**2. Clinical study: enkephalin regulation of IL-17 in multiple sclerosis**

A retrospective clinical study was designed to examine serum levels of cytokines and endogenous peptides, including [Met<sup>5</sup> ]-enkephalin.

### **2.1 Methods**

Patients were identified through the Institute of Personalized Medicine at the Pennsylvania State University College of Medicine and had a clinically definitive diagnosis of relapsing–remitting multiple sclerosis [17, 18, 21, 36]. Selected individuals were between 18 and 70 years of age, of which 17 were males and 36 were females. Cohorts were established based on the disease-modifying therapy each of these patients was receiving. The five groups included multiple sclerosis patients receiving dimethyl fumarate, glatiramer acetate, low-dose naltrexone, or no disease-modifying therapy and a control group of non-multiple sclerosis patients. The 13 non-multiple sclerosis patients were recruited from the Neuroscience Institute and were age and gender matched to the study population. All patients were de-identified to the study team.

Blood was collected and stored in non-heparinized ethylenediaminetetraacetic acid (EDTA) tubes to prevent formation of a clot [65, 66]. Whole blood was centrifuged at 4°C for 10 min at 2500 rpm [65, 66]. Serum was aliquoted and stored long term at −80°C in 200 μL vials to prevent repeated freeze and thaw cycles.

Serum was analyzed using commercially produced sandwich or competitive enzyme labeled immunosorbent assay (ELISA). Opioid growth factor (MBS990622), β-endorphin (MBS770600), IL17A (MBS00565) and IL17 (MBS772095) ELISA kits were manufactured by MyBioSource (San Diego, CA); whereas the TNFα ELISA kit (EK0525) was purchased from BosterBio (Pleasanton, CA). Assays were completed following the manufacturer's recommendations; all samples and standards were run in duplicate and averaged for the data analyses. Chemiluminescence was measured using a BioTek microplate spectrophotometer and Gen5 software at 450 nm.

**77**

*Enkephalin Therapy Improves Relapsing-Remitting Multiple Sclerosis*

To ensure redundancy and reproducibility, duplicate samples were run on multiple

Data analysis was performed using GraphPad Prism 8.0 software. Parametric data were analyzed using one-way analysis of variance (ANOVA) with post-hoc comparisons made using Newman-Keuls. Correlations were determined using Pearson's Correlation Coefficient (R). Significance was determined with a p value

A cohort of 53 patients was analyzed in this study, 40 of whom were individuals with an established diagnosis of relapsing-remitting (RR) multiple sclerosis and 13 who did not have multiple sclerosis. Patients with multiple sclerosis who received no disease-modifying therapy were designated as controls along with the 13 individuals with no diagnosis of multiple sclerosis. Within the control population, 17 were females and 7 males, whereas the study population had 19 females and 10 males. The age distribution between the cohorts of patients on treatment ranged between 25 and 69 years of age while the control cohort ranged in age from 25 to 78 years. Length of disease in the multiple sclerosis patient group was similar between the no-drug, dimethyl fumarate and LDN group which had mean lengths of 14.3, 10.4

Serum OGF levels differed substantially between multiple sclerosis patients who declined therapy and control subjects considered non-multiple sclerosis (**Figure 1A**). OGF levels were more than three-fold higher in non-multiple sclerosis cohort relative to the no-drug cohort (93 ± 26 pg/ml). Because there is no diagnosis provided for the no-drug volunteers, it is difficult to more fully assess these data. Mean serum levels for RR-multiple sclerosis subjects prescribed disease-modifying therapy were comparable to serum values for the no-drug cohort, and significantly less than serum values for the non-multiple sclerosis cohort (**Figure 1B**). RR-multiple sclerosis patients receiving dimethyl fumarate had serum OGF values of 125 ± 22 pg/ml whereas those individuals receiving glatiramer acetate had serum levels of 136 ± 30 pg/ml. RR-multiple sclerosis subjects receiving LDN had mean OGF serum levels of 217 ± 29 mg/ml; these values did not differ from the non-multiple sclerosis cohort. Correlations between serum OGF levels and age or length of disease for cohorts of no drug, DMTs, or LDN are presented in **Figure 2A** and **B**, respectively. The association between age and serum OGF had an overall significance, with dimethyl fumarate subjects revealing a corresponding decrease in OGF serum values with increasing age (p = 0.03). Regarding the length of disease, RR-multiple sclerosis subjects taking LDN had comparable OGF serum values irrespective of having disease for 2 months or 33 years (**Figure 2B**). RR-multiple sclerosis subjects not taking any drugs (no-drug cohort) also displayed a biphasic response, suggesting that length of disease does not impact OGF serum. Analyses of OGF serum values by gender indicated that females had a pattern similar to that in **Figure 1B**. A majority of the males in this study received dimethyl fumarate and had serum OGF levels that ranged from 25 to 200 pg/ml; the small number of male subjects in the other cohorts

Serum β-endorphin levels were also measured in three cohorts of RR-multiple sclerosis subjects and non-multiple sclerosis subjects. Levels of serum β-endorphin ranged between ~1 ng/ml (no-drug and dimethyl fumarate cohorts) to the mean of 2.3 ng/ml (non-multiple sclerosis cohorts); individual samples ranged as high as 4 ng/ml in the non-multiple sclerosis cohort. Despite the variation, significant two-fold increases were recorded for non-multiple sclerosis and glatiramer acetate cohorts relative to no drug and dimethyl fumarate RR-multiple sclerosis

*DOI: http://dx.doi.org/10.5772/intechopen.91010*

ELISA kits from the same manufacturer.

less than 0.05.

and 16.5 years, respectively.

prevented data analyses.

**2.2 Results**

### *Enkephalin Therapy Improves Relapsing-Remitting Multiple Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.91010*

To ensure redundancy and reproducibility, duplicate samples were run on multiple ELISA kits from the same manufacturer.

Data analysis was performed using GraphPad Prism 8.0 software. Parametric data were analyzed using one-way analysis of variance (ANOVA) with post-hoc comparisons made using Newman-Keuls. Correlations were determined using Pearson's Correlation Coefficient (R). Significance was determined with a p value less than 0.05.

## **2.2 Results**

*An Overview and Management of Multiple Chronic Conditions*

patients on disease-modifying therapies.

and endogenous peptides, including [Met<sup>5</sup>

were de-identified to the study team.

restored to normal levels.

**2.1 Methods**

government agencies are reluctant to support new trials. Three clinical trials have suggested that LDN increased the quality of life of patients with relapse-remitting or secondary progressive multiple sclerosis, without serious adverse effects [42, 43, 45]. Two retrospective studies examining charts of patients prescribed LDN alone, as well as in combination with the disease-modifying therapy Copaxone®, revealed no exacerbation of the disease or any substantial side effect for patients in either cohort [44]. In this study, the average length of disease was 14 years, with an average of 3 years on LDN alone. Clinical laboratory data revealed that patients on LDN alone had no significant differences in their blood chemistry, nutrition or liver data from

However, these studies did not measure serum enkephalins, endorphins, or cytokines in an effort to gain more information on the mechanism of action for this biotherapeutic. A small study obtained stored serum from relapsing-remitting multiple sclerosis subjects and reported that enkephalin (i.e., OGF) levels were depressed relative to controls [51]. In the few individuals on LDN alone, serum OGF levels were elevated 2-fold in comparison to multiple sclerosis subjects on Copaxone® alone, suggesting that LDN may be effective at restoring serum enkephalin. Given this information, studies on both the mouse model of EAE and multiple sclerosis have been pursued to evaluate select cytokines that may be dysregulated in multiple sclerosis and possibly modulated by LDN (and enkephalin levels) and

**2. Clinical study: enkephalin regulation of IL-17 in multiple sclerosis**

A retrospective clinical study was designed to examine serum levels of cytokines

Patients were identified through the Institute of Personalized Medicine at the Pennsylvania State University College of Medicine and had a clinically definitive diagnosis of relapsing–remitting multiple sclerosis [17, 18, 21, 36]. Selected individuals were between 18 and 70 years of age, of which 17 were males and 36 were females. Cohorts were established based on the disease-modifying therapy each of these patients was receiving. The five groups included multiple sclerosis patients receiving dimethyl fumarate, glatiramer acetate, low-dose naltrexone, or no disease-modifying therapy and a control group of non-multiple sclerosis patients. The 13 non-multiple sclerosis patients were recruited from the Neuroscience Institute and were age and gender matched to the study population. All patients

Blood was collected and stored in non-heparinized ethylenediaminetetraacetic acid (EDTA) tubes to prevent formation of a clot [65, 66]. Whole blood was centrifuged at 4°C for 10 min at 2500 rpm [65, 66]. Serum was aliquoted and stored long

Serum was analyzed using commercially produced sandwich or competitive enzyme labeled immunosorbent assay (ELISA). Opioid growth factor (MBS990622), β-endorphin (MBS770600), IL17A (MBS00565) and IL17 (MBS772095) ELISA kits were manufactured by MyBioSource (San Diego, CA); whereas the TNFα ELISA kit (EK0525) was purchased from BosterBio (Pleasanton, CA). Assays were completed following the manufacturer's recommendations; all samples and standards were run in duplicate and averaged for the data analyses. Chemiluminescence was measured using a BioTek microplate spectrophotometer and Gen5 software at 450 nm.

term at −80°C in 200 μL vials to prevent repeated freeze and thaw cycles.

]-enkephalin.

**76**

A cohort of 53 patients was analyzed in this study, 40 of whom were individuals with an established diagnosis of relapsing-remitting (RR) multiple sclerosis and 13 who did not have multiple sclerosis. Patients with multiple sclerosis who received no disease-modifying therapy were designated as controls along with the 13 individuals with no diagnosis of multiple sclerosis. Within the control population, 17 were females and 7 males, whereas the study population had 19 females and 10 males. The age distribution between the cohorts of patients on treatment ranged between 25 and 69 years of age while the control cohort ranged in age from 25 to 78 years. Length of disease in the multiple sclerosis patient group was similar between the no-drug, dimethyl fumarate and LDN group which had mean lengths of 14.3, 10.4 and 16.5 years, respectively.

Serum OGF levels differed substantially between multiple sclerosis patients who declined therapy and control subjects considered non-multiple sclerosis (**Figure 1A**). OGF levels were more than three-fold higher in non-multiple sclerosis cohort relative to the no-drug cohort (93 ± 26 pg/ml). Because there is no diagnosis provided for the no-drug volunteers, it is difficult to more fully assess these data. Mean serum levels for RR-multiple sclerosis subjects prescribed disease-modifying therapy were comparable to serum values for the no-drug cohort, and significantly less than serum values for the non-multiple sclerosis cohort (**Figure 1B**). RR-multiple sclerosis patients receiving dimethyl fumarate had serum OGF values of 125 ± 22 pg/ml whereas those individuals receiving glatiramer acetate had serum levels of 136 ± 30 pg/ml. RR-multiple sclerosis subjects receiving LDN had mean OGF serum levels of 217 ± 29 mg/ml; these values did not differ from the non-multiple sclerosis cohort. Correlations between serum OGF levels and age or length of disease for cohorts of no drug, DMTs, or LDN are presented in **Figure 2A** and **B**, respectively. The association between age and serum OGF had an overall significance, with dimethyl fumarate subjects revealing a corresponding decrease in OGF serum values with increasing age (p = 0.03). Regarding the length of disease, RR-multiple sclerosis subjects taking LDN had comparable OGF serum values irrespective of having disease for 2 months or 33 years (**Figure 2B**). RR-multiple sclerosis subjects not taking any drugs (no-drug cohort) also displayed a biphasic response, suggesting that length of disease does not impact OGF serum. Analyses of OGF serum values by gender indicated that females had a pattern similar to that in **Figure 1B**. A majority of the males in this study received dimethyl fumarate and had serum OGF levels that ranged from 25 to 200 pg/ml; the small number of male subjects in the other cohorts prevented data analyses.

Serum β-endorphin levels were also measured in three cohorts of RR-multiple sclerosis subjects and non-multiple sclerosis subjects. Levels of serum β-endorphin ranged between ~1 ng/ml (no-drug and dimethyl fumarate cohorts) to the mean of 2.3 ng/ml (non-multiple sclerosis cohorts); individual samples ranged as high as 4 ng/ml in the non-multiple sclerosis cohort. Despite the variation, significant two-fold increases were recorded for non-multiple sclerosis and glatiramer acetate cohorts relative to no drug and dimethyl fumarate RR-multiple sclerosis

### **Figure 1.**

*Scatterplots of serum OGF levels (pg/ml) in (A) control subjects who were either non-multiple sclerosis subjects or (B) RR-MS patients who were not receiving any therapy (no drug) or RR-multiple sclerosis patients receiving dimethyl fumarate, glatiramer acetate, or low dose naltrexone (LDN). One-way ANOVA or t-tests were used to show the differences between groups. Significantly different from non-multiple sclerosis values at p < 0.01 (\*\*).*

individuals. Associations between age and β-endorphin revealed that endorphin levels were relatively stable within the population of RR-multiple sclerosis subjects on DMT.

With regard to serum levels of inflammatory cytokines, expression levels of two cytokines in serum are presented in **Figure 3**. Serum levels of IL-17 (A) and TNFα (B) were measured in RR-multiple sclerosis subjects on DMTs, LDN, or no-drug, as well as non-multiple sclerosis controls. Mean IL-17A (not shown) concentrations ranged from 27 ± 3 pg/ml (dimethyl fumarate) to 39 ± 10 pg/ml (glatiramer acetate group). Only one sample of blood from RR-multiple sclerosis patients on LDN was assayed; serum IL-17A value was 46 pg/ml. No differences in serum levels were noted between any therapeutic treatment and controls. IL-17 levels ranged from the mean 0.12 ng/ml recorded for non-multiple sclerosis cohort to 0.92 ± 0.35 ng/ml for the dimethyl fumarate group (**Figure 3A**); levels of the IL-17 cytokine recorded for these subjects differed significantly from glatiramer acetate and no-drug cohorts. RR-multiple sclerosis patients had a mean IL-17 cytokine level of near 0, whereas some individuals using dimethyl fumarate had more than 3 pg/ml IL-17 levels.

**79**

**Figure 2.**

with OGF serum values.

**2.3 Discussion and summary**

*P values less than 0.05 were considered significant.*

*Enkephalin Therapy Improves Relapsing-Remitting Multiple Sclerosis*

Non-multiple sclerosis and RR-multiple sclerosis patients not taking any DMT had comparable TNFα values (16 ± 10 and 22 ± 3 pg/ml, respectively) (**Figure 3B**). RR-multiple sclerosis cohorts on DMTs had TNFα serum values that were at least 6-fold higher and differed significantly from the non-multiple sclerosis levels. Correlation analyses indicated that OGF (**Figure 4A**) and IL-17A (**Figure 4B**) were positively associated (R = 0.82), whereas TNFα values did not correlate (R = 0.20)

*Expression levels of serum OGF (pg/ml) measured by ELISA as a function of the age (years) of subject (A) or the length of disease (years) (B). Correlations were determined by Pearson's Correlation Coefficient (R) tests.* 

Cohorts of RR-multiple sclerosis patients on disease-modifying therapy, as well as a group of individuals diagnosed with RR-multiple sclerosis and taking LDN only were compared with control volunteers. in an assessment of peripheral serum levels of two endogenous neuropeptides—OGF (chemically termed methionine enkephalin) and β-endorphin, as well as 3 cytokines that are known to be involved

*DOI: http://dx.doi.org/10.5772/intechopen.91010*

*Enkephalin Therapy Improves Relapsing-Remitting Multiple Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.91010*

### **Figure 2.**

*An Overview and Management of Multiple Chronic Conditions*

individuals. Associations between age and β-endorphin revealed that endorphin levels were relatively stable within the population of RR-multiple sclerosis subjects

*Scatterplots of serum OGF levels (pg/ml) in (A) control subjects who were either non-multiple sclerosis subjects or (B) RR-MS patients who were not receiving any therapy (no drug) or RR-multiple sclerosis patients receiving dimethyl fumarate, glatiramer acetate, or low dose naltrexone (LDN). One-way ANOVA or t-tests were used to show the differences between groups. Significantly different from non-multiple sclerosis values at* 

With regard to serum levels of inflammatory cytokines, expression levels of two cytokines in serum are presented in **Figure 3**. Serum levels of IL-17 (A) and TNFα (B) were measured in RR-multiple sclerosis subjects on DMTs, LDN, or no-drug, as well as non-multiple sclerosis controls. Mean IL-17A (not shown) concentrations ranged from 27 ± 3 pg/ml (dimethyl fumarate) to 39 ± 10 pg/ml (glatiramer acetate group). Only one sample of blood from RR-multiple sclerosis patients on LDN was assayed; serum IL-17A value was 46 pg/ml. No differences in serum levels were noted between any therapeutic treatment and controls. IL-17 levels ranged from the mean 0.12 ng/ml recorded for non-multiple sclerosis cohort to 0.92 ± 0.35 ng/ml for the dimethyl fumarate group (**Figure 3A**); levels of the IL-17 cytokine recorded for these subjects differed significantly from glatiramer acetate and no-drug cohorts. RR-multiple sclerosis patients had a mean IL-17 cytokine level of near 0, whereas some individuals using dimethyl fumarate had more than 3 pg/ml IL-17 levels.

**78**

on DMT.

*p < 0.01 (\*\*).*

**Figure 1.**

*Expression levels of serum OGF (pg/ml) measured by ELISA as a function of the age (years) of subject (A) or the length of disease (years) (B). Correlations were determined by Pearson's Correlation Coefficient (R) tests. P values less than 0.05 were considered significant.*

Non-multiple sclerosis and RR-multiple sclerosis patients not taking any DMT had comparable TNFα values (16 ± 10 and 22 ± 3 pg/ml, respectively) (**Figure 3B**). RR-multiple sclerosis cohorts on DMTs had TNFα serum values that were at least 6-fold higher and differed significantly from the non-multiple sclerosis levels. Correlation analyses indicated that OGF (**Figure 4A**) and IL-17A (**Figure 4B**) were positively associated (R = 0.82), whereas TNFα values did not correlate (R = 0.20) with OGF serum values.

### **2.3 Discussion and summary**

Cohorts of RR-multiple sclerosis patients on disease-modifying therapy, as well as a group of individuals diagnosed with RR-multiple sclerosis and taking LDN only were compared with control volunteers. in an assessment of peripheral serum levels of two endogenous neuropeptides—OGF (chemically termed methionine enkephalin) and β-endorphin, as well as 3 cytokines that are known to be involved

### **Figure 3.**

*Scatterplots of serum cytokines IL-17 (A) and TNFα (B) measured by ELISA tests (pg/ml) in RR-multiple sclerosis patients receiving DMTs or no drug; controls include volunteer non-multiple sclerosis subjects. Data was analyzed using one-way ANOVA with subject comparisons made by Newman-Keuls tests. Significantly different at p < 0.05 (\*) and p < 0.01 (\*\*).*

in RR-multiple sclerosis, specifically IL-17, IL-17A, and TNFα [67–71]. Inclusion criteria restricted our analyses to RR-MS subjects to those receiving glatiramer acetate or dimethyl fumarate. A few RR-multiple sclerosis subjects in this study were only prescribed LDN as an off-label product.

The restricted access to blood samples and corresponding patient data in this retrospective study limited the findings, as did inclusion of RR-multiple sclerosis patients on no drug. No rationale was reported in the REDCap database to explain why the RR-multiple sclerosis patients had denied therapy. It is conjured that some multiple sclerosis patients who have stabilized over a long period of time request to be removed from therapy.

The focus of this study was to determine the relationship between serum expression levels of endogenous neuropeptides, specifically OGF, and a select group of pro-inflammatory cytokines. OGF levels in the serum of RR-multiple sclerosis subjects declined with age and were not associated with the length of disease. In general, the OGF levels of RR-multiple sclerosis patients using DMTs, or those on no therapy, were substantially lower than RR-multiple sclerosis subjects on LDN. This relationship between OGF and RR-multiple sclerosis has been reported previously [51], suggesting that the biotherapeutic LDN treatment is producing enkephalins. Clinical studies have reported that LDN treatment resulted in perceived increased

**81**

**Figure 4.**

RR-multiple sclerosis.

*Enkephalin Therapy Improves Relapsing-Remitting Multiple Sclerosis*

quality of life [42, 43] and stabilization of disease symptomatology [44]. A second neuropeptide, β-endorphin, is known as the "feel-good" hormone and has been reported to increase during exercise and pregnancy [72] and to increase during multiple sclerosis remission [73]. In this study, β-endorphin levels were comparable in all subjects suggesting that these RR-multiple sclerosis patients had stabilized endorphins. Based on the scatter gram plots of individual expression levels of endorphin, some of the individuals in the glatiramer acetate group may have been in remission. Moreover, whereas OGF levels fluctuate with RR-multiple sclerosis and different therapies, β-endorphin does not appear to be altered by DMTs in

*Associations of cytokine expression between IL-17A (pg/ml) and OGF (pg/ml) (A) or TNFα (pg/ml) and OGF (B) for RR-multiple sclerosis subjects receiving a DMT. Data were analyzed by Pearson's Correlation* 

*Coefficient (R) tests. P values less than 0.05 were considered significant.*

The proinflammatory cytokine IL-17 may be a prime indicator of RR-multiple sclerosis progression [69, 71]. The IL-17 family of cytokines is produced by CD4<sup>+</sup> T-helper cells known as Th17 cells. At least 7 polymorphisms have been identified, with IL-17A and IL-17F most frequently associated with neutrophils activation in

*DOI: http://dx.doi.org/10.5772/intechopen.91010*

*Enkephalin Therapy Improves Relapsing-Remitting Multiple Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.91010*

**Figure 4.**

*An Overview and Management of Multiple Chronic Conditions*

in RR-multiple sclerosis, specifically IL-17, IL-17A, and TNFα [67–71]. Inclusion criteria restricted our analyses to RR-MS subjects to those receiving glatiramer acetate or dimethyl fumarate. A few RR-multiple sclerosis subjects in this study were only

*Scatterplots of serum cytokines IL-17 (A) and TNFα (B) measured by ELISA tests (pg/ml) in RR-multiple sclerosis patients receiving DMTs or no drug; controls include volunteer non-multiple sclerosis subjects. Data was analyzed using one-way ANOVA with subject comparisons made by Newman-Keuls tests. Significantly* 

The restricted access to blood samples and corresponding patient data in this retrospective study limited the findings, as did inclusion of RR-multiple sclerosis patients on no drug. No rationale was reported in the REDCap database to explain why the RR-multiple sclerosis patients had denied therapy. It is conjured that some multiple sclerosis patients who have stabilized over a long period of time request to

The focus of this study was to determine the relationship between serum expression levels of endogenous neuropeptides, specifically OGF, and a select group of pro-inflammatory cytokines. OGF levels in the serum of RR-multiple sclerosis subjects declined with age and were not associated with the length of disease. In general, the OGF levels of RR-multiple sclerosis patients using DMTs, or those on no therapy, were substantially lower than RR-multiple sclerosis subjects on LDN. This relationship between OGF and RR-multiple sclerosis has been reported previously [51], suggesting that the biotherapeutic LDN treatment is producing enkephalins. Clinical studies have reported that LDN treatment resulted in perceived increased

prescribed LDN as an off-label product.

*different at p < 0.05 (\*) and p < 0.01 (\*\*).*

be removed from therapy.

**80**

**Figure 3.**

*Associations of cytokine expression between IL-17A (pg/ml) and OGF (pg/ml) (A) or TNFα (pg/ml) and OGF (B) for RR-multiple sclerosis subjects receiving a DMT. Data were analyzed by Pearson's Correlation Coefficient (R) tests. P values less than 0.05 were considered significant.*

quality of life [42, 43] and stabilization of disease symptomatology [44]. A second neuropeptide, β-endorphin, is known as the "feel-good" hormone and has been reported to increase during exercise and pregnancy [72] and to increase during multiple sclerosis remission [73]. In this study, β-endorphin levels were comparable in all subjects suggesting that these RR-multiple sclerosis patients had stabilized endorphins. Based on the scatter gram plots of individual expression levels of endorphin, some of the individuals in the glatiramer acetate group may have been in remission. Moreover, whereas OGF levels fluctuate with RR-multiple sclerosis and different therapies, β-endorphin does not appear to be altered by DMTs in RR-multiple sclerosis.

The proinflammatory cytokine IL-17 may be a prime indicator of RR-multiple sclerosis progression [69, 71]. The IL-17 family of cytokines is produced by CD4<sup>+</sup> T-helper cells known as Th17 cells. At least 7 polymorphisms have been identified, with IL-17A and IL-17F most frequently associated with neutrophils activation in

autoimmunity [67, 69]. IL-17A has been identified in the CNS following migration of Th17 cells in response to a pro-inflammatory event [74]. Clinical studies have reported that IL-17A may be responsible for breakdown of the blood-brain barrier facilitating the entrance of other inflammatory cytokines into the CNS [71]. IL-17A has been shown in preclinical studies to accelerate glial activation leading to neuroinflammation and neurodegeneration [68]. Thus, it would be expected that IL-17A levels may be lowest in RR-multiple sclerosis individuals who are in remission or most responsive to therapy [67]. Moreover, recent preclinical studies have reported that both IL-17A and IL-17F increase pro-inflammatory cytokine IL-6 secretion [70].

In the present study, TNFα, a proinflammatory marker has been shown to increase in RR-multiple sclerosis [75, 76] and to be consistently elevated regardless of the status of disease type (i.e., clinically isolated syndrome, primary progressive, or relapsing-remitting), with serum levels 40–50% higher in multiple sclerosis subjects relative to controls. Preclinical studies have reported an association between TNFα and impaired memory in mice with EAE [77] and implicated astrocyte signaling as the downstream target of overexpression of the cytokine. Despite studies whereby TNFα knockdown mice had more severe EAE [69] and therapeutic administration of the cytokine protected against the disease [74], this cytokine is reported to be a major contributor to cognitive deficits related to late stages of multiple sclerosis. In the present study, age, gender, number of relapses, or length of disease had no effect of TNFα levels, but in general, the expression levels of this cytokine were elevated in RR-multiple sclerosis subjects relative to cohorts of RR-multiple sclerosis patients presumably in remission or non-multiple sclerosis.

LDN is an off-label therapeutic used in substantially lower dosages than prescribed for drug overdose or alcohol use (3 vs. 50 mg). Its use is increasing worldwide [78, 79] and is consistently reported to be well-tolerated over extended periods of time [46]. Small clinical trials have reported few, if any, side effects [42, 43]. In this study, OGF serum levels appear to be correlated with IL-17A. Alternatively, whereas β-endorphin has been associated with the propensity to avoid alcohol or opioids, it was not related to RR-multiple sclerosis. The serum levels of cytokines IL-17A and TNFα did not appear to be discriminating biomarkers at least with the current population of RR-multiple sclerosis subjects. Perhaps with additional study, and possibly other DMTs, these cytokines will be able to discern progression of disease.

## **3. Conclusions**

In summary, the preclinical and clinical data illustrate that enkephalin levels (i.e., OGF) are decreased in animals with EAE and humans with relapsing remitting multiple sclerosis. LDN as a biotherapy is associated with elevating enkephalins and from all aspects, appears to reduce symptomatology of multiple sclerosis. LDN therapy to upregulate the body's own production of enkephalins has been shown by a number of clinical trials to be a safe adjuvant, or primary, treatment for RR-multiple sclerosis. LDN continues to be associated with stabilizing multiple sclerosis and does not appear to interfere with other disease-modifying therapies. Whether enkephalin levels directly or indirectly alter the therapeutic pathways is unclear at this time. Moreover, it is difficult to determine at this point whether specific disease-modifying therapies are more advantageous for manipulating enkephalin levels. However, it is evident that restored serum enkephalin expression is associated with reduced inflammatory cytokines and better patient outcome. Moving forward, studies will be conducted to determine the mechanistic role of

**83**

*Enkephalin Therapy Improves Relapsing-Remitting Multiple Sclerosis*

OGF in modulating both pro and anti-inflammatory cytokines. Collectively the data from published studies as well as the new data presented in this report demonstrate that the biotherapeutic LDN and resulting enkephalin (specifically OGF) levels play a role in disease progression of multiple sclerosis. By integrating animal model work and patient serum analysis, future studies will try and understand the role of OGF during initiation of disease and definitive diagnosis of disease. The efficacy of LDN alone needs to be evaluated in prospective, randomized, controlled studies, but unfortunately this design is not forthcoming as most physicians will prescribe the

This research was supported in part by a generous gift from The Paul K. and Anna E. Shockley Family Foundation, and private donations to the Penn State LDN

The authors acknowledge the support of Indira Purushothaman from the McLaughlin and Zagon lab as well as Syndi Reed and Trang Doan from the Institute

At the time of preparation of this manuscript, PJM, ISZ, and CLP had no personal or financial conflicts or disclosures, and GAT was on the speaker bureau

Chirag L. Patel, Ian S. Zagon, Gary A. Thomas and Patricia J. McLaughlin\* The Pennsylvania State University College of Medicine, Hershey, Pennsylvania,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: pxm9@psu.edu

provided the original work is properly cited.

of Personalized Medicine at Penn State College of Medicine.

for Biogen, but had no equity interest in the product (Tecfidera®).

*DOI: http://dx.doi.org/10.5772/intechopen.91010*

off-label drug based on its safety record.

**Acknowledgements**

**Author declaration**

**Author details**

United States

Fund.

*Enkephalin Therapy Improves Relapsing-Remitting Multiple Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.91010*

OGF in modulating both pro and anti-inflammatory cytokines. Collectively the data from published studies as well as the new data presented in this report demonstrate that the biotherapeutic LDN and resulting enkephalin (specifically OGF) levels play a role in disease progression of multiple sclerosis. By integrating animal model work and patient serum analysis, future studies will try and understand the role of OGF during initiation of disease and definitive diagnosis of disease. The efficacy of LDN alone needs to be evaluated in prospective, randomized, controlled studies, but unfortunately this design is not forthcoming as most physicians will prescribe the off-label drug based on its safety record.
