Multiple sclerosis (MS) is a debilitating disease with a multifactorial etiology that remains to be fully defined. In the United States alone it is estimated that there are one million individuals living with MS [1,2]. Development of MS involves inflammatory reactions against self-antigens and progressive demyelination in the brain and spinal cord. Epidemiological data on MS indicate a 2:1 ratio of women to men with a worsening disease prognosis for men than for women [1]. In addition to a genetic predisposition [3], geography [4] and sun exposure increasing melatonin [5] and vitamin D levels [6] are risk factors for the development of MS. The disorder is prevalent in younger individuals of Northern European ancestry who may have an accumulation of genetic and biological risk factors, one or more of which may be involved in pathways that have become dysregulated.

Clinical reports first described a relationship between enkephalins, endorphins, and MS in individuals receiving low doses of naltrexone (LDN) who reported that they “felt” better following this non-traditional treatment [7-9]. A retrospective chart review of more than 50 MS patients, 23 receiving LDN alone, revealed that in comparison to the diseasemodifying therapy (DMT) Copaxone, LDN maintained stable physiological signs, with no change in MRIs, and health in the RR-MS individuals [9]. Evaluation of MRIs, blood laboratory data (e.g., white cell counts, platelet count, hemoglobin levels, liver enzymes), and behavior as assessed by a timed 25-foot walk revealed no differences in values for patients on Copaxone and those on LDN only [9].

Because of the variability in patient profiles, as well as the broad array of DMTs that are now available, it is difficult to perform rigorous and reproducible studies on humans. Given our understanding of the Opioid Growth Factor (OGF) - OGF receptor (OGFr) axis, and the role that naltrexone plays in modulating this pathway, we investigated the role of OGF and LDN utilizing the experimental autoimmune encephalomyelitis (EAE) mouse model [10-18]. Different antigens and strains of mice can be used to develop chronic progressive EAE (Ch-EAE) or relapsing-remitting EAE (RREAE). Accumulated evidence from our research supports the hypothesis that down-regulation of the OGF-OGFr pathwayl disease and central nervous system pathology [12-20]. resulting in decreased blood levels of OGF are associated with a poor prognosis for MS [10,11,19] or EAE [19,20]. Although animal models of MS are imperfect, they provide a reliable platform to assess independent variables in a controlled setting. Manipulation of the OGF-OGFr axis in Ch-EAE mice by injecting OGF or intermittent blockade of the pathway with LDN provides a reproducible animal model enabling rigorous investigations. Data demonstrated that increased blood levels of OGF resulted in positive outcomes and reduction in clinical disease and central nervous system pathology [12-20].

Initial preclinical work utilized the EAE model that was established by immunization with myelin oligodendrocytic glycoprotein (MOG) of female C57Bl/6J mice. The regimen produced behavioral patterns that resembled primary progressive MS or a chronic course of behavioral decline until full paralysis was observed and animals were humanely euthanized. This Ch-EAE model presents with a caudal to rostral pathology detected by demyelination of the spinal cord and glial cell activation [e.g., 12,13]. Daily treatment with OGF beginning at the time of disease induction delayed, and even prevented in some mice, the onset of EAE which was otherwise present in all saline-treated mice inoculated with MOG within 3 weeks of immunization [13]. Behavioral scores of limp tail, wobbly gait, and hind limb paralysis were markedly reduced in OGF-treated EAE mice relative to saline-treated EAE mice.

Recognizing that clinical therapy routinely begins after symptoms and signs of MS have appeared for a number of weeks or months, another mouse paradigm was established to study a more clinically relevant model. Mice were immunized with MOG and treatment began 2 days after the first appearance of disease (i.e., limp tail or wobbly gait). OGF therapy reversed the disease progression; disease scores of the OGF-treated EAE mice declined significantly from those of saline-treated EAE mice. Moreover, spinal cord pathology and astrocyte activation were substantially reduced in the EAE mice receiving OGF [14].

RR-MS represents more than 85% of all individuals with MS [1], warranting preclinical investigations in a mouse model of RR-EAE. Female SJL mice were immunized with proteolipid proteins to establish such a model. Several studies conducted on RR-EAE have documented that induction of this form of disease is variable, and lends itself to having incomplete responses [15,16]. Some animals are responders to therapy and others are non-responders. While this phenomenon is not unique to EAE, and is particularly evident in addiction studies, it presents some difficulty in the interpretation of data. Several studies that established RR-EAE showed repeatedly that mice injected daily with OGF had lower behavioral scores and reduced disease severity [15]. The length of remissions as measured by a return to a disease score of 0.5 or less was significantly greater in terms of days, as was the length of time mice had mild disease for mice receiving OGF.

Investigations on the treatment of OGF using established RR-EAE mouse models were completed with daily injections of OGF beginning 2 days after behavioral signs of disease were observed [16] and continued once daily for 40 days. OGF treated RR-EAE mice had markedly reduced clinical signs of disease, more periods of remission with each one being longer that those recorded for saline-injected RR-EAE mice. OGF inhibited proliferation of T lymphocytes, reduced the number of Iba-1+ cells and CD3+ cells, and decreased proliferating astrocytes. Exploration of the mechanism of OGF and LDN therapy in Ch-EAE mice revealed that these treatments delayed CNS infiltration of CD4+ T lymphocytes, another sign of inflammation [17]. Thus, multiple studies demonstrated that daily injections of OGF could reduce the severity of both relapsing remitting and chronic forms of established EAE.

Once we established that manipulation of the OGF-OGFr axis was involved in EAE progression, we focused on determining the serum levels of endogenous peptides, OGF and β-endorphin, in Ch- EAE mice. Within 5 days of immunization of MOG to 6 week old C57BL/6J female mice, OGF levels were significantly reduced (~70 pg/ml) from normal levels of approximately 150 pg/ml [18]. The deficits in OGF preceded the appearance of any clinical behavioral signs by at least 4 days. Treatment of EAE mice with injections of 10 mg/kg OGF daily increased the blood levels of OGF above those of saline-treated EAE mice, but still below those in normal mice. However, the serum OGF levels correlated with reduced open field movement and von Frey sensitivity, and were inversely correlated with disease severity. Thus, the progression of disease corresponded with declining OGF blood levels.

Investigations examined whether the timing of OGF administration to mice altered their immune response. In a well-controlled set of experiments, female mice were immunized with MOG and received OGF prophylactically beginning at the time of disease induction or as traditional therapy once clinical behavior was observed for 2 days [20]. Prophylactic OGF delayed the onset of disease behavior and suppressed lymphocyte and neutrophils replication. Traditional treatment regimens of OGF resulted in a reversal of the clinical behavior, restored OGF serum levels, and inhibited microglial activation within an 8 day period of time. Longer studies were aborted due to the pandemic. This work suggests that treatments to increase serum OGF levels should begin as early as possible following diagnosis of MS, and that maintenance of blood levels of OGF is a positive biomarker of disease progression.

Clinical studies on MS are complicated by a number of factors including length of MS disease, age of patient at the onset of disease, and the patient’s self-reported symptoms. Moreover, OGF is not FDA approved for clinical use in humans. An alternative therapy is low dosages of an opioid receptor antagonist such as naltrexone that invokes a biofeedback mechanism to increase production and/or release of endogenous opioids including β-endorphin and [Met5]- enkephalin. In clinical studies, elevated levels of enkephalins, specifically OGF, as well as β-endorphin, were measured following either disease modifying therapy or LDN leading to self-reported higher scores on the MS-QoL survey [9,10]. A clinical study (IRB protocol 9784) conducted with consented volunteer patients at the Penn State Hershey Neurology Clinic revealed that serum [Met5]-enkephalin (i.e., OGF) levels were lower in MS patients relative to values from non-MS control humans [10]. LDN therapy restored OGF levels to near normal, but small sample size and demographic variability in length of disease and treatment were too large for reliable comparisons.

In a follow-up study, a larger cohort of individuals with diagnosed RR-MS was consented and blood samples assayed for OGF levels, IL-17A and TNFα cytokines; comparisons were made to blood samples for age and sex matched non- MS individuals [10]. Serum samples were analyzed using commercial ELISA kits for OGF, β-endorphin, IL17A, 1L17, and TNFα. Data revealed that MS diagnosis resulted in lower OGF serum values, even in patients on disease modifying therapy relative to OGF levels in non-MS individuals; a small cohort of RR-MS individuals on LDN had OGF comparable to those of non-MS individuals, but sample size was too small for statistical reliability. Serum β-endorphin levels ranged between 1 and 4 ng/ml, and were elevated in RR-MS individuals receiving glatiramer acetate therapy. A direct correlation was found between OGF levels and IL- 17A cytokine values, the pro-inflammatory T-helper cell marker, but no correlation was noted between OGF and TNFα values. Sample sizes and treatment variability prevented additional reliable comparisons. Additional research is needed to expand on the observations that OGF inhibits activated T and B cells in EAE with a focus on whether this mechanism can be modulated by LDN [21].

Preclinical and clinical data reveal that OGF serum levels are significantly decreased in mouse models of EAE and in RR-MS. Treatment with OGF for mice, or LDN for humans, appears to increase serum OGF leading to improved disease outcomes. In Ch-EAE and RR-EAE mice models, OGF treatment reduced behavioral signs, decreased spinal cord pathology, and correlated with a cytokine profile supporting immunological repair. In humans, restored serum enkephalin is associated with a reduced inflammatory cytokine profile. Data distinguishing whether different disease modifying therapies are more likely to alter OGF serum levels are warranted. Collectively, higher serum OGF levels are predictive of better patient outcome. The addition of OGF serum values to the diagnostic panel for MS may be warranted. This would enable physicians to begin early treatment with LDN or another disease modifying therapy.

The authors declare no conflict of interest.

All research studies on MS and/or EAE were supported in part by the Paul K. and Anna E. Shockey Family Foundation and the LDN Research Fund.

The authors acknowledge the contributions to this research by former graduate students in our laboratory.

Authors have read and edited drafts and the final version of this review.