Commentary: "A systems view on the future of medicine: inspiration from Chinese medicine?".

Chinese medicine could serve as a source of inspiration for drug development. Using systems biology in combination with reverse pharmacology is a novel way for the discovery of novel biological active compounds and targets as well as for proving the occurrence of synergy and prodrugs. A key factor for coming to evidence-based Chinese medicine will be the quality control. Metabolomics is a very promising tool for this purpose.

Liposomes as sustained release system for human interferon-gamma: biopharmaceutical aspects.

Interferon-gamma (IFNgamma) has proven to be a promising adjuvant in vaccines against cancer and infectious diseases. However, due to its rapid biodegradation and clearance, its efficacy is severely reduced. Liposomal association might prolong the residence time of IFNgamma, but no efforts have been made to optimize the biopharmaceutical characteristics of liposomal IFNgamma for its application in therapy or as vaccine immunoadjuvant. In the present study, various liposomal formulations of recombinant human IFNgamma (hIFNgamma), differing in lipid composition, were prepared via the film hydration method and characterized in vitro regarding association efficiency and bioactivity, and in vivo regarding cytokine release kinetics after subcutaneous (s.c.) administration into mice. Human IFNgamma can be formulated in large, multilamellar liposomes with high association efficiency (>80%) and preservation of bioactivity. A critical parameter is the inclusion of negatively charged phospholipids to obtain a high liposome association efficiency, which is dominated by electrostatic interactions. The fraction of externally adsorbed protein compared to the total associated protein can be minimized from 74+/-9% to 8+/-3% by increasing the ionic strength of the dispersion medium. After injection of free (125)I-hIFNgamma, the radiolabel was detectable up to 48 h at the injection site. Liposomal encapsulation of (125)I-hIFNgamma increased the local area under the curve 4-fold, and the presence of the radiolabeled hIFNgamma at the injection site was prolonged to 7 days. The release kinetics and overall residence time of the cytokine at the s.c. administration site was influenced by depletion of the externally adsorbed IFNgamma, reducing the initial burst release. Increasing the rigidity of the liposome bilayer also resulted in a more pronounced reduction of the burst release and a 19-fold increase in the residence time of the protein at the s.c. administration site, compared to the free cytokine. As adjuvanticity of liposomal IFNgamma may strongly depend on the release kinetics of cytokines in vivo, the findings in this paper may contribute to a rational design of liposomal-cytokine adjuvants in vaccines against cancer and infectious diseases.

Subcutaneous administration of superoxide dismutase entrapped in long circulating liposomes: in vivo fate and therapeutic activity in an inflammation model.

PURPOSE: We are exploring liposomal delivery with the aim to change the pharmacokinetics and biodistribution of SOD to increase its therapeutic activity. From a practical point of view, a convenient route of administration would be the subcutaneous (s.c.) route. Liposomal size has been shown to be the most important factor influencing the rate and extent of drainage of liposomes from the s.c. injection site. METHODS: To monitor the in vivo fate of the subcutaneous administered SOD-containing liposomes in rats with a chronic arthritis inflammation, the liposomes were labeled by the co-encapsulation of the 111In-DTPA complex in the internal water space. RESULTS: Over the initial 10h-observation period post-injection, the small-sized poly(ethyleneglycol)-liposomes (mean size about 110 nm) left the site of injection to a 2-fold higher extent (45% of the injected dose) as compared to large-sized poly(ethyleneglycol)-liposomes (mean size about 450 nm). Small-sized liposomes gave a 17-fold higher uptake in the inflamed foot than the large-sized liposomes. Comparing the localization in the inflamed foot with the non-inflamed foot, uptake was more than 15-fold higher for the small-sized liposomes as compared to the large-sized liposomes. After s.c. administration, small-sized SOD-liposomes showed substantial higher activity than large-sized SOD-liposomes. S.C. administration of small-sized SOD-liposomes is equally effective as i.v. administration of the same liposomes. I.V. administration of the large-sized SOD-liposomes yielded a significantly higher activity as compared to s.c. administration. CONCLUSIONS: These results indicate that small-sized poly(ethyleneglycol)-liposomes can be used for the targeting of SOD to arthritic sites after subcutaneous administration.

Intravenous administration of superoxide dismutase entrapped in long circulating liposomes. II. In vivo fate in a rat model of adjuvant arthritis.

Rheumatoid arthritis (RA) is a prevalent and debilitating autoimmune disease that affects the joints. RA is characterized by an infiltration of the affected joint by blood-derived cells. In response to activation, these cells generate reactive oxygen species, resulting in an oxidative stress situation. One approach to counteract this oxidative stress situation is the use of antioxidants as therapeutic agents. The free radical scavenger enzyme superoxide dismutase (SOD) may be used as a therapeutic agent in rheumatoid arthritis, but its rapid elimination from the circulation is a major limitation. Targeted delivery of SOD may overcome this limitation. In this study, the utility of PEGylated liposomes (PEG-liposomes) for targeting SOD to arthritic sites was explored. The targeting of SOD to arthritic sites following intravenous administration of both PEG-liposomes and positively charged liposomes lacking PEG but containing stearylamine (SA-liposomes) in rats with adjuvant arthritis was studied. At 24 h post injection, the blood levels of long circulating liposomes with a mean size of 0.11 micrometer and 0.20 micrometer were 8- and 3-fold higher, respectively, as compared to the SA-liposomes. The majority of SOD administered in liposomal form remains within the liposomes when they circulate in the bloodstream. The highest target uptake was observed with PEG-liposomes with a mean size of 0.11 micrometer and the lowest uptake with the SA-liposomes. These results demonstrate that SOD can be targeted to inflamed sites most efficiently via small-sized PEG-liposomes. Small-sized PEG-coated liposomes are to be preferred if prolonged circulation and enhanced localization of SOD at arthritic sites are desired.

Thrombolytic treatment with tissue-type plasminogen activator (t-PA) containing liposomes in rabbits: a comparison with free t-PA.

In this study, we aimed at improving the therapeutic index of tissue-type Plasminogen Activator (t-PA) as thrombolytic agent in the treatment of myocardial infarction. Liposome-encapsulated t-PA was tested in a rabbit jugular vein thrombosis model: administration of free t-PA (t-PA) as a bolus injection in the ear vein was compared to a similar administration of liposomal t-PA (t-PA-lip), liposomal t-PA in plasminogen-coated liposomes (Plg-t-PA-lip), a mixture of free t-PA and empty liposomes (t-PA+ empty lip) and a saline-blank (blank) in terms of thrombolytic activity and side effects. Liposomal t-PA (t-PA-lip/Plg-t-PA-lip) showed a significantly better thrombolysis efficiency than equimolar doses of free t-PA (t-PA/ t-PA+ empty lip): about 0.24 mg/kg of liposomal t-PA practically equalled the lysis-activity of a dose of free t-PA of 1.0 mg/kg (t-PA1mg/kg). On the other hand, liposome encapsulation did not affect the systemic activation of alpha 2-antiplasmin and plasminogen by t-PA. We conclude that for this model an improvement in thrombolytic efficacy of t-PA is achieved by liposome encapsulation of t-PA. As t-PA-lip and Plg-t-PA-lip -treatment induced similar results, targeting of liposomal t-PA by coupled glu-Plg remains a topic to be optimized in future studies.

Immunoliposomes in vivo.

Attachment of antibodies to the surface of liposomes was performed to confer specificity for a certain cell or organ expressing the targeted antigenic determinant. These so-called immunoliposomes are expected to be applied as targeted drug carriers. In this article, the literature concerning in vivo studies of the targeting of immunoliposomes to various sites in the body is reviewed. The anatomical, physiological, and pathological constraints and current progress are described. Moreover, perspectives on the therapeutic feasibility of this drug targeting system are discussed.

Reconstituted membranes of tumour cells (proteoliposomes) induce specific protection to murine lymphoma cells.

Antigens presented on cell membranes or on liposomes are usually more immunogenic than antigens in soluble form, this being one of the reasons for the weak immunogenicity of extracted tumour-associated transplantation antigens (TATA). The main objective of this study is to solubilize TATA from tumour cells and to present them on a membrane-like structure to the immune system. Crude tumour cell membranes of SL2 lymphosarcoma cells (a spontaneously arising, weakly immunogenic tumour) were solubilized with octylglucoside or sodium deoxycholate, and reconstituted membranes (proteoliposomes) were prepared by detergent removal. Mice immunized s.c. with reconstituted membranes were protected against an i.p. challenge with tumour cells. Although octylglucoside solubilized only 41% of the membrane proteins, the reconstituted membranes were as immunoprotective as crude membranes. (Glyco)proteins were probably the major membrane components in the reconstituted membranes that induce immunoprotection, as mice immunized with preparations constituted of (glyco)lipids from SL2 cells could not reject SL2 cells. If Freund's complete adjuvant was used with the first immunization injection, no potentiation of the elicited immune responses was observed. Besides the membrane TATA, SL2 cells contained an apparently non-membrane-bound TATA, which was found in the cytoplasm. It is concluded that detergent solubilization of membranes and subsequent preparation of reconstituted membranes can be used to obtain membrane tumour-associated antigens that retain activity for induction of protective tumour immunity. The major advantage of this method is that membrane proteins are solubilized and are subsequently presented on a membrane-like structure that resembles the tumour cell membrane. On theoretical and practical grounds it provides a promising alternative for whole-cell vaccines.

Intestinal absorption of drugs. I: The influence of taurocholate on the absorption of dantrolene in the small intestine of the rat.

The influence of sodium taurocholate (1) on the intestinal absorption of the lipophilic drug dantrolene (2) was studied in vivo in a chronically isolated internal loop in the rat. Concentrations of 2 were kept below the saturation concentration in saline. Absorption kinetics of 2 were evaluated on the basis of steady-state blood levels, which develop during single-pass perfusions, and on the basis of the rate of disappearance of the drug from the perfusate during recirculating perfusions. Compound 1 at a concentration of 10 mM in the perfusate induced a twofold reduction of the absorption rate compared with the same concentration of 2 in saline. Pretreatment of the absorptive surface with a 10 mM solution of 1 had no detectable effect on the absorption rate of 2 in saline. After perfusions with 10 mM solutions of 1, the perfusate concentration of proteins, phosphorus, and hexoses in the effluent was increased. The reduction of the absorption rate can be ascribed mainly to a reduction of the thermodynamically active concentration of 2 as calculated from the phase-separation model. In addition, 10 mM 1 seems to temporarily increase the barrier function of the mucous layer.

Incorporation of the major outer membrane protein of Neisseria gonorrhoeae in saponin-lipid complexes (iscoms): chemical analysis, some structural features, and comparison of their immunogenicity with three other antigen delivery systems.

We incorporated the major outer membrane protein (PI) of Neisseria gonorrhoeae into immunostimulating complexes (iscoms) and examined some analytical, physicochemical, and immunological properties of these structures. The immunogenicity was compared with that of three other PI-containing structures, i.e., liposomes, outer membrane complexes produced by the bacterium, and protein-detergent-adjuvant complexes. AIPO4 and dioctadecyldimethylammonium bromide were used as adjuvants. Our results show that iscoms are much more immunogenic than liposomes and protein-detergent complexes but are also much more toxic. The localization of PI in iscoms was investigated. Therefore, the chymotrypsin susceptibility of PI in iscoms was tested, and the incorporation of fragments of PI was determined. Amphiphilic fragments of PI were incorporated in iscoms, but hydrophilic and hydrophobic fragments were not. Chymotrypsin degradation of PI in iscoms indicated that the protein is exposed to the environment in a similar manner as PI in outer membrane complexes, i.e., with both termini anchored in the iscom.

The interaction of cytostatic drugs with adsorbents in aqueous media. The potential implications for liposome preparation.

A new method that involves the use of the cation exchange resin Dowex 50W-X4 to remove non-encapsulated drugs from liposome dispersions was investigated. Cytostatic drugs widely varying in their molecular structure can be removed from aqueous solutions by Dowex 50W-X4. The applicability of the resin to separate free from liposome-bound drugs was illustrated for a number of cytostatic drugs (cisplatin, doxorubicin, vincristine). The technique presented allows for a rapid, efficient and convenient procedure for the free drug removal from liposome dispersions without dilution of the liposomal preparation. Studies with liposome-encapsulated drugs will be facilitated by the use of this method, since it avoids many of the problems introduced by conventional methods as dialysis, gel filtration and centrifugation/washing. To elucidate the interaction mechanism of doxorubicin with Dowex 50W-X4, alternative adsorbents were studied for their doxorubicin binding properties. In the adsorption process of doxorubicin onto Dowex 50W-X4 both electrostatic (ion exchange) and hydrophobic effects play a role. The results indicate that hydrophobic contributions to the interaction are responsible for the high resistance offered by the binding forces against desorption of adsorbed doxorubicin. For other adsorbents the interactions are either mainly of an electrostatic or a hydrophobic nature.