Beate Bittner

Tetracycline-HCl-loaded poly(dl-lactide-co-glycolide) microspheres prepared by a spray drying technique: influence of γ-irradiation on radical formation and polymer degradation

Tetracycline-HCl (TCH)-loaded microspheres were prepared from poly(lactide-co-glycolide) (PLGA) by spray drying. The drug was incorporated in the polymer matrix either in solid state or as w/o emulsion. The spin probe 4-hydroxy-2,2,6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) and the spin trap tert-butyl-phenyl-nitrone (PBN) were co-encapsulated into the TCH-loaded and placebo particles. We investigated the effects of gamma-irradiation on the formation of free radicals in polymer and drug and the mechanism of chain scission after sterilization. Gamma-Irradiation was performed at 26.9 and 54.9 kGy using a 60Co source. The microspheres were characterized especially with respect to the formation of radicals and in vitro polymer degradation. Electron paramagnetic resonance (EPR) spectroscopy, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectroscopy (GC-MS), and scanning electron microscopy (SEM) were used for characterization of the microspheres. Using EPR spectroscopy, we successfully detected gamma-irradiation induced free radicals within the TCH-loaded microspheres, while unloaded PLGA did not contain radicals under the same conditions. The relatively low glass transition temperature of the poly(dl-lactide-co-glycolide) (37-39 degrees C) seems to favor subsequent reactions of free radicals due to the high mobility of the polymeric chains. Because of the high melting point of TCH (214 degrees C), the radicals can only be stabilized in drug loaded microspheres. In order to determine the mechanism of polymer degradation after exposure to gamma-rays, the spin trap PBN and the spin probe TEMPOL were encapsulated in the microspheres. gamma-Irradiation of microspheres containing PBN resulted in the formation of a lipophilic spin adduct, indicating that a polymeric radical was generated by random chain scission. Polymer degradation by an unzipping mechanism would have produced hydrophilic spin adducts of PBN and monomeric radicals of lactic or glycolic acid. These degradation products were not detected by EPR. This result is confirmed by the observation that possible diamagnetic reaction products of low molecular weight, consisting of TEMPOL and lactide or glycolide monomers, could not be detected by GC-MS. While an irradiation dose-dependent decrease in molecular weight of PLGA could be verified in agreement with the literature, TCH content of the microspheres was not affected by the exposure to gamma-rays. It can be concluded that EPR spectroscopy in combination with GPC, DSC, and HPLC allows a detailed characterization of the impact of gamma-sterilization on biodegradable parenteral drug delivery systems.

Recombinant human erythropoietin (rhEPO) loaded poly(lactide-co-glycolide) microspheres: influence of the encapsulation technique and polymer purity on microsphere characteristics

Recombinant human erythropoietin (EPO) and fluorescein isothiocyanate-labelled dextran (FITC-dextran) loaded biodegradable microspheres were prepared from poly(lactide-co-glycolide) (PLG) by a modified spray-drying technique. This microencapsulation method was compared with the water-in-oil-in-water (w/o/w) double-emulsion method. As expected, microsphere morphology, particle size and particle size distribution strongly depended on the production process. The spray-drying method was found to have a number of advantages compared to the w/o/w double-emulsion technique. The content of residual dichloromethane (DCM) in the final product was significantly lower in case of the microspheres prepared by spray-drying. Concerning EPO loaded microspheres, spray-drying yielded higher encapsulation efficiencies. Although the microspheres obtained by spray-drying are subjected to intensive mechanical and thermal stress during the preparation, the amount of aggregates of EPO in PLG microspheres were not increased compared to the w/o/w technique. Depending on the manufacturing method, addition of cyclic DL-lactide dimers (referred to as monomers in the following) affected the in vitro release profiles of EPO and FITC-dextran from PLG microspheres. Using differential scanning calorimetry it was shown that these low molecular weight substances only seem to be present inside the microspheres produced by spray-drying. DL-Lactide significantly reduced the initial burst release of both EPO and FITC-dextran. While the following release period of EPO was not affected by the DL-lactide content, a more linear FITC-dextran release pattern could be achieved. It can be concluded that the spray-drying technique provides a number of advantages compared to the w/o/w method. The modulation of protein release using low molecular weight additives is of particular interest for parenteral depot systems.

Monitoring Microviscosity and Microacidity of the Albumin Microenvironment Inside Degrading Microparticles from Poly(lactide-co-glycolide) (PLG) or ABA-triblock Polymers Containing Hydrophobic Poly(lactide-co-glycolide) A Blocks and Hydrophilic Poly(ethyleneoxide) B Blocks

AbstractPurpose. The purpose of this study was to monitor the microenvironment of an encapsulated model protein during the release from biodegradable microparticles (MP) made from three different polymers, namely poly(lactide-co-glycolide) (PLG) and ABA-triblock polymers containing hydrophobic poly(lactide-co-glycolide) A blocks and hydrophilic poly(ethyleneoxide) B blocks with an A:B ratio of 90:10 (ABA10) and 70:30 (ABA30). Methods. MP loaded with spin labeled albumin were prepared by a w/o/w technique. The particles were characterized by light scattering and electron microscopy. In vitro release of albumin was determined by size exclusion chromatography. Light microscopic experiments were conducted to visualize water penetration in the matrix. The protein microenvironment inside the degrading microparticles was characterized noninvasively by 2 GHz EPR spectroscopy. Results. Water penetrated rapidly into all MP in the range of few minutes. A burst release was observed for PLG. The release from ABA block-polymers continued for over 14 days despite the rapid solubilization of the protein inside the microparticles. The initial microviscosity of the protein environment inside the ABA particles after exposure to buffer was 2 mm2/s and increased with time. A gradual decrease of the pH to a value of 3.5 was observed within the MP. Conclusions. The data indicate that the microviscosity and microacidity inside protein loaded microparticles can be studied nondestructively by EPR spectroscopy. Our results clearly demonstrate that ABA-block polymers are superior to PLG allowing a controlled release of proteins from swollen microspheres.

Potential inhibitory effects of formulation ingredients on intestinal cytochrome P450

The use of common formulation ingredients (categorized into six groups) for preclinical animal studies has been assessed with respect to cytochrome P450 (CYP) inhibition, specifically CYP3A inhibition, in expressed human CYP3A4, human liver microsomes, dog- and cynomolgus monkey intestinal microsomes. Results indicated a wide range of inhibition potentials and there appeared to be species differences with inhibition of CYP3A activity. Generally, greater inhibition of CYP3A activity was observed with amphiphilic ingredients (for example mixed micellar solutions, Tween 80, and oleic acid). From the data presented, it can be predicted that the majority of the ingredients tested would not have a significant impact on the potential inhibition, by the formulation, on any apparent first pass metabolism in the intestinal tract for new drug entities being tested in the preclinical environment. However a number of common ingredients will require further investigation based on the estimated concentration within the gastrointestinal tract.

Degradation and protein release properties of microspheres prepared from biodegradable poly(lactide-co-glycolide) and ABA triblock copolymers: influence of buffer media on polymer erosion and bovine serum albumin release

The aim of the present study was to investigate the influence of the chemical insertion of poly(ethylene oxide), PEO, into a poly(lactide-co-glycolide), PLG, backbone on the mechanisms of in vitro degradation and erosion of the polymer. For this purpose microspheres prepared by a modified W/O/W double emulsion technique using ABA triblock copolymers, consisting of PLG A-blocks attached to central PEO B-blocks were compared with microspheres prepared from PLG. Due to their molecular architecture the ABA triblock copolymers differed in their erosion and degradation behavior from PLG. Degradation occurred faster in the ABA polymers by cleavage of ester bonds inside the polymer backbone. Even erosion was shown to start immediately after incubation in different buffer media. By varying pH and ionic strength of the buffer it was found that both mass loss and molecular weight decay were accelerated in alkaline and acidic pH in the case of the ABA triblock copolymers. Although the pH of the medium had a moderate influence on the degradation of PLG, the molecular weight decay was not accompanied by a mass loss during the observation time. In a second set of experiments we prepared bovine serum albumin, BSA, loaded microspheres from both polymers. The release of BSA from ABA microspheres under in vitro conditions parallels the faster swelling and erosion rates. This could be confirmed by electron paramagnetic resonance, EPR, measurements with spin labeled albumin where an influx of buffer medium into the ABA microspheres was already observed within a few minutes. In contrast, PLG microspheres revealed a burst release without any erosion. The current study shows that the environmental conditions affected the degradation and erosion of the pure polymer microspheres in the same way as the release of the model protein. This leads to the conclusion that the more favorable degradation profile of the ABA triblock copolymers was responsible for the improvement of the release profile.

Comparison of subcutaneous and intravenous administration of trastuzumab: a phase I/Ib trial in healthy male volunteers and patients with HER2-positive breast cancer.

Trastuzumab is a key component of treatment for human epidermal growth factor receptor 2 (HER2)–positive breast cancer in both the early and metastatic settings. It is administered intravenously, with between 17 and 52 infusions in standard regimens over 1 year. Intravenous administration of trastuzumab requires substantial time commitments for patients and health care professionals and can result in patient discomfort. A subcutaneous formulation of trastuzumab, containing recombinant human hyaluronidase to overcome subcutaneous absorption barriers, would reduce the administration duration and remove the need to establish intravenous access, thus improving the overall convenience of trastuzumab administration. This open‐label, 2‐part, phase I/Ib study (NCT00800436) was undertaken in healthy male volunteers and female patients with HER2‐positive early breast cancer to identify the dose of subcutaneous trastuzumab that resulted in exposure comparable with the approved intravenous trastuzumab dose. A subcutaneous trastuzumab dose of 8 mg/kg was found to result in exposure comparable with the intravenous trastuzumab dose of 6 mg/kg. The subcutaneous formulation was well tolerated, with a trend toward fewer adverse events versus intravenous administration; most adverse events were mild in intensity. These results support an ongoing phase III efficacy and safety study comparing a fixed subcutaneous trastuzumab dose with intravenous trastuzumab administration.

Biodegradable recombinant human erythropoietin loaded microspheres prepared from linear and star-branched block copolymers: influence of encapsulation technique and polymer composition on particle characteristics.

Recombinant human erythropoietin (EPO) and fluorescein isothiocyanate labeled dextran (FITC-dextran) loaded microspheres were prepared by a modified W/O/W double-emulsion technique. Biodegradable linear ABA block copolymers consisting of poly(L-lactide-co-glycolide) A blocks attached to central poly(ethyleneoxide) (PEO) B blocks and star-branched AB block copolymers containing A blocks of poly(L-lactide) or poly(L-lactide-co-glycolide) and star-branched poly(ethyleneoxide) B blocks were investigated for their potential as sustained release drug delivery systems. Microsphere characteristics were strongly influenced by the polymer composition. In the case of the linear block copolymers, a reduced lactic acid content in a linear block copolymer yielded smaller particles, a lower encapsulation efficiency, and a higher initial drug release both in the case of EPO and FITC-dextran. The investigation of the effects of several manufacturing parameters on microsphere formation showed that the process temperature plays an important role. Microsphere formation in a +1 degrees C environment resulted in higher drug loadings without increasing the amount of residual dichloromethane inside the particles. Other parameters such as the homogenization of the primary W/O emulsion and of the W/O/W double-emulsion have less impact on microsphere characteristics. Branched block copolymers containing star-shaped PEO also showed potential for the preparation of drug loaded microspheres. A certain amount of glycolic acid in the copolymer was necessary for the successful preparation of non-aggregating microspheres at room temperature. Again, the processing temperature strongly affected particle characteristics. Microsphere preparation at +1 degrees C allows the formation of microspheres from a polymer not containing glycolic acid, a result which could not be achieved at room temperature. Moreover, compared to microsphere formation at room temperature, the effective FITC-dextran loading was increased. Concerning the EPO loaded microspheres, the amount of EPO aggregated was comparable to that using the linear ABA polymers. A continuous release of the protein from these star-shaped polymers could not be achieved. In conclusion, apart from microsphere preparation in a +1 degrees C environment the choice of the polymer represents the main factor for a successful entrapment of proteins into biodegradable microspheres.

Pharmacokinetics and safety of subcutaneous rituximab in follicular lymphoma (SABRINA): stage 1 analysis of a randomised phase 3 study

BACKGROUND Intravenous rituximab is a mainstay of treatment for follicular lymphoma. A subcutaneous formulation that achieves equivalent rituximab serum concentrations might improve convenience and save health-care resources without sacrificing clinical activity. We aimed to assess pharmacokinetic non-inferiority of 3 week cycles of fixed-dose subcutaneous rituximab versus standard intravenous rituximab. METHODS In our two-stage, randomised, open-label, phase 3 trial, we enrolled patients with previously untreated grade 1-3a, CD20-positive follicular lymphoma at 67 centres in 23 countries. In stage 1, we randomly allocated patients 1:1 with the Pocock and Simon algorithm to intravenous rituximab (375 mg/m(2)) or fixed-dose subcutaneous rituximab (1400 mg), stratified by induction chemotherapy regimen (cyclophosphamide, doxorubicin, vincristine, prednisone or cyclophosphamide, vincristine, prednisone), Follicular Lymphoma International Prognostic Index score, and region. After randomisation, patients received one induction dose of intravenous rituximab in cycle 1 and then allocated treatment for cycles 2-8. Patients with a complete or partial response following induction therapy continued intravenous or subcutaneous rituximab as maintenance every 8 weeks. The primary endpoint was the ratio of observed rituximab serum trough concentrations (Ctrough) between groups at cycle 7 (before cycle 8 dosing) of induction treatment in a per-protocol population. Patients were analysed as treated for safety endpoints. Stage 2 follow-up is ongoing and is fully accrued. This study is registered with ClinicalTrials.gov, number NCT01200758. FINDINGS Between Feb 4, 2010, and Oct 21, 2011, we enrolled 127 patients. Pharmacokinetic data were available for 48 (75%) of 64 patients randomly allocated intravenous rituximab and 54 (86%) of 63 patients randomly allocated subcutaneous rituximab. Geometric mean Ctrough was 83·13 μg/mL in the intravenous group and 134·58 μg/mL in the subcutaneous group (ratio 1·62, 90% CI 1·36-1·94), showing non-inferiority of subcutaneous rituximab. 57 (88%) of 65 patients in the intravenous rituximab safety population had adverse events (30 [46%] grade ≥3), as did 57 (92%) of 62 patients in the subcutaneous rituximab safety population (29 [47%] grade ≥3). The most common grade 3 or worse adverse event in both groups was neutropenia (14 [22%] patients in the intravenous group and 16 [26%] patients in the subcutaneous group). Adverse events related to administration were mostly grade 1-2 and occurred in 21 (32%) patients in the intravenous group and 31 (50%) patients in the subcutaneous group. INTERPRETATION Stage 1 data show that the pharmacokinetic profile of subcutaneous rituximab was non-inferior to intravenous rituximab and was not associated with new safety concerns. Stage 2 will provide data for efficacy and safety of the subcutaneous administration. FUNDING F Hoffmann-La Roche.

Comparison of Subcutaneous Versus Intravenous Administration of Rituximab As Maintenance Treatment for Follicular Lymphoma: Results From a Two-Stage, Phase IB Study

PURPOSE This two-stage phase IB study investigated the pharmacokinetics and safety of subcutaneous (SC) versus intravenous (IV) administration of rituximab as maintenance therapy in follicular lymphoma. PATIENTS AND METHODS In stage 1 (dose finding), 124 patients who responded to rituximab induction were randomly assigned to SC rituximab (375 mg/m2, 625 mg/m2, or an additional group at 800 mg/m2) or IV rituximab (375 mg/m2). The objective was to determine an SC dose that would yield a rituximab serum trough concentration (Ctrough) in the same range as that of IV rituximab. In stage 2, 154 additional patients were randomly assigned (1:1) to SC rituximab (1,400 mg) or IV rituximab (375 mg/m2) given at 2- or 3-month intervals. The objective was to demonstrate noninferior rituximab Ctrough of SC rituximab relative to IV rituximab 375 mg/m2. RESULTS Stage 1 data predicted that a fixed dose of 1,400 mg SC rituximab would result in a serum Ctrough in the range of that of IV rituximab. Noninferiority (ie, meeting the prespecified 90% CI lower limit of 0.8) was then confirmed in stage 2, with geometric mean Ctrough SC:Ctrough IV ratios for the 2- and 3-month regimens of 1.24 (90% CI, 1.02 to 1.51) and 1.12 (90% CI, 0.86 to 1.45), respectively. Overall safety profiles were similar between formulations (in stage 2, 79% of patients experienced one or more adverse events in each group). Local administration-related reactions (mainly mild to moderate) occurred more frequently after SC administration. CONCLUSION The fixed dose of 1,400 mg SC rituximab predicted by using stage 1 results was confirmed to have noninferior Ctrough levels relative to IV rituximab 375 mg/m2 dosing during maintenance, with a comparable safety profile. Additional investigation will be required to determine whether the SC route of administration for rituximab provides equivalent efficacy compared with that of IV administration.

Improved oral bioavailability of cyclosporin A in male Wistar rats. Comparison of a Solutol HS 15 containing self-dispersing formulation and a microsuspension.

Oral bioavailability of the highly lipophilic and poorly water-soluble immunosuppressive agent cyclosporin A (CyA) in two different formulations was investigated in male Wistar rats. An aqueous microsuspension and a self-dispersing formulation composed of the surface-active ingredients Solutol HS 15:Labrafil M2125CS:oleic acid=7:2:1 (v/v/v) were administered to the animals at a dose level of 20 mg/kg. In order to calculate the absolute oral bioavailability, CyA was additionally administered intravenously at 10 mg/kg as microsuspension. It was found that the oral bioavailability of CyA in the Solutol HS 15-based formulation was twofold higher as compared to the microsuspension (69.9+/-2.8 vs. 35.7+/-3.3%, P=0.001). By contrast, the time to reach maximum plasma concentration (t(max)) and the terminal half-life (t(1/2)) did not differ significantly with the different formulations (t(max): 7.0+/-1.0 vs. 6.3+/-1.7 h; t(1/2): 20.5+/-2.9 vs. 16.7+/-4.7 h). In vitro solubility experiments demonstrated a marked increase in the aqueous solubility of CyA in the presence of the self-dispersing formulation as compared to the micronized powder alone (solubility after 120 min at 37 degrees C: 136 vs. 23.2 microg/ml in human gastric juice; 133 vs. 10.8 microg/ml in simulated intestinal juice). Most likely, the enhanced systemic exposure of CyA in the self-dispersing formulation was caused by improved solubility of CyA in the gastrointestinal fluids in the presence of the surface-active ingredients. Additional factors that may have contributed to increased oral bioavailability are inhibition of metabolism and/or transport processes as well as permeability enhancement by the co-administered excipients.