RESUMEN
Hyaluronan (HA) is a glycosaminoglycan that forms a gel-like barrier in the subcutaneous (SC) space, limiting bulk fluid flow and the dispersion of SC-administered therapeutics. Recombinant human hyaluronidase PH20 (rHuPH20) facilitates the rapid delivery of co-administered therapeutics by depolymerizing HA in the SC space. Administration of rHuPH20 can induce the formation of anti-rHuPH20 antibodies, or anti-drug antibodies (ADAs), with the potential to bind endogenous PH20 hyaluronidase in the adult testes and epididymis. Using a variety of relevant animal models and multiple dose regimens of rHuPH20 across the full spectrum of animal development, we demonstrated that rHuPH20 administration resulted in the formation of ADAs. Although these ADAs can bind both the recombinant rHuPH20 enzyme and recombinant versions of animal model-specific hyaluronidases, they had no impact on fertility parameters (as measured by sperm concentration and motility, litter size, and litter viability) or fetal development. We present the result of our nonclinical studies in order of the developmental lifecycle, beginning with adults. Toxicology studies that extend beyond the standard package are also presented. These studies demonstrate the favorable safety profile of rHuPH20 and ADAs in nonclinical models. Additionally, we identified substantial safety margins for clinically relevant doses of rHuPH20.
RESUMEN
Subcutaneous (SC) infusion of large volumes at rapid flow rates has historically been limited by the glycosaminoglycan hyaluronan (HA), which forms a barrier to bulk fluid flow in the SC space. Recombinant human hyaluronidase PH20 (rHuPH20) depolymerizes HA, temporarily eliminating this barrier to rapid SC delivery of large volume co-administered therapeutics. Using a miniature pig model, in-line pressure and applied force to the delivery hardware were measured when subcutaneously infusing a representative macromolecule (human polyclonal immunoglobulin [Ig]), at varying concentrations and viscosities (20-200 mg/mL), co-formulated with and without rHuPH20 (2000 U/mL and 5000 U/mL). Maximal flow rate (Qmax) was calculated as the flow rate producing a statistically significant difference in mean applied force between injections administered with or without rHuPH20. There was a significant reduction in mean applied force required for SC delivery of 100 mg/mL Ig solution with 5000 U/mL rHuPH20 versus Ig solution alone. Similar significant reductions in mean applied force were observed for most Ig solution concentrations, ranging from 25-200 mg/mL when administered with or without 2000 U/mL rHuPH20. Qmax was inversely proportional to Ig solution viscosity and Qmax for solutions co-formulated with 5000 U/mL rHuPH20 was approximately double that of 2000 U/mL rHuPH20 solutions. Mathematical simulation of a hypothetical 800 mg Ig dose co-formulated with rHuPH20 showed that delivery times <30 s could be achieved across a broad range of concentrations. Addition of rHuPH20 can help overcome volume and time constraints associated with SC administration across a range of concentrations in a dose-dependent manner.
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Anticuerpos , Hialuronoglucosaminidasa , Humanos , Porcinos , Animales , Porcinos Enanos , Viscosidad , Simulación por Computador , Ácido HialurónicoRESUMEN
Multiple FDA-approved and clinical-development stage therapeutics include recombinant human hyaluronidase PH20 (rHuPH20) to facilitate subcutaneous administration. As rHuPH20-reactive antibodies potentially interact with endogenous PH20, we investigated rHuPH20 immunogenicity risk through hyaluronidase tissue expression, predicted B cell epitopes, CD4+ T cell stimulation indices and related these to observed clinical immunogenicity profiles from 18 clinical studies. Endogenous hyaluronidase PH20 expression in humans/mice was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative RT-PCR, and deep RNA-Seq. rHuPH20 potential T cell epitopes were evaluated in silico and confirmed in vitro. Potential B cell epitopes were predicted for rHuPH20 sequence in silico, and binding of polyclonal antibodies from various species tested on a rHuPH20 peptide microarray. Clinical immunogenicity data were collected from 2643 subjects. From 57 human adult and fetal tissues previously screened by RT-PCR, 22 tissue types were analyzed by deep RNA-Seq. Hyaluronidase PH20 messenger RNA expression was detected in adult human testes. In silico analyses of the rHuPH20 sequence revealed nine T cell epitope clusters with immunogenic potential, one cluster was homologous to human leukocyte antigen. rHuPH20 induced T cell activation in 6-10% of peripheral blood mononuclear cell donors. Fifteen epitopes in the rHuPH20 sequence had the potential to cross-react with B cells. The cumulative treatment-induced incidence of anti-rHuPH20 antibodies across clinical studies was 8.8%. Hyaluronidase PH20 expression occurs primarily in adult testes. Low CD4+ T cell activation and B cell cross-reactivity by rHuPH20 suggest weak rHuPH20 immunogenicity potential. Restricted expression patterns of endogenous PH20 indicate low immunogenicity risk of subcutaneous rHuPH20.
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Epítopos de Linfocito T , Hialuronoglucosaminidasa , Humanos , Adulto , Masculino , Ratones , Animales , Hialuronoglucosaminidasa/genética , Hialuronoglucosaminidasa/metabolismo , Epítopos de Linfocito T/genética , Epítopos de Linfocito B , Leucocitos Mononucleares , Proteínas Recombinantes/metabolismo , Testículo/metabolismo , Anticuerpos , Factores de Riesgo , ARN Mensajero , ADN Polimerasa Dirigida por ARNRESUMEN
PURPOSE: To evaluate the duration of effect of rHuPH20 on SC absorption of cetuximab and to develop a mechanistic pharmacokinetic model linking the kinetics of rHuPH20 action with hyaluronan (HA) homeostasis and absorption of cetuximab from the SC space. METHODS: Serum pharmacokinetics of cetuximab was evaluated after IV and SC dosing at 0.4 and 10 mg/kg (control groups). In test groups, SC cetuximab was administered simultaneously with rHuPH20 (Co-Injection) or 12 h after injection of rHuPH20 (Pre-Injection). Mechanistic pharmacokinetic model was developed to simultaneously capture cetuximab kinetics in all groups. RESULTS: Administration of rHuPH20 resulted in a faster absorption of cetuximab; the difference between co-injection and pre-injection groups appeared to be dependent on the dose level. The model combined three major components: kinetics of rHuPH20 at SC site; HA homeostasis and its disruption by rHuPH20; and cetuximab systemic disposition and the effect of HA disruption on cetuximab SC absorption. The model provided good description of experimental data obtained in this study and collected previously. CONCLUSIONS: Proposed model can serve as a potential translational framework for capturing the effect of rHuPH20 across multiple preclinical species and in human studies and can be used for optimization of SC delivery of biotherapeutics.
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Ácido Hialurónico , Hialuronoglucosaminidasa , Animales , Cetuximab/farmacología , Humanos , Inyecciones Subcutáneas , Ratas , Proteínas RecombinantesRESUMEN
INTRODUCTION: The glycosaminoglycan hyaluronan forms a gel-like substance, which presents a barrier to bulk fluid flow in the subcutaneous (SC) space, limiting SC drug delivery volume and administration rates. Recombinant human hyaluronidase PH20 (rHuPH20) acts locally to temporarily remove this barrier, facilitating rapid SC delivery of large volumes and/or high doses of sequentially or co-administered therapeutics. AREAS COVERED: An extensive clinical and post-marketing dataset of safety and immunogenicity of rHuPH20 in its current applications with approved therapeutics demonstrates that rHuPH20 acts locally, without measurable systemic absorption at the SC doses used in the approved products, and is well tolerated in combination with several co-administered therapeutic agents across diverse patient groups. The immunogenicity profile demonstrates no adverse effects associated with treatment-emergent rHuPH20 antibody responses. Immunogenicity to monoclonal antibodies co-formulated with rHuPH20 shows no clinical difference between SC and intravenous administration. Safety assessments of patient subsets for special populations, including children, elderly patients, and pregnant women, raise no additional safety concerns. EXPERT OPINION: The benefits of SC administration for patients and healthcare systems often outweigh those of intravenous administration, driving future initiation of SC-only drug development programs. Injection devices allowing large-volume SC administration could be facilitated by incorporating co-formulated biologics containing rHuPH20.
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Hialuronoglucosaminidasa , Preparaciones Farmacéuticas , Anciano , Niño , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inyecciones Subcutáneas , Embarazo , Proteínas RecombinantesRESUMEN
BACKGROUND: Scarce drug penetration in solid tumours is one of the possible causes of the limited efficacy of chemotherapy and is related to the altered tumour microenvironment. The abnormal tumour extracellular matrix (ECM) together with abnormal blood and lymphatic vessels, reactive stroma and inflammation all affect the uptake, distribution and efficacy of anticancer drugs. METHODS: We investigated the effect of PEGylated recombinant human hyaluronidase PH20 (PEGPH20) pre-treatment in degrading hyaluronan (hyaluronic acid; HA), one of the main components of the ECM, to improve the delivery of antitumor drugs and increase their therapeutic efficacy. The antitumor activity of paclitaxel (PTX) in HA synthase 3-overexpressing and wild-type SKOV3 ovarian cancer model and in the BxPC3 pancreas xenograft tumour model, was evaluated by monitoring tumour growth with or without PEGPH20 pre-treatment. Pharmacokinetics and tumour penetration of PTX were assessed by HPLC and mass spectrometry imaging analysis in the same tumour models. Tumour tissue architecture and HA deposition were analysed by histochemistry. RESULTS: Pre-treatment with PEGPH20 modified tumour tissue architecture and improved the antitumor activity of paclitaxel in the SKOV3/HAS3 tumour model, favouring its accumulation and more homogeneous intra-tumour distribution, as assessed by quantitative and qualitative analysis. PEGPH20 also reduced HA content influencing, though less markedly, PTX distribution and antitumor activity in the BxPC3 tumour model. CONCLUSION: Remodelling the stroma of HA-rich tumours by depletion of HA with PEGPH20 pre-treatment, is a potentially successful strategy to improve the intra-tumour distribution of anticancer drugs, increasing their therapeutic efficacy, without increasing toxicity.
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Antineoplásicos Fitogénicos/uso terapéutico , Hialuronoglucosaminidasa/uso terapéutico , Neoplasias/tratamiento farmacológico , Paclitaxel/uso terapéutico , Animales , Antineoplásicos Fitogénicos/farmacología , Femenino , Humanos , Hialuronoglucosaminidasa/farmacología , Ratones , Paclitaxel/farmacología , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Recombinant human hyaluronidase PH20 (rHuPH20) facilitates the dispersion and absorption of subcutaneously administered therapeutic agents. This study aimed to characterize the transient, local action of rHuPH20 in the subcutaneous (SC) space using focused biodistribution and dye dispersion studies conducted in mice. MATERIALS AND METHODS: To evaluate the biodistribution of rHuPH20, mice were intradermally administered rHuPH20 (80 U). The enzymatic activity of rHuPH20 was analyzed in the skin, lymph nodes, and plasma. Animal model sensitivity was determined by intravenous administration of rHuPH20 (80 U) to the tail vein. To evaluate local dispersion, mice received an intradermal injection of rHuPH20 followed by an intradermal injection of Trypan Blue dye at a contralateral site 45 minutes later. Dye dispersion was measured using a digital caliper. RESULTS: After intradermal rHuPH20 injection, enzymatic activity was detected within the skin near the injection site with levels decreasing rapidly after 15 minutes. There was no clear evidence of systemic exposure after administration of rHuPH20, and no discernible rHuPH20 activity was observed in lymph or plasma as a function of time after dosing. In the dye dispersion study, delivery of rHuPH20 at one site did not impact dye dispersion at a distal skin site. CONCLUSION: These observations support the classification of rHuPH20 as a transiently active and locally acting permeation enhancer.
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Antígenos de Neoplasias , Histona Acetiltransferasas , Hialuronoglucosaminidasa , Animales , Antígenos de Neoplasias/farmacología , Femenino , Histona Acetiltransferasas/farmacocinética , Histona Acetiltransferasas/farmacología , Humanos , Hialuronoglucosaminidasa/farmacocinética , Hialuronoglucosaminidasa/farmacología , Inyecciones Subcutáneas , Ratones , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Distribución TisularRESUMEN
Aim: Delivery of nanoparticles (NPs) to tumors can be impeded by high levels of hyaluronan (HA) in the stroma. Enzymatic depolymerization of HA with PEGylated hyaluronidase (PEGPH20) improves the delivery of antibodies to tumors. However, it is unknown whether NP delivery is enhanced by this strategy. Methods: The impact of PEGPH20 pretreatment on the uptake and tumor penetration of model PEGylated polystyrene NPs was studied in mice with orthotopic breast cancers. Results: Tumor oxygenation and NP penetration, but not overall tumor uptake, of 50 nm NPs, was significantly enhanced by PEGPH20 pre-administration. Conclusion: PEGPH20 has the potential to improve intratumoral penetration of NP-based drug delivery systems and enhance access to cancer cells in poorly vascularized regions of the tumor.
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Neoplasias de la Mama , Nanopartículas , Animales , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Ácido Hialurónico , Hialuronoglucosaminidasa , Ratones , PolietilenglicolesRESUMEN
Recombinant human hyaluronidase PH20 (rHuPH20) facilitates subcutaneous (SC) delivery of co-administered therapeutic agents by locally and transiently degrading hyaluronan in the SC space, and can be administered with therapeutics using a variety of devices. Two SC delivery studies were carried out to assess auto-injector (AI) performance, each in 18 Yucatan miniature pigs. Abdominal injections were administered using three auto-injectors of 1 mL (AI1) and 2 mL (AI2 and sAI2) with different injection speeds and depths (5.5-7.5 mm) and two pre-filled syringe (PFS) devices of 1 and 2 mL. The injection included a placebo buffer with and without rHuPH20 to evaluate the effect of rHuPH20 on SC injection performance. The feasibility of using similar devices to deliver a placebo buffer in humans was investigated. rHuPH20 was not studied in humans. In miniature pigs, postinjection swelling was evident for most PFS/AI injections, particularly 2 mL. Swelling heights and back leakage were typically lower with rHuPH20 co-administration versus placebo for most device configurations (1 or 2 mL PFS or AI). Auto-injections with versus without rHuPH20 also resulted in reduced swelling firmness and faster swelling resolution over time. Slow injections with rHuPH20 had shorter and more consistent injection time versus placebo. In humans, minimal injection site swelling and negligible back leakage were observed for 2-mL injections of placebo, while more erythema was observed in humans versus miniature pigs. Even at high delivery rates with PFS or AI, the addition of rHuPH20 resulted in improved SC injection performance versus placebo in miniature pigs.
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Hialuronoglucosaminidasa/administración & dosificación , Animales , Humanos , Inyecciones Subcutáneas , Masculino , Proteínas Recombinantes/administración & dosificación , Porcinos , Porcinos EnanosRESUMEN
INTRODUCTION: Subcutaneous (SC) formulations of therapeutics with recombinant human hyaluronidase PH20 (rHuPH20) are currently approved across various disease indications. The rHuPH20-mediated enzymatic degradation of SC hyaluronan (HA) facilitates bulk fluid flow and dispersion of co-administered therapeutics. However, current methods of quantifying dispersion in the SC space are limited. Here, a novel method is outlined to quantify and follow rapid SC volumetric dispersion of a representative therapeutic fluid in the presence of rHuPH20 using computed tomography (CT). METHODS: Ten Yucatan miniature swine were randomized to three groups. Animals received simultaneous infusions of contrast agent (CA) alone (left side of the animal) or in combination with rHuPH20 (right side) at infusion rates of 2.5, 5, or 10 mL/min. Spiral CT scans (1.5 mm thickness) were conducted before and after the infusion and at regular time intervals throughout. Scans were used to create three-dimensional (3D) reconstructions of the fluid pockets and analyze surface area, volume, and sphericity. RESULTS: 3D reconstruction showed increased dispersion of CA with rHuPH20 compared with CA alone, with fenestration and increased dispersion in the craniocaudal and lateromedial directions. The CA with rHuPH20 fluid pockets showed an average increase of 46% in surface area (p = 0.001), a 35% increase in volume (p = 0.001) and a 17% decrease in sphericity post-infusion compared with CA alone at 30 min post-infusion. DISCUSSION: This exploratory study confirms the value of CT imaging as a non-invasive method of assessing real-time spatial and temporal behavior of SC-administered fluids. This technique could help to assess the dispersion pattern of novel rHuPH20 SC co-formulations.
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Moléculas de Adhesión Celular/farmacología , Tomografía Computarizada de Haz Cónico , Portadores de Fármacos/farmacología , Hialuronoglucosaminidasa/farmacología , Tejido Subcutáneo/diagnóstico por imagen , Distribución Tisular/efectos de los fármacos , Animales , Medios de Contraste/administración & dosificación , Medios de Contraste/farmacocinética , Estudios de Factibilidad , Femenino , Humanos , Infusiones Subcutáneas , Modelos Animales , Proteínas Recombinantes/farmacología , Análisis Espacio-Temporal , Tejido Subcutáneo/metabolismo , Porcinos , Porcinos EnanosRESUMEN
BACKGROUND: Recombinant human hyaluronidase PH20 (rHuPH20) is used in subcutaneous formulations (eg, RITUXAN HYCELA [rituximab and hyaluronidase human], HERCEPTIN HYLECTA [trastuzumab and hyaluronidase-oysk], PHESGO [pertuzumab/trastuzumab/hyaluronidase-zzxf], and Darzalex FASPRO [daratumumab and hyaluronidase-fihj]) to increase the dispersion and absorption of coadministered therapeutics. Although unlikely, subcutaneous products that include rHuPH20 could be mistaken for the intravenous formulation of the corresponding drugs (eg, RITUXAN [rituximab], HERCEPTIN [trastuzumab], and DARZALEX [daratumumab]). To understand the potential effects of inadvertent intravenous injection of rHuPH20, we investigated the safety profile, pharmacokinetics (PK), and pharmacodynamics (PD) of rHuPH20 administered intravenously. OBJECTIVES: This Phase I, open-label, single-center study in healthy volunteers was designed to assess the safety profile, tolerability, PK, and PD of rHuPH20 administered intravenously. METHODS: Healthy volunteers received 5 mL intravenous infusion of either 10,000 U (nâ¯=â¯12) or 30,000 U (nâ¯=â¯12) rHuPH20 over 5 minutes. Blood samples for PK and PD analysis were obtained at baseline and at various times after initiation of infusion. Adverse events and laboratory parameters were measured to assess the safety profile and tolerability of the intravenous infusion. The PK of rHuPH20 was assessed using both an enzymatic assay and a mass-based immunoassay, and plasma hyaluronan concentrations were measured as a PD marker using an HPLC-MS/MS disaccharide assay. RESULTS: All 24 volunteers (mean ageâ¯=â¯36.5 years) completed the study, and no serious adverse events were reported in either treatment group. Overall, 2 adverse events (both Grade 1) were reported; catheter site pain in the 10,000 U group and hypotension in the 30,000 U group. Plasma concentrations of rHuPH20 increased during the 5-minute intravenous infusion (median tmaxâ¯=â¯6 minutes from intravenous initiation) followed by a rapid plasma clearance (t1/2 â¼10 minutes from intravenous initiation). Plasma hyaluronan concentrations increased with dose and time (tmax rangeâ¯=â¯45â120 minutes from intravenous initiation) and returned to baseline within 1 week of administration. Changes in both PK and PD measurements appeared proportional to dose. CONCLUSIONS: The study demonstrated that intravenous administration of up to 30,000 U rHuPH20 was well tolerated, rapidly cleared from the plasma, and did not appear to be associated with any serious adverse effects at doses used in subcutaneous therapeutic products. (Curr Ther Res Clin Exp. 2020; 81).
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INTRODUCTION: There is currently no consensus in the literature concerning the impact of aging on the properties of hyaluronan (HA) in the subcutaneous (SC) space. Recombinant human hyaluronidase PH20 (rHuPH20) facilitates SC administration of injected therapeutics by depolymerizing SC HA, facilitating bulk fluid flow, dispersion and absorption. This study assessed the impact of intrinsic aging on HA in the SC space and thus the ability of rHuPH20 to enhance delivery of co-administered therapeutics. METHODS: Histologic evaluations of HA levels and degradation were performed on human skin samples from six age groups, aged from 20 to 100 years. HA levels were evaluated by HA staining and degradation by staining samples for HA following incubation with rHuPH20. HA was extracted from samples and HA size determined by gel electrophoresis. Dermal reconstitution was assessed in young (aged 1.5 months) and elderly (aged > 16 months) mice. Baseline dye dispersion was measured at 5 and 20 min post-intradermal dye injection. Following treatment with rHuPH20, dye dispersion was measured again at 2, 24, 48, 72 and 96 h. RESULTS: Distribution of HA was confined to the interstitial space between adipocytes, with similar pericellular presence and levels of HA found across all age groups. Substantial levels of high-molecular-weight HA were observed in all age groups at baseline. Incubation with a clinically relevant dose of rHuPH20 resulted in degradation of all SC HA and similar degradation profiles independent of age. No difference in dye dispersion time was observed between young and elderly mice across the range of time points assessed, with dye dispersion returning to baseline levels by 24 h after rHuPH20 treatment. CONCLUSIONS: Subcutaneous delivery of approved therapeutics facilitated by co-administration with rHuPH20 should not be impacted by intrinsic aging, with this study providing no evidence for an effect of aging on HA distribution, structure or a loss of rHuPH20 efficacy.
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Interstitial, e.g. subcutaneous (SC) or intradermal (ID), administration of monoclonal antibodies (mAb) is less invasive than intravenous administration and leads to mAb uptake into both lymphatic and blood capillaries draining the injection site. Interstitial administration, however, is hindered by the presence of hyaluronan (HA), a glycosaminoglycan that is a major fluid barrier in the interstitial space. The transient removal of HA with recombinant human hyaluronidase (rHuPH20) helps facilitate the interstitial administration of often high therapeutic doses of mAb in the clinic. rHuPH20's impact on the systemic pharmacokinetics of several mAbs has been previously described, however effects on route of absorption (lymph vs blood) are unknown. The current study has therefore explored the lymphatic transport and bioavailability of cetuximab and trastuzumab after SC and ID coadministration in the presence and absence of rHuPH20 in rats. After SC administration cetuximab absolute bioavailability increased from 67 % to 80 % in the presence of rHuPH20. Cetuximab recovery in the lymphatics also increased after SC (35.8 % to 49.4 %) and ID (26.7 % to 58.8 %) administration in the presence of rHuPH20. When the injection volume (and therefore dose) was increased 10-fold in the presence of rHuPH20 cetuximab plasma exposure increased approximately linearly (12- and 8.9-fold respectively after SC and ID administration), although the proportional contribution of cetuximab lymphatic transport reduced slightly (6.2-fold increase for both administration routes). In contrast, co-administration with rHuPH20 did not lead to increases in plasma exposure for trastuzumab after SC or ID administration, most likely reflecting the fact that the reported absolute bioavailability of trastuzumab (in the absence of rHuPH20) is high (â¼77-99 %). However, lymphatic transport of trastuzumab did increase when coadministered ID with rHuPH20 in spite of the lack of change to overall bioavailability. The data suggest that co-administration with rHuPH20 is able to increase the volume of mAb that can be administered interstitially, and in some instances can increase the amount absorbed into both the blood and the lymph. In the current studies the ability of rHuPH20 to enhance interstitial bioavailability was higher for cetuximab where intrinsic interstitial bioavailability was low, when compared to trastuzumab where interstitial bioavailability was high.
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Cetuximab/farmacocinética , Ácido Hialurónico/metabolismo , Hialuronoglucosaminidasa/farmacología , Trastuzumab/farmacocinética , Animales , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/farmacocinética , Disponibilidad Biológica , Cetuximab/administración & dosificación , Femenino , Humanos , Hialuronoglucosaminidasa/administración & dosificación , Inyecciones Intradérmicas , Inyecciones Subcutáneas , Linfa/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Trastuzumab/administración & dosificaciónRESUMEN
The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidinâ biotin bond. Implications for the molecular mechanism of unbinding of HAâ hyaladherin bonds under force are discussed, which underpin the mechanical properties of HAâ hyaladherin complexes and HA-rich extracellular matrices.
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Ácido Hialurónico/metabolismo , Fenómenos Mecánicos , Receptores de Superficie Celular/metabolismo , Fenómenos Biomecánicos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Receptores de Superficie Celular/química , Análisis EspectralRESUMEN
Many patients with primary immunodeficiency disease (PIDD) require lifelong immunoglobulin (Ig) replacement therapy. Home-based subcutaneous (SC) infusion provides advantages to patients with PIDD compared to hospital-based intravenous infusion. One limitation of current practice with SCIg infusion is the need for small-volume infusions at multiple injection sites on a frequent basis. A method was developed for large-volume SC infusion that uses preinfusion of recombinant human hyaluronidase (rHuPH20) to facilitate fluid dispersion. Miniature swine was used as a preclinical model to assess the effects of rHuPH20-facilitated infusions, of a single monthly dose, on fluid dispersion, infusion-related pressure, swelling, induration, and tissue damage. Preinfusion of vehicle (control) or rHuPH20 (75 U/g Ig) was performed simultaneously on contralateral abdominal sites on each animal, followed by infusion of 300 mL 10 % Ig (30 g) at each site. Compared to control infusions, rHuPH20 significantly reduced infusion pressure and induration (p < 0.05) and accelerated postinfusion Ig dispersion. Histological evaluation of infusion site tissue showed moderate to severe swelling for the control. Swelling after rHuPH20-facilitated infusion was mild on day 1 and had completely resolved shortly thereafter. Laser Doppler imaging of control infusion sites revealed local cutaneous hypoperfusion during Ig infusion, which was reduced almost 7-fold (p < 0.05) with the use of rHuPH20. These results demonstrate that rHuPH20-facilitated Ig infusion is associated with improved dispersion of Ig, resulting in reduced tissue pressure, induration, and reduced risk of tissue damage from mechanical trauma or local ischemia, thus enabling SC administration of large volumes of Ig at a single site.
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Excessive cutaneous scarring is an important clinical disorder resulting in adverse tissue growth and function as well as undesirable cosmetic appearance. p21WAF-1/Cip-1 is a cyclin-dependent kinase inhibitor that blocks cell cycle progression and inhibits cell proliferation. We used a recombinant adenovirus containing the human p21WAF-1/Cip-1 cDNA (rAd-p21) to evaluate proliferative responses in skin models. In vitro dose-response studies using primary human dermal fibroblasts resulted in a dose-dependent expression of p21WAF-1/Cip-1 protein and a 3- to 80-fold reduction in cell proliferation as measured by 5-bromodeoxyuridine incorporation. Further, rAd-p21 reduced type I procollagen production when compared to control virus. A rat polyvinyl alcohol sponge model was used to determine rAd-p21 effects on granulation tissue formation in vivo. Sponges pretreated with a granulation tissue stimulator, rAd-PDGF-B and subsequently rAd-p21 on a second injection, showed a p21WAF-1/Cip-1 specific dose-dependent decrease in percent granulation fill as the rAd-p21 dose increased (p < 0.001). Immunohistochemistry identified human p21WAF-1/Cip-1 expression in sponges treated with rAd-p21 5 days postinjection. Additionally, 5-bromodeoxyuridine and Ki67 staining in sponges treated with rAd-p21 showed a significant decrease in proliferation when compared to rAd-platelet-derived growth factor-B alone or vehicle control groups (p < 0.01). These data support the utility of p21WAF-1/Cip-1 in targeting hyperproliferative disorders of the skin.
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Adenoviridae , Cicatriz/terapia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Vectores Genéticos , Cicatrización de Heridas/genética , Adulto , Animales , Proliferación Celular , Células Cultivadas , Cicatriz/genética , Fibroblastos , Técnicas de Transferencia de Gen , Humanos , Modelos Animales , Procolágeno/genética , Ratas , Piel/fisiopatologíaRESUMEN
The localized, sustained delivery of growth factors for wound healing therapy is actively being explored by gene transfer to the wound site. Biocompatible matrices such as bovine collagen have demonstrated usefulness in sustaining gene therapy vectors that express growth factors in local sites for tissue repair. Here, new synthetic biocompatible materials are prepared and shown to deliver a protein to cultured cells via the use of an adenoviral delivery vector. The synthetic construct consists of a linear, beta-cyclodextrin-containing polymer and an adamantane-based cross-linking polymer. When the two polymers are combined, they create an extended network by the formation of inclusion complexes between the cyclodextrins and adamantanes. The properties of the network are altered by controlling the polymer molecular weights and the number of adamantanes on the cross-linking polymer, and these modifications and others such as replacement of the beta-cyclodextrin (host) and adamantane (guest) with other cyclodextrins (hosts such as alpha, gamma, and substituted members) and inclusion complex forming molecules (guests) provide the ability to rationally design network characteristics. Fibroblasts exposed to these synthetic constructs show proliferation rates and migration patterns similar to those obtained with collagen. Gene delivery (green fluorescent protein) to fibroblasts via the inclusion of adenoviral vectors in the synthetic construct is equivalent to levels observed with collagen. These in vitro results suggest that the synthetic constructs are suitable for in vivo tissue repair applications.