vetrugno, exp op orph drugs, 15

1. Introduction

2. Past therapeutic attempts and

rationales

3. Past clinical trials: strategies

and difficulties

4. Ongoing or future possible

trials

5. Conclusions

6. Expert opinion

Review

The future for treatingCreutzfeldt--Jakob diseaseVito Vetrugno, Maria Puopolo, Franco Cardone, Fiorentino Capozzoli,Anna Ladogana & Maurizio Pocchiari†

Istituto Superiore di Sanit�a, Department of Cell Biology and Neurosciences, Roma, Italy

Introduction: Creutzfeldt--Jakob disease (CJD) is a rare, transmissible and fatal

neurodegenerative illness that affects people worldwide with a prevalence of

about 1 -- 2 cases per million people. Early diagnosis of CJD is still difficult

despite recent development of novel assays for detecting the pathological

prion protein, the only reliable marker of disease found in the cerebrospinal

fluid, urine and mucosa olfactoria.

Areas covered: This analysis covers attempts of therapy in CJD and related dis-

eases. It looks at problems encountered in designing and interpreting the few

available trials with the aim that learning from past experiences would

improve future clinical experimentations.

Expert opinion: The future of therapeutic intervention in CJD should begin

with the identification of novel compounds with strong antiprion effects.

These need validating in animal models of disease before their use in humans.

Improvement of preclinical studies according to internationally recognized

guidelines should cover critical aspects that have been poorly followed in

the past. The use of more than one model of prion infection should also be

encouraged. Novel international diagnostic criteria for including CJD patients

in clinical trials in an early stage are needed and consensus should be reached

for determining the most meaningful criteria to evaluate the progression

of disease.

Keywords: Creutzfeldt--Jakob disease, dementia, neurodegenerative disorders, therapy

Expert Opinion on Orphan Drugs (2015) 3(1):57-74

1. Introduction

Clinical and pathological features of Creutzfeldt--Jakob disease (CJD) weredescribed about one century ago, but understanding the mechanisms of pathologicalprocesses only begun in the late 60s when the disease was successfully transmitted tonon-human primates and thought to be caused by a slow and unconventionalvirus [1]. This finding neared CJD to kuru, an exotic disease of the Fore-speakingpeople in Papua New Guinea and now disappearing [2], and scrapie, an endemic dis-ease affecting herds of sheep and goats in most of the world [3]. However, until thelate 70s to early 80s, there was limited knowledge on clinical and epidemiologicaldata, and factors influencing disease duration were unknown or poorly under-stood [4,5]. A better understanding of the natural history of the disease came inthe 90s from the establishment of national surveillance systems in European andnon-European countries [6,7], and from understanding the pathogenic mechanismof disease, which basically consists in the poorly known process that misfolds thecellular prion protein (PrPc) into different pathological conformers (type 1, 2Aand 2B PrPTSE) with high beta-chain contents [8].

The majority of CJD cases apparently occur spontaneously (sporadic CJD),about 10 -- 20% within families (genetic CJD; Gerstmann--Straussler--Scheinkerdisease [GSS] and fatal familial insomnia [FFI]) in people carrying a pathogenicpoint or insert mutation in one allele of the prion protein gene (PRNP), and a

10.1517/21678707.2015.994605 © 2015 Informa UK, Ltd. e-ISSN 2167-8707 57All rights reserved: reproduction in whole or in part not permitted

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http://informahealthcare.com/journal/EOD

few cases are related to medical procedures (iatrogenicCJD) [7,9] or to the consumption of bovine spongiformencephalopathy-contaminated food (variant CJD) [10].Sporadic CJD occurs with different clinical phenotypes and

it is usually difficult to make a correct early diagnosis [11].Dementia usually occurs in the early phase of disease, butthere are cases where patients present other neurologicalsymptoms (i.e., ataxia or visual disturbances) at onset. Thedisease usually progresses rapidly with death occurringbetween 3 and 6 months in about 60% of cases [12]. Supportfrom clinical diagnosis comes from the electroencephalogram(EEG), cerebrospinal fluid (CSF) exams and brain MRI, butso far no disease-specific markers are included in the interna-tional diagnostic criteria for human prion diseases (Table 1) [13].EEG shows a characteristic generalized bi- or triphasic peri-odic sharp wave complexes with a frequency of around1 -- 2 per second in about 60% of sporadic CJD but it mightappear late in the course of disease and, occasionally, in otherconditions [14,15]. Typical EEG is present in some forms ofgenetic prion diseases (genetic CJD), but not in others(GSS, FFI) nor in variant CJD [16]. Routine CSF analysesare usually normal, but in the majority of sporadic CJD casesthe 14-3-3 proteins test results positive and tau is highly ele-vated [17]. The presence of 14-3-3 proteins in the CSF, how-ever, is not specific for prion diseases and it acquires adiagnostic value only in combination with CJD-specific

neurological signs [18]. Moreover, in about 50% of variantCJD this test is negative, therefore of no diagnostic value [19].Finally, brain MRI shows signal abnormalities in the anteriorbasal ganglia (caudate/putamen) and often in the cortex insporadic CJD, while signal abnormalities are observed inabout 90% of variant CJD in the posterior thalamic region(pulvinar) [13,20]. Brain MRI is also useful for excluding otherneurological conditions, some of which might be susceptibleto pharmacological or surgical treatment [21]. The geneticforms of prion diseases are easily recognized by the combina-tion of neurological signs in patients presenting specific insertor point mutations in the open reading frame of the PRNPgene [22]. The analysis of the PRNP gene in people belongingto affected families is also a powerful tool for the identifica-tion of potentially high-risk subjects, although it is usually dif-ficult to predict when people carrying a PRNP mutation willdevelop disease [22,23] and in several occasions carriers do notdevelop disease at all [23,24].

In the last 10 years, there have been substantial improve-ments in the detection of the only disease-specific marker ofdisease (i.e., PrPTSE) that finally translated into the develop-ment of novel diagnostic tools that have achieved enough sen-sitivity for assessing PrPTSE in easily accessible tissues or bodyfluids [25]. Among them, the in vitro amplification of the mis-folded prion protein has, in preliminary studies, successfullydetected PrPTSE in CSF [26,27], in nasal swab samples of spo-radic CJD [28] and urine of variant CJD [29] with no false-positive signals, while an adapted solid-state binding matrixassay is apparently able to detect PrPTSE in blood samples ofvariant CJD patients with over 70% sensitivity and 100%specificity [30]. As soon as these methods are fully validated,it would be possible to improve the correctness of an earlydiagnosis, thus allowing a faster enrolment of CJD patientsin clinical trials.

Attempts of therapy in patients with CJD and related dis-eases have been extensively reviewed in recent years [31-37],with the conclusion that none of the tested drugs has convinc-ingly shown a significant impact on the natural history of thedisease with the exception of the intraventricular infusion ofthe pentosan polysulfate (PPS) in variant CJD patients [38].To date, this conclusion still holds. This updated reviewfocuses on the appropriateness of these treatments with thehope that learning from past weaknesses will improve thefuture of therapy in prion diseases.

2. Past therapeutic attempts and rationales

Except for several and inconclusive reports on isolated casestreated with a variety of drugs (see [31] for a comprehensivereview), there are only five compounds that have been testedin comparative studies in patients with prion diseases.

2.1 AmantadineIn the early 70s, soon after the successful transmission of CJDto non-human primates suggested a viral origin of the disease,

Article highlights.

. Four randomized double-blind placebo-controlled clinicaltrial studies were attempted in Creutzfeldt--Jakobdisease (CJD) patients showing no efficacy, but provingthat clinical trials are feasible in CJD, though withsome difficulties.

. Analyses of past clinical trials revealed the importance ofrecruiting patients at national or international levelbecause of the rarity of disease, the difficulty inrecruiting patients at an early stage of disease and someannoyance by patients or caregivers in acceptingrandomization because of the rapidlyprogressive disease.

. Preclinical studies were often overinterpreted. Mostcompounds showed preventive rather than therapeuticeffects in preclinical studies but this finding was oftennot fully considered.

. There is only one preventive treatment in people at riskof developing prion disease. This ongoing trial withdoxycycline is in healthy carriers of the pathogenicmutation of the PRNP gene responsible for the fatalfamilial insomnia.

. Internationally recognized guidelines for performing validand relevant preclinical studies should be required forplanning future therapy in humans.

. It is necessary to develop international diagnostic criteriafor including CJD patients in clinical trials in a very earlystage by using new PrPTSE amplification techniques inbody fluids or easily accessible relevant tissues.

This box summarizes key points contained in the article.

V. Vetrugno et al.

58 Expert Opinion on Orphan Drugs (2015) 3(1)

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Table

1.History

andfuture

developments

ofdiagnostic

criteriaforsp

oradic

Creutzfeldt--Jakobdisease.

*Clinicalsignsaddedin

Europeancriteria.

CJD:Creutzfeldt--Jakobdisease;CSF:

Cerebrospinalfluid;EEG:Electroencephalogram;OM:Olfactory

mucosa;RT-Q

uIC:Realtimequackinginducedconversion.

The future for treating Creutzfeldt--Jakob disease

Expert Opinion on Orphan Drugs (2015) 3(1) 59

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therapy with the antiviral drug amantadine was comprehensi-bly attempted in a bunch of single CJD cases with somereported beneficial effects [39-41]. The rationale of two observa-tional studies [42,43] was to clarify the previously questionableeffect of the drug in CJD patients, despite the failure of pro-longing survival in experimental models of prion diseasewere already available (Table 2) [44,45]. Both studies concludedthat treatment did not substantially modify the course of dis-ease, although Terzano et al. [42] reported some clinicalimprovements. Formal clinical trials were likely not per-formed because of the great difficulties to recruit a reasonablenumber of patients in those years when only a small percent-age of CJD cases were recognized and no neurological centrehad the chance to observe more than a handful number ofpatients in years of activity. Because no clinical trials haveever been performed and preclinical studies with amantadinein animal models are limited, it is difficult to draw any defin-itive conclusion on the efficacy of this and other antiviraldrugs, such as acyclovir [46,47], vidarabine [48] and IFN [49] inCJD patients. However, with the current knowledge of thepathogenic mechanisms of prions and considering the avail-able, although scanty clinical studies, it is reasonable to con-clude that available antiviral therapies are ineffective in thetreatment of CJD patients.

2.2 FlupirtineIn 2004, Otto et al. [50] tested the effect of the non-opioid anal-gesic flupirtine versus placebo in 28 patients with CJD with theaim of determining the influence of the drug in the rate of cog-nitive decline. The study showed a transient beneficial effect offlupirtine in delaying the cognitive decline of CJD patients butno changes in survival times (Table 2). In in vitro studies, flupir-tine prevented apoptosis in neural cell culture exposed to a vari-ety of toxic agents, including A-beta and a short (21 aminoacids) fragment of PrP (PrP106-126) [51-53], but it had neverbeen tested in animal models of prion or other neurodegenera-tive disorders, making disputable its use in CJD patients. Theauthors did not comment on the rationale for the possible ben-eficial effect of flupirtine in delaying cognitive decline in CJDapart from reporting the in vitro cytoprotective activity.Otto et al., however, had the great merit to show that double-blind randomized controlled studies were feasible in CJD,despite the rarity of disease, the difficulties in getting a correctclinical diagnosis and the relative short clinical duration.

2.3 QuinacrineCompassionate therapy with quinacrine began > 10 years agosoon after the group of the Nobel Laureate Stanley Prusinerconfirmed a previous study [54], showing that this tricyclicderivative of acridine potently inhibits PrPTSE formation inscrapie-infected neuroblastoma cell cultures [55]. Becausequinacrine was used for decades in humans against malariaand crosses the blood--brain-barrier, Korth et al. [55] suggestedits immediate use in patients with CJD and related diseaseswithout waiting for confirmatory preclinical studies. In several

countries, the compassionate use of quinacrine in CJDpatients was initially done under the great pressure of familieswith the authorization of competent government authorities;in the USA, it was advertised in neurology journals, officialwebsites and by letters to the US neurologists. Overall, therationale for these observational studies were based on theantiprion effect of quinacrine in cell culture, its long andsafety use in humans and because it reaches the CNS. Treat-ment of isolated CJD or FFI patients with quinacrine showedeither no benefit [56-59] or a transient improvement of clinicalconditions [60-63], and observational studies gave ambiguousresults (Tables 2 and 3). Haik et al. [64] reported neither clinicalimprovement nor prolonged survival, Collinge et al. [65]

reported a transient response on neurological rating scales in afew patients but no effect in survival, while Geschwind et al.[66] reported a prolonged survival. All observational studieswith quinacrine started to recruit patients between 2000 and2001, just before the reported failure of quinacrine to prolongthe incubation periods in mice intracerebrally injected with aCJD-adapted strain [67]. Quinacrine inefficacy in animalmodelsof prion diseases was further confirmed in the followingyears [68-70]. Finally, in 2013 Geschwind et al. reported theonly randomized double-blind placebo-controlled clinical trial(RCT-DB) to quinacrine or placebo [66]. Patients were recruitedbetween 2005 and 2009, with the rationale that the sameauthors previously observed a positive effect of quinacrine inprolonging survival in CJD patients in an observationalstudy [66]. This study finally showed the inefficacy of quinacrineto improve survival in sporadic CJD patients (Tables 2 and 3)and likely represents the end of the therapeutic use of quinacrinein prion diseases.

2.4 DoxycyclineTetracycline antibiotics revert in vitro the protease resistanceof PrPTSE extracted from brain tissue of patients withCJD [71]. Doxycycline, a tetracycline derivative, shares thesame antiprion effect with the advantage of poor toxicityand of efficiently crossing the blood--brain barrier [72]. Basedon these observations, the group of Fabrizio Tagliavini atCarlo Besta Neurological Institute in Milan, Italy, pioneeredin 2002 the compassionate therapy with doxycycline forCJD patients (Tagliavini, pers. commun.). A few years later,De Luigi et al. [73] reported that doxycycline efficiently pro-longs the incubation period of hamsters peripherally inocu-lated with the 263K strain of scrapie and that theintracerebroventricular infusion of liposome-entrapped doxy-cycline in hamsters with early clinical signs delays diseaseduration confirming its potential use for human therapy.Data from observational studies in Italy [74] andGermany [33,75] also showed that treatment with doxycyclineextends between 4 and 7 months the survival of CJD patientscompared with historical controls (Tables 2 and 4). Thesefindings prompted an Italian-French RCT-DB Phase II studythat, however, did not confirm the positive findings of theobservational studies [76].

V. Vetrugno et al.


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Table

2.Attempts

oftherapyin

patients

withpriondiseases.

Drug

Studydesign

Ref.

Year

Country

Disease

Treated:

controls

Authorco

nclusion

Authors’rationale

Comments

Amantadine

Observational

[42]

1983

Italy

CJD

(likely

sporadic)

4:5

Noeffect

onsurvival

Partialclinicalbenefit

Antiviraleffect

Possible

beneficialeffects

oncase

reports

Absence

ofefficacy

on

incubationperiodsin

anim

almodelswas

alreadyavailable

atthe

timeofthestudies

[43]

1984

Italy

CJD

(likely

sporadic)

4:4

Noeffect

onsurvival

Noclinicalbenefit

Uncertainness

ofTerzano

etal.data

andpossible

beneficialeffectsoncase

reports

Flupirtine

RCT-DB

[50]

2004

Germ

any

Sporadic

and

geneticCJD

13:15

Noeffect

onsurvival

Benefitofcognitive

functions

Cytoprotectiveactivityin

vitroandin

vivo

on

neuronsinducedto

undergoapoptosis

Reductionofin

vitro

PrP106-126neurotoxicity

Nopreclinicalstudiesin

anim

almodels

Quinacrine

Observational

[64]

2004

France

Sporadic

and

variantCJD

32:125

(historical)

Noeffect

onsurvival

Invitroantiprionactivity

Possible

effect

inpatients

withvariantCJD

Thisstudybegunwhen

preclinicalstudiesin

anim

alstudieswere

not

yetreported

Open-label,

patient

preference

trial

(Observational)

[65]

2009

UK

Sporadic,iatrogenic,

variant,and

geneticCJD

32:69

Noeffect

onsurvival

Transientresponse

on

neurologicalrating

scalesin

4patients


RequestedbytheBritish

ChiefMedicalOfficer

Absence

ofefficacy

on

incubationperiodsin

anim

almodelswas

alreadyavailable

atthe

timeofthestudy

Observational

[66]

2013

USA

Sporadic

CJD

29:58

Increase

insurvival


Thisstudybegunwhen

preclinicalstudiesin

anim

alstudieswere

not

yetreported

RCT-DB(Phase

IIB)

[66]

2013

USA

Sporadic

CJD

23:28

Noim

provementin

2-m

onthssurvival

Noclinicalim

provement

Increase

survivalin

the

observationalstudy

previouslyconductedby

theAuthors

Absence

ofefficacy

on

incubationperiodsin

anim

almodelswas

alreadyavailable

atthe

timeofthestudy

Doxycycline

Observational

[74]

2008

Italy

CJD

(likely

sporadic)

21:78

Increase

survival

Extendedsurvivalin

experimentalmodels

-

[33]

2009

Germ

any

Sporadic

and

geneticCJD

51:not

reported

Increase

survival

Extendedsurvivalin

experimentalmodels

Controlswere

takenfrom

historicalcollections

RCT-DB(Phase

II)[76]

2014

Italyand

France

Sporadic,

iatrogenic,

variantand

geneticCJD

62:59

Noeffect

onsurvival

Extendedsurvivalin

observationalstudies

-

CJD:Creutzfeldt--Jakobdisease;GSS:Gerstm

ann--Straussler--Scheinkerdisease;PPS:Pentosanpolysulfate;RCT-DB:Randomizeddouble-blindplacebo-controlledclinicaltrial.



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2.5 Intraventricular infusion of PPSAttempts of therapy with intraventricular infusion of PPS(iPPS) began in January 2003 [77] soon after Doh-Ura et al.reported in an international meeting in Paris (2002 [78],then published in 2004 [69]) the successful effect of this treat-ment in rodents experimentally infected with different strainsof prions. The antiprion efficacy of intraperitoneal adminis-tration of PPS and other polyanionic compounds was estab-lished since 1984 [79] and then confirmed in other studies inperipherally or intracerebrally scrapie-infected mice or ham-sters [80], but its efficacy was limited to treatment given justbefore or after prion inoculation, a situation non-applicablefor human therapy. Despite these experimental data, therapywith oral administration of low molecular weight PPS wasreported in eight Japanese CJD patients [81] with no success.

The finding that iPPS was modestly effective even whengiven at the beginning of clinical signs in prion-infectedmice reawaken interest in this drug for human therapy [69].In 2003, Dealler and Rainov [82] reported on the potentialtherapeutic use of iPPS for CJD patients based on the exper-imental findings and on the poor side effects of PPS infusion.Treatment was then given to several other patients with vari-ous forms of CJD. In 2007, Rainov et al. [83] in reviewing26 attempts of therapy with iPPS (4 variant, 8 sporadic,7 iatrogenic and 1 genetic CJD; 5 GSS and 1 unspecifiedcase) reported that it appears reasonably safe but survivalwas convincingly prolonged only in variant CJD. Bone et al.[84] and Tsuboi et al. [85] reached similar conclusions in British(3 variant, 2 iatrogenic CJD; 2 GSS) and Japanese (6 sporadic,2 iatrogenic and 2 genetic CJD; 1 GSS) patients. A recentoverall picture of the effect of iPPS in prolonging diseaseduration in variant British CJD patients showed that four offive treated patients survived well beyond untreated patients(Tables 2 and 5) [38].

3. Past clinical trials: strategies anddifficulties

Only 4 RCT-DB studies were attempted in CJD patientsdespite 14 drugs were occasionally tested in the last 40 years [31].All RCT-DB studies showed no efficacy of the investigateddrugs, but they prove that clinical trials are also feasible inCJD, though with some difficulties. The analysis of the prob-lems encountered in designing and interpreting the trialsmight contribute to improve future clinical experimentations.

In the following pages, we present the major difficultiesthat challenged the design of RCT-DB studies for prion dis-eases, such as the definition of the end points, the approachfor recruiting a significant number of patients, the diagnosticcriteria for including or excluding patients to the study, thedesigning of the randomized trial and the ethical considera-tions for obtaining the consent to the experimentation or forusing a placebo in an otherwise deadly disease.T

able

2.Attempts

oftherapyin

patients

withpriondiseases(continued).

Drug

Studydesign

Ref.

Year

Country

Disease

Treated:

controls

Authorco

nclusion

Authors’rationale

Comments

Intraventricular

infusionofPPS

Review

[83]

2007

-Sporadic,iatrogenic,

variantandgenetic

CJD;GSS

26:0

Increase

survivalin

variantCJD

Extendedsurvivalin

experimentalmodelswith

direct

PPSbrain

infusions

Nocontrolpopulation

Observational

[84]

2008

UK

Iatrogenicand

variantCJD;GSS

7:0

Possible

positive

effect

onsurvival

invariantCJD

Extendedsurvivalin


direct

PPSbrain

infusions

Increase

survivalin

other

observationalstudies

Controlswere

takenfrom

historicalcollections

Observational

[85]

2009

Japan

Sporadic,iatrogenic

andgeneticCJD;GSS

11:0

Noeffect

on

neurological

deficits;possible

extendedsurvival

Extendedsurvivalin


direct

PPSbrain

infusions

Nocontrolpopulation

Case

report

andreview

[38]

2014

UK

VariantCJD

5:171

Extendedsurvival

on4/5

Extendedsurvivalin


direct

PPSbrain

infusions

4of5treatedpatients

hadasurvivallongerthat

allotherhistoricalcases

CJD:Creutzfeldt--Jakobdisease;GSS:Gerstm

ann--Straussler--Scheinkerdisease;PPS:Pentosanpolysulfate;RCT-DB:Randomizeddouble-blindplacebo-controlledclinicaltrial.

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3.1 End point(s) and minimum clinically relevant

differencesPrimary end points represent the bases for determining thesample size of a clinical trial. Except for the flupirtine trial [50],which was designed to detect cognitive (ADAS-Cog test) dif-ferences between baseline and best score under treatment, theprimary end points of the other three trials were time ofsurvival, though with some differences among studies in termsof minimum clinically relevant differences (Table 6).Collinge et al. (quinacrine) [65] were looking at a reductionin 2-year mortality from 50 to 22%; Geschwind et al. (quin-acrine) [66] at doubling of mean survival from 0.9 to1.8 months after 2 months from randomization; Haik et al.(doxycycline) [76] at doubling survivors after 1 year of ran-domization from 30 to 60% (Italian patients) or increasingmean survival from 6.2 to 11 months (French patients). Thecalculated sample sizes varied accordingly, but overall itranged between 30 and ‡ 160 patients and took between2 and 4 years for recruiting this significant number of patients(Table 6). Patients were followed-up in a single neurologicalcenter for the flupirtine [50] and both the US [66] andUK [65] quinacrine studies, while for the doxycycline study [76]

patients were recruited and followed-up in either three centers(Italian patients) or by multiple and not predetermined neu-rological units for French patients. Secondary end pointsaim to assess possible clinical improvements related to therapywere considered in all studies but were finally difficult to eval-uate mostly because of the serious and advanced clinical phaseof enrolled patients.

Considering the rarity of CJD, the number of patients thatneeds to be recruited requires national, or better international-based studies for speeding the completion of clinical trials. Onthe other hand, the recruitment of patients on national orinternational base might prevent simultaneous investigationsof different drugs with delay in testing novel and promisingtherapeutic approaches.

Sample size is strongly influenced by the choice of the min-imum clinically relevant differences; conspicuous clinicaleffects require small sample size but might be unrealistic, beresponsible for closing the trial for futility (improbable todetect prefixed effects) and therefore undermining future eval-uations of that specific treatment. On the other hand, minorbut realistic clinical improvements would require a large andunfeasible number of patients. On top of that, the compute

Table 3. Quinacrine.

BSE: Bovine spongiform encephalopathy; CJD: Creutzfeldt--Jakob disease; ic: Intracerebral; ip: Intraperitoneal; RCT-DB: Randomized double-blind placebo-controlled

clinical trial; RML: Rocky mountain laboratory; Ukn: Unknown.



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Table

4.Tetracyclic

compounds.

BSE:Bovinespongiform

encephalopathy;

CJD:Creutzfeldt--Jakobdisease;GSH:GoldenSyrianhamster;ic:Intracerebral;im

:Intramuscular;IDX:4’-iodo-4’-deoxy-doxorubicin;RCT-DB:Randomizeddouble-blind

placebo-controlledclinicaltrial;sc:Subcutaneous;

Ukn:Unknown.

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of the sample size needs to account for multiple testing ininterim analyses for an ethical assessment on whether to ter-minate the study earlier than planned because of consistentevidences of futility, efficacy or safety.

3.2 Diagnostic criteriaThe presence of sporadic, genetic, iatrogenic and variantprion diseases as well as criteria for diagnostic accuracy (possi-ble, probable and definite) based upon clinical, laboratory andinstrumental analyses (Table 1) require the a priori selection ofpatients based on disease variability and diagnostic accuracy.

Two studies (UK quinacrine [65] and doxycycline [76])included all forms of probable or definite prion diseases, thoughwith minor differences, while the other two (flupirtine [50] andUS quinacrine [66]) selected only probable or definite sporadicCJD patients. However, in these two last studies, > 5% ofenrolled sporadic CJD patients finally were classified as geneticprion diseases. This is because the result of the genetic screeningrequires time and is therefore available only after randomizationand family history is often unremarkable and does not reveal thesuspicion of a genetic trait in these patients.

The authors of the flupirtine study [50] finally decided toinclude genetic patients in the analyses of the study whilethose of the US quinacrine [66] did not. Because clinical fea-tures of genetic prion diseases are heterogeneous, mostlydepending on the type of PRNP mutation, it is reasonableto include only genetic forms of CJD (i.e., the E200K and

V210I mutations) strongly resembling sporadic CJD for clin-ical presentation, age at onset and disease duration, andexclude others (i.e., GSS or FFI) that significantly differfrom the sporadic forms. This last approach was adopted inthe doxycycline study [76] for the Italian patients.

All studies included only patients with high (> 90%) diag-nostic accuracy (i.e., probable or definite cases according tointernational classifications). This obligatory strategy, how-ever, has the great limitation of including most of the patientsin an advanced stage of disease and, therefore, with short nat-ural survival [86]. In our data set from over 1000 definite spo-radic CJD cases collected since 1993 during the nationalprogram of surveillance, the median interval between onsetand in vitam diagnosis of ‘probable’ CJD was 3 months(interquartile range, 3 months) and from the diagnosis ofprobable to death only 1 month (interquartile range,3 months). The two major determinants of survival, that is,codon 129 polymorphism and PrPTSE isotype [12], influencethe interval between onset and diagnosis of ‘probable’ butnot from here to death. These data indicate the urgency ofrevising diagnostic criteria for the inclusion of CJD patientsin future clinical trials (Table 1).

3.3 RandomizationThe golden standard for clinical trials is the allocation ofpatients to experimental arms by a randomized procedurefor minimizing biases caused by unknown confounding

Table 5. Intraventricular infusion of pentosan polysulfate.

CJD: Creutzfeldt--Jakob disease; GSS: Gerstmann--Straussler--Scheinker disease; RML: Rocky mountain laboratory.



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Table 6. Clinical trials in prion diseases.

Drug (vs placebo) Flupirtine Quinacrine Quinacrine Doxycycline

Ref. and year ofpublication

[50], 2004 [65], 2009 [66], 2013 [76], 2014

Aim Efficacy Efficacy and safety Efficacy Efficacy and tolerabilityPrimary end points Difference in cognitive

performance (ADAS-Cog)between baseline andtreatment

Survival and clinicalimprovement or lack ofdeterioration

Survival at 2 monthsfrom randomization

Survival time fromrandomization

Type of study RCTNational (Germany)

Patient-preference trial-partially randomized butfinally an observationalstudyNational (UK)

RCTNational (USA)

RCTInternational (Italy andFrance)

Inclusion criteria Probable sporadic CJDwho scored ‡ 50% in atleast 2 of 12 subtests ofADAS-Cog andGoettingen CJD dementiatests

All definite or probablehuman prion diseases

Definite or probablesporadic CJD

Italy: Definite or probablesporadic and genetic CJDwith disease duration£ 6m;France: All definite orprobable human priondiseases

Randomization/blinding Yes/double-blinding Choice betweenrandomization or norandomization/double-blinding

Yes/double-blindingfor the first 2 months,then open

Yes/double-blinding

Procedure ofrandomization

Simple Stratified by type ofdisease

Stratified by theBarthel Index Score

Italy: Minimizationmethod by sex, age atonset, months since CJDclinical onset, codon 129PRNP genotype.France: Simple

Randomization ratio 1:1 1:1 1:1 1:1Informed consent(provided by)

Yes (unspecified) Yes (patients or familymembers or independentneurologists)

Yes (patients or legallyauthorizedrepresentative)

Yes (patients orcaregivers)

Minimum clinicalsignificant difference

Difference (‡ 4) betweenbaseline and treatment(ADAS-Cog)

Reduction in 2 yearmortality from 50 to 22%

Increase in meansurvival time sincerandomization from0.9 to 1.8 months

Italy: Doubling thepercentage of survivors at1 year (from 30 to 60%);France Increase in meansurvival time since onsetfrom 6.2 to 11 months

a; statistical power 0.05; 0.70 0.05; 0.80 0.05; 0.80 0.05; 0.80;Calculated sample size 30 160 60 Italy: 63

France: sequential design,interim analysis after first80 patients enrolled andevery 20 patientsthereafter

Number of patientsassessed for eligibility orreferred to the centres

682 (assessed foreligibility)

221 (referred to thenational Prion Clinic)

425 (referred to theUCSF)

103 in Italy and 560 inFrance (screened foreligibility)

Number of patientsenrolled

28 84 54 121 (Italy: 55; France: 66)

% of enrolled patients 5% 38% 47% Italy: 53%; France:12%Number of patients perarm

Flupirtine: 13Placebo: 15

Chose immediatequinacrine: 24Chose no quinacrine: 59Chose random allocation:1

Quinacrine: 23Placebo: 28

Doxycycline: 62Placebo: 59

Status Completed Closed Completed Stopped for futility at firstinterim analysis

CJD: Creutzfeldt--Jakob disease; RCT: Randomized controlled trial.

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factors, immeasurable variables or both. Informed consent isasked to patients, often caregivers, when patients have seriouscognitive deficits, who need to accept the gamble of beingassigned to the arm of the putative active drug. This difficultchoice is clearly exemplified in the UK quinacrine study [65],where patients (or the caregivers) had the choice to take thedrug, not take it or accept randomization to immediate quin-acrine versus 6-months deferred quinacrine. Only 2 of84 enrolled patients chose randomization, 23 immediatequinacrine and 59 no quinacrine resulting in an observationalrather than a RCT study. Interestingly, the most and leastseverely affected patients did not choose treatment with quin-acrine. In the US quinacrine [66], flupirtine [50] and doxycy-cline [76] studies, however, randomization was successfulsuggesting that RCT-DB studies are feasible, though withsome difficulties. In the US quinacrine study [66], 87% of sur-viving patients in the placebo arm and 100% in the quina-crine arm returning for the 2-month visit and preferredtreatment with quinacrine in contrast with the trend of theBritish study [65], where only 27% of recruited patients askedfor the putative active drug.

A variety of randomization procedures were adopted inCJD trials; simple random procedures in the flupirtine [50]

and doxycycline (French patients) [76] studies, stratified bytype of disease in the UK quinacrine [65], stratified by perfor-mance in daily living activities (Barthel Index) in the US quin-acrine [66], and minimization by sex, age at onset, monthssince CJD clinical onset and codon 129 PRNP genotype inthe doxycycline (Italian patients) study [76]. This last random-ization procedure was adopted for improving the control ofknown predictors of survival [12]. Despite differences in therandomization procedures, all studies adopted the question-able choice, in a fatale and untreatable disease, of a 1:1 ratiobetween the active drug arm and the placebo arm.

Overall, these data indicate caution in oversimplifying theexperience of a single study in terms of feasibility in conduct-ing an RCT-DB study or in accepting treatment with anexperimental drug, though with uncertain efficacy, by patientswith CJD or their caregivers.

4. Ongoing or future possible trials

Despite the appearance of increasing number of scientificarticles reporting novel therapeutic approaches in in vitro orin vivomodels of prion diseases [35,87,88], the only ongoing clin-ical trials in human prion diseases is limited to the doxycyclinepreventive treatment in healthy members of a large Italian fam-ily affected by FFI [36,89]. The only other treatments in advancedstage for a first-in-human safety study are, to the best of ourknowledge, the MRC Prion Unit’s PRN100 antibody [90,91]

and the oligomer modulator Anle138b [92].

4.1 Doxycycline preventive treatment in FFIFFI is a rare genetic prion disease linked to the presence of theD178N mutation coupled with methionine at the

polymorphic codon 129 (D178N/M129) of the PRNP geneand characterized by severe insomnia, autonomic disturbances,cognitive changes, ataxia and endocrine manifestations [93,94].Most of mutated subjects develop disease during their lifetime,usually in the 50s, and inevitably die in about 12months [93,94].Because therapy is unavailable, Forloni et al. proposed tomembers of one large affected kindred in northern Italy alife-long preventive therapy with doxycycline [36,89], which inexperimental models of prion diseases efficiently delay onsetof disease if given before onset of clinical signs [73]. Currently,11 healthy carriers and 19 non-carriers belonging to the abovekindred and born between 1958 and 1969 are blinded treatedwith a daily dose of 100 mg doxycycline (mutated carriers) orplacebo (non-mutated subjects) for 11 years. Young familymembers will be included in the study as soon as they reach41 years of age. Participants of the study are requested toundertake a complete neurological examination every 2 yearsincluding neuropsychological assessment, autonomic nervoussystem assessment, polysomnographic EEG recording, actigra-phy, brain MRI and FDG-PET [95]. The aim of this study is toassess the delay (or prevention) of disease onset in people whoare predestined to develop FFI in their middle age. The ratio-nale of this trial is based on the observation in animal modelsthat therapy with doxycycline and all other antiprion drugs ismuch more effective when given before clinical onset [73].The ambitious result of the study is to observe a 50% reduc-tion of mortality in treated carriers over 10 years of treatmentwith respect to historical familial data. The success of thisstudy will open the possibility of preventive treatment ingenetic prion diseases [22] and eventually in people at highrisk of developing iatrogenic CJD [9].

4.2 MRC Prion Unit’s PRN100 antibodyIn 2003, White et al. [96] showed that ICSM35 andICSM18 mAbs directed against the a-helix-1 of PrP [97]

strongly delay onset of scrapie disease in intraperitoneallyinfected mice. Although treatment was only effective if startedbefore clinical onset, the finding that these antibodies did notcause any brain damages fostered the possibility of their use forhuman therapy [98]. The MRC Pion Unit followed this line ofthought and developed a human analogue of ICSM18 (namedPRN100), which showed no CNS toxicity in cynomolgusmacaques at intravenous doses (up to 200 mg/kg) suggestinga safe use for human studies [90]. With these bases, the MRCPrion Unit (UK) has designed a clinical trial, waiting for finalapprovals, to simultaneously evaluate safety and efficacy on asmall number of CJD patients [91]. There are, however, issuesthat need clarification. One is on the real efficacy of passiveimmunization therapy in prion diseases. Petsch et al. [99]

showed that treatments with the mAb W226, which binds tothe same epitopes of ICSM18, is unable to protect micefrom an intraperitoneal prion infection, arguing that the ther-apeutic efficacy likely depends also on the genetic backgroundof the host (Petsch et al. [99] and White et al. [96] used differentstrains of mice) or differences in the antibody sequences



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(ICSM18 and W226 show significant differences in thesequence of the hypervariable complementarity determiningregions), which might influence the half-life of the compoundor its efficacy to reach the CNS. The other issue is safety,which remains unsettled because of contrasting experimentaldata between mAbs binding overlapping epitopes on a-helix-1of PrP. ICSM18 [100], D18 [101] and PRN100 [90] look safewhile POM1 shows severe neuronal loss [102]. Understandingthe mechanisms that differentiate mAbs with the same bindingepitopes on PrP in terms of efficacy and safety is mandatorybefore starting any clinical trial in patients with CJD.

4.3 3-(1,3-Benzodioxol-5-yl)-5-(3-bromophenyl)-1H-

pyrazole (anle138b)Anle138b is a synthetic compound with a di-phenyl-pyrazolestructure that selectively inhibits pathological oligomer accu-mulation of PrPTSE and a-synuclein without interferingwith the physiological functions of the non-pathologicalmonomeric isoforms [103]. The finding that anle138b is effec-tive in preventing PrPTSE formation from different prionstrains and a-synuclein suggests that it targets commonstructure-dependent epitopes responsible for the oligomericformation of pathological proteins. Experimental data alsoshowed that anle138b prolongs the incubation period ofscrapie-infected mice and inhibits disease progression in aParkinson mouse model, even when treatment is started afterdisease onset [103,104]. The spin-off company (MODAGGmbH) from the Ludwig-Maximilians-Universitat Munchenand the Max Planck Society plans to develop anle138b up tomarket maturity for a Phase I/II clinical trial in CJD andother neurodegenerative diseases [105]. Although the com-pound has an excellent oral bioavailability, efficiently bypassesthe blood--brain barrier and shows no toxicity at therapeuticdoses, it would be wise to improve preclinical data (i.e., prov-ing efficacy in mice with different genetic backgrounds andinoculated with a variety of prion strains, including sporadicCJD) before its use in human therapy.

5. Conclusions

In the last few years, treatment of patients with CJD has beensuccessfully approached with RCTs [50,66,76]. Although theoutcome of these initial trials was substantially negative,they establish the feasibility of RCTs in rare and rapidly pro-gressive diseases such as CJD. The failure of these trialsdepends on several factors, which include the difficulty of anearly clinical diagnosis and the consequent short intervalbetween treatment and death [86], the variability of clinicalduration of illness [12], the absence of reliable disease-modifiermarkers [25] and validated clinical scales [106,107] and often theoverinterpretation of preclinical data.Patients are usually recruited when clinical signs and instru-

mental features fulfill the diagnosis of probable CJD (Table 1),which occurs relatively late in the course of illness and it isinevitably accompanied by an irreversible brain damage.

Novel diagnostic procedures, based on the signal amplifica-tion of the misfolded prion protein in body fluids, that is,CSF [26,27] and urine [29], or easily accessible tissues, such asnasal olfactory mucosa [28] would likely anticipate the timefor an accurate diagnosis of CJD. Whether PrPTSE in bodyfluids and nasal olfactory mucosa would be a useful markerfor monitoring the effect of future therapy is still unknown.

Variability in disease duration depends on known factors(codon 129 polymorphism, type of PrPTSE deposition in theCNS, gender and age at onset) [12] that were partially consid-ered during randomization [76], but survival significantly varieseven within these categories [12] for yet unknown reasons,likely including the poorly controllable supportive care of fam-ily members [108]. A boost in searching for such factors is com-ing from genome-wide association studies, which might soonidentify novel genes able to modulate disease duration [109,110].

Finally, the rationales leading to the use of compoundsentering into human therapy were either poorly supportedby preclinical data or these studies were overinterpreted. Quin-acrine, for example, was initially given to CJD patients basedonly on in vitro studies [54,55] without waiting for confirmatoryefficacy in animal models, which finally did not confirm theantiprion effect [67,68] and doxycycline was given to patientslate in the course of disease despite preclinical studies showedonly modest, if any, therapeutic effect in early diseasedprion-infected rodents [73].

6. Expert opinion

The future of therapeutic intervention in CJD starts from theidentification of novel compounds with strong antiprioneffects. Based on the only undisputed pathogenic mechanismof disease, novel drugs are usually selected for their action onhalting PrPTSE formation or increasing its clearance byin silico technology [111] or through the screening of largechemical libraries containing hundreds of thousands ofcompounds [111-115]. These ongoing activities have alreadyidentified a number of potential antiprion compounds thatwould now being further tested for determining their efficacyin animal models of prion disease, CNS bioavailability andtoxicity before their possible use in humans. Other potentialantiprion candidates are biological products, either in termsof passive or active immunization with mAbs targeting spe-cific PrPC epitopes [116-118], by using small interfering RNAsthat downregulate PrPC expression [119,120], or targeting novelpathway of neurodegeneration [121,122]. Immunological thera-pies in Alzheimer’s disease, another protein misfolding CNSpathology, yield some positive results, which were, however,so far overcome by serious safety issues [123,124]. The futureof immunological treatments in neurodegenerative diseasesis promising but some adjustments of the molecules are likelyneeded to minimize adverse effects.

Prion diseases have the great advantage over other neurode-generative diseases of excellent animal models that mimic clin-ical, pathological and biochemical features of human diseases,

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but their use for the design and interpretation of preclinicalstudies were often poorly performed or misinterpreted.A boost in getting valid and relevant preclinical studies for plan-ning future therapy in humans would come from the develop-ment of an ad hoc internationally recognized guideline forpreclinical studies in prion diseases based on available check-lists [125]. This guideline should cover critical aspects that havebeen poorly followed in past preclinical prion studies, such asthe adoption of a randomized procedure for the allocation ofanimals, the use of appropriate controls, the blindness of theinvestigators during the experiment and a power calculationperformed a priori to determine sample size. Other importantaspects should include the use of more than one model of prioninfection considering that available data showed that drugsmight be effective in one model and not in others [126-129], thechoice of relevant models, for example, mice [130] or bankvoles [131] infected with and reproducing human PrPTSE

type 1 or type 2 [8] rather than animal strains, the route of infec-tion and the need of confirming efficacy by independentresearch groups. Finally, it is important to define the relevanttiming and route of treatment administration in relation tothe aim of the study. Researchers often inferred that drugswith strong preventive effects in preclinical studies, that is,given during the preclinical phase of disease, would have thera-peutic effects despite countless evidences that treatments areineffective or poorly effective when given at early clinical phasesor even close to onset of disease [69,73,80]. Overall, these preclin-ical studies show that brain damage is already too severe at onsetof clinical signs for attempting any possible therapy and there-fore suggest that any potential treatment in CJD patientsshould begin as soon as possible. This is in contrast with pastclinical trials where patients were enrolled only when they ful-filled the diagnostic criteria of probable CJD, which inevitablyoccurs at a late stage of disease [86]. Thus, it would be necessaryto develop novel international diagnostic criteria for includingCJD patients in clinical trials in a very early stage by using thenew approaches of PrPTSE amplification and identification inthe CSF [26,27], urine [29] or olfactory mucosa (Table 1) [28].Although these assays need further validations before their usein diagnostic criteria for surveillance, they might be adequatefor the selection and inclusion of patients in clinical trials atan early stage of disease. Autopsy should be performed in allenrolled patients to confirm clinical diagnosis and for deter-mining PrPTSE type accumulation, which is an importantdeterminant of survival in sporadic CJD [12].

Future clinical studies in CJD need some rethinking interms of the choice of end points, the design of randomized

trials and the ethical considerations for obtaining the consentto the experimentation or for using a placebo in an otherwisedeadly disease [132,133].

Survival is a robust end point in prion diseases but drugs,such as antibiotics (e.g., doxycycline), able to improve thegeneral conditions of terminally ill patients, might give false-positive outcomes. The identification of disease-modified bio-markers, the use of internationally validated prion ratingscales or both might represent valid alternatives [106,107]. How-ever, consensus is needed on the minimal clinically importantchanges because a high expectancy [134] might be unrealisticand will make future trials ending for futility, or be ethicallycontroversial [132,133].

Improvement in the design of trials for rare, fatal and rap-idly progressive diseases such as CJD is strongly needed, espe-cially if treatments with striking efficacy become available. Insuch circumstances, study designs should rapidly prove or dis-prove efficacy. Randomized trials have the advantage of pre-venting biases but there are still uncertainties whetherrandomization is always necessary [135] or ethicallyacceptable [133,136-138]. It is of note that 33% of the pivotal tri-als of orphan drugs for neurological diseases approved by theUS FDA did not use placebo controls, 27% were not doubleblind and 12% were not randomized [139]. Properly collectedhistorical controls might be a valid alternative [133], but biasesin terms of selection of patients based on those who consent totake the active drugs and assessment of primary end pointsshould be considered.

Acknowledgments

We acknowledge funding from the Ministry of Health,National Centre for Disease Prevention and Control, CentralActions ‘Sorveglianza della Malattia di Creutzfeldt--Jakob’.We thank D Meloni for critical reading of the manuscriptand A Garozzo for technical support.

Declaration of interest

The Italian Ministry of Health has supported this work. Theauthors have no other relevant affiliations or financial involve-ment with any organization or entity with a financial interestin or financial conflict with the subject matter or materials dis-cussed in themanuscript. This includes employment, consultan-cies, honoraria, stock ownership or options, expert testimony,grants or patents received or pending or royalties.



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AffiliationVito Vetrugno PhD, Maria Puopolo DStat,

Franco Cardone PhD, Fiorentino Capozzoli MD,

Anna Ladogana MD & Maurizio Pocchiari† MD†Author for correspondence

Istituto Superiore di Sanit�a, Department of Cell

Biology and Neurosciences, Viale Regina Elena

299, Roma 00161, Italy

E-mail: [email protected]

V. Vetrugno et al.


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