PE1296/N

Jill P. Smith, MD Penn State College of Medicine Tel: (717) 531-3694 Professor of Medicine Penn State Milton S Hershey Medical Center Fax: (717) 531-6770 Gastroenterology & Hepatology, H-045 [email protected] 500 University Drive, PO Box 850 Hershey, PA 17033-0850

Franck David Assistant Clerk to the Public Petitions Committee Parliament of Scotland, UK TG.01 Edinburgh

January 8, 2010

Dear Mr. David: I am writing in response to your inquiry regarding a petition by Robert Thomson on behalf of LDN to the Scottish Parliament requesting information. The petition information you provided is included in the paragraph below: Petition by Robert Thomson on behalf of LDN Now Scotland calling on the Scottish Parliament to urge the Scottish Government to make Low Dose Naltrexone readily available on the NHS to auto-immune disease sufferers as well as other conditions not classified as auto-immune such as HIV/AIDS, cancer and fertility, in each NHS board area thereby reducing the danger of sufferers having to access riskier alternatives and also incurring higher costs by purchasing the drug through private medical providers and to provide guidance to all GPs on LDN protocol and require them to collect LDN clinical data.

You have asked me to respond to specifics in the petition and answer the questions below:

™ What is the clinical evidence of the effectiveness of LDN (providing that the clinical trials/tests you carried out were for Naltrexone in 4.5mg or lower dosage)? ™ Is there any clinical and scientific evidence that Naltrexone in 4.5mg or lower dosage is unsafe? ™ Has any guidance to clinicians in the USA about LDN and its clinical uses been issued?

I am a board certified gastroenterologist at a University hospital (Pennsylvania State University) and I have conducted research in both animals and human subjects using naltrexone for Crohn’s disease. I have not conducted research on any of the other disorders listed in the petition and therefore, I am not eligible to comment on them. We carried out a study in mice where we induced colitis with a chemical and treated the mice with injections of naltrexone. We found that PE1296/N

the naltrexone decreased inflammatory cytokine expression and improved the histology of the colon lining under the microscope. This work was published in the J Immunotoxicology 15:179- 187, 2008 (attached) and was the basis for the dose used in the human trials. Our first clinical human trial published in the Am J Gastroenterology in 2007 (attached) showed that naltrexone orally at 4.5 mg per day improved active Crohn’s disease in 89% and induced remission in 67% of those treated. We have extended our research and recently completed a clinical double blind placebo controlled trial testing the efficacy of naltrexone versus placebo in 40 adult subjects with active Crohn’s disease. Although the research is complete, it has not yet been published; therefore, I cannot release any of these results yet. We are currently also conducting a clinical trial in children with active Crohn’s disease where we treat with either capsules or liquid at 0.1 mg/kg not to exceed 4.5 mg. I have applied to the Food and Drug Administration (FDA) in the USA for FDA Orphan Drug Status for naltrexone in children. Currently naltrexone is approved by the US FDA at a dosage of 50mg for alcohol withdrawal syndromes. Our animal studies suggest that the higher dose may not work as effectively at reversing inflammation as the lower dose. Naltrexone is not approved in the USA for Crohn’s disease therapy or at the 4.5 mg dosing. In order for me to treat patients with this medication I had to apply for a license from the FDA and be given an Investigator New Drug number. I am required to keep accurate records and file annual reports to the FDA regarding subjects treated with naltrexone in our clinical trials. I also have submitted a patent for the use of naltrexone in inflammatory bowel disease using the lower dosing. This application was filed in the USA, UK and many other international countries and is currently under review. We have also applied to the National Institutes of Health for additional research funding to answer your question about dosing; ie., to try both higher and lower doses. There is no scientific data on the use of naltrexone as doses different from our current publications to accurate answer your second question. Regarding your 3rd question about guidance provided to US physicians prescribing naltrexone, my comment is that most physicians are not prescribing it because it is not yet approved and is considered experimental. Although some physicians may prescribe it ‘off-label’, this practice is not supported by the FDA or the American Medical Association.

Because the current treatments available for Crohn’s patients include biological agents and immunomodulating drugs that suppress the immune system, there is a serious risk of developing infections and even malignancies with these drugs. Because of the increased incidence of leukemias and hepatosplenic lymphomas using infliximab, the UDA FDA has issued a black box warning on the use of biologics in patients. Also the cost of biologics is extreme, and that along with the potential risks is why there is a desperate need for safer drugs to treat patients with Crohn’s disease. Naltrexone is an inexpensive medication that has the advantage of being given orally. In our human studies the side effects were minimal and the results very good. Although we believe more studies using this compound will improve our understanding and use of it in the future, I cannot encourage its use outside of medically monitored trials at the present time. If the FDA supports our application for approval for this indication and at this dose, that will open the door for new treatments of patients with Inflammatory bowel disease and also spark research in other areas of autoimmune conditions as mentioned in the petition above.

PE1296/N

I hope this communication will help you in responding to the petition. Sincerely Jill P. Smith, MD Professor of Medicine Penn State University College of Medicine H-045; 500 University Drive Hershey, PA 17033 717-531-3694

Journal of Immunotoxicology, 15:179–187, 2008 Copyright c Informa Healthcare USA, Inc. ISSN: 1547-691X print / 1547-6901 online DOI: 10.1080/15476910802131469 The Opioid Antagonist Naltrexone Improves Murine Inflammatory Bowel Disease

Gail L. Matters Departments of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA John F. Harms and Christopher McGovern Department of Medicine, Pennsylvania State University College of Medicine, Hershey, Pennsylvania Leo Fitzpatrick Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA Anuj Parikh, Nicholas Nilo, and Jill P. Smith Department of Medicine, Pennsylvania State University College of Medicine, Hershey, Pennsylvania

less weight loss, lower DAI scores, and less histologic evidence of Inflammatory bowel disease (IBD) is a condition of the intes- inflammation compared to controls. Significantly, elevated levels tine with significant morbidity. Although hereditary, environmen- of colonic RNA for pro-inflammatory cytokines interleukin (IL)-6 tal, immunologic, and bacterial factors have been implicated, the and IL-12 were also decreased toward normal with NTX. Similar etiology of IBD remains unknown. Since opioid peptides modu- to patients with severe and unresponsive disease, animals in the se- late inflammatory cytokine production and opioid antagonists pro- vere colitis study did not significantly respond to treatment. Thus, mote tissue growth and repair, we hypothesized the opioid antag- NTX therapy reverses physical symptoms, histologic evidence, and onist naltrexone could reduce inflammation of the bowel. Using a molecular markers of inflammation in moderate colitis. The mech- chemically-induced mouse model of IBD, C57BL/6J mice received anism by which NTX acts to reverse colitis is related in part to the either untreated drinking water or water containing 2% dextran decreased expression of pro-inflammatory cytokines. sulfate sodium (DSS) in two parallel regimens modeling moder- ate and severe colitis. After colitis was established, animals in the Keywords Naltrexone, inflammatory bowel disease, opioids, moderate colitis study were administered either saline (control) or antagonists naltrexone (NTX; 8 or 400 µg/kg) daily, while those in the severe colitis study received 0.1 or 10 mg/kg NTX. DSS-treated animals INTRODUCTION had significant weight loss (p = 0.006) and higher disease activ- ity index (DAI) scores (p < 0.001) compared to water controls. Inflammatory bowel disease (IBD) includes two idiopathic However, NTX treatment of mice with moderate colitis resulted in conditions termed ulcerative colitis and Crohn’s disease which affect approximately 1 million people in North America Received 12 December 2007; accepted 21 February 2008. (Cominelli, 2004). The pathogenesis of IBD appears to in- Authors Matters and Harms contributed equally. Gail Matters is volve the dysregulation of the immune system in the intes- also affiliated with the Department of Medicine, Penn State, and John tine in response to either commensal bacteria or environment F. Harms is also affiliated with the Department of Biological Sciences, in a genetically predisposed host (Cominelli, 2004; Sands, Messiah College, Grantham, Pennsylvania. The authors wish to thank Dan Krissinger and Rob Brucklacker 2007). Cytokines have been implicated in the pathogenesis of of the Functional Genomics Core Facility of the Section of Research Crohn’s disease, in particular the pro-inflammatory/TH1cy- Resources, Pennsylvania State College of Medicine for assistance with tokines interleukin-1 (IL-1), IL-2, IL-6, IL-12, IL-18, interferon the Real Time RT-PCR, and Dr. David Mauger, Department of Public (INF)-γ , and tumor necrosis factor-alpha (TNFα) (Pizarro and Health Sciences, Pennsylvania State College of Medicine, for statistical consultation. Lynn Budgeon, Melissa Nelson, and Evan Gilius are ac- Cominelli, 2007). knowledged for their expert technical help. Grant Support: Supported Medical treatment of this condition has focused on target- by a gift fund at Pennsylvania State University to JPS. ing the inflammatory response with immunosuppressive drugs Abbreviations: Dextran sulfate sodium, DSS; Disease activity index, (i.e., corticosteroids, azathioprine) or immune-specific biologi- DAI; Inflammatory bowel disease, IBD; Naltrexone, NTX. α Address correspondence to Jill P. Smith, M.D., H-045 GI and Hep- cal drugs, such as monoclonal antibodies against TNF (Targan atology, Pennsylvania State University, 500 University Drive, Hershey, et al., 1997; Sandborn and Hanauer, 1999; Hanauer and Present, PA 17033, USA; e-mail: [email protected] 2003; Navarro and Hanauer, 2003; Bell and Kamm, 2000; 179 180 MATTERS ET AL.

Kamm, 2006). Unfortunately, chronic immunosuppression may research protocol was approved by the Institutional Animal Care increase the risk of developing infections such as tuberculosis and Usage Committee of the Pennsylvania State University Col- (Keane et al., 2001) and lymphoma (Kandiel et al., 2005). Since lege of Medicine and animals were housed in accordance with the risks may at times outweigh the benefits when immunomodu- the AAACCR guidelines for veterinary medicine. lating drugs are combined with biologic agents (Hanauer, 2007), In the moderate colitis study, 6- to 8-wk-old male C57BL/6J novel therapeutic approaches to treat IBD are needed. mice (Charles River, Wilmington, MA) were randomly allo- Accumulating evidence points to roles for endogenous opi- cated into one of two groups of 24 mice each. Food and wa- oid peptides in the development or perpetuation of inflamma- ter were provided ad libitum. Individual mice were housed tion. Immune cells have been shown to express µ, κ, and δ- in separate cages for accurate measurement of food and wa- opioid receptors that bind both opioid agonists and antagonists ter intake to determine whether NTX alone affected animal (McCarthy et al., 2001). In vivo treatments with opioids have weight, water intake, and food consumption, each measured been shown to induce the release of pro-inflammatory cytokines, daily. The first group (Normal) received untreated drinking wa- such as IL-12 and TNFα,bymouse peritoneal macrophages ter and the second group (DSS) received water containing 2% (Tomassini et al., 2003). Studies have verified similar roles dextran sulfate sodium (DSS; TDB Consultancy AB, Uppsala, with endogenous opioids, demonstrating that opioids, including Sweden; molecular weight, 40,000) for six days followed by un- [Met5]-enkephalin, stimulate peritoneal macrophages in rodents treated water for three additional days (See treatment schedule, systems (Vujic et al., 2004). Opioid receptors are found through- Figure 1A). out the gastrointestinal tract in the myenteric and submucosal Mice in each group (Normal or DSS) were randomly subdi- plexus as well as in epithelial cells (Jimenez et al., 2006). vided into three treatment groups of 8 mice each. Starting on Day Furthermore, in these studies, cytokine production was de- 3, mice were treated once daily for six consecutive days with creased by simultaneous treatment with the opioid receptor an- a subcutaneous (SC) injection (0.1 ml) of one of the follow- tagonist, naltrexone, indicating the effects were mediated by ing: saline (control), 8.0 µg/kg naltrexone (NTX), or 400 µg/kg opioid receptors expressed by peripheral and intestinal immune NTX. These doses were selected to bracket the approximate cells. Pre-treatment with naltrexone (10 mg/kg) has also been clinical dosing reported with efficacy in humans with Crohn’s shown to block TNFα synthesis and induction of septic shock disease (Smith et al. 2007). On Day 9, all animals were necrop- in LPS/D-galactosamine-treated mice (Greeneltch et al., 2004). sied and their colons resected and analyzed. Recently, a clinical study in human subjects with active Crohn’s In the severe colitis study, C57BL/6J mice were placed into disease demonstrated that naltrexone therapy improved Crohn’s one of four groups of 10 mice each. The first group received inflammatory scores and quality of life (Smith et al., 2007). normal water (control). The remaining groups received water Given the role of endogenous opioids in inflammation and containing 2% DSS (30 mice) for the duration of the study (9 the inhibition of these effects by the opioid receptor antagonist, days). Beginning on Day 3, the normal water mice and 10 of naltrexone, we hypothesized that naltrexone therapy could im- the DSS-treated mice were injected with vehicle (water; 0.1 prove colitis in mice. In order to test this hypothesis we used ml SC daily). For NTX treatment in this study, an intermedi- the DSS (dextran sulfate sodium) model of experimental col- ate dose (100 µg/kg NTX) and an escalated dose (10 mg/kg itis in mice to examine symptoms, tissue histology, and RNA NTX) were used to test efficacy against the more severe symp- profiling of cytokines in response to naltrexone. The addition of toms anticipated with extended DSS treatment. Mice were in- DSS to the drinking water induces hematochezia, weight loss, jected (0.1 mg/kg or 10 mg/kg; 0.1 ml SC daily; n = 10) until intestinal shortening, and infiltration of neutrophils, and thus the end of the experiment on Day 9 (See treatment schedule, serves as a model for human inflammatory bowel diseases, par- Figure 2B). ticularly ulcerative colitis (Okayasu et al., 1990). Breakdown The colitis disease activity index (DAI) was calculated for of epithelial barrier function in DSS-treated mice leads to an each mouse according to the system established by Murthy and induction of pro-inflammatory cytokines, which are thought to colleagues (1993) using animal weight, stool occult blood, and play a central role in disease progression (Strober et al., 2002). stool consistency. Overt changes in stool consistency were rarely Treatments aimed at reducing this excessive inflammatory re- discerned in the moderate colitis study so a modified DAI was sponse have demonstrated therapeutic promise in DSS models; calculated based on percent weight loss and stool hemoccult or therefore, DSS-treated mice are particularly suitable for proof- presence of gross bleeding. of-concept studies of novel IBD therapeutics and treatments that may reduce the cytokine-induced inflammatory state (Pizarro Histologic Evaluation et al., 2003). At necropsy, the entire colon was excised, measured in length, METHODS and bisected into proximal and distal portions. The proximal and distal colons were additionally divided for RNA extrac- Animals and Experimental Procedures tion and histology. Each histology specimen was fixed in 10% Two separate studies were performed to test the effects of neutral buffered formalin, paraffin embedded and sectioned naltrexone: chemically induced moderate or severe colitis. The for hematoxylin and eosin (H&E) staining. Specimens were LOW-DOSE NALTREXONE AS A TREATMENT FOR IBD 181

FIG. 1. Daily weight, food and water consumption. (A) Mean weight (expressed as % starting weight ± SEM) of animals receiving either normal drinking water (upper panel) or 2% DSS in drinking water (lower panel) is shown. DSS-induced weight loss was lessened by treatment with naltrexone (NTX) in moderate colitis by Day 6. (B) Mean daily food intake in grams per mouse and (C) total water intake in ml per mouse over the 9-day course of study is shown. Asterisks indicate significantly different values between corresponding DSS and Normal (No DSS) treatment groups (*p < 0.025; **p < 0.005). Significant differences in water intake between saline- and 8 µg/kg NTX-treated mice are indicated by a † (p = 0.022). examined microscopically and scored based upon the criteria tiple comparisons to controls. Statistical comparisons were established by Williams et al. (2001) by an investigator blinded performed between NTX treatment sub-groups and their cor- to the treatment groups. Briefly, a representative longitudinal responding vehicle control, as well as between the Normal section from each mouse was scored at six random fields for and DSS groups, comparing corresponding vehicle or NTX inflammation severity, extent of inflammation (mucosa, sub- treatments. mucosa, transmural) and crypt damage. Each of these scores was weighted to reflect the percent involvement of the over- Quantitative Real-Time PCR all section and the weighted scores from each of the six fields Total RNA was extracted from the distal colon samples (Tri- were averaged to achieve an overall inflammation score for each zol; Invitrogen, Carlsbad, CA) and subjected to analysis by Real- mouse. Time RT-PCR. RNA (18S and 28S bands) was visualized using the Agilent 2100 Bioanalyzer (Agilent Technologies) and con- Statistical Analysis centrations were adjusted. First strand cDNA was then produced Results were calculated as mean ± SEM. To eliminate undo from 1.0 µgoftotal RNA using random hexamer primers and influence of abnormally sick or resistant mice, statistical out- the SuperScript III Reverse Transcription kit (Invitrogen). The liers from normally distributed data in weight loss and his- concentration and quality of resulting cDNA was quantified and tological sectioning were determined (Minitab 13, State Col- analyzed using the Agilent 2100 Bioanalyzer or spectroscopi- lege, PA; below Q1 – 1.5×IQR, above Q3 + 1.5×IQR) and cally with the NanoDrop ND-1000 (NanoDrop Technologies, excluded. Pairwise Student t-tests were performed (Minitab Wilmington, DE). Samples were standardized to 30 ng/µl, and 13) using a modified Bonferroni method to correct for mul- 60 ng of cDNA per sample was then utilized as a template 182 MATTERS ET AL.

FIG. 2. Naltrexone treatment reduces Disease Activity Index (DAI) scores. Mice that received normal drinking water (saline or NTX-treated) showed noevidence of colitis by DAI score (A, upper panel; mean ± SEM). DAI scores increased over time in both the moderate (A, lower panel) and the severe colitis (B) studies. Treatment with 400 µg/kg NTX significantly lowered DAI scores in moderate colitis on Day 6 († p = 0.015). In contrast, NTX failed to improve DAI scores of the severe colitis model (B). for Real-Time RT-PCR using a SYBR Green Master Mix (Qi- RESULTS agen, Valencia, CA). 18S rRNA primers (Eurogentec, San Diego, CA) and the following gene-specific primer sequences Effects of Naltrexone on Animal Weight, Food and Water obtained from PrimerBank (Wang and Seed, 2003) were uti- Consumption lized: β-actin, 6671509a; IL-5, 6754336a; IL-6, 13642311a; IL- Over the 9-day course of the moderate colitis study, Normal 12, 6680395a; STAT3, 13277852a; STAT4, 6755670a; Muc2, control animals given untreated drinking water exhibited steady 3452503a2; TFF3, 6755773a1; Palladin, 9828173a1; TGF-β weight gain (Figure 1A, upper panel) while DSS mice showed BP, 7305243a1; and, TNFα, 202093a3. To exclude the possi- weight loss beginning between Days 4 and 6 (Figure 1A, lower bility of genomic DNA contamination, control reactions with panel) and continuing until necropsy (Day 9). Animals treated no cDNA template were also performed for each gene-specific with naltrexone exhibited less weight loss compared to DSS primer set. PCR amplification and analysis were performed with + saline mice, reaching statistical significance on Day 6 (p = the Applied Biosystems Sequence Detection System 7300 us- 0.02). NTX-treated mice also had a tendency toward decreased ing the Relative Quantification (ddCt) Plate setup. At least six weight loss on Days 7 and 8, although they did not reach statis- replicates were performed for each target gene set. Amplifi- tical significance. cation data for the target genes were calibrated using the 18S Food consumption was significantly decreased in the animals rRNA endogenous control within each sample. The resulting with DSS-induced colitis compared to animals without colitis mean CT values were then normalized to the β-actin expres- (Figure 1B). Naltrexone treatment alone had no effect on food sion (corresponding treatment groups) to ensure observed dif- consumption in both normal animals and DSS-treated animals. ferences were biologically robust. Pairwise Student t-tests were Animals treated with NTX (8 µg/kg) had slightly decreased wa- performed on the normalized mean CT values for each group ter consumption (Figure 1C). DSS-treated mice drank less water using a modified Bonferroni method to correct for multiple that their treatment counterpart, but there was no difference comparisons. between the DSS groups treated with saline or NTX (Figure 1C). LOW-DOSE NALTREXONE AS A TREATMENT FOR IBD 183

Disease Activity Index (DAI) Scores are Reduced by Expression of DSS-Induced, Pro-inflammatory Cytokine Naltrexone Treatment Genes is Decreased by Naltrexone Treatment To monitor disease progression, a disease activity index Because naltrexone reduced the inflammatory histology of (DAI) was assessed daily for each mouse including weight and DSS-induced colitis, the expression of several genes of inter- stool hemoccult. DAI scores for all Normal mice (no DSS), est, including both cytokines and downstream mediators, was regardless of saline or NTX treatment, showed no clinical evi- examined by Real-Time RT-PCR. Expression of β-actin, cy- dence of colitis (Figure 2A, upper panel), suggesting that NTX tokines IL-5, IL-6, IL-12, and transcription factors STAT3 and alone has no deleterious effects on the colon. All DSS-treated STAT4were assessed using 18S rRNA as an endogenous control. mice developed colitis symptoms (hemoccult-positive stools and After normalizing to β-actin, IL-5 levels were not significantly increased DAI scores) by Day 4, which continued to increase changed by either DSS + saline or by DSS + NTX (Figure through the end of each study (moderate colitis, Figure 2A lower 4A). Similarly, levels of TNFα were not significantly changed panel; severe colitis, Figure 2B). A reduction in the DAI scores in DSS-treated animals compared to normal, uninflamed tissue wasevident with NTX treatment in animals with moderate coli- (data not shown). Because TNFα is an early mediator of inflam- tis; on Day 6, DSS + 400 µg/kg NTX animals had significantly mation, transcript levels may have already subsided during the lower (55%) DAI scores than DSS + saline mice (p = 0.015). post-DSS recovery that preceded necropsy. DAI scores for 400 µg/kg NTX mice at Day 7 were also im- By contrast, mRNA encoding the cytokines IL-6 and IL-12, proved, but did not reach significance (p = 0.038). In contrast, known to be up-regulated in IBD, were increased in DSS + neither dose of NTX (0.1 mg/kg or 10 mg/kg) improved the DAI saline animals in comparison to Normal controls (Figures 4B score in severe colitis (Figure 2B). and 4C). The increase in IL-6 mRNA was 66-fold, while the increase in IL-12 was more modest (2.7-fold). Upon treatment with naltrexone, levels of IL-6 and IL-12 were reduced and, for Colon Length IL-12, naltrexone treatment restored mRNA expression to that Reduced colon length, another indicator of colitis, was also seen in the colitis-free, Normal mice. The reduction in IL-6 was evident in all DSS-treated animals. In the moderate colitis study, also significant, although levels were not completely restored to control mice drinking normal water and injected with saline had those seen in the colitis-free animals. colon length 26% longer than DSS-treated animals (9.32 ± 0.39 The mRNA for cytokine signaling intermediates STAT3, cm vs. 6.95 ± 0.43 cm, respectively; p = 0.002). DSS-mice downstream of IL-6, and STAT4, downstream of IL-12 (Mudter treated with naltrexone had more typical colon lengths (7.86 ± et al., 2005), also were increased in DSS + saline animals 0.31 cm; p = 0.1), although they were still 15% shorter than in (2.20- and 8.03-fold, respectively) (Figures 4B and 4C). How- the Normal (no DSS) animals. ever, STAT3 and STAT4 mRNA levels were not decreased to as great an extent by naltrexone treatment. This may reflect Histological Evidence for Reduced Colonic Inflammation the fact that STAT 3 and STAT4 activities are regulated both in Naltrexone-Treated Mice at the level of transcription and post-transcriptionally by phos- Histologic inflammation scores and H&E stains of the distal phorylation and nuclear relocalization in response to cytokine colon in untreated and treated animals are shown in Figure 3. No signaling (Mudter et al., 2005). In the severe colitis study, inflam- inflammation was observed in control animals drinking normal matory cytokine markers, as measured by Real-Time RT-PCR, water (saline or NTX-treated), indicating that naltrexone alone were also significantly elevated by DSS treatment. However, did not alter the mucosal integrity of the colon (Figure 3A). All in these animals exhibiting severe, rather than moderate colitis, DSS animals had increased inflammation scores, and exhibited neither NTX dose significantly affected RNA levels (data not crypt damage and increased leukocyte infiltration. In moderate shown). colitis (Figures 3A and 3B), the DSS + NTX animals had a significant decrease in inflammation and damage. The DSS + DISCUSSION 400 µg/kg NTX treated mice had reduced histology scores (p = This study is the first to report improvement of colitis in a 0.018; Figure 3A), with improved crypt architecture and fewer murine model upon treatment with an opioid antagonist. In both invading leukocytes than were observed in the DSS + saline the moderate and severe colitis studies, we administered DSS for mice (Figure 3B). three days prior to either vehicle or naltrexone injections, emu- Animals with severe colitis had no improvement in their his- lating a condition of established bowel inflammation preceding tology scores with NTX. Although those mice treated with the treatment. Naltrexone treatment resulted in a rapid mitigation of lower dose of NTX (0.1 mg/kg) had less inflammation than moderate colitis symptoms in DSS mice, including weight loss saline-treated mice and high-dose NTX (10 mg/kg) mice, this and bleeding. Furthermore, the modest differences in histology difference was not statistically significant (Figure 3C and 3D). (DSS ± NTX) may have been even greater if the diseased an- No histologic changes consistent with colitis were observed imals were not allowed potential time for recovery (post-DSS) in the proximal colons of the mice (data not shown). prior to necropsy. 184 MATTERS ET AL.

FIG. 3. Histologic scores are improved in moderate colitis mice treated with naltrexone. At necropsy, longitudinal sections of the distal colon were H&E stained and evaluated using the scoring system of Williams and colleagues (2001). Histology scores are expressed as the mean ± SEM. Normal histology was observed in animals drinking untreated water compared to high inflammatory scores in DSS-treated animals (A). Naltrexone treatment improved the histologic scores in animals with moderate colitis (A). Representative H&E sections of distal colon from animals with moderate colitis are shown in B. Compared to the normal appearance of healthy murine colon (Normal + saline; top), leukocyte infiltration and an absence of normal crypt architecture are evident in the DSS + saline mice (middle). Improved architecture and less inflammation are clearly discernable in DSS mice treated with 400 µg/kg NTX (bottom). Cross indicates significantly different values between DSS + saline and DSS + 400 µg/kg NTX (p = 0.018). In contrast, NTX was unable to reverse the inflammatory response observed in the severe colitis model (C, D).

A robust, impact on pro-inflammatory gene expression by significantly decreased their expression in DSS mice to nor- naltrexone was also confirmed. Characteristic of IBD, signif- mal or near-normal levels. This suggests a possible pathway by icant elevations in the gene expression of IL-6 and IL-12 cy- which NTX improves colitis. Pro-inflammatory NF-κB signal- tokines were induced with DSS. However, naltrexone treatment ing is associated with opioid receptor activity in immune cells LOW-DOSE NALTREXONE AS A TREATMENT FOR IBD 185

FIG. 4. Gene expression of DSS-induced pro-inflammatory cytokines is decreased by naltrexone. Relative mRNA levels for (A) beta-actin and the cytokine IL-5; (B) cytokine IL-12 and downstream mediator STAT4; and (C) cytokine IL-6 and downstream mediator STAT3, were determined by Real-time RT-PCR using total RNA from the distal colon. Target genes were calibrated to 18S rRNA, and means were normalized to beta-actin expression for each corresponding treatment group. Histogram columns represent the mean relative quantity (RQ = 2−CT) and bars represent a 95% confidence interval (CI; RQ = 2−(CT±CI)). DSS induced significant elevations in the RNA of pro-inflammatory cytokines IL-6 and IL-12. However, mice treated with naltrexone exhibited restoration toward normal levels. Asterisks indicate significantly different values between corresponding Normal + saline and each DSS treatment group (*p < 0.0008). Significant differences between DSS-Saline and DSS-NTX mice are indicated by a † (IL-12, p < 0.005; STAT4, p < 0.024; IL-6, p < 0.0008).

(Chen et al., 2006). When opioid receptor signaling is blocked a defect in T-lymphocyte activation and a reduced ability of T- by naltrexone, the over-stimulation of immune system is mod- lymphocytes to proliferate (Hook et al., 2003). erated, cytokine levels are restored, and a more normal mucosal Both endogenous and exogenous opioids have been shown structure reappears. to intensify the production of pro-inflammatory cytokines by There is ample evidence that opioid peptides, particularly immune cells and, in some cases, this opioid-stimulated in- delta opioid receptor agonists, can regulate immune responses crease in cytokines has been abrogated by opioid receptor antag- (House et al., 1996). First, immune cells secrete opioid pep- onists (Vujic et al., 2004). Third, while opioid peptides do not, tides (Cabot, 2001; McCarthy et al., 2001) and activation of in themselves, induce inflammatory responses, they can sensi- murine T-lymphocytes increases the synthesis and secretion of tize T-lymphocytes and macrophages to other pro-inflammatory [Met5]-enkephalin (Zurawski et al., 1986). Second, immune stimuli (Hucklebridge et al., 1990; Kamphuis et al., 1998). cells express delta, mu, and kappa opioid receptors that respond Thus, because opioids can enhance the immune response to pro- to endogenous and synthetic opioids (Cabot, 2001; McCarthy inflammatory stimuli and thereby contribute to the escalation of et al., 2001). In fact, [Met5]-enkephalin knock-out mice exhibit inflammation, it is reasonable to suggest that naltrexone’s action 186 MATTERS ET AL. on both T-lymphocytes and macrophages could effectively mod- arguably complicated by the induction of more severe colitis erate the excessive pro-inflammatory response in IBD. Consis- than in the moderate study. This reflects current care for patients tent with this notion, the DSS-induced pro-inflammatory re- with severe colitis, in which the inflammation at times exceeds sponse in this study was down-regulated by opioid receptor medical management, necessitating surgery. While naltrexone blockade. has been approved by the Food and Drug Administration for Although naltrexone is a competitive antagonist at delta, mu alcohol withdrawal syndromes (Petrakis et al., 2007) at a dose and kappa opioid receptors, the effects on immune function re- of 50 mg daily, it is unknown how this higher dose affects in- ported herein are consistent with delta receptor blockade. In flammatory responses and cytokines. a broad sense, while delta opioid receptor stimulation is pro- Precedence for greater efficacy of low-dose therapies al- inflammatory, signaling through mu opioid receptors has been ready exists. Others have shown that micromolar concentrations shown to be immunosuppressive (McCarthy et al., 2001). In- of dextromethorphan, a D-isomer of the codeine analog levor- deed, the use of specific mu receptor agonists for treatment of phanol, induces a neuroprotective effect in the brain by suppres- IBD has shown therapeutic benefit both in mouse models and sion of proinflammatory factors superoxide, NO and TNFα (Liu in human mucosal explants (Philippe et al., 2003, 2006). Stud- et al., 2003, 2005). This inhibitory effect on free radical gener- ies with delta and mu receptor-specific antagonists will further ation by ultra-low concentrations of dynorphins is shared by clarify the role of these receptors in colonic inflammation. Con- another opioid peptide, enkephalin (Zaitsev et al., 1991; Efanov ceivably, a delta receptor antagonist and mu receptor agonist et al., 1994), where femtomolar concentrations of an enkephalin may function synergistically with maximal therapeutic benefit. analog inhibited the reactive burst from human neutrophils and It has yet to be determined whether naltrexone blocks an over- mouse macrophages. Thus manipulating the opioid-opioid re- active opioid peptide system which is aggravating the inflamma- ceptor axis with small doses of these compounds plays a role in tory response, or whether it augments the release of endogenous modulating inflammatory mechanisms. opioids to alter diarrhea and pain through specific receptors in These data demonstrate a role of endogenous opioids in the the gut. In addition to inflammatory modulation, naltrexone may development and progression of IBD and the effectiveness of elicit other complementary effects, potentially mediated through opioid receptor blockade in reducing of colonic inflammation blockade of a second type of opioid receptor, the opioid growth and damage. On the whole-animal, tissue, and molecular lev- factor (OGF) receptor. [Met5]-enkephalin binding to the OGF re- els, naltrexone treatment alleviated the effects of DSS-induced ceptor generally inhibits growth and repair. Naltrexone blockade colitis. 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Coll. of Gastroenterology doi: 10.1111/j.1572-0241.2007.01045.x Published by Blackwell Publishing ORIGINAL ARTICLES Low-Dose Naltrexone Therapy Improves Active Crohn’s Disease Jill P. Smith, M.D.,1 Heather Stock, M.D.,1 Sandra Bingaman, R.N.,1 David Mauger, Ph.D.,2 Moshe Rogosnitzky,3 and Ian S. Zagon, Ph.D.4 Departments of 1Medicine and 2Health Evaluation Sciences, Pennsylvania State University College of Medicine, Hershey, Pennsylvania; 3Consultant, Telz Stone, Israel; and 4Neural and Behavioral Sciences, Pennsylvania State University, Hershey, Pennsylvania

OBJECTIVES: Endogenous opioids and opioid antagonists have been shown to play a role in healing and repair of tissues. In an open-labeled pilot prospective trial, the safety and efficacy of low-dose naltrexone (LDN), an opioid antagonist, were tested in patients with active Crohn’s disease. METHODS: Eligible subjects with histologically and endoscopically confirmed active Crohn’s disease activity index (CDAI) score of 220–450 were enrolled in a study using 4.5 mg naltrexone/day. Infliximab was not allowed for a minimum of 8 wk prior to study initiation. Other therapy for Crohn’s disease that was at a stable dose for 4 wk prior to enrollment was continued at the same doses. Patients completed the inflammatory bowel disease questionnaire (IBDQ) and the short-form (SF-36) quality of life surveys and CDAI scores were assessed pretreatment, every 4 wk on therapy and 4 wk after completion of the study drug. Drug was administered by mouth each evening for a 12-wk period. RESULTS: Seventeen patients with a mean CDAI score of 356 ± 27 were enrolled. CDAI scores decreased significantly (P = 0.01) with LDN, and remained lower than baseline 4 wk after completing therapy. Eighty-nine percent of patients exhibited a response to therapy and 67% achieved a remission (P < 0.001). Improvement was recorded in both quality of life surveys with LDN compared with baseline. No laboratory abnormalities were noted. The most common side effect was sleep disturbances, occurring in seven patients. CONCLUSIONS: LDN therapy appears effective and safe in subjects with active Crohn’s disease. Further studies are needed to explore the use of this compound. (Am J Gastroenterol 2007;102:1–9)

INTRODUCTION cells suggesting that the bowel is either reacting immunolog- ically to a stimulus or the endogenous immune system of the Chronic relapsing and remitting inflammation of the gas- gastrointestinal track is off balance (4). trointestinal tract is the hallmark of ulcerative colitis and Although there has been progress in defining the patho- Crohn’s disease, conditions termed inflammatory bowel dis- genesis of these diseases, their cause remains obscure. The eases (IBD) (1). The peak age of onset of this disease is be- current most comprehensive hypothesis is that IBD is a het- tween the first and fourth decades of life, with a prevalence of erogeneous group of diseases that have a final manifesta- 100–200 per 100,000 in Europe and North America. IBD ac- tion, which is mucosal inflammation, and that several genetic counts for significant morbidity and lower quality of life, and and environmental factors are implicated in the pathogene- is responsible for nearly $2.0 billion in annual medical costs sis of the disease (5–8). The result of these events in some in the United States (2). Crohn’s disease is characterized by way leads to a disordered immune response to one or more transmural, patchy, granulomatous inflammation of any part mucosal antigens or bacteria in a genetically determined of the gastrointestinal tract, although it is most common in host (9, 10). the ileocecal area (3). The major symptoms of Crohn’s dis- Traditionally, treatment of Crohn’s disease includes com- ease include abdominal pain, diarrhea, gastrointestinal bleed- pounds designed to reduce the inflammatory response, such ing, malabsorption, and weight loss. Although the etiology of as corticosteroids, cyclosporine, and azathioprine, which of- Crohn’s disease is unknown, research suggests it involves a ten lead to serious side effects (11, 12). Major advances in complex interplay of environmental, genetic, microbial, im- the understanding of the pathogenesis of IBD have led to the mune, and nonimmune factors. Biopsies obtained from the development of novel immunotherapies. Such treatments in- bowel in subjects with Crohn’s disease reveal inflammatory clude the administration of chimeric antibodies specific for

1 2 Smith et al. molecules such as cytokines known to be central to the patho- by either endocopic or radiographic procedures. Patients had genesis of mucosal inflammation (antitumor necrosis factor moderate to severely active disease as defined by a CDAI [TNF], interleukin [IL]-2, IL-10) (13, 14). Although this spe- score of >220 and <450(27). Patients taking stable doses of cific immunotherapy has helped those with Crohn’s disease, aminosalicylates, immunomodulators, corticosteroids, or an- still about 20% do not respond to this treatment (14) and tibiotics were permitted to enter the study, and were continued many cannot continue this therapy due to untoward side ef- at the same dosage throughout the trial. Women of childbear- fects (15, 16). Additionally, treatment with the monoclonal ing age were permitted to enroll and, if not surgically sterile, antibody infliximab is expensive with each infusion costing were required to use adequate contraception (defined as oral thousands of dollars. or depot contraceptive, IUD, or barrier plus spermicide) for Endogenous opioid systems (i.e., opioids and opioid re- the duration of the study. These women were required to ceptors) have been shown to participate in a wide variety continue adequate contraception for 3 months after the com- of functions including growth and immunity (17). [Met5]- pletion of the study. Exclusion criteria included: women who enkephalin is an endogenous pentapeptide that is located were pregnant or breastfeeding, subjects with an ileostomy, throughout the gastrointestinal tract (18). In addition to the colostomy, ileorectal anastomosis, or short bowel syndrome growth characteristics of [Met5]-enkephalin, this endogenous from surgery, and patients with abnormal liver function tests. opioid has also been shown to influence the immune system Subjects taking tacrolimus, cyclosporine, mycophenolate, or with effects on OK10 cells, Leu11, and natural killer cells infliximab within 8 wk of enrollment were excluded. (19). Acetorphan is an oral enkephalinase inhibitor that ele- Approval for the study was granted by the Institutional vates endogenous enkephalin blood levels and has been used Review Board of the Human Subjects Protection Office at in Europe and elsewhere to treat diarrheal disorders such the Pennsylvania State University Milton S. Hershey Med- as cholera (20) and AIDS diarrhea (21). In a clinical study ical Center. The LDN was assigned an Investigational New (22), 193 subjects with diarrhea received either acetorphan Drug Number 67,442 by the Food and Drug Administration or placebo for 10 days, and the incidence of diarrhea was (FDA). reduced by 30%. Additionally, the symptoms of abdominal pain, anorexia, and nausea were also significantly reduced Study Design compared with placebo (22). The study was designed as an open-labeled pilot investigation Zagon and McLaughlin (23) have reported in an animal to evaluate response, safety, and toxicity to LDN in subjects model that a low dose of naltrexone can produce an intermit- with active Crohn’s disease. Eligibility was assessed by tele- tent blockade of the opioid receptor. This short-term blockade phone, and potential candidates were scheduled for a screen- resulted in a rise in the endogenous tissue levels of [Met5]- ing visit in the General Clinical Research Center (GCRC). enkephalin and endorphins and results in the same effects on At the screening visit, patients were subjected to a history growth as exogenous enkephalin (23). It is presumed that too and physical examination and laboratory testing (chemistry high a dose of receptor antagonist would block the receptor panel, complete blood count [CBC], urinalysis, and erythro- completely and obliterate the effects of the endogenous opi- cyte sedimentation rate [ESR]). Patients were dispensed a oids (24). In fact, naltrexone therapy has been used to aid 7-day diary to record symptoms of frequency of diarrhea, in the healing of corneal abrasions (25). Naltrexone has also abdominal pain, and general well-being. Within 14 days, pa- been shown to block TNF-α synthesis and induction of septic tients returned for assessment and calculation of the CDAI shock in LPS/D-galactosamine-treated mice (26), suggesting score. Qualifying subjects were dispensed medication and that perhaps naltrexone itself may have anti-inflammatory ef- given a new diary in order to calculate the subsequent month’s fects. CDAI score at the conclusion of this visit (baseline). Patients In this pilot study, we investigated the effects of low-dose returned after 2 wk for an interim visit to evaluate side ef- naltrexone (LDN) in patients with active Crohn’s disease. fects and perform a CBC. Follow-up visits were scheduled It was hypothesized that LDN would improve activity of for weeks 4, 8, 12, and 16. Crohn’s disease in patients by showing a decline in the Crohn’s disease activity index (CDAI) scores and blood inflammatory Treatment markers (C-reactive protein and ESR), and improve quality Naltrexone hydrochloride was compounded into capsules of life. It is proposed that the mecahnism by which LDN will containing 4.5 mg by GMP-approved standards at Williams improve Crohn’s disease will be by causing an elevation in Apothecary in Lancaster, PA.Because the dosage used in this endogenous opioid levels. study was lower than the FDA-approved dose of 50 mg, it will be referred to as “low-dose naltrexone” or LDN. Quality assurance of packaging and purity were confirmed by Ana- PATIENTS AND METHODS lytical Research Laboratories (Oklahoma City, OK). Patients were treated with LDN orally each evening for 3 months. Patient Selection A monthly supply of medication was dispensed to patients Eligible patients were both male and female, at least 18 yr by the Investigational Pharmacy of the Pennsylvania State of age, and with the confirmed diagnosis of Crohn’s disease University Milton S. Hershey Medical Center. On the first Naltrexone and Crohn’s Disease 3 visit, an additional 10-day supply of LDN was provided in tained monthly and 4 wk post-therapy. Data from laboratory the event of an appointment delay. Subjects were required to results and quality of life surveys were entered into an Excel bring the vials to each appointment for counting and drug spreadsheet. A longitudinal data analysis, based on the linear accountability; extra capsules were returned to the Investi- mixed-effects model was applied using PROC MIXED pro- gational Pharmacy the day of the visit and another month’s gram. The Bonferroni statistical method was used to adjust supply dispensed. significance, where analysis including multiple comparisons to the baseline were made. P values for binary outcomes of Assessments response and remission were calculated using the exact test In order to assess LDN’s effect on disease activity, patients for binomial proportions. kept a Crohn’s symptom diary for the 7 days preceding each visit for calculation of the CDAI score (27). A response to therapy was defined as a 70-point decline in the CDAI RESULTS score and remission was defined as attaining a CDAI score Patients and Demographics of 150 or less. To assess quality of life, patients completed Twenty-one subjects were screened for the study and sev- two standardized quality of life surveys, the inflammatory enteen were eligible to participate. Of the four who were bowel disease questionnaire (IBDQ) (28) and SF-36 health screened that did not participate: one was a screening failure survey (29). Appropriate licensure was purchased through due to elevated liver enzymes, one failed the screen secondary contractual agreement for the use of these surveys from each to severe psychiatric illness, and the other two subjects opted facility. for other therapy and declined before receiving drug. Of the Routine blood work including CBC, chemistry panel, and seventeen subjects who enrolled in the study, only one subject ESR were assessed monthly. In addition, urine tests and preg- terminated before wk 12 secondary to a flare-up in Crohn’s nancy tests were done for monitoring and safety purposes disease when she discontinued her concomitant medications. pretreatment and at each monthly visit. C-reactive protein This subject was followed and data included throughout the (C-RP) was measured at baseline and at week 12. [Met5]- study as an intent-to-treat subject. The characteristics of the enkephalin levels were determined by radioimmunoassay patients at enrollment are shown in Table 1, including age, (RIA) at baseline and wk 4, 8, 12, and 16 (Peninsula Lab- gender, and body weight. Most patients had both small bowel oratories, San Carlos, CA). and colonic disease, and two patients had active perianal fis- tulas. Eight patients had prior surgical resection performed Safety Measures for their Crohn’s disease. Seventy-six percent of patients had The study was monitored by the data safety monitoring board prior treatment with anti-TNF-α therapy, and were either al- (DSMB) at the Pennsylvania State University Milton S. Her- lergic, intolerant, or unresponsive to this medication. Con- shey Medical Center. The safety and toxicity of LDN were comitant medications for Crohn’s disease taken by patients assessed by adverse events, laboratory parameters, and vital throughout the study are also shown in Table 1. signs. Nonhematologic and hematologic toxicities were de- termined by the WHO criteria (30). All adverse events were General reported to the Institutional Review Board according to the Statistical analysis showed that there was no significant guidelines established by the Pennsylvania State University change in body weight from screening visit through wk 16 of Milton S. Hershey Medical Center. Patients who required rescue medication based on an in- crease in CDAI score of 100 points were terminated from the Table 1. Patient Demographics study. These subjects were given a tapering regime of LDN, Mean age ± SEM (yr) 42.1 ± 2.6 involving dose reduction to every other day for 10 days before (range) (23–63) discontinuing the medication. Patients necessitating discon- Gender, N (% of patients) tinuation from the study were required to return for follow-up Male 3 (18%) visits and analyzed as intent-to-treat subjects. Female 14 (82%) Mean body weight ± SEM (kg) 72 ± 4 (range) (53–101) Statistical Analysis Disease site Data were entered into a secure computer in the GCRC by Small bowel 2 (12%) a nurse assigned to this project and analyzed by the De- Small bowel & colon 10 (59%) partment of Health Evaluation Sciences. An intent-to-treat Colon 5 (29%) Past resection performed, N (% of patients) 8 (47%) analysis was performed in which the available data from all Prior anti-TNF-α therapy, N (% of patients) 13 (76%) evaluable patients were included in the statistical analysis. Concomitant meds for Crohn’s, N (% of patients) The parameters of measurement (CDAI scores, laboratory Aminosalicylates 11 (65%) values, and quality of life surveys) were analyzed by SAS Immunomodulators 8 (47%) statistical software system (version 8.1) computer program Glucocorticoids 4 (24%) Antibiotics 1 (6%) by the biostatistician comparing baseline values to those ob- 4 Smith et al. the study (data not shown). Two patients elected to discon- When the components of the CDAI scores were evaluated tinue taking routine medications for Crohn’s disease prior to separately, the number of bowel movements and pain assess- wk 12 and symptoms of Crohn’s disease recurred in one of ment both independently improved significantly (P < 0.01) them. Data from both patients were analyzed with an intent- from baseline at each 4-wk interval on LDN and 4 wk after to-treat paradigm. The two subjects with entercutaneous and dicontinuing LDN. In addition, the CDAI score minus the rectovaginal fistulas had closure of the fistulas with LDN ther- number of bowel movements and pain was also statistically apy. Unexpectedly, one study subject with Crohn’s disease improved with LDN therapy (P < 0.01). These results indi- and multiple sclerosis was found also to have improvement cate that both pain and number of bowel movements are im- in her neuological symptoms and manifestations of multiple portant markers in the CDAI score; however, they were not sclerosis with LDN. the only parameters contributing to the improved response found. Inflammatory Response (CDAI Scores) CDAI scores were used to measure the patient’s disease activ- Quality of Life ity and inflammatory response to LDN therapy. Mean CDAI Two standardized quality of life surveys, the IBDQ (Fig. 3) scores (Fig. 1) at wk 4, 8, and 12 following the initiation of and the SF-36 health survey (Fig. 4), were administered to pa- LDN therapy were 41, 55, and 49%, respectively, decreased tients receiving LDN treatment. By both measures, patients from baseline. Four weeks after discontinuation of therapy experienced a significant improvement in their quality of life (wk 16), the mean CDAI score was 45% less than baseline on LDN therapy. With regard to the IBDQ survey, signifi- and not statistically different from the mean scores measured during the therapy. Figure 2 shows the percentage of patients responding to therapy (Fig. 2A), as well as the percentage A Response LDN of subjects achieving a remission of disease (Fig. 2B). At 1 4 wks post-LDN month after treatment, 76% had achieved a response to ther- 100 apy (a decrease in the CDAI score by 70 points), and at 8 and 12 wk, 88% showed a response. Four weeks after discon- 80 tinuation of LDN, 73% continued to show a response. At 1 month after starting LDN therapy, 29% of the patients had 60 achieved a remission (a CDAI score of 150 points or less), and at wk 8 and 12 of LDN therapy, 53 and 47%, respectively, 40 had achieved remission (Fig. 2B). Four weeks after discontin- % Patients uation of LDN therapy, 33% of the subjects were in clinical 20 remission. Therefore, at some point during the 16-wk trial, 89% of patients exhibited a response (P < 0.001), and 67% 0 achieved a remission (P = 0.07) with LDN. 4 8 12 16 Week B Remission 500 Baseline 60 LDN 400 4 wks post-LDN 50

40 300 **** **** **** 30 200 **** Patients % % Patients

CDAI Score 20

100 10

0 0 4 8 12 16 0481216 Week Week Figure 2. The percent of patients responding with a decline in CDAI Figure 1. Mean Crohn’s disease activity index (CDAI) scores ± score of at least 70 points (A), and the percent of patients achieving SEM are shown at baseline (wk 0), wk 4, 8, and 12 after initiation remission by a CDAI score of 150 or less (B), to LDN therapy are of LDN therapy and 4 wk after discontinuation of LDN therapy (wk shown at wk 4, 8, and 12 and 4 wk after discontinuation of LDN 16). ∗∗∗∗Significantly different from baseline at P < 0.0001. therapy (wk 16). Naltrexone and Crohn’s Disease 5

of 23.3 ± 0.4 mm/h to 17.9 ± 0.3 mm/h, which was also sig- Baseline = LDN nificant (P 0.04). Baseline plasma enkephalin levels were 4 wks post-LDN 9.5 ± 2.8 pg/mL, and decreased to a value of 3.6 ± 1.0 pg/mL 200 at wk 12 of LDN therapy, but this difference in plasma **** **** enkephalin levels was not statistically significant. ** *** 150 Side Effects The most frequently reported side effect with LDN therapy was sleep disturbances, and this was noted in seven patients; 100 one reported unusual dreams. Five subjects changed the tim- ing of LDN from the evening to morning due to insomnia. In no instance was a dose reduction necessary for sleep 50 disturbances. Other rare reported events included nausea (N = 1), hair thinning (N = 1), blurred vision (N = 1), IBD Questionnaire Score irritability (N = 1), mood swings (N = 1), and mild dis- 0 orientation (N = 1). 0481216 Week DISCUSSION Figure 3. Mean inflammatory bowel disease questionnaire (IBDQ) scores ± SEM are shown at baseline (wk 0), wk 4, 8, and 12 after The results of this pilot study are the first to show that LDN initiation of LDN therapy, and 4 wk after discontinuation of treat- therapy significantly decreases symptoms and improves qual- ∗∗ ment (wk 16). Significantly different from baseline at P < 0.01, ity of life in patients with active Crohn’s disease. In fact, ∗∗∗ < ∗∗∗∗ < P 0.001, and P 0.0001. two-thirds of enrolled patients achieved remission at some point during LDN treatment. It is known in a condition such cant improvement in quality of life was noted compared with as Crohn’s disease that remissions of activity occur sponta- baseline at wk 4, 8, and 12 on LDN, as well as 1 month after neously (31); therefore, it is possible the remission occurred completion of treatment. by chance. In a recent large randomized placebo-controlled Patients experienced a significant improvement in quality trial for Crohn’s disease, the remission rate with a placebo was of life in a variety of parameters as measured by the SF-36 recorded at 23% at wk 12 with even lower placebo remission health survey (Fig. 4A–H). At wk 4, 8, and 12 of therapy with rates earlier in the study (32). Therefore, in the present study, LDN there was, a five- to eightfold improvement in physical with 67% achieving remission, it would appear that LDN is role scores (A) and a 61–65% improvement in bodily pain effective; however, a randomized placebo-controlled trial is (B). Energy scores at wk 8 and 12 of LDN treatment (C) warranted. were at least twofold greater than at the time of initiation of Another finding in this trial was the fairly rapid onset of therapy, whereas the scores for health perception (D) were effect from LDN in that by 4 wk there was significant im- 33% and 49%, respectively, greater than baseline. At 4 and provement. Corticosteroids may be effective in decreasing 8 wk of LDN therapy, the physical function (E) was 23% symptoms of Crohn’s patients in 7–10 days, but other med- greater than baseline values. Social function (F) was 70% ications such as the immunomodulators (azathioprine and greater than baseline at wk 4, 8, and 12, but was only statis- 6-mercaptopurine) may take 3–4 months to demonstrate im- tically different at wk 8. Role–emotional (G) and emotional provement in symptoms (33). Often symptoms recur within 1 health (H) were comparable to baseline values at wk 4, 8, and month after discontinuing corticosteroids or aminosalicylates 12 of LDN treatment. (31, 33). However, in the present study, continued improve- At 4 wk after termination of LDN (i.e., wk 16), all param- ment in CDAI scores and quality of life was reported even eters except emotional health showed improvement ranging 4 wk after discontinuing LDN. Longer studies are needed to from 27% to an eightfold improvement over baseline. evaluate the long-term effects of LDN and whether it can be used for maintenance therapy as well as induction therapy. Laboratory Data Another finding in this pilot study was that LDN improved At wk 4, 8, and 12 of LDN therapy, there was no change from the quality of life of subjects with active Crohn’s disease. The baseline in CBC or chemistry values. Liver panels were not baseline value on the IBDQ was similar to that reported in altered from baseline levels at wk 12. Assessment of CBC other clinical trials (32), indicating that our subject group did at wk 2 of LDN therapy was comparable to baseline values. not differ from those used in other studies. Statistical analysis C-reactive protein levels decreased from a median value of indicated that for two separate quality of life surveys, a signif- 2.6 (normal <0.8) at baseline to a value of 0.9 by the 12th icant difference from baseline occurred in those individuals week of treatment, and this change was statistically significant on LDN. Moreover, even 1 month after discontinuation of (P = 0.03). The ESR decreased from a mean baseline value LDN therapy, the quality of life remained better in almost 6 Smith et al.

Baseline LDN 4 wks post-LDN

A Role - Physical B Bodily Pain 80 80 **** ** *** ** * *** 60 ** ** 60

40 40 Score Score

20 20

0 0 0481216 0481216 Week Week C Energy D General Health Perception 50 60 **** 50 *** 40 *** **** * ** 40 30 30 Score Score 20 20

10 10

0 0 0 4 8 12 16 0481216 Week Week E Physical Functioning Social Functioning 100 F 100 ** ** * * 80 80 *

60 60 Score 40 Score 40

20 20

0 0 0481216 Week G Role - Emotional H Emotional Health 120 100

100 * 80 80 60 60 Score Score 40 40

20 20

0 0 0481216 0481216 Week Week

Figure 4. Mean SF-36 health survey scores ± SEM are shown at baseline (wk 0), wks 4, 8, 12 of LDN therapy, and 4 wk after discontinuation of treatment (wk 16) for each of the parameters measured by the SF-36 health survey. Significantly different from baseline values included the following: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. all parameters measured for these patients. It is unknown at Treatment with LDN may provide some advantages over this time, how long the quality of life benefit of LDN persists other standard therapy for Crohn’s disease. Although the after discontinuing therapy, but this observation merits fur- long-term safety profile of LDN in Crohn’s patients is un- ther investigation for duration of response. known, the safety profile of LDN appears to be excellent in Naltrexone and Crohn’s Disease 7 this short-term study, with infrequent and minor side effects fonic acid) murine model of colitis (47). Plasma enkephalin and no known suppression of immunity or greater risk of levels were not altered in this study; however, the enkephalin secondary infections. Cortiocosteroids have short-term side levels were only obtained monthly at the time of the AM clinic effects of weight gain, emotional laibility, glucose intoler- appointment. Because enkephalin levels have been shown to ance, and risk of secondary infections, especially fungal (34). increase in animals administered LDN through transient re- Acute complications with some immunomodulators (azathio- ceptor blockade, it is possible that the plasma enkephalin prine, 6-mercaptopurine) include idiopathic pancreatitis and levels were increased by the compound, but the time of the neutopenia (35). Acute allergic reactions have been reported blood sampling was inappropriate. Peptide levels usually are with the new anti-TNF-α compounds; these drugs can also short-lived in the peripheral blood and frequent sampling increase the risk of reactivation of tuberculosis (36) and in- postingestion would be necessary to perform accurate phar- duce a lupus-like reaction, serum sickness syndrome, and/or macokinetic assays. Another possible explanation for the un- anaphylaxis (37). Higher doses of naltrexone (i.e., 50 mg) changed enkephalin levels in this study may be that perhaps used for alcohol and opioid abuse have been reported to el- the dose used in this study was too low and did not effect a suf- evate liver transaminases (38). In contrast, the use of LDN ficient blockade to stimulate upregulation of [Met5]-enkep- herein at 4.5 mg daily did not change liver transaminases halin. during treatment. LDN may also be acting by another mechanism unrelated Infliximab has become the standard medical therapy for pa- to changes in enkephalin levels such as through a reduction in tients with fistulizing disease associated with Crohn’s disease cytokine activity or promotion of direct growth and mucosal (39). It is of interest that the two subjects in our study with repair. In fact, opioids have been shown to increase release enterocutaneous fistulas noted closure with LDN when they of peritoneal cytokines (45) and naltrexone has been shown had not previously responded to infliximab. Perhaps closures to block TNF-α production in a murine model (26). Another of the fistulas may be related to lower intestinal secretions or possibility is that therapy with the low-dose opioid antagonist mucosal healing. Naltrexone has been reported to promote may have affected a different endogenous opioid substance, healing of corneal abrasions and epithelial wound healing by such as endorphins, which were not measured. stimulating DNA synthesis (25); therefore, this compound Naltrexone may be playing a role in direct mucosal heal- may promote healing. Perhaps, the fistulas closed as a result ing unrelated to its effects on cytokines. Opioids have been of a lower number of bowel movements and improved mu- shown to decrease cell growth (48) through the interaction cosal fluid absorption as reported in diarrheal disorders of with the nuclear opioid growth factor receptor (49), and in- other etiologies that respond to enkephalins (22). deed blockade of the opioid receptor with naltrexone has been It was of interest that one study subject with multiple scle- shown to promote DNA synthesis and healing of corneal ul- rosis and Crohn’s disease in our study also had improvement cers (25). Lastly, there are opioid receptors throughout the of her neurologic symptoms with LDN. Although the eti- gastrointestinal tract that are involved in analgesia, fluid, and ology of both disease processes is unknown, another mon- water absorption. Because the CDAI scores are partially cal- oclonal antibody, natalizumab, has been useful in treating culated with the patient’s number of liquid bowel movements both of these conditions (40), suggesting perhaps a similar and perceived pain, naltrexone may have improved the CDAI underlying defect. If so, perhaps evaluation of LDN in other scores in these individuals through another opioid-mediated inflammatory conditions such as multiple sclerosis would be mechanism. warranted. The results of this feasibility study support the need for Medical care for IBD is costly (41, 42). Aminosalicylate further investigation with a randomized controlled Phase 2 therapy can cost several hundred dollars per month, and an in- trial of LDN therapy and comparison to a placebo group. fliximab infusion generally exceeds several thousand dollars Because the present study found that subjects were still un- (not to mention the time away from the workplace for IV ad- changed and improved 4 wk after stopping naltrexone therapy, ministration) (43). Naltrexone is a generic medication and the a longer follow-up period should be observed to determine the cost is therefore inexpensive. Moreover, effective mesalamine durability of response. Extended treatment periods in future therapy (Pentasa) may require up to 8–16 tables per day. An- studies would further define optimal naltrexone treatment pa- other advantage of LDN is the once-a-day dosing, which may rameters. It is unknown whether naltrexone may be beneficial improve patient compliance. in reducing the amount of additional Crohn’s medications The mechanism by which LDN improves symptoms and required, i.e., steroid sparing. Future studies are needed to reduces inflammation of those individuals with active IBD is further explore these interesting results and perhaps provide unknown. Opioid receptors for µ, κ, and δ have been identi- hope for a common frequently debilitating disease. fied on immune cells (44) and morphine has been shown to induce the release of proinflammatory cytokines from mouse 5 peritoneal marcrophages (45). [Met ]-enkephalin has simi- ACKNOWLEDGMENTS larly been shown to stimulate peritoneal macrophages in ro- dents (46). In contrast, Philippe and coworkers have shown We acknowledge Mr. Moshe Rogosnitzky for his suggestion that stimulation of the µ opioid receptor with elective agonists of using low-dose naltrexone for the treatment of Crohn’s dis- reduces inflammation in the TNBS (2,4,6-trinitrobenzene sul- ease. Funding was provided by NIH grants #M01 RR010732 8 Smith et al. and C06-RR016499 to the General Clinical Research Center 9. Papadakis KA, Targan SR. The role of chemokines and and an Institutional Dean’s Feasibility Grant. chemokine receptors in mucosal inflammation. Inflamm Bowel Dis 2000;6:303–13. 10. Targan SR, Murphy LK. Clarifying the causes of Crohn’s. STUDY HIGHLIGHTS Nat Med 1995;1:1241–3. 11. Hanauer SB, Present DH. The state of the art in the man- What Is Current Knowledge agement of inflammatory bowel disease. Rev Gastroenterol r Disord 2003;3:81–92. The current medical therapy of Crohn’s disease in- 12. Navarro F, Hanauer SB. 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