma. Investigation carried out using murine myeloid cell line showed that the polymeric prodrug maintains partial pharmacological activity of paclitaxel. The DL50 of the conjugate (over 40 ng/ml) as compared to free paclitaxel (about 1 ng/ml) was correlated to the slow drug release. Finally a pharmacokinetic study carried out by intravenous inoculation of the macromolecular prodrug to mice demonstrated that the polymer conjugation modify dramatically the in vivo fate of the drug. The conjugate disappeared from the bloodstream much more quickly as compared to both free drug and naked polymer. Massive accumulation of bioconjugate in the liver (80% of the dose) was found to persist throughout 1 week.

PEGylation of growth hormone-releasing hormone (GRF) analogues.

Synthetically produced GRF1-29 (Sermorelin) has an amino acid composition identical to the N-terminal 29 amino acids sequence of the natural hypothalamic GHRH1-44 (Figure 1). It maintains bioactivity in vitro and is almost equally effective in eliciting secretion of endogenous growth hormone in vivo. The main drawbacks associated with the pharmaceutical use of hGRF1-29 relate to its short half-life in plasma, about 10-20 min in humans, which is caused mostly by renal ultrafiltration and enzymatic degradation at the N terminus. PEGylation has been considered as one valid approach to obtain more stable forms of the peptide, with a longer in vivo half-life and ultimately with increased pharmacodynamic response along the somatotropic axis (endogenous GH, IGF-1 levels). Different PEGylated GRF conjugates were obtained and their bioactivity was tested in vitro and in vivo by monitoring endogenous growth hormone (GH) serum levels after intravenous (i.v.) injection in rats, and intravenous and subcutaneous (s.c.) injection in pigs. It was found that GRF-PEG conjugates are able to bind and activate the human GRF receptor, although with different potency. The effect of PEG molecular weight, number of PEG chains bound and position of PEGylation site on GRF activity were investigated. Mono-PEGylated isomers with a PEG5000 polymer chain linked to Lys 12 or Lys 21 residues, showed high biological activity in vitro, which is similar to that of hGRF1-29, and a higher pharmacodynamic response as compared to unmodified GRF molecule.

Pretargeted adjuvant radioimmunotherapy with yttrium-90-biotin in malignant glioma patients: a pilot study.

In a previous study we applied a three-step avidin-biotin pretargeting approach to target 90Y-biotin to the tumour in patients with recurrent high grade glioma. The encouraging results obtained in this phase I-II study prompted us to apply the same approach in an adjuvant setting, to evaluate (i) time to relapse and (ii) overall survival. We enrolled 37 high grade glioma patients, 17 with grade III glioma and 20 with glioblastoma, in a controlled open non-randomized study. All patients received surgery and radiotherapy and were disease-free by neuroradiological examinations. Nineteen patients (treated) received adjuvant treatment with radioimmunotherapy. In the treated glioblastoma patients, median disease-free interval was 28 months (range=9-59); median survival was 33.5 months and one patient is still without evidence of disease. All 12 control glioblastoma patients died after a median survival from diagnosis of 8 months. In the treated grade III glioma patients median disease-free interval was 56 months (range=15-60) and survival cannot be calculated as only two, within this group, died. Three-step radioimmunotherapy promises to have an important role as adjuvant treatment in high grade gliomas, particularly in glioblastoma where it impedes progression, prolonging time to relapse and overall survival. A further randomized trial is justified.

Production of insulin-loaded poly(ethylene glycol)/poly(l-lactide) (PEG/PLA) nanoparticles by gas antisolvent techniques.

Insulin and insulin/poly(ethylene glycol) (PEG)-loaded poly(l-lactide) (PLA) nanoparticles were produced by gas antisolvent (GAS) CO(2) 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.

Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers.

Four new poly(hydroxyethylaspartamide)-based copolymers bearing (a) poly(ethylene glycol) 2000, (b) poly(ethylene glycol) 5000, (c) poly(ethylene glycol) 2000 and hexadecylalkyl, (d) poly(ethylene glycol) 5000 and hexadecylalkyle, as pendant groups were synthesised. The copolymers were obtained by partial aminolysis of polysuccinimide with poly(ethylene glycol) and hexadecylalkyl amino derivatives followed by reaction with ethanolamine. Naked polyhydroxyaspartamide was obtained by polysuccinimide reaction with ethanolamine. The nuclear magnetic resonance, infrared, light scattering and elemental analysis allowed for the extensive physico-chemical characterisation of the carriers. The molecular mass of all the polymers was in the range of 27000-34000 Da, and the polydispersivity was in the range of 1.5-1.7. By intravenous injection to mice bearing a solid tumour, all the polymeric carriers displayed a bi-compartmental pharmacokinetic behaviour. Both the poly(ethylene glycol) and the hexadecylalkyle conjugation prolonged and enhanced the distribution phase of poly(hydroxyethylaspartamide). The poly(ethylene glycol) conjugation was found to promote the carrier elimination by kidney ultrafiltration and to prevent partially the accumulation in the spleen and in the liver. The poly(ethylene glycol)/hexadecylalkyle conjugates localised preferentially in the liver were over 30% of the dose/g of tissue was determined after 144 h from administration. In the tumour all the polymers displayed a relevant accumulation that significantly increased throughout the time to reach high concentrations after 24 h. In particular, the poly(ethylene glycol)/hexadecylalkyle conjugates achieved a concentration of 15-25% of the dose/g of tissue after 24 h from administration that was maintained up to 144 h.

Calcitonin release system in the treatment of experimental osteoporosis. Histomorphometric evaluation.

Bioabsorbable polyphosphazene matrices charged with 250 microg calcitonin were evaluated as calcitonin delivery systems for the controlled release of this drug in an in vivo experimental model. Matrices were implanted under general anesthesia in osteopenic female rats, while a group of osteopenic animals (control group) received unloaded matrices. After 30 days a second series of matrices, loaded and unloaded was, respectively, implanted in both groups. In a third group of osteopenic female rats (positive control), 10 IU calcitonin were injected daily for two months. A baseline group and a sham operated group of animals were also included in the study. The explanted matrices were histologically evaluated together with the surrounding tissues, and bone histomorphometry was performed on undecalcified sections from femurs. The results showed the good biocompatibility of the matrices. Bone histomorphometry revealed that bone architecture in the treatment group was improved and results were not different from those observed after calcitonin injection. These data confirm a good in vivo behavior of the system, as well as a therapeutic effect of the released calcitonin in the osteopenic condition.

Immunological properties of uricase conjugated to neutral soluble polymers.

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) > or = 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.

Controlled release of biomolecules from temperature-sensitive hydrogels prepared by radiation polymerization.

Poly(acryloyl-L-proline methyl ester)-based hydrogels containing 1 and 5% of a crosslinking agent were studied as drug delivery systems. The drug loading properties were investigated by matrix incubation into solutions containing biomolecules with molecular weight ranging between 300 and 65,000 Da. The loading yield was found to depend on both the crosslinking degree and the molecular weight of the drug. In vitro release studies were carried out with both swollen and dry matrices loaded with gentamicin, isoniazid and insulin. Gentamicin and isoniazid were released by a bimodal Fickian diffusion with a remarkable burst that was found to depend on both matrix crosslinking degree and physical state. In vivo, the subcutaneous implantation into mice of the isoniazid loaded matrices allowed for an efficient drug release for 800 h. In vitro insulin was released from the swollen matrices for 1500 h by diffusional Fickian mechanism while the dry ones displayed a lag time followed by Fickian diffusion release. The subcutaneous implantation of the insulin-loaded matrices into diabetic mice induced a remarkable decrease in the glucose concentrations in blood. In particular, the dry 1% matrices were found to maintain a low glucose level for 700 h.

Pre-targeted locoregional radioimmunotherapy with 90Y-biotin in glioma patients: phase I study and preliminary therapeutic results.

The aim of this study was to determine the maximum-tolerated dose, of a pre-targeting three-step (3-S) method employing 90Y-biotin in the locoregional radioimmunotherapy (RIT) of recurrent high grade glioma, and to investigate the antitumor efficacy of this new treatment. Twenty-four patients with recurrent glioma underwent second surgical debulking and implantation of a catheter into the surgical resection cavity (SRC), in order to introduce the radioimmunotherapeutic agents [biotinylated monoclonal antibody (MoAb), avidin and 90Y-biotin]. Eight patients with anaplastic astrocytoma (AA) and 16 patients with glioblastoma (GBM) were injected with biotinylated anti-tenascin MoAb (2 mg), then with avidin (10 mg; 24 h later) and finally 90Y-biotin (18 h later). Each patient received two of these treatments 8-10 weeks apart. The injected activity ranged from 0.555 to 1.110 GBq (15-30 mCi). Dosage was escalated by 0.185 GBq (5 mCi) in four consecutive groups. The treatment was well tolerated without acute side effects up to 0.740 GBq (20 mCi). The maximum tolerated activity was 1.110 GBq (30 mCi) limited by neurological toxicity. None of the patients developed hematologic toxicity. In three patients infection occurred around the catheter. The average absorbed dose to the normal brain was minimal compared with that received at the SRC interface. At first control (after 2 months), partial (PR) and minor (MR) responses were observed in three GBM (1 PR; 2 MR) and three AA patients (1 PR; 2 MR) with an overall objective response rate of 25%. Stable disease (SD) was achieved in seven GBM and five AA patients (50%). There was disease progression in six GBM patients (25%), but in none of the AA patients. At the dosage of 0.7-0.9 GBq per cycle, locoregional 3-S-RIT was safe and produced an objective response in 25% of patients. Based on these encouraging results, phase II studies employing 3-S-RIT soon after first debulking are justified.

New PEGs for peptide and protein modification, suitable for identification of the PEGylation site.

New PEG derivatives were studied for peptide and protein modification, based upon an amino acid arm, Met-Nle or Met-beta Ala, activated as succinimidyl ester. PEG-Met-Nle-OSu or PEG-Met-beta Ala-OSu react with amino groups in protein-yielding conjugates with stable amide bond. From these conjugates PEG may be removed by BrCN treatment, leaving Nle or beta Ala as reporter amino acid, at the site where PEG was bound. The conjugation of PEG and its removal by BrCN treatment was assessed on a partial sequence of glucagone and on lysozyme as model peptide or protein. Furthermore, insulin, a protein with three potential sites of PEGylation, was modified by PEG-Met-Nle, and the PEG isomers were separated by HPLC. After removal of PEG, as reported above, the sites of PEGylation were identified by characterization of the two insulin chains obtained after reduction and carboxymethylation. Mass spectrometry, amino acid analysis and Edman sequence, could reveal the position of the reporter norleucine that corresponds to the position of PEG binding.

Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine).

Uricase from Bacillus fastidiosus (UC) was covalently linked to linear PEG (PEG-1) (Mw 5 kDa), branched PEG (PEG-2) (Mw 10 kDa) and to poly(N-acryloylmorpholine) (PAcM) (Mw 6 kDa). The conjugation of UC with linear PEG and PAcM was accompanied by complete loss of enzymatic activity but, if uric acid as site protecting agent was included in the reaction mixture, the conjugate protein retained enzymatic activity. On the other hand, the modification with PEG-2 gave a conjugate that also maintained enzymatic activity in the absence of any active site protection. This behaviour must be related to hindrance of the branched polymer in reaching the enzyme active site. The UC conjugates exhibited increased resistance to proteolytic digestion while minor variations in the inhibitory constant, optimal pH, heat stability, affinity for substrate, were observed. Pharmacokinetic investigations in mice demonstrated increased residence time in blood for all the conjugates as compared with native uricase. Uricase conjugated with linear PEG was longer lasting in blood UC derivative, followed by branched PEG and the PAcM conjugates. Unconjugated uricase was rapidly removed from circulation. All these data are in favour of the use of the less known amphiphilic polymer PAcM as an alternative to PEGs in modification of enzymes devised for therapeutic applications.

Polyphosphazene microspheres for insulin delivery.

Polyphosphazene based microspheres for insulin delivery were prepared following three different procedures: (A) suspension-solvent evaporation; (B) double emulsion-solvent evaporation; (C) suspension/double emulsion-solvent evaporation. Methods A and C allowed for higher protein loading than procedure B. Scanning electron microscopy showed that all preparation procedures achieve microparticles with spherical shape, porous surface and internal honeycomb structure. In all cases insulin was released 'in vitro' by a bi-modal behaviour: fast release during the first 2 hours followed by a slow release. However, both the physical properties and the 'in vitro' release profiles were found to depend upon the preparation conditions. Subcutaneous administration to diabetic mice of microspheres obtained with methods A and C rapidly reduced the glucose levels of about 80% but most of activity was lost in 100 hours. Both preparations B induced a remarkable decrease in glucose levels and the activity was maintained throughout 1000 h. Finally all preparations stimulated anti-insulin antibody production that constantly increased over a period of 8 weeks.

Plain and drug loaded polyphosphazene membranes and microspheres in the treatment of rabbit bone defects.

The healing of periodontal surgical defects was studied in rabbits, using polyphosphazenes (POP) membranes and microspheres, both plain or drug-enriched. POP polymers having amino acid ester as backbone substituents, are used since they resorb and undergo hydrolytic degradation to ammonia, phosphate and amino acids. Fourteen animal were operated in tibia, and other fourteen at angle of the mandible, that was reached by extraoral access. Bone defects were performed in tibiae, and covered either with POP or with poly-tetrafluoroethylene (PTFE) membranes, while other rabbits served as controls. The animals were sacrificed after one and two months, and the tibiae taken and processed for optical microscopy. Similar surgical defects were made in mandible, and POP membranes were placed over the breaches, some of which were filled with POP microspheres, both alone or mixed with granular hydroxyapatite. For comparison, two rabbits were treated with PTFE membranes, while other two served as controls. The animals operated at the mandible were all sacrificed after one month, and the operated bones taken and processed for histology. It was found that POP membranes were very effective in promoting the healing in tibiae, while less satisfactory results were found in the animals treated with PTFE membranes and in controls. In mandible, the healing occurred without a clear relationship with the grafted microgranular material or the membrane, since repairing bone was found also in controls. In any case, both POP membranes and microspheres showed excellent biocompatibility, as no inflammatory cells or macrophages were found in the surrounding tissue. This property was completely independent from the presence of drug, since the matrix-entrapped drugs, released in the tissue, did not hamper the bone healing. It was also found that POP, by itself, has a positive effect in stimulating the bone repair.

Design and in vivo evaluation of an oral delivery system for insulin.

PURPOSE: To develop an oral controlled release system for insulin. METHODS: The polymer-inhibitor conjugates carboxymethylcellulose (CMC)-Bowman-Birk inhibitor and CMC-elastatinal were homogenized with polycarbophil-cysteine conjugate, insulin, and mannitol, compressed to 2 mg microtablets and enteric coated with a polymethacrylate. The protective effect of this delivery system for insulin towards enzymatic degradation, as well as the release profile, was evaluated in vitro. In addition, the effect of the dosage form on glucose levels of diabetic mice was determined. RESULTS: Tablets containing the CMC-inhibitor conjugates showed a strong protective effect for insulin. Whereas 91.6 +/- 7.4% (mean +/- SD, n = 3) of insulin in the dosage form without the inhibitor conjugates has been degraded within 3 h of incubation in an artificial intestinal fluid containing physiological concentrations of trypsin, chymotrypsin, and elastase, 49.7 +/- 5.5% (mean +/- SD, n = 3) of insulin remained stable in the delivery system containing the polymer-inhibitor conjugates. Additionally, polycarbophil-cysteine (PCP-Cys) provides high cohesiveness of the dosage form, due to the formation of interas well as intramolecular disulfide bonds within the polymer matrix. According to this, a controlled release of insulin could be achieved over a time period of 10 h. Furthermore, in vivo studies in diabetic mice showed a decrease in basal glucose levels of 20% to 40% during a time period of 80 h. CONCLUSIONS: Mucoadhesive polymer-inhibitor conjugates might represent a promising excipient in delivery systems for oral (poly)peptide delivery.

Immunogenic and tolerogenic properties of monomethoxypoly(ethylene glycol) conjugated proteins.

An immunogenic and tolerogenic characterisation of monomethoxypoly(ethylene glycol) conjugated proteins was carried out using, as immunogen models, an anti-malaria chimera monoclonal antibody (PfChMab) and a macrophage colony stimulating factor (M-CSF). Two conjugates of PfChMab were prepared by polymer derivatisation of 19 and 33% protein amino groups and one conjugate of M-CSF was obtained by modification of 24% amino groups. In mice M-CSF was found to elicit rapidly high IgG and IgM levels whereas the monomethoxypoly(ethylene glycol) derivatised M-CSF stimulated a significantly lower immunoresponse. Native PfChMab was found to induce a delayed immunoresponse with high IgM levels but low production of IgG. Furthermore, similar immunogenic profiles were obtained with the native and modified protein forms. The pre-administration of polymer conjugated M-CSF to mice subsequently treated with the native protein was found to suppress up to 75% of anti-native M-CSF IgG, while IgM production was not affected. On the other hand the pre-administration of monomethoxypoly(ethylene glycol) derivatised PfChMab was found to reduce significantly the generation of anti-native PfChMab IgM. Such suppression depended on the degree of modification: the conjugate with the higher number of polymer chains was more effective in suppressing the immunoresponse.

Biopharmaceutical properties of uricase conjugated to neutral and amphiphilic polymers.

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).