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Negatively charged human serum albumins as inhibitors of HIV-1 replication: Mechanism of action and in vivo efficacy in mice
Podium Presentations-Drug
Delivery
III
Intradlctkn: The purposa of thii study was to axamna tha possibilities of livar call specific delivery of tha an&iiflnmatory drug naproren (Nap1 using a human sarm albumin lHSAl conjugate. Pmvioua studii have showntb cell specifii delivery of nagativaly charged HSA rnolacub IMeijar er d, Sem in Liar Di 15202. 19951. Liver andothalial calls and Kupffar cefls play an important role in the pathoganaais of acute and chronic inflammatory liver diseases. Targeting Nap to thasa calfs might incmase the efficacy of this drug in the treatment of inflammatory liver diseases and mduca its side affects. The HSA-conjugats lNap,HSAI consisted of 23 Nap-mole&s directly coupled to the lysinic amino groups of HSA through their carboxylii groups, which causes an increased net rwgative charge and adds extra hydrophabiiity to tha HSA mobcule. Rae&r: During acute inflarrvnation, livers showed an increased uptake of Nap when coupled to HSA as compared to administrating fme Nap. After 3 hours 28% of tha administered dose of Nap,.HSA is found in tha liver versus 1.1% for the 50 mglkp Nap dose (p
Porous bead cellulose (BC) fipophilic solvent to prepare
was loaded with isopropyl myristate as a a carrier system for lipophilic drugs as
well as for retarded drug release. The products were manufactured by a special dissolution-wprecipitatkm process in connection with infrared solvent evaporation. Up to the threefold weight ratio of EC to isopropyl myristate the coprecipitates wBre spherical, flowable and only slightly agglomerated. Isopropyl myristate was adsorbed at the surface of the beads forming a transparent film, but it was also incorporated into the pores preventing the beads from shrinking during coprecipitation. The contact angle amounted to 58’ indicating wettability and adsorption forces between isopropyl myristate and cellulose compared to the higher contact angle of 71’ for pure BC and water. On the other side hydrophobic properties of the BC isopropyl myristate coprecipitates wBre evident from a contact angle of 90’ for water. Prednisolon and griseofulvin are only slightly soluble in isopropyl myristate and therefore used as model drugs.for retarded release investigations. In dependence of weight ratio of isopropyl myristate to prednisolone and resulting dispersed state the drug release was fast in the case of amorphous and retarded et crystalline state. In comparison, from cqxecipitates of pure BC and of BC with hydrophilic solubilizer and prednisolone the release was complete “ I during the first 10 minti’\ The release of crysta me dispersed griseofulvin from coprecipitates was retarded with increasing content of isopropyl myristate. By variation of the weight ratio of BC to isopropyl myristate to drug the release rate can be changed in a tide range. Coprecipitates of BC and isopropyl myristate can be used as spherical, flowable carriers of hiih loading capacity with retarded drug release. Application is envisaged as pure granules or as hard gelatine capsule filling material.
I Eur. J. Phnrm. Sci. 4 Suppl. (1996) S87-SN8
Objaatiw: Negatively chargad albumins (NCAs). with the prototypas SucHSA and Aco-HSA. am polyanionic proteins with a potent antivbal activity on HIV-l laboratory strains. In the present study the mechanism of their antiviral action was addrassad. Resulted Formation of syncytia of infected and uninfected T-lymphecytas was inhibited by the NCAs. Earlyproducts of reverse transcription, in cells inoculated with HIV-l in the prasance of NCAs. were also absent. Interaction between NCAs and irranobiiad HIV.1 peptidas was only observed for the positively charged V3 loop and the C.tarminal part of gpl20. This binding is predominantly caused by electrostatic interactions as indicated with cmnptition studies with heparin and dextran sulfate, although hydrophobic domains of the NCAs warn also involved. Furthermore protaolytic cbavage of tha tip of tha V3 loop IGPGRAF saquance) caused a complete loss of binding affinity of the NCAs for the V3 loop. In addition. it was observed that the NCAs bind to uninfected human lymphocytes and that prastimulation of lhase lymphocytes with PHA markedly increased the binding of tha NCAs to the calls. The LC, vakres of the NCAs against the priiary HIV-1 isolates were all in the nanomalar range. Furthermore, tha NCAs showed equipotent inhibition of rapfication of a mecrophegstropic HIV.1 variant in either PEW or pnmary macrophagas. I.p. injections of 3 and 300 mg/kg SWHSA were sufficient to complataly protect knmunosupmssadmica grafted with human bbed calls against infection wrth HIV.1, whils avan an i.p. injection of 0.3 mgikg partially protected the grafts. Coecluriana: These results indicate that tha NCAs intarfara at an early level in the virus replication cycle and suggest that the antiviral activity of the NCAs is based on shielding of the positively charged V3 loop from a fwKtional interaction with tha target call surface, both by binding to the HIV-l particbs end by bindii to inducabb racaptors on the target calls. Preliminary results indicate that NCAs a!sa pmvant protaolyti cleavega of tha V3 loop that is supposed to pm&e tha initiation of the actual fusion prowsa. These thma eapasts may explain tha prafemntial influanca of the NCAs on viruslcafl fuaian instead of gpl20jCD4 binding. This combined with the potent h v&o anti-HIV-1 activity in various HIV.1 infected cell types and tha antiviral affieaccy in tha HN-1 mousa modal, mndar thaaa conjugates promising candidates for the tasting of their anti-HIV activity in humans for tha use as intriisically active carriars for otfmr anti-HIV agents (dual targeting).
Lectins are proteins or glycoproteins of non-immune nature capable of binding to one or more speclic sugar residues. Some lectins have been reported to be mitogenic, inflammatory and/or oytotoxic (Shier 1979). Previous work has investigated the potent&l for using la&Ins as a means of ‘anchoring’ a drug delivery system IO the mucosal surfaces of eye or mouth, and the lectins from Manurn tuberosum and Helixpomatia. have shown particular promise. The aim of this study was to consider the acute toxicity of these lacttns. in terms of their potential to cause inflammation and tissue necrosis. Three female New Zealand white rabbits had their backs shaved 24 h prior to the study. These were then anaesthetised with an intraperitoneal injection of pentobarbitone sodium (2.6 mg Kg-l), then 1 mLof2%EvansbluesolutioninjectedIntoamarginalearvein. The lectin solutions were prepared in normal sellne at a range of concentrations from 50 to 500 pg ml-1 and sterilii by filtration. Solutions of 1% carageenan and 20% ethanol were used as positive controls and normal saline as a negative control. 50 pL volumes oi the test solutions were injected intradermalty at regular intervals across each rabbii’s back. The rabbits were maintained under anaesthesia for 3 h then killed with an overdose of anaesthetic. An inflammatory response was visualised by a weal of Evans blue accompanied by oedema at the site of injection. and this was evident with the positive controls. There was however no evidence of an inflammatory reaction wtth any of the iectin solutions tested. On histological sectioning slightly more heterophils wetwapparent in the tissue sections with only the 500 kg ml-l concentration of both lectins. It can be concluded that these lectins do not show any significant acute toxicity, and may therefore be taken forward for further in-vivo studies. Shier W.T. in: Drug carriers in Biology and Medicine G.). Academic Press, London (1979) pp. 43-70.
(EdGregordiadis
Delivery
III
Intradlctkn: The purposa of thii study was to axamna tha possibilities of livar call specific delivery of tha an&iiflnmatory drug naproren (Nap1 using a human sarm albumin lHSAl conjugate. Pmvioua studii have showntb cell specifii delivery of nagativaly charged HSA rnolacub IMeijar er d, Sem in Liar Di 15202. 19951. Liver andothalial calls and Kupffar cefls play an important role in the pathoganaais of acute and chronic inflammatory liver diseases. Targeting Nap to thasa calfs might incmase the efficacy of this drug in the treatment of inflammatory liver diseases and mduca its side affects. The HSA-conjugats lNap,HSAI consisted of 23 Nap-mole&s directly coupled to the lysinic amino groups of HSA through their carboxylii groups, which causes an increased net rwgative charge and adds extra hydrophabiiity to tha HSA mobcule. Rae&r: During acute inflarrvnation, livers showed an increased uptake of Nap when coupled to HSA as compared to administrating fme Nap. After 3 hours 28% of tha administered dose of Nap,.HSA is found in tha liver versus 1.1% for the 50 mglkp Nap dose (p
Porous bead cellulose (BC) fipophilic solvent to prepare
was loaded with isopropyl myristate as a a carrier system for lipophilic drugs as
well as for retarded drug release. The products were manufactured by a special dissolution-wprecipitatkm process in connection with infrared solvent evaporation. Up to the threefold weight ratio of EC to isopropyl myristate the coprecipitates wBre spherical, flowable and only slightly agglomerated. Isopropyl myristate was adsorbed at the surface of the beads forming a transparent film, but it was also incorporated into the pores preventing the beads from shrinking during coprecipitation. The contact angle amounted to 58’ indicating wettability and adsorption forces between isopropyl myristate and cellulose compared to the higher contact angle of 71’ for pure BC and water. On the other side hydrophobic properties of the BC isopropyl myristate coprecipitates wBre evident from a contact angle of 90’ for water. Prednisolon and griseofulvin are only slightly soluble in isopropyl myristate and therefore used as model drugs.for retarded release investigations. In dependence of weight ratio of isopropyl myristate to prednisolone and resulting dispersed state the drug release was fast in the case of amorphous and retarded et crystalline state. In comparison, from cqxecipitates of pure BC and of BC with hydrophilic solubilizer and prednisolone the release was complete “ I during the first 10 minti’\ The release of crysta me dispersed griseofulvin from coprecipitates was retarded with increasing content of isopropyl myristate. By variation of the weight ratio of BC to isopropyl myristate to drug the release rate can be changed in a tide range. Coprecipitates of BC and isopropyl myristate can be used as spherical, flowable carriers of hiih loading capacity with retarded drug release. Application is envisaged as pure granules or as hard gelatine capsule filling material.
I Eur. J. Phnrm. Sci. 4 Suppl. (1996) S87-SN8
Objaatiw: Negatively chargad albumins (NCAs). with the prototypas SucHSA and Aco-HSA. am polyanionic proteins with a potent antivbal activity on HIV-l laboratory strains. In the present study the mechanism of their antiviral action was addrassad. Resulted Formation of syncytia of infected and uninfected T-lymphecytas was inhibited by the NCAs. Earlyproducts of reverse transcription, in cells inoculated with HIV-l in the prasance of NCAs. were also absent. Interaction between NCAs and irranobiiad HIV.1 peptidas was only observed for the positively charged V3 loop and the C.tarminal part of gpl20. This binding is predominantly caused by electrostatic interactions as indicated with cmnptition studies with heparin and dextran sulfate, although hydrophobic domains of the NCAs warn also involved. Furthermore protaolytic cbavage of tha tip of tha V3 loop IGPGRAF saquance) caused a complete loss of binding affinity of the NCAs for the V3 loop. In addition. it was observed that the NCAs bind to uninfected human lymphocytes and that prastimulation of lhase lymphocytes with PHA markedly increased the binding of tha NCAs to the calls. The LC, vakres of the NCAs against the priiary HIV-1 isolates were all in the nanomalar range. Furthermore, tha NCAs showed equipotent inhibition of rapfication of a mecrophegstropic HIV.1 variant in either PEW or pnmary macrophagas. I.p. injections of 3 and 300 mg/kg SWHSA were sufficient to complataly protect knmunosupmssadmica grafted with human bbed calls against infection wrth HIV.1, whils avan an i.p. injection of 0.3 mgikg partially protected the grafts. Coecluriana: These results indicate that tha NCAs intarfara at an early level in the virus replication cycle and suggest that the antiviral activity of the NCAs is based on shielding of the positively charged V3 loop from a fwKtional interaction with tha target call surface, both by binding to the HIV-l particbs end by bindii to inducabb racaptors on the target calls. Preliminary results indicate that NCAs a!sa pmvant protaolyti cleavega of tha V3 loop that is supposed to pm&e tha initiation of the actual fusion prowsa. These thma eapasts may explain tha prafemntial influanca of the NCAs on viruslcafl fuaian instead of gpl20jCD4 binding. This combined with the potent h v&o anti-HIV-1 activity in various HIV.1 infected cell types and tha antiviral affieaccy in tha HN-1 mousa modal, mndar thaaa conjugates promising candidates for the tasting of their anti-HIV activity in humans for tha use as intriisically active carriars for otfmr anti-HIV agents (dual targeting).
Lectins are proteins or glycoproteins of non-immune nature capable of binding to one or more speclic sugar residues. Some lectins have been reported to be mitogenic, inflammatory and/or oytotoxic (Shier 1979). Previous work has investigated the potent&l for using la&Ins as a means of ‘anchoring’ a drug delivery system IO the mucosal surfaces of eye or mouth, and the lectins from Manurn tuberosum and Helixpomatia. have shown particular promise. The aim of this study was to consider the acute toxicity of these lacttns. in terms of their potential to cause inflammation and tissue necrosis. Three female New Zealand white rabbits had their backs shaved 24 h prior to the study. These were then anaesthetised with an intraperitoneal injection of pentobarbitone sodium (2.6 mg Kg-l), then 1 mLof2%EvansbluesolutioninjectedIntoamarginalearvein. The lectin solutions were prepared in normal sellne at a range of concentrations from 50 to 500 pg ml-1 and sterilii by filtration. Solutions of 1% carageenan and 20% ethanol were used as positive controls and normal saline as a negative control. 50 pL volumes oi the test solutions were injected intradermalty at regular intervals across each rabbii’s back. The rabbits were maintained under anaesthesia for 3 h then killed with an overdose of anaesthetic. An inflammatory response was visualised by a weal of Evans blue accompanied by oedema at the site of injection. and this was evident with the positive controls. There was however no evidence of an inflammatory reaction wtth any of the iectin solutions tested. On histological sectioning slightly more heterophils wetwapparent in the tissue sections with only the 500 kg ml-l concentration of both lectins. It can be concluded that these lectins do not show any significant acute toxicity, and may therefore be taken forward for further in-vivo studies. Shier W.T. in: Drug carriers in Biology and Medicine G.). Academic Press, London (1979) pp. 43-70.
(EdGregordiadis