Mechanism and Effects of Manipulating Chloride Homeostasis in Acute Heart Failure
Phase: EARLY_PHASE1
Conditions: Heart Failure
Sponsor: Yale University

ClinicalTrials.gov ID: NCT03446651 
Oﬃcial Title: Mechanism and Eﬀects of ManipulaƟng Chloride Homeostasis in Acute Heart Failure   
Date: 9/28/2022 
Document: Protocol w SAP 

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Page 1 of 33
HRP-503B – BIOMEDICAL RESEARCH PROTOCOL
Protocol Title: Mechanism and Effects of Manipulating Chloride Homeostasis in Acute Heart Failure 
Principal Investigator: Jeffrey M. Testani, MD, MTR, Associate Professor of Medicine: Cardiology 
Version Date: Version #13: 09.15..2022
Clinicaltrials.gov Registration #: NCT03446651
Does this study have a Clinical Trials Agreement? No
Will this study have a billable service? No
A billable service is defined as any service rendered to a study subject that, if he/she was not on a study, 
would normally generate a bill from either Yale-New Haven Hospital (YNHH) or Yale Medical Group 
(YMG) to the patient or the patient’s insurer. The service may or may not be performed by the research 
staff on your study, but may be provided by professionals within either YNHH or YMG (examples 
include x-rays, MRIs, CT scans, specimens sent to central labs, or specimens sent to pathology). Notes: 
1. There is no distinction made whether the service is paid for by the subject or their insurance 
(Standard of Care) or by the study’s funding mechanism (Research Sponsored). 2. This generally 
includes new services or orders placed in EPIC for research subjects.
If answered, “yes”, this study will need to be set up in OnCore, Yale’s clinical research 
management system, for Epic to appropriately route research related charges. Please 
contact   oncore.support@yale.edu
Are there any procedures involved in this protocol that will be performed at YNHH or one of its 
affiliated entities? Yes
Does your YNHH privilege delineation currently include the specific procedure that you will 
perform? Yes
Will you be using any new equipment or equipment that you have not used in the past for this 
procedure? No
Will a novel approach using existing equipment be applied? No
IMPORTANT REMINDER ABOUT RESEARCH AT YNHH
Please note that if this protocol includes YNHH patients, including patients at the HRU, the Principal 
Investigator (PI) and any co-investigators who are physicians or mid-level practitioners (includes PAs, 
APRNs, psychologists and speech pathologists) who may have direct patient contact with patients on 
YNHH premises must have medical staff appointment and appropriate clinical privileges at YNHH. If you 
are uncertain whether the study personnel meet the criteria, please telephone the Physician Services 
Department at 203-688-2615. By submitting this protocol as a PI, you attest that you and any 
coinvestigator who may have patient contact has a medical staff appointment and 
appropriate clinical privileges at YNHH
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SECTION I: RESEARCH PLAN
1.0
Statement of Purpose: The overarching goal of this proposal is to develop a comprehensive 
understanding of the biology and therapeutic potential of sodium-free chloride supplementation. 
While sodium homeostasis has been the focus of substantial investigation, very little research has 
been devoted to understanding chloride homeostasis. Thus, this proposal is designed to obtain the 
full spectrum of information pertaining to chloride, such as novel areas with great interest by the 
scientific community (i.e. modulation of the WNK-kinase system and the use of exosomes), to more 
practical/basic questions (i.e. what happens to sodium chloride balance when a patient is 
challenged with chloride). The proposed multi-site inpatient study has been designed to serve as a 
real world efficacy study. With extensive biobanking and analysis of samples in the proposed 
setting, we will be able to deliver a great wealth of information on the biology and therapeutic 
potential of manipulating chloride homeostasis in heart failure.
We have seen that many heart failure therapies demonstrate measurable benefit in highly controlled 
environments, but lack effectiveness when studied in decompensated patients receiving standard 
decongestive therapies. As such, this study seeks to understand the effects of chloride 
supplementation on volume-overloaded patients concomitantly treated with IV diuretics.
2.0  Probable Duration of Project: Subject enrollment is planned to last 5 years, with a target rate of 
25 patients per year. Enrollment of up to 5 patients into the open-label arm will take place over the 
course of 12 months. Biomarker measurement will occur on a rolling basis staggered from specimen 
collection by approximately 6 months. Completion of enrollment, statistical analysis, and 
publication is planned to be complete within 6 years.
3.0  Scientific Background and Study Rationale
Scope of the problem. Nearly 7 million Americans are affected with heart failure (HF), and its 
prevalence is expected to increase to nearly 10 million by 2030.1 At age 40, an American’s lifetime 
risk of developing HF is 1 in 5.1 HF causes tremendous morbidity and mortality, with over 1 million 
hospitalizations in the U.S. each year and with HF listed on 1 in 9 death certificates respectively.1 
While HF is generally regarded as the inability of the heart to pump sufficient blood, on a 
population  level, congestion is the primary driver of symptoms leading to hospitalization.2-5
Our understanding of sodium homeostasis and its management in HF is incomplete: It 
has been recognized for some time that HF is a sodium avid state, and that retained sodium is the 
primary driver of volume overload.6 The simplistic paradigm has followed that salt should be avoided 
or minimized as much as possible. However, this fails to consider that the normal (appropriate) 
response of the kidney to a restriction in salt is compensatory neurohormonal activation and 
increased sodium avidity.6 While it is known that administering saline to an otherwise healthy person 
with hypovolemia (e.g., dehydration from diarrheal illness) will correct the neurohormonal 
activation and sodium avid state, it has been assumed that this physiology would not apply to HF. 
This is due, in part, to the fact that many HF patients with overt volume overload continue to display 
sodium avid physiology. Thus, it is commonly believed that the ability of salt to “quench” the sodium 
avid state in HF is defective and thus salt administration would only lead to worsening volume 
overload. However, it is important to stress that we have limited understanding of the actual 
sensor(s) for sodium avidity in HF beyond difficult to measure concepts such as effective arterial 
under-filling.7
Accumulating evidence suggests more salt can sometimes be beneficial in HF: A growing 
body of recent evidence challenges the assumption that salt is universally detrimental in HF. Dr. John 
Burnett’s group recently conducted an experiment where they varied the sodium intake from
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Page 3 of 33
0.25g/day to 5.8g/day in ~20kg dogs undergoing HF induction with rapid pacing.8 Not surprisingly, 
they found massive suppression of the renin angiotensin aldosterone system (RAAS) with high salt. 
However, what was remarkable was that filling pressures and body weight were not different 
between groups and ejection fraction and clinical HF symptoms were significantly better in the dogs 
on high salt diet.8 There have also been multiple observational and more than 10 randomized trials 
of modifying sodium intake in human HF.9-25 In these studies, there was substantial heterogeneity 
in the effects of high vs. low salt on biomarkers with some studies showing both improved volume 
status (natriuretic peptides) and RAAS activation, some showing improved RAAS activation, but at 
the expense of worsened volume status, and some showing worsening in all parameters. Several 
studies in acute decompensated HF have shown that high salt diet or administration of sodium 
chloride intravenously can improve diuresis, renal function, and outcomes.17, 26-28 In studies 
reporting hospitalization and mortality, the effect was also heterogeneous, but many studies 
actually demonstrated substantial benefit from a high salt. These conflicting data on the 
risk/benefit of salt  restriction motivated the American Heart Association/American College of 
Cardiology and European  Society of Cardiology guideline committees to remove the specific 
recommendation on the quantity of sodium intake for HF patients.29, 30
A critical advance would be disassociating the beneficial from harmful effects of salt: 
From the existing data on salt in HF we can conclude: 1) Increasing salt intake is a double-edged 
sword with competing benefit and harm. 2) Whether a harmful or beneficial effect will predominate 
varies greatly across study populations. 3) It is unclear what factors drive this variability. Although 
further research to determine the optimal salt intake in HF is clearly warranted, disassociating the 
beneficial sodium avidity “quenching” aspects from the volume retaining aspects of salt would be a 
more direct path.
It has been known for decades that the kidney “sees” salt through chloride rather 
than sodium. Sodium is unequivocally the primary driver of extracellular volume, and thus 
responsible for volume overload in HF.6 However, renal salt sensing appears primarily driven by the 
chloride anion rather than sodium cation. It has been known for decades that salt sensitive renal 
responses such as tubuloglomerular feedback and renin release are determined by chloride rather 
than sodium. For example, loading rats with various chloride-free salts, (NaHCO3, NaAcetate, 
NaNO3, KHCO3, CaGluconate) failed to suppress renin secretion. However, when rats were loaded 
with sodium chloride or various sodium free chloride salts (KCl, cholineCl, CaCl2) significant 
suppression of renin occurred.31 Nearly identical observations have been made for tubuloglomerular 
feedback.32, 33 Furthermore, with selective depletion of either chloride or sodium by dialysis with 
maintenance of euvolemia, only chloride depletion causes an increase in plasma renin activity 
(PRA).34
The WNK-Kinases are the molecular sensors for chloride and the “master regulators” 
of sodium-chloride transport: Recently, a family of serine-threonine kinases (With-No-Lysine 
(K), WNK) has been demonstrated to play a key role in the regulation of electrolyte homeostasis, the 
actions of the renin-angiotensin-aldosterone system, and the transporters upon which loop and 
thiazide diuretics work.35-38 Notably, chloride appears to bind directly to the catalytic site of at least 
two of the WNK kinases, inhibiting their auto-phosphorylation and activation.39-43 In the setting of 
low chloride, auto-phosphorylated WNK kinases regulate solute transport through phosphorylation 
of SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress–responsive  
kinase 1). These kinases then phosphorylate highly conserved residues of major sodium transporters 
in the kidney such as Na-K-2 chloride co-transporter (NKCC2) and electro-neutral sodium chloride 
cotransporter (NCC).40, 41, 44-57 NKCC2 is the target of loop diuretics and NCC the target of thiazide 
diuretics, transporters that mediate >25% of renal salt reabsorption (>140 grams of sodium a day in 
health).49, 58, 59 Thus, small aberrations in their regulation can have substantial influence on sodium 
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balance.60, 61 In addition to direct regulation by phosphorylation, the WNK system modulates protein 
abundance of its targets and constituents by multiple factors such as clathrin-mediated endocytosis, 
altering intracellular trafficking, and gene expression.53, 62-83
Chloride is important in human HF: The constellation of observations and biology described 
in the paragraphs above led us to hypothesize that chloride may play a significant role in human 
HF. Over a series of 7 peer-reviewed publications, we have demonstrated several key findings:84-90 1) 
Chloride and sodium levels are much less closely linked than one would assume (r2=0.19), and 
many patients have isolated low chloride or isolated low sodium (Figure 1A) 2) Low chloride, 
rather than low sodium, is strongly associated with diuretic responsiveness (Figure 1B). 3) 
Chloride is linked to renin levels (r=-0.46, p<0.001), but sodium has no incremental association 
(p=0.49). 4) Chloride depletion appeared to be an important contributory mechanism to 
hypochloremia in HF patients as the fractional excretion of chloride was higher [1.1% (0.6-3.4) vs. 
0.6 (0.3-1.5), p=0.04)] and urine osmolality was lower (301±54 vs. 366±139 osmol/L, p=0.002) in 
hypo vs. normochloremic patients. 5) Chloride levels were prognostically very important, whereas 
sodium had no independent association with mortality (Figure 1C) 6) The prognostic importance 
of low chloride appeared to be primarily restricted to patients with depletional, rather than 
dilutional, hypochloremia (p interaction <0.0001).
Investigation of new compounds that often take decades before they will be available 
at the bedside, and when available, are generally very expensive. Given that chloride is 
a macronutrient and available as the counter ion in a number of different FDA approved 
formulations, amino acids, and dietary supplements, additional research and deployment to 
patients can occur rapidly and with a fraction of the expense of new drug development.
Efficacy trials of decongestive therapies are conducted in hospitalized HF patient. In 
this setting, intercepting the patient before usual care is initiated is remarkably challenging as 
patients arrive via multiple portals of entry 24 hours a day. Furthermore, there is great 
heterogeneity in therapy with respect to type, intensity, and duration of treatment. These factors 
introduce substantial heterogeneity into the measurements and makes it difficult to keep 
participants on protocol.
A
B
C
D
Transitional Care Clinic (YTCC) is a 5-bed nurse 
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practitioner 
run/physician 
supervised 
outpatient 
diuretic 
treatment center where protocolized care is 
delivered 
to 
decompensated 
HF 
https://www.ynhh.org/services/heart-
failure/transitional-  
). 
Here HF patients with volume overload are 
referred as an 
alternative to hospitalization. Patients present in the 
morning to a single location and are treated with 
protocol driven 
IV) loop diuretics, monitoring, follow-up, etc. The 
diuretic treatment protocol involves aggressive stepwise escalation of 
IV diuretics every few hours until urine output is adequate, with 
addition of metolazone if loop monotherapy fails. These protocols were based upon the NHLBI 
Heart Failure Network CARRESS and DOSE trial findings/algorithms.94, 95 As such, “usual care” in 
the YTCC is state-of- the-art aggressive diuretic 
therapy. The YTCC allows us to eliminate many of obstacles to the study of decompensated HF 
and dramatically reduce resources required to complete a study of decongestive therapy. As a 
result of these advantages, we have studied over 200 patients in the YTCC since its creation.
Preliminary data with relevance to all aims: A pilot  study of sodium free chloride 
administration was performed in 10 stable HF outpatients.2 ,52 The primary purpose of the pilot was 
to determine feasibility, to ensure volume status wouldn’t worsen with the chloride load (this was 
unlikely, but unknown), and to see if any biologic effects plausibly related to chloride dependent 
processes could be detected. Details of inclusion/exclusion have been previously published2. Briefly, 
enrollment required a diagnosis of HF, a loop diuretic dose of ≥80 mg/day furosemide equivalents, 
clinical stability and optimal volume status per an advanced HF physician. The median diuretic 
dose in those enrolled was 160 mg/day and the median NT-proBNP was 298 pg/ml, indicating a 
euvolemic population on high dose diuretics. There was no specific inclusion related to serum 
chloride levels, but the median level was 98 mmol/L and all patients had a chloride ≤100 mmo/L. 
Seven grams of lysine chloride was administered three times a day, providing 115 mmol/day of 
sodium free chloride daily. 115 mmol/day chloride supplementation was chosen as this should 
approximately double daily chloride intake. Patients were instructed to consume a 2-gram sodium 
diet for the week prior to and during the intervention period. All diuretics and HF medications were 
maintained constant over the duration of the study.
Compliance with lysine chloride appeared to be good as serum chloride improved in 8 of 10 
patients, (p=0.01, Figure 2A), however compliance with the 2-gram sodium diet in these 
outpatients was poor as the pre-intervention median 24-hour sodium excretion was over 5 grams. 
Given the uncontrolled diet rich in sodium chloride we anticipated null findings. However, we 
were quite surprised to find striking signals supporting a meaningful change in volume status in 
these euvolemic patients. Hemoconcentration (a surrogate for improvement in blood volume) 
occurred in 8 of 10 patients (p=0.036), which is remarkable as this occurs in less than 50% of 
hospitalized HF patients receiving multiple days of IV diuretics.98, 99 An improvement in body 
weight occurred in the majority of patients with 3 patients losing more than 5 pounds. We also 
saw an improvement in natriuretic peptide levels in 8 of 10 patients, with a relative improvement 
of 25% in those that did improve (Figure 2B, p=0.01). Further support for a reduction in blood 
volume in these euvolemic patients came from the fact that the blood urea nitrogen to creatinine 
ratio (p=0.007, Figure 2C) and PRA (Figure 2D, p=0.005) also increased. Despite the change 
in volume status in these euvolemic HF patients, kidney function remained stable with no change 
in creatinine, cystatin C, serum potassium, or the markers of kidney injury neutrophil 
gelatinaseassociated lipocalin, kidney injury molecule-1, and interleukin-18, p=NS for all. As 
The Yale 
patients.( care-
center.aspx
Figure 2: Changes in various 
parameters with lysine chloride 
administration
intravenous (
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described in detail in the preliminary data section of Aim 3, we observed a remarkable 72% ± 33% 
reduction in the level of NCC (p=0.0005). This is contrary to the expectation that a reduction in 
volume and increase in PRA would be associated with significant upregulation of sodium 
transporters. Thus finding the strong signal of reduction in NCC in this setting is in support of the 
hypothesis that sodium free chloride supplementation is in fact suppressing WNK-kinase 
mediated sodium avidity.
Rationale for blood volume as an endpoint throughout this proposal: Change in blood 
volume was selected as the primary endpoint to measure the effect of lysine chloride on volume 
for several reasons: 1) The majority of metrics of volume status are either imprecise (e.g., physical 
examination, change in weight or fluid loss), invasive (e.g., right heart catheterization), or of 
unclear clinical relevance (e.g., bioimpedance or ultrasound derived parameters). 2) A normal 
blood volume or a change in blood volume in the setting of decongestive therapy is prognostically 
very important.98-104 Notably, a change in body weight is not reproducibly linked to improved  
outcomes.105 Furthermore, significant change in total body water with tolvaptan did not translate 
into improved outcomes.92 Thus change in intravascular volume appears to be an important 
parameter. 3) An FDA approved semi-automated test with very high precision is available to  
measure blood volume (http://www.daxor.com/wp-  
content/uploads/2014/10/bva100brochure.pdf). 4) Although the cost of measuring blood volume 
is substantially greater to that of a surrogate marker of intravascular volume (i.e., NT-proBNP), 
the increased variability in surrogate markers is dramatically greater than that of blood volume 
measurement. Notably, the average coefficient of variation in NT-proBNP levels obtained one 
week apart in stable HF, is remarkably large (>20% in some studies).106 Directly measured blood 
volume appears to have a very narrow variance in stable HF. In n=26 stable outpatient HF 
patients with blood volume measured twice for research purposes 1 year apart, the coefficient of 
variation was only 7.6% (personal communication Dr. Wayne Miller, Mayo Clinic). Since the cost 
of the blood volume test will represent only a fraction of the cost of the overall per patient costs of 
the proposed prospective randomized trials, the smaller sample size possible the low variance 
blood volume endpoint will actually substantially reduce total costs.
4.0
Study Design: We propose a multi-site, randomized, double blind, placebo 
controlled study of 200 patients with decompensated heart failure receiving treatment 
with IV diuretics for volume overload. Participants will be enrolled at Yale New Haven 
Hospital and St. Francis hospital. This study was designed to be more pragmatic than 
the outpatient study (HIC2000022016) proposed in a separate protocol, with a parallel 
group randomization to 7 days of therapy with either 115 mmol/day of lysine chloride or 
placebo. 115 mmol/day of lysine chloride was chosen as this represents a similar 
cumulative chloride load to the higher dose 5-day inpatient study (separate HIC 
application). The primary endpoint will be the change in blood volume from baseline to 
7 days (the range of days may vary between 5-8 days to minimize missing data). 
Due to the current pandemic of COVID-19 (SARS-coV2) and to remain in place for the 
future, the number of days between the V1 and V2 may be shortened (less than 5 days).   
This will allow for the completion of the study while the patient remains admitted to the 
hospital. The shortened duration will be utilized if the patient will be discharged before 
the 5-8-day window and allowing for complete data collection in the safest way possible.  
Thus, potentially eliminating the need to bring the patient back as an outpatient to 
complete the V2 in times of a pandemic or reduced visit capacity.
We propose an additional study arm to be completed in up to 5 additional patients. This 
arm would be an open-label study in patients with decompensated heart failure receiving 
treatment with IV diuretics for volume overload. The proposed arm would consist of 7 
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days (5-8 days to minimize missing data) of therapy with 115 mmol/day of lysine 
chloride. Patients enrolled into this arm will undergo identical study procedures to the 
randomized arm, with the exception of receiving only lysine chloride.
i.1
Study Protocol
A: Study Visit 1
1. Patient Arrival & Enrollment
a. Enrollment: Patients that meet inclusion/exclusion criteria will be 
approached during their admission at YNHH (York St 5-1, 5-2, 5-3, 5-4, 5-7, 58 
or St. Raphael’s Campus Verdi 5 East and Verdi 3 East).  If they agree to 
participate, the patient will sign an Informed Consent form prior to any study 
procedures taking place.
b.
Vital Signs, Weight and Height: Baseline vital signs, bladder scan, weight 
and height are measured at the start of the study visit. Vital signs and weight 
measurements are repeated at the end of the study visit.
c.
Pregnancy Testing: A point-of-care pregnancy test is performed for women 
under the age of 65 who have not had a hysterectomy.
d. IV Line Placement & Patient Acclimation
1.
IV Line Placement: A peripheral IV line is placed as part of the Standard of 
Care for receiving IV diuretics during admission at YNHH. If a suitable IV for 
the blood volume procedure is not available, one will be placed.
2.
If a KVO is required to keep the IV line patent, Dextrose 5% fluid will 
be used.
e.
Acclimation: Patient rests in a semi-recumbent position for approximately 60 
minutes prior to undergoing blood volume determination and randomization.
f.
Screening: After the peripheral IV line is placed, blood will be drawn for 
determination of venous pH via iSTAT and/or YNHH laboratory. If the patient’s 
venous pH is less than 7.25, they will be withdrawn from the study.
g. Patients will receive one of the following tracers depending on 
availability:
1.
Iothalamate  Administration
1
IV iothalamate: 1 cc of Iothalamate meglumine is administered 
intravenously in patients without a contrast allergy 
2 Iohexol Administration
2
IV Iohexol: 1 cc of Iohexol will be administered intravenously in 
patients without a contrast allergy
i.
Volumex Administration and Daxor Blood Volume Sample Collection
i.1 Baseline Blood Collection: Blood is collected prior to the Volumex 
injection to establish a baseline that reflects background radiation.
i.2 Volumex Injection: Up to 25 microcuries of the Volumex tracer 
(radiolabeled albumin) is injected as an intravenous bolus (IV-push).
i.3 Blood Volume Serial Blood Collection A series of 5 postVolumex 
blood samples is collected after tracer injection, allowing for complete 
mixing in the bloodstream.
i.4 Blood Volume Sample Timing: Samples are ideally spaced ~6 
minutes apart, and are collected approximately 12, 18, 24, 30 and 36 minutes 
after Volumex administration. Dextrose 5% fluid will be used to flush the IV 
line with administration of Volumex and in between blood draws for the 
blood volume.
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ii.
Standard YTCC or Inpatient IV Diuretic Therapy Protocol ii.1 The IV diuretic 
therapy prescribed by their treating clinician if inpatient.
iii. Randomization & 1st Dose of Study Drug (Lysine Chloride or Placebo) 1 After 
receiving the Standard IV Diuretic Therapy Protocol, the patient will then receive the 
first dose of study drug (lysine chloride or placebo). If patient is enrolled into the open-
label arm, they will not be randomized but will receive the first dose of lysine chloride. 
2 At this time, if a patient typically uses metformin, it will be discontinued for the 
duration they are on study medication (5-8 days).
•
Only if holding metformin is clinically appropriate (per study PI and 
patient provider).
•
Patients that have their metformin held while inpatient will be under 
the care of their treating physicians who will monitor their glucometer 
values and treat accordingly.
•
Patients that have their metformin and are discharged from the 
hospital while still taking the study medication will be asked to check 
their glucose twice daily or per their usual schedule (whichever is 
greater) for the duration of the time metformin is held.
•
If the participant does not own a glucometer, they will be provided a 
home blood glucose monitoring kit.
•
Any consistent glucometer readings greater than 200 mg/dL while 
outpatient and still on study medication, the patient will be instructed 
to contact their study physician.
iv. Blood Samples Collected iv.1 The total amount of blood samples collected at 
each visit (1 and visit 2) is 100cc. This includes 36cc for the blood volume and an 
additional 64 cc collected during the study visits  ( 1 and 2).
v. Timed-Void Spot Urine Samples and Bladder Scans
v.1
Timed spot urine samples will be collected periodically during the visit
v.2
Post-Void Residual Volume Bladder Scans: A post-void residual (PVR) 
volume is measured with a bladder scanner after each timed-void urine 
sample is collected.
vi. Deuterium Oxide (D20) Administration
Oral D20: 10.0 grams of liquid D20 is administered orally.
11.Imodium AD or Imodium(Loperamide)
a.
Once on Lysine Chloride/ Placebo if the participant experiences any abdominal upset 
as unformed stools (loose stools, diarrhea) from the Lysine Chloride/ Placebo. They 
will 
have 
the 
option 
of 
taking 
over 
the 
counter 
Imodium 
AD 
or 
Imodium(loperamide)”as needed” to relieve those symptoms.
•
The participant will be instructed to inform their treating physician when 
inpatient and the study team ( if discharged) of any abdominal upset, the Imodium 
will be built into the epic order set(PRN order) for the care team to administer. If 
the patient is discharged, the PI or sub- I will be notified, and it is deemed 
appropriate; The study team will provide the participant with the Imodium.
•
The participant will be assessed daily for additional or worsening unformed stools, 
if appropriate the patient will receive additional doses of Imodium according to 
the manufacturer’s instructions in conjunction with approval from the treating 
physician( inpatient) 0r study PI or sub- I if (discharged).
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•
Imodium over the counter: initial: Take 2 tablets/ capsules (4 mg) once.  May take 
1 additional tablet/ capsules after another episode of unformed stool.  Up to a 
maximum of (8) 2 mg tablets or capsules or 16mg daily.
•
The study team will provide the over the counter Imodium (once the patient is 
discharged) and the study team will record the number of doses taken per day
Patient Discharge vi.1 Vital Signs: Vital signs are obtained at the 
end of the study visit prior to discharge, to ensure patient safety.
vi.2 Home Urine Collection: patients will be given 24-hour urine collection 
containers to continue collecting urine at home.
B: Daily Monitoring and Dose Adjustment If a patient has a serum bicarbonate level 
less than 22mmol/L at screening, a venous pH and basic blood chemistries will be obtained 
via iSTAT and/or YNHH laboratory each day during the study period. 1cc of blood will be 
drawn each day to perform the test. If the patient has been discharged from YNHH and 
these tests are indicated, additional safety visits will take place (up to 5). For all patients, 
the results of any safety tests will be reviewed by the study physician and study nephrologist 
(Dr. Jeffrey Turner). If necessary, the study drug dose will be adjusted to once daily or no 
doses. The standard dosage is twice a day.
C: Continued IV Diuretic Therapy: The patient will remain admitted until the treating 
practitioner feels the patient no longer requires IV therapy, at which point they will be 
transitioned to oral diuretics.
D: Transition to Oral Diuretics: Upon transition to oral diuretics, the patients 
will be seen every other day (except for weekends) for laboratory monitoring of 
electrolytes and renal function. 10cc of blood will be drawn at each of these safety 
visits (which are separate from the additional safety visits that will be conducted 
based on serum bicarbonate), and a urine sample will also be collected.
E: Study Visit 2: After 7 days of randomized therapy (5-8 to minimize missing data), 
the patient will return or if still admitted have repeat blood volume assessment and 
bio-samples collection. The number of days of randomized therapy may be less than 
5 days in order to allow for the completion of the visit 2 while the patient remains 
hospitalized, this would be utilized only during times of a pandemic or when there is 
an reduced visit capacity. 
 Patients enrolled into the open-label arm will return after 7 days of therapy with lysine 
chloride (5-8 to minimize missing data).
F: Follow Up Visit/ Phone Call: One week after the completion of Visit 2, 
subjects will be given the option to either come in for a follow up visit or complete a 
follow up phone call. The purpose of this follow up visit is to make sure they are not 
experiencing any side effects or adverse events after taking the study 
medication/placebo as well as obtain a current weight, vital signs, a 10cc blood 
sample and a urine sample. If the subject is not able to come in for the follow up visit, 
a follow up phone can be done in its place. During the phone call, the subject will be 
asked their current weight, how they are feeling and will be assessed for any adverse 
events.
G: Study Locations: This is a multi-site study. Depending on space availability and 
other logistics, the patient may be asked to complete any daytime study procedures 
(either study visits or follow up visits) at either the YNHH Hospital Research Unit 
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(HRU), at the research group’s outpatient clinic space at 135 College Street in New 
Haven, CT, or at St. Francis Hospital.  The St. Francis site will seek IRB approval 
from their local IRB.
6.1
Genetic Testing & Maintaining Subjects’ Privacy
6.2
Types of future research to be conducted using the materials: Subjects’ genetic 
material will be collected for genome sequencing. One of the goals of this study is to 
determine if variations in human genome influence HF risk, progression and response to 
treatment. The accrued genetic material from subjects will permit potential future 
Genome Wide Association Studies (GWAS) regarding the interaction between genetic 
material and HF disease progression, phenotype and prognosis. No immortalized cell 
lines or animal studies are planned for this study.
6.3 Plan for collecting materials or the conditions under which material will be 
received: Subjects’ genetic material will be collected and processed at YNHH Park 
Street clinical laboratory, our study’s laboratory facilities at 300 George Street, or at 
YNHH-SRC campus. Each subject’s specimens will be processed, labeled with unique 
study IDs and stored in cryovials in a -80˚C freezer.
6.4 Types of Protected Health Information (PHI) about the individual contributors 
that will be entered into a database: Name, address, telephone number, medical 
record number (MRN), medical history and allergies, current and past medications and 
therapies, vital signs, family medical history, results of blood, urine and imaging tests (ex: 
x-rays, CT scans, MRIs, or ultrasound) and pathology tests (biopsies), DNA, tissue and 
blood.
6.5 Conditions or procedures for sharing of materials and/or distributing for  future 
research projects: Data collected by the PI and other study personnel listed in this 
protocol will be distributed for secondary research purposes only after the recipient 
investigator has obtained HIC approval for the proposed research objective, received an 
exemption or determination by the HIC the study is not considered human subject research.
Data will be distributed for research projects of the same nature and similar purpose 
specified in this protocol, as agreed to by the subjects by signing the compound informed 
consent and HIPAA authorization form upon enrollment to the study. The PI is 
responsible for receiving appropriate attestation by recipient investigators prior to 
permitting access to the database for activities considered preparatory to research. 
Attestation will be obtained using the Request for Access to PHI for a Research Purpose 
Form found at http://www.yale.edu/hrpp/forms-templates/hipaa.html.
6.6 Widespread sharing of materials: Samples and data collected during the research 
study will be shared with other investigators for research projects of the same nature 
and similar purpose specified in this protocol, as described above.
6.7 Stripping materials of all identifiers: Samples will be immediately stripped of all 
identifiers, and given a unique study ID as soon as deposited in the AVA-HF bio-repository.
6.8 Can donor-subjects withdraw their materials at any time, and/or withdraw the 
identifiers that connect them to their materials? Yes; researchers may still use the 
material or data that was collected before the subject withdrew permission/authorization 
in order to complete the research that has already commenced. All specimens and data  
retained in the AVA-HF biorepository will be destroyed upon receiving notice of a subject’s 
withdrawal of permission for continued use of their specimens.
6.9 Provisions for protecting participants’ privacy: The PI will ensure patients’ anonymity 
is maintained. Each subject is assigned a unique study identification number and is tracked 
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through this number. Subjects’ clinical data will be entered into password protected 
software and stored on a secure server. A log of subjects’ names, subject ID numbers and 
pertinent registration information (ex: address, phone number and emergency contact 
information) is maintained on a password-protected computer, to allow reidentification of 
subjects when necessary.
6.10 Methods for the security of storage and sharing of materials: Specimens are  labeled 
only with unique study ID numbers. When certain samples are shipped and analyzed by 
appropriate collaborating laboratories, samples will be sent anonymously, ensuring 
laboratories will not have access to any clinical or phenotype data, thus assessments will be 
made in a blinded fashion. The specific laboratories will vary depending on the biochemical 
test performed; specimens may be analyzed at individual or core laboratories, academic 
institutions or industry collaborators.
7.1
Research Subject Population
Subject Recruitment and Screening: Patients with a clinical diagnosis of HF who have 
decompensated and require treatment with IV diuretics at YNHH (York St 5-1, 5-2, 5-3, or 
5- 4, 5-7, 5-8 or St. Raphael’s Campus Verdi 5 East and Verdi 3 East) will be identified from 
their electronic medical record by study coordinators in coordination with treating 
clinicians.
Patients identified from electronic medical records whose providers feel they are 
appropriate for the study will be approached and recruited to participate in this research 
study. Patients will be approached by doctors, cardiac surgeons, residents, fellows, nurse 
practitioners, physician assistants and registered nurses.
Research staff will have a one-on-one conversation with the patient regarding the study 
and what participating would involve. All patients approached will be 18 years or older and 
capable of providing written, informed consent. Pursuant to HIPAA regulations, a log of 
disclosures of protected health information for recruitment/screening purposes will be 
kept.
7.2      Subject Classification: Will subjects who may require additional safeguards or other 
considerations be enrolled in the study? N/A Is this research proposal designed to 
enroll children who are wards of the state as potential subjects? N/A 
7.3 Subject Eligibility Determination: The study coordinator, working in conjunction with 
the PI, will determine subject eligibility.
7.4     Subject Inclusion and Exclusion Criteria
Inclusion Criteria
a. Clinical diagnosis of decompensated heart failure with at least one objective sign 
of volume overload (rales, edema, elevated JVP, or weight gain of at least 
5 pounds)
b.
A projected need by the treating clinician for continued treatment with IV 
diuretics 
c.
 Chronic loop diuretic use OR no chronic loop diuretic use and serum chloride 
<100 mmol/l
d. 18 years old
Exclusion Criteria
a. Inability to commit to or comply with serial visits
b.
History of severe metabolic or respiratory acidosis within 30 days of enrollment
c.
Use of metformin, acetazolamide, or any other agent that could predispose to 
acidosis. Patients who are on metformin may be enrolled if their metformin can 
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be discontinued safely for the duration of the study. Any participants who have 
consistently elevated Blood glucose readings > 200 mg/dL while inpatient will 
not be enrolled.
d. Serum bicarbonate level <20mmol/L
e.
Estimated glomerular filtration rate <20 mL/min or renal replacement therapy
f.
Urine output of <500 ml/24 hours despite use of IV loop diuretic therapy
g. Appears unlikely, or unable to participate in the required study procedures, as 
assessed by the study PI or research RN (ex: clinically-significant psychiatric, 
addictive, or neurological disease)
h.
Inability to give written informed consent or follow study protocol
Exclusion criteria for randomization:
Prior to randomization, a venous pH level will be determined. If the venous pH is less 
than 7.25, the patient will not be randomized.
8.1
Risks and Minimizing Risks
8.2
Risks
a. Data collection: We anticipate minimal risk related to collection of data from the 
clinical record, including paper charts, EMR, physical exams, and blood and urine 
samples. We also anticipate minimal risk associated with data entry into the 
electronic database, which will be password-protected, and stored on a secure 
server.
b.
Emotional, psychological, economic risk: In terms of economic expenditure, 
patients who are admitted to the hospital will not have to drive to the study site or 
pay for parking to participate in the study. Patients who are not currently inpatient 
will have to drive to the study site, park at their own expense, and spend up to 8 
hours there for the study protocol and thus the research study will only modestly 
increase the inconvenience for inpatients.
c.
Urine collection and bladder scanning: There are virtually no risks to the 
patient related to urine collection and bladder scanning, other than mild discomfort 
when the ultrasound probe is briefly pressed on the abdomen.
d. Blood draws: Blood drawing is generally associated with minimal risk, such as 
bleeding at the site, pain, bruising at the site, rare transient dizziness, anemia, 
infection, emboli or phlebitis.
e.
IV drug administration: IV injections generally present minimal risk to the 
patient, though the injection site can become infected, there can be bruising, 
inflammation of the vein, leaking of fluid into the surrounding tissues, or an 
injection site reaction depending on the type of medication injected.
f.
Lysine Chloride: Lysine Chloride is freely available to the public as a dietary 
supplement and food additive and is on the FDA Generally Recognized as Safe 
(GRAS) list. Every day  we eat large quantities of lysine and chloride in our diet. No 
significant side effects are anticipated to occur from lysine chloride based on a 
multitude of animal and human studies done with this supplement and the brevity 
of this intake (8 days). There was a rare incidence of minor GI upset associated with 
taking lysine chloride as a supplement in some of these studies, including nausea, 
abdominal pain and diarrhea. However, no side effects necessitating the 
discontinuation of lysine chloride supplementation were reported.a, b, c, d This 
research group previously performed a preliminary study with 11 patients involving 
supplementation of lysine chloride at 21 grams per day, with no adverse events 
reported (HIC #1308012508, minimal risk study). The doses of lysine chloride 
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administered in this study are higher than the majority of prior studies, which did 
not study patients with heart failure. As a result, new, unexpected, or exaggerated 
side effects are possible.
i.
Acidosis: One possible side effect that we will follow closely will be the 
buildup of acid in the patients’ blood, a condition that in very severe cases 
can cause illness or even be fatal. This condition would slowly develop if it 
were to happen and thus we will closely monitor serum pH and bicarbonate 
over the course of the study to be sure this does not occur. Importantly, we 
did not see any evidence of acid buildup in our pilot study of heart failure 
patients were similar doses were used.
ii.
Volume depletion: An additional possibility is that lysine chloride will 
work “too well” in removing fluid which could cause dehydration and 
possible kidney damage. As a result, we will closely follow kidney function 
throughout the course of the study and adjust diuretics dosage and the study 
medication if too much fluid is removed.
References
a. Rubin, A.L., et al., The Use of L-Lysine Monohydrochloride in Combination with 
Mercurial 
Diuretics in the Treatment of Refractory Fluid Retention. Circulation, 1960. 21(3): p. 
332-336.
b.Spencer, H., Samachson, J., 1963. Effect of lysine on calcium metabolism in man. J. 
Nutr. 81:301-6.
c. Griffith, R. S., D. E. Walsh, et al. (1987). "Success of L-lysine therapy in frequently 
recurrent 
herpes simplex infection. Treatment and prophylaxis." Dermatologica 175(4): 183-190.
d.Thein, D J, and W C CHurt. "Lysine as a prophylactic agent in the treatment of recurrent 
herpes simplex labialis." Oral surgery, oral medicine, oral pathology 58.6 (1984):659-
666.
g. Iothalamate meglumine (for patients without a contrast allergy): The 
most common adverse reactions to iothalamate meglumine are allergic reactions 
(which can be severe), headache, dizziness, low BP, nausea, vomiting, flushed 
sensation, transient taste perversion, and local injection site reactions. While 
iothalamate carries the risk of contrast associated kidney injury, the risk with this 
very-low dose (1cc) is exceedingly small; doses in this range have been proven to be 
exceptionally safe, even in the highest risk patients.
h.
Iohexol: The most common adverse reactions are allergic reactions (which can be 
severe), contrast induced kidney injury, pain, vision abnormalities including blurred 
vision, headache, taste perversion, arrhythmias including premature ventricular 
contractions (PVCs) and premature atrial contractions (PACs), angina/chest pain, 
nausea
i.
D2O: Deuterium oxide is a naturally-occurring, stable, non-radioactive isotope of 
hydrogen that is well-tolerated by humans. Also known as “heavy water,” D2O has 
twice the mass of a hydrogen atom and occurs naturally in human blood at 
concentrations of ~150 ppm. D2O has been widely used to measure water space in 
both animals and humans. For an adult human, 10mg of D20 is swallowed, 
increasing the blood’s D2O content from 150 to 300 ppm. There is thought to be 
essentially no risk with ingesting this amount of D2O.
j.
Volumex (radiolabeled albumin): Volumex is albumin labeled with the iodine 
isotope I-131 and is an FDA-approved method used to determine total blood 
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volume. The labeled albumin is available from Volumex, Daxor Corporation, NY, in 
a specialized, pre-filled flow chamber syringe designed to ensure >99.8% delivery of 
the radioisotope dose. A single 1cc dose of Volumex contains an ultra-low-dose of up 
to 25 micro-curies of activity. Although each organ will receive a different dose, the 
amount of radiation exposure the patient will receive from 2 doses of Volumex over 
the course of this study will be equivalent to the whole body being uniformly 
exposed to 0.2 rem of radiation. This amount of radiation is well below the dose 
guidelines established by the federal government and adhered to by the YNHH 
Radiation Safety Committee for research subjects. To give you an idea about how 
much radiation the patient will be exposed to, we will compare it to an every-day 
situation. Everyone receives a small amount of unavoidable radiation each year. 
Some of this radiation comes from space and some from naturally- occurring 
radioactive forms of water and minerals. The Volumex given during this study will 
give patients the equivalent of about 8 extra months’ worth of natural radiation.
k.
Potassium Iodide (KI): While the risks of 8 days of chronic exposure have been 
deemed greater than the potential benefits, with respect to acute administration, 
there may be some potential value for younger patients, given the FDA 
recommendations on the use of KI. As such, patients under the age of 40 who do not 
have a known iodine hypersensitivity will be given the option of taking a one- time 
dose of KI to further reduce the risk of radiation  exposure from Volumex. The 
major exposure of the thyroid to unbound radioactive iodine will be the day the 
dose of Volumex is injected. As such, the option will be given to patients to take the 
KI after explaining that taking a dose of KI could potentially reduce the already low 
chances of damage to the thyroid by radiation, at the expense of the known, short-
term effects of the KI. While side effects are unlikely when Potassium Iodide is used 
at the recommended dose and for a short period of time, the following are possible 
side effects: cardiac arrhythmias, myxedema, confusion, fatigue, metallic taste, 
numbness, tingling sensation, skin rash, goiter, hyperthyroidism, hypothyroidism, 
diarrhea, salivary gland swelling or tenderness, gastric distress, gastrointestinal 
hemorrhage, nausea, stomach pain, vomiting, adenopathy, thyroid adenoma, 
hypersensitivity reaction (angioedema, cutaneous and mucosal hemorrhage, serum- 
sickness-like symptoms), arthralgia, weakness,  pharyngeal edema, fever, iodism.
l.
Dextrose 5% Fluid: There is risk of fluid retention, hyperglycemia or 
hyperosmolar hyperglycemic state, hyponatremia, hypophosphatemia, 
hypersensitivity reactions and refeeding syndrome. We anticipate these risks to be 
minimal as the Dextrose 5% infusion will be used during the I 131 administration 
and the thirty-six-minute blood collection for the blood volume to flush the IV line. 
The Dextrose 5% may also be used as a KVO fluid should the participants IV line 
require this to maintain patency. The maximum infusion amount is 10ml/hr over 
the 8 hour study period(for KVO) and IV line flushes or 100 ml total per visit 1 and 
2. Although the participant will be receiving 5% Dextrose during the I 131 
administration and some participants may also receive 10 ml/hr to keep their IV 
patent, there is a minimal risk of hyperglycemia in diabetic and non-diabetic 
patients as the amount received will be a maximum of 100 ml over 8 hours which 
equates to only 5 grams of glucose.
m. Imodium AD or Imodium (Loperamide): There are risks of allergic reaction 
to this loperamide hydrochloride. Use cautiously in patients with heart arrhythmias, 
liver impairments, mucus in their stool and fever. Contraindicated in patients that 
have abdominal pain without diarrhea. May cause drowsiness, dizziness, and 
tiredness, may need caution when operating heavy machinery. If clinical 
improvement is not observed in 48 hours or they develop abdominal pain or 
bulging, the Imodium may be stopped by the study PI or sub I.
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8.3 Minimizing Risks
a. Data collection: Identifying data will be available only to the study PI and study 
coordinator, and will be used only to facilitate medical record review, and prevent 
duplicate screenings of patients while looking for subjects to enroll in the study. All data 
sheets, electronic data entries, and laboratory samples will be labeled with a unique 
identifier code. These numbers will be unique to the patient and will be the common link 
between specimens and the data. No identifying data will be reported to the public or in 
any publications that may result from this research.
b.
Emotional, psychological, economic risk: Blood samples will be drawn off of the IV 
line to reduce patient discomfort, and to avoid the patient being subjected to multiple 
straight needle sticks.
c.
Urine collection and bladder scanning: Collecting urine and performing bladder 
scans during the study presents minimal risk to the patient.
d. IV Insertion & Blood draws: The risks of blood draws will be minimized by drawing 
the minimum volume necessary for study purposes. The risks of pain and infection will be 
minimized by having blood drawn by qualified, experienced staff who have achieved 
competency with phlebotomy. All standard precautions will be maintained during the 
procedure. Patients’ discomfort will be minimized by drawing blood from the IV line when 
possible, instead of subjecting the patient to multiple straight sticks.
e.
IV drug administration: Injections will be performed by qualified, experienced staff 
who have demonstrated competency with the technique. The sterility of the IV line will be 
diligently maintained by research staff to help prevent infections. Sites will be closely 
monitored for warmth, redness, swelling, pain and leaking. The IV line will be flushed 
before and after each medication is administered
f.
Lysine Chloride: Patients will be seen every other day (except for weekends) for 
laboratory monitoring of electrolytes and renal function to ensure the absence of any 
adverse effects, and to ensure that the supplement is being taken in the correct manner.
i.
Acidosis: If laboratory results provide any indication of acidification, the dose of 
lysine chloride will be adjusted (either 2 doses/day, 1 dose/day or 0 dose/day) at 
the discretion of the study physician and study nephrologist.
ii.
Volume depletion: Daily lab values will be monitored by the study team. In 
addition, vital signs will be measured daily. Patients who are admitted at YNHH 
will additionally be monitored by their treatment teams. Daily lab values will be 
reviewed by the study physician and nephrologist and, at their discretion, study 
medication or diuretic dosage may be adjusted.
g. Iothalamate meglumine (for patients without a contrast allergy): The dose 
administered is the smallest effective dose possible to minimize any medication side 
effects.
h.
Iohexol (for patients without a contrast allergy): The dose administered is the 
smallest effective dose possible to minimize any medication side effects.
i.
D2O: Deuterium oxide is a safe, natural isotope found in everyone’s tap water. It is a 
frequently used tracers in studies determining TBW. We do not anticipate any risk to the 
patients from its use.
j.
Volumex (radiolabeled albumin): We do not anticipate any significant risks from 
patients receiving 2 doses of Volumex. This will expose patients to 0.2 rem of radiation, 
which equals about 8 extra months’ worth of natural radiation exposure. Patients under 
the age of 40 will be given the option of taking a one-time dose of Potassium Iodide to 
further reduce the risk of radiation exposure from Volumex.
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k.
Potassium Iodide (KI): To reduce the risks of taking KI, the lowest effective, one-time 
dose (135 mg) will be administered to patients under the age of 40 who have opted to take 
this medication to further protect against radiation exposure from Volumex.
l.
Dextrose 5% Fluid: The volume of Dextrose 5% will be administered during the blood 
volume procedures to flush the line and given continuously throughout the study visit only 
if a KVO fluid is required to keep the IV-line patent. At these volumes, we anticipate the 
potential of these risks to be minimal. In addition, the participant’s vital signs will be 
measured.
m. Imodium AD or Imodium(Loperamide): This medication is generally well tolerated; 
it will be administered in smallest effective dose to maximize the benefit of the medication 
and to minimize any medication side effects. We expect minimal risk by giving the 
smallest effective dose, initial dose is 4mg and maximum daily dose is 16mg.
9.0 Moderate Risk Data and Safety Monitoring Plan (DSMP) with Data Safety Monitoring 
Board 
(DSMB)
Personnel responsible for the safety review and its frequency: The PI will be 
responsible for monitoring the data, assuring protocol compliance, and conducting the safety 
reviews at the specified frequency which must be conducted at a minimum of every 6 months 
(including when re-approval of the protocol is sought). During the review process, the PI 
(monitor) will evaluate whether the study should continue unchanged, require modification 
&/or amendment, or close to enrollment. Either the PI, the IRB, or the Yale Data Safety 
Monitoring Board (DSMB) have the authority to stop or suspend the study or require 
modifications.
The overall risk associated with the proposed study is deemed moderate for the 
following reasons: We believe there are minimal risks to the subjects from the lysine chloride 
and D2O administered during the study. Lysine chloride is freely available to the public as a dietary 
supplement. No significant side effects are anticipated to occur from supplementing lysine chloride 
based on a multitude of animal and human studies done with this supplement and the brevity of 
this intake (8 days). This research group previously performed a preliminary study with 11 patients 
involving supplementation of lysine chloride at 21 grams per day, with no adverse events reported 
(HIC #1308012508). D2O occurs naturally, is widely used in various water space studies, and is 
very well tolerated, especially in such small, one-time doses. Iothalamate and Iohexol have been 
used extensively in diagnostic radiology, and while it can cause transient nausea, vomiting, facial 
flushing and a feeling of body warmth, these side effects are typically only seen in much higher doses 
than that proposed for this study. We view the risks associated with collecting up to 105cc of blood 
during each Study Visit as moderate, as the proposed volume to be drawn is larger than the amount 
traditionally drawn for a basic complete count and chemistry. The IV radiolabeled albumin presents 
moderate risk to the study subjects. Each ultra-low dose proposed will be equivalent to whole body, 
uniform exposure of 0.1 rem of radiation, which is well below the dose guidelines established by the 
federal government, and adhered to by the YNHH Radiation Safety Committee for research 
subjects.
Although we have assessed the proposed study as one of moderate risk, the potential exists for 
anticipated and/or unanticipated adverse events (AE), serious or otherwise, to occur since it is 
not possible to predict with certainty the absolute risk in any given individual or in advance of 
first-hand experience with the proposed study methods. Therefore, we provide a plan for 
monitoring the data and safety of the proposed study as follows:
Attribution of Adverse Events (AE): AE will be monitored for each study participant and 
attributed to the study procedures / design by the PI according to the following categories:
-Definite: AE is clearly related to investigational procedures(s)/agent(s)
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-Probable: AE is likely related to investigational procedures(s)/agent(s)
-Possible: AE may be related to investigational procedures(s)/agent(s)
-Unlikely: AE is likely not related to the investigational procedures(s)/agent(s) -Unrelated: 
AE is clearly not related to investigational procedures(s)/agent(s)
Plan for Grading Adverse Events: AE noted during the study will be graded by severity as 
Mild, Moderate or Severe.
Plan for Determining Seriousness of Adverse Events: In addition to grading the AE, 
the PI will determine whether the AE meets the criteria for a Serious Adverse Event (SAE). 
An AE is considered serious if it:
-is life-threatening OR
-results in in-patient hospitalization or prolongation of existing hospitalization OR
-results in persistent or significant disability or incapacity OR
-results in a congenital anomaly or birth defect OR
-results in death OR
-based upon appropriate medical judgment, may jeopardize the subject’s health and may 
require medical or surgical intervention to prevent one of the other outcomes listed in 
this definition, OR -adversely affects the risk/benefit ratio of the study
An AE may be graded as severe but still not meet the criteria for an SAE. Similarly, an AE may be 
graded as moderate but still meet the criteria for an SAE. It is important for the PI to consider the 
grade of the event as well as its “seriousness” when determining whether reporting to the IRB is 
necessary.
Plan for reporting Reportable AE and other unanticipated problems involving risks 
to subjects or others to the IRB and DSMB: The PI will report the following types of events 
to  the IRB:
A. AE that are serious or life-threatening AND unanticipated (or anticipated but occurring with a 
greater frequency than expected) AND possibly, probably or definitely related to the 
drug/device/intervention; and
B. Other unanticipated problems  involving  risks  to  subjects or others.  These AE or unanticipated 
problems involving risks to subjects or others will be reported to the IRB in accordance with IRB 
Policy 710, using the appropriate forms found on the website.
C. Any untoward or unexpected occurrence in this study (physical or psychological harm)  temporally 
associated with the individual’s participation in the research (whether or not considered related to 
participation in the research) will be reported to the IRB within 5 calendar days and  would be 
presented to the DSMB meetings/calls. For the randomized portion of the study, the side effects 
described that were described previously in this submission that are untoward or unexpected will 
be reported within the window between randomization and the final visit or phone call at the end 
of the two weeks.
D. Reportable Events (which are events that are serious or life-threatening and unanticipated (or 
anticipated but occurring with a greater frequency than expected) and possibly, probably, or 
definitely related) or Unanticipated Problems Involving Risks to Subjects or Others that may 
require a temporary or permanent interruption of study activities will be reported immediately (if 
possible), followed by a written report within 5 calendar days of the Principal Investigator 
becoming aware of the event to the IRB (using the appropriate forms from the website) and any 
appropriate funding and regulatory agencies.
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Plan for reporting AE as appropriate to the following individuals or group entities: 
For this study, the following individuals and regulatory agencies will be notified: All 
coinvestigators listed on the study protocol and the Yale DSMB. The PI will conduct a review of 
all AE upon completion of every study subject. AE frequency and severity will be evaluated to 
determine if modifications to the protocol or informed consent form (ICF) are required.
For multi-site studies for which the Yale PI serves as the lead investigator: 
A. How will adverse events and unanticipated problems involving risks to subjects or 
others be reported, reviewed and managed? 
Adverse events and unanticipated problems will be recorded and reported in 
accordance with local institutional and IRB policies.
B. What provisions are in place for management of interim results? 
Given the observational nature of this study, no interim data analysis is planned. 
C. What will the multi-site process be for protocol modifications? When a protocol 
modification is suggested or required at any of the three clinical sites, each site will 
receive the proposed changes. 
Modifications will be assessed for relevancy to the clinical site and for their impacts on 
the study design. When necessary, protocol modifications will be submitted in 
accordance with local IRB policy.
PI’s assessment of the overall risk level if children are participating in this study? N/A
Statistical and Analytical Considerations
Endpoints: Change in blood volume over the 7-day intervention will serve as the primary 
endpoint. Secondary endpoints will include the change in log NTpro-BNP, serum creatinine, 
cystatin C, chloride and bicarbonate.
Analysis plan: A linear mixed effect model will be used to analyze the trial with class variables 
of time and treatment group. The mixed model will be able to accommodate the incomplete data 
by using all available observations. In this model, the fixed effects include the main effect of the 
treatment, the main effect of time, and the interaction between the them and the random effects 
include random intercept. Residual diagnosis for the mixed model will be performed to check 
whether model assumption is seriously violated. Statistical significance will be set at an alpha of 
0.05. Secondary endpoints will be analyzed with the same approach.
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.
Power and sample size: The change in blood volume in hospitalized patients undergoing several 
days of IV diuresis was reported by Miller and Mullan to be on average -0.7L (a 9% reduction), with a 
standard deviation of 1.1L.107 The correlation between pre and post treatment blood volumes was high 
at r=0.87 (personal communication with Dr. Miller). Although some of this variability is driven by 
heterogeneity in treatment (which conducting the trial in the YTCC will address) some of it is also driven 
by heterogeneity in individual response to standard therapy and the relative importance of each is 
unknown.
Additionally, the variability in blood volume response to lysine chloride (if one exists) is unknown. As 
such, given the multiple sources of variance in order to detect a 0.5L difference in change in blood 
volume between lysine chloride and placebo, we need to power for a standard deviation as high as 
1.5L. Figure 5 illustrates that we will have an 80% power to detect an effect size 1/3 the standard 
deviation with n=100 participants.
SECTION II: RESEARCH INVOLVING DRUGS, BIOLOGICS, RADIOTRACERS, PLACEBOS AND 
DEVICES
11.1 RADIOTRACERS: FDA-approved Volumex (Iodinated I-131 Albumin)
If an exemption from IND filing requirements is sought for a clinical investigation of a 
drug product that is lawfully marketed in the United States, review the following categories and 
complete the category that applies:
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Exempt Category 1: The clinical investigation of a drug product that is lawfully marketed in the 
United States can be exempt from IND regulations if all of the following are yes:
1)
The intention of the investigation is NOT to report to the FDA as a well-controlled study in 
support of a new indication for use or to be used to support any other significant change in 
the labeling for the drug. YES
2) The drug that is undergoing investigation is lawfully marketed as a prescription drug product, 
and the intention of the investigation is NOT to support a significant change in the advertising 
for the product. YES
3) The investigation does NOT involve a route of administration or dosage level or use in 
populations or other factor that significantly increases the risks (or decreases the acceptability 
of the risks) associated with the use of the drug product. YES
4) The investigation will be conducted in compliance with the requirements for institutional 
(HIC) review and with the requirements for informed consent of the FDA regulations (21 CFR 
Part 50 and 21 CFR Part 56). YES
5) The investigation will be conducted in compliance with the requirements regarding 
promotion and charging for investigational drugs. YES
11.2Drugs/Biologics: Background Information on Study Medications and Tracers
Previous 
Human Use
Known risks
Study 
Dose (x 2 
Visits)
General 
Dose 
Range
Route 
of 
Admin
Lysine 
Chloride
Lysine Chloride has 
been used in a 
multitude of human 
studies, including a 
preliminary study 
with 11 patients at 
Yale involving 
supplementation 
with 21 grams/day 
of Lysine 
Chloride, with no 
adverse 
events reported 
(HIC
May cause nausea, 
abdominal pain and 
diarrhea.
165 
mmol/day 
(30.1 g/day)
1 gram 
2X/day
PO
(powder)
Volumex 
(Iodinated 
I-131
Albumin)
FDA-approved 
radio- 
pharmaceutical for 
the determination 
total blood & 
plasma volumes 
studies. & protein 
turnover
Immune
virtually unaltered by 
iodination process, 
but an allergic 
reaction could occur 
after multiple dose
1.0cc (up to 
25 
microcurie 
s)
blood & plasma 
volume studies 
= 
0.2cc- 
2.0cc
IV-Push
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Iothalamate 
Meglumine*
Non- isotopic 
(cold) contrast 
agent. Cost 
effective tracer to 
measure GFR, 
allowing 
researchers to 
avoid exposing 
patients and 
themselves to 
isotopes.
Most frequently: 
nausea, vomiting, 
facial flush and a 
feeling of body 
warmth but generally 
occur at far higher 
doses than used in 
current study.
1 ml
30-60 ml
IV
Iohexol*
Cost effective 
tracer to measure 
GFR, allowing 
researchers to 
avoid exposing 
patients and 
themselves to 
isotopes
Contrast induced 
kidney injury 
pain, vision 
abnormalities 
including blurred 
vision, headache, 
taste perversion, 
arrhythmias 
including 
premature 
ventricular 
contractions 
(PVCs) and 
premature atrial 
contractions 
(PACs), 
angina/chest 
pain, nausea
1 ml
50 ml-200 ml
IV
D2O
Standard 
reagent used in 
common 
clinical 
technique to 
determine 
TBW.
Naturally occurring, 
stable isotope of 
hydrogen found in 
all tap water. Known 
to be a safe, non- 
radioactive 
technique. Exact 
toxicity amount not 
determined; animal 
studies have shown 
D20 concentrations 
must reach 30-35% 
of TBW to exert toxic 
effects (required 
dose would be ~15L 
for a 70kg patient).
10.0 grams
9-11mg
PO
(liquid)
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Potassium 
Iodide**
Used to block 
thyroidal uptake of 
radioactive 
isotopes of iodine.
Can cause 
arrhythmias, 
confusion, fatigue, 
fever, numbness, 
tingling, 
hyperthyroidism 
(prolonged use), 
hypothyroidism 
(prolonged use), 
diarrhea, nausea, 
vomiting, 
eosinophilia, 
lymphedema, 
hypersensitivity 
reaction, arthralgia, 
weakness, dyspnea, 
wheezing.
130 mg
130 mg
PO
(Liquid)
Dextrose 5 
% Fluid
FDA approved 
nonpyrogenic 
salt free solution 
for fluid and 
calorie 
replenishment for 
intravenous 
administration
Can cause fluid 
retention, 
hyperglycemia or 
hyperosmolar 
hyperglycemic state, 
hyponatremia, 
hypophosphatemia, 
hypersensitivity 
reactions and 
refeeding syndrome
200ml
500ml-
1000ml
IV
Imodium 
AD
or 
Imodium***
FDA approved
to treat diarrhea
Can cause dizziness, 
drowsiness and 
Tiredness
Taken only
is the 
participant 
experience 
diarrhea 
after taking 
Lysine 
Chloride/ 
placebo.
Maximum 
dose 16 mg 
/day
PO
* Iothalamate or Iohexol will only be administered to patients without a contrast dye allergy
**Potassium Iodide will be offered to patients under 40 to further reduce the radiation risks of Volumex 
to the thyroid.
*** Imodium will only be administered to participants who experience loose stools and are randomized on 
the study medication.
**** Unforeseen changes in the treatment plan may preclude the study team from continuing with study 
procedures after the iothalomate/iohexol and D2O are given.  In this case the team may plan to initiate study on a 
subsequent day and administer an additional dose of iothalomate/iohexol and D2O on that day.  
Iodide will be sourced from YNHH IDS. The Volumex will be sourced from the Daxor Corporation.
11.3 Is the drug provided free of charge to subjects? Yes, by the research group
11.4  Storage, Preparation & Use: Study medications will be stored by and obtained from YNHH 
          IDS.
11.5
Investigational Drug Service utilized: YNHH IDS
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11.6
Use of a Placebo
a. Describe the safety and efficacy of other available therapies. If there are  
no other available therapies, state this. There are several other therapies 
available to help control volume status in patients with HF: diuretics, vasodilators 
(angiotensin converting enzyme inhibitors, angiotensin receptor blockers, 
nitrodilators, etc.), cardiostimulatory or ionotropic medications (digitalis, beta- 
agonists, and phosphodiesterase inhibitors), and cardio-inhibitory medications 
(beta- blockers and calcium-channel blockers).
b.
Maximum time study subjects may receive placebo: 8 days
c.
Greatest potential harm that may come to a subject as a result of 
receiving placebo: Enrolled patients will receive either the study drug (lysine 
chloride) or the matched placebo for an 8-day period. Patients will continue taking 
their home loop diuretics, and will be monitored closely by YTCC and the research 
study for signs of volume overload.
d. Procedures in place to safeguard subjects receiving placebo: After 
transitioning to oral diuretics per the YTCC protocol, the patient will be seen every 
other day (except for weekends) for laboratory monitoring of electrolytes and renal 
function. Vital signs and weight will be measured. Patients will be monitored just 
as closely if taking the placebo as they would be monitored if taking the study 
medication (lysine chloride).
11.7
Continuation of Drug Therapy After Study Closure: No, subjects will not be 
provided the opportunity to continue receiving the study drugs after the study ends. After 
completing the research study, the patient will continue taking their regularly prescribed 
medications, including oral diuretics, if appropriate.
11.8
DEVICES: N/A
SECTION III: RECRUITMENT AND CONSENT PROCEDURES
12.1
Recruitment and Consent Procedures
12.2  Targeted Enrollment: 200 patients are anticipated to be enrolled across all study sites and 
complete all study procedures over the course of 6 years. Up to 5 additional patients enrolled 
into the open label arm are anticipated to complete all study procedures over the course of 1 
year.
12.4  Recruitment Procedures
a.
Recruitment Methods to be used: Medical Record Review
b.
Subject Recruitment and Screening: Potential subjects will be identified by study 
coordinators in conjunction with treating clinicians by screening the EMR of patients with a 
clinical diagnosis of HF receiving outpatient loop diuretics. Patients whose providers feel they are 
appropriate for the study will be approached. Pursuant to HIPAA regulations, a log of disclosures 
of protected health information for recruitment/screening purposes will be kept.
c.
Who is recruiting potential subjects? The PI or study coordinator will first approach the 
patient’s treating clinician (ex: physician, nurse practitioner, or physician’s assistant) regarding 
possible involvement in the study. If the provider feels the patient is appropriate, they will ask if 
the patient would be interested in hearing about a research project they might be able to 
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participate in. If the patient expresses interest, the PI or study coordinator will then approach the 
patient
12.5
Assessment of Current Health Provider Relationship for HIPAA Consideration: 
Does the PI or any member of the research team have a direct existing clinical relationship 
with any potential subject? Yes, some of the subjects. Jeffrey Testani, Tariq Ahmad, Lavanya 
Bellumkonda, Henry Cabin, Matthew Griffin, Daniel Jacoby, Christopher Maulion, Edward Miller, 
Jeffrey Turner and Albert Sinusas are all physicians at YNHH, and therefore could potentially be the 
attending physician for some of the enrolled patients. Dr. Ralph Riello is a pharmacist at YNHH, and 
therefore could be the attending pharmacist for some of the enrolled patients.
12.6
Request for waiver of HIPAA authorization for Recruitment/Screening Purposes 
Only: Describe why it would be impracticable to obtain the subject’s authorization for 
use/disclosure of this data: Due to the inclusion criteria that involves PHI, without a waiver of 
consent it would be extremely challenging to identify patients to approach for possible study enrollment.
The PI assures that the PHI for which a Waiver of Authorization has been requested will 
not be reused or disclosed to any person or entity other than those listed in this 
application, except as required by law, for authorized oversight of this research study, or 
as specifically approved for use in another study by an IRB.
Researchers are reminded that unauthorized disclosures of PHI to individuals outside of the Yale 
HIPAACovered entity must be accounted for in the “accounting for disclosures log,” by subject 
name, purpose, date, recipients, and a description of information provided. Logs are to be 
forwarded to the Deputy HIPAA Privacy Officer.
12.7
Process of Consent: Proper written, informed consent and HIPAA authorization will be 
obtained prior to patient participation in this trial. The overall protocol (including objectives, procedures 
and duration), potential risks and benefits, voluntary nature and ability to withdraw will be discussed 
with each patient. The patient will be given a copy of the IRB-approved ICF to review and will have all 
questions answered before being asked to sign the ICF. Verification of comprehension of the consent will 
be obtained by asking the subject to describe in their words the purpose and risks of the study. The patient 
will be instructed that his or her care will not be affected by his or her decision to participate, or not. If 
the patient voluntarily agrees to participate in the trial, he or she will be asked to sign the ICF. The original 
ICF will be kept with the study documents and a copy will be made for the patient’s chart. The subject 
will be given a signed copy of the ICF and HIPAA authorization form. All study subjects will be 18 years 
or older, with the capacity and ability to provide informed consent, thus making parental or surrogate 
permission not applicable.
12.8
Evaluation of Subjects’ Capacity to Provide Informed Consent: Patients will be screened 
within EPIC for study eligibility, including mental status and ability to provide informed consent. Study 
coordinators will confirm with the patient’s provider that the patient has the requisite ability and capacity 
to give informed consent prior to approaching the patient regarding the study. If at any time during the 
study there is concern regarding a patient’s mental status, study staff will both notify the patient’s 
provider, and re-evaluate whether the patient is still appropriate for participation in the study. After the 
study objectives and procedures are explained to the subject, they will be asked to describe in their words 
what the purpose and risks of the study are.
12.9
Non-English Speaking Subjects: N/A. Only English-speaking subjects will be enrolled.  12.10  
Requesting a Waiver of Consent for Recruitment/Screening only
a.
Does the research pose greater than minimal risk to subjects? NO
b.
Will the waiver adversely affect subjects’ rights and welfare? NO
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c.
Why would the research be impracticable to conduct without the 
waiver? Determining which patients meet inclusion/exclusion criteria would 
be impossible without this waiver.
d.
Where appropriate, how will pertinent information be returned  to, 
or shared with subjects at a later date? All screened information will be 
present in the patient’s electronic medical record (EMR), and thus the patient 
will already have full access to any information involved in the 
screening/recruitment by contacting YNHH’s medical records department.
SECTION IV: PROTECTION OF RESEARCH SUBJECTS
13.1
Confidentiality & Security of Data
13.2
Study subject PHI (medical information along with HIPAA identifiers)  
collected and used for the research: Name, address, telephone number, MRN, 
medical history, allergies, current and past medications or therapies, family medical 
history, information from a physical examination (ex: BP, heart rate, respiratory rate and 
temperature), results of blood and urine tests, pathology tests (biopsies), results of imaging 
tests (ex: x-rays, CT scans, MRI scans, or ultrasound), DNA, tissue and blood.
13.3
Research data collection, recording, and storage: Research data will be collected 
from patients’ clinical record (paper charts and EMR, blood and urine samples and cardiac 
pressure monitoring data collected during the study). Data will be recorded in electronic 
databases (RedCap & Oncore), stored in password-protected software on a secure server.  
Yale University will serve as the Data Collection Center (DCC) for this study.
13.4
Digital data storage: ITS-approved computer on a secured server
13.5
What methods and procedures will be used to safeguard the confidentiality  
and security of the identifiable study data and the storage media indicated 
above during and after the subject’s participation in the study? Each subject’s 
samples will be given a unique study ID number and cryovials will be labeled with barcodes. 
There will be no PHI or information directly linking the patient to these samples. Subjects’ 
clinical data will be stored on password-protected software maintained on a secure server. 
A “linker file” with PHI will be maintained on a separate, password- protected system, 
which only the study PI and study coordinator will have access to.
13.6
What will be done with the data when the research is completed? Are there 
plans to destroy the identifiable data? If yes, describe how, by whom and when 
identifiers will be destroyed. If no, describe how the data and/or identifiers 
will be secured. After study enrollment is complete continued analysis of samples in the 
AVAHF biorepository will be undertaken, and thus destruction of identified data is not 
planned until completion of all analyses potentially requiring the linkers to remain in place.
13.7
PHI Access: The PI, co-investigators, and study coordinators will have access to the 
complete data set, including all PHI collected.  Study documents and records may be 
accessed by the Food and Drug Administration.
13.8
Certificate of Confidentiality: N/A
13.9
Study procedures likely to yield information subject to mandatory reporting 
requirements: N/A
SECTION V: POTENTIAL BENEFITS
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14.0
Potential Benefits: Although there are no direct benefits that a participant can expect from this 
study, there are significant potential benefits to society at large. Given the tremendous public 
health importance of HF and the management of volume overload in HF, any incremental 
knowledge in this area has great potential impact.
SECTION VI: RESEARCH ALTERNATIVES AND ECONOMIC CONSIDERATIONS
15.0 Alternatives: What other alternatives are available to study subjects outside of the 
research? Patients may choose to proceed with the standard of care for heart failure, without 
choosing to participate in the research study.
16.1 Payments for Participation (Economic Considerations): Patients will be compensated for 
each Study Visit, Safety Visit and Home Urine Collection. This compensation includes the 
inconveniences involved in serial collection of urine and blood samples, and home urine 
collections. Patients completing all study and safety visits and urine collections will receive a total 
of up to $575.
i.
$500 for first Study Visit (8 hours)
ii.
$20 for patients who must be withdrawn after IV placement and screening due 
to venous pH
iii.
$25 for each Safety Visit (up to 5) 
iv.
$15 for each 24 Hr Urine Collection (x7) 
v.
$500 for last Study Visit (8 hours) if in the hospital, $1,000 if the study takes 
place outpatient 
vi.
$ 25 for follow up visit (1) or $10 for follow up phone call
vii. $50 if a study is initiated but cannot be completed
17.0 Costs for Participation (Economic Considerations): Clearly describe the subject’s 
costs associated with participation in the research, and the interventions or 
procedures of the study that will be provided at no cost to subjects. The following will 
be provided to the patient while participating in the study: blood draw supplies, urine sample 
collection supplies, and all study medications and placebo.
18.1
In Case of Injury: Required for any research involving more than minimal risk, and for minimal 
risk research that presents the potential for physical harm (e.g., research involving blood draws).
a.
Will medical treatment be available if research-related injury occurs? Yes
b.
Where and from whom may treatment be obtained? Patient’s medical provider
c.
Are there any limits to the treatment being provided? Unknown
d.
Who will pay for this treatment? Patient’s own insurance
e.
How will the medical treatment be accessed by subjects? Through standard 
contact with their health care provider.
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