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VIROSOMES Roshan M A Pharmaceutics DEPT. Srinivas College of Pharmacy Valachill , Mangalore




INTRODUCTION A virosome is a drug or vaccine delivery mechanism consisting of unilamellar phospholipid vesicle incorporating virus derived proteins to allow the virosomes t o fuse with target cells. V irosomes are not able to replicate but are pure fusion active vesicles. These are reconstituted viral envelops that can serve as vaccines and as vesicles for cellular delivery of macromolecules. Influenza virus is the most common virus of choice. The success of virosomal drug delivery depends on the methods used to prepare the encapsulated bioactive materials and incorporate them into the virosomes , as are characterization and formulation of finished preparation. Virosomes protect pharmaceutically active substance from proteolytic degradation and low pH within endosomes, allowing their contents to remain intact when they reach the cytoplasm 4/24/2014 3

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In contrast to liposomes, virosomes contain functional viral envelope glycoproteins: influenza virus hemagglutinin (HA) and neuraminidase (NA) are intercalated within the phospholipid bilayer membrane Virosomal Ultrastructure and Modifications: 4/24/2014 4

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Fusion activity of virosomal carriers Virosomes has the unique properties of fusion because of the existence of influenza HA in their membrane. HA is responsible for the structural stability and virosomal formulation homogeneity, also significantly contributes to the fusion activity of virosomes . Virosomal HA promotes binding at the target cell surface followed by receptor-mediated endocytosis. The acidic environment of the endosome responsible for stimulation of HA-mediated membrane fusion, and the therapeutically active substance escapes from the endosome into the cytoplasm of the target cell. Thus , virosomal HA significantly enhances cytosolic delivery . This is a major advantage of virosomes over liposomes and proteoliposomal carrier systems, which provide less protection for therapeutic macromolecules from different compartmental unadoptable micro-environments. 4/24/2014 5

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Advantages of Virosomal Drug Delivery Virosomal technology is approved by the FDA for use in humans, and has a high safety profile. Virosomes are biodegradable, biocompatible, and non-toxic. No disease-transmission risk. Patent protected. No autoimmunogenity or anaphylaxis. Broadly applicable with almost all important drugs (anticancer drugs, proteins, peptides, nucleic acids, antibiotics, fungicides ). Enables drug delivery into the cytoplasm of target cell. Promotes fusion activity in the endolysosomal pathway. 4/24/2014 7

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Protects drugs against degradation. Encapsulation of drug protects patient against side effects. Target-specific delivery of antigens and amplification of the immune response. Extended uptake, distribution and elimination of the drug in the body. Virosomes allow patient specific modular vaccine regimen. Up-scaling according to standard procedure. The fully functional fusion-activity of virosomes enables receptor mediated uptake and natural intracellular of the antigen, which leads to stimulation of both arms of the immune system such as humoral and cellular immune responses . The antigen is partially protected from extracellular degradation and the resulting depot effect greatly facilitates immune potentiation. 4/24/2014 8

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Mode of action: Virosomes operate both as a carrier and as an adjuvant, with multiple functions for the duration of initiation of an immune response. The carrier function comprises the affirmative effects of embedding the antigen into a privileged structure, the virosome particle. The adjuvant task relates to immune stimulating properties of the virosomes and their components on the immune system. Most importantly, virosomes accomplish something in stimulating specific immunity without causing nonspecific inflammation. 4/24/2014 9

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4/24/2014 10 Carrier function: The integration of the antigen into the higher structure of the virosome particle stabilizes the antigen, preserves the native status of B cell epitopes , and protects the antigen from degradation. The antigen displayed on the virosomal surface mimics the original pathogen or target cell and thereby favors the generation of antibodies relevant for protection. Moreover, the presentation of the antigen as a repetitive surface structure enhances its recognition by antibody-producing B cells. Finally, the size and surface structure of the virosome particles make them an attractive target for uptake and processing by immune cells, which is a crucial step in the initiation of an immune response.

adjuant function: :

adjuant function : The adjuvant function of virosome relies on the presence of influenza derived envelope proteins, in particular the predominant HA. Pre-existing antibodies against influenza bind to virosome and tag them efficiently for rapid uptake and processing by antigen presenting cells. Also pre-existing influenza – specific helper T cells are activated by those APC displaying the processed fragments of the influenza proteins. Activated helper cells rapidly proliferate and secrete cytokines to support and enhance the induction of effect of immune cells. 4/24/2014 11

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4/24/2014 12 Mechanism of Virosomes to Stimulate the Immune Systems


METHOD OF PREPARATION 1. Selection of virosomes : Virosome are reconstituted viral envelope that can be derived from different virus. Influenza virus envelope is the most commonly used to produce virosome but virosomes can be made from Sendai virus, Epstein burr- virus, HIV, Sindbis , Semlikiforest , virus Friend murine leukemia virus , herpes simplex virus . 2. Selection of antigen: Antigen is selected as per requirement. Antigen such a parasite, carcinogenic cell, bacterium or whole cell is used. As antigen such as cell component DNA, RNA or plasmid can also used as antigen. This antigen is coupled to lipid anchor, so antigen will ready to load on virosomes 3. Reconstitution of virosome : Virosome solubilised with detergent ( octaglucoside , triton x-100, nonidert p-40 ). Due to solubilization with detergent, internal viral protein and genetic material will sediment. Then detergent is removed by different method such as dialysis and hydrobhobic resins from supernatant. 4/24/2014 13

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Then using ultracentrifugation process viral matrix protein and nucleiocapsid is removed. Viral phospholipid (82%) and viral protein is recovered. Now antigen which is already coupled to lipid anchor is mixed with polymer or surfactant solution and this solution is processed with virosome carrier so that antigen bound virosome is obtained. 4/24/2014 14


CHARACTERIZATION OF VIROSOMES Protein detection : Virosome preparation should generally result in a relatively uniform protein-to-lipid ratio. Sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) can confirm the presence of HA protein in the virosomes . Structure and size : Negative-stain electron microscopy can generally be used to determine the ultra structure and size of virosomes . The staining solutions should preferably be of neutral pH, to avoid acid-induced conformational changes of HA.18 Fusion activity : Generally virosomes exhibit pH- dependent membrane fusion activity similar to native influenza virus. Virosomal fusion with biological or artificial target membranes can be assessed in vitro with an excimer assay using pyrene-labeled lipids, where the decrease of surface density of the pyrene - phosphatidylcholine -label on fusion with an unlabeled membrane corresponds to a reduction of excimer fluorescence . Fusion activity also can be indirectly monitored by determining hemolytic activity, which corresponds closely to fusion activity and exhibits pH dependence identical with that of fusion. 4/24/2014 15

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Surface charge: free flow electrophoresis Electrical surface potential and surface pH : zeta potential measurements and pH sensitive probes lamellarity : small angle x-ray scattering, freeze fracture electron microscopy, 13p- nmr Phase behavior : freeze fracture electron microscopy, differential scanning colorimetry Percent of free drug : mini column centrifugation, gel exclusion, ion exchange chromatography, protamine aggregation, radiolabel ling Drug release : diffusion cell/ dialysis Pyrogenicity : rabbit fever response test or limulus ambeocyte lysate ( lal ) test Animal toxicity : monitoring survival rates, histology and pathology Chemical analysis of surface : static secondary ion mass spectrometry, spectrometer 4/24/2014 16


Applications Cancer treatment : Virosome have been also used in oncology field to carry peptide corresponding to tumour associated antigen as in case of peptide from parathyroid hormone related protein or from recombinant proteins such as her -2 neu Fab combined the anti Fab – doxovirosome combined the anti proliferate properties of the monoclonal antibodies and cyototoxic effect of doxorubicin in vivo. Gene delivery : Haemagglutinin the membrane fusion protein of influenza virus is known to mediate a low pH dependent fusion reaction between the viral envelope and the limiting membrane of endosomal cell compartment following cellular uptake of virus particle by receptor mediated endocytosis. 4/24/2014 17

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Virosomes associated with plasmid DNA, antisense oligonucleotides given by IV,IM,IA,IT, topical and oral used in gene and antisense therapy desiring high yield DNA incorporation, fusogenic ( cationic,virosome,phsensitive ), and targeted . Malaria therapy : Virosome represent an innovative drug delivery system for various biologically active molecules, but especially nucleic acid or genes and for numerous indications. The surface of virosomes can be suitably modified to facilitate targeted drug delivery. 4/24/2014 18

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General Applications: Blood substitutes for hemoglobin Immunoadjuvat , immunomodulator , immunodiagnosis Artificial blood surrogates Pharmaceutical pigments or dyes In tumor therapy a carrier of small cytotoxic molecules Vehicle for macromolecules as cytokines or genes As biological response modifiers Virosomal drugs Virosomes as drug/protein drug delivery vehicles As radiopharmaceutical and radio diagnosis carriers 4/24/2014 19

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As gene therapy genetic targeted intracellular material delivery Enzyme replacement therapy and lysosomal storage disorders. In cosmetics and dermatology Enhanced drug solubilization In antifungal (lung therapy), antiviral and anti microbial therapy Altered pharmacokinetics and biodistribution Masking action Drug overdose treatment Separation and extraction technique In fabrication of microcapsulated dosage form Enzyme immobilization 4/24/2014 20

Marketed products:

Marketed products S. No. Virosomal preparations Application A Virosomes antigen based products   1 1 Hepatitis A virus envelope proteins (EpaxalW) Hepatitis a 2 Influenza virus (InflexalW V) Influenza B Virosomal antigen preparations under clinical trials   1 Diphtheria/tetanus toxoid virus envelope proteins Diphtheria, Tetanus 2 Peptidomimetic of loop I from domain III of Plasmodium falciparum AMA-1 Malaria 3 PEV6 Breast cancer C Virosomal antigen preparations under pre clinical trials   1 Doxorubicin Cancer 2 Doxorubicin Ovarian carcinoma 3 L-myc antisense ODNs Cancer 4 DNA-encoded TAA Prostate Carcinoma 5 DNA-encoded mumps antigen Mumps 6 Melan A/Mart-1 peptides Melanoma 7 RSV F protein RSV 8 Hepatitis C peptides Hepatitis C 9 VSV G protein Vesicular stomatitis 10 NDV envelope proteins Newcastle disease 11 HIV proteins AIDs 12 PEV4 RSV 13 PEV7 Candida 14 PEV 8 Universal influenza 4/24/2014 21


references Sowmya G, Mandanapu C. Virosomal Drug Delivery System: A Novel Vaccination Technology. American J PharmTech Res. 2013; 3(3): 35-54 Rajat S, Mohd Y. Virosomes : A Novel Carrier for Drug Delivery. Int J PharmTech Res. 2010; 2(4): 2327-2339 Kapoor D, Vyas RB, Lad C, Patel M. A multipurpose and novel carrier for drug delivery and targetting - virosomes . J Drug Delivery & Therapeutics.2013; 3(5):143-147 Sylvain Fleury . Virosomes : Enveloped VLP-based vaccines for eliciting mucosal response as front line defense against HIV-1 and RSV entry and infection Christian Moser. Virosome technology. Pevion Biotech. Priyanka R, Gaurav S. Virosomes : A novel vaccination technology. Int J Ph Sci Res. 2012; 3(10): 3591-3597 4/24/2014 22

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