FDA and industry collaboration: Identifying opportunities to further reduce reliance on nonhuman primates for nonclinical safety evaluations.

The nonhuman primate (NHP) has always been a limited resource for pharmaceutical research with ongoing efforts to conserve. This is due to their inherent biological properties, the growth in biotherapeutics and other modalities, and their use in small molecule drug development. The SARS-CoV-2 pandemic has significantly impacted the availability of NHPs due to the immediate need for NHPs to develop COVID-19 vaccines and treatments and the China NHP export ban; thus, accelerating the need to further replace, reduce and refine (3Rs) NHP use. The impact of the NHP shortage on drug development led DruSafe, BioSafe, and the United States (U.S.) Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) to discuss this issue at their 2021 annual meeting. This meeting identified areas to further the 3Rs in NHP use within the current nonclinical safety evaluation regulatory framework and highlighted the need to continue advancing alternative methods towards the aspirational goal to replace use of NHPs in the long term. Alignment across global health authorities is necessary for implementation of approaches that fall outside existing guidelines. This article captures the proceedings from this meeting highlighting current best practices and areas for 3Rs in NHP use.

Parsaclisib Is a Next-Generation Phosphoinositide 3-Kinase δ Inhibitor with Reduced Hepatotoxicity and Potent Antitumor and Immunomodulatory Activities in Models of B-Cell Malignancy.

The clinical use of first-generation phosphoinositide 3-kinase (PI3K)δ inhibitors in B-cell malignancies is hampered by hepatotoxicity, requiring dose reduction, treatment interruption, and/or discontinuation of therapy. In addition, potential molecular mechanisms by which resistance to this class of drugs occurs have not been investigated. Parsaclisib (INCB050465) is a potent and selective next-generation PI3Kδ inhibitor that differs in structure from first-generation PI3Kδ inhibitors and has shown encouraging anti-B-cell tumor activity and reduced hepatotoxicity in phase 1/2 clinical studies. Here, we present preclinical data demonstrating parsaclisib as a potent inhibitor of PI3Kδ with over 1000-fold selectivity against other class 1 PI3K isozymes. Parsaclisib directly blocks PI3K signaling-mediated cell proliferation in B-cell lines in vitro and in vivo and indirectly controls tumor growth by lessening immunosuppression through regulatory T-cell inhibition in a syngeneic lymphoma model. Diffuse large B-cell lymphoma cell lines overexpressing MYC were insensitive to proliferation blockade via PI3Kδ signaling inhibition by parsaclisib, but their proliferative activities were reduced by suppression of MYC gene transcription. Molecular structure analysis of the first- and next-generation PI3Kδ inhibitors combined with clinical observation suggests that hepatotoxicity seen with the first-generation inhibitors could result from a structure-related off-target effect. Parsaclisib is currently being evaluated in multiple phase 2 clinical trials as a therapy against various hematologic malignancies of B-cell origin (NCT03126019, NCT02998476, NCT03235544, NCT03144674, and NCT02018861). SIGNIFICANCE STATEMENT: The preclinical properties described here provide the mechanism of action and support clinical investigations of parsaclisib as a therapy for B-cell malignancies. MYC overexpression was identified as a resistance mechanism to parsaclisib in DLBCL cells, which may be useful in guiding further translational studies for the selection of patients with DLBCL who might benefit from PI3Kδ inhibitor treatment in future trials. Hepatotoxicity associated with first-generation PI3Kδ inhibitors may be an off-target effect of that class of compounds.

Nonclinical safety assessment of epigenetic modulatory drugs: Current status and industry perspective.

Pharmaceutic products designed to perturb the function of epigenetic modulators have been approved by regulatory authorities for treatment of advanced cancer. While the predominant effort in epigenetic drug development continues to be in oncology, non-oncology indications are also garnering interest. A survey of pharmaceutical companies was conducted to assess the interest and concerns for developing small molecule direct epigenetic effectors (EEs) as medicines. Survey themes addressed (1) general levels of interest and activity with EEs as therapeutic agents, (2) potential safety concerns, and (3) possible future efforts to develop targeted strategies for nonclinical safety assessment of EEs. Thirteen companies contributed data to the survey. Overall, the survey data indicate the consensus opinion that existing ICH guidelines are effective and appropriate for nonclinical safety assessment activities with EEs. Attention in the framework of study design should, on a case by case basis, be considered for delayed or latent toxicities, carcinogenicity, reproductive toxicity, and the theoretical potential for transgenerational effects. While current guidelines have been appropriate for the nonclinical safety assessments of epigenetic targets, broader experience with a wide range of epigenetic targets will provide information to assess the potential need for new or revised risk assessment strategies for EE drugs.

INCB040093 Is a Novel PI3Kδ Inhibitor for the Treatment of B Cell Lymphoid Malignancies.

Phosphatidylinositol 3-kinase delta (PI3Kδ) is a critical signaling molecule in B cells and is considered a target for development of therapies against various B cell malignancies. INCB040093 is a novel PI3Kδ small-molecule inhibitor and has demonstrated promising efficacy in patients with Hodgkin's lymphoma in clinical studies. In this study, we disclose the chemical structure and the preclinical activity of the compound. In biochemical assays, INCB040093 potently inhibits the PI3Kδ kinase, with 74- to >900-fold selectivity against other PI3K family members. In vitro and ex vivo studies using primary B cells, cell lines from B cell malignancies, and human whole blood show that INCB040093 inhibits PI3Kδ-mediated functions, including cell signaling and proliferation. INCB040093 has no significant effect on the growth of nonlymphoid cell lines and was less potent in assays that measure human T and natural killer cell proliferation and neutrophil and monocyte functions, suggesting that the impact of INCB040093 on the human immune system will likely be restricted to B cells. INCB040093 inhibits the production of macrophage-inflammatory protein-1ß (MIP-1beta) and tumor necrosis factor-ß (TNF-beta) from a B cell line, suggesting a potential effect on the tumor microenvironment. In vivo, INCB040093 demonstrates single-agent activity in inhibiting tumor growth and potentiates the antitumor growth effect of the clinically relevant chemotherapeutic agent, bendamustine, in the Pfeiffer cell xenograft model of non-Hodgkin's lymphoma. INCB040093 has a favorable exposure profile in rats and an acceptable safety margin in rats and dogs. Taken together, data presented in this report support the potential utility of orally administered INCB040093 in the treatment of B cell malignancies.

Results from oral gavage carcinogenicity studies of ruxolitinib in Tg.rasH2 mice and Sprague-Dawley (Crl:CD) rats.

Ruxolitinib is a selective and potent inhibitor of Janus kinase (JAK) 1 and JAK2. It is approved for the treatment of patients with intermediate or high-risk myelofibrosis, or those with polycythemia vera who have had an inadequate response to or are intolerant of hydroxyurea. To investigate its carcinogenic potential, ruxolitinib was administered by oral gavage once daily to Tg.rasH2 mice for 6 months at doses of 15, 45 or 125 mg/kg/day, and to Sprague-Dawley (Crl:CD) rats for 2 years at 10, 20 or 60 mg/kg/day. Ruxolitinib had no effect on survival, and did not increase the incidence of any neoplastic findings in either species. Exposure (AUC) was similar to or exceeded that associated with therapeutic use. Lymphoid depletion and a decrease in extramedullary hematopoiesis in the spleen occurred in rats, which were attributed to the pharmacologic activity of ruxolitinib. In Tg.rasH2 mice, increased inflammation in the nasal cavity was observed. Dose-dependent decreases in a number of spontaneous neoplastic/preneoplastic lesions were observed in rats, including mammary tumors in females, adrenal pheochromocytomas in males, hepatocellular adenomas/carcinomas in males, and hepatic basophilic (males and females) and eosinophilic (males) foci. Peribiliary fibrosis was also decreased. Clear cell foci in the liver were increased in females. Based on the results of these studies, ruxolitinib is not considered to be carcinogenic.

Extending (Q)SARs to incorporate proprietary knowledge for regulatory purposes: A case study using aromatic amine mutagenicity.

Statistical-based and expert rule-based models built using public domain mutagenicity knowledge and data are routinely used for computational (Q)SAR assessments of pharmaceutical impurities in line with the approach recommended in the ICH M7 guideline. Knowledge from proprietary corporate mutagenicity databases could be used to increase the predictive performance for selected chemical classes as well as expand the applicability domain of these (Q)SAR models. This paper outlines a mechanism for sharing knowledge without the release of proprietary data. Primary aromatic amine mutagenicity was selected as a case study because this chemical class is often encountered in pharmaceutical impurity analysis and mutagenicity of aromatic amines is currently difficult to predict. As part of this analysis, a series of aromatic amine substructures were defined and the number of mutagenic and non-mutagenic examples for each chemical substructure calculated across a series of public and proprietary mutagenicity databases. This information was pooled across all sources to identify structural classes that activate or deactivate aromatic amine mutagenicity. This structure activity knowledge, in combination with newly released primary aromatic amine data, was incorporated into Leadscope's expert rule-based and statistical-based (Q)SAR models where increased predictive performance was demonstrated.

Society for birth defects research and prevention's multidisciplinary research needs workshop 2022: A call to action.

The Society for Birth Defects Research and Prevention (BDRP) strives to understand and protect against potential hazards to developing embryos, fetuses, children, and adults by bringing together scientific knowledge from diverse fields. The theme of 62nd Annual Meeting of BDRP, "From Bench to Bedside and Back Again", represented the cutting-edge research areas of high relevance to public health and significance in the fields of birth defects research and surveillance. The multidisciplinary Research Needs Workshop (RNW) convened at the Annual Meeting continues to identify pressing knowledge gaps and encourage interdisciplinary research initiatives. The multidisciplinary RNW was first introduced at the 2018 annual meeting to provide an opportunity for annual meeting attendees to participate in breakout discussions on emerging topics in birth defects research and to foster collaboration between basic researchers, clinicians, epidemiologists, drug developers, industry partners, funding agencies, and regulators to discuss state-of-the-art methods and innovative projects. Initially, a list of workshop topics was compiled by the RNW planning committee and circulated among the members of BDRP to obtain the most popular topics for the Workshop discussions. Based on the pre-meeting survey results, the top three discussion topics selected were, A) Inclusion of pregnant and lactating women in clinical trials. When, why, and how? B) Building multidisciplinary teams across disciplines: What cross-training is needed? And C) Challenges in applications of Artificial Intelligence (AI) and machine learning for risk factor analysis in birth defects research. This report summarizes the key highlights of the RNW workshop and specific topic discussions.

Overview: developmental toxicology: new directions.

Since regulatory agencies began implementing the use of standardized developmental toxicology protocols in the mid-1960s, our knowledge base of embryo-fetal development and technologies for experimentation has grown exponentially. These developmental toxicology protocols were a direct result of the thalidomide tragedy from earlier that decade, when large numbers of women were exposed to the drug and over 10,000 cases of phocomelia resulted. In preventing a recurrence of such tragedies, the testing protocols are immensely successful and the field of toxicology has been dedicated to using them to advance safety and risk assessment of chemicals and pharmaceuticals. Recently, our perspectives on toxicity testing have been challenged by a growing awareness that while we have excelled in hazard identification, we are in dire need of improved methodologies for human health risk assessment, particularly with respect to the large numbers of environmental chemicals for which we have little toxicology data and to the growing sentiment that better alternatives to whole animals tests are needed. To provide a forum for scientists, researchers, and regulators, the Developmental and Reproductive Toxicology Technical Committee of the Health and Environmental Sciences Institute organized a 2-day workshop titled "Developmental Toxicology-New Directions" to evaluate lessons learned over the past 30 years and discuss the future of toxicology testing. The following four articles describe different presentations and discussions that were held over the course of those 2 days.

Developmental toxicology: new directions workshop: refining testing strategies and study designs.

In April 2009, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute's (HESI) Developmental and Reproductive Toxicology Technical Committee held a two-day workshop entitled "Developmental Toxicology-New Directions." The third session of the workshop focused on ways to refine animal studies to improve relevance and predictivity for human risk. The session included five presentations on: (1) considerations for refining developmental toxicology testing and data interpretation; (2) comparative embryology and considerations in study design and interpretation; (3) pharmacokinetic considerations in study design; (4) utility of genetically modified models for understanding mode-of-action; and (5) special considerations in reproductive testing for biologics. The presentations were followed by discussion by the presenters and attendees. Much of the discussion focused on aspects of refining current animal testing strategies, including use of toxicokinetic data, dose selection, tiered/triggered testing strategies, species selection, and use of alternative animal models. Another major area of discussion was use of non-animal-based testing paradigms, including how to define a "signal" or adverse effect, translating in vitro exposures to whole animal and human exposures, validation strategies, the need to bridge the existing gap between classical toxicology testing and risk assessment, and development of new technologies. Although there was general agreement among participants that the current testing strategy is effective, there was also consensus that traditional methods are resource-intensive and improved effectiveness of developmental toxicity testing to assess risks to human health is possible. This article provides a summary of the session's presentations and discussion and describes some key areas that warrant further consideration.

Effects of the opioid analgesic oxymorphone hydrochloride on reproductive function in male and female rats.

BACKGROUND: Endogenous opioids seem to regulate hypothalamic gonadotropin release in both males and females, as evidenced by the effects of opioid agonists and antagonists on LHRH release and reproductive hormone levels. The effects of long-term oral administration of opioid analgesics on reproductive function have not been well characterized. METHODS: The reproductive effects of oxymorphone, a potent opioid agonist, were investigated in male and female Crl:CD(SD) IGS BR rats at oral doses of 0, 5, 10, and 25 mg/kg/day (25 animals/sex/group). Males were treated for approximately 9 weeks (mated after 4 weeks of dosing). Females were treated for 14 days before mating, and through Gestation Day (GD) 7. Estrous cycling was evaluated during the premating period. On GD15, pregnancy status and the numbers of corpora lutea, implantation sites, live and dead embryos were determined. Epididymal and testicular sperm counts and epididymal sperm motility and morphology were evaluated in males. RESULTS: Two males given 25 mg/kg/day died. Behavioral changes and deficits in body weight gain occurred at all doses. There were no effects of oxymorphone on reproductive function or sperm parameters in males. The estrous cycle was prolonged in females given 25 mg/kg/day (mean of 5.3 vs. 4.3 days in controls). A small, but consistent decrease in the numbers of corpora lutea (with associated decreases in implantation sites and embryos) occurred in females given > or =10 mg/kg/day. There were no effects on mating or fertility in females. CONCLUSIONS: Oxymorphone seems to partially inhibit ovulation in female rats, with no significant effects on male reproductive outcome.

Evidence that oxymorphone-induced increases in micronuclei occur secondary to hyperthermia.

Oxymorphone is a potent opioid analgesic. Oral administration of oxymorphone to rats at doses >or= 20 mg/kg and mice at 500 mg/kg produced an increase in micronucleated polychromatic erythrocytes (MPCEs). Oxymorphone does not produce chromosome aberrations in vitro, suggesting that the increased MPCEs in vivo may involve indirect mechanisms. Opioids are known to affect thermoregulatory mechanisms. Changes in body temperature can increase the incidence of MPCEs in rodents. Studies were conducted to examine the relationship between increased MPCEs in rats given oxymorphone and changes in body temperature. Single oral doses of oxymorphone associated with increased MPCEs (20 and 40 mg/kg) also produced a marked, rapid increase in body temperature. When animals were pretreated with sodium salicylate, peak body temperature was lower and returned to baseline more quickly than when oxymorphone was given alone. MPCEs were evaluated in rats after administration of oxymorphone (40 mg/kg) alone or following pretreatment with an oral dose of sodium salicylate. Oxymorphone alone produced a statistically significant increase in the incidence of MPCEs (3.6 per 1000 polychromatic erythrocytes vs. 0.4 in controls). The number of MPCEs in animals pretreated with sodium salicylate was similar to controls. Sodium salicylate alone had no effect on the number of MPCEs. Systemic oxymorphone exposure was not affected by sodium salicylate pretreatment; maximum plasma concentration (C(max)) and area-under-the-curve values were similar after administration of oxymorphone alone or following pretreatment with sodium salicylate. These results indicate that the increased incidence of MPCEs following oxymorphone administration is directly related to increased body temperature.

Oxymorphone hydrochloride, a potent opioid analgesic, is not carcinogenic in rats or mice.

Despite their long history of chronic use, little information is available regarding the carcinogenicity of opioid analgesics. Oxymorphone is a potent morphinan-type mu-opioid analgesic used for treatment of moderate-to-severe pain. Oxymorphone was tested for carcinogenicity in Crl:CD IGS BR rats and CD-1 mice. Oxymorphone hydrochloride was administered orally once daily for 2 years to rats at doses of 2.5, 5 and 10 mg/kg/day (males) and 5, 10 and 25 mg/kg/day (females), and mice at 10, 25, 75 and 150 mg/kg/day (65 animals per sex per group; 100 animals per sex in controls). In rats, survival was generally higher than controls in oxymorphone-treated groups, attributable to lower body weight gain. In mice, survival was generally higher than controls in females at all doses and males given < or = 25 mg/kg/day but lower in males given > or = 75 mg/kg/day due to a high incidence of obstructive uropathy. Opioid-related clinical signs and reduced body weight gain occurred in both species throughout the study. Nonneoplastic findings associated with oxymorphone pharmacology included ocular and pulmonary changes in rats considered secondary to inhibition of blinking and mydriasis, and antitussive activity, respectively, and urinary tract and renal findings in mice considered secondary to urinary retention. There was no target organ toxicity, and no increase in any neoplastic lesions attributed to oxymorphone. Plasma oxymorphone levels achieved in these studies exceeded those in patients taking high therapeutic doses of oxymorphone (Area under the curve [AUC(0-24 h)] values up to 5.6-fold and 64-fold in rats and mice, respectively). Oxymorphone is not considered to be carcinogenic in rats or mice under the conditions of these studies.

Neurobehavioral assessment: a survey of use and value in safety assessment studies.

This report describes the results of a survey designed to evaluate the contribution of F1 neurobehavioral testing to hazard identification and characterization in safety assessment studies. (To review the details of the distributed survey, please see the supplementary data for this article on the journal's Web site.) The survey provided information about studies completed in industrial laboratories in the United States, Europe, and Japan since 1990 on 174 compounds. The types of compounds included were pharmaceutical (81%), agricultural (7%), industrial (1%), or were undefined (10%). Information collected included the intended use of the test agent, general study design and methodology, the types and characteristics of F1 behavioral evaluations, and the frequency with which agents affected neurobehavioral parameters in comparison to other F0 and F1 generation parameters. F1 general toxicology parameters such as mortality, pre- and postweaning body weight, and food intake were assessed in most studies and were affected more frequently than other parameters by the test agents. F1 behavioral parameters were assessed less consistently across studies, and were less frequently affected by the agents tested. Although affected by agents less often than general toxicology parameters, F1 behavioral parameters along with other parameters defined the no-observed-effect level (NOEL) in 17/113 (15%) of studies and solely defined the NOEL in 3/113 (2.6%) of studies. Thus, F1 behavioral parameters sometimes improved on the standard toxicological measures of hazard identification. While not detecting agent effects as readily as some measures, the F1 behavioral parameters provide information about agent effects on specialized functions of developing offspring not provided by other standard measures of toxicity. The survey results emphasize the need for further research into the methods of behavioral assessment as well as the mechanisms underlying the neurobehavioral alterations.