Metformin and Aging: A Review.

Metformin is sometimes proposed to be an "anti-aging" drug, based on preclinical experiments with lower-order organisms and numerous retrospective data on beneficial health outcomes for type 2 diabetics. Large prospective, placebo-controlled trials are planned, in pilot stage or running, to find a new use (or indication) for an aging population. As one of the metformin trials has "frailty" as its endpoint, similar to a trial with a plant-derived senolytic, the latter class of novel anti-aging drugs is briefly discussed. Concerns exist not only for vitamin B12 and B6 deficiencies, but also about whether there are adverse effects of metformin on individuals who try to remain healthy by maintaining cardiovascular fitness via exercise.

Novel pharmacological chaperones that correct phenylketonuria in mice.

Phenylketonuria (PKU) is caused by inherited phenylalanine-hydroxylase (PAH) deficiency and, in many genotypes, it is associated with protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin (BH(4)), can act as a pharmacological chaperone (PC) that rescues enzyme function. However, BH(4) shows limited efficacy in some PKU genotypes and its chemical synthesis is very costly. Taking an integrated drug discovery approach which has not been applied to this target before, we identified alternative PCs for the treatment of PKU. Shape-focused virtual screening of the National Cancer Institute's chemical library identified 84 candidate molecules with potential to bind to the active site of PAH. An in vitro evaluation of these yielded six compounds that restored the enzymatic activity of the unstable PAHV106A variant and increased its stability in cell-based assays against proteolytic degradation. During a 3-day treatment study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)-uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice (Pah(enu1)). Notably, benzylhydantoin was twice as effective as tetrahydrobiopterin. In conclusion, we identified two PCs with high in vivo efficacy that may be further developed into a more effective drug treatment of PKU.

Targeting the Kv1.3 potassium channel for immunosuppression in vascularized composite allotransplantation - a pilot study.

Kv1.3-channels are critically involved in activation and function of effector memory T cells. Blocking Kv1.3-channels was investigated for its effect on skin rejection in a rat limb-transplantation-model. Animals received the Kv1.3-blocker correolide C systemically or locally as intra-graft-treatment in combination with tacrolimus. Systemic (intraperitoneal) administration of correolide C resulted in slight, but significant prolongation of allograft survival compared with untreated and placebo treated controls. In 4/6 correolide C treated animals, histology showed an intact epidermis and a mild infiltrate by day 10. High correolide C plasma trough levels correlated with prolonged allograft survival. A decrease in CD4+ and CD8+ effector memory T cells was observed in allograft skin, peripheral blood and the spleen on day 5. When applied subcutaneously in combination with systemic tacrolimus (30 days+/-anti-lymphocyte serum) detectable, but insignificant prolongation of graft survival was achieved. 2/5 animals showed an intact epidermis and a mild infiltrate until day 45. Tapering systemic tacrolimus and weaning on day 50 resulted in rejection by day 55, regardless of local correolide C treatment. Subcutaneous injection did not lead to systemic plasma levels. The Kv1.3-channel is a potential drug target worth exploring in more detail for immunosuppression in vascularized composite allotransplantation.

Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling.

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo.

The recent approval of sapropterin dihydrochloride, the synthetic form of 6[R]-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), for the treatment of phenylketonuria (PKU) as the first pharmacological chaperone drug initiated a paradigm change in the treatment of monogenetic diseases. Symptomatic treatment is now replaced by a causal pharmacological therapy correcting misfolding of the defective phenylalanine hydroxylase (PAH) in numerous patients. Here, we disclose BH(4) responsiveness in Pah(enu1), a mouse model for PAH deficiency. Loss of function resulted from loss of PAH, a consequence of misfolding, aggregation, and accelerated degradation of the enzyme. BH(4) attenuated this triad by conformational stabilization augmenting the effective PAH concentration. This led to the rescue of the biochemical phenotype and enzyme function in vivo. Combined in vitro and in vivo analyses revealed a selective pharmaceutical action of BH(4) confined to the pathological metabolic state. Our data provide new molecular-level insights into the mechanisms underlying protein misfolding with loss of function and support a general model of pharmacological chaperone-induced stabilization of protein conformation to correct this intracellular phenotype. Pah(enu1) will be essential for pharmaceutical drug optimization and to design individually tailored therapies.

Vitamin D, UV, and skin cancer in the elderly: to expose or not to expose?

There is mounting concern about vitamin D insufficiency, especially in the ageing population. Increases in indoor lifestyle, obesity, car travel, clothing habits, the use of photo-protective makeup, and campaigns driven by dermatologists, governments, and the cosmetic industry to avoid or protect against the sun as much as possible are contributing to this trend. In a recent article in Gerontology, Barysch et al. [1] recommend that the ageing population avoid any 'intentional' UV radiation as well as 'minimize sun exposure' based on known dangers of developing skin cancer. They warn that 'studies with vitamin D supplements reported increased risks of prostate and esophagus cancer as well as atopy in childhood' and concluded that 'adequate levels of vitamin D are essential for the elderly', but do not suggest which level is optimal. I will argue that the ageing population should keep their serum 25-(OH)-vitamin D(3) (25-(OH)-D) levels in the 75-100 nmol/l range. An oral cholecalciferol intake of ∼2,000 IU/day is recommended as a supplement throughout the year for those who cannot enjoy the sun in summer or only during 'vitamin D winter' for the others.

Pharmacology of metformin - An update.

Despite being a successful diabetes type 2 drug for more than a half-century in Europe, the mode of action of metformin is still debated. It is the purpose of this review to inform the reader about most recent findings for metformin with respect to its antidiabetic activity as well as proposed benefits beyond glucose control in humans. Clinical evidence now suggests that most of metformin benefits originate from its actions in the gut, involving hormone signaling by glucagon-like peptide 1 and peptide YY. Growth differentiation factor 15, also mainly produced in the gut, was first identified as a biomarker for metformin use but is now suggested to play a significant role in e.g. weight loss of prediabetics. The pharmacokinetics of the drug in humans as basis for pharmacodynamics, resulting in high tissue levels of the intestinal wall, including the colon, proven by biopsies, is presented. A critical survey of metformin actions on mitochondria, increasing the AMP/ATP ratio but also acting as a mild uncoupler, and of postulated new cellular targets (lysosomes) is included.

Torpor: The Rise and Fall of 3-Monoiodothyronamine from Brain to Gut-From Gut to Brain?

3-Monoiodothyronamine (T1AM), first isolated from rat brain, is reported to be an endogenous, rapidly acting metabolite of thyroxine. One of its numerous effects is the induction of a "torpor-like" state in experimental animals. A critical analysis of T1AM, to serve as an endogenous cryogen, is given. The proposed biosynthetic pathway for formation of T1AM, which includes deiodinases and ornithine decarboxylase in the upper intestinum, is an unusual one. To reach the brain via systemic circulation, enterohepatic recycling and passage through the liver may occur. The possible role of gut microbiota is discussed. T1AM concentrations in human serum, measured by a specific monoclonal assay are up to three orders of magnitude higher compared to values obtained by MS/MS technology. The difference is explained by the presence of a high-affinity binder for T1AM (Apolipoprotein B-100) in serum, which permits the immunoassay to measure the total concentration of the analyte but limits MS/MS technology to detect only the unbound (free) analyte, a view, which is contested here.

Discovery of high-affinity ligands of sigma1 receptor, ERG2, and emopamil binding protein by pharmacophore modeling and virtual screening.

ERG2, emopamil binding protein (EBP), and sigma-1 receptor (sigma(1)) are enzymes of sterol metabolism and an enzyme-related protein, respectively, that share high affinity for various structurally diverse compounds. To discover novel high-affinity ligands, pharmacophore models were built with Catalyst based upon a series of 23 structurally diverse chemicals exhibiting K(i) values from 10 pM to 100 microM for all three proteins. In virtual screening experiments, we retrieved drugs that were previously reported to bind to one or several of these proteins and also tested 11 new hits experimentally, of which three, among them raloxifene, had affinities for sigma(1) or EBP of <60 nM. When used to search a database of 3525 biochemicals of intermediary metabolism, a slightly modified ERG2 pharmacophore model successfully retrieved 10 substrate candidates among the top 28 hits. Our results indicate that inhibitor-based pharmacophore models for sigma(1), ERG2, and EBP can be used to screen drug and metabolite databases for chemically diverse compounds and putative endogenous ligands.

Prospective virtual screening in a sparse data scenario: design of small-molecule TLR2 antagonists.

Toll-like receptors (TLRs) are critical signaling molecules with roles in various severe clinical conditions such as sepsis and rheumatoid arthritis, and have therefore been advocated as promising drug targets for the treatment of these diseases. The aim of this study was to discover small-molecule antagonists of TLR2 by computer-aided drug design. This goal poses several challenges due to the lack of available data on TLR2 modulators. To overcome these hurdles we developed a combined structure- and ligand-based virtual screening approach. First, we calculated molecular interaction fields of the TLR2 binding site to derive a structure-based 3D pharmacophore, which was then used for virtual screening. We then performed a two-step shape- and feature-based similarity search using known TLR2 ligands as query structures. A selection of virtual screening hits was biologically tested in a cell-based assay for TLR2 signaling inhibition, leading to the identification of several compounds with antagonistic activity (IC50 values) in the low-micromolar range.

A marriage of two "Methusalem" drugs for the treatment of psoriasis?: Arguments for a pilot trial with metformin as add-on for methotrexate.

In this article we present arguments that the "antidiabetic" drug metformin could be useful as an add-on therapy to methotrexate for the treatment of psoriasis and, perhaps, for rheumatoid arthritis as well. Biochemical data suggest that both drugs may share a common cellular target, the AMP-activated protein kinase (AMPK). This enzyme is a master regulator of metabolism and controls a number of downstream targets, e.g., important for cellular growth or function in many tissues including T-lymphocytes. Clinical observations as well as experimental results argue for anti-inflammatory, antineoplastic and antiproliferative activities of metformin and a case-control study suggests that the drug reduces the risk for psoriasis. Patients with psoriasis have higher risk of metabolic syndrome, type 2 diabetes and cardiovascular mortality. Metformin has proven efficacy in the treatment of prediabetes and leads to a pronounced and sustained weight loss in overweight individuals. We expect that addition of metformin to methotrexate can lead to positive effects with respect to the PASI score, reduction of the weekly methotrexate dose and of elevated cardiovascular risk factors in patients with metabolic syndrome and psoriasis. For reasons explained later we suggest that only male, overweight patients are to be included in a pilot trial. On the other side of the coin are concerns that the gastrointestinal side effects of metformin are intolerable for patients under low dose, intermittent methotrexate therapy. Metformin has another side effect, namely interference with vitamin B12 and folate metabolism, leading to elevated homocysteine serum levels. As patients must receive folate supplementation and will be controlled with respect to their B12 status increased hematological toxicity is unlikely to result.

Does rosuvastatin increase serum levels of 25-hydroxy-vitamin D?

An observational study and a "clinical trial" seem to prove that rosuvastatin (but not fluvastatin) dramatically increases serum levels of 25-(OH)-D3 (three-fold above starting values). A critical analysis of the two publications, presented below, raises serious concerns. Conclusions from these two studies have already been drawn in the scientific literature.It is argued that claiming or believing in a "novel pleiotropic effect of rosuvastatin" may be misleading and premature.

New insights into tetrahydrobiopterin pharmacodynamics from Pah enu1/2, a mouse model for compound heterozygous tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency.

Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH(4)) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah(enu1/2) bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH(4) treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH(4) pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using (13)C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah(enu1/1), and Pah(enu1/2) mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response. Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah(enu1/1) and Pah(enu1/2) indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH(4). In conclusion, our findings show a significant impact of the genotype on the response to BH(4) in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects.

Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol delta8-delta7 isomerase activity.

EBP (emopamil-binding protein) is a high-affinity binding protein for [3H]emopamil and belongs to the family of so-called sigma receptors. Mutations that disrupt EBP's 3beta-hydroxysteroid sterol delta8-delta7 isomerase activity (EC 5.3.3.5) impair cholesterol biosynthesis and cause X-chromosomal dominant chondrodysplasia punctata. We identified a human cDNA for a novel EBPL (EBP-like protein) with a calculated mass of 23.2 kDa. Amino acid sequence alignments and phylogenetic analysis revealed that EBPL is distantly related to EBP (31% identity and 52% similarity) and found in animals but not in plants. EBPL is encoded by four exons on human chromosome 13q14.2 covering 30.7 kb, and a partially processed EBPL pseudogene was found on 16q21. The EBPL mRNA was expressed ubiquitously and most abundant in liver, lung and kidney. Upon heterologous expression in yeast EBPL had no detectable 3beta-hydroxysteroid sterol delta8-delta7 isomerase and sigma-ligand-binding activity. Nine out of ten amino acid residues essential for catalytic activity of EBP were conserved in EBPL. Replacement of the only differing residue (EBP-Y111W) reduced catalytic activity of EBP. Transfer of the divergent residue from EBP to EBPL (EBPL-W91Y) and chimaerization of EBP and EBPL at various positions failed to restore catalytic activity of EBPL. Chemical cross-linking induced homodimerization of EBPL and EBP. Whereas mevinolin increased the mRNA for EBP and DHCR7 (delta7-sterol reductase) in HepG2 cells, it had no effect on mRNAs for EBPL and sigma1 receptor, indicating that EBP and EBPL expression are not co-ordinated. We propose that EBPL has a yet-to-be-discovered function other than cholesterol biosynthesis.

Functional consequences of P/Q-type Ca2+ channel Cav2.1 missense mutations associated with episodic ataxia type 2 and progressive ataxia.

We have investigated the functional consequences of three P/Q-type Ca(2+) channel alpha1A (Ca(v)2.1alpha(1)) subunit mutations associated with different forms of ataxia (episodic ataxia type 2 (EA-2), R1279Stop, AY1593/1594D; progressive ataxia (PA), G293R). Mutations were introduced into human alpha1A cDNA and heterologously expressed in Xenopus oocytes or tsA-201 cells (with alpha(2)delta and beta1a) for electrophysiological and biochemical analysis. G293R reduced current density in both expression systems without changing single channel conductance. R1279Stop and AY1593/1594D protein were expressed in tsA-201 cells but failed to yield inward barium currents (I(Ba)). However, AY1593/1594D mediated I(Ba) when expressed in oocytes. G293R and AY1593/1594D shifted the current-voltage relationship to more positive potentials and enhanced inactivation during depolarizing pulses (3 s) and pulse trains (100 ms, 1 Hz). Mutation AY1593/1594D also slowed recovery from inactivation. Single channel recordings revealed a change in fast channel gating for G293R evident as a decrease in the mean open time. Our data support the hypothesis that a pronounced loss of P/Q-type Ca(2+) channel function underlies the pathophysiology of EA-2 and PA. In contrast to other EA-2 mutations, AY1593/1594D and G293R form at least partially functional channels.