[Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase].

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

Encapsulation of mitoxantrone into pegylated SUVs enhances its antineoplastic efficacy.

Mitoxantrone (MIT) was encapsulated into 60, 80 and 100nm pegylated hydrogenated soy phosphatidylcholine/cholesterol (HSPC/chol) vesicles using a transmembrane (NH(4))(2)SO(4) gradient. In-vitro release studies revealed that small-sized formulation had fast drug-release rate. Acute toxicity studies performed in c57 mice proved that all pegylated liposomal MIT (plm) formulations could be well-tolerated at a dose of 9mg/kg, significantly compared to severe toxicity induced by free mitoxantrone (f-M). In KM mice, plm60 was at least 2- to 3-fold less toxic than f-M. After intravenous injection, plm60 was slowly eliminated from plasma relative to f-M, resulting in about 6459-fold increase in AUC and its plasma kinetics exhibited dose dependence. In S-180 bearing KM mice, plm60 preferentially accumulated into tumor zone, with a approximately 12-fold increase in AUC and approximately 10-fold increase in C(max) Furthermore, the accumulation of plm60 in almost all normal tissues markedly decreased. The antitumor efficacy of plm60 was also considerably enhanced. In L1210 ascitic tumor model, plm60 was the most efficacious which led to a approximately 70% long-term survival, significantly compared to 16-33% survival rate in plm80, plm100 and f-M groups at the same dose level (4mg/kg). The antitumor efficacy of plm60 was more encouraging in L1210 liver metastasis model. At a dose of 6mg/kg, approximately 90% animals receiving plm60 treatment could survive 60 days; however, in f-M group at the same dose, all the mice died at approximately 14 days post inoculation. Similarly, plm60 could effectively inhibit the growth of RM-1 tumor in BDF1 mice, resulting in marked increase in tumor doubling time at different dose levels relative to f-M. The improved antineoplastic effects could be ascribed to its small vesicle size, which allowed more drug release after the accumulation into tumor zone. Theoretical considerations revealed that the reduction of vesicle size could increase the specific area of MIT/sulfate precipitate inside the vesicle and the release constant K, which is inversely proportional to vesicle volume (K=pA(m)k(2)k(2)(')/([H(+)](i)(2)V(i))).

Copper ion-mediated liposomal encapsulation of mitoxantrone: the role of anions in drug loading, retention and release.

Besides pH gradient, other transmembrane gradients such as metal ion gradient could be also employed to load drugs into liposomes. In pH gradient method, anions have an important role since they could form specific aggregates with drugs, and then affect drug release kinetics from vesicles. To explore the role of anions in metal ion gradient method, copper ion-mediated mitoxantrone (MIT) loading was investigated systematically. When empty liposomes exhibiting a transmembrane copper ion gradient (300 mM) were mixed with MIT in a molar ratio of 0.2:1, after 5 min incubation at 60 degrees C, >95% MIT could be loaded into vesicles and the encapsulation was stable, regardless of the kinds of anions and initial intraliposomal pH values. The encapsulation ratio decreased with increased MIT/lipid molar ratio. But even when the molar ratio increased to 0.4, >90% encapsulation could still be achieved. In the presence of nigericin and ammonium, the drug loading profiles were affected to different degree with respect to both drug loading rate and encapsulation ratio. Relative to CuSO(4)-containing systems, CuCl(2) mediated MIT loading was unstable. Both nigericin and ammonium could alter the absorption spectra of liposomal MITs loaded with CuSO(4) gradient. In vitro release studies were performed in glucose/histidine buffer and in 50% human plasma using a dialysis method. In both of release media, CuCl(2)-containing vesicles displayed rapid release kinetics in comparison with CuSO(4) systems; and during the experiment period, MIT was lost from the vesicles continuously. When the formulations were injected into BDF1 mice at a dose of 4 mg/kg, all the liposomal formulations exhibited enhanced blood circulation time, with half-life values of 6.8-7.2h, significantly compared to the rapid clearance of free-MIT. In L1210 ascitic model, CuCl(2) formulation was more therapeutically active than CuSO(4) formulation. At a dose of 6 mg/kg, the treatment with CuCl(2) formulation resulted in a median survival time of 21 days, considerably larger than that of CuSO(4) groups (15 days). Based on these data, it was concluded that during the drug loading process, a dynamic transmembrane pH gradient is generated and intraliposomal pH might affect the complexation manner in which Cu(2+) binds MIT. Owing to the presence of pH gradient, after the accumulation within vesicles, a part of MIT will be protonated and precipitated by sulfate. Accordingly, the aggregation status of MIT inside CuSO(4) system was more complicated than that in CuCl(2) vesicles. The difference in physical status of MIT aggregates affects not only the drug release rate, but also their therapeutic effects.