Setting impurity limits for endogenous substances: Recommendations for a harmonized procedure and an example using fatty acids.

Endogenous substances, such as fatty, amino, and nucleic acids, are often purposefully used in parenterally pharmaceuticals, but may be present as impurities. Currently, no consensus guidance exists on setting impurity limits for these substances. Specific procedures are needed, as the amount and types of toxicity data available for endogenous substances are typically far less than those for other chemical impurities. Additionally, the parenteral route of administration of these substances is inherently non-physiological, resulting in potentially different or increased severity of toxicity. Risk Assessment Process Maps (RAPMAPs) are proposed as a model to facilitate the development of health-based exposure limits (HBELs) for endogenous substances. This yielded a framework that was applied to derive HBELs for several fatty acids commonly used in parenteral pharmaceuticals. This approach was used to derive HBELs with further vetting based on anticipated perturbations in physiological serum levels, impacts of dose-rate, and consideration of intermittent dosing. Parenteral HBELs of 100-500 mg/day were generated for several fatty acids, and a proposed class-based limit of 50 mg/day to be used in the absence of chemical-specific data. This default limit is consistent with the low toxicity of this chemical class and ICH Q3C value for Class 3 solvents.

A longitudinal study of naloxone opioid overdose awareness and reversal training for first-year medical students: specific elements require reinforcement.

BACKGROUND: The opioid epidemic is a progressively worsening public health crisis that continues to impact healthcare system strategies such as overdose reversal and destigmatization. Even among healthcare professionals, there remains a lack of confidence in naloxone administration and a prevalence of stigma. While training can play a major impact in reducing these shortcomings, the long-term effectiveness has yet to be characterized in training healthcare professionals. This study examined the long-term retention of opioid overdose awareness and reversal training (OOART) by evaluating performance at two-time intervals, immediately post-training and at a 3-month follow-up. METHODS: Voluntary training was offered to first-year (M1) medical students at the Drexel University College of Medicine in 2021. At this training, 118 students completed training, 95 completed the post-training survey, and 42 completed the 3-month follow-up. RESULTS: Opioid reversal knowledge questions assessed significantly increased scores post-training and at the 3-month follow-up. In three of the attitude questions, scores were improved at both follow-up timepoints. In addition, three attitude questions indicating a participant's confidence to respond to an opioid overdose situation increased directly after the training, but regressed at the 3-month follow-up. The remaining questions did not show any statistical difference across the survey intervals. CONCLUSIONS: This study establishes that while OOART provides participants with the knowledge of how to respond to an opioid overdose, the retention of this knowledge at a 3-month interval is reduced. The results were mixed for longitudinal assessment of participant's attitudes toward people with opioid use disorder. Some positive increases in attitudes were retained at the 3-month interval, while others trended back toward pre-training levels. These results support the effectiveness of the training but also provide evidence that OOART must be reinforced often.

The utility of hERG channel inhibition data in the derivation of occupational exposure limits.

Inhibition of the human ether-à-go-go (hERG) channel may lead to QT prolongation and fatal arrhythmia. While pharmaceutical drug candidates that exhibit potent hERG channel inhibition often fail early in development, many drugs with both cardiac and non-cardiac indications proceed to market. In this study, the relationship between in vitro hERG channel inhibition and published occupational exposure limit (OEL) was evaluated. A total of 23 cardiac drugs and 44 drugs with non-cardiac indications with published hERG channel IC50 and published OELs were identified. There was an apparent relationship between hERG IC50 potency and the OEL for cardiac and non-cardiac drugs. Twenty cardiac and non-cardiac drugs were identified that had a potent hERG IC50 (≤25 µM) and a contrastingly large OEL value (≥100 µg/m3). OELs or hazard banding corresponding to ≤100 µg/m3 should be sufficiently protective of effects following occupational exposure to the majority of APIs with hERG IC50 values ≤ 100 µM. It is important to consider hERG IC50 values and possible cardiac effects when deriving OEL values for drugs, regardless of indication. These considerations may be particularly important early in the drug development process for establishing exposure control bands for drugs that do not yet have full clinical safety data.

In silico approaches in organ toxicity hazard assessment: Current status and future needs for predicting heart, kidney and lung toxicities.

The kidneys, heart and lungs are vital organ systems evaluated as part of acute or chronic toxicity assessments. New methodologies are being developed to predict these adverse effects based on in vitro and in silico approaches. This paper reviews the current state of the art in predicting these organ toxicities. It outlines the biological basis, processes and endpoints for kidney toxicity, pulmonary toxicity, respiratory irritation and sensitization as well as functional and structural cardiac toxicities. The review also covers current experimental approaches, including off-target panels from secondary pharmacology batteries. Current in silico approaches for prediction of these effects and mechanisms are described as well as obstacles to the use of in silico methods. Ultimately, a commonly accepted protocol for performing such assessment would be a valuable resource to expand the use of such approaches across different regulatory and industrial applications. However, a number of factors impede their widespread deployment including a lack of a comprehensive mechanistic understanding, limited in vitro testing approaches and limited in vivo databases suitable for modeling, a limited understanding of how to incorporate absorption, distribution, metabolism, and excretion (ADME) considerations into the overall process, a lack of in silico models designed to predict a safe dose and an accepted framework for organizing the key characteristics of these organ toxicants.

A harmonization effort for acceptable daily exposure application to pharmaceutical manufacturing - Operational considerations.

A European Union (EU) regulatory guideline came into effect for all new pharmaceutical products on June 1st, 2015, and for all existing pharmaceutical products on December 1st, 2015. This guideline centers around the use of the Acceptable Daily Exposure (ADE) [synonymous with the Permitted Daily Exposure (PDE)] and operational considerations associated with implementation are outlined here. The EU guidance states that all active pharmaceutical ingredients (API) require an ADE; however, other substances such as starting materials, process intermediates, and cleaning agents may benefit from an ADE. Problems in setting ADEs for these additional substances typically relate to toxicological data limitations precluding the ability to establish a formal ADE. Established methodologies such as occupational exposure limits or bands (OELs or OEBs) and the threshold of toxicological concern (TTC) can be used or adjusted for use as interim ADEs when only limited data are available and until a more formal ADE can be established. Once formal ADEs are derived, it is important that the documents are routinely updated and that these updates are communicated to appropriate stakeholders. Another key operational consideration related to data-poor substances includes the use of maximum daily dose (MDD) in setting cross-contamination limits. The MDD is an important part of the maximum allowable/safe concentration (MAC/MSC) calculation and there are important considerations for its use and definition. Finally, other considerations discussed include operational aspects of setting ADEs for pediatrics, considerations for large molecules, and risk management in shared facilities.

Framework for human health risk assessment of non-cancer effects resulting from short-duration and intermittent exposures to chemicals.

Durations of exposure to chemicals, whether for single, repeated or intermittent periods, may vary from those upon which most guidance values are normally based. Because it is presently not feasible to conduct toxicity studies or develop toxicity reference values (TRVs) specific to each scenario of interest, methods are needed to address these various durations, drawing as much as possible on existing TRVs. A working framework was developed to address the potential for non-cancer effects resulting from continuous short-duration and intermittent exposures to chemicals. The framework presents an integrated, tiered approach that assists the user in identifying when existing TRVs can be applied directly, and the adaptations needed to assess the acceptability of short-duration or intermittent exposure scenarios. Descriptions of when and how toxicokinetic and toxicodynamic aspects need to be taken into consideration are also presented. The framework incorporates the use of TRVs based on exposure periods as similar as possible to the "actual" exposure periods and application of dose averaging under limited, specified conditions. This framework has been developed to aid in improving the scientific basis for the evaluation of short-duration and intermittent exposures in a variety of settings. Copyright © 2016 John Wiley & Sons, Ltd.

Toxicokinetic and toxicodynamic considerations when deriving health-based exposure limits for pharmaceuticals.

The purpose of this paper is to describe the use of toxicokinetic (TK) and toxicodynamic (TD) data in setting acceptable daily exposure (ADE) values and occupational exposure limits (OELs). Use of TK data can provide a more robust exposure limit based on a rigorous evaluation of systemic internal dose. Bioavailability data assist in extrapolating across different routes of exposure to be protective for route-based differences of exposure. Bioaccumulation data enable extrapolation to chronic exposures when the point of departure (PoD) is from a short-term critical study. Applied in the context of chemical-specific adjustment factors (CSAFs), TK data partially replace traditional default adjustment factors for interspecies extrapolation (extrapolation from studies conducted in animals to humans) and intraspecies variability (to account for human population variability). Default adjustments of 10-fold each for interspecies and intraspecies extrapolation are recommended in several guidelines, although some organization recommend other values. Such default factors may overestimate variability for many APIs, while not being sufficiently protective for variability with other APIs. For this reason, the use of chemical specific TK and TD data are preferred. Making full use of existing TK and TD data reduces underlying uncertainties, increases transparency, and ensures that resulting ADEs reflect the best available science.

The regulatory framework for preventing cross-contamination of pharmaceutical products: History and considerations for the future.

Cross-contamination in multi-product pharmaceutical manufacturing facilities can impact both product safety and quality. This issue has been recognized by regulators and industry for some time, leading to publication of a number of continually evolving guidelines. This manuscript provides a historical overview of the regulatory framework for managing cross-contamination in multi-product facilities to provide context for current approaches. Early guidelines focused on the types of pharmaceuticals for which dedicated facilities and control systems were needed, and stated the requirements for cleaning validation. More recent guidelines have promoted the idea of using Acceptable Daily Exposures (ADEs) to establish cleaning limits for actives and other potentially hazardous substances. The ADE approach is considered superior to previous methods for setting cleaning limits such as using a predetermined general limit (e.g., 10 ppm or a fraction of the median lethal dose (LD50) or therapeutic dose). The ADEs can be used to drive the cleaning process and as part of the overall assessment of whether dedicated production facilities are required. While great strides have been made in using the ADE approach, work remains to update good manufacturing practices (GMPs) to ensure that the approaches are clear, consistent with the state-of-the-science, and broadly applicable yet flexible enough for adaptation to unique products and situations.

Reciprocal regulation of angiopoietin-1 and angiopoietin-2 following myocardial infarction in the rat.

OBJECTIVE: This study sought to characterize changes in the angiopoietin system in a rat model of myocardial infarction (MI). BACKGROUND: Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) bind to the endothelial-specific receptor tyrosine kinase, TIE-2. Ang-2 has been suggested to be an antagonist of TIE-2, possibly acting to release endothelial cells from the tonic stabilizing influence of Ang-1. However, on prolonged exposure, Ang-2 has been shown to acquire agonistic activity at TIE-2, raising the possibility that this isoform may play a direct role in neovascularization. METHODS: Sprague-Dawley rats were subjected to left coronary ligation and myocardial tissues were harvested from the infarct and peri-infarct regions, or from non-infarcted myocardium. Changes in gene expression were determined by RT-PCR and confirmed by Northern analysis. Changes in protein expression were confirmed by Western analysis and immunocytochemistry, and TIE-2 activity was determined by immunoprecipitation with anti-TIE-2 and antiphosphotyrosine immunoblotting. RESULTS: At 24 h, Ang-1 mRNA and protein expression within the infarct and peri-infarct regions were decreased compared to non-infarcted myocardium, whereas Ang-2 mRNA levels were markedly increased and TIE-2 expression was unchanged. Immunohistochemical staining revealed Ang-1 and TIE-2 immunoreactivity localized to vascular endothelium. In the infarct territory, Ang-2 immunostaining was localized primarily to invading leukocytes at 24 h. At 1 week, Ang-1 expression was partially restored, whereas Ang-2 expression remained elevated. At the time of peak elevation in Ang-2, Tie2 phosphorylation was found to be markedly increased, consistent with receptor activation. CONCLUSIONS: Thus, myocardial ischemia induced by left coronary artery ligation resulted in a sustained increase in Ang-2 expression and a reciprocal decrease in Ang-1, consistent with a predominant role for Ang-2 in the angiogenic response to MI.

The intravascular persistence and methemoglobin formation of Hemolink (hemoglobin raffimer) in dogs.

Hemoglobin raffimer (HEMOLINK, Hemosol Inc, Mississauga, Canada) is an o-raffinose cross-linked, purified human hemoglobin-based oxygen therapeutic that is currently being evaluated in late stage clinical trials. It is composed of several molecular weight (MW) species, comprising principally of stabilized tetramers (34-42%) and oligomers (54-62%). The objective of this study was to determine the in vivo circulating half-life (T1/2) of hemoglobin raffimer (Hb raffimer) and of its individual MW components in dogs subjected to a topload infusion of 25% of the estimated blood volume (18 mL/kg). Sampling was done over a 64-hour period that was expected to be equivalent to approximately two-and-half to three half-lives. Methemoglobin (MetHb) levels were also measured at intervals over the same period. The mean circulating half-life of Hb raffimer was 25.4 +/- 3.9 hours. The T1/2 for the individual MW components (determined by size exclusion chromatography) of Hb raffimer was 11 +/- 2 hours for the cross-linked tetramer and 35 +/- 7 hours for the fraction of oligomers. The apparent volume of distribution for Hb raffimer was estimated at 78 mL/kg. There was no difference in the apparent volumes of distribution of the tetrameric and oligomeric components of Hb raffimer. Throughout the course of the experiment (in which MetHb could be measured), plasma MetHb concentration, expressed as a percentage of the total plasma hemoglobin concentration, remained at 10% or less, and the mass concentration of MetHb in plasma remained at about 1 g/L. Thus, in the dog subjected to an estimated 25% topload infusion, the T1/2 of the infused Hb raffimer is approximately one day with the intravascular retention of the individual Hb raffimer components being dependent on the MW. Furthermore, oxidation of Hb raffimer to MetHb is limited under these conditions.

The effects of hemodilution with Hemolink upon hemodynamics and blood flow distribution in anesthetized dogs.

Hemoglobin based oxygen-carrying solutions (HBOCs) as hemoglobin replacement therapeutics are being tested for clinical use. Some of these products are associated with elevations in both systemic and pulmonary vascular resistances but their effect on the distribution of blood flow to major organs in larger animals have not been extensively described. We tested two formulations of o-raffinose cross-linked human hemoglobin, Hemolink (frozen-Hemolink-1 and refrigerated Hemolink-2) and compared them to Pentaspan, a colloid volume expander in extensive clinical use. Cardiovascular measurements and the distribution of blood flow (radionuclide-labeled microspheres) to the major organs were determined in Beagle dogs (n = 5 per group). After baseline measurements, either Hemolink-1, Hemolink-2, or Pentaspan was exchange transfused in an isovolemic manner (resulting in hematocrit reduction to approximately 20-25%); measurements were made 30, 60, 120 and 180 min post-exchange. There was no significant difference in cardiac output, mean arterial pressures and systemic or pulmonary vascular resistances after exchange in any of the three groups. Myocardial blood flow increased in all three groups post-exchange but the increase was more sustained in the Hemolink groups. Endocardial/epicardial flow ratios were also maintained after exchange in all groups. Thus, Hemolink is ideally suited for volume replacement when used in conjunction with acute normovolemic hemodilution because under these circumstances, the adverse hemodynamic effects are alleviated while extra hemoglobin is added to the blood.

DNA integrity and transgene expression after passage through the NOGA needle catheter used for therapeutic myocardial angiogenesis.

BACKGROUND: The NOGA (Biosense Webster, Markham, ON, Canada) injection catheter is an innovative navigational device that provides an ideal platform for intra-myocardial injection material. However, injection through a long (1.91 m), narrow (27G) nitinol needle could result in deterioration in the integrity and functionality of DNA. METHODS: To test this possibility, DNA in plasmid form (pcDNA3.1) containing the Lac Z transgene (250 micro l) was passed through the NOGA needle using a hand-held 1 cc syringe at a gentle hand injection pressure (43 +/- 3 PSI, 3.0 +/- 0.2 kg/cm(2)) or at maximal manual pressure (90 +/- 6 PSI, 6.3 +/- 0.4 kg/cm(2)), either once or 20 times. This DNA, compared to DNA not passed through the NOGA needle (control), was then used to transfect primary cultures of rat skin fibroblasts (FB) from Fisher 344 rats and the cells were subsequently stained for beta galactosidase (betagal). RESULTS: Transfection efficiency was significantly reduced by passing the DNA through the needle at both 43 +/- 3 PSI (78 +/- 4% of control, n = 10, P < 0.05 versus control) and 90 +/- 6 PSI (66 +/- 4 % of control, n = 10, P < 0.01 versus control, P < 0.02 versus 43 +/- 3 PSI). Passage of the DNA through the NOGA needle 20 times resulted in a transfection efficiency of only 5 +/- 1% of control (n = 20, P < 0.1 x 10(-11) versus control). Capillary Electrophoresis revealed that the reduction in transfection efficiency was due to a conformational change in the DNA from predominantly supercoiled to nicked and linearized DNA. Transfection efficiency as compared with control decreased as the concentration of the DNA solution which was passed through the needle was increased from 0.3 micro g/ micro l to 2.4 micro g/ micro l. Recovery experiments confirmed that the reduction in transfection efficiency was not due to loss of DNA by binding to the NOGA needle. CONCLUSION: These results suggest that DNA is susceptible to shear forces when injected through the NOGA needle even at nominal clinical injection pressures, suggesting that careful and controlled injections will be required to achieve optimal gene integrity and expression.