The -aminobutyric acid type A (GABA_A) receptor is modulated by a number of neuroactive steroids. Sulfated steroids and 3β-hydroxy steroids inhibit, while 3α-hydroxy steroids typically potentiate the receptor. Here, we have investigated inhibition of the α1β32L GABA_A receptor by the endogenous neurosteroid 3α-hydroxy-5β-pregnan-20-one (3α5βP) and the synthetic neuroactive steroid 3α-hydroxy-5α-androstane-17β-carbonitrile (ACN). The receptors were expressed in Xenopus oocytes. All experiments were done using two-electrode voltage-clamp electrophysiology. In the presence of low concentrations of GABA, 3α5βP and ACN potentiate the GABA_A receptor. To reveal inhibition, we conducted the experiments on receptors activated by the combination of a saturating concentration of GABA and propofol to fully activate the receptors and mask potentiation, or on mutant receptors in which potentiation is ablated. Under these conditions, both steroids inhibited the receptor with IC₅₀s of ~13 μM and maximal inhibitory effects of 70–90%. Receptor inhibition by 3α5βP was sensitive to substitution of the α1 transmembrane domain (TM) 2-2' residue, previously shown to ablate inhibition by pregnenolone sulfate. However, results of coapplication studies and the apparent lack of state dependence suggest that pregnenolone sulfate and 3α5βP inhibit the GABA_A receptor independently and through distinct mechanisms. Mutations to the neurosteroid binding sites in the α1 and β3 subunits statistically significantly, albeit weakly and incompletely, reduced inhibition by 3α5βP and ACN.

The orexigenic gut peptide ghrelin is an endogenous ligand for the growth hormone secretagogue receptor type 1a (GHSR1a). Systemic ghrelin administration has previously been shown to increase gastric motility and emptying. While these effects are known to be mediated by the vagus nerve, the cellular mechanism underlying these effects remains unclear. Therefore, the purpose of the present study was to investigate the signaling mechanism by which GHSR1a inhibits voltage-gated Ca²⁺ channels in isolated rat gastric vagal afferent neurons using whole-cell patch-clamp electrophysiology. The ghrelin pharmacological profile indicated that Ca²⁺ currents were inhibited with a log (Ic₅₀) = –2.10 ± 0.44 and a maximal inhibition of 42.8 ± 5.0%. Exposure to the GHSR1a receptor antagonist (D-Lys3)-GHRP-6 reduced ghrelin-mediated Ca²⁺ channel inhibition (29.4 ± 16.7% vs. 1.9 ± 2.5%, n = 6, P = 0.0064). Interestingly, we observed that activation of GHSR1a inhibited Ca²⁺ currents through both voltage-dependent and voltage-independent pathways. We also treated the gastric neurons with either pertussis toxin (PTX) or YM-254890 to examine whether the Ca²⁺ current inhibition was mediated by the Gα_i/o or Gα_q/11 family of subunits. Treatment with both PTX (Ca²⁺ current inhibition = 15.7 ± 10.6%, n = 8, P = 0.0327) and YM-254890 (15.2 ± 11.9%, n = 8, P = 0.0269) blocked ghrelin’s effects on Ca²⁺ currents, as compared with control neurons (34.3 ± 18.9%, n = 8). These results indicate GHSR1a can couple to both Gα_i/o and Gα_q/11 in gastric vagal afferent neurons. Overall, our findings suggest GHSR1a-mediated inhibition of Ca²⁺ currents occurs through two distinct pathways, offering necessary insights into the cellular mechanisms underlying ghrelin’s regulation of gastric vagal afferents.

The antitumor effect of cardiotonic steroids (CTS) has stimulated the search for new methods to evaluate both kinetic and thermodynamic aspects of their binding to Na⁺/K⁺-ATPase (IUBMB Enzyme Nomenclature). We propose a real-time assay based on a chromogenic substrate for phosphatase activity (pNPPase activity), using only two concentrations with an inhibitory progression curve, to obtain the association rate (k_on), dissociation rate (k_off), and equilibrium (K_i) constants of CTS for the structure-kinetics relationship in drug screening. We show that changing conditions (from ATPase to pNPPase activity) resulted in an increase of K_i of the cardenolides digitoxigenin, essentially due to a reduction of k_on. In contrast, the K_i of the structurally related bufadienolide bufalin increased much less due to the reduction of its k_off partially compensating the decrease of its k_on. When evaluating the kinetics of 15 natural and semisynthetic CTS, we observed that both k_on and k_off correlated with K_i (Spearman test), suggesting that differences in potency depend on variations of both k_on and k_off. A rhamnose in C3 of the steroidal nucleus enhanced the inhibitory potency by a reduction of k_off rather than an increase of k_on. Raising the temperature did not alter the k_off of digitoxin, generating a H (k_off) of –10.4 ± 4.3 kJ/mol, suggesting a complex dissociation mechanism. Based on a simple and inexpensive methodology, we determined the values of k_on, k_off, and K_i of the CTS and provided original kinetics and thermodynamics differences between CTS that could help the design of new compounds.

Aberrant type 2 inflammatory responses are the underlying cause of the pathophysiology of allergic asthma, allergic rhinitis, and other atopic diseases, with an alarming prevalence in relevant parts of the Western world. A bulk of evidence points out the important role of the DP2 receptor in these inflammation processes. A screening of different polyunsaturated fatty acids at a fluorescence resonance energy transfer–based DP2 receptor conformation sensor expressed in human embryonic kidney (HEK) cells revealed an agonistic effect of the prostaglandin (PG)-D₂ precursor arachidonic acid on DP2 receptor activity of about 80% of the effect induced by PGD₂. In a combination of experiments at the conformation sensor and using a bioluminescence resonance energy transfer–based G protein activation sensor expressed together with DP2 receptor wild type in HEK cells, we found that arachidonic acid acts as a direct activator of the DP2 receptor, but not the DP1 receptor, in a concentration range considered physiologically relevant. Pharmacological inhibition of cyclooxygenases and lipoxygenases as well as cytochrome P450 did not lead to a diminished arachidonic acid response on the DP2 receptor, confirming a direct action of arachidonic acid on the receptor.

G protein–coupled receptors (GPCRs) couple to heterotrimeric G proteins, comprised of α and β subunits, to convert extracellular signals into activation of intracellular signaling pathways. Canonically, GPCR-mediated activation results in the exchange of GDP for GTP on G protein α subunits (Gα) and the dissociation of Gα-GTP and G protein β subunits (Gβ), both of which can regulate a variety of signaling pathways. Hydrolysis of bound GTP by Gα returns the protein to Gα-GDP and allows reassociation with Gβ to reform the inactive heterotrimer. Naturally occurring mutations in Gα have been found at conserved glutamine and arginine amino acids that disrupt the canonical G protein cycle by inhibiting GTP hydrolysis, rendering these mutants constitutively active. Interestingly, these dysregulated Gα mutants are found in many different cancers due to their ability to sustain aberrant signaling without a need for activation by GPCRs. This review will highlight an increased recognition of the prevalence of such constitutively activating Gα mutations in cancers and the signaling pathways activated. In addition, we will discuss new knowledge regarding how these constitutively active Gα are regulated, how different mutations are biochemically distinct, and how mutationally activated Gα are unique compared with GPCR-activated Gα. Lastly, we will discuss recent progress in developing inhibitors directly targeting constitutively active Gα mutants.

ABSTRACTDespite global consensus on the importance of male involvement in family planning, disparities persist in low- and middle-income countries, where women continue to bear the responsibility for these initiatives. The Philippines, with a high fertility rate and unmet family planning needs, exemplifies this challenge. We present the experiences and lessons learned from implementing the no-scalpel vasectomy (NSV) program in Davao City, showcasing its potential for increasing male engagement in family planning decisions. Launched in 2008, the program aimed to address gender disparity by promoting NSV as a safe and effective contraceptive alternative to female-centric methods. Through the use of culturally sensitive information campaigns and couple-focused counseling, the program challenged traditional notions of masculinity and encouraged shared decision-making. Strong local government commitment and public-private partnerships played key roles in driving the program’s success. Results showed an average annual increase of 80% in NSV clients over the past 3 years compared to before the COVID-19 pandemic, underscoring its effectiveness. The program presents a compelling intervention model for similar initiatives, highlighting how overcoming cultural barriers, infrastructure limitations, and budgetary constraints through policy advocacy, strategic partnerships, and tailored approaches can significantly boost male involvement in family planning and improve reproductive health outcomes within communities.

ABSTRACTIntroduction:Family Planning 2020 (FP2020) was established in 2012 with the goal of expanding contraceptive access. By 2020, 46 countries had made commitments to FP2020. A sustained focus on adolescents and youth (AY) began in 2016. During the commitment formulation process, substantial support was offered to countries to develop AY commitments based on sound data, research evidence, and programmatic experience. This study assesses how country commitments under FP2020 and FP2030 have evolved over time with respect to improving attention to and focus on the needs of adolescents and youth sexual and reproductive health (AYSRH).Methods:We analyzed the content of FP2020 and FP2030 country commitments focusing on AY (aged 10–24 years) using a scoring guideline we developed to measure the AY commitments in terms of completeness, clarity, and quality.Results:This analysis shows that FP2030 commitments better articulate strategies and activities to reach AY with contraceptive information and services when compared to FP2020 commitments.Conclusion:FP2030 commitments are stronger in some areas on AYSRH, such as commitment to establish national or local policies, strategies, and guidance for AY programming, specifying the target audience of the AY commitment, and partnering with AY or youth-led organizations in commitments. However, more work remains to be done by countries to dedicate a budget for achieving AY objectives, including measurable targets for monitoring progress, identifying and addressing the root causes that impact AY access to and use of contraception, including child marriage and gender-based violence, and reducing financial barriers to access contraception.

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ABSTRACTThe private health care sector is an important source of service delivery in low- and middle-income countries (LMICs). Yet, the private sector remains fragmented, making it difficult for health system actors to support and ensure the availability of quality health care services. In global health programs, social franchising is one model used to engage and organize the private health care sector. Two social franchise networks, ProFam in West Africa and Tunza in East and Central Africa, provide health care through branded networks of facilities. However, these social franchise networks include a limited number of private health care facilities, and in fragile contexts, like Burundi and Mali, they have faced challenges in integrating with national health systems. The MOMENTUM Private Healthcare Delivery (MPHD) project in Burundi and Mali sought to expand the number of health facilities it engaged beyond the existing ProFam and Tunza networks. The expansion aimed to help improve service quality in more private facilities while advancing localization and reducing fragmentation for improved stewardship by health system actors. MPHD achieved this expansion by removing barriers for private health facilities to join inclusive, nonbranded networks and engaging local partners to build and maintain these networks. We share lessons learned regarding the growing role of local organizations as actors within mixed health systems and provide insights on strengthening stewardship of the increasingly heterogeneous private health care delivery sector in LMICs, particularly in fragile settings.

ABSTRACTIntroduction:Since the health information system (HIS) in public health care services in Serbia was introduced in 2009, it has gradually expanded. However, it is unclear how well the HIS components have developed and the whole system’s stage of maturity.Method:In June–September 2021, a maturity assessment of the Serbian HIS was conducted for the first time using the HIS Stages of Continuous Improvement (SOCI) toolkit. The toolkit measures HIS status across 5 HIS domains: leadership and governance, management and workforce, information and communication technology (ICT), standards and interoperability, and data quality and use. The domains were further divided into 13 components and 39 subcomponents whose maturity stage was assessed on a 5-point Likert scale, indicating the level of development: (1) emerging/ad hoc; (2) repeatable; (3) defined; (4) managed; and (5) optimized. The toolkit was applied in a working group of 32 professionals and experts who were engaged in developing the new national eHealth strategy and action plan.Results:The overall maturity score of the Serbian HIS was 1.6, which indicates a low level. The highest baseline score (2) was given to the standards and interoperability domain, and the lowest (1.1) was given to ICT infrastructure. The remaining 3 domains (leadership and governance, Management and Workforce, and Data Quality and Use) were similarly rated (1.7, 1.7, and 1.6, respectively).Conclusion:A baseline assessment of the maturity level of Serbian HIS indicates that the majority of components are between the emerging/ad hoc stage and repeatable, which represent isolated, ad hoc efforts, with some basic processes in place and existing and accessible policies. This exercise provided an opportunity to address identified weaknesses in the upcoming national eHealth strategy.

ABSTRACTIntroduction:Emergency contraceptive pills (ECPs) are effective and can be used safely at any age repeatedly within the same cycle. They are often favored by youth yet are underutilized. Private facilities can increase ECP access but present barriers including cost. Identifying effective public-sector ECP distribution models can help ensure equitable access. The Malawi Ministry of Health developed a strategy to improve ECP access in 2020. We documented ECP provision through select public, youth-serving channels recommended by the strategy: general and youth-specific outreach, paid and unpaid community health workers (CHWs), and youth clubs.Methods:We conducted this mixed methods study from November 2022–March 2023 in 2 rural districts (Mchinji and Phalombe) implementing the strategy. We conducted qualitative interviews with 10 national stakeholders, 46 providers, and 24 clients aged 15–24 years about ECP service delivery. Additionally, 25 providers collected quantitative tally data about clients seeking ECPs. We analyzed qualitative data using grounded theory and quantitative data descriptively.Results:Stakeholders and providers reported ECP uptake increased in geographies where the strategy was implemented, especially among youth. Providers documented 3,988 client visits for ECPs over 3 months. Of these visits, 26% were from male clients, 36% were from clients aged younger than 20 years, and 64% received ECPs for the first time. Across channels, youth club leaders and unpaid CHWs reported the most client visits per provider and served the youngest clients. However, no ECPs were dispensed during 29% of visits due to stock-outs. While many providers were supportive of youth accessing ECPs, most held unfavorable attitudes toward repeat use.Conclusion:ECP access should be expanded through provision in the studied channels, especially youth clubs and CHWs. However, to meet demand, the supply chain must be strengthened. We recommend addressing providers’ attitudes about repeat use to ensure informed method choice.

ABSTRACTIntroduction:Health risks associated with short interpregnancy intervals, coupled with women’s desires to avoid pregnancy following childbirth, underscore the need for effective postpartum family planning programs. The antenatal period provides an opportunity to intervene; however, evidence is limited on the effectiveness of interventions aimed at reaching women in the antenatal period to increase voluntary postpartum family planning in low- and middle-income countries (LMICs). This systematic review aimed to identify and describe interventions in LMICs that attempted to increase postpartum contraceptive use via contacts with pregnant women in the antenatal period.Methods:Studies published from January 2012 to July 2022 were considered if they were conducted in LMICs, evaluated an intervention delivered during the antenatal period, were designed to affect postpartum contraceptive use, were experimental or quasi-experimental, and were published in French or English. The main outcome of interest was postpartum contraceptive use within 1 year after birth, defined as the use of any method of contraception at the time of data collection. We searched EMBASE, Global Health, and Medline and manually searched the reference lists from studies included in the full-text screening.Results:We double-screened 771 records and included 34 reports on 31 unique interventions in the review. Twenty-three studies were published from 2018 on, with 21 studies conducted in sub-Saharan Africa. Approximately half of the study designs (n=16) were randomized controlled trials, and half (n=15) were quasi-experimental. Interventions were heterogeneous. Among the 24 studies that reported on the main outcome of interest, 18 reported a positive intervention effect, with intervention recipients having greater contraceptive use in the first year postpartum.Conclusion:While the studies in this systematic review were heterogeneous, the findings suggest that interventions that included a multifaceted package of initiatives appeared to be most likely to have a positive effect.

ABSTRACTIn the Democratic Republic of the Congo (DRC), male engagement, social norms, and social networks mitigate family planning behavior. We discuss the adaptation of the Social Norms Exploration Tool (SNET), which identifies relevant social norms and community members upholding these norms, to inform the development of family planning interventions in the DRC. The SNET provides activity tools and templates to guide users through the following steps: (1) plan and prepare, (2) identify reference groups, (3) explore social norms, (4) analyze results, and (5) apply findings.The SNET approach resulted in discussion of social norms, particularly around birth spacing and gender norms framing the man as the decision-maker. However, despite applying a methodology specifically designed to identify social norms, other factors limiting use of contraceptive methods were identified in the process, including lack of education, rumors, and misconceptions. Adaptations were needed to include the full range of reference groups due to narrow phrasing of primary questions, and some of the participatory methods were overly complicated. Feedback from experienced data collectors suggested that the social norms framework is not intuitive, is difficult to apply correctly, and may require that data collectors have a stronger foundation in the relevant concepts to produce valid and actionable results.Although the SNET provides language for discussing normative factors and techniques to identify reference groups and social norms, modifications to the implementation process are recommended when adapting the tool for research.

ABSTRACTMore than half of births among Indigenous women in Guatemala are still being attended at home by providers with no formal training. We describe the incorporation of comadronas (traditional midwives) into casas maternas (birthing centers) in the rural highlands of western Guatemala. Although there was initial resistance to the casa, comadronas and clients have become increasingly enthusiastic about them. The casas provide the opportunity for comadronas to continue the cultural traditions of prayers, massages, and other practices that honor the vital spiritual dimension of childbirth close to home in a home-like environment with extended family support while at the same time providing a safer childbirth experience in which complications can be detected by trained personnel at the casa, managed locally, or promptly referred to a higher-level facility. Given the growing acceptance of this innovation in an environment in which geographical, financial, and cultural barriers to deliveries at higher-level facilities lead most women to deliver at home, casas maternas represent a feasible option for reducing the high level of maternal mortality in Guatemala.This article provides an update on the growing utilization of casas and provides new insights into the role of comadronas as birthing team members and enthusiastic promotors of casas maternas as a preferable alternative to home births. Through the end of 2023, these casas maternas had cared for 4,322 women giving birth. No maternal deaths occurred at a casa, but 4 died after referral.The Ministry of Health of Guatemala has recently adopted this approach and has begun to implement it in other rural areas where home births still predominate. This approach deserves consideration as a viable and feasible option for reducing maternal mortality throughout the world where home births are still common, while at the same time providing women with respectful and culturally appropriate care.

ABSTRACTTraining health workers is one of the biggest challenges and cost drivers when introducing a new contraceptive method or service delivery innovation. PATH developed a digital training curriculum for family planning providers who are learning to offer subcutaneous DMPA (DMPA-SC), including through self-injection, as an option among a range of contraceptive methods. The DMPA-SC eLearning course for health workers includes 10 lessons with an emphasis on informed choice counseling and training clients to self-inject. In partnership with Ministries of Health in Senegal and Uganda, the course was rolled out in select areas in 2019–2020, including during the COVID-19 pandemic when physical distancing requirements restricted in-person training. We conducted evaluations in both countries to assess the practical application of this digital training approach for contraceptive introduction. The evaluation consisted of a post-training survey, an observational assessment conducted during post-training supportive supervision, and an estimation of training costs.In both countries, a majority (88.6% in Uganda and 64.3% in Senegal) scored above 80% on a DMPA-SC knowledge test following the training. In Senegal, where there was a comparison group of providers trained in person, those providers scored similar on the post-test to eLearners. Providers in both groups and in both countries felt more prepared to administer DMPA-SC or offer self-injection to clients after receiving a supervision visit (93%–98% of eLearners felt very prepared after supervision as compared to 45%–72% prior). The evaluation results suggest that digital approaches offer a number of benefits, can be cost-effective, and are most optimal when blended with in-person training and/or supportive supervision.

ABSTRACTIntroduction: Nigeria has the highest number of children who have not received any vaccines in Africa. The training-of-trainers (TOT) model used to train program managers (PMs) and health care workers (HCWs) is ineffective for adult learning and limits immunization programs’ success. We incorporated adult learning principles (ALPs) in designing and delivering TOT for immunization PMs and HCWs to use data to engage communities for tailored immunization strategies.Methods: Our study was implemented in 3 local government areas (LGAs) of the Federal Capital Territory, Nigeria. A training curriculum was developed, integrating ALPs and technical and operational content based on best practices in delivering immunization training and the training needs assessment findings. State PMs (n=10), LGA PMs (n=30), and HCWs (n=42) were trained on the human-centered design for tailoring immunization programs (HCD-TIP) approaches using ALPs. We used interviews and surveys with purposively and conveniently sampled PMs and HCWs, respectively, and observations to assess participants’ satisfaction, knowledge and competence, behavior changes, and results. The interviews were analyzed thematically, and surveys were statistically.Results: There was a high level of satisfaction with the training among LGA PMs (100%), state PMs (91%), and HCWs (85%), with significant knowledge and competence improvements post-training (P<.001). The trained participants conducted 2 HCD sessions with 24 undervaccinated communities and co-designed 24 prototype solutions for testing. Results showed increased coverage of the pentavalent vaccine first dose (54%) and third dose (188%) across 12 participating communities. Improved community colaboration, communication skills, and data-driven approaches were the most cited behavior changes in practice.Conclusion: The application of ALPs in training, use of HCD-TIP approaches and tools, and supportive supervision enhanced PMs’ and HCWs’ capacity for tailored interventions. Countries should consider adopting a holistic approach that focuses on using these approaches in immunization programs to strengthen the health system for equitable vaccine coverage.

ABSTRACTIntroduction:Postpartum hemorrhage (PPH) remains the leading cause of maternal mortality. A new clinical intervention (E-MOTIVE) holds the potential to improve early PPH detection and management. We aimed to develop and pilot implementation strategies to support uptake of this intervention in Kenya, Nigeria, South Africa, and Tanzania.Methods:Implementation strategy development: We triangulated findings from qualitative interviews, surveys and a qualitative evidence synthesis to identify current PPH care practices and influences on future intervention implementation. We mapped influences using implementation science frameworks to identify candidate implementation strategies before presenting these at stakeholder consultation and design workshops to discuss feasibility, acceptability, and local adaptations. Piloting: The intervention and implementation strategies were piloted in 12 health facilities (3 per country) over 3 months. Interviews (n=58), case report forms (n=1,269), and direct observations (18 vaginal births, 7 PPHs) were used to assess feasibility, acceptability, and fidelity.Results:Implementation strategy development: Key influences included shortages of drugs, supplies, and staff, limited in-service training, and perceived benefits of the intervention (e.g., more accurate PPH detection and reduced PPH mortality). Proposed implementation strategies included a PPH trolley, on-site simulation-based training, champions, and audit and feedback. Country-specific adaptations included merging the E-MOTIVE intervention with national maternal health trainings, adapting local PPH protocols, and PPH trollies depending on staff needs. Piloting: Intervention and implementation strategy fidelity differed within and across countries. Calibrated drapes resulted in earlier and more accurate PPH detection but were not consistently used at the start. Implementation strategies were feasible to deliver; however, some instances of limited use were observed (e.g., PPH trolley and skills practice after training).Conclusion:Systematic intervention development, piloting, and process evaluation helped identify initial challenges related to intervention fidelity, which were addressed ahead of a larger-scale effectiveness evaluation. This has helped maximize the internal validity of the trial.

ABSTRACTPolitics is one of the critical factors that influence health policy agendas. However, scholarly efforts, especially in low- and middle-income countries, rarely focus on how politics influence health policy agenda-setting. We conducted a qualitative document review to examine the factors that led to developing the free primary health care policy for maternal health in Papua New Guinea. We also discuss mechanisms through which national politics, as an overriding factor, influenced the development of the policy. The review draws on Kingdon’s multiple-stream model for agenda-setting and incorporates theoretical insights from Fox and Reich’s framework for analyzing the politics of health reform for universal health coverage in low- and middle-income countries.

Mitochondrial dysfunction is a hallmark of many genetic neurodegenerative diseases, but therapeutic options to reverse mitochondrial dysfunction are limited. While recent studies support the possibility of improving mitochondrial fusion/fission dynamics and motility to correct mitochondrial dysfunction and resulting neurodegeneration in Charcot-Marie-Tooth disease (CMT) and other neuropathies, the clinical utility of reported compounds and relevance of preclinical models are uncertain. Here, we describe motor and sensory neuron dysfunction characteristic of clinical CMT type 2 A in a CRISPR/Casp-engineered Mfn2 Thr105Met (T105M) mutant knock-in mouse. We further demonstrate that daily oral treatment with a novel mitofusin activator derived from the natural product piperine can reverse these neurologic phenotypes. Piperine derivative 8015 promoted mitochondrial fusion and motility in Mfn2-deficient cells in a mitofusin-dependent manner and reversed mitochondrial dysfunction in cultured fibroblasts and reprogrammed motor neurons from a human CMT2A patient carrying the MFN2 T105M mutation. Like previous mitofusin activators, 8015 exhibited stereospecific functionality, but the more active stereoisomer, 8015-P2, is unique in that it has subnanomolar potency and undergoes entero-hepatic recirculation which extends its in vivo half-life. Daily administration of 8015-P2 to Mfn2 T105M knock-in mice for 6 weeks normalized neuromuscular and sensory dysfunction and corrected histological/ultrastructural neurodegeneration and neurogenic myoatrophy. These studies describe a more clinically relevant mouse model of CMT2A and an improved mitofusin activator derived from piperine. We posit that 8015-P2 and other piperine derivatives may benefit CMT2A or other neurodegenerative conditions wherein mitochondrial dysdynamism plays a contributory role.

Glioblastoma (GBM) is a disease of the whole brain, with infiltrative tumor cells protected by an intact blood-brain barrier (BBB). GBM has a poor prognosis despite aggressive treatment, in part due to the lack of adequate drug permeability at the BBB. Standard of care GBM therapies include radiation and cytotoxic chemotherapy that lead to DNA damage. Subsequent activation of DNA damage response (DDR) pathways can induce resistance. Various DDR inhibitors, targeting the key regulators of these pathways such as ataxia telangiectasia mutated and Rad3-related (ATR), are being explored as radio- and chemosensitizers. Elimusertib, a novel ATR kinase inhibitor, can prevent repair of damaged DNA, increasing efficacy of DNA-damaging cytotoxic therapies. Robust synergy was observed in vitro when elimusertib was combined with the DNA-damaging agent temozolomide; however, we did not observe improvement with this combination in in vivo efficacy studies in GBM orthotopic tumor-bearing mice. This in vitro–in vivo disconnect was explored to understand factors influencing central nervous system (CNS) distribution of elimusertib and reasons for lack of efficacy. We observed that elimusertib is rapidly cleared from systemic circulation in mice and would not maintain adequate exposure in the CNS for efficacious combination therapy with temozolomide. CNS distribution of elimusertib is partially limited by P-glycoprotein efflux at the BBB, and high binding to CNS tissues leads to low levels of pharmacologically active (unbound) drug in the brain. Acknowledging the potential for interspecies differences in pharmacokinetics, these data suggest that clinical translation of elimusertib in combination with temozolomide for treatment of GBM may be limited.

The development of transforming growth factor βreceptor inhibitors (TGFβRi) as new medicines has been affected by cardiac valvulopathy and arteriopathy toxicity findings in nonclinical toxicology studies. PF-06952229 (MDV6058) selected using rational drug design is a potent and selective TGFβRI inhibitor with a relatively clean off-target selectivity profile and good pharmacokinetic properties across species. PF-06952229 inhibited clinically translatable phospho-SMAD2 biomarker (≥60%) in human and cynomolgus monkey peripheral blood mononuclear cells, as well as in mouse and rat splenocytes. Using an optimized, intermittent dosing schedule (7-day on/7-day off/cycle; 5 cycles), PF-06952229 demonstrated efficacy in a 63-day syngeneic MC38 colon carcinoma mouse model. In the pivotal repeat-dose toxicity studies (rat and cynomolgus monkey), PF-06952229 on an intermittent dosing schedule (5-day on/5-day off cycle; 5 cycles, 28 doses) showed no cardiac-related adverse findings. However, new toxicity findings related to PF-06952229 included reversible hepatocellular (hepatocyte necrosis with corresponding clinically monitorable transaminase increases) and lung (hemorrhage with mixed cell inflammation) findings at ≥ targeted projected clinical efficacious exposures. Furthermore, partially reversible cartilage hypertrophy (trachea and femur in rat; femur in monkey) and partially to fully reversible, clinically monitorable decreases in serum phosphorus and urinary phosphate at ≥ projected clinically efficacious exposures were observed. Given the integral role of TGFβ in endochondral bone formation, cartilage findings in toxicity studies have been observed with other TGFβRi classes of compounds. The favorable cumulative profile of PF-06952229 in biochemical, pharmacodynamic, pharmacokinetic, and nonclinical studies allowed for its evaluation in cancer patients using the intermittent dosing schedule (7-day on/7-day off) and careful protocol-defined monitoring.

Despite a significant decrease in the number of prescriptions for opioids, the opioid crisis continues, fueled in large part by the availability of the phenylpiperidine mu opioid receptor (MOR) agonist fentanyl. In contrast, the number of prescriptions for and the off-label use of gabapentinoids (gabapentin and pregabalin) has increased dramatically, with gabapentinoids commonly detected in opioid overdose victims. Although gabapentinoids can decrease the potency of the opioid receptor antagonist naloxone to reverse heroin-induced hypoventilation in male rats, the specificity and nature of interaction between gabapentinoids and MOR agonists and any potential sex difference in those interactions are not well characterized. Gabapentinoids were studied in female and male rats discriminating fentanyl (0.0032 mg/kg, i.p.) or cocaine (3.2 mg/kg, i.p.). Alone, neither gabapentin nor pregabalin significantly increased fentanyl- or cocaine-appropriate responding. In rats discriminating fentanyl, each gabapentinoid dose-dependently shifted the fentanyl and heroin discrimination dose-effect functions to the left, whereas naloxone dose-dependently shifted the fentanyl and heroin discrimination dose-effect functions to the right. Each gabapentinoid (100 mg/kg) significantly decreased the potency of naloxone to antagonize the discriminative stimulus effect of fentanyl or heroin. In contrast, each gabapentinoid dose-dependently shifted the cocaine and d-methamphetamine discrimination dose-effect functions to the right. There were no significant sex differences in this study. These results suggest that gabapentinoids impact the misuse of opioids, the co-use of opioids and stimulant drugs, and the increasing number of overdose deaths in individuals using opioids, stimulant drugs, and gabapentinoids in mixtures.

Previous studies demonstrated that sigma receptor (R) antagonists alone fail to alter cocaine self-administration despite blocking various other effects of cocaine. However, R antagonists when combined with dopamine transporter (DAT) inhibitors substantially decrease cocaine self-administration. To better understand the effects of this combination, the present study examined the effects of R antagonist and DAT inhibitor combinations in male rats discriminating cocaine (10 mg/kg, i.p.) from saline injections. The DAT inhibitors alone [(–)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate monohydrate (WIN 35,428) and methylphenidate] at low (0.1-mg/kg) doses that were minimally active failed to shift the dose-effect function for discriminative-stimulus effects of cocaine to the left more than 2-fold. At 0.32 mg/kg the DAT inhibitors alone shifted the cocaine dose-effect function leftward 24- or 6.6-fold, respectively. The R antagonists (BD1008, BD1047, and BD1063) failed to fully substitute for cocaine, although BD1008 and BD1047 substituted partially. At 10 mg/kg, BD1008, BD1047, or BD1063 alone shifted the cocaine dose-effect function leftward less than 6.0-fold. In combination with 0.1 mg/kg WIN 35,428, the 10 mg/kg doses of R antagonists shifted the cocaine dose-effect function from 12.3- to 36.7-fold leftward, and with 0.32 mg/kg WIN 35,428 from 14.3- to 440-fold leftward. In combination with 0.1 mg/kg methylphenidate, those R antagonist doses shifted the cocaine dose-effect function from 5.5- to 55.0-fold leftward, and with 0.32 mg/kg methylphenidate from 10.5- to 48.1-fold leftward. The present results suggest that dual DAT/R inhibition produces agonist-like subjective effects that may promote decreases in self-administration obtained in previous studies.

Clinical reports suggest that the most effective strategies for managing opioid use disorder comprise a comprehensive treatment program of both pharmacological and nonpharmacological approaches. However, the conditions under which these combinations are most effective are not well characterized. This study examined whether the presence of an alternative reinforcer could alter the efficacy of Food and Drug Administration–approved opioid antagonist or agonist medications, as well as the nonopioid flumazenil, in decreasing oxycodone choice self-administration in nonhuman primates. Adult squirrel monkeys (n = 7; four females) responded under concurrent second-order fixed-ratio (FR)-3(FR5:S);TO45s schedules of reinforcement for intravenous oxycodone (0.1 mg/kg) or saline on one lever and 30% sweetened condensed milk or water on the other. Doses of naltrexone (0.00032–1.0 mg/kg), nalbuphine (0.32–10 mg/kg), buprenorphine (0.0032–0.032 mg/kg), methadone (0.32–1.0 mg/kg), or flumazenil (1–3.2 mg/kg) were administered intramuscularly prior to oxycodone self-administration sessions that occurred with either milk or water as the alternative. Naltrexone, a μ-opioid receptor antagonist, was >30-fold more potent when milk was available compared with water and abolished oxycodone intake (injections/session) while concomitantly increasing milk deliveries at the highest dose tested. Pretreatment with the low-efficacy μ-agonist nalbuphine was most effective in the presence of milk compared with water, decreasing oxycodone preference to <50% of control values. The higher efficacy μ-agonists, methadone and buprenorphine, and the benzodiazepine antagonist flumazenil did not appreciably alter the reinforcing potency of oxycodone under either condition. These results suggest that antagonist medications used in combination with alternative reinforcers may be an effective strategy to curtail opioid abuse–related behaviors.

Although acute inflammation serves essential functions in maintaining tissue homeostasis, chronic inflammation is causally linked to many diseases. Macrophages are a major cell type that orchestrates inflammatory processes. During inflammation, macrophages undergo polarization and activation, thereby mobilizing pro-inflammatory and anti-inflammatory transcriptional programs that regulate ensuing macrophage functions. Fatty acid binding protein 5 (FABP5) is a lipid chaperone highly expressed in macrophages. FABP5 deletion is implicated in driving macrophages toward an anti-inflammatory phenotype, yet signaling pathways regulated by macrophage-FABP5 have not been systematically profiled. We leveraged proteomic and phosphoproteomic approaches to characterize pathways modulated by FABP5 in M1 and M2 polarized bone marrow-derived macrophages (BMDMs). Stable isotope labeling by amino acids-based analysis of M1 and M2 polarized wild-type and FABP5 knockout BMDMs revealed numerous differentially regulated proteins and phosphoproteins. FABP5 deletion impacted downstream pathways associated with inflammation, cytokine production, oxidative stress, and kinase activity. Toll-like receptor 2 (TLR2) emerged as a novel target of FABP5 and pharmacological FABP5 inhibition blunted TLR2-mediated activation of downstream pathways, ascribing a novel role for FABP5 in TLR2 signaling. This study represents a comprehensive characterization of the impact of FABP5 deletion on the proteomic and phosphoproteomic landscape of M1 and M2 polarized BMDMs. Loss of FABP5 altered pathways implicated in inflammatory responses, macrophage function, and TLR2 signaling. This work provides a foundation for future studies seeking to investigate the therapeutic potential of FABP5 inhibition in pathophysiological states resulting from dysregulated inflammatory signaling.

Cannabinoid and opioid receptor activities can be modulated by a variety of post-translational mechanisms including the formation of interacting complexes. This study examines the involvement of endogenous and exogenous chaperones in modulating the abundance and activity of cannabinoid CB₁ receptor (CB₁R), opioid receptor (DOR), and CB₁R-DOR interacting complexes. Focusing on endogenous protein chaperones, namely receptor transporter proteins (RTPs), we examined relative mRNA expression in the mouse spinal cord and found RTP4 to be expressed at higher levels compared with other RTPs. Next, we assessed the effect of RTP4 on receptor abundance by manipulating RTP4 expression in cell lines. Overexpression of RTP4 causes an increase and knock-down causes a decrease in the levels of CB₁R, DOR, and CB₁R-DOR interacting complexes; this is accompanied by parallel changes in signaling. The ability of small molecule lipophilic ligands to function as exogenous chaperones was examined using receptor-selective antagonists. Long-term treatment leads to increases in receptor abundance and activity with no changes in mRNA supporting a role as pharmacological chaperones. Finally, the effect of cannabidiol (CBD), a small molecule ligand and a major active component of cannabis, on receptor abundance and activity in mice was examined. We find that CBD administration leads to increases in receptor abundance and activity in mouse spinal cord. Together, these results highlight a role for chaperones (proteins and small molecules) in modulating levels and activity of CB₁R, DOR, and their interacting complexes potentially through mechanisms including receptor maturation and trafficking.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of chemotherapy treatment, routinely manifesting as increased pain sensitivity (allodynia) in distal extremities. Despite its prevalence, effective treatment options are limited. Cannabinoids are increasingly being evaluated for their ability to treat chronic pain conditions, including CIPN. While previous studies have revealed sex differences in cannabinoid-mediated antinociception in acute and chronic pain models, there is a paucity of studies addressing potential sex differences in the response of CIPN to cannabinoid treatment. Therefore, we evaluated the long-term antiallodynic efficacy of cannabinoid receptor type 1 (CB₁)-selective, cannabinoid receptor type 2 (CB₂)-selective, and CB₁/CB₂ mixed agonists in the cisplatin CIPN model, using both male and female mice. CB₁ selective agonism was observed to have sex differences in the development of tolerance to antiallodynic effects, with females developing tolerance more rapidly than males, while the antiallodynic effects of selective CB₂ agonism lacked tolerance development. Compound-specific changes to the female estrous cycle and female plasma estradiol levels were noted, with CB₁ selective agonism decreasing plasma estradiol while CB₂ selective agonism increased plasma estradiol. Chronic administration of a mixed CB₁/CB₂ agonist resulted in increased mRNA expression of proinflammatory cytokines and endocannabinoid regulatory enzymes in female spinal cord tissue. Ovarian tissue was noted to have proinflammatory cytokine mRNA expression following administration of a CB₂ acting compound while selective CB₁ agonism resulted in decreased proinflammatory cytokines and endocannabinoid regulatory enzymes in testes. These results support the need for further investigation into the role of sex and sex hormones signaling in pain and cannabinoid-mediated antinociceptive effects.

Oxidative stress, fibrosis, and inflammasome activation from advanced glycation end product (AGE)–receptor of advanced glycation end product (RAGE) interaction contribute to diabetic cardiomyopathy (DCM) formation and progression. Our study revealed the impact of β-caryophyllene (BCP) on activating cannabinoid type 2 receptors (CB2Rs) against diabetic complication, mainly cardiomyopathy and investigated the underlying cell signaling pathways in mice. The murine model of DCM was developed by feeding a high-fat diet with streptozotocin injections. After the development of diabetes, the animals received a 12-week oral BCP treatment at a dose of 50 mg/kg/body weight. BCP treatment showed significant improvement in glucose tolerance and insulin resistance and enhanced serum insulin levels in diabetic animals. BCP treatment effectively reversed the heart remodeling and restored the phosphorylated troponin I and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a expression. Ultrastructural examination showed reduced myocardial cell injury in DCM mice treated with BCP. The preserved myocytes were found to be associated with reduced expression of AGE/RAGE in DCM mice hearts. BCP treatment mitigated oxidative stress by inhibiting expression of NADPH oxidase 4 and activating phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor erythroid 2–related factor 2 (Nrf2) signaling. Also, BCP suppressed cardiac fibrosis and endothelial-to-mesenchymal transition in DCM mice by inhibiting transforming growth factor β (TGF-β)/suppressor of mothers against decapentaplegic (Smad) signaling. Further, BCP treatment suppressed nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome activation in DCM mice and alleviated cellular injury to the pancreatic tissues evidenced by significant elevation of the number of insulin-positive cells. To demonstrate a CB2R-dependent mechanism of BCP, another group of DCM mice were pretreated with AM630, a CB2R antagonist. AM630 was observed to abrogate the beneficial effects of BCP in DCM mice. Taken together, BCP demonstrated the potential to protect the myocardium and pancreas of DCM mice mediating CB2R-dependent mechanisms.

Neuropathic pain is a form of chronic pain that develops because of damage to the nervous system. Treatment of neuropathic pain is often incompletely effective, and most available therapeutics have only moderate efficacy and present side effects that limit their use. Opioids are commonly prescribed for the management of neuropathic pain despite equivocal results in clinical studies and significant abuse potential. Thus, neuropathic pain represents an area of critical unmet medical need, and novel classes of therapeutics with improved efficacy and safety profiles are urgently needed. The cannabidiol structural analog and novel antagonist of GPR55, KLS-13019, was screened in rat models of neuropathic pain. Tactile sensitivity associated with chemotherapy exposure was induced in rats with once-daily 1-mg/kg paclitaxel injections for 4 days or 5 mg/kg oxaliplatin every third day for 1 week. Rats were then administered KLS-13019 or comparator drugs on day 7 in an acute dosing paradigm or days 7–10 in a chronic dosing paradigm, and mechanical or cold allodynia was assessed. Allodynia was reversed in a dose-dependent manner in the rats treated with KLS-13019, with the highest dose reverting the response to prepaclitaxel injection baseline levels with both intraperitoneal and oral administration after acute dosing. In the chronic dosing paradigm, four consecutive doses of KLS-13019 completely reversed allodynia for the duration of the phenotype in control animals. Additionally, coadministration of KLS-13019 with paclitaxel prevented the allodynic phenotype from developing. Together, these data suggest that KLS-13019 represents a potential new drug for the treatment of neuropathic pain.

Patients with arthritis report using cannabis for pain management, and the major cannabinoid delta-9-tetrahydrocannabinol (⁹-THC) has anti-inflammatory properties, yet the effects of minor cannabinoids on arthritis are largely unknown. The goal of the present study was to determine the antiarthritic potential of the minor cannabinoid delta-8-tetrahydrocannabinol (⁸-THC) using the collagen-induced arthritis (CIA) mouse model. Adult male DBA/1J mice were immunized and boosted 21 days later with an emulsion of collagen and complete Freund’s adjuvant. Beginning on the day of the booster, mice were administered twice-daily injections of ⁸-THC (3 or 30 mg/kg), the steroid dexamethasone (2 mg/kg), or vehicle for two weeks. Dorsal-ventral paw thickness and qualitative measures of arthritis were recorded daily, and latency to fall from an inverted grid was measured on alternating days, to determine arthritis severity and functional impairment. On the final day of testing, spontaneous wire-climbing behavior and temperature preference in a thermal gradient ring were measured to assess CIA-depressed behavior. The ⁸-THC treatment (30 mg/kg) reduced paw swelling and qualitative signs of arthritis. ⁸-THC also blocked CIA-depressed climbing and CIA-induced preference for a heated floor without producing locomotor effects but did not affect latency to fall from a wire grid. In alignment with the morphologic and behavioral assessments in vivo, histology revealed that ⁸-THC reduced synovial inflammation, proteoglycan loss and cartilage and bone erosion in the foot joints in a dose-dependent manner. Together, these findings suggest that ⁸-THC not only blocked morphologic changes but also prevented functional loss caused by collagen-induced arthritis.

⁹-Tetrahydrocannabinol (THC) is a psychoactive phytocannabinoid found in the Cannabis sativa plant. THC is primarily metabolized into 11-hydroxy-⁹-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-⁹-tetrahydrocannabinol (COOH-THC), which may themselves be psychoactive. There is very little research-based evidence concerning the pharmacokinetics and pharmacodynamics of 11-OH-THC as an individual compound. Male C57BL/6 mice were treated with THC or 11-OH-THC via intraperitoneal injection, tail vein intravenous injection, or oral gavage, and whole-blood compound levels were measured to determine pharmacokinetic parameters [C_max, time to C_max (T_max), elimination half-life, area under the curve, apparent volume of distribution, systemic clearance, terminal rate constant, and absolute bioavailability] while also monitoring changes in catalepsy, body temperature, and nociception. 11-OH-THC achieved a T_max at 30 minutes for all routes of administration. The maximum concentration at 30 minutes was not different between intravenous and intraperitoneal routes, but the oral gavage C_max was significantly lower. THC had a 10-minute time to the maximum concentration, which was the first blood collection time point, for intravenous and intraperitoneal and 60 minutes for oral gavage, with a lower C_max for intraperitoneal and oral gavage compared with intravenous. When accounting for circulating compound levels and ED₅₀ responses, these data suggest that 11-OH-THC was 153% as active as THC in the tail-flick test of nociception and 78% as active as THC for catalepsy. Therefore, 11-OH-THC displayed equal or greater activity than the parent compound THC, even when accounting for pharmacokinetic differences. Thus, the THC metabolite 11-OH-THC likely plays a critical role in the bioactivity of cannabis; understanding its activity when administered directly will aid in the interpretation of future animal and human studies.

People with sickle cell disease (SCD) often experience chronic pain as well as unpredictable episodes of acute pain, which significantly affects their quality of life and life expectancy. Current treatment strategies for SCD-associated pain primarily rely on opioid analgesics, which have limited efficacy and cause serious adverse effects. Cannabis has emerged as a potential alternative, yet its efficacy remains uncertain. In this study, we investigated the antinociceptive effects of ⁹-tetrahydrocannabinol (THC), cannabis’ intoxicating constituent, in male HbSS mice, which express >99% human sickle hemoglobin, and male HbAA mice, which express normal human hemoglobin A, as a control. Acute THC administration (0.1–3 mg/kg^–1, i.p.) dose-dependently reduced mechanical and cold hypersensitivity in human sickle hemoglobin (HbSS) but not human normal hemoglobin A (HbAA) mice. In the tail-flick assay, THC (1 and 3 mg/kg^–1, i.p.) produced substantial antinociceptive effects in HbSS mice. By contrast, THC (1 mg/kg^–1, i.p.) did not alter anxiety-like behavior (elevated plus maze) or long-term memory (24-hour novel object recognition). Subchronic THC treatment (1 and 3 mg/kg^–1, i.p.) provided sustained relief of mechanical hypersensitivity but led to tolerance in cold hypersensitivity in HbSS mice. Together, the findings identify THC as a possible therapeutic option for the management of chronic pain in SCD. Further research is warranted to elucidate its mechanism of action and possible interaction with other cannabis constituents.

The National Center for Complementary and Integrative Health (NCCIH), which is part of the US National Institutes of Health (NIH), has a broad interest in studying the biologic activities of natural products, especially those for which compelling evidence from preclinical research suggests biologic activities that may be beneficial to health or have a potential role in disease treatment, as well as products used extensively by the American public. As of 2023, use of cannabis for medical purposes is legal in 38 states and Washington, D.C. Such use continues to climb generally without sufficient knowledge regarding risks and benefits. In keeping with NCCIH’s natural product research priorities and recognizing this gap in knowledge, NCCIH formally launched a research program in 2019 to expand research on the possible benefits for pain management of certain substances found in cannabis: minor cannabinoids and terpenes. This Viewpoint provides additional details and the rationale for this research priority at NCCIH. In addition, NCCIH’s efforts and initiatives to facilitate and coordinate an NIH research agenda focused on cannabis and cannabinoid research are described.

Cannabis and its products have been used for centuries for both medicinal and recreational purposes. The recent widespread legalization of cannabis has vastly expanded its use in the United States across all demographics except for adolescents. Meanwhile, decades of research have advanced our knowledge of cannabis pharmacology and particularly of the endocannabinoid system with which the components of cannabis interact. This research has revealed multiple targets and approaches for manipulating the system for therapeutic use and to ameliorate cannabis toxicity or cannabis use disorder. Research has also led to new questions that underscore the potential risks of its widespread use, particularly the enduring consequences of exposure during critical windows of brain development or for consumption of large daily doses of cannabis with high content ⁹-tetrahydrocannabinol. This article highlights current neuroscience research on cannabis that has shed light on therapeutic opportunities and potential adverse consequences of misuse and points to gaps in knowledge that can guide future research.

Low-efficacy mu opioid receptor (MOR) agonists may serve as novel candidate analgesics with improved safety relative to high-efficacy opioids. This study used a recently validated assay of pain-depressed behavior in mice to evaluate a novel series of MOR-selective C9-substituted phenylmorphan opioids with graded MOR efficacies. Intraperitoneal injection of dilute lactic acid (IP acid) served as a noxious stimulus to depress locomotor activity by mice in an activity chamber composed of two compartments connected by an obstructed door. Behavioral measures included (1) crosses between compartments (vertical activity over the obstruction) and (2) movement counts quantified as photobeam breaks summed across compartments (horizontal activity). Each drug was tested alone and as a pretreatment to IP acid. A charcoal-meal test and whole-body-plethysmography assessment of breathing in 5% CO₂ were also used to assess gastrointestinal (GI) inhibition and respiratory depression, respectively. IP acid produced a concentration-dependent depression in crosses and movement that was optimally alleviated by intermediate- to low-efficacy phenylmorphans with sufficient efficacy to produce analgesia with minimal locomotor disruption. Follow-up studies with two low-efficacy phenylmorphans (JL-2-39 and DC-1-76.1) indicated that both drugs produced naltrexone-reversible antinociception with a rapid onset and a duration of ~1 h. Potency of both drugs increased when behavior was depressed by a lower IP-acid concentration, and neither drug alleviated behavioral depression by a non-pain stimulus (IP lithium chloride). Both drugs produced weaker GI inhibition and respiratory depression than fentanyl and attenuated fentanyl-induced GI inhibition and respiratory depression. Results support further consideration of selective, low-efficacy MOR agonists as candidate analgesics.

In nuclear medicine, estimating the number of radioactive decays that occur in a source organ per unit administered activity of a radiopharmaceutical (i.e., the time-integrated activity coefficient [TIAC]) is an essential task within the internal dosimetry workflow. TIAC estimation is commonly derived by least-squares fitting of various exponential models to organ time–activity data (radiopharmaceutical biodistribution). Rarely, however, are methods used to objectively determine the model that best characterizes the data. Additionally, the uncertainty associated with the resultant TIAC is generally not evaluated. As part of the MIRDsoft initiative, MIRDfit has been developed to offer a biodistribution fitting software solution that provides the following essential features and advantages for internal dose assessment: nuclear medicine–appropriate fit functions; objective metrics for guiding best-fit selection; TIAC uncertainty calculation; quality control and data archiving; integration with MIRDcalc software for dose calculation; and a user-friendly Excel-based interface. For demonstration and comparative validation of MIRDfit’s performance, TIACs were derived from serial imaging studies involving ¹⁸F-FDG and ¹⁷⁷Lu-DOTATATE using MIRDfit. These TIACs were then compared with TIAC estimates obtained using other software. In most cases, the TIACs agreed within approximately 10% between MIRDfit and the other software. MIRDfit has been endorsed by the MIRD Committee of the Society of Nuclear Medicine and Molecular Imaging and has been integrated into the MIRDsoft suite of free dosimetry software; it is available for download at no user cost (https://mirdsoft.org/).

The aim of this study was to validate a previously developed deep learning model in 5 independent clinical trials. The predictive performance of this model was compared with the international prognostic index (IPI) and 2 models incorporating radiomic PET/CT features (clinical PET and PET models). Methods: In total, 1,132 diffuse large B-cell lymphoma patients were included: 296 for training and 836 for external validation. The primary outcome was 2-y time to progression. The deep learning model was trained on maximum-intensity projections from PET/CT scans. The clinical PET model included metabolic tumor volume, maximum distance from the bulkiest lesion to another lesion, SUV_peak, age, and performance status. The PET model included metabolic tumor volume, maximum distance from the bulkiest lesion to another lesion, and SUV_peak. Model performance was assessed using the area under the curve (AUC) and Kaplan–Meier curves. Results: The IPI yielded an AUC of 0.60 on all external data. The deep learning model yielded a significantly higher AUC of 0.66 (P < 0.01). For each individual clinical trial, the model was consistently better than IPI. Radiomic model AUCs remained higher for all clinical trials. The deep learning and clinical PET models showed equivalent performance (AUC, 0.69; P > 0.05). The PET model yielded the highest AUC of all models (AUC, 0.71; P < 0.05). Conclusion: The deep learning model predicted outcome in all trials with a higher performance than IPI and better survival curve separation. This model can predict treatment outcome in diffuse large B-cell lymphoma without tumor delineation but at the cost of a lower prognostic performance than with radiomics.

The Registry of Fast Myocardial Perfusion Imaging with Next-Generation SPECT (REFINE SPECT) has been expanded to include more patients and CT attenuation correction imaging. We present the design and initial results from the updated registry. Methods: The updated REFINE SPECT is a multicenter, international registry with clinical data and image files. SPECT images were processed by quantitative software and CT images by deep learning software detecting coronary artery calcium (CAC). Patients were followed for major adverse cardiovascular events (MACEs) (death, myocardial infarction, unstable angina, late revascularization). Results: The registry included scans from 45,252 patients from 13 centers (55.9% male, 64.7 ± 11.8 y). Correlating invasive coronary angiography was available for 3,786 (8.4%) patients. CT attenuation correction imaging was available for 13,405 patients. MACEs occurred in 6,514 (14.4%) patients during a median follow-up of 3.6 y (interquartile range, 2.5–4.8 y). Patients with a stress total perfusion deficit of 5% to less than 10% (unadjusted hazard ratio [HR], 2.42; 95% CI, 2.23–2.62) and a stress total perfusion deficit of at least 10% (unadjusted HR, 3.85; 95% CI, 3.56–4.16) were more likely to experience MACEs. Patients with a deep learning CAC score of 101–400 (unadjusted HR, 3.09; 95% CI, 2.57–3.72) and a CAC of more than 400 (unadjusted HR, 5.17; 95% CI, 4.41–6.05) were at increased risk of MACEs. Conclusion: The REFINE SPECT registry contains a comprehensive set of imaging and clinical variables. It will aid in understanding the value of SPECT myocardial perfusion imaging, leverage hybrid imaging, and facilitate validation of new artificial intelligence tools for improving prediction of adverse outcomes incorporating multimodality imaging.

Recent studies have demonstrated promising results of fibroblast activation protein (FAP) inhibitor (FAPI) PET in prognosticating and monitoring interstitial lung diseases (ILDs). As a first step toward successful translation, our primary aim was to validate the FAPI PET uptake through immunohistochemistry in patients with advanced ILD who underwent lung transplantation after a FAPI PET scan. Methods: This is a preliminary analysis of a single-center, open-label, single-arm, prospective exploratory biodistribution study of ⁶⁸Ga-FAPI-46 PET imaging in patients with ILD (NCT05365802). Patients with ILD confirmed by high-resolution CT and scheduled for lung transplant were included. Tissue samples of explanted lungs were obtained from both the central and peripheral lung parenchyma of each lobe. Additional samples were obtained from areas of the lung corresponding to regions of FAPI PET activity. Immunohistochemical staining was performed with an anti-FAP antibody. Percentages of FAP immunohistochemistry-positive area were measured semiautomatically using QuPath software. SUVs in the areas of pathologic samples were measured on FAPI PET/CT by referencing the gross photomap of the explanted lung. A Spearman correlation coefficient test was used to assess the relationship between FAPI PET uptake and FAP immunohistochemical expression in each specimen. Results: Four patients with advanced ILD who underwent FAPI PET/CT before lung transplantation were included. The types of ILD were idiopathic pulmonary fibrosis (n = 2), rheumatoid arthritis–associated ILD (n = 1), and nonspecific interstitial pneumonia (n = 1). FAPI uptake was visualized mainly in the fibrotic area on CT. Twenty-nine surgical pathology samples from 3 patients were analyzed. FAP staining was predominantly positive in fibroblastic foci. FAPI PET SUV_max and SUV_mean showed a positive correlation with the immunohistochemical FAP expression score (SUV_max: r = 0.57, P = 0.001; SUV_mean: r = 0.54, P = 0.002). Conclusion: In this analysis conducted in patients who underwent lung transplantation after a FAPI PET scan, FAPI PET uptake was positively correlated with FAP immunohistochemistry. These findings provide a rationale for further investigation of FAPI PET as a potential imaging biomarker for ILD.

The diagnostic work-up of patients with fever of unknown origin (FUO) begins with a thorough history and physical examination, complete blood count with differential, chest x-ray, urinalysis and culture, electrolyte panel, liver enzymes, erythrocyte sedimentation rate, and C-reactive protein level. Additional imaging procedures, including nuclear medicine tests, are generally used as second-line procedures, with ¹⁸F-FDG PET and PET/CT assuming increasingly important roles in the diagnostic work-up. The Society of Nuclear Medicine and Molecular Imaging, the Infectious Diseases Society of America, and the American College of Nuclear Medicine convened an autonomous expert work group to comprehensively review the published literature for nuclear imaging in adults and children with FUO and establish appropriate use criteria (AUC). This process was performed in accordance with the Protecting Access to Medicare Act of 2014, which requires that all referring physicians consult AUC by using a clinical decision support mechanism before ordering advanced diagnostic imaging services. The complete findings and discussions of the work group were published on January 8, 2023, and are available at https://www.snmmi.org/ClinicalPractice/content.aspx?ItemNumber=15666. The AUC in the final document are intended to assist referring health care providers in appropriate use of nuclear medicine imaging procedures in patients with FUO. The work group noted limitations in the current literature on nuclear medicine imaging for FUO, with the need for well-designed prospective multicenter investigations. Consensus findings from published data and expert opinions were used to create recommendations in common clinical scenarios for adults and children. Included in the complete document is a discussion of inflammation of unknown origin (IUO), a recently described entity. In view of the fact that the criteria for FUO and IUO are similar (except for fever > 38.3°C [100.9°F]) and that the most common etiologies of these 2 entities are similar, it is the expert opinion of the work group that the recommendations for nuclear medicine imaging of FUO are also applicable to IUO. These recommendations are included in the full guidance document. This summary reviews rationale, methodology, and main findings and refers the reader to the complete AUC document.

Simplified methods of acquisition and quantification would facilitate the use of synaptic density imaging in multicenter and longitudinal studies of Alzheimer disease (AD). We validated a simplified tissue-to-reference ratio method using SUV ratios (SUVRs) for estimating synaptic density with [¹¹C]UCB-J PET. Methods: Participants included 31 older adults with AD and 16 with normal cognition. The distribution volume ratio (DVR) using simplified reference tissue model 2 was compared with SUVR at short scan windows using a whole-cerebellum reference region. Results: Synaptic density was reduced in AD participants using DVR or SUVR. SUVR using later scan windows (60–90 or 70–90 min) was minimally biased, with the strongest correlation with DVR. Effect sizes using SUVR at these late time windows were minimally reduced compared with effect sizes with DVR. Conclusion: A simplified tissue-to-reference method may be useful for multicenter and longitudinal studies seeking to measure synaptic density in AD.

Chronic hypertension leads to injury and fibrosis in major organs. Fibroblast activation protein (FAP) is one of key molecules in tissue fibrosis, and ⁶⁸Ga-labeled FAP inhibitor-46 (FAPI46) PET is a recently developed method for evaluating FAP. The aim of this study was to evaluate FAP expression and fibrosis in a hypertension model and to test the feasibility of ⁶⁸Ga-FAPI46 PET in hypertension. Methods: Hypertension was induced in mice by angiotensin II infusion for 4 wk. ⁶⁸Ga-FAPI46 biodistribution studies and PET scanning were conducted at 1, 2, and 4 wk after hypertension modeling, and uptake in the major organs was measured. The FAP expression and fibrosis formation of the heart and kidney tissues were analyzed and compared with ⁶⁸Ga-FAPI46 uptake. Subgroups of the hypertension model underwent angiotensin receptor blocker administration and high-dose FAPI46 blocking, for comparison. As a preliminary human study, ⁶⁸Ga-FAPI46 PET images of lung cancer patients were analyzed and compared between hypertension and control groups. Results: Uptake of ⁶⁸Ga-FAPI46 in the heart and kidneys was significantly higher in the hypertension group than in the sham group as early as week 1 and decreased after week 2. The uptake was specifically blocked in the high-dose blocking study. Immunohistochemistry also revealed FAP expression in both heart and kidney tissues. However, overt fibrosis was observed in the heart, whereas it was absent from the kidneys. The angiotensin receptor blocker–treated group showed lower uptake in the heart and kidneys than did the hypertension group. In the pilot human study, renal uptake of ⁶⁸Ga-FAPI46 significantly differed between the hypertension and control groups. Conclusion: In hypertension, FAP expression is increased in the heart and kidneys from the early phases and decreases over time. FAP expression appears to represent fibrosis activity preceding or underlying fibrotic tissue formation. ⁶⁸Ga-FAPI46 PET has potential as an effective imaging method for evaluating FAP expression in progressive fibrosis by hypertension.