The goal of this study is to develop a controlled methotrexate delivery system that releases its content by a target specific stimulus. To accomplish this, both methotrexate and an engineered channel protein (MscL) were incorporated into DOPC/CH/DSPE-PEG liposomes, providing a controlled drug delivery system. Reconstitution and encapsulation methods were optimized in order to ensure optimal channel gating activity and high drug:lipid ratio. At the optimal conditions, a 95% release of MTX could be reached.

Deemter, M. van,

Meijberg, W.,

Robillard, G.,

Schaaf, G. van der,

Šmisterová, J.,

University of Groningen Digital Archive

Controlled Delivery of Methotrexate from Channel-Protein Containing Liposomes

The goal of this study is to develop a controlled methotrexate delivery system that releases its content by a target specific stimulus. To accomplish this, both methotrexate and an engineered channel protein (MscL) were incorporated into DOPC/CH/DSPE-PEG liposomes, providing a controlled drug delivery system. Reconstitution and encapsulation methods were optimized in order to ensure optimal channel gating activity and high drug:lipid ratio. At the optimal conditions, a 95% release of MTX could be reached

Deemter, M. van

Šmisterová, J.

Schaaf, G. van der

Meijberg, W.

Robillard, G.

ARTS repository - University of Groningen

   University of GroningenControlled Delivery of Methotrexate from Channel-Protein Containing LiposomesDeemter, M. van; Šmisterová, J.; Schaaf, G. van der; Meijberg, W.; Robillard, G.Published in:Journal of Controlled ReleaseIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Deemter, M. V., Šmisterová, J., Schaaf, G. V. D., Meijberg, W., & Robillard, G. (2005). Controlled Deliveryof Methotrexate from Channel-Protein Containing Liposomes. Journal of Controlled Release, 101(1-3), 342-343.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 13-02-2023CONTROLLED DELIVERY OF METHOTREXATE FROM CHANNEL-PROTEIN CONTAINING LIPOSOMESM. van Deemter, J. Šmisterová, G. van der Schaaf, W . Meijberg, G. RobillardBiomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The NetherlandsSummaryThe goal of this study is to develop a controlled methotrexate delivery system that releases its content by a target specific stimulus. Toaccomplish this, both methotrexate and an engineered channel protein (MscL) were incorporated into DOPC/CH/DSPE-PEG liposomes,providing a controlled drug delivery system. Reconstitution and encapsulation methods were optimized in order to ensure optimal channelgating activity and high drug:lipid ratio. At the optimal conditions, a 95% release of MTX could be reached.IntroductionIn tumor therapy, many potential drugs cannot be used because they are toxic to several vital organs. For these drugs, encapsulationin liposomes can be a solution. Further improvements in the design of liposomes resulting in specific release of drug at the tumorsite will make the utilization of liposomes more feasible. To accomplish this, we are developing a liposomal system containing a pH-sensitive proteinacious valve that opens as a response to lowered pH values, such as these reported, e.g., for solid tumors [1]. Thisproteinacious valve is the mechanosensitive channel of large conductance (MscL) from E.coli. The glycine at the 22nd position of theMscL is mutated to a cysteine, which is used to target either pH-sensitive chemical groups or [2-(trimethylamonium)ethyl]methanethiosulfonate bromide (MTSET) to the protein. Binding of MTSET introduces a positive charge into the hydrophobic channelpore leading to opening of the channel. MscL-G22C was reconstituted in therapeutically optimized liposomes composed of DOPC/Cholesterol/DSPE-PEG2000 (70:20:10), which was shown to be compatible with the channel protein activity. The encapsulationmethod for MTX has been optimized.Experimental methodsThe preparation of MscL containing liposomes with encapsulated MTX combines the detergent-mediated reconstitution of MscL in liposomesby using BioBeads and the encapsulation of MTX by one of the following methods: (a) lipid film rehydration in a drug-containing solution, (b)reverse phase evaporation and (c) addition of the drug in the presence of detergent during protein reconstitution. For the analysis of drug:lipidratio, MTX-proteoliposomes were separated from the free drug on a Sephadex G50 column. Total lipid content was determined from a sensitivefluorescence assay, using diphenylhexatriene as the probe (excitation at 355 nm and emission at 440 nm). MTX content was measured by HPLC(detection at 294/356 nm [2]). Drug:lipid ratio was calculated as Ag drug per mg total lipid.Release of MTX after activation with MTSET (pH nonsensitive) was determined after separation of liposomes and free drug ona desalting PD10 column followed by the measurement of total lipid and drug concentrations.Results and discussionIn order to see which MTX encapsulation method gives the highest drug:lipid ratio in combination with high drug release afteractivation, three methods were compared. The results can be seen in Table 1. All three encapsulation methods tested gave similardrug:lipid ratios (between 5 and 7 Ag/mg) and also similar release (about 95%) upon the activation of the channel. For practical reasons,the method in which the drug was encapsulated during protein reconstitution was used in further experiments.Table 1Drug:lipid ratios and drug release after different MTX encapsulation methodsEncapsulation method Drug:lipid (Ag/mg) Released drug afteractivation (%)Reverse phase evaporation 6.99 95During protein reconstitution 6.39 95Lipid film rehydration 5.13 94Poster Abstracts342ConclusionA proteoliposomal delivery system for MTX has been developed, which can release almost all encapsulated drug upon the activation of thechannel protein by chemical labeling. We are currently busy with the development of pH-sensitive proteoliposomes.References[1] G.R. Martin, K.R. Jain, Nonevasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imagingmicroscopy, Cancer Res. 54 (1994) 5670–5674.[2] S. V ezmar, U. Bode, U. Jaehde, Monitoring of methotrexate and reduced folates in the cerebrospial fluid of cancer patients, Int. J. Clin.Pharmacol. Ther. 40:582–583.TARGETED NORFLOXACIN IS ACTIVE IN VIVO AGAINST PERSISTENT MYCOBACT ERIUM BOVIS BCGD. Domurado1, A.-M. Balazuc2, M. Lagranderie2, P. Pescher2, P. Chavarot2, E. Roseeuw3, V . Coessens3, S. Stern4, E. Schacht3, G. Marchal21Groupe de Pharmacocinétique des Prodrogues et Conjugués Macromoléculaires (INSERM), CRBA-UMR 5473 CNRS, Faculté de Pharmacie,15 avenue Charles Flahault, BP 14491, 34093 Montpellier cedex 5, France2Laboratoire de Référence des Mycobactéries, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France3Biomaterial and Polymer Research Group, Department of Organic Chemistry, University of Ghent, Krijgslaan 281, S4 bis, 9000 Ghent,Belgium4Laboratoire de Radiobiologie Cellulaire, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 V illejuif cedex, FranceSummaryTuberculosis is difficult to treat because Mycobacterium tuberculosis persistent forms resist the usual antituberculous agents. This results in 2–3million deaths each year. In order to improve tuberculosis treatment, we established a short-term in vivo model of persistent mycobacteria. Also,we synthetized a macromolecular prodrug targeted toward macrophages and demonstrated its in vivo efficacy against persistent mycobacteria.To our knowledge, this is the first report of a drug active in vivo against persistent mycobacteria.IntroductionTuberculosis infects 2 billion people worldwide. Even if most of them present no symptoms and will never develop the disease, the eventualdevelopment of an overt tuberculosis, up to decades after contamination [1,2], results in 2–3 million deaths each year.The possible late development of tuberculosis results from the induction of Mycobacterium tuberculosis persistence by different conditions,such as nutrient starvation [3] or hypoxia [4]. This means that M. tuberculosis keeps its infectious capacity intact while it no longer multiplies.Persistent bacilli resist antituberculous agents, such as isoniazid, although the same antibiotics rapidly eliminate actively multiplying M.tuberculosis. Consequently, tuberculosis is very difficult to treat: the usual regimen requires four antibiotics for two months, then two antibioticsfor the next four months, watching out for the return of persistent bacteria to normal metabolism.In patients, persistent bacilli survive in macrophages after phagocytosis where they are rather well protected from immune response andantibiotic treatment. It is expected that antibiotics targeted toward macrophages improve treatment efficacy by concentrating drugs close tobacteria. The aim of this work was to design a conjugate able to deliver an antibiotic into macrophages phagosomal vacuole in close contact withthe intracellular bacteria.We synthesized targeted macromolecular prodrugs composed of a macromolecular carrier, of a targeting device and of an antibiotic activein vitro against M. tuberculosis. The carrier, dextran, bears both mannose, the homing device intended to target the macrophage, andnorfloxacin, to be delivered into the phagosomal vacuole [5]. Norfloxacin was linked to the macromolecular carrier through two differentpeptide arms.M. bovis BCG was used as a mycobacterial model because this bacterium has a physiology closely related to the physiology of M.tuberculosis, while being at the same time much less pathogenic than the latter, and because the immune response of the mice rapidly controlledextracellular infection and left only intracellular bacilli, i.e., the goal of the targeted norfloxacin.This model takes advantage of different pO2 levels in different mouse organs, hence of different persistence statuses of bacteria in theseorgans, and it therefore allowed us to test the in vivo antibiotic activity of our macromolecular prodrugs against persistent M. bovis BCG inmice. In this paper, we report the measurement of pO2 in liver and spleen of mice. We study the therapeutic activity of our macromolecularprodrugs versus isoniazid and ofloxacin used as controls against well oxygenated and hypoxic, hence persistent, bacilli by counting them inlungs, spleen and liver. These organs are rich in macrophages and possess different oxygenation statuses.Poster Abstracts 343

English

   University of GroningenControlled Delivery of Methotrexate from Channel-Protein Containing LiposomesDeemter, M. van; Šmisterová, J.; Schaaf, G. van der; Meijberg, W.; Robillard, G.Published in:Journal of Controlled ReleaseIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Deemter, M. V., Šmisterová, J., Schaaf, G. V. D., Meijberg, W., & Robillard, G. (2005). Controlled Deliveryof Methotrexate from Channel-Protein Containing Liposomes. Journal of Controlled Release, 101(1-3), 342-343.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-2023CONTROLLED DELIVERY OF METHOTREXATE FROM CHANNEL-PROTEIN CONTAINING LIPOSOMESM. van Deemter, J. Šmisterová, G. van der Schaaf, W . Meijberg, G. RobillardBiomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The NetherlandsSummaryThe goal of this study is to develop a controlled methotrexate delivery system that releases its content by a target specific stimulus. Toaccomplish this, both methotrexate and an engineered channel protein (MscL) were incorporated into DOPC/CH/DSPE-PEG liposomes,providing a controlled drug delivery system. Reconstitution and encapsulation methods were optimized in order to ensure optimal channelgating activity and high drug:lipid ratio. At the optimal conditions, a 95% release of MTX could be reached.IntroductionIn tumor therapy, many potential drugs cannot be used because they are toxic to several vital organs. For these drugs, encapsulationin liposomes can be a solution. Further improvements in the design of liposomes resulting in specific release of drug at the tumorsite will make the utilization of liposomes more feasible. To accomplish this, we are developing a liposomal system containing a pH-sensitive proteinacious valve that opens as a response to lowered pH values, such as these reported, e.g., for solid tumors [1]. Thisproteinacious valve is the mechanosensitive channel of large conductance (MscL) from E.coli. The glycine at the 22nd position of theMscL is mutated to a cysteine, which is used to target either pH-sensitive chemical groups or [2-(trimethylamonium)ethyl]methanethiosulfonate bromide (MTSET) to the protein. Binding of MTSET introduces a positive charge into the hydrophobic channelpore leading to opening of the channel. MscL-G22C was reconstituted in therapeutically optimized liposomes composed of DOPC/Cholesterol/DSPE-PEG2000 (70:20:10), which was shown to be compatible with the channel protein activity. The encapsulationmethod for MTX has been optimized.Experimental methodsThe preparation of MscL containing liposomes with encapsulated MTX combines the detergent-mediated reconstitution of MscL in liposomesby using BioBeads and the encapsulation of MTX by one of the following methods: (a) lipid film rehydration in a drug-containing solution, (b)reverse phase evaporation and (c) addition of the drug in the presence of detergent during protein reconstitution. For the analysis of drug:lipidratio, MTX-proteoliposomes were separated from the free drug on a Sephadex G50 column. Total lipid content was determined from a sensitivefluorescence assay, using diphenylhexatriene as the probe (excitation at 355 nm and emission at 440 nm). MTX content was measured by HPLC(detection at 294/356 nm [2]). Drug:lipid ratio was calculated as Ag drug per mg total lipid.Release of MTX after activation with MTSET (pH nonsensitive) was determined after separation of liposomes and free drug ona desalting PD10 column followed by the measurement of total lipid and drug concentrations.Results and discussionIn order to see which MTX encapsulation method gives the highest drug:lipid ratio in combination with high drug release afteractivation, three methods were compared. The results can be seen in Table 1. All three encapsulation methods tested gave similardrug:lipid ratios (between 5 and 7 Ag/mg) and also similar release (about 95%) upon the activation of the channel. For practical reasons,the method in which the drug was encapsulated during protein reconstitution was used in further experiments.Table 1Drug:lipid ratios and drug release after different MTX encapsulation methodsEncapsulation method Drug:lipid (Ag/mg) Released drug afteractivation (%)Reverse phase evaporation 6.99 95During protein reconstitution 6.39 95Lipid film rehydration 5.13 94Poster Abstracts342ConclusionA proteoliposomal delivery system for MTX has been developed, which can release almost all encapsulated drug upon the activation of thechannel protein by chemical labeling. We are currently busy with the development of pH-sensitive proteoliposomes.References[1] G.R. Martin, K.R. Jain, Nonevasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imagingmicroscopy, Cancer Res. 54 (1994) 5670–5674.[2] S. V ezmar, U. Bode, U. Jaehde, Monitoring of methotrexate and reduced folates in the cerebrospial fluid of cancer patients, Int. J. Clin.Pharmacol. Ther. 40:582–583.TARGETED NORFLOXACIN IS ACTIVE IN VIVO AGAINST PERSISTENT MYCOBACT ERIUM BOVIS BCGD. Domurado1, A.-M. Balazuc2, M. Lagranderie2, P. Pescher2, P. Chavarot2, E. Roseeuw3, V . Coessens3, S. Stern4, E. Schacht3, G. Marchal21Groupe de Pharmacocinétique des Prodrogues et Conjugués Macromoléculaires (INSERM), CRBA-UMR 5473 CNRS, Faculté de Pharmacie,15 avenue Charles Flahault, BP 14491, 34093 Montpellier cedex 5, France2Laboratoire de Référence des Mycobactéries, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France3Biomaterial and Polymer Research Group, Department of Organic Chemistry, University of Ghent, Krijgslaan 281, S4 bis, 9000 Ghent,Belgium4Laboratoire de Radiobiologie Cellulaire, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 V illejuif cedex, FranceSummaryTuberculosis is difficult to treat because Mycobacterium tuberculosis persistent forms resist the usual antituberculous agents. This results in 2–3million deaths each year. In order to improve tuberculosis treatment, we established a short-term in vivo model of persistent mycobacteria. Also,we synthetized a macromolecular prodrug targeted toward macrophages and demonstrated its in vivo efficacy against persistent mycobacteria.To our knowledge, this is the first report of a drug active in vivo against persistent mycobacteria.IntroductionTuberculosis infects 2 billion people worldwide. Even if most of them present no symptoms and will never develop the disease, the eventualdevelopment of an overt tuberculosis, up to decades after contamination [1,2], results in 2–3 million deaths each year.The possible late development of tuberculosis results from the induction of Mycobacterium tuberculosis persistence by different conditions,such as nutrient starvation [3] or hypoxia [4]. This means that M. tuberculosis keeps its infectious capacity intact while it no longer multiplies.Persistent bacilli resist antituberculous agents, such as isoniazid, although the same antibiotics rapidly eliminate actively multiplying M.tuberculosis. Consequently, tuberculosis is very difficult to treat: the usual regimen requires four antibiotics for two months, then two antibioticsfor the next four months, watching out for the return of persistent bacteria to normal metabolism.In patients, persistent bacilli survive in macrophages after phagocytosis where they are rather well protected from immune response andantibiotic treatment. It is expected that antibiotics targeted toward macrophages improve treatment efficacy by concentrating drugs close tobacteria. The aim of this work was to design a conjugate able to deliver an antibiotic into macrophages phagosomal vacuole in close contact withthe intracellular bacteria.We synthesized targeted macromolecular prodrugs composed of a macromolecular carrier, of a targeting device and of an antibiotic activein vitro against M. tuberculosis. The carrier, dextran, bears both mannose, the homing device intended to target the macrophage, andnorfloxacin, to be delivered into the phagosomal vacuole [5]. Norfloxacin was linked to the macromolecular carrier through two differentpeptide arms.M. bovis BCG was used as a mycobacterial model because this bacterium has a physiology closely related to the physiology of M.tuberculosis, while being at the same time much less pathogenic than the latter, and because the immune response of the mice rapidly controlledextracellular infection and left only intracellular bacilli, i.e., the goal of the targeted norfloxacin.This model takes advantage of different pO2 levels in different mouse organs, hence of different persistence statuses of bacteria in theseorgans, and it therefore allowed us to test the in vivo antibiotic activity of our macromolecular prodrugs against persistent M. bovis BCG inmice. In this paper, we report the measurement of pO2 in liver and spleen of mice. We study the therapeutic activity of our macromolecularprodrugs versus isoniazid and ofloxacin used as controls against well oxygenated and hypoxic, hence persistent, bacilli by counting them inlungs, spleen and liver. These organs are rich in macrophages and possess different oxygenation statuses.Poster Abstracts 343

Proceedings - University of Groningen

   University of GroningenControlled Delivery of Methotrexate from Channel-Protein Containing LiposomesDeemter, M. van; Šmisterová, J.; Schaaf, G. van der; Meijberg, W.; Robillard, G.Published in:Journal of Controlled ReleaseIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Deemter, M. V., Šmisterová, J., Schaaf, G. V. D., Meijberg, W., & Robillard, G. (2005). Controlled Deliveryof Methotrexate from Channel-Protein Containing Liposomes. Journal of Controlled Release, 101(1-3), 342-343.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 31-10-2023CONTROLLED DELIVERY OF METHOTREXATE FROM CHANNEL-PROTEIN CONTAINING LIPOSOMESM. van Deemter, J. Šmisterová, G. van der Schaaf, W . Meijberg, G. RobillardBiomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The NetherlandsSummaryThe goal of this study is to develop a controlled methotrexate delivery system that releases its content by a target specific stimulus. Toaccomplish this, both methotrexate and an engineered channel protein (MscL) were incorporated into DOPC/CH/DSPE-PEG liposomes,providing a controlled drug delivery system. Reconstitution and encapsulation methods were optimized in order to ensure optimal channelgating activity and high drug:lipid ratio. At the optimal conditions, a 95% release of MTX could be reached.IntroductionIn tumor therapy, many potential drugs cannot be used because they are toxic to several vital organs. For these drugs, encapsulationin liposomes can be a solution. Further improvements in the design of liposomes resulting in specific release of drug at the tumorsite will make the utilization of liposomes more feasible. To accomplish this, we are developing a liposomal system containing a pH-sensitive proteinacious valve that opens as a response to lowered pH values, such as these reported, e.g., for solid tumors [1]. Thisproteinacious valve is the mechanosensitive channel of large conductance (MscL) from E.coli. The glycine at the 22nd position of theMscL is mutated to a cysteine, which is used to target either pH-sensitive chemical groups or [2-(trimethylamonium)ethyl]methanethiosulfonate bromide (MTSET) to the protein. Binding of MTSET introduces a positive charge into the hydrophobic channelpore leading to opening of the channel. MscL-G22C was reconstituted in therapeutically optimized liposomes composed of DOPC/Cholesterol/DSPE-PEG2000 (70:20:10), which was shown to be compatible with the channel protein activity. The encapsulationmethod for MTX has been optimized.Experimental methodsThe preparation of MscL containing liposomes with encapsulated MTX combines the detergent-mediated reconstitution of MscL in liposomesby using BioBeads and the encapsulation of MTX by one of the following methods: (a) lipid film rehydration in a drug-containing solution, (b)reverse phase evaporation and (c) addition of the drug in the presence of detergent during protein reconstitution. For the analysis of drug:lipidratio, MTX-proteoliposomes were separated from the free drug on a Sephadex G50 column. Total lipid content was determined from a sensitivefluorescence assay, using diphenylhexatriene as the probe (excitation at 355 nm and emission at 440 nm). MTX content was measured by HPLC(detection at 294/356 nm [2]). Drug:lipid ratio was calculated as Ag drug per mg total lipid.Release of MTX after activation with MTSET (pH nonsensitive) was determined after separation of liposomes and free drug ona desalting PD10 column followed by the measurement of total lipid and drug concentrations.Results and discussionIn order to see which MTX encapsulation method gives the highest drug:lipid ratio in combination with high drug release afteractivation, three methods were compared. The results can be seen in Table 1. All three encapsulation methods tested gave similardrug:lipid ratios (between 5 and 7 Ag/mg) and also similar release (about 95%) upon the activation of the channel. For practical reasons,the method in which the drug was encapsulated during protein reconstitution was used in further experiments.Table 1Drug:lipid ratios and drug release after different MTX encapsulation methodsEncapsulation method Drug:lipid (Ag/mg) Released drug afteractivation (%)Reverse phase evaporation 6.99 95During protein reconstitution 6.39 95Lipid film rehydration 5.13 94Poster Abstracts342ConclusionA proteoliposomal delivery system for MTX has been developed, which can release almost all encapsulated drug upon the activation of thechannel protein by chemical labeling. We are currently busy with the development of pH-sensitive proteoliposomes.References[1] G.R. Martin, K.R. Jain, Nonevasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imagingmicroscopy, Cancer Res. 54 (1994) 5670–5674.[2] S. V ezmar, U. Bode, U. Jaehde, Monitoring of methotrexate and reduced folates in the cerebrospial fluid of cancer patients, Int. J. Clin.Pharmacol. Ther. 40:582–583.TARGETED NORFLOXACIN IS ACTIVE IN VIVO AGAINST PERSISTENT MYCOBACT ERIUM BOVIS BCGD. Domurado1, A.-M. Balazuc2, M. Lagranderie2, P. Pescher2, P. Chavarot2, E. Roseeuw3, V . Coessens3, S. Stern4, E. Schacht3, G. Marchal21Groupe de Pharmacocinétique des Prodrogues et Conjugués Macromoléculaires (INSERM), CRBA-UMR 5473 CNRS, Faculté de Pharmacie,15 avenue Charles Flahault, BP 14491, 34093 Montpellier cedex 5, France2Laboratoire de Référence des Mycobactéries, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France3Biomaterial and Polymer Research Group, Department of Organic Chemistry, University of Ghent, Krijgslaan 281, S4 bis, 9000 Ghent,Belgium4Laboratoire de Radiobiologie Cellulaire, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 V illejuif cedex, FranceSummaryTuberculosis is difficult to treat because Mycobacterium tuberculosis persistent forms resist the usual antituberculous agents. This results in 2–3million deaths each year. In order to improve tuberculosis treatment, we established a short-term in vivo model of persistent mycobacteria. Also,we synthetized a macromolecular prodrug targeted toward macrophages and demonstrated its in vivo efficacy against persistent mycobacteria.To our knowledge, this is the first report of a drug active in vivo against persistent mycobacteria.IntroductionTuberculosis infects 2 billion people worldwide. Even if most of them present no symptoms and will never develop the disease, the eventualdevelopment of an overt tuberculosis, up to decades after contamination [1,2], results in 2–3 million deaths each year.The possible late development of tuberculosis results from the induction of Mycobacterium tuberculosis persistence by different conditions,such as nutrient starvation [3] or hypoxia [4]. This means that M. tuberculosis keeps its infectious capacity intact while it no longer multiplies.Persistent bacilli resist antituberculous agents, such as isoniazid, although the same antibiotics rapidly eliminate actively multiplying M.tuberculosis. Consequently, tuberculosis is very difficult to treat: the usual regimen requires four antibiotics for two months, then two antibioticsfor the next four months, watching out for the return of persistent bacteria to normal metabolism.In patients, persistent bacilli survive in macrophages after phagocytosis where they are rather well protected from immune response andantibiotic treatment. It is expected that antibiotics targeted toward macrophages improve treatment efficacy by concentrating drugs close tobacteria. The aim of this work was to design a conjugate able to deliver an antibiotic into macrophages phagosomal vacuole in close contact withthe intracellular bacteria.We synthesized targeted macromolecular prodrugs composed of a macromolecular carrier, of a targeting device and of an antibiotic activein vitro against M. tuberculosis. The carrier, dextran, bears both mannose, the homing device intended to target the macrophage, andnorfloxacin, to be delivered into the phagosomal vacuole [5]. Norfloxacin was linked to the macromolecular carrier through two differentpeptide arms.M. bovis BCG was used as a mycobacterial model because this bacterium has a physiology closely related to the physiology of M.tuberculosis, while being at the same time much less pathogenic than the latter, and because the immune response of the mice rapidly controlledextracellular infection and left only intracellular bacilli, i.e., the goal of the targeted norfloxacin.This model takes advantage of different pO2 levels in different mouse organs, hence of different persistence statuses of bacteria in theseorgans, and it therefore allowed us to test the in vivo antibiotic activity of our macromolecular prodrugs against persistent M. bovis BCG inmice. In this paper, we report the measurement of pO2 in liver and spleen of mice. We study the therapeutic activity of our macromolecularprodrugs versus isoniazid and ofloxacin used as controls against well oxygenated and hypoxic, hence persistent, bacilli by counting them inlungs, spleen and liver. These organs are rich in macrophages and possess different oxygenation statuses.Poster Abstracts 343

NARCIS 

Dissertations of the University of Groningen

   University of GroningenControlled Delivery of Methotrexate from Channel-Protein Containing LiposomesDeemter, M. van; Šmisterová, J.; Schaaf, G. van der; Meijberg, W.; Robillard, G.Published in:Journal of Controlled ReleaseIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Deemter, M. V., Šmisterová, J., Schaaf, G. V. D., Meijberg, W., & Robillard, G. (2005). Controlled Deliveryof Methotrexate from Channel-Protein Containing Liposomes. Journal of Controlled Release, 101(1-3), 342-343.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-10-2022CONTROLLED DELIVERY OF METHOTREXATE FROM CHANNEL-PROTEIN CONTAINING LIPOSOMESM. van Deemter, J. Šmisterová, G. van der Schaaf, W . Meijberg, G. RobillardBiomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The NetherlandsSummaryThe goal of this study is to develop a controlled methotrexate delivery system that releases its content by a target specific stimulus. Toaccomplish this, both methotrexate and an engineered channel protein (MscL) were incorporated into DOPC/CH/DSPE-PEG liposomes,providing a controlled drug delivery system. Reconstitution and encapsulation methods were optimized in order to ensure optimal channelgating activity and high drug:lipid ratio. At the optimal conditions, a 95% release of MTX could be reached.IntroductionIn tumor therapy, many potential drugs cannot be used because they are toxic to several vital organs. For these drugs, encapsulationin liposomes can be a solution. Further improvements in the design of liposomes resulting in specific release of drug at the tumorsite will make the utilization of liposomes more feasible. To accomplish this, we are developing a liposomal system containing a pH-sensitive proteinacious valve that opens as a response to lowered pH values, such as these reported, e.g., for solid tumors [1]. Thisproteinacious valve is the mechanosensitive channel of large conductance (MscL) from E.coli. The glycine at the 22nd position of theMscL is mutated to a cysteine, which is used to target either pH-sensitive chemical groups or [2-(trimethylamonium)ethyl]methanethiosulfonate bromide (MTSET) to the protein. Binding of MTSET introduces a positive charge into the hydrophobic channelpore leading to opening of the channel. MscL-G22C was reconstituted in therapeutically optimized liposomes composed of DOPC/Cholesterol/DSPE-PEG2000 (70:20:10), which was shown to be compatible with the channel protein activity. The encapsulationmethod for MTX has been optimized.Experimental methodsThe preparation of MscL containing liposomes with encapsulated MTX combines the detergent-mediated reconstitution of MscL in liposomesby using BioBeads and the encapsulation of MTX by one of the following methods: (a) lipid film rehydration in a drug-containing solution, (b)reverse phase evaporation and (c) addition of the drug in the presence of detergent during protein reconstitution. For the analysis of drug:lipidratio, MTX-proteoliposomes were separated from the free drug on a Sephadex G50 column. Total lipid content was determined from a sensitivefluorescence assay, using diphenylhexatriene as the probe (excitation at 355 nm and emission at 440 nm). MTX content was measured by HPLC(detection at 294/356 nm [2]). Drug:lipid ratio was calculated as Ag drug per mg total lipid.Release of MTX after activation with MTSET (pH nonsensitive) was determined after separation of liposomes and free drug ona desalting PD10 column followed by the measurement of total lipid and drug concentrations.Results and discussionIn order to see which MTX encapsulation method gives the highest drug:lipid ratio in combination with high drug release afteractivation, three methods were compared. The results can be seen in Table 1. All three encapsulation methods tested gave similardrug:lipid ratios (between 5 and 7 Ag/mg) and also similar release (about 95%) upon the activation of the channel. For practical reasons,the method in which the drug was encapsulated during protein reconstitution was used in further experiments.Table 1Drug:lipid ratios and drug release after different MTX encapsulation methodsEncapsulation method Drug:lipid (Ag/mg) Released drug afteractivation (%)Reverse phase evaporation 6.99 95During protein reconstitution 6.39 95Lipid film rehydration 5.13 94Poster Abstracts342ConclusionA proteoliposomal delivery system for MTX has been developed, which can release almost all encapsulated drug upon the activation of thechannel protein by chemical labeling. We are currently busy with the development of pH-sensitive proteoliposomes.References[1] G.R. Martin, K.R. Jain, Nonevasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imagingmicroscopy, Cancer Res. 54 (1994) 5670–5674.[2] S. V ezmar, U. Bode, U. Jaehde, Monitoring of methotrexate and reduced folates in the cerebrospial fluid of cancer patients, Int. J. Clin.Pharmacol. Ther. 40:582–583.TARGETED NORFLOXACIN IS ACTIVE IN VIVO AGAINST PERSISTENT MYCOBACT ERIUM BOVIS BCGD. Domurado1, A.-M. Balazuc2, M. Lagranderie2, P. Pescher2, P. Chavarot2, E. Roseeuw3, V . Coessens3, S. Stern4, E. Schacht3, G. Marchal21Groupe de Pharmacocinétique des Prodrogues et Conjugués Macromoléculaires (INSERM), CRBA-UMR 5473 CNRS, Faculté de Pharmacie,15 avenue Charles Flahault, BP 14491, 34093 Montpellier cedex 5, France2Laboratoire de Référence des Mycobactéries, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France3Biomaterial and Polymer Research Group, Department of Organic Chemistry, University of Ghent, Krijgslaan 281, S4 bis, 9000 Ghent,Belgium4Laboratoire de Radiobiologie Cellulaire, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 V illejuif cedex, FranceSummaryTuberculosis is difficult to treat because Mycobacterium tuberculosis persistent forms resist the usual antituberculous agents. This results in 2–3million deaths each year. In order to improve tuberculosis treatment, we established a short-term in vivo model of persistent mycobacteria. Also,we synthetized a macromolecular prodrug targeted toward macrophages and demonstrated its in vivo efficacy against persistent mycobacteria.To our knowledge, this is the first report of a drug active in vivo against persistent mycobacteria.IntroductionTuberculosis infects 2 billion people worldwide. Even if most of them present no symptoms and will never develop the disease, the eventualdevelopment of an overt tuberculosis, up to decades after contamination [1,2], results in 2–3 million deaths each year.The possible late development of tuberculosis results from the induction of Mycobacterium tuberculosis persistence by different conditions,such as nutrient starvation [3] or hypoxia [4]. This means that M. tuberculosis keeps its infectious capacity intact while it no longer multiplies.Persistent bacilli resist antituberculous agents, such as isoniazid, although the same antibiotics rapidly eliminate actively multiplying M.tuberculosis. Consequently, tuberculosis is very difficult to treat: the usual regimen requires four antibiotics for two months, then two antibioticsfor the next four months, watching out for the return of persistent bacteria to normal metabolism.In patients, persistent bacilli survive in macrophages after phagocytosis where they are rather well protected from immune response andantibiotic treatment. It is expected that antibiotics targeted toward macrophages improve treatment efficacy by concentrating drugs close tobacteria. The aim of this work was to design a conjugate able to deliver an antibiotic into macrophages phagosomal vacuole in close contact withthe intracellular bacteria.We synthesized targeted macromolecular prodrugs composed of a macromolecular carrier, of a targeting device and of an antibiotic activein vitro against M. tuberculosis. The carrier, dextran, bears both mannose, the homing device intended to target the macrophage, andnorfloxacin, to be delivered into the phagosomal vacuole [5]. Norfloxacin was linked to the macromolecular carrier through two differentpeptide arms.M. bovis BCG was used as a mycobacterial model because this bacterium has a physiology closely related to the physiology of M.tuberculosis, while being at the same time much less pathogenic than the latter, and because the immune response of the mice rapidly controlledextracellular infection and left only intracellular bacilli, i.e., the goal of the targeted norfloxacin.This model takes advantage of different pO2 levels in different mouse organs, hence of different persistence statuses of bacteria in theseorgans, and it therefore allowed us to test the in vivo antibiotic activity of our macromolecular prodrugs against persistent M. bovis BCG inmice. In this paper, we report the measurement of pO2 in liver and spleen of mice. We study the therapeutic activity of our macromolecularprodrugs versus isoniazid and ofloxacin used as controls against well oxygenated and hypoxic, hence persistent, bacilli by counting them inlungs, spleen and liver. These organs are rich in macrophages and possess different oxygenation statuses.Poster Abstracts 343

University of Groningen Research Database

  
 University of Groningen
Controlled Delivery of Methotrexate from Channel-Protein Containing Liposomes
Deemter, M. van; Šmisterová, J.; Schaaf, G. van der; Meijberg, W.; Robillard, G.
Published in:
Default journal
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2005
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Deemter, M. V., Šmisterová, J., Schaaf, G. V. D., Meijberg, W., & Robillard, G. (2005). Controlled Delivery
of Methotrexate from Channel-Protein Containing Liposomes. Default journal.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
CONTROLLED DELIVERY OF METHOTREXATE FROM CHANNEL-PROTEIN CONTAINING LIPOSOMES
M. van Deemter, J. Sˇmisterova´, G. van der Schaaf, W . Meijberg, G. Robillard
Biomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands
Summary
The goal of this study is to develop a controlled methotrexate delivery system that releases its content by a target specific stimulus. To
accomplish this, both methotrexate and an engineered channel protein (MscL) were incorporated into DOPC/CH/DSPE-PEG liposomes,
providing a controlled drug delivery system. Reconstitution and encapsulation methods were optimized in order to ensure optimal channel
gating activity and high drug:lipid ratio. At the optimal conditions, a 95% release of MTX could be reached.
Introduction
In tumor therapy, many potential drugs cannot be used because they are toxic to several vital organs. For these drugs, encapsulation
in liposomes can be a solution. Further improvements in the design of liposomes resulting in specific release of drug at the tumor
site will make the utilization of liposomes more feasible. To accomplish this, we are developing a liposomal system containing a pH-
sensitive proteinacious valve that opens as a response to lowered pH values, such as these reported, e.g., for solid tumors [1]. This
proteinacious valve is the mechanosensitive channel of large conductance (MscL) from E.coli. The glycine at the 22nd position of the
MscL is mutated to a cysteine, which is used to target either pH-sensitive chemical groups or [2-(trimethylamonium)ethyl]
methanethiosulfonate bromide (MTSET) to the protein. Binding of MTSET introduces a positive charge into the hydrophobic channel
pore leading to opening of the channel. MscL-G22C was reconstituted in therapeutically optimized liposomes composed of DOPC/
Cholesterol/DSPE-PEG2000 (70:20:10), which was shown to be compatible with the channel protein activity. The encapsulation
method for MTX has been optimized.
Experimental methods
The preparation of MscL containing liposomes with encapsulated MTX combines the detergent-mediated reconstitution of MscL in liposomes
by using BioBeads and the encapsulation of MTX by one of the following methods: (a) lipid film rehydration in a drug-containing solution, (b)
reverse phase evaporation and (c) addition of the drug in the presence of detergent during protein reconstitution. For the analysis of drug:lipid
ratio, MTX-proteoliposomes were separated from the free drug on a Sephadex G50 column. Total lipid content was determined from a sensitive
fluorescence assay, using diphenylhexatriene as the probe (excitation at 355 nm and emission at 440 nm). MTX content was measured by HPLC
(detection at 294/356 nm [2]). Drug:lipid ratio was calculated as Ag drug per mg total lipid.
Release of MTX after activation with MTSET (pH nonsensitive) was determined after separation of liposomes and free drug on
a desalting PD10 column followed by the measurement of total lipid and drug concentrations.
Results and discussion
In order to see which MTX encapsulation method gives the highest drug:lipid ratio in combination with high drug release after
activation, three methods were compared. The results can be seen in Table 1. All three encapsulation methods tested gave similar
drug:lipid ratios (between 5 and 7 Ag/mg) and also similar release (about 95%) upon the activation of the channel. For practical reasons,
the method in which the drug was encapsulated during protein reconstitution was used in further experiments.
Table 1
Drug:lipid ratios and drug release after different MTX encapsulation methods
Encapsulation method Drug:lipid (Ag/mg) Released drug after
activation (%)
Reverse phase evaporation 6.99 95
During protein reconstitution 6.39 95
Lipid film rehydration 5.13 94
Poster Abstracts342
Conclusion
A proteoliposomal delivery system for MTX has been developed, which can release almost all encapsulated drug upon the activation of the
channel protein by chemical labeling. We are currently busy with the development of pH-sensitive proteoliposomes.
References
[1] G.R. Martin, K.R. Jain, Nonevasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imaging
microscopy, Cancer Res. 54 (1994) 5670–5674.
[2] S. V ezmar, U. Bode, U. Jaehde, Monitoring of methotrexate and reduced folates in the cerebrospial fluid of cancer patients, Int. J. Clin.
Pharmacol. Ther. 40:582–583.
TARGETED NORFLOXACIN IS ACTIVE IN VIVO AGAINST PERSISTENT MYCOBACT ERIUM BOVIS BCG
D. Domurado1, A.-M. Balazuc2, M. Lagranderie2, P. Pescher2, P. Chavarot2, E. Roseeuw3, V . Coessens3, S. Stern4, E. Schacht3, G. Marchal2
1Groupe de Pharmacocine´tique des Prodrogues et Conjugue´s Macromole´culaires (INSERM), CRBA-UMR 5473 CNRS, Faculte´ de Pharmacie,
15 avenue Charles Flahault, BP 14491, 34093 Montpellier cedex 5, France
2Laboratoire de Re´fe´rence des Mycobacte´ries, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France
3Biomaterial and Polymer Research Group, Department of Organic Chemistry, University of Ghent, Krijgslaan 281, S4 bis, 9000 Ghent,
Belgium
4Laboratoire de Radiobiologie Cellulaire, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 V illejuif cedex, France
Summary
Tuberculosis is difficult to treat because Mycobacterium tuberculosis persistent forms resist the usual antituberculous agents. This results in 2–3
million deaths each year. In order to improve tuberculosis treatment, we established a short-term in vivo model of persistent mycobacteria. Also,
we synthetized a macromolecular prodrug targeted toward macrophages and demonstrated its in vivo efficacy against persistent mycobacteria.
To our knowledge, this is the first report of a drug active in vivo against persistent mycobacteria.
Introduction
Tuberculosis infects 2 billion people worldwide. Even if most of them present no symptoms and will never develop the disease, the eventual
development of an overt tuberculosis, up to decades after contamination [1,2], results in 2–3 million deaths each year.
The possible late development of tuberculosis results from the induction of Mycobacterium tuberculosis persistence by different conditions,
such as nutrient starvation [3] or hypoxia [4]. This means that M. tuberculosis keeps its infectious capacity intact while it no longer multiplies.
Persistent bacilli resist antituberculous agents, such as isoniazid, although the same antibiotics rapidly eliminate actively multiplying M.
tuberculosis. Consequently, tuberculosis is very difficult to treat: the usual regimen requires four antibiotics for two months, then two antibiotics
for the next four months, watching out for the return of persistent bacteria to normal metabolism.
In patients, persistent bacilli survive in macrophages after phagocytosis where they are rather well protected from immune response and
antibiotic treatment. It is expected that antibiotics targeted toward macrophages improve treatment efficacy by concentrating drugs close to
bacteria. The aim of this work was to design a conjugate able to deliver an antibiotic into macrophages phagosomal vacuole in close contact with
the intracellular bacteria.
We synthesized targeted macromolecular prodrugs composed of a macromolecular carrier, of a targeting device and of an antibiotic active
in vitro against M. tuberculosis. The carrier, dextran, bears both mannose, the homing device intended to target the macrophage, and
norfloxacin, to be delivered into the phagosomal vacuole [5]. Norfloxacin was linked to the macromolecular carrier through two different
peptide arms.
M. bovis BCG was used as a mycobacterial model because this bacterium has a physiology closely related to the physiology of M.
tuberculosis, while being at the same time much less pathogenic than the latter, and because the immune response of the mice rapidly controlled
extracellular infection and left only intracellular bacilli, i.e., the goal of the targeted norfloxacin.
This model takes advantage of different pO2 levels in different mouse organs, hence of different persistence statuses of bacteria in these
organs, and it therefore allowed us to test the in vivo antibiotic activity of our macromolecular prodrugs against persistent M. bovis BCG in
mice. In this paper, we report the measurement of pO2 in liver and spleen of mice. We study the therapeutic activity of our macromolecular
prodrugs versus isoniazid and ofloxacin used as controls against well oxygenated and hypoxic, hence persistent, bacilli by counting them in
lungs, spleen and liver. These organs are rich in macrophages and possess different oxygenation statuses.
Poster Abstracts 343


University of Groningen

   University of GroningenControlled Delivery of Methotrexate from Channel-Protein Containing LiposomesDeemter, M. van; Šmisterová, J.; Schaaf, G. van der; Meijberg, W.; Robillard, G.Published in:Default journalIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2005Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Deemter, M. V., Šmisterová, J., Schaaf, G. V. D., Meijberg, W., & Robillard, G. (2005). Controlled Deliveryof Methotrexate from Channel-Protein Containing Liposomes. Default journal.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019CONTROLLED DELIVERY OF METHOTREXATE FROM CHANNEL-PROTEIN CONTAINING LIPOSOMESM. van Deemter, J. Sˇmisterova´, G. van der Schaaf, W . Meijberg, G. RobillardBiomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The NetherlandsSummaryThe goal of this study is to develop a controlled methotrexate delivery system that releases its content by a target specific stimulus. Toaccomplish this, both methotrexate and an engineered channel protein (MscL) were incorporated into DOPC/CH/DSPE-PEG liposomes,providing a controlled drug delivery system. Reconstitution and encapsulation methods were optimized in order to ensure optimal channelgating activity and high drug:lipid ratio. At the optimal conditions, a 95% release of MTX could be reached.IntroductionIn tumor therapy, many potential drugs cannot be used because they are toxic to several vital organs. For these drugs, encapsulationin liposomes can be a solution. Further improvements in the design of liposomes resulting in specific release of drug at the tumorsite will make the utilization of liposomes more feasible. To accomplish this, we are developing a liposomal system containing a pH-sensitive proteinacious valve that opens as a response to lowered pH values, such as these reported, e.g., for solid tumors [1]. Thisproteinacious valve is the mechanosensitive channel of large conductance (MscL) from E.coli. The glycine at the 22nd position of theMscL is mutated to a cysteine, which is used to target either pH-sensitive chemical groups or [2-(trimethylamonium)ethyl]methanethiosulfonate bromide (MTSET) to the protein. Binding of MTSET introduces a positive charge into the hydrophobic channelpore leading to opening of the channel. MscL-G22C was reconstituted in therapeutically optimized liposomes composed of DOPC/Cholesterol/DSPE-PEG2000 (70:20:10), which was shown to be compatible with the channel protein activity. The encapsulationmethod for MTX has been optimized.Experimental methodsThe preparation of MscL containing liposomes with encapsulated MTX combines the detergent-mediated reconstitution of MscL in liposomesby using BioBeads and the encapsulation of MTX by one of the following methods: (a) lipid film rehydration in a drug-containing solution, (b)reverse phase evaporation and (c) addition of the drug in the presence of detergent during protein reconstitution. For the analysis of drug:lipidratio, MTX-proteoliposomes were separated from the free drug on a Sephadex G50 column. Total lipid content was determined from a sensitivefluorescence assay, using diphenylhexatriene as the probe (excitation at 355 nm and emission at 440 nm). MTX content was measured by HPLC(detection at 294/356 nm [2]). Drug:lipid ratio was calculated as Ag drug per mg total lipid.Release of MTX after activation with MTSET (pH nonsensitive) was determined after separation of liposomes and free drug ona desalting PD10 column followed by the measurement of total lipid and drug concentrations.Results and discussionIn order to see which MTX encapsulation method gives the highest drug:lipid ratio in combination with high drug release afteractivation, three methods were compared. The results can be seen in Table 1. All three encapsulation methods tested gave similardrug:lipid ratios (between 5 and 7 Ag/mg) and also similar release (about 95%) upon the activation of the channel. For practical reasons,the method in which the drug was encapsulated during protein reconstitution was used in further experiments.Table 1Drug:lipid ratios and drug release after different MTX encapsulation methodsEncapsulation method Drug:lipid (Ag/mg) Released drug afteractivation (%)Reverse phase evaporation 6.99 95During protein reconstitution 6.39 95Lipid film rehydration 5.13 94Poster Abstracts342ConclusionA proteoliposomal delivery system for MTX has been developed, which can release almost all encapsulated drug upon the activation of thechannel protein by chemical labeling. We are currently busy with the development of pH-sensitive proteoliposomes.References[1] G.R. Martin, K.R. Jain, Nonevasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imagingmicroscopy, Cancer Res. 54 (1994) 5670–5674.[2] S. V ezmar, U. Bode, U. Jaehde, Monitoring of methotrexate and reduced folates in the cerebrospial fluid of cancer patients, Int. J. Clin.Pharmacol. Ther. 40:582–583.TARGETED NORFLOXACIN IS ACTIVE IN VIVO AGAINST PERSISTENT MYCOBACT ERIUM BOVIS BCGD. Domurado1, A.-M. Balazuc2, M. Lagranderie2, P. Pescher2, P. Chavarot2, E. Roseeuw3, V . Coessens3, S. Stern4, E. Schacht3, G. Marchal21Groupe de Pharmacocine´tique des Prodrogues et Conjugue´s Macromole´culaires (INSERM), CRBA-UMR 5473 CNRS, Faculte´ de Pharmacie,15 avenue Charles Flahault, BP 14491, 34093 Montpellier cedex 5, France2Laboratoire de Re´fe´rence des Mycobacte´ries, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France3Biomaterial and Polymer Research Group, Department of Organic Chemistry, University of Ghent, Krijgslaan 281, S4 bis, 9000 Ghent,Belgium4Laboratoire de Radiobiologie Cellulaire, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 V illejuif cedex, FranceSummaryTuberculosis is difficult to treat because Mycobacterium tuberculosis persistent forms resist the usual antituberculous agents. This results in 2–3million deaths each year. In order to improve tuberculosis treatment, we established a short-term in vivo model of persistent mycobacteria. Also,we synthetized a macromolecular prodrug targeted toward macrophages and demonstrated its in vivo efficacy against persistent mycobacteria.To our knowledge, this is the first report of a drug active in vivo against persistent mycobacteria.IntroductionTuberculosis infects 2 billion people worldwide. Even if most of them present no symptoms and will never develop the disease, the eventualdevelopment of an overt tuberculosis, up to decades after contamination [1,2], results in 2–3 million deaths each year.The possible late development of tuberculosis results from the induction of Mycobacterium tuberculosis persistence by different conditions,such as nutrient starvation [3] or hypoxia [4]. This means that M. tuberculosis keeps its infectious capacity intact while it no longer multiplies.Persistent bacilli resist antituberculous agents, such as isoniazid, although the same antibiotics rapidly eliminate actively multiplying M.tuberculosis. Consequently, tuberculosis is very difficult to treat: the usual regimen requires four antibiotics for two months, then two antibioticsfor the next four months, watching out for the return of persistent bacteria to normal metabolism.In patients, persistent bacilli survive in macrophages after phagocytosis where they are rather well protected from immune response andantibiotic treatment. It is expected that antibiotics targeted toward macrophages improve treatment efficacy by concentrating drugs close tobacteria. The aim of this work was to design a conjugate able to deliver an antibiotic into macrophages phagosomal vacuole in close contact withthe intracellular bacteria.We synthesized targeted macromolecular prodrugs composed of a macromolecular carrier, of a targeting device and of an antibiotic activein vitro against M. tuberculosis. The carrier, dextran, bears both mannose, the homing device intended to target the macrophage, andnorfloxacin, to be delivered into the phagosomal vacuole [5]. Norfloxacin was linked to the macromolecular carrier through two differentpeptide arms.M. bovis BCG was used as a mycobacterial model because this bacterium has a physiology closely related to the physiology of M.tuberculosis, while being at the same time much less pathogenic than the latter, and because the immune response of the mice rapidly controlledextracellular infection and left only intracellular bacilli, i.e., the goal of the targeted norfloxacin.This model takes advantage of different pO2 levels in different mouse organs, hence of different persistence statuses of bacteria in theseorgans, and it therefore allowed us to test the in vivo antibiotic activity of our macromolecular prodrugs against persistent M. bovis BCG inmice. In this paper, we report the measurement of pO2 in liver and spleen of mice. We study the therapeutic activity of our macromolecularprodrugs versus isoniazid and ofloxacin used as controls against well oxygenated and hypoxic, hence persistent, bacilli by counting them inlungs, spleen and liver. These organs are rich in macrophages and possess different oxygenation statuses.Poster Abstracts 343

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