Arco Y. Jeng

Pharmacokinetics and Pharmacodynamics of LCZ696, a Novel Dual-Acting Angiotensin Receptor−Neprilysin Inhibitor (ARNi)

Angiotensin receptor blockade and neprilysin (NEP) inhibition together offer potential benefits for the treatment of hypertension and heart failure. LCZ696 is a novel single molecule comprising molecular moieties of valsartan and NEP inhibitor prodrug AHU377 (1:1 ratio). Oral administration of LCZ696 caused dose‐dependent increases in atrial natriuretic peptide immunoreactivity (due to NEP inhibition) in Sprague‐Dawley rats and provided sustained, dose‐dependent blood pressure reductions in hypertensive double‐transgenic rats. In healthy participants, a randomized, double‐blind, placebo‐controlled study (n = 80) of single‐dose (200–1200 mg) and multiple‐dose (50–900 mg once daily for 14 days) oral administration of LCZ696 showed that peak plasma concentrations were reached rapidly for valsartan (1.6–4.9 hours), AHU377 (0.5–1.1 hours), and its active moiety, LBQ657 (1.8–3.5 hours). LCZ696 treatment was associated with increases in plasma cGMP, renin concentration and activity, and angiotensin II, providing evidence for NEP inhibition and angiotensin receptor blockade. In a randomized, open‐label crossover study in healthy participants (n = 56), oral LCZ696 400 mg and valsartan 320 mg were shown to provide similar exposure to valsartan (geometric mean ratio [90% confidence interval]: AUC0‐∞ 0.90 [0.82–0.99]). LCZ696 was safe and well tolerated. These data support further clinical development of LCZ696, a novel, orally bioavailable, dual‐acting angiotensin receptor—NEP inhibitor (ARNi) for hypertension and heart failure.

The Design, Structure, and Therapeutic Application of Matrix Metalloproteinase Inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes involved in the degradation and remodeling of extracellular matrix proteins. The activities of these enzymes are well regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Chronic stimulation of MMP activities due to an imbalance in the levels of MMPs and TIMPs has been implicated in the pathogenesis of a variety of diseases such as cancer, osteoarthritis, and rheumatoid arthritis. Thus, MMP inhibitors are expected to be useful for the treatment of these disorders. This article reviews briefly the biochemistry of MMPs and evidence for their pathogenic roles using molecular biology approaches. Biomolecular structures used in the design of MMP inhibitors are thoroughly covered. Major emphasis is on recently published potent, small molecular weight MMP inhibitors and their pharmacological properties. Finally, available clinical results of compounds in development are summarized.

Aldosterone Synthase Inhibitor Ameliorates Angiotensin II–Induced Organ Damage

Background—Aldosterone and angiotensin (Ang) II both may cause organ damage. Circulating aldosterone is produced in the adrenals; however, local cardiac synthesis has been reported. Aldosterone concentrations depend on the activity of aldosterone synthase (CYP11B2). We tested the hypothesis that reducing aldosterone by inhibiting CYP11B2 or by adrenalectomy (ADX) may ameliorate organ damage. Furthermore, we investigated how much local cardiac aldosterone originates from the adrenal gland. Methods and Results—We investigated the effect of the CYP11B2 inhibitor FAD286, losartan, and the consequences of ADX in transgenic rats overexpressing both the human renin and angiotensinogen genes (dTGR). dTGR-ADX received dexamethasone and 1% salt. Dexamethasone-treated dTGR-salt served as a control group in the ADX protocol. Untreated dTGR developed hypertension and cardiac and renal damage and had a 40% mortality rate (5/13) at 7 weeks. FAD286 reduced mortality to 10% (1/10) and ameliorated cardiac hypertrophy, albuminuria, cell infiltration, and matrix deposition in the heart and kidney. FAD286 had no effect on blood pressure at weeks 5 and 6 but slightly reduced blood pressure at week7 (177±6 mm Hg in dTGR+FAD286 and 200±5 mm Hg in dTGR). Losartan normalized blood pressure during the entire study. Circulating and cardiac aldosterone levels were reduced in FAD286 or losartan-treated dTGR. ADX combined with dexamethasone and salt treatment decreased circulating and cardiac aldosterone to barely detectable levels. At week 7, ADX-dTGR-dexamethasone-salt had a 22% mortality rate compared with 73% in dTGR-dexamethasone-salt. Both groups were similarly hypertensive (190±9 and 187±4 mm Hg). In contrast, cardiac hypertrophy index, albuminuria, cell infiltration, and matrix deposition were significantly reduced after ADX (P<0.05). Conclusions—Aldosterone plays a key role in the pathogenesis of Ang II–induced organ damage. Both FAD286 and ADX reduced circulating and cardiac aldosterone levels. The present results show that aldosterone produced in the adrenals is the main source of cardiac aldosterone.

Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action

The effects of inhibitors of protein kinase C on the activities of the intact enzyme, the proteolytically-generated catalytic domain, and [3H]phorbol 12,13-dibutyrate (PDBu) binding were compared in an effort to evaluate this approach for clarifying mechanisms of inhibitor action. Staurosporine, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], and quercetin inhibited the catalytic fragment with similar potencies as for the intact enzyme while having little or no effect on binding, consistent with reports that they are competitive with ATP. Adriamycin, trifluoperazine, and tamoxifen, suggested to disrupt hydrophobic interactions between the regulatory domain of protein kinase C and phospholipid, were all most effective on the intact enzyme. They appear to possess a mixed mechanism, however, inhibiting activity of the catalytic domain with approximately 3-fold lower potencies. Gossypol inhibited intact enzyme, catalytic fragment, and PDBu binding with similar potencies. In light of multiple apparent sites of action for such protein kinase C inhibitors, comparison of their activities on the individual functional domains of the kinase may provide a useful complement to studies with the intact enzyme.

Mechanism of action of the phorbol ester tumor promoters: Specific receptors for lipophilic ligands

Cells and tissue preparations specifically bind the phorbol ester tumor promoters. The agreement in structure-activity relationships between binding and biological response strongly argues that these binding sites function as phorbol ester receptors. Upon subcellular fractionation, the phorbol ester binding activity is particulate. In addition, a phorbol ester apo-receptor can be detected in cytosol which requires phospholipids for reconstitution. This apo-receptor appears to correspond to protein kinase C. Diacylglycerols, the probable natural activators of protein kinase C, competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogs. In certain systems, heterogeneity of phorbol ester binding is found. An outstanding issue therefore is whether protein kinase C is the phorbol ester receptor or whether it is only the most abundant class of receptor. Although this question remains unresolved, we can demonstrate heterogeneity of phorbol ester binding by reconstitution of apo-receptor into a heterogeneous lipid environment.

The Design, Structure, and Clinical Update of Small Molecular Weight Matrix Metalloproteinase Inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes involved in the degradation and remodeling of extracellular matrix proteins. Under normal physiological conditions, the activities of these enzymes are well-regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Chronic stimulation of MMP activities due to an imbalance in the levels of MMPs and TIMPs has been implicated in the pathogenesis of a variety of diseases such as cancer, osteoarthritis, and rheumatoid arthritis. Thus, MMP inhibitors are expected to be useful for the treatment of these disorders. Because of their importance in a variety of pathological conditions, a number of small molecular weight MMP inhibitors have entered clinical trials in humans. However, the results of these trials have been extremely disappointing and have led many investigators to conclude that MMP inhibitors have no therapeutic benefit in human cancer. To date, the first generation MMP inhibitors exhibited poor bioavailability while second-generation compounds revealed that prolonged treatment caused musculoskeletal pain and inflammation or had a lack of efficacy. This article describes the design of small molecular weight MMP inhibitors, a brief description of available three-dimensional MMP structures, a review of the proposed therapeutic utility of MMP inhibitors, and a clinical update of compounds that have entered clinical trials in humans. The experimentally determined structures used in the structure-based design of MMP inhibitors are thoroughly covered. Major emphasis is on recently published and/or patented potent MMP inhibitors, from approximately January 2000 to April 2003, and their pharmacological properties. Protein inhibitors of these proteolytic enzymes, i.e. TIMPs, will not be discussed.

Effect of Matrix Metalloproteinase Inhibition on Progression of Atherosclerosis and Aneurysm in LDL Receptor‐Deficient Mice Overexpressing MMP‐3, MMP‐12, and MMP‐13 and on Restenosis in Rats after Balloon Injury

ABSTRACT: The broad‐spectrum MMP inhibitor CGS 27023A was tested to determine its potential as a therapy for atherosclerosis, aneurysm, and restenosis. LDL receptor‐deficient (LDLr −/−) mice fed a high‐fat, cholic acid‐enriched diet for 16 weeks developed advanced aortic atherosclerosis with destruction of elastic lamina and ectasia in the media underlying complex plaques. Lesion formation correlated with a 4.6‐ to 21.7‐fold increase in MMP‐3, ‐12, and ‐13 expression. Treatment with CGS 27023A (p.o., b.i.d. at 50 mg/kg) had no effect on the extent of aortic atherosclerosis (36 ± 4% versus 30 ± 2% in controls), but both aortic medial elastin destruction and ectasia grade were significantly reduced (38% and 36%, respectively, p < 0.05). In the rat ballooned‐carotid‐artery model, CGS 27023A (12.5 mg/kg/day via osmotic minipump) reduced smooth muscle cell migration at 4 days by 83% (p < 0.001). Intimal lesions were reduced by 85% at 7 days (p < 0.001), but intimal smooth muscle proliferation was unaffected, and inhibitory efficacy was lost with time. At 12 days, intimal lesion reduction was less potent (52%, p < 0.01). At 3 and 6 weeks, reductions of 11% and 4%, respectively, were not significant. This demonstrates that it is essential to include late time points when the ballooned‐carotid‐artery model is employed to ensure that lesion size does not “catch up” when a compound solely inhibits smooth muscle cell migration. In summary, MMP inhibitor therapy delayed but did not prevent intimal lesions, thereby demonstrating little promise to prevent restenosis. In contrast, MMP inhibitor therapy may prove useful to retard progression of aneurysm.

Role of central nervous system aldosterone synthase and mineralocorticoid receptors in salt-induced hypertension in Dahl salt-sensitive rats

In Dahl salt-sensitive (S) rats, high salt intake increases cerebrospinal fluid (CSF) Na(+) concentration ([Na(+)]) and blood pressure (BP). Intracerebroventricular (ICV) infusion of a mineralocorticoid receptor (MR) blocker prevents the hypertension. To assess the role of aldosterone locally produced in the brain, we evaluated the effects of chronic central blockade with the aldosterone synthase inhibitor FAD286 and the MR blocker spironolactone on changes in aldosterone and corticosterone content in the hypothalamus and the increase in CSF [Na(+)] and hypertension induced by high salt intake in Dahl S rats. After 4 wk of high salt intake, plasma aldosterone and corticosterone were not changed, but hypothalamic aldosterone increased by approximately 35% and corticosterone tended to increase in Dahl S rats, whereas both steroids decreased by approximately 65% in Dahl salt-resistant rats. In Dahl S rats fed the high-salt diet, ICV infusion of FAD286 or spironolactone did not affect the increase in CSF [Na(+)]. ICV infusion of FAD286 prevented the increase in hypothalamic aldosterone and 30 mmHg of the 50-mmHg BP increase induced by high salt intake. ICV infusion of spironolactone fully prevented the salt-induced hypertension. These results suggest that, in Dahl S rats, high salt intake increases aldosterone synthesis in the hypothalamus and aldosterone acts as the main MR agonist activating central pathways contributing to salt-induced hypertension.

MODULATION OF CARRAGEENAN-INDUCED HIND PAW EDEMA BY SUBSTANCE P

Substance P has been implicated as a mediator of inflammation. The involvement of this neuropeptide in carrageenan-induced hind paw edema in the rat was assessed. Subcutaneous injection of carrageenan into the rat paw caused a significant increase in substance P levels, which preceded the onset of inflammation. While injection of substance P alone caused mild edema, coadministration of submaximal doses of carrageenan and substance P resulted in a synergistic exacerbation in the degree of inflammation. This synergistic response was not detected when the nonamidated precursor of substance P was coinjected with carrageenan. The effects of substance P depletion on inflammation were also evaluated. In animals pretreated with capsaicin followed by injection with carrageenan, no significant increase in either the levels of substance P or the extent of edema was observed when compared to capsaicin-treated controls. These results indicate that substance P may play an important role in the early stages of carrageenan-induced paw edema and that a reduction in the biosynthesis of substance P may lessen the severity of this inflammatory response.

Phosphorylation of neurofilament proteins by protein kinase C.

The low molecular mass (70 kDa) subunit of neurofilaments (NF‐L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site‐specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two‐dimensional α‐chymotryptic phosphopeptide map analyses of the NF‐L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse‐radiolabeled with [32P]orthophosphate in vivo.