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1.
ACS Biomater Sci Eng ; 10(6): 3806-3812, 2024 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-38709857

RESUMEN

In this work, for the first time, we demonstrate light control of a therapeutic protein's release from a depot in the subcutaneous layer of the skin. The subcutaneous layer is a standard location for therapeutic protein depots due to its large size and ease of access, but prior attempts to utilize this space failed because insufficient light can reach this deeper layer. An analysis of existing biophysical literature suggested that an increase of photoactivation wavelength from 365 to 500 nm could allow an increase of depot irradiation in the subcutaneous by >100-fold. We therefore used a green light-activated thio-coumarin-based material and demonstrated robust release of a therapeutic, insulin, in response to skin illumination with an LED light source. We further demonstrated that this release is ultrafast, as fast or faster than any commercially used insulin, while maintaining the native insulin sequence. This release of insulin was then accompanied by a robust reduction in blood glucose, demonstrating the retention of bioactivity despite the synthetic processing required to generate the material. In addition, we observed that the material exhibits slow basal release of insulin, even in the absence of light, potentially through biochemical or photochemical unmasking of insulin. Thus, these materials can act much like the healthy pancreas does: releasing insulin at a slow basal rate and then, upon skin irradiation, releasing an ultrafast bolus of native insulin to reduce postprandial blood glucose excursions.


Asunto(s)
Insulina , Luz , Animales , Glucemia/metabolismo , Glucemia/efectos de los fármacos , Humanos , Piel/metabolismo , Piel/efectos de la radiación , Piel/efectos de los fármacos , Cumarinas/química , Tejido Subcutáneo/efectos de los fármacos , Tejido Subcutáneo/metabolismo , Masculino , Luz Verde
2.
Bioorg Med Chem Lett ; 92: 129388, 2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37369330

RESUMEN

Previously we have demonstrated that light can be used to control the release of insulin in diabetic animals, followed by a reduction in blood glucose. This is accomplished using a photoactivated depot (PAD) of insulin injected into the skin, and irradiated by a small external LED light source. In this work for the first time we demonstrate dose-response, showing that we can vary insulin release and commensurate blood glucose reduction by varying the amount of light administered. In addition to demonstrating dose-response, we have shown multi-day depot response, with insulin being released on two different days from the same depot. The material used in these studies was CD-insulin, a form of insulin that has a highly non-polar cyclododecyl group attached, markedly reducing the solubility of the modified material, and allowing it to form a depot upon injection. Upon photolysis, the cyclododecyl group is removed, releasing fully native, soluble insulin. Variable response and multi-day response as demonstrated strongly support the potential utility of the PAD approach for the variable and extended release of therapeutic peptides.


Asunto(s)
Glucemia , Insulina , Animales , Piel , Solubilidad , Fotólisis
3.
ACS Biomater Sci Eng ; 7(4): 1506-1514, 2021 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-33703874

RESUMEN

In this work, we have brought the release of glucagon under the control of light. The aim of this approach is to allow minimally invasive, two-hormone control of blood glucose. Glucagon has two major challenges associated with its therapeutic application: (1) the required amount and timing of glucagon release is highly variable, and (2) glucagon rapidly fibrillates in solution, forming aggregates that are inactive. We have developed a light activated glucagon trimer, in which we have joined three glucagon molecules via light cleaved linkers. We demonstrated that this material can be stimulated by light to release glucagon in a predictable manner. In addition, we demonstrated that in the absence of light, the trimer does not form fibrils and thus releases normal unfibrillated glucagon upon irradiation. These qualities make this material ideal for incorporation into a two hormone light-activated artificial pancreas system.


Asunto(s)
Glucagón , Páncreas Artificial , Glucemia , Insulina
5.
Curr Diab Rep ; 19(11): 122, 2019 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-31696345

RESUMEN

PURPOSE OF REVIEW: The aim of this review is to summarize the development of the photoactivated depot (PAD) approach for the minimally invasive and continuously variable delivery of insulin. RECENT FINDINGS: Using an insulin PAD, we have demonstrated that we can release native, bioactive insulin into diabetic animals in response to light signals from a small external LED light source. We have further shown that this released insulin retains bioactivity and reduces blood glucose. In addition, we have designed and constructed second generation materials that have high insulin densities, with the potential for multiple day delivery. The PAD approach for insulin therapy holds promise for addressing the pressing need for continuously variable delivery methods that do not rely on pumps, and their myriad associated problems.


Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Implantes de Medicamentos/administración & dosificación , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Luz , Fármacos Fotosensibilizantes/administración & dosificación , Animales , Glucemia/análisis , Preparaciones de Acción Retardada/administración & dosificación , Diabetes Mellitus Tipo 1/sangre , Inyecciones Subcutáneas
6.
Mol Pharm ; 16(11): 4677-4687, 2019 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-31647241

RESUMEN

In this work, we describe the synthesis, characterization, and ultimate in vivo assessment of second-generation insulin photoactivated depot (PAD) materials. These are the first to use visible light to stimulate insulin release and have an in vivo performance that is 28-fold improved relative to first-generation materials. This improvement is due to two major factors linked to the utilized chemistry: (1) we have incorporated the coumarin photocleavable group, which increases the photorelease wavelength into the visible range, enhancing tissue penetration of the light; (2) phototoggling of insulin solubility is produced by linking three insulin molecules to a central bridge via light cleaved groups, and not by bonding to a large polymer. The resulting trimer is, therefore, highly dense (87% insulin dry w/w) but retains the insolubility required of the approach. Only after irradiation with visible light is native, soluble insulin is released from the dermal depot. This high density increases the amount and ease of insulin release, as the density of photolytic groups is 10-20-fold higher than in polymer-based first-generation materials. We have synthesized new azide-terminated coumarin linkers that we react with the amine groups of insulin. Using mass spectrometry methods, we identify the sites of reaction and purify individual isomers, which we demonstrate have in vitro photolysis rates that are within a factor of 2 of each other. We then reacted these terminal azide groups with a tridentate strained alkyne linker. We show that the resulting insulin trimer is highly insoluble, but can be milled into injectable particles that release insulin only in response to light from a 406 nm light source. Finally, we demonstrate that these materials have a significantly improved in vivo performance, releasing 28-fold more insulin on a per energy basis than first-generation materials.


Asunto(s)
Insulina/química , Azidas/química , Cumarinas/química , Luz , Fotólisis , Polímeros/química , Solubilidad
7.
Methods Enzymol ; 624: 113-128, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31370926

RESUMEN

In this work, we describe methods for synthesizing and incorporating a wide range of photocleavable groups into proteins. These are based on the di-methoxyl nitro phenyl ethyl (DMNPE) group. Using a common ketone starting material, we have modified the DMNPE core with different peptides and small molecules. We describe how these can be incorporated into DMNPE either by solution or solid phase methods. In addition, we show how the ketone group can be effectively converted into a hydrazone group and ultimately into a diazo. The potential pitfall of azine formation is also delineated, as are the strategies for avoiding this side product. We then show how these modified diazo groups can then be reacted with the carboxyl groups of the protein to make the final ester product. Finally, we show how the ultimate product can be purified, and the products identified using 280 and 345nm ratios, as well as ESI-MS characterization. The combined methods should allow the incorporation of many possible photocleavable groups into a range of proteins, and allow the ultimate properties of the modified protein to be subsequently toggled with light.


Asunto(s)
Compuestos Azo/química , Técnicas de Química Sintética/métodos , Nitrobencenos/química , Proteínas/química , Compuestos Azo/síntesis química , Humanos , Hidrazonas/síntesis química , Hidrazonas/química , Luz , Nitrobencenos/síntesis química , Péptidos/síntesis química , Péptidos/química , Procesos Fotoquímicos , Proteínas/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos
8.
Mol Pharm ; 16(7): 2922-2928, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-31117739

RESUMEN

We have previously described the photoactivated depot (PAD) approach for the light-stimulated release of therapeutic proteins such as insulin. The aim of this method is to release insulin from a shallow dermal depot in response to blood glucose information, using transcutaneous irradiation. Our first approach utilized a photocleavable group that linked insulin to an insoluble but injectable polymer bead. The bead conferred insolubility, ensuring that the injected material stayed at the site of injection, until light cleaved the link, and allowed insulin to be absorbed systemically. While this proved to be effective, the use of a polymer to ensure insolubility introduces two major design problems: (1) low concentration of insulin, as a majority of the material is composed of polymer, and (2) upon release of the insulin, the polymer has to be cleared from the system. To address these two problems, in this work, we have pursued "hydrophobic tags", photocleavable small nonpolar molecules that confer insolubility to the modified insulin prior to irradiation without the bulk or need for biodegradation required of polymers. We developed a combined solid- and solution-phase synthetic approach that allowed us to incorporate a range of small nonpolar moieties, including peptides, into the final depot materials. The resulting materials are >90% w/w insulin and have sharply decreased solubilities relative to unmodified insulin (≤1000 × lower). We demonstrated that they can be milled into low micron-sized particles that can be readily injected through a 31G needle. These suspensions can be prepared at an effective concentration of 20 mM insulin, a concentration at which 120 µL contains 7 days of insulin for a typical adult. Finally, upon photolysis, the insoluble particles release soluble, native insulin in a predictable fashion. These combined properties make these new modified insulins nearly ideal as candidates for PAD materials.


Asunto(s)
Liberación de Fármacos/efectos de la radiación , Interacciones Hidrofóbicas e Hidrofílicas/efectos de la radiación , Insulina Regular Humana/química , Insulina Regular Humana/efectos de la radiación , Luminiscencia , Adulto , Humanos , Inyecciones , Cinética , Concentración Osmolar , Tamaño de la Partícula , Fotólisis/efectos de la radiación , Polímeros/administración & dosificación , Polímeros/química , Proteínas Recombinantes/química , Solubilidad , Suspensiones/química
9.
Bioorg Med Chem Lett ; 29(3): 424-429, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30579791

RESUMEN

In this work we have developed specific inhibitors of HIV-1 reverse transcriptase by targeting the RNA/DNA duplex that is a principal substrate of the enzyme. To accomplish this, we have developed what we are calling the "weak intercalator" approach, wherein we attempt to simultaneously bind multiple weak intercalators to critical polymerase nucleic acids. We define weak intercalators as planar sp2 hybridized molecules with only two cycles, that have poor binding affinity individually and can only bind with high affinity if two or more weak intercalation events can take place. Using this approach, we have identified linear and cyclic molecules that present two weak intercalators that can inhibit HIV-1-RT 50 to 100 times more effectively than single weak intercalators. Specifically, a cyclic peptide motif that presents two quinoxaline rings inhibits HIV-1-RT at low µM concentration, shows no inhibition of DNA polymerase and in addition maintains a majority of its inhibitory power in the presence of 90,000 fold excess duplex DNA. These results suggest that the weak intercalator approach may prove effective as a way of targeting increasingly complex nucleic acid structures in a highly specific manner.


Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Quinoxalinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Dosis-Respuesta a Droga , Transcriptasa Inversa del VIH/metabolismo , Humanos , Estructura Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Quinoxalinas/síntesis química , Quinoxalinas/química , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Relación Estructura-Actividad
10.
Int J Pept Res Ther ; 24(4): 535-542, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30416404

RESUMEN

We have developed a straightforward and robust strategy for synthesizing a family of cyclic peptide scaffolds for the presentation of defined moieties in a wide range of orientations. Specifically we are exploring quinoxaline as the moiety, as a potential nucleic acid binding motif. The method requires the use of four degrees of orthogonality, which in turn allow the extension of the main chain, incorporation of the target side chains, on-resin cyclization, and the revelation of an amino group upon cleavage to increase solubility. We show that related approaches fail for a range of reasons, including the failure of cyclization. Following the optimization of the approach with a single cyclic peptide, we synthesized a family of all possible bis and tris quinoxaline adducts showing by ESI-MS that the desired full length cyclic product is produced in a majority of cases.

11.
Chembiochem ; 19(12): 1264-1270, 2018 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-29516677

RESUMEN

There is a need for methods to chemically incorporate photocleavable labels into synthetic and biologically sourced nucleic acids in a chemically defined and reversible manner. We have previously demonstrated that the light-cleaved diazo di-methoxy nitro phenyl ethyl (diazo-DMNPE) group has a remarkable regiospecificity for modifying terminally phosphorylated siRNA. Building on this observation, we have identified conditions under which a diazo-DMNPE reagent that we designed (diazo-DMNPE-azide or DDA) is able to singly modify any nucleic acid (RNA, DNA, single-stranded, double-stranded, 3' or 5' phosphate). It can then be modified with any clickable reagent to incorporate arbitrary labels such as fluorophores into the nucleic acid. Finally, native nucleic acid can be regenerated directly through photolysis of the reagent. Use of the described approach should allow for the tagging of any nucleic acid, from any source-natural or unnatural-while allowing for the light-induced regeneration of native nucleic acid.


Asunto(s)
Azidas/química , Compuestos Azo/química , Química Clic/métodos , ADN/química , Nitrocompuestos/química , ARN/química , Azidas/síntesis química , Compuestos Azo/síntesis química , ADN/síntesis química , Indicadores y Reactivos , Nitrocompuestos/síntesis química , Fosforilación , Fotólisis , ARN/síntesis química , Coloración y Etiquetado/métodos , Estereoisomerismo
12.
J Am Chem Soc ; 139(49): 17861-17869, 2017 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-29192764

RESUMEN

We previously described the photoactivated depot or PAD approach that allows for the light control of therapeutic protein release. This approach relies on the ability to use light to change a protein's solubility. Traditionally this was accomplished by linking the protein to an insoluble but injectable polymer via a light cleaved linker. This allows the injected material to remain at the site of injection, until transcutaneous irradiation breaks the link between polymer and protein, permitting the protein to be absorbed. However, there are multiple problems associated with polymer based approaches: The polymer makes up a majority of the material, making it inefficient. In addition, after protein release, the polymer has to be cleared from the body, a significant design challenge. In this work, we create materials that form photoactivated depots of insulin without the need for polymers, by linking photolysis to an isoelectric point shift, which itself is linked to a solubility shift. Specifically, we linked basic groups to insulin via a light cleaved linker. These shift the normal pI of insulin from 5.4 to approximately 7. The result of this incorporation are materials that are completely soluble in mildly acidic solutions but precipitate upon injection into a pH 7 environment, i.e., the skin. We successfully synthesized four such modified insulins, demonstrating that their pI values were shifted in the expected manner. We then analyzed one of them, P2-insulin, in detail, demonstrating that it behaves as designed: It is soluble in a formulation pH of 4, but precipitates at pH 7.2, its approximate pI value. Upon irradiation, the photocleavable link to insulin is broken, and completely native and soluble insulin is released from the depot in a well behaved, first order fashion. These materials are 90% therapeutic, form completely soluble and injectable formulations in mildly acidic conditions, form insoluble depots at neutral pH, efficiently release soluble protein from these depots when irradiated, and leave behind only small easily absorbed molecules after irradiation. As such they approach ideality for photoactivated depot materials.


Asunto(s)
Insulina/química , Insulina/efectos de la radiación , Punto Isoeléctrico , Luz , Precipitación Química , Humanos , Concentración de Iones de Hidrógeno , Piel , Solubilidad/efectos de la radiación
13.
Mol Pharm ; 13(11): 3835-3841, 2016 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-27653828

RESUMEN

In this work we demonstrate that blood glucose can be controlled remotely through light stimulated release of insulin from an injected cutaneous depot. Human insulin was tethered to an insoluble but injectable polymer via a linker, which was based on the light cleavable di-methoxy nitrophenyl ethyl (DMNPE) group. This material was injected into the skin of streptozotocin-treated diabetic rats. We observed insulin being released into the bloodstream after a 2 min trans-cutaneous irradiation of this site by a compact LED light source. Control animals treated with the same material, but in which light was blocked from the site, showed no release of insulin into the bloodstream. We also demonstrate that additional pulses of light from the light source result in additional pulses of insulin being absorbed into circulation. A significant reduction in blood glucose was then observed. Together, these results demonstrate the feasibility of using light to allow for the continuously variable control of insulin release. This in turn has the potential to allow for the tight control of blood glucose without the invasiveness of insulin pumps and cannulas.


Asunto(s)
Glucemia/efectos de los fármacos , Insulina/química , Luz , Fotoquímica/métodos , Animales , Ensayo de Inmunoadsorción Enzimática , Insulina/farmacología , Masculino , Ratas , Ratas Sprague-Dawley
14.
Macromol Biosci ; 16(8): 1250, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27491330

RESUMEN

Back Cover: The authors have created polymers which release insulin using light. These materials utilize insulin itself as the primary monomer and therefore achieve a very high density (85% w/w insulin). This near perfect density makes them ideal as photoactivated depots of insulin. Further details can be found in the article by Bhagyesh R. Sarode, Piyush K. Jain, Simon H. Friedman on page 1138.

15.
Macromol Biosci ; 16(8): 1138-46, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27171861

RESUMEN

The use of light-sensitive polymers for the release of therapeutics is an important approach allowing the timing and amount of the release to be controlled precisely. The use of light has been pioneered to control insulin release from a dermal photoactivated depot, or PAD. One of the main impediments to the use of light-sensitive polymers in this context is the density of the materials: The large majority of the material is the carrier polymer, with the minority being the therapeutic. In this work, the feasibility of using insulin itself as a monomer in the polymerization process is demonstrated. Insulin modified with either one or two light cleavable azide groups is polymerized with a tridentate alkyne-bridging monomer using a click reaction. The resulting material called a "macropolymer" is ≈85% insulin, is insoluble in aqueous solvent, and releases native, soluble insulin upon irradiation.


Asunto(s)
Insulina/metabolismo , Luz , Polimerizacion , Polímeros/química , Dimetilsulfóxido/química , Ensayo de Inmunoadsorción Enzimática , Humanos , Cinética , Fotólisis/efectos de la radiación , Polímeros/síntesis química
16.
Nat Protoc ; 9(1): 11-20, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24309973

RESUMEN

Light-activated RNA interference (LARI) is an effective way to control gene expression with light. This, in turn, allows for the spacing, timing and degree of gene expression to be controlled by the spacing, timing and amount of light irradiation. The key mediators of this process are siRNA or dsRNA that have been modified with four photocleavable groups of dimethoxy nitro phenyl ethyl (DMNPE), located on the four terminal phosphate groups of the duplex RNA. These mediators can be easily synthesized and purified using two readily available products: synthetic RNA oligonucleotides and DMNPE-hydrazone. The synthesis of the tetra-DMNPE-modified duplex RNA is made possible by a remarkable regiospecificity of DMNPE for terminal phosphates (over internal phosphates or nucleobases) that we have previously identified. The four installed DMNPE groups effectively limit RNAi until irradiation cleaves them, releasing native, active siRNA. By using the described protocol, any process that is mediated by RNAi can be controlled with light. Although other methods exist to control gene expression with light by using specialized reagents, this method requires only two commercially available products. The protocol takes ∼3 d in total for the preparation of modified RNA.


Asunto(s)
Luz , Interferencia de ARN/efectos de la radiación , ARN Bicatenario/química , ARN Interferente Pequeño/química , Técnicas Genéticas , Células HeLa , Humanos , Nitrobencenos/química
18.
Bioorg Med Chem Lett ; 22(14): 4844-8, 2012 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-22695131

RESUMEN

We have previously demonstrated that polymerases such as telomerase can be inhibited by molecules (e.g., intercalators) that target the key RNA/DNA duplex substrate. In this work we show that this also holds true for reverse transcriptase, and show that the lead intercalators can be modified to increase inhibition efficacy. Specifically, we use the strategy of multiple simultaneous intercalation, by linking two intercalators with a variable linker. The rationale behind this design is that a specific linker has the potential to increase affinity and specificity for the target duplex. We have synthesized a library of 45 ethidium bis-intercalators in which the distance between intercalators is systematically varied. We observe that members of the dimer library have improved telomerase and reverse transcriptase inhibition, relative to the monomeric leads. We show that this improvement in inhibition over mono-intercalators is most prominent when non-productive sites of inhibitor binding are limited in the assay mix. When this is done, a 400-fold increase in inhibition efficacy is observed.


Asunto(s)
Inhibidores Enzimáticos/química , Sustancias Intercalantes/química , Inhibidores Enzimáticos/farmacología , Sustancias Intercalantes/farmacología , Estructura Molecular , ADN Polimerasa Dirigida por ARN/metabolismo , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Telomerasa/antagonistas & inhibidores
19.
Nat Chem ; 4(5): 426, 2012 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-22522265

Asunto(s)
Carbono
20.
Pharm Res ; 28(12): 3050-7, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21739319

RESUMEN

PURPOSE: To improve light-activated RNA interference by incorporating phosphorothioate linkages into the dsRNA used. The rationale behind this approach is that the groups have the potential to improve nuclease stability and therefore prevent cleavage of photolabile groups from the RNA termini prior to photolysis. METHODS: Photolabile groups (di-methoxy nitro phenyl ethyl or DMNPE) were incorporated into multiple double-stranded precursors of siRNA (dsRNA) that had six, two or no phosphorothioate linkages at the 3' and 5' ends of the strands. They were analyzed for their ability to toggle light-activated RNA interference with light and for serum stability. RESULTS: Incorporation of phosphorothioate linkages increased serum stability of all dsRNA examined. Presence of DMNPE groups reduced overall stability of the modified RNA relative to the analogous species without DMNPE modification. DMNPE-modified dsRNA with two phosphorothioate linkages in each strand significantly improved the window of expression toggled by light. CONCLUSIONS: Incorporating phosphorothioate groups into dsRNA both stabilizes them towards degradation by serum enzymes, as well as improves them as the basis for light-activated RNA interference.


Asunto(s)
Fosfatos/química , Interferencia de ARN , ARN Bicatenario/química , ARN Bicatenario/genética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Secuencia de Bases , Células HeLa , Humanos , Luz , Nitrobencenos/química , ARN Bicatenario/metabolismo , ARN Interferente Pequeño/metabolismo , Suero/metabolismo , Transfección
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