John C. Anthes

Biochemical characterization of desloratadine, a potent antagonist of the human histamine H1 receptor

We have characterized desloratadine (5H-benzo[5,6]cyclohepta[1,2-b]pyridine, 8-chloro-6,11-dihydro-11-(4-piperidinylidene), CAS 100643-71-8) as a potent antagonist of the human histamine H(1) receptor. [3H]Desloratadine bound to membranes expressing the recombinant human histamine H(1) receptor in Chinese hamster ovary cells (CHO-H(1)) in a specific and saturable manner with a K(d) of 1.1+/-0.2 nM, a B(max) of 7.9+/-2.0 pmol/mg protein, and an association rate constant of 0.011 nM(-1) x min(-1). The K(d) calculated from the kinetic measurements was 1.5 nM. Dissociation of [3H]desloratadine from the human histamine H(1) receptor was slow, with only 37% of the binding reversed at 6 h in the presence of 5 microM unlabeled desloratadine. Seventeen histamine H(1)-receptor antagonists were evaluated in competition-binding studies. Desloratadine had a K(i) of 0.9+/-0.1 nM in these competition studies. In CHO-H(1) cells, histamine stimulation resulted in a concentration-dependent increase in [Ca(2+)](i) with an EC(50) of 170+/-30 nM. After a 90-min preincubation with desloratadine, the histamine-stimulated increase in [Ca(2+)](i) was shifted to the right, with a depression of the maximal response at higher concentrations of antagonist. The apparent K(b) value was 0.2+/-0.14 nM with a slope of 1.6+/-0.1. The slow dissociation from the receptor and noncompetitive antagonism suggests that desloratadine may be a pseudoirreversible antagonist of the human histamine H(1) receptor. The mechanism of desloratadine antagonism of the human histamine H(1) receptor may help to explain the high potency and 24-h duration of action observed in clinical studies.

Cloning and pharmacological characterization of mouse TRPV1.

The Transient Receptor Potential cation channel V1 (TRPV1) is expressed in peripheral nociceptive neurons and is subject to polymodal activation via various agents including capsaicin, noxious heat, low extracellular pH, and direct phosphorylation by protein kinase C (PKC). We have cloned and heterologously expressed mouse TRPV1 (mTRPV1) and characterized its function utilizing FLIPR-based calcium imaging to measure functional responses to various small molecule agonists, low pH and direct phosphorylation via PKC. The various TRPV1 agonists activated mTRPV1 with a rank order of agonist potency of (resiniferatoxin (RTX) = arvanil > capsaicin = olvanil > OLDA > PPAHV) (EC50 values of 0.15+/-0.04 nM, 0.27+/-0.07 nM, 9.1+/-1.2 nM, 3.7+/-0.3 nM, 258+/-105 nM, and 667+/-151 nM, respectively). Additionally, mTRPV1 was activated by either low pH or with addition of the PKC activator phorbol 12-myristate 13-acetate (PMA). The TRPV1 antagonists iodinated-resiniferatoxin (I-RTX) or BCTC were both able to block capsaicin, pH and PKC-induced responses of mTRPV1 (IC50 (I-RTX) = 0.35+/-0.12 nM, 1.9+/-0.7 nM, and 0.80+/-0.68 nM, IC50 (BCTC) = 1.3+/-0.36 nM, 0.59+/-0.16 nM, and 0.37+/-0.15 nM, respectively). However, the antagonist capsazepine was only able to inhibit a capsaicin-evoked response of mTRPV1 with an IC50 of 1426+/-316 nM. Comparable results were achieved with rat TRPV1, while capsazepine blocked all modes of human TRPV1 activation. Thus, the mTRPV1 cation channel has a molecular pharmacological profile more akin to rat TRPV1 than either human or guinea pig TRPV1 and the molecular pharmacology suggests that capsazepine may be an ineffective TRPV1 antagonist for in vivo models of inflammatory pain in the mouse.

The Toxicity of SCH 351591, a Novel Phosphodiesterase-4 Inhibitor, in Cynomolgus Monkeys

SCH351591, a novel phosphodiesterase-4 inhibitor under investigation as a potential therapeutic for asthma and chronic obstructive pulmonary disease (COPD), was evaluated in a 3-month rising-dose study in Cynomolgus monkeys. Four groups, containing four monkeys/sex, received vehicle control or rising doses up to 12, 24, or 48 mg/kg of SCH351591 daily. Although initial exposure produced clinical signs of emesis, reduced food intake, and reduced body weight, tachyphylaxis to the emesis allowed dose escalation up to 48 mg/kg/day. Two monkeys died and 3 were sacrificed in moribund condition over the course of the study. Early mortality, involving monkeys dosed with 12 or 24 mg/kg, was attributed to sepsis (2 monkeys) or colon inflammation (3 monkeys). Leukocyte function assays on low- and mid-dose group survivors revealed an inhibition of T lymphocyte proliferation for 12 mg/kg group males and 24 mg/kg group monkeys of both sexes. Necropsy findings, unassociated with early mortality, included reduced size and weight of the thymus, depletion of body fat, red discoloration of the gastric mucosa, and perivascular hemorrhage of the stomach and heart. Stomach and heart gross findings were present in the high-dose group only. Histopathologic lesions, in addition to those attributed to concurrent bacterial infection, included thymic atrophy, serous atrophy of fat, myocardial degeneration and acute to chronic inflammation of small to medium-sized arteries in various organs and tissues including the heart, kidneys, stomach, salivary glands, pancreas, esophagus, gallbladder, and mesentery. The findings of this study demonstrate the potential of a PDE4 inhibitor to alter immunologic response as well as to produce arteriopathy in nonhuman primates.

Phospholipase D in homogenates from HL-60 granulocytes: Implications of calcium and G protein control

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263, 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[32P]phosphatidylcholine (alkyl-[32P]PC), 1-0-[3H]alkyl-2-oleoyl-phosphatidylcholine [( 3H]alkyl-PC) and [14C]arachidonyl-phospholipids as substrates. In the presence of Ca2+ and GTP gamma S, post-nuclear homogenates degrade alkyl-[32P]PC to produce 1-0-alkyl-[32P]phosphatidic acid (alkyl-[32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). By comparing the 3H/32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn-1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca2+ and GTP for activation.

Granulocyte phospholipase D is activated by a guanine nucleotide dependent protein factor

When post-nuclear homogenates from HL-60 granulocytes are incubated in the presence of CaCl2, GTP gamma S and ethanol, phospholipase D (PLD) metabolizes both exogenous 2-[14C]arachidonyl-phosphatidylcholine and endogenous phosphatidyl[3H]choline to produce 2-[14C]arachidonyl-phosphatidic acid, 2-[14C]arachidonyl-phosphatidylethanol and [3H]choline. Fractionation of the homogenate by ultracentrifugation into cytosolic and membrane fractions results in the loss of PLD activity. However, when these two fractions are combined in the same proportion as found in the unfractionated homogenate, PLD activity is completely restored. This activity is proportional to the concentration of both the cytosol and the particulate fractions. Release of [14C]arachidonate by PLA2 that occurs under these assay conditions does not require the combined presence of cytosol and membrane fractions. We conclude that, in granulocyte homogenates, PLD activity but not arachidonate release, exhibits an essential requirement for a heat-labile factor whose activity depends on the presence of GTP gamma S.

TRPV1 Antagonists as Potential Antitussive Agents

Cough is an important defensive pulmonary reflex that removes irritants, fluids, or foreign materials from the airways. However, when cough is exceptionally intense or when it is chronic and/or nonproductive it may require pharmacologic suppression. For many patients, antitussive therapies consist of OTC products with inconsequential efficacies. On the other hand, the prescription antitussive market is dominated by older opioid drugs such as codeine. Unfortunately, “codeine-like” drugs suppress cough at equivalent doses that also often produce significant ancillary liabilities such as GI constipation, sedation, and respiratory depression. Thus, the discovery of a novel and effective antitussive drug with an improved side effect profile relative to codeine would fulfill an unmet clinical need in the treatment of cough. Afferent pulmonary nerves are endowed with a multitude of potential receptor targets, including TRPV1, that could act to attenuate cough. The evidence linking TRPV1 to cough is convincing. TRPV1 receptors are found on sensory respiratory nerves that are important in the generation of the cough reflex. Isolated pulmonary vagal afferent nerves are responsive to TRPV1 stimulation. In vivo, TRPV1 agonists such as capsaicin elicit cough when aerosolized and delivered to the lungs. Pertinent to the debate on the potential use of TRPV1 antagonist as antitussive agents are the observations that airway afferent nerves become hypersensitive in diseased and inflamed lungs. For example, the sensitivity of capsaicin-induced cough responses following upper respiratory tract infection and in airway inflammatory diseases such as asthma and COPD is increased relative to that of control responses. Indeed, we have demonstrated that TRPV1 antagonism can attenuate antigen-induced cough in the allergic guinea pig. However, it remains to be determined if the emerging pharmacologic profile of TRPV1 antagonists will translate into a novel human antitussive drug. Current efforts in clinical validation of TRPV1 antagonists revolve around various pain indications; therefore, clinical evaluation of TRPV1 antagonists as antitussive agents will have to await those outcomes.

Phospholipase C and phospholipase D are activated independently of each other in chemotactic peptide-stimulated human neutrophils.

When cytochalasin B‐treated neutrophils were stimulated with fMet‐Leu‐Phe (fMLP) in the presence of Ca2+, phospholipase C (PLC) activity, as measured by inositol‐1,4,5‐trisphosphate (IP3) formation, preceded phospholipase D (PLD)‐catalyzed breakdown of choline‐ containing phosphoglycerides to form choline and dirady 1‐sn ‐glycero‐3‐phosphate (phosphatidic acid), suggesting a possible link between PLC and PLD. However, in the absence of cytochalasin B or extracellular Ca2+, PLC was fully activated by fMLP with minimal activation of PLD, indicating that PLC activation alone is not sufficient for PLD activation. Full activation of PLD by fMLP required the simultaneous presence of both Ca2+ and cytochalasin B, a condition that caused no further enhancement of PLC. This result suggests that PLD products are not involved in the regulation of PLC activation. Furthermore, under conditions of complete inhibition of PLC by phorbol 12‐myristate 13‐acetate (PMA), there was no inhibition of PLD, showing that fMLP can activate PLD in the absence of PLC. Treatment of intact neutrophils with pertussis toxin inhibited both PLC and PLD, with PLC inhibition occurring at lower concentrations than PLD inhibition. These differential effects of pertussis toxin and the observed lack of inhibition of fMLP‐stimulated PLD by PMA, which is believed to inactivate G‐proteins involved in PLC activation, imply that PLC and PLD are linked to fMLP receptors through distinct G‐proteins. Taken together, these observations suggests that, in fMLP‐stimulated neutrophils, PLC and PLD are activated through independent mechanisms.

Desloratadine Inhibits Constitutive and Histamine-Stimulated Nuclear Factor-κB Activity Consistent with Inverse Agonism at the Histamine H1 Receptor

Background: The human histamine H1 receptor is constitutively active and exhibits basal activation of nuclear factor-ĸB (NF-ĸB), an important modulator of allergic inflammation. Certain H1 antihistamines have recently been shown to inhibit basal NF-ĸB activity by stabilizing the H1 receptor in an inactive state, a phenomenon called ‘inverse agonism’. Methods: We evaluated the effect of the new H1 antihistamine, desloratadine, on basal and histamine-stimulated NF-ĸB activity and compared it with the activities of other H1 antihistamines. Results: Transiently transfected COS-7 cells co-expressing NF-ĸB-luciferase and the H1 receptor exhibited constitutive NF-ĸB activity. H1 antihistamines reduced basal NF-ĸB activity (rank order of potency: desloratadine > pyrilamine > cetirizine > loratadine > fexofenadine). Histamine stimulated basal NF-ĸB activity 8-fold, which was blocked by H1 antihistamines (rank order of potency: desloratadine > cetirizine > pyrilamine > loratadine > fexofenadine). Neither histamine nor antihistamines had any effect on NF-ĸB activity in the absence of the H1 receptor. Conclusions: Desloratadine, acting through the histamine H1 receptor, inhibited basal NF-ĸB activity and can thus be classified as an inverse agonist. Inhibition of basal and histamine-stimulated NF-ĸB activity may help to explain previously reported inhibitory effects of desloratadine on allergic inflammatory mediators.

Chemotactic peptide, calcium and guanine nucleotide regulation of phospholipase C activity in membranes from DMSO-differentiated HL60 cells

Membranes prepared from DMSO-differentiated HL60 cells labeled with [3H]inositol hydrolyze polyphosphoinositides in a Ca2+-dependent manner, generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). Incubation of membranes with GTP or GTP gamma S reduces the concentration of Ca2+ required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP3 formation by GTP or GTP gamma S. These results suggest that differentiated HL60 cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to FMLP receptors and is pertussis toxin sensitive.

Pharmacological characterization of postjunctional α-adrenoceptors in human nasal mucosa

Background Functional α1- and α2-adrenoreceptor subtype pharmacology was characterized in an in vitro human nasal mucosa contractile bioassay. Methods Nasal mucosa was obtained from 49 donor patients and mucosal strips were placed in chambers filled with Krebs–Ringer solution and attached to isometric force transducers. Results Nonselective α-adrenoreceptor agonists epinephrine, norepinephrine, and oxymetazoline produced concentration-dependent contractions of isolated human nasal mucosa (pD2= 5.2, 4.9, and 6.5, respectively). The α2-adrenoreceptor agonist BHT-920 (10 μM)–induced contractions were blocked by yohimbine (0.01–1 μM) and prazosin (0.01–1 μM) inhibited the contractile response to the α1-adrenoreceptor agonist phenylephrine (10 μM). Histological analysis showed that phenylephrine and BHT-920 differentially contracted the arteries and veins of human nasal mucosa, respectively. Conclusion Our results indicate that functional α1- and α2-adrenoceptors are present and functional in human nasal mucosa. The a 2-adrenoceptors display a predominant role in contracting the veins and the α1-adrenoceptors appear to preferentially constrict the human nasal arteries.