Inhibition of Acetyl-CoA Carboxylase Causes Malformations in Rats and Rabbits: Comparison of Mammalian Findings and Alternative Assays.

Acetyl-CoA carboxylase (ACC) is an enzyme within the de novo lipogenesis (DNL) pathway and plays a role in regulating lipid metabolism. Pharmacologic ACC inhibition has been an area of interest for multiple potential indications including oncology, acne vulgaris, metabolic diseases such as type 2 diabetes mellitus, and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. A critical role for ACC in de novo synthesis of long-chain fatty acids during fetal development has been demonstrated in studies in mice lacking Acc1, where the absence of Acc1 results in early embryonic lethality. Following positive predictions of developmental toxicity in the alternative in vitro assays (positive in murine embryonic stem cell [mESC] assay and rat whole embryo culture, but negative in zebrafish), developmental toxicity (growth retardation and dysmorphogenesis associated with disrupted midline fusion) was observed with the oral administration of the dual ACC1 and 2 inhibitors, PF-05175157, in Sprague Dawley rats and New Zealand White rabbits. The results of these studies are presented here to make comparisons across the assays, as well as mechanistic insights from the mESC assay demonstrating high ACC expression in the mESC and that ACC-induced developmental toxicity can be rescued with palmitic acid providing supportive evidence for DNL pathway inhibition as the underlying mechanism. Ultimately, while the battery of alternative approaches and weight-of-evidence case were useful for hazard identification, the embryo-fetal development studies were necessary to inform the risk assessment on the adverse fetal response, as malformations and/or embryo-fetal lethality were limited to doses that caused near-complete inhibition of DNL.

Airborne particulate concentration during laser hair removal: A comparison between cold sapphire with aqueous gel and cryogen skin cooling.

BACKGROUND: High concentrations of sub-micron nanoparticles have been shown to be released during laser hair removal (LHR) procedures. These emissions pose a potential biohazard to healthcare workers that have prolonged exposure to LHR plume. OBJECTIVE: We sought to demonstrate that cold sapphire skin cooling done in contact mode might suppress plume dispersion during LHR. METHODS: A total of 11 patients were recruited for laser hair removal. They were treated on the legs and axilla with a 755 or 1064 nm millisecond-domain laser equipped with either (i) cryogen spray (CSC); (ii) refrigerated air (RA); or (iii) contact cooling with sapphire (CC). Concentration of ultrafine nanoparticles <1 µm were measured just before and during LHR with the three respective cooling methods. RESULTS: For contact cooling (CC), counts remained at baseline levels, below 3,500 parts per cubic centimeter (ppc) for all treatments. In contrast, the CSC system produced large levels of plume, peaking at times to over 400,000 ppc. The CA cooled system produced intermediate levels of plume, about 35,000 ppc (or about 10× baseline). CONCLUSIONS: Cold Sapphire Skin cooling with gel suppresses plume during laser hair removal, potentially eliminating the need for smoke evacuators, custom ventilation systems, and respirators during LHR. Lasers Surg. Med. 50:280-283, 2018. © 2017 Wiley Periodicals, Inc.

Use of Rat Primary Mesenteric Cells for the Prediction of PDE4 Inhibitor Drug-Induced Vascular Injury.

Drug-induced vascular injury (DIVI) in preclinical studies can delay, if not terminate, a drug development program. Clinical detection of DIVI can be very difficult as there are no definitive biomarkers known to reliably detect this disorder in all instances. The preclinical identification of DIVI requires detailed microscopic examination of a wide range of tissues although one of the most commonly affected areas in rats is the mesenteric vasculature. The reason for this predisposition of mesenteric arteries in rats as well as the exact mechanism and cell types involved in the initial development of these lesions have not been fully elucidated. We hypothesized that by using a mixed culture of cells from rat mesenteric tissue, we would be able to identify an RNA expression signature that could predict the invivo development of DIVI. Five compounds designed to inhibit Phosphodiesterase 4 activity (PDE4i) were chosen as positive controls. PDE4i's are well known to induce DIVI in the mesenteric vasculature of rats and there is microscopic evidence that this is associated, at least in part, with a proinflammatory mechanism. We surveyed, by qRT-PCR, the expression of 96 genes known to be involved in inflammation and using a Random-Forest model, identified 12 genes predictive of invivo DIVI outcomes in rats. Using these genes, we were able to cross-validate the ability of the Random-Forest modeling to predict the concentration at which PDE4i caused DIVI invivo.

Cervical & thoracic manipulations: Acute effects upon pain pressure threshold and self-reported pain in experimentally induced shoulder pain.

BACKGROUND: Emerging evidence suggests that cervical and thoracic joint manipulations may be advocated in treating patients with shoulder pain. OBJECTIVES: To determine the acute effects of cervical, cervicothoracic, and thoracic joint manipulations on outcomes of self-reported pain and pain pressure threshold in experimentally induced shoulder pain. DESIGN: Repeated measures. METHODS: Twenty (20) healthy volunteers were tested on two sessions. Session 1 consisted on baseline assessment of pain pressure threshold testing over the infraspinatus bilaterally and self-reported shoulder pain using the shoulder pain and disability index (SPADI) pain scale. An isokinetic exercise protocol was used to induce delayed onset muscle soreness. In session 2 (24-48 h later), all variables were reassessed before and immediately after a combination of cervical, cervicothoracic and thoracic manipulations. RESULTS: SPADI pain scale scores were significantly different between time points (p < 0.001): the exercise protocol significantly increased reported pain [mean increase 14.1, p < 0.001] while the manipulation significantly decreased reported pain (mean decrease 5.60, p < 0.001)) although pain remained higher than baseline levels. Pain pressure threshold differences were also found between time points (p = 0.001): manipulation significantly increased pain threshold bilaterally (p < 0.001) similar to baseline levels. CONCLUSIONS: Cervical, cervicothoracic, and thoracic joint manipulations acutely increased pain pressure threshold and decreased self-reported shoulder pain in participants with experimentally induced shoulder pain. Physiotherapists may consider the combination of such techniques to achieve short-term hypoalgesic effects and facilitate the application of more active interventions.

Primary cell cultures for understanding rat epididymal inflammation.

Treatment-induced epididymal inflammation and granuloma formation is only an occasional problem in preclinical drug development, but it can effectively terminate the development of that candidate molecule. Screening for backup molecules without that toxicity must be performed in animals (generally rats) that requires at least 2 to 3 weeks of in vivo exposure, a great deal of specially synthesized candidate compound, and histologic examination of the target tissues. We instead hypothesized that these treatments induced proinflammatory gene expression, and so used mixed-cell cultures from the rat epididymal tubule to monitor the induction of proinflammatory cytokines. Cells were exposed for 24 hr and then cytotoxicity was evaluated with the MTS assay and mRNA levels of Interleukin-6 (IL-6) and growth-related oncogene (GRO) were measured. We found that compounds that were more toxic in vivo stimulated a greater induction of IL-6 and GRO mRNA levels in vitro. By relating effective concentrations in vitro with the predicted C(eff), we could rank compounds by their propensity to induce inflammation in rats in vivo. This method allowed the identification of several compounds with very low inflammatory induction in vitro. When tested in rats, the compounds produced small degrees of inflammation at an acceptable margin (approximately 20×), and have progressed into further development.

Embryo-fetal transfer of bevacizumab (Avastin) in the rat over the course of gestation and the impact of neonatal Fc receptor (FcRn) binding.

BACKGROUND: There is concern about embryo-fetal exposure to antibody-based biopharmaceuticals based on the increase of such therapies being prescribed to women of childbearing potential. Therefore, there is a desire to better characterize embryo-fetal exposure of these molecules. The pregnant rat is a standard model for evaluating the potential consequences of exposure but placental transfer of antibody-based biopharmaceuticals is not well understood in this model. METHODS: The relative embryo-fetal distribution of an antibody-based biopharmaceutical was evaluated in the rat. Bevacizumab (Avastin) was chosen as a tool antibody since it does not have significant target binding in the rat that might influence embryo-fetal biodistribution. Avastin was labeled with a fluorescent dye, characterized, and injected into pregnant rats at different gestation ages. Labeled Avastin in fetal tissues was visualized ex vivo using an IVIS 200 (Caliper, A PerkinElmer Company, Alameda, CA). RESULTS: Avastin localized to the fetus as early as 24-hr post intravenous injection of the dam, and was taken up by the fetus in a dose-dependent manner. Avastin was detectable in the developing embryo as early as gestation day 13 and continued to be transferred until the end of gestation. Fetal transfer of Avastins mutated in the portion of the antibody that binds the neonatal Fc receptor (FcRn) was tested in late gestation and was found to correlate with affinities of the mutant Avastin antibody to FcRn. CONCLUSIONS: The novel application of this imaging technology was used to characterize the onset and duration of Avastin maternal-fetal transfer in rats and the importance of FcRn binding.

Sensitive windows of skeletal development in rabbits determined by hydroxyurea exposure at different times throughout gestation.

The critical periods of axial skeletal development in rats and mice have been well characterized, however the timing of skeletal development in rabbits is not as well known. It is important to have a more precise understanding of this timing of axial skeletal development in rabbits due to the common use of this species in standard nonclinical studies to assess embryo-fetal developmental toxicity. Hydroxyurea, a teratogen known to induce a variety of fetal skeletal malformations, was administered to New Zealand White rabbits as a single dose (500 mg/kg) on individual days during gestation (gestation day, GD 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 19) and fetal external, visceral, and skeletal morphology was examined following cesarean sections on GD 29. A wide range of fetal skeletal effects was observed following hydroxyurea treatment, with a progression of malformations from anterior to posterior structures over time, as well as from proximal to distal structures over time. The sensitive window of axial skeletal development was determined to be GD 8 to 13, while disruption of appendicular and cranio-facial skeletal development occurred primarily from GD 11 to 16 and GD 11 to 12, respectively. The results of this study provide a better understanding of the critical developmental window for different segments of the rabbit skeleton, which will aid in the design of window studies to investigate teratogenicity in rabbits.

Full-face treatments with the 2790-nm erbium:YSGG laser system.

BACKGROUND: Traditional full-face resurfacing has been limited to erbium-doped yttrium aluminium garnet (Er:YAG) and carbon dioxide (CO2) lasers. These devices offer wavelength-specific advantages and disadvantages. METHODS: Nine patients were enrolled in a pilot study of a resurfacing system using a 2790-nm erbium:yttrium-scandium-gallium-garnet (Er:YSGG) laser system. Two treatments were carried out 1 month apart over the entire face. Test spots were performed prior to the full-face sessions to determine the optimal fluence for 1-pass laser resurfacing. Biopsies were performed at the time of treatment and at the final follow-up visit one month after the second treatment. Clinical endpoints included changes in pigment dyschromias, wrinkles, and skin tone. All outcomes were graded by blinded observers. RESULTS: Eight patients completed the 2 treatments. Biopsies showed thermal damage extending as deep as 80 microm below the stratum corneum. Reepithelialization was complete within 4 days. No scarring, post inflammatory hyperpigmentation (PIH), or infections were observed. CONCLUSION: A 2790-nm laser can be used for skin rejuvenation with a 4 day recovery window.

Ultrastructural effects of an infrared handpiece on forehead and abdominal skin.

BACKGROUND: Collagen fibril contraction has been shown to be associated with tissue tightening by nonablative skin rejuvenation. Transmission electron microscopy has proven to be an effective method for characterizing collagen contraction delivered by ablative and nonablative devices used on human skin. OBJECTIVE: The purpose of this two-part study was to evaluate ultrastructural changes in cadaveric forehead skin and live abdominal skin by transmission electron microscopy for different fluence levels using the Titan infrared handpiece (Cutera, Inc., Brisbane, CA). This device is a noncoherent selectively filtered infrared device operating in the 1,100- to 1,800-nm bandwidth, intended to provide dermal heating. METHODS AND MATERIALS: Cadaveric forehead skin at 37 degrees C was treated with a 1x1.5-cm spot at fluences of 50 and 100 J/cm2. Informed consent was obtained and abdominal skin of one patient (before abdominoplasty) was treated in vivo with a 1x1.5-cm spot at fluences of 30, 45, and 65 J/cm2. Punch biopsies of the treatment areas and a control area were obtained immediately after treatment. Transmission electron microscopy at depths of 0 to 1 and 1 to 2 mm was performed for each biopsy to evaluate morphologic alterations of collagen fibrils in treated areas compared to the control area. RESULTS: In the cadaveric forehead skin samples, the collagen fibril alteration was greatest in the depth range of 1 to 2 mm for both fluence settings. In the abdominal skin samples, collagen fibril alteration was not seen in the control site but was observed at all treatment levels at both the 0 to 1 and the 1 to 2-mm depths, with the least alteration seen at the shallow depth and the lowest fluence. CONCLUSIONS: Our findings suggest that collagen fibril denaturation, consistent with fibril thermocontraction, occurs immediately after infrared tissue tightening. Collagen denaturation occurs at a depth range appropriate for deep dermal treatments. The peak in collagen fibril alteration at 1 to 2 mm is consistent with contact cooling protecting the more superficial layers of the skin.