A Novel C-C Chemoattractant Cytokine (Chemokine) Receptor 6 (CCR6) Antagonist (PF-07054894) Distinguishes between Homologous Chemokine Receptors, Increases Basal Circulating CCR6+ T Cells, and Ameliorates Interleukin-23-Induced Skin Inflammation.

Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a ß-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.

Increasing the Reuse of Protein Non-Naïve Nonhuman Primates in Pharmaceutical Drug Discovery and Development: An Overview and Industry Position on the Challenges and Benefits.

The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper's content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.

Immediate Weight-Bearing Protocol for the Determination of Ankle Stability in Patients With Isolated Distal Fibular Fractures.

OBJECTIVE: To evaluate an alternative protocol for allowing immediate weight-bearing (WB) as tolerated in a functional walking boot in patients with a medial clear space (MCS) of less than 4 mm on nonstressed initial radiographs with subsequent WB radiographs at 1-week follow-up to determine if this can differentiate stable from unstable distal fibular fractures. DESIGN: Retrospective case series. SETTING: Level 1 trauma center. PATIENTS: Seventy-nine patients who sustained an isolated distal fibular fracture with an MCS less than 4 mm on initial non-weight-bearing radiographs during a 6-year period. INTERVENTION: Patients with MCS less than 4 mm on 1-week radiographs were treated nonoperatively. Patients with MCS greater than or equal to 4 mm were treated operatively. MAIN OUTCOME MEASUREMENTS: Medial clear space measurements on WB ankle radiographs at the time of radiographic bony union. RESULTS: Two of the 79 (2.5%) patients had an MCS greater than 4 mm at 1-week follow-up with WB radiographs and underwent operative fixation. The remaining 77 patients were treated nonoperatively. All 77 patients had an MCS less than 4 mm on WB radiographs at the time of radiographic healing. CONCLUSION: These results suggest that our immediate weight-bearing protocol may be an effective method for determination of functional ankle stability only in the setting of an isolated distal fibula fracture with MCS less than 4 mm. However, it should be cautioned that careful evaluation of WB radiographs for joint asymmetry and/or MCS widening is mandatory to avoid poor outcomes. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Is It Safe to Prep the External Fixator In Situ During Second-Stage Pilon Surgical Treatment?

OBJECTIVE: To evaluate the infection rate of our protocol of prepping the external fixator in situ during definitive second-stage pilon fracture open reduction internal fixation. DESIGN: Retrospective clinical investigation. SETTING: Academic Level 1 Trauma Center. PATIENTS/PARTICIPANTS: Out of 229 patients with distal tibia fractures presenting to our institution from 1999 to 2014, 100 were treated in a 2-stage fashion utilizing this protocol. INTERVENTION: Prepping the external fixator into the surgical field during the second-stage/definitive open reduction internal fixation procedure. MAIN OUTCOME MEASUREMENT: The rates of deep and superficial infections after definitive fixation. RESULTS: The deep infection rate was 13%, and the superficial infection rate was 11%. CONCLUSIONS: Infection rates using this protocol are comparable to previously reported infection rates for two-stage surgical treatment of pilon fractures. This protocol provides the treating surgeon information about an alternative method to streamline definitive fixation. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Ritual drinks in the pre-Hispanic US Southwest and Mexican Northwest.

Chemical analyses of organic residues in fragments of pottery from 18 sites in the US Southwest and Mexican Northwest reveal combinations of methylxanthines (caffeine, theobromine, and theophylline) indicative of stimulant drinks, probably concocted using either cacao or holly leaves and twigs. The results cover a time period from around A.D. 750-1400, and a spatial distribution from southern Colorado to northern Chihuahua. As with populations located throughout much of North and South America, groups in the US Southwest and Mexican Northwest likely consumed stimulant drinks in communal, ritual gatherings. The results have implications for economic and social relations among North American populations.

Toxicity of hydroxyurea in rats and dogs.

The toxicity of hydroxyurea, a treatment for specific neoplasms, sickle-cell disease, polycythemia, and thrombocytosis that kills cells in mitosis, was assessed in repeat-dose, oral gavage studies in rats and dogs and a cardiovascular study in telemetered dogs. Hydroxyurea produced hematopoietic, lymphoid, cardiovascular, and gastrointestinal toxicity with steep dose response curves. In rats dosed for 10 days, 50 mg/kg/day was tolerated; 500 mg/kg/day produced decreased body weight gain; decreased circulating leukocytes, erythrocytes, and platelets; decreased cellularity of thymus, lymph nodes, and bone marrow; and epithelial degeneration and/or dysplasia of the stomach and small intestine; 1,500 mg/kg/day resulted in deaths on day 5. In dogs, a single dose at ≥ 250 mg/kg caused prostration leading to unscheduled euthanasia. Dogs administered 50 mg/kg/day for 1 month had decreased circulating leukocytes, erythrocytes, and platelets; increased bone marrow cellularity with decreased maturing granulocytes; increased creatinine kinase activity; and increased iron pigment in bone marrow and hepatic sinusoidal cells. In telemetered dogs, doses ≥ 15 mg/kg decreased systolic blood pressure (BP); 50 mg/kg increased diastolic BP, heart rate, and change in blood pressure over time (+dP/dt), and decreased QT and PR intervals and maximum left ventricular systolic and end diastolic pressures with measures returning to control levels within 24 hr.

Sequence motifs associated with hepatotoxicity of locked nucleic acid--modified antisense oligonucleotides.

Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure-toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure-toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.

Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice.

Distal tibia nonunions.

Although tibia metaphyseal nonunion is rare, its treatment is often complex. The merits of related management techniques are discussed. These techniques include: intramedullary nailing, fine wire fixation, and blade plate reconstruction, which is the method preferred by the authors.

Cherokee Choices: a diabetes prevention program for American Indians.

In 1999, the Centers for Disease Control and Prevention (CDC) provided Racial and Ethnic Approaches to Community Health 2010 (REACH 2010) funds to the Eastern Band of Cherokee Indians to develop a community-based intervention to improve the health of this rural, mountainous community in North Carolina. During the first year of the Cherokee Choices program, team members conducted formative research, formed coalitions, and developed a culturally appropriate community action plan for the prevention of type 2 diabetes, particularly among children. The Eastern Band of Cherokee Indians has higher rates of obesity and type 2 diabetes than the U.S. and North Carolina general populations. The Cherokee Choices program includes three main components: elementary school mentoring, worksite wellness for adults, and church-based health promotion. A social marketing strategy, including television advertisements and a television documentary series, supports the three components. School policy was altered to allow Cherokee Choices to have class time and after-school time devoted to health promotion activities. School staff have shown an interest in improving their health through attendance at fitness sessions. The credibility of the program has been validated through multiple invitations to participate in school events. Participants in the worksite wellness program have met dietary and physical activity goals, had reductions in body fat, and expressed enthusiasm for the program. A subcoalition has been formed to expand the worksite wellness component and link prevention efforts to health care cost reduction. Participants in the church program have walked more than 31,600 miles collectively.

Regulation of muscle growth by multiple ligands signaling through activin type II receptors.

Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn(-/-) mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo.

Functional outcome after blade plate reconstruction of distal tibia metaphyseal nonunions: a study of 11 cases.

OBJECTIVES: To present the functional outcome of a cohort of 11 patients prospectively followed before and after blade plate reconstruction of a distal tibia metaphyseal nonunion. DESIGN: Prospective case series. SETTING: University hospital tertiary referral center. PATIENTS: Eleven patients with an average age of 48 years. Average duration of nonunion was 11 months. Patients had undergone an average of 3.1 procedures before the index surgery. Three patients had prior deep infections, and one patient had an active infection. INTERVENTION: A precontoured 4.5-mm cannulated blade plate was applied to the medial tibial surface through a posteromedial approach. Autograft was added in eight patients to fill bone voids. MAIN OUTCOME MEASURES: AOFAS scores were assigned to each patient preoperatively and at most recent follow-up. RESULTS: All patients healed their nonunions after the index surgery. Average time to radiographic union was 16 weeks. Average time to full weight bearing was 12 weeks. AOFAS scores improved in all patients from an average preoperative score of 29 to an average postoperative score of 89. The only complication was a deep infection, which was treated successfully with one irrigation and débridement and 6 weeks of intravenous antibiotics. CONCLUSION: Blade plate reconstruction of distal tibia metaphyseal nonunion is a safe and reliable method that results in a high union and low complication rate.