Nuts and health in aging

Nuts and health in aging

Larry H. Bernstein, MD, FCAP, Curator




Nut consumption and age-related disease

Giuseppe GrossoRamon Estruch

MATURITAS · OCT 2015     http://dx.doi.org/10.1016/j.maturitas.2015.10.014

Current knowledge on the effects of nut consumption on human health has rapidly increased in recent years and it now appears that nuts may play a role in the prevention of chronic age-related diseases. Frequent nut consumption has been associated with better metabolic status, decreased body weight as well as lower body weight gain over time and thus reduce the risk of obesity. The effect of nuts on glucose metabolism, blood lipids, and blood pressure are still controversial. However, significant decreased cardiovascular risk has been reported in a number of observational and clinical intervention studies. Thus, findings from cohort studies show that increased nut consumption is associated with a reduced risk of cardiovascular disease and mortality (especially that due to cardiovascular-related causes). Similarly, nut consumption has been also associated with reduced risk of certain cancers, such as colorectal, endometrial, and pancreatic neoplasms. Evidence regarding nut consumption and neurological or psychiatric disorders is scarce, but a number of studies suggest significant protective effects against depression, mild cognitive disorders and Alzheimer’s disease. The underlying mechanisms appear to include antioxidant and anti-inflammatory actions, particularly related to their mono- and polyunsaturated fatty acids (MUFA and PUFA, as well as vitamin and polyphenol content. MUFA have been demonstrated to improve pancreatic beta-cell function and regulation of postprandial glycemia and insulin sensitivity. PUFA may act on the central nervous system protecting neuronal and cell-signaling function and maintenance. The fiber and mineral content of nuts may also confer health benefits. Nuts therefore show promise as useful adjuvants to prevent, delay or ameliorate a number of chronic conditions in older people. Their association with decreased mortality suggests a potential in reducing disease burden, including cardiovascular disease, cancer, and cognitive impairments.


Global life expectancy has increased from 65 years in 1990 to about 71 years in 2013 [1]. As life expectancy has increased, the number of healthy years lost due to disability has also risen in most countries, consistent with greater morbidity [2]. Reduction of mortality rates in developed countries has been associated with a shift towards more chronic non-communicable diseases [1]. Cardiovascular diseases (CVDs) and related risk factors, such as hypertension, diabetes mellitus, hypercholesterolemia, and obesity are the top causes of death globally, accounting for nearly one-third of all deaths worldwide [3]. Equally, the estimated incidence, mortality, and disability- adjusted life-years (DALYs) for cancer rose to 14.9 million incident cancer cases, 8.2 million deaths, and 196.3 million DALYs, with the highest impact of prostate and breast cancer in men and women, respectively [4]. Depression is a leading cause of disability worldwide (in terms of total years lost due to disability), especially in high-income countries, increasing from 15th to 11th rank (37% increase) and accounting for 18% of total DALYs (almost 100 million DALYs) [5]. Overall, the global rise in chronic non-communicable diseases is congruent with a similar rise in the elderly population. The proportion of people over the age of 60 is growing faster than any other age group and is estimated to double from about 11% to 22% within the next 50 years [6]. Public health efforts are needed to face this epidemiological and demographic transition, both improving the healthcare systems, as well as assuring a better health in older people. Accordingly, a preventive approach is crucial to dealing with an ageing population to reduce the burden of chronic disease.

In this context, lifestyle behaviors have demonstrated the highest impact for older adults in preventing and controlling the morbidity and mortality due to non- communicable diseases [7]. Unhealthy behaviors, such as unbalanced dietary patterns, lack of physical activity and smoking, play a central role in increasing both cardiovascular and cancer risk [7]. Equally, social isolation and depression in later life may boost health decline and significantly contribute to mortality risk [8]. The role of diet in prevention of disability and death is a well-established factor, which has an even more important role in geriatric populations. Research has focused on the effect of both single foods and whole dietary patterns on a number of health outcomes, including mortality, cardiovascular disease (CVD), cancer and mental health disorders (such as cognitive decline and depression) [9-13]. Plantbased dietary patterns demonstrate the most convincing evidence in preventing chronic non-communicable diseases [14-17]. Among the main components (including fruit and vegetables, legumes and cereals), only lately has attention focused on foods such as nuts. Knowledge on the effect of nut consumption on human health has increased rapidly in recent years. The aim of this narrative review is to examine recent evidence regarding the role of nut consumption in preventing chronic disease in older people.

Tree nuts are dry fruits with an edible seed and a hard shell. The most popular tree nuts are almonds (Prunus amigdalis), hazelnuts (Corylus avellana), walnuts (Juglans regia), pistachios (Pistachia vera), cashews (Anacardium occidentale), pecans (Carya illinoiensis), pine nuts (Pinus pinea), macadamias (Macadamia integrifolia), Brazil nuts (Bertholletia excelsa), and chestnuts (Castanea sativa). When considering the “nut” group, researchers also include peanuts (Arachis hypogea), which technically are groundnuts. Nuts are nutrient dense foods, rich in proteins, fats (mainly unsaturated fatty acids), fiber, vitamins, minerals, as well as a number of phytochemicals, such as phytosterols and polyphenols [18]. Proteins account for about 10-25% of energy, including individual aminoacids, such as L-arginine, which is involved in the production of nitric oxide (NO), an endogenous vasodilatator [19].

The fatty acids composition of nuts involves saturated fats for 415% and unsaturated fatty acids for 30-60% of the content. Unsaturated fatty acids are different depending on the nut type, including monounsaturated fatty acids (MUFA, such as oleic acid in most of nuts, whereas polyunsaturated fatty acids (PUFA, such as alpha-linolenic acid) in pine nuts and walnuts [20]. Also fiber content is similar among most nut types (about 10%), although pine nuts and cashews hold the least content. Vitamins contained in nuts are group B vitamins, such as B6 (involved in many aspects of macronutrient metabolism) and folate (necessary for normal cellular function, DNA synthesis and metabolism, and homocysteine detoxification), as well as tocopherols, involved in anti-oxidant mechanisms [21]. Among minerals contained in vegetables, nuts have an optimal content in calcium, magnesium, and potassium, with an extremely low amount of sodium, which is implicated on a number of pathological conditions, such as bone demineralization, hypertension and insulin resistance[22]. Nuts are also rich in phytosterols, non-nutritive components of certain plant-foods that exert both structural (at cellular membrane phospholipids level) and hormonal (estrogen-like) activities [23]. Finally, nuts have been demonstrated to be a rich source of polyphenols, which account for a key role in their antioxidant and anti-inflammatory effects.


Metabolic disorders are mainly characterized by obesity, hypertension, dyslipidemia, and hyperglycemia/ hyperinsulinemia/type-2 diabetes, all of which act synergistically to increase morbidity and mortality of aging population.

Obesity Increasing high carbohydrate and fat food intake in the last decades has contributed significantly to the rise in metabolic disorders. Nuts are energy-dense foods that have been thought to be positively associated with increased body mass index (BMI). As calorie-dense foods, nuts may contain 160–200 calories per ounce. The recommendation from the American Heart  Association to consume 5 servings per week (with an average recommended serving size of 28 g) corresponds to a net increase of 800–1000 calories per week, which may cause weight gain. However, an inverse relation between the frequency of nut consumption and BMI has been observed in large cohort studies [24]. Pooling the baseline observations of BMI by category of nut consumption in 5 cohort studies found a significant decreasing trend in BMI values with increasing nut intake [24]. While the evidence regarding nut consumption and obesity is limited, findings so far are encouraging [25, 26]. When the association between nut consumption and body weight has been evaluated longitudinally over time, nut intake was associated with a slightly lower risk of weight gain and obesity [25]. In the Nurses’ Health Study II (NHS II), women who eat nuts ≥2 times per week had slightly less weight gain (5.04 kg) than did women who rarely ate nuts (5.55 kg) and marginally significant 23% lower risk of obesity after 9-year follow-up [25]. Further evaluation of the NHS II data and the Physicians’ Health Study (PHS) comprising a total of 120,877 US women and men and followed up to 20 years revealed that 4-y weight change was inversely associated with a 1-serving increment in the intake of nuts (20.26 kg) [27]. In the “Seguimiento Universidad de Navarra” (SUN) cohort study, a significant decreased weight change has been observed over a period of 6 years [26]. After adjustment for potential confounding factors the analysis was no longer significant, but overall no weight gain associated with >2 servings per week of nuts has been observed. Finally, when considering the role of the whole diet on body weight, a meta-analysis of 31 clinical trials led to the conclusion of a null effect of nut intake on body weight, BMI, and waist circumference [28].

Glucose metabolism and type-2 diabetes The association between nut consumption and risk of type-2 diabetes in prospective cohort studies is controversial [29-32]. A pooled analysis relied on the examination of five large cohorts, including the NHS, the Shanghai Women’s Health Study, the Iowa Women’s Health Study, and the PHS, and two European studies conducted in Spain (the PREDIMED trial) and Finland including a total of more than 230,000 participants and 13,000 cases, respectively. Consumption of 4 servings per week was associated with 13% reduced risk of type-2 diabetes without effect modification by age [29]. In contrast, other pooled analyses showed non-significant reduction of risk for increased intakes of nuts, underlying that the inverse association between the consumption of nuts and diabetes was attenuated after adjustment for confounding factors, including BMI [30]. However, results from experimental studies showed promising results. Thus, nut consumption has been demonstrated to exert beneficial metabolic effects due to their action on post-prandial glycemia an insulin sensitivity. A number of RCTs have demonstrated positive effects of nut consumption on post-prandial glycemia in healthy individuals [33-38]. Moreover, a meta-analysis of RCTs on the effects of nut intake on glycemic control in diabetic individuals including 12 trials and a total of 450 participants showed that diets with an emphasis on nuts (median dose = 56 g/d) significantly lowered HbA1c (Mean Difference [MD] : -0.07%; 95% confidence interval [CI]: -0.10, -0.03%; P = 0.0003) and fasting glucose (MD : -0.15 mmol/L; 95% CI: -0.27, -0.02 mmol/L; P = 0.03) compared with control diets [39]. No significant treatment effects were observed for fasting insulin and homeostatic model assessment (HOMA-IR), despite the direction of effect favoring diet regimens including nuts.

Blood lipids and hypertension Hypertension and dyslipidemia are major risk factors for CVD. Diet alone has a predominant role in blood pressure and plasma lipid homeostasis. One systematic review [40] and 3 pooled quantitative analyses of RCTs [41-43] evaluated the effects of nut consumption on lipid profiles. A general agreement was relevant on certain markers, as daily consumption of nuts (mean = 67 g/d) induced a pooled reduction of total cholesterol concentration (10.9 mg/dL [5.1% change]), low-density lipoprotein cholesterol concentration (LDL-C) (10.2 mg/dL [7.4% change]), ratio of LDL-C to high-density lipoprotein cholesterol concentration (HDL-C) (0.22 [8.3% change]), and ratio of total cholesterol concentration to HDL-C (0.24 [5.6% change]) (P <0.001 for all) [42]. All meta-analyses showed no significant effects of nut (including walnut) consumption on HDL cholesterol or triglyceride concentrations in healthy individuals [41], although reduced plasma triglyceride levels were found in individuals with hypertriglyceridemia [42]. Interestingly, the effects of nut consumption were dose related, and different types of nuts had similar effects on blood lipid concentrations.

There is only limited evidence from observational studies to suggest that nuts have a protective role on blood pressure. A pooled analysis of prospective cohort studies on nut consumption and hypertension reported a decreased risk associated with increased intake of nuts [32]. Specifically, only a limited number of cohort studies have been conducted exploring the association between nut consumption and hypertension (n = 3), but overall reporting an 8% reduced risk of hypertension for individuals consuming >2 servings per week (Risk Ratio [RR] = 0.92, 95% CI: 0.87-0.97) compared with never/rare consumers, whereas consumption of nuts at one serving per week had similar risk estimates (RR = 0.97, 95% CI: 0.83, 1.13) [32]. These findings are consistent with results obtained in a pooled analysis of 21 experimental studies reporting the effect of consuming single or mixed nuts (in doses ranging from 30 to 100 g/d) on systolic (SBP) and diastolic blood pressure (DBP) [44]. A pooled analysis found a significant reduction in SBP in participants without type2 diabetes [MD: -1.29 mmHg; 95% CI: -2.35, -0.22; P = 0.02] and DBP (MD: -1.19; 95% CI: -2.35, -0.03; P = 0.04), whereas subgroup analyses of different nut types showed that pistachios, but not other nuts, significantly reduced SBP (MD: -1.82; 95% CI: -2.97, -0.67; P = 0.002) and SBP (MD: -0.80; 95% CI: -1.43, -0.17; P = 0.01) [44].

Nut consumption and CVD risk Clustering of metabolic risk factors occurs in most obese individuals, greatly increasing risk of CVD. The association between nut consumption and CVD incidence [29-31] and mortality [24] has been explored in several pooled analyses of prospective studies. The overall risk calculated for CVD on a total of 8,862 cases was reduced by 29% for individuals consuming 7 servings per week (RR = 0.71, 95% CI: 0.59, 0.85) [30]. A meta-analysis including 9 studies on coronary artery disease (CAD) including 179,885 individuals and 7,236 cases, reporting that 1-serving/day increment would reduce risk of CAD of about 20% (RR = 0.81, 95% CI: 0.72, 0.91) [31]. Similar risk estimates were calculated for ischemic heart disease (IHD), with a comprehensive reduced risk of about 25-30% associated with a daily intake of nuts [29, 30]. Findings from 4 prospective studies have been pooled to estimate the association between nut consumption and risk of stroke, and a non-significant/borderline reduced risk was found [29-31, 45]. CVD mortality was explored in a recent meta-analysis including a total of 354,933 participants, 44,636 cumulative incident deaths, and 3,746,534 cumulative person-years [24]. One serving of nuts per week and per day resulted in decreased risk of CVD mortality (RR = 0.93, 95% CI: 0.88, 0.99 and RR =0.61, 95% CI: 0.42, 0.91, respectively], primarily driven by decreased coronary artery disease (CAD) deaths rather than stroke deaths [24]. Overall, all pooled analyses demonstrated a significant association between nut consumption and cardiovascular health. However, it has been argued that nut consumption was consistently associated with healthier background characteristics reflecting overall healthier lifestyle choices that eventually lead to decreased CVD mortality risk.

Nut consumption and cancer risk Cancer is one of the leading causes of death in the elderly population. After the evaluation of the impact on cancer burden of food and nutrients, it has been concluded that up to one third of malignancies may be prevented by healthy lifestyle choices. Fruit and vegetable intake has been the focus of major attention, but studies on nut consumption and cancer are scarce. A recent metaanalysis pooled together findings of observational studies on cancer incidence, including a total of 16 cohort and 20 casecontrol studies comprising 30,708 cases, compared the highest category of nut consumption with the lowest category and found a lower risk of any cancer of 25% (RR = 0.85, 95% CI: 0.86, 0.95) [46]. When the analysis was conducted by cancer site, highest consumption of nuts was associated with decreased risk of colorectal (RR = 0.76, 95% CI: 0.61, 0.96), endometrial (RR = 0.58, 95% CI: 0.43, 0.79), and pancreatic cancer (RR = 0.71, 95% CI: 0.51, 0.99), with only one cohort study was conducted on the last [46]. The potential protective effects of nut consumption on cancer outcomes was supported also by pooled analysis of 3 cohort studies [comprising the PREDIMED, the NHS, the HPS, and the Health Professionals Follow-Up Study (HPFS) cohorts] showing a decreased risk of cancer death for individuals consuming 3-5 servings of nuts per week compared with never eaters (RR = 0.86, 95% CI: 0.75, 0.98) [24]. The analysis was recently updated by including results from the Netherlands Cohort Study reaching a total of 14,340 deaths out of 247,030 men and women observed, confirming previous results with no evidence of between-study heterogeneity (RR = 0.85, 95% CI: 0.77, 0.93) [47]. However, a dose- response relation showed the non-linearity of the association, suggesting that only moderate daily consumption up to 5 g reduced risk of cancer mortality, and extra increased intakes were associated with no further decreased risk.

Nut consumption and affective/cognitive disorders Age-related cognitive decline is one of the most detrimental health problems in older people. Cognitive decline is a paraphysiological process of aging, but timing and severity of onset has been demonstrated to be affected by modifiable lifestyle factors, including diet. In fact, the nature of the age- related conditions leading to a mild cognitive impairment (MCI) differs by inflammation-related chronic neurodegenerative diseases, such as dementia, Alzheimer’s disease, Parkinson’s disease and depression. Evidence restricted to nut consumption alone is scarce, but a number of studies have been conducted on dietary patterns including nuts as a major component. A pooled analysis synthesizing findings of studies examining the association between adherence to a traditional Mediterranean diet and risk of depression (n = 9), cognitive decline (n = 8), and Parkinson’s disease (n = 1) showed a reduction of risk of depression (RR = 0.68, 95% CI: 0.54, 0.86) and cognitive impairment (RR = 0.60, 95% CI: 0.43, 0.83) in individuals with increased dietary adherence [10].

The study that first found a decreased risk of Alzheimer’s disease in individuals highly adherent to the Mediterranean diet was conducted in over 2,000 individuals in the Washington/Hamilton Heights-Inwood Columbia Aging Project (WHICAP), a cohort of non-demented elders aged 65 and older living in a multi-ethnic community of Northern Manhattan in the US (Hazard Ratio [HR] = 0.91, 95% CI: 0.83, 0.98) [48]. These results have been replicated in further studies on the Mediterranean diet, however nut consumption was not documented [49, 50]. A number of observational studies also demonstrated a significant association between this dietary pattern and a range of other cognitive outcomes, including slower global cognitive decline [51]. However, evidence from experimental studies is limited to the PREDIMED trial, providing interesting insights on the association between the Mediterranean diet supplemented with mixed nuts and both depression and cognitive outcomes. Regarding depression, the nutritional intervention with a Mediterranean diet supplemented with nuts showed a lower risk of about 40% in participants with type-2 diabetes (RR = 0.59, 95% CI: 0.36, 0.98) compared with the control diet [52]. However the effect was not significant in the whole cohort overall [52]. Regarding cognitive outcomes after a mean follow-up of 4.1 years, findings from the same trial showed significant improvements in memory and global cognition tests for individuals allocated to the Mediterranean diet supplemented with nuts [adjusted differences: -0.09 (95% CI: -0.05, 0.23), P = 0.04 and -0.05 (95% CI: -0.27, 0.18), P = 0.04, respectively], compared to control group, showing that Mediterranean diet plus mixed nuts is associated with improved cognitive function [53].


Potential mechanisms of protection of nut consumption Despite the exact mechanisms by which nuts may ameliorate human health being largely unknown, new evidence has allowed us to start to better understand the protection of some high-fat, vegetable, energy-dense foods such as nuts. Non- communicable disease burden related with nutritional habits is mainly secondary to exaggerated intakes of refined sugars and saturated fats, such as processed and fast- foods. Nuts provide a number of nutrient and non-nutrient compounds and it is only recently that scientists have tried to examine their effects on metabolic pathways.

Metabolic and cardiovascular protection With special regard to body weight and their potential effects in decreasing the risk of obesity (or weight gain, in general), nuts may induce satiation (reduction in the total amount of food eaten in a single meal) and satiety (reduction in the frequency of meals) due to their content in fibers and proteins, which are associated with increased release of glucagon-like protein 1 (GLP-1) and cholecystokinin (CCK), gastrointestinal hormones with satiety effects [54, 55]. The content in fiber of nuts may also increase thermogenesis and resting energy expenditure, and reduce post- prandial changes of glucose, thus ameliorating inflammation and insulin resistance. Moreover, the specific content profile of MUFA and PUFA provides readily oxidized fats than saturated or trans fatty acids, leading to reduced fat accumulation [56, 57]. The beneficial effects of nuts toward glucose metabolism may be provided by their MUFA content that improves the efficiency of pancreatic beta-cell function by enhancing the secretion of GLP1, which in turn helps the regulation of postprandial glycemia and insulin sensitivity [58]. MUFA and PUFA are also able to reduce serum concentrations of the vasoconstrictor thromboxane 2, which might influence blood pressure regulation. Together with polyphenols and anti-oxidant vitamins, nuts may also ameliorate inflammatory status at the vascular level, reducing circulating levels of soluble cellular adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, which are released from the activated endothelium and circulating monocytes [59]. Moreover, nuts may improve vascular reactivity due to their content in L-arginine, which is a potent precursor of the endogenous vasodilator nitric oxide. Nuts content in microelements is characterized by a mixture that may exert a direct effect in modulating blood pressure, including low content of sodium and richness in magnesium, potassium and calcium, which may interact to beneficially influence blood pressure
Despite the exact mechanisms by which nuts may ameliorate human health being largely unknown, new evidence has allowed us to start to better understand the protection of some high-fat, vegetable, energy-dense foods such as nuts. Non- communicable disease burden related with nutritional habits is mainly secondary to exaggerated intakes of refined sugars and saturated fats, such as processed and fast- foods. Nuts provide a number of nutrient and non-nutrient compounds and it is only recently that scientists have tried to examine their effects on metabolic pathways.

Cancer protection The potential mechanisms of action of nuts that may intervene in the prevention of cancer have not been totally elucidated. Numerous hypotheses have been proposed on the basis of basic research exploring the antioxidant and anti-inflammatory compounds characterizing nuts [61]. Vitamin E can regulate cell differentiation and proliferation, whereas polyphenols (particularly flavonoids such as quercetin and stilbenes such as resveratrol) have been shown to inhibit chemically-induced carcinogenesis [62]. Polyphenols may regulate the inflammatory response and immunological activity by acting on the formation of the prostaglandins and pro-inflammatory cytokines, which may be an important mechanism involved in a number of cancers, including colorectal, gastric, cervical and pancreatic neoplasms [62]. Among other compounds contained in nuts, dietary fiber may exert protective effects toward certain cancers (including, but not limited to colorectal cancer) by the aforementioned metabolic effects as well as increasing the volume of feces and anaerobic fermentation, and reducing the length of intestinal transit. As a result, the intestinal mucosa is exposed to carcinogens for a reduced time and the carcinogens in the colon are diluted [62]. Finally, there is no specific pathway demonstrating the protective effect of PUFA intake against cancer, but their interference with cytokines and prostaglandin metabolism may inhibit a state of chronic inflammation that may increase cancer risk [63].

Cognitive aging and neuro-protection There is no universal mechanism of action for nuts with regard to age-related conditions. A number of systemic biological conditions, such as oxidative stress, inflammation, and reduced cerebral blood flow have been considered as key factors in the pathogenesis of both normal cognitive ageing and chronic neurodegenerative disease [64]. Nuts, alone or as part of healthy dietary patterns, may exert beneficial effects due to their richness in antioxidants, including vitamins, polyphenols and unsaturated fatty acids, that may be protective against the development of cognitive decline and depression [65, 66]. Both animal studies and experimental clinical trials demonstrated vascular benefits of nuts, including the aforementioned lowering of inflammatory markers and improved endothelial function, which all appear to contribute to improved cognitive function [67]. The antioxidant action may affect the physiology of the ageing brain directly, by protecting neuronal and cell-signaling function and maintenance. Moreover, certain compounds contained in nuts may directly interact with the physiology and functioning of the brain. For instance, walnuts are largely composed of PUFA, especially ALA, which have been suggested to induce structural change in brain areas associated with affective experience [66]. Moreover, PUFA have been associated with improved symptoms in depressed patients, suggesting an active role in the underlying pathophysiological mechanisms [68]. Thus, the mechanisms of action of nut consumption on age-related cognitive and depressive disorders are complex, involving direct effects on brain physiology at the neuronal and cellular level and indirect effects by influencing inflammation.


Summary From an epidemiological point of view, nut eaters have been associated with overall healthier lifestyle habits, such as increased physical activity, lower prevalence of smoking, and increased consumption of fruits and vegetables [24]. These variables represent strong confounding factors in determining the effects of nuts alone on human health and final conclusions cannot be drawn. Nevertheless, results from clinical trials are encouraging. Nuts show promise as useful adjuvants to prevent, delay or ameliorate a number of chronic conditions in older people.

Role of Inflammation in Disease

Larry H. Bernstein, MD, FCAP, Curator





The debate over the latest cure-all craze.


Medical Dispatch NOVEMBER 30, 2015 ISSUE     http://www.newyorker.com/magazine/2015/11/30/inflamed


The National Institutes of Health recently designated inflammation a priority.


The National Institutes of Health recently designated inflammation a priority.


Several years ago, I fell at the gym and ripped two tendons in my wrist. The pain was excruciating, and within minutes my hand had swollen grotesquely and become hot to the touch. I was reminded of a patient I’d seen early in medical school, whose bacterial infection extended from his knee to his toes. Latin was long absent from the teaching curriculum, but, as my instructor examined the leg, he cited the four classic symptoms of inflammation articulated by the Roman medical writer Celsus in the first century: rubor, redness; tumor, swelling; calor, heat; and dolor, pain. In Latin, inflammatio means “setting on fire,” and as I considered the searing pain in my injured hand I understood how the condition earned its name.

Inflammation occurs when the body rallies to defend itself against invading microbes or to heal damaged tissue. The walls of the capillaries dilate and grow more porous, enabling white blood cells to flood the damaged site. As blood flows in and fluid leaks out, the region swells, which can put pressure on surrounding nerves, causing pain; inflammatory molecules may also activate pain fibres. The heat most likely results from the increase in blood flow.
The key white blood cell in inflammation is called a macrophage, and for decades it has been a subject of study in my hematology laboratory and in many others. Macrophages were once cast as humble handmaidens of the immune system, responsible for recognizing microbes or debris and gobbling them up. But in recent years researchers have come to understand that macrophages are able to assemble, within themselves, specialized platforms that pump out the dozens of molecules that promote inflammation. These platforms, called inflammasomes, are like pop-up factories—quickly assembled when needed and quickly dismantled when the crisis has passed.

For centuries, scientists have debated whether inflammation is good or bad for us. Now we believe that it’s both: too little, and microbes fester and spread in the body, or wounds fail to heal; too much, and nearby healthy tissue can be degraded or destroyed. The fire of inflammation must be tightly controlled—turned on at the right moment and, just as critically, turned off. Lately, however, several lines of research have revealed that low-level inflammation can simmer quietly in the body, in the absence of overt trauma or infection, with profound implications for our health. Using advanced technologies, scientists have discovered that heart attacks, diabetes, and Alzheimer’s disease may be linked to smoldering inflammation, and some researchers have even speculated about its role in psychiatric conditions.

As a result, understanding and controlling inflammation has become a central goal of modern medical investigation. The internal research arm of the National Institutes of Health recently designated inflammation a priority, mobilizing several hundred scientists and hundreds of millions of dollars to better define its role in health and disease; in 2013, the journal Science devoted an entire issue to the subject. This explosion in activity has captured the public imagination. In best-selling books and on television and radio talk shows, threads of research are woven into cure-all tales in which inflammation is responsible for nearly every malady, and its defeat is the secret to health and longevity. New diets will counter the inflammation simmering in your gut and restore your mental equilibrium. Anti-inflammatory supplements will lift your depression and ameliorate autism. Certain drugs will tamp down the silent inflammation that degrades your tissues, improving your health and extending your life. Everything, and everyone, is inflamed.

Such claims aside, there is genuine evidence that inflammation plays a role in certain health conditions. In atherosclerosis, blood flow to the heart or the brain is blocked, resulting in a heart attack or a stroke. For a long time, atherosclerosis was thought to result mainly from eating fatty foods, which clogged the arteries. “Atherosclerosis was all about fats and grease,” Peter Libby, a professor at Harvard Medical School and a cardiologist at Brigham and Women’s Hospital, in Boston, told me recently. “Most physicians saw atherosclerosis as a straight plumbing problem.”

During his cardiology training, Libby studied immunology, and he became fascinated with the work of Rudolf Virchow, a nineteenth-century German pathologist. Virchow speculated that atherosclerosis might be an active process, caused by inflamed blood vessels, not one caused simply by the accumulation of fat. In the mid-nineteen-nineties, in studies with mice, Libby, working in parallel with other groups of scientists, found that low-density lipoproteins—LDLs, those particles of “bad” cholesterol—can work their way into the lining of arteries. There, they sometimes trigger an inflammatory response, which can cause blood clots that block the artery. Libby and others began to understand that atherosclerosis wasn’t a mere plumbing problem but also an immune disease—“our body’s defenses turned against ourselves,” he told me.

Paul Ridker, a cardiovascular expert and a colleague of Libby’s at Harvard and Brigham and Women’s, moved the research beyond the laboratory. He found that many patients who’d had heart attacks, despite lacking factors such as high blood pressure, high cholesterol, and a history of smoking, had an elevated level of C-reactive protein, a molecule produced in response to inflammation, in their blood. After demonstrating, in a separate study, that cholesterol-reducing statins could also reduce C-reactive-protein levels, Ridker launched the Jupiter trial, in which people with elevated levels of C-reactive protein but normal cholesterol levels were given a placebo or a statin medication. In 2008, the published results showed that the subjects who received the statin saw their levels of C-reactive protein drop and were less likely three and a half years later to suffer a heart attack. This suggested that elevated cholesterol isn’t the only factor at work in cardiovascular disease, and that in some cases statins, acting as anti-inflammatory agents, could be used to treat the condition.
The benefit was modest: the statin treatment reduced the risk of heart attack in only about one per cent of the patients. Still, that figure is statistically significant, and for one in every hundred patients—a hundred in every ten thousand—it’s meaningful. An independent safety-monitoring board ended the study early, saying that it was unethical to continue once it was clear that statins provided a benefit not available to the subjects on the placebo. (Critics argue that shortening the trial, which was funded by a drug company, exaggerated the potential benefits and underestimated long-term harm, but the researchers strongly disagree.) The N.I.H. and other scientific groups are funding new studies to further explore whether anti-inflammatory drugs—for example, low doses of immunomodulatory agents that are used for treating severe arthritis—can help prevent cardiovascular disease.
Another chronic condition that has been linked to inflammation is Type II diabetes. People with this condition can’t adequately use insulin, a molecule that enables the body’s cells to take glucose out of the bloodstream and derive energy from it. Their organs fail and glucose builds to dangerous levels in the blood. Recently, researchers have found macrophages in the pancreases of people with Type II diabetes. The macrophages release inflammatory molecules that are thought to impair insulin activity. One of these inflammatory molecules is called interleukin-1, and in 2007 the New England Journal of Medicine reported on a clinical trial in which an interleukin-1 blocker proved to be modestly effective at lowering blood-sugar levels in Type II diabetics. This suggests that, by blocking inflammation, it might be possible to restore insulin activity and alleviate some of the symptoms of diabetes.

Alzheimer’s disease, too, seems to show a link to inflammation. Alzheimer’s results from the buildup of amyloid and tau proteins in the brain; specialized cells called glial cells, which are related to macrophages, recognize these proteins as debris and release inflammatory molecules to get rid of them. This inflammation is thought to further impair the working of neurons, worsening Alzheimer’s. The connection is tantalizing, but it’s important to note that it doesn’t mean that inflammation causes Alzheimer’s. Nor is there strong evidence that inflammation contributes to other forms of dementia where the brain isn’t filled with protein debris. And in clinical trials anti-inflammatory drugs like naproxen and ibuprofen have failed to ameliorate or prevent Alzheimer’s.


On September 18, 2015, scientists at the N.I.H. convened a meeting to publicly present their research priorities, one of which is to decipher the consequences of inflammation. It’s increasingly apparent that inflammation plays some role in many health conditions, but scientists are far from grasping the nature of that relationship, the mechanisms involved, or the extent to which treating inflammation is helpful.

“We really don’t know how much inflammation contributes to diabetes, Alzheimer’s, depression, and other disorders,” Michael Gottesman, a director of research at the N.I.H., told me. “We know a lot about the mouse and its immune response. Much, much less is understood in humans. As we learn more, we see how much more we need to learn.” Gottesman pointed out that, of the thousand or so proteins circulating in our bloodstream, about a third are involved in inflammation and in our immune response, so simply detecting their presence doesn’t reveal much about their potential involvement in any particular disease. “Correlation is not causation,” he emphasized. “Because you find an inflammatory protein in a certain disorder, it doesn’t mean that it is causing that disorder.”
This lack of certainty hasn’t dampened the enthusiasm of a growing number of doctors who believe that inflammation is the source of a wide range of conditions, including dementia, depression, autism, A.D.H.D., and even aging. One of the most prominent such voices is that of Mark Hyman, whose books—including “The Blood Sugar Solution 10-Day Detox Diet”—are best-sellers. Hyman serves as a personal health adviser to Bill and Hillary Clinton and to the King and Queen of Jordan. Recently, he was recruited by the Cleveland Clinic with millions of dollars in funding to establish a center based on his ideas. Trained in family medicine, Hyman told me that he considers himself a new type of doctor. “I am a doctor who treats root causes and addresses the body as a dynamic system,” he wrote in an e-mail. “Being an inflammalogist is part of that.”

Studies with human subjects clearly indicate that, in cases where inflammation underlies a chronic condition, the inflammation is local: in the arteries (heart disease); or in the brain (Alzheimer’s); or in the pancreas (diabetes). And though there are associations between various forms of inflammatory disease—for example, people with psoriasis or periodontal disease have a somewhat higher risk of heart disease—it has not been proved that there is a causal connection. Hyman and other doctors, such as the neurologist David Perlmutter, promote a more radical idea: that certain foods and environmental toxins cause smoldering inflammation, which somehow spreads to other areas of the body, including the brain, degrading one’s health, mental acuity, and life span.

The notion of a gut-brain connection seems to derive from studies with mice, including one that showed that introducing a bacterium into a mouse’s gastrointestinal tract led to behavioral changes, such as a reluctance to navigate mazes. But there’s scant evidence that anything similar happens in people, or any rigorous study to show that “anti-inflammatory diets” reduce depression. Earlier this year, the journal Brain, Behavior, and Immunity published a meta-analysis of more than fifty clinical studies that found inflammatory molecules in patients with depression. The paper revealed that there was little consistency from study to study about which molecules correlated to the condition. Steven Hyman, a former director of the National Institute of Mental Health and now the head of the Stanley Center at the Broad Institute (and no relation to Mark Hyman), in Cambridge, Massachusetts, noted that depression is “one of those topics where exuberant theorization vastly outstrips the data.”

Nonetheless, Mark Hyman holds fast to his view. “Inflammation is the final common pathway for pretty much all chronic diseases,” he told me. His recommended solution is an “anti-inflammatory diet”—omitting sugar, caffeine, beans, dairy, gluten, and processed foods, as well as taking a variety of supplements, including probiotics, fish oil, Vitamins C and D, and curcumin, a key molecule in turmeric. Hyman introduced me to one of the patients he had treated with his anti-inflammatory diet and supplements, a forty-seven-year-old hedge-fund manager in Cambridge named Jim Silverman. Two decades ago, Silverman began noticing blood in his stool. A colonoscopy resulted in a diagnosis of ulcerative colitis. In the ensuing years, Silverman was treated by gastroenterologists with aspirin-based medication, anti-inflammatory suppositories, and even corticosteroids, but the problem persisted. Then, five years ago, on a flight home from a business conference, he happened to sit next to Hyman, who told him that he could cure colitis.
“I thought, What a bullshitter,” Silverman said. He travelled anyway to Hyman’s UltraWellness Center, in Lenox, Massachusetts, to consult with him. Hyman told him that dairy was inflaming his bowel. Silverman was skeptical, but he kept track of his diet and bleeding episodes, and ultimately concluded that restricting dairy products resulted in long periods without bleeding. He now thinks that he could be suffering from a dairy allergy. In addition to avoiding dairy products, he continues to follow the anti-inflammatory regimen of supplements prescribed by Hyman. “I’m just taking it because I’m doing well,” he said. “I have no idea if it’s doing anything, but I don’t want to rock the boat.”

I asked Gary Wu, a professor of gastroenterology at the Perelman School of Medicine, at the University of Pennsylvania, and one of the world’s experts on the gut microbiome, about the alleged value of treating inflammatory bowel disease by restricting specific foods. Recently, in the journal Gastroenterology, Wu and his colleagues published a comprehensive review of scientific studies on diet and inflammatory bowel disease. They found only two dietary interventions that had been proved to reduce inflammation: an “elemental diet,” which is a liquid mixture of amino acids, simple sugars, and triglycerides, and a slightly more complex liquid diet. The liquid mixtures are typically administered with a tube placed through the nose. “The diet is not palatable,” Wu said. “And you don’t eat during the day. There is no intake of whole foods at all.”

David Agus, a cancer specialist and a professor of medicine and engineering at the University of Southern California, is equally skeptical of Hyman’s claims for the anti-inflammation diet. Agus, who is perhaps best known for being the doctor on “CBS This Morning,” recently received a multimillion-dollar grant from the National Cancer Institute to study how inflammation may spur the growth of tumors. “This notion that foods cause inflammation and foods can block inflammation, there’s zero data that it changes clinical outcomes,” he told me. “If the idea gets people to eat fruits and vegetables, I love it, but it’s not real.” Agus noted that vitamins don’t counter inflammation, and that it’s been shown, in rigorous clinical trials, that they may increase one’s risk of developing cancer.
Still, Agus views inflammation as a component not only of cancer but also of chronic diseases like diabetes and dementia. Rather than special diets, he supports preventively taking approved anti-inflammatory medications, such as aspirin and statins, and scrupulously scheduling the standard vaccinations in order to prevent infections. In “The End of Illness,” Agus encourages the reader to “reduce your daily dose of inflammation” by, among other things, not wearing high heels, since these can inflame your feet and the inflammation could possibly affect your vital organs. When I pressed him on that suggestion, he told me, “What I meant is that if your feet hurt all day it’s probably not a good thing. The downside is you just wear a different pair of shoes. The upside is it gave you an understanding of inflammation and its role in disease.”

Mark Hyman, at times, acknowledges the possible limits of his paradigm. When I asked him about the alleged link among gut inflammation, diet, and psychological disorders, he conceded that some of his evidence was anecdotal, derived from his own clinical practice. He mentioned the case of a child with asthma, eczema, and A.D.H.D., whom he treated with “an elimination diet, taking him off processed foods, and giving him supplements.” The child’s allergic problems improved and his behavior was markedly better, Hyman said: “It was a light-bulb moment. I saw secondary effects on the brain that came out of treating physical problems.”

He also cited studies of patients with rheumatoid arthritis, a painful and debilitating auto-immune condition that inflames and erodes the joints, who became less depressed after being treated with inflammatory blockers. But had the anti-inflammatory treatment directly lifted their depression, or had their mood improved simply because they were more mobile and in less pain? I told Hyman that it was hard to connect the dots. “For sure,” he said.


Connecting the dots is a challenge even for scientists who are actively involved in inflammation research. One afternoon, I visited Ramnik Xavier, the chair of gastroenterology at Massachusetts General Hospital and an expert in Crohn’s disease and ulcerative colitis. The bowel is inflamed in both conditions: ulcerative colitis affects the colon, whereas Crohn’s disease can affect any part of the digestive system. But the nature of inflammation varies almost from person to person and involves interactions among DNA, many kinds of gastrointestinal cells, and the peculiarities of the gut microbiome. “Lots of cells, lots of genes, lots of bugs,” Xavier said.

Xavier, a compact man with a laconic manner and thick black hair marked by streaks of gray, initially studied the role of specialized white blood cells, known as T-cells and B-cells, in defending the body against the development of colitis. Eventually, with Mark Daly, a geneticist at the Broad Institute, Xavier began to search for genes that predispose people to inflammatory bowel disease and for genes that might protect them against it. The two scientists, as part of an international consortium, have identified at least a hundred and sixty areas of DNA that are associated with an increased risk of inflammatory bowel disease; Xavier’s lab has zeroed in on about two dozen genes within these regions of DNA.
One of the frustrations of treating inflammation is that our weapons against it are so imprecise. Drugs like naproxen and ibuprofen are the equivalent of peashooters. At the other extreme, cannon-like steroids shut down the immune system, raising the risk of infection, eroding the bones, predisposing the patient to diabetes, and causing mood swings. Even the peashooters can cause collateral damage: aspirin may help to protect against colon cancer, heart attack, and stroke, but it also raises the risk of gastrointestinal bleeding. Ibuprofen, naproxen, and similar drugs were labelled by the F.D.A. as increasing the risk of heart attack and stroke in people who’ve never suffered either condition, and clinical trials failed to show that they prevent or ameliorate dementia. (Although these drugs reduce inflammation, they may also alter the lining of blood vessels and increase the risk of clots.) Statins lower the chance of a heart attack, but there is growing concern not only about the side effect of muscle pain but also about increasing the likelihood of diabetes. And the absolute benefits of these preventive medications is slight, measured in single digits.

In the lab at the Broad Institute, Xavier and his team were trying to discover new treatments that can block inflammation in a targeted manner. The day I visited, they were assessing molecules associated with colitis, especially one called interleukin-10, or IL-10, which is known to decrease inflammation. In a cavernous room, I watched as a robotic arm moved among racks of plastic plates, each containing hundreds of small wells in which chemical compounds were being tested. Some people with Crohn’s disease have genetic mutations that disable the salubrious effects of IL-10. Xavier is trying to identify molecules that can compensate for this deficiency, in the hope that such molecules might eventually be turned into drugs to treat this subset of patients.

But other patients suffer from a different manifestation of Crohn’s—they can’t fully clear debris from cells in their gut, so it builds up, triggering inflammation. In a neighboring lab, members of Xavier’s research team were trying to develop drugs for that condition, too. A robotic arm was handling plates that contained genetically engineered cells and moving them under a fluorescent microscope. The images appeared on a computer screen—fields of cells studded with yellow and green dots, like the sky in van Gogh’s “Starry Night.”

On another visit, Xavier took me to his clinic at Mass General. Patients, ranging from the very young to the elderly, were reclining in Barcaloungers as nurses and physicians intravenously administered potent anti-inflammatory drugs. Later, I spoke by phone to one of Xavier’s patients, a forty-nine-year-old woman named Maria Ray, who received a diagnosis of colitis in 1998. She was treated with sulfa drugs and corticosteroids, which controlled the problem for several years, but in 2004, after a series of flare-ups, she underwent surgery to remove her colon. Soon after, she developed ulcers on her skin, arthritis of her knees and elbows, and inflammation in both eyes. Xavier prescribed other drugs, and for two years her condition improved, but lately her skin lesions and eye inflammation have returned. “We hoped surgery would cure her ulcerative colitis,” Xavier said. “But we don’t really understand why there is such an overactive immune system now inflaming these other parts of her body.”
At the very least, the fact that Ray has symptoms in many organs, despite the removal of her colon, complicates the simplistic view that treating the gut will suppress inflammation elsewhere. Moreover, there’s no evidence that patients with Crohn’s or colitis are more likely than average to develop dementia and other cognitive disorders. “What we see in mice is not always reproduced in people,” Xavier said.


Perhaps no aspect of inflammation is more compelling, or illusory, than the idea that it may be responsible for aging. An internist friend in Manhattan told me that healthy patients occasionally come in to her office carrying Mark Hyman’s books, eager to live longer by following his anti-inflammation life style. When I asked Hyman if he could introduce me to someone who follows his longevity regimen, he readily offered himself. “I’m aiming to live to a hundred and twenty,” he said.

The notion stems from grains of evidence, such as studies that have shown an increase in inflammation with age. The genesis of aging is still a mystery. It may occur for a host of reasons—a waning of the energy generated by the mitochondria within cells, the tendency of DNA to grow fragile and more mutation-prone over time—and it’s much too simplistic to attribute the process to inflammation alone. Luigi Ferrucci, the scientific director of the National Institute on Aging, conducted some of the early research on inflammation and aging, and for a while, he told me, he believed the avenue held promise. On the morning we spoke, he had just finished his daily six-mile run. Sixty-one years old, born in Livorno, on the coast of Tuscany, Ferrucci is an animated man with a stubbly beard who favors crew-neck sweaters. In the past four decades, he has studied thousands of people in order to identify the biological processes that result in aging. He measured scores of molecules in the blood, hoping to find clues that would lead him to the cause of aging’s hallmarks, particularly sarcopenia, or loss of muscle mass, and cognitive decline.

His most illuminating studies involved people in late middle age who showed no sign of heart disease, diabetes, dementia, or other conditions that might be associated with inflammation. He found that a single inflammatory molecule, called interleukin-6, was the most powerful predictor of who would eventually become disabled. Healthy patients with high levels of the IL-6 molecule aged more quickly and grew sicker than those without the inflammatory molecule. “I thought I had discovered the cause of aging and was going to win the Nobel Prize,” Ferrucci said, laughing.
But then he found other subjects with no evidence of inflammation, and without elevated levels of IL-6 or other inflammatory molecules, whose bodies nevertheless began to decline. “We are looking at the layer, not at the core of the problem,” he said. “Inflammation may accelerate aging in some people—but it is a manifestation of something that is occurring underneath.” He reiterated the point that correlation is not causation. “If you have the curiosity of the scientist, you can’t stop there, because you want to know why,” he said. “You want to break the toy so you can see how it’s working inside.”

Toward that end, Ferrucci recently organized a large team of collaborators and launched a new clinical study, GESTALT, which stands for Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing. Groups of healthy people will be studied intensively as they age, with detailed analyses of their DNA, RNA, proteins, metabolic capacity, and other sophisticated parameters, every two years for at least a decade. “Then we can say what mechanisms account for increased inflammation with aging, and the loss of muscle mass, or the loss of memory, or the loss of energy capacity or fitness,” Ferrucci said. “These have never really been addressed on a deep level in humans.”

In the meantime, he sticks to a Mediterranean diet, mainly out of fealty to his heritage. (Ferrucci is known among his N.I.H. colleagues as a gourmet Italian cook.) The media recently gave much attention to a study, published in 2013 in the New England Journal of Medicine, on the benefits of a Mediterranean diet in preventing heart attack or stroke. But, as Ferrucci noted, the benefits weren’t clearly related to inflammation and they accrued to a very small percentage of the subjects on the diet. “Believe me, if there were a diet that prevented aging, I would be on it,” he said.

We’d all like a simple solution for complex medical problems. We’re desperate to feel in command of our lives, particularly as we age and see friends and family afflicted by Alzheimer’s, stroke, and heart failure. “My patients, understandably, are very focussed on the foods they eat, wanting control, hoping they won’t have to take immune-suppressive treatments,” Gary Wu, the University of Pennsylvania gastroenterologist, told me.

Some years ago, I became obsessed with a restrictive diet—no bread, cheese, ice cream, cookies—in an attempt to lower my cholesterol levels. (My father died of a heart attack in his fifties, and I was haunted by his fate.) After nearly six months, I’d lost some fifteen pounds, but my cholesterol level had hardly budged, and I’d become so vigilant about everything I ate that I stopped enjoying meals. Gradually, I resumed a balanced and more reasonable diet and regained an appreciation for one of life’s fundamental pleasures.

Scientists may yet discover that inflammation contributes to disease in unexpected ways. But it’s important to remember, too, that inflammation serves a vital role in the body. “We are playing with one of the primary mechanisms selected by nature to maintain the integrity of our body against the thousand environmental attacks that we receive every day,” Ferrucci said. “Inflammation is part of our maintenance and repair system. Without it, we can’t heal.”

follow-on complex drugs

follow-on complex drugs

Larry H. Bernstein, MD, FCAP, Curator




Clinical development, immunogenicity, and interchangeability of follow-on complex drugs

Generics and Biosimilars Initiative Journal (GaBI Journal). 2014;3(2):71-8.       DOI: http://dx.doi.org:/10.5639/gabij.2014.0302.020


Although not derived from living sources, non-biological complex drug (NBCD) products have the immunogenicity and molecular complexity of biological drugs. NBCDs typically contain heterogenous mixtures of closely related nanoparticulate components that cannot be isolated, quantified, or entirely characterized physicochemically. Development of follow-on versions of NBCDs poses many of the same scientific challenges associated with biosimilar drugs. Like biologicals, the manufacturing methods used by the innovator to produce NBCDs ensure their identity, and consistent quality and activity. Some variation in alternate-sourced products is inevitable. Because of their complexity and because biological activity is often not correlated with serum pharmacokinetics, follow-on NBCDs can be shown to be similar, but not identical, to the originator product. Even slight variations in a follow-on NBCD can increase the risk of unwanted immunogenicity, safety problems, and/or reduced therapeutic effects. Issues related to follow-on versions of liposomal formulations, iron-carbohydrate complexes, and glatiramoids are described here to illustrate aspects of NBCDs that render the abbreviated pathway for approval of small-molecule drugs unsuitable for follow-on NBCDs. The US Food and Drug Administration has made ‘equivalence of complex drugs’ a Generic Drug User Fee Amendment priority initiative for fiscal year 2014. Experience suggests the same enhanced pre-approval scrutiny of biosimilar drugs should be applied to follow-on NBCDs. Preclinical and/or clinical data may be required to establish similar quality, immunogenicity, safety, and efficacy between a follow-on NBCD and a reference drug, and automatic switching or substitution of a follow-on NBCD for the originator should be contingent on demonstration of therapeutic equivalence.


The Biologics Price Competition and Innovation (BPCI) Act of 2009 was instituted to create an abbreviated pathway for approval of biosimilar drugs [1]. In 2014, the biosimilar pathway is still evolving; at this writing, the US Food and Drug Administration (FDA) has issued three draft guidelines for manufacturers seeking approval of biosimilar drugs [24]. Regulatory authorities agree that pre-approval evaluation of biosimilar drugs must be held to a higher standard than generic versions of small-molecule drugs because of their complexity and immunogenicity [57]. Non-biological complex drugs (NBCDs) have the molecular complexity of biological drugs, are immunogenic, and developing follow-on NBCDs poses many of the same scientific challenges associated with biosimilar drugs [810]. NBCDs typically contain heterogenous mixtures of closely related, macromolecular, nanoparticulate components that cannot be isolated, quantified, and/or entirely characterized physicochemically using available analytical technology [11]. As is true for biological drugs, consistent NBCD activity and quality typically rely on strictly controlled manufacturing procedures [1214], such that even small differences in the manufacture of a follow-on NBCD from that of the originator product can increase the risk of safety problems or reduced therapeutic efficacy [13, 15, 16].

Currently, follow-on NBCDs can be approved under the generics pathway established for traditional small-molecule drugs via an abbreviated new drug application (ANDA [505(j) application]), or under section 505(b)(2) of the Federal Food, Drug, and Cosmetic Act (FFDCA) [17]. Acknowledging that this pathway may not address the scientific challenges of ensuring the safety and efficacy of follow-on NBCDs [11, 18], FDA has made ‘equivalence of complex drugs’ a Generic Drug User Fee Amendment (GDUFA) Regulatory Science Priority Initiative for fiscal year 2014 [19].

A key aspect of pending legislation for biosimilars and follow-on NBCDs will be the development of science-based policies for interchangeability and drug substitution. The BPCI Act makes clear that biosimilarity does not imply interchangeability or substitutability [1]. Unlike generic copies of small-molecule drugs, biosimilars and follow-on NBCDs will not be identical to the innovator products. Because of their complexity and because the manufacturing method used to produce the innovator drug is often proprietary, some variation in alternate-sourced products is inevitable. Slight but clinically meaningful differences between originator and follow-on NBCDs may make interchangeability unfeasible. By law, to gain approval for interchangeability for biological drugs, the risk in terms of safety or diminished efficacy of alternating or switching between the generic product and the reference product must be no greater than continuing to use the reference product [1].

Scientific issues related to therapeutic equivalence of NBCDs

The ANDA generic drug pathway for small-molecule drugs requires proof of therapeutic equivalence of the generic to the innovator product, i.e. pharmaceutical equivalence (identical active substances, dosage form, strength, route of administration, labelling, quality, performance characteristics, and intended use), and bioequivalence (comparable pharmacokinetics in healthy humans) [17]. For some NBCDs, full proof of pharmaceutical equivalence is impossible, since two drugs cannot be shown to have identical active substances if the active substance has not been identified and the mechanism of action of the reference drug remains unknown [8, 10, 13]. Gross characterization of drug composition showing similarities in certain vectors, e.g. molar ratio or molecular weight distribution of constituents, does not guarantee similarity of other product characteristics [13, 20]. Similarly, bioequivalence cannot be established for many NBCDs because their biological activity is not correlated to serum pharmacokinetics [10, 21, 22]. These drugs typically comprise nanoparticle-size substructures that release or form the active ingredient, which is then transported to the targeted tissue.

Additionally, follow-on NBCDs cannot be presumed to have the same immunogenic profiles as innovator complex drugs [23]. The ability to predict drug-induced immunogenicity of uncharacterized NBCDs is limited, because immunogenicity is subject to influence by many variables. Patient-related factors such as genetic background, immune status, and the disease under treatment will influence the immunogenic response to treatment [24]. Autoimmune diseases can augment the immune response to immunogenic drugs. Product- and manufacturing-related factors also influence immunogenicity [5, 25, 26]; minor but key changes to the synthesis or manufacture of follow-on protein- and peptide-based NBCDs can lead to formation of aggregates or other impurities that can enhance drug-related immunogenicity and be immunogenic in their own right [26, 27].

Two products purported to be the same drug can produce antibodies with varying specificity such that one drug produces neutralizing antibodies (NABs) and the other does not [14]. When switching between a follow-on drug and the reference product (or among follow-on products), pre-existing antibodies to one NBCD could neutralize the efficacy of an analogous product. NABs that decrease drug efficacy can develop months or years after beginning treatment [28, 29]. For example, clinically important NABs associated with interferon-beta (IFNβ) therapy for MS generally develop after 12 to 18 months of treatment [30], and the clinical effects of decreased efficacy may take years to detect, resulting in irreversible disability progression that might have been avoided by performing regular antibody assessments [31].

Liposomes, iron-carbohydrate complexes, and glatiramoids

The 2014 GDUFA initiative regarding equivalence of complex drugs specifically mentions generic versions of (among others) liposomal drug formulations, e.g. Doxil (doxorubicin HCl liposome); iron-carbohydrate complexes, e.g. Venofer (iron sucrose); and products that contain complex peptide mixtures and peptides, e.g. Copaxone (glatiramer acetate) [19]. These NBCD classes exemplify challenges to the classic abbreviated pathway for generic drug approval and indicate a need for increased pre-approval assessment for follow-on NBCDs.

Liposomal drug formulations
Liposomal drug formulations act as carrier vehicles to deliver active agents to a specific body site. Nanoparticles of the bioactive agent encapsulated in vesicles composed of a phospholipid bilayer act as targeted antigen delivery systems to induce therapeutic humoral and cell-mediated immune responses [22]. As vaccines, synthetic antigenic peptides in liposomal formulation induce autoantibodies for prophylaxis of chronic conditions, such as hypertension [32]. Liposomal formulations of anticancer drugs allow antibody- or ligand-mediated targeting specifically to tumour cells, to increase therapeutic effects while reducing toxicity [33].

The physicochemical properties of liposomal vaccines – method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties – strongly influence the immune responses to them [22, 34], see Table 1. Thus, small differences in particles or complex attributes in follow-on versions of liposomal drugs could alter the activity of the drug, its distribution profile, and/or its persistence at tissue/cellular or subcellular levels to a clinically meaningful extent [35]. Currently, little is known about the cellular distribution of lipid-modified
peptides [22].


Consistent plasma concentrations of the active substance in two liposomal formulations does not guarantee similar efficacy or safety of the two products, since nanoparticles of active drug may distribute differently in tissues and cells [13, 35]. To investigate whether a conventional bioequivalence approach could ensure therapeutic equivalence of liposomal products, the pharmacokinetics, efficacy, and toxicity of six formulation variants of the originator PEGylated liposomal doxorubicin product (Doxil/Caelyx, Janssen-Cilag Pty Ltd) were prepared differing in composition and liposome size and evaluated in preclinical models for antitumour activity and toxicity [36]. Although some formulations demonstrated similar plasma pharmacokinetics and systemic exposure of doxorubicin, they exhibited different antitumour activity and toxicity profiles. Investigators concluded that a conventional bioequivalence approach is not appropriate for establishing therapeutic equivalence of a generic product.

Augmenting immunogenicity is key to the therapeutic activity of many liposomal preparations. However, some therapeutic liposomes are recognized by the immune system as foreign, likely because the phospholipid vesicles of the liposome mimic the size and shape of pathogenic microbes [37], leading to a variety of adverse immune reactions. Hypersensitivity reactions to liposomal drugs appear to be primarily mediated through complement activation triggered by an immune reaction to liposome surface charge or topography [37].

Detecting clinically meaningful differences in the therapeutic activity, toxicity, and immunogenicity of a follow-on liposomal drug may require nonclinical and clinical studies. A reflection paper issued by the European Medicines Agency (EMA) on data requirements for follow-on versions of liposomal products indicates clinical data for these products will be considered on a case-by-case basis [38]. Currently, EMA has not approved any follow-on versions of liposomal drugs.

Iron-carbohydrate drugs
Intravenous (IV) iron products are used to treat iron deficiency anaemia in patients undergoing chronic haemodialysis and receiving supplemental EPO therapy and in people with iron-deficiency anaemia associated with chronic blood loss or impaired iron absorption. The chemical structures of parenteral iron agents have not been characterized in full detail. Venofer (iron sucrose, Vifor Inc) and Ferrlecit (iron gluconate, Sanofi) comprise nanoparticle-sized iron cores surrounded by a complex carbohydrate layer. Because the physicochemical and biological properties of iron-carbohydrate compounds depend on their manufacturing processes, subtle structural modifications during manufacture may affect drug stability; if weakly bound iron dissociates prematurely it can catalyse the generation of reactive oxygen species leading to oxidative stress and inflammation [39]. Moreover, any variation in mean/median size and size distribution of the iron-carbohydrate nanoparticles can result in a generic product with different physicochemical properties and different biopharmaceutical profile with respect to pharmacokinetics and biodistribution compared with the originator drug [21]. In fact, animal studies show differences between the originator iron sucrose product (Venofer) and iron sucrose similar (ISS) products with increased markers of inflammation and increased serum iron and transferrin saturation levels in animals receiving the ISS [13, 40].

Despite these differences and the inability to completely characterize these drugs, and with no nonclinical or clinical studies to establish their therapeutic equivalence to the innovator drug, ISS products gained marketing approval via the small-molecule drug generic pathway [41]. Subsequently, in controlled trials in anaemic patients undergoing haemodialysis, ISS use was associated with reduced efficacy and the potential for increased safety risk related to iron overload [42, 43]. Clinically meaningful differences have been demonstrated when patients were switched to an ISS from Venofer. A switching study in which 75 stable haemodialysis patients taking Venofer for at least six months switched to an ISS product for six months resulted in decreased haemoglobin levels and reduced iron indices despite higher doses of the ISS [42], see Figure 1.


Iron-carbohydrate products can cause life-threatening or fatal hypersensitivity reactions, especially in pregnant women [44, 45]. The immunologic basis of allergic hypersensitivity to iron agents remains unknown [44]. Substitution of Venofer with an ISS at the pharmacy level (without physician or patient knowledge) was associated with hypersensitivity reactions and hospitalization in subjects who previously tolerated the originator drug [41]. Safety concerns surrounding all IV iron products led to recommendations of stronger measures to manage and minimize the risk of hypersensitivity [45]. The recommendations state that every dose of IV iron administered should be monitored for potential hypersensitivity reactions, even if previous administrations were well tolerated.

Both FDA and EMA have indicated that follow-on versions of iron sucrose (FDA) and nanoparticulate iron medicinal products (EMA) are not suitable for approval through the classic generic approval pathway [21, 46]. Neither agency has indicated what clinical evaluation will be required for approval of follow-on products.

The prototype glatiramoid, Copaxone, (Teva Pharmaceutical Industry) is a complex heterogenous mixture of synthetic proteins and polypeptide nanoparticles with immunomodulatory activity approved for treatment of relapsing-remitting multiple sclerosis (RRMS) [10,4750]. The active ingredient in Copaxone, glatiramer acetate, comprises a potentially incalculable number of unidentified active peptide moieties that are not characterizable with available technology [10], although the amino acid sequences in Copaxone are not entirely random [8]). The mechanism of action of Copaxone is not fully elucidated but the drug is thought to act as an antigen-based therapeutic vaccine [5153]. Pharmacokinetic data are uninformative for glatiramoids because the polypeptides in a glatiramoid mixture are hydrolysed at the drug injection site into unidentifiable peptide fragments that stimulate proliferation of glatiramoid-specific immune cells, which migrate to the central nervous system where they ameliorate auto-immune destruction of myelin [54, 55]. Therefore, blood levels of the glatiramoid or its hydrolysis products are not indicative of drug activity.

Glatiramoids appears to act as altered peptide ligands (APL) of encephalitogenic epitopes within myelin basic protein (MBP), an autoantigen implicated in MS [56]. Decades of clinical use demonstrate that Copaxone does not contain encephalitogenic epitopes and does not induce auto-reactive antibodies [48]. However, the same cannot be assumed for a follow-on glatiramer acetate product. In the last two decades, other APLs of MBP epitopes have been studied for use as therapeutic vaccines in MS. Clinical development of at least two APLs of MBP antigenic peptides was halted due to adverse events indicative of auto-reactive antibodies (i.e. immediate-type hypersensitivity reactions [57]) or substantial expansion of pro-inflammatory T cells that were cross-reactive with the MBP autoantigens [58].

Because Copaxone works as a therapeutic vaccine, anti-drug antibodies are detectable in all treated patients [48, 49, 52]. These antibodies, however, do not neutralize biological activity or clinical efficacy and are not associated with local or systemic adverse effects in RRMS patients receiving chronic treatment [49]. Anti-Copaxone antibody titers and isotypes change over time with repeated drug administration [48, 49, 59]. Although anti-Copaxone antibodies are predominantly of the IgG subclasses over time [48, 49, 60, 61], there have been rare reports of anti-Copaxone IgE antibodies associated with anaphylactic reactions that can arise up to 10 months to a year after treatment initiation, with no symptomology beforehand to signal hypersensitivity [62, 63].

A purported follow-on product of glatiramer acetate is currently marketed in India and the Ukraine (Glatimer, Natco Pharma Ltd, Hyderabad, India). There are no published data of the safety, efficacy, or immunogenicity of this product at this writing. In analytical tests, this product demonstrated physicochemical differences from Copaxone and poor batch-to-batch reproducibility [8, 20, 64]. When activatedex vivo with Glatimer, splenocytes from GA-treated mice showed distinctly different gene transcription profiles among different batches, and between Glatimer and Copaxone, see Figure 2 [64].


A comparability trial of a generic glatiramer acetate product (GTR, Synthon VB) and Copaxone is currently underway in RRMS patients [65]. The GATE trial (ClinicalTrials.gov NCT01489254) is a 24-month study comprising a 9-month, placebo-controlled, active-comparator phase followed by a 15-month open-label phase in which all participants remaining in the study receive GTR. Characterizing the immunogenicity of GTR is not an objective of the study because the protocol suggests that anti-GTR antibodies will be the same as anti-Copaxone antibodies; specifically, that because anti-Copaxone antibodies are not neutralizing, anti-GTR antibodies will not be neutralizing either [65]. This assumption requires verification: GTR may be shown to be a close approximation to Copaxone at best and small differences in amino acid sequences or of protein folding in GTR could generate an antibody repertoire with different, isotopes, specificities, and affinities from those of anti-Copaxone antibodies, with variable consequences on patient safety and response to therapy [10, 13, 66, 67]. Accordingly, experts in the field of MS agree that the immunogenicity of follow-on versions of Copaxone cannot be assumed and should be established for each formulation [23].

According to the study protocol, the assessment of the immunogenicity of GTR is to compare proportions of subjects who develop anti-drug antibodies after receiving GTR or Copaxone [65]. As antigen-based therapeutic vaccines, antibody development to either drug would be expected in 100% of treated participants. Thus, it will not be possible to compare efficacy outcomes in patients free from anti-GTR antibodies with outcomes in GTR-antibody-positive patients to determine potential formation of NABs.

In the US, at least three manufacturers have filed ANDAs for follow-on glatiramer acetate products with FDA under the small-molecule generic pathway [6871]. The manufacturers maintain that these products will be interchangeable with Copaxone, despite the fact that the first clinical exposure to these products in MS patients will occur post-approval. Given the complexity, unknown mechanism of action, uncharacterized epitopes, and strong immunogenicity of Copaxone; the variable nature of RRMS disease activity; and the inter- and intra-patient variability of antibody responses to immunogenic drugs; adequate testing of the immunogenicity of uncharacterized follow-on glatiramoid products in MS patients should precede approval and marketing of these products.

Considerations for approval of follow-on NBCDs

The generic approach should be limited to products that can be fully characterized and allow prediction of biological effects with pharmacokinetics data as surrogates for clinical efficacy [11]. Because NBCDs have many of the same features as biologicals, it seems prudent to extend guidelines for biosimilar products to follow-on NBCDs [2, 5, 24, 72]. When it is not possible to prove bioequivalence of follow-on NBCDs, requiring non-clinical and clinical testing can ensure therapeutic equivalence between NBCDs and the reference drug. Comparability evaluations for a follow-on NBCD should include physicochemical properties, impurities, biological activity, pharmacokinetics, efficacy, and safety, see Table 2. The extent of testing needed to establish adequate similarity between an originator drug and a follow-on product will likely depend on NBCD complexity, mode of action (if known), and the potential for toxicity. The risks of free substitution between an uncharacterized, immunogenic NBCD and a follow-on product will remain unknown without a clinical crossover study that provides direct evidence that repeated switching between the reference and the generic drug has no negative impact.


For immunogenic NBCDs, it may be necessary to ensure that the immunologic and immunogenic safety of the follow-on NBCD is comparable to that of the reference drug in clinical studies in patients with the disease under study. Considerable inter-individual variability in antibody responses warrants assessment in a sufficient number of patients to characterize variability in antibody responses. Additionally, evaluation of a follow-on NBCD should ensure that anti-drug antibodies do not neutralize drug efficacy or bind to endogenous proteins; and characterize the immunologic effects of switching between a reference NBCD and a generic product.

In many countries generic approval of a follow-on product allows automatic substitution at the pharmacy level. While there is continued pressure worldwide to reduce drug costs, a major concern is whether patient safety and well-being are compromised by automatic substitution or interchange with follow-on products [73]. Commonly, clinicians, caregivers and patients are not aware of the change in medication [11], often to the frustration of prescribing physicians [7477]. At minimum, substitution of NBCDs without the involvement of a healthcare professional should be discouraged. Generally, patients should not be automatically switched to a generic NBCD if they are doing well. If a switch is unavoidable, the safety and efficacy of the new product should be monitored [78].

In some instances, substituting a lower priced generic for an innovator drug has resulted in higher healthcare utilization and overall costs [16, 17, 25] due to decreased efficacy or adverse events. Overall, drug product replacement that is guided by acquisition cost only may increase other costs and not be cost-effective from the patient’s and payer’s perspective [11].


Patients, physicians, and third-party payers expect generic products to be equally safe and comparably effective to the reference drug. For follow-on NBCDs, this will likely require more thorough assessment than the current generic drug approval process. Ultimately, regulatory requirements for approval and interchangeability of follow-on NBCDs will probably require a ‘case-by-case’ approach.

As FDA approaches the challenge of developing guidelines for follow-on NBCDs, it will be important to include a variety of constituents in the process. Members of the medical community have expressed concerns about the safety and efficacy of biosimilar drugs that indicate an increasing lack of trust of the drug regulatory process, primarily due to ‘an absence of the organized medical community in the public process of creating and updating the guidelines’ [74]. The same may hold true for follow-on NBCDs. Regulators must operate in different worlds to balance legal, scientific, and public health considerations as legislation for approval of follow-on NBCD products evolves. Scientific discussion and multidisciplinary research between experts from academia, industry, the medical community, and regulatory bodies; and consensus discussions with all stakeholders on an international level will aid in development of meaningful regulatory guidelines to ensure the safety and effectiveness of follow-on NBCD products.


1. Biologics Price Competition and Innovation Act of 2009, Public Law 111-148, Sec. 7001-7003, 124 Stat. 119. Mar. 23, 2010.
2. U.S. Department of Health and Human Services. Guidance for industry scientific considerations in demonstrating biosimilarity to a reference product. Draft guidance. Februray 2012 [homepage on the Internet]. 2012 Feb [cited 2014 Feb 24]. Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291128.pdf
3. U.S. Department of Health and Human Services. Guidance for industry biosimilars questions and answers regarding implementation of the Biologics Price Competition Act of 2009. Draft guidance. Februray 2012 [homepage on the Internet]. 2012 Feb [cited 2014 Feb 24]. Available from: http://www.fda.gov/downloads/Drugs/Guidances/UCM273001.pdf
4. U.S. Department of Health and Human Services. Guidance for industry quality considerations in demonstrating biosimilarity to a reference protein product. Draft guidance. Februray 2012 [homepage on the Internet]. 2012 Feb [cited 2014 Feb 24]. Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM291134.pdf
5. European Medicines Agency. Guideline on immunogenicity assessment of biotechnology-derived therapeutic proteins. EMEA/CHMP/BMWP/14327/2006. 13 December 2007 [homepage on the Internet]. 2008 Jan [2014 Feb 24]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003946.pdf
6. European Medicines Agency. Guideline on similar biological medicinal products. CHMP/437/04. 30 October 2005 [homepage on the Internet]. 2005 Nov [cited 2014 Feb 24]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003517.pdf
7. European Medicines Agency. Guideline on similar biological medicinal products containing biotechnology-derived proteins as active substance: non-clinical and clinical issues. EMEA/CHMP/BMWP/42832/2005 Rev. 1. 3 June 2013 [homepage on the Internet]. 2013 Jun [cited 2014 Feb 24]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2013/06/WC500144124.pdf
8. Nicholas JM. Complex drugs and biologics: scientific and regulatory challenges for follow-on products. Drug Inf J. 2012;46(2):197-206.
9. Schellekens H, Klinger E, Mühlebach S, Brin JF, Storm G, Crommelin DJ. The therapeutic equivalence of complex drugs. Regul Toxicol Pharmacol. 2011;59(1):176-83.
10. Varkony H, Weinstein V, Klinger E, et al. The glatiramoid class of immunomodulator drugs. Expert Opin Pharmacother. 2009;10(4):657-68.




moving beyond incremental innovation towards re-innovation

Larry H. Berntein, MD, FCAP, Curator




The generic pharmaceutical industry: moving beyond incremental innovation towards re-innovation
Generics and Biosimilars Initiative Journal (GaBI Journal). 2013;2(1):13-9.   DOI: http://dx.doi.org:/10.5639/gabij.2013.0201.011

Background: Due to the declining innovativeness of the classic R & D model in the original pharmaceutical industry, the generic pharmaceutical industry is aiming to become an innovation generator itself.
Objective: The objective of this article is to gain insight into the re-innovation model in some of the innovative generic pharmaceutical firms. To this effect, we show how some of the generic pharmaceutical firms attempt to achieve competitive advantages either by improving existing product attributes or by replacing new components, reshaping their configuration, and using new technology platforms to produce new innovative products.
Methods: We used a qualitative method to examine re-innovation at several levels within these companies, in their management systems, business models and product portfolios. The research was conducted by a series of semi-structured interviews with chief executive officers, consultants, researchers, patent attorneys, pharmacists and medics in different countries.
Results: Those generic pharmaceutical firms that implement new competitive strategies have integrated re-innovation design into their product portfolio to provide more personalized, cost-effective products to meet the healthcare systems’, policymakers’ and patients’ demand for high quality accessible treatments. This re-orientation hopes to better face the changing competition challenges in both mature and developing markets.
Conclusion: A new approach to innovativeness together with a value proposition strategy aims to deliver high quality products to patients.


Innovation is widely regarded as an instrument to create competitive advantage. Different types of innovation exist, including incremental innovation, re-innovation and radical innovation. Incremental innovation deals with creating minor improvements or simple adjustments in a product’s current state [1, 2]. Re-innovation has been defined as: ‘the process of innovation and product development that occurs after a new product is launched, building upon early success but improving the next generation with revised and refined features’ [3]. Finally, radical innovation refers to radical, new inventions that produce milestones, new products or services, and as a result lead to the development of new industries [4]. Today there is less radical product innovation in the original pharmaceutical industry. Moreover, the concept of ‘new product’ has also evolved by the application of strategies such as incremental innovation and re-innovation. In the past, radical or disruptive innovation changed the pharmaceutical market, whereas today generic pharmaceutical firms attempt to innovate in a less costly way in a shorter time with less regulatory obstacles due to the substantial R & D costs to achieve a radical new product. Incremental innovation and re-innovation meet these objectives.

The generic pharmaceutical industry is now evolving in an innovative way. Some firms are applying strategic changes in their management systems and business models and creating new product portfolios fortified with ‘super generics’, new chemical entities and novel drug delivery systems. A super generic drug is an improved version of an original drug which has lost product patent protection. The product patent for the original drug will have expired or have been circumvented by the company developing the super generics. The nature of the improvement may include drug delivery, manufacturing or reformulation technology. This kind of value-added version is manufactured in a re-innovation framework. This innovative design is between incremental and radical innovation. Companies producing super generics have a greater regulatory risk in gaining marketing approval compared to strict generics manufacturers [5]. Without getting into the details, there are three regulatory pathways for drug approval in Europe and the US.

The US Food and Drug Administration (FDA) does not recognize the term ‘super generics’. These products are also referred to as ‘added value generics, new therapeutic entities or hybrids’. These products differ from the original product in formulation or method of delivery. These products are improved formulation of a known product.

This group of generics needs a completely New Drug Application (NDA) in order to gain FDA approval. The regulatory pathway in Europe appears to be very similar to that in the US and was introduced within the Directive 2001/83/EC in November 2001 and in the Regulation (EC) No 726/2004. These products are not interchangeable with the brand-name drugs. Those regulatory pathways are summarized inTable 1.


With a NDA, innovative drug therapies are reaching the market in a specific dosage form for one or more clinically proven indications of which, after expiration of the patent or the data exclusivity, copies are launched using Abbreviated New Drug Applications (ANDA). Advanced therapies that emerged from launched molecules during their product life cycle have gained considerable attention as clinical practice provides evidence for additional therapeutic values; patient centric delivery systems show improved therapeutic outcomes or emerging technologies offer efficiency gains in manufacturing or access to emerging markets. The US and European regulatory framework has set reasonable regulations in place for these super generics or hybrid applications. While these regulations are relatively recent the pharmaceutical industry is just starting to use this route for its product development [6, 7].

However, super generics take an average of three to four years development time to registration, and enjoy reduced development and regulatory risks compared to new chemical entities. The end product may gain a significant price premium to conventional generics once marketing approval is received. Depending on the type of modification to the original formulation and whether the super generic drug is being developed for the same or a different indication will also have an impact on the level of additional research that is needed to gain approval for the reformulated product [8]. The quantity of issued patents highlights the technical knowledge and skill sets that are available in generic pharmaceutical firms. The success of these pharmaceutical firms has illustrated the possibility of changing from the classic model of ‘copy maker’ towards a model of creating new value-added products, manufacturing strategies and new business models [9, 10].

Meanwhile, the demand side for pharmaceutical treatments has also evolved. ‘New’ customers have emerged, i.e. a better informed, web data empowered generation of patients searching for cost-effective treatments. The generic pharmaceutical industry is reacting to this by applying new business models.

By applying a patient-centred and quality-based perspective into their business models, the generic pharmaceutical industry is attempting to offer new less risky and cost-effective products. The most important aspect is that innovation is no longer just about the product itself, it is also centred on how a company contributes to improving the health of patients. This process has required the out-licensing of innovative generic drug products and has also involved the establishment of new partnerships and alliances to better utilize technological platforms and manufacturing facilities [11]. As an example, Teva Pharmaceuticals acquired Ivax in 2006, Barr Laboratories in 2008, and Ratiopharm in 2010.

The aim of this article is to gain insight into re-innovation in the generic pharmaceutical industry by focusing on product innovation, and a business model based on value proposition employed by some of the innovative generic pharmaceutical firms. This is an alternative model between hybrid and classic R & D companies.




Producing novel products is defined as the part of new product development strategy which explores the extension of existing innovations, which can only happen after the first generation of a new product is launched [13]. This is, for example, the case with the development of super generics and bio-superior products that follow on from reference biopharmaceutical products. Being built upon early successful products, re-innovative products are created through applying new platforms, new components, or new configurations with breakthrough technologies to previous products or manufacturing processes [14, 15]. The new re-innovated medicines are focusing on improving health outcomes for patients.

‘… In the past, successful pharmaceuticals stemmed from having good clinical trial data which companies owned and controlled. In the future, their success in the market will instead be evaluated by post marketing data resulting from patients’ satisfaction, of which they will no longer have sole possession …’ (Pharma Researcher, UK)

At the industrial level, through re-innovation attempts, generic pharmaceutical firms aim to minimize the new product failure rate [7], reduce the cost of developing a new product and decrease the lead time in bringing it to market. A pharmaceutical product developed and manufactured with less excipients and unit operation, while maintaining the product therapeutic performance compared to the originator, could be considered as an improved therapeutic entity as it reduces the overall costs of manufacturing that could lead to reduced healthcare spending [16].

Innovative generic pharmaceutical firms may apply QbD and design of experiment methods to optimize their production outcome and minimize the risk. Quality by design means designing and developing a product and associated manufacturing processes that will be used during product development to ensure that the product consistently attains a predefined quality at the end of the manufacturing process [17]. Statistical methods are becoming increasingly vital for pharmaceutical firms. Design of experiments is a tool for determining the relationship between the factors that have an effect on a process and the response of that process [18].

The re-innovative product (as compared to an incremental new product) can be defined as a product that provides new features, benefits, or improvements through existing technology. As such, re-innovation and incremental innovation are different in two aspects: 1) incremental products are improved only by incremental technologies while breakthrough technologies can be used in re-innovative products; and 2) incremental products must be based on the current platform but re-innovative products are either (mostly) based on a new platform or (occasionally) based on an existing platform [19].

‘… As to technology platforms, if for example you consider aerosolization as a platform, then using such a platform to create new, better forms of an existing entity are part of re-innovation …’ (US Manager, 2012)

Re-innovation by the generic pharmaceutical industry can be observed in drug product design, formulation, process development and manufacturing processes going back to the early stages of the product development cycle.

Some product examples are:
1-Abraxane, super generic form of Taxol (FDA, 2005), which uses albumin to deliver the chemotherapy, not Cremophor, and so avoids hypersensitivity and claims a greater tumour response rate than Taxol. The drug Abraxane (nanoparticle albumin bound paclitaxel) uses the approach of coating Taxol with albumin to reduce the side effects associated with standard Taxol (paclitaxel), making it possible to give it without steroids (which can be a rather bothersome issue for many patients, causing problems from severe insomnia to very high blood sugars and more) and also reducing some other Taxol-associated side effects like joint and muscle aches [20, 21].

2-SUBACAP is an improved version of the conventional itraconazole formulation used to treat fungal infections. In June 2012, Mayne Pharma announced that the UK Medicines and Healthcare products Regulatory Agency (MHRA) had reversed its previous decision on SUBACAP and advised that the SUBACAP marketing authorization application was approvable in the UK. Mayne Pharma is in the process of submitting the response to re-activate the ‘Decentralized Procedure’ to seek approval in Germany, Spain and Sweden. Following approval in these countries, the company will seek a second round of approvals in other European countries, including Belgium, Italy, Greece, Portugal and The Netherlands. The total European market sales of itraconazole in 2011 were US$85 million (companies communication and annual report 2012). SUBACAP provides enhancements to patients and prescribers with reduced inter- and intra-patient variability and therefore a more predictable clinical response enabling a reduction in active drug quantity to deliver therapeutic blood levels. Itraconazole is one of the broadest spectrum antifungal drugs on the market and can be used to treat both superficial fungal infections such as onychomycosis (nail infection) and systemic fungal infections such as histoplasmosis, aspergillosis and candidiasiswhich can be life threatening to immunocompromised patients [22].

Another example of novel technology platform used in super generic drug manufacturing is the application of nanoparticle technology to address challenges associated with the delivery of poorly soluble compounds. Re-innovation has, for example, led to the development of a tablet dosage form that incorporated candesartan cilexetil nanoparticles [2328] to reduce dosage, reduce toxicity, improve bioavailability and enhance solubility. The original candesartan cilexetil is used for the treatment of hypertension. The major drawback in the therapeutic efficacy of candesartan cilexetil is its very low aqueous solubility leading to low and variable bioavailability. Low bioavailability may lead to variability in therapeutic response. The formulation change resulting from Design of Experiments and nanoparticle technology resulted in better solubility. Using Design of Experiments for process optimization resulted in a robust scalable manufacturing process with design space established for critical process parameters that can balance milling time, particles size and yield. Design of Experiments studies indicated that, out of the three parameters tested in the experimental design, disc speed, pump speed and bead volume were found to affect the critical product attributes either through non-linear, quadratic or interaction effects [29, 30].

The robustness of the model was validated based on confirmatory trials that indicated statistically no difference between predicted and experimental values. The rate and extent of drug dissolution from tablet dosage form incorporating drug nanoparticles was significantly higher than in the tablet containing micronized drug and marketed product.

The increase in drug dissolution resulted in significant enhancement in rate (Cmax) and extent of drug absorption (AUC).

The manufacturing process used is simple and scalable indicating general applicability of the approach to develop oral dosage forms of sparingly soluble drug.

The formulation approach used provides a viable approach to enhance dissolution and bioavailability of sparingly soluble compounds (BCS class II) that may translate into improved therapeutic outcome [23].

This innovative change is also illustrated by the following quote:

‘Super-generic [drug] products, mostly nano- and micro-sized drug delivery systems, focus on improving active principles which were previously commercialized in another formulation. These new formulations are certainly not bioequivalent in the generic [drug] industry’s sense of the term, they are therefore not generics. They are new, i.e. innovative, drugs, which can replace treatment with the previous entity.’ (Drug Delivery Manager, USA)

Another example of re-innovation in super generic drugs relates to the development of a per oral [29] dosage form for a sparingly soluble camptothecin analogue. This was achieved by formulating it as a drug complex [30]. This formulation approach addressed limitations of the currently marketed product that is only amenable for intravenous administration. The drug complex following oral administration demonstrated safety and efficacy comparable to marketed product in athymic mice with implanted tumours. The manufacturing process used is simple and scalable indicating general applicability of the approach to develop oral dosage forms of sparingly soluble drugs. An oral dosage form should result in lower treatment cost, better patient compliance and improved therapeutic outcome for better disease management [23].

In a recent compliance review for antihypertensive drug treatments it was found that some drug classes have significantly poorer adherence performance by patients than other drug classes. Only one third of patients were adherent to b-blockers and diuretics, while two thirds of patients were adherent to angiotension converting enzyme inhibitors and angiotensin II Receptor blockers [31]. Even an adherence of two-thirds of patients still remains at an unsatisfactory low level and leaves considerable room for improvement.

Modifying the release of drugs that have a short biological half-life by extending their release, circumvents high plasma peaks, reduces fluctuations in plasma levels and allows for a once-daily intake that can optimize therapy. This can avoid the daily oral intake for people with dysphagia or dementia. In this new business model, therapy is moving away from a clinical parameter oriented treatment to an outcome oriented disease management programme [32].


The low price of generic drugs threatens to undermine the sustainability of the generic pharmaceutical industry in regards to its low margins, number of competitors, increased requirements for pharmacovigilance, the mature markets in developed countries, and the post-patent cliff arena after 2015 [8]. Meanwhile, medical and technological changes push the pharmaceutical industry to implement new business models. These changes coincide with a growing demand from ageing populations, and better-informed patients who have a substantial need for individualized cost-effective treatments.

Several generic pharmaceutical firms have evolved their traditional business models into innovative models. These models are key to maintaining market position. They are focused on patients’ unmet medical needs and a high quality approach to the manufacturing process.

The innovative business models emerge from new management systems. The challenge of new management systems in these innovative generic pharmaceutical firms is on product innovation: how to manage a better organization to achieve a maximum product differentiation through value proposition to patients? How to optimize product quality? How to reduce manufacturing costs? How to reduce the time to market?

The generic pharmaceutical industry is evolving into a less generic, but more innovative format. In this respect, it should be noted that many generic pharmaceutical firms have the capacity to re-innovate. They have experience, good knowledge and the technical possibility to re-innovate. Alternatively, new alliances can provide the necessary financial resources for technical and marketing requirements.

Implementing re-innovation as a strategy strives to convert price-focused competition into product quality competition, this is central to an innovative business model. Some generic pharmaceutical firms are re-innovating their product portfolio by using new technology platforms, new components and new configurations. These attempts have mainly resulted in super generic drugs; value added products or hybrid products and biosimilars. These super generic drugs and improved therapeutic entities are an important source for innovation in drug therapy in the coming decades.

The so-called super generics are a promising alternative. The value added products resulting from re-innovation strategy by using new ‘technology platforms’, new components and new services will be a strategic element for affordable and individualized medicines.

According to our findings:

  1. The classic innovation model of R & D in Big Pharma is no longer able to provide sufficient results, because it is too costly, too time-consuming and too risky. There are more regulatory barriers, changing demographic and economic features, and Big Pharma is becoming too big to manage innovation. Generic pharmaceutical company aims to provide innovative products to meet: price pressure, low margins, government’s pressure, competition, mature markets, and tendering.
  2. The generic pharmaceutical industry is facing now unmet medical needs of a new generation of patients (demand-side is evolved), there is a real demand for high quality geriatric pharmaceuticals for a rapidly ageing population in some developed countries such as Japan.
  3. Better results will be obtained by using novel technology platforms to achieve new formulations, reducing costs and time by applying QbD.
  4. These new products produced by some generics companies are only one example; they try to switch to biosimilars, and new chemical entities. The future is related to a new kind of disease management requiring more value for more affordable treatments.

This research may be followed by further investigation in innovative business models adapted by an evolving generic pharmaceutical industry that has not yet been studied. A quantitative study of R & D investment and strategic alliances in an innovative generic pharmaceutical industry will reveal more.


Due to evolution in the pharmaceutical industry landscape, some generic pharmaceutical companies are restructuring their business models. In this new industrial design, some of the generics manufactures are re-inventing their product portfolio through a re-innovation strategy. New technology platforms, new components and new configurations are adopted to provide patient compliance and increase patient quality of life. Super generics, biosimilars, bio-superiors and value added versions are some of the new product alternatives resulting from this innovative evolution. The product itself is not the only target; the conversion of competition from price to product quality ensures the value proposition and provides product differentiation. New innovative product portfolios are the evidence that innovative generics companies are not only mastering incremental innovation but are also adopting re-innovation in their new strategies. In this perspective, biotechnology, nanosciences and nanotechnology are ‘strategic’ areas for its scientific and commercial development.

For patients

For patients, the innovative changes in product portfolios struggle to improve patient’s quality of life, reduce side effects and enhance efficiency by new product alternatives. These new product alternatives are developed by applying new technology platforms like nanotechnology. Enhancing drug solubility is often key to improving a product’s formulation. New nanotechnologiesa are now being used to solubilize drugs with the aim of improving bioavailability and activity, and reducing in vivo variabilityb.

Retromer in neurological disorders

Larry H. Bernstein, MD, FCAP, Curator




Retromer in Alzheimer disease, Parkinson disease and other neurological disorders.

Scott A. Small and Gregory A. Petsko
Nature Reviews Neuroscience 16; 126–132 (2015)      http://dx.doi.org:/10.1038/nrn3896


As discussed in the forum (see video here), there are many cellular pathways which are believed to be perturbed in Alzheimer’s Disease. Recent work has suggested that deficits in retromer complex function may underlie impairment of endosomal trafficking in neurons and may contribute to AD pathogenesis. This recent review illustrates the function of the retromer complex and discusses how its dysfunction may contribute to neurodegeneration.

By Tim Spencer on 24 Nov, 2015


Retromer is a protein assembly that has a central role in endosomal trafficking, and retromer dysfunction has been linked to a growing number of neurological disorders. First linked to Alzheimer disease, retromer dysfunction causes a range of pathophysiological consequences that have been shown to contribute to the core pathological features of the disease. Genetic studies have established that retromer dysfunction is also pathogenically linked to Parkinson disease, although the biological mechanisms that mediate this link are only now being elucidated. Most recently, studies have shown that retromer is a tractable target in drug discovery for these and other disorders of the nervous system.


Yeast has proved to be an informative model organism in cell biology and has provided early insight into much of the molecular machinery that mediates the intracellular transport of proteins1,2. Indeed, the term ‘retromer’ was first introduced in a yeast study in 1998 (Ref. 3). In this study, retromer was referred to as a complex of proteins that was dedicated to transporting cargo in a retrograde direction, from the yeast endosome back to the Golgi.

By 2004, a handful of studies had identified the molecular4 and the functional5, 6 homologies of the mammalian retromer, and in 2005 retromer was linked to its first human disorder, Alzheimer disease (AD)7. At the time, the available evidence suggested that the mammalian retromer might match the simplicity of its yeast homologue. Since then, a dramatic and exponential rise in research focusing on retromer has led to more than 300 publications. These studies have revealed the complexity of the mammalian retromer and its functional diversity in endosomal transport, and have implicated retromer in a growing number of neurological disorders.

New evidence indicates that retromer is a ‘master conductor’ of endosomal sorting and trafficking8. Synaptic function heavily depends on endosomal trafficking, as it contributes to the presynaptic release of neurotransmitters and regulates receptor density in the postsynaptic membrane, a process that is crucial for neuronal plasticity9. Therefore, it is not surprising that a growing number of studies are showing that retromer has an important role in synaptic biology10, 11, 12, 13. These observations may account for why the nervous system seems particularly sensitive to genetic and other defects in retromer. In this Progress article, we briefly review the molecular organization and the functional role of retromer, before discussing studies that have linked retromer dysfunction to several neurological diseases — notably, AD and Parkinson disease (PD).


The endosome is considered a hub for intracellular transport. From the endosome, transmembrane proteins can be actively sorted and trafficked to various intracellular sites via distinct transport routes (Fig. 1a). Studies have shown that the mammalian retromer mediates two of the three transport routes out of endosomes. First, retromer is involved in the retrieval of cargos from endosomes and in their delivery, in a retrograde direction, to the trans-Golgi network (TGN)5,6. Retrograde transport has many cellular functions but, as we describe, it is particularly important for the normal delivery of hydrolases and proteases to the endosomal–lysosomal system. The second transport route in which retromer functions is the recycling of cargos from endosomes back to the cell surface14, 15 (Fig. 1a). It is this transport route that is particularly important for neurons, as it mediates the normal delivery of glutamate and other receptors to the plasma membrane during synaptic remodelling and plasticity10, 11, 12, 13.

Figure 1: Retromer’s endosomal transport function and molecular organization.
Retromer's endosomal transport function and molecular organization.

a | Retromer mediates two transport routes out of endosomes via tubules that extend out of endosomal membranes. The first is the retrograde pathway in which cargo is retrieved from the endosome and trafficked to the trans-Golgi network (TGN). The second is the recycling pathway in which cargo is trafficked back from the endosome to the cell surface. The degradation pathway, which is not mediated by retromer, involves the trafficking of cargo from endosomes to lysosomes for degradation. b | The retromer assembly of proteins can be organized into distinct functional modules, all of which work together as part of retromer’s transport role. The ‘cargo-recognition core’ is the central module of the retromer assembly and comprises a trimer of proteins, in which vacuolar protein sorting-associated protein 26 (VPS26) and VPS29 bind VPS35. The ‘tubulation’ module includes protein complexes that bind the cargo-recognition core and aid in the formation and stabilization of tubules that extend out of endosomes, directing the transport of cargos towards their final destinations. The ‘membrane-recruiting’ proteins recruit the cargo-recognition core to the endosomal membrane. The WAS protein family homologue (WASH) complex of proteins also binds the cargo-recognition core and is involved in endosomal ‘actin remodelling’ to form actin patches, which are important for directing cargos towards retromer’s transport pathways. Retromer cargos includes a range of receptors — which bind the cargo-recognition core — and their ligands. PtdIns3P, phosphatidylinositol-3-phosphate.

As well as extending the endosomal transport routes, recent studies have considerably expanded the number of molecular constituents and what is known about the functional organization of the mammalian retromer. Following this expansion in knowledge of the molecular diversity and organizational complexity, retromer might be best described as a multimodular protein assembly. The protein or group of proteins that make up each module can vary, but each module is defined by its distinct function, and the modules work in unison in support of retromer’s transport role.

Two modules are considered central to the retromer assembly. First and foremost is a trimeric complex that functions as a ‘cargo-recognition core’, which selects and binds to the transmembrane proteins that need to be transported and that reside in endosomal membranes5, 6. This trimeric core comprises vacuolar protein sorting-associated protein 26 (VPS26), VPS29 and VPS35; VPS35 functions as the core’s backbone to which the other two proteins bind16. VPS26 is the only member of the core that has been found to have two paralogues, VPS26a and VPS26b17,18, and studies suggest that VPS26b might be differentially expressed in the brain19, 20. Some studies suggest that VPS26a and VPS26b are functionally redundant21, whereas others suggest that they might form distinct cargo-recognition cores20, 22.

The second central module of the retromer assembly is the ‘tubulation’ module, which is made up of proteins that work together in the formation and the stabilization of tubules that extend out of endosomes and that direct the transport of cargo towards its final destination (Fig. 1b). The proteins in this module, which directly binds the cargo-recognition core, are members of the subgroup of the sorting nexin (SNX) family that are characterized by the inclusion of a carboxy-terminal BIN–amphiphysin–RVS (BAR) domain23. These members include SNX1, SNX2, SNX5 and SNX6 (Refs 24,25). As part of the tubulation module, these SNX-BAR proteins exist in different dimeric combinations, but typically SNX1 interacts with SNX5 or SNX6, and SNX2 interacts with SNX5 or SNX6 (Refs 26,27). The EPS15-homology domain 1 (EHD1) protein can be included in this module, as it is involved in stabilizing the tubules formed by the SNX-BAR proteins28.

A third module of the retromer assembly functions to recruit the cargo-recognition core to endosomal membranes and to stabilize the core once it is there (Fig. 1b). Proteins that are part of this ‘membrane-recruiting’ module include SNX3 (Ref. 29), the RAS-related protein RAB7A30, 31,32 and TBC1 domain family member 5 (TBC1D5), which is a member of the TRE2–BUB2–CDC16 (TBC) family of RAB GTPase-activating proteins (GAPs)28. In addition, the lipid phosphatidylinositol-3-phosphate (PtdIns3P), which is found on endosomal membranes, contributes to recruiting most of the retromer-related SNXs through their phox homology domains33. Interestingly, another SNX with a phox homology domain, SNX27, was recently linked to retromer and its function15, 34. SNX27 functions as an adaptor for binding to PDZ ligand-containing cargos that are destined for transport to the cell surface via the recycling pathway. Thus, according to the functional organization of the retromer assembly, SNX27 belongs to the module that engages in cargo recognition and selection.

Recent studies have identified a fourth module of the retromer assembly. The five proteins in this module — WAS protein family homologue 1 (WASH1), FAM21, strumpellin, coiled-coil domain-containing protein 53 (CCDC53) and KIAA1033 (also known as WASH complex subunit 7) — form the WASH complex and function as an ‘actin-remodelling’ module28, 35, 36 (Fig. 1b). Specifically, the WASH complex functions in the rapid polymerization of actin to create patches of actin filaments on endosomal membranes. The complex is recruited to endosomal membranes by binding VPS35 (Ref. 28), and together they divert cargo towards retromer transport pathways and away from the degradation pathway.

The cargos that are transported by retromer include the receptors that directly bind the cargo-recognition core and the ligands of these receptors that are co-transported with the receptors. The receptors that are transported by retromer that have so far been identified to be the most relevant to neurological diseases are the family of VPS10 domain-containing receptors (including sortilin-related receptor 1 (SORL1; also known as SORLA), sortilin, and SORCS1, SORCS2 and SORCS3)7; the cation-independent mannose-6-phosphate receptor (CIM6PR)6, 5; glutamate receptors10; and phagocytic receptors that mediate the clearing function of microglia37. The most disease-relevant ligand to be identified that is trafficked as retromer cargo is the β-amyloid precursor protein (APP)7, 38, 39, 40, 41, which binds SORL1 and perhaps other VPS10 domain-containing receptors42 at the endosomal membrane.

Retromer dysfunction

Guided by retromer’s established function, and on the basis of empirical evidence, there are three well-defined pathophysiological consequences of retromer dysfunction that have proven to be relevant to AD and nervous system disorders. First, retromer dysfunction can cause cargos that typically transit rapidly through the endosome to reside in the endosome for longer than normal durations, such that they can be pathogenically processed into neurotoxic fragments (for example, APP, when stalled in the endosome, is more likely to be processed into amyloid-β, which is implicated in AD43 (Fig. 2a)). Second, by reducing endosomal outflow via impairment of the recycling pathway, retromer dysfunction can lead to a reduction in the number of cell surface receptors that are important for brain health (for example, microglia phagocytic receptors37 (Fig. 2b)).

Figure 2: The pathophysiology of retromer dysfunction.
The pathophysiology of retromer dysfunction.

Retromer dysfunction has three established pathophysiological consequences. In the examples shown, the left graphic represents a cell with normal retromer function and the right graphic represents a cell with a deficit in retromer function. a | Retromer dysfunction causes increased levels of cargo to reside in endosomes. For example, in primary neurons, retromer transports the β-amyloid precursor protein (APP) out of endosomes. Accordingly, retromer dysfunction increases APP levels in endosomes, leading to accelerated APP processing, resulting in an accumulation of neurotoxic fragments of APP (namely, β-carboxy-terminal fragment (βCTF) and amyloid-β) that are pathogenic in Alzheimer disease. b | Retromer dysfunction causes decreased cargo levels at the cell surface. For example, in microglia, retromer mediates the transport of phagocytic receptors to the cell surface and retromer dysfunction results in a decrease in the delivery of these receptors. Studies suggest that this cellular phenotype might have a pathogenic role in Alzheimer disease. c | Retromer dysfunction causes decreased delivery of proteases to the endosome. Retromer is required for the normal retrograde transport of the cation-independent mannose-6-phosphate receptor (CIM6PR) from the endosome back to the trans-Golgi network (TGN). It is in the TGN that this receptor binds cathepsin D and other proteases, and transports them to the endosome, to support the normal function of the endosomal–lysosomal system. By impairing the retrograde transport of the receptor, retromer dysfunction ultimately leads to reduced delivery of cathepsin D to this system. Cathepsin D deficiency has been shown to disrupt the endosomal–lysosomal system and to trigger tau pathology either within endosomes or secondarily in the cytosol.

The third consequence (Fig. 2c) is a result of the established role that retromer has in the retrograde transport of receptors, such as CIM6PR5, 6 or sortilin44, after these receptors transport proteases from the TGN to the endosome. Once at the endosome, the proteases disengage from the receptors, are released into endosomes and migrate to lysosomes. These proteases function in the endosomal–lysosomal system to degrade proteins, protein oligomers and aggregates45. Retromer functions to transfer the ‘naked’ receptor from the endosome back to the TGN via the retrograde pathway5, 6, allowing the receptors to continue in additional rounds of protease delivery. Accordingly, by reducing the normal retrograde transport of these receptors, retromer dysfunction has been shown to reduce the proper delivery of proteases to the endosomal–lysosomal system5,6, which, as discussed below, is a pathophysiological state linked to several brain disorders.

Although requiring further validation, recent studies suggest that retromer dysfunction might be involved in two other mechanisms that have a role in neurological disease. One study suggested that retromer might be involved in trafficking the transmembrane protein autophagy-related protein 9A (ATG9A) to recycling endosomes, from where it can then be trafficked to autophagosome precursors — a trafficking step that is crucial in the formation and the function of autophagosomes46. Autophagy is an important mechanism by which neurons clear neurotoxic aggregates that accumulate in numerous neurodegenerative diseases47. A second study has suggested that retromer dysfunction might enhance the seeding and the cell-to-cell spread of intracellular neurotoxic aggregates48, which have emerged as novel pathophysiological mechanisms that are relevant to AD49, PD50 and other neurodegenerative diseases.

Alzheimer disease

Retromer was first implicated in AD in a molecular profiling study that relied on functional imaging observations in patients and animal models to guide its molecular analysis7. Collectively, neuroimaging studies confirmed that the entorhinal cortex is the region of the hippocampal circuit that is affected first in AD, even in preclinical stages, and suggested that this effect was independent of ageing (as reviewed in Ref. 51). At the same time, neuroimaging studies identified a neighbouring hippocampal region, the dentate gyrus, that is relatively unaffected in AD52. Guided by this information, a study was carried out in which the two regions of the brain were harvested post mortem from patients with AD and from healthy individuals, intentionally covering a broad range of ages. A statistical analysis was applied to the determined molecular profiles of the regions that was designed to address the following question: among the thousands of profiled molecules, which are the ones that are differentially affected in the entorhinal cortex versus the dentate gyrus, in patients versus controls, but that are not affected by age? The final results led to the determination that the brains of patients with AD are deficient in two core retromer proteins — VPS26 and VPS35 (Ref. 7).

Little was known about the receptors of the neuronal retromer, so to understand how retromer deficiency might be mechanistically linked to AD, an analysis was carried out on the molecular data set that looked for transmembrane molecules for which expression levels correlated with VPS35 expression. The top ‘hit’ was the transcript encoding the transmembrane protein SORL1 (Ref. 43). As SORL1 belongs to the family of VPS10-containing receptors and as VPS10 is the main retromer receptor in yeast3, it was postulated that SORL1 and the family of other VPS10-containing proteins (sortillin, SORCS1, SORCS2 and SORCS3) might function as retromer receptors in neurons7. In addition, SORL1 had recently been reported to bind APP53, so if SORL1 was assumed to be a receptor that is trafficked by retromer, then APP might be the cargo that is co-trafficked by retromer. This led to a model in which retromer traffics APP out of endosomes7, which are the organelles in which APP is most likely to be cleaved by βAPP-cleaving enzyme 1 (BACE1; also known as β-secretase 1)43; this is the initial enzymatic step in the pathogenic processing of APP.

Subsequent studies were required to further establish the pathogenic link between retromer and AD, and to test the proposed model. The pathogenic link was further supported by human genetic studies. First, a genetic study investigating the association between AD, the genes encoding the components of the retromer cargo-recognition core and the family of VPS10-containing receptors found that variants of SORL1 increase the risk of developing AD38. This finding was confirmed by numerous studies, including a recent large-scale AD genome-wide association study54. Other genetic studies identified AD-associated variants in genes encoding proteins that are linked to nearly all modules of the retromer assembly55, including genes encoding proteins of the retromer tubulation module (SNX1), genes encoding proteins of the retromer membrane-recruiting module (SNX3 and RAB7A) and genes encoding proteins of the retromer actin-remodelling module (KIAA1033). In addition, nearly all of the genes encoding the family of VPS10-containing retromer receptors have been found to have variants that associate with AD56. Finally, a study found that brain regions that are differentially affected in AD are deficient in PtdIns3P, which is the phospholipid required for recruiting many sorting nexins to endosomal membranes57. Thus, together with the observation that the brains of patients with AD are deficient in VPS26a and VPS35 (Refs 7,37), all modules in the retromer assembly are implicated in AD.

Studies in mice39, 58, 59, flies39 and cells in culture34, 40, 41, 60, 61 have investigated how retromer dysfunction leads to the pathogenic processing of APP. Although rare discrepancies have been observed among these studies62, when viewed in total, the most consistent findings are that retromer dysfunction causes increased pathogenic processing of APP by increasing the time that APP resides in endosomes. Moreover, these studies have confirmed that SORL1 and other VPS10-containing proteins function as APP receptors that mediate APP trafficking out of endosomes.

Retromer has unexpectedly been linked to microglial abnormalities37 — another core feature of AD — which, on the basis of recent genetic findings, seem to have an upstream role in disease pathogenesis54, 63. A recent study found that microglia harvested from the brains of individuals with AD are deficient in VPS35 and provided evidence suggesting that retromer’s recycling pathway regulates the normal delivery of various phagocytic receptors to the cell surface of microglia37, including the phagocytic receptor triggering receptor expressed on myeloid cells 2 (TREM2) (Fig. 2b). Mutations in TREM2 have been linked to AD63, and a recent study indicates that these mutations cause a reduction in its cell surface delivery and accelerate TREM2 degradation, which suggests that the mutations are linked to a recycling defect64. While they are located at the microglial cell surface, these phagocytic receptors function in the clearance of extracellular proteins and other molecules from the extracellular space65. Taken together, these recent studies suggest that defects in the retromer’s recycling pathway can, at least in part, account for the microglial defects observed in the disease.

The microtubule-associated protein tau is the key element of neurofibrillary tangles, which are the other hallmark histological features of AD. Although a firm link between retromer dysfunction and tau toxicity remains to be established, recent insight into tau biology suggests several plausible mechanisms that are worth considering. Tau is a cytosolic protein, but nonetheless, through mechanisms that are still undetermined, it is released into the extracellular space from where it gains access to neuronal endosomes via endocytosis66, 67. In fact, recent studies suggest that the pathogenic processing of tau is triggered after it is endocytosed into neurons and while it resides in endosomes67. Of note, it still remains unknown which specific tau processing step — its phosphorylation, cleavage or aggregation — is an obligate step towards tau-related neurotoxicity. Accordingly, if defects in microglia or in other phagocytic cells reduce their capacity to clear extracellular tau, this would accelerate tau endocytosis in neurons and its pathogenic processing.

A second possibility comes from the established role retromer has in the proper delivery of cathepsin D and other proteases to the endosomal–lysosomal system via CIM6PR or sortilin (Fig. 2c). Studies in sheep, mice and flies68 have shown that cathepsin D deficiency can enhance tau toxicity and that this is mediated by a defective endosomal–lysosomal system68. Whether this mechanism leads to abnormal processing of tau within endosomes or in the cytosol via caspase activation68 remains unclear. As discussed above, retromer dysfunction will lead to a decrease in the normal delivery of cathepsin D to the endosome and will result in endosomal–lysosomal system defects. Retromer dysfunction can therefore be considered as a functional phenocopy of cathepsin D deficiency, which suggests a plausible link between retromer dysfunction and tau toxicity. Nevertheless, although these recent insights establish plausibility and support further investigation into the link between retromer and tau toxicity, whether this link exists and how it may be mediated remain open and outstanding questions.

Parkinson disease

The pathogenic link between retromer and PD is singular and straightforward: exome sequencing has identified autosomal-dominant mutations in VPS35 that cause late-onset PD69, 70, one of a handful of genetic causes of late-onset disease. However, the precise mechanism by which these mutations cause the disease is less clear.

Among a group of recent studies, all46, 48, 71, 72, 73, 74, 75, 76 but one77 strongly suggest that these mutations cause a loss of retromer function. At the molecular level, the mutations do not seem to disrupt mutant VPS35 from interacting normally with VPS26 and VPS29, and from forming the cargo-recognition core. Rather, two studies suggest that the mutations have a restricted effect on the retromer assembly but reduce the ability of VPS35 to associate with the WASH complex46, 75. Studies disagree about the pathophysiological consequences of the mutations. Four studies suggest that the mutations affect the normal retrograde transport of CIM6PR71, 73, 75, 76 from the endosome back to the TGN (Fig. 2c). In this scenario, the normal delivery of cathepsin D to the endosomal–lysosomal system should be reduced and this has been empirically shown73. Cathepsin D has been shown to be the dominant endosomal–lysosomal protease for the normal processing of α-synuclein76, and mutations could therefore lead to abnormal α-synuclein processing and to the formation of α-synuclein aggregates, which are thought to have a key pathogenic role in PD.

A separate study suggested that the mutation might cause a mistrafficking of ATG9, and thereby, as discussed above, reduce the formation and the function of autophagosomes46. Autophagosomes have also been implicated as an intracellular site in which α-synuclein aggregates are cleared. Thus, although future studies are needed to resolve these discrepant findings (which may in fact not be mutually exclusive), these studies are generally in agreement that retromer defects will probably increase the neurotoxic levels of α-synuclein aggregates48.

Several studies in flies71, 74 and in rat neuronal cultures71 provide strong evidence that increasing retromer function by overexpressing VPS35 rescues the neurotoxic effects of the most common PD-causing mutations in leucine-rich repeat kinase 2 (LRRK2). Moreover, a separate study has shown that increasing retromer levels rescues the neurotoxic effect of α-synuclein aggregates in a mouse model48. These findings have immediate therapeutic implications for drugs that increase VPS35 and retromer function, as discussed in the next section, but they also offer mechanistic insight. LRRK2 mutations were found to phenocopy the transport defects caused either by theVPS35 mutations or by knocking down VPS35 (Ref. 71). Together, this and other studies78suggest that LRRK2 might have a role in retromer-dependent transport, but future studies are required to clarify this role.

Other neurological disorders

Besides AD and PD, in which a convergence of findings has established a strong pathogenic link, retromer is being implicated in an increasing number of other neurological disorders. Below, we briefly review three disorders for which the evidence of the involvement of retromer in their pathophysiology is currently the most compelling.

The first of these disorders is Down syndrome (DS), which is caused by an additional copy of chromosome 21. Given the hundreds of genes that are duplicated in DS, it has been difficult to identify which ones drive the intellectual impairments that characterize this condition. A recent elegant study provides strong evidence that a deficiency in the retromer cargo-selection protein SNX27 might be a primary driver for some of these impairments79. This study found that the brains of individuals with DS were deficient in SNX27 and that this deficiency may be caused by an extra copy of a microRNA (miRNA) encoded by human chromosome 21 (the miRNA is produced at elevated levels and thereby decreases SNX27 expression). Consistent with the known role of SNX27 in retromer function, decreased expression of this protein in mice disrupted glutamate receptor recycling in the hippocampus and led to dendritic dysfunction. Importantly, overexpression of SNX27 rescued cognitive and other defects in animal models79, which not only strengthens the causal link between retromer dysfunction and cognitive impairment in DS but also has important therapeutic implications.

Hereditary spastic paraplegia (HSP) is another disorder linked to retromer. HSP is caused by genetic mutations that affect upper motor neurons and is characterized by progressive lower limb spasticity and weakness. Although there are numerous mutations that cause HSP, most are unified by their effects on intracellular transport80. One HSP-associated gene in particular encodes strumpellin81, which is a member of the WASH complex.

The third disorder linked to retromer is neuronal ceroid lipofuscinosis (NCL). NCL is a young-onset neurodegenerative disorder that is part of a larger family of lysosomal storage diseases and is caused by mutations in one of ten identified genes — nine neuronal ceroid lipofuscinosis (CLN) genes and the gene encoding cathepsin D82. Besides cathepsin D, for which the link to retromer has been discussed above, CLN3 seems to function in the normal trafficking of CIM6PR83. However, the most direct link to retromer has been recently described for CLN5, which seems to function, at least in part, as a retromer membrane-recruiting protein84.

Retromer as a therapeutic target

As suggested by the first study implicating retromer in AD7, and in several subsequent studies71,85, increasing the levels of retromer’s cargo-recognition core enhances retromer’s transport function. Motivated by this observation and after a decade-long search86, we identified a novel class of ‘retromer pharmacological chaperones’ that can bind and stabilize retromer’s cargo-recognition core and increase retromer levels in neurons61.

Validating the motivating hypothesis, the chaperones were found to enhance retromer function, as shown by the increased transport of APP out of endosomes and a reduction in the accumulation of APP-derived neurotoxic fragments61. Although there are numerous other pharmacological approaches for enhancing retromer function, this success provides the proof-of-principle that retromer is a tractable therapeutic target.

As retromer functions in all cells, a general concern is whether enhancing its function will have toxic adverse effects. However, studies have found that in stark contrast to even mild retromer deficiencies, increasing retromer levels has no obvious negative consequences in yeast, neuronal cultures, flies or mice40, 48, 61, 71. This might make sense because unlike drugs that, for example, function as inhibitors, simply increasing the normal flow of transport through the endosome might not be cytotoxic.

If retromer drugs are safe and can effectively enhance retromer function in the nervous system — which are still outstanding issues — there are two general indications for considering their clinical application. One rests on the idea that these agents will only be efficacious in patients who have predetermined evidence of retromer dysfunction. The most immediate example is that of individuals with PD that is caused by LRRK2 mutations. As discussed above, several ‘preclinical’ studies in flies and neuronal cultures have already established that increasing retromer levels71, 74can reverse the neurotoxic effects of such mutations and, thus, if this approach is proven to be safe, LRRK2-linked PD might be an appropriate indication for clinical trials.

Alternatively, the pathophysiology of a disease might be such that retromer-enhancing drugs would be efficacious regardless of whether there is documented evidence of retromer dysfunction. AD illustrates this point. As reviewed above, current evidence suggests that retromer-enhancing drugs will, at the very least, decrease pathogenic processing of APP in neurons and enhance microglial function, even if there are no pre-existing defects in retromer.

More generally, histological studies comparing the entorhinal cortex of patients with sporadic AD to age-matched controls have documented that enlarged endosomes are a defining cellular abnormality in AD87, 88. Importantly, enlarged endosomes are uniformly observed in a broad range of patients with sporadic AD, which suggests that enlarged endosomes reflect an intracellular site at which molecular aetiologies converge87. In addition, because they are observed in early stages of the disease in regions of the brain without evidence of amyloid pathology87, enlarged endosomes are thought to be an upstream event. Mechanistically, the most likely cause of enlarged endosomes is either too much cargo flowing into endosomes — as occurs, for example, with apolipoprotein E4 (APOE4), which has been shown to accelerate endocytosis89, 90 — or too little cargo flowing out, as observed in retromer dysfunction40, 61 and related transport defects57. By any mechanism, retromer-enhancing drugs might correct this unifying cellular defect and might be expected to be beneficial regardless of the specific aetiology.


The fact that retromer defects, including those derived from bona fide genetic mutations, seem to differentially target the nervous system suggests that the nervous system is differentially dependent on retromer for its normal function. We think that this reflects the unique cellular properties of neurons and how synaptic biology heavily depends on endosomal transport and trafficking. Although plausible, future studies are required to confirm and to test the details of this hypothesis.

However, currently, it is the clinical rather than the basic neuroscience of retromer that is much better understood, with the established pathophysiological consequences of retromer dysfunction providing a mechanistic link to the disorders in which retromer has been implicated. Nevertheless, many questions remain. The two most interesting questions, which are in fact inversions of each other, relate to regional vulnerability in the nervous system. First, why does retromer dysfunction target specific neuronal populations? Second, how can retromer dysfunction cause diseases that target different regions of the nervous system? Recent evidence hints at answers to both questions, which must somehow be rooted in the functional and molecular diversity of retromer.

The type and the extent of retromer defects linked to different disorders might provide pathophysiological clues as well as reasons for differential vulnerability. As discussed, in AD there seem to be across-the-board defects in retromer, such that each module of the retromer assembly as well as multiple retromer cargos have been pathogenically implicated. By contrast, the profile of retromer defects in PD seems to be more circumscribed, involving selective disruption of the interaction between VPS35 and the WASH complex. These insights might agree with histological87, 88 and large-scale genetic studies54 that suggest that endosomal dysfunction is a unifying focal point in the cellular pathogenesis of AD. In contrast, genetics and other studies91suggest that the cellular pathobiology of PD is more distributed, implicating the endosome but other organelles as well, in particular the mitochondria.

Interestingly, studies suggest that the entorhinal cortex — a region that is differentially vulnerable to AD — has unique dendritic structure and function92, which are highly dependent on endosomal transport. We speculate that it is the unique synaptic biology of the entorhinal cortex that can account for why it might be particularly sensitive to defects in endosomal transport in general and retromer dysfunction in particular, and for why this region is the early site of disease. Future studies are required to investigate this hypothesis, as well as to understand why the substantia nigra or other regions that are differentially vulnerable to PD would be particularly sensitive to the more circumscribed defect in retromer.

Perhaps the most important observation for clinical neuroscience is the now well-established fact that increasing levels of retromer proteins enhances retromer function and has already proved capable of reversing defects associated with AD, PD and DS in either cell culture or in animal models. The relationships between protein levels and function are not always simple, but emerging pharmaceutical technologies that selectively and safely increase protein levels are now a tractable goal in drug discovery93. With the evidence mounting that retromer has a pathogenic role in two of the most common neurodegenerative diseases, we think that targeting retromer to increase its functional activity is an important goal that has strong therapeutic promise.


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Neurovascular pathways to neurodegeneration

Larry H. Bernstein, MD, FCAP, Curator




In addition to the many cellular insults which may contribute to neurodegeneration, there is also a wealth of evidence which suggests that dysfunction of the blood-brain barrier and other CNS vascular insults may also play a key role in Alzheimer’s Disease pathogenesis. This review from Berislav Zlokovic describes much of the recent work into understand how BBB dysfunction contributes to neurodegeneration.    By Tim Spencer on 24 Nov, 2015


Neurovascular pathways to neurodegeneration in Alzheimer’s disease and other disorders.

Berislav V. Zlokovic    About the author

Nat Rev Neurosci. 2011 Nov 3;12(12):723-38.        http://dx.doi.org:/10.1038/nrn3114


The neurovascular unit (NVU) comprises brain endothelial cells, pericytes or vascular smooth muscle cells, glia and neurons. The NVU controls blood–brain barrier (BBB) permeability and cerebral blood flow, and maintains the chemical composition of the neuronal ‘milieu’, which is required for proper functioning of neuronal circuits. Recent evidence indicates that BBB dysfunction is associated with the accumulation of several vasculotoxic and neurotoxic molecules within brain parenchyma, a reduction in cerebral blood flow, and hypoxia. Together, these vascular-derived insults might initiate and/or contribute to neuronal degeneration. This article examines mechanisms of BBB dysfunction in neurodegenerative disorders, notably Alzheimer’s disease, and highlights therapeutic opportunities relating to these neurovascular deficits.



Neurons depend on blood vessels for their oxygen and nutrient supplies, and for the removal of carbon dioxide and other potentially toxic metabolites from the brain’s interstitial fluid (ISF). The importance of the circulatory system to the human brain is highlighted by the fact that although the brain comprises ~2% of total body mass, it receives up to 20% of cardiac output and is responsible for ~20% and ~25% of the body’s oxygen consumption and glucose consumption, respectively1. To underline this point, when cerebral blood flow (CBF) stops, brain functions end within seconds and damage to neurons occurs within minutes2.

Neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) are associated with microvascular dysfunction and/or degeneration in the brain, neurovascular disintegration, defective blood–brain barrier (BBB) function and/or vascular factors1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Microvascular deficits diminish CBF and, consequently, the brain’s supply of oxygen, energy substrates and nutrients. Moreover, such deficits impair the clearance of neurotoxic molecules that accumulate and/or are deposited in the ISF, non-neuronal cells and neurons. Recent evidence suggests that vascular dysfunction leads to neuronal dysfunction and neurodegeneration, and that it might contribute to the development of proteinaceous brain and cerebrovascular ‘storage’ disorders. Such disorders include cerebral β-amyloidosis and cerebral amyloid angiopathy (CAA), which are caused by accumulation of the peptide amyloid-β in the brain and the vessel wall, respectively, and are features of Alzheimer’s disease1.

In this Review, I will discuss neurovascular pathways to neurodegeneration, placing a focus on Alzheimer’s disease because more is known about neurovascular dysfunction in this disease than in other neurodegenerative disorders. The article first examines transport mechanisms for molecules to cross the BBB, before exploring the processes that are involved in BBB breakdown at the molecular and cellular levels, and the consequences of BBB breakdown, hypoperfusion, and hypoxia and endothelial metabolic dysfunction for neuronal function. Next, the article reviews evidence for neurovascular changes during normal ageing and neurovascular BBB dysfunction in various neurodegenerative diseases, including evidence suggesting that vascular defects precede neuronal changes. Finally, the article considers specific mechanisms that are associated with BBB dysfunction in Alzheimer’s disease and ALS, and therapeutic opportunities relating to these neurovascular deficits.

The neurovascular unit

The neurovascular unit (NVU) comprises vascular cells (that is, endothelium, pericytes and vascular smooth muscle cells (VSMCs)), glial cells (that is, astrocytes, microglia and oliogodendroglia) and neurons1, 2, 13 (Fig. 1). In the NVU, the endothelial cells together form a highly specialized membrane around blood vessels. This membrane underlies the BBB and limits the entry of plasma components, red blood cells (RBCs) and leukocytes into the brain. The BBB also regulates the delivery into the CNS of circulating energy metabolites and essential nutrients that are required for proper neuronal and synaptic function. Non-neuronal cells and neurons act in concert to control BBB permeability and CBF. Vascular cells and glia are primarily responsible for maintenance of the constant ‘chemical’ composition of the ISF, and the BBB and the blood–spinal cord barrier (BSCB) work together with pericytes to prevent various potentially neurotoxic and vasculotoxic macromolecules in the blood from entering the CNS, and to promote clearance of these substances from the CNS1.

Figure 1 | Cerebral microcirculation and the neurovascular unit.

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders

In the brain, pial arteries run through the subarachnoid space (SAS), which contains the cerebrospinal fluid (CSF). These vessels give rise to intracerebral arteries, which penetrate into brain parenchyma. Intracerebral arteries are separated from brain parenchyma by a single, interrupted layer of elongated fibroblast-like cells of the pia and the astrocyte-derived glia limitans membrane that forms the outer wall of the perivascular Virchow–Robin space. These arteries branch into smaller arteries and subsequently arterioles, which lose support from the glia limitans and give rise to pre-capillary arterioles and brain capillaries. In an intracerebral artery, the vascular smooth muscle cell (VSMC) layer occupies most of the vessel wall. At the brain capillary level, vascular endothelial cells and pericytes are attached to the basement membrane. Pericyte processes encase most of the capillary wall, and they communicate with endothelial cells directly through synapse-like contacts containing connexins and N-cadherin. Astrocyte end-foot processes encase the capillary wall, which is composed of endothelium and pericytes. Resting microglia have a ‘ramified’ shape and can sense neuronal injury.

Transport across the blood–brain barrier. The endothelial cells that form the BBB are connected by tight and adherens junctions, and it is the tight junctions that confer the low paracellular permeability of the BBB1. Small lipophilic molecules, oxygen and carbon dioxide diffuse freely across the endothelial cells, and hence the BBB, but normal brain endothelium lacks fenestrae and has limited vesicular transport.

The high number of mitochondria in endothelial cells reflects a high energy demand for active ATP-dependent transport, conferred by transporters such as the sodium pump ((Na++K+)ATPase) and the ATP-binding cassette (ABC) efflux transporters. Sodium influx and potassium efflux across the abluminal side of the BBB is controlled by (Na++K+)ATPase (Fig. 2). Changes in sodium and potassium levels in the ISF influence the generation of action potentials in neurons and thus directly affect neuronal and synaptic functions1, 12.

Figure 2 | Blood–brain barrier transport mechanisms.

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders

Small lipophilic drugs, oxygen and carbon dioxide diffuse across the blood–brain barrier (BBB), whereas ions require ATP-dependent transporters such as the (Na++K+)ATPase. Transporters for nutrients include the glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)), the lactate transporter monocarboxylate transporter 1 (MCT1) and the L1 and y+ transporters for large neutral and cationic essential amino acids, respectively. These four transporters are expressed at both the luminal and albuminal membranes. Non-essential amino acid transporters (the alanine, serine and cysteine preferring system (ASC), and the alanine preferring system (A)) and excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are located at the abluminal side. The ATP-binding cassette (ABC) efflux transporters that are found in the endothelial cells include multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) and solute carrier organic anion transporter family member 1C1 (OATP1C1). Finally, transporters for peptides or proteins include the endothelial protein C receptor (EPCR) for activated protein C (APC); the insulin receptors (IRs) and the transferrin receptors (TFRs), which are associated with caveolin 1 (CAV1); low-density lipoprotein receptor-related protein 1 (LRP1) for amyloid-β, peptide transport system 1 (PTS1) for encephalins; and the PTS2 and PTS4–vasopressin V1a receptor (V1AR) for arginine vasopressin.

Brain endothelial cells express transporters that facilitate the transport of nutrients down their concentration gradients, as described in detail elsewhere1, 14 (Fig. 2). Glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)) — the BBB-specific glucose transporter — is of special importance because glucose is a key energy source for the brain.

Monocarboxylate transporter 1 (MCT1), which transports lactate, and the L1 and y+ amino acid transporters are expressed at the luminal and abluminal membranes12, 14. Sodium-dependent excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are expressed at the abluminal side of the BBB15 and enable removal of glutamate, an excitatory neurotransmitter, from the brain (Fig. 2). Glutamate clearance at the BBB is essential for protecting neurons from overstimulation of glutaminergic receptors, which is neurotoxic16.

ABC transporters limit the penetration of many drugs into the brain17. For example, multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) controls the rapid removal of ingested toxic lipophilic metabolites17 (Fig. 2). Some ABC transporters also mediate the efflux of nutrients from the endothelium into the ISF. For example, solute carrier organic anion transporter family member 1C1 (OATP1C1) transports thyroid hormones into the brain. MCT8 mediates influx of thyroid hormones from blood into the endothelium18 (Fig. 2).

The transport of circulating peptides across the BBB into the brain is restricted or slow compared with the transport of nutrients19. Carrier-mediated transport of neuroactive peptides controls their low levels in the ISF20, 21, 22, 23, 24 (Fig. 2). Some proteins, including transferrin, insulin, insulin-like growth factor 1 (IGF1), leptin25, 26, 27 and activated protein C (APC)28, cross the BBB by receptor-mediated transcytosis (Fig. 2).

Circumventricular organs. Several small neuronal structures that surround brain ventricles lack the BBB and sense chemical changes in blood or the cerebrospinal fluid (CSF) directly. These brain areas are known as circumventricular organs (CVOs). CVOs have important roles in multiple endocrine and autonomic functions, including the control of feeding behaviour as well as regulation of water and salt metabolism29. For example, the subfornical organ is one of the CVOs that are capable of sensing extracellular sodium using astrocyte-derived lactate as a signal for local neurons to initiate neural, hormonal and behavioural responses underlying sodium homeostasis30. Excessive sodium accumulation is detrimental, and increases in plasma sodium above a narrow range are incompatible with life, leading to cerebral oedema (swelling), seizures and death29.

Vascular-mediated pathophysiology

The key pathways of vascular dysfunction that are linked to neurodegenerative diseases include BBB breakdown, hypoperfusion–hypoxia and endothelial metabolic dysfunction (Fig. 3). This section examines processes that are involved in BBB breakdown at the molecular and cellular levels, and explores the consequences of all three pathways for neuronal function and viability.

Figure 3 | Vascular-mediated neuronal damage and neurodegeneration.

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders

a | Blood–brain barrier (BBB) breakdown that is caused by pericyte detachment leads to leakage of serum proteins and focal microhaemorrhages, with extravasation of red blood cells (RBCs). RBCs release haemoglobin, which is a source of iron. In turn, this metal catalyses the formation of toxic reactive oxygen species (ROS) that mediate neuronal injury. Albumin promotes the development of vasogenic oedema, contributing to hypoperfusion and hypoxia of the nervous tissue, which aggravates neuronal injury. A defective BBB allows several potentially vasculotoxic and neurotoxic proteins (for example, thrombin, fibrin and plasmin) to enter the brain. b | Progressive reductions in cerebral blood flow (CBF) lead to increasing neuronal dysfunction. Mild hypoperfusion, oligaemia, leads to a decrease in protein synthesis, whereas more-severe reductions in CBF, leading to hypoxia, cause an array of detrimental effects.


Blood–brain barrier breakdown. Disruption to tight and adherens junctions, an increase in bulk-flow fluid transcytosis, and/or enzymatic degradation of the capillary basement membrane cause physical breakdown of the BBB.

The levels of many tight junction proteins, their adaptor molecules and adherens junction proteins decrease in Alzheimer’s disease and other diseases that cause dementia1, 9, ALS31, multiple sclerosis32 and various animal models of neurological disease8, 33. These decreases might be partly explained by the fact that vascular-associated matrix metalloproteinase (MMP) activity rises in many neurodegenerative disorders and after ischaemic CNS injury34, 35; tight junction proteins and basement membrane extracellular matrix proteins are substrates for these enzymes34. Lowered expression of messenger RNAs that encode several key tight junction proteins, however, has also been reported in some neurodegenerative disorders, such as ALS31.

Endothelial cell–pericyte interactions are crucial for the formation36, 37 and maintenance of the BBB33, 38. Pericyte deficiency can lead to a reduction in expression of certain tight junction proteins, including occludin, claudin 5 and ZO1 (Ref. 33), and to an increase in bulk-flow transcytosis across the BBB, causing BBB breakdown38. Both processes can lead to extravasation of multiple small and large circulating macromolecules (up to 500 kDa) into the brain parenchyma33, 38. Moreover, in mice, an age-dependent progressive loss of pericytes can lead to BBB disruption and microvasular degeneration and, subsequently, neuronal dysfunction, cognitive decline and neurodegenerative changes33. In their lysosomes, pericytes concentrate and degrade multiple circulating exogenous39and endogenous proteins, including serum immunoglobulins and fibrin33, which amplify BBB breakdown in cases of pericyte deficiency.

BBB breakdown typically leads to an accumulation of various molecules in the brain. The build up of serum proteins such as immunoglobulins and albumin can cause brain oedema and suppression of capillary blood flow8, 33, whereas high concentrations of thrombin lead to neurotoxicity and memory impairment40, and accelerate vascular damage and BBB disruption41. The accumulation of plasmin (derived from circulating plasminogen) can catalyse the degradation of neuronal laminin and, hence, promote neuronal injury42, and high fibrin levels accelerate neurovascular damage6. Finally, an increase in the number of RBCs causes deposition of haemoglobin-derived neurotoxic products including iron, which generates neurotoxic reactive oxygen species (ROS)8, 43 (Fig. 3a). In addition to protein-mediated vasogenic oedema, local tissue ischaemia–hypoxia depletes ATP stores, causing (Na++K+)ATPase pumps and Na+-dependent ion channels to stop working and, consequently, the endothelium and astrocytes to swell (known as cytotoxic oedema)44. Upregulation of aquaporin 4 water channels in response to ischaemia facilitates the development of cytotoxic oedema in astrocytes45.

Hypoperfusion and hypoxia. CBF is regulated by local neuronal activity and metabolism, known as neurovascular coupling46. The pial and intracerebral arteries control the local increase in CBF that occurs during brain activation, which is termed ‘functional hyperaemia’. Neurovascular coupling requires intact pial circulation, and for VSMCs and pericytes to respond normally to vasoactive stimuli33, 46, 47. In addition to VSMC-mediated constriction and vasodilation of cerebral arteries, recent studies have shown that pericytes modulate brain capillary diameter through constriction of the vessel wall47, which obstructs capillary flow during ischaemia48. Astrocytes regulate the contractility of intracerebral arteries49, 50.

Progressive CBF reductions have increasingly serious consequences for neurons (Fig. 3b). Briefly, mild hypoperfusion — termed oligaemia — affects protein synthesis, which is required for the synaptic plasticity mediating learning and memory46. Moderate to severe CBF reductions and hypoxia affect ATP synthesis, diminishing (Na++K+)ATPase activity and the ability of neurons to generate action potentials9. In addition, such reductions can lower or increase pH, and alter electrolyte balances and water gradients, leading to the development of oedema and white matter lesions, and the accumulation of glutamate and proteinaceous toxins (for example, amyloid-β and hyperphopshorylated tau) in the brain. A reduction of greater than 80% in CBF results in neuronal death2.

The effect of CBF reductions has been extensively studied at the molecular and cellular levels in relation to Alzheimer’s disease. Reduced CBF and/or CBF dysregulation occurs in elderly individuals at high risk of Alzheimer’s disease before cognitive decline, brain atrophy and amyloid-β accumulation10, 46, 51, 52, 53, 54. In animal models, hypoperfusion can induce or amplify Alzheimer’s disease-like neuronal dysfunction and/or neuropathological changes. For example, bilateral carotid occlusion in rats causes memory impairment, neuronal dysfunction, synaptic changes and amyloid-β oligomerization55, leading to accumulation of neurotoxic amyloid-β oligomers56. In a mouse model of Alzheimer’s disease, oligaemia increases neuronal amyloid-β levels and neuronal tau phosphophorylation at an epitope that is associated with Alzheimer’s disease-type paired helical filaments57. In rodents, ischaemia leads to the accumulation of hyperphosphorylated tau in neurons and the formation of filaments that resemble those present in human neurodegenerative tauopathies and Alzheimer’s disease58. Mice expressing amyloid-β precursor protein (APP) and transforming growth factor β1 (TGFβ1) develop deficient neurovascular coupling, cholinergic denervation, enhanced cerebral and cerebrovascular amyloid-β deposition, and age-dependent cognitive decline59.

Recent studies have shown that ischaemia–hypoxia influences amyloidogenic APP processing through mechanisms that increase the activity of two key enzymes that are necessary for amyloid-β production; that is, β-secretase and γ-secretase60, 61, 62, 63. Hypoxia-inducible factor 1α (HIF1α) mediates transcriptional increase in β-secretase expression61. Hypoxia also promotes phosphorylation of tau through the mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase (ERK)) pathway64, downregulates neprilysin — an amyloid-β-degrading enzyme65 — and leads to alterations in the expression of vascular-specific genes, including a reduction in the expression of the homeobox protein MOX2 gene mesenchyme homeobox 2 (MEOX2) in brain endothelial cells5 and an increase in the expression of the myocardin gene (MYOCD) in VSMCs66. In patients with Alzheimer’s disease and in models of this disorder, these changes cause vessel regression, hypoperfusion and amyloid-β accumulation resulting from the loss of the key amyloid-β clearance lipoprotein receptor (see below). In addition, hypoxia facilitates alternative splicing of Eaat2 mRNA in Alzheimer’s disease transgenic mice before amyloid-β deposition67 and suppresses glutamate reuptake by astrocytes independently of amyloid formation68, resulting in glutamate-mediated neuronal injury that is independent of amyloid-β.

In response to hypoxia, mitochondria release ROS that mediate oxidative damage to the vascular endothelium and to the selective population of neurons that has high metabolic activity. Such damage has been suggested to occur before neuronal degeneration and amyloid-β deposition in Alzheimer’s disease69, 70. Although the exact triggers of hypoxia-mediated neurodegeneration and the role of HIF1α in neurodegeneration versus preconditioning-mediated neuroprotection remain topics of debate, mitochondria-generated ROS seem to have a primary role in the regulation of the HIF1α-mediated transcriptional switch that can activate an array of responses, ranging from mechanisms that increase cell survival and adaptation to mechanisms inducing cell cycle arrest and death71. Whether inhibition of hypoxia-mediated pathogenic pathways will delay onset and/or control progression in neurodegenerative conditions such as Alzheimer’s disease remains to be determined.

When comparing the contributions of BBB breakdown and hypoperfusion to neuronal injury, it is interesting to consider Meox2+/− mice. Such animals have normal pericyte coverage and an intact BBB but a substantial perfusion deficit5 that is comparable to that found in pericyte-deficient mice that develop BBB breakdown33 Notably, however,Meox2+/− mice show less pronounced neurodegenerative changes than pericyte-deficient mice, indicating that chronic hypoperfusion–hypoxia alone can cause neuronal injury, but not to the same extent as hypoperfusion–hypoxia combined with BBB breakdown.

Endothelial neurotoxic and inflammatory factors. Alterations in cerebrovascular metabolic functions can lead to the secretion of multiple neurotoxic and inflammatory factors72, 73. For example, brain microvessels that have been isolated from individuals with Alzheimer’s disease (but not from neurologically normal age-matched and young individuals) and brain microvessels that have been treated with inflammatory proteins release neurotoxic factors that kill neurons74, 75. These factors include thrombin, the levels of which increase with the onset of Alzheimer’s disease76. Thrombin can injure neurons directly40 and indirectly by activating microglia and astrocytes73. Compared with those from age-matched controls, brain microvessels from individuals with Alzheimer’s disease secrete increased levels of multiple inflammatory mediators, such as nitric oxide, cytokines (for example, tumour necrosis factor (TNF), TGFβ1, interleukin-1β (IL-1β) and IL-6), chemokines (for example, CC-chemokine ligand 2 (CCL2; also known as monocyte chemoattractant protein 1 (MCP1)) and IL-8), prostaglandins, MMPs and leukocyte adhesion molecules73. Endothelium-derived neurotoxic and inflammatory factors together provide a molecular link between vascular metabolic dysfunction, neuronal injury and inflammation in Alzheimer’s disease and, possibly, in other neurodegenerative disorders.

Neurovascular changes

This section examines evidence for neurovascular changes during normal ageing and for neurovascular and/or BBB dysfunction in various neurodegenerative diseases, as well as the possibility that vascular defects can precede neuronal changes.

Age-associated neurovascular changes. Normal ageing diminishes brain circulatory functions, including a detectable decay of CBF in the limbic and association cortices that has been suggested to underlie age-related cognitive changes77. Alterations in the cerebral microvasculature, but not changes in neural activity, have been shown to lead to age-dependent reductions in functional hyperaemia in the visual system in cats78 and in the sensorimotor cortex in pericyte-deficient mice33. Importantly, a recent longitudinal CBF study in neurologically normal individuals revealed that people bearing the apolipoprotein E (APOE) ɛ4 allele — the major genetic risk factor for late-onset Alzheimer’s disease79, 80, 81 — showed greater regional CBF decline in brain regions that are particularly vulnerable to pathological changes in Alzheimer’s disease than did people without this allele82.

A meta-analysis of BBB permeability in 1,953 individuals showed that neurologically healthy humans had an age-dependent increase in vascular permeability83. Moreover, patients with vascular or Alzheimer’s disease-type dementia and leucoaraiosis — a small-vessel disease of the cerebral white matter — had an even greater age-dependent increase in vascular permeability83. Interestingly, an increase in BBB permeability in brain areas with normal white matter in patients with leukoaraiosis has been suggested to play a causal part in disease and the development of lacunar strokes84. Age-related changes in the permeability of the blood–CSF barrier and the choroid plexus have been reported in sheep85.

Vascular pathology. Patients with Alzheimer’s disease or other dementia-causing diseases frequently show focal changes in brain microcirculation. These changes include the appearance of string vessels (collapsed and acellular membrane tubes), a reduction in capillary density, a rise in endothelial pinocytosis, a decrease in mitochondrial content, accumulation of collagen and perlecans in the basement membrane, loss of tight junctions and/or adherens junctions3, 4, 5, 6, 9, 46, 86, and BBB breakdown with leakage of blood-borne molecules4, 6, 7, 9. The time course of these vascular alterations and how they relate to dementia and Alzheimer’s disease pathology remain unclear, as no protocol that allows the development of the diverse brain vascular pathology to be scored, and hence to be tracked with ageing, has so far been developed and widely validated87. Interestingly, a recent study involving 500 individuals who died between the ages of 69 and 103 years showed that small-vessel disease, infarcts and the presence of more than one vascular pathological change were associated with Alzheimer’s disease-type pathological lesions and dementia in people aged 75 years of age87. These associations were, however, less pronounced in individuals aged 95 years of age, mainly because of a marked ageing-related reduction in Alzheimer’s disease neuropathology relative to a moderate but insignificant ageing-related reduction in vascular pathology87.

Accumulation of amyloid-β and amyloid deposition in pial and intracerebral arteries results in CAA, which is present in over 80% of Alzheimer’s disease cases88. In patients who have Alzheimer’s disease with established CAA in small arteries and arterioles, the VSMC layer frequently shows atrophy, which causes a rupture of the vessel wall and intracerebral bleeding in about 30% of these patients89, 90. These intracerebral bleedings contribute to, and aggravate, dementia. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type have accelerated VSMC degeneration resulting in haemorrhagic strokes and dementia91. Duplication of the gene encoding APP causes early-onset Alzheimer’s disease dementia with CAA and intracerebral haemorrhage92.

Early studies of serum immunoglobulin leakage reported that patients with ALS had BSCB breakdown and BBB breakdown in the motor cortex93. Microhaemorrhages and BSCB breakdown have been shown in the spinal cord of transgenic mice expressing mutant variants of human superoxide dismutase 1 (SOD1), which in mice cause an ALS-like disease8, 94, 95. In mice with ALS-like disease and in patients with ALS, BSCB breakdown has been shown to occur before motor neuron degeneration or brain atrophy8, 11, 95.

BBB breakdown in the substantia nigra and the striatum has been detected in murine models of Parkinson’s disease that are induced by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)96, 97, 98. However, the temporal relationship between BBB breakdown and neurodegeneration in Parkinson’s disease is currently unknown. Notably, the prevalence of CAA and vascular lesions increases in Parkinson’s disease99,100. Vascular lesions in the striatum and lacunar infarcts can cause vascular parkinsonism syndrome101. A recent study reported BBB breakdown in a rat model of Huntington’s disease that is induced with the toxin 3-nitropropionic acid102.

Several studies have established disruption of BBB with a loss of tight junction proteins during neuroinflammatory conditions such as multiple sclerosis and its murine model, experimental allergic encephalitis. Such disruption facilitates leukocyte infiltration, leading to oliogodendrocyte death, axonal damage, demyelination and lesion development32.

Functional changes in the vasculature. In individuals with Alzheimer’s disease, GLUT1 expression at the BBB decreases103, suggesting a shortage in necessary metabolic substrates. Studies using 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) have identified reductions in glucose uptake in asymptomatic individuals with a high risk of dementia104, 105. Several studies have suggested that reduced glucose uptake across the BBB, as seen by FDG PET, precedes brain atrophy104, 105, 106, 107, 108.

Amyloid-β constricts cerebral arteries109. In a mouse model of Alzheimer’s disease, impairment of endothelium-dependent regulation of neocortical microcirculation110, 111occurs before amyloid-β accumulation. Recent studies have shown that CD36, a scavenger receptor that binds amyloid-β, is essential for the vascular oxidative stress and diminished functional hyperaemia that occurs in response to amyloid-β exposure112. Neuroimaging studies in patients with Alzheimer’s disease have shown that neurovascular uncoupling occurs before neurodegenerative changes10, 51, 52, 53. Moreover, cognitively normal APOE ɛ4 carriers at risk of Alzheimer’s disease show impaired CBF responses to brain activation in the absence of neurodegenerative changes or amyloid-β accumulation54. Recently, patients with Alzheimer’s disease as well as mouse models of this disease with high cerebrovascular levels of serum response factor (SRF) and MYOCD, the two transcription factors that control VSMC differentiation, have been shown to develop a hypercontractile arterial phenotype resulting in brain hypoperfusion, diminished functional hyperaemia and CAA66, 113. More work is needed to establish the exact role of SRF and MYOCD in the vascular dysfunction that results in the Alzheimer’s disease phenotype and CAA.

PET studies with 11C-verapamil, an ABCB1 substrate, have indicated that the function of ABCB1, which removes multiple drugs and toxins from the brain, decreases with ageing114 and is particularly compromised in the midbrain of patients with Parkinson’s disease, progressive supranuclear palsy or multiple system atrophy115. More work is needed to establish the exact roles of ABC BBB transporters in neurodegeneration and whether their failure precedes the loss of dopaminergic neurons that occurs in Parkinson’s disease.

In mice with ALS-like disease and in patients with ALS, hypoperfusion and/or dysregulated CBF have been shown to occur before motor neuron degeneration or brain atrophy8, 116. Reduced regional CBF in basal ganglia and reduced blood volume have been reported in pre-symptomatic gene-tested individuals at risk for Huntington’s disease117. Patients with Huntington’s disease display a reduction in vasomotor activity in the cerebral anterior artery during motor activation118.

Vascular and neuronal common growth factors. Blood vessels and neurons share common growth factors and molecular pathways that regulate their development and maintenance119, 120. Angioneurins are growth factors that exert both vasculotrophic and neurotrophic activities121. The best studied angioneurin is vascular endothelial growth factor (VEGF). VEGF regulates vessel formation, axonal growth and neuronal survival120. Ephrins, semaphorins, slits and netrins are axon guidance factors that also regulate the development of the vascular system121. During embryonic development of the neural tube, blood vessels and choroid plexus secrete IGF2 into the CSF, which regulates the proliferation of neuronal progenitor cells122. Genetic and pharmacological manipulations of angioneurin activity yielded various vascular and cerebral phenotypes121. Given the dual nature of angioneurin action, these studies have not been able to address whether neuronal dysfunction results from a primary insult to neurons and/or whether it is secondary to vascular dysfunction.

Increased levels of VEGF, a hypoxia-inducible angiogenic factor, were found in the walls of intraparenchymal vessels, perivascular deposits, astrocytes and intrathecal space of patients with Alzheimer’s disease, and were consistent with the chronic cerebral hypoperfusion and hypoxia that were observed in these individuals73. In addition to VEGF, brain microvessels in Alzheimer’s disease release several molecules that can influence angiogenesis, including IL-1β, IL-6, IL-8, TNF, TGFβ, MCP1, thrombin, angiopoietin 2, αVβ3 and αVβ5 integrins, and HIF1α73. However, evidence for increased vascularity in Alzheimer’s disease is lacking. On the contrary, several studies have reported that focal vascular regression and diminished microvascular density occur in Alzheimer’s disease4, 5, 73 and in Alzheimer’s disease transgenic mice123. The reason for this discrepancy is not clear. The anti-angiogenic activity of amyloid-β, which accumulates in the brains of individuals with Alzheimer’s disease and Alzheimer’s disease models, may contribute to hypovascularity123. Conversely, genome-wide transcriptional profiling of brain endothelial cells from patients with Alzheimer’s disease revealed that extremely low expression of vascular-restricted MEOX2 mediates aberrant angiogenic responses to VEGF and hypoxia, leading to capillary death5. This finding raises the interesting question of whether capillary degeneration in Alzheimer’s disease results from unsuccessful vascular repair and/or remodelling. Moreover, mice that lack one Meox2allele have been shown to develop a primary cerebral endothelial hypoplasia with chronic brain hypoperfusion5, resulting in secondary neurodegenerative changes33.

Does vascular dysfunction cause neuronal dysfunction? In summary, the evidence that is discussed above clearly indicates that vascular dysfunction is tightly linked to neuronal dysfunction. There are many examples to illustrate that primary vascular deficits lead to secondary neurodegeneration, including CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts), an hereditary small-vessel brain disease resulting in multiple small ischaemic lesions, neurodegeneration and dementia124; mutations in SLC2A1 that cause dysfunction of the BBB-specific GLUT1 transporter in humans resulting in seizures; cognitive impairment and microcephaly125; microcephaly and epileptiform discharges in mice with genetic deletion of a single Slc2a1 allele126; and neurodegeneration mediated by a single Meox2 homebox gene deletion restricted to the vascular system33. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type provide another example showing that primary vascular dysfunction — which in this case is caused by deposition of vasculotropic amyloid-β mutants in the arterial vessel wall — leads to dementia and intracerebral bleeding. Moreover, as reviewed in the previous sections, recent evidence suggests that BBB dysfunction and/or breakdown, and CBF reductions and/or dysregulation may occur in sporadic Alzheimer’s disease and experimental models of this disease before cognitive decline, amyloid-β deposition and brain atrophy. In patients with ALS and in some experimental models of ALS, CBF dysregulation, BSCB breakdown and spinal cord hypoperfusion have been reported to occur before motor neuron cell death. Whether neurological changes follow or precede vascular dysfunction in Parkinson’s disease, Huntington’s disease and multiple sclerosis remains less clear. However, there is little doubt that vascular injury mediates, amplifies and/or lowers the threshold for neuronal dysfunction and loss in several neurological disorders.

Disease-specific considerations

This section examines how amyloid-β levels are kept low in the brain, a process in which the BBB has a central role, and how faulty BBB-mediated clearance mechanisms go awry in Alzheimer’s disease. On the basis of this evidence and the findings discussed elsewhere in the Review, a new hypothesis for the pathogenesis of Alzheimer’s disease that incorporates the vascular evidence is presented. ALS-specific disease mechanisms relating to the BBB are then examined.

Alzheimer’s disease. Amyloid-β clearance from the brain by the BBB is the best studied example of clearance of a proteinaceous toxin from the CNS. Multiple pathways regulate brain amyloid-β levels, including its production and clearance (Fig. 4). Recent studies127,128, 129 have confirmed earlier findings in multiple rodent and non-human primate models demonstrating that peripheral amyloid-β is an important precursor of brain amyloid-β130, 131, 132, 133, 134, 135, 136. Moreover, peripheral amyloid-β sequestering agents such as soluble LRP1 (ref.137), anti-amyloid-β antibodies138, 139,140, gelsolin and the ganglioside GM1 (Ref. 141), or systemic expression of neprilysin142, 143 have been shown to reduce the amyloid burden in Alzheimer’s disease mice by eliminating contributions of the peripheral amyloid-β pool to the total brain pool of this peptide.

Figure 4 | The role of blood–brain barrier transport in brain homeostasis of amyloid-β.

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders

Amyloid-β (Aβ) is produced from the amyloid-β precursor protein (APP), both in the brain and in peripheral tissues. Clearance of amyloid-β from the brain normally maintains its low levels in the brain. This peptide is cleared across the blood–brain barrier (BBB) by the low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 mediates rapid efflux of a free, unbound form of amyloid-β and of amyloid-β bound to apolipoprotein E2 (APOE2), APOE3 or α2-macroglobulin (not shown) from the brain’s interstitial fluid into the blood, and APOE4 inhibits such transport. LRP2 eliminates amyloid-β that is bound to clusterin (CLU; also known as apolipoprotein J (APOJ)) by transport across the BBB, and shows a preference for the 42-amino-acid form of this peptide. ATP-binding cassette subfamily A member 1 (ABCA1; also known as cholesterol efflux regulatory protein) mediates amyloid-β efflux from the brain endothelium to blood across the luminal side of the BBB (not shown). Cerebral endothelial cells, pericytes, vascular smooth muscle cells, astrocytes, microglia and neurons express different amyloid-β-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme (IDE), tissue plasminogen activator (tPA) and matrix metalloproteinases (MMPs), which contribute to amyloid-β clearance. In the circulation, amyloid-β is bound mainly to soluble LRP1 (sLRP1), which normally prevents its entry into the brain. Systemic clearance of amyloid-β is mediated by its removal by the liver and kidneys. The receptor for advanced glycation end products (RAGE) provides the key mechanism for influx of peripheral amyloid-β into the brain across the BBB either as a free, unbound plasma-derived peptide and/or by amyloid-β-laden monocytes. Faulty vascular clearance of amyloid-β from the brain and/or an increased re-entry of peripheral amyloid-β across the blood vessels into the brain can elevate amyloid-β levels in the brain parenchyma and around cerebral blood vessels. At pathophysiological concentrations, amyloid-β forms neurotoxic oligomers and also self-aggregates, which leads to the development of cerebral β-amyloidosis and cerebral amyloid angiopathy.

The receptor for advanced glycation end products (RAGE) mediates amyloid-β transport in brain and the propagation of its toxicity. RAGE expression in brain endothelium provides a mechanism for influx of amyloid-β144, 145 and amyloid-β-laden monocytes146 across the BBB, as shown in Alzheimer’s disease models (Fig. 4). The amyloid-β-rich environment in Alzheimer’s disease and models of this disorder increases RAGE expression at the BBB and in neurons147, 148, amplifying amyloid-β-mediated pathogenic responses. Blockade of amyloid-β–RAGE signalling in Alzheimer’s disease is a promising strategy to control self-propagation of amyloid-β-mediated injury.

Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder149 have shown that diminished amyloid-β clearance occurs in brain tissue in this disease. LRP1 plays an important part in the three-step serial clearance of this peptide from brain and the rest of the body150 (Fig. 4). In step one, LRP1 in brain endothelium binds brain-derived amyloid-β at the abluminal side of the BBB, initiating its clearance to blood, as shown in many animal models151, 152, 153, 154, 155, 156 and BBB models in vitro151, 157, 158. The vasculotropic mutants of amyloid-β that have low binding affinity for LRP1 are poorly cleared from the brain or CSF151, 159, 160. APOE4, but not APOE3 or APOE2, blocks LRP1-mediated amyloid-β clearance from the brain and, hence, promotes its retention161, whereas clusterin (also known as apolipoprotein J (APOJ)) mediates amyloid-β clearance across the BBB via LRP2 (Ref. 153). APOE and clusterin influence amyloid-β aggregation162, 163. Reduced LRP1 levels in brain microvessels, perhaps in addition to altered levels of ABCB1, are associated with amyloid-β cerebrovascular and brain accumulation during ageing in rodents, non-human primates, humans, Alzheimer’s disease mice and patients with Alzheimer’s disease66,151, 152, 164, 165, 166. Moreover, recent work has shown that brain LRP1 is oxidized in Alzheimer’s disease167, and may contribute to amyloid-β retention in brain because the oxidized form cannot bind and/or transport amyloid-β137. LRP1 also mediates the removal of amyloid-β from the choroid plexus168.

In step two, circulating soluble LRP1 binds more than 70% of plasma amyloid-β in neurologically normal humans137. In patients with Alzheimer’s disease or mild cognitive impairment (MCI), and in Alzheimer’s disease mice, amyloid-β binding to soluble LRP1 is compromised due to oxidative changes137, 169, resulting in elevated plasma levels of free amyloid-β isoforms comprising 40 or 42 amino acids (amyloid-β1–40 and amyloid-β1–42). These peptides can then re-enter the brain, as has been shown in a mouse model of Alzheimer’s disease137. Rapid systemic removal of amyloid-β by the liver is also mediated by LRP1 and comprises step three of the clearance process170.

In brain, amyloid-β is enzymatically degraded by neprilysin171, insulin-degrading enzyme172, tissue plasminogen activator173 and MMPs173, 174 in various cell types, including endothelial cells, pericytes, astrocytes, neurons and microglia. Cellular clearance of this peptide by astrocytes and VSMCs is mediated by LRP1 and/or another lipoprotein receptor66, 175. Clearance of amyloid-β aggregates by microglia has an important role in amyloid-β-directed immunotherapy176 and reduction of the amyloid load in brain177. Passive ISF–CSF bulk flow and subsequent clearance through the CSF might contribute to 10–15% of total amyloid-β removal152, 153, 178. In the injured human brain, increasing soluble amyloid-β concentrations in the ISF correlated with improvements in neurological status, suggesting that neuronal activity might regulate extracellular amyloid-β levels179.

The role of BBB dysfunction in amyloid-β accumulation, as discussed above, underlies the contribution of vascular dysfunction to Alzheimer’s disease (see Fig. 5 for a model of vascular damage in Alzheimer’s disease). The amyloid hypothesis for the pathogenesis of Alzheimer’s disease maintains that this peptide initiates a cascade of events leading to neuronal injury and loss and, eventually, dementia180, 181. Here, I present an alternative hypothesis — the two-hit vascular hypothesis of Alzheimer’s disease — that incorporates the vascular contribution to this disease, as discussed in this Review (Box 1). This hypothesis states that primary damage to brain microcirculation (hit one) initiates a non-amyloidogenic pathway of vascular-mediated neuronal dysfunction and injury, which is mediated by BBB dysfunction and is associated with leakage and secretion of multiple neurotoxic molecules and/or diminished brain capillary flow that causes multiple focal ischaemic or hypoxic microinjuries. BBB dysfunction also leads to impairment of amyloid-β clearance, and oligaemia leads to increased amyloid-β generation. Both processes contribute to accumulation of amyloid-β species in the brain (hit two), where these peptides exert vasculotoxic and neurotoxic effects. According to the two-hit vascular hypothesis of Alzheimer’s disease, tau pathology develops secondary to vascular and/or amyloid-β injury.

Figure 5 | A model of vascular damage in Alzheimer’s disease.

Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders

a | In the early stages of Alzheimer’s disease, small pial and intracerebral arteries develop a hypercontractile phenotype that underlies dysregulated cerebral blood flow (CBF). This phenotype is accompanied by diminished amyloid-β clearance by the vascular smooth muscle cells (VSMCs). In the later phases of Alzheimer’s disease, amyloid deposition in the walls of intracerebral arteries leads to cerebral amyloid angiopathy (CAA), pronounced reductions in CBF, atrophy of the VSMC layer and rupture of the vessels causing microbleeds. b | At the level of capillaries in the early stages of Alzheimer’s disease, blood–brain barrier (BBB) dysfunction leads to a faulty amyloid-β clearance and accumulation of neurotoxic amyloid-β oligomers in the interstitial fluid (ISF), microhaemorrhages and accumulation of toxic blood-derived molecules (that is, thrombin and fibrin), which affect synaptic and neuronal function. Hyperphosphorylated tau (p-tau) accumulates in neurons in response to hypoperfusion and/or rising amyloid-β levels. At this point, microglia begin to sense neuronal injury. In the later stages of the disease in brain capillaries, microvascular degeneration leads to increased deposition of basement membrane proteins and perivascular amyloid. The deposited proteins and amyloid obstruct capillary blood flow, resulting in failure of the efflux pumps, accumulation of metabolic waste products, changes in pH and electrolyte composition and, subsequently, synaptic and neuronal dysfunction. Neurofibrillary tangles (NFTs) accumulate in response to ischaemic injury and rising amyloid-β levels. Activation of microglia and astrocytes is associated with a pronounced inflammatory response. ROS, reactive oxygen species.


Amyotrophic lateral sclerosis. The cause of sporadic ALS, a fatal adult-onset motor neuron neurodegenerative disease, is not known182. In a relatively small number of patients with inherited SOD1 mutations, the disease is caused by toxic properties of mutant SOD1 (Ref. 183). Mutations in the genes encoding ataxin 2 and TAR DNA-binding protein 43 (TDP43) that cause these proteins to aggregate have been associated with ALS182, 184. Some studies have suggested that abnormal SOD1 species accumulate in sporadic ALS185. Interestingly, studies in ALS transgenic mice expressing a mutant version of human SOD1 in neurons, and in non-neuronal cells neighbouring these neurons, have shown that deletion of this gene from neurons does not influence disease progression186, whereas deletion of this gene from microglia186 or astrocytes187substantially increases an animal’s lifespan. According to an emerging hypothesis of ALS that is based on studies in SOD1 mutant mice, the toxicity that is derived from non-neuronal neighbouring cells, particularly microglia and astrocytes, contributes to disease progression and motor neuron degeneration186, 187, 188, 189, 190, whereas BBB dysfunction might be critical for disease initiation8, 43, 94, 95. More work is needed to determine whether this concept of disease initiation and progression may also apply to cases of sporadic ALS.

Human data support a role for angiogenic factors and vessels in the pathogenesis of ALS. For example, the presence of VEGF variations (which were identified in large meta-analysis studies) has been linked to ALS191. Angiogenin is another pro-angiogenic gene that is implicated in ALS because heterozygous missense mutations in angiogenin cause familial and sporadic ALS192. Moreover, mice with a mutation that eliminates hypoxia-responsive induction of the Vegf gene (Vegfδ/δ mice) develop late-onset motor neuron degeneration193. Spinal cord ischaemia worsens motor neuron degeneration and functional outcome in Vegfδ/δ mice, whereas the absence of hypoxic induction of VEGF in mice that develop motor neuron disease from expression of ALS-linked mutant SOD1G93A results in substantially reduced survival191.

Therapeutic opportunities

Many investigators believe that primary neuronal dysfunction resulting from an intrinsic neuronal disorder is the key underlying event in human neurodegenerative diseases. Thus, most therapeutic efforts for neurodegenerative diseases have so far been directed at the development of so-called ‘single-target, single-action’ agents to target neuronal cells directly and reverse neuronal dysfunction and/or protect neurons from injurious insults. However, most preclinical and clinical studies have shown that such drugs are unable to cure or control human neurological disorders2, 181, 183, 194, 195. For example, although pathological overstimulation of glutaminergic NMDA receptors (NMDARs) has been shown to lead to neuronal injury and death in several disorders, including stroke, Alzheimer’s disease, ALS and Huntington’s disease16, NMDAR antagonists have failed to show a therapeutic benefit in the above-mentioned human neurological disorders.

Recently, my colleagues and I coined the term vasculo-neuronal-inflammatory triad195to indicate that vascular damage, neuronal injury and/or neurodegeneration, and neuroinflammation comprise a common pathological triad that occurs in multiple neurological disorders. In line with this idea, it is conceivable that ‘multiple-target, multiple-action’ agents (that is, drugs that have more than one target and thus have more than one action) will have a better chance of controlling the complex disease mechanisms that mediate neurodegeneration than agents that have only one target (for example, neurons). According to the vasculo-neuronal-inflammatory triad model, in addition to neurons, brain endothelium, VSMCs, pericytes, astrocytes and activated microglia are all important therapeutic targets.

Here, I will briefly discuss a few therapeutic strategies based on the vasculo-neuronal-inflammatory triad model. VEGF and other angioneurins may have multiple targets, and thus multiple actions, in the CNS120. For example, preclinical studies have shown that treatment of SOD1G93A rats with intracerebroventricular VEGF196 or intramuscular administration of a VEGF-expressing lentiviral vector that is transported retrogradely to motor neurons in SOD1G93A mice197 reduced pathology and extended survival, probably by promoting angiogenesis and increasing the blood flow through the spinal cord as well as through direct neuronal protective effects of VEGF on motor neurons. On the basis of these and other studies, a phase I–II clinical trial has been initiated to evaluate the safety of intracerebroventricular infusion of VEGF in patients with ALS198. Treatment with angiogenin also slowed down disease progression in a mouse model of ALS199.

IGF1 delivery has been shown to promote amyloid-β vascular clearance and to improve learning and memory in a mouse model of Alzheimer’s disease200. Local intracerebral implantation of VEGF-secreting cells in a mouse model of Alzheimer’s disease has been shown to enhance vascular repair, reduce amyloid burden and improve learning and memory201. In contrast to VEGF, which can increase BBB permeability, TGFβ, hepatocyte growth factor and fibroblast growth factor 2 promote BBB integrity by upregulating the expression of endothelial junction proteins121 in a similar way to APC43. However, VEGF and most growth factors do not cross the BBB, so the development of delivery strategies such as Trojan horses is required for their systemic use25.

A recent experimental approach with APC provides an example of a neurovascular medicine that has been shown to favourably regulate multiple pathways in non-neuronal cells and neurons, resulting in vasculoprotection, stabilization of the BBB, neuroprotection and anti-inflammation in several acute and chronic models of the CNS disorders195 (Box 2).

Box 2 | A model of multiple-target, multiple-action neurovascular medicine

The recognition of amyloid-β clearance pathways (Fig. 4), as discussed above, opens exciting new therapeutic opportunities for Alzheimer’s disease. Amyloid-β clearance pathways are promising therapeutic targets for the future development of neurovascular medicines because it has been shown both in animal models of Alzheimer’s disease1 and in patients with sporadic Alzheimer’s disease149 that faulty clearance from brain and across the BBB primarily determines amyloid-β retention in brain, causing the formation of neurotoxic amyloid-β oligomers56 and the promotion of brain and cerebrovascular amyloidosis3. The targeting of clearance mechanisms might also be beneficial in other diseases; for example, the clearance of extracellular mutant SOD1 in familial ALS, the prion protein in prion disorders and α-synuclein in Parkinson’s disease might all prove beneficial. However, the clearance mechanisms for these proteins in these diseases are not yet understood.

Conclusions and perspectives

Currently, no effective disease-modifying drugs are available to treat the major neurodegenerative disorders202, 203, 204. This fact leads to a question: are we close to solving the mystery of neurodegeneration? The probable answer is yes, because the field has recently begun to recognize that, first, damage to neuronal cells is not the sole contributor to disease initiation and progression, and that, second, correcting disease pathways in vascular and glial cells may offer an array of new approaches to control neuronal degeneration that do not involve targeting neurons directly. These realizations constitute an important shift in paradigm that should bring us closer to a cure for neurodegenerative diseases. Here, I raise some issues concerning the existing models of neurodegeneration and the new neurovascular paradigm.

The discovery of genetic abnormalities and associations by linkage analysis or DNA sequencing has broadened our understanding of neurodegeneration204. However, insufficient effort has been made to link genetic findings with disease biology. Another concern for neurodegenerative research is how we should interpret findings from animal models202. Genetically engineered models of human neurodegenerative disorders inDrosophila melanogaster and Caenorhabditis elegans have been useful for dissecting basic disease mechanisms and screening compounds. However, in addition to having much simpler nervous systems, insects and avascular species do not have cerebrovascular and immune systems that are comparable to humans and, therefore, are unlikely to replicate the complex disease pathology that is found in people.

For most neurodegenerative disorders, early steps in the disease processes remain unclear, and biomarkers for these stages have yet to be identified. Thus, it is difficult to predict whether mammalian models expressing human genes and proteins that we know are implicated in the intermediate or later stages of disease pathophysiology, such as amyloid-β or tau in Alzheimer’s disease7, 181, will help us to discover therapies for the early stages of disease and for disease prevention, because the exact role of these pathological accumulations during disease onset remains uncertain. According to the two-hit vascular hypothesis of Alzheimer’s disease, incorporating vascular factors that are associated with Alzheimer’s disease into current models of this disease may more faithfully replicate dementia events in humans. Alternatively, by focusing on the comorbidities and the initial cellular and molecular mechanisms underlying early neurovascular dysfunction that are associated with Alzheimer’s disease, new models of dementia and neurodegeneration may be developed that do not require the genetic manipulation of amyloid-β or tau expression.

The proposed neurovascular triad model of neurodegenerative diseases challenges the traditional neurocentric view of such disorders. At the same time, this model raises a set of new important issues that require further study. For example, the molecular basis of the neurovascular link with neurodegenerative disorders is poorly understood, in terms of the adhesion molecules that keep the physical association of various cell types together, the molecular crosstalk between different cell types (including endothelial cells, pericytes and astrocytes) and how these cellular interactions influence neuronal activity. Addressing these issues promises to create new opportunities not only to better understand the molecular basis of the neurovascular link with neurodegeneration but also to develop novel neurovascular-based medicines.

The construction of a human BBB molecular atlas will be an important step towards understanding the role of the BBB and neurovascular interactions in health and disease. Achievement of this goal will require identifying new BBB transporters by using genomic and proteomic tools, and by cloning some of the transporters that are already known. Better knowledge of transporters at the human BBB will help us to better understand their potential as therapeutic targets for disease.

Development of higher-resolution imaging methods to evaluate BBB integrity, key transporters’ functions and CBF responses in the microregions of interest (for example, in the entorhinal region of the hippocampus) will help us to understand how BBB dysfunction correlates with cognitive outcomes and neurodegenerative processes in MCI, Alzheimer’s disease and related disorders.

The question persists: are we missing important therapeutic targets by studying the nervous system in isolation from the influence of the vascular system? The probable answer is yes. However, the current exciting and novel research that is based on the neurovascular model has already begun to change the way that we think about neurodegeneration, and will continue to provide further insights in the future, leading to the development of new neurovascular therapies.

  1. Zlokovic, B. V. The blood–brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008).

  2. Moskowitz, M. A., Lo, E. H. & Iadecola, C. The science of stroke: mechanisms in search of treatments. Neuron 67, 181–198 (2010).
    A comprehensive review describing mechanisms of ischaemic injury to the neurovascular unit.

  3. Zlokovic, B. V. Neurovascular mechanisms of Alzheimer’s neurodegeneration.Trends Neurosci. 28, 202–208 (2005).

  4. Brown, W. R. & Thore, C. R. Review: cerebral microvascular pathology in ageing and neurodegeneration. Neuropathol. Appl. Neurobiol. 37, 56–74 (2011).

  5. Wu, Z. et al. Role of the MEOX2 homeobox gene in neurovascular dysfunction in Alzheimer disease. Nature Med. 11, 959–965 (2005).
    A study demonstrating that low expression of MEOX2 in brain endothelium leads to aberrant angiogenesis and vascular regression in Alzheimer’s disease.


antimicrobial evaluation of some novel bis-heterocyclic chalcones

Larry H. Bernstein, MD, FCAP, Curator



Synthesis and antimicrobial evaluation of some novel bis-heterocyclic chalcones from cyclic imides under microwave irradiation
Ravindra S. Dhivare1* and Shankarsing S. Rajput2
Chem Sci Rev Lett 2015; 4(15): 937-944     Article CS05204609    http://chesci.com/articles/csrl/v4i15/19_CS05204609.pdf


Synthesis of a new series of bis-heterocyclic chalcone derivatives has been reported. The condensation proceeded by the one pot reaction of N-phenyl succinimides and 4-hydroxy-3-methoxy benzaldehyde in presence of neutral alumina under microwave irradiation. The advantages of this method are high yield, shorter reaction time and simple workup procedure. Most of the synthesized compounds have shown moderate to significant antimicrobial activity against different microbial strains.


Chalcones and cyclic imides perform a significant title role in the heterocyclic synthesis containing oxygen, nitrogen and sulphur groups. Cyclic imides [1] like succinimides [2] [3] [4], maleimides [5], glutarimide [6], itaconimide[7] and phthalimides[8] showed the defensive antibacterial [9], antifungal activities. Cyclic imides exhibited the CNS anxiolytic and anti-depressive [10], brain metabolism [11], nephrotoxic [12], antiviral [13], anticonvulsant [14] electroshocks [15] , muscle relaxant [16], anti-mutagenic [17], analgesic [18], anxiety and depression [19], myeloperoxidase induction [20], antiproliferative [21], seedling growth [22] activities. The substituted heterocyclic imides were developed from cyclic anhydrides [23], formamide [24], trifluoroacetylation [25], triethylamine [26], hydroxylamine [27], thalidomide biotin [28] , pyrolidine triones [29] reagents and bis-heterocyclic analogs [30] by microwave synthesis. Bis-chalcones are the pioneer flavonoids of heterocycle ancestor containing carbon stuck between α, β- unsaturated aromatic rings and carbonyl carbons. These are prepared by the condensation [31] of the substituted ketones and aldehyde groups [32] [33]. The chalcone showed significant cytotoxic activities against antimicrobial [34] , cell line and breast cancer [35], anti-oxidant [36], bovine lens aldose reductase [37], tumor-genesis activities [38]. The chalcones are synthesized by utilizing a number of synthetic routes like solid phase Claisen-Schemdit, Cross-Aldol condensation, acid catalyst [39], coupling reaction [40], Knoevenagel condensation [41] and microwave assisted synthesis.


Preparation of substituted bis-heterocyclic chalcones: The N-substituted Phenylpyrrolidine-2, 5-diones or N-phenyl succinimides are conventionally synthesized by succinic anhydride and substituted anilines. Then afforded succinimides were employed for the preparation of bis-chalcone derivatives by microwave synthesis. The experimental method of conventional to microwave synthesis is schematically represented in figure 1

Figure 1 Experimental design of conventional to microwave method

General Procedure for the Synthesis of N-phenyl-pyrrolidine-2, 5-dione or N-Phenyl Succinimides

To accomplish the work succinic anhydride (0.1 moles) benzene was added and heated under reflux with constant stirring for 15 to 20 min till the solution becomes clear. Into this solution the primary aromatic amines (0.2 moles) in 5 ml benzene was slowly poured with constant stirring for 15- 20 min till the solution becomes homogenized. On the vaporization of benzene amorphous powder of 3-(N-phenyl) propanoic acid was obtained. Further the mixture of 3- (N-phenyl) propanoic acid and acetyl chloride (0.9 moles) was reflux for 15-20 min by thoroughly evolution of HCl fumes. The reaction mixture was cooled at room temp the solid product was obtained and recrystallized by ethanol as shown in the scheme 1

General procedure for the synthesis of bis-chalcones derived from N-phenyl succinimides

The bis-chalcones (6a-j) derivatives were synthesized by the mixture of 0.1 moles of N-phenyl succinimides (4a-j) and 0.2 mole of 4-hydroxy-3-methoxy benzaldehyde in 2 gm of neutral Al2O3 under microwave supported solvent free condition on 640W power for 5-8 min. The developed compounds were recrystallized from ethanol (Scheme 2)


Chemistry: The starting compounds of bis-chalcones 6a-j were prepared by the reaction of substituted N-phenyl-pyrrolidine-2, 5- dione 4a-j using 4-hydroxy-3-methoxy benzaldehyde. The series of 3,4-bis(4-hydroxy-3-methoxy benzylidene)-Nphenylpyrrolidine-2,5-diones 6a-j were synthesized in reasonable yields by the microwave irradiation of cyclic imides 4a-j with vanillin in presence of neutral alumina in solvent free condition. The structure of bis-chalcones was confirmed by IR, 1H NMR and elemental analysis.

Antimicrobial activities (4a-j and 6a-j): All the synthesized compounds 4a-j and 6a-j were screened for their antibacterial activity against gram positive bacteria Bacillus subtilis (MCMB-310) and gram negative bacteria Escherichia coli (MCMB-301) using DMF solvent as shown in the graph -1. And antifungal activities against Candida albicans (NCIM-3471) and Aspergillus niger (NCIM- 545) strains using DMSO solvent revealed in the graph – 2. All the results of the synthesized compounds were carried out by the triplicate format N=3 with Mean ± SD. The statistical significance was carried out by one way ANOVA and confirmed by Dunnett multiple comparisons test performed the standard drugs against synthesized compounds. P value < 0.05 was considered as statistically significant remarked by *p<0.05, **p<0.01, ***p<0.001 compared to standard groups. The calculated data were tabulated (not shown).

Table 1 Antimicrobial activities of Bis-chalcones      Bacillus subtilis, Escherichia coli, Candida albicans, Aspergillus niger

Graph 1 Antibacterial activities of 4a-j and 6a-j (B.S. and E.C.) Mean±SD

Graph 2 Antifungal activities of 4a-j and 6a-j (C.A. and A.N.) Mean±SD


Conclusion An entire new series of bis-heterocyclic chalcones containing 4-hydroxy-3-methoxy benzylidene nucleus have been synthesized in one pot and facile manner from cyclic imides in good yield. A good number of the synthesized bischalcone 6a-j showed noticeable synergistic antifungal activities against Candida albicans and Aspergillus niger fungal strains.


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