Measurement of Relaxin in the Serum and Cerebrospinal Fluid of Subjects With and Without the Relapsing Form of Multiple Sclerosis
Conditions: Multiple Sclerosis, Relapsing-Remitting
Sponsor: Providence Health & Services

 
 
 
 
 
 
STUDY TITLE: 
 
 
Measurement of Relaxin in the Serum and Cerebrospinal 
Fluid of Subjects With and Without the Relapsing Form of 
Multiple Sclerosis 
 
 
PRINCIPAL 
INVESTIGATOR: 
 
 
Stanley Cohan, M.D., Ph.D. 
 
 
INSTITUTION: 
 
 
Providence Brain and Spine Institute  
Providence Health & Services  
 
 
PROTOCOL VERSION & 
DATE: 
 
 
Amendment 5, 04JAN2023 
 
 
NCT: 
 
 
NCT01909492 
  

13-089A: Amendment 5, 04JAN2023 
 
 
Measurement of Relaxin in the Serum and Cerebrospinal Fluid of Subjects With 
and Without the Relapsing Form of Multiple Sclerosis 
 
 
INTRODUCTION: 
MS is an auto-immune disease of the central nervous system (CNS), with more 
than 400,000 cases in the United States. It is the cause of severe disability in 75-
85% of subjects developing the disease1. Recognition that MS is  largely an auto-
reactive lymphocyte-driven disease has led to the development of therapeutic 
strategies which have resulted in decreased risk of clinical relapses, reduction in 
progression of lesion burden on MRI scans, and, in some cases, reduced disability 
progression over the short term, generally for 2 to 3 years2-5. It is not known 
whether there is significant therapy-induced long term reduction in disability 
progression risk, although some recent data suggests that relapse reduction, 
particularly early in the disease course, may be associated with less risk of 
disability progression6. 
 
The current FDA approved therapeutic agents pose significant difficulties. The first-
line 
medications, 
interferon 
beta, 
glatiramer 
acetate, 
teriflunomide,   
monomethyl/dimethyl fumarate esters and diroximel, provide modest efficacy2-5, 
and despite excellent safety profiles, have side effects that are a major obstacle to 
compliance by many patients. The second line medications, natalizumab, 
fingolimod, ozanimod, siponimod, ponesimod, ocrelizumab and ofatumumab, have 
greater efficacy, and are better-tolerated from the perspective of side effects, but 
carry much greater risk of serious adverse events7-10. The need for new therapeutic 
agents continues, with emphasis being placed not only upon improved efficacy and 
tolerability, but also greater safety. 
 
It is well-known that MS clinical disease activity abates during pregnancy, although 
the mechanism(s) of these changes are not understood. Investigations of 
estrogens and progesterone have not provided consistent data supporting their 
potential role in MS protection during pregnancy. Relaxin (RLX), a peptide member 
of the insulin superfamily, is dramatically up-regulated and plays an important role 
in pregnancy. It also is an immune regulator and may contribute to the auto-
tolerance that seems to develop during pregnancy in females with MS. The data 
that supports a potential role for RLX as an auto-immune mediator, and a potential 
therapeutic agent in multiple sclerosis, is presented in the paragraphs that follow. 
 
Perioxisomal Proliferator-activated Receptor-Gamma (PPARG): 
PPARG is part of a superfamily of nuclear hormone, ligand-activated transcription 
factors, that are receptors with DNA-linked and C-terminal ligand-linked domains 
11. Ligand activation of these receptors results in reduced pro-inflammatory and 
increased anti-inflammatory gene expression. PPARG is also constitutionally 
expressed on T-cell lymphocytes and macrophages in the periphery12, on 

Page 2 of 9 
astrocytes and microglia within the CNS, and may not only regulate systemic 
immune responses, but also regulate inflammatory responses within the CNS13.  
PPARG agonists reduce T-cell proliferation 14, inhibit production of the pro-
inflammatory cytokines IL-1B, IL-2,  IL-6, IL-12 15, interferon gamma (IFNG) and 
TNFa 14, and promote T-cell apoptosis 14, 16. PPARG ligand agonists also inhibit 
expression of IFNG-stimulated pro-inflammatory chemokine production by 
endothelial cells17, thus potentially inhibiting inflammatory cell trafficking.  
 
Thiazolidinedone (TZD) anti-glycemic agents, such as pioglitazone and 
rosiglitazone, are PPARG agonist ligands that have both antiglycemic and anti-
inflammatory properties 16. Pioglitazone and rosiglitazone inhibit mitogen-
stimulated T-cell proliferation, as well as TNFa and IFNG production14.   
 
In experimental allergic encephalomyelitis (EAE), used as an animal model of MS, 
TZDs reduce lymphocyte proliferation and infiltration into the CNS18, block IL-12-
induced TH0 differentiation into pro-inflammatory TH1 T cells19,  reduce gene 
expression for IL-1B and IL-6, reduce production of TNFa and IFNG, and reduce 
clinical disease expression 14. 
 
PPARG knockout mice are more susceptible to developing EAE15, and PPARG 
antagonists block the neuroprotective effects of the TZDs in EAE20. In limited 
studies in MS patients, PPARG expression is reduced in peripheral immune cells 
and these cells have reduced responsiveness to PPARG agonists14. 
 
Relaxin biology overview:  
Relaxins (RLX) are members of the insulin-relaxin peptide family21, 22, that do not 
bind to insulin receptors and have no effects on glucose metabolism 23. RLXs have 
been shown to regulate uterine contraction and cervical growth and softening 
during pregnancy24,  are vasodilators and promoters of angiogenesis and may be 
protective in ischemic cardiovascular disease 25. RLX may also affect immune 
function, promoting anti-inflammatory mechanisms. There are 3 known isoforms 
of RLX, RLX 2 being the most abundant circulating form in humans 24.  The gene 
responsible for RLX2 production is on chromosome 926, and  is produced primarily 
in the corpus luteum of females, and  heart, lung and kidney in males 27.  RLX 
acting via its receptor RXFP-1, increases cAMP production22 and nitric oxide 
production via NO synthetase25. 
 
Relaxin as an immune-modulating/regulating agent: 
RLX increases PPARG transcription activity via the receptor Relaxin Family 
Petide-1 (RXFP-1)21, 23, 28, without increasing PPARG mRNA or PPARG protein 
levels28.  RLX independently promotes PPARG via RXRP-1. In addition to its direct 
PPARG promoting effects, it also increases responsiveness PPARG to TZDs such 
as rosiglitazone28.  Given the known anti-inflammatory effects of PPARG 
promotion, this is one possible mechanism by which RLX impacts immune 
function.  A second mechanism likely involves promotion of the glucocorticoid 
receptor (GCR) gene29. Although found in cell nucleus, exogenous RLX can also 

Page 3 of 9 
enter the cell nucleus and up-regulate GCR gene, leading to increased production 
of GCR protein, whereas corticosteroids down-regulate the production of GCR 
protein, thus creating a negative feedback that can be opposed by RLX 
competition for the GCR gene site. RLX blocks mitogen-induced production of IL-
1, IL-6 and TNFa by macrophages via GCR gene activation, which can be blocked 
by the GCR inhibitor RU-48629. 
 
Despite laboratory data demonstrating the potential for RLX as an immune 
regulating molecule via PPARG28 and GCR29 pathways, and its safety in the limited 
settings in which it has been given to humans for treatment of systemic sclerosis30 
and ischemic heart disease 31, there have been no published preclinical or clinical 
studies addressing potential efficacy of RLX in inflammatory CNS disease, and 
only one presented abstract in which it was reported that patients diagnosed with 
MS had higher circulating levels of RLX, and reduced binding affinity to RXFP-1, 
when compared to normal controls, suggesting that the increase in circulating RLX 
was the result of loss of RLX-RXFP-1 regulatory feedback mechanisms.  Further 
analysis revealed that there was structural abnormality in the MS patients’ RLX 
due to a mutation in the B- chain binding cassette of RX (Garvin and Burns, 
personal communication). 
  
 
 
RESEARCH STRATEGY 
1. Because of the potential benefits of using a human molecule, with an 
encouraging safety profile, capable of regulating immune responses, we 
intend, in the future,  to develop a therapeutic trial to assess the efficacy 
and safety of RLX in the treatment of patients with the relapsing form of 
MS.  However there is no information available on such fundamental 
questions as what serum and CSF levels of RLX are observed in patients 
with active versus stable relapsing MS, how these values compare to that 
found in human volunteers without MS or other inflammatory diseases, 
and whether RLX functions normally in subjects with active or stable MS.  
It is for this latter reason that we are proposing research to obtain 
baseline information on serum and CSF RLX levels in patients with MS, 
as well as to further study RXFP-1 receptor binding affinity for RLX in 
patients with active and clinically stable MS. Fortunately there are 
reliable, commercially available SIMOA assays to measure RLX serum 
and CSF concentrations and available cell-based RXFP-1 affinity assay 
systems. 
 
PROTOCOL 
Subjects:  
All subjects will be age 18 to 60, non-pregnant females, since 65 to 80% of MS 
patients are female, and there are known gender differences in circulating RLX 
levels. Patients with known history of diabetes will also be excluded. 
 

Page 4 of 9 
There will be 3 groups of subjects: 
 
Group 1 will consist of 10 subjects with suspected MS, who have  had a clinical 
attack within the last 12 weeks, have at least one gadolinium-enhancing lesion on 
brain or spinal cord MRI taken within the prior 4 weeks, and for which they have 
not received any immunomodulating or immunosupressant medication. Because 
a subject in this group may have had a lumbar puncture in an emergency dept or 
in-patient setting for diagnostic purposes, prior to the investigators becoming 
aware of the patient, consent may be obtained post LP in this circumstance, and 
the analyses performed on residual CSF and serum, provided it had been handled 
in a manner not to interfere with the validity of the analyses.  These subjects will 
also have had a urine pregnancy test performed prior to any relaxin analyses being 
performed on their samples. 
 
Group 2 will consist of 10 subjects with clinically stable definite MS, with no 
evidence of clinical relapse for at least the past 12 weeks, and have no gadolinium 
enhancing lesions on MRI in the prior 12 weeks. These subjects will fulfill the 
Revised (2017) McDonald’s Criteria for the Diagnosis of MS. 
 
Group 3 will consist of 10 subjects without evidence of inflammatory systemic or 
inflammatory CNS disease, who require CSF removal for some other cause, such 
treatment of benign intracranial hypertension or as part of the procedure for 
insertion of an intrathecal medication delivery system. 
 
Study Procedures: 
All subjects will sign an IRB-approved informed consent prior to undergoing any 
study procedures. For patients with reproductive potential, urine sample will be 
collected and processed with the priority of STAT for qualitative pregnancy testing.  
The results of the STAT urine pregnancy test will be reviewed by research staff 
and routed to the study physician. The LP will not proceed unless a negative urine 
pregnancy test is confirmed. The one exception to this will be in Group 1 patients 
who had already had LP performed on an emergency basis and in whom consent 
to analyze residual CSF and serum was obtained. In this latter instance pregnancy 
test result will be obtained before any relaxin analysis is performed.  Every patient 
will provide blood sample within 24 hours before, or 24 hours after the LP. Whole 
blood (8-ml) will be processed and serum (5-mL) collected for study analysis. In 
the case of CSF removal for suspected MS (Group 1), lumbar puncture and CSF 
removal is not a departure from standard of diagnosis/care, however an additional 
5 mL of fluid will be collected for the purpose of this study during the standard 
procedure. In the case of the non-MS controls (Group 3), CSF removal is a routine 
part of their therapy and no additional fluid will be removed (5 mL of their fluid will 
be used for this study). Lumbar puncture and CSF removal is not a routine part of 
the diagnosis or care of patients with stable MS (Group 2), and thus their informed 
consent would reflect the fact that they are volunteering for a clinically unnecessary 
invasive procedure. No patient in Group 2, with clinically stable MS, will be 
responsible for the costs of performing the lumbar puncture, whereas the patients 

Page 5 of 9 
in groups 1 and 3 would be having lumbar puncture in the routine course of the 
management of their disease, and thus the cost of the procedure should be borne 
by their insurer. The total volume of CSF for Group 2 is also 5 mL. 
 
All study samples will be stored frozen before batch transport to Quanterix for 
testing. 
 
Approximately 24 hours after the LP procedure research staff will contact the 
patient by phone to discuss any symptoms or serious adverse events. The 
outcome of the phone call will be routed to the physician and the patient will be 
encouraged to contact the study doctor with any additional questions or concerns. 
All SAEs will be reported the Institutional Review Board per institutional policy and 
guidelines. 
 
Analysis: 
Descriptive statistics will be applied to each variable show the distribution of the 
data. T-tests or nonparametric tests, depending on the type and distribution of the 
data, will be performed to compare the differences between groups. 
 
Results: 
Patients may be informed of the results of their study testing only after the 
completion and analysis of all study data. 
 
CONFIDENTIALITY: 
Each volunteer subject will be assigned a unique identification number, and this 
will be used to identify all samples sent to the diagnostic laboratory. The subjects’ 
names, or any other personal identification information will never be available to 
the diagnostic laboratory or its members. 
 
The results of the data will be returned to the primary investigators for analysis, 
identified by unique code only, and only after the data has been received will the 
group that each sample belongs to be identified. The sole individual who knows 
both personal identification and code numbers will always be blinded to the analytic 
results and will not share the identity of any subject with the other investigators. 
 
ASSAYS 
RLX 1 is measured in serum and CSF by DuoSet (R & D Systems # DY3257) 
sandwich ELISA. This method is RLX 1 specific and does not cross react with RLX 
2, The range of sensitivity of the assay is from 100-10000 pg/mL., and is linear 
within this range.  
 
RLX 2 is measured in serum and CSF by Quantikine (R & D Systems # DRL 200) 
sandwich ELISA.  This method is RLX 2 specific and does not cross react with 
RLX 1. The range of sensitivity of the assay has been demonstrated to be 7.8 to 
500 pg/mL and is linear within this range. 
 

Page 6 of 9 
RLX-RXFP-1 affinity is measured as a function of cAMP production in human 
monocyte THP-1 cells expressing RXFP-1, following incubation with RLX 1 or RLX 
2, both of which act as agonist ligands for this G protein-coupled receptor.  
 
The cAMP levels will also be determined by a commercially available cAMP assay 
(Flash Plate RAI 125 cAMP assay-Perkin Elmer) in the presence and absence RX 
neutralizing antibodies.  All RLX assays will be performed at Quanterix  Labs, 
Lexington MA. . 
 
These analyses will provide us with quantitative data re: baseline RLXlevels in 
serum and CSF in subjects with and without known inflammatory CNS disease, as 
well as alterations in RLX receptor affinity that may occur, and determine whether 
there are differences in serum/CSF RLX levels and RLX receptor affinities during 
acute inflammatory and stable states in patients with the relapsing form of MS. 
 
INSTITUTIONAL SUPPORT 
The Providence Brain and Spine Institute’s clinical research team consists of a 
manager, two full-time research nurses, five full-time clinical research coordinators 
(CRC), and 3 part-time clinical research assistants.  In 2012, the team supported 
more than 30 active clinical trials in Alzheimer’s disease, ALS, multiple sclerosis, 
Parkinson’s disease, and stroke. The research staff has the experience and skills 
in conducting cooperative group trials, industry-sponsored trials, and investigator-
initiated studies. For each trial, the research staff works closely with the 
investigators to coordinate study activities, collect clinical research data, track 
patient and study status, and perform quality assurance activities regularly. The 
research team is familiar with clinical trials that require multiple specimen 
collections, diagnostic testing, and neurological and other clinical assessments. 
The MS Center has 4 full-time providers (5 MS neurologist and 1 NP) and a team 
of clinical support staff that take care of close to 3,000 MS patients. The group has 
successfully collaborated with industry and federally-funded sponsors, clinical 
research organizations, investigators, and ancillary services facilities on more than 
30 MS studies in the past 5 years. 
 
For the performance of the serum and CSF RLX and Sema4A levels and RXFP-1 
RLX receptor affinity analyses, commercial and university laboratories will be 
employed as noted in the protocol.  

Page 7 of 9 
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