Scientific and Regulatory Policy Committee Points to Consider: Fixation, Trimming, and Sectioning of Nonrodent Eyes and Ocular Tissues for Examination in Ocular and General Toxicity Studies.

A Working Group of the Society of Toxicologic Pathology's Scientific and Regulatory Policy Committee conducted a technical and scientific review of current practices relating to the fixation, trimming, and sectioning of the nonrodent eye to identify key points and species-specific anatomical landmarks to consider when preparing and evaluating eyes of rabbits, dogs, minipigs, and nonhuman primates from ocular and general toxicity studies. The topics addressed in this Points to Consider article include determination of situations when more comprehensive evaluation of the globe and/or associated extraocular tissues should be implemented (expanded ocular sampling), and what constitutes expanded ocular sampling. In addition, this manuscript highlights the practical aspects of fixing, trimming, and sectioning the eye to ensure adequate histopathological evaluation of all major ocular structures, including the cone-dense areas (visual streak/macula/fovea) of the retina for rabbits, dogs, minipigs, and nonhuman primates, which is a current regulatory expectation for ocular toxicity studies.[Box: see text].

Selected Aspects of Ocular Toxicity Studies With a Focus on High-Quality Pathology Reports: A Pathology/Toxicology Consultant's Perspective.

Ocular toxicity studies are the bedrock of nonclinical ocular drug and drug-device development, and there has been an evolution in experience, technologies, and challenges to address that ensures safe clinical trials and marketing authorization. The expectations of a well-designed ocular toxicity study and the generation of a coherent, integrative ocular toxicology report and subreports are high, and this article provides a pathology/toxicology consultant's perspective on achieving that goal. The first objective is to cover selected aspects of study designs for ocular toxicity studies including considerations for contract research organization selection, minipig species selection, unilateral versus bilateral dosing, and in-life parameters based on fit-for-purpose study objectives. The main objective is a focus on a high-quality ocular pathology report that includes ocular histology procedures to meet regulatory expectations and a report narrative and tables that correlate microscopic findings with key ophthalmic findings and presents a clear interpretation of test article-, vehicle-, and procedure-related ocular and extraocular findings with identification of adversity and a pathology peer review. The last objective covers considerations for a high-quality ophthalmology report, which in concert with a high-quality pathology report, will pave the way for a best quality toxicology report for an ocular toxicity study.

Toxicology Evaluation of Drugs Administered via Uncommon Routes: Intranasal, Intraocular, Intrathecal/Intraspinal, and Intra-Articular.

As the need for nasal, ocular, spinal, and articular therapeutic compounds increases, toxicology assessments of drugs administered via these routes play an important role in human safety. This symposium outlined the local and systemic evaluation to support safety during the development of these drugs in nonclinical models with some case studies. Discussions included selection of appropriate species for the intended route; conducting nonclinical studies that closely mimic the intended use with adequate duration; functional assessment, if deemed necessary; evaluation of local tissues with special histological staining procedure; and evaluations of safety margins based on local and systemic toxicity.

Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.

Society of Toxicologic Pathology position paper on best practices on recovery studies: the role of the anatomic pathologist.

This article reviews the regulatory guidelines that provide for the inclusion of recovery groups in toxicology studies, presents the challenges in the design and interpretation of nonclinical recovery studies, and summarizes the best practices for the role of an anatomic pathologist regarding toxicology studies with recovery groups. Evaluating the potential recovery of histopathologic findings induced by a biopharmaceutical requires the active participation of one or more anatomic pathologists. Their expertise is critical in risk assessment regarding the potential for recovery as well as providing scientific guidance in the design and evaluation of studies with recovery groups.

RNAi-based treatment for neovascular age-related macular degeneration by Sirna-027.

PURPOSE: To assess the safety, tolerability, pharmacokinetics, and dose-limiting toxicity of single intravitreal injection of Sirna-027, a small interfering RNA targeting vascular endothelial growth factor receptor-1, in patients with choroidal neovascularization (CNV) resulting from neovascular age-related macular degeneration (AMD). Secondary objectives included assessment of anatomic changes in retinal thickness, size of CNV, and changes in visual acuity. DESIGN: Prospective, open-label, single-dose, dose-escalation phase 1 study. METHODS: Twenty-six eyes of 26 patients with a median age of 82 years and CNV resulting from AMD who had previous treatments with other therapies were treated at 2 academic retinal practices. Patients received a single dose of Sirna-027 (100, 200, 400, 800, 1200, or 1600 microg/eye). Blood was sampled for pharmacokinetic analysis at 1, 4, and 24 hours after injection and on day 7. Patients underwent ophthalmic examinations including visual acuity, fluorescein angiography, and optical coherence tomography at screening and days 7, 14, 28, and 84. The main outcome measures were adverse reactions and dose-limiting toxicities. RESULTS: Intravitreal injection of a single dose of Sirna-027 from 100 to 1600 microg was well tolerated in patients with AMD, with no dose-limiting toxicity found. Adverse events were mild to moderate in severity. Adjusted mean foveal thickness decreased within 2 weeks after study treatment. The decrease was most pronounced in the 100- and 200-microg doses. CONCLUSIONS: A single intravitreal dose of Sirna-027 up to 1600 microg/eye was well tolerated in patients with CNV resulting from neovascular AMD that had been refractory to other therapies. Stabilization or improvement in visual acuity and foveal thickness was observed. No dose-response or dose-limiting effects were noted.

Carcinogenicity assessments of biotechnology-derived pharmaceuticals: a review of approved molecules and best practice recommendations.

An important safety consideration for developing new therapeutics is assessing the potential that the therapy will increase the risk of cancer. For biotherapeutics, traditional two-year rodent bioassays are often not scientifically applicable or feasible. This paper is a collaborative effort of industry toxicologists to review past and current practice regarding carcinogenicity assessments of biotherapeutics and to provide recommendations. Publicly available information on eighty marketed protein biotherapeutics was reviewed. In this review, no assessments related to carcinogenicity or tumor growth promotion were identified for fifty-one of the eighty molecules. For the twenty-nine biotherapeutics in which assessments related to carcinogenicity were identified, various experimental approaches were employed. This review also discusses several key principles to aid in the assessment of carcinogenic potential, including (1) careful consideration of mechanism of action to identify theoretical risks, (2) careful investigation of existing data for indications of proliferative or immunosuppressive potential, and (3) characterization of any proliferative or immunosuppressive signals detected. Traditional two-year carcinogenicity assays should not be considered as the default method for assessing the carcinogenicity potential of biotherapeutics. If experimentation is considered warranted, it should be hypothesis driven and may include a variety of experimental models. Ultimately, it is important that preclinical data provide useful guidance in product labeling.

Alternative mouse models for carcinogenicity assessment: industry use and issues with pathology interpretation.

The Carcinogenicity Alternative Mouse Models (CAMM) Working Group of the Society of Toxicologic Pathology (STP) surveyed the membership to define current practices and opinions in industry regarding the use of alternative mouse models for carcinogenicity testing. The results of the survey indicated that CAMM are used most often to fulfill a regulatory requirement (e.g., to replace the two-year mouse bioassay) and are being accepted by regulatory agencies. Alternative models are also sometimes used for internal decision making or to address a mechanistic question. The CAMM most commonly used are the p53+/- and rasH2. The rasH2 appears to be the currently accepted model for general carcinogenicity testing. Problems with study interpretation included lack of historic background data, unexpected tumor finding, and tumor identification/characterization of early lesions. Problems with implementation or conduct of the study included extent of the pathology evaluation, numbers of animals, survival, and study duration. Recommendations were developed for, frequency and type of positive control testing, extent of histopathologic examination of test article-treated and positive control animals, current use and future development of diagnostic criteria; increased availability and use of historic data, and use of other genetically modified mice in carcinogenicity testing.

Safety evaluation of ocular drug delivery formulations: techniques and practical considerations.

Development of new drug candidates and novel delivery techniques for treatment of ocular diseases has recently accelerated. Treatment of anterior-segment diseases has witnessed advances in prodrug formulations and permeability enhancers. Intravitreal, subconjunctival, and periocular routes of administration and sustained-release formulations of nanoparticles and microparticles, as well as nonbiodegradable and biodegradable implants to deliver drugs to the posterior segment of the eye, are becoming popular therapeutic approaches. Without adequate regulatory guidance for ocular drugs, such routes of administration and novel formulations can pose unique challenges to those involved in designing nonclinical programs, including considering clinical and nonclinical factors and choosing species, strains, and ocular toxicity parameters. Toxicologic pathologists also contribute practical experience to evaluating morphological effects of these novel formulations. Lastly, understanding species' anatomical differences is useful for interpreting toxicological and pathological responses to the eye and is important for human risk assessment of these important new therapies for ocular diseases.

Maggot therapy in "lower-extremity hospice" wound care: fewer amputations and more antibiotic-free days.

We sought to assess, in a case-control model, the potential efficacy of maggot debridement therapy in 60 nonambulatory patients (mean +/- SD age, 72.2 +/- 6.8 years) with neuroischemic diabetic foot wounds (University of Texas grade C or D wounds below the malleoli) and peripheral vascular disease. Twenty-seven of these patients (45%) healed during 6 months of review. There was no significant difference in the proportion of patients healing in the maggot debridement therapy versus control group (57% versus 33%). Of patients who healed, time to healing was significantly shorter in the maggot therapy than in the control group (18.5 +/- 4.8 versus 22.4 +/- 4.4 weeks). Approximately one in five patients (22%) underwent a high-level (above-the-foot) amputation. Patients in the control group were three times as likely to undergo amputation (33% versus 10%). Although there was no significant difference in infection prevalence in patients undergoing maggot therapy versus controls (80% versus 60%), there were significantly more antibiotic-free days during follow-up in patients who received maggot therapy (126.8 +/- 30.3 versus 81.9 +/- 42.1 days). Maggot debridement therapy reduces short-term morbidity in nonambulatory patients with diabetic foot wounds.

Comparison of ophthalmic gatifloxacin 0.3% and ciprofloxacin 0.3% in healing of corneal ulcers associated with Pseudomonas aeruginosa-induced ulcerative keratitis in rabbits.

OBJECTIVE: Evaluation of gatifloxacin 0.3% ophthalmic solution efficacy in a corneal ulcer model of Pseudomonas keratitis. METHODS: Heptanol-induced corneal ulcers in New Zealand White rabbits (n = 41; 8 females/group) were inoculated with 10(6) CFU of Pseudomonas aeruginosa. Gatifloxacin 0.3% dosing varied among 4 groups with frequencies of 16-48 doses/day (days 1-2), 3-16 doses/day (days 3-7), and maintenance dosing of 3-4 doses/day (days 8-22). Ciprofloxacin 0.3% was administered as labeled for corneal ulcers, with 44 doses on day 1, 16 doses on day 2, and 4 doses/day on days 3-21. RESULTS: All eyes showed evidence of infection by 48 hours postinoculation with 36 of 41 eyes (87.8%) exhibiting moderate-to-severe keratitis. All eyes exhibited corneal healing by day 15, with no significant differences among groups. Three of 4 groups receiving gatifloxacin tended to have smaller fluorescein retention area scores than did the ciprofloxacin group. No eyes tested positive for Pseudomonas at the end of the study. No corneal precipitates were found following as many as 48 doses/day of gatifloxacin. CONCLUSION: Ophthalmic gatifloxacin 0.3% is at least as effective as ciprofloxacin at healing corneal ulcers infected with Pseudomonas aeruginosa when gatifloxacin is administered less frequently than ciprofloxacin. Trends favored gatifloxacin in fluorescein retention scores.

Use of a maggot motility index to evaluate survival of therapeutic larvae.

Maggot debridement therapy is rapidly increasing in popularity at major diabetic foot and wound care centers worldwide. However, we are unaware of specific guidelines on the short-term storage of larvae. We sought to evaluate differences in maggot motility over time in larvae refrigerated versus those stored at room temperature. We also introduce a simple surrogate method for evaluating maggot vitality that may be useful for in vivo studies if validated in future works. We randomly selected ten larvae from the same shipment at ten different times in 9 days. Larvae were placed on a translucent acetate grid, and their total excursion in 30 sec was measured. This was converted into a Maggot Motility Index. In the refrigerated group, the index remained at or above 40 mm/min for approximately 60 hours from baseline, when there was a significant decrease. This same phenomenon occurred during the first 12 hours in the nonrefrigerated group. There were significant differences in motility between refrigerated and nonrefrigerated larvae immediately after baseline until day 8. Larvae are more practical for repeated clinical use if kept refrigerated between applications.

Clinical efficacy of the first metatarsophalangeal joint arthroplasty as a curative procedure for hallux interphalangeal joint wounds in patients with diabetes.

OBJECTIVE: To evaluate the safety and efficacy of first metatarsophalangeal joint arthroplasty compared with standard, nonsurgical management of wounds at the plantar hallux interphalangeal joint in patients with diabetes. RESEARCH DESIGN AND METHODS: We evaluated 41 patients with ulcers classified as University of Texas Grade 1A or 2A at the plantar aspect of the hallux interphalangeal joint using a case-control model [correction]. Case subjects were patients treated with resectional arthroplasty and control subjects received standard nonsurgical care. Both groups received standard off-loading and wound care. Outcomes included time to healing, reulceration, infection, and amputation. RESULTS: The surgery group healed significantly faster than patients in the standard therapy group (standard 67.1 +/- 17.1 versus surgery 24.2 +/- 9.9 days, P = 0.0001), and they had fewer recurrent ulcers (standard 35.0 versus surgery 4.8%, P = 0.02, odds ratio 7.6, 95% CI 1.1-261.7) Both groups had similar rates of infection (standard 38.1 versus surgery 40.0%, P = 0.9) and amputation (standard 10.0 versus surgery 4.8%, P = 0.5). CONCLUSIONS: Results suggest that resectional arthroplasty is a safe and effective procedure to treat wounds of the plantar hallux compared with nonsurgical therapy.

Outcomes of hyaluronan therapy in diabetic foot wounds.

The purpose of this study was to evaluate outcomes of persons with neuropathic diabetic foot wounds treated with a hyaluronan-containing dressing. Data were abstracted for 36 patients with diabetes, 72.2% male, aged 60.0+/-10.7 years and a mean glycated hemoglobin (HbA(1c)) of 9.5+/-2.5% presenting for care at two large, multidisciplinary wound care centers. All patients received surgical debridement for their diabetic foot wounds and were placed on therapy consisting of hyaluronan dressing (Hyalofill, Convatec, USA) with dressing changes taking place every other day. Outcomes evaluated included time to complete wound closure and proportion of patients achieving wound closure in 20 weeks. Hyalofill therapy was used until the wound bed achieved 100% granulation tissue. Therapy was then followed by a moisture-retentive dressing until complete epithelialization. In total, 75.0% of wounds measuring a mean 2.2+/-2.2 cm(2) healed in the 20-week evaluation period. Of those that healed in this period, healing took place in a mean 10.0+/-4.8 weeks. The average duration of Hyalofill therapy in all patients was 8.6+/-4.2 weeks. Deeper (UT Grade 2A) wounds were over 15 times less likely to heal than superficial (1A) wounds (94.7 vs. 52.9%, Odds Ratio=15.9, 95% Confidence Interval=1.7-142.8, P=0.006). We conclude that a regimen consisting of moist wound healing using hyaluronan-containing dressings may be a useful adjunct to appropriate diabetic foot ulcer care. We await the completion of a multicenter randomized controlled trial in this area to either support or refute this initial assessment.

Maggot debridement therapy: a primer.

Treatment of chronic wounds of the lower extremity requires a systematic, multidisciplinary approach as well as flexibility in order to achieve acceptable, consistent short-term and long-term results. Maggots, once considered an obsolete therapeutic modality, can be a useful addition to the armamentarium of the foot and ankle specialist. This article describes the use of maggot debridement therapy for intractable wounds of the lower extremity.

Technique for fabrication of an "instant total-contact cast" for treatment of neuropathic diabetic foot ulcers.

Addressing pressure reduction in the treatment of diabetic foot wounds is a critical component of therapy. The total-contact cast has proven to be the gold standard of treatment because of its ability to reduce pressure and facilitate patient adherence to the off-loading regimen. Removable cast walkers have proven to be as effective as total-contact casts in pressure reduction, but this has not translated into equivalent time to healing. A simple technique to convert the removable cast walker into a device that is not as easily detached from the lower extremity, thereby encouraging the use of this device over a 24-hour period, is presented in this article. The procedure involves wrapping the cast walker with cohesive bandage or plaster of Paris. In the authors' opinion, this technique addresses many of the disadvantages of the total-contact cast, resulting in an adequate compromise in this aspect of care.