Aims. Glucagon-like peptide-1 (GLP-1) analog promotes insulin secretion by acting on pancreatic β-cells. This antihyperglycemic treatment for type 2 diabetes mellitus (DM) has attracted increased clinical attention not only for its antihyperglycemic action but also for its potential extrapancreatic effects. We investigated whether liraglutide, a GLP-1 analog, could enhance insulin sensitivity as assessed by the hyperinsulinemic-euglycemic clamp in type 2 DM patients. Materials. We prospectively enrolled 31 uncontrolled type 2 DM patients who were hospitalized and equally managed by guided diet- and exercise-therapies and then introduced to either liraglutide- or intensive insulin-therapy for 4 weeks. Insulin sensitivity was assessed by the glucose infusion rate (GIR) using hyperinsulinemic-euglycemic clamp before and after the therapies. Results. Values of HbA1c, postprandial plasma glucose, and body mass index (BMI) were significantly decreased by hospitalized intensive insulin-therapy or liraglutide-therapy. GIR was significantly increased by liraglutide-therapy but not by insulin-therapy, indicating that liraglutide-therapy significantly enhanced insulin sensitivity. BMI decreased during liraglutide-therapy but was not significantly correlated with changes in GIR. Multivariate logistic regression analysis demonstrated that liraglutide-therapy significantly correlated with increased insulin sensitivity in uncontrolled DM patients. Conclusions. Liraglutide may exhibit favorable effects on diabetes control for type 2 DM patients by increasing insulin sensitivity as an extrapancreatic action. Clinical trial registration Unique Identifier is UMIN000015201. New pharmacological agents have been developed for the management of hyperglycemia and type 2 DM . Incretiemptying . A detaiemptying , 6. in vitro experiments [Investigations of impaired insulin sensitivity are mainly involved in the pathogenesis of type 2 DM. Several animal studies have demonstrated that GLP-1 and GLP-1 analog exhibit the ability of improving insulin sensitivity . A GLP-1eriments . The AMPeriments . An earleriments . Regardieriments –13. As rWe investigated whether liraglutide, a GLP-1 analog, could enhance insulin sensitivity as assessed by the hyperinsulinemic-euglycemic clamp in patients with uncontrolled type 2 DM.We prospectively recruited stable but uncontrolled type 2 diabetes patients  at the outpatient clinic of the Diabetes Care Center in Jinnouchi Hospital between March 2011 and June 2014. The patients who agreed to get treatment in hospital for diabetes were admitted to Jinnouchi Hospital and treated for 4 weeks. Exclusion criteria were type 1 DM, age > 80 years, unstable cardiovascular diseases, active inflammation, severe liver diseases, dementia, chronic kidney disease stage ≥4, disability in bed, and cancer. Patients with newly diagnosed diabetes without any treatments and patients treated with thiazolidinediones were also excluded. For lifestyle modification, all patients were instructed for dietary-therapy by nutritionists and exercise-therapy by exercise trainers. Before starting the hospitalized intensive insulin- or liraglutide-therapies, fasting and postprandial blood samples were collected from the antecubital vein in the morning and the hyperinsulinemic-euglycemic clamp was performed. On admission, the ongoing oral medications for diabetes treatments  were adjusted and dipeptidyl peptidase-4 inhibitors were stopped but metformin was continued with the same doses as before admission. At the same time, all patients nonrandomly started with treatments of either gradually increasing doses of liraglutide  or multiple daily subcutaneous insulin injections (intensive insulin-therapy). The intensive insulin-therapy group was treated with insulin aspart (Novo Nordisk), insulin lispro (Eli Lilly), or insulin glulisine (Sanofi Aventis) before each meal and insulin degludec (Novo Nordisk) or insulin glargine (Sanofi Aventis) at bedtime. During the hospitalization therapies, frequent adjustments were made to the intensive insulin regimens to achieve fasting plasma glucose (FPG) of approximately 100 mg/dL and a postprandial glucose of <180 mg/dL. After the 4-week hospitalized treatment, fasting and postprandial blood samples and a second hyperinsulinemic-euglycemic clamp were performed.This study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the Human Ethics Review Committee of Jinnouchi Hospital, and a signed consent form was obtained from each patient.The hospitalized lifestyle modification therapy was provided by physicians, nutritionists, nurses, and exercise trainers on admission . All patFasting and postprandial blood samples were collected from the antecubital vein in the morning before starting the hospitalized therapies and after the 4 weeks of treatment with liraglutide or insulin. Blood analyses were conducted in the hospital laboratory for the measurement of blood glucose, HbA1c, insulin, and C-peptide immunoreactivity (CPR).At hospital admission before starting the therapies and at 4 weeks following each treatment, insulin sensitivity was evaluated by a hyperinsulinemic-euglycemic clamp examination using an artificial pancreas  with adjustment for the baseline measure of body mass index (BMI) and HbA1c. Logistic regression analysis was used to evaluate the association between the increased insulin sensitivity (GIR increase > 0.5 mg/kg/min) and the baseline clinical variables including liraglutide-therapy allocation. Associations between groups and all other parameters were analyzed by univariate logistic regression analysis followed by multivariate logistic regression analysis with the forced inclusion model, and the Hosmer-Lemeshow goodness-of-fit statistic was calculated. To determine the relation between changes in various clinical parameters and the changes in GIR as the insulin-sensitivity measure in the liraglutide group, correlations between variables of interest were analyzed using Pearson's correlation coefficient. A P value <0.05 was considered statistically significant. Statistical analyses were performed using the Statistical Package for Social Sciences version 19  and SAS version 9.4 .The results of the normally distributed continuous variables (determined by the Shapiro-Wilk test) were expressed as the mean , while those of the continuous variables with skewed distributions were expressed as median values (interquartile ranges). Differences in the baseline characteristics of the two groups were analyzed by Student's2, HbA1c was 8.8%, and FPG was 151.6 mg/dL. At enrollment, 29.7% were treated with insulin. The baseline characteristics of patients in the intensive insulin group were similar to those of the liraglutide group. The levels of BMI, HbA1c, FPG, postprandial plasma glucose (PPPG), duration of diabetes, and fasting blood CPR were not significantly different between the groups. There was no significant difference in the insulin sensitivity assessed by GIR values in the hyperinsulinemic-euglycemic clamp examination between the groups at baseline , FPG , PPPG , and BMI . As shown in At the end of the hospitalized therapies, 21.4 ± 9.6 units/day of insulin was used for the intensive insulin-therapy and 0.9 mg/day of liraglutide was administered to all the allocated patients. Using the hospitalized multidisciplinary approach including the appropriate dietary- and exercise-therapies, all patients of both treatment groups presented with significant improvements in HbA1c , indicating that the significant association between the liraglutide-therapy and the increased insulin sensitivity was independent of the baseline BMI and HbA1c levels in patients with uncontrolled type 2 DM.In the ANCOVA model using the baseline BMI and HbA1c levels as covariates, the liraglutide-treatment demonstrated significantly greater change in GIR compared with the intensive insulin-therapy (P = 0.047). The Hosmer-Lemeshow statistic was appropriate (P = 0.19). Because weight loss effects of liraglutide are well known, we included changes in BMI in the univariate logistic regression analysis and found that reduction in BMI was not significantly associated with improved GIR  in the whole population. In the multivariate logistic regression analysis with the forced inclusion model for reduction in BMI and therapies, liraglutide-therapy was still significantly associated with improved GIR . The Hosmer-Lemeshow statistic was appropriate (P = 0.14).As shown in To determine the associated factors between the liraglutide-induced insulin sensitivity and the changes in clinical parameters, we investigated the correlation coefficient between GIR increase and the changes in variables during the liraglutide-therapy. As shown in We demonstrated that the 4-week short-term liraglutide-therapy enhanced insulin sensitivity in patients with uncontrolled type 2 DM attending a clinical practice. The treatment-related decrease in BMI by liraglutide was not significantly correlated with the increase in insulin sensitivity.Impaired insulin sensitivity and increased insulin resistance are profoundly involved in the pathogenesis and pathophysiology of type 2 DM and its associated cardiovascular complications . TherapiPreviously, studies have shown that metformin and thiazolidinedione can clinically be effective in improving insulin sensitivity . In the In patients with uncontrolled diabetes, the use of glucose-lowering therapies associated with relieving high glucose-mediated glucotoxicity might potentially bring amelioration of insulin resistance . The preCellular and molecular mechanisms of the liraglutide-induced insulin sensitivity augmentation are still under investigation. Chen et al.  reportedThe present study has several limitations. It included a relatively small number of patients and the study protocol was prospective in design and not a randomized clinical trial. Patients with larger BMI tended to be treated by liraglutide and this selection bias might have affected the results. However, the multivariate logistic regression analysis and ANCOVA demonstrated the independent significance of clinically based liraglutide-therapy on the increased insulin sensitivity. Thus, we believe that liraglutide could in part exhibit beneficial effects on insulin sensitivity in uncontrolled type 2 DM patients. Another limitation of the study was that assessment of hospitalized lifestyle intervention could not have been performed in a quantitative manner. Because the present results were achieved by hospitalized short-term intervention, it is necessary to investigate and confirm the long-term and chronic effects of liraglutide on insulin sensitivity.In this study, the short-term use of liraglutide enhanced insulin sensitivity in patients with type 2 diabetes. Liraglutide could exhibit favorable effects on the pathogenesis of metabolic disorders in patients with type 2 DM by increasing insulin sensitivity as an extrapancreatic action.


The incidence of deep venous thrombosis (DVT) related to a central venous catheter varies considerably in ICUs depending on the population included. The aim of this study was to determine subclavian central venous catheter (SCVC)-related DVT risk factors in severely traumatized patients with regard to two kinds of polyurethane catheters.Critically ill trauma patients needing a SCVC for their usual care were prospectively included in an observational study. Depending on the month of inclusion, patients received one of the two available products in the emergency unit: either an aromatic polyurethane SCVC or an aliphatic polyurethane SCVC. Patients were screened weekly by ultrasound for SCVC-related DVT. Potential risk factors were collected, including history-related, trauma-related and SCVC-related characteristics.A total of 186 patients were included with a median Injury Severity Sore of 30 and a high rate of severe brain injuries . Incidence of SCVC-related DVT was 37%  in patients or 20/1,000 catheter-days. SCVC-related DVT occurred within 8 days in 65% of cases. There was no significant difference in DVT rates between the aromatic polyurethane and aliphatic polyurethane SCVC groups (38% vs. 36%). SCVC-related DVT independent risk factors were age >30 years, intracranial hypertension, massive transfusion (>10 packed red blood cell units), SCVC tip position in the internal jugular or in the innominate vein, and ipsilateral jugular catheter.SCVC-related DVT concerned one-third of these severely traumatized patients and was mostly clinically silent. Incidence did not depend on the type of polyurethane but was related to age >30 years, intracranial hypertension or misplacement of the SCVC. Further studies are needed to assess the cost-effectiveness of routine screening in these patients in whom thromboprophylaxis may be hazardous. The incidence of central venous catheter (CVC)-related deep venous thrombosis (DVT) varies considerably in the ICU, depending on the population included and the detection methods ,2. The rWith regard to insertion sites, a femoral insertion is known to increase the risk of infection or thrombosis. The incidence of CVC-related DVT in the upper extremities is estimated to be 2 to 6% when symptomatic and 11 to 19% when asymptomatic . In theiTrauma patients have specific risk factors and the use of anticoagulant is often problematic in the initial phase because of the risk of bleeding. Studies in trauma patients have found widely varying rates of overall DVT from 0.36 to 58% depending on the patients studied and the means used to diagnose DVT . HoweverThe aim of this study was to identify SCVC-related DVT risk factors in ICU trauma patients and to determine whether the type of polyurethane used could be an independent risk factor. The study was performed regardless of the patient's clinical signs with systematic venous ultrasound screening.This prospective, single-center study took place in a 25-bed surgical trauma ICU at Bordeaux University Hospital. This level 1 trauma center, serving the southwest of France (2.8 million inhabitants), takes care especially of severe brain or spinal cord injuries. The institutional review board at the University Hospital of Bordeaux approved the study and waived the need for informed consent because the study was observational and did not interfere in the treatment of patients .We included adult patients (≥18 years), with trauma (Injury Severity Sore (ISS) >8), needing at least one CVC for an estimated length >5 days. Subclavian insertion was the only access route considered in this study. We excluded patients with known DVT risk factors, such as thromboembolic history, thrombophilia, malignancy, patients with anticoagulant treatment at the time of their injury, and patients needing immediate therapeutic anticoagulation for their injury.® catheter; Arrow International, Reading, PA, 19605 USA) or an Al SCVC , To test the kind of CVC as an independent risk factor, the type of available CVC (Ar or Al) was established on a monthly randomized basis.Two kinds of catheter are available in our hospital: an Ar SCVC  using an Acuson CV70, Siemens Medical Solution, Mountain View, CA 94043 USA with a high-frequency transducer dedicated to vascular imaging (5 to 10 MHz). A DVT was defined as partial if the vein was still partly compressible, or as complete when no venous flow could be detected. Thrombosis was considered SCVC related when a partial or complete thrombus was found in the subclavian or axillary or humeral veins with the SCVC in this territory. The examination concerned the superior vena caval territory ipsilateral and contralateral to the SCVC and the lower limbs .The following data were collected: age, sex, date of admission, comorbidities, injury mechanism, and Simplified Acute Physiologic Score II. Injuries were recorded and their severity coded according to the Abbreviated Injury Score (AIS) maximum for each body region and to the ISS. Other data were collected concerning difficulties at the time of insertion, parenteral nutrition, medications infused through the catheter, transfusion, microbiologically documented concomitant infections (bacteremia or pneumonia), length of catheter maintenance, reason for removal, and results of catheter culture.Coagulation screening tests performed at the time of each examination included standard tests  and specific assays for D-dimer, fibrinogen, factor II, factor V, and factor X.A clinical examination was performed by a senior medical doctor before the first ultrasound examination in order to determine whether a DVT was likely or very likely based upon signs such as warmth, edema and collateral circulation.The SCVC type was considered the main potential risk factor. A preliminary study showed that the Ar-SCVC-related DVT incidence was 27%. A sample size of 112 patients in each group would make it possible to detect a 15% difference in the percentage of DVT incidence  with a power of 80%.t test or the Mann-Whitney test for quantitative variables according to their distribution. The value of the clinical examination to determine a SCVC-related DVT was estimated by calculating positive and negative predictive values. Rates were calculated in patients and also reported as DVTs/1,000 catheter-days. Univariate and multivariate logistic regression analysis were performed to identify independent risk factors for development of SCVC-related DVT. All variables associated with a SCVC-related DVT risk with P <0.20 in the univariate analysis were investigated using multivariate analysis, with SCVC-related DVT as the dependent factor and other factors as the independent factors. Statistical significance was set at P <0.05 for all analysis.Quantitative data are presented as mean ± standard deviation or as median with 25 to 75th quartiles (interquartile range (IQR)) according to their distribution. Comparisons between patients were performed using the chi-square test or Fisher's exact test as appropriate, and the Student Four hundred and eighty-six patients were screened during 21 months. Two hundred and ninety-five patients were not included for various reasons: cancer history, associated anticoagulation, CVC set up before transfer to our hospital, CVC in a femoral vein, estimated required duration of CVC <5 days, or logistic reasons (impossible to follow more than 10 patients at the same time on a weekly basis). Five patients were secondarily excluded . Finally, 186 patients were included in the analysis (84 in the Ar group and 102 in the Al group). Most were male (80%). Known DVT risk factors related to the patients' medical history were uncommon (Table Sixty-two patients developed a DVT diagnosed while the SCVC was in place (incidence 33%) and seven more were found 1 to 5 days after ablation of the SCVC  . They were found at the same examination in six cases, before SCVC-related DVT in two cases and after SCVC-related DVT in five cases. The overall incidence of thrombosis in this sample of very severe trauma patients was thus 44%.There were 22 cases of above-knee DVT in the 186 patients (12%) and the femoral vein was involved in 20 out of 22. They also occurred early: 45% were found at the first ultrasound examination. Thirteen occurred in patients with SCVC-related DVT  (Table The Ar group and the Al group were comparable concerning demographic and trauma characteristics Table . The ove3) Table .P <0.05). SCVC-related DVT risk factors in relation to trauma characteristics are shown in Table Mean age was not statistically different between the two groups Table . HoweverP = NS). In patients who were free of DVT at the first examination, the mean C-reactive protein at the end of the second week was 153 ± 87 mg/l in patients who had a thrombosis at this second examination versus 82 ± 61 mg/l in patients still unaffected (P <0.005). We found no relationship between the C-reactive protein level measured weekly during the first 3 weeks and the occurrence of pneumonia or septicemia during the duration of catheterization. Nor did we find any relation between the highest C-reactive protein level during the study in each patient and the occurrence of any infectious episode during the same period.At the first examination, no difference could be found in coagulation standard tests between patients with or without a CVC-related DVT. D-dimers were very high in both groups . Finally, a more prolonged inflammatory status seemed to be associated with a higher risk of SCVC-related DVT. At the first examination, the mean C-reactive protein was 160 ± 89 mg/l in patients whose examination showed a thrombosis and 151 ± 79 mg/l in the other patients (P <0.001). SCVC-related DVT occurring while the patients were receiving LMWH occurred later  than in SCVC-related DVT without prophylaxis  days). In the event of SCVC-related DVT, anticoagulant treatment was given immediately in 17 cases and later in 25 others.Introduction of thromboprophylaxis (subcutaneous low molecular weight heparin (LMWH)) or curative heparin (mainly LMWH) was decided on a case-by-case basis. Thromboprophylaxis was prescribed in 117 patients during the duration of the study with a median delay of 13 days after the trauma. This delay was different between brain-injured and other patients: 15 ± 6 days when maximum head AIS >2  versus 11 ± 6 days in other patients. Five patients developed SCVC-related DVT while anticoagulant therapy or prophylaxis was ongoing: for 5 days in one patient and for >10 days in four others . The rate of SCVC-related DVT was 41% in patients without any prophylaxis during their follow-up versus 7.5% in patients with heparin prophylaxis .P <0.0001). Administration of parenteral nutrition was associated with a higher incidence of SCVC thrombosis while the rate of SCVC-related DVT was lower in patients who received propofol via the SCVC. The occurrence of SCVC-related DVT was not different in patients with a septic episode during catheterization: 28% of DVTs if pneumonia during catheterization versus 33% if not; 11% if bacteremia versus 31% if not.The rate of DVT did not depend on the number of lumens Table . A SCVC 3 colony-forming units/ml) was found in 2.9% of thrombotic SCVCs versus 2.5% without (P = NS). Colonization did not depend on the length of catheterization (15 ± 11days in colonized SCVCs vs. 14 ± 9 days in others). Among the 71 SCVC-related DVTs, 18% were found after SCVC removal; 38% SCVCs still in place were removed within 0 to 3 days after discovery of SCVC-related DVT, while 48% were left in place longer.Upon removal, the catheter tips were systematically collected for bacteriological analysis. This analysis was available in 225 cases: catheter colonization . Healing had occurred at the last examination in 12 out of 27 (44%) patients with LWMH and in 11 out of 22 (50%) patients without this treatment (P = NS). Two patients presented a pulmonary embolism (PE) confirmed by radiological imagery. One was found to have occlusive thrombi both around the SCVC and in the leg. This patient was treated with heparin and the thrombi disappeared. He had a PE 20 days later. In another patient no thrombus was found at the time of the last ultrasound examination when the CVC was removed. A PE was then diagnosed 4 days later and another ultrasound examination showed an occlusive thrombus at the site of the former SCVC. A PE was suspected in three more patients who presented a brief hypoxic episode at the time of CVC removal. Lastly, a patient suddenly died 1 month after having developed a CVC thrombosis, despite receiving curative anticoagulant treatment. This could have been due to a PE, although no radiological examination was available to confirm this hypothesis. One patient was discharged with occlusive SCVC-related DVT under anticoagulant therapy. She was hospitalized again 1 year later and the DVT was still occlusive.Forty-nine SCVC-related DVTs were revised after the date of thrombosis. SCVC-related DVTs disappeared in 23 cases (47%). Healing was observed in 50% of cases where the SCVC was left in place and 44% where it was removed , transfusion, surgery, spinal cord injury, pelvic fracture, head injury, and mobility score. Our patients were particularly vulnerable to DVT. First, the patients were in an ICU for severe polytrauma and during the first week they were all immobilized by sedation to allow iterative surgical treatment and mechanical ventilation. Secondly, 70% of these patients had severe brain injury. A brain injury was demonstrated to increase threefold to fourfold the risk of developing a clinically diagnosed DVT, whatever the pharmacological prophylaxis, in two large cohorts of patients admitted to trauma centers ,20. In aSCVC-related DVT is a particularly underestimated complication. Clinical signs of SCVC-related thrombosis are neither sensitive nor specific ,5,26,27.The second complication may be a post-thrombotic syndrome. Prandoni and colleagues found a 17% cumulative incidence of post-thrombotic syndrome in patients with vein catheter at 1 year but their study concerned only symptomatic upper-arm DVT . Few stuHigh-risk patients should benefit from improved preventive measures. Clinicians are hesitant to use chemoprophylaxis because of the risk of intracranial hemorrhage. LMWH has proved effective as pharmacologic thromboprophylaxis in ICU trauma patients ,9,17. HoThese findings have led us to implement preventive measures to decrease SCVC thrombosis. An educational film about good practices in CVC insertion has been made, which is now available for both junior and senior practitioners. Second, any SCVC whose tip is misplaced in the internal jugular vein or in the innominate vein is removed. The use of ultrasound for SCVC placement decreases the number of punctures and the failure rate, and is now encouraged ,38. ThirThis study demonstrates that SCVC-related DVT is a frequent and early complication in severe polytraumatized patients, and its incidence does not depend on the type of polyurethane. SCVC-related DVT concerns one-third of patients and occurs early within the first week. The main risk factors were intracranial hypertension, a misplaced SCVC, or a massive transfusion. SCVC-related DVT is often underdiagnosed because it is mostly asymptomatic. Further large prospective studies are required to confirm our findings, to identify other risk factors, to study long-term morbidity linked to these SCVC-related DVTs and to evaluate the cost-effectiveness of systematic screening by venous ultrasound in these patients in whom pharmacologic thromboprophylaxis may be hazardous.• The incidence of SCVC-related DVT does not depend on the kind of polyurethane.• The main risk factors are intracranial hypertension and misplaced SCVC.• One-half of CVC-related DVTs were found at the end of the first week after trauma.• Routine coagulation tests do not help to diagnose these DVTs.• The long-term consequences of these asymptomatic SCVC-related DVTs would be investigated to determine their clinical impact.AIS: Abbreviated Injury Score; Al: aliphatic polyurethane; Ar: aromatic polyurethane;CVC: central venous catheter; DVT: deep venous thrombosis; ICHT: intracranial hypertension;IQR: interquartile range; ISS: Injury Severity Score; LMWH: low molecular weight heparin; PE: pulmonary embolism; SCVC: subclavian central venous catheter.The authors declare that they have no competing interests.LP, FM, VC, JB-B and GF contributed to the study conception. AG, LP, FM, VC and FS contributed to the data acquisition, and writing. LP and VC performed ultrasonographic examinations. CP, AL and J-FC contributed to clinical examinations of patients to determine the probability of thrombosis. FM contributed to the data analysis. All authors have made substantial contributions to the conception, the design and interpretation of results, have been involved in drafting the manuscript or revising it critically for important intellectual content, and have given final approval of the version to be published.


MMN fortified beverage interventions could have major programmatic implications for reducing the burden of anemia and iron deficiency in school-aged children in low-middle income countries. Additional research is needed to investigate effects on other biochemical outcomes and population subgroups.Multiple-micronutrient (MMN) fortification of beverages may be an effective option to deliver micronutrients to vulnerable populations. The aim of the present systematic review and meta-analysis is to evaluate the nutritional impacts of MMN fortified beverages in the context of low-middle income countries. A systematic search of published literature yielded 1022 citations, of which 10 randomized controlled trials (nine in school-aged children and one in pregnant women) met inclusion criteria. Results of school-aged children were included in the meta-analysis. Compared to iso-caloric controls, children who received MMN fortified beverages for 8 weeks to 6 months showed significant improvements in hemoglobin (+2.76 g/L, 95% CI ,  In resource poor settings, vulnerable population groups including children and women of reproductive age are prone to multiple micronutrient (MMN) deficiencies primarily due to relatively high requirements, poor dietary quality  and/or iThe two most widely used strategies for delivering additional micronutrients are: (1) supplementation—defined as the provision of micronutrients in capsule, tablet, or syrup form; and (2) fortification—for the purposes of the current paper defined as micronutrients added to processed food vehicles, or at point-of-use (home fortification) ,7. The aThe aim of the present systematic review and meta-analysis is to review the nutritional impacts of non-dairy fortified beverages in the context of low-middle income countries. The primary focus of the review is on school aged children due to the paucity of studies in other population groups. Dairy beverages were excluded to limit potential confounding effects of other nutritional factors present in milk products in the absence of non-dairy MMN fortified beverage control groups. The systematic review was conducted in line with the guidelines for Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). A systematic search of published literature from 1 January 1966 through 28 February 2013 was undertaken, and subsequently updated on 15 February 2015, using the US national Library of medicine’s MEDLINE/PubMed bibliographic search engine. Multiple PubMed searches were conducted using various combinations of Medical Subject Heading (MeSH) and Title/Abstract (TIAB) keywords . A filteIn order to identify any additional articles not found by the PubMed searches, the reference sections for full articles reviewed were searched, and subject matter experts were consulted. An external scientific advisory board consisting of two subject matter experts was consulted to ensure that no related articles were missed in our analyses.Studies considered for selection assessed the effects of non-dairy MMN fortified beverages (RTC or powders for reconstitution) on micronutrient status, anthropometry, morbidity, and/or functional outcomes. Only single or double blind randomized controlled trials (RCT) with treatment and placebo groups conducted in the context of low-middle income countries, on apparently healthy children and women of reproductive age were considered. No restrictions were made for the number of study arms, micronutrient formulations, or study duration. For continuous outcomes, pooled SDs of the change were calculated if the change from baseline to end line and their SDs were reported. Final sample sizes were used to calculate the pooled SD if the number of participants decreased during the course of the study. If the change was not reported, the difference in final value between the intervention and control group was calculated and the SD of the final values from each group was used to calculate pooled SD. If the SD was not reported, 95%-CI of mean values were converted to SD; or medians with 25th and 75th percentiles were converted to mean and SD. For binary data, risk ratios and 95%-CI were calculated. Differences were considered significant at α= 0.05.vs. iso-caloric control was selected for the analysis [For trials with more than two groups, the comparison group most similar to MMN-fortified beverage analysis ,14. One analysis . For com2 statistic calculated for each meta-analysis. Heterogeneity cutoffs were classified as: low < 25%; moderate 25%–75%; and high > 75% [vs. high) and subgroup analyses.A random-effects model was used to account for heterogeneity across studies, with Igh > 75% . Meta-ren = 136), titles and abstracts of 1022 citations were screened. Of these citations, 996 studies were excluded for various reasons (The PubMed literature searches yielded 1158 citations updated on 15 February 2015. After removing duplicates ( reasons . Full ten = 9); ferritin (n = 9); retinol (n = 6); zinc (n = 4); vitamin B12 (n = 3); folate (n = 3); vitamin C (n = 2); riboflavin (n = 2); vitamin B6 (n = 1); thiamin (n = 1); niacin (n = 1); iodine (n = 1); anthropometry (n = 8); cognition (n = 3); and physical performance (n = 2).The final set of 10 studies included a total of 4645 participants who had both baseline and end line measurements . Studiesn = 1) and high (n = 2)  . At the  (n = 2) .n = 9, total sample size = 4094) were included in the meta-analyses. Compared with controls who received a non-fortified beverage, children who received MMN-fortified beverages for a duration of 8 weeks to 6 months demonstrated significantly greater improvements in circulating hemoglobin  and ferritin . Exclusion of an anomalous study that found no change in hemoglobin or ferritin in either intervention or control groups did not significantly alter these results [p = 0.16; 5 studies), zinc , or vitamin B12 . Results of all trials conducted in children , while neither height gain nor weight-for-age or height-for-age Z-scores differed significantly between groups . Although the number of data points precluded meta-analysis, we found some evidence of a favorable effect of supplementation with MMN-fortified beverages on riboflavin status [A moderate but significant effect was found for weight gain in the intervention compared with the control groups , iron deficiency , and iron deficiency anemia   . Fortifip = 0.02) with all studies included in analysis. The association was attenuated when total iron dose was substituted for daily iron dose . However, the significance of this relationship did not persist after excluding the study by Aaron et al. (p > 0.90) [Meta-regression analyses were conducted for outcomes with at least five data points from different trials . Considered independently, daily iron dose, total iron dose (daily dose x duration), intervention duration, baseline status, and deworming (binary Y/N) were not significantly associated with the difference in ferritin change between groups. A significant inverse association was found between daily iron dose and hemoglobin change  . Baselinet al., differences were no longer significant.Study duration was not significantly associated with any of the biochemical or anthropometric outcomes measured. When comparing reconstituted powder to RTC beverages, there were no significant differences in any index except for ferritin, which demonstrated significantly less change in interventions with RTC beverages. However, after excluding the study by Aaron p = 0.015) and serum ferritin (p = 0.009) but not in serum retinol in women in the intervention group. Consumption of iron/folic acid tablets did not differ significantly between the two groups. A single placebo-controlled trial evaluated an 8-week intervention with a non-dairy MMN-fortified beverage in Tanzanian women who were 12–34 weeks gestation at baseline . Only woTo the best of our knowledge, this is the first systematic review and meta-analysis to focus on nutritional outcomes related to consumption of MMN fortified beverages in the context of low-middle income countries. Among school children who consumed non-dairy MMN fortified beverages compared with controls consuming iso-caloric non-fortified placebo beverages, results from the meta-analysis showed significant improvements in hemoglobin and ferritin, but not in other biochemical indices. Consequently, consumption of fortified beverages significantly reduced the risk of anemia, iron deficiency, and iron deficiency anemia, but did not significantly alter the risk of other micronutrient deficiencies. In the meta-regression analyses, baseline status, intervention dose, study duration, and deworming were not significant predictors of differences in hemoglobin, ferritin, or weight change between groups. vs. control groups despite beverages in both groups being iso-caloric. No effects were found on other anthropometric indices. Morbidity and functional outcomes, such as cognition and physical performance, could not be adequately assessed due to insufficient data points. Although meta-analysis was precluded by the scarcity of data, findings from a single MMN-fortified beverage trial conducted in pregnant women in Tanzania were consistent with results of the meta-analysis in school children.A small but significant effect was found for weight gain in micronutrient Results from the present analysis are in general agreement with the findings from a previous systematic review on MMN fortification of foods or beverages in school children . Eleven Findings from the present study were similar to a recently published Cochrane review on the effects of micronutrient powders (MNP) among infants and children (6–23 months at the time of the intervention) in low-income countries ; retinolThe greater impact of MMN-fortified beverages on anemia and iron status compared with other biochemical outcomes in this as well as previous reviews ,23 may hDespite the stronger grade of evidence for hemoglobin and iron status outcomes in the present analysis, evidence suggests that combined MMN supplementation may have a greater effect than iron supplementation alone. A meta-analysis on the effects of micronutrients on growth in children below age 18 years found that iron alone had no significant effect but that MMN interventions improved linear and possibly ponderal growth . This phZ-score [Several considerations should be made with regard to implementing beverage fortification strategies. Firstly, as with any nutrition strategy, the justification for fortifying a vehicle should be based on demonstrated micronutrient needs and health parameters of the population, and should take into account issues such as safe administration of micronutrient interventions . In highZ-score .Strengths of the present systematic review and meta-analysis include a comprehensive and targeted search strategy to identify relevant publications and a thorough quantitative analysis of available data. Limitations were the small number of beverage fortification RCT, multiple reported outcomes with insufficient data points for inclusion in meta-analysis, and incomplete information on quantities and chemical forms of micronutrients, quantities of macronutrients (particularly energy and protein), and compliance. However, despite the aforementioned limitations, results from the analyses showed a clear benefit of MMN-fortified beverage fortification on anemia and iron status, both of which are high burden global public health issues. In summary, evidence from the present study suggests that the administration of non-dairy MMN-fortified beverages in the context of school settings in low-middle income countries is effective at improving hemoglobin and ferritin, and reducing the prevalence of anemia, iron deficiency, and iron deficiency anemia. While only one study evaluated the impacts on women, results were consistent with the broader findings in meta-analyses among children. Despite the positive impacts found in the present review, considerations to limit the caloric content of beverages selected for fortification are important if used in programs or market-based settings. Additional research is needed to investigate the effects of MMN-fortified beverages on other biochemical outcomes and population subgroups, as well as to evaluate effectiveness in programmatic settings.


Most of the current approved RTK–TKIs in preclinical and clinical settings are multi-targeted inhibitors with several side effects. Only a few specific/selective RTK–TKIs have been developed for the treatment of cancer patients. Specific/selective RTK–TKIs have shown less deleterious effects compared to multi-targeted inhibitors. This review intends to highlight the importance of specific/selective TKIs for future development with less side effects and more manageable agents. This article provides an overview of: (1) the characteristics and function of RTKs and TKIs; (2) the recent advances in the improvement of specific/selective RTK–TKIs in preclinical or clinical settings; and (3) emerging RTKs for targeted cancer therapies by TKIs.Chemotherapeutic and cytotoxic drugs are widely used in the treatment of cancer. In spite of the improvements in the life quality of patients, their effectiveness is compromised by several disadvantages. This represents a demand for developing new effective strategies with focusing on tumor cells and minimum side effects. Targeted cancer therapies and personalized medicine have been defined as a new type of emerging treatments. Small molecule inhibitors (SMIs) are among the most effective drugs for targeted cancer therapy. The growing number of approved SMIs of receptor tyrosine kinases (RTKs)  Cancer is a complex disorder of the uncontrolled proliferation of cells. Currently, eight hallmarks explain the typical capabilities acquired by tumor cells in the process of tumorigenesis . These pMultiple cytotoxic drugs and combination regimens have resulted in significant improvement of prognosis and survival of cancer patients. However, their therapeutic uses have been blunted by several disadvantages. This represents a request for developing more specific drugs.Currently, rapid clinical application of targeted cancer therapy agents has dominated drug development. Targeted cancer therapy agents have significantly progressed during recent years by developing and receiving approval from authorities for use in cancer treatment. The aim of targeted cancer treatment is to destroy cancer cells by targeting tumor-specific (TSA) or -associated antigens (TAA) without affecting normal cells. In fact, tumor cells of different origins display unusual molecules that are either inappropriate for normal cells and their environment or are usually present during the organogenesis. For instance, receptor tyrosine kinase-like orphan receptor 1 (ROR1), a recently identified oncofetal protein, expresses in neuronal cells and other fetal tissues during embryogenesis. However, this receptor tyrosine kinase (RTK) has been found to be over-expressed in various tumors but not in normal adult cells ,6,7,8,9.For targeted cancer therapies, monoclonal antibodies (mAbs) and small-molecule inhibitors (SMIs) of tyrosine kinase activity (TKIs) are ideal candidates which target tumor cells via binding to cell-surface antigens and intracellular molecules, respectively .An SMI is a type of compound that interferes with specific molecules required for tumor cell growth and function. SMIs selectively target molecules with a unique structure and are different from the non-specific destruction associated with traditional chemotherapy. They have been used for the treatment of a wide range of diseases, including infectious diseases, as well as autoimmune and malignant disorders.Among the various targets for SMIs, receptor tyrosine kinases (RTKs) have attractive features. RTKs have distinct structural and biological properties that make them capable of transducing extracellular signals into the intracellular compartments . Most onMost of the current RTK–TKIs are multi-targeted and inhibit a variety of molecules in a non-specific manner. Multi-targeted inhibition has been shown to have several disadvantages due to targeting various RTKs compared to selective/specific TKIs . Only a In recent years, there has been a significant progress in developing specific/selective TKIs for targeted cancer therapy. Due to the lack of cumulative data on selective/specific TKIs, this review summarizes the recent findings regarding more specific/selective RTK–TKIs that are approved by authorities or are in preclinical and clinical settings. Moreover, approaching RTKs that might be proper candidates for targeted therapy will also be overviewed.The term “oncogenic addiction” established by Weinstein  describeProtein kinases are enzymes that are involved in phosphorylation and transfer of a phosphate group from adenosine 3 phosphates (ATP) to tyrosine, serine or threonine residues. Protein phosphorylation is one of the most important events in regulating cell activities. Some oncoproteins need phosphorylation for regulation and activation .Among different protein kinases, RTKs comprise a well-known group and consist of a transmembrane receptor linked to the intracellular kinase domain. These proteins have emerged as key pharmacological targets in oncology . PhosphoSimilar to transmembrane proteins, RTKs’ structure consists of three different parts: extracellular, transmembrane and cytoplasmic regions . The extThe cytoplasmic region contains tyrosine residues that are phosphorylated upon ligand binding and activation, regulate catalytic function, and also serve as docking sites for SRC Homology 2 (SH2) domain-containing proteins .Deregulation of RTK activity is the major mechanism by which tumor cells escape from physiological constraints on survival and growth . AberranIdentifying new oncogenic RTKs that are over-expressed by tumor cells and regulate the growth, survival, invasion, and communication of tumor cells with their microenvironment have facilitated the development of new anti-cancer agents and have revolutionized treatment options. Therefore, due to the interesting biological features, RTKs are of the main focus for developing new TKIs for therapeutic interventions in cancer patients.TKIs, as well as other small inhibitors, are low molecular weight organic compounds. A cut off at 500 Daltons is recommended based on the observation that clinical attrition rates are significantly reduced when the molecular weight falls below 500 Daltons  23,24].,24.23,24in vivo use ..27].TKIs are classified into three main groups. Most of the current TKIs are ATP-competitive inhibitors and are classified as type I inhibitors. Due to the highly conservative ATP-binding sites in TK domains and a high rate of competition with intracellular ATP, several difficulties obstruct the development of specific/selective TKIs of type I. Therefore, TKIs might target other kinases, thereby suggesting that the anti-tumor effects may be due to the effects on other signaling molecules. Types II and III are non-ATP competitors and act through induction of structural changes in the RTKs. The conformational shifts modify the TK domain in a way that the TK domain loses its kinase activity . MoreoveMost of the FDA approved TKIs for the treatment of cancer are multi-targeted inhibitors of several intracellular tyrosine kinases , and a fEGFR (ErbB) is a family of four structurally related RTKs: ErbB-1/ EGFR, ErbB-2/HER2/neu, ErbB-3/HER3, and ErbB-4/HER4. This family plays critical roles in the regulation of normal cell proliferation, differentiation and survival. Under physiologic circumstances, specific soluble ligand (EGF) binds to the extracellular region of EGFR and following homo/heterodimerization with other members lead to phosphorylation at specific tyrosine residues within the intracellular domain .EGFR members are abnormally activated by several mechanisms like receptor over-expression, mutation, ligand-dependent receptor dimerization, ligand-independent stimulation, and are associated with the development of tumors of epithelial origin, including non-small cell lung cancer (NSCLC) , breast Gefitinib   and erlo+ tumors, including gastric ..30].These EGFR–TKIs are dual-inhibitors of EGFR and HER2. They bind to the EGFR and HER2 receptors and reduce autophosphorylation in cells by targeting a cysteine residue (Cys) in the ATP-binding pocket of the receptors.The activity of gefitinib and erlotinib has been shown to be impressive in patients with EGFR mutations. However, most patients have developed acquired resistance to these TKIs. To overcome the problem, afatinib was developed as an irreversible EGFR–TKI.Afatinib has been investigated as a first-line treatment for patients with EGFR mutation-driven NSCLC and as a second- or third-line treatment in patients with NSCLC, who have received prior treatment with an EGFR–TKI . It has Preclinical studies have shown the promising activity of neratinib in NSCLC and breast cancer. Cell lines transformed with HER2 have shown to be resistant to the erlotinib; however, these cells as well as NSCLC cell line, Calu-3, over-expressing HER2, were highly sensitive to the neratinib . Moreovein vitro and in vivo in xenografted mice with cell lines over-expressing EGFR and HER2 [Lapatinib  is a reversible inhibitor of EGFR/HER2 and was developed in 2002 for dual-targeted therapies  55]. La. La55]. Phase I clinical studies started at 2002 and the safety, tolerability and pharmacokinetics were assessed in healthy controls . BiologiIn combination with capecitabine, lapatinib has shown anti-tumor activity and prolongation of the time to progression of the disease in breast cancer patients who have previously been treated with trastuzumab, an anthracycline and a taxane . Lapatinin vivo in xenografted mice. It has been tested in phase I trials in patients with head and neck, breast and NSCLC cancer. The water-soluble analog of canertinib has been shown to inhibit EGFR activity in vitro and suppressed human A431 cells xenografts in nude mice . It. It72].  in vivo .in vitro. In concentrations up to 1 μM, vatalanib did not have any cytotoxic or anti-proliferative effect on cells that do not express VEGFR. It induced dose-dependent inhibition of VEGF-induced angiogenesis in a growth factor implanted and a tumor cell-driven angiogenesis model. Moreover, it inhibited the growth of various human carcinomas in nude mice as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Microvessel formation has revealed to be inhibited by vatalanib in the interior of the tumor . In. In50 vaIn a pilot study, combined imaging, microcomputed tomography and histologic tumor evaluation with a xenografted model of breast cancer cell line, cediranib induced significant prevention of tumor growth and regression of MCF7–VEGF cells (transfected with VEGF) in xenografted athymic mice. Moreover, blood flow and microvessel density and proliferation have been shown to decrease significantly in MCF7–VEGF tumors .Several preclinical studies demonstrated the significant reduction of tumor growth in various cancers including kidney, breast, lung, prostate, ovarian, colorectal cancer, sarcomas, glioblastoma, renal cell carcinoma, and NSCLC . Based oPDGFRs belong to the type III tyrosine kinase family. They are not expressed in normal tissues but only in fibroblasts, smooth muscle cells in lungs and pericytes of the vascular wall. PDGFR over-expression leading to constitutive PDGFR activation has been reported in a number of malignancies, including NSCLC and gliomas . PDGFRs All approved PDGFR–TKIs are multi-targeted and a few specific/selective inhibitors are in preclinical and clinical settings.In vitro experiments have demonstrated that CP-673451 inhibited cell growth of mesenchymal-like NSCLC cell line H1703 with high expression of PDGFRα, but not in the epithelial cell line H292 lacking PDGFR . T. T117].  cancers . Treatme cancers .The first clinical evaluation of tivantinib started in 2010 and the safety and tolerability, dose-limiting toxicities (DLT) and maximum tolerated dose (MTD), anti-tumor activity, pharmacokinetic, and pharmacodynamic profiles were evaluated in patients with advanced or metastatic solid tumors . A maximSeveral phase II and III clinical trials are ongoing in patients with solid tumors and promising results have been achieved .50 value of LY2801653 for inhibition of MET autophosphorylation in HGF-stimulated H460 and S114 cells was shown to be 35.2 ± 6.9 and 59.2 nM, respectively. This TKI shows more anti-proliferative activity in cells with MET over-expression e.g., H1993 than the cells without MET gene amplification. It strongly inhibited MET phosphorylation and tumor growth in S114, U-87MG and NCI-H441 xenografted mice models [LY2801653 was developed in China in 2013 and is a type II MET/RON–TKI  120]. T. T120]. This TKI inhibited NSCLC cell lines and patient-derived tumor xenografts. It prevented the constitutive activation of MET signaling and NCI-H441 cell proliferation, anchorage-independent growth, migration, and invasion. Treatment of the NCI-H441 orthotopic model significantly inhibited both primary tumor growth and metastasis. Moreover, treatment of tumor-bearing mice showed a significantly higher survival time compared to controls .50 of 627.6, 72.9 and 9.28 nmol/L, respectively. It also prevented the growth of A549 and H1993 tumor xenografts in nude mice and growth of A549-luc-C8 lung orthotopic tumor xenografts in SCID mice . I. I131]. poptosis .Treatment of FLT3-mutated xenografted model displayed dose-dependent and significant tumor cell growth inhibition. Furthermore, KW-2449 showed anti-proliferative activity on primary AML cells and imatinib-resistant cells .in vitro and in vivo [Crenolanib is also a selective type I pan-FLT3 inhibitor with inhibitory effects on other TKs with higher concentrations  135]. C. C135]. Overall, a few selective FLT3–TKIs have been developed and have shown promising activity against leukemic cells of AML patients. More investigations in clinical trials and on the other malignancies are necessary to explore the potential anti-tumor activity of FLT3–TKIs.AXL and MER RTKs belong to the TAM family, including TYRO-3, AXL and MER. The TAM receptors are defined by the presence of two Ig-like domains and two fibronectin type III repeats in the extracellular part and a cytoplasmic domain with kinase activity . The vitCurrently, among a few selective AXL–TKIs, R428 has shown encouraging results in preclinical assays and might be a proper AXL–TKI for treatment.in vitro [R428 (BGB324) is a first-in-class, highly selective AXL–TKI and has been tested in preclinical and clinical settings. R428 dephosphorylates AXL in a dose-dependent manner and prevents the invasion of both human MDA-MB-231 and murine 4T1 breast cancer cell lines in vitro .In mice models, R428 showed acceptable plasma stability and pharmacologically proper doses have been achieved in oral administration. It inhibited breast cancer cell metastasis and angiogenesis following inhibition of AKT and ERK phosphorylation. R428 has shown proper activity in combination with cisplatin to inhibit liver and lung metastases in a breast cancer mouse model .in vitro and xenografted models, both in combination with other TKIs or chemotherapeutic agents. R428 has shown a synergistic anti-proliferation effect on NCI-H1299  human NSCLC cells in combination with erlotinib. In NCI-H1299 cells xenografted mice R428 has significantly enhanced the anti-tumor activity of Docetaxel. Moreover, treatment of A549 NSCLC cell line with R428 and in combination with either erlotinib or anti-VEGF human antibody bevacizumab displayed additive anti-tumor activity. Treatment of HCC827 xenografted model with R428 has delayed the development of resistance to erlotinib [The effects of R428 have been tested on NSCLC cells rlotinib . R428 serlotinib .ROR1 belongs to ROR–RTK family and is evolutionarily conserved between different species . ROR1 coThere is little information about ROR1–TKIs and they are presently under intensive investigation in preclinical settings.p ˂ 0.001) compared to PCI-32765, CAL-101, R406, R788, STK-156485 and STK-156133 inhibitors [Biochemical studies have shown that ROR1–TKIs IN0439365 and KAN0438063 are highly selective and killed leukemic CLL cells with high specificity. Among several compounds, the best (IN0439365) killed 50 times more CLL cells than normal blood lymphocytes, dephosphorylated ROR1 and inactivated PI3K/AKT/mTOR proteins ,149. KANhibitors ,149.Similar effects were observed on pancreatic adenocarcinoma cell lines. Incubation of PaCa2 and PANC1 cells with IN0439365 induced a significant cell death following dephosphorylation of ROR1 and inactivation of SRC, PI3K, AKT, mTOR, and CREB ,150.Several TKIs have been developed against other oncogenic RTKs, including anaplastic lymphoma kinase (ALK), EPHA/B, IGF2R, KIT, among others . Most ofResistance to the current targeted therapies, including SMIs, can be divided into the intrinsic and acquired resistance . IntrinsDrug resistance has been shown in various malignancies. Resistance to BCR–ABL-targeted therapies (imatinib) has been noted in AML and is associated with the intrinsic resistance of leukemic stem cells . Unexpecin vivo or in vitro [Dual blockade of extra- and intra-cellular parts of RTKs or targeting more than one RTK by mAbs and TKIs is likely to represent improvement in the treatment and decrease the rate of drug resistance. A combination of trastuzumab and lapatinib in xenografted mice with HER2-over-expressing cells has shown remarkable inhibition of tumor growth and survival . Dual tain vitro ,90.in vivo studies; (5) a deeper understanding of the tumor heterogeneity in different individuals and the role of cancer stem cells; and (6) developing specific RTK–TKIs to target cancer stem cells. All factors are crucial to a better understanding of the nature of tumors and the raise of drug resistance.Most of the available targeted cancer therapy agents have significantly improved patients’ progression-free survival, but none of them has yet proven to cure the disease. Several RTK–TKIs and other inhibitors have been developed. Despite the considerable efforts from screening to clinical trials, which are expensive and time-consuming, only a few TKIs have entered clinical trials or have been approved by authorities for cancer treatment. Moreover, most TKIs as research tools or in the clinic are multi-targeted drugs. Multi-targeted property has several disadvantages, including side effects, a complication of the interpretation of results, and inducement of early resistance. Therefore, developing more specific/selective TKIs are necessary to overcome the current problems. On the other hand, tumor cells and their microenvironments are more complicated than in normal situations. Tumor cells simultaneously over-express several RTKs Targeting a specific RTK using a specific/selective TKI might give us a better understanding of the mechanism of tumor cell resistance. This may lead to select proper agents or prevent second-line of resistance. Therefore, identification and development of more specific SMIs will be critical for the successful treatment. To achieve this goal, several factors are required to be considered, including: (1) proper knowledge of the effects and characteristics of each TKI in preclinical studies; (2) TKIs with the highest selectivity and specificity are preferred to prevent or delay side effects and drug resistance; (3) developing more type II and III inhibitors than type I inhibitors; (4) using different animal models for preclinical


Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) are near universally fatal conditions if untreated. Although effective therapeutic options are available for these diseases, treatment regimens are associated with both short- and long-term adverse effects. The recent identification of effective B-cell-targeted therapy with an anti-CD20 monoclonal antibody has transformed the treatment landscape of AAV. Questions, nevertheless, remain regarding the appropriate timing, dose, frequency, duration, and long-term effects of treatment. The aim of this article is to provide an overview of the current information, recent advances, ongoing clinical trials, and future treatment possibilities in AAV. The systemic vasculitides are a heterogeneous group of diseases characterized by inflammation of blood vessels, resulting in tissue damage and end-organ dysfunction. Although relatively rare, if left untreated, these conditions lead to significant morbidity and mortality. While the etiology of the primary vasculitides is still unknown, a better understanding of their pathogenic mechanisms in combination with an expanded armamentarium of targeted therapeutics has led to significant advances in treatment. Among the primary systemic vasculitides, the treatment of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) has most notably benefited from these recent advances. Landmark studies in AAV showing the efficacy of B cell depletion have led to rapid clinical utilization of rituximab (RTX), an anti-CD20 monoclonal antibody which targets and depletes premature and mature B lymphocytes. However, once patients are in remission, there is controversy surrounding the appropriate timing, dose, frequency, duration, and long-term effects of this treatment. The purpose of this review is to provide an overview of the current information, recent advances, ongoing clinical trials, and future treatment possibilities in AAV.The AAVs comprise granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). These multisystem, immune-mediated conditions primarily affect small blood vessels, most commonly small arteries, arterioles, capillaries, and venules of the lung and kidney. For patients with severe systemic AAV, the mainstay of remission induction treatment has included high-dose glucocorticoids and oral or intravenous (IV) pulse cyclophosphamide (CYC).1, the use of CYC, particularly in refractory and relapsing disease, is limited owing to cumulative toxicity and serious adverse events which can include infertility, bladder hemorrhage, severe cytopenias, serious infection, and increased risk of malignancy. Because of these associated side effects, reducing the overall cumulative exposure to CYC during remission induction has been advocated. De Groot and colleagues demonstrated that pulse dose CYC induced remission of AAV as well as daily oral regimens while reducing the cumulative CYC dose by half and significantly decreasing the frequency of treatment-related leukopenia2. Despite limited power to determine the effect of the evaluated induction regimens on relapse, longer-term follow-up identified that patients receiving pulse dose CYC had a twofold increased risk of relapse compared to those receiving daily oral CYC3. This was particularly pronounced among those with anti-proteinase 3 antibodies (PR3-ANCA). As such, limiting CYC treatment may not be as beneficial for some patient subsets.Although effective in 70–90% of patients with AAV4. B cells and their progeny appear to be central in the pathogenesis of AAV through the production of ANCA, as well as T cell co-stimulation and cytokine production5. Furthermore, the number of circulating activated B cells correlates with disease activity6. The identification of these pathogenic roles provided the framework for two landmark randomized trials to evaluate B-cell-targeted therapy for remission induction in patients with GPA and MPA.In AAV, CYC appears to exert a greater effect on B cells compared to T cells2) weekly for 4 weeks with two IV pulses of CYC or IV CYC pulses for 3–6 months followed by azathioprine (AZA) maintenance7. Sustained remissions at 12 months were similar between the RTX (76%) and CYC/AZA (82%) groups, demonstrating the efficacy of RTX for remission induction in patients with AAV and significant renal dysfunction. Surprisingly, no difference in adverse events or mortality was observed. Extended trial follow-up data at 24 months8 showed that the RTX-based induction without maintenance therapy had similar longer-term outcomes to the CYC/AZA regimen with regard to relapse (21% vs. 18%) and mortality (18% vs. 27%).The Rituximab vs. Cyclophosphamide in ANCA-associated Renal Vasculitis (RITUXVAS) trial included 44 patients with newly diagnosed GPA or MPA and renal involvement. In addition to receiving high-dose glucocorticoids, patients were randomized (3:1) to induction treatment with either RTX  of patients reaching remission at 6 months compared to 42% (21/50) with CYC/AZA. Extended follow-up results showed similar long-term efficacy between the two regimens with 48% and 39% of patients in sustained remission at 12 and 18 months in the RTX arm and 39% and 33% in sustained remission at 12 and 18 months in the CYC/AZA arm, respectively10.The Rituximab vs. Cyclophosphamide for ANCA-associated Vasculitis (RAVE) trial was a randomized, double-blind, double-dummy, placebo-controlled, non-inferiority trial evaluating a total of 197 patients with GPA or MPA9. Potential reasons may include identification of treatment-related complications due to concomitant high-dose glucocorticoids and short duration of follow-up preventing an observed difference in known long-term CYC-associated adverse events.Though it was anticipated that induction with RTX might be safer than CYC, it is of note that the number of adverse events in both the RITUXVAS and RAVE studies was similar between the two treatment armsTaken together, the results of RITUXVAS and RAVE demonstrate RTX to be equivalent to CYC for remission induction of AAV among treatment-naïve patients and likely superior to CYC for relapsing disease.1. However, a slight numerical increase in relapse was observed in longer-term follow-up among those receiving shorter CYC courses11. Further research is needed to determine if certain subsets of patients would benefit from a duration of CYC treatment tailored to their risk of relapse and likelihood of treatment-related morbidity11. While AZA is the most commonly used immunosuppressive agent for remission maintenance, methotrexate has also shown similar efficacy12, provided patients have adequate renal function. Mycophenolate mofetil, on the other hand, has been shown to be inferior to AZA for remission maintenance but remains an alternative for patients intolerant of other immunosuppressive options13. In addition to conventional oral immunosuppressive medications, repeated administration of RTX may also be a viable option for remission maintenance.GPA and MPA, however, are chronic diseases, and up to 40–50% of patients can have a relapsing course despite immunosuppressive treatment. Traditionally, patients treated with a CYC induction regimen are transitioned to an oral conventional immunosuppressive agent, while glucocorticoids are gradually tapered. Trial data from the Cyclophosphamide vs. Azathioprine for Early Remission Phase of Vasculitis (CYCAZAREM) study found that early substitution with AZA following a minimum of 3 months of oral CYC therapy had similar overall outcomes to a 12-month oral CYC regimen16. Several open-label studies have evaluated the use of RTX to prevent relapse; unfortunately, heterogeneity in the timing, dose, and duration of treatment has prevented consensus on a preferred maintenance regimen. Smithet al. retrospectively studied relapsing GPA/MPA patients induced with RTX and then either observed until relapse or treated with fixed-interval RTX maintenance dosing of 1 g every 6 months17. Over 2 years, 73% of the observed group had relapsed compared to only 12% receiving fixed-interval doses of RTX. Additional investigators have observed similar efficacy of pre-emptive dosing for reducing relapse rates and prolonging relapse-free survival21, leading many clinicians to employ such treatment approaches.The long-term follow-up data from the RITUXVAS and RAVE studies confirm that a single course of RTX is beneficial for induction. Unfortunately, relapse will occur in the majority of patients if they are not provided with maintenance treatment. This appears particularly true among those with upper respiratory tract disease, granulomatous infiltrates, or PR3-ANCA positivity22. This open-label study randomized 115 patients in complete remission following standard induction with CYC and glucocorticoids to receive either RTX  or AZA . At month 28, major relapse occurred in 29% (17/58) of patients receiving AZA maintenance compared to only 5% (3/57) with RTX. Although this study demonstrates RTX may be superior to AZA in maintaining remission at 2 years, it should be noted that the AZA dose was reduced starting  at 12 months, a schedule not frequently employed in clinical practice. Indeed, 41% of the relapses in the AZA group occurred after treatment cessation. Therefore, it is unknown if the difference in relapse rates would have been less striking if higher doses of AZA were maintained throughout the entire study period. In order to address this question, an international collaborative trial  is ongoing. RITAZAREM will evaluate relapsing AAV patients randomized to either RTX 1 g every 4 months for five doses or AZA 2 mg/kg/day for 24 months, more closely paralleling typical clinical practice.Several prospective randomized trials are currently aimed at understanding the utility of RTX as maintenance therapy . MAINRIT21, others demonstrate notably increased adverse events, including serious infection, late-onset neutropenia, and hypogammaglobulinemia23. A tailored approach to guide pre-emptive treatment decisions based on serial B lymphocyte and ANCA titer monitoring has been suggested by Cartin-Ceba and colleagues19. However, other investigators have demonstrated that these parameters are not uniformly consistent in relapse prediction24. MAINRITSAN 2  was organized to assess this critical knowledge gap and has been designed to compare two treatment regimens for RTX maintenance; 500 mg every 6 months vs. 500 mg whenever CD19+ lymphocytes re-populate or when ANCA titers become positive or rise. Extended follow-up of this trial with an additional 18 months of fixed dosing RTX at 6-month intervals vs. placebo is also planned . These two trials will greatly advance the understanding of RTX use in remission maintenance, and results are expected in the next 3–5 years.Although considered to be a potentially safer alternative to CYC, the delayed adverse effects of long-term RTX maintenance in AAV are unknown. While some studies show that repeat dosing is well tolerated25. In this trial, dialysis-independent patients improved with either standard drug therapy with adjunctive PLEX or drug therapy alone, regardless of initial serum creatinine. However, patients who were dialysis dependent at study entry experienced greater likelihood of renal recovery with the addition of PLEX compared to standard drug therapy. In a larger trial, Jayneet al.26 evaluated the effect of adjunctive therapy with PLEX or IV methylprednisolone (MP) in 137 patients with severe renal vasculitis  receiving standard treatment with oral CYC and oral prednisone. Those randomized to PLEX had a higher rate of renal recovery and independence from dialysis compared to those treated with IV MP at both 3 months  and 12 months .The overall benefit of plasma exchange (PLEX) in severe AAV remains uncertain. Initial evidence of efficacy with PLEX was seen in a small randomized controlled trial by Pusey and colleagues evaluating patients with anti-GBM-negative renal vasculitis27. A meta-analysis comprising 387 patients with AAV from nine randomized trials found that patients treated with PLEX had a significant reduction in dialysis dependence  but not mortality 28. However, the authors note that the cumulative evidence was inadequate to conclude that PLEX effectively decreases ESRD because more than 1478 patients would have been needed to determine a 25% RR reduction with appropriate confidence28.Although short-term outcomes were encouraging, long-term benefits are unclear, as subsequent follow-up of this cohort for a median of 3.95 years did not identify a significant difference between PLEX and IV MP among the outcomes of end-stage renal disease (ESRD) or mortality29 found that prompt initiation of PLEX in addition to standard induction therapy for AAV led to resolution of DAH in all 20 patients retrospectively reviewed. Resolution occurred after an average of six treatments, and no treatment-associated complications were seen. Nevertheless, the absence of a control group prevents concluding whether PLEX itself led to such improvements. In a recent retrospective study, Cartin-Cebaet al. evaluated the outcomes of 73 patients with AAV and DAH and did not observe a benefit in achieving complete remission at 6 months between adjunctive PLEX and standard induction therapy 30. The authors further evaluated 11 uncontrolled studies, including their own, and found resolution of DAH and survival to hospital discharge was achieved in 66% (69/104) of patients treated with PLEX compared to 75% (51/68) without, tempering the likelihood that PLEX is necessary in this population30.In addition to its use for severe renal vasculitis, PLEX has been considered for the treatment of AAV with pulmonary complications from diffuse alveolar hemorrhage (DAH). The use of PLEX in DAH, however, is limited to uncontrolled observational studies and results are conflicting. Klemmer and colleaguesTo address these uncertain roles of PLEX, a randomized controlled trial recruiting patients with severe renal vasculitis and/or diffuse alveolar hemorrhage secondary to AAV has been developed and is underway . Currently, the precise role of PLEX as adjunctive therapy for remission induction in AAV remains unclear. However, given the severity of disease, it is still considered by some experts to be a reasonable option for patients with severe renal disease or dialysis dependence at the time of diagnosis. The results of PEXIVAS are highly awaited to provide further insight and recommendations.Until this past decade, CYC was considered the only reliable induction agent for AAV. The success of non-selective B cell depletion with RTX in AAV has opened the door for the next generation of targeted therapies focusing on the innate and adaptive immune system .31, and have been associated with autoimmune conditions such as systemic lupus erythematosus32. Elevated BAFF levels have also been seen in patients with GPA, most notably among active untreated patients33, and ANCA-stimulated neutrophils promote B cell survival through the release of BAFF34. Interestingly, researchers have observed BAFF levels increasing after B cell depletion with RTX in AAV models34. This finding demonstrates that BAFF may be an integral factor in the survival of autoreactive B cells and likely facilitates disease chronicity and relapse.B-cell-activating factor (BAFF), also known as B lymphocyte stimulator (BLyS), appears to have a key role in the stimulation of B cell proliferation and the promotion of immature B cell survival. Increased BAFF levels favor the selection of autoreactive B cells, leading to autoantibody productionBelimumab is a monoclonal antibody directed against BAFF that has been approved for the treatment of systemic lupus erythematosus. This targeted BAFF inhibitor is currently being investigated in a phase III multicenter randomized trial evaluating the efficacy and safety of this medication in combination with AZA for the maintenance of remission in GPA and MPA . Additional B cell survival factors are also under investigation in AAV. Blisibimod, a fusion protein that binds both soluble and membrane-bound BAFF, is currently being considered for a phase II trial for remission induction in non-severe AAV patients receiving concomitant methotrexate.35. An open-label study evaluating abatacept, a fusion protein that blocks the co-stimulatory signal needed for T cell activation, showed disease improvement in 90% (18/20) and steroid discontinuation in 73% (11/15) of patients with non-severe AAV. However, 30% (6/20) had to terminate the study owing to increases in disease activity36, portending the likelihood that T cell activation alone is unlikely to be the critical pathway for disease control. A multicenter, phase III, double-blind, placebo-controlled trial further evaluating the use of abatacept in relapsing non-severe AAV is ongoing .Although B cell dysregulation has gained center stage in the treatment of AAV, abnormal circulating and lesional T cell activation may also play a role in pathogenesis37. Two open-label studies have noted disease improvement among 70–95% of patients with refractory GPA39. Follow-up data have also demonstrated continued efficacy and minimal toxicity with up to 5 years of treatment41. A clinical trial comparing gusperimus to conventional therapy is currently being designed.Gusperimus, a synthetic immunosuppressive drug derived from the antitumor antibiotic spergualin, also modulates lymphocyte function in addition to other poorly understood pathways and is a candidate drug for use in AAV42. C5a acts as a priming agent for neutrophils, resulting in increased surface expression of the auto-antigens proteinase 3 (PR3) and myeloperoxidase (MPO). These auto-antigens interact with ANCA and subsequently stimulate the release of factors that activate the alternative complement pathway, further perpetuating cleavage of C5 and increases in C5a levels, leading to an amplification loop of ANCA-mediated neutrophil activation43. In animal models, both C5a knockout and blockade of the neutrophil C5a receptor have been shown to be protective against ANCA-induced glomerulonephritis44.Complement activation, particularly component C5a, has also been recently implicated in the pathogenesis of AAV. C5a is a potent inflammatory mediator as well as a strong chemoattractant and neutrophil activatorAn orally administered inhibitor of C5a receptor (CCX168) has recently completed phase II investigation in Europe . The CLEAR study evaluated three arms . All pat45. Furthermore, higher serum IL-6 levels appear to be associated with patients with frequent relapse and more severe organ damage45. IL-6 blockade with tocilizumab has shown promise in limited case reports46 and requires further evaluation. Similar to IL-6, increased serum levels of IL-17 and IL-23 have been observed in patients with AAV compared to healthy controls47. Additionally, AAV patients with higher IL-23 levels had more active disease and higher ANCA titers47. Both cytokines are associated with T helper 17 (Th17) cells, a T cell subset critical in mediating autoimmune disease. Specifically, IL-23 enhances the differentiation of T cells towards the Th17 subset and further assists in maintaining the production of IL-17, which itself is a proinflammatory cytokine with pleiotropic action. Ustekinumab (anti-IL-12/23) and secukinumab (anti-IL-17A) are both currently commercially available for the treatment of plaque psoriasis. These targeted anti-inflammatory cytokine therapies have yet to be trialed in AAV, but plans for future research are being considered.Finally, evidence is accumulating that inflammatory cytokines may play a role in the pathogenesis and activity of AAV. Interleukin (IL)-6 levels are increased in both serum and histopathologic samples from patients with active AAVThe success of RTX in AAV is unequivocal, and its rapid utilization in these conditions highlights the four-decade-long unmet need for reliable alternatives to CYC. Induction with RTX can be considered a first-line option for both treatment-naïve and relapsing patients, particularly in young patients wanting to preserve fertility and for patients with a history, or at increased risk, of malignancy. The benefit of PLEX in induction management is still unclear but is considered reasonable in patients presenting with severe renal dysfunction. Although prospective trial evidence is limited, it is clear that maintenance treatment is needed following RTX induction in the majority of patients. In the next 5 years, the results from ongoing clinical trials will provide guidance and clarity to several clinical questions that have arisen in the post-RTX era.Paralleling the advances made in rheumatoid arthritis treatment over the past two decades, the treatment of vasculitis in general, and AAV in particular, has now entered the increasingly expansive arena of targeted therapeutics. While phenotypically similar, the immune pathway targets at the individual level in immune-mediated diseases are extraordinarily complex and likely the reason for the variability in clinical outcome among large therapeutic trials. Significant advances have been made in understanding AAV at a population level, and this will ultimately lead to better understanding at the individual level to allow tailoring of treatments and a more personalized approach to management. In addition to generating novel therapeutics to newly identified pathway targets, the next decade will also likely bring combined therapeutic approaches where biologic agents are trialed in combination or succession.