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Improving treatment and liver fibrosis outcomes with metformin in HCV-HIV co-infected and HCV mono-infected… Doyle, Mary-Anne; Singer, Joel; Lee, Terry; Muir, Miriam; Cooper, Curtis Jul 20, 2016

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STUDY PROTOCOL Open AccessImproving treatment and liver fibrosisoutcomes with metformin in HCV-HIV co-infected and HCV mono-infected patientswith insulin resistance: study protocol for arandomized controlled trialMary-Anne Doyle1,3,4*, Joel Singer4, Terry Lee4, Miriam Muir3 and Curtis Cooper2,3,4AbstractBackground: Approximately 180 million people worldwide, (3 % of the world’s population) are infected with hepatitisC (HCV). Insulin resistance (IR) and type 2 diabetes (T2DM) are common extrahepatic manifestations of chronic HCVinfection and associated with poor treatment and liver-related outcomes. The presence of these metabolic complicationshave been associated with poor response to interferon-based HCV antiviral therapy and increased risk of liver-relatedoutcomes. Metformin, an insulin sensitizer is known to improve HCV treatment response and has been associated with areduced risk of developing hepatocellular carcinoma (HCC). This study will evaluate the effect of metformin on preventingprogression or promoting regression of liver fibrosis, rate of virologic cure (SVR) and other metabolic measures in HCV-HIVco-infected and HCV mono-infected study participants who have IR and are planning on initiating HCV treatment.Methods: This study is a prospective 48-week single-centre, randomized, open-label, controlled trial of HIV-HCVco-infected and HCV mono-infected patients with IR (HOMA-IR ≥ 2.0) who are planning to initiate HCV antiviraltherapy. Sixty participants will be recruited from The Ottawa Hospital Viral Hepatitis Clinic. Participants will berandomized in a 1:1 ratio to either arm 1, metformin 2 g (1 g twice daily) plus lifestyle, or to arm 2, lifestyle alone.The primary outcome will be the change in FibroScan® score (kPa) from baseline to week 12 (start of HCV treatment),the end of HCV treatment (week 24) and 24 weeks post HCV treatment (week 48). Secondary outcomes includechanges in liver fibrosis using AST to platelet ratio index, changes in glucose and lipid levels, anthropometric measures,changes in alpha-fetoprotein levels, patient acceptability, and changes in dietary and physical activity parameters.Discussion: This pilot study will be the first to evaluate the role of metformin on liver fibrosis in HCV-HIV co-infectedand HCV mono-infected patients with IR receiving DAA HCV treatment. If metformin is effective in reducing liverfibrosis in this patient population, this will represent a well-tolerated, easy-to-administer, inexpensive therapy that willprotect against negative HCV outcomes. This study will also be an opportunity to evaluate the impact of insulinresistance and hyperglycemia on viral clearance in HCV-infected patients treated with interferon-free regimens.Trial registration: ClinicalTrials.gov NCT02306070 version 4.0 (June 29, 2015)Keywords: HCV, HIV, Liver fibrosis, Insulin resistance, Insulin sensitizer, Metformin, HCV antiviral therapy* Correspondence: madoyle@toh.ca1Division of Endocrinology and Metabolism, Department of Medicine,University of Ottawa, Ottawa, ON, Canada3Ottawa Hospital Research Institute, Ottawa, ON, CanadaFull list of author information is available at the end of the article© 2016 Doyle et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Doyle et al. Trials  (2016) 17:331 DOI 10.1186/s13063-016-1454-6BackgroundApproximately 180 million people worldwide (3 % of theworld’s population) are infected with hepatitis C (HCV)[1]. Although HCV mainly affects the liver, the virus isalso associated with several extrahepatic manifestationsincluding hematologic, autoimmune, dermatologic, andendocrine disorders [2]. Insulin resistance (IR) and type2 diabetes (T2DM) are the most common endocrinedisorders in HCV-infected individuals. This associationwas first demonstrated in HCV-infected individuals withcirrhosis compared with individuals with cirrhosis fromother causes (prevalence of T2DM 50 % versus 9 %,respectively) [3]. HCV is believed to directly impact in-sulin signaling by interacting with specific proteins suchas serine/threonine kinases that subsequently inhibit insu-lin signaling molecules [4]. HCV is also believed to indir-ectly cause insulin resistance by inducing the productionof pro-inflammatory cytokines that impair insulin signal-ing pathways in uninfected tissues [5].The presence of IR and T2DM in HCV has been associ-ated with poor HCV antiviral treatment response [6–8],acceleration of liver fibrosis [9, 10], increased risk forhepatocellular carcinoma (HCC) [11], higher transplantcomplication rates [12], and possibly increased morbidityfrom cardiovascular and metabolic complications [13]. Atleast one observational study identified improved IR ingenotype 1 HCV-infected patients achieving viral clear-ance with HCV antiviral therapy [14].Hepatic fibrosis is a response to liver injury that arisesfrom the activation of hepatic stellate cells (HSCs) by in-flammatory cytokines. Once HSCs are activated, theyfurther release cytokines that promote inflammation,fibrosis, contraction and mitosis [15]. Insulin resistanceis a major determinant of liver fibrosis, however, themechanism by which IR promotes liver fibrosis is notwell established.In recent years, the treatment of HCV has evolved withthe development of direct-acting antiviral (DAA) therapies(protease inhibitors, NS5a inhibitors, and nucleotide andnon-nucleotide polymerase inhibitors). Compared to inter-feron and ribavarin therapies alone, these new treatmentsare associated with significantly improved sustainedvirological response (SVR) rates, shorter treatment dur-ation and more favourable side effect profile.The effect of DAA HCV treatments on insulin resistanceand long-term risk of type 2 diabetes has yet to be clearlyestablished. A randomized controlled trial of HCV mono-infected study participants receiving 14 days of mono-therapy with the protease inhibitor danoprevir, foundthat serum HCV RNA and homeostatic model assess-ment of insulin resistance (HOMA-IR) correlated sig-nificantly (Spearman rho = 0.379, p < 0.0001) [16]. Atthe end of 14 days of danoprevir monotherapy themean decrease in HCV RNA was 2.2 ± 1.3 log10 IU/ml(p < 0.0001) in patients who received the active drug (n =40), which correlated with a decrease in mean HOMA-IRscore by 1.6 ± 1.1 (p < 0.0001). In contrast, HCV-RNA andHOMA-IR remained unchanged in placebo recipients.The role of insulin resistance and hyperglycemia as pre-dictors of SVR with DAA HCV treatment has not beenclearly established. There are also no studies to date thathave examined the role insulin resistance or hypergly-cemia play in promoting progression or preventing regres-sion of liver fibrosis in patients that have achieved SVRwith HCV antiviral therapy.In high-risk populations, metformin, an insulin sensitizer,has been shown to delay or prevent the onset of T2DM[17]. While it is unknown whether this specific benefit ex-tends to those infected with HCV, metformin does improveHCV treatment and liver-related outcomes. At least onestudy showed treatment with metformin resulted in higherSVR rates when compared with the control group (59.2 %vs. 38.8 %, chi-square 4.083, p = 0.043) [18]. HCV-infectedpatients with cirrhosis and T2DM who were taking metfor-min had a decreased risk of developing HCC [19, 20].At the cellular level, metformin is highly concentratedin the liver and improves IR through the activation ofAMP-activated protein kinase (AMPK) which decreasesgluconeogenesis in the liver and increases glucose uptake inthe skeletal muscle [21]. In vitro studies have demonstratedthat pharmacologic activation of AMPK with metforminmay have antifibrotic effects by inhibiting the transforminggrowth factor beta (TGF-β1)-induced fibrogenic propertyof HSCs via transcriptional coactivator p300 [22]. TGF-β1is the most characterized of the fibrotic cytokines and hasbeen found to be increased in HCV-HIV co-infectedpatients [23]. It has also been proposed that the anticancereffects of metformin in HCV-infected patients may also bevia AMPK and the inhibition of mammalian target of rapa-mycin (mTOR), which in turn regulates cell cycle progres-sion and cell growth [24]. There are no studies to date thathave looked at the effect of metformin in preventing pro-gression of liver fibrosis or accelerating regression fibrosisin patients treated with HCV antiviral therapy.Alpha-fetoprotein (AFP) is an oncofetal protein associ-ated with hepatic malignancies and liver regeneration[25, 26]. HCV core protein, inflammation, necrosis andhepatocellular injury have all been suggested as causes forelevated AFP levels in chronic HCV infection [25–28]. Al-though type 2 diabetes and insulin resistance have beenidentified as risk factors for the progression of liver fibrosisand development of hepatocellular carcinoma the mechan-ism by which this occurs is not clear [9, 11, 29, 30]. The re-lationship between AFP and insulin resistance was recentlyexamined in a retrospective analysis of 300 HCV-infectedpatients [31]. This study demonstrated that whole-body in-sulin resistance and hepatic fibrosis correlated directly withelevated levels of AFP lifestyle modification over a 3-monthDoyle et al. Trials  (2016) 17:331 Page 2 of 10period correlated with improved insulin resistance and a re-duction in AFP levels. This study draws attention to theneed for further prospective studies to understand the rela-tionship between insulin resistance, AFP, hepatic fibrosisand hepatocarcinogenesis. No studies have examined theeffect of metformin, an insulin sensitizer, on AFP levels.While SVR is associated with improved liver out-comes, the rate of liver fibrosis regression with SVR isvariable and predictors of regression are not well estab-lished [32]. In addition, achieving SVR in patients withcirrhosis does not necessarily prevent decompensationor eliminate the risk of HCC. A better understanding ofthe role insulin resistance and impaired glucose metab-olism have on these outcomes in HCV patients whoachieve SVR are needed.Identifying and targeting potentially modifiable risk fac-tors such as IR may be of significant importance in pre-venting progression of and promoting regression of liverfibrosis, reducing mortality and improving outcomes forHCV-HIV co-infected and HCV mono-infected patients.Given the above body of evidence and the establishedsafety of metformin in HCV and HIV, we developed aprotocol to evaluate the effect of metformin on prevent-ing progression or promoting regression of liver fibrosis,rate of virologic cure (SVR) and other metabolic mea-sures in HCV-HIV co-infected and HCV mono-infectedstudy participants who have IR and are planning on ini-tiating HCV treatment.MethodsHypothesisThe use of metformin will slow the progression and pro-mote regression of liver fibrosis in HCV-HIV co-infectedand HCV mono-infected patients with impaired insulinsensitivity (defined as HOMA-insulin resistance > 2.0).ObjectivesThe primary objective of this study will be to evaluatethe role of metformin in preventing progression andpromoting regression of liver fibrosis in HCV-HIV co-infected and HCV mono-infected participants with IRreceiving antiviral HCV treatment as assessed by transi-ent elastography (FibroScan®, Echosens, Paris, France).As secondary objectives we will evaluate the effectmetformin has on virologic response rates based on SVR(i.e. 12 weeks post HCV antiviral treatment and its effecton the progression and regression of liver fibrosis usingaspartate aminotransferase (AST)-to-platelet ratio index(APRI). We will additionally consider the effect of metfor-min and lifestyle modification have on metabolic parame-ters (fasting insulin, fasting glucose and fasting lipid levels),inflammatory markers [interleukin 6 (IL-6), interleukin8 (IL-8), tumor necrosis factor alpha (TNF-α), TGF-β,C-reactive protein (CRP], anthropometric measurements[weight, body mass index (BMI), waist circumference], AFPlevels, HCV viral status, human immunodeficiency virus(HIV) viral status and liver enzymes. Patient acceptability ofmetformin will be evaluated in arm 1 using a patient ac-ceptability questionnaire. All participants will receive life-style and dietary counselling at baseline and at 24-weekintervals. The effects of lifestyle modification will be evalu-ated using dietary and physical activity questionnaires.Study design and settingThis study is a prospective 48-week single-centre, random-ized, open-label, controlled trial of HIV-HCV co-infectedand HCV mono-infected patients with IR (HOMA-IR ≥2.0) who are planning to initiate HCV antiviral therapy.Sixty (60) participants meeting eligibility criteria willbe randomized in a 1:1 ratio using variable block sizes toeither arm 1, metformin 2 g [1 g twice a day (BID)] pluslifestyle, or to arm 2, lifestyle alone (the control arm).Randomization will be stratified according to HIV statusand according to stage of liver fibrosis (F0–2 vs. F3–4)and will be conducted utilizing a web-based randomizationsystem. The allocations will be generated by a statisticianunassociated with the study using the SAS statistical pro-gram (SAS Institute, Inc., Cary, NC, USA) and uploadedinto the randomization system. A log of all transactionsincluding date and time of randomization, stratum andtreatment allocation will be recorded. The randomizationsystem will be accessed by the study coordinator at thestudy site when they have identified consenting eligible par-ticipants ready to be randomized.Arm 1 (Metformin group) will receive metformin andlifestyle treatment during a 12-week period prior tostarting HCV therapy (week 0–12), during the 12-weektreatment phase (week 12–24) and 24 weeks post HCVtreatment (week 24–48). Participants who require >12 weeks of HCV treatment will be excluded. If initiationof HCV treatment is delayed beyond 12 weeks, participantswill continue on metformin during this time period and willreceive less metformin post HCV treatment. Arm 2 (Con-trol group) will receive lifestyle treatment alone during a12-week period prior to starting HCV therapy (week 0–12),during the 12-week treatment phase (week 12–24) and24 weeks post HCV treatment (week 24–48). A detailedstudy flow chart is depicted in Fig. 1.Selection of participantsParticipants will be recruited from The Ottawa HospitalViral Hepatitis Clinic. Study recruitment will begin inJuly 2016. All adult patients between the ages of 18 and79 years of age, who have provided informed consent,have documented history of chronic HCV RNA infectionwith evidence of fibrosis on FibroScan® > 8.0 kilopascals(kPa) or liver biopsy score > 2 (Batts-Ludwig System [33]within 2 years, insulin resistance as determined by aDoyle et al. Trials  (2016) 17:331 Page 3 of 10HOMA-IR of > 2.0 at screening and intend to start an8–12 week interferon (IFN)-free HCV antiviral therapy.If participants are HIV-infected and not on HIV antiviraltherapy, they will require a CD4 count of at least 200 tobe included in the study.Individuals will be excluded from the study if they are:pregnant, suspected to be pregnant, planning to becomepregnant or breastfeeding, have a chronic HBV infec-tion, an HbA1c > 8.0 %, are using immune-suppressingmedications, have an active malignancy, are currentlyor previously treated with metformin or other oral diabetesmedications or insulin, have a history of pre-existingdiabetes (type 1, type 2 or gestational diabetes), clinicalevidence of decompensated cirrhosis (ascites, esophagealvarices, hepatic encephalopathy, hepatocellular carcinoma),renal impairment [serum creatinine levels > = 136 umol/L(males), > = 124 umol/L (females)], history of congestiveheart failure requiring pharmacologic therapy, Wilson’sdisease, alpha-1 antitrypsin, hemochromatosis, biliary cir-rhosis, alcohol consumption > 50 g/day on average, partici-pation in other clinical investigations during the study, or ahistory of lactic acidosis irrespective of precipitating factors.Active illicit drug use (IDU) and stable health illnesswill not be exclusionary assuming it is unlikely to com-promise study adherence to protocol and study drug. InHIV-infected participants, HIV antiretroviral use andsuppressed HIV viral load will not be required forparticipation.Informed consent and patient confidentialityAll participants will be given detailed oral and written infor-mation about the trial. Consent forms describing in detailRandomization: 1:1 Stratified based on HIV status and stage of liver fibrosisArm 1: Metformin 2 g (1 g bid)* + Lifestyle modificationN=3012 weeks pre HCV treatment (weeks 0-12)*(or highest tolerated dose)Arm 2: Lifestyle modification (Control)N=3012 weeks pre HCV treatment (weeks 0-12)Primary Outcome:Progression/regression of liver fibrosis from baseline to start of HCV therapy (week 12), during treatment (week 12-24) and post HCV treatment (week 24 to48) as measured by FibroScan® Score (kPa)Eligible HCV-HIV co-infected and HCV mono-infected participants with IR who are initiating HCV antiviral therapy (N=60)Assessments at 0, 4, 8, 12, 16, 24, 36, 48 weeksArm 1: Metformin 2 g (1 g bid)* + Lifestyle modification+ HCV DAA Treatment (weeks 12-24)*(or highest tolerated dose)Arm 1: Metformin 2 g (1 g bid)* + Lifestyle modification24 weeks post treatment (weeks 24-48)*(or highest tolerated dose)Arm 2: Lifestyle modification (Control) + HCV DAA Treatment (weeks 12 to 24)Arm 2: Lifestyle modification (Control)N=3024 weeks post treatment (weeks 24-48)Fig. 1 Flow diagram of study processes. Sixty participants meeting eligibility criteria will be randomized in a 1:1 ratio using variable block sizes toeither arm 1, metformin 2 g (1 g BID) plus lifestyle (Metformin group), or to arm 2, lifestyle alone (Control group). Abbreviations: HCV hepatitis, HIVhuman immunodeficiency virus, IR insulin resistance, BID twice daily, DAA direct-acting antiviral therapy, kPa kilopascalsDoyle et al. Trials  (2016) 17:331 Page 4 of 10the study medication/intervention(s), study proceduresand risks will be given to each participant and writtendocumentation of informed consent is required priorto starting study medication/intervention. Participantsmay withdraw consent at any time during the courseof the trial.All participant-related information including clinicalrecords, laboratory specimens, evaluation forms, reports,will be kept strictly confidential. All records will be keptin a secure, locked location and only research staff willhave access to the records. Participants will be identifiedonly by means of a coded number specific to each par-ticipant and the site’s alphabetic letter code. All computer-ized databases will identify participants by numeric andalphabetic codes only, and will be password protected.Metformin dosingArm 1 participants will be provided with the followingrecommended dose adjustments and advised to increasethe dose accordingly: week 1: 500 mg once daily (QD);week 2: 500 mg BID; week 3: 500 mg three times daily(TID); week 4: 1 g BID or 500 mg QD. Participants willalso be encouraged to administer their study treatmentwith food whenever possible to minimize the gastric intol-erances. If participants are unable to tolerate an increasein dose, they will continue on the last dose tolerated.Metformin dosing will additionally be based on renalfunction and dosed as follows: if creatinine clearance (CrCl) 30–60 mL/min,metformin will be reduced to 50 % of the dose or500 mg BID if CrCl < 30 mL/min, metformin will be stopped.The potential side effects associated with metforminwill be reviewed with each participant.Lifestyle modification interventionParticipants in both treatment arms will be given counsel-ling on lifestyle and dietary modifications that may help toprevent progression of liver fibrosis. Participants will begiven instructions on recommended types and duration ofphysical activity as per Canadian Diabetes Associationpractice guidelines. They will be encouraged to aim for150 minutes of moderate to vigorous aerobic exerciseand two to three sessions of resistance training eachweek. They will additionally be provided with tips onhow to become more active.Participants will further be provided with recommenda-tions for dietary modifications and tips for healthy eatingas provided by the Canadian Diabetes Association practiceguidelines. Recommended limits for alcohol consumptionwill be reviewed. The educational sessions will be repeatedduring 6-month follow-up visits.Primary outcomeAs the primary outcome we will evaluate the change inFibroScan® score (kPa) from baseline to week 12 (start ofHCV treatment), the end of HCV treatment (week 24)and 24 weeks post HCV treatment (week 48) (Table 1).Secondary outcomesThe following will be evaluated as secondary outcomes:(i) virological response rates (SVR 12 weeks post HCV anti-viral therapy) will be compared between treatment groups;(ii) the change in APRI measurements from baseline tostart of HCV treatment, to the end of HCV treatment(week 24) and 24 weeks post HCV treatment (week 48) willbe compared between treatment groups; (iii) changes inglucose metabolism (HOMA-IR, fasting insulin, glucoselevels) from baseline to 4, 8, 12, 24, 36 and 48 weeks; (iv)changes in lipid levels [total cholesterol, low-density lipo-protein cholesterol (LDL-c), high-density lipoprotein chol-esterol (HDL-c), triglycerides] from baseline to 0, 12, and36 and 48 weeks; (v) changes in anthropometric measures(waist circumference, body weight and BMI) from baselineto 0, 4, 8, 12, 24, 36 and 48 weeks; (vi) changes in liver-related inflammatory markers by (IL-6, IL-8, TNF-α, TGF-β, C-reactive protein (CRP)) from baseline to 0, 4, 8, 12, 24and 36 weeks; (vii) changes in AFP levels from baseline to0, 12, 24, 36 and 48 weeks; (viii) participant acceptability tostudy medication dosing will be assessed (in arm 1 only) atweeks 8, 24, and 48 weeks by questionnaire. Changes inscores from week 8 to end of study will be evaluated; (ix)changes in diet and physical exercise parameters from base-line to 24 and 48 weeks using dietary and physical activityquestionnaires (Table 1).Safety assessmentsMetformin has been studied in both HCV and HIVmono-infected patients with no increase in adverseevents (AE) [18, 34]. Metformin does have the potentialrisk of lactic acidosis. A reduced dose (50 % of maximaldose) is used for patients with a CrCl < 60 mL/min. It isnot recommended for use in patients with a creatinineclearance of < 30 mL/min. Patients will be cautionedagainst excessive alcohol intake, either acute or chronicwhen taking metformin, since alcohol intake potentiatesthe effect of metformin on lactate metabolism [35, 36].A Data and Safety Monitoring Committee (DSMC)will be implemented to safeguard participant safety. Allmembers will be independent from the study investigators.This committee will conduct regularly scheduled meetingsto review the study progress including recruitment andconduct, and will identify any potential safety signals in thestudy population.At each contact with the participant, information re-garding adverse events (AEs) will be elicited byDoyle et al. Trials  (2016) 17:331 Page 5 of 10appropriate questioning and examinations. Any AE thatoccurs between the time that the participant is random-ized and the time that s/he departs the study at the endof the final follow-up visit (or at the time of earlywithdrawal of the participant from the study for any rea-son) is to be recorded. Participants will also be moni-tored during the 48-week study period for seriousadverse events (SAEs). If an SAE is ongoing at the time aTable 1 Schedule of events and data collectionVisit window +/- 2 daysWeeks -4 (Screen) 0 (Baseline) 4 8 12 16 24 36 48Informed consent XMedical history XInclusion/exclusion criteria X XDrug, alcohol and smoking history XPregnancy testa X XaMedication review X X X X X X X X XFibroScan®b X X X X X XPhysical examinationc and vital signs X Xc Xc Xc Xc Xc Xc Xc XcHepatitis B serologyd XHCV RNA viral loade Xe X X X X X X X XHCV genotyped XHIV viral loadd,e (as per SOC) Xe X X X X XCD4 counte (as per SOC) X5 X X X X XHematologyf and chemistryg X X X X X X X X XHbA1c X X X X X XFasting insulin and glucose X X X X X X X X XFasting lipidsh X X X X XInflammatory markers/research blood X X X X X X X XTSH X X X XAFP levels X X X X X2-hr OGTT X XAdverse events X X X X X X XAnthropometric measuresi Xi X X X X XCounselling on lifestyle modification X X XBlock food frequency questionnaire X X XInternational physical activity questionnaire X X XAudit-C and illicit drug use questionnaires X X X X XTSQM for patient acceptability (arm 1) X X XStudy drug accountability/dispensation (arm 1) X X X X X X XBirth control review X X X X X X X XHCV hepatitis C, HIV human immunodeficiency virus, HbA1c glycated hemoglobin, TSH thyroid-stimulating hormone, AFP alpha-fetoprotein, OGTT oral glucosetolerance test, TSQM Treatment Satisfaction Questionnaire for MedicationaIf randomization occurs > = 4 weeks from last pregnancy test, perform pregnancy test at baselinebFibroScan® can be done at screening if necessary for inclusion; baseline test can be done on different day than rest of visit if necessary (within 4 weeks ofbaseline). FibroScan® window can be +/− 7 days of the week 12 visit, +/− 21 days of week 24 and 36 visits, and up to 21 days before the week 48 visitcTargeted physical exam: vitals, cardiac, respiratory and abdominal exams; exam as pertinent to patient complaintsdTests to be done if not already in participant medical records [hepatitis B (HBV) needs to be within 6 months of screening visit; HIV needs to be within 1 monthof screening]eIf results available within 1 month of screening visit, no need to retestfHematology: complete blood count (CBC) with differential, platelets, international normalized ratio (INR)gChemistry: electrolytes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), lipase, albumin, direct bilirubin, lactate,creatinine, estimated glomerular filtration rate (eGFR)hLipids: total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceridesiAnthropometric measures: height, weight, body mass index (BMI), waist circumference, hip circumference, waist-to-hip ratio. At screening, only height andweight documentedDoyle et al. Trials  (2016) 17:331 Page 6 of 10participant discontinues/completes the trial, the SAE willbe followed until the investigator agrees that the event issatisfactorily resolved, becomes chronic, or that no furtherfollow-up is required (Table 1).Study treatment adherenceThe following criteria will be used to define complianceduring this study:1. Adherence with study treatment will be assessedbased on participant-provided information and tabletcount, i.e., drug accountability, during follow-up visits.Non-adherence will be considered for participantswho have an average daily intake of less than 80 % oftheir maximum tolerated dose.2. Attend baseline visit and all follow-up visits.3. Adherence with HIV antiretroviral therapy (ART)(if applicable) and HCV antiviral therapy will bedocumented as standard of care.Sample size calculationTo date there have been no studies that have investi-gated the benefits of improved IR in the HIV-HCV co-infected population or in HCV mono-infected patients.Studies that have investigated the effects of HCV anti-viral therapy on FibroScan® score reported a 32 % reduc-tion in mean score (10.6 +/- 4.8 kPa) compared withbaseline in patients that obtained a treatment response[37]. Based on these study results, a sample size of 24participants in each group would provide approximately80 % power to detect a 4 kPa difference between the twotreatment arms assuming a similar standard deviation.We anticipate a 20 % loss to follow-up, so an additionalsix participants will be randomized per group to accountfor loss to follow-up and to allow secondary outcomeanalyses. Using regression of the final measurement onthe initial measurement will likely improve the powerrelative to using simple difference scores.All analyses will be based on intention-to-treat basisand per protocol analysis. Intention-to-treat popula-tion will be defined as the set of all patientsrandomized.Statistical analysisAs the primary outcome of this study, the influence ofmetformin on preventing and promoting regression ofliver fibrosis in HCV antiviral treatment recipients willbe assessed by transient elastography. The change intransient elastography scores (kPa) at week 48 (24 weekspost HCV therapy) between groups will be assessedusing linear regression adjusted for baseline score. Wewill also compare the trend on fibrosis progression overthe course of the study (12, 24, 36 and 48 weeks) betweengroups using mixed-effects regression analysis. All analyseswill be based on intention-to-treat basis. A sensitivity ana-lysis of the change at 48 weeks will be conducted usingmultiple imputations to include those participants who donot have 48-week follow-up scores.The same set of analyses based on a per protocol basiswill also be performed. For the analysis, which examinesthe outcome longitudinally, participants who terminatedthe assigned treatment prematurely will be censored atthe time that they went off treatment.Secondary outcome measures include: virological re-sponse rates, changes from baseline to week 48 in indirectmeasures of liver fibrosis (APRI), inflammatory markers,metabolic measures, anthropometric measurements, HCVand HIV viral loads, AFP levels and liver function. Linearregression adjusted for baseline score will be used to com-pare the outcome at week 48 between groups. Mixed-effects regression analysis will also be used to compare thetrend over time between groups.To account for potential confounding factors (i.e., HCV/HIV viral status, alcohol consumption, IR, BMI) associatedwith changes in liver fibrosis, a sensitivity analysis basedon multivariate regression analysis will be performed.Patient acceptability of daily long-term study treatmentuse will be evaluated (in arm 1 only) using the TreatmentSatisfaction Questionnaire for Medication. Patient accept-ability will be assessed based on change in median scorefrom week 8 (first administration of questionnaire) toweek 24 and week 48. Descriptive statistics will be used tosummarize the data.Baseline characteristics will be expressed as mean andstandard deviation (SD) for continuous variables and per-centages for categorical variables. Baseline variables to beconsidered include: age, gender, ethnicity, HCV genotypeand viral load, HIV viral load and CD4 count (as per SOC),HbA1c, HOMA-IR, FibroScan® scores, smoking history, al-cohol consumption, and illicit drug use.Lifestyle modification will be evaluated through changesin physical activity (changes in median MET/week, percent-age change in participants classified as low, medium andhigh level of activity) and dietary changes (change in me-dian daily caloric intake, daily consumption of fat, protein,carbohydrates and micro/macro nutrients). Linear regres-sion adjusted for baseline score will be used to compare theoutcome at week 48 between groups. Mixed-effects regres-sion analysis will also be used to compare the trend overtime between groups.Data will be collected on liver-related complications(progression of liver fibrosis stage, HCC, transplantation)and mortality. We anticipate that the event rate will below given the small sample size and short duration offollow-up.Subgroup analyses will be conducted based on SVR tocompare the effect virological response influences liver fi-brosis and the benefit of metformin in reducing progressionDoyle et al. Trials  (2016) 17:331 Page 7 of 10of fibrosis. Subgroup analyses will also be conducted basedon HIV status to compare the influence of HIV infectionon progression of liver fibrosis and benefit of metformin inreducing the progression of fibrosis.Subgroup analyses based on metformin doses (fulldose versus not) in the metformin group and weight lossin patients in both treatment arms will also be con-ducted. These additional analyses will be useful in deter-mining if lower doses can achieve the same benefit andthe impact that potential weight loss associated with thismedication may have on steatosis and improving liveroutcomes respectively.Subgroup analyses will also be conducted based onend of study HOMA-IR score (<2 vs. > 2) to evaluate ifthe benefits of metformin are dependent or independentof the insulin-sensitizing effects of this treatment.As a sensitivity analysis, we will also consider usingmultiple imputation techniques to impute missing datafor the primary outcome at 48 weeks. Linear regressionadjusted for baseline score will then be used to comparethe groups.When half (n = 30) of the participants have completedtheir 48-week visit, an interim analysis of efficacy will beconducted. The same technique and analytic strategywill be used to analyze the primary end point, transientelastography score (kPa) at 48 weeks. An intention-to-treat approach will be taken, linear regression adjustingfor baseline score, and missing imputations to accountfor missing data will be used. The O’Brien-Fleming ap-proach using the Lan-DeMets alpha spending functionwill be used, so that the criterion for statistical signifi-cance at the first analysis will be 0.005 and the criterionat the final analysis will be 0.048 to maintain 80 % poweroverall. This analysis will be supplemented by a mixed-effects regression model examining the outcome at alltime points for all patients available, and adjusting forbaseline values.Early terminationThe criteria for premature discontinuation of furtherstudy medication for an individual participant in arm 1are as follows: treatment-related toxicity, requirementfor prohibited concomitant medications, clinical reasonsbelieved to be life-threatening by the physician, even ifnot addressed in the toxicity section of the protocol.The participant will continue to be followed with theparticipant’s permission if the study medication is prema-turely discontinued. If the participant discontinues studymedication between scheduled visits, the investigator mayrequest that the participant visit the clinic as soon aspossible for lab and safety assessments and to returnthe study medication for accountability purposes. Therewill be no changes to the follow-up visit schedule,except no study medication will be administered. If theparticipant chooses not to remain in the study, then theparticipant will be withdrawn from the study and par-ticipant will be asked to come in for an early termin-ation visit.The criteria for permanent withdrawal from the studyfor an individual participant are as follows:loss to follow-up, pregnancy or suspected pregnancy,HCV treatment required for >12 weeks, request of theparticipant to withdraw from the trial, any clinical AE,laboratory abnormality, intercurrent illness, other med-ical condition or situation occurs such that continuedparticipation in the study would not be in the best inter-est of the participant, the participant is judged by theinvestigator to be at significant risk of failing to complywith the provisions of the protocol as to cause harm toself or seriously interfere with the validity of the trial results,progression of diabetes requiring additional diabetes medi-cation (i.e. oral diabetes medications or insulin therapy).In the event that the participant is withdrawn from thestudy due to an AE, this must be recorded on the casereport form (CRF). The subject should be followed andtreated by the investigator until the abnormal parameteror symptom has resolved or stabilized.Trial registration and disseminationThis study has been registered with ClinicalTrials.gov(Identifier Number NCT02306070, https://clinicaltrials.gov/ct2/show/NCT02306070). A SPIRIT checklist is pro-vided as an additional file (see Additional file 1). Thefindings of this trial will be submitted to a peer-reviewedjournal and abstracts will be presented at relevant nationaland international conferences.DiscussionHCV antiviral therapy has evolved rapidly in recent yearsand access to these medications has improved. WhileSVR is associated with improved liver outcomes, the rateof liver fibrosis regression with SVR is variable and predic-tors of regression are not well established [32]. In addition,achieving SVR in patients with cirrhosis does not neces-sarily prevent decompensation or eliminate the risk ofHCC. A better understanding of the role insulin resistanceand impaired glucose metabolism have on these outcomesin HCV patients who achieve SVR are needed.Identifying and targeting potentially modifiable riskfactors such as IR and T2DM may be of significant im-portance in preventing progression of and promotingregression of liver fibrosis, reducing mortality and im-proving outcomes for HCV-HIV co-infected and HCVmono-infected patients.This pilot study will be the first to evaluate the role ofmetformin on liver fibrosis in HCV-HIV co-infected andHCV mono-infected patients with IR receiving DAAHCV treatment. If metformin is effective in reducing liverDoyle et al. Trials  (2016) 17:331 Page 8 of 10fibrosis in this patient population, this will represent a well-tolerated, easy-to-administer, inexpensive therapy that willprotect against negative HCV outcomes. This study willalso be an opportunity to evaluate the impact of insulin re-sistance and hyperglycemia on viral clearance in HCV-infected patients treated with interferon-free regimens. Inaddition, the study will further explore the relation-ship between HCV, insulin resistance and AFP levels.This knowledge will inform patient care, translate intoimproved therapeutic outcomes for liver and meta-bolic diseases, and guide further innovative researchin this area.Trial statusRecruitment for this study will begin in July 2016.Additional fileAdditional file 1: SPIRIT 2013 checklist: recommended items to addressin a clinical trial protocol and related documents. (DOC 121 kb)AbbreviationsAE, adverse event; AFP, alpha-fetoprotein; AMPK, AMP-activated protein kinase;APRI, AST to platelet ratio index; ART, antiretroviral therapy; BID, twice daily; BMI,body mass index; CBC, complete blood count; CrCl, creatinine clearance; CRF,case report form; CRP, C-reactive protein; CTN, Canadian HIV Trials Network;DAA, direct-acting antivirals; DSMC, Data and Safety Monitoring Committee;eGFR, estimated glomerular filtration rate; HBV, hepatitis B; HCC, hepatocellularcarcinoma; HCV, hepatitis C; HDL-c, high density lipoprotein cholesterol; HIV,human immunodeficiency virus; HOMA-IR, homeostatic model assessment ofinsulin resistance; HSC, hepatic stellate cells; ICH, International Conference onHarmonization; IDU, illicit drug use; IFN, interferon; IL-6, interleukin 6; IL-8,interleukin 8; INR, international normalized ratio; IR, insulin resistance; IRB/REB, institutional review board; kPa, kilopascal; LDL-c, low-density lipoproteincholesterol; mTOR, mammalian target of rapamycin; N, number; OGTT,oral glucose tolerance test; PI, principal investigator; QD, once daily; REB,research ethics board; SAE, serious adverse event; SOP, standard operatingprocedure; SVR, sustained virological response; T2DM, type 2 diabetes;TGF-β, transforming growth factor beta; TID, three times daily; TNF-α,tumour necrosis factor alpha; WHO, World Health OrganizationAcknowledgementsThe authors would like to acknowledge the support and contributions of Jo-AnnDonner, Dana Nohynek, Jayamarx Jayaraman from the Canadian HIV TrialsNetwork (CTN) in guiding protocol development.FundingThis project is funded through support from the Canadian HIV Trials Network(CTN). Mary-Anne Doyle also received support through Gilead/CTN Postdoc-toral Fellowship Award.Availability of data and materialsNot applicable.Authors’ contributionsMD made substantial contributions to the conception, design of the study,protocol development and writing the manuscript. MM participated in theprotocol development and study coordination. JS and TL contributed to thestudy development by providing advice and guidance on randomizationand statistical analysis. CC is the principal investigator (PI), responsible forrecruitment and trial coordination. He made substantial contributions tostudy design, protocol development, writing and revising of the manuscript.All authors will participate in the implementation and/or analysis of thestudy. All authors have read and approved this manuscript.Competing interestsThe authors declare that they have no competing interests.Consent for publicationNot applicable.Ethical approval and consent to participateThe conduct of this study will conform with the International Conference forHarmonization Good Clinical Practice (ICH-GCP) regulations and guidelines andthe current revision of the Declaration of Helsinki. Approval was obtained fromthe Ottawa Health Science Network Research Ethics Board (REB) and HealthCanada. Written consent will be obtained from all participants before inclusionin the study. Participants may withdraw consent at any time.Author details1Division of Endocrinology and Metabolism, Department of Medicine,University of Ottawa, Ottawa, ON, Canada. 2Division of Infectious Diseases,Department of Medicine, University of Ottawa, Ottawa, ON, Canada. 3OttawaHospital Research Institute, Ottawa, ON, Canada. 4CIHR Canadian HIV TrialsNetwork, Vancouver, BC, Canada.Received: 31 March 2016 Accepted: 17 June 2016References1. Global surveillance and control of hepatitis C. Report of a WHO Consultationorganized in collaboration with the Viral Hepatitis Prevention Board, Antwerp,Belgium. J Viral Hepat. 1999;6(1):35–47.2. Antonelli A, Ferri C, Ferrari SM, Colaci M, Sansonno D, Fallahi P. Endocrinemanifestations of hepatitis C virus infection. Nat Clin Pract EndocrinolMetab. 2009;5(1):26–34.3. Allison ME, Wreghitt T, Palmer CR, Alexander GJ. Evidence for a link betweenhepatitis C virus infection and diabetes in a cirrhotic population. J Hepatol.1994;21:1135–9.4. 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The efficacy and safety of insulin-sensitizing drugs inHIV-associated lipodystrophy syndrome: a meta-analysis of randomizedtrials. BMC Infect Dis. 2010;10:183.35. Salpeter S, Greyber E, Pasternak G, Salpeter E. Risk of fatal and nonfatal lacticacidosis with metformin use in type 2 diabetes. Cochrane Database SystRev. 2006;1:CD002967.36. Valeant Canada LP. PrGlycol® Product Monograph. Revision date December05, 2008.37. Ogawa E, Furusyo N, Toyoda K, Takeoka H, Maeda S, Hayashi J. The longitudinalquantitative assessment by transient elastography of chronic hepatitis C patientstreated with pegylated interferon alpha-2b and ribavirin. Antiviral Res.2009;83(2):127–34.•  We accept pre-submission inquiries •  Our selector tool helps you to find the most relevant journal•  We provide round the clock customer support •  Convenient online submission•  Thorough peer review•  Inclusion in PubMed and all major indexing services •  Maximum visibility for your researchSubmit your manuscript atwww.biomedcentral.com/submitSubmit your next manuscript to BioMed Central and we will help you at every step:Doyle et al. Trials  (2016) 17:331 Page 10 of 10


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