Synthesis and Pharmacological Characterization of Subtype-Selective Ligands, Including Radio- and Fluorescence Labeled Ligands, for the Histamine H₂ Receptor

The H₂R is one of four receptor subtypes which mediate the action of the biogenic amine histamine. However, the (patho-) physical role of the H₂R, especially in the brain, is still far from being understood. Therefore this work aimed at the development of selective high affinity molecular tools for the H₂R, including agonists, antagonists and radiolabeled as well as fluorescent H₂R ligands. The number of high affinity tritiated radioligands for the H₂R is very limited. Guanidinothiazole containing ligands such as famotidine represent a class of surmountable H₂R antagonists. The synthesized high affinity hH₂R antagonist N-[8-(2-[2-(2-guanidinothiazol-4-ylmethylthio)ethylamino]-3,4-dioxo¬cyclobut-1-ene-1-ylamino)octyl]propionamide (3.25, pKi: 7.65) showed selectivity over the other subtypes and was also synthesized in radiolabeled form. [3H]3.25 bound in a saturable manner (Kd: 15-22 nM) to membrane preparations of Sf9 cells and intact Hek cells both recombinantly expressing hH₂Rs. Although a part of [3H]3.25 bound in (pseudo)irreversible manner, the kinetic Kd value of 26 nM was comparable to that determined at equilibrium, and the radioligand [3H]3.25 was completely displacable by standard ligands. Aminopotentidine and it´s derivatives are reported as high affinity H₂R antagonists. Aminopotentidine and its analogs with different substituents (e.g. iodine, bromine, chlorine, trifluoromethyl) in position 3, were prepared and propionylated. Within the series of propionylated derivatives the brominated ligand (4.37) and the iodinated ligand (4.38) showed the highest hH₂R affinities (pKi: 8.5 and 8.18) along with excellent selectivities over the hH₃R (6900- or 2500-fold). Fluorescent ligands have become an attractive alternative to radioligands for the investigation of ligand-receptor interactions. In order to expand the range of applications and avoid the high cellular autofluorescence, fluorescent ligands labeled with red-emitting fluorophores are required. Recently, a series of fluorescent ligands with a piperidinomethylphenoxy¬propylamino pharmacophore was reported. In this work, the fluorescent labeled antagonists UR-DE229 (5.12) and UR-DE56 (5.18) were prepared and investigated in different assay systems. Furthermore, a small library of fluorescent ligands was synthesized for the exploration of the impact of linker length and the net charge of the fluorophores. The highest affinities to the hH₂R (pKi: > 7.0) in radioligand competition assays were obtained in case of the pyridinium labeled ligands 5.12-5.14 and the cyanine labeled ligands 5.16 and 5.18. Despite the low selectivity towards the hH₃R the investigated fluorescent ligands proved to be useful tools for binding studies using different techniques (flow cytometry, high content imaging and confocal microscopy), when genetically engineered cells, expressing the H₂R were used. Investigated ligands bound in a saturable manner to the hH₂R and the determined Kd values were in good agreement with the corresponding Ki values from radiolligand binding. Kinetic Kd(kin) values were consistent with the Kd values in equilibrium even though they showed an incomplete dissociation (insurmountable antagonism). Nonetheless, the fluorescent ligands 5.14 and 5.18 also proved to be useful for the determination of binding affinities of unlabeled ligands in competition binding assays. N(G)-acylated amino(methyl)thiazolepropylguanidines are reported as potent and selective histamine H₂R agonists, but the acylguanidine group is prone to hydrolytic cleavage in aqueous solution. A bioisosteric approach, replacing the acylguanidine structure with a carbamoylguanidine, led in many cases to more stable compounds. For exploration of the structure-activity and the structure-selectivity relationships of this class of compounds, a series of carbamoylguanidines with various aminothiazole-based substructures, i.e., the 3-(2-amino-4-methylthiazol-5-yl)propyl moiety, a conformationally constrained (aminothiazolyl)phenyl and a 2-amino-4,5,6,7-tetrahydrobenzothiazol-6-yl portion were prepared. Compounds containing the conformationally constrained aminothiazole headgroup were weak antagonists/ inverse agonists or weak partial agonists. Amino(methyl)thiazolyl¬ propyl containing compounds achieved up to 80-fold the potency of histamine (pEC50: 6.3-7.76) and partial to full agonistic activities with bell-shaped concentration-response curves. Additionally, representative ligands showed affinity to the hD₂R(long) (pKi: 5.6-6.97) and hD₃R (pKi: 5.3-7.6)

A case of dengue type 3 virus infection imported from Africa to Italy, October 2009.

In October 2009, a traveller returning from Africa to Italy was hospitalised with symptoms suggestive of a haemorrhagic fever of unknown origin. The patient was immediately placed in a special biocontainment unit until laboratory investigations confirmed the infection to be caused by a dengue serotype 3 virus. This case reasserts the importance of returning travellers as sentinels of unknown outbreaks occurring in other countries, and highlights how the initial symptoms of dengue fever resemble those of other haemorrhagic fevers, hence the importance of prompt isolation of patients until a final diagnosis is reached

3HUR-DE257: Development of a tritium-labeled squaramide-type selective histamine H2 receptor antagonist

A series of new piperidinomethylphenoxypropylamine-type histamine H2 receptor (H2R) antagonists with different substituted “urea equivalents” was synthesized and characterized in functional in vitro assays. N-6-(3,4-Dioxo-2-{3-3-(piperidin-1-ylmethyl)phenoxy]-propylamino}cyclobut-1-enylamino)hexyl]-(2,3-3H2)propionic amide (3HUR-DE257, 24b) was selected for radiosynthesis. The radioligand (specific activity: 63 Ci • mmol-1) had a high affinity for the human, the rat and the guinea pig H2R (hH2R, Sf9 cells: Kd, saturation binding: 31 nM, kinetic studies: 20 nM). UR-DE257 (24a) revealed high H2R selectivity on membranes of Sf9 cells, expressing the respective hHxR subtype (Ki values: hH1R: >10000 nM, hH2R: 28 nM, hH3R: 3800 nM, hH4R: >10000 nM). Regardless of insurmountable antagonism, characteristic for this class of compounds depending on the type of functional assay, and of rebinding of 24b to the H2R (extended residence time), the title compound proved to be a valuable pharmacological tool for the determination of H2R affinities in competition binding assays

Pharmacological characterization of a new series of carbamoylguanidines reveals potent agonism at the H2R and D3R

Even today, the role of the histamine H2 receptor (H2R) in the central nervous system (CNS) is widely unknown. In previous research, many dimeric, high-affinity and subtype-selective carbamoylguanidine-type ligands such as UR-NK22 (5, pKi = 8.07) were reported as H2R agonists. However, their applicability to the study of the H2R in the CNS is compromised by their molecular and pharmacokinetic properties, such as high molecular weight and, consequently, a limited bioavailability. To address the need for more drug-like H2R agonists with high affinity, we synthesized a series of monomeric (thio)carbamoylguanidine-type ligands containing various spacers and side-chain moieties. This structural simplification resulted in potent (partial) agonists (guinea pig right atrium, [35S]GTPγS and β-arrestin2 recruitment assays) with human (h) H2R affinities in the one-digit nanomolar range (pKi (139, UR-KAT523): 8.35; pKi (157, UR-MB-69): 8.69). Most of the compounds presented here exhibited an excellent selectivity profile towards the hH2R, e.g. 157 being at least 3800-fold selective within the histamine receptor family. The structural similarities of our monomeric ligands to pramipexole (6), a dopamine receptor agonist, suggested an investigation of the binding behavior at those receptors. The target compounds were (partial) agonists with moderate affinity at the hD2longR and agonists with high affinity at the hD3R (e.g. pKi (139, UR-KAT523): 7.80; pKi (157, UR-MB-69): 8.06). In summary, we developed a series of novel, more drug-like H2R and D3R agonists for the application in recombinant systems in which either the H2R or the D3R is solely expressed. Furthermore, our ligands are promising lead compounds in the development of selective H2R agonists for future in vivo studies or experiments utilizing primary tissue to unravel the role and function of the H2R in the CNS

Mimicking of arginine by functionalized Nω-carbamoylated arginine as a new broadly applicable approach to labeled bioactive peptides: high affinity angiotensin, neuropeptide Y, neuropeptide FF and neurotensin receptor ligands as examples

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N-omega-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with K-i values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described

Fluorescent H2 Receptor Squaramide-Type Antagonists: Synthesis, Characterization, and Applications

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH(2)R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19-27) was synthesized and characterized. The highest hH(2)R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pK(i): 7.71-7.76) and cyanine labeled antagonists 23 and 25 (pK(i): 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed pK(d) values comparable to the pK(i) values. The fluorescent probes 21, 23, and 25 could be used to localize H-2 receptors in HEK cells and to determine the binding affinities of unlabeled compounds

Immunogenicity of Viral Vaccines in the Italian Military

Military personnel of all armed forces receive multiple vaccinations and have been doing so since long ago, but relatively few studies have investigated the possible negative or positive interference of simultaneous vaccinations. As a contribution to fill this gap, we analyzed the response to the live trivalent measles/mumps/rubella (MMR), the inactivated hepatitis A virus (HAV), the inactivated trivalent polio, and the trivalent subunits influenza vaccines in two cohorts of Italian military personnel. The first cohort was represented by 108 students from military schools and the second by 72 soldiers engaged in a nine-month mission abroad. MMR and HAV vaccines had never been administered before, whereas inactivated polio was administered to adults primed at infancy with a live trivalent oral polio vaccine. Accordingly, nearly all subjects had baseline antibodies to polio types 1 and 3, but unexpectedly, anti-measles/-mumps/-rubella antibodies were present in 82%, 82%, and 73.5% of subjects, respectively (43% for all of the antigens). Finally, anti-HAV antibodies were detectable in 14% and anti-influenza (H1/H3/B) in 18% of the study population. At mine months post-vaccination, 92% of subjects had protective antibody levels for all MMR antigens, 96% for HAV, 69% for the three influenza antigens, and 100% for polio types 1 and 3. An inverse relationship between baseline and post-vaccination antibody levels was noticed with all the vaccines. An excellent vaccine immunogenicity, a calculated long antibody persistence, and apparent lack of vaccine interference were observed

Safety of Multiple Vaccinations and Durability of Vaccine-Induced Antibodies in an Italian Military Cohort 5 Years after Immunization

We previously examined the safety and immunogenicity of multiple vaccines administered to a military cohort, divided into two groups, the first composed of students at military schools, thus operating inside the national borders for at least 3 years, and the other formed of soldiers periodically engaged in a 9-month-long mission abroad (Lebanon). In the current study, we analyzed 112 individuals of this cohort, 50 pertaining to the first group and 62 to the second group, in order to examine the possible late appearance of side effects and to calculate the half-life of the induced antibodies. Moreover, the possible involvement of B-cell polyclonal activation as a pathogenetic mechanism for long term antibody persistence has even been explored. No late side effects, as far as autoimmunity and/or lymphoproliferation appearance, have been noticed. The long duration of the vaccine induced anti-HAV antibodies has been confirmed, whereas the antibodies induced by tetravalent meningococcal polysaccharide vaccine have been found to persist above the threshold for putative protection for a longer time, and anti-tetanus, diphtheria, and polio 1 and 3 for a shorter time than previously estimated. No signs of polyclonal B-cell activation have been found, as a possible mechanism to understand the long antibody persistence