text: EMBARGOED FOR RELEASE: 11 A.M. (ET), MONDAY, JANUARY 11, 2016 
   
   Media Advisory: To contact study corresponding author Morgan E. Grams, M.D., call Lauren Nelson at 410-955-8725 or email Laurennelson@jhmi.edu. To contact editorial corresponding author Adam Jacob Schoenfeld, M.D., call Scott Maier at 415-476-3595 or email scott.maier@ucsf.edu. 
   
   To place an electronic embedded link in your story: Links are live at the embargo time: http://archinte.jamanetwork.com/article.aspx?doi=10.1001/jamainternmed.2015.7193; http://archinte.jamanetwork.com/article.aspx?doi=10.1001/jamainternmed.2015.7927 
   
   JAMA Internal Medicine 
   
   Proton pump inhibitors (PPIs), which are commonly used drugs to reduce acid in the stomach, appear to be associated with an increased risk of chronic kidney disease but more research is needed to determine whether PPI use causes kidney damage, according to an article published online by JAMA Internal Medicine. 
   
   PPIs are one of the most commonly prescribe medications in the United States and an estimated 25 percent to 70 percent of these prescriptions may have no appropriate indication for use. Other observational studies have linked PPIs to serious adverse health outcomes. However, the authors note that no population-based studies, to their knowledge, have looked at the association between PPI use and the risk of chronic kidney disease (CKD). 
   
   Morgan E. Grams, M.D., Ph.D., of Johns Hopkins University, Baltimore, and coauthors quantified the association between PPI use and incident CKD in the general population using data on self-reported PPI use in the Atherosclerosis Risk in Communities (ARIC) study (10,482 participants followed up for a median of nearly 14 years) or an outpatient PPI prescription in the Geisinger Health System in Pennsylvania (248,751 participants followed up for a median of six years). The results were replicated at Geisinger. 
   
   At baseline, PPI users in both groups were more likely to have a higher body mass index and take antihypertensive, aspirin or statin medications. 
   
   In the ARIC group, there were 56 incident CKD events among 322 baseline PPI users (14.2 per 1,000-person years) and 1,382 events among 10,160 baseline nonusers (10.7 per 1,000 person-years). PPI use was associated with risk of incident CKD in unadjusted and adjusted analyses. The 10-year estimated absolute risk of CKD among the 322 baseline PPI users was 11.8 percent while the expected risk had they not used PPIs was 8.5 percent, according to the results. 
   
   In the replication group at Geisinger, there were 1,921 incident CKD events among 16,900 baseline PPI users (20.1 per 1,000 person-years) and 28,226 events among 231,851 baseline nonusers (18.3 per 1,000 person-years). PPI use was associated with risk of incident CKD in analyses. The 10-year absolute risk of CKD among the 16,900 baseline PPI users was 15.6 percent and the expected risk had they not used PPIs was 13.9 percent, results indicate. 
   
   The authors note several study limitations, including that participants who are prescribed PPIs may be at higher risk of CKD for reasons unrelated to their PPI use. 
   
   “We note that our study is observational and does not provide evidence of causality. However, a causal relationship between PPI use and CKD could have a considerable public health effect given the widespread extent of use. More than 15 million Americans used prescription PPIs in 2013, costing more than $10 billion. Study findings suggest that up to 70 percent of these prescriptions are without indication and that 25 percent of long-term PPI users could discontinue therapy without developing symptoms. Indeed, there are already calls for the reduction of unnecessary use of PPIs,” the study concludes. 
   
   (JAMA Intern Med. Published online January 11, 2016. doi:10.1001/jamainternmed.2015.7193. Available pre-embargo to the media at http://media.jamanetwork.com.) 
   
   Editor’s Note: The study includes funding/support disclosures. Please see the article for additional information, including other authors, author contributions and affiliations, financial disclosures. 
   
   Editorial: Adverse Effects Associated with Proton Pump Inhibitors 
   
   Adam Jacob Schoenfeld, M.D., and Deborah Grady, M.D., M.P.H., of the University of California, San Francisco, wrote a related editorial summarizing recent data on the adverse effects of PPI use. 
   
   “A large number of patients are taking PPIs for no clear reason – often remote symptoms of dyspepsia or “heartburn” that have since resolved. In these patients, PPIs should be stopped to determine if symptomatic treatment is needed,” they conclude. 
   
   (JAMA Intern Med. Published online January 11, 2016. doi:10.1001/jamainternmed.2015.7927. Available pre-embargo to the media at http://media.jamanetwork.com.) 
   
   Editor’s Note: Please see the article for additional information, including other authors, author contributions and affiliations, financial disclosures. 
   
   # # # 
   
   For more information, contact JAMA Network Media Relations at 312-464-JAMA (5262) or email mediarelations@jamanetwork.org. ||||| Popular Heartburn Pills Can Be Hard To Stop, And May Be Risky 
   
   Enlarge this image Katherine Streeter for NPR Katherine Streeter for NPR 
   
   When Marcella Lafayette started having really bad heartburn, she went to her doctor to see if there was anything that might help. 
   
   "I was experiencing a lot of chest pain, back pain caused from heartburn," says Lafayette, 62, of Portland, Ore. 
   
   Her doctor diagnosed her with gastroesophageal reflux disease, or GERD, and prescribed a drug called a proton pump inhibitor, or PPI. The drug worked, but Lafayette soon started having other problems, such as muscle weakness and severe leg cramping. 
   
   Lafayette discovered she was suffering from a magnesium deficiency, a side effect of the drug. She also learned that PPIs can cause not just deficiencies of nutrients but other side effects, such as an increased risk for infections. 
   
   So Lafayette decided to try to stop using her PPI. But whenever she did, her heartburn returned. And the symptoms were much worse. 
   
   "I can't seem to get off the drug, because when I do, I experience severe stomach pain. I can't eat anything without experiencing stomach pain," Lafayette says. "It just feels like you've got a knife in your gut. It's just really painful." 
   
   Lafayette is not alone. Many people have trouble discontinuing PPIs because the amount of acid in their digestive system surges when they stop taking the drug. 
   
   Experiences like Lafayette's, and rising evidence that the drugs may be associated with a variety of increased risks, are making doctors increasingly worried about their wide use. 
   
   "The teaching for many years was that these drugs were quite safe," says John Clarke, a gastroenterologist at Johns Hopkins Hospital in Baltimore. "But there is data that's emerging that suggests PPIs may not be as safe as we think they are." 
   
   An estimated 15 million Americans use PPIs, which are sold by prescription and over the counter under a variety of brand names, including Nexium, Prilosec and Prevacid. 
   
   They work by blocking production of stomach acid. And that could be the root of the problem, according to Clarke. Stomach acid helps digest food and also has a "barrier function against different pathogens which are ingested," he says. 
   
   So when there's less stomach acid, it leaves people vulnerable to nutritional deficiencies and infections, including food poisoning like salmonella, a serious, sometimes life-threatening digestive system infection called Clostridium difficile, and perhaps pneumonia. 
   
   Stomach acid also helps digest food. So if you don't have any of that acid, it can make it hard to get the vitamins, minerals and other nutrients you need from your food — including nutrients that keep bones strong and prevent fractures. 
   
   In addition, one recent study suggested people who take PPIs may be at greater risk of heart disease; another suggested the drugs could increase the risk for chronic kidney disease. 
   
   Updated Feb. 15, 11 a.m. ET: The latest concern is that PPIs might increase the risk for dementia. Britta Haenisch and colleagues at the German Center for Neurodegenerative Diseases in Bonn studied 73,679 people ages 75 and older. The researchers found regular PPI users had at least a 44 percent increased risk of dementia compared with those not using the drugs. 
   
   The researchers caution that the risk could only be considered an "association," until more research could be conducted to produce more conclusive evidence. But the findings indicate "the avoidance of PPI medication may contribute to the prevention of dementia." 
   
   How PPIs might increase the risk for dementia is unclear. But other researchers recently reported that, in the brains of mice, PPIs seem to increase levels of a damaging protein that accumulates in the brains of dementia patients, known as beta-amyloid. 
   
   In an editorial accompanying the study in the journal JAMA Neurology, Lewis Kuller of the University of Pittsburgh wrote that the findings "provided an important and interesting challenge to evaluate the possible association," which is a "very important issue given" how commonly the drugs are used by the elderly, who are already at increased risk for dementia. 
   
   Our original post continues: As this evidence has emerged, Clarke says, "I think it's imperative that people who take these drugs really make sure that they are looking at the risks versus benefit and people don't use these drugs lightly." 
   
   Many people take PPIs when they don't really need them, Clarke says. They could get rid of their heartburn by making lifestyle changes, such as losing weight and cutting back on alcohol, caffeine and spicy and fatty foods. And many people stay on them a lot longer than they need them, he says. PPIs are usually supposed to be taken for two to eight weeks, although doctors may recommend more. 
   
   The companies that make PPIs say they're safe for most people if they use them the way they're supposed to. And doctors say many people really need to take a PPI for severe heartburn. 
   
   "Proton pump inhibitors do have some very positive benefits to patients," says Kenneth DeVault, a gastroenterologist at the Mayo Clinic who is president of the American College of Gastroenterology. "They relieve symptoms better than any other medication that has ever been developed." 
   
   The most important "positive effect of proton pump inhibitors is restoration of a quality of life," DeVault says. "This is probably the big one." 
   
   PPIs may also reduce the risk for esophageal cancer for some people, he says. 
   
   DeVault says if someone really needs a PPI, they should take one. But they should try everything else first, keep an eye out for any side effects, and talk to their doctor about how long they should stay on it. ||||| A key event in the pathogenesis of Alzheimer’s disease (AD) is the accumulation of amyloid-β (Aβ) species in the brain, derived from the sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Based on a systems biology study to repurpose drugs for AD, we explore the effect of lansoprazole, and other proton-pump inhibitors (PPIs), on Aβ production in AD cellular and animal models. We found that lansoprazole enhances Aβ37, Aβ40 and Aβ42 production and lowers Aβ38 levels on amyloid cell models. Interestingly, acute lansoprazole treatment in wild type and AD transgenic mice promoted higher Aβ40 levels in brain, indicating that lansoprazole may also exacerbate Aβ production in vivo. Overall, our data presents for the first time that PPIs can affect amyloid metabolism, both in vitro and in vivo. 
   
   Competing interests: Albert Pujol and Mireia Coma are employed by Anaxomics Biotech. However, there are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. 
   
   Funding: The present work has been supported by the Spanish Ministerio de Ciencia e Innovación (PSE-010000-2009-1; BIO2010-22073) and by the own fund of each institute, and the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 
   
   Copyright: © 2013 Badiola et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 
   
   In this study, we explore the effects of lansoprazole, and other PPIs, on β-amyloid production in a well-established cellular model of amyloid pathology, with special attention to the effect over the different Aβ species. We assess the in vivo relevance of our findings in wild-type (wt) and AD triple transgenic (3xTg-AD) mice and we ultimately speculate about the potential mechanisms underlying the observed alterations. 
   
   Lansoprazole is a proton pump inhibitor (PPI) drug widely used in the treatment of peptic ulcer disease and other conditions where inhibition of gastric secretion may be beneficial [26] , [27] . PPIs are generally well tolerated, and adverse effects are relatively infrequent [28] , [29] . Yet, chronic administration of PPIs is becoming increasingly common, and there is a growing concern about potential unexplored adverse effects from such long-term therapy [30] . 
   
   Despite all the efforts put on AD research over the past years, there are no effective treatments to prevent, halt or cure the disease. Indeed, there are only four FDA-approved drugs for AD treatment, although they mainly provide a symptomatic improvement and are unable to stop the disease progression [22] . This prompted us to use the therapeutic performance mapping system (TPMS) [23] to explore potential novel indications of marketed drugs to modify the biology of AD. In brief, the TPMS is a top-down systems biology approach with potential applications in drug repositioning [24] . Starting from the clinical effects produced by different therapeutic compounds (both, positive and negative), we first split them into causative physiological motifs and identified the responsible molecular effectors, which were then mapped onto the disease-related cell network. We afterwards established different relationships between drug targets and effector proteins in the network that are used for training a classifier, with capacity for predicting and scoring novel potential indications on AD of totally unrelated drugs. The complete results of this study will be published elsewhere [25] . Interestingly, the TPMS analysis suggested that lansoprazole could act as a strong potential modulator of the processes involved in amyloid-β pathology. 
   
   More recently, an alternative hypothesis has been growing and gaining support, based on the idea that the toxic component is within the soluble fraction. There is data showing that soluble forms of Aβ correlate more closely with dementia severity than fibrillar Aβ [9] , [10] , [11] , [12] and that Aβ oligomers alter dendritic spine density and affect hippocampal synaptic plasticity in vivo [13] , [14] , [15] , [16] , [17] . Furthermore, it has been demonstrated that brain oligomeric Aβ, but not total amyloid plaque burden, correlates with neuronal loss and astrocyte inflammatory response in APP/Tau transgenic mouse model [18] . In this context, several studies show the presence of Aβ oligomers in CSF of AD patients, while not in healthy individuals, and also the direct correlation of oligomers with cognitive impairment [19] , [20] , [21] . 
   
   Several studies support the hypothesis that classic fibrillar amyloid plaques are deleterious to the brain, showing that the subpopulation of dense-core Aβ plaques in particular, the so-called neuritic plaques, are intimately associated with local dendritic spine loss, changes in neurites, and gliosis in AD and mouse models [5] , [6] . However, the total number of amyloid plaques do not correlate well with the severity of illness [7] or with loss of neurons [8] , arguing against a direct causal effect of plaques on cognition or neuronal cell death in AD. 
   
   Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide. Many molecular lesions have been detected in AD, but one of the major pathological hallmarks is the extracellular deposition of amyloid-β (Aβ) peptides in the brain [1] , that results in oxidative and inflammatory damage, which in turn leads to energy failure and synaptic dysfunction [2] . Formation of Aβ species is caused by the sequential cleavage of amyloid precursor protein (APP) by two proteases, β-secretase, also called β-site APP-cleaving enzyme 1 (BACE1), and γ-secretase. Aβ40 is the major secreted form while Aβ42 has been suggested to be the main pathological species in AD pathogenesis [3] , although other truncated Aβ peptides might also contribute substantially to toxicity and amyloidogenesis [4] . 
   
   We determined lysate protein concentration using the Bio-Rad D C protein assay (Bio-Rad Laboratories). We loaded 20–40 µg of each cell lysate and electrophoresed in 10% Tris-glycine gels for Western blot analysis. For Aβ species detection, supernatants we run in 11% urea gels. For the immunoblotting we incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-C-terminal APP (Sigma-Aldrich), rabbit polyclonal anti-BACE (catalytic domain, Abcam), rabbit polyclonal anti-sAPPβ (IBL), 6E10 (against Aβ 1–16, Covance) or mouse monoclonal anti-actin (Sigma-Aldrich) antibodies. Afterwards, we incubated with either an HRP-conjugated secondary antibody plus enhanced chemiluminescence substrate (Millipore), or with an infrared fluorescent-labelled secondary antibody (IRDye, Rockland Immunochemicals, Gilbertsville, PA) for 1 h at room temperature. 
   
   We thawed 3xTg-AD mouse brains on ice in 3× (w/v) 2% SDS with a protease inhibitor cocktail (Complete® EDTA-free, Roche) and then we homogenized them. Next, we centrifuged homogenates at 100.000 g for 1 h at 4°C. Finally, we collected the supernatants and we stored samples at −80°C as SDS-soluble Aβ fractions. 
   
   We thawed non-transgenic mouse brains on ice in 3× (w/v) 0.2% of diethylamine (DEA) and 50 mM of NaCl buffer with a protease inhibitor cocktail (Complete® EDTA-free, Roche), and then we homogenized them. We subsequently centrifuged homogenates at 100.000 g for 1 h at 4°C. Finally, we collected the supernatants, and neutralized them by addition of 1∶10 volume of 0.5 M Tris-HCl pH 6.8. We stored samples at −80°C as DEA-soluble Aβ fractions. 
   
   We treated both female 3xTg-AD mice (Charles River) and non-transgenic mice (B6129SF1/J) at 7 month of age for 5 consecutive days with an intraperitoneal injection of lansoprazole. We diluted and administered lansoprazole in 10% DMSO and 18% of encapsin (2-hydroxipropil beta-cyclodextrine), at 20 mg/kg or 100 mg/kg, respectively. At the end point, we sacrificed mice 5 h after the last treatment and we froze each hemisphere in liquid nitrogen and stored them at −80°C. 
   
   All animals were housed in an animal facility that is fully compliant with the European policy on the use of Laboratory Animals. Experimental protocols were approved by the Parc Científic of Barcelona Committee and meet the European and Spanish guidelines of animal experimentation. 
   
   We used W0-2antibody and Protein-G Sepharose beads to immunoprecipitate human Aβ from conditioned media. We washed Sepharose beads twice in PBS and twice in 100 mM ammonium acetate. We then eluted Aβ twice with 300 µl of 50% acetic acid and vacuum-dried. Finally, we resuspended the samples in 10 µl of 33% acetonitrile containing 0.1% tri-fluoric acetic acid and ultrasonicated. Afterwards we analyzed Aβ species by MALDI-MS on sinapinic acid matrix with an UltraflexII TOF/TOF (BrukerDaltonics). 
   
   We quantified human Aβ42 and Aβ40 levels by human β-amyloid (1–42) and (1–40) ELISA kits (Wako Pure Chemical Industries), respectively. We loaded 100 µL of conditioned media on every assay. For 3xTg-AD mouse brain extracts we previously diluted them 1/40 in standard buffer and then we loaded following manufacturer’s protocol. We measured rodent Aβ40 and Aβ42 levels with ELISA kits (IBL International) according to manufacturer’s protocol. 
   
   For Aβ production analysis, we seeded PS70 cells in 12-well plates at a density of 150,000 cells per well and we subsequently treated for 24 h with lansoprazole, omeprazole, pantoprazole or esomeprazole (Sigma-Aldrich) at different concentrations. We used the γ–secretase inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, Sigma-Aldrich) at 2 µM as a positive control. Lastly, we collected and stored cell culture supernatants at −80°C until further use. 
   
   Results and Discussion 
   
   Lansoprazole and Other PPIs Increase Aβ Levels in AD-like Cellular Models To investigate the potential effect of lansoprazole on Aβ production, we treated PS70 Chinese hamster ovary (CHO) cells stably expressing both wild-type human APP and presenilin 1 (PS1) with increasing concentrations of lansoprazole for 24 h. We first used the MTT reduction assay as a proxy to determine whether the tested concentrations caused cell toxicity, which was not the case below 50 µM (data not shown), in agreement with previous studies [32]. We subsequently measured the Aβ40 and Aβ42 levels in conditioned media of cultures by specific ELISA immunoassays. We observed that Aβ40 levels in the conditioned media increased after treatments above 10 µM (Figure 1A), up to 2-fold increase in the amount of Aβ40 with respect to the vehicle-treated cultured cells. Furthermore, we observed a dose-dependent increase in Aβ42 between 5 µM and 50 µM (Figure 1A), with more than 200% Aβ42 increase at 25 µM and over 300% when treated with 50 µM. As expected, cells treated with the γ–secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) showed negligible levels of Aβ. These results indicate that lansoprazole treatment increase Aβ production at commonly used concentrations [32], [33], [34], [35], [36], which in turn are comparable to other Aβ inducers such as the calcium ionophore A23187, which increases the production of Aβ approximately 3-fold [37], or caffeine (at millimolar concentrations), that increases Aβ levels until 4-fold [38]. In addition, we have observed an Aβ42 increase of over 250% compared to vehicle in cell cultures treated at 50 µM lansoprazole, while fenofibrate, a potent Aβ42 raising Aβ38 lowering compound, generates an increase of 125% at the same concentration [39]. Overall, these findings provide further evidence about the accuracy of the TPMS to predict pharmacological alterations in Aβ metabolism. PPT PowerPoint slide 
   
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   larger image TIFF original image Download: Figure 1. Lansoprazole and similar PPIs increase Aβ levels at 5 µM–50 µM range in AD-like cells. A, Treatment of PS70 cells with lansoprazole at different concentrations (250 nM–50 µM) for 24 h increased Aβ40 and Aβ42 levels, as measured by ELISA immunoassays (n = 6± SD) p<0.05 (+), p<0.01 (*). B, Similar treatment with omeprazole, pantoprazole and esomeprazole at different concentrations (5 µM–50 µM) also increased Aβ40 and Aβ42 levels (n = 4± SD) p<0.05 (+), p<0.01 (*). https://doi.org/10.1371/journal.pone.0058837.g001 To check whether these results could also be extrapolated to other PPIs, we examined the effect of compounds of the same drug class on Aβ production. Omeprazole is one of the most widely prescribed drugs worldwide, which is administered as a racemic mixture of its two enantiomers, S-omeprazole (esomeprazole) and R-omeprazole. Similarly, pantoprazole is also broadly used because of its relatively long duration of action compared with other PPIs, and its lower propensity to become activated in slightly acidic body compartments [40]. Since the chemical structures and pharmacological targets of the four PPIs are very similar, our TPMS computational analyses also predicted an effect of these compounds on the Aβ metabolism. Thus, we decided to investigate the effect of omeprazole, pantoprazole and esomeprazole within 5 µM and 50 µM range, based on our previous results. Although the increase in Aβ40 was non-homogeneous for the 3 drugs, they did show a dose-dependent increase in Aβ42 levels (Fig. 1B), achieving up to 175% increase. Hence, these results suggest that the modulation of Aβ production is a shared feature among the PPI drug class. 
   
   Lansoprazole Might Modulate the γ-secretase Complex To gain a deeper insight in the relative formation of Aβ species induced by lansoprazole, we conducted an Aβ-immunoprecipitation coupled with mass spectrometry analysis of high dose lansoprazole-conditioned media. Different Aβ species are generated because γ–secretase has multiple APP cleavage sites. The main produced species is Aβ40 and, to a lower extent, Aβ38 and Aβ42. Intriguingly, small molecule drugs called γ–secretase modulators (GSM) are able to shift the γ–secretase cleavage site, being classified as straight GSMs when they lower Aβ42 and rise Aβ38 [41], or as inverse GSMs (iGSM) when they do the opposite [39], [42], [43]. Interestingly, our MALDI-MS analysis revealed a considerably altered Aβ peptide pattern in cells treated with lansoprazole at 50 µM. The relative levels of Aβ42 increased whereas the relative levels of Aβ38 decreased (Figure 2A). Intriguingly, there was also an increase in Aβ37. As expected, the γ-secretase inhibitor DAPT completely abrogated Aβ production, showing no Aβ peaks. We further confirmed the Aβ42 increase and Aβ38 decrease by Western blot (Figure 2B). Therefore, attending to the results obtained with lansoprazole in the Aβ species production shift, one possible explanation would be that lansoprazole might act as an inverse GSM. PPT PowerPoint slide 
   
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   larger image TIFF original image Download: Figure 2. Lansoprazole changes the production levels of several Aβ species. A, MALDI-MS analysis of Aβ-immunoprecipitated species from conditioned PS70 supernatants. Cells treated with lansoprazole at 50 µM for 24 h showed a significant increase on Aβ42 compared to vehicle spectrum. DAPT treated cells were used as a negative control, showing none Aβ species as expected. B, Western blot analysis of the different Aβ species present in treated PS70 supernatants. A decrease on Aβ38 and increase on Aβ42 was detected in conditioned media of treated cells with lansoprazole at 50 µM for 24 h. Short and long exposures are shown for better visualization. C, Aβ40/42 levels in conditioned media from treated PS70 cells were measured by ELISA immunoassays (n = 3± SD) p<0.01 (*). Cells treated with both lansoprazole at 50 µM and R-flurbiprofen at 200 µM for 24 h displayed reduced Aβ42 levels when compared to lansoprazole-only treated cells, indicating the lansoprazole is capable to counteract the R-flurbiprofen Aβ42 lowering effect. D, Western blot analysis of APP and BACE1 protein levels in total lysates, showing no differences between treated and non-treated cells. A representative experiment is shown (n = 3 independent experiments). E, Western blot analysis of sAPPβ and sAPPα protein levels in conditioned media. The immunoblot shows an increase in sAPPβ in conditioned media from cells treated with lansoprazole. A representative experiment is shown (n = 3 independent experiments). https://doi.org/10.1371/journal.pone.0058837.g002 To further investigate this hypothesis, we wanted to test if lansoprazole was able to counterbalancing the Aβ42-lowering capability of R-flurbiprofen, a well characterized non-steroidal anti-inflammatory drug (NSAID) that acts as straight GSM [44], [45]. NSAIDs are widespread used due to the prevalence of diseases in the aging population and to their crucial role as effective antipyretic analgesics in a wide spectrum of conditions and diseases ranging from a common cold to rheumatoid arthritis [46]. However, they are known to disrupt the mucosal resistance to gastric acid through several mechanisms including suppression of prostaglandin production and are thus associated with adverse events such as gastric or duodenal ulcers. For that reason, the co-administration with PPIs is strongly recommended in certain circumstances [47], [48]. Notably, albeit we can find straight and inverse GSM modulators within NSAIDs, the former have been considered as very interesting therapeutic agents in AD, since they can lower Aβ42 without perturbing the cyclooxygenase (COX) activity, the principal pharmacological target of NSAIDs [41], [49]. As expected, cells treated with R-flurbiprofen showed decreased Aβ42 levels while cells treated with lansoprazole increased Aβ42 levels (Figure 2C). Interestingly, when the two drugs were combined, lansoprazole blocked the Aβ42 decrease induced by R-flurbiprofen. Therefore, these results suggest that concomitant administration of PPIs with NSAIDs may neutralize the Aβ42 lowering effect provided by NSAIDs, at least in cell culture. Taken together, these findings suggest that lansoprazole increase Aβ42 production similarly to other described iGSMs. Nevertheless, alternative mechanisms related to APP dimerization processes could also play a role in the observed changes of Aβ42 levels [50]. 
   
   Lansoprazole Might Increase BACE1 and Meprin β Protease Activities The putative iGSM activity of lansoprazole could explain the increase of Aβ42 coupled to a decrease of Aβ38, but it cannot account for the rise of Aβ40. Hence, we wanted to investigate other possible effects of lansoprazole on Aβ production. The APP can be processed by two different pathways: the non-amyloidogenic pathway, involving α-secretase and γ-secretase activities, and the amyloidogenic pathway, requiring BACE1 and γ-secretase [2]. If the first cleavage is performed by α-secretase, soluble APPα (sAPPα) and APP C83 C-terminal fragments are generated, and the consecutive cleavage by γ-secretase produces the p3 peptides, which are non-amylodogenic. On other hand, when APP is first cleaved by BACE1, soluble APPβ (sAPPβ) and APP C99 fragments are otherwise generated, and γ-secretase cleavage ultimately generates Aβ peptides and the amyloid precursor protein intracellular domain (AICD) fragments. To explore how lansoprazole was able to increase Aβ37 and Aβ40 levels, we first tested whether it increased APP or BACE1 protein levels, since higher amounts of Aβ substrate or processing enzyme would certainly explain the increase in Aβ production [1], [51]. However, we did not observe any significant change in the protein expression levels of either protein (Figure 2D). We then interrogated if lansoprazole could enhance BACE1 activity instead, and measured the generation of sAPPβ, a BACE1 cleavage product. Interestingly, we observed that lansoprazole promoted sAPPβ production, suggesting an increase of BACE1 activity. In contrast, sAPPα remained unaffected by lansoprazole treatment (Figure 2E). In addition, the mass spectra also revealed a slight increase of the Aβ2-37, Aβ2-40 and Aβ2-42 species (Fig. 2A), which could be accounted for the meprin β metalloprotease, recently identified as an APP cleaving enzyme at the p2 position [52]. Variations in the media pH induced by lansoprazole could thus boost this protease activity, generating Aβ2-x peptides. Overall, these findings suggest that lansoprazole may not only modulate γ-secretase, but also seems to increase BACE1 activity, boosting the Aβ species production that would be eventually reflected in Aβ40 and Aβ37 increased levels. Nevertheless, since the PPIs are known to be irreversible inhibitors of H+/K+ ATPase, lansoprazole-induced variations in the media pH may also affect the activity of other proteases, such as meprin β, and ultimately affecting Aβ production, particularly Aβ2-x species. 
   
   Acute Treatment with Lansoprazole Increases Aβ Levels in Both wt and AD Mouse Models Our results would not go beyond a cellular curiosity if lansoprazole could not cross the blood brain barrier. However, it has been reported that it can indeed cross it and exert its effects in brain tissue [53]. Thus, to determine whether lansoprazole is capable of altering Aβ production in the brain, we conducted short-term intraperitoneal administration in wt and AD triple-transgenic (3xTg-AD) mouse models, like in previous studies [39] (Figure 3). 3xTg-AD mice overexpress human tau and APP in a mutant PS1 knock-in background, and present both plaque and tangle pathologies in an age-related manner [54]. Even though, acute treatments do not enable an evaluation of histopathological or cognitive alteration, since changes in Aβ plaque burden or cognitive impairment usually occur after a long and sustained treatment of at least 2–3 months. Yet, 8-month age transgenic mice typically contain few Aβ plaques but they have significant amounts of intracellular and soluble Aβ [54], being a suitable age to test changes in soluble Aβ. PPT PowerPoint slide 
   
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   larger image TIFF original image Download: Figure 3. Lansoprazole raises Aβ40 production in mice. A, Non-transgenic mice were treated 5 consecutive days with 100 kg/mg of lansoprazole (n = 10). Soluble Aβ40 and Aβ42 from brain extracts were measured by ELISA (n = 10± SD) p<0.05 (+). Lansoprazole increased Aβ40 levels in non-transgenic mice. B, 3xTg-ADwere treated 5 consecutive days with 100 kg/mg of lansoprazole (n = 6). Soluble Aβ40 and Aβ42 from brain extracts were measured by ELISA (n = 6± SD) p<0.05 (+), p<0.01 (*). Lansoprazole increased soluble Aβ40 levels in 3xTg-AD mice in a dose-dependent manner. https://doi.org/10.1371/journal.pone.0058837.g003 We used administration doses of 20 mg/kg/day and 100 mg/kg/day. When compared to the equivalent doses in human [55], the administered concentrations are far below the LC50 (5000 mg/Kg) and comparable to the doses prescribed to treat certain pathologies, such as the Zollinger-Ellison syndrome, 180 mg/day [56]. We found that, indeed, acute treatment with 100 mg/kg/day intraperitoneally administered lansoprazole increased soluble Aβ40 levels in healthy, non-transgenic mice (Figure 3A). Levels of soluble Aβ42 were also slightly increased, although they did not reach statistical significance. In the case of 3xTg-AD mice, short-term treatment with 20 mg/kg/day or 100 mg/kg/day of lansoprazole dramatically raised soluble Aβ40 levels in a dose-dependent manner (Figure 3B). Interestingly, Aβ40 production was higher than in non-transgenic mice, attaining almost to the 250% rise at 100 mg/kg/day. Similarly to the non-transgenic mice, we also observed a moderate increase in soluble Aβ42 levels, although they were not statistically significant either. In order to contextualize the relevance of the effects shown in mice at doses used in this study, it is worth to mention that a recent patent proposes a daily dose up to 400 mg of lansoprazole to inhibit tumor growth in humans [33]. Using the body surface area (BSA) normalization method [55], the conversion of the 20 mg/Kg and 100 mg/Kg mouse doses into equivalent human doses results in around 100 mg/day and 486 mg/day, respectively. Interestingly, we found an increase of Aβ levels in 3xTg-AD mice treated with 20 mg/Kg/day of lansoprazole, providing evidence of a clear effect at human prescribed equivalent doses. The differences observed in the Aβ40/Aβ42 levels between cellular and mouse models could be partially explained by the Aβ quantization, since we only measured extracellular Aβ in cells, while we measured both extracellular and intracellular Aβ species in brain homogenates. Despite these differences, our findings demonstrate that lansoprazole is able to augment Aβ production both in vitro and in vivo models, with an exacerbated effect in AD models. ||||| WASHINGTON, Feb. 15 (UPI) -- The number of potential side effects from the use of proton-pump inhibiting drugs to control heartburn continues to grow, with new research in Germany linking them to the development of dementia. 
   
   A new study links the widely used PPIs -- which include Nexium, Prevacid and Prilosec -- to an increased risk for cognitive decline, though researchers caution the study has limitations, and does not show a definite cause. 
   
   PPIs have recently been linked to kidney disease, heart disease, and deficiencies of B12 and other vitamins. While patients have reported side effects of the drugs, not taking them often results in stomach pains and worse heartburn as the drug leaves their systems. 
   
   "It does not tell us anything that should change medical practice right now," Keith Fargo, director of scientific programs and outreach for the Alzheimer's Association, told CBS News. "I don't think there's going to be an uprising among doctors telling patients not to take their PPIs. This doesn't rise anywhere near the level of evidence you would need for that." 
   
   For the study, published in JAMA Neurology, researchers analyzed patient data collected from 2004 to 2011 by the German health insurer Allgemeine Ortskrankenkassen for 73,679 of its customers. 
   
   The patients, who were 75 years or older and did not have dementia, on PPI drugs showed a 44 percent increased risk of dementia compared with those who did not use them. Researchers said in the study the data is supported by recent studies with mice that found rodents on PPIs had increased levels of amyloid plaques in their brains, the buildup of which contributed to dementia. 
   
   Researchers and doctors note that these studies have not accounted for lifestyle or diet, both of which affect the risk for dementia, or other diseases for which PPIs may increase risk. 
   
   "The teaching for many years was that these drugs were quite safe," John Clarke, a gastroenterologist at Johns Hopkins Hospital, told NPR. "But there is data that's emerging that suggests PPIs may not be as safe as we think they are." ||||| Importance Medications that influence the risk of dementia in the elderly can be relevant for dementia prevention. Proton pump inhibitors (PPIs) are widely used for the treatment of gastrointestinal diseases but have also been shown to be potentially involved in cognitive decline. 
   
   Objective To examine the association between the use of PPIs and the risk of incident dementia in the elderly. 
   
   Design, Setting, and Participants We conducted a prospective cohort study using observational data from 2004 to 2011, derived from the largest German statutory health insurer, Allgemeine Ortskrankenkassen (AOK). Data on inpatient and outpatient diagnoses (coded by the German modification of the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision) and drug prescriptions (categorized according to the Anatomical Therapeutic Chemical Classification System) were available on a quarterly basis. Data analysis was performed from August to November 2015. 
   
   Exposures Prescription of omeprazole, pantoprazole, lansoprazole, esomeprazole, or rabeprazole. 
   
   Main Outcomes and Measures The main outcome was a diagnosis of incident dementia coded by the German modification of the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision. The association between PPI use and dementia was analyzed using time-dependent Cox regression. The model was adjusted for potential confounding factors, including age, sex, comorbidities, and polypharmacy. 
   
   Results A total of 73 679 participants 75 years of age or older and free of dementia at baseline were analyzed. The patients receiving regular PPI medication (n = 2950; mean [SD] age, 83.8 [5.4] years; 77.9% female) had a significantly increased risk of incident dementia compared with the patients not receiving PPI medication (n = 70 729; mean [SD] age, 83.0 [5.6] years; 73.6% female) (hazard ratio, 1.44 [95% CI, 1.36-1.52]; P < .001). 
   
   Conclusions and Relevance The avoidance of PPI medication may prevent the development of dementia. This finding is supported by recent pharmacoepidemiological analyses on primary data and is in line with mouse models in which the use of PPIs increased the levels of β-amyloid in the brains of mice. Randomized, prospective clinical trials are needed to examine this connection in more detail. ||||| A popular class of heartburn medications might raise a senior's risk of dementia, a new study suggests. 
   
   Called proton pump inhibitors (PPIs), this group of drugs includes Prilosec, Nexium and Prevacid. They work by lowering the amount of acid produced by the stomach. 
   
   But German researchers found that people 75 or older who regularly take the medications had a 44 percent increased risk of dementia, compared with seniors not using the drugs. The study only found an association, however, and not a cause-and-effect link. 
   
   "To evaluate cause-and-effect relationships between long-term PPI use and possible effects on cognition in the elderly, randomized, prospective clinical trials are needed," said corresponding author Britta Haenisch, from the German Center for Neurodegenerative Diseases in Bonn. 
   
   In the meantime, "Clinicians should follow guidelines for PPI prescription, to avoid overprescribing PPIs and inappropriate use," Haenisch said. 
   
   The report was published Feb. 15 in the journal JAMA Neurology. 
   
   The results are surprising enough that at least one leading expert on aging, Dr. Malaz Boustani, plans to share the findings with older patients who are using PPIs. 
   
   Boustani said earlier studies have linked another type of antacid, H2 blockers, with an increased risk of dementia. Up to now, he's recommended that patients use PPIs to treat acid reflux and steer clear of H2 blockers like Tagamet, Pepcid and Zantac. 
   
   "I'm going to disclose the finding to my patients and then let them decide whether they will take the risk or not," said Boustani, a professor of medicine with the Indiana University Center for Aging Research and a spokesman for the American Federation for Aging Research. "On Monday I have clinic, and if I have patients taking a PPI or an H2 blocker I will tell them exactly what I'm telling you, and then they can decide." 
   
   More than 15 million Americans used prescription PPIs in 2013, at a total cost of more than $10 billion, according to a report by the IMS Institute for Healthcare Informatics. Several popular PPIs -- Prilosec, Prevacid and Zegerid -- also are available over-the counter, further boosting their use. 
   
   Concern has been increasing that Americans might be overusing PPIs to treat minor cases of heartburn or acid reflux. 
   
   As many as 70 percent of PPI prescriptions in the United States have been inappropriately handed out by doctors, and 25 percent of long-term users could stop taking the medication without suffering increased heartburn or acid reflux, according to a study published in January in the journal JAMA Internal Medicine. 
   
   Overuse of PPIs could have drastic effects on health, that study found. For example, the medications have been linked to a 20 percent to 50 percent higher risk of chronic kidney disease. 
   
   And now the German researchers report there also is some evidence that PPI use might affect a person's ability to reason. 
   
   PPIs appear to effect levels of amyloid beta and tau, which are proteins associated with Alzheimer's disease, the German authors said. PPI use can also lead to vitamin B12 deficiency, which has been associated with cognitive decline. 
   
   To test the possible association between PPIs and dementia, the German researchers collected data from a large German health insurance firm on almost 74,000 seniors aged 75 or older. The data ran from 2004 to 2011, and included diagnoses and drug prescriptions. 
   
   About 2,950 patients regularly used PPIs, which for this study was defined as at least one PPI prescription in each quarter of an 18-month interval. 
   
   Regular users of PPIs had a 44 percent increased risk of dementia compared with those not receiving PPI medications. 
   
   However, the study doesn't make clear whether PPIs are also available over the counter in Germany, as they are in the United States, said Dr. Arun Swaminath, director of the inflammatory bowel disease program at Lenox Hill Hospital in New York City. 
   
   If over-the-counter PPIs are available, then more people might have been taking them and the dementia risk described in this paper could be overestimated, Swaminath said. 
   
   "However, I think the point here is that for some patients, weaning off PPIs is reasonable and they should make this decision with their doctors," he said. 
   
   People who want to ease off PPIs can take a number of steps to reduce excess acid or prevent acid reflux, Boustani said. They can eat smaller meals, lay off chocolate and caffeine, and stay upright for a few hours following each meal. 
   
   Boustani added that his group has received federal funding to design clinical trials in which people would be weaned off PPIs, to see how it affects their ability to remember and think. 
   
   Dr. Gisele Wolf-Klein, director of geriatric education at North Shore-LIJ Health System in New Hyde Park, N.Y., added that even the researchers aren't sure why PPIs would affect the aging brain. 
   
   "We don't know what is causing the brain to deteriorate. Until we know this, there's no reason for people who are taking PPIs to be too concerned about that and stop taking those agents if they are needed," Wolf-Klein said. 
   
   Another expert agreed. 
   
   While the study is intriguing, it's not enough for doctors to warn patients off PPI use, said Keith Fargo, director of scientific programs and outreach for the Alzheimer's Association. 
   
   "It does not tell us anything that should change medical practice right now," Fargo said. "I don't think there's going to be an uprising among doctors telling patients not to take their PPIs. This doesn't rise anywhere near the level of evidence you would need for that." 
   
   One of the paper's main flaws is that researchers could not control for diet and body weight as risk factors, Fargo said. 
   
   "Both of those things, we know, are risk factors for developing cognitive decline and dementia in later life, and both of those are reasons why a person might need to take a proton pump inhibitor," Fargo explained.
target: – Certain heartburn drugs have already been linked to heath woes including heart disease and kidney disease. Now a study in JAMA Neurology says those drugs—proton-pump inhibitors like Prilosec, Prevacid, and Nexium—may also boost the chance of dementia, UPI reports. Analyzing a German health insurer's data on nearly 74,000 customers aged 75 and up from 2004 to 2011, researchers found a 44% higher dementia risk among "PPI" users. The finding also coincides with research showing that mice on PPIs had higher amyloid plaque levels in their brains, which has been linked to dementia. So what to do, when roughly 15 million Americans use prescription PPIs and other patients take over-the-counter versions? For now, nothing. More clinical trials are needed to analyze the possible PPI-dementia link, senior study author Britta Haenisch tells CBS News. For one thing, the JAMA Neurology study didn't account for lifestyle and diet, which can influence dementia risk. But, she adds, "clinicians should follow guidelines for PPI prescription, to avoid overprescribing PPIs and inappropriate use." Up to 70% of PPI prescriptions may be needless reactions to minor cases of acid reflux or heartburn, per a recent study. Big picture: PPIs are being seen in a whole new light. "The teaching for many years was that these drugs were quite safe," a gastroenterologist tells NPR. "But there is data that's emerging that suggests PPIs may not be as safe as we think they are." (Turns out vegetable oils may cause dementia when heated.)