Clinical trial NCT03283566
Conditions: Chronic Pancreatitis Insulin Dependent Diabetes
Keywords: Pancreatectomy Autologous Islet Transplantation Pancreatitis Diabetes Hydroxychloroquine
Sponsor: The Cleveland Clinic
Start date: 2017-10-03; Completion date: 2020-05-31
Enrollment: 9 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This will be a pilot, 12-month phase II, open label, randomized, two-arm, single-blinded, placebo-controlled, parallel clinical trial of individuals undergoing TPAIT (Total Pancreatectomy and Autologous Islet Transplantation) for treatment of chronic pancreatitis (CP). The two study arms consist of HCQ-treated (Hydroxychloroquine) and placebo-treated individuals. The purpose of this study is to investigate the effects of HCQ administration compared to placebo on islet cell function post-autologous transplantation.

Detailed description:
A compelling level of evidence exists on the effects of the innate immunity-driven inflammation on the decline of functional beta cell mass in the autologous transplant setting. The investigators hypothesize that HCQ administration during the peri-transplant period will preserve islet mass and improve islet cell function in TPAIT by reducing inflammation. The investigators specifically aim to demonstrate a higher stimulated C-peptide level as well as greater glucose control in response to mixed meal tolerance testing (MMTT) at 6 and 12 months following TPAIT in patients treated with HCQ compared to placebo. A better response in the HCQ arm suggests improved islet survival and metabolic performance, potentially facilitating higher rates of insulin independence.

HCQ administration:

Arm 1 (n=5): Subjects will receive a pre-transplant HCQ 200 mg daily dose 30 days prior TPAIT followed by HCQ use for an additional 3 months post-surgery.

Arm 2 (n=5) subjects will receive placebo treatment following the same schedule as in Arm 1.

Exploratory mechanistic studies:

All subjects will undergo a MMTT to assess islet cell function at 6 and 12 months following TPAIT (in addition to MMTT pre-surgery performed as standard of care, and whose results will be used for pre-randomization in this pilot). Baseline metabolic tests obtained too early after surgery may not be indicative of islet function, due to insulin supporting therapy administered for several weeks after transplantation. Also, compelling data indicate that stabilization of islet function may require up to 1 year to occur. Blood glucose and C-peptide serum levels will be measured in peripheral blood samples immediately prior and subsequent to MMTT. The research coordinator will contact the subjects at 3, 6 and 12 months for interview on the course of follow up and will assist in scheduling the 6 and 12-month appointments for MMTT.

Mitochondrial Function and Metabolic Outcomes in TPAIT:

Mitochondrial efficiency is important in the setting of TPAIT, where increase in metabolic demand and decrease in oxygenation have been established. The investigators will assess mitochondrial efficiency by measuring rates of mitochondrial respiration and glycolysis. These measures will be obtained on islets procured for donation and after islet isolation. Small amounts of digest left after islet isolation, that would normally be discarded, will be used for this portion of the study. The islets from the digest will be collected and will undergo extracellular efflux analysis through the Seahorse XF analyzer for mitochondrial function assessment. Commercially available normal human islet cells for experiments will be used as control. Controls will be compared simultaneously with islets isolated from study subjects.

Genome-wide Gene Expression in TPAIT Patients:

On the genomic level, several genetic pathways have been implicated in islet cell function and survival. The genetic profiles of islet cells from CP patients undergoing TPAIT have not been evaluated yet. The investigators aim to build an RNA-gene sequence database for islet cells of CP patients undergoing TPAIT, specifically targeting genes previously identified as key players in islet function. Small amounts of digest from the procedure used for isolating islets, and what remains in the circuit after the isolation process is complete, that would normally be discarded, will also be used for islet gene expression assessment.

Arms: arm 1: Administered pre-transplant through 3 months after surgery. arm 2: Placebo treatment following the same schedule as Arm 1 (i.e. Hydroxychloroquine).
Interventions: intervention 1: Hydroxychloroquine intervention 2: Placebo
Intervention descriptions: intervention 1: Subjects will receive a pre-transplant 200 mg daily dose of HCQ 30 days before TPAIT and will continue on the drug for 3 months after surgery. intervention 2: Subjects will receive a pre-transplant placebo 30 days before TPAIT and will continue on the placebo for 3 months after surgery.

Protocol documents:
Prot_SAP_000.pdf: 
1 
 
Study Protocol: Hydroxychloroquine and Metabolic 
Outcomes in Patients Undergoing Total Pancreatectomy and 
Autologous Islet Transplantation: A Clinical, Molecular, 
and Genomic Study 
 
Principle Investigators  
Betul A. Hatipoglu 
Co-Investigators 
Khawla F. Ali, Yu Kuei Lin, John P. Kirwan, Seung K. Kim, Rita 
Bottino, R. Matthew Walsh 
Protocol Authors 
Khawla F. Ali, Yu Kuei Lin, John P. Kirwan, Seung K. Kim, Rita 
Bottino, R. Matthew Walsh, Betul A. Hatipoglu 
NCT:  
NCT03283566    
IRB: 
17-912 
Date 
Nov 25th, 2016 
 
 
 
 
 
 
 
 
 
 
 


2 
 
 
 
1. Introduction: 
Total pancreatectomy (TP) has been used as the therapy of last resort in patients with chronic pancreatitis 
(CP). The procedure is often coupled with transplantation of islets of Langerhans recovered from the 
resected pancreas (autologous islet transplantation, AIT) intended to prevent or attenuate the effects of 
surgically-induced diabetes in select patients based on pre-surgical metabolic assessments [1-3]. AIT is 
not significantly affected by the stress of post-transplant cellular rejection that one encounters in 
allotransplantation. However, islet loss and altered beta cell functionality have been demonstrated to be 
inevitable [4-6]. A compelling level of evidence exists on the effects of the innate immunity-driven 
inflammation on the decline of functional beta cell mass in the autologous transplant setting [5]. 
Rationally-selected anti-inflammatory approaches for patients undergoing total pancreatectomy and 
autologous islet transplantation (TPAIT) may be a potential approach for the protection of islets from the 
initial inflammatory challenge resulting in preservation of beta cell function and leading to sustained 
long-term insulin independence. 
 
2. Background and Significance: 
An extensive amount of preclinical and clinical data support the viability of islet transplantation as 
therapy for treating insulin-dependent diabetes. Islet transplantation has become an increasingly 
acceptable approach to reverse diabetes and has been largely tested in the context of autologous as well as 
allogeneic donor-recipient pairs [2, 7-9]. The outcomes of AIT in diabetes remission differ across patients 
that undergo TPAIT. Recent literature has shown at a median follow-up of 28-months post transplantation 
that about a third of patients was insulin independent, one third was fully insulin-dependent, and one third 
showed some islet graft function but still required exogenous insulin [1, 2]. In the latter case, even a 
partial graft function confers benefit to patient’s health and quality-of-life. 
A critical factor in predicting successful islet transplantation which translates into insulin-independence, 
is the islet equivalent (IEQ) count [1, 2]. Recent data from the largest TPIAT cohort reported a 3-year 
insulin independence rates that range from 12% in those receiving <2,500 IEQ/kg to 72% in those 
receiving >5,000 IEQ/kg of body weight [2]. Despite compelling evidence of the latter, about a third of 
patients receiving more than double IEQ/kg remain insulin dependent post-TPAIT. More interestingly, a 
recent report on a subject receiving as few as 954 IEQ/kg achieved and maintained insulin independence 


3 
 
up to 4 years after transplantation [10]. Therefore, additional factors besides the IEQ count, contribute to 
and limit islet survival and function post-AIT.  
Islet cell survival and function, both short- and long-term are governed by the degree of cellular damage: 
both pre-existing from the underlying inflammation seen in chronic pancreatitis, and inflicted damage by 
the isolation and transplantation processes. Reports on beta cell mass among CP patients estimate 29% 
loss of the fractional beta cell area as a result of chronic inflammation and fibrosis, coupled with an 
additional 21% reduction in overall pancreatic mass resulting in more pronounced deficit in total islet 
mass [11]. As for inflicted damage, the islet isolation procedure is a key pre-transplant factor in islet mass 
loss [12, 13]. Harmful events also occur in the early post-AIT period and are induced by stressors such as 
hypoxia, hyperglycemia, and the infiltrating innate immune cell-derived cytokines and other pro-
inflammatory factors [5, 14, 15]. A pro-coagulatory and pro-inflammatory cascade is activated within 
minutes after islet infusion back into the host and actively accelerate the injury to transplanted islets [16]. 
The levels of inflammatory cytokines such as IL-1Ra, IL-6, IL-8 and IL-10 have been reported to peak 
within the first 6 hours of islet infusion and gradually subside to pre-transplant levels days from islet 
transplantation [5, 16].  
Stemming from experimental and clinical transplantation data, there is general agreement that providing 
anti-inflammatory cover during the early post-islet infusion period can protect the graft [15, 16]. 
Protection of the islet graft from the initial inflammatory challenge should prevent cell loss and preserve 
beta cell function leading to sustained long-term insulin independence and delay of complications 
associated with uncontrolled hyperglycemia [17, 18]. Recent studies have proposed the use of TNF-α 
blockers/inhibitors to short-circuit the innate inflammation known to be detrimental to beta cell function 
and survival [19-25]. Etanercept, a TNF-a blocker, has been incorporated into a number of 
immunosuppressive protocols for islet allotransplantation which have proven successful [19, 20, 23-26]. 
In the multicenter Clinical Islet Transplantation (CIT) study, the use of etanercept resulted in 
improvement in beta cell secretory capacity of more than 40% of normal and a three-fold gain in islet 
engraftment efficiency [20]. When these patients were evaluated at 1 year post-transplant, they remained 
free of insulin use with a trend towards further improvement in beta cell secretory capacity to more than 
50% of normal [20]. Even though one cannot conclude that etanercept alone was an important factor in 
improving the outcomes, its inclusion in the immunosuppressive regimens is believed to have made some 
contribution that could be relevant in preserving beta cell mass and function consequent to TNF-alpha 
blockade. This same mechanism of islet loss is also relevant in the autologous islet transplantation 
settings. 
Hydroxychloroquine (HCQ) is a quinolone anti-malarial agent, FDA-approved for the treatment of 
autoimmune diseases. HCQ is considered the first-line agent for the treatment of rheumatoid arthritis, 


4 
 
systemic lupus erythematosus and sjogren syndrome [27]. The immune effects of HCQ are modulated 
though several mechanisms, particularly through decrease in pro-inflammatory cytokine production such 
as IL-1 and IL-6 and reduction of proteolysis and antigen presentation. Compared to TNF-a blockers, 
HCQ is administered orally, is relatively inexpensive, and has excellent tolerability with minimal side 
effect profile.  
Previous studies have demonstrated that patients treated with HCQ have a lower incidence of diabetes 
[28-32]. Hypoglycemia and/or reduction in HbA1c levels have been encountered in HCQ-treated patients, 
both in those with or without diabetes [33-37]. In three randomized, controlled trials conducted with type 
2 DM patients, the use of HCQ resulted in reduced insulin need and improvement in HbA1c levels and 
beta cell function in all three trials [38-40]. A few mechanisms have been proposed regarding the effects 
of HCQ in improving glycemic profiles, most notably through the inhibition of inflammatory cytokines 
resulting in decreased beta cell loss as demonstrated in a rat model of pancreatic endocrine failure [41]. 
Another proposed mechanism is through improvement of insulin metabolism at the level of peripheral 
tissues [42].  
We hypothesize that HCQ administration during the peri-transplant period will preserve islet mass and 
improve beta cell function in TPAIT by attenuating inflammation. The aim of our proposed study is to 
demonstrate a higher stimulated C-peptide level as well as better glucose control in response to a mixed 
meal tolerance test (MMTT) at 6 and 12 months following TPAIT in patients treated with HCQ compared 
to controls. A better response in the HCQ arm would suggest improved islet survival and metabolic 
performance, potentially facilitating higher rates of insulin independence. 
We foresee that that the use of HCQ will help facilitate engraftment and preservation of the transplanted 
islet mass, both increasing the likelihood of graft survival initially and fostering long-term engraftment 
and improved outcomes in functional beta cell mass for patients who undergo TPAIT.  If the study goals 
are achieved, and more controlled clinical studies confirm a potential positive outcome, anti-inflammatory 
treatments may become part of the standard protocol in patients undergoing TPAIT, and long-term 
complications of surgical diabetes may be attenuated. It is also anticipated that if the proposed treatment 
demonstrates improved metabolic function, higher rates and longer duration of insulin independence, this 
may encourage patients suffering from CP and qualifying for TP, to undergo surgery at a time in which a 
significant islet mass is still retrievable.  
Data obtained from this pilot study will be used to inform a future phase I/II clinical trial to determine the 
optimum dosage and timing of this anti-inflammatory treatment to maximize improvement in beta cell 
function. In particular, the mechanisms of inflammation in the early phase after transplant may differ 


5 
 
from those associated with long-term islet loss, which we believe is an important variable to better 
understand.  
2.1 Innovation: 
2.1.1 Part I: Mitochondrial Function and Metabolic Outcomes in TPAIT 
From a molecular standpoint, mitochondrial function of beta cells is directly related to the ability of beta 
cells to secret insulin [43]. Physiologically, as the insulin release is regulated by cytoplasmic adenosine 
triphosphate (ATP)/adenosine diphosphate (ADP) ratio, a mitochondrial defect will lead to defect in 
insulin release. Mitochondrial efficiency is important for the survival of transplanted islets due to increase 
in metabolic demand [44], and decrease in oxygenation [45]. 
In patients with chronic pancreatitis, as there are several factors influencing the islet health and function, 
such as inflammation, fibrosis, limited blood flow, compartment-like effect and loss of innervation [46], 
the mitochondrial function is probably compromised by this pathological microenvironment. 
Extracellular flux analyzer is a method to assess mitochondrial respiration and glycolysis through the 
measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), 
respectively. With a microchamber system, the analyzer allows the measurement of the change in the 
concentration of dissolved oxygen and the free protons. As the analyzer is equipped with drug 
introducers, a physiological stress can be imposed to the cells to test their respiration capacity in such 
situations. A method to assess the human islet with this analyzer has been validated and used in research 
[47]. 
In this pilot study, we are investigating the degree mitochondrial dysfunction in islets of patients with 
chronic pancreatitis receiving TPIAT, reflected by measured OCR and whether the latter is associated 
with poorer metabolic outcome compared with control, normal islets. This information will further 
contribute to the knowledge of autoislet function, and will identify other factors that are related to 
metabolic outcome that can be targeted for islet therapy in the future.  
We hypothesize that mitochondrial dysfunction in islets of chronic pancreatitis patients is associated with 
a poor metabolic outcome after TP-IAT compared with control, normal islets. We also hypothesize that a 
trend towards less mitochondrial oxidative stress would be seen in patients receiving the anti-
inflammatory drug HCQ prior to transplantation. 
2.1.2 Part II: Genome-wide Gene Expression in TPAIT Patients 


6 
 
Previous investigations have identified pathways that regulate human beta-cell proliferation, function and 
maturation [48-51]. The genes regulating beta-cell proliferation and function may change dramatically 
with various factors, such as age [52]. Little is known about the gene signatures for islets from an 
inflammatory environment, particularly long-term CP patients, whose pancreata have been altered by the 
disease over time. The gene expression may influence the islet engraftment, survival, function and 
proliferation and impact the metabolic outcomes in islet transplantation.  
We hypothesize that the gene expression signature plays a significant role in the clinical outcomes in 
TPAIT in patients with CP. Specifically, this study would help to determine: 
1. Whether there is heterogeneity in the autoislet gene expression profiles prepared for TP-AIT 
2. How the heterogeneity in the gene expression profiles is related to clinical outcomes of AIT 
3. Study Design 
3.1 Methods 
This will be a pilot, 12-month phase II, open label, randomized, two-arm, single-blinded, placebo-
controlled, parallel clinical trial of individuals undergoing TPAIT. The two study arms consist of HCQ-
treated and placebo-treated individuals. To take into consideration an expected more favorable outcome in 
beta cell function when higher IEQ/Kg body weight is infused, MMTT and C-peptide measurement prior 
to surgery will be used to assign potential subjects to either a predicted lower or higher IEQ/Kg group. 
This “distinguo” will be useful in the analysis of the results. Subjects from each group will be evenly 
distributed and randomized into the two treatment arms.   
3.2 Sample 
All patients undergoing TPIAT for CP in the Cleveland Clinic Foundation (CCF) will be under the auto 
islet registry (Institutional Review Board (IRB) number 08-340) and are potential candidates for the 
study.  
A total of 10 patients scheduled for TPAIT at CCF, who meet the inclusion/exclusion criteria for TPAIT, 
confirmed by a multidisciplinary team, consisting of a hepatobiliary surgeon, gastroenterologist, 
endocrinologist, diabetes educator, social worker, nutrition therapist, psychologist, and pain management 
team will be recruited into the study. The inclusion/exclusion criteria for entry into the trial, which 
consists of two parts (criteria for TPAIT and then criteria to be enrolled into this pilot trial), are 
summarized in Table 1. Historically, gender representation has been equal in the cohort of patients 
undergoing TPAIT at CCF [1]. Although a trend to better outcome in females was observed even though 
the mechanisms are unknown [1], the most significant predictor of metabolic function in patients 


7 
 
undergoing TPAIT is the islet mass infused, which will be factored into this trial in two ways: 1) by pre-
testing and assigning patients to predicted islet yield groups as has been described previously and 2) by 
normalizing parameters of metabolic function per IEQ/Kg infused.  
Arm 1 (n=5) subjects will receive a pre-transplant 200 mg daily dose of HCQ 30 days before TPAIT and 
will continue on the drug for 3 months after surgery. This dosage will not exceed a daily dose of 5 
mg/kg/day using actual body weight due to potential risk of retinal toxicity; patients weighing less than 
40 kg will be excluded from study.  
Arm 2 (n=5) subjects will receive placebo treatment following the same schedule as in Arm 1.  
3.3 Feasibility of study 
Since the program of TPAIT at CCF has become fully operational, approximately 10-13 patients undergo 
TPAIT within a 12-month period. It is anticipated that the requisite number of subjects proposed for the 
study will be enrolled within the time period allowed for the study. 
3.4 Pace of enrollment 
We anticipate that 12 months will be needed to recruit study subjects and 1-year for trial, totaling a study 
period of up to 24 months. 
3.5 Benefits and risks 
Hydroxychloroquine is considered to be among the safest antirheumatic medications with the potential for 
any serious adverse event being extremely rare [53]. Gastrointestinal symptoms are the most common 
side effects, nausea being the most common. The relative safety of HCQ has resulted in no regular 
laboratory testing required to monitor for toxicities. Monitoring for adverse events is clinical. Retinopathy 
is the most notable complication of HCQ, though any serious ophthalmologic toxicity is rarely seen with 
regular follow up. The risk of retinopathy increases with higher dosages and longer durations of therapy. 
In a study of 2,361 patients on HCQ for at least 5 years, the prevalence of retinal toxicity was less than 2 
percent during the first 10 years of therapy in patients treated with 4.0 to 5.0 mg/kg of real body weight 
[54]. The current 2016 revised recommendations of the American Academy of Ophthalmology 
recommend that all patients undergo a baseline eye examination before or within a year of beginning the 
treatment with HCQ [55]. Given that that the likelihood of HCQ-induced retinopathy is rare with short-
term use (i.e. 4 months in this pilot study), no baseline ophthalmological evaluation will be mandated. 
However, patients with preexisting retinopathy will be disqualified from enrollment.  
 Table 1: Inclusion/Exclusion Criteria  
Inclusion criteria for TPAIT [1, 56] 
Exclusion criteria for TPAIT [1, 56] 


8 
 
Clinically confirmed diagnosis of CP 
Intractable abdominal pain 
History of failed operation(s) for CP 
Recurrent acute pancreatitis 
HbA1c <8.0% 
Sustained alcohol remission 
Chronic narcotic use 
Insulin dependence 
Pancreatic carcinoma 
Pancreatic mass suspicious for carcinoma 
Cirrhosis 
Portal hypertension 
Continued alcohol abuse 
 
Inclusion criteria for HCQ phase 
Exclusion criteria for HCQ phase 
Acceptance for TPAIT at CC 
Age range 18-65 
Manufacturer’s product label-contraindicated use of 
HCQ 
History of retinopathy 
Actual weight at enrollment <40 Kg 
Inclusion/exclusion criteria for TPAIT unmet 
 
4. Research Procedures  
4.1 Total pancreatectomy and islet isolation and transplantation 
Pylorus-preserving TP with duodenectomy will be performed in all cases usually associated with 
splenectomy to preserve perfusion to body and tail.  Warm ischemia time will be minimized. Finally, the 
pancreas and duodenum will be removed intact and flushed/drained via the splenic artery and vein 
respectively with UW solution [56]. Islet isolation and purification will be performed as described [56-
58]. The islet Isolation Laboratory located at the Institute of Cellular Therapeutics at Allegheny General 
Hospital (AGH) has performed more than 60 islet isolating procedures for the Cleveland Clinic and will 
perform all of the islet isolation for the study [58]. The number of islets will be quantified as islet 
equivalents (IEQ). The isolated islet through standard method will be transported back to the Cleveland 
Clinic and transplanted as previously described [56]. 
4.2 Randomization  
Fasting blood glucose (FPG) level <100mg/dl with stimulated C-peptide >4 ng/ml during MMTT pre-
surgery, will be the criteria to assign subjects to groups with predicted lower versus higher post-isolation 
islet yield. If either condition is not met, patients will be assigned to the lower yield group. With a 
representation from both groups, study subjects will be then randomized to HCQ treatment Arm 1 or 
placebo Arm 2 in a 1:1 allocation before the pancreatectomy procedure without any bias on age, gender, 
body weight, or IEQ isolated.   
4.3 Blinding  


9 
 
The study will be single-blinded. The PI, biostatistician who will analyze the data, consultants, and 
technicians running assays will be blinded to the study arm into which the subjects have been 
randomized. An alphanumeric identifier that refers to the study subject without any indicators of study 
arm allocation will be used. Only the surgeons and the research coordinator, but not the personnel 
conducting the metabolic studies, will be un-blinded as to the study arm randomization.  
4.4 HCQ administration 
Arm 1 (n=5): Subjects will receive a pre-transplant HCQ 200 mg daily dose 30 days prior TPAIT 
followed by HCQ use for an additional 3 months post-surgery. Oral medications are continued peri-
operatively. Patients are typically ordered for “NPO except for medications” starting from post-operative 
day 1 which will not preclude the administration of HCQ in the peri-transplant period.  
Arm 2 (n=5) subjects will receive placebo treatment following the same schedule as in Arm 1.  
4.5 Exploratory mechanistic studies 
All subjects will undergo a MMTT to assess islet cell function at 6 and 12 months following TPAIT (in 
addition to MMTT pre-surgery performed as standard of care, and whose results will be used for pre-
randomization in this pilot). Baseline metabolic tests obtained too early after surgery may not be 
indicative of islet function, due to insulin supporting therapy administered for several weeks after 
transplantation. Also, compelling data indicate that stabilization of islet function may require up to 1 year 
to occur.  Blood glucose and C-peptide serum levels will be measured in peripheral blood samples 
immediately prior and subsequent to MMTT. Laboratory tests will be measured in CCF hospital 
laboratory. 
The research coordinator will contact the subjects at 3, 6 and 12 months for interview on the course of 
follow up and will assist in scheduling the 6 and 12-month appointments for MMTT.     
4.6 Innovation: 
4.6.1 Part I: Mitochondrial Function and Metabolic Outcomes in TPAIT 
Small amounts of digest left after islet isolation, that would normally be discarded, will be used for this 
study. The islets from the digest will be collected and will undergo extracellular efflux analysis through 
the Seahorse XF analyzer located in the Kirwan Laboratory of the Cleveland Clinic Lerner Research 
Institute for mitochondrial function assessment. Commercially available normal human islet cells for 
experiments will be used as control. Controls will be shipped within 1 week of planned TPAIT date and 
will be compared simultaneously with islets isolated from study subjects. A protocol developed by 
Seahorse Bioscience in collaboration with the Mitochondrial Advancing Research through Collaborations 


10 
 
at Boston University School of Medicine will be used for the Seahorse XF analyzer islet analysis [59].The 
OCR of isolated islets will be analyzed with metabolic outcomes from post-transplantation follow-up.  
4.6.2 Part II: Genome-wide Gene Expression in TPAIT Patients 
Small amounts of digest from the procedure used for isolating islets, and what remains in the circuit after 
the isolation process is complete, that would normally be discarded, will also be used for islet gene 
expression assessment. These collected islets will be shipped to Seung Kim Laboratory at Stanford 
University School of Medicine for gene expression assessment.  
For the flow cytometry, RNA isolation and RNA-sequence library preparation, islets or acinar tissues will 
be dispersed into single cells by enzymatic digestion and incubated. Cells will be then stained with 
conjugated primary antibodies and sorted, followed by cytometry data collection. The cells then will be 
lysed, and total RNA will be isolated. RNA quality and quantity will be measured, and RNA-Sequence 
libraries will be built. Sequencing RNA reads will be aligned with default parameters. Transcript counts 
will be obtained using a collapsed Ref-Seq reference transcriptome list. 
4.7 Potential problems and alternative strategies: 
The TPAIT procedure carries with it a risk of bacterial contamination from the pancreatic organ itself 
along with islet infusion, however CCF has one of lowest incidences (38%) than reported [60, 61]. 
Patients undergoing TPAIT are fully immunocompetent, and will be receiving antibiotics treatment as 
part of the post-operative protocols. Routine microbiological analysis will allow germ identification and 
prompt target therapy, if required.  Patients will remain in the hospital for post-operative care (median 10 
days) [1], housed in ICU after surgery. Hospitalization will be followed by outpatient daily visits for 
additional 5-7 days, which will allow for control of any possible event associated with the administration 
of HCQ. Blood glucose levels will be monitored and eventually corrected if potential hypoglycemic 
events associated to HCQ use should occur. Post-operatively, patients will be under continuous glucose 
monitoring and insulin drips and on an IV line, which will allow for a quick response to changes in 
glycemic levels.   
Metabolic test such as arginine stimulation [62] would have been more informative than the MMTT, al-
lowing for better control of variables such as glucagon responses. If the data support the hypothesis, in the 
future, a larger and more controlled phase I/II study could consider the more cumbersome, but better 
informative arginine stimulation test.   
Dr. Hatipoglu and her clinical staff will monitor the subjects for HCQ-emergent adverse events prior to 
discharge and the research coordinator will follow-up by phone contact throughout the duration of the 
trial. 


11 
 
Table 2: Study Timeline  
 
T= -1 months 
T= 0 (TPAIT 
day) 
T = +3 months 
T = +6 months 
T = +12 months 
HCQ 
group 
(n=5) 
- MMTT #1 
- Start of HCQ 
- Seahorse XF 
analyzer for 
isolated islets 
(Kirwan Lab) 
- Genomic 
analysis (Kim 
Lab) 
- Discontinue 
HCQ 
 
- MMTT #2 
 
- MMTT #3 
 
Placebo 
group 
(n=5) 
- MMTT #1 
- Start of 
placebo 
- Seahorse XF 
analyzer for 
isolated islets 
(Kirwan Lab) 
- Genomic 
analysis (Kim 
Lab) 
- Discontinue 
placebo 
- MMTT #2 
- MMTT #3 
 
Figure 1: Study Timeline 
 
5. Data Analysis 
5.1 Study endpoints 
5.1.1 Primary endpoints:  
- 
Quotient of MMTT stimulated (90min) C-peptide/glucose/IEQ/Kg at 6 and 12 months after 
TPAIT. 
Rationale for the proposed endpoint: MMTT is a common metabolic test used to assess the insulin and C-
peptide response to a mixed meal challenge. It triggers physiologic pathways to induce maximal insulin 
and C-peptide secretion. It is simple to conduct, and the outcome is widely believed to represent beta cell 
insulin secretion capacity. In recipients of islet allografts, the ratio of MMTT C-peptide/glucose at 90min 
correlates with the islet mass infused and it proves a valuable indicator of islet function. Whereas IVGTT 
  
  
HCQ 
PLACEBO 
T= -1 m 
T= 0 
M
M
T
T 
M
M
T
T 
M
M
T
T 
T
P
A
I
T 
T= +6 m 
T= +12 m 
Arm 1  
 
n=5 
Arm 2 
 
 n=5 
T= +3 m 


12 
 
is also broadly utilized to assess islet function, our preference has been given to a test that shows 
correlation with predicted islet yield (used in a pre-randomization step) and infused islet mass.  
5.1.2 Secondary endpoints:  
- 
Stimulated C-peptide area under the curve (AUC) in response to MMTT at 6 and 12 months after 
TPAIT. 
- 
Stimulated glucose AUC in response to MMTT at 6 and 12 months after TPAIT. 
- 
Stimulated C-peptide and glucose levels in response to MMTT at 6 and 12 month after TPAIT. 
5.1.3 Innovative endpoints: 
5.1.3.1 Part I: Mitochondrial Function and Metabolic Outcomes in TPAIT 
Oxygen consumption rate (OCP) and extracellular acidification rate (ECAR) of isolated islets’ 
mitochondria in subjects receiving HCQ and placebo.  
5.1.3.2 Part II: Genome-wide Gene Expression in TPAIT Patients 
Differential gene expression calls will be done on isolated islets. These will be compared to 
appropriate control gene expression sets [52]. 
5.2 Study outcomes: 
5.2.1 Primary outcomes: 
- 
Increased quotient of stimulated C-peptide/glucose level normalized for IEQ/Kg infused in 
response to MMTT at 12 months in the HCQ-treated compared to placebo arm.   
5.2.2 Secondary outcomes: 
- 
Higher C-peptide AUC in response to MMTT 12 months in the HCQ-treated compared to 
placebo arm.  
- 
Higher ratio of C-peptide AUC to glucose AUC in response to MMTT at 12 months in the HCQ-
treated compared to placebo arm.  
5.2.3 Innovative outcomes: 
- 
Lower rates of oxygen consumption within isolated islets from CP patients compared to 
commercial controls 
- 
Higher rates of oxygen consumption within isolated islets of HCQ-treated subjects compared to 
placebo.  
- 
Full RNA sequence library for isolated islets from CP patients  


13 
 
5.3 Data collection: 
The clinical data will be collected under auto islet registry stored in REDCap. All data in the Islet 
Isolation Laboratory at the Institute of Cellular Therapeutics, Allegheny Health Network and Kirwan 
Laboratory of the Cleveland Clinic Lerner Research Institute will be de-identified and collected into 
Microsoft Excel as medium, then transferred to REDCap by the investigators. All investigators will have 
received proper HIPPA training.  
5.4 Data access and confidentiality:  
All data will be collected either directly into REDCap or indirectly through Microsoft Excel on CCF 
Desktop computer as medium. The data will contain no personal health identifier (PHI) when it is being 
shared with investigators outside of the CCF.  A study ID will be assigned to each subject. The de-
identified clinical data will be shared for the purpose of manuscript construction under the standard user 
data agreement from the CCF IRB Committee. All investigators will have received proper HIPPA 
training.  
5.5 Statistical Analysis 
Measurement of serum C-peptide levels during MMTT and, specifically, the quotient of C-peptide 
(CP)/glucose (G) (ng/ml x100/mg/dl) at 90min during the test, have shown to be indicators of islet 
function and islet mass after allogeneic islet transplantation. Increments in the quotient according to islet 
mass (IEQ)/kg have also been identified. We expect this quotient to be applicable to the TPAIT setting in 
line with several studies showing that MMTT is a valuable assay to assess islet function after TPAIT. We 
applied the proposed increments to estimate levels of the CP/G (0.35±0.07) based on our historical 
TPAIT data, indicating on average IEQ/Kg infused of 4500±1000. Our hypothesis is that higher survival 
of beta cell mass due to the treatment will result in higher CP/G quotient in the HCQ-treated arm, 
compared to the placebo group, after normalization for IEQ/Kg infused. A one-sided, 2 sample t-test was 
used for sample size calculation with the assumption of equal variance between the two study arms. 
Assuming a 20-30% difference in outcome (CP/G quotient) between study arms, which is equivalent to 
0.08-0.12 CP/G difference and a 0.07 standard deviation, a sample size of 6 subjects (1:1 randomization) 
will detect a CP/G difference of at least 0.12, assuming 80% power, with a 0.05 significance level. We 
propose that a sample size of 10 subjects (5 for each arm) with the same assumptions but at 75% power, 
(Table 2) will be justified, given the expected feasibility of subject enrollment and the pilot nature of this 
trial.  
Table 3: Statistical Analysis 


14 
 
 
 
 
 
 
6. Adverse Events and Data Monitoring Committee (DMC) 
7. Consent 
Patients will be consented to participate in this study. Those whom agree to participate will be enrolled. A 
nurse coordinator whom performs the consent for TPIAT will perform the consenting process for this 
study. A copy of the consent form is attached to this IRB application.  
8. Budget 
An outline of our budget is found in table 4. Data collection and entry will be performed by the study’s 
investigators at no cost. MMTT testing at 6 and 12 months post-TPAIT is part of the standard-of-care 
testing in all centers performing TPAIT and thus no additional resources will be allocated for MMTT 
conduction. The cost of the RNA genomic analysis will be covered by the Seung Kim Laboratory at 
Stanford University School of Medicine at no additional cost to us.  
An application will be submitted for the Research Program Committee (RPC) Grant to cover half of the 
study expenses (around $11,000). The planned date of submission of the application is February 6th, 
2017. The remainder of the budget will be covered internally from EMI funding and the study’s PI.  
 
Table 4: Preliminary proposed budget  
 Item  
Units 
needed 
Estimated expense of items 
Total 
expenses 
HCQ (4 month supply) and Placebo (4 
month supply) 
600 HCQ 
pills, 600 
matching 
placebo 
- $1,200 for capsule 
manufacturing 
- $1,750 onetime pharmacy 
initiation fee 
- $60 monthly drug storage and 
inventory maintenance fee  
- $40 dispensing fee per 
occasion 
- $2,452 for generic HCQ 
(#600) 
 
$1,200 
$1,750 
$240 
$1,600 
$2,452 
 
 
Sample size (per arm) 
 
Δ=0.08 
Δ=0.1 
Δ=0.12 
SD=0.07 
n=9 
n=7 
n=5 
SD=0.08 
n=12 
n=8 
n=6 
SD=0.09 
n=15 
n=10 
n=7 


15 
 
Seahorse XF24 Islet Capture 
Microplates from Agilent Technologies 
2 
$369 
$738 
Islet controls from Lonza 
Pharma&Biotech 
11  
$945  
$10,395 
Genomic Analysis 
 
 $0 (to be covered by Kim’s Lab) $0 
MMTT testing 
 
$0 (to be covered by insurance 
providers) 
$0 
Expense of islet transport and care from 
CCF to AGH, from AGH to both 
Stanford and CCF 
 
$0 (to be covered by Kim’s Lab, 
AGH, and insurance providers) 
$0 
Statistical support 
 
~$2,000 
~$2,000 
Data collection and entry 
 
$0 (to be performed by study 
investigators)  
$0 
 
~$20,375 
 
9. Extramural Grant Application Plan 
An application will be submitted for the Research Program Committee (RPC) Grant to cover the 
remainder cost of the study. The planned date of submission of the application is February 6th, 2017.  
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