potential future therapies for x-ald.docx

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Potentia l future therapies for X-ALD

B y G a v i n M e t c a l f , B S c . ( H o n s ) , A M S B .

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A few potential future therapies have been investigated in the following document.These include:

Pharmacological intervention by Fibrates (Page 1) Modified Gene Therapy (Pages 2 - 3) Enzymatic Controls (Page 4) Antioxidant intervention (Pages 5 - 6)

Fibrate Drugs:

M Engelen, et al. (2012). Bezafibrate lowers very long-chain fatty acids in X-linked adrenoleukodystrophy fibroblasts by inhibiting fatty acid elongation.

Journal of Inherited Metabolic Diseases. (Ahead of print)

[Freely available online at:http://www.springerlink.com/content/p0819uu81w81518j/]

Within this paper, the researchers begin by describing that VLCFAs are primarily derivedfrom endogenous synthesis via elongation of very long fatty acid (ELOVL) enzymes.Seven of these enlongases have been denoted in mammals (ELOVL1 ELOL7).Pharmacological inhibition by Fibrate drugs (including Benzafibrate; BF), a class ofamphipathic carboxylic acids which are used for a range of metabolic diseases, havebeen described to have potential to reduce VLCFAs in individuals with malfunctioning

ABC proteins e.g. X-ALD sufferers.

A daily 400M (micromolar) amount of the BF displayed reductions of up to 75% inC26:0 levels within human fibroblasts (a type of specialised skin cell) isolated from X-

ALD patients.

The key researchers of this paper have suggested two possible means of howpharmacological intervention could have facilitated these results:

1 - Indirect modification of other cellular processes, such as gene expression (how muchof a particular gene is read and used to produce proteins, such as the enlongase enzymes)

via epigenetics for example. Epigenetics is a way the body can control what genes areexpressed simply by attaching methane groups to particular areas of the DNA, a bit likeputting a set of bollards in a roadit stops cars traveling along the road; methane groupsblock transcribing enzymes from traveling along that particular part of DNA.

2 - Direct inhibition of the Elongase enzymes (ELOVL1 ELOL7) themselves. Thiswould mean that BF, or its metabolites (broken down BF

products), fit into the active site of the enzymes blocking them, a bit like a key in a lock,and reducing synthesis of C26:0 Fatty acids.

OVERALL COMMENTS: The work described in the paper suggests that inhibition ofVLCFA synthesis by pharmacological means could be a feasible treatment option for X-ALD. BF is a good candidate for this approach, with a proven safety profile for (long-term) use in humans. With a maximum daily dose of BF therapeutic levels might bereached in plasma. A small-scale proof of principle clinical trial is currently ongoing in

the US to evaluate the effect in X-ALD patients, if this trial proves successful it may notbe long before we see clinical trials involving BF in the United Kingdom.
http://www.springerlink.com/content/p0819uu81w81518j/http://www.springerlink.com/content/p0819uu81w81518j/http://www.springerlink.com/content/p0819uu81w81518j/http://www.springerlink.com/content/p0819uu81w81518j/


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Modi f i ed Gene Ther apy :

Car t i e r , e t a l . ( 2012 ) Lentiviral hematopoietic cell gene therapy for x-

linked adrenoleukodystrophy. Methods in Enzymology. Vol. 50 7: pp1 87 -98 .

[Abs t r ac t a t : h t tp ://www.ncb i . n lm .n ih . gov/pubmed/22365775]

A l l oge ne i c he ma topo i e t i c c e l l g e ne the ra py ha s bee n s how n tor e su l t i n l ong - t e r m bene f i t s b y a r r e s t i ng the p r og r e s s ion o f c e r eb r a ldemye l i n a t i on i n boys w i th X-AL D. When pe r f o r med a t an e a r l y s t age HCT i s t hough t t o be one o f t he on l y t he r apeu t i c app r oachestha t c an s t ab i l i z e c e r eb r a l d emye l i n a t i on . Howeve r , i t i s no t knownas o f y e t i f HCT can p r even t , o r r e scue , ad r enomye loneu r opa thy i na f f ec t ed i nd iv idua l s .

Th i s s tudy de sc r ibed the u se o f a ge ne de l iv e ry ve h i c l e - Len t i v i r a lv ec t o r s . T hey have r ece n t l y bee n ad ap te d tha nks to the i r a b i l i t y toin t eg r a t e i n to the genome (DNA) o f non -d iv id ing ce l l s . ( S ee imagebe low)

(h t tp ://en .w ik iped i a .o r g/w ik i/F i l e :L en t i v i r a l _vec to r .png )

The a bove imag e d i sp l a y s how the d e f ec t iv e gene , ABC D1 inCD34+ ce l l s ( a t ype o f c e l l i nvo lved i n t he immune sy s t em) f r ompa t i en t s w i th X-AL D, c an be r ep l aced by the f unc t ion ing gene f r omhea l thy donor ce l l s . CD34+ ce l l s we r e t aken f r om the X-AL Dpat i en t s b lood , and e s sen t i a l l y m ixed w i th the L en t i v i r a l v ec to r scon t a in ing the un -mut a t ed DNA w i th in a l abo r a to r y s e t t i ng ( i nv i t r o) . Th e vec t o r , be ing a p a r t i c u l a r t y pe o f v i ru s , i s a b l e tot r an s f e r t he hea l t hy DNA sec t i on i n to the t a r ge t c e l l s g enome v i a P O L enzymes ( a t ype o f po l ymer a se enzyme) .

The tw o pa t i e n t s in th i s s t udy we re age d 7 and 7 . 5 , a nd both hado lde r s i b l i ng s who d i ed o f AL D. The ce l l s t h a t we r e deno t ed a shav ing i n co r po r a t ed the new (hea l t hy ) DNA in to the i r g enomes
http://www.ncbi.nlm.nih.gov/pubmed/22365775http://www.ncbi.nlm.nih.gov/pubmed/22365775http://en.wikipedia.org/wiki/File:Lentiviral_vector.pnghttp://en.wikipedia.org/wiki/File:Lentiviral_vector.pnghttp://en.wikipedia.org/wiki/File:Lentiviral_vector.pnghttp://www.ncbi.nlm.nih.gov/pubmed/22365775


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we re r e in t roduce d in to t h e pa t i en t s . The se a l t e r e d ce l l s wou l d thenbe l e f t t o mu l t i p l y and r e in t r oduce some f unc t iona l ABC p r o t e in s .

F o l l ow-up t e s t s we r e comp le t ed w i th bo th pa t i en t s . In t e r e s t i ng l y

enough , t he i r MRIs i nd i c a t ed tha t p r og r e s s ion o f c e r eb r a lmye l i n i s a t i ng l e s ions was a r r e s t ed a t 12 -14 mon ths a f t e r HCT.F ur the r mor e , up to 36 mon ths no add i t i ona l l e s i ons o r changes i nthe MRI had been no t ed . These f i nd ing s sugges t t h a t t hep r oduc t ion o f f unc t iona l ABC p r o t e in s may have r educed the bu i l dup o f h a r mf u l VL CF As , wh i ch may have r educed the neu r o log i c a le f f ec t s o f t he se F As .

OVERALL COMMENTS: Th i s p r oce s s , a l t hough t r i cky and w i thpo t en t i a l d ange r s , h a s been shown be a po t en t i a l l y i n f l u en t i a l

t h e r apy f o r boys w i th X-AL D. Howeve r , t h i s was a sma l l - s c a l es t udy , w i th on l y two pa r t i c i p an t s ( n=2 ) . Th i s means th a t wha t may have wor ked f o r t he two pa t i en t s t e s t ed , may no t wor k f o r o the r s .

A dd i t i ona l l y , o the r u nknown fac to r s may ha ve c on t r ib u te d t o thea l t e r a t i on s i n MRIs - t h e pa t i en t s genomes may have con t r ibu t edin some way to the i r a l t e r ed ce r eb r a l d emye l i n a t i on? F u r the r mor e ,i t wou ld be s en s ib l e t o sugges t t h a t l onge r pos t -HCT t e s t s bepe r f o r med to check i f t h e changes i n c e r eb r a l l e s i ons wer e i ndeed ac onc r e t e change , no t a b l i p t h a t may a l t e r a t a l a t e r d a t e .

*Addi t iona l in fo r mat ion on HCT can be found a t the fo l lowingl ink : h t tp ://www.x-a ld .n l/ t r ea tment -op t ions/hsc t/*
http://www.x-ald.nl/treatment-options/hsct/http://www.x-ald.nl/treatment-options/hsct/


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Enzymat ic con t r o l s :

S ingh , e t a l . ( 2011 ) Histone deacetylase inhibitor suberoylanilide

hydroxamic acid normalizes the levels of very long chain fatty acids in humanskin fibroblasts from X-Adrenoleukodystrophy patients and downregulates theexpression of proinflammatory cytokines in Abcd1/2 silenced mouse astrocytes.

Journal of Lipid Research.

[Available from:http://www.jlr.org/content/early/2011/09/04/jlr.M017491.full.pdf]

This study underlines the importance of a potent histone deacetylase (HDAC) inhibitor -Suberoylanilide hydroxamic acid (SAHA) in inducing the expression of various ABCDgenes - producing ALD proteins, and normalizing peroxisomal beta-oxidation (the breakdown, or catabolism, of VLCFAs) in cultured human cells from X-ALD patients.

The cells used in this study (Fibroblasts and Astrocytes) were obtained from securestorage (Coriell Cell Repositories:www.ccr.coriell.org), after being donated by various X-

ALD patients.

The researchers report that at the highest dose of SAHA (5M) significantly increasedABCD2 gene expression by up to 11.8-fold in three days of drug administration incultured fibroblasts (See page 1 for information on these cells). Additionally, a 10-foldincrease in the expression of ABCD3 genes was also seen within fibroblasts.

Suberoylanilide hydroxamic acid was also denoted to significantly increase peroxisomal

beta-oxidation in fibroblast cells, meaning there was an increase in VLCFA breakdown.Therefore, the levels of VLCFAs decreased, which would conceivably lead to a reductionor at least a slow-down of demyelination in the cerebrum.

Furthermore, SAHA reduced the production of Reactive Oxygen Species (ROS) inAstrocyte cells (a type of cell found only in the brain), as well as reducing the productionof Tumour Necrosis Factor - alpha (TNF-), a pro-inflammatory cytokine thought toinfluence cerebral demyelination.

OVERALL COMMENTS: SAHA has been shown to provide a favorable outcome ina variety of animal models of inflammatory disease conditions including LPS-inducedendotoxemia, lupus, graft-versus-host disease, ischemia, TLR activation, septic shock andinflammatory hyperalgesia. Moreover, SAHA is presently in numerous clinical trials;more than 100 clinical trials with SAHA are in progress at the NIH(http://www.clinicaltrials.gov). This indicates that SAHA is a potential candidate drugfor correction of the metabolic defect as well as the secondary neuroinflammatorydisease in X-ALD.
http://www.jlr.org/content/early/2011/09/04/jlr.M017491.full.pdfhttp://www.jlr.org/content/early/2011/09/04/jlr.M017491.full.pdfhttp://www.jlr.org/content/early/2011/09/04/jlr.M017491.full.pdfhttp://www.ccr.coriell.org/http://www.ccr.coriell.org/http://www.ccr.coriell.org/http://www.clinicaltrials.gov/http://www.clinicaltrials.gov/http://www.clinicaltrials.gov/http://www.clinicaltrials.gov/http://www.ccr.coriell.org/http://www.jlr.org/content/early/2011/09/04/jlr.M017491.full.pdf


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A nt i ox idant in ter ven t ion:

A s d i s cu s sed i n t he p rev i ous s ec t i on , ox ida t i ve dama ge by Reac t i ve

Oxygen Spec i e s (ROS) a r e t hough t t o con t r ibu t e t owar ds thece r eb r a l d emye l i n a t i on i n X-AL D pa t i en t s , due to the bu i l d up o f V LC FAs in c e l l s i n c l ud ing As t rocy te s .

A va r i e t y o f an t i ox i da nt re l a t e d the rap i e s a r e ou t th e re , bu t t he sea r e two o f wh i ch I t h ink co l l e c t i v e l y d i sp l a y a pos i t i v e t ake homemessage abou t t he po t en t i a l o f an t iox idan t i n t e r ven t ion a s athe r apy .

Lopez-Er ausk in , e t a l . ( 2011 ) Antioxidants halt axonal degeneration in

a mouse model of X adrenoleukodystrophy.Ann Neurol. Vol. 70: pp. 84-92.

[Available from:http://onlinelibrary.wiley.com/doi/10.1002/ana.22363/full]

The study used mouse models, with the ABCD1 gene knocked out (removed) (ABCD1 -

models). The mice models displayed a variety of late onset neurological phenotypes withlocomotor disability and axonal degeneration in the brain and spinal cord.

The researchers focused upon three antioxidants; N-acetyl-cysteine (NAC), alpha-lipoicacid (LA), and alpha-tocopherol. These three antioxidants were described as scavengersof VLCFA-dependant ROS, generated out of the body in laboratory conditions (in vitro).

The three antioxidants were administered (LA, and alpha-tocopherol in food chow,and NAC in water) in a pre-clinical setting. This cocktail of antioxidants displayed threeremarkable reversals in the mice models:

1) Oxidative stress and lesions to proteins reduced.2) Signs of axonal degeneration reversed.3) Locomotor impairment in a variety of physical/behavioural tests performed with

the mice reduced also.

Therapeutic implications derived from this work could also be extrapolated to otherdiseases that share both axonal degeneration and oxidative stress as a main or early

contributing pathogenic factor, e.g. motor neurone disease.

OVERAL COMMENTS: These three factors displayed that the impact of oxidativedamage to the brain and spinal cord resulting from the build up of VLCFAs is aparticularly major one. This also suggests that antioxidant intervention could be yetanother potential therapy for X-ALD. However, this treatment would only appear to bea possible therapy in combination with other drugs that would reduce proinflammatorycytokines in patients with severe neuroinflammatory demyelination present.
http://onlinelibrary.wiley.com/doi/10.1002/ana.22363/fullhttp://onlinelibrary.wiley.com/doi/10.1002/ana.22363/fullhttp://onlinelibrary.wiley.com/doi/10.1002/ana.22363/fullhttp://onlinelibrary.wiley.com/doi/10.1002/ana.22363/full


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Fourcade, et al. (2010) Valproic acid induces antioxidant effects in X-linkedAdrenoleukodystrophy. Human Molecular Genetics. Vol. 19: pp. 2005-2014.

[Available from:http://www.ncbi.nlm.nih.gov/pubmed/20179078]

This was a six-month pilot study, whereby Valproic acid (VPA), a drug already used as atherapeutic agent for epilepsy, was administered both in body (in vivo) and out of body ina laboratory setting (in vitro). The drug has previously been shown to be able to freelycross the blood-brain barrier, i.e. diffuse from the blood circulatory system into thebrain, a process that is very difficult to obtain. This gave the drug a big head start as apossible therapeutic agent involving X-ALD as a neurological disorder.

As described in the previous study on antioxidants, the researchers of this paperinvestigated X-ALD fibroblasts, which were cultured in the presence of VPA (2mM) for

2 and 12 days. A 2.8-fold increase in ABCD2 gene expression was seen in X-ALDfibroblasts after 2 days treatment. Also a 1.4-fold increase in ABCD3 expression was alsodenoted after treatment with VPA. In contrast, ABCD4 expression was not affected by

VPA administration.

It was denoted that VPA did not alter C26:0 levels, nor C24:0. However, in the presenceof VPA the production of C26:1w9, a monounsaturated VLCFA fell by over 40% - a bigdrop. This raises the possibility that the antioxidant Valproic Acid (VPA) might, at leastpartially, mediate the decrease in C26:1w9.

As the half-life (the time of the drug circulating in the body prior to excretion of break

down) is very short, only 2.5 hours in rodents compared to 8-10 hours in humans an ex-vivo system rather than in-vivo was used. Whereby rodents were sacrificed and theirhippocampus (part of the brain) finely sliced at 200 uM thickness and exposed to the

VPA. A small number of human hippocampal tissue samples, acquired from epilepsypatients who had brain surgery, were also treated the same and exposed to VPA to see if

ABCD2 expression increased.

ABCD2 expression increased in both rodent and human samples, but was higher inhumans compared to the rodents, but this may have been due to interspecies differences.

OVERALL COMMENTS: VPA, although not effective upon C26:0 or C24:0, wasseen to reduce C26:1w9 levels by an increased expression of ABCD2 and ABCD3. Thissuggests that the drug could be combined with other antioxidants in a cocktail method tonot only increase the amount of functional ABC proteins, but also reduce the levels ofharmful VLCFAs in the brain and spinal axons.

* Please note that all information documented has come from above referenced sources, none of the studyoutcomes nor the information they provide is owned by me, G. Metcalf, or ALD Life *
http://www.ncbi.nlm.nih.gov/pubmed/20179078http://www.ncbi.nlm.nih.gov/pubmed/20179078http://www.ncbi.nlm.nih.gov/pubmed/20179078

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