Treating Chronic Fatigue Syndrome (CFS) & Fibromyalgia (FM) by

Targeting the Methylation Cycle

Derek Enlander, MD860 Fifth Avenue

New York, NY 10065 USA1-212-794-2000

denlander@aol.com

Overview of basic cell processes

Basic cell process

Chronic Fatigue Syndrome (CFS)

Studies of PathogenesisImmune system - IC’s, IgG, B cells, NK

Th2 phenotypecytokine dysregulation / chronic immune activation

Infection - virus, bacteriumNervous system - paresis, visual loss, ataxia, confusion

abnormal metabolism of 5-HIAA, A-V, 5-HT, PRLbrain scan abnormalities

Endocrine system - slight HPA axisCardiovascular system - vasodilatationPsychological function - depression & anxietyGenetic predisposition - deduced from twin studies

Pathogenisis

Virus as Causative Agent•CFS often starts with a flu-like episode

•CFS symptoms wax and wane

•Antiviral pathways are activated

•CFS symptoms are similar to many viral conditions including:oEBV mononucleosis oRoss River virus

•Geographic outbreaks have been reported

•Gene expression profiling found genetic variants that impact antiviral defenses

•Antiviral treatments have been effective in in small studiesoAmpligenoisoprinosineobeta interferonovalganiclovir

Viruses Implicated In CFS

HHV6 virus• Discovered in 1986 by Ablashi and

Salahuddin at NCI in patients with lymphoproliferative disease( slide by kind permission of Dharam Ablashi)

• HHV-6 is an enveloped, double-stranded DNA virus with an icosahedral capsid and virion size of 160-200 nm. The genome contains 70 proteins. Some of these proteins (early antigen and immediate early antigen) can be used to detect active infections.

• Two very distinct variants with 90% nucleotide sequence homology. The genome of variant A or B ranges from 159 – 170 kb.

• Beta herpesvirus and genus roseola virus

• Over 90% of adults seropositive

• Predominantly CD4 lymphotropic

•HHV-6 has two variants, A and B.  Each variant is associated with a subset of illnesses. 

•HHV-6A infection comes later in life and has been linked to the pathogenesis of CFS, rhomboencephalitis, and MS. It also plays a role in HIV, causing more immunosuppression in AIDS patients. 

•HHV-6B is the causative agent of Exanthem subitum, is strongly linked to infections in transplant patients and is also associated with epilepsy and post-transplant acute limbic encephalitis.

•HHV-6A is more lytic, and readily infects a variety of neural and other cells such as

astrocytes , and is also more neurotropic, leading to cognitive disorders in CFS and MS patients.

•Both A & B strains cause encephalitis, amnesia, facial paralysis, chronic myelitis and transverse myelitis.

•HHV-6A can lead to enhanced production of EBV, HSV and HHV-8. Both variants also induce expression of human endogenous retrovirus K-18 encoded super antigen.

HHV6

•Both strains establish latency in monocytes, macrophages and target CD4 cells for infection.

•Since HHV-6, like other human herpesviruses, is ubiquitous, its role in the pathogenesis is established when the virus is in the active state of infection.

•Anti-viral agents foscarnet, cidofovir and ganciclovir inhibit CMV infection do not block HHV-6 infection, especially of the A variant as efficiently.

HHV-6 infection contributes to immune suppression by:

•Disturbing key immune activation pathways and cytokine networks.

•Depleting CD4 T lymphocytes via direct infection and induction of apoptosis (Lusso)

•Upregulating TNF alpha, TNF- gamma, IL-1beta and IL-10.(Flamand, Dockerell, Li) and IL-21.•Downregulating complement activity through the CD46 receptorsuppressing macrophages to produce IL-12 stimulated with gamma interferon.(Lusso 2004)

HHV6 (cont.)

www.ikdt.com

HHV-6 B HHV-6 A

Detection of interstitial cells IHC Labeling of cardiomyocyte

Localization of HHV-6 variants in myocardial tissue by IHC

Kühl et al., 2008, submitted

Myocardial hhv6

Infected astrocyte

Astrocytes obtained from lateral temporal lobe infected with HHV-6

Astrocyte hhv6

Antiviral / immuno used for CFS

Antiviral Study effective?

ampligen Strayer 1994 yes ???isoprinosine (Immunovir) Pinching 2002 yesalpha 2a interferon See, 1996 yes ?acyclovir (Zovirax) Straus 1990 (5 weeks) novalacyclvir (Valtrex) Lerner, 2001 (6 mos) yes ??valganciclovir (Valcyte) Lerner, 2006 (6 mos) yesvalganciclovir (Valcyte) Montoya, 2006 (6 mos) yesHepapressin, Immunoprop Enlander 2007 (12 mos) yes

Methylation cycle

Reasons to Target Methylation Cycle

• Paul Cheney, M.D. reported “almost universal” glutathione depletion in CFS in 1999

• Rich Van Konynenburg, Ph.D. reported that CFS symptoms can be explained by glutathione depletion [AACFS 7th Intl. Conf.]

• Personal experience with glutathione supplementation and methylation targeting

Current Treatment part 1

Current Treatment part 2

Current Treatment part 3

Major Pathways - Methylation Cycle

Diet Choline

Betaine TMGZn

DHF

THF

DNA, RNAMethionine

Protein synthesis, CarnitineSAM

Methyl Synthase (MS)

SAH

Homocysteine

Cystathionine Glutathione

Zn, B6

BHMT B12

MAT (methionine adenosyltransferase)

Main Role of Methylation Cycle

• Methyl Group Source for the body

• Coordinate sulfur metabolism

• Coordinate production of DNA

Methionine-Homocysteine Pathway

Methionine

S-Adenosylmethionine

S-Adenosylhomocysteine

Homocysteine

Cystathionine

Cysteine Toxic Sulphites

SulphatesGlutathione

B12, B6, Mg

Role of Hepapressin Injection

Methionine

S-Adenosylmethionine

S-Adenosylhomocysteine

Homocysteine

Cystathionine

Cysteine Toxic Sulphites

SulphatesGlutathione

B12, B6, Mg

Folic Acid

Hepapressin / Kutapressin / NexavirBovine/Porcine Liver Extract once weekly

Phase I clinical trial 1994

Invivo antiviral activity

Valganciclovir ..Valcyte 450mg twice dailyMontoya, 2006

Case Study, Retinal Uveitis + CFS

Modest Effect

Important to identify appropriate CFS patient titer 1:640

Antiviral treatment

Hypothesis: that abnormalities of gene regulation occur in CFS

25 CFS patients & 25 normal controlsGene levels determined by Microarray analysis (9,522 genes) & real-time PCR

Study of Gene Expression in Chronic fatigue syndrome

Pilot study - 2005

Pilot study

Gene expression 2005

CLINICAL COLLABORATORSDr Selwyn Richards, Dorset CFS ServiceDr Janice Main, Imperial College LondonProfessor Terry Daymond, SunderlandProfessor Andrew Smith, University of CardiffDr David Honeybourne, Birmingham Dr Amolak Bansal, St Helier Hospital, SurreyProfessor Jon Ayres, Aberdeen UniversityProfessor Robert Peveler, University of Southampton Professor David Nutt, University of BristolDr John Axford, St George’s University of LondonDr Russell Lane, Charing Cross Hospital, LondonDr John K Chia, UCLA Medical Centre, CA, USADr Derek Enlander, NY, USADr Paul Langford, Imperial College LondonProfessor Mike Levin, Imperial College London

FUNDINGCFS Research Foundation, Hertfordshire, UK

Acknowledgements

STUDY DESIGN & LABORATORY WORKDeepikaDevanur, St George’s University of LondonRobert Petty, St George’s University of LondonBeverley Burke, St George’s University of LondonNarendraKaushik, Imperial College LondonRob Wilkinson, Imperial College LondonClare McDermott, Dorset CFS ServiceJane Montgomery, Dorset CFS ServiceDavid Fear, Kings College LondonTim Harrison, UCLPaul Kellam, UCLDavid AJ Tyrrell, CFS Research Foundation Stephen T Holgate, University of SouthamptonEmile Nuwaysir, Nimblegen Inc, USA.Don Baldwin, University of Pennsylvania, USAPeter Rogers, NBSDiana Carr, NBSJulie Williams, NBSFrank Boulton, NBSAndrew Bell, Poole Hospital

Kerr collaborators

Microarray kind permission of Jonathan Kerr

CFS Gene microarray

Microarray

Gene Microarray

CFS Associated genes

16 CFS-associated Genes

Immune IL-10RA IL-10 receptor alphaCD2BP2 CD2 antigen binding protein 2

Neurological PRKCL1 Protein kinaseC-like 1GABARAPL1 GABA(A) receptor associated protein like-1KHSRP KH-type splicing regulatory proteinNTE Neuropathy target esteraseGSN Gelsolin

Mitochondrion MRPL23 Mitochondrial ribosomal protein L23EIF2B4 Euk. translation initiation factor 2B, subunit 4δ, tv-1EIF4G1 Euk. Translation initiation factor 4G, subunit 1, tv-5

Apoptosis / cell cycle PDCD2 Programmed cell death 2, tv-1ANAPC11 Anaphase promoting complex subunit 11homologBRMS1 Breast cancer metastasis suppressor 1

Peroxisome ABCD4 ATP-binding cassette subfamily D, member 4PEX16 Peroxisomal biogenesis factor 16

Transcription POLR2G RNA polymerase II (DNA-directed) polypeptide G

Pilot study Kerr,

Enlander et al.

Diagnostic test for CFS

Biomarkers

**Collaboration with Dept of Paediatrics, Imperial College London

Diagnostic slide test

GcMaf

• GcMaf protein.. Gc Macrophage Activation Factor original work Yamamoto

• Reduced by action of Nagalase• Nagalase if increased GcMaf is decreased • Research into ME/CFS serum nagalase

levels• Administration of GcMaf injection in

research protocol• Cheney researches MAF 314 probiotic

yogurt• ME/CFS center researches MAF 878

probiotic

Ampligen

• Research treatment protocol • Thought to act on the immune system• 5 centers chosen in US• California, New York, Utah, Carolina,

Florida• Hemispherx part funding • Patient cost for 6 months $12,000• Not yet FDA approved

Retro Virus XMRV• New retrovirus XMRV testing in Nevada….

Judy Mikovits• Exciting press releases Oct 2009

• Helps patients and doctors stress the physiological aspect of CFS / ME

• Harvey Alter, NIH confirms MLV results 2010

• Small number of patients tested, not adequately replicated as yet

• Moderation in determining this as causative agent

• Researcher battle ensues• Science requests withdrawal of original paper

May 2011• WPI dismisses Dr Mikovits , sues her for stealing her own notebooks , jails her for 4

days

XMRV retro virus

• Whitmore Pederson Institute, Reno, Nevada

• XMRV retro virus study• Science journal

publishes article 8th Oct 2009

• Battle ensues by other researchers some confirm, other deny

• May 2011 Science asks Judy Miklovits to withdraw article

Dr Judy Mikovits

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 2922-2926, April 1991 Medical Sciences

Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome (Epstein-Barr virus syndrome/infectious mononucleosis/myalgic encephalomyelitis/polymerase chain reaction/in situ hybridization)

ELAINE DEFREITAS*, BRENDAN HILLIARD, PAUL R. CHENEY, DAVID S. BELLS, EDWARD KIGGUNDU, DIANE SANKEY, ZOFIA WROBLEWSKA, MARIA PALLADINO, JOHN P. WOODWARD§, AND HILARY KOPROWSKI

The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104 Contributed by Hilary Koprowski, Nov13, 1990

ABSTRACT Chronic fatigue immune dysfunction syndrome (CFIDS) is a recently recognized illness characterized by debilitating fatigue as well as immunological and neurological abnormalities [Straus, S. E. (1988) J. Inf. Dis. 157, 405412]. Once thought to be caused by Epstein-Barr virus, it is now thought to have a different but unknown etiology. We evaluated 30 adult and pediatric CFIDS patients from six eastern states for the presence of human T-lymphotropic virus (HTLV) types I and II by Western immunoblotting, polymerase chain reaction,

and in situ hybridization of blood samples. The majority of patients were positive for HTLV antibodies by Western

blotting and for HTLV-II gag sequences by polymerase chain reaction and in situ hybridization. Twenty nonexposure healthy controls were negative in all assays. These data support an association between an HTLV-ll-like virus and CFIDS.

Cohort population Initiation 3 months 6 months 9 months

ControlPlacebo

215 48 (2.4) 49 (2.6) 53 (2.4) 50 (2.3)

Kutapressin /Hepapressin

alone

209 46 (2.6) 57 (2.4) 55 (1.8) 62 (1.5)

Kutapressin /Hepapressin complexed

210 47 (2.1) 47 (1.9) 64 (1.3) 74 (0.8)

Kutapressin /Hepapressin complexed

ImmunopropImmunoplus

216 45 (2.5) 52 (1.7) 73 (0.9) 81 (0.5)

Hepapressin complex treatment Karnofsky & Fatigue scores

GcMaf / Nagalase…. Collaboration with Dr. Kenny deMeirleir

GcMaf Study in treatment of CFIDS/ME patients

Ampligen treatment study

Part Sponsored by Hemispherx Inc

B12 changes with BetaMax (sublingual methyl B12)

Average increase 56% after avg. 3 months of use

Retrospective analysis of Carnitine in CFS

120 patients, randomly selected serum Carnitine Low

Analysis of urinary H2S with Lead Arsenate

70 patients, randomly selected 35% positive Lead arsenate analysis

Ongoing research

Proposed Research

• Retuximab • CMX 1000• Enbrel• Pre and post Exertional Malaise study

Retuximab Study

• Norwegian Study Oct 2011 • ME/CFS Patients with lymphoma • ME/CFS seemed to improve after chemo• Initial study 5 patients• Followup with 15 patients 15 controls

Retuximab Study

• Patients have to be hospitalised overnight• Retuximab is a chemotherapeutic infusion • Administered monthly for 6 months• Not FDA / GMC approved• Past side effects including fatal outcome• Relapse 6 months after infusion completion• Reversed after second infusion

The UK and Ireland scene• Problems in finding the diagnosis• If diagnosis is made here or elsewhere problem in

finding suitable care and treatment• Doctors are only given psychiatric methods of

treatment CBT & GET• Research into physical aspect of ME/CFS is

neglected• Disability is undercompensated• National cost in GNP is estimated at 10,000

million pound annually

The UK and Ireland scene

• Provide research funds to physical diagnosis and treatment

• Provide training to young doctors • Provide information to old doctors• Set up Central and regional ME/CFS centres to

diagnose and treat this physical disease• Reverse old ideas of psychiatric treatment

Immediate Provisional solution• Initiation and funding of two fellowships in the

diagnosis and treatment of ME/CFS by the Government and/or Private sources

• We have set up the fellowship training for 12 months for two post Doc physicians

• The training will be provided at the ME/CFS Centre in New York

• Dr Eric Schadt will supervise the research part of the fellowship

• Dr Derek Enlander will supervise the clinical part of the fellowship

ME/CFS Center Mount Sinai Medical Center, New York

set up in Nov 2011 with a donation of one million dollars by a patient of Dr Enlander

First ME/CFS centre in a major School of medicine• Research in Genomics Eric Schadt• Immunology Miriam Merad• Virology Ila Singh• Pulmonology Christian Becker• Clinical diagnosis and treatment Derek Enlander

and David Bell