Progress toward a Glycoprotein VI Modulator for the Treatment of Thrombosis.

Pathogenic thrombus formation accounts for the etiology of many serious conditions including myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. Despite the development of numerous anticoagulants and antiplatelet agents, the mortality rate associated with these diseases remains high. In recent years, however, significant epidemiological evidence and clinical models have emerged to suggest that modulation of the glycoprotein VI (GPVI) platelet receptor could be harnessed as a novel antiplatelet strategy. As such, many peptidic agents have been described in the past decade, while more recent efforts have focused on the development of small molecule modulators. Herein the rationale for targeting GPVI is summarized and the published GPVI modulators are reviewed, with particular focus on small molecules. A qualitative pharmacophore hypothesis for small molecule ligands at GPVI is also presented.

Synthesis and Evaluation of [18F]FEtLos and [18F]AMBF3Los as Novel 18F-Labelled Losartan Derivatives for Molecular Imaging of Angiotensin II Type 1 Receptors.

Losartan is widely used in clinics to treat cardiovascular related diseases by selectively blocking the angiotensin II type 1 receptors (AT1Rs), which regulate the renin-angiotensin system (RAS). Therefore, monitoring the physiological and pathological biodistribution of AT1R using positron emission tomography (PET) might be a valuable tool to assess the functionality of RAS. Herein, we describe the synthesis and characterization of two novel losartan derivatives PET tracers, [18F]fluoroethyl-losartan ([18F]FEtLos) and [18F]ammoniomethyltrifluoroborate-losartan ([18F]AMBF3Los). [18F]FEtLos was radiolabeled by 18F-fluoroalkylation of losartan potassium using the prosthetic group 2-[18F]fluoroethyl tosylate; whereas [18F]AMBF3Los was prepared following an one-step 18F-19F isotopic exchange reaction, in an overall yield of 2.7 ± 0.9% and 11 ± 4%, respectively, with high radiochemical purity (>95%). Binding competition assays in AT1R-expressing membranes showed that AMBF3Los presented an almost equivalent binding affinity (Ki 7.9 nM) as the cold reference Losartan (Ki 1.5 nM), unlike FEtLos (Ki 2000 nM). In vitro and in vivo assays showed that [18F]AMBF3Los displayed a good binding affinity for AT1R-overexpressing CHO cells and was able to specifically bind to renal AT1R. Hence, our data demonstrate [18F]AMBF3Los as a new tool for PET imaging of AT1R with possible applications for the diagnosis of cardiovascular, inflammatory and cancer diseases.

Cytotoxicity of Graphene Oxide (GO) and Graphene Oxide Conjugated Losartan Potassium (GO-LP) on Neuroblastoma (NB41A3) Cells.

Despite several advancements in the biomedical sciences, an efficient cancer therapy still remains a challenge. Nanomedicines have shown potential to overcome certain roadblocks faced in the existing treatment modalities. Losartan potassium (LP) which is a known vasodilator also exhibits anti fibrolytic and anti-metastatic properties altogether. Further, also being a potential angiotensin II type 1 receptor antagonist, it has been well explored for down regulating tumourogenic biomarkers like VEGF-A (Vascular endothelial growth factor A) and suppression of neovascularization, making it a suitable drug to target for cancer treatment. Besides this, it too reflected the stimulation of pro apoptotic signaling pathways. But due to its lower bioavailability and extensive hepatic metabolism its therapeutic index reduces down. Thus, the present study is focused on designing a nano-delivery system using graphene oxide (GO) as a nano-vehicle and conjugated the LP with it. Then, the successful synthesis of GO and GO-LP nano conjugates were characterized by high-resolution transmission electron microscopy, X-ray diffraction, FTIR and UV visible spectroscopy, confirming the formation of nanosheets. The qualitative morphological evaluation of NB41A3 neuroblastoma cell line treated with bare GO, LP and GO-LP using microscopy and DAPI staining revealed the inhibitory action of GO-LP nano conjugate on cell proliferation. Additionally, the cytotoxicity was also estimated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Nitric oxide (NO) and Lactate dehydrogenase (LDH) assays. The results show that GO-LP significantly suppresses the cell viability in comparison to control and bare GO suggesting that the designed system may express its potential to be used with existing chemo drugs for the treatment of neural cancers.

Mechanisms underlying the cardiac antifibrotic effects of losartan metabolites.

Excessive myocardial collagen deposition and cross-linking (CCL), a process regulated by lysyl oxidase (LOX), determines left ventricular (LV) stiffness and dysfunction. The angiotensin II antagonist losartan, metabolized to the EXP3179 and EXP3174 metabolites, reduces myocardial fibrosis and LV stiffness in hypertensive patients. Our aim was to investigate the differential influence of losartan metabolites on myocardial LOX and CCL in an experimental model of hypertension with myocardial fibrosis, and whether EXP3179 and EXP3174 modify LOX expression and activity in fibroblasts. In rats treated with NG-nitro-L-arginine methyl ester (L-NAME), administration of EXP3179 fully prevented LOX, CCL and connective tissue growth factor (CTGF) increase, as well as fibrosis, without normalization of blood pressure (BP). In contrast, administration of EXP3174 normalized BP and attenuated fibrosis but did not modify LOX, CCL and CTGF. In TGF-ß1-stimulated fibroblasts, EXP3179 inhibited CTGF and LOX expression and activity with lower IC50 values than EXP3174. Our results indicate that, despite a lower antihypertensive effect, EXP3179 shows higher anti-fibrotic efficacy than EXP3174, likely through its ability to prevent the excess of LOX and CCL. It is suggested that the anti-fibrotic effect of EXP3179 may be partially mediated by the blockade of CTGF-induced LOX in fibroblasts.

Blockade of the renin-angiotensin system prevents acute and immunologically relevant colitis in murine models.

BACKGROUND: Blockade of the renin-angiotensin system (RAS) has been shown to alleviate inflammatory processes in the gastrointestinal tract. The aim of this study was to determine if blockade of the RAS would be effective in an immunologically relevant colitis model, and to compare outcome with an acute colitis model. METHODS: A losartan analog, CCG-203025 (C23H26ClN3O5S) containing a highly polar sulfonic acid moiety that we expected would allow localized mucosal antagonism with minimal systemic absorption was selected as an angiotensin II type 1a receptor antagonist (AT1aR-A). Two colitis models were studied: (1) Acute colitis was induced in 8- to 10-week-old C57BL/6J mice by 2.5 % dextran sodium sulfate (DSS, in drinking water) for 7 days. (2) IL10-/-colitis Piroxicam (200 ppm) was administered orally in feed to 5-week-old IL-10-/-mice (C57BL/6J background) for 14 days followed by enalaprilat (ACE-I), CCG-203025 or PBS administered transanally for 14 days. RESULTS: In the DSS model, weight loss and histologic score for CCG-203025 were better than with placebo. In the IL10-/-model, ACE-I suppressed histologic damage better than CCG-203025. Both ACE-I and CCG-203025 reduced pro-inflammatory cytokines and chemokines. CONCLUSIONS: This study demonstrated the therapeutic efficacy of both ACE-I and AT1aR-A for preventing the development of both acute and immunologically relevant colitis.

Liquid chromatography-mass spectrometric determination of losartan and its active metabolite on dried blood spots.

A simple and rapid quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for simultaneous determination of losartan and its active metabolite, losartan carboxylic acid on rat dried blood spots was developed and validated as per regulatory guidelines. Losartan and its metabolite were extracted from dried blood spots using 50% aqueous methanol and separated on Waters XTerra(®) RP18 (250 mm × 4.6 mm, 5 µm) column using mobile phase composed of 40% acetonitrile and 60% aqueous ammonium acetate (10mM). The eluents were monitored using ESI tandem mass spectrometric detection with negative polarity in MRM mode using ion transitions m/z 421.2→179.0, m/z 435.3→157.0 and m/z 427.3→193.0 for losartan, losartan carboxylic acid and Irbesartan (internal standard), respectively. The method was validated over the linear range of 1-200 ng/mL and 5-1000 ng/mL with lower limits of quantification of 1.0 ng/mL and 5.0 ng/mL for losartan and losartan carboxylic acid, respectively. Inter and intra-day precision and accuracy (Bias) were below 5.96% and between -2.8 and 1.5%, respectively. The mean recoveries of the analytes from dried blood spots were between 89% and 97%. No significant carry over and matrix effects were observed. The stability of stock solution, whole blood, dried blood spot and processed samples were tested under different conditions and the results were found to be well within the acceptable limits. Additional validation parameters such as influence of hematocrit and spot volume were also evaluated and found to be well within the acceptable limits.

1-pentanoyl-N-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-pyrrolidine-2-carboxamide: investigation of structural variations.

We recently reported a series of 1-acyl-N-(biphenyl-4-ylmethyl)pyrrolidine-2-carboxamides as AT(1) receptor ligands. The most potent compound of the series, 1-pentanoyl-N-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-pyrrolidine-2-carboxamide, showed an interesting affinity for the receptor. To investigate the influence of structure variations on affinity, the synthesis of additional compounds belonging to this series has been performed. Biological tests run on the newly synthesized compounds on CHO-hAT(1) cells stably expressing the human AT(1) receptor confirm our previous hypothesis, i.e. that, within this series, the length of the acyl chain, the substitution of the amidic group and the nature of the acidic one are crucial for the receptor interaction, being a valeric chain, a secondary amidic function and the tetrazole moiety, respectively, the optimal ones.

Use of enterally delivered angiotensin II type Ia receptor antagonists to reduce the severity of colitis.

BACKGROUND: Renin-angiotensin system blockade reduces inflammation in several organ systems. Having found a fourfold increase in angiotensin II type Ia receptor expression in a dextran sodium sulfate colitis model, we targeted blockade with angiotensin II type Ia receptor antagonists to prevent colitis development. Because hypotension is a major complication of angiotensin II type Ia receptor antagonists use, we hypothesized that use of angiotensin II type Ia receptor antagonists compounds which lack cell membrane permeability, and thus enteric absorption, would allow for direct enteral delivery at far higher concentrations than would be tolerated systemically, yet retain efficacy. METHODS: Based on the structure of the angiotensin II type Ia receptor antagonist losartan, deschloro-losartan was synthesized, which has extremely poor cell membrane permeability. Angiotensin II type Ia receptor antagonist efficacy was evaluated by determining the ability to block NF-κB activation in vitro. Dextran sodium sulfate colitis was induced in mice and angiotensin II type Ia receptor antagonist efficacy delivered transanally was assessed. RESULTS: In vitro, deschloro-losartan demonstrated near equal angiotensin II type Ia receptor blockade compared to losartan as well as another angiotensin II type Ia receptor antagonist, candesartan. In the dextran sodium sulfate model, each compound significantly improved clinical and histologic scores and epithelial cell apoptosis. Abundance of TNF-α, IL-1ß, and IL6 mRNA were significantly decreased with each compound. In vitro and in vivo intestinal drug absorption, as well as measures of blood pressure and mucosal and colonic blood flow, showed significantly lower uptake of deschloro-losartan compared to losartan and candesartan. CONCLUSIONS: This study demonstrated efficacy of high-dose angiotensin II type Ia receptor antagonists in this colitis model. We postulate that a specially designed angiotensin II type Ia receptor antagonist with poor oral absorption may have great potential as a new therapeutic agent for inflammatory bowel disease in the future.

An efficient synthesis of a rationally designed 1,5 disubstituted imidazole AT(1) angiotensin II receptor antagonist: reorientation of imidazole pharmacophore groups in losartan reserves high receptor affinity and confirms docking studies.

A new 1,5 disubstituted imidazole AT(1) Angiotensin II (AII) receptor antagonist related to losartan with reversion of butyl and hydroxymethyl groups at the 2-, 5-positions of the imidazole ring was synthesized and evaluated for its antagonist activity (V8). In vitro results indicated that the reorientation of butyl and hydroxymethyl groups on the imidazole template of losartan retained high binding affinity to the AT(1) receptor concluding that the spacing of the substituents at the 2,5- positions is of primary importance. The docking studies are confirmed by binding assay results which clearly show a comparable binding score of the designed compound V8 with that of the prototype losartan. An efficient, regioselective and cost effective synthesis renders the new compound as an attractive candidate for advanced toxicological evaluation and a drug against hypertension.

Chronic treatment with losartan results in sufficient serum levels of the metabolite EXP3179 for PPARgamma activation.

The losartan metabolite EXP3174 exhibits angiotensin II receptor 1 (AT1R)-blocking properties, whereas the metabolite EXP3179 potently induces the activity of the insulin-sensitizing peroxisome proliferator-activated receptor gamma (PPARgamma) as a partial agonist in vitro. We investigated whether chronic treatment with losartan leads to sufficient serum levels of EXP3179 to activate PPARgamma in monocytes derived from losartan-treated patients. Hypertensive patients (n=15) treated with losartan (100 mg/daily for at least the past 2 months) and untreated control patients (n=7) were included. Monocytes were extracted by negative isolation using a Dynal Monocyte Kit, followed by analysis of PPARgamma target gene expression (CD36, ABC transporter G1 [ABCG1]) by quantitative real-time RT-PCR. Serum was prepared before, 2, 4, and 6 hours after losartan (100 mg) ingestion for HPLC-based determination of losartan, EXP3174, and EXP3179. Chronic treatment with losartan resulted in basal levels of losartan, EXP3174, and EXP3179 of 348.3+/-101.8 ng/mL, 115.3+/-56.1 ng/mL, and 176.2+/-143.4 ng/mL, respectively. Levels of both EXP3174 and EXP3179 were time-dependently increased in serum with a maximum 2 hours after drug intake (1706.0+/-760.1 ng/mL, 808.9+/-618.2 ng/mL, respectively). In consonance with detectable PPARgamma-activating EXP3179 serum levels, monocytic PPARgamma target gene expression was significantly upregulated in patients treated with losartan by 3.75+/-0.95- and 252.02+/-46.86-fold for CD36 and ABCG1 (P=0.043, P=0.0045 versus control patients, respectively). This is the first clinical description of monocytic PPARgamma-target gene regulation by chronic treatment with losartan, which likely is mediated by its metabolite EXP3179. Our data show that sufficient serum levels of EXP3179 are present under losartan treatment. PPARgamma activation by AT1R-blockers may translate into synergistic beneficial actions in monocytes.

Losartan metabolite EXP3179 blocks NADPH oxidase-mediated superoxide production by inhibiting protein kinase C: potential clinical implications in hypertension.

Oxidative stress plays a critical role in the pathogenesis of hypertension. The NADPH oxidase constitutes a major source of superoxide anion in phagocytic cells, and its activation is associated with matrix metalloproteinase (MMP)-9 secretion by these cells. We investigated the effects of the angiotensin II type 1 receptor antagonist losartan and its metabolites (EXP3174 and EXP3179) on NADPH oxidase activity and MMP-9 secretion in human phagocytic cells. EXP3179, but not losartan and EXP3174, dose-dependently inhibited (P<0.05) phorbol myristate acetate and insulin-stimulated NADPH oxidase activity. EXP3179 also inhibited phorbol myristate acetate-induced NADPH oxidase in endothelial cells. In addition, EXP3179 inhibited (P<0.05) both phorbol myristate acetate-stimulated p47phox translocation from cytosol to membranes and protein kinase C activity. Affinity experiments and enzymatic assays confirmed that EXP3179 inhibited several protein kinase C isoforms. EXP3179 also inhibited (P<0.05) phorbol myristate acetate-stimulated MMP-9 secretion. In a study performed in 153 hypertensive patients, phagocytic NADPH oxidase activity was lower (P<0.05) in losartan-treated compared with untreated patients and in patients treated with other angiotensin II type 1 receptor antagonists or with angiotensin-converting enzyme inhibitors. Plasma levels of MMP-9 were lower (P<0.05) in losartan-treated hypertensives compared with the other group of patients. Thus, EXP3179 acts as a blocker of the NADPH oxidase in phagocytic cells by a potential mechanism that targets the protein kinase C signaling pathway. This effect can be involved in reduced MMP-9 secretion by these cells. It is proposed that the EXP3179 metabolite may confer to losartan the specific capacity to reduce oxidative stress mediated by phagocytic cells in hypertensive patients.

Comparative antihypertensive activities of losartan and HM70186 in rats with hepatic dysfunction.

HM70186, a medoxomil ester of EXP3174 which is an active metabolite of angiotensin II receptor blocker losartan, was synthesized, and its antihypertensive efficacy was evaluated in rats with hepatic dysfunction. Male Wistar rats were intraperitoneally injected with 0.5 mL/kg of carbon tetrachloride to cause hepatic injury, and implanted with an osmotic minipump containing angiotensin II (0.4 mg/kg/day) to induce hypertension. After confirmation of both hepatic damage and hypertension, the rats were orally administered losartan or HM70186, and then blood pressure and heart rate were monitored for 24 h. In normal animals, angiotensin II-induced hypertension was lowered by losartan, resulting in an ED(-30 mmHg) of 9.05 mg/kg. HM70186 also immediately decreased the blood pressure in a dose-dependent manner, exhibiting an ED(-30 mmHg) of 0.89 ng/kg (10,000 times the potency observed with losartan). Moreover, HM70186 (3 ng/kg) exerted a strong antihypertensive effect even in rats with hepatic injury, while losartan (10 microg/kg) was ineffective. These results suggest that HM70186 could be a promising candidate for the treatment of hypertension accompanied by hepatic dysfunction.

AT1 receptor ligands: virtual-screening-based design with TOPP descriptors, synthesis, and biological evaluation of pyrrolidine derivatives.

As a continuing effort to establish the structure-activity relationships (SARs) within the series of the angiotensin II antagonists (sartans), a pharmacophoric model was built by using novel TOPP 3D descriptors. Statistical values were satisfactory (PC4: r(2)=0.96, q(2) ((5) (random) (groups))=0.84; SDEP=0.26) and encouraged the synthesis and consequent biological evaluation of a series of new pyrrolidine derivatives. SAR together with a combined 3D quantitative SAR and high-throughput virtual screening showed that the newly synthesized 1-acyl-N-(biphenyl-4-ylmethyl)pyrrolidine-2-carboxamides may represent an interesting starting point for the design of new antihypertensive agents. In particular, biological tests performed on CHO-hAT(1) cells stably expressing the human AT(1) receptor showed that the length of the acyl chain is crucial for the receptor interaction and that the valeric chain is the optimal one.

EXP3179 inhibits collagen-dependent platelet activation via glycoprotein receptor-VI independent of AT1-receptor antagonism: potential impact on atherothrombosis.

OBJECTIVE: Thrombus formation after atherosclerotic plaque rupture critically involves the platelet collagen receptor glycoprotein (GP) VI. We investigated the impact of EXP3179, an active metabolite of the angiotensin II type 1 (AT1)-receptor antagonist Losartan (LOS) on GPVI-dependent platelet activation. METHODS AND RESULTS: EXP3179 and LOS but not EXP3174--the major AT1-receptor blocking metabolite of LOS--dose-dependently inhibited collagen-I (P<0.01) and GPVI-dependent platelet aggregation (P<0.01) analyzed by optical aggregometry. Platelet activation was further determined by flow cytometry measuring the expression of platelet PAC-1, an epitope of the activated fibrinogen-receptor complex. EXP3179 and LOS inhibited collagen-I (P<0.01) and GPVI-dependent PAC-1 expression (P<0.01). EXP3179 and LOS but not EXP3174 decreased the adhesion of GPVI-receptor expressing Chinese hamster ovarian cells on collagen-I under arterial shear conditions determined by flow chamber analysis (P<0.01 and P<0.05). EXP3179 also reduced human atherosclerotic plaque material-induced platelet aggregation (P<0.01) in vitro and murine platelet adhesion after acute vessel injury in vivo as determined by intravital microscopy (P<0.01). CONCLUSION: EXP3179 acts as a specific inhibitor of the platelet collagen receptor GPVI independent of AT1-receptor antagonism. Further investigations may clarify its individual potential as a novel pharmacological approach to specifically inhibit atherothrombotic events by GPVI-receptor blockade.

Regulation of peroxisome proliferator-activated receptor gamma activity by losartan metabolites.

Two active metabolites of the angiotensin type 1 (AT1) receptor blocker losartan have been described previously, EXP3174 and EXP3179. Whereas EXP3174 is the main antihypertensive AT1 receptor-blocking metabolite, the role of EXP3179 is widely unknown. Recently, a subgroup of AT1 receptor blockers has been identified as ligands for the peroxisome proliferator-activated receptor gamma (PPAR-gamma). Here we characterize the PPAR-gamma-activating properties of the 2 active losartan metabolites. PPAR-gamma activity was measured with a chimeric Gal4-DNA-binding domain-hPPARgamma-ligand-binding domain (LBD) fusion protein on a Gal4-dependent luciferase reporter system. EXP3179 prominently induced the activation of the PPAR-gamma-LBD reaching a maximum at 100 micromol/L with a 7.1+/-1-fold induction (P<0.05 versus vehicle-treated cells). Maximum PPAR-gamma-LBD activation by EXP3179 reached 51% of the maximum response induced by the full PPAR-gamma agonist pioglitazone, identifying EXP3179 as a partial PPAR-gamma agonist. EXP3174 did not induce PPAR-gamma-LBD activation. EC50 values were calculated for PPAR-gamma-LBD activity (pioglitazone EC50: 0.88 micromol/L; EXP3179 EC50: 17.1 micromol/L; losartan EC50: >50 micromol/L). Consistent with the activation of PPAR-gamma, EXP3179 potently induced 3T3-L1 adipocyte differentiation, a typical PPAR-gamma-dependent cell function, and markedly stimulated PPAR-gamma target gene expression. EXP3174 failed to regulate differentiation or PPAR-gamma target gene expression. The present study characterizes the active losartan metabolite EXP3179 as a partial PPAR-gamma agonist. PPAR-gamma activation by EXP3179 may help us to understand the beneficial metabolic effects of losartan observed in clinical trials.

Structure elucidation and conformational study of V8: a novel synthetic non peptide AT(1) antagonist.

AT(1) antagonists constitute the most recent class of antihypertensive drugs which act through the Renin Angiotensin System (RAS). In an effort to comprehend their stereoelectronic features, a study was initiated to compare the conformational properties of drugs already marketed for the treatment of hypertension with synthetic ones, possessing common structural characteristics. In this study, the synthetic AT(1) antagonist V8 is structurally elucidated and its conformational properties are studied through a combination of NMR spectroscopy and computational analysis. Its conformational properties are compared with those of the structurally similar prototype AT(1) antagonist losartan.

NO-sartans: a new class of pharmacodynamic hybrids as cardiovascular drugs.

The aim of this work was to develop lead pharmacodynamic hybrids, NO-sartans, possessing the characteristics of a typical AT1-antagonist and of a "slow NO donor", by adding NO-donor side chains to losartan. These new compounds, 2a and 2b, displayed vasorelaxing effects, due to the release of NO, and antagonized the vasocontractile effects of angiotensin II, with potency values similar to that of losartan. In vivo, the antihypertensive effects of 2a were similar to those of losartan and captopril.

Conformation and bioactivity. Design and discovery of novel antihypertensive drugs.

Peptidomimitism is applied to the medicinal chemistry in order to synthesize drugs that devoid of the disadvantages of peptides. AT1 antagonists constitute a new generation of drugs for the treatment of hypertension designed and synthesized to mimic the C-terminal segment of Angiotensin II and to block its binding action on AT1 receptor. An effort was made to understand the molecular basis of hypertension by studying the conformational analysis of Ang II and its derivatives as well as the AT1 antagonists belonging to SARTANs class of molecules. Such studies offer the possibility to reveal the stereoelectronic factors responsible for bioactivity of AT1 antagonists and to design and synthesize new analogs. An example will be given which proves that drugs with better pharmacological and financial profiles may arise based on this rational design.