Drug discovery faces an efficacy crisis to which ineffective mainly single-target and symptom-based rather than mechanistic approaches have contributed. We here explore a mechanism-based disease definition for network pharmacology. Beginning with a primary causal target, we extend this to a second using guilt-by-association analysis. We then validate our prediction and explore synergy using both cellular in vitro and mouse in vivo models. As a disease model we chose ischemic stroke, one of the highest unmet medical need indications in medicine, and reactive oxygen species forming NADPH oxidase type 4 (Nox4) as a primary causal therapeutic target. For network analysis, we use classical protein–protein interactions but also metabolite-dependent interactions. Based on this protein–metabolite network, we conduct a gene ontology-based semantic similarity ranking to find suitable synergistic cotargets for network pharmacology. We identify the nitric oxide synthase (Nos1 to 3) gene family as the closest target to Nox4. Indeed, when combining a NOS and a NOX inhibitor at subthreshold concentrations, we observe pharmacological synergy as evidenced by reduced cell death, reduced infarct size, stabilized blood–brain barrier, reduced reoxygenation-induced leakage, and preserved neuromotor function, all in a supraadditive manner. Thus, protein–metabolite network analysis, for example guilt by association, can predict and pair synergistic mechanistic disease targets for systems medicine-driven network pharmacology. Such approaches may in the future reduce the risk of failure in single-target and symptom-based drug discovery and therapy.

https://www.pnas.org/content/pnas/116/14/7129.full.pdf

From single drug targets to synergistic network pharmacology in ischemic stroke.

This uncontrolled open label study evaluated the effect of dipeptidyl peptidase-4 inhibitors (DPP-4i): sitagliptin and vildagliptin on augmentation index standardized for 75 beats per minute (cAiX@75), blood pressure (BP), lipid profile and high-sensitivity C-reactive protein (hsCRP) in patients with type 2 diabetes mellitus (T2DM). Fifty-one well-regulated T2DM patients were randomly assigned to either sitagliptin or vildagliptin (100 mg/day) for 3 months continuing their previous treatment. Lipid profile, cAiX@75, hsCRP, glycated hemoglobin (HbA1c) were measured at baseline at 4, 8 and 12th week were accessed. cAiX@75 and pulse wave velocity (PWV) were determined by SphygmoCor device. Following DPP-4 treatment there was a significant reduction in total serum cholesterol (5.18 vs 4.62 mmol/L), low-density lipoprotein (2.89 vs 2.54 mmol/L), hsCRP (3.21 vs 1.95 mg/L), cAiX@75 (24.5 vs 22.3) and central systolic BP (131.8 vs 119.5 mmHg). The sitagliptin treated group reached cAiX@75 reduction earlier in the study period while neither sitagliptin or vildagliptin use resulted in the significant HbA1c reduction. The treatment with DPP-4i: sitagliptin and vildagliptin provides favorable metabolic and vascular effects beyond glucose-control. Further studies are required to elucidate their implication in metabolic pathways.

/pdf/dipeptidyl-peptidase-4-inhibitors-improve-arterial-stiffness-fetgb1f2fl.pdf

Dipeptidyl peptidase-4 inhibitors improve arterial stiffness, blood pressure, lipid profile and inflammation parameters in patients with type 2 diabetes mellitus

Targeting epigenetic machinery: Emerging novel allosteric inhibitors

The 1998 Nobel Prize in Medicine and Physiology for the discovery of nitric oxide, a nitrogen containing reactive oxygen species (also termed reactive nitrogen or reactive nitrogen/oxygen species) stirred great hopes. Clinical applications, however, have so far pertained exclusively to the downstream signaling of cGMP enhancing drugs such as phosphodiesterase inhibitors and soluble guanylate cyclase stimulators. All clinical attempts, so far, to inhibit NOS have failed even though preclinical models were strikingly positive and clinical biomarkers correlated perfectly. This rather casts doubt on our current way of target identification in drug discovery in general and our way of patient stratification based on correlating but not causal biomarkers or symptoms. The opposite, NO donors, nitrite and enhancing NO synthesis by eNOS/NOS3 recoupling in situations of NO deficiency, are rapidly declining in clinical relevance or hold promise but need yet to enter formal therapeutic guidelines, respectively. Nevertheless, NOS inhibition in situations of NO overproduction often jointly with enhanced superoxide (or hydrogen peroxide production) still holds promise, but most likely only in acute conditions such as neurotrauma (Stover et al., J Neurotrauma 31(19):1599-1606, 2014) and stroke (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016; Casas et al., Proc Natl Acad Sci U S A 116(14):7129-7136, 2019). Conversely, in chronic conditions, long-term inhibition of NOS might be too risky because of off-target effects on eNOS/NOS3 in particular for patients with cardiovascular risks or metabolic and renal diseases. Nitric oxide synthases (NOS) and their role in health (green) and disease (red). Only neuronal/type 1 NOS (NOS1) has a high degree of clinical validation and is in late stage development for traumatic brain injury, followed by a phase II safety/efficacy trial in ischemic stroke. The pathophysiology of NOS1 (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016) is likely to be related to parallel superoxide or hydrogen peroxide formation (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016; Casas et al., Proc Natl Acad Sci U S A 114(46):12315-12320, 2017; Casas et al., Proc Natl Acad Sci U S A 116(14):7129-7136, 2019) leading to peroxynitrite and protein nitration, etc. Endothelial/type 3 NOS (NOS3) is considered protective only and its inhibition should be avoided. The preclinical evidence for a role of high-output inducible/type 2 NOS (NOS2) isoform in sepsis, asthma, rheumatic arthritis, etc. was high, but all clinical development trials in these indications were neutral despite target engagement being validated. This casts doubt on the role of NOS2 in humans in health and disease (hence the neutral, black coloring).

Nitric Oxide Synthase Inhibitors into the Clinic at Last

After publication of our article [1] the authors have notified us that one of the names has been incorrectly spelled.

/pdf/correction-to-efficacy-of-ronopterin-vas203-in-patients-with-2o7xid2jng.pdf

Correction to: Efficacy of Ronopterin (VAS203) in Patients with Moderate and Severe Traumatic Brain Injury (NOSTRA phase III trial): study protocol of a confirmatory, placebocontrolled, randomised, double blind, multi-centre study.

Aims Reduced nitric oxide (NO) availability may adversely affect renal perfusion and glomerular filtration. The aim of the present study was to characterize in detail the pharmacological effects of VAS203, an inhibitor of NO synthase, on renal haemodynamics in humans. Methods This double-blind, randomized, placebo-controlled, cross-over phase-I-study comprised 18 healthy men. Renal haemodynamics were assessed with constant-infusion input-clearance technique with p-aminohippurate and inulin for renal plasma flow (RPF) and glomerular filtration rate (GFR), respectively. After baseline measurement, a constant infusion of the tetrahydrobiopterin analogue ronopterin (VAS203, total 10 mg/kg body weight) or placebo was administered at random order for 6 hours additionally. After a wash-out phase of 28 days, the second course was applied. In parallel, markers of early kidney injury and renal function were assessed repeatedly up to 48 hours after starting VAS203/placebo-infusion. Results VAS203-infusion resulted in a significant decrease of RPF (P .20) have been observed. Conclusions Our phase-I-study in healthy humans indicates that VAS203 (10 mg/kg body weight) reduces renal perfusion and glomerular function within the physiological range mainly due to vasoconstriction at the preglomerular site.

https://bpspubs.onlinelibrary.wiley.com/doi/pdf/10.1111/bcp.13870

Effects of the nitric oxide synthase inhibitor ronopterin (VAS203) on renal function in healthy volunteers.

In type-2 diabetes early vascular changes (among others) are hyperperfusion in the retinal circulation (like in the kidney) and increased pulse wave reflection leading to increased aortic pressure. We analyzed whether the DPP4-inhibitor saxagliptin reverses early vascular and haemodynamic changes in type-2 diabetes.

In this double-blind randomized controlled clinical cross-over trial 42 patients with type-2 diabetes (age 60.3, 13 were females, BMI 30.6 kg/m2, mean duration of diabetes 4 years, HbA1c 7.0%, blood pressure 132/79 mmHg) were consecutively [PB1] included and randomized to placebo or saxagliptin 5 mg for 6 weeks each. Retinal capillary flow (RCF) was assessed at baseline and after flickerlight exposure (as a vasodilatory test) by scanning laser Doppler flowmetry. Central (aortic) systolic blood pressure (SBP), central pulse pressure (PP), augmentation index and pulse wave velocity were determined with the SphygmoCor device.

Following treatment with saxagliptin (as opposed to placebo) saxagliptin effected a better glycemic control, a reduced retinal capillary flow (p=0.033) and, in parallel, reduced central systolic augmentation and pulse pressure (see table). In accordance, Flicker light induced increment of RCF (indicative of vasodilatory capacity of the retinal circulation) was numerically 2-fold greater, although not significant.

View this table:

Table 1





Our data suggest that treatment with saxagliptin for 6 weeks normalizes retinal capillary flow and improves central haemodynamics in type 2 diabetes.

/pdf/effects-of-the-dpp-4-inhibitor-saxagliptin-on-early-vascular-4s56503whz.pdf

Effects of the DPP-4 inhibitor saxagliptin on early vascular changes in the retinal and systemic circulation

In type-2 diabetes early vascular changes (among others) are hyperperfusion in the retinal circulation (like in the kidney) and increased pulse wave reflection leading to increased aortic pressure. We analyzed whether the DPP4-inhibitor saxagliptin reverses early vascular and haemodynamic changes in type-2 diabetes. In this double-blind randomized controlled clinical cross-over trial 42 patients with type-2 diabetes (age 60.3, 13 were females, BMI 30.6 kg/m2, mean duration of diabetes 4 years, HbA1c 7.0%, blood pressure 132/79 mmHg) were consecutively [PB1] included and randomized to placebo or saxagliptin 5 mg for 6 weeks each. Retinal capillary flow (RCF) was assessed at baseline and after flickerlight exposure (as a vasodilatory test) by scanning laser Doppler flowmetry. Central (aortic) systolic blood pressure (SBP), central pulse pressure (PP), augmentation index and pulse wave velocity were determined with the SphygmoCor device. Following treatment with saxagliptin (as opposed to placebo) saxagliptin effected a better glycemic control, a reduced retinal capillary flow (p=0.033) and, in parallel, reduced central systolic augmentation and pulse pressure (see table). In accordance, Flicker light induced increment of RCF (indicative of vasodilatory capacity of the retinal circulation) was numerically 2-fold greater, although not significant.

S Friedrich

Papers

Effects of the nitric oxide synthase inhibitor ronopterin (VAS203) on renal function in healthy volunteers.

Effects of the DPP-4 inhibitor saxagliptin on early vascular changes in the retinal and systemic circulation

Effects of the DPP-4 inhibitor saxagliptin on early vascular changes in the retinal and systemic circulation