RESUMEN
Prodrugs engineered for preferential activation in diseased versus normal tissues offer immense potential to improve the therapeutic indexes (TIs) of preclinical and clinical-stage active pharmaceutical ingredients that either cannot be developed otherwise or whose efficacy or tolerability it is highly desirable to improve. Such approaches, however, often suffer from trial-and-error design, precluding predictive synthesis and optimization. Here, using bromodomain and extra-terminal (BET) protein inhibitors (BETi)-a class of epigenetic regulators with proven anticancer potential but clinical development hindered in large part by narrow TIs-we introduce a macromolecular prodrug platform that overcomes these challenges. Through tuning of traceless linkers appended to a "bottlebrush prodrug" scaffold, we demonstrate correlation of in vitro prodrug activation kinetics with in vivo tumor pharmacokinetics, enabling the predictive design of novel BETi prodrugs with enhanced antitumor efficacies and devoid of dose-limiting toxicities in a syngeneic triple-negative breast cancer murine model. This work may have immediate clinical implications, introducing a platform for predictive prodrug design and potentially overcoming hurdles in drug development.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Profármacos/farmacología , Proteínas/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Sustancias Macromoleculares/síntesis química , Sustancias Macromoleculares/química , Sustancias Macromoleculares/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Estructura Molecular , Profármacos/síntesis química , Profármacos/química , Proteínas/metabolismoRESUMEN
At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection, and gene-expression analyses. In rats and dogs, the prodrugs are retained long-term in liver tissue and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Diseño de Fármacos , Cirrosis Hepática/tratamiento farmacológico , Profármacos/uso terapéutico , Telmisartán/uso terapéutico , Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacocinética , Animales , Tetracloruro de Carbono/toxicidad , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Semivida , Hígado/metabolismo , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Polímeros/química , Profármacos/química , Profármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , Telmisartán/químicaRESUMEN
In the version of this Article originally published, the author Peter Blume-Jensen was not denoted as a corresponding author; this has now been amended and the author's email address has been added. The 'Correspondence and requests for materials' statement was similarly affected and has now been updated with the author's initials 'P.B-J.'
RESUMEN
Tissue engineering strategies for cartilage defect repair require technology for local targeted delivery of chondrogenic and anti-inflammatory factors. The objective of this study was to determine the release kinetics of transforming growth factor ß1 (TGF-ß1) from self-assembling peptide hydrogels, a candidate scaffold for cell transplant therapies, and stimulate chondrogenesis of encapsulated young equine bone marrow stromal cells (BMSCs). Although both peptide and agarose hydrogels retained TGF-ß1, fivefold higher retention was found in peptide. Excess unlabeled TGF-ß1 minimally displaced retained radiolabeled TGF-ß1, demonstrating biologically relevant loading capacity for peptide hydrogels. The initial release from acellular peptide hydrogels was nearly threefold lower than agarose hydrogels, at 18% of loaded TGF-ß1 through 3 days as compared to 48% for agarose. At day 21, cumulative release of TGF-ß1 was 32-44% from acellular peptide hydrogels, but was 62% from peptide hydrogels with encapsulated BMSCs, likely due to cell-mediated TGF-ß1 degradation and release of small labeled species. TGF-ß1 loaded peptide hydrogels stimulated chondrogenesis of young equine BMSCs, a relevant preclinical model for treating injuries in young human cohorts. Self-assembling peptide hydrogels can be used to deliver chondrogenic factors to encapsulated cells making them a promising technology for in vivo, cell-based regenerative medicine.
Asunto(s)
Condrogénesis/efectos de los fármacos , Hidrogeles/farmacología , Células Madre Mesenquimatosas/citología , Péptidos/farmacología , Factor de Crecimiento Transformador beta1/farmacología , Adsorción , Animales , Bovinos , Células Inmovilizadas/citología , Células Inmovilizadas/efectos de los fármacos , Células Inmovilizadas/metabolismo , Preparaciones de Acción Retardada , Caballos , Humanos , Radioisótopos de Yodo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , SefarosaRESUMEN
BACKGROUND: The best strategy for delivering growth factors to cells for the purpose of cartilage tissue engineering remains an unmet challenge. Tethering biotinylated insulin-like growth factor-1 (bIGF-1) to the self-assembling peptide scaffold (RADA)(4) effectively delivers bioactive bIGF-1 to cardiac tissue. QUESTIONS/PURPOSES: We therefore asked whether: (1) soluble bIGF-1 could stimulate proteoglycan production by chondrocytes; (2) bIGF-1 could be adsorbed or tethered to the self-assembling peptide scaffold (KLDL)(3); (3) adsorbed or tethered bIGF-1 could stimulate proteoglycan production; and (4) transforming growth factor-ß1 (TGF-ß1) could be adsorbed or tethered and stimulate proteoglycan production by bone marrow stromal cells (BMSCs). METHODS: Chondrocytes or BMSCs were encapsulated in (KLDL)(3). The growth factors were (1) delivered solubly in the medium; (2) adsorbed to (KLDL)(3); or (3) tethered to (KLDL)(3) through biotin-streptavidin bonds. Fluorescently tagged streptavidin was used to determine IGF-1 kinetics; sGAG and DNA content was measured. RESULTS: Soluble bIGF-1 stimulated comparable sGAG accumulation as soluble IGF-1. Tethering IGF-1 to (KLDL)(3) increased retention of IGF-1 in (KLDL)(3) compared with adsorption, but neither method increased sGAG or DNA accumulation above control. Adsorbing TGF-ß1 increased proteoglycan accumulation above control, but tethering did not affect sGAG levels. CONCLUSIONS: Although TGF-ß1 can be effectively delivered by adsorption to (KLDL)(3), IGF-1 cannot. Additionally, although tethering these factors provided long-term sequestration, tethering did not stimulate proteoglycan production. CLINICAL RELEVANCE: Tethering growth factors to (KLDL)(3) results in long-term delivery, but tethering does not necessarily result in the same bioactivity as soluble delivery, indicating presentation of proteins is vital when considering a delivery strategy.
Asunto(s)
Portadores de Fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Péptidos/química , Ingeniería de Tejidos/métodos , Andamios del Tejido , Factor de Crecimiento Transformador beta1/farmacología , Adsorción , Animales , Biotinilación , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Bovinos , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , ADN/metabolismo , Fluorometría , Glicosaminoglicanos/metabolismo , Hidrogeles , Factor I del Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/metabolismo , Cinética , Péptidos/metabolismo , Conformación Proteica , Proteoglicanos/metabolismo , Solubilidad , Estreptavidina/metabolismo , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Técnicas de Cultivo de Tejidos , Factor de Crecimiento Transformador beta1/química , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
We describe analysis of suspensory ligaments from horses with advanced degenerative suspensory ligament desmitis (DSLD) to identify the major proteoglycans (PGs), ADAMTS-aggrecanases and inter-alpha-trypsin inhibitor (IαI) components associated with ligament degeneration. Specific anatomical regions of suspensory ligaments from two normal horses and four diagnosed with DSLD were analyzed by Western blot and immunohistochemistry for the following: aggrecan, aggrecan fragments, decorin, ADAMTS4, ADAMTS5, and IαI components. When compared to normal, DSLD ligaments showed about a 15-fold increase (P < 0.0014) in aggrecan levels and markedly enhanced staining with Safranin O. The aggrecan was composed of two distinct high molecular weight core protein species. The largest species was found only in DSLD samples and it co-migrated with aggrecan synthesized by equine mesenchymal stem cells (MSC). Many of the DSLD samples also contained abnormally high concentrations of ADAMTS4, ADAMTS5, and IαI. Notably, the ADAMTS5 in DSLD samples, but not normals, was present largely as a high molecular weight complex. We conclude that ligament degeneration in DSLD is associated with matrix changes characteristic of an inflammatory nonhealing wound, specifically containing chondrogenic progenitor cells. Since aggrecan accumulation is a major feature of incomplete healing in tendon and skin of the ADAMTS5 knockout mouse, we propose that ligament failure in DSLD results from a process involving tissue inflammation and the complexation of ADAMTS5.
Asunto(s)
Proteínas ADAM/metabolismo , Agrecanos/metabolismo , alfa-Globulinas/metabolismo , Enfermedades del Tejido Conjuntivo/metabolismo , Enfermedades de los Caballos/metabolismo , Ligamentos/metabolismo , Animales , Western Blotting , Enfermedades del Tejido Conjuntivo/patología , Endopeptidasas/metabolismo , Femenino , Enfermedades de los Caballos/patología , Caballos , Inmunohistoquímica , Ligamentos/patología , MasculinoRESUMEN
Self-assembling peptide hydrogels were modified to deliver transforming growth factor ß1 (TGF-ß1) to encapsulated bone-marrow-derived stromal cells (BMSCs) for cartilage tissue engineering applications using two different approaches: (i) biotin-streptavidin tethering; (ii) adsorption to the peptide scaffold. Initial studies to determine the duration of TGF-ß1 medium supplementation necessary to stimulate chondrogenesis showed that 4 days of transient soluble TGF-ß1 to newborn bovine BMSCs resulted in 10-fold higher proteoglycan accumulation than TGF-ß1-free culture after 3 weeks. Subsequently, BMSC-seeded peptide hydrogels with either tethered TGF-ß1 (Teth-TGF) or adsorbed TGF-ß1 (Ads-TGF) were cultured in the TGF-ß1-free medium, and chondrogenesis was compared to that for BMSCs encapsulated in unmodified peptide hydrogels, both with and without soluble TGF-ß1 medium supplementation. Ads-TGF peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like extracellular matrix accumulation, whereas Teth-TGF did not stimulate chondrogenesis. In parallel experiments, TGF-ß1 adsorbed to agarose hydrogels stimulated comparable chondrogenesis. Full-length aggrecan was produced by BMSCs in response to Ads-TGF in both peptide and agarose hydrogels, whereas medium-delivered TGF-ß1 stimulated catabolic aggrecan cleavage product formation in agarose but not peptide scaffolds. Smad2/3 was transiently phosphorylated in response to Ads-TGF but not Teth-TGF, whereas medium-delivered TGF-ß1 produced sustained signaling, suggesting that dose and signal duration are potentially important for minimizing aggrecan cleavage product formation. Robustness of this technology for use in multiple species and ages was demonstrated by effective chondrogenic stimulation of adult equine BMSCs, an important translational model used before the initiation of human clinical studies.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Hidrogeles/química , Péptidos/química , Factor de Crecimiento Transformador beta1/farmacología , Animales , Western Blotting , Células de la Médula Ósea/metabolismo , Bovinos , Células Cultivadas , Caballos , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Ingeniería de Tejidos , Factor de Crecimiento Transformador beta1/químicaRESUMEN
Our objective was to test the hypothesis that self-assembling peptide hydrogel scaffolds provide cues that enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs). BMSCs were encapsulated within two unique peptide hydrogel sequences, and chondrogenesis was compared with that in agarose hydrogels. BMSCs in all three hydrogels underwent transforming growth factor-beta1-mediated chondrogenesis as demonstrated by comparable gene expression and biosynthesis of extracellular matrix molecules. Expression of an osteogenic marker was unchanged, and an adipogenic marker was suppressed by transforming growth factor-beta1 in all hydrogels. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher glycosaminoglycan content and more spatially uniform proteoglycan and collagen type II deposition. The G1-positive aggrecan produced in peptide hydrogels was predominantly the full-length species, whereas that in agarose was predominantly the aggrecanase product G1-NITEGE. Unique cell morphologies were observed for BMSCs in each peptide hydrogel sequence, with extensive cell-cell contact present for both, whereas BMSCs in agarose remained rounded over 21 days in culture. Differences in cell morphology within the two peptide scaffolds may be related to sequence-specific cell adhesion. Taken together, this study demonstrates that self-assembling peptide hydrogels enhance chondrogenesis compared with agarose as shown by extracellular matrix production, DNA content, and aggrecan molecular structure.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Hidrogeles/farmacología , Péptidos/farmacología , Actinas/metabolismo , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Agrecanos/metabolismo , Animales , Western Blotting , Células de la Médula Ósea/metabolismo , Bovinos , Forma de la Célula/efectos de los fármacos , Condrogénesis/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , ADN/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glicosaminoglicanos/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
Bone marrow mesenchymal stem cells (BM-MSCs) and adipose-derived progenitor cells (ADPCs) are potential alternatives to autologous chondrocytes for cartilage resurfacing strategies. In this study, the chondrogenic potentials of these cell types were compared by quantifying neo-tissue synthesis and assaying gene expression and accumulation of extracellular matrix (ECM) components of cartilage. Adult equine progenitor cells encapsulated in agarose or self-assembling peptide hydrogels were cultured in the presence or absence of TGFbeta1 for 3 weeks. In BM-MSCs-seeded hydrogels, TGFbeta1 stimulated ECM synthesis and accumulation 3-41-fold relative to TGFbeta1-free culture. In ADPC cultures, TGFbeta1 stimulated a significant increase in ECM synthesis and accumulation in peptide (18-29-fold) but not agarose hydrogels. Chromatographic analysis of BM-MSC-seeded agarose and peptide hydrogels cultured in TGFbeta1 medium showed extensive synthesis of aggrecan-like proteoglycan monomers. ADPCs seeded in peptide hydrogel also synthesized aggrecan-like proteoglycans, although to a lesser extent than seen in BM-MSC hydrogels, whereas aggrecan-like proteoglycan synthesis in ADPC-seeded agarose was minimal. RT-PCR analysis of TGFbeta1 cultures showed detectable levels of type II collagen gene expression in BM-MSC but not ADPC cultures. Histological analysis of TGFbeta1-cultured peptide hydrogels showed the deposition of a continuous proteoglycan- and type II collagen rich ECM for BM-MSCs but not ADPCs. Therefore, this study showed both protein and gene expression evidence of superior chondrogenesis of BM-MSCs relative to ADPCs.