Comparative studies of polyethylene glycol-modified liposomes prepared using different PEG-modification methods.

To address the issue of excess polyethylene glycol (PEG)-lipid degradation observed when PEG-modified liposomes are prepared using the pH-gradient method, a concept using a novel PEG-modification method, called the post-modification method, was proposed and evaluated. To assess the proof concept, a preservation-stability study and a pharmacokinetic study were performed that compared the conventional PEG-modification method, called the pre-modification method, with the post-modification method. The results show that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, the post-modification method could be used without any manufacturing process difficulties, especially with high PEG-lipid content. In addition, a higher blood circulation capability was observed in the post-modification method. Through comparative studies, it was found that the post-modification method was advantageous compared to the pre-modification method. In conclusion, the post-modification method has the potential to be a novel PEG-modification method that can achieve a higher preservation stability of PEG-lipid, a greater ease of manufacturing, and a higher blood circulation capability, especially in the manufacturing of pH-gradient liposomal products.

Comparative studies of irinotecan-loaded polyethylene glycol-modified liposomes prepared using different PEG-modification methods.

Recently, a polyethylene glycol (PEG)-modification method for liposomes prepared using pH-gradient method has been proposed. The differences in the pharmacokinetics and the impact on the antitumor effect were examined; however the impact of PEG-lipid molar weight has not been investigated yet. The main purpose of this study is to evaluate the impact of PEG-lipid molar weight against the differences in the pharmacokinetics, the drug-release profile, and the antitumor effect between the proposed PEG-modification method, called the post-modification method, and the conventional PEG-modification method, called the pre-modification method. Various comparative studies were performed using irinotecan as a general model drug. The results showed that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, prolonged circulation time was observed in the post-modification method. The sustained drug-release was observed in the post-modification method by the results of the drug-releasing test in plasma. Moreover, a higher antitumor effect was observed in the post-modification method. It was also confirmed that the same behaviors were observed in all comparative studies even though the PEG molecular weight was lower. In conclusion, the post-modification method has the potential to be a valuable PEG-modification method that can achieve higher preservation stability of PEG-lipid, prolonged circulation time, and higher antitumor effect with only half the amount of PEG-lipid as compared to the pre-modification method. Furthermore, it was demonstrated that PEG(5000)-lipid would be more desirable than PEG(2000)-lipid since it requires much smaller amount of PEG-lipid to demonstrate the same performances.

[Efficacy of chemotherapy combined with bevacizumab for unresectable advanced or recurrent colorectal cancer].

Systemic treatment for metastatic or advanced colorectal cancer(mCRC)has remarkably progressed during recent years. All previously untreated mCRC patients at our institution between November, 2007 and June, 2010 were retrospectively evaluated. Of 72 patients, 39 were treated with chemotherapy alone, and 33 were treated with chemotherapy plus bevacizumab(BV). The median progression-free survival(mPFS)was 329 days in the group given chemotherapy plus BV, compared with 209 days in the group given chemotherapy alone(p=0. 0189). In sub-group analysis of those treated with chemotherapy plus BV, mPFS between 70 y/o

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs.

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.

Room-Temperature Pentacene Fluids: Oligoethylene Glycol Substituent-Controlled Morphologies and Singlet Fission.

We report the first synthesis of solvent-free pentacene fluids at room temperature together with observation of singlet fission (SF). Three pentacenes with different number of ethylene glycol (EG) side chains (n) were employed (denoted as (EG)n-Pc-(EG)n: n = 2, 3, and 4). The morphologies of these pentacenes largely depend on the lengths of EG chains (n). (EG)3-Pc-(EG)3 and (EG)4-Pc-(EG)4 indicate fluid compounds at room temperature, whereas (EG)2-Pc-(EG)2 is a solid compound. Microscopic clustering with short-range interactions between pentacene chromophores was confirmed in X-ray diffraction profiles of solvent-free fluids. Such a structural trend is an important origin of SF and consistent with the steady-state spectroscopic results. To one's surprise, femtosecond transient absorption spectroscopy demonstrated that SF occurred in thin films prepared from solvent-free fluids of (EG)3-Pc-(EG)3 and (EG)4-Pc-(EG)4 in spite of such excessive EG chains.

[A case of breast reconstruction surgery using flap of latissimus dorsi after neoadjuvant chemotherapy].

We performed breast reconstruction surgery with mastectomy after neoadjuvant chemotherapy(NAC)for a patient with NAC indication desiring breast conservation. The case was a 34-year-old single woman. In March, 2007, she was aware of a lump in her left breast and visited our hospital. The diagnosis was solid-tubular carcinoma 3 cm in diameter from close examination. We performed preoperative chemotherapy with EC(epirubicin 90 mg/m(2), cyclophosphamide 600 mg/m(2))x4, followed by 3w-paclitaxel 175 mg/m(2)x4, and then performed mastectomy with axillary dissection and breast reconstruction surgery using the flap of latissimus dorsi at the same time. Pathologically, pCR was provided. We thought that there are many advantages to both treatment of breast cancer and the cosmetic characteristics. The patient was very satisfied. But further cumulative examinations are awaited because there is not much evidence at present.

Impact of Sterilization Method on Protein Aggregation and Particle Formation in Polymer-Based Syringes.

The effects of sterilization methods on the storage stability of erythropoietin (EPO) in polymer-based syringes were assessed by quantifying protein oxidation, aggregation, and particle formation. Micro-particle counting and size exclusion chromatography coupled with a multi-angle light scattering detector demonstrated much lower levels of protein particles and aggregates for EPO stored for 12 weeks in steam-sterilized than in radiation (Rad)-sterilized syringes. Intermediate levels of damage were observed for EPO stored in ethylene oxide-sterilized syringes. HPLC analysis documented that the Rad-sterilized syringes caused increased oxidation of the protein during storage. In contrast, in the steam- and ethylene oxide-sterilized syringes EPO oxidation did not change. Analysis with electron spin resonance revealed that only Rad-sterilized syringes formed radicals in the syringe body, which persisted over the 12-week storage period. These results demonstrated that Rad-sterilization generated radicals in the syringes which in turn caused increased EPO oxidation, particle formation, and protein aggregation. Therefore, steam sterilization was shown to be a preferable sterilization method for the polymer-based syringe system when using biopharmaceutical drugs highly sensitive to oxidation, and particle formation and aggregation.

Low Leachable Container System Consisting of a Polymer-Based Syringe with Chlorinated Isoprene Isobutene Rubber Plunger Stopper.

UNLABELLED: A 36 month leachable study on water for injection in direct contact within a polymer-based prefillable syringe consisting of a cyclo olefin polymer barrel, a chlorinated isoprene isobutene rubber plunger stopper, a polymer label attached on the barrel, and a secondary packaging was conducted at 25 ± 2 °C and 60 ± 5% relative humidity. Through the various comparison studies, no difference in the leachable amounts was observed between this polymer-based prefilled syringe and a glass bottle as a blank sample reference by 36 months. No influence on the leachables study outcome was noted from the printed label and/or label adhesive or from the secondary packaging. In an additional study, no acrylic acid used as the label adhesive leachable was detected by an extended storage for 45 months at 25 ± 2 °C and 60 ± 5% relative humidity as a worst case. To obtain more details, a comparison extractable study was conducted between a cyclo olefin polymer barrel and a glass barrel. In addition, chlorinated isoprene isobutene rubber and bromo isoprene isobutene rubber were compared. As a result, no remarkable difference was found in the organic extractables for syringe barrels. On the other hand, in the case of element extractable analysis, the values for the cyclo olefin polymer barrel were lower than that for the glass barrel. For the plunger stoppers, the chlorinated isoprene isobutene rubber applied in this study was showing a lower extractable profile as compared to the bromo isoprene isobutene rubber, both for organic and element extractables. In conclusion, the proposed polymer-based prefillable syringe system has great potential and represents a novel alternative that can achieve very low level extractable profiles and can bring additional value to the highly sensitive biotech drug market. LAY ABSTRACT: A 36 month leachable study on water for injection in direct contact within a cyclo olefin polymer barrel and chlorinated isoprene isobutene rubber plunger stopper that has a polymer label attached to the barrel and is wrapped into a secondary packaging was conducted at 25 °C and 60% relative humidity. Through the various comparison studies, no difference in the leachable amounts was observed between polymer-based prefilled syringes and a glass bottle as a blank sample reference by 36 months. No influences on the leachables study outcome were noted from the secondary packaging. To obtain more details, a comparison extractable study was conducted between the cyclo olefin polymer and the glass barrel. In addition, chlorinated isoprene isobutene rubber and bromo isoprene isobutene rubber plunger stoppers were compared as well. As a result, no remarkable difference was found in the organic extractables for barrels. As for element extractable analysis, the values for the cyclo olefin polymer barrel were lower than that for the glass barrel. For the plunger stoppers, the chlorinated isoprene isobutene rubber applied in this study was showing a lower extractable profile as compared to the bromo isoprene isobutene rubber, both for organic and element extractables. In conclusion, the proposed polymer-based prefillable syringe system has great potential and represents a novel alternative that can achieve very low level extractable profiles and can bring additional value to the highly sensitive biotech drug market.

Assessment of the effects of sterilization methods on protein drug stability by elucidating decomposition mechanism and material analysis.

The effects of different sterilization methods on the stability of highly sensitive protein drugs were assessed by elucidating mechanism involved in the process of protein decomposition. Results demonstrated that the steam sterilized syringes produced less protein oxidation compared with sterilization by the electron beam method. Electron spin resonance analysis showed that while considerably high levels of radicals were observed in the electron beam-sterilized syringes, no radicals were detected with steam sterilization. To identify the factor involved in protein oxidation, stability of the chemical composition of the syringe material was investigated using various analytical methods. Results showed that the syringe material itself was oxidized and two forms of oxidation products were identified with electron beam sterilization. Protein oxidation was shown to increase over time, and this was thought to be as a result of persistent exposure to the oxidized syringe barrel surface, which induced further protein oxidation. These results suggest that compared to electron beam sterilization, steam sterilization is a preferable method for the plastic prefilled syringe system, particularly for biopharmaceutical drug products that are highly sensitive to oxidization.

A strategy for the prevention of protein oxidation by drug product in polymer-based syringes.

UNLABELLED: Recently, new and advanced ideas have been presented on the value of polymer-based syringes for improved safety, better strength, reduced aggregation, and the prevention of drug degradation. In this report, our findings on drug degradation from protein oxidation will be presented and discussed. Commonly, dissolved oxygen is one of the factors for causing protein degradation. Due to the nature of higher gas permeability in polymer-based syringes, it was thought to be difficult to control the oxygen level during storage. However, this report demonstrates the appropriateness of combining the use of an oxygen absorber within the secondary packaging as a deoxygenated packaging system. In addition, this report suggests that another factor to enhance protein oxidization is related to radicals on the syringe barrel from sterilization by irradiation. We demonstrate that steam sterilization can minimize protein oxidization, as the protein filled in steam sterilized syringe is much more stable. In conclusion, the main oxidation pathway of a protein has been identified as dissolved oxygen and radical generation within a polymer container. Possible solutions are herewith presented for controlling oxidation by means of applying a deoxygenated packaging system as well as utilizing steam sterilization as a method of sterilization for prefillable polymer syringes. LAY ABSTRACT: There have been many presentations and discussions about the risks associated with glass prefilled syringes. Advanced ideas are being presented on the value of polymer-based syringes for improved safety, better strength, reduced protein aggregation, and the prevention of drug degradation. Drug degradation based on protein oxidation is discussed in this report. Identification of the main factors causing this degradation and possible solutions available by using polymer-based syringes will be presented. The causes of protein oxidation have been identified as dissolved oxygen and radicals generated by the applied method of sterilization. The oxidation reaction created by dissolved oxygen within the drug product can be effectively inhibited by controlling the removal of the oxygen through the use of a deoxygenated packaging system. This packaging system can control the level or complete removal of oxygen from the primary container and the secondary packaging system. Protein oxidation induced by the formation of radicals from sterilization by irradiation is another critical aspect where it was thought that various sterilization methods were acceptable without loosing drug product quality. However, this report is first to demonstrate that gamma sterilized polymer-based syringes accelerated protein oxidation by radical generation; this effect can be prevented by means of steam sterilization.

Effect of poly(ethylene glycol) grafts on temperature-sensitivity of thermosensitive polymer-modified liposomes.

A copolymer of N-isopropylacrylamide and N-acryloylpyrrolidine, which exhibits a lower critical solution temperature (LCST) at 38 degrees C, having a didodecyl group at the chain terminal was synthesized and calcein-loaded liposomes were prepared from a mixture of dioleoylphosphatidylethanolamine and the copolymer. While the contents release from the copolymer-modified liposomes was strongly accelerated around the LCST of the copolymer, some extent of the release was also observed below the LCST, probably because the partly-dehydrated copolymer chains interacted with the liposome membrane. When poly(ethylene glycol) with the number average molecular weight of 550 having a didodecyl group at the chain end was introduced into the copolymer-modified liposomes, the contents release from the copolymer-modified liposomes was effectively suppressed below the LCST but hardly affected above the LCST. As a result, the liposomes exhibited much sharper enhancement of the contents release in a narrow temperature region. The temperature-sensitive contents release property of the copolymer-modified liposomes became less significant in the presence of serum. However, inclusion of the anchor-bearing poly(ethylene glycol) derivative improved the contents release behavior of the copolymer-modified liposomes in the presence of serum.

Functional evaluation and characterization of a newly developed silicone oil-free prefillable syringe system.

The functionality of a newly developed silicone oil-free (SOF) syringe system, of which the plunger stopper is coated by a novel coating technology (i-coating™), was assessed. By scanning electron microscopy observations and other analysis, it was confirmed that the plunger stopper surface was uniformly covered with the designed chemical composition. A microflow imaging analysis showed that the SOF system drastically reduced both silicone oil (SO) doplets and oil-induced aggregations in a model protein formulation, whereas a large number of subvisible particles and protein aggregations were formed when a SO system was used. Satisfactory container closure integrity (CCI) was confirmed by means of dye and microorganism penetration studies. Furthermore, no significant difference between the break loose and gliding forces was observed in the former, and stability studies revealed that the SOF system could perfectly show the aging independence in break loose force observed in the SO system. The results suggest that the introduced novel SOF system has a great potential and represents an alternative that can achieve very low subvisible particles, secure CCI, and the absence of a break loose force. In particular, no risk of SO-induced aggregation can bring additional value in the highly sensitive biotech drug market.

Designing a novel in vitro drug-release-testing method for liposomes prepared by pH-gradient method.

In order to evaluate the drug-release behavior from pH-gradient liposomal formulation, a simple release-testing method without using biological components was newly designed on the basis of inversed ammonia gradient principle. Various factors influencing drug-release (releasing factor) were examined. As a result, releasing factor's concentration, pH, osmolarity in test fluid, and releasing factor's structure were found to be the critical factors to be optimized. Various vincristine-loaded liposomes with different lipid compositions or with different lipid/cholesterol ratio were tested for drug-release behavior and successfully obtained drug-release profiles reflecting differences in the physicochemical properties of individual liposomes. Furthermore, since the comparative release study of vincristine-loaded liposomes and doxorubicin-loaded liposomes could reproduce the phenomena as other researchers recently reported, a possibility was suggested for the proposed method to estimate the physicochemical status of drug inside of liposomes. Proof of concept study concluded, as a whole, that the novel release-testing method would be useful for a formulation study and also useful as a tool for the quality assurance or quality control in the manufacturing of pH-gradient liposomal products.

Temperature sensitization of liposomes by use of N-isopropylacrylamide copolymers with varying transition endotherms.

Three kinds of copolymers of N-isopropylacrylamide (NIPAM) with the same conformational transition temperature and varying transition endotherms were synthesized with N-acryloylpyrrolidine (APr), N,N-dimethylacrylamide (DMAM), and N-isopropylmethacrylamide (NIPMAM) as the comonomers. Two dodecyl groups were incorporated into the termini of these copolymers as an anchor for the fixation to a liposomal membrane. Egg yolk phosphatidylcholine liposomes having these copolymers were prepared and their temperature-sensitive contents release and association properties were investigated. While these copolymer exhibited a conformational transition at ca. 40 degrees C, DeltaH for the transition increased in the order of poly(APr-co-NIPAM) < poly(DMAM-co-NIPAM) < poly(NIPMAM-co-NIPAM). The liposomes containing poly(NIPMAM-co-NIPAM) showed a drastic release enhancement of entrapped calcein above the transition temperature, whereas the liposomes with poly(DMAM-co-NIPAM) and those with poly(APr-co-NIPAM) exhibited moderate and slight enhancement of calcein release above that temperature, respectively. On the contrary, the liposomes containing poly(APr-co-NIPAM) showed significant aggregation above the transition temperature, but the aggregation was hardly observed for the liposomes having poly(NIPMAM-co-NIPAM), indicating that poly(APr-co-NIPAM) more efficiently made the liposome surface hydrophobic. Thus, we concluded that the copolymer with a large DeltaH is suitable for obtaining functional liposomes with a temperature-sensitive contents release property, whereas the copolymer with a small DeltaH is appropriate for preparing functional liposomes with a temperature-sensitive surface property.