Evaluation of the Antioxidant Capacity and Protective Effects of a Comprehensive Topical Antioxidant Containing Water-soluble, Enzymatic, and Lipid-soluble Antioxidants.

Objectives: Investigators sought to evaluate the antioxidant capacity of a comprehensive topical antioxidant (WEL-DS), its ability to protect skin against the oxidizing effects of UVA/UVB radiation, and to assess the effectiveness and tolerability of WEL-DS for visible improvements in facial photodamage. Study Designs: In-vitro testing utilized a hydrogen peroxide assay to detect activity in human skin explants following application with WEL-DS, a leading antioxidant serum (L-AOX), and a saline control. Clinical studies included a minimal erythema dose (MED) trial in female subjects, aged 35 to 60 years. Skin was initially irradiated to determine each subject's MED. WEL-DS was applied for four days to one site on the lower back of subjects; the other site remained untreated. Both sites were irradiated with 1X, 2X and 3X each subject's MED, digital images were obtained, and punch biopsies were collected from the 3X MED irradiated areas for histological analysis. A second clinical study evaluated efficacy and tolerability of twice daily application of WEL-DS in female subjects, aged 25 to 65 years with mild-to-moderate photodamage. Changes in fine lines/ wrinkles, dyschromia, erythema, skin tone, pores, and tolerability were assessed at baseline and Weeks 4, 8, and 12. A subset of subjects were evaluated through Week 16. Results: Skin treated with WEL-DS neutralized up to 53 percent more oxidative stress relative to L-AOX. WEL-DS-treated skin demonstrated significantly less UV-induced erythema at 1X, 2X, and 3X MED and demonstrated cellular protective effects versus untreated irradiated skin (N=5). WEL-DS demonstrated average improvements from baseline of 37 percent, fine lines/ wrinkles; 17 percent, skin tone; 13 percent, dyschromia; 18 percent, erythema; and four percent, pores (N=21; Week 12). Continued improvements were demonstrated in all parameters in an extension study (n=14; week 16). WEL-DS was well-tolerated. Conclusion: These studies demonstrate WEL-DS's innate ability to quench free radicals, protect skin from the oxidizing effects of UV radiation, and reduce the visible effects of facial photodamage.

Local Stent-Based Release of Transforming Growth Factor-ß1 Limits Arterial In-Stent Restenosis.

The long-term success of intra-arterial stenting remains limited by in-stent restenosis (ISR). Transforming growth factor-ß1 (TGF-ß1) can inhibit smooth muscle cell (SMC) proliferation and migration and convert SMCs into extracellular matrix (ECM)-synthesizing cells. Here, we evaluate the effects of stent-based delivery of TGF-ß1 on ISR in a rabbit model. Channeled stents loaded with TGF-ß1 or control microspheres were deployed in rabbit aortas. Stented aortas were harvested at 7 and 28 d and evaluated for Ki-67-positive cells, collagenous ECM production, and intima-to-media (I/M) ratio. At 7 d, the TGF-ß1 group exhibited fewer Ki-67-positive cells were found for the TGF-ß1 group (17.87 ± 2.18 cells per mm(2)) relative to control (25.07 ± 2.65 cells per mm(2), p = 0.04), but increased collagen content (31.4 ± 2.5 percentage area) compared with control (29.3 ± 1.2 percentage area, p = 0.019). The I/M ratio in the TGF-ß1 group was reduced by 50% and 9.1% versus control at 7 d (0.13 ± 0.02 vs. 0.26 ± 0.02, p = 0.0001) and 28 d (1.80 ± 0.05 vs. 1.98 ± 0.08, p = 0.0038), respectively. Stent-based controlled release of TGF-ß1 limits ISR and is associated with inhibition of SMC proliferation but an increase in ECM production.

Experiences with CPP-Based Self Assembling Peptide Systems for Topical Delivery of Botulinum Toxin.

Considerations in rational designs of CPP-based transcutaneous delivery systems are described. Impact of design considerations of nonclinical and clinical results are presented in detail.

Safety and efficacy of RT002, an injectable botulinum toxin type A, for treating glabellar lines: results of a phase 1/2, open-label, sequential dose-escalation study.

BACKGROUND: RT002 is a new injectable BoNTA product. This formulation limits the spread of BoNTA, potentially permitting safe administration of larger doses and possibly extending its duration of action. OBJECTIVE: This phase 1/2 clinical study was designed to establish the safety of RT002 for the treatment of moderate-to-severe glabellar lines. MATERIALS AND METHODS: Subjects were randomized into four groups (N=12/group). Cohorts 1-3 received escalating doses of RT002 ranging from half the equivalent dose of approved toxins up to twice the current dose levels. The safety of each dose was confirmed prior to administering the next dose. Subjects in Cohort 4 were treated with the highest dose and observed for 36 weeks or until GLSS returned to baseline. RESULTS: All doses of RT002 were well-tolerated. Common AEs were headache and mild injection site reactions. At Week 4, all doses of RT002 were highly effective at maximum frown. Cohort 4 achieved 7 month median duration. At 6 months, 80% of subjects maintained a ≥1-point improvement in Investigator GLSS and 60% maintained WSS of None or Mild. Study limitations include an open-label design and modest number of subjects. CONCLUSION: RT002 is a safe and effective BoNTA product with an extended duration of action.

Development and validation of a new clinically-meaningful rating scale for measuring lateral canthal line severity.

BACKGROUND: Several scales have been employed for evaluating the effects of cosmetic treatments in the periorbital area. The Food and Drug Administration (FDA) has recently issued new recommendations specifying a rigorous process to validate new aesthetic scales. OBJECTIVES: The authors describe and validate a new clinical rating scale: the Investigator's Global Assessment of Lateral Canthal Line (IGA-LCL) severity scale. METHODS: The new FDA recommendations were utilized to validate the new scale. The first step was concept elicitation (based on direct input from clinicians, patients, and literature) and evaluation of content validity (appropriateness of concepts). The resulting five-point scale provided detailed descriptions of the lateral canthal lines (LCL), including quantitative assessment of LCL length and depth. Performance parameters, including intra- and interrater reproducibility and construct validity, were then evaluated in clinical studies. Finally, the scale's threshold for clinically-meaningful benefit and the ability of the scale to detect change were confirmed in two Phase 2b clinical studies involving a total of 270 subjects. RESULTS: Content validity was established and the IGA-LCL scale showed excellent interrater reliability (weighted Kappa = 0.89) and interrater reliability (weighted Kappa = 0.77; Kendall's coefficient of concordance = 0.89). In clinical trials, the scale was sensitive enough to detect clinically-meaningful one- and two-point changes in LCL severity following treatment with topical botulinum toxin type A (BoNT-A). The authors observed statistically-significant correlations between the physician-rated IGA-LCL results and patient-reported outcomes. CONCLUSIONS: The IGA-LCL scale was shown to be reliable, appropriate, and clinically meaningful for measuring LCL severity.

Results of a randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of a botulinum toxin type A topical gel for the treatment of moderate-to-severe lateral canthal lines.

BACKGROUND: Injections of botulinum toxin type A are commonly used to treat facial wrinkles; however, undesirable effects are associated with injections (e.g., pain, bruising, ptosis, immunogenicity, and needle aversion). To address these issues, RT001 Botulinum Toxin Type A Topical Gel is being developed for the treatment of lateral canthal lines. OBJECTIVES: To assess the safety and efficacy of RT001 for the treatment of lateral canthal lines in a randomized, double-blind, placebo-controlled study. MATERIALS & METHODS: Adult subjects were enrolled to receive a single treatment of RT001 (n=45) or placebo (n=45) applied topically in the lateral canthal area. The primary endpoint was the composite of the Investigator Global Assessment of Lateral Canthal Line Severity (IGA-LCL) and the Patient Severity Assessment of lateral canthal line severity (PSA) defined as a 2-point or greater improvement on both scales. RESULTS: At four weeks, 44.4 percent of subjects treated with RT001 achieved a 2-point or greater improvement on a rigorous composite of both the IGA-LCL and PSA scales compared to 0.0% for the placebo subjects (P<0.0001). At four weeks, 88.9 percent of subjects achieved clinically relevant improvement by investigator assessment. Adverse events were mild in severity and unrelated to study treatment. CONCLUSIONS: RT001 appears to be a safe and well-tolerated treatment for improvement of lateral canthal lines.

Nonclinical and clinical experiences with CPP-based self-assembling peptide systems in topical drug development.

Considerations in rational designs of CPP-based transcutaneous delivery systems are described. Impact of design considerations of nonclinical and clinical results are presented in detail.

Efficacy and safety evaluation of a novel botulinum toxin topical gel for the treatment of moderate to severe lateral canthal lines.

BACKGROUND: Botulinum toxin type A (BoNTA) is commonly injected to treat facial wrinkles. Complications include pain, erythema, bruising, and potential infection. RT001 Botulinum Toxin Type A Topical Gel (RT001) is under development for the treatment of lateral canthal lines (LCLs). OBJECTIVE: To assess the efficacy and safety of RT001 for the treatment of LCLs using a randomized, double-blind, repeat-dose, placebo-controlled study design. METHODS & MATERIALS: Healthy adult subjects were randomized to receive RT001 (N=19) or placebo (N=17) applied to their lateral canthal areas (LCAs). To evaluate safety of repeat exposure, treatment was administered at baseline and week 4. The primary efficacy measure was improvement in baseline LCL severity using the Investigator's Global Assessment of Lateral Canthal Line at Rest (IGA-LCL) Severity Scale. RESULTS: At 8 weeks, 19 (50%) LCAs treated with RT001 showed a 2-point or greater improvement in baseline IGA-LCL severity, versus none (0%) of the placebo-treated subjects (p<.001); 36 (94.7%) LCAs treated with RT001 showed a 1-point or more improvement in baseline IGA-LCL severity, versus five (14.7%) placebo-treated LCAs (p<.001). There were no treatment-related adverse events. CONCLUSION: RT001 was well tolerated and demonstrated an improvement in LCLs.

Directed migration of smooth muscle cells to engineer plaque-resistant vein grafts.

PURPOSE: To test the hypothesis that controlled perivascular release of tissue plasminogen activator (tPA) can generate cleaved extracellular matrix (ECM) chemotactic gradients to guide the migration of vascular smooth muscle cells (SMCs) away from the lumen, thereby limiting neointima formation. METHODS: This hypothesis was tested in rabbit models in which the perivascular surface of vein bypass grafts was treated with microspheres releasing tPA (MS-tPA), microspheres containing no drug (MS-blank), or phosphate buffered saline (PBS). Vein graft segments harvested after 7 days were then evaluated for elastin content, proliferating SMCs, intima-to-media (I/M) ratio, and inflammation; late impact on neointima formation was also examined. RESULTS: The 7-day results demonstrated cleaved elastin gradients and proliferating SMCs that assumed a more peripheral distribution in the MS-tPA group than MS-blank and PBS controls (p<0.05). At 28 days, vein grafts treated with MS-tPA showed a mean I/M ratio (0.35+/-0.04) that was 63.5% lower than PBS controls (0.96+/-0.07, p<0.005) and 43.5% lower than MS-blank specimens (0.62+/-0.08, p<0.05). CONCLUSIONS: Perivascular release of tPA modifies ECM gradients, directionally guides SMC migration away from the lumen, and limits neointima formation.

Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases.

Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth-Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naive liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naive liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders.

Efficient inhibition of in-stent restenosis by controlled stent-based inhibition of elastase: a pilot study.

PURPOSE: It is proposed that local elastase inhibition could suppress the extracellular matrix (ECM) degradation and subsequent smooth muscle cell migration and limit subsequent in-stent restenosis. This study evaluated the effect of stent-based controlled elastase inhibition on restenosis after stent implantation in a rabbit model. MATERIALS AND METHODS: Biodegradable microspheres containing the potent elastase inhibitor alpha-1-antitrypsin (AAT) were prepared. Daily release of AAT from the microspheres was confirmed in vitro. The microspheres were loaded into stents with an abluminal polymer reservoir. Implantation of the stent with AAT microspheres and blank microspheres (control) was performed in the abdominal aortae of six rabbits in each group. After stent deployment, all stents were overdilated to 125% diameter. Stent-implanted arteries were harvested after 7 days (n = 3 each) or 28 days (n = 3 each). To assess the effect of local delivery of AAT, elastase activity and elastin content of the stent-implanted aortae were analyzed. As an endpoint, intima-to-media (I/M) ratio was determined in the 7-day and 28-day specimens. RESULTS: Significant inhibition of elastase was confirmed in treated vessels versus controls at 7 days after stent implantation (P < .05). This reduction in elastase activity was sufficient to afford early and late reduction of in-stent neointima. Plaque progression in the 28-day specimens decreased to 67% with elastase inhibition relative to controls (P < .05). CONCLUSION: Stent-based controlled release of elastase inhibitor may significantly reduce ECM degradation and might limit in-stent restenosis.

Stent-based controlled release of intravascular angiostatin to limit plaque progression and in-stent restenosis.

PURPOSE: To evaluate the importance of angiogenesis in plaque progression after stent placement, this study examines stent-based controlled delivery of the antiangiogenic agent, angiostatin, in a rabbit model. MATERIALS AND METHODS: Controlled release biodegradable microspheres delivering angiostatin or polymer-only microspheres (polylactic-co-glycolic-acid-polyethylene glycol; PLGA/PEG) were loaded in channeled stents, anchored, and deployed in the aorta of adult New Zealand white rabbits (n = 6 animals per group, three each per time point). The stented aortas were harvested at 7 days and 28 days and evaluated for neovascularization, local inflammation, vascular smooth muscle cell proliferation, and in-stent plaque progression. RESULTS: At 7 days, neovascularization was significantly decreased in the angiostatin groups (1.6 +/- 1.6 neovessels per mm(2) plaque) versus the control group (15.4 +/- 2.6 neovessels per mm(2) plaque; P =.00081), as were local inflammation where angiostatin-treated groups demonstrated significantly lower macrophage recruitment per cross section (34.9 +/- 4.9 cells per cross section) relative to the control group (55.2 +/- 3.84 cells per cross section; P =.0037). And a significant decrease in the overall vascular smooth muscle cell proliferation (143.8 +/- 26.3 Ki-67 positive cells per mm(2)) relative to the control group (263.2 +/- 16.6 Ki-67 positive cells per mm(2); P =.00074). At both 7 and 28 days, in-stent plaque progression in the angiostatin groups was successfully limited relative to the control group by 54% (0.255 +/- 0.019% of cross section; P =.00016) and 19% (1.981 +/- 0.080; P =.0033) respectively and resulted in reduction of in-stent restenosis relative to the control group. CONCLUSION: Angiostatin-eluting stents may limit neovascularity after arterial implantation, offer insight into in-stent restenosis, and allow future refinement of bioactive stent designs and clinical strategies, particularly in light of evidence that intimal smooth muscle cells may in part be marrow-derived.

In-stent restenosis limitation with stent-based controlled-release nitric oxide: initial results in rabbits.

PURPOSE: To evaluate effect of controlled stent-based release of an NO donor to limit in-stent restenosis in rabbits. MATERIALS AND METHODS: Bioerodable microspheres containing NO donor or biodegradable polymer (polylactide-co-glycolide-polyethylene glycol) were prepared and loaded in channeled stents. Daily concentrations of NO release from NO-containing microspheres were assayed in vitro. NO- and polymer-containing (control) microsphere-loaded stents were deployed in aortas of New Zealand white rabbits (n = 8). Aortas with stents were harvested at 7 (n = 5) and 28 days (n = 3) and evaluated for cyclic guanosine monophosphate (cGMP) levels (7 days), number of proliferating cell nuclear antigen-positive cells (7 days), and intima-to-media ratio (7 and 28 days), with statistical significance evaluated by using one-way analysis of variance. RESULTS: NO-containing microspheres released NO with an initial bolus in the 1st week, followed by sustained release for the remaining 3 weeks. Significant increase in cGMP levels and decrease in proliferating cell nuclear antigen-positive cells were found at 7 days for the NO-treated group relative to controls (P <.05). Intima-to-media ratio in the NO-treated group was reduced by 46% and 32% relative to controls at 7 and 28 days, respectively (mean, 0.14 +/- 0.01 [standard error] vs 0.26 +/- 0.02 at 7 days, P <.01; 1.34 +/- 0.05 vs 1.98 +/- 0.08 at 28 days, P <.01). CONCLUSION: Stent-based controlled release of NO donor significantly reduces in-stent restenosis and is associated with increase in vascular cGMP and suppression of proliferation.

Inhibition of vascular endothelial growth factor-mediated neointima progression with angiostatin or paclitaxel.

PURPOSE: Therapeutic angiogenesis represents a new paradigm for treatment of ischemic vascular syndromes. However, vascular endothelial growth factor (VEGF) enhances the rate and degree of plaque formation. This study evaluates the potential to block these effects nonspecifically with paclitaxel or specifically with angiostatin. MATERIALS AND METHODS: Recombinant human VEGF(165) (rhVEGF) was administrated intramuscularly (2-microg/kg single injection) in combination with adventitial delivery of paclitaxel, angiostatin, or vehicle alone at the site of femoral arterial balloon overdilation injury in New Zealand White rabbits (n = 5 per treatment). Five additional animals with no rhVEGF and no adventitial delivery served as procedural controls. All rabbits were fed according to a 0.25% cholesterol diet beginning 28 days before angioplasty. Treated arteries were harvested after 7 days and evaluated to determine intima-to-media (I/M) ratios, macrophage infiltrate, and endothelial cell density. RESULTS: On histologic analysis, the rhVEGF/gel control group exhibited a mean I/M ratio of 0.337 +/- 0.028, a 77% increase over procedural controls, which exhibited a mean I/M of 0.190 +/- 0.010. rhVEGF/paclitaxel reduced I/M ratios to 0.151 +/- 0.007. In contrast, specific antiangiogenic therapy (rhVEGF/angiostatin) reduced I/M ratios to 0.032 +/- 0.003, a 91% decrease relative to rhVEGF/gel and an 83% decrease relative to procedural controls (P =.001 for each comparison). Local macrophages and endothelial cells also decreased with treatment. CONCLUSIONS: This study shows that paclitaxel and angiostatin each afford local protection against rhVEGF-mediated increases in neointima. Angiostatin further prevents progression of underlying neointima. These local therapies may allow broader use of therapeutic angiogenesis while avoiding and treating potentially undesirable effects.

In vivo vascular engineering of vein grafts: directed migration of smooth muscle cells by perivascular release of elastase limits neointimal proliferation.

PURPOSE: Saphenous vein bypass grafting for coronary revascularization procedures remains limited by accelerated neointima formation. It was hypothesized that creation of a modified chemotactic gradient in vivo could guide migration of smooth muscle cells (SMCs) peripherally instead of in a luminal direction and reduce intimal hyperplasia during vein graft arterialization. MATERIALS AND METHODS: Surgical bypass vein grafting to femoral arteries was performed in adult male New Zealand White rabbits (n = 8 per treatment group; five for 7 d and three for 28 d). Controlled-release microspheres delivering elastase or buffered polymer only were administered perivascularly at the vein graft site. At 7 days, five vein grafts per group were harvested and cross-sections were immunostained with anti-proliferating cell nuclear antigen (PCNA) to determine the number and distribution of proliferating SMCs. At 28 days, three vein grafts per group were harvested and intima-to-media (I/M) ratios were calculated after staining with Verhoeff von Gieson-Masson trichrome stain. RESULTS: Significant early outward-directed elastin degradation resulted from elastase treatment. Concurrently, proliferating SMCs migrated peripherally. PCNA(+) cells in the outer half of the wall increased 2.37 fold compared to procedural controls (P <.0001). Directional shifts in SMC migration underlie these results because overall SMC proliferation was not significantly different. At 28 days after vein graft surgery, a 38% reduction (P =.0008) in neointima was observed relative to procedural controls. CONCLUSION: Directional guidance of SMC responses through perivascular elastase release achieves favorable vein graft remodeling characteristics, including limited neointima development. This represents practical evidence that SMC migration can be directionally guided in vivo in a vein graft model and that plaque progression can be prevented by redistributing elastin without decreasing functional vein graft wall stability.

In vivo vascular engineering: directed migration of smooth muscle cells to limit neointima.

Pathologic neointima formation requires directional smooth muscle cell (SMC) migration from media to intima. The very direction of SMC migration thus becomes a potential therapeutic target. Here, we hypothesize that proliferating SMC after injury can be redirected using engineered chemotactic gradients of elastin degradation to limit late pathologic neointima formation. Buffered bioerodible polymeric microspheres (MS) were constructed to provide 4-week sustained release of elastase, heat-killed elastase, or polymer only. In vitro elastase function and timecourse of release at 37 degrees C, physiologic pH, and shear was determined. Curves revealed an initial bolus followed by sustained linear release for elastase MS, while controls exhibited baseline hydrolysis of substrate. We then employ controlled perivascular release of elastase after angioplasty to engineer modified in vivo gradients of elastin degradation in rabbit femoral arteries. NZW rabbits (n = 8 each) underwent balloon angioplasty of the common femoral artery followed by perivascular distribution of MS. Significant early perivascular elastin degradation resulted. Concurrently, proliferating SMC were guided peripherally (further from lumen) with treatment without significant changes in total proliferation or inflammation. At 28 days, treatment significantly reduces neointima by 42% relative to controls. These results confirm that directionally guiding SMC responses after injury achieves favorable arterial remodeling and limits development of pathologic neointima. Thus, a potential class of therapeutics and the paradigm of in vivo vascular engineering emerge from this work.