INVITED REVIEW

Therapy development for ALS: Lessons learned and path forward

VEENA LANKA & MERIT CUDKOWICZ

Neurology Clinical Trials Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown,

Massachusetts, USA

AbstractSeveral therapies have shown promise in preclinical models of motor neuron disease. Several of these treatment approaches,however, failed in human studies. In moving forward with new promising therapies, it is important to first identify whetherthe past trials were unsuccessful due to wrong therapy and biological target or because of flaws in trial design and conduct.We review treatment development in ALS and discuss the strengths and limitations of past clinical trials. Better biomarkersof disease and markers of biological activity of the therapies under development are urgently needed. Obtaining informationregarding dosage, pharmacokinetics, short-term safety and biological activity in well designed phase I and II studies iscritical to the design of phase III trials that will yield meaningful results.

Key words: Amyotrophic lateral sclerosis, clinical trials, study design

Introduction

There have been tremendous advances in under-

standing the biology of motor neuron disorders

including amyotrophic lateral sclerosis (ALS). This

has led directly to the development of one marketed

treatment, improved clinical management of people

with ALS, and many clinical trials of novel therapies.

ALS is a rare disorder with an incidence of 1�3/

100,000 per annum. A few treatments are now

available including riluzole, respiratory care and

nutritional support that produce important benefits

in survival, function, and quality of life for people

with ALS (1,2). However, there remains a critical

need to develop additional treatments that will slow

disease progression and ultimately turn ALS into a

long-term treatable illness. Several new and exciting

therapies are under development for ALS (Table I).

Encouraging pilot data are available from human

studies for several of these agents including talam-

panel, tamoxifen, sodium phenylbutrate, arimoclo-

mol and lithium. A critical review of past experiences

in clinical trials in ALS and suggested strategies for

future trials are presented.

Pathogenesis

Amyotrophic lateral sclerosis is a heterogeneous

disease with a complex etiology. Several pathways

have been implicated in disease pathogenesis includ-

ing glutamate mediated excitotoxicity, mitochon-

drial dysfunction, neuro-inflammation, apoptosis,

oxidative stress, protein aggregation, aberrant axonal

transport, and autoimmunity (3,4). New hypotheses

include disruption of the vascular endothelial growth

factor (VEGF) response to hypoxia (3), autophagy

(5,6), and retroviral infection (7�9). Drawing from

these hypotheses, several different therapies have

been tested in people with ALS (Table II). Approxi-

mately 5�10% of ALS cases are inherited (3), of

which approximately 20% are caused by dominant

inheritance of mutations in the superoxide dismutase

1 gene (SOD1) (10). Novel approaches to shut

down mutant SOD1 production are under develop-

ment for the subset of patients with these mutations

(11,12). Other mutations identified in families

with motor neuron disorders include genes encoding

the proteins alsin (13), dynactin (14), senataxin

(15) vesicle-associated membrane protein-associated

Correspondence: M. Cudkowicz, Massachusetts General Hospital, 149 13th Street, Room 2274, Charlestown, MA 02129, USA. Fax: 617 724 7290.

E-mail: mcudkowicz@partners.org

(Received 4 April 2008; accepted 7 April 2008)

Amyotrophic Lateral Sclerosis. 2008; 9: 131�140

ISSN 1748-2968 print/ISSN 1471-180X online # 2008 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)

DOI: 10.1080/17482960802112819

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proteins, and TAR DNA binding protein (TDP-43)

gene (16,17). The discovery of genetic mutations

that lead to motor neuron disease has led to the

development of genetically engineered models of

motor neuron disease (18).

Pre-clinical lessons

Valid disease models to study efficacy and biological

activity of therapy are critical to the development of

new agents for ALS. Currently, a wide variety of in

vitro and in vivo models are available to both study

the biology and screen therapeutic compounds

(Table III). However, there is not enough informa-

tion available to either validate or invalidate these

models as useful preclinical screen to accurately

predict therapies that will succeed in humans. The

models are invaluable as tools to test proof of

concept that the proposed therapy has desired

biological activity. This type of pre-clinical informa-

tion is critical to obtain prior to testing a novel

therapy in patients. While some therapies such as

riluzole were effective in both animal and human

studies (19�21), several others were not (22�31).

Numerous factors can account for the current

mismatch between results of in vivo models and

the human studies. Due to pharmacokinetic differ-

ences in mice and humans, it is challenging to

accurately extrapolate the dosage and pharmacoki-

netics from mouse to man. Therapies can have

different biodistribution and effects in rodents versus

humans. For example, celecoxib lowered prostaglan-

din E2 levels in cerebrospinal fluid (CSF) of mice

but not in human CSF (24). It is unknown whether

the transgenic mutant SOD1 mouse models

(mSOD1-G93A) accurately reflect sporadic ALS,

particularly models with higher copy numbers of the

mutation that develop a much more severe form of

the disease (32). A systematic analysis of 85 pub-

lished animal studies in ALS found that most of the

study designs did not include randomization or

blinding, even when outcome measures were re-

corded subjectively. A recent study suggests that

most published results in the murine model were

reflections of noise in survival distribution and the

authors recommend a minimum study design for

future mSOD1-G93A therapeutic studies (33).

Practical lessons from clinical trials

The community has gained valuable experience in

the design and conduct of clinical trials in ALS.

Several treatments with solid preclinical supportive

data have failed in human trials. It is essential to

critically assess the reasons for the failed trials in

ALS and in particular to assess whether the experi-

mental treatment did not show efficacy because the

therapy and biological target were wrong or due to

of trial design and conduct flaws (Table IV). It is

crucial to make this distinction by carefully review-

ing past trials so that a potential good therapy and

biological target is not dismissed because of a flawed

trial design or conduct. A common trial design flaw

in ALS studies has been conducting phase III studies

with inadequate information on drug dosage and

biological activity in humans.

Dosage selection

Identification of study medication dosage with the

best benefit-risk ratio is an important goal in early

phase trials. Studies that explore dosage response

relationships can provide important information for

selecting dosage for efficacy studies. Several efficacy

studies in ALS were conducted without knowledge

of dosage response relationships. For example, in the

clinical trial of topiramate, one dosage, selected

based on approved dosages for epilepsy, was tested

in ALS. Participants received the maximum toler-

ated dosage of 800 mg/day of topiramate and had a

faster decline in arm strength and higher risk for

several adverse events compared to a placebo group

(26). It is unknown if a lower dosage would have

been better tolerated or shown efficacy. In phase II

safety studies of minocycline, 200 mg/day was well

tolerated, while 400 mg/day was associated with an

accelerated decline in the Amyotrophic Lateral

Sclerosis Functional Rating Scale (ALSFRS) (34).

Despite these findings, the phase III trial tested the

maximum tolerated dose 400 mg/day for nine

months. A faster decline in function as measured

by ALSFRS-R was similarly found in the treatment

group (23). High dosages of study medication can be

associated with more adverse events and poor

tolerability with increased participant dropout rates,

thus reducing the power to detect benefit.

Testing a single dosage that is too low to produce

desired biological activity also may lead to erro-

neous rejection of a potentially beneficial therapy.

This may have occurred in trials of creatine and

celecoxib (22,24). Efficacy trials of creatine in

Table I. Current and future ALS treatment trials.

Compound Potential mechanism of action

Ceftriaxone Increase astrocyte glutamate

transport (EAAT2/GLT1 activity)

ONO-2506 Prevents reactive astrocytosis;

glutamate antagonism

Diaphragm pacing Provide respiratory support and

muscle training

Arimoclomol Heat shock protein inducer

Antisense oligonucleotide

SOD1 (ISIS)

Decrease production of SOD1

protein

Talampanal AMPA receptor antagonist

TRO19622 Improved mitochondrial function

KNS-760704 Improved mitochondrial function,

antioxidant

Lithium Increase in autophagy

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patients with ALS were conducted at 5 and 10 g/day.

However, body weight based conversions from the

effective dosage used in animal studies suggest a

corresponding human dosage of 30�35 g/day. Recent

studies of creatine in Parkinson’s disease and Hun-

tington’s disease also suggest that higher dosages

(20�30 g/day) are needed (35,36). Performing do-

sage-ranging studies early in drug development can

help overcome these challenges and lead to im-

proved phase III studies.

Pharmacodynamic markers

Another important goal of early phase studies is

to obtain evidence of biological activity of the

experimental therapy. This information supports

decisions on the appropriateness of therapy, its route

of administration, and dosage. Very few trials in ALS

have successfully employed pharmacodynamic mar-

kers to study the actions of the experimental therapy.

One example of a study that successfully used a

pharmacodynamic marker was the clinical trial of

the antioxidant alpha-tocopherol. In this trial an

effect of therapy on oxidative stress markers was

found with decreases in plasma thiobarbituric acid

reactive species and an increase in glutathione three

months after drug initiation (27). Incorporating

pharmcodynamic markers in studies provides more

opportunity to understand why a treatment fails or

works in humans. Preclinical studies can often

Table II. Past clinical trials in ALS.

Therapy Sample size Primary outcome measure Refs

Riluzole 155 Survival and rate of change of functional status (51)

959 Survival without tracheostomy (20)

Gabapentin 152 Slope of arm megascore using MVIC (25)

204 Slope of arm megascore using MVIC (52)

Topiramate 296 Slope of arm megascore using MVIC (26)

Lamotrigine 67 Clinical scores (age of onset, bulbar and respiratory involvement,

ambulation and functional disability)

(53)

39 Functional decline (Norris, Plaitakis and Bulbar scales) (54)

Dextromethorphan 45 Survival (55)

Talampanel 60 ALSFRS-R

Ciliary Neurotrophic Factor(rhCNTF) 730 Isometric muscle dynamometry (56)

570 Combination megascore of MVIC & FVC (57)

Insulin-like growth factor-1 183 Rate of change in Appel ALS score (58)

266 Rate of change in Appel ALS score

Brain-derived neurotrophic factor 1135 FVC & Survival (59)

Thyrotropin 36 Tufts quantitative neuromuscular exam (60)

Releasing hormone 30 Muscle strength decline (61)

Xaliproden 867 (I) Time to death, tracheostomy or permanent assisted ventilation (47)

1210 (II)

Vitamin E 289 Rate of deterioration of function by modified Norris limb scale (27)

160 Survival (62)

N-acetyl-L-cysteine 110 Survival (63)

Selegeline 133 Rate of change of Appel ALS total score (64)

Coenzyme Q10 31 Slope of arm megascore using MVIC (65)

Creatine 175 Death, permanent assisted ventilation and tracheostomy (42)

102 Slope of arm megascore using MVIC (66)

Branched chain amino acids 95 Clinical muscle strength, maximal isometric muscle torque (67)

126 Disability scales (68)

Nimodipine 87 Isometric muscle strength (69)

Verapamil 72 FVC and limb megascores (70)

TCH346 591 ALSFRS-R (46)

Pentoxifylline 400 Survival (71)

Minocycline 42 Safety/tolerability measures (34)

412 ALSFRS-R (72)

Sodium phenylbutyrate 40 Safety and tolerability (38)

Cyclophosphamide 44 Neurological function score (73)

Bovine gangliosides 40 Neuromuscular function (74)

40 Various objective tests of muscle strength (75)

Interferon-beta (IF1a) 61 Non self-supporting status (Medical Research Council Scale,

Norris Scale, Bulbar scores)

(76)

Celecoxib 300 Slope of arm megascore using MVIC (24)

Glutathione 32 Manual muscle testing (77)

Oxandrolone 19 Slope of arm megascore using MVIC (78)

Tamoxifen 50 Safety, slope of arm megascore (79)

Lithium 44 Survival (21)

ALSFRS-R: Amyotrophic Lateral Sclerosis Functional Rating Scale; FVC: Forced Vital Capacity; MVIC: Maximum Voluntary Isometric

Contraction.

Therapy development for ALS 133

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provide a basis for identifying suitable markers of

biological activity. For example, administration of

sodium phenylbutrate, a histone deacetylase inhibi-

tor, to mG93A-SOD1 mice resulted in increased

tissue histone acetylation and prolonged survival

(37). In a phase II human trial of sodium phenylbu-

tyrate, safety and biological activity of several

dosages were tested (38). Sodium phenylbutyrate

at dosages of 9 g and higher increased histone

acetylation whereas dosages greater than 15 g/day

were not well tolerated. Therefore, in this study, the

dosages that were safe and had desired biological

activity were identified. This type of study design

allows selection of appropriate dosages for a sub-

sequent phase III study. Administration of celecoxib

to mG93A-SOD1 mice decreased spinal cord and

CSF levels of prostaglandin E2 in (31). For this

reason, as a marker of biological effect, CSF was

collected in the human celecoxib trial for measure-

ment of PGE2 levels. However, celecoxib did not

lower CSF PGE2 levels in the treated participants

(24). This suggests that, in humans, celecoxib at 800

mg/day (the maximum dosage that could be admi-

nistered) did not have the desired biological effect.

This information is critical to deciding whether to

pursue additional trials with celecoxib at higher

dosages or other perhaps more potent cyclooxygen-

ase 2 inhibitors.

Biomarkers of disease

Several potential biomarkers of disease presence and

disease progression are under study including ge-

netic markers, proteins and metabolites in plasma or

CSF, and imaging markers (Table V). The develop-

ment of a validated diagnostic marker of ALS could

lead to faster diagnosis and earlier initiation of

experimental therapy. Panels of proteomic markers

are promising diagnostic tools, as they may have

higher sensitivity and specificity than an individual

marker (39). Biomarkers that correlate well dynami-

cally with the disease process and reflect disease

modification by a study drug are also needed. An

emerging biomarker, especially in familial ALS

patients, is neurofilament light protein (NF-L) levels

in the CSF, which are elevated in ALS patients and

correlate inversely with the duration of disease (40).

Table III. Pre-clinical models.

Models Refs

In vitro

1. Mature cells+ Normal or mutant rodent or avian motor neurons in pure/mixed cultures (80,81)+ Organotypic spinal cord cultures from postnatal rats (82)+ Glutamate excitotoxicity based models (83)

2. Embryonic cells+ Embryonic stem cell based cultures and cocultures (84)+ Organotypic slice cultures from wild-type/G93A embryonic spinal cords (85)+ Purified human motor neurons and astrocytes from human embryonic spinal cord anterior horns (86)

3. Neuroblastoma and other cell lines+ Cultured neuroblastoma cells expressing normal or mutant SOD Example: SHSY5Y human neuroblastoma cultures (87)+ Motor neuron-neuroblastoma hybrid cells (VSC4.1) constitutively expressing a mutant (G93A) SOD1 (88)+ PC12 cell lines (89)

In vivo

1. Rodent models+ Transgenic rodent models: G93A SOD1 mouse, G85RSOD1, G37R SOD rodents. Dynamitin overexpression

model with disrupted dynein/dynactin complex

(18,90�92)

+ Model of adult motor neuron degeneration from proximal axonal injury of peripheral nerves (93)+ Spontaneous mutation based: pmn mouse, wobbler mouse, Ham-spastic wistar rat, VEGF d/d mouse (94�96)

3. Drosophila melanogaster transgenic and knockout models of neurodegeneration (97,98)

4. Caenorhabditis elegans transgenic and knockout models of neurodegeneration (99)

Table IV. ALS clinical trials: potential limitations.

Potential Trial Limitations Trial Examples

Insufficient power Vitamin E, Selegiline, Nimodipine, Verapamil, N-acetylcysteine, Dextromethorphan,

Lamotrigine, Creatine

Dosage too low Creatine, Celecoxib

Dosage too high Minocycline, Topiramate

No dosage ranging Pentoxifylline, Creatine, Topiramate, Minocycline

No pharmacodynamic markers Minocycline, Creatine, Dextromethorphan, Nimodipine, Verapamil, TCH346, BDNF,

IGF-1, Topiramate

Drug interactions Xaliproden, Minocycline, Pentoxifylline, CNTF

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Such a marker could be a valuable objective measure

of the effect of experimental therapy in future

clinical trials.

Sample size considerations

Because of the variability of disease progression

between patients with ALS, sample size estimates

for proof of concept phase II designs are often quite

large (41). The development of validated surrogate

markers for survival could shorten the duration of

phase II studies and decrease sample size. The use of

sequential study designs (creatine, 2003) (42) and

adaptive designs (coenzyme Q10 and ceftriaxone)

(43,44) can help decrease the overall sample size and

the time between different study phases compared to

traditional designs. A potentially effective strategy

for early phase trials for a disease with constrained

resources may be to screen multiple agents or

multiple dosages of a single agent, followed by

rigorous testing of the winning agent (or dosage).

This could decrease the time to drug development

substantially. For example, testing five drugs against

each other first and testing the winner at pB0.05

decreases the sample size requirement by 50% (45).

An example of an effective phase II trial design in

ALS is the study of TCH-346 (46). This study

included a 16-week lead-in period to determine each

participant’s rate of disease progression, tested four

doses of TCH-346, and was powered to detect a

25% reduction in the rate of decline of ALSFRS-R

compared to placebo. The sample size requirement

was significantly reduced by including a lead-in

period of observation of the participant’s natural

disease progression and comparing this to the

subject’s own post-treatment progression (46). No

effect of treatment at any tested dosages was found.

Drug interactions

The majority of people with ALS are taking riluzole

and several other prescription and non-prescription

treatments. Since riluzole is a proven effective

therapy for ALS, participants enrolling in a trial

are allowed to take riluzole. The use of off-label

drugs, such as coenzyme Q10, vitamin E, and

creatine, is very common in participants who enroll

in trials (Table VI). Drug interactions between new

investigational agents and these treatments are im-

portant potential confounders of effect in clinical

trials in ALS. These agents may alter the disease

progression, decrease potency of experimental ther-

apy or cause an increased number of adverse events.

For example, in the phase III trial of xaliproden,

beneficial effect was observed in forced vital capacity

(p�0.009) of subjects on xaliproden 2 mg/day alone

but no effect on subjects concomitantly taking

riluzole (47). It is therefore essential to consider

relevant drug interactions in the design and safe

conduct of clinical trials in ALS.

Outcome measures

Survival remains the gold standard primary endpoint

for phase III trials in ALS. However, trials with

survival as the primary outcome measures are

typically 18 months in duration and require large

sample sizes, depending on the desired effect size

(48). Survival can be influenced by the individual’s

rate of progression of disease, site of onset, nutrition

and improved by treatments including riluzole use,

ventilation (invasive and non-invasive), and gastro-

stomy (20,49,50). There is currently no validated

surrogate marker for survival. It is also important

to have reliable, clinically relevant measures of

Table V. Potential diagnostic biomarkers of ALS.

Diagnostic source Biomarkers Sample size Refs

Plasma Homocysteine� 150 (100)

Reverse transcriptase� 88 (8,9)

Matrix metalloproteinase 9 (MMP-9) 25 (101)

TGFbeta1� 24 (102)

Cerebrospinal fluid Cytokine Flt3 ligand� 46 (103)

Transthyretin¡ 54 (39)

Cystatin C¡ 54 (39)

Carboxy-terminal fragment of neuroendocrine protein 7B2� 54 (39)

3 protein panel 13.8-kDa (Cystatin C), 6.7-kDa and 4.8-kDa(peptide

fragment of neuroendocrine specific VGF)¡57 (104)

Neurofilament light levels (NF-L)� 325 (40)

C4d complement protein� 27 (105)

Interleukin 6� 105 (106)

MCP1/VEGF ratio� 57 (107)

Plasma and CSF Monocyte chemoattractant protein-1 57 (107,108)

(MCP-1)� 8-hydroxy-2?-deoxyguanosine(8OH2’dG)� 65 (109)

Hydroxynonenal (HNE)� 122 (110)

Prostaglandin E2� 40 (111)

Muscle biopsy Nogo-A protein� 33 (112)

Therapy development for ALS 135

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function. Commonly used functional measures in

ALS trials include those of respiratory function

(Forced Vital Capacity (FVC)), muscle strength

(Manual Muscle Testing (MMT)), Maximum Vo-

luntary Isometric Contraction (MVIC), Hand Held

Dynamometry (HHD), electrophysiological indices

(Motor Unit Number Estimation (MUNE)) or

comprehensive rating scales (Norris scale, Appel

scale, ALSFRS-R). The ALSFRS-R is frequently

used as a primary outcome measure because it is

clinically relevant, has good inter-rater reliability and

is easy to administer. The assumption that its slope

of decline is linear may not be valid (30,34).

Study conduct factors

Experiences with clinical trials in ALS have high-

lighted some of the successes and the challenges in

trial conduct. Patients with ALS and their families

on the whole are actively engaged in clinical

research. Enrollment rates are modest, with sites

enrolling 1�2 participants per month (Northeast

Amyotrophic Lateral Sclerosis Consortium

(NEALS) database). Early study drug discontinua-

tion has been modestly high in ALS trials and is

related to adverse events, study duration, study

complexity and burden. As all of these studies were

analyzed by intent to treat, the impact on study

power of a high percentage of participants stopping

study medication early can be quite large. Review of

data from three clinical trials conducted by the

NEALS Consortium demonstrates that sites vary

greatly in their enrollment rates and slightly less than

50% of sites meet the target enrollment for a trial

(Figure 1). Taking care to design a trial convenient

to patients and caregivers, including reimbursement

of travel and other expenses where appropriate,

patient education on absence of any proven bene-

ficial drug agents other than riluzole and including

an open label phase after study completion may

improve participant enrollment and retention.

Conclusions

The urgent need for a successful disease modifying

therapy for ALS remains despite remarkable pro-

gress made recently in understanding the underlying

Table VI. Most frequently used concomitant medications

(�10%).

Medication

Frequency (%)

(n�782)

Riluzole 510 (65.2)

Vitamin E 482 (61.8)

Vitamin C 392 (50.19)

Creatine 348 (44.62)

Non-steroidal anti-inflammatory agents 291 (37.21)

Anti-depressants* 225 (28.81)

Coenzyme Q10 158 (20.23)

Calcium 109 (13.96)

Beta-carotene 107 (13.68)

Aspirin 98 (12.53)

Baclofen 91 (11.64)

Anti-depressants*: Celexa, Doxepin, Effexor, Elavil, Lexapro,

Paxil, Prozac, Remeron, Trazodone, Wellbutrin, Zoloft.

Source: Northeast ALS Consortium database.

(Medications listed taken by �10% subjects at baseline; n�782).

Selected NEALS trials in ALS from 1999�2006.

Trial Sites

Site 1

Site 2

Site 3

Site 4

Site 5

Site 6

Site 7

Site 8

Site 9

Site 10

Site 11

Site 12

Site 13

Site 14

Site 15

Site 16

Site 17

Site 18

Site 19

Site 20

Site 21

Site 22

Site 23

Site 24

Site 25

Site 26

Site 27

En

rollm

ent

0

5

10

15

20

25

30

Figure 1. Total enrollment by trial site. (Target enrollment�11 subjects/site) (NEALS trial conducted 2004�2006).

136 V. Lanka & M. Cudkowicz

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biology. Several promising therapies identified in

preclinical studies have not been successful in hu-

man studies. In retrospect, in the case of most

therapies, there was not enough information on the

biological activity of the therapy or dosage related

information in humans to conclude whether the

therapy failed or the trial design failed. Before

dismissing a particular therapy or a class of drugs,

it is essential to critically review data from clinical

and preclinical trials to rule out trial limitations. To

better translate from preclinical studies to human

trials, it is critical to gather information on the

dosage response relationships and proof of proposed

biological activity before starting a large efficacy

trial. Developing biomarkers of disease and markers

of biological activity of therapy, innovative study

designs and learning from past trials will improve

future clinical trials. With improved clinical stan-

dards of care, availability of rigorously trained

clinical trial sites and better understanding of

challenges in trial design and conduct in ALS, the

chances of success are greatly improved.

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