Wachter-Et-Al-LSM-32(2003)101-110.Pdf
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Lasers in Surgery and Medicine 32:101–110 (2003) Topical Rose Bengal: Pre-Clinical Evaluation of Pharmacokinetics and Safety 1 1 1 1 2 Eric Wachter, PhD, * Craig Dees, PhD, Jay Harkins, Timothy Scott, PhD, Mark Petersen, DVM, 3 4 Rusty E. Rush, MS, and Amy Cada, PhD 1Provectus Pharmaceuticals, Inc., Knoxville, Tennessee 37931 2College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37916 3Springborn Laboratories, Inc., Spencerville, Ohio 45887 4Therimmune Research Corporation, Gaithersburg, Maryland 20879 Background and Objectives: Rose bengal (RB) is a should have minimal potential for side effects, such as potent photosensitizer that has largely been overlooked as a prolonged photosensitivity. potential photodynamic therapy (PDT) agent. In this study, Since RB readily photobleaches [29], its photodynamic the feasibility of topical delivery of RB to the epidermis has effects may be self-limiting. This is particularly relevant for been evaluated. treatment of many dermatologic conditions, such as psoria- Study Design/Materials and Methods: Topical formu- sis and actinic keratosis, since precise light dosimetry is lations of RB were assessed on murine and rabbit skin for impractical over the large surface areas typically involved pharmacokinetic properties, cutaneous toxicity, and photo- in these diseases: a PDT regimen that exhibits self-limiting sensitization. effects would avoid the need for complex light dosimetry. Results: Hydrophilic formulations (1% RB) exhibited The combination of photodynamic potential, substantial rapid, selective, uniform delivery to the epidermis, with regulatory precedent, minimal known side-effects, and no significant acute cutaneous toxicity in normal skin. likelihood of self-limiting therapeutic effect, motivated us Illumination (532 nm) elicited no acute phototoxicity for to evaluate key pharmacokinetic and safety aspects of light intensities 100 mW/cm2 at a light dose of 100 J/cm2; topical RB with green light activation, where the mini- use of higher intensities resulted in superficial thermal mally-penetrating nature of such green light matches a damage. Repeat treatment of rabbit skin (weekly for four desire to restrict photodynamic action to the epidermis. weeks) elicited minor phototoxicity only at the highest concentration (1% RB). MATERIALS AND METHODS Conclusions: These results indicate that RB is safe for Reagents and Vehicles PDT treatment of skin disorders, exhibiting negligible RB was obtained from Sigma Chemicals (St. Louis, MO) effects in normal skin. Lasers Surg. Med. 32:101–110, or Akorn Inc.(Decatur, IL). Carboxymethylcellulose, U.S.P. 2003. ß 2003 Wiley-Liss, Inc. medium viscosity sodium salt (CMC), dimethylsulfoxide Key words: psoriasis; actinic keratosis; photodynamic (DMSO), ethanol, isopropanol, and propylene glycol (PG) therapy; photosensitizer; dermatology; laser were purchased from Sigma Chemicals (St. Louis, MO). Saline (sodium chloride, 0.9% U.S.P.), was obtained from Abbott Laboratories (North Chicago, IL) or B. Braun (Burns Veterinary Supply, Farmers Branch, TX). AquaGel INTRODUCTION Rose bengal (4,5,6,7-tetrachloro-20,40,50,70-tetraiodo- fluorescein disodium, or RB) is a well known type-II Abbreviations: CMC, carboxymethylcellulose; D, density; DE, photosensitizer exhibiting facile photocatalytic conversion dermis; DMSO, dimethylsulfoxide; dT, temperature rise; ED, 3 1 of triplet oxygen ( O2) to singlet oxygen ( O2*) [1–7]. It has epidermis; H&E, hematoxylin and eosin; LoD, limit of detection; LSM, laser scanning microscopy; NF, National Formulary; an extremely large cross-section in the green (EM ¼ 99,800 1 O2*, singlet oxygen; PDT, photodynamic therapy; PG, propylene MÀ1 cmÀ1 at 549 nm in water) [4] that is only mildly affected glycol; PI, penetration index; RB, rose bengal; SC, stratum by local environment [6]. RB has an intersystem crossing corneum; Tmax, maximum surface temperature; U, uniformity; w/v, weight-to-volume. quantum yield approaching unity (Fisc ¼ 0.98) and a high Eric Wachter, Craig Dees, and Timothy Scott have disclosed a 1 singlet oxygen yield (r( O2*) > 0.75), indicating that RB is potential conflict of interest with this study. 1 *Correspondence to: Eric Wachter, PhD, Provectus Pharma- capable of highly efficient O2* production upon irradiation ceuticals, Inc., 7327 Oak Ridge Highway, Knoxville, TN 37931. with green light [8]. Its long history of safe use as a systemic E-mail: [email protected] diagnostic of hepatic function [9–21] as well as a topical Accepted 27 September 2002 Published online in Wiley InterScience ophthalmic diagnostic [22–28] suggests that, in marked (www.interscience.wiley.com). contrast to many photodynamic therapy (PDT) agents, RB DOI 10.1002/lsm.10138 ß 2003 Wiley-Liss, Inc. 102 WACHTER ET AL. TABLE 1. Rose Bengal (RB) Cutaneous Penetration Results, 30 Minutes Topical Application to Murine Skin Formulation Penetration index Composition Properties N PISC PIED PIDE 1% RB in saline Hydrophilic aqueous liquid 7 2.4 2.5 0.0 1% RB in saline þ 2% CMC Hydrophilic aqueous hydrogel 2 2.5 2.8 0.0 1% RB in AquaGel Hydrophilic aqueous hydrogel 2 2.0 2.3 0.0 1% RB in LiquaGel Hydrophilic aqueous hydrogel 3 2.2 2.0 0.0 1% RB in PG Moderately lipophilic liquid 4 1.5 0.0 0.0 1% RB in LiquaDerm A Lipophilic liquid 1 1.5 1.0 0.0 1% RB in Dermabase Emulsion (oil-in-water) 1 1.5 2.0 0.0 1% RB in DMSO Universal penetrant 3 3.0 3.0 3.0 N indicates number of replicate treatments conducted for each formulation. No local or systemic adverse effects were noted for any tests. was purchased from Parker Laboratories (Minneapolis, below). Specimens of treated skin were collected upon MN). LiquaDerm A, LiquaGel, and Dermabase were pur- sacrifice 24 hours after final topical administration of RB chased from Paddock Laboratories (Fairfield, NJ). Addi- in carbomer hydrogel, then processed by frozen sectioning tional RB hydrogel formulations (ca. 0.2% Carbomer 934P (as described above for murine specimens). Blood samples NF with 5% PG U.S.P.) were prepared by Midwest Institute collected 24 hours prior to final treatment with RB and of Research and Technology (Edmund, OK). All materials at 1, 4, and 24 hours following such treatment were were used as received. Topical formulations are summar- centrifuged for 15 minutes at 3,000 rpm. The resultant ized in Table 1. separated plasma was collected and analyzed for RB con- tent using fluorimetry (Cytofluor 2350, Millipore, Bedford, Animals and Animal Husbandry MA); the limit of detection (LoD) for this assay was ca. 20 ng Animal housing and care were based on standards RB/ml plasma. established by the Association for Assessment and Accreditation of Laboratory Animal Care, International Microscopy and Fluorescence Microscopy (AAALAC) and guidelines set forth in the Guide for the An Olympus BX60 microscope (Olympus America, Inc. Care and Use of Laboratory Animals, NIH Publication Melville, NY) was used to examine and photograph No. 96-03, 1996. processed tissue sections (skin and underlying tissue) using transmitted light brightfield observation (halogen Pharmacokinetic Studies illumination). Specimens were photographed using Fuji- Topical delivery of RB was evaluated in hairless chrome 64 Professional T slide film (Fuji Photo Film Co., female mice (Crl:SKH-1-hrBR, Charles River Laboratories, Ltd, Tokyo, Japan). Additionally, specimens that were not Wilmington, MA). Mice were anesthetized via continuous counterstained during processing were uniformly illumi- inhalation using 2% Isofluorane (Baxter Healthcare Cor- nated with green light and the resultant fluorescence poration, Deerfield, IL). A single patch of skin on one flank photographed using a BX-FLA reflected light fluorescence was cleaned with ethanol, and an absorbent patch (2 cm  attachment fitted with a U-MWG fluorescence filter cube 2 cm section of Kimwipe, Kimberly-Clark, Roswell, GA) (510–550 nm excitation, >590 nm emission, Olympus). applied (for all vehicles except Dermabase and saline-CMC, This approach allowed RB present in tissue to be readily wherein no patch was used). The patch was saturated with imaged (as orange RB fluorescence on an otherwise dark one of the study formulations, then covered with an background). Control sections (having received no applied occlusive dressing (Handiwrap, Dow, Indianapolis, IN or RB nor H&E counterstaining) exhibited no detectable Tegaderm, 3M, St. Paul, MN). After 30 minutes contact, the autofluorescence under such illumination. covering and patch were removed, the skin blotted dry with Additional imaging was performed using a Zeiss LSM- a gauze pad, and the animal euthanized by carbon dioxide 510 laser scanning confocal microscope (Carl Zeiss Micro- inhalation. Tissue specimens from the treated area, and of Imaging, Inc., Thornwood, NY). Excitation at 543 nm using untreated skin, were immediately collected and processed a40Â/1.30 Plan-NEOFLUAR oil objective, coupled with by frozen sectioning. A portion of the resultant slides confocal detection using a 560 nm long-pass detection filter, underwent H&E staining, while the remainder were pro- allowed sub-cellular distribution of RB to be imaged against cessed without counterstaining. an otherwise non-fluorescent background. Additional pharmacokinetic data were obtained using tissue and blood samples from New Zealand white rabbits Histologic Assessment of RB Penetration (collected at the end of a four-week multiple-treatment Fluorescence micrographs were examined to assess RB regimen with topical RB, as described in greater detail staining; micrographs were scored by two blinded readers. TOPICAL ROSE BENGAL 103 A scale of 0 (low) to 3 (high) was used to assign uniformity 24 hours, at which point it was removed and the test site (U) and density (D) scores for RB staining of each specimen. rinsed with gauze moistened in deionized water. Individual scores for U and D were assigned for stra- General health/mortality checks were performed twice tum corneum (SC), viable epidermis (ED, comprising the daily, and detailed clinical observation of each animal was Malpighian layers), and dermis (DE). Separate scores were performed daily. Any post-treatment abnormalities at the then combined into a single metric (penetration index, PI) test site were recorded.