Transcatheter Left Atrial Appendage Exclusion: Preclinical and Early Clinical Results With the Laminar Device.

BACKGROUND: The Laminar device rotates and closes the left atrial appendage (LAA) using an integrated ball and lock that excludes and eliminates the LAA pouch. There is a low device surface area, minimizing the risk of peridevice leak (PDL) and device-related thrombus (DRT) formation. OBJECTIVES: This study evaluates the safety and efficacy of the Laminar LAA exclusion device in healthy animals and human subjects with nonvalvular atrial fibrillation at risk of ischemic stroke and systemic thromboembolism. METHODS: The preclinical study implanted the Laminar device into canine subjects that underwent transesophageal echocardiography (TEE) and fluoroscopic evaluation, followed by necropsy and histological assessment at 45 and 150-days post-implant. The early clinical study implanted the device in human subjects, followed to 12 months postimplantation. Procedural success was defined as device implantation in the intended location without residual LAA leak >5 mm as seen by TEE. Safety endpoints included freedom from stroke, systemic embolism, pericardial effusion, or tamponade, life-threatening/major bleeding, or death. RESULTS: The Laminar device was successfully implanted in 10 canines. In all animals at 45 days and 150 days, no PDL or DRT was found, and histological examination showed fully closed LAAs covered with neo-endocardium. The device was successfully implanted in 15 human subjects with no safety events out to 12 months postimplantation. All subjects had successful protocol-defined LAA closure without DRT at 45 days by TEE and computed tomography, which remained stable through 12 months' follow-up. CONCLUSIONS: The preclinical and early clinical results demonstrate a promising safety and efficacy profile for the Laminar LAA exclusion device.

In vivo Hemodynamic Evaluation of an Implantable Left Ventricular Assist Device in a Long-term Anti-coagulation Regimen.

Left ventricular assist devices (LVADs) are used to treat patients with severe (New York Heart Association class IV) heart failure. Thrombosis and bleeding are severe LVAD-related complications; thus, an effective anticoagulation regimen is crucial for successful postoperative management. The CH-VAD™ (CH Biomedical, Inc.) is a small, implantable, full-support (>5 L/min) LVAD with a centrifugal flow pump that has a fully magnetically levitated rotor, which confers superior hemocompatibility. In this study, the CH-VAD™ was implanted in two calves to evaluate its hemocompatibility and to establish an anticoagulation regimen for future GLP (good laboratory practice) studies. Heparin infusion was used during the surgery, and during postoperative management, the proper dosage of warfarin was given orally to maintain an international normalized ratio (INR) between 2.0 and 3.0. Pump performance, animal condition, and hematology results were recorded throughout the study (approximately 60 days). The results show that under the established anticoagulation regimen, the CH-VAD™ was well tolerated in the bovine model, with no significant thrombus or thromboembolic lesion formation in distal end organs. Low plasma free hemoglobin levels suggest that the device did not cause hemolysis. These results and the experience gained pave the way for future GLP studies.

Investigation of the role of infusate properties related to midline catheter failure in an ovine model.

PURPOSE: Infusate osmolarity, pH, and cytotoxicity were investigated as risk factors for midline catheter failure. METHODS: An experimental, randomized, controlled, blinded trial was conducted using an ovine model. Two 10-cm, 18-gauge single-lumen midline catheters were inserted into the cephalic veins of sheep. The animals were divided into 6 study arms and were administered solutions of vancomycin 4 mg/mL (a low-cytotoxicity infusate) or 10 mg/mL (a high-cytotoxicity infusate), doxycycline 1 mg/mL (an acidic infusate), or acyclovir 3.5 mg/mL (an alkaline infusate) and 0.9% sodium chloride injection; or 1 of 2 premixed Clinimix (amino acids in dextrose; Baxter International) products with respective osmolarities of 675 mOsm/L (a low-osmolarity infusate) and 930 mOsm/L (a mid-osmolarity infusate). Contralateral legs were infused with 0.9% sodium chloride injection for control purposes. Catheter failure was evaluated by assessment of adverse clinical symptoms (swelling, pain, leakage, and occlusion). A quantitative vessel injury score (VIS) was calculated by grading 4 histopathological features: inflammation, mural thrombus, necrosis, and perivascular reaction. RESULTS: Among 20 sheep included in the study, the overall catheter failure rate was 95% for test catheters (median time to failure, 7.5 days; range, 3-14 days), while 60% of the control catheters failed before or concurrently (median time to failure, 7 days; range, 4.5-14 days). Four of the 6 study arms (all but the Clinimix 675-mOsm/L and acyclovir 3.5-mg/mL arms) demonstrated an increase in mean VIS of ≥77% in test vs control legs (P ≤ 0.034). Both pain and swelling occurred at higher rates in test vs control legs: 65% vs 10% and 70% vs 50%, respectively. The mean difference in rates of occlusive pericatheter mural thrombus between the test and control arms was statistically significant for the vancomycin 10-mg/mL (P = 0.0476), Clinimix 930-mOsm/L (P = 0.0406), and doxycycline 1-mg/mL (P = 0.032) arms. CONCLUSION: Administration of infusates of varied pH, osmolarity, and cytotoxicity via midline catheter resulted in severe vascular injury and premature catheter failure; therefore, the tested infusates should not be infused via midline catheters.

Epicardial left atrial appendage occlusion with a new medical device: assessment of procedural feasibility, safety and efficacy in a large animal model.

BACKGROUND: Left atrial appendage occlusion (LAAO) represents a treatment alternative to anticoagulation in patients with atrial fibrillation. We evaluate a novel device for epicardial LAAO in a translational canine model. METHODS: Nine hounds (n = 9) were used to assess usability, safety, and efficacy of the TigerPaw Pro (TPP) device for epicardial LAAO. Following baseline imaging (intra-cardiac echocardiography (ICE) and angiography) and intraoperative visual inspection, usability was tested via a ``closure/re-opening`` maneuver followed by deployment of a total of twenty TPP devices (n = 20) on the left and right atrial appendages respectively. Procedural safety was evaluated by assessing for adverse-events via direct Epicardial inspection and endocardial imaging. Efficacy evaluation included assessment of device positioning, presence of residual stumps and completeness of closure. Post-mortem evaluation was performed to confirm safety and efficacy. RESULTS: Usability testing of all TPP devices was successful (n = 20;100%, delivery-time range 22-120 s) without any procedural adverse-events (tissue damage or tears, bleeding, vessel-impingement, structural impact). All devices fully traversed the ostium (n = 18) or appendage body (n = 2), and conformed smoothly to adjacent cardiac anatomy. In nineteen deployments (n = 19;95%), all device connector pairs were fully engaged, while in one TPP device the most distal pair remained unengaged. ICE and post-mortem inspections revealed complete closure of all appendage ostia (n = 18;100%) and only in one case a small residual stump was detected. Intraoperative safety findings were further confirmed post-mortem. Devices created a nearly smooth line of closure via symmetric endocardial tissue-coaptation. CONCLUSIONS: In this preclinical model, the TPP demonstrated good ease of use for ostial access, ability to re-position (after engagement) and rapid deployment, while achieving safe and effective LAAO.

Pathological Safety Assessment in Preclinical Neurothrombectomy Studies.

The design, production, and preclinical testing of neurothrombectomy devices is in a burgeoning phase as the demand escalates for safe and reliable treatment options following neurovascular stroke. Currently, there is a paucity of published data describing the development of iatrogenic vascular lesions occurring secondary to neurothrombectomy procedures. In an effort to test new devices, demonstrate device safety, satisfy regulatory requirements, and develop an understanding of the potential for associated vascular pathology, investigators are establishing appropriate methodology in suitable animal models. Significant challenges exist in identifying a single animal species that can be consistently utilized in all phases of device development. These aforementioned challenges are underscored by the intricacies of neurovascular pathology, thrombovascular interactions, and vascular responses to injury.

Chlorhexidine-coated peripherally inserted central catheters reduce fibroblastic sleeve formation in an in vivo ovine model.

PURPOSE:: This study compared an antimicrobial and anti-thrombogenic peripherally inserted central catheter treated with a chlorhexidine-based technology, a peripherally inserted central catheter with bulk distributed fluoro-oligomers, and a poly 2-methoxyethyl acrylate-based peripherally inserted central catheter to an untreated peripherally inserted central catheter (control) in an ovine model at 14 and 30 days post-implant. METHODS:: One of four types of peripherally inserted central catheters was surgically implanted into the left jugular vein of each of 18 sheep for 14 or 30 days. Blood analysis consisted of complete blood counts, serum chemistries, and coagulation (fibrinogen, prothrombin time, and partial thromboplastin time) profiles. Sheep were sacrificed to examine the vein and thorax. Histological analysis was performed on serial catheter sections using standard microscopy on hematoxylin and eosin-stained tissues. RESULTS:: All catheters developed fibroblastic sleeves at both 14 and 30 days. The chlorhexidine-peripherally inserted central catheter showed a 64% lower mean fibroblastic sleeve weight and a 66% shorter mean fibroblastic sleeve length compared to the untreated control at 14 days. By 30 days, compared to untreated control, the chlorhexidine-peripherally inserted central catheter showed 81% lower mean fibroblastic sleeve weight with 75% shorter mean fibroblastic sleeve length, the fluoro-oligomer-peripherally inserted central catheter showed 54% lower mean sheath weight with 40% shorter mean fibroblastic sleeve length, and the poly 2-methoxyethyl acrylate-peripherally inserted central catheter had 41% lower mean fibroblastic sleeve weight with 57% lower fibroblastic sleeve length. CONCLUSION:: Among the three anti-thrombogenic peripherally inserted central catheter technologies, the chlorhexidine-peripherally inserted central catheter had the smallest fibroblastic sleeves, followed by the fluoro-oligomer-peripherally inserted central catheter, poly 2-methoxyethyl acrylate-peripherally inserted central catheter, and control peripherally inserted central catheter.

Efficacy and Safety of a Bovine-Associated Staphylococcus aureus Phage Cocktail in a Murine Model of Mastitis.

Overuse of antibiotics is a major problem in the treatment of bovine mastitis, and antibiotic treatment is frequently non-curative, thus alternative treatments are necessary. The primary aim of this study was to evaluate the efficacy of a purified phage cocktail for treatment of bovine Staphylococcus aureus mastitis in a well-defined mouse model. Candidate phages were selected based on their in vitro performance and subsequently processed into an optimally composed phage cocktail. The highest scoring phages were further tested for efficacy and resistance suppression in broth and raw milk, with and without supplemental IgG. As these in vitro results displayed significant decreases in CFU, the cocktail was purified for testing in vivo. Lactating mice were intramammarily inoculated with S. aureus N305 (ATCC 29740), a clinical bovine mastitis isolate commonly used for experimental infection of dairy cows. The phage cocktail was applied via the same route 4 h post-inoculation. Treated mammary glands were graded for gross pathological appearance and excised for bacterial and phage load quantification as well as histopathology. Observation of gross macroscopic and histopathological changes and CFU quantification demonstrated that the phage cocktail treatment significantly improved mastitis pathology and decreased bacterial counts. Phage PFU quantification indicated that the tested phage cocktail treatment was able to maintain high intramammary phage titers without spreading systemically. The in vivo results complement the in vitro data and support our concept of phage therapy as an innovative alternative or supplementation therapy to antibiotics for the treatment of bovine mastitis.

The miniaturized pediatric continuous-flow device: Preclinical assessment in the chronic sheep model.

BACKGROUND: The Infant Jarvik 2015 is an implantable axial-flow ventricular assist device (VAD) that has undergone the major evolutionary design modifications to improve hemocompatibility. This study was conducted in anticipation of data submission to the US Food and Drug Administration to obtain Investigational Device Exemption approval. METHODS: The VAD was implanted via a left thoracotomy in Barbado sheep (n = 10, 26 (19-34] kg). Anticoagulation was maintained with coumadin, with a target international normalized ratio of greater than the individual sheep's baseline values. The VAD was managed at the highest possible speed as clinically tolerable. Complete necropsy was performed at the end of the study. RESULTS: There were 2 early mortalities: tension pneumothorax (n = 1) and shower emboli of the fragmented myocardium (n = 1). The remaining 8 sheep (2 with 30-day and 6 with 60-day protocols) completed the anticipated study duration in excellent condition, with the 6 completing 60-day sheep showing appropriate weight gain during support. There were no signs of clinically significant hemolysis, with the final plasma-free hemoglobin of 2 (1-17) mg/dL. Necropsy showed old renal infarction in 7 sheep. Although thromboembolism can be the potential etiology, given the mild anticoagulation regimen, other sources of emboli were identified in 2 sheep (graft coating material and fragmented myocardium). Flow study demonstrated favorable increase in flow (up to 3.0 L/min) in proportion to change in pump speed. CONCLUSIONS: This study has demonstrated that the Infant Jarvik 2015 VAD is capable of maintaining its functionality for an extended period of time with minimal hemolysis.

Comparison of Topical Hemostatic Agents in a Swine Model of Extremity Arterial Hemorrhage: BloodSTOP iX Battle Matrix vs. QuikClot Combat Gauze.

BloodSTOP iX Battle Matrix (BM) and QuikClot Combat Gauze (CG) have both been used to treat traumatic bleeding. The purpose of this study was to examine the efficacy and initial safety of both products in a swine extremity arterial hemorrhage model, which mimics combat injury. Swine (37.13 ± 0.56 kg, NBM = 11, NCG = 9) were anesthetized and splenectomized. We then isolated the femoral arteries and performed a 6 mm arteriotomy. After 45 s of free bleeding, either BM or CG was applied. Fluid resuscitation was provided to maintain a mean arterial pressure of 65 mmHg. Animals were observed for three hours or until death. Fluoroscopic angiography and wound stability challenge tests were performed on survivors. Tissue samples were collected for histologic examination. Stable hemostasis was achieved in 11/11 BM and 5/9 CG subjects, with recovery of mean arterial pressure and animal survival for three hours (p < 0.05, Odds Ratio (OR) = 18.82 (0.85-415.3)). Time to stable hemostasis was shorter for the BM-treated group (4.8 ± 2.5 min vs. 58 ± 20.1 min; Median = 2, Interquartile Range (IQR) = 0 min vs. Median = 60, IQR = 120 min; p < 0.05) and experienced longer total stable hemostasis (175.2 ± 2.5 min vs. 92.4 ± 29.9 min; Median = 178, IQR = 0 min vs. Median = 120, IQR = 178 min; p < 0.05). Post-treatment blood loss was lower with BM (9.5 ± 2.4 mL/kg, Median = 10.52, IQR = 13.63 mL/kg) compared to CG (29.9 ± 9.9 mL/kg, Median = 29.38, IQR = 62.44 mL/kg) (p = 0.2875). Standard BM products weighed less compared to CG (6.9 ± 0.03 g vs. 20.2 ± 0.4 g) (p < 0.05) and absorbed less blood (3.4 ± 0.8 g vs. 41.9 ± 12.3 g) (p < 0.05). Fluoroscopic angiography showed recanalization in 5/11 (BM) and 0/5 (CG) surviving animals (p = 0.07, OR = 9.3 (0.41-208.8)). The wound stability challenge test resulted in wound re-bleeding in 1/11 (BM) and 5/5 (CG) surviving animals (p < 0.05, OR = 0.013 (0.00045-0.375)). Histologic evidence indicated no wound site, distal limb or major organ damage in either group. BM is more effective and portable in treating arterial hemorrhage compared to CG. There was no histologic evidence of further damage in either group.

Beyond the "B": a new concept of the surgical staple enabling miniature staplers.

BACKGROUND: Surgical staplers currently all rely on the same staple form-the "B" which necessitates a high delivery profile (12 mm). A novel "D" shape staple allows for an extremely low profile of the applicator. The acute and long-term efficacy of a D-shaped staple (Cardica, Redwood City, CA, USA) was compared to conventional B-form staples (Covidien, Norwalk, CN, USA) in an animal model for intestinal transections and anastomoses. METHODS: Jejunojejunal anastomoses (JJ) were performed via mini-laparotomy in a swine model. White & blue D- and B-shaped staples were studied in three groups (planned survival 14-84 days). Intraoperative assessment included completeness of staple line, hemostasis, and need for intervention. Postoperatively, animals were evaluated for complications. At the time of sacrifice, gross pathological and histological assessments were performed. RESULTS: Twenty-three animals had 40 anastomoses (23 "D" and 17 "B" staple anastomoses) with no intraoperative mortalities. One "D" staple application required a manual extension of the cut. Acute hemostasis was 100%. Group 1 (n = 5) compared white staples in JJs (D staple n = 5; B staple n = 5; 14-day survival = 100%). Group 2 (n = 12) compared white staples in JJs (D staple n = 12; B staple n = 6; 34-day survival = 92 %). One animal died on day 4 for a non-staple related cause. Group 3 (n = 6) compared blue staples in JJs (D staple n = 6; B staple n = 6; 84 day survival = 84%). One animal died on day 18 due to an obstruction at the B staple JJ caused by stricture. There were no other bleeding, leaks or strictures in any of the groups. Gross pathology and histology were unremarkable in all JJs. CONCLUSIONS: This study showed no difference in intraoperative performance and the chronic healing response in JJs between D- and B-shaped staples. Based on these findings, the D-shaped staple elicits a normal healing response in jejunostomies and offers the possibility of clinical use of this advance in staple design.

Mynx vascular closure device achieves reliable closure and hemostasis of percutaneous transfemoral venous access in a porcine vascular model.

AIMS: Vascular closure device (VCD)-based venous closure has been anecdotally reported, but systematic evaluation of the reparative response of the vessel wall to venous closure is lacking. The need to control groin complications, and minimize risks associated with postponed sheath removal under conditions of persistent anticoagulation, has generated interest in the role of VCDs for venous access closure. We sought to characterize the vessel wall response to venous closure, both acutely and in delayed fashion at 30 days using angiography, ultrasound, and histology. METHODS: Ten venous 7 Fr sheaths were deployed in the femoral veins of swine. Bilateral venous access sites were subsequently closed utilizing manual compression (MC; control arm: n = 4) or a closure device utilizing an extravascular polyethylene glycol sealant (MynxGrip treatment arm: n = 6). Acute (post closure), 3-day, and 30-day vascular ultrasound, as well as venography (internal jugular approach) were used to assess outcomes. Gross pathology and histology were obtained at the 30-day endpoint for all femoral venous closure sites. RESULTS: Hemostasis was successfully achieved in all cases without access-site complications. Venography and ultrasound confirmed normal ilio-femoral anatomy and flow at all study time points. Gross pathology and histopathology revealed no evidence of deep vein thrombosis, and no abnormalities were seen in the vena cava, heart, or lungs. Histology at 30 days showed complete healing of the vein wall access site, with a small focus of chronic inflammation and fibrosis in the perivascular adventitial tissue of the access tract. There was no microscopic evidence of the sealant. The tissue tract showed mild discrete inflammation (foamy macrophages, lymphocytes, plasma cells) with microgranulomas centered on residual red cells in both treatment and control groups. CONCLUSIONS: This study characterizes the angiographic, ultrasound, and histopathology outcomes of femoral vein closure, and provides insight into the healing mechanisms following venotomy. The bio-resorptive role of MynxGrip extravascular sealant in achieving effective venous closure and preserved long-term vessel patency without venous thromboembolism is demonstrated.

Discovery of novel selective norepinephrine inhibitors: 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides (WYE-114152).

Sequential modification of the previously identified 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols led to the identification of a new series of 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides that are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound 10b (WYE-114152) had low nanomolar hNET potency (IC(50) = 15 nM) and good selectivity for hNET over hSERT (>430-fold) and hDAT (>548-fold). 10b was additionally bioavailable following oral dosing and demonstrated efficacy in rat models of acute, inflammatory, and neuropathic pain.

Discovery of novel selective norepinephrine reuptake inhibitors: 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols (WYE-103231).

Structural modification of a virtual screening hit led to the identification of a new series of 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols which are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound S-17b (WYE-103231) had low nanomolar hNET potency (IC(50) = 1.2 nM) and excellent selectivity for hNET over hSERT (>1600-fold) and hDAT (>600-fold). S-17b additionally had a good pharmacokinetic profile and demonstrated oral efficacy in rat models of ovariectomized-induced thermoregulatory dysfunction and morphine dependent flush as well as the hot plate and spinal nerve ligation (SNL) models of acute and neuropathic pain.

Pharmacological characterization of the muscarinic agonist (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983) in in vitro and in vivo models of chronic pain.

Here, we have investigated the in vitro pharmacology of a muscarinic agonist, (3R,4R)-3-(3-hexylsulfanyl-pyrazin-2-yloxy)-1-aza-bicyclo[2.2.1]heptane (WAY-132983), and we demonstrated its activity in several models of pain. WAY-132983 had a similar affinity for the five muscarinic receptors (9.4-29.0 nM); however, in calcium mobilization studies it demonstrated moderate selectivity for M(1) (IC(50) = 6.6 nM; E(max) = 65% of 10 muM carbachol-stimulation) over the M(3) (IC(50) = 23 nM; E(max) = 41%) and M(5) receptors (IC(50) = 300 nM; E(max) = 18%). WAY-132983 also activated the M(4) receptor, fully inhibiting forskolin-induced increase in cAMP levels (IC(50) = 10.5 nM); at the M(2) receptor its potency was reduced by 5-fold (IC(50) = 49.8 nM). In vivo, WAY-132983 demonstrated good systemic bioavailability and high brain penetration (>20-fold over plasma levels). In addition, WAY-1329823 produced potent and efficacious antihyperalgesic and antiallodynic effects in rodent models of chemical irritant, chronic inflammatory, neuropathic, and incisional pain. It is noteworthy that efficacy in these models was observed at doses that did not produce analgesia or ataxia. Furthermore, a series of antagonist studies demonstrated that the in vivo activity of WAY-132983 is mediated through activation of muscarinic receptors primarily through the M(4) receptor. The data presented herein suggest that muscarinic agonists, such as WAY-132983, may have a broad therapeutic efficacy for the treatment of pain.

Adverse effect of complete thoracic evacuation during chronic pneumothorax in a dog.

OBJECTIVE: To describe an unusual response to evacuation of a chronic pneumothorax in a dog. CASE: Resolution of a pneumothorax is usually associated with lung expansion and a marked improvement in pulmonary function. In the case presented here, evacuation of a chronic pneumothorax improved oxygenation, but markedly impaired ventilation. CLINICAL RELEVANCE: This is a previously unreported complication associated with the treatment of pneumothorax in dogs. It suggests that in some cases of chronic pneumothorax, total evacuation of the pleural air pocket may improve oxygenation but ventilation deteriorates and increases PCO2, causing further complications.

Studies toward the discovery of the next generation of antidepressants. 3. Dual 5-HT1A and serotonin transporter affinity within a class of N-aryloxyethylindolylalkylamines.

N-aryloxylethylindolealkylamines (5) having dual 5-HT transporter and 5-HT(1A) affinity are described. These compounds represent truncated analogues of our previously reported piperidinyl derivatives (3). Compounds in this investigation were found to have more similar affinities and functional activities for the 5-HT(1A) receptor and 5-HT transporter. Though 5-HT(1A) antagonism is not consistently observed throughout series 5, several molecular features were found to be essential to obtain high and balanced activities. The proper placement of a heteroatom in the aryl ring and the length of the linkage used to tether the indole moiety had significant influence on 5-HT(1A) and 5-HT transporter affinities. Introduction of a halogen into the aryl ring usually lowered intrinsic activity and in some cases led to full 5-HT(1A) antagonists. Compounds 33 and 34 were observed to be full 5-HT(1A) antagonists with K(i) values of approximately 30 nM for the 5-HT(1A) receptor and K(i) values of 5 and 0.5 nM for the 5-HT transporter, respectively. Unfortunately, similar to our previous series (3), compounds in this report also had high affinity for the alpha(1) receptor.

Characterization of the 5-HT6 receptor coupled to Ca2+ signaling using an enabling chimeric G-protein.

We examined the feasibility of coupling the 5-HT(6) receptor to a Ca(2+) signaling read-out using a chimeric G-protein, comprising of G(alphaq) with the C-terminal five amino acids from G(alphas), to facilitate assays on the fluorometric imaging plate reader (FLIPR). Using a transient transfection assay in human embryonic kidney (HEK) cells, Ca(2+) signaling in response to serotonin (5-HT) was facilitated by co-transfection of the 5-HT(6) receptor with the G(alphaq)/G(alphas) chimera, but not with the 5-HT(6) receptor alone or with a similar chimera incorporating the C-terminal five amino acids of G(alphai3). A series of agonist concentration-response curves were constructed using the 5-HT(6)-G(alphaq)/G(alphas) signaling assay generating the following rank order of agonist potency; 5-methoxytryptamine (EC(50), 9 nM)=5-HT (12 nM)=2-methyl 5-HT (13 nM)>tryptamine (86 nM)=5-carboxamidotryptamine (5-CT) (119 nM)>>lisuride (>1 microM). In comparison, essentially identical EC(50) values were observed for the stimulation of cAMP accumulation with the same compounds; 5-methoxytryptamine (EC(50), 6 nM)=5-HT (6 nM)=2-methyl 5-HT (15 nM)>tryptamine (91 nM)=5-CT (153 nM)>lisuride (>350 nM). Clozapine and SB 271046 both produced a concentration-dependent antagonism of the 5-HT-stimulated Ca(2+) response with IC(50) values of 45 and 11 nM, respectively. In contrast, aripiprazole, a recently launched atypical anti-psychotic with a novel mechanism of action described as a dopamine/serotonin stabilizer, was essentially devoid of 5-HT(6) receptor antagonist activity. Our results demonstrate that a FLIPR-based Ca(2+) signaling assay is a feasible approach to the functional characterization of 5-HT(6) receptor ligands. Moreover, the equivalent coupling efficiency, as indexed by agonist potency, observed using this system compared with the native coupling assay to cAMP suggests that the C-terminal five amino acids of G(alphas) are the major determinant for the receptor/G-protein interaction of the 5-HT(6) receptor subtype.

The role of transmembrane helix 5 in agonist binding to the human H3 receptor.

We have used alanine scanning mutagenesis to identify residues in transmembrane domain 5 of the histamine H3 receptor that are important for agonist binding. All of the mutants generated were functionally expressed as demonstrated by their ability to bind [(125)I]iodoproxyfan with comparable affinity to the wild-type receptor and their ability to inhibit forskolin-stimulated cAMP formation when activated by histamine. Many mutations produced small changes in the potency of histamine, but the most pronounced reduction in potency and affinity of the agonists, histamine, R-alpha-methylhistamine, imetit, and impentamine, was seen with mutation of glutamate 206. Our modeling suggests that this residue plays a key role in ligand binding by interacting with the imidazole ring of histamine. Interestingly, L199A greatly reduced agonist potency in functional assays but had only minor effects on agonist affinity, implicating a role for this residue in the mechanism of receptor activation. We also studied the functional effects of the mutations by linking the receptor to calcium signaling using a chimeric G protein. A comparison of the two functional assays demonstrated contrasting effects on agonist activity. Histamine, imetit, and impentamine were full agonists in the cAMP assay, but imetit exhibited only partial agonist activity through the chimeric G protein. Furthermore, impentamine, another potent agonist in the cAMP assay, was only able to activate the E206A mutant in the calcium assay despite being inactive at the wild-type receptor. These observations suggest that the agonist receptor complexes formed by these three different H3 agonists are not conformationally equivalent.