Category Archives: Nanomedicine

Development of ketoprofen lysine nanomedicine

Drug Design and Action, Nanomedicine, nanoparticles, Pain Management, Pharmaceutical Technology, Pharmacy
J Pharm Pharmacogn Res 7(1): 47-58, 2019.

Original Article | Artículo Original

Development of nano-sized ketoprofen lysine incorporated Eudragit® S100 nanomedicine by double emulsion solvent evaporation and in vitro characterization

[Desarrollo de un nanofármaco de ketoprofeno lisina de tamaño nanométrico con Eudragit® S100 incorporado mediante doble evaporación del disolvente de emulsión y su caracterización in vitro]

A. Alper Öztürk1*, Nur İlge Çinar2, Evrim Yenilmez1

1Department of Pharmaceutical Technology, Faculty of Pharmacy, Anadolu University, Eskişehir, Turkey.
2Department of Pharmaceutical Technology, Graduation School of Health Sciences, Anadolu University, Eskişehir, Turkey.

*E-mail: aaozturk@anadolu.edu.tr

Abstract

Context: Pain has a very important effect on the biological, psychological, sociological and economic situation of a patient. Nanoparticles (NPs) are being extensively investigated as drug delivery systems worldwide for pharmaceutical applications.

Aims: To design and compare the release characteristics of sustained-release formulations of ketoprofen lysine (KL) NPs.

Methods: KL-Eudragit® S100 NPs were produced by double emulsion solvent evaporation method. The physicochemical characteristics of NPs were studied.

Results: Particle size of NPs prepared was in the range of 99 and 141 nm. Encapsulation efficiency (%) was obtained (76%) for NP formulations prepared. Weibull models were determined to be the most appropriate kinetic models for NP containing KL. KL-loaded NPs demonstrated nanostructural character and NPs showed extended release of KL.

Conclusions: NPs developed were found to be stable and representing a promising system for sustained delivery of KL.

Keywords: Eudragit® S100; ketoprofen lysine; nanoparticle.

Resumen

Contexto: El dolor tiene un efecto muy importante en la situación biológica, psicológica, sociológica y económica de un paciente. Las nanopartículas (NP) están siendo ampliamente investigadas como sistemas de administración de fármacos en todo el mundo para aplicaciones farmacéuticas.

Objetivos: Diseñar y comparar las características de liberación de formulaciones de liberación sostenida de NP de ketoprofeno lisina (KL).

Métodos: Las NP de KL-Eudragit® S100 se produjeron por el método de evaporación con doble emulsión de disolvente. Se estudiaron las características fisicoquímicas de las NP.

Resultados: El tamaño de partícula de las NP preparadas estaba en el intervalo de 99 y 141 nm. Se obtuvo la eficiencia de encapsulación (76%) para las formulaciones de NP preparadas. Se determinó que los modelos cinéticos de Weibull son los más apropiados para NP que contienen KL. Las NP cargadas con KL demostraron carácter nanoestructural y una liberación extendida de KL.

Conclusiones: Se encontró que las NP desarrolladas son estables y representan un sistema prometedor para la administración sostenida de KL.

Palabras Clave: Eudragit® S100; ketoprofeno lisina; nanopartícula.

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Citation Format: Öztürk AA, Çinar NI, Yenilmez E (2019) Development of nano-sized ketoprofen lysine incorporated Eudragit® S100 nanomedicine by double emulsion solvent evaporation and in vitro characterization. J Pharm Pharmacogn Res 7(1): 47–58.

© 2019 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

Herbal polymeric nanoformulations to contest depression

Brain, Nanomedicine, nanoparticles, Neuroprotection, Pharmaceutical Technology
J Pharm Pharmacogn Res 5(3): 187-199, 2017.

Original Article | Artículo Original

Nanoparticulated formulations of St. John’s wort (Hypericum perforatum L.) as smart drug delivery system combating depression incited in mice models

[Formulas nanoparticuladas de hierba de San Juan (Hypericum perforatum L.) como sistema inteligente de administración de fármacos para combatir la depresión inducida en modelos de ratones]

Violet Dhayabaran, Anita Margret*

Department of Biotechnology and Bioinformatics, Bishop Heber College, Vayallor Road, Puthur, Tiruchirappalli-620017, Tamilnadu, India.

*E-mail: anitamargret@gmail.com; anita.bt@bhc.edu.in

Abstract

Context: Hypericum perforatum L., commonly known as St. John’s wort, is practised as an alternative medicine against depression. Conversely, its remedial efficacy is indulged by various adverse effects that are recuperated in formulating nanoscaled commercial capsules encased by the biopolymer chitosan. A potential application of nanoencapsulation with regards to polymer enhances a slow controlled release of the targeted drug to achieve the desired delay until the right stimulus is obtained.

Aims: To value synthesizing biopolymeric nanocomposites encapsulating St. John’s wort commercial capsules substantiating it with a study of animal model of depression to endorse the effect of nanocapsulated drug as an effective brain drug.

Methods: The nanoparticulated suspension was prepared by ionic gelation technique and characterized to attest its antidepressant activity by in vivo studies.

Results: The drug binding efficiency was endorsed by FT-IR studies and the nanoparticles were characterized by an average particle size of 211.4 nm with a positive zeta potential of 45.9 mV. The animal despair studies on depression induced mice models displayed a significant difference in the immobility time during force swimming and tail suspension test. The commercial capsules were administed orally (p.o., 50 and 100 mg/kg). The animal despair studies were substantiated with affirmative biochemical assessments like SOD, CAT, GPx, GSH and LPO and compared with control groups.

Conclusions: The outcomes of this work manifest the calibre of St. John’s wort nanocomposites in a lower dosage that can alleviate depression and reduce side effects.

Keywords: biopolymer; chitosan; depression; Hypericum perforatum; nanoparticles.

Resumen

Contexto: Hypericum perforatum L., comúnmente conocida como hierba de San Juan, es usada como una medicina alternativa contra la depresión. Por el contrario, su eficacia terapéutica se acompaña de diversos efectos adversos que se aminoran con la formulación de nanocápsulas con el biopolímero quitosano. Una aplicación potencial de la nanoencapsulación con respecto al polímero mejora una lenta liberación controlada del fármaco dirigido para conseguir el retardo deseado hasta que se obtenga el estímulo correcto.

Objetivos: Evaluar nanocompuestos biopoliméricos sintéticos que encapsulan las cápsulas comerciales de hierba de San Juan que los justifican con un estudio en modelos animales de depresión para endosar el efecto del medicamento nanocapsulado como fármaco eficaz para el cerebro.

Métodos: La suspensión nanoparticulada se preparó mediante una técnica de gelificación iónica y caracterizada por atestiguar su actividad antidepresiva mediante estudios in vivo.

Resultados: La eficacia de unión a fármacos fue respaldada por estudios FT-IR y las nanopartículas se caracterizaron por un tamaño medio de partícula de 211,4 nm, con un potencial zeta positivo de 45,9 mV. Los estudios de desesperación animal en modelos de depresión en ratones mostraron una diferencia significativa en el tiempo de inmovilidad durante la natación forzada y la prueba de suspensión de cola. Las cápsulas comerciales se administraron por vía oral (p.o., 50 y 100 mg/kg). Los estudios de desesperación animal se confirmaron con valoraciones bioquímicas positivas como SOD, CAT, GPx, GSH y LPO y se compararon con los grupos de controles.

Conclusiones: Los resultados de este trabajo manifiestan el calibre de los nanocompuestos de la hierba de San Juan, en una dosis más baja, que puede aliviar la depresión y reducir los efectos secundarios.

Palabras Clave: biopolímero; depresión; Hypericum perforatum; nanopartículas; quitosano.

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Citation Format: Dhayabaran V, Margret A (2017) Nanoparticulated formulations of St. John’s wort (Hypericum perforatum L.) as smart drug delivery system combating depression incited in mice models. J Pharm Pharmacogn Res 5(3): 187–199.

© 2017 Journal of Pharmacy & Pharmacognosy Research (JPPRes)

CO 061: NANOSTRUCTURE: SURFACE MODIFICATIONS BY LASER BEAM

Nanomedicine
J Pharm Pharmacogn Res 2(Suppl. 1): S37, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 061: NANOSTRUCTURE: SURFACE MODIFICATIONS BY LASER BEAM

Guastaldi AC.

Grupo de Biomateriais. Instituto de Química de Araraquara, UNESP, Brasil. Email: guastald@iq.unesp.br
Abstract

This presentation is directed related to surface modifications by laser beam comparing to various existing processes, such as: mechanical (machining and jetting with abrasives), chemistries (acid attack and passivation) and thermal processes (plasma-spray). The modification of the surface obtained by the laser beam incidence in the surface of the implant cause micro/nanomorphologic structure assembles the equivalent characteristic without leaving vestige of contamination for being a clean, reproducible process and to make possible a larger control of the variables of the process. Commercially pure titanium implants with surfaces modified by laser beam with and without chemical deposition of apatite applied biomechanical and topographical analysis in rabbits. The contemporaneous implantology looks for advances on implants’ surfaces which allow a safer rehabilitation treatment in a lower period of time. Once the implantation used is subjected to the action of complex mechanical efforts and variations of the physical-chemical properties of the way in which is applied. Literature shows that the implant needs a micro/nanomorphologic superficial structure, not only to assure the mechanic anchorage of the bone on its surface, but also to provide bone-interaction, which can be understood as the direct connection between the new-formed bone and the surface of an implant, without the fibrous tissue interposition. However, it is difficult to conclude precisely what causes the increase in bone response to a modified surface, particularly in the HA coated implants, as this process alters the chemistry by the addition of HA, but simultaneously, it may alter the physical-chemical properties, micro and nanoroughness. Moreover, depending on the application process of HA, alteration may occur in the surface area. The research considered here validates its development; thereafter it is characterized with great potential to generate and to transfer Brazilian technology, from a process of ample commercial use. It is treated, thus, of a clear possibility of application of an innovative chemical covering process of bioactive layers in the surfaces of implants, which will facilitate the access to a population socially less attended, reverting, thus, with a notable social character. Finally, the physical chemical properties of the surface are responsible by osseointegration phenomenon and the nanoroughness or nanomorphologic will supply more or less area to occur osseointegration.

CO 060: PEGYLATION: AN EFFECTIVE TOOL FOR BIOMASKING

Nanomedicine, Pharmaceutical Technology
J Pharm Pharmacogn Res 2(Suppl. 1): S36, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 060: PEGYLATION: AN EFFECTIVE TOOL FOR BIOMASKING

Ramón JA1, Saez V1, Peniche C1, Hardy E2.

1Center for Biomaterials, University of Havana, P.O. Box 6130, Havana, Cuba. Email: jose@biomat.uh.cu
2Institute for Science and Technology of Materials, University of Havana, Havana, Cuba.
Abstract

Nowadays, a large group of medical treatments are based on new substances. These were obtained due to strong development of science and technology in last few decades and include: (i) proteins and peptides used as replacement therapy and as inhibitors or regulators of the immune system, (ii) materials used in drug delivery systems like phospholipids and biodegradable polymers, and (iii) metals and their alloys, polymers, ceramics and composites of them used in various devices such as prostheses, stents, heart valves, etc.

Unfortunately the behaviour of these materials in the body is sometimes negative for the initial purpose. For example, proteins and peptides have low stability in vivo, a short half-life time and immunogenicity. Among the problems associated with other materials are: a) thrombus formation on artificial surfaces in contact with living tissues, b) damages to the tissues (e.g. vascular weakening produced by the liposomes) and c) recognition and elimination by the reticuloendothelial system of micro/nano-devices used as DDS like liposomes and biodegradable polymer microspheres.

In general, all these phenomena are due to unwanted interactions that occur at the interfaces between biodrugs or synthetic biomaterials and biological medium. Consequently, any agent that mediates this interaction, and become to this in a more “natural” fashion, promotes the acceptance by living organisms of these “foreign bodies”. An ideal substance for this mediation, it might be call “biomasking”, is the polyethylene glycol (PEG).

This presentation is about advantages of PEGylation (conjugation to PEG) for biomasking. PEGylation is a well-established technology used to transform proteins, peptides, small molecules and oligonucleotides into more potent drugs than their corresponding unmodified native molecules. Furthermore, PEGylated liposomes had received approval for improve the delivery of encapsulated drugs, such as the anticancer agent doxorubicin, and PEG-modified polymers (such as nhexadecylcyanoacrylate and PLGA) are studied extensively to obtain enhanced particulate delivery systems.

CO 059: NOVEL PEGYLATION TECHNOLOGIES FOR THE DEVELOPMENT OF NEXT GENERATION BIODRUGS

Nanomedicine, Pharmaceutical Technology
J Pharm Pharmacogn Res 2(Suppl. 1): S36, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 059: NOVEL PEGYLATION TECHNOLOGIES FOR THE DEVELOPMENT OF NEXT GENERATION BIODRUGS

Ikeda Y1, Katamachi J1, Kawasaki H1, Nagasaki Y2.

1Department of Materials Science, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan
2 Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan. E-mail: ikeda@ims.tsukuba.ac.jp
3Satellite Laboratory, International Center for Materials Nanoarchitectonics (MANA), National Institute of Materials Science (NIMS), Tennodai 1-1-1, Tsukuba, Ibaraki 305-8573, Japan.
Abstract

Introduction: PEGylation refers to the covalent attachment of poly(ethylene glycol) on the biomolecules. PEGylation is recognized as a promising method to increase the therapeutic efficacy of medicines in clinical settings. A variety of molecules have been modified with PEG and several PEGylated drugs have been approved in clinical settings. In this presentation, our recent developments of novel PEGylation technologies on biomolecules will be shown.

Material and methods: For the construction of PEGylated oligonucleotide by solid phase synthesis, a novel solid phase which was pre-installed with PEG was prepared. In the case of protein PEGylation, a novel PEG derivative which possesses glutaraldehyde at one end has been synthesized.

Results: Solid phase synthesis of PEGylated oligonucleotide. A novel solid-phase synthesis method for poly(ethylene glycol) (PEG)oligonucleotide conjugates was developed to increase the stability of therapeutic oligonucleotides such as antisense oligonucleotides and siRNA. A prepared solid phase was pre-installed with PEG to provide oligonucleotides modified with PEG at the 3′ terminus. Compared with the conventional liquid-phase synthesis method, the developed solid-phase method is simple and reproducible. PEGylation at the 3′ terminus was confirmed to stabilize not only DNA but also RNA more than PEGylation at the 5′ terminus, which has been widely used thus far. A novel chemistry for the PEGylated protein with a high activity. Several PEGylated proteins have been approved as therapeutic drugs. In many cases, PEGylated protein has been synthesized by the conjugation reaction between PEG possessing activated ester and amine(s) in the protein. This reaction, however, often causes inactivation of PEGylated proteins.

Conclusions: In this report, we present a novel chemistry which enables the PEGylation of proteins under the mild reaction condition. PEGylated protein prepared by the method developed exhibited much higher biological activity than the PEGylated protein prepared by the conventional method.

CO 058: NANOPARTICLES AS DRUG CARRIERS: CHARACTERISTICS AND PERSPECTIVES

Nanomedicine, Pharmaceutical Technology
J Pharm Pharmacogn Res 2(Suppl. 1): S35, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 058: NANOPARTICLES AS DRUG CARRIERS: CHARACTERISTICS AND PERSPECTIVES

Oropesa-Nuñez R1, Jáuregui-Haza U2.

1Centro de Estudios Avanzados de Cuba (CEAC). Carretera San Antonio, Km 1 1/2, Valle Grande, La Lisa, La Habana, Cuba
2Instituto Superior de Tecnologías y Ciencias Aplicadas (InSTEC). Ave. Salvador Allende y Luaces, Plaza de la Revolución, La Habana, Cuba. E-mail: ulises.jauregui@infomed.sld.cu
Abstract

Nanoparticles can copy or modify biological processes because they propose solutions to the old problems associated with solubility, bioavailability, immunocompatibility and cytotoxicity of many traditional drugs. Carbon nanotubes are an example of nanoparticles and nanotechnology due to their small diameters. They can be manipulated chemically and physically. Besides, they are mainly used in nanomedicine as carriers and as excipients to obtain different drug delivery systems. In this work, the state of the art of the research on nanoparticles as drug carriers for medical applications, with emphasis on their properties, determination of physico-chemical properties and carbon nanotubes applications is analyzed. It is demonstrated that drug formulation and administration has been changed with the advances of nanotechnology. The application of nanoparticles in medicine includes their use as carriers through the functionalization or drug encapsulation. With the use of nanoformulations, an important amount of pharmaceuticals have improved their therapeutic action. The biomedical applications of carbon nanotubes have open a way to a new field in therapy and medical diagnosis. The main part of these applications might consist on the implant of nanotubes or functionalized nanotubes in patients. However, the use of carbon nanotubes in medicine depends on the evaluation of their toxicity in humans.

CO 057: COMBINATION EFFECT OF SONOPHORESIS AND IONTOPHORESIS ON ANTIPYRINE TRANSDERMAL PENETRATION

Nanomedicine, Pharmaceutical Technology
J Pharm Pharmacogn Res 2(Suppl. 1): S35, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 057: COMBINATION EFFECT OF SONOPHORESIS AND IONTOPHORESIS ON ANTIPYRINE TRANSDERMAL PENETRATION

Aoyagi T1, Watanabe S2, Ga K2, Yamamoto K3.

1National Institute for Materials Science, 1-1, Namiki, Tsukuba, Ibaraki 305-0044, Japan. E-mail: aoyagi.takao@nims.go.jp
2Yutoku Pharmaceutical Ind. Co., Japan.
3Kagoshima University, Japan.
Abstract

Introduction: Transdermal drug delivery is promising method to administrate unstable drugs in digestive organs or avoids first pass effect. To promote the drug penetration, sonophoresis and iontophoresis are very effective as physical enhancing methods. The each enhancing effects have been widely studied and actually there are many reports. In this study, we investigated the combination effect of sonophoresis and iontophoresis of transdermal penetration of antipyrene as model drug.

Material and method: We used Sonoion® was used to achieve the objective. It is developed by Kagoshima Supersonic Laboratory Co., Ltd. The excised back and abdominal skin of hairless mouse was used to estimate the enhanced condition of Sonoion® such as current, voltage and order of application (sono. to ionto. or ionto. to sono.) and so on. The animal is established by Kyudo Co., in Kumamoto, Japan. The excised skin was set in Franz-type cell and the Sonoion® was applied. The temperature was kept in 37°C.

Results: The combination of sonophoresis and iontophoresis showed synergy effect on the antipyrine skin permeation. By Field-Emission scanning electron microscopy (FE-SEM), the some small holes in stratum corneum was observed by sonophoresis apply. In terms of the order of application, the higher effect was obtained by first sonophoresis application. Moreover, the long drug release was observed by only short time application of Sonoion®.

Conclusion: The combination of sonophoresis andiontophoresis is very effective to enhance drug permeation and long-time drug release. Such effect would be based on the small holes formation and deposition of the drug molecules in stratum corneum.

CO 056: DEVELOPMENT AND CHARACTERIZATION OF MANGIFERIN NANOCAPSULES

Nanomedicine, Natural Products, Pharmaceutical Technology
J Pharm Pharmacogn Res 2(Suppl. 1): S34, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 056: DEVELOPMENT AND CHARACTERIZATION OF MANGIFERIN NANOCAPSULES

Moura UJ, Barbosa MG, Genro C, Seibel D, Gomes P, Raffin PR.

Centro Universitário Franciscano. Rua dos Andradas, 1614, Santa Maria-RS, Brasil.
Abstract

Introduction: Manginferin (1,3,6,7-tetrahydroxi-xantona-C2-b-Dglucosilada) is a natural bioactive, more specifically a glycosylated xanthone isolated from Mangifera indica L. Mangiferin is the main component of mango extract that presents pharmacological activities on different organs and tissues, promoting preventive and therapeutic effects against a considerable number of diseases. However, one of the problems of mangiferin is its low solubility in water and the poor chemical stability. In this way, nanoencapsulation can be a promising tool to improve mangiferin bioavailability and stability in order to develop new medicines.

The objectives of this study were developed and characterize a nanoencapsulated formulation containing mangiferin and evaluate the physico-chemical characteristics of this nanosystem.

Material and methods: The interfacial deposition of preformed polymer technique was used to produce nanocapsules. It was necessary to add small amounts of DMSO in order to solubilize mangiferin in the organic phase. Mangifrin was encapsulated at 0.025%. Particle size, polydispersity index and zeta potential were evaluated in ZetaSizer NanoSeries. pH was assessed directly into the colloidal suspensions. Drug content and drug release experiments (dialysis) were analyzed in HPLC through a validated method.

Results: Nanocapsules were successfully obtained using Eudragit S100 as polymer. Particle size was 96.37 nm, polydispersity index was 0.202 and zeta potential was -16.63 mV. pH was 3.42. The results showed no significant difference (p < 0.05) between nanocapsules with and without mangiferin. Encapsulation efficiency was obtained by ultrafiltration/centrifugation and it was approximately 80.0%. Drug release profiles showed that mangiferin was released from nanocapsules half of the amount than free mangiferin, demonstrating a controlled profile.

Conclusions: Based on these results, we can conclude that mangiferin nanocapsules were developed with adequate characteristics for biological experiments.

This article has been cited by:
  • Rajneet Kaur Khurana, Ranjot Kaur, Manninder Kaur, Rajpreet Kaur, Jasleen Kaur, Harpreet Kaur & Bhupinder Singh (2017) Exploring and validating physicochemical properties of mangiferin through GastroPlus® software. Future Science OA. doi: 10.4155/fsoa-2016-0055

CO 055: NANOEMULSION PREPARATIONS FROM A PLANT LIPID EXTRACT WITH WIDE RANGE OF APPLICATIONS IN MEDICINE

Nanomedicine, Natural Products, Pharmaceutical Technology
J Pharm Pharmacogn Res 2(Suppl. 1): S33, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 055: NANOEMULSION PREPARATIONS FROM A PLANT LIPID EXTRACT WITH WIDE RANGE OF APPLICATIONS IN MEDICINE

Turiño L1, González E2, Nogueira A3, López O3.

1Centro de Estudios Avanzados de Cuba (CEAC). Carretera de San Antonio km 1½, La Lisa, La Habana, Cuba. E-mail: lazaro.td@cea.cu
2Centro Nacional de Biopreparados (BIOCEN). Carretera a Beltrán km 1½, Bejucal, Mayabeque, Cuba. E-mail: btj@biocen.cu
3Centro de Investigación y Desarrollo de Medicamentos (CIDEM). Ave. 26 No. 1605, e/ Puentes Grandes y Boyeros, Plaza de la Revolución, La Habana, Cuba. Email: oredloher@yahoo.es
Abstract

Introduction: The lipid extracts from vegetal origin have shown a very effective activity on the different mechanisms related to Benign Prostatic Hyperplasia. The oil obtained from the seeds of the Cuban pumpkin species Cucurbita pepo L., is an example of natural components useful for men who suffer this common hypertrophy. The administration of oily components by oral way as nanocapsules would be a variant of minimal invasion consumption and would also represent a strategy for favoring the absorption and bioavailability of the product.

Material and methods: In this work, the main objective was to obtain nanoemulsions by means of a high-energy method called High-speed Homogenization, using lipid extract from the seeds of Cucurbita pepo L. as a dispersed phase. The droplet size determination was performed by the diffraction method, Induced Grating. Through an experimental design, the influence of three speed levels (9500, 13500 and 20500/min) and three distance from the disperser to the bottom of the vessel, where the nanoemulsions were prepared (5, 10 and 15 mm) on droplet diameter were evaluated.

Results: It was shown statistically that for the studied values related to the distance from the disperser to the bottom, any significant difference was obtained, not being this the case the effect shown by the speed, since for the highest level of speed, a diameter 10 times lower (11.64 nm) relative to the one achieved with the minimum operation speed was reached.

Conclusions: The result obtained in this investigation showed that by subjecting the system to a high degree of shear, a significant reduction of the droplet size in the oil phase is guaranteed, which has an impact on absorption, due to the small size reached.

CO 054: AFFINITY MATURATION AND FINE FUNCTIONAL MAPPING OF AN ANTIBODY FRAGMENT AGAINST A NOVEL NEUTRALIZING EPITOPE ON HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR

Immunotherapy, Nanomedicine
J Pharm Pharmacogn Res 2(Suppl. 1): S33, 2014

Special supplement with the abstract book of LATINFARMA 2013

Oral Communication

CO 054: AFFINITY MATURATION AND FINE FUNCTIONAL MAPPING OF AN ANTIBODY FRAGMENT AGAINST A NOVEL NEUTRALIZING EPITOPE ON HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR

Lamdan Ha, Gavilondo JVa, Muñoz Ya, Pupo Ab, Huerta Va, Musacchio Aa, Pérez La, Ayala Ma, Rojas Gb, Balint RFc, Larrick JWd.

aCenter for Genetic Engineering and Biotechnology, La Habana 10600, Cuba. Email: humberto.lamdan@cigb.edu.cu
bCenter of Molecular Immunology, La Habana 11600, Cuba.
cCytoDesign, Inc., Palo Alto, CA 94306, USA.
dPanorama Research Inc., Sunnyvale, CA 94089, USA.
Abstract

Vascular endothelial growth factor (VEGF) is a major mediator of pathological angiogenesis. VEGF antagonists represent attractive candidates as therapeutic agents in the treatment of tumors and other angiogenesis-dependent diseases. Despite the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between VEGF and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Soluble 2H1 inhibited VEGF biological activity but exhibited a moderate binding affinity. We performed the affinity maturation of 2H1 antibody fragment. Two phage-displayed libraries were constructed by diversification of the third complementarity-determining regions (CDRs) of the light (VL) and heavy (VH) chain variable domains of 2H1 using parsimonious mutagenesis. A competitive phage-selection strategy in the presence of 2H1 as a competitor was used to eliminate low affinity binders. High affinity variants were retrieved from both libraries. An optimized VL variant was constructed by combining recurrent replacements found among selected variants, resulting in an additional affinity increase. Further affinity improvements were achieved by combining this optimized VL with the best VH variants. The final variant, L3H6, showed an overall affinity improvement of 18-fold over the parental antibody and exhibited an enhanced potency to block the binding of VEGF to its receptor. Functional mapping studies of L3H6 using phage display and extensive mutagenesis of VEGF revealed a novel neutralizing epitope on human VEGF.