Skin sensitization testing needs and data uses by US regulatory and research agencies.

United States regulatory and research agencies may rely upon skin sensitization test data to assess the sensitization hazards associated with dermal exposure to chemicals and products. These data are evaluated to ensure that such substances will not cause unreasonable adverse effects to human health when used appropriately. The US Consumer Product Safety Commission, the US Environmental Protection Agency, the US Food and Drug Administration, the Occupational Safety and Health Administration, the National Institute for Occupational Safety and Health, and the US Department of Defense are member agencies of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). ICCVAM seeks to identify opportunities for the use of non-animal replacements to satisfy these testing needs and requirements. This review identifies the standards, test guidelines, or guidance documents that are applicable to satisfy each of these agency's needs; the current use of animal testing and flexibility for using alternative methodologies; information needed from alternative tests to fulfill the needs for skin sensitization data; and whether data from non-animal alternative approaches are accepted by these US federal agencies.

In chemico skin sensitization risk assessment of botanical ingredients.

Skin sensitization risk assessment of botanical ingredients is necessary for consumers' protection and occupational hazard identification. There are currently very few available alternative methods that can assist in the evaluation of complex mixtures. Chemical methods can provide essential information in a timely manner and thus help to reduce the need for in vivo testing, and they can complement and facilitate targeted in vitro assays. In the present work, the applicability of the high-throughput screening with dansyl cysteamine (DCYA) method for the systematic evaluation of skin sensitization of complex botanicals was explored. Botanical ingredients of four unrelated plant species were obtained and tested with the high-throughput fluorescence method at three concentrations. To illustrate the minimal matrix effects of the tested extracts on the developed method, the least DCYA-reactive extract (Rosa canina) was spiked with known sensitizers at different concentrations. The data obtained from the four plant extracts and the spiking experiments with known sensitizers, suggest that the high-throughput screening-DCYA method can be successfully applied for estimating the skin sensitization potential of complex botanical matrices. This is the first report of an attempt to develop a versatile in chemico method for the rapid detection of reactive skin sensitizers in complex botanical extracts, which could complement the battery of existing validated, non-animal methods.

Dihydroxyacetone: A Review.

The sunless tanning industry has experienced rapid growth due to public education on the dangers of ultraviolet radiation on skin and improvements in products. Dihydroxyacetone (DHA) is a 3-carbon sugar allowed by the Food and Drug Administration (FDA) as a color additive in sunless tanning products. Bronzers, a product removed with soap and water, may also contain DHA. We aim to review the literature on DHA. DHA is intended for external application, not including the mucous membranes or in or around the eye area. DHA has been used in spray-tan booths and by airbrushing it onto consumers, although these are unapproved uses, as contact with the color additive is not restricted to the external part of the body. Consequently, the FDA recommends customers shield their eyes, lips, and mucous membranes, as well as refrain from ingestion or inhalation of DHA. Unlike sunscreens, products that protect against ultraviolet radiation and are regulated by the FDA as non-prescription drugs, sunless tanning products are regulated as cosmetics and cannot provide any protection from exposure to ultraviolet radiation. There are reports of non-cosmetic uses of DHA that are not FDA approved. With the wide-spread use of DHA, additional studies on its safety are warranted.

J Drugs Dermatol. 2018;17(4):387-391.

Skin sensitizers in cosmetics and beyond: potential multiple mechanisms of action and importance of T-cell assays for in vitro screening.

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity (DTH) reaction induced by repeated contact with sensitizers. The ability of a chemical to act as a sensitizer has most frequently been tested in animals. As the use of animals for these purposes is gradually and globally being phased out, there is a need for reliable in vitro surrogate assays. Currently proposed in vitro assays are designed to test four key events of the adverse outcome pathway (AOP) involving covalent modification of self-proteins by sensitizers (haptenation) and presentation of new antigens (hapten/carrier complexes) to the immune system. There appears to be imperfect alignment of in vitro assays with clinical and/or animal data, suggesting possibly additional mechanisms of ACD development. Indeed, studies on allergies to small drugs, small chemical-induced HLA-peptide exchange for vaccination purposes and cosmetic ingredient-induced exposure of autoantigens suggest a possibility of DTH response promotion by hapten/carrier-independent mechanisms. Therefore, there is a need for additional appropriate in vitro assays, in order to achieve maximal concordance between clinical and/or animal data and in vitro assays. In this paper, we will review evidence supporting the idea of diverse mechanisms of ACD development. We will also discuss the impact of these multiple mechanisms, on the AOP and on the in vitro assays that should be used for allergen detection. We will propose alloreactivity-like reactions, aided by computer modeling and biochemical tests of compound-HLA binding, as additional tools for better prediction of DTH reactions, resulting from exposure to ingredients in cosmetic products. The combination of the proposed tests, along with the existing assays, should further enhance animal-free assessment of sensitizing potential of individual chemicals.

Formulation and performance of danazol nano-crystalline suspensions and spray dried powders.

PURPOSE: This study focuses on the formulation optimization, in vitro and in vivo performance of differently sized nano-crystalline liquid suspensions and spray-dried powders of a poorly soluble BCS class II compound i.e. Danazol. METHODS: A DoE approach was utilized to optimize stabilizer concentration and formulate danazol (BCS class II) nano-crystalline suspensions and dry powders via wet milling followed by spray drying. Solubility studies were performed to select best stabilizers. Particle size, PXRD, contact angle measurement and in vitro dissolution were utilized in characterization of the liquid and spray-dried powder formulations. RESULTS: The liquid nano-crystalline suspensions followed particle size-dependent dissolution rates i.e. faster dissolution for smaller crystals. The spray-dried nano-crystal powders did not show fast dissolution profiles compared to the liquid nano-crystalline suspension. The poor dissolution of the spray-dried powder correlated to its high LogP value (i.e. LogP 4.53) and poor wetting (or polar surface-area). In vivo bioavailability studies showed superior performance of the liquid nano-crystalline suspensions compared to other milled and un-milled formulations. CONCLUSION: Wet-milling and spray-drying optimization for danazol nano-crystalline suspension was performed. This study indicates that drug candidates with high LogP values and low polar surface area may not be suitable for formulation as dry nano-crystals.

Trust your gut: Establishing confidence in gastrointestinal models - An overview of the state of the science and contexts of use.

The webinar series and workshop titled "Trust Your Gut: Establishing Confidence in Gastrointestinal Models ­ An Overview of the State of the Science and Contexts of Use" was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and pro­cesses occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from dif­ferent countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.

A novel QSAR model of Salmonella mutagenicity and its application in the safety assessment of drug impurities.

As indicated in ICH M7 draft guidance, in silico predictive tools including statistically-based QSARs and expert analysis may be used as a computational assessment for bacterial mutagenicity for the qualification of impurities in pharmaceuticals. To address this need, we developed and validated a QSAR model to predict Salmonella t. mutagenicity (Ames assay outcome) of pharmaceutical impurities using Prous Institute's Symmetry(SM), a new in silico solution for drug discovery and toxicity screening, and the Mold2 molecular descriptor package (FDA/NCTR). Data was sourced from public benchmark databases with known Ames assay mutagenicity outcomes for 7300 chemicals (57% mutagens). Of these data, 90% was used to train the model and the remaining 10% was set aside as a holdout set for validation. The model's applicability to drug impurities was tested using a FDA/CDER database of 951 structures, of which 94% were found within the model's applicability domain. The predictive performance of the model is acceptable for supporting regulatory decision-making with 84±1% sensitivity, 81±1% specificity, 83±1% concordance and 79±1% negative predictivity based on internal cross-validation, while the holdout dataset yielded 83% sensitivity, 77% specificity, 80% concordance and 78% negative predictivity. Given the importance of having confidence in negative predictions, an additional external validation of the model was also carried out, using marketed drugs known to be Ames-negative, and obtained 98% coverage and 81% specificity. Additionally, Ames mutagenicity data from FDA/CFSAN was used to create another data set of 1535 chemicals for external validation of the model, yielding 98% coverage, 73% sensitivity, 86% specificity, 81% concordance and 84% negative predictivity.

In silico modeling to predict drug-induced phospholipidosis.

Drug-induced phospholipidosis (DIPL) is a preclinical finding during pharmaceutical drug development that has implications on the course of drug development and regulatory safety review. A principal characteristic of drugs inducing DIPL is known to be a cationic amphiphilic structure. This provides evidence for a structure-based explanation and opportunity to analyze properties and structures of drugs with the histopathologic findings for DIPL. In previous work from the FDA, in silico quantitative structure-activity relationship (QSAR) modeling using machine learning approaches has shown promise with a large dataset of drugs but included unconfirmed data as well. In this study, we report the construction and validation of a battery of complementary in silico QSAR models using the FDA's updated database on phospholipidosis, new algorithms and predictive technologies, and in particular, we address high performance with a high-confidence dataset. The results of our modeling for DIPL include rigorous external validation tests showing 80-81% concordance. Furthermore, the predictive performance characteristics include models with high sensitivity and specificity, in most cases above ≥80% leading to desired high negative and positive predictivity. These models are intended to be utilized for regulatory toxicology applied science needs in screening new drugs for DIPL.

Microphysiological Models for Mechanistic-Based Prediction of Idiosyncratic DILI.

Drug-induced liver injury (DILI) is a major contributor to high attrition rates among candidate and market drugs and a key regulatory, industry, and global health concern. While acute and dose-dependent DILI, namely, intrinsic DILI, is predictable and often reproducible in preclinical models, the nature of idiosyncratic DILI (iDILI) limits its mechanistic understanding due to the complex disease pathogenesis, and recapitulation using in vitro and in vivo models is extremely challenging. However, hepatic inflammation is a key feature of iDILI primarily orchestrated by the innate and adaptive immune system. This review summarizes the in vitro co-culture models that exploit the role of the immune system to investigate iDILI. Particularly, this review focuses on advancements in human-based 3D multicellular models attempting to supplement in vivo models that often lack predictability and display interspecies variations. Exploiting the immune-mediated mechanisms of iDILI, the inclusion of non-parenchymal cells in these hepatoxicity models, namely, Kupffer cells, stellate cells, dendritic cells, and liver sinusoidal endothelial cells, introduces heterotypic cell-cell interactions and mimics the hepatic microenvironment. Additionally, drugs recalled from the market in the US between 1996-2010 that were studies in these various models highlight the necessity for further harmonization and comparison of model characteristics. Challenges regarding disease-related endpoints, mimicking 3D architecture with different cell-cell contact, cell source, and the underlying multi-cellular and multi-stage mechanisms are described. It is our belief that progressing our understanding of the underlying pathogenesis of iDILI will provide mechanistic clues and a method for drug safety screening to better predict liver injury in clinical trials and post-marketing.

Parallel evaluation of alternative skin barrier models and excised human skin for dermal absorption studies in vitro.

Skin permeation is a primary consideration in the safety assessment of cosmetic ingredients, topical drugs, and human users handling veterinary medicinal products. While excised human skin (EHS) remains the 'gold standard' for in vitro permeation testing (IVPT) studies, unreliable supply and high cost motivate the search for alternative skin barrier models. In this study, a standardized dermal absorption testing protocol was developed to evaluate the suitability of alternative skin barrier models to predict skin absorption in humans. Under this protocol, side-by-side assessments of a commercially available reconstructed human epidermis (RhE) model (EpiDerm-200-X, MatTek), a synthetic barrier membrane (Strat-M, Sigma-Aldrich), and EHS were performed. The skin barrier models were mounted on Franz diffusion cells and the permeation of caffeine, salicylic acid, and testosterone was quantified. Transepidermal water loss (TEWL) and histology of the biological models were also compared. EpiDerm-200-X exhibited native human epidermis-like morphology, including a characteristic stratum corneum, but had an elevated TEWL as compared to EHS. The mean 6 h cumulative permeation of a finite dose (6 nmol/cm2) of caffeine and testosterone was highest in EpiDerm-200-X, followed by EHS and Strat-M. Salicylic acid permeated most in EHS, followed by EpiDerm-200-X and Strat-M. Overall, evaluating novel alternative skin barrier models in the manner outlined herein has the potential to reduce the time from basic science discovery to regulatory impact.

Considerations from an International Regulatory and Pharmaceutical Industry (IQ MPS Affiliate) Workshop on the Standardization of Complex In Vitro Models in Drug Development.

In May 2022, there is an International Regulatory and Pharmaceutical Industry (Innovation and Quality [IQ] Microphysiological Systems [MPS] Affiliate) Workshop on the standardization of complex in vitro models (CIVMs) in drug development. This manuscript summarizes the discussions and conclusions of this joint workshop organized and executed by the IQ MPS Affiliate and the United States Food and Drug Administration (FDA). A key objective of the workshop is to facilitate discussions around opportunities and/or needs for standardization of MPS and chart potential pathways to increase model utilization in the context of regulatory decision making. Participation in the workshop included 200 attendees from the FDA, IQ MPS Affiliate, and 26 global regulatory organizations and affiliated parties representing Europe, Japan, and Canada. It is agreed that understanding global perspectives regarding the readiness of CIVM/MPS models for regulatory decision making and potential pathways to gaining acceptance is useful to align on globally. The obstacles are currently too great to develop standards for every context of use (COU). Instead, it is suggested that a more tractable approach may be to think of broadly applicable standards that can be applied regardless of COU and/or organ system. Considerations and next steps for this effort are described.

Regulation of Cosmetics in the United States.

In the United States, cosmetics are regulated under the Food, Drug, and Cosmetic Act and the Fair Packaging and Labeling Act. Accordingly, cosmetic ingredients, with the exception of color additives, are not subject to premarket approval. However, they must not be adulterated or misbranded. This article describes the statutes and regulations relevant to cosmetic regulation by the Food and Drug Administration (FDA). It also describes relevant domestic programs of the FDA (Voluntary Cosmetic Registration Program, Good Manufacturing Practice guidance, Adverse Event Reporting System, Recalls) and international efforts regarding cosmetics regulation.

Comparative profile of commercially available urinary biomarkers in preclinical drug-induced kidney injury and recovery in rats.

We designed a study to provide reversibility and comparative injury data for several candidate urinary biomarkers of kidney injury in the United States Food and Drug Administration biomarker qualification process. The nephrotoxin gentamicin was given to rats once on each of three days and the animals were killed during dosing or over the following 42 days. Between days one and three, all biomarkers except albumin were elevated, peaked at day 7, and returned to control levels by day 10 (µ- and α-glutathione S-transferases, and renal papillary antigen-1) or day 15 (kidney injury molecule-1, lipocalin-2, osteopontin, and clusterin). All biomarkers performed better during injury than during recovery except osteopontin, which performed equally well in both time periods. During the evolution of injury, kidney injury molecule-1, renal papillary antigen-1, and clusterin best mirrored the histopathologic lesions. During injury resolution, kidney injury molecule-1, osteopontin, and blood urea nitrogen best reflected recovery. Based on histopathology, necrosis, or apoptosis scoring, kidney injury molecule-1 was the best biomarker of overall renal injury. Evaluation by regeneration score showed that renal papillary antigen-1 best reflected tubular and/or collecting duct regeneration, especially during recovery. Thus, these biomarkers performed with different effectiveness when evaluated by individual pathological processes such as necrosis, apoptosis, and regeneration.

Differences in immunolocalization of Kim-1, RPA-1, and RPA-2 in kidneys of gentamicin-, cisplatin-, and valproic acid-treated rats: potential role of iNOS and nitrotyrosine.

The present study compared the immunolocalization of Kim-1, renal papillary antigen (RPA)-1, and RPA-2 with that of inducible nitric oxide synthase (iNOS) and nitrotyrosine in kidneys of gentamicin sulfate (Gen)- and cisplatin (Cis)-treated rats. The specificity of acute kidney injury (AKI) biomarkers, iNOS, and nitrotyrosine was evaluated by dosing rats with valproic acid (VPA). Sprague-Dawley (SD) rats were injected subcutaneously (sc) with 100 mg/kg/day of Gen for six or fourteen days; a single intraperitoneal (ip) dose of 1, 3, or 6 mg/kg of Cis; or 650 mg/kg/day of VPA (ip) for four days. In Gen-treated rats, Kim-1 was expressed in the epithelial cells, mainly in the S1/S2 segments but less so in the S3 segment, and RPA-1 was increased in the epithelial cells of collecting ducts (CD) in the cortex. Spatial expression of iNOS or nitrotyrosine with Kim-1 or RPA-1 was detected. In Cis-treated rats, Kim-1 was expressed only in the S3 segment cells, and RPA-1 and RPA-2 were increased in the epithelial cells of medullary CD or medullary loop of Henle (LH), respectively. Spatial expression of iNOS or nitrotyrosine with RPA-1 or RPA-2 was also identified. These findings suggest that peroxynitrite formation may be involved in the pathogenesis of Gen and Cis nephrotoxicity and that Kim-1, RPA-1, and RPA-2 have the potential to serve as site-specific biomarkers for Gen or Cis AKI.

Energy dispersive X-ray analysis of titanium dioxide nanoparticle distribution after intravenous and subcutaneous injection in mice.

In an effort to understand the disposition and toxicokinetics of nanoscale materials, we used EDS (energy dispersive X-ray spectroscopy) to detect and map the distribution of titanium dioxide (TiO2) in tissue sections from mice following either subcutaneous (s.c.) or intravenous (i.v.) injection. TiO2 nanoparticles were administered at a dose of 560 mg/kg (i.v.) or 5600 mg/kg (s.c.) to Balb/c female mice on two consecutive days. Tissues (liver, kidney, lung, heart, spleen, and brain) were examined by light microscopy, TEM (transmission electron microscopy), SEM (scanning electron microscopy), and EDS following necropsy one day after treatment. Particle agglomerates were detected by light microscopy in all tissues examined, EDS microanalysis was used to confirm that these tissues contained elemental titanium and oxygen. The TEM micrographs and EDS spectra of the aggregates were compared with in vitro measurements of TiO2 nanoparticle injection solution (i.e., in water). The nanoparticles were also characterized using dynamic light scattering in water, 10 mM NaCl, and phosphate buffered saline (PBS). In low ionic strength solvents (water and 10 mM NaCl), the TiO2 particles had average hydrodynamic diameters ranging from 114-122 nm. In PBS, however, the average diameter increases to 1-2 microm, likely due to aggregation analogous to that observed in tissue by TEM and EDS. This investigation demonstrates the suitability of energy dispersive X-ray spectroscopy (EDS) for detection of nanoparticle aggregates in tissues and shows that disposition of TiO2 nanoparticles depends on the route of administration (i.v. or s.c.).

Particle size determination of sunscreens formulated with various forms of titanium dioxide.

BACKGROUND: There has been some apprehension expressed in the scientific literature that nanometer-sized titanium dioxide (TiO(2)) and other nanoparticles, if able to penetrate the skin, may cause cytotoxicity. In light of a lack of data regarding dermal penetration of titanium dioxide from sunscreen formulations, the Food and Drug Administration Center for Drug Evaluation and Research initiated a study in collaboration with the National Center for Toxicology Research using minipigs to determine whether nanoscale TiO(2) in sunscreen products can penetrate intact skin. Four sunscreen products were manufactured. METHOD: The particle size distribution of three TiO(2) raw materials, a sunscreen blank (no TiO(2)) and three sunscreen formulations containing uncoated nanometer-sized TiO(2), coated nanometer-sized TiO(2) or sub-micron TiO(2) were analyzed using scanning electron microscopy (SEM), laser scanning confocal microscopy (LSCM), and X-ray diffraction (XRD) to determine whether the formulation process caused a change in the size distributions (e.g., agglomeration or deagglomeration) of the TiO(2). RESULTS: SEM and XRD of the formulated sunscreens containing nanometer TiO(2) show the TiO(2) particles to have the same size as that observed for the raw materials. This suggests that the formulation process did not affect the size or shape of the TiO(2) particles. CONCLUSION: Because of the resolution limit of optical microscopy, nanoparticles could not be accurately sized using LSCM, which allows for detection but not sizing of the particles. LSCM allows observation of dispersion profiles throughout the sample; therefore, LSCM can be used to verify that results observed from SEM experiments are not solely surface effects.

Immunolocalization of Kim-1, RPA-1, and RPA-2 in kidney of gentamicin-, mercury-, or chromium-treated rats: relationship to renal distributions of iNOS and nitrotyrosine.

Immunohistochemical studies for kidney injury molecule-1 (Kim-1), renal papillary antigen-1 (RPA-1), and renal papillary antigen-2 (RPA-2) were conducted to explore their relationship to inducible nitric oxide synthase (iNOS) and nitrotyrosine expression. Male Sprague-Dawley rats were exposed to gentamicin (100 mg/kg/day Gen, sc, for 3 days), mercury (0.25 mg Hg/kg, iv, single dose), or chromium (5 mg Cr/kg, sc, single dose) and kidney tissue was examined 24 hours or 72 hours after the last dose of the nephrotoxicant. Another group of kidneys was evaluated 24 hours after rats were administered 3 daily doses (50, 100, 150, 200, or 300 mg/kg/day) of Gen. Gen- and Cr-treated rats exhibited increased immunoreactivity of Kim-1, RPA-1, and RPA-2 largely in the S1/S2 segments and to a lesser extent in the S3 segments of the proximal tubule of the kidney, whereas Hg-treated rats showed increased immunoreactivity of Kim-1, RPA-1, and RPA-2 in the S3 segments. Up-regulation of Kim-1, RPA-1, and RPA-2 expression correlated with injured tubular epithelial cells and also correlated with immunoreactivity of iNOS and nitrotyrosine. It is possible that iNOS activation with nitrotyrosine production in injured nephron segments may be involved in the induction of Kim-1, RPA-1, and RPA-2 following exposure to nephrotoxicants.

Quantitative determination of thallium binding to ferric hexacyanoferrate: Prussian blue.

Ferric hexacyanoferrate, (Fe(4)(III)[Fe(II)(CN)(6)](3)), also known as insoluble Prussian blue (PB), is the active pharmaceutical ingredient (API) of Radiogardase which is the first approved drug product (DP) for treatment of thallium and radiocesium poisoning. The aim of this study is (1) to determine the in vitro thallium binding capacity and binding rates of insoluble PB; and (2) to evaluate the effect of physiological pH conditions, PB particle size and storage conditions on the binding to PB. Experimental pH levels from 1.0 to 7.5 were used to cover the range of pH levels that PB may encounter when traveling through the gastrointestinal (GI) tract in humans. Measurements of thallium binding were made between 1 and 24h, to cover gastric and intestinal tract residence time. PB was found to have a binding capacity of approximately 1400 mg/g at pH 7.5. When the pH decreased, the binding decreased as well. The results indicated that the hydration state of PB influences the thallium binding process. It was also found that there exits a direct correlation between the moisture loss in PB and the thallium binding rate constant. The PB with 17 mol of water had a binding rate constant of 0.52, which was reduced to 0.32 when PB was dehydrated to 2.5 mol of water. Significant differences were observed in both binding capacity and binding rate constant among PB fractions with different particle size ranges. PB fraction with particle size of 220-1000 microm had a binding rate constant of 0.43, which increased to 0.64 when the particle size was reduced to 32-90 microm. Batch-to-batch variation in thallium binding was also observed among the APIs and the DPs and this was related to particle size and hydration state. These findings can be utilized to evaluate and predict drug product quality under certain manufacturing and dry storage conditions.

Quantitative determination of cesium binding to ferric hexacyanoferrate: Prussian blue.

Ferric hexacyanoferrate (Fe4III[FeII(CN)6]3), also known as insoluble Prussian blue (PB) is the active pharmaceutical ingredient (API) of the drug product, Radiogardase. Radiogardase is the first FDA approved medical countermeasure for the treatment of internal contamination with radioactive cesium (Cs) or thallium in the event of a major radiological incident such as a "dirty bomb". A number of pre-clinical and clinical studies have evaluated the use of PB as an investigational decorporation agent to enhance the excretion of metal cations. There are few sources of published in vitro data that detail the binding capacity of cesium to insoluble PB under various chemical and physical conditions. The study objective was to determine the in vitro binding capacity of PB APIs and drug products by evaluating certain chemical and physical factors such as medium pH, particle size, and storage conditions (temperature). In vitro experimental conditions ranged from pH 1 to 9, to cover the range of pH levels that PB may encounter in the gastrointestinal (GI) tract in humans. Measurements of cesium binding were made between 1 and 24h, to cover gastric and intestinal tract residence time using a validated atomic emission spectroscopy (AES) method. The results indicated that pH, exposure time, storage temperature (affecting moisture content) and particle size play significant roles in the cesium binding to both the PB API and the drug product. The lowest cesium binding was observed at gastric pH of 1 and 2, whereas the highest cesium binding was observed at physiological pH of 7.5. It was observed that dry storage conditions resulted in a loss of moisture from PB, which had a significant negative effect on the PB cesium binding capacity at time intervals consistent with gastric residence. Differences were also observed in the binding capacity of PB with different particle sizes. Significant batch to batch differences were also observed in the binding capacity of some PB API and drug products. Our results suggest that certain physiochemical properties affect the initial binding capacity and the overall binding capacity of PB APIs and drug products during conditions that simulated gastric and GI residence time. These physiochemical properties can be utilized as quality attributes to monitor and predict drug product quality under certain manufacturing and storage conditions and may be utilized to enhance the clinical efficacy of PB.

Validation of an in vitro method for the determination of cyanide release from ferric-hexacyanoferrate: Prussian blue.

Prussian blue (PB), ferric hexacyanoferrate, Fe(4)[Fe(CN)(6)](3) is indicated for the treatment of known or suspected internal contamination with radioactive cesium, radioactive thallium, or non-radioactive thallium. Owing to the molecular properties, cyanide is likely dissociated from PB under physiologically relevant pH conditions, thus raising a concern for the safety of the product. The objective of this study was to calibrate and validate a cyanide assay over a wide pH range (from 0.5 to 12) on the basis of Spectroquant cyanide test method (Merck). Merck's photometric method requires that the measurement solution be within pH 5.5-6.0, hence samples and standards need to be adjusted to this pH range. Since the process of pH adjustment may have significant impact on the determination of cyanide, the analysis method needs to be optimized, calibrated and validated under each pH condition in the study. The validation characteristics included accuracy, precision, quantification limit, linearity, and stability. The intra-day accuracy ranged from 90% to 109% for the deionized water and solutions of pH 0.5-12. The intra-day precision (R.S.D.) ranged from 2.4% to 8.1% for the deionized water and solutions of pH 0.5-12. The analytical range was linear from 0.05 to 0.5 ppm (mg/L). The R(2) ranged from 0.9925 to 0.9998. This validated method was successfully implemented to determine cyanide release from PB under various pH conditions (from 1.0 to 12) at different time-points (from 1 to 24 h).