WO2011117267A1

WO2011117267A1 - Cladribine treatment of multiple sclerosis in patient groups defined by genotype

Info

Publication number: WO2011117267A1
Application number: PCT/EP2011/054388
Authority: WO
Inventors: Jérôme Wojcik; Emmanuel Monnet
Original assignee: Merck Serono Sa
Priority date: 2010-03-24
Filing date: 2011-03-23
Publication date: 2011-09-29
Also published as: WO2011117267A9

Abstract

The invention relates to a method for determining the capacity of a subject suffering from Relapsing-Remitting Multiple Sclerosis (RRMS) to respond to treatment with Cladribine, said method comprising determining the polymorphism genotype of the subject in at least one SNP located on chromosome X, particularly an SNP located in the Duchenne Muscular Dystrophy (DMD) gene, for example rs5971598. The invention also relates to Cladribine for use in treating Relapsing-Remitting Multiple Sclerosis (RRMS) in a subject, wherein the treatment is specifically adapted to the capacity of the subject to respond to Cladribine treatment, as predicted by genotype.

Description

Cladribine treatment of Multiple Sclerosis in Patient Groups defined by Genotype

The present invention relates to Cladribine for use in therapy of Multiple Sclerosis (MS) in specific patient populations defined by genotype, and to methods for optimizing patient response to Cladribine in MS treatment on the basis of genotype. The invention further concerns the use of molecular markers, particularly SNPs located in the Duchenne Muscular Dystrophy (DMD) gene on the X chromosome, in the definition of MS patient populations with a view to specifically adapting Cladribine therapy, particularly in terms of dose regimen, treatment schedule and combination therapy, to that patient population.

Multiple Sclerosis MS is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) and is one of the most common causes of neurological disability in young adults. The neuropathology of the disease is marked by accumulation of leukocytes in the CNS, oligodendrocyte loss, demyelination, axonal atrophy, and neuronal loss. Clinically it is characterized by multi-focal recurrent attacks of neurological symptoms and signs with variable recovery. Eventually, the majority of patients develop a progressive clinical course. The exact cause of MS is unknown, although an autoimmune process has been implicated. Genetic susceptibility plays a role in disease initiation, but unidentified environmental factors may also be involved.

Four courses of the disease are individualized: relapsing-remitting (RR), secondary progressive (SP), primary progressive (PP) and progressive relapsing (PR) multiple sclerosis. More than 80% of patients with MS will initially display a RR course with clinical exacerbation of neurological symptoms, followed by a recovery that may or may not be complete.

During RRMS, accumulation of disability results from incomplete recovery from relapses. Approximately, half of the patients with RRMS switch to a progressive course, called SPMS, 10 years after the disease onset. During the SP phase, worsening of disability results from the accumulation of residual symptoms after exarcerbation but also from insidious progression between exacerbations. 10% of MS patients have PPMS which is characterized by insidious progression of the symptoms from the disease onset. Less than 5 % of patients have PRMS and are often considered to have the same prognosis as PPMS. It is suggested that distinct pathogenic mechanisms may be involved in different patient sub-groups and have wide-ranging implications for disease classification MS onset is defined by the occurrence of the first neurological symptoms of CNS dysfunction. Advances in cerebrospinal fluid (CSF) analysis and magnetic resonance imaging (MRI) have simplified the diagnostic process and facilitated early diagnostic. The International Panel on the Diagnosis of MS issued revised criteria facilitating the diagnosis of MS and including MRI together with clinical and para-clinical diagnostic methods (Mc Donald et al, 2001 , Ann. Neurol, 50: 121-127).

Treatment benefits and disease modification can be obtained with the currently approved parenteral immunomodulatory and immunosuppressant therapies, namely interferon beta, glatiramer acetate, mitoxantrone and natalizumab. However, treatment responses are often less than complete and safety and tolerability concerns may limit their general utilization. Parenteral administration may present relative or absolute barriers to access, all limiting treatment adherence and long-term outcomes.

Cladribine is a chlorinated purine analog, 2-chloro-2'-deoxyadenosine (2-CdA), that differs in structure from the naturally occurring nucleoside, deoxyadenosine, by the substitution of a chlorine for hydrogen in the 2-position of the purine ring. This substitution renders it resistant to deamination by adenosine deaminase. Cells with high levels of deoxycytidine kinase and low levels of deoxynucleotidase activity (e.g., lymphocytes and monocytes), phosphorylate Cladribine to the triphosphate form, impairing DNA (deoxyribonucleic acid) synthesis and cellular metabolism and causing death in dividing and quiescent cells.

An oral formulation of cladribine has recently been developed for the treatment of multiple sclerosis (MS) studies (see for example International patent application WO 2006/067141 ). Indeed, results from a placebo-controlled trial of an oral therapy in MS demonstrated that annual short-course dosing with Cladribine tablets provides rapid and sustained treatment benefits. In this study, patients received oral Cladribine initially over an induction period, and then over a maintenance period, the induction and maintenance periods being separated by a cladribine-free period, and the maintenance period being followed by a Cladribine-free period. The total study period was 96 weeks. The combined duration of the induction period and the first Cladribine-free period was twelve months, and the combined duration of the maintenance period and the second Cladribine-free was also twelve months. Patients received Cladribine in either a low or high dose, those in the low-dose group receiving a total target dose of 1.75 mg/kg both in the induction period and in the maintenance period. Patients in the high-dose group received a total target dose of 3.5 mg/kg in the induction period and a total target dose of 1.75 mg/kg in the maintenance period. Patients in the placebo group received placebo over the whole study period. The results of this study showed that annualized relapse rate over 96 weeks was significantly reduced in both low and high-dose groups compared with the placebo treatment Group (0.14 and 0.15 versus 0.33, respectively; relative reduction 57.6% and 54.5%, respectively, both p<0.001 ). Significantly higher proportions of patients remained relapse-free over 96 weeks in both cladribine groups relative to placebo (odds ratios approximately 2.5; both p<0.001 ). There was a 33% and 31 % reduction in the risk of developing 3-month sustained disability progression over 96 weeks in both low and high-dose groups versus placebo (hazard ratio = 0.67, 95% confidence interval [0.48, 0.93] p=0.018; hazard ratio = 0.69, 95% confidence interval [0.49, 0.96] p=0.026, respectively), and 55% and 46% increases in the odds for not progressing (p=0.016 and p=0.032, respectively). Cladribine treatment resulted in significant reductions in MRI activity measures versus placebo. Patients in the low-dose group and in the high-dose group had 85.7% and 87.9% relative reductions in T1 gadolinium-enhancing lesions per patient per scan (mean 0.12 and 0.1 1 versus 0.91 ); 73.4% and 76.9% relative reductions in active T2 lesions per patient per scan (mean 0.38 and 0.33 versus 1.43); and 74.4% and 77.9% relative reductions in combined unique lesions per patient per scan (mean 0.43 and 0.38 versus 1.72), respectively; all p<0.001 versus placebo. Treatment benefits on MRI were evident as early as 24 weeks.

The study thus concluded that treatment with cladribine tablets in the above described total target doses and regimen resulted in greater than 50% reductions in clinical relapse rates, reductions in the risk of disability progression by more than 30%, and suppression of active inflammatory lesions visualized by MRI by up to 88% over the study duration.

It has thus been demonstrated that oral Cladribine significantly reduces relapse rates, risk of disability progression, and MRI measures of disease activity in RRMS. Moreover, safety and tolerance of oral Cladribine is acceptable. Oral short-course treatment with cladribine tablets thus provides an important new option in relapsing-remitting multiple sclerosis therapy.

The significant and sustained benefits of oral Cladribine in MS therapy are exhibited by the majority of patients treated. However, for a given treatment regimen, the extent of Cladribine response may vary among individuals, even though the individuals may present symptoms of comparable severity at the outset of the treatment. For example, some patients show highly positive response to relatively low doses of oral Cladribine, whereas others require higher doses. Likewise, some patients are totally relapse free after a series of short courses of Cladribine given over a period of two years whilst others, although showing an increase in the length of time to first relapse, nevertheless experience relapse during treatment over this period, and therefore may benefit from the addition of an immunomodulatory / immunosuppressive therapeutic agent to Cladribine therapy.

It is therefore an object of the present invention to identify response factors associated with, and predictive of, different clinical outcomes in Cladribine treatment of MS. It is also an object of the invention to define patient populations on the basis of such response factors, and to provide Cladribine treatment regimens specifically adapted to these different patient populations.

The present inventors have investigated a number of different factors potentially linked to patient response to Cladribine. In particular, molecular genetics analysis has been undertaken to identify DNA polymorphisms or gene expression profiles associated with certain traits (i.e. response, adverse events) of cladribine used in the treatment of multiple sclerosis as well as potential susceptibility loci for multiple sclerosis. Analyses were performed by comparison of allele frequency of single nucleotide polymorphisms (SNPs) spread throughout the human genome. Methodology used included DNA amplification, genotyping using Affymetrix DNA chip 500K, Affymetrix DNA chip DMET, TaqMan and sequencing for candidate markers, and statistical analysis.

The inventors have surprisingly discovered that a number of genetic markers not previously known to be associated with Cladribine response or MS are associated with Cladribine efficacy. These markers are single nucleotide polymorphisms (SNPs) located on the X chromosome, more particularly within the Duchenne Muscular Dystrophy (DMD) gene.

Summary of the Invention:

The invention relates to a method for determining the capacity of a subject suffering from Relapsing-Remitting Multiple Sclerosis (RRMS) to respond to treatment with Cladribine, said method comprising determining the polymorphism genotype of the subject in at least one SNP located on chromosome X, particularly an SNP located in the Duchenne Muscular Dystrophy (DMD) gene, for example rs5971598.

The invention also relates to Cladribine for use in treating Relapsing-Remitting Multiple Sclerosis (RRMS) in a subject, wherein the subject has undergone an analysis of genotype for rs5971598 resulting in confirmation of a genotype of TT for a female subject and T for a male subject and wherein Cladribine is administered in a low-dose regimen, preferably in the absence of combination therapy with beta-interferon.

The invention further relates to Cladribine for use in treating Relapsing-Remitting Multiple Sclerosis (RRMS) in a subject, wherein the subject has undergone an analysis of genotype for rs5971598 resulting in confirmation of a genotype having at least one C allele, and wherein Cladribine is administered in a high-dose regimen, possibly as part of a combination therapy with beta-interferon.

The invention also relates to a kit for adapting the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS) to a subject's response profile to Cladribine, comprising :

a pharmaceutical composition comprising cladribine, for example in an oral dosage form, and

means for determining the polymorphism genotype of the subject in at least one SNP located on chromosome X, for example rs5971598,

optionally, instructions for adaptation of the dosage of Cladribine for the different genotypes of the at least one SNP,

optionally, a pharmaceutical composition comprising beta-interferon and instructions for separate, sequential or simultaneous administration of combination therapy with beta- interferon for the different genotypes of the at least one SNP.

In addition, the invention also relates to the use of at least one SNP located on chromosome X, preferably an SNP within the DMD gene such as rs5971598, as a marker for Cladribine efficacy in subjects suffering from Relapsing-Remitting Multiple Sclerosis (RRMS). This aspect of the invention also includes a method for determining or predicting efficacy of Cladribine treatment of Relapsing-Remitting Multiple Sclerosis (RRMS) in a subject in need of such treatment by use of at least one SNP located on chromosome X, preferably an SNP within the DMD gene such as rs5971598, as a marker for Cladribine efficacy.

In accordance with the present invention, the following terms are defined as follows :

The "total dose" or "cumulative dose" refers to the total dose of Cladribine administered during the treatment, i.e. the dose reached at the end of the treatment that is calculated by adding the daily doses. For example, the total dose of Cladribine corresponding to a treatment of 0.7 mg/kg Cladribine per day during 5 days is 3.5 mg/kg or the total dose of Cladribine corresponding to a treatment of 0.35 mg/kg Cladribine per day during 5 days is 1.75 or about 1.7 mg/kg. "The total effective dose" or "cumulative effective dose" refers to the bioavailable dose of Cladribine after a given administration period, i.e. the bioavailable dose reached at the end of the treatment that is calculated by adding the daily doses reduced by the bioavailability coefficient. For example, the total effective dose of Cladribine corresponding to a treatment of 0.7 mg/kg Cladribine per day during 5 days wherein the bioavailability of Cladribine is of about 40% is about 1.4 mg/kg or the total effective dose of Cladribine corresponding to a treatment of 0.35 mg/kg Cladribine per day during 5 days wherein the bioavailability of Cladribine is of about 40% is about 0.7 mg/kg. Typically, the bioavailability of Cladribine or of a Cladribine formulation used in the context of this invention is from about 30% to about 90%, preferably from about 40% to about 60%, such as about 50%.

"A week" refers to a period of time of or about 5, about 6 or about 7 days.

"A month" refers to a period of time of or about 28, about 29, about 30 or about 31 days. "Treatment" comprises the sequential succession of an "induction treatment" and at least a "maintenance treatment". Typically, a treatment according to the invention comprises an "induction treatment" and about one or about two or about three maintenance treatments. Typically, a treatment according to the invention is of about 2 years (about 24 months) or about 3 years (about 36 months) or about 4 years (about 48 months).

An "Induction Treatment" consists in the sequential succession of (i) an induction period wherein the Cladribine or the Cladribine pharmaceutical preparation of the invention is orally administered and (ii) a Cladribine-free period. An induction period lasts up to about 4 months or up to about 3 month or up to about 2 months. For example, an induction period lasts for about 2 to about 4 months. An induction period consists in the oral administration of Cladribine or a pharmaceutical preparation thereof during about 1 to about 7 days each month.

A "Cladribine-free period" is a period wherein no Cladribine is administered to the patient. During a Cladribine-free period, the patient can be free of any administration or be dosed with a placebo-pill or a drug other than Cladribine . A Cladribine-free period lasts at least about 8 months. Preferably it lasts up to about 10 months or up to 9 months or up to about 8 months. For example, a Cladribine-free period lasts from about 8 to about 10 months, typically at least about 8 months. In another embodiment of the present invention, the Cladribine-free period lasts as long as necessary for reducing lymphopenia caused by the induction and/or maintenance treatment. Preferably, in this embodiment it lasts as long as necessary to achieve about 50%, about 75% or about 90% of the original lymphocyte count before the induction treatment.

A "Maintenance Treatment" consists in the sequential succession of (i) a maintenance period wherein the Cladribine or the Cladribine pharmaceutical preparation of the invention is orally administered at the same or at a lower dose than the Cladribine dose orally administered during the induction treatment and (ii) a Cladribine-free period. A maintenance period lasts for up to about 4 months, or up to about 3 months, or up to about 2 months, preferably up to about 2 months. For example, a maintenance period lasts for about 2 to about 4 months, preferably for about 2 months. A maintenance period consists in the oral administration of Cladribine or of a pharmaceutical preparation thereof during about 1 to about 7 days each month.

Within the context of this invention, the beneficial effect, including but not limited to an attenuation, reduction, decrease or diminishing of the pathological development after onset of the disease, may be seen after one or more a "treatments", after an "induction treatment", after a "maintenance treatment" or during a Cladribine-free period.

"Daily dose" refers to the total dose of Cladribine orally administered to the patient each day of administration. The daily dose can be reached through a single or several administrations per day, such as for example once a day, twice a day or three times a day.

The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.

Patients suffering from MS can be defined for example as having clinically definite or laboratory-definite MS according to Schumacher or Poser criteria (Schumacher et al, 1965, Ann. NY Acad. Sd. 1965; 122:552-568; Poser et al, 1983, Ann. Neurol. 13(3): 227-31 ). "Relapses" involve neurologic problems that occur over a short period, typically days but sometimes as short as hours or even minutes. These attacks most often involve motor, sensory, visual or coordination problems early in the disease. Later, bladder, bowel, sexual and cognitive problems may be shown. Sometimes the attack onset occurs over several weeks. Typical MS relapse involves a period of worsening, with development of neurological deficits, then a plateau, in which the patient is not getting any better but also not getting any worse followed by a recovery period. Recovery usually begins within a few weeks.

"Efficacy" of a treatment according to the invention can be measured based on changes in the course of disease in response to a use according to the invention. For example, treatment of MS efficacy can be measured by the frequency of relapses in RRMS and the presence or absence of new lesions in the CNS as detected using methods such as MRI technique {Miller et al, 1996, Neurology, 47(Suppl 4): S217; Evans et al, 1997, Ann. Neurology, 41 : 125-132). The observation of the reduction and/or suppression of MRI T1 gadolinium-enhanced lesions (thought to represent areas of active inflammation) gives a primary efficacy variable.

Secondary efficacy variables include MRI T1 enhanced brain lesion volume, MRI T1 enhanced lesion number, MRI T2 lesion volume (thought to represent total disease burden, i.e. demyelination, gliosis, inflammation and axon loss), MRI T1 enhanced hypointense lesion volume (thought to represent primarily demyelination and axon loss), time-to- progression of MS, frequency and severity of exacerbations and time-to-exacerbation, Expanded Disability Status Scale score and Scripps Neurologic Rating Scale (SNRS) score (Sipe et al., 1984, Neurology, 34, 1368-1372). Methods of early and accurate diagnosis of multiple sclerosis and of following the disease progression are described in Mattson, 2002, Expert Rev. Neurotherapeutics, 319-328.

Patients according to the invention are patients suffering from multiple sclerosis, preferably RRMS or early SPMS. Patients are selected from human males or females and are preferably between 18 and 65 years age, most preferably between 18 and 55 years old. Typical patients had at least one relapse within the 12 months prior to the treatment. Degree of disability of MS patients can be for example measured by Kurtzke Expanded Disability Status Scale (EDSS) score {Kurtzke, 1983, Neurology, 33, 1444-1452). Typically a decrease in EDSS score corresponds to an improvement in the disease and conversely, an increase in EDSS score corresponds to a worsening of the disease.

Detailed Description of the Invention :

A first aspect of the invention relates to the use of at least one SNP located on chromosome X, preferably within the Duchenne Muscular Dystrophy (DMD) gene, as a marker for Cladribine efficacy in subjects suffering from Relapsing-Remitting Multiple Sclerosis (RRMS). The invention thus includes a method for determining or predicting Cladribine efficacy in a subject suffering from Relapsing-Remitting Multiple Sclerosis (RRMS), said method comprising the use of an SNP located on the X chromosome, particularly in the DMD gene, as a marker.

According to a preferred variant of this aspect of the invention, there is provided a method for determining the capacity of a subject suffering from Relapsing-Remitting Multiple Sclerosis (RRMS) to respond to treatment with Cladribine, wherein the method comprises determining the polymorphism genotype of the subject in at least one SNP located on the X chromosome, particularly in the DMD gene. Examples of suitable SNPs located on the X chromosome are indicated in Chart A below, in particular rs5971598, rs17338758, rs6628643, rs5935310, rs7892480, rs593531 1. Particularly preferred SNPs are those located within the Duchenne Muscular Dystrophy (DMD) gene, namely rs5971598, rs17338758, rs6628643. SNP rs5971598 is most particularly preferred. The DMD gene is a very large gene thought to participate in the signal transduction in synapses through anchorage of receptors within the membrane. The rs5971598 is located in intron 49 of DMD and rs17338758 and rs6628643 in intron 50. DMD is not located in the pseudo-autosomal region (PAR) of the chromosome X. Therefore males carry only one allele for these markers. Accordingly, the two possible alleles for an A B SNP on males' X chromosome is hereinafter noted as AA and BB, which allows the comparison with AA and BB homozygote genotypes on females' X chromosome.

Other suitable markers, namely rs5935310, rs7892480, rs593531 1 , are located in the FRMPD4 gene.

Each of these markers presents a single nucleotide polymorphism. The genotype of a subject for each of these markers has been found, in phase III clinical trials, to be predictive of the subject's response to Cladribine treatment of MS in so far as each of the markers is linked to one or more specific therapeutic endpoints, namely reduced number of relapses in comparison to placebo treatment, relapse-free status and / or progression-free status. By determining the genotype of a subject for one or more of these markers, it can thus be reliably predicted whether the subject is likely to achieve such endpoint.

More specifically, this aspect of the invention relates to a method for determining the capacity of a subject suffering from RRMS to respond to treatment with Cladribine, comprising

i) contacting a sample of genomic DNA obtained from a subject suffering from RRMS with a means for determining the polymorphism genotype of at least one SNP selected from the group consisting of rs5971598, rs17338758, rs6628643, rs5935310, rs7892480, rs593531 1 , ii) determining the genotype of the subject for the at least one SNP,

iii) correlating the genotype with the capacity of the subject to respond to Cladribine treatment,

wherein

- a genotype comprising at least one T allele in any one of rs5971598, rs17338758 and rs593531 1 , or

- a genotype comprising at least one C allele in any one of rs6628643 and rs7892480 or

- a genotype comprising at least one A allele in rs5935310, is indicative of the subject's capacity to respond positively to Cladribine.

In the context of the invention, the determination of the "capacity of a subject to respond to treatment with Cladribine" means the determination of whether the subject in question is more likely than not to show a positive response to Cladribine.

In the context of the invention, a "positive response" to Cladribine means that a subject, male or female, achieves the therapeutic endpoint characteristic of a particular SNP marker over a two year period, preferably a 96-week period, following initiation of Cladribine treatment. These endpoints are in particular :

- for rs5971598 : relapse-free status, or reduced number of relapses compared to placebo treatment or progression-free status ;

- for rs 17338758 and rs6628643 : relapse-free status ;

- for rs5935310, rs7892480 and rs593531 1 : combined progression-free and relapse status.

The Cladribine treatment to which reference is made in this context is preferably an oral treatment comprising alternating periods of Cladribine administration and Cladribine-free periods. In particular, the treatment comprises sequentially, an initial treatment period (so-called induction period), a Cladribine-free period, a re-treatment period (so-called maintenance period), and a second Cladribine-free period. Cladribine is preferably administered at a dosage such that the total dose achieved at the end of the induction period is between 1 .5 mg/kg and 4.5 mg/kg, preferably 1.75 mg/kg to 3.5mg/kg and the total dose achieved at the end of the maintenance period is between 1.5 mg/kg and 2.25 mg/kg, preferably 1.75mg/kg. The sum of the durations of the two treatment periods and the two Cladribine-free periods is preferably about 22 to 26 months, in particular about 24 months for example 96 to 100 weeks in total.

Alternatively, the Cladribine treatment to which reference is made in the context of the invention can be a non-oral treatment, i.e. parenteral administration. In this case, the doses administered to the patient are calculated such that the total effective dose reached at the end of each treatment period (i.e. the bioavailable dose reached at the end of the treatment period) is approximately the same as the total effective dose achieved by the above oral treatment, according to which the bioavailability of Cladribine is about 40% to about 60%, for example about 50%. Chart A

BO. According to this aspect of the invention, use of the marker rs5971598 is most preferred for determining a patient's capacity to respond to Cladribine. A positive response to Cladribine as shown by SNP rs5971598, means that a subject (male or female) shows no relapses over a two year period following initiation of Cladribine treatment, or less relapses over the two year period following initiation of Cladribine treatment as compared to a subject having the same genotype treated with a placebo.

It has been found that for the rs5971598 marker, a subject having a TT (i.e. a female subject) or a T genotype (i.e. a male subject), has the greatest probability of showing a positive response to Cladribine. Indeed, around 85% of treated TT / T patients show a positive response to the oral Cladribine treatment defined above. The CC (female) or C genotype (male) has the least probability of showing a positive response, around 55% of CC/C patients showing a positive response. The CT genotype (female) is intermediate between the TT and CC genotype in terms of probability of showing a positive response, around 75% of subjects having the CT genotype showing a positive response (see for example Table 14 below). More particularly, in terms of an individual patient :

- the likelihood that a subject having the TT (female) or T (male) genotype of rs5971598 will have no relapses over the two year period following initiation of Cladribine treatment is increased by about 25% compared to a subject treated with a placebo. Moreover, the likelihood that this subject will have one relapse, or two relapses, over the two year period following initiation of treatment with Cladribine is decreased by about 15%, or about 5%, respectively, in comparison to a subject treated with placebo ;

- the likelihood that a subject having a CC genotype (female) or a C genotype (male) of rs5971598 will have no relapses, or one relapse over the two year period following initiation of Cladribine treatment is approximately comparable to that of a subject treated with a placebo. The likelihood that this subject will have two relapses over the two year period following initiation of treatment with Cladribine is decreased by about 10% in comparison to a subject treated with placebo;

- for the TC genotype of rs5971598 (i.e. a female subject), the likelihood that this subject will have no relapses over the two year period following initiation of Cladribine treatment is increased by about 5% compared to a subject treated with a placebo. The likelihood that this subject will have one relapse is decreased by approximately 5% in comparison to the placebo-treated subject, and the likelihood that this subject will have two relapses over the two year period is comparable to that of a subject taking placebo. The rs5971598 marker is thus linked to gender, and the most prevalent genotype (TTVT : female / male respectively) is associated with positive response, whereas heterozygotes have a lesser probability of showing a positive response and the less prevalent genotype (CC/C) presents a response similar to patients in the placebo arm, suggesting a dose effect of the genotype. Furthermore, this SNP is moderately associated with a reduced disease progression, a trend being apparent between presence of the TT or T genotype and the absence of MRI activities. Therefore, this marker is particularly suitable for efficacy stratification.

Interestingly, rs5971598 is part of a T-C-T haplotype (rs5971598, rs17338758 and rs6628643) which has also been found to be associated with Cladribine efficacy. Consequently, according to the invention, a particularly preferred method for predicting subject response to Cladribine comprises determining the genotype of each of rs5971598, rs17338758 and rs6628643, a T-C- T haplotype indicating maximum probability of a positive response to Cladribine.

According to the method of the invention, the biological sample used for the genotyping may be any type of sample which contains genomic DNA. Particularly preferred samples include blood.

The means for determining the polymorphism genotype may comprise any means suitable in the art, for example means for nucleic acid amplification, sequencing and / or DNA chip technology. Details of such means are provided in the Examples below.

The correlation between the clinical response which a subject is likely to show to Cladribine and the genotype of the markers, permits adaptation of the Cladribine treatment regimen, depending on the subject's genotype, in order to optimise chances of a positive response. In particular, for rs5971598, the inventors have established not only that the different genotypes reflect different probabilities of positive response in an individual, but also that the different genotypes react differently to different treatment regimens, particularly changes in dose of Cladribine, and combination with a second therapeutic agent. On the basis of these observations, a treatment regimen can be specifically adapted to a particular patient to enhance efficacy of Cladribine treatment of RRMS.

More specifically, it has been established by the inventors that a TT / T genotype for rs5971598 is indicative that the patient's response to oral Cladribine will not be dose dependent or will be less dose dependent. Consequently, such a subject will not benefit from higher doses of Cladribine and can be treated optimally at lower doses, i.e. can be treated highly effectively, with lower risk of side-effects, for example at a total dose of about 1 .5 to 2.25 mg/kg over both induction and maintenance periods. Thus, in such subjects, a higher dose does not necessarily result in increased efficacy.

This aspect of the invention thus relates to a method of treating Relapsing-Remitting Multiple Sclerosis (RRMS) in a subject, wherein said method comprises

a) carrying out an analysis of genotype for rs5971598 on the subject

b) if the analysis results in confirmation of a genotype of TT for a female subject or T for a male subject, administering Cladribine orally to the subject, wherein the treatment regimen comprises the sequential steps :

i) an induction period during which Cladribine is administered such that the total dose reached at the end of the induction period is between 1.5 and 2.25 mg/kg, followed by ii) a first cladribine-free period in which no Cladribine is administered.

iii) a maintenance period during which Cladribine is administered such that the total dose reached at the end of the maintenance period is between 1.5 and 2.25 mg/kg, and iv) a second cladribine-free period in which no Cladribine is administered.

This regimen, specifically adapted for the TT/T rs5971598 genotype, will be referred to hereinafter as the 'low-dose TT/T genotype regimen', wherein 'TT/T' signifies a homozygous TT genotype for a female subject and a T genotype for a male subject. According to this aspect of the invention, the induction period (i) has a duration of up to 4 months, preferably two or three months. For example, oral Cladribine is given as a series of short courses each consisting of one or two 10 mg cladribine tablets given once daily for the first 4 or 5 days of a 28-day period, patients receiving four consecutive courses in the initial treatment period (starting at Day 1 and Weeks 5, 9 and 13).

Preferably, according to this treatment regimen, the cumulative dose reached at the end of the induction period (i) is between 1.6 to 2.0 mg/kg, most preferably 1.75 mg/kg.

The induction period is followed by a first Cladribin-free period (ii) which may have a duration of 8 to 12 months, most preferably 10 months. During this period, no Cladribine is administered to the subject.

After the first Cladribine-free period, a period of Cladribine re-treatment, referred to as the maintenance period (iii), is applied. During this maintenance period (iii), which, according to this low-dose TT/T genotype regimen, has a duration of from two to 4 months, preferably two months, Cladribine is administered at a dose such that the total dose reached at the end of the maintenance period is approximately equal to that administered during the induction period, i.e. approximately 1.6 to 2.0 mg/kg, most preferably 1.75 mg/kg. Cladribine is normally administered as a series (two, three or four courses) of short courses over the maintenance period, for example once daily for the first four or 5 days of a 28-day period, depending on the weight of the subject. Typically, the maintenance period is composed of two short courses for example starting at weeks 48 and 52 after initiation of the induction period.

Finally, the maintenance period is followed by a second Cladribine-free period (iv), which has a duration of about 8 to 12 months, preferably about 10 months.

The low-dose TT/T genotype regimen is thus typically composed of a total of two administration periods (I) and (iii) and two Cladribine-free periods (ii) and (iv), the periods (i), (ii), (iii) and (iv) together having a total duration of about 2 years, for example 94 to 108 weeks, particularly about 96 weeks.

A significant feature of the low-dose TT/T genotype regimen is that the subject does not require rescue therapy by beta interferon. Specifically, according to this aspect of the invention, the subject does not receive rescue therapy by beta interferon at any time during periods (i), (ii), (iii) or (iv). It has indeed been established by the inventors that the responsiveness of the TT/T rs5971598 genotype patients is sufficiently high to be able to dispense with any type of combination therapy with beta interferon, such as Rebif, throughout the Cladribine treatment. This finding is particularly advantageous.

Moreover, in the framework of the present invention, it has been found that response to Cladribine is not associated with efficacy candidate markers to beta-interferon response, particularly Rebif, rs2229207 (in gene IFNAR2), rs2071543 (in gene PSMB8) and rs469304 (in gene MX1 ) (see for example International patent application WO 2010/010057). Thus response to beta interferon (for example Rebif) in a subject is independent of the subject's response to oral Cladribine. Consequently, if the subject is non-responsive to beta-interferon (Rebif), the subject may be treated with Cladribine as an alternative. Since subjects having a TT/T genotype for rs5971598 are particularly likely to have a positive response to oral Cladribine, and are particularly likely not to need combination therapy with beta-interferon, it follows that Cladribine treatment is ideally suited for subjects having a TT/T genotype for rs5971598 who are non- responders to beta-interferon (Rebif) and / or who are beta-interferon (Rebif) naive. The invention thus extends to methods of treating such patients with the low-dose TT/T genotype regimen. Contrary to the findings for the TT/T genotype for rs5971598, it has been established by the present inventors that the CT or CC / C genotype for rs5971598 is indicative that the patient's response to oral Cladribine will be dose dependent. Consequently, such a subject will benefit from higher doses of Cladribine, for example at a total dose of about 3.0 to 4.5 mg/kg over the induction period.

a) carrying out an analysis of genotype for rs5971598 on the subject

b) if the analysis results in confirmation of a genotype comprising at least one C, that is a CC/C genotype or a C/T genotype, administering Cladribine orally to the subject, wherein the treatment regimen comprises the sequential steps :

i) an induction period during which Cladribine is administered such that the total dose reached at the end of the induction period is between 3.0 and 4.5 mg/kg, followed by ii) a first cladribine-free period in which no Cladribine is administered,

This regimen, specifically adapted for the CC/C or C/T rs5971598 genotype, will be referred to hereinafter as the 'high-dose CC/C genotype regimen', wherein 'CC/C signifies a homozygous CC genotype for a female subject and a C genotype for a male subject, and C/T signifies a heterozygous female subject. According to this aspect of the invention, the induction period (i) has a duration of up to 4 months, preferably two or three months. For example, oral Cladribine is given as a series of short courses each consisting of one or two 10 mg cladribine tablets given once daily for the first 4 or 5 days of a 28-day period, patients receiving four consecutive courses in the initial treatment period (starting at Day 1 and Weeks 5, 9 and 13).

According to this aspect of the invention, the cumulative dose reached at the end of the induction period (i) is between 3.0 and 4.5 mg/kg, for example 3.25 to 3.75 mg/kg, more preferably about 3.5 mg/kg.

After the induction period, the high-dose CC/C genotype regimen includes a first Cladribin-free period (ii) which may have a duration of 8 to 12 months, most preferably 10 months. During this period, no Cladribine is administered to the subject. There then follows, a period of Cladribine re-treatment, referred to again as the maintenance period (iii). During this maintenance period (iii), which, according to this high-dose CC/C genotype regimen, has a duration of from two to 4 months, preferably two months, Cladribine is administered at a dose such that the total dose reached at the end of the maintenance period is less than that administered during the induction period, for example approximately 1.6 to 2.0 mg/kg, most preferably 1.75 mg/kg. Over the maintenance period, Cladribine is usually administered as a series (two, three or four courses) of short courses, for example once daily for the first four or 5 days of a 28-day period, depending on the weight of the subject. Typically, the maintenance period is composed of two short courses for example starting at weeks 48 and 52 after initiation of the induction period.

The high dose CC/C genotype regimen is thus typically composed of a total of two administration periods (i) and (iii) and two Cladribine-free periods (ii) and (iv), the periods (i), (ii), (iii) and (iv) together having a total duration of about 2 years, for example 94 to 108 weeks, particularly about 96 weeks. This regimen thus differs from the low-dose TT/T genotype regimen only in terms of dose, the administrations schedules being otherwise entirely comparable.

Importantly, it has been established by the inventors that subjects having a CC/C or C/T genotype are particularly likely to benefit from combination or rescue therapy with beta- interferon, for example Rebif. The term "interferon-beta (IFN-[beta])", as used herein, is intended to include fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells, as well as its salts, functional derivatives, variants, analogs and active fragments. IFN-[beta] suitable in accordance with the present invention is commercially available e.g. as Rebif(R) (MerckSerono), Avonex(R) (Biogen) or Betaferon(R) (Schering). The use of interferons of human origin is also preferred in accordance with the present invention. The term interferon, as used herein, is intended to encompass salts, functional derivatives, variants, analogs and active fragments thereof.

In accordance with the present invention, such combination therapy may involve administration of beta interferon simultaneously, subsequently or separately from Cladribine administration. For example the beta interferon treatment may be concomitant with the Cladribine treatment, or adjunctive to the Cladribine treatment or alternating with the Cladribine treatment, or sequential to the Cladribine treatment.

According to a preferred embodiment of the invention, there is provided a method for treating Relapsing-Remitting Multiple Sclerosis (RRMS) in a subject, said method comprising

a) carrying out an analysis of genotype for rs5971598 on the subject

b) if the analysis results in confirmation of a genotype comprising at least one C, that is a CC/C genotype or a C T genotype, administering Cladribine orally to the subject, wherein the treatment regimen comprises the sequential steps :

i) an induction period during which Cladribine is administered such that the total dose reached at the end of the induction period is between 1.5 and 4.5 mg/kg, followed by ii) a first cladribine-free period in which no Cladribine is administered,

iii) a maintenance period during which Cladribine is administered such that the total dose reached at the end of the maintenance period is between 1.5 and 2.25 mg/kg, and iv) a second cladribine-free period in which no Cladribine is administered,

and wherein beta interferon is administered during at least the induction period (i) and / or the maintenance period (iii).

According to this aspect of the invention beta interferon is administered at a dose of approximately 20 to 50 meg sc tiw (subcutaneously thrice weekly) by use of Rebif or at a dose of approximately 30 meg (once weekly intramuscularly) by use of Avonex or at a dose of approximately 300 meg every other day by use of Betaferon, during both the induction and the maintenance period, for example 22 to 44 meg sc tiw. Alternatively, the beta interferon may be given only during the induction period or only during the maintenance period. The beta interferon may or may not be continued over the Cladribine-free periods, or may be given exclusively during the Cladribine-free periods. The subject may have been receiving beta interferon prior to the first administration of Cladribine, in which case the Cladribine is an "addon" to the beta interferon treatment. Alternatively the subject may be beta-interferon naive.

According to this combination therapy aspect of the invention, the dose of Cladribine which is given during the induction period can be a low or high dose, i.e. can be a low dose between 1.5 and 2.25 mg/kg, or alternatively a high dose between 3.0 and 4.5 mg/kg. The low dose is particularly preferred. A further embodiment of the invention relates to a method of treatment of RRMS in a subject by administering oral Cladribine according to the high-dose CC/C genotype regimen described above, wherein the subject is interferon-beta naive or beta-interferon non-responsive.

Further aspects of the present invention include a kit for the adaptation of the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS) to a subject's response profile to Cladribine, comprising :

- a pharmaceutical composition comprising cladribine for oral administration,

- means for determining the polymorphism genotype of the subject in at least one SNP located on chromosome X, particularly in the Duchenne Muscular Dystrophy (DMD) gene,

- optionally, instructions for adaptation of the dosage of Cladribine for the different genotypes of the at least one SNP, and

- optionally, a pharmaceutical composition comprising beta-interferon and instructions for separate, sequential or simultaneous administration of combination therapy with beta-interferon for the different genotypes of the at least one SNP.

According to this embodiment of the invention, the at least one SNP is chosen from the group consisting of rs5971598, rs17338758, rs6628643 rs5935310, rs7892480, rs593531 1 or a combination thereof. Preferably, the at least one SNP comprises rs5971598.

The kit of the invention preferably contains, as the means for determining the polymorphism genotype, any suitable means in the art for example means for nucleic acid amplification, sequencing and / or whole genome scan chip technology.

According to this embodiment, the pharmaceutical composition comprising Cladribine for oral administration may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, colouring agents, flavouring agents, adjuvants, and the like. The compositions may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate). Tablets may be coated according to methods well known in the art. Compositions of this invention may also be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, nonaqueous vehicles and preservatives. Suspending agent include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p- hydroxybenzoate and sorbic acid.

The invention also relates to the use of at least one SNP located on chromosome X as a marker for Cladribine efficacy in subjects suffering from Relapsing-Remitting Multiple Sclerosis (RRMS). Preferably the at least one SNP is selected from rs5971598, rs 17338758, rs6628643 or a combination thereof, or alternatively is selected from rs5935310, rs7892480, rs593531 1 or a combination thereof.

Different aspects of the invention are illustrated in the Figures :

Figure 1 - Patient Enrollment and Disposition.

Withdrawal data shown are study discontinuations.

Other reasons for discontinuation in the placebo and cladribine 3.5 and 5.25 mg/kg groups comprise consent withdrawal for administrative, convenience and personal reasons.

Note: Two deaths occurred after patients withdrew from study, one in each cladribine group.

Figure 2 - Efficacy Outcome Measures Relating to Relapse and Disability Over the 96-week Study by Treatment Group (Intent-to-Treat Population).

(A) Annualized Relapse Rates : error bars indicate upper limit of 95% confidence interval

(B) Kaplan-Meier Plot of Time to First Relapse : a solid line indicates placebo ; an intermittent line represents Cladribine 3.5 mg/kg ; a dotted line represents Cladribine 5.25 mg/kg,

(C) Cumulative Relapses Over Time : a solid line indicates placebo ; an intermittent line represents Cladribine 3.5 mg/kg ; a dotted line represents Cladribine 5.25 mg/kg and (D) Kaplan-Meier Plot of Time to 3-Month Sustained Progression of Disability : a solid line indicates placebo ; an intermittent line represents Cladribine 3.5 mg/kg ; a dotted line represents Cladribine 5.25 mg/kg.

P-values shown in figures 2(B) and 2(D) relate to the hazard ratio and 95% confidence intervals versus placebo for improvement over 96 weeks, estimated using Cox proportional hazards model with fixed effects for treatment group and region.

Information relative to Figure 2(C) :

Number of patients contributing qualifying relapses at each timepolnt

Placebo 50 39 33 30 33 34 1? 12

Cladribine 3,5 mg/kg 21 13 19 19 15 9 8 4

Cladribine 5.25 mg/kg 30 11 13 16 14 15 10 5

Information relative to Figure 2(D) :

Number of patients at risk

Piacebo 437 424 399 373 355 333 315 304 304

Cladribine 3.5 mg/kg 433 424 407 389 373 364 355 347 347

Cladribine 5,25 mg/kg 456 447 425 404 388 375 363 350 350

Figure 3 - Distribution of the number of qualifying relapses in the whole BM/PGx population. Dark bars represent high dose ; lightly shaded bars represent low dose ; white bars represent placebo.

Figure 4 - Primary endpoint stratification by HLA-DRB1*15 presence and absence.

Figure 5 - Time to first qualifying relapse Kaplan-Meier curves on HLA-DRB1*15 presence and absence stratified subpopulation vs. whole BM/PGx population.

Figure -6 - QQ-plot of p-values of association with relapse-free status in treated subgroups.

Figure 7 - Association of rs5971598 with relapse-free status in treated subgroups.

Figure 8 - Association of rs5971598 with qualifying relapse (QR)-free status by treatment subgroup.

Figure 9 - Association of rs5971598 with relapse-free status by sex (M/F) in treated subgroups. Genotype frequencies are indicated relative to female (f_F), male (f_M) and whole populations (f). Figure 10 - Association of rs5971598 with qualifying relapse (QR)-free status by region (Americas, East Europe, Rest of World, Russia, West Europe) in treated subgroups.

Figure 11 - Association of rs5971598 with qualifying relapse-free status by dose group.

Figure 12 - Association of rs5971598 with qualifying relapse-free status. Presence of allele C ("Pres. C"), respectively T, means genotype CC or CT, respectively CT or TT.

Figure 13 - Association of rs5971598 with primary endpoint (number of qualifying relapses at 96 weeks). Boxed percentages indicate the average reduction of the number of relapses at 96 weeks compared to Placebo subgroup.

Figure 14 - Stratification by rs5971598 CC in terms of time to first qualifying relapse.

Figure 15 - Stratification by rs5971598 CT in terms of time to first qualifying relapse.

Figure 16 - Stratification by rs5971598 TT in terms of time to first qualifying relapse.

Figure 17 - Association of rs5971598 with disability progression-free status in placebo and treated groups.

Figure 18 - Association of rs5971598 with MRI activity in placebo and treated groups.

Figure 19 - Association of rs593531 1 with absence of progression and relapse in placebo and treated groups.

Figure 20 - Association of rs5971598 with absence of progression and qualifying relapse in placebo and treated groups.

EXAMPLES

EXAMPLE 1 Randomized, Placebo-controlled Trial of Cladribine Tablets for Relapsing-Remitting Multiple Sclerosis

Cladribine is a synthetic purine nucleoside analog that is resistant to deamination by the enzyme adenosine deaminase. Intracellular accumulation of the active metabolite, 2- chlorodeoxyadenosine triphosphate, results in disruption of cellular metabolism, inhibition of DNA synthesis and repair, and subsequent apoptosis. Cladribine preferentially affects lymphocytes because these cells possess relatively high ratios of deoxycytidine kinase to 5'- nucleotidase activities, and are dependent on adenosine deaminase activity to maintain equilibrium of cellular concentrations of triphosphorylated nucleotides. Accumulation of the cladribine nucleotide produces rapid and sustained reductions in CD4+ and CD8+ cells and rapid, though more transient, effects on CD19+ B cells. Cladribine is relatively sparing of other immune cells. Cladribine also has been shown to cause reductions in pro-inflammatory cytokines, serum and CSF chemokine levels, adhesion molecule expression, and mononuclear cell migration.

The efficacy and safety of cladribine tablets in a 96-week, phase III, double-blind, placebo- controlled, multicenter trial in patients with relapsing-remitting multiple sclerosis (RRMS): the CLARITY (CLAdRlbine tablets Treating multiple sclerosis orallY) study, were investigated.

Methods

i) STUDY DESIGN

The study was randomized, double-blind, placebo-controlled for two years (i.e. 24 months with 4 weeks each [see WO 2006/067141 , page 7, lines 5 to 13]). Medication was administered orally in form of tablets containing cladribine in a cyclodextrine formulation. To maintain the double- blind nature of the study, all patients within a weight range received the same number of tablets (cladribine or matched placebo). Patients (1326) had at least one relapse within 12 months of study entry and were randomized (1 : 1 : 1 ) into three groups, i.e. Group 1 , Group 2 and Group 3.

Group 1

Group 1 received placebo tablets over the whole of the study period, in accordance with Table 1 of WO 2006/067141.

Group 2

Group 2 received a cladribine tablet treatment regimen in accordance with WO 2006/067141 Table 1 , which consisted of : a) a period of 2 months, wherein cladribine was administered (induction period), followed by b) a period of 10 months (cladribine free period) consisting of 2 months, wherein placebo was administered and a period of 8 months of no treatment, followed by

c) a period of 2 months, wherein cladribine was administered again (maintenance period), followed by

d) a period of 10 months of no treatment (cladribine free period).

Cladribine was administered, dependent on the weight of the patient, as one or two 10-mg cladribine tablets given daily for the first 4 or 5 days of a month, in order to achieve in Group 2 a total target dose of 1 .75 mg/kg in the induction period and in the maintenance period (Group 2 is also referred to herein as the 3.5 mg/kg group : 1.75 + 1 .75 = 3.5 mg/kg)

Group 3

Group 3 received a cladribine tablet treatment regimen in accordance with Table 1 of WO 2006/067141 , which consisted of :

a) a period of 4 months, wherein cladribine was administered (induction period), followed by b) a period of 8 months of no treatment (cladribine free period), followed by

d) a period of 10 months of no treatment (cladribine free period).

Cladribine was administered, dependent on the weight of the patient, as one or two 10-mg cladribine tablets given daily for the first 4 or 5 days of a month, in order to achieve in Group 3 a total target dose of 3.5 mg/kg in the induction period and of 1.75 mg/kg in the maintenance period (Group 3 is also referred to herein as the 5.25 mg/kg group : 3.5 + 1 .75 = 5.25 mg/kg). ii) SAFETY

Overall, the frequency of reported treatment-emergent adverse events was comparable across treatment groups, with some exceptions related to the mechanism of action of cladribine. Lymphopenia was reported more frequently with cladribine tablets than placebo treatment, and mostly graded as mild or moderate. Severe thrombocytopenia was reported as an adverse event only for 1 patient (in Group 3), and severe neutropenia reported in only 3 patients (1 in Group 2 and 2 in Group 3). Severe pancytopenia was reported for only 1 patient in Group 3. There were no cases of severe anemia. Infections/infestations were reported in 47.7% and 48.9% versus 42.5% patients in Group 2 and Group 3 versus the placebo group, with most events graded as mild or moderate by investigators. Adverse events led to treatment discontinuation for only 3.5%, 7.9% and 2.1 % patients in Group 2 and Group 3 and the placebo group, with 0.5% and 2.0% versus 0% discontinuing due to lymphopenia, respectively. Groups were comparable for other events leading to treatment discontinuation. The incidence of serious adverse events in Group 2 and Group 3 and the placebo group were 8.4%, 9.1 % and 6.4% patients, respectively. Infections/infestations were reported as serious adverse events in 2.3%, 2.9% and 1.6% patients in Group 2 and Group 3 and the placebo group, respectively.

Hi) STUDY PROCEDURES AND END POINTS

To maintain the double-blind nature of the study, all patients within a weight range received the same number of tablets (cladribine or matched placebo); in addition, at each study site a treating physician reviewed clinical laboratory results and assessed treatment-emergent adverse events and safety information, and an independent blinded evaluating physician performed neurological examinations and was responsible for determining if a clinical event fulfilled criteria consistent with a qualifying relapse. A central neuroradiology center, also blinded to treatment, assessed MRI evaluations.

Neurological examinations included the EDSS, ⁶ conducted at pre-study evaluation and at Study Day 1 and at Weeks 13, 24, 36, 48, 72, 84 and 96. MRI scans were obtained at pre-study evaluation and at Weeks 24, 48 and 96. Clinical laboratory tests, including chemistry, hematology and urinalysis, were performed by a central laboratory at frequent intervals over the 96-week study (see Appendix). For suspected relapses occurring between study visits, patients were required to attend the study site within 7 days of onset of the neurological symptoms for objective evaluation by the blinded evaluating physician. Relapses within study could be treated with intravenous steroids at the discretion of the treating physician.

The primary endpoint was the qualifying relapse rate at 96 weeks. A qualifying relapse was defined as a 2-grade increase in >1 functional system of the EDSS or a 1-grade increase in >2 functional systems, excluding changes in bowel/bladder or cognition, in the absence of fever, lasting for >24 hours, and preceded by >30 days of clinical stability or improvement. Hereafter, the term relapse will indicate qualifying relapse.

Key clinical secondary efficacy endpoints were the proportion of relapse-free patients and the time to sustained disability progression, defined as the time to a sustained change (for >3 months) in EDSS of >1 point, or >1.5 points if baseline EDSS was 0. Additional clinical efficacy endpoints included the time to first relapse and the proportion of patients receiving rescue therapy with interferon beta-1a. Secondary MRI endpoints, which will be reported in full elsewhere, were the mean number of lesions per patient per scan over 96 weeks for: T1 gadolinium-enhancing lesions; active T2 lesions; and combined unique lesions, defined as new T1 gadolinium-enhancing and/or new T2 non-enhancing or enlarging lesions (without double- counting).

Safety assessment included the incidence of treatment-emergent adverse events in each treatment group, physical examination and laboratory parameters. A strict protocol was established for the management of hematological events (see Appendix). iv) STATISTICAL METHODS

A total of 1290 patients (approximately 430 in each group) was required to provide 90% power to detect a clinically meaningful 25% relative reduction in relapse rate to Week 96 (primary endpoint) with cladribine compared with placebo. This was calculated using a 2-sided t-test assuming a mean of 2.1 relapses at 96 weeks in the placebo group, a standard deviation of 2.02 for the number of relapses in each group, a 10% non-evaluable rate and a Type I error rate of 2.5% for the comparison of each cladribine group versus placebo.

The primary efficacy analysis population was the intent-to-treat population, which included all randomized patients, while the safety population included all patients who received at least one dose of study treatment and had follow-up safety data. The primary efficacy parameter was analyzed using a Poisson regression model including effects for treatment and region, and the log of time on study as the offset variable. Treatment groups were compared using an approximate chi-square test based on Wald statistics. For patients who received rescue therapy, the primary and secondary efficacy analyses included the imputed data based on a pre-specified methodology formalized in the statistical analysis plan.

In the analysis of secondary endpoints, the proportion of relapse-free patients was analyzed using a logistic regression model including treatment and region effects, and the odds ratio and 95% confidence intervals for being relapse-free were estimated for each treatment group. The time to first relapse and time to 3-month sustained change in EDSS were analyzed using a Cox proportional hazards model including treatment and region effects. Treatment groups were compared using an approximate chi-square test based on Wald statistics, and the hazard ratio of time to 3-month sustained change in EDSS score in each group and associated 95% confidence intervals were estimated. Kaplan-Meier plots of time to first relapse and 3-month sustained change in EDSS score were also generated.

Secondary endpoints related to lesion counts on MRI were analyzed using a non-parametric analysis of covariance model on ranked data with effects for treatment and region adjusted for baseline T1 gadolinium-enhancing lesion counts. RESULTS

i) PATIENTS

The intent-to-treat population of 1326 patients was well balanced across treatment groups (Table 1 ). Almost one third of patients had previously received disease-modifying therapy. Overall, 1 184 (89.3%) patients completed the 96-week study (87.0%, 91.9% and 89.0% in the placebo, cladribine 3.5 and 5.25 mg/kg groups) (Figure 1 ), and 1 165 completed treatment (86.3%, 91 .2% and 86.2%, respectively). For the placebo and the cladribine tablets 3.5 and 5.25 mg/kg treatment groups, the mean time on study was comparable at 87.8, 91.0 and 89.4 weeks, respectively. ii) EFFICACY

The annualized relapse rate over 96 weeks was significantly reduced in the 3.5 and 5.25 mg/kg cladribine groups compared with placebo treatment (relative reduction 57.6% and 54.5%, respectively, both P<0.001 ) (Table 2, Figure 2a). Significantly higher proportions of patients remained relapse-free over 96 weeks in both cladribine groups relative to placebo (odds ratios approximately 2.5; both P<0.001 ; Table 2 and Figure 2b, 2c). These improvements were achieved with a 60% and 69% reduction in the odds for requiring interferon beta-1a rescue therapy in the cladribine 3.5 and 5.25 mg/kg groups relative to placebo (P=0.01 1 and P=0.003, respectively; Table 2).

There was a 33% and 31 % reduction in the risk of developing 3-month sustained disability progression over 96 weeks in the cladribine tablets 3.5 or 5.25 mg/kg groups versus placebo (hazard ratio = 0.67, 95% confidence interval [0.48, 0.93] P=0.018; hazard ratio = 0.69, 95% confidence interval [0.49, 0.96] P=0.026, respectively) (Table 2 and Figure 2d), and 55% and 46% increases in the odds for not progressing (P=0.016 and P=0.032, respectively; Table 2).

Cladribine treatment resulted in significant reductions in MRI activity measures versus placebo. Patients in the 3.5 or 5.25 mg/kg cladribine groups group had 85.7% and 87.9% relative reductions in T1 gadolinium-enhancing lesions per patient per scan (mean 0.12 and 0.1 1 versus 0.91 ); 73.4% and 76.9% relative reductions in active T2 lesions per patient per scan (mean 0.38 and 0.33 versus 1.43); and 74.4% and 77.9% relative reductions in combined unique lesions per patient per scan (mean 0.43 and 0.38 versus 1.72), respectively; all P<0.001 versus placebo. Treatment benefits on MRI were evident as early as 24 weeks. Table 1. Baseline Patient Demographics and Disease Characteristics (Intent-to-Treat Population).

Cladribine Cladribine

Placebo 3.5 mg/kg 5.25 mg/kg (N=437) (N=433) (N=456)

Age (yr)

Mean ± SD 38.7 ± 9.9 37.9 ± 10.3 39.1 ± 9.9

Range 18-64 18-65 18-65 Gender, N female (%) 288 (65.9) 298 (68.8) 312 (68.4) Weight, mean ± SD (kg) 70.3 ± 15.4 68.1 ± 14.6 69.3 ± 14.8 Race, N (%)

White 429 (98.2) 425 (98.2) 446 (97.8)

Black 1 (0.2) 2 (0.5) 4 (0.9)

Other 7 (1.6) 6 (1.4) 6 (1.3)

Prior treatment with any disease- modifying drug*, N (%) 142 (32.5) 1 13 (26.1 ) 147 (32.2) Disease duration from first attack,

median (range) years 7.1 (0.4-39.5) 5.8 (0.3-42.3) 7.2 (0.4-35.2) EDSS category, N (%)

0 13 (3.0) 12 (2.8) 1 1 (2.4)

1 70 (16.0) 75 (17.3) 80 (17.5)

2 127 (29.1 ) 133 (30.7) 1 19 (26.1 )

3 96 (22.0) 108 (24.9) 108 (23.7)

4 83 (19.0) 71 (16.4) 84 (18.4)

>5 48 (1 1.0) 34 (7.9) 54 (1 1.8)

Mean ± SD 2.9 ± 1.3 2.8 ± 1.2 3.0 ± 1.4 T1 gadolinium-enhancing lesions

Patients with lesions, N (%) 128 (29.3) 138 (31.9) 147 (32.2)

Number of lesions, mean ± SD 0.8 ± 2.1 1.0 ± 2.7 1.0 ± 2.3 T2 lesion volume, mm³

Mean ± SD 14287.6 ± 13104.8 14828.0 ± 16266.8 17202.1 ± 17467.7

EDSS=Expanded Disability Status Scale; SD=standard deviation

*Most commonly: intramuscular interferon beta-1 a (Avonex, 1 1.2%), subcutaneous interferon beta-1 b (Betaseron, 10.6%), subcutaneous interferon beta-1 a (Rebif, 9.4%) and subcutaneous glatiramer acetate (Copaxone, 6.5%) Table 2. Clinical Efficacy Outcomes at 96 Weeks (Intent-to-Treat Population).

Cladribine Cladribine

Placebo 3.5 mg/kg 5.25 mg/kg

End Point (N=437) (N=433) (N=456)

Relapse rate - primary end point

Annualized relapse rate (95%

0.33 (0.29, 0.38) 0.14 (0.12, 0.17) 0.15 (0.12, 0.17) confidence interval)

Relative reduction in annualized

relapse rate for cladribine versus 57.6% 54.5% placebo

P-value^† <0.001 <0.001

Relapse-free rate, N (%) 266 (60.9%) 345 (79.7%) 360 (78.9%)

Odds ratio for cladribine vs placebo,

point estimate (95% confidence 2.53 (1.87, 3.43) 2.43 (1.81 , 3.27) interval) *

P-value^§ <0.001 <0.001

Patients requiring rescue therapy, N (%) 27 (6.2) 1 1 (2.5) 9 (2.0) Odds ratio for cladribine vs placebo,

0.40 (0.19 ,

point estimate (95% confidence 0.31 (0.14, 0.66)

0.81 )

interval)*

P-value^§ 0.01 1 0.003

Time to first relapse, 15^th percentile in

141 (4.6) 408 (13.4) 406 (13.3) days (months)¹¹

Hazard ratio for cladribine vs

placebo, point estimate (95% 0.44 (0.34, 0.58) 0.46 (0.36, 0.60) confidence interval)¹

P-value¹ <0.001 <0.001

Time to 3-month sustained change in

EDSS score, 10^th percentile in days 330 (10.8) 414 (13.6) 414 (13.6) (months)¹¹

Hazard ratio for cladribine vs

placebo, point estimate (95% 0.67 (0.48, 0.93) 0.69 (0.49, 0.96) confidence interval)¹

P-value¹ 0.018 0.026

Patients without a 3-month sustained

347 (79.4) 371 (85.7) 387 (84.9) change in EDSS score, N (%)

Odds ratio for cladribine vs placebo,

point estimate (95% confidence 1.55 (1.09, 2.22) 1.46 (1.03, 2.07) interval) *

P-value^§ 0.016 0.032

EDSS=Expanded Disability Status Scale; SD=standard deviation

Calculated as the ratio of the difference in annualized relapse rate (placebo - cladribine) relative to the annualized relapse rate in the placebo group ^TP-value based on Wald Chi-square test from analysis of number of relapses using a Poisson regression model with fixed effects for treatment and region, and using log time on study as an offset variable *Odds ratio and associated 95% confidence intervals were estimated using a logistic regression model with fixed effects for treatment group and region

^§P-value based on Wald Chi-square test from analysis of endpoint using a logistic regression model with fixed effects for treatment group and region

^The 10^th and 15^th percentile values are estimated from the Kaplan-Meier survival curve

The hazard ratio, 95% confidence intervals and P-values were estimated using Cox proportional hazards model with fixed effects for treatment group and region

Hi) SAFETY

Overall, the frequency of reported treatment-emergent adverse events was comparable across treatment groups, with some exceptions related to the mechanism of action of cladribine (Table 3). Lymphopenia was reported more frequently with cladribine tablets 3.5 or 5.25 mg/kg than placebo treatment, and mostly graded as mild or moderate (Table 3). Severe neutropenia was reported in three patients (1 in the 3.5 mg/kg and two in the 5.25 mg/kg group), with one of these patients in the 5.25 mg/kg group experiencing severe thrombocytopenia and pancytopenia (see appendix). There were no cases of severe anemia. Infections/infestations were reported in 47.7% and 48.9% versus 42.5% patients in the 3.5 and 5.25 mg/kg versus placebo groups, with most of these events graded as mild or moderate by investigators (99.6% and 98.6% versus 99% respectively). Adverse events led to treatment discontinuation for only 3.5%, 7.9% and 2.1 % patients in the 3.5, 5.25 mg/kg and placebo groups, with 0.5% and 2.0% versus 0% discontinuing due to lymphopenia, respectively. Groups were comparable for other events leading to treatment discontinuation.

The incidence of serious adverse events in cladribine 3.5, 5.25 mg/kg and placebo groups were 8.4%, 9.0% and 6.4% patients, respectively. Infections/infestations were reported as serious adverse events in 2.3%, 2.9% and 1.6% patients in the 3.5, 5.25 mg/kg and placebo groups, respectively. Herpes zoster was reported as a serious adverse event for 1 [0.2%] patient in the 3.5 mg/kg group, and for 2 [0.4%] patients in the 5.25 mg/kg group. However, all cases of herpes zoster were self-limiting and dermatomal. Neoplasms, benign/malignant/unspecified were reported as serious adverse events in 1 .4%, 0.9% and 0% patients in the 3.5, 5.25 mg/kg and placebo groups, respectively. There were three isolated cases of single malignancies across different organ systems: a malignant melanoma, a pancreatic and an ovarian carcinoma (in the 3.5 mg/kg group). A precancerous cervical in situ case (stage 0) was also reported in the 5.25 mg/kg group. Time from last treatment course to diagnosis in these patients was: 2, 6, unknown and 7 months, respectively. Relevant medical history included an HPV16+ test 3 years prior to diagnosis for the cervical in situ case. In addition, one case of choriocarcinoma was diagnosed approximately 9 months post-study completion in the 5.25 mg/kg group. The standardized incidence ratio (SIR) for malignancies observed in cladribine-treated patients during study compared with the expected incidence of malignancies in the general population (matched for country, gender and age) was calculated to be 0.99 (95% confidence interval: 0.25, 2.70).

There were 4 deaths during the study and 2 deaths occurred after patients were withdrawn from study (withdrawn due to adverse events); the 6 deaths were equally distributed across the 3 treatment groups.

Table 3. Treatment-Emergent Adverse Events and Investigator-Assessed Severity over 96

Weeks in Each Treatment Group (Safety Population).

Cladribine Cladribine Overall

Placebo 3.5 mg/kg 5.25 mg/kg Cladribine

Treatment-Emergent Adverse

Event (N=435) (N=430) (N=454) (N=884)

Any treatment-emergent

319 (73.3) 347 (80.7) 381 (83.9) 728 (82.4) adverse event, N patients (%)

Most common events,* N

patients (%)

Headache 75 (17.2) 104 (24.2) 94 (20.7) 198 (22.4)

Lymphopenia 8 (1.8) 93 (21.6) 143 (31 .5) 236 (26.7)

Nasopharyngitis 56 (12.9) 62 (14.4) 58 (12.8) 120 (13.6)

Upper respiratory tract

42 (9.7) 54 (12.6) 52 (1 1.5) 106 (12.0) infection

Nausea 39 (9.0) 43 (10.0) 50 (1 1.0) 93 (10.5)

Most common events,* N

mild/moderate : severe

Headache 186 : 3 258 : 6 260 : 5 518 : 1 1

Lymphopenia 1 1 : 0 1 18 : 5 180 : 15 298 : 20

Nasopharyngitis 95 : 0 107 : 0 91 : 0 198 : 0

Upper respiratory tract

80 : 0 1 18 : 0 99 : 1 217 : 1 infection

Nausea 48 : 1 73 : 1 68 : 1 141 : 2

*Preferred terms, reported by >10% patients in any group CONCLUSIONS OF EXAMPLE 1

Results from this placebo-controlled, phase-Ill trial of an oral therapy in RRMS, convincingly demonstrated that annual short-course dosing with cladribine tablets provides rapid and sustained treatment benefits for patients with RRMS over 96 weeks. Treatment with cladribine tablets resulted in greater than 50% reductions in clinical relapse rates, reductions in the risk of disability progression by more than 30%, and suppression of active inflammatory lesions visualized by MRI by up to 88% over the study duration.

The reductions in annualized relapse rate observed in this study are clinically relevant in comparison to the levels of clinical efficacy achieved by currently approved first line agents for RRMS. Although direct comparisons between studies are known to be problematic, it is generally accepted that interferon- and glatiramer-based first-line therapies reduce the clinical attack rate over 2 years by 18-34%. ⁷ These results therefore suggest that cladribine tablets could offer greater benefits to RRMS patients, with the added advantage of short treatment courses and a convenient mode of administration.

The overall incidence of adverse events with cladribine tablets in this study was comparable to placebo with some exceptions related to the mechanism of action of cladribine. The most commonly reported adverse event was lymphopenia, with the vast majority of events considered mild to moderate. The overall incidence of infections was comparable across treatment groups, suggesting that first-line immune defense mechanisms remained adequate, perhaps due to a sparing of immune cells such as neutrophils. Activation of latent herpes zoster occurred in a small number of cladribine treated patients; notably all herpes zoster cases were dermatomal and uncomplicated. In addition, the incidence of serious adverse events within the study was relatively low and similar across treatment groups. Malignancies were isolated cases across different organ systems and there are currently too few events to conclude on a difference in malignancy risk between treated and reference populations. Treatment was well tolerated, as shown by approximately 90% of cladribine treated subjects completing treatment, and only 1.2-2% of cladribine treated subjects discontinuing study due to adverse events. This compares very favorably with discontinuation rates recently reported in controlled studies with currently available first-line disease modifying treatments.^18,19

Although the 3.5 and 5.25 mg/kg cumulative cladribine doses were equally efficacious, there was a trend suggesting that 3.5 mg/kg may be better tolerated. Additional analyses and results from the pharmacogenomics study reported in Example 2 below yield additional insights in relation to dose effects. In conclusion, the results of this double blind, randomized, placebo-controlled, study demonstrate that treatment with cladribine tablets for only 8-20 days per year provides rapid, significant and sustained benefits for patients with RRMS on relapse rate, disability progression and MRI disease activity measures. Taken together with the observed safety and tolerability profile, these results suggest that cladribine tablets could represent a significant advance in the therapy of RRMS patients.

EXAMPLE 2 : PHARMACOGENOMICS ANALYSIS

1. INTRODUCTION

Molecular genetics analysis has been undertaken to identify DNA polymorphisms or gene expression profiles associated with certain traits (i.e. response, adverse events) of cladribine used in the treatment of multiple sclerosis as well as potential susceptibility loci for multiple sclerosis. Analyses were performed by comparison of allele frequency of single nucleotide polymorphisms (SNPs) spread throughout the human genome. Methodology used included DNA amplification, genotyping using Affymetrix DNA chip 500K, Affymetrix DNA chip DMET, TaqMan and sequencing for candidate markers, and statistical analysis.

2. MATERIAL AND METHODS

2.1 GENETICS LABORATORY OPERATIONS

2.1.1. SAMPLE MAN A GEMENT

2.1.1.1. Sample handling and storage

For patients who consented to participate in the pharmacogenomics investigation, blood samples for DNA extraction were collected in EDTA tubes and shipped for storage from one hundred and twenty-four different investigator sites for the duration of the clinical trial.

A total of 758 blood samples from 754 patients were received (four patients had two blood samples collected instead of one).

Upon receipt, blood samples were double coded using a specific PGx identification code (PGx ID) and stored at -80°C until DNA extraction. The key linking the patient identification code and the PGx ID was maintained in a restricted location. All subsequent analysis performed with the PGx samples were carried out and tracked using the PGx ID. DNA was extracted from 758 blood samples using a Qiagen extraction kit (QiaAmp DNA Blood Maxi Kit).

2.1.1.2. Sample preparation

Measures of samples absorbance at wavelengths of 260 nm and 280 nm using a spectrophotometer and electrophoresis of DNA samples on agarose gels were performed to estimate the quality and quantity of DNA after extraction from blood.

Two Batches of samples were prepared according to different laboratory analysis:

A first set of 748 DNA samples (corresponding to 747 patients, one of whom had 2 DNA samples and 2 PGx Ids, 00-M-40984 & 00-M-40997). An aliquot of 3 g for each DNA sample was distributed into 9 micro-plates (96 wells) noted from 50-PL10 to 50-PL18, each micro-plate containing 84 DNA samples.

A second set of 126 DNA samples undergoing PK analyses (corresponding to 125 patients, one of whom had 2 DNA samples and 2 PGx Ids, 00-M-41413_1 & 00-M-41413_2) was created for analysis of ADME genes using the Affymetrix DMET chip ("ADME subset").

2.1.2. AFFYMETRIX WHOLE GENOME SCAN

The genotyping was performed to screen the whole genome (process called Whole Genome Scan). There was no hypothesis concerning any of the variants (Single Nucleotide Polymorphisms or SNPs). The SNPs were randomly distributed in all the chromosomes and were used as tagging markers of the corresponding genomic area. Affymetrix Genome Wide SNP 5.0 Assays were used to perform the Whole Genome Scan. The Affymetrix technology is based on a DNA chip allowing the genotyping of approximately 500,000 SNPs per patient. Seven hundred and forty-eight samples and nine positive controls (commercial DNA samples) have been processed. .

2.1.2.1. DNA sample amplification, fragmentation and labeling

For each plate, genomic DNA samples were digested with Nsp I and Sty I restriction endonucleases, ligated with specific adaptors (Nsp I or Sty I). Ligation products were amplified by Polymerase Chain Reaction (PCR), in triplicate for Sty I digestion products and in quadruplicate for Nsp I digestion products, in order to optimize the following steps. All the PCR products were pooled, purified, quantified, fragmented and labelled. 2.1.2.2. Hybridization and raw data analysis with Genotyping Console algorithm

The 757 labelled products (748 samples and 9 reference genomic DNA controls) were hybridized onto the Affymetrix Genome Wide SNP 5.0 arrays. Five different lots of chips have been used for the study. .

The 757 hybridization cocktails, corresponding to each sample have been hybridized in several batches of 32 chips or less.

After hybridization and staining, the Affymetrix GeneChips (or arrays) have been scanned to create image data (DAT) files. Then, the AGCC Software automatically aligned a grid on the DAT files and computed the Cell Intensity data (CEL) file. Finally, the .CEL. data is read by the Genotyping Console software that generated Probe Analysis (CHP) data.

100% of samples of the study passed the acceptance criteria of all the quality controls of the process.

2.1.3. HLA-DRB1*15 TYPING

Genetic variation detection in the major histocompatiblity complex genes on chromosome 6p21.3, was carried out for HLA-DRB1*15 extended haplotypes.

The genotyping was performed using Olerup SSP™ Kit, PCR-based method (Olerup SSP™ DRB1*15 high resolution, part number 101.125-24, version 35E).

Twenty-four PCR reactions were performed to detect all variable combinations of the HLA- DRB1*15 allele. The HLA Olerup kits contained the primer sets pre-aliquoted into PCR tubes, the PCR Master Mix (nucleotides, buffer, glycerol and cresol red included) and PCR lids. The PCR reaction mix was prepared by adding the Master Mix, the water, the basic Taq DNA polymerase (from Perkin Elmer) and the sample genomic DNA, calculated for twenty four reactions. The PCR reaction mix was distributed into each PCR tube of the kit prior PCR cycles.

The PCR products were separated by agarose gel electrophoresis (Invitrogene pre-cast E-Gel, 4%).

The allele determination was interpreted using a combination table (supplied with the kit). Seven hundred and forty eight (748) samples were HLA-DRB1 *15 genotyped in parallel by one operator.

The interpretation of the electrophoresis was carried out by one operator and one supervisor. For each sample, the internal control band of each PCR was checked and the specific PCR product was observed at the expected size . All samples passed acceptance criteria of quality controls. . 2.1.4. TaqMan SNP GENOTYPING

TaqMan SNP Genotyping was performed to detect selected markers..

In a TaqMan® SNP Genotyping assay, two locus-specific PCR primers surrounding the SNP are used to amplify a ~ 100 bp fragment. Two allele-specific probes are then hybridized to their specific SNP sequence. Each probe was labeled at its 5^' extremity with either a fluorescent reporter dye (FAM), either the VIC reporter dye. Each probe also has a non-fluorescent quencher dye, MGB, at the 3^' end. In each PCR cycle, if the target sequence of the allele- specific probe is amplified, the probe will hybridize to the DNA during the annealing step and extend. When the DNA polymerase comes into contact with this hybridized probe, the reporter dye of the probe is cleaved from the probe leaving the quencher dye behind. In each cycle of the PCR, cleavage of the reporter dyes from one or both of the allele-specific probes causes an exponential increase in the fluorescent intensity. At PCR completion, the total fluorescence of each sample is read on the ABI 9700 (384-well format). If fluorescence is observed from only one probe, the sample is homozygous for this allele. If fluorescence is observed for both allele- specific probes, the sample is heterozygous for both alleles. If the probe does not hybridize, the fluorescence of the dye is "quenched" or reduced by the quencher dye, and thus minimal fluorescence is observed, indicating a failed genotype.

Seven hundred and forty eight samples were genotyped with three SNPs distributed on three candidate genes by one operator. The details of SNP Id are described in Table 4. Specifically, seven hundred and forty eight genomic DNA samples and twenty "No Template Controls" (NTCs) were distributed in eight quadrants of two 384-wells plates. Three 384-well plates, containing the same genomic DNA sample distribution, were prepared simultaneously for the three TaqMan SNP assays.

One hundred and twenty six PK samples were genotyped with twenty-two SNPs distributed on nine candidate genes by one operator .

Specifically, one hundred and twenty six genomic DNA samples and six NTCs were distributed in two quadrants of one 384-wells plate. Twenty-two 384-well plates, containing the same genomic DNA sample distribution, were prepared simultaneously for the twenty-two TaqMan SNP assays. For each TaqMan SNP assay, the reaction mix containing the PCR master mix and the SNP assay (primers and probes) was added into each reaction well and PCR amplification was run using MJ Research PTC-225 tetrad DNA engine.

After PCR cycles, the plate was read using ABI 7900HT instrument then analyzed with the End Point Allelic Discrimination method. Batches from one to three TaqMan SNP assays were performed per day.

2.1.5. SNP genotyping by sequencing

For two selected SNPs that were un-processable with TaqMan SNP genotyping assays (i.e. unsuccessful probe design), the sequencing method was used for detection of the variant alleles.

The sequencing technology used was based on primer extension with unlabelled primer and dye terminators. After extension, separation of the reaction product was performed by capillary electrophoresis.

Short genomic DNA fragment (about 1 kb), containing the SNP locus, was amplified by PCR. The design of the oligonucleotides for the PCR primers was carried using the Oligo6 software. The two strands of the purified PCR product was sequenced using Applied Biosystems PRISM v3.1 Kit and both primers used for PCR amplification. The purified reaction product was separated, scanned and analyzed using ABI 3730 XL instrument and ABI Sequencing Analysis software. The sequences were then compiled using Sequencher 4.7 software (Gene CodeS Corporation) to determine the SNP genotype.

Two SNPs were performed by one operator.

The two close or neighbouring SNPs in one gene were genotyped using the same PCR fragment and the same sequencing reaction.

One hundred and twenty-six genomic DNA samples were amplified by PCR. The PCR products were controlled on agarose gel electrophoresis to check the single amplification and the specific band size.

One hundred and twenty-six samples were sequenced in forward and reverse strands to detect the polymorphisms. Table 4: Details of TaqMan SNP Id screened on all samples

Hugo Gene SNP ID Allele (NCBI) TaqMan assay type

IFNAR2 rs2229207 (=rs4986956) C/T Custom Taqman(R) SNP Genotyp

Assay

rs2071543 A/C TaqMan(R) Genotyping Assays

PSMB8

rs469304 A/G TaqMan(R) Genotyping Assays

MX1

2.2 STATISTICS

2.2.1. Descriptive analyses

Descriptive statistics are provided for the whole BM/PGx population and detailed by treatment arms.

^■ For efficacy candidate markers: alleles, genotype and allele counts, Minor Allele Frequency (MAF), chromosomic position, Hardy-Weinberg Disequilibrium p-value (using the SAS ALLELE procedure).

^■ For whole genome scan markers: number of Single Nucleotide Polymorphisms (SNPs), MAF and MGF distributions.

^■ For ADME genotype data:

- Per gene: gene name, number of markers investigated, numbers of polymorphic markers among them)

- Per marker (for every marker within gene): marker name, number of genotyped individuals, number of observed alleles, and the details of allele and genotype frequencies for each observed allele and genotype respectively.

Candidate efficacy polymorphisms are all analyzed. For whole-genome scan and ADME candidates, rare polymorphisms (MAF smaller than 5%) and polymorphisms with insufficient genotyping quality (missing data rate equal or greater than 5%) are not analyzed.

2.2.2. Stratification analyses

2.2.2.1. Primary efficacy endpoint

The imputed number of relapse during 96 weeks on treatment (RELQSD96 variable) is analyzed on the whole BM/PGx population by means of a Poisson regression model including effects for treatment and region and the log of time on study as an offset variable (using the SAS GENMOD procedure). The analysis is repeated for two contrasts ("High dose vs. placebo" and "Low dose vs. placebo") for every of the 4 sub-populations defined per marker, namely for a A/B polymorphism: AA, AA+AB ("A"), AB+BB ("B") and BB (see SAP). For every test, the following were reported :

The Bonferroni corrected Chi-square p-value of the Wald test.

The frequency of the marker.

The effect size E = the diminution of the relapse rate in treated arm compared to placebo.

The relative risk RR and its 95% confidence interval (RR = 1 - E)

Significant markers are then selected as follows:

Number of patients per sub-population equal or greater than 30.

Bonferroni-corrected p-value smaller than 5%.

RR is greater than 1.

Number of patients per sub-population is equal or greater than the minimal number required to ensure π = 90% power at a combined type-l error threshold of a' = 4% (which means a individual type I error rate for each of the two tests of a = 1 - l -OC ' ~ 2.0202 ):

where μ₀ = 0.527 observed relapses on average in the placebo BM/PGx subgroup, σ₀ = 0.873 common standard deviation in all subgroups (it corresponds to the value observed on the placebo BM/PGx subgroup), and Z_x is the Gaussian distribution percentile at the precision x (Zo.9 = 1.28155 and Z_{0 0202} = 2.32254).

2.2.2.1. Secondary efficacy endpoints and safety endpoints

The imputed following status (phenotypes) are used for secondary efficacy endpoint analyses:

Relapse-free status at 96 weeks, QRID96CD.

Progression-free status at 96 weeks, NAPD96CD.

MRI-activity free status at 96 weeks, i.e. Gd-enhancing T1 lesion free status (LRNT1 GCD) and T2 lesion free status (LRT2ACCD) and Combined Unique lesion free status (LRCULECD).

Relapse-free and progression-free status at 96 weeks (QRID96CD and NAPD96CD). The clinical trial team provided the following list of AE, for which a significant superior frequency was observed in treated groups as compared to Placebo, to be considered for safety stratification analyses:

Presence of at least one "Lymphopenia" adverse event.

Presence of at least one "Neutropenia" adverse event.

Presence of at least one "Herpes zoster" adverse event. For each marker and each 0/1 status described above, three 2x3 (2 status, 3 genotypes: AA, AB, BB) contingency tables are derived in the three treatment arms. The association of alleles A vs. B, AA vs. AB+BB and AA+AB vs. BB are measured by the mean of an exact Fisher test (using the SAS FREQ procedure). For every test, we report:

The exact Fisher p-value.

The Bonferroni corrected p-value.

The Benjamini-Hochberg estimated False Discovery Rate (FDR)

The frequency of the marker.

The proportion of observed phenotypes in the stratified sub-population.

Significant markers are first selected as follows:

P-value smaller than 5% in the High dose treatment arm.

P-value smaller than 5% in the Low dose treatment arm.

P-value greater than 5% in the Placebo treatment arm.

Marker passes the following multiple-testing correction:

o Candidate efficacy markers: no multiple-testing correction,

o Whole genome scan markers: Bonferroni-corrected p-value in the Low dose treatment arm smaller than 5% or estimated FDR in the Low dose treatment arm smaller than 20%; Exception for safety endpoint: top-ranked markers are reported even if they do not meet this requirement.

o ADME candidate markers: Per-gene Bonferroni-corrected p-value in the treated arms (Low Dose + High Dose) smaller than 5% (the p-value of a SNP in a given gene is adjusted by the number of tested SNPs in that gene).

Selected stratified sub-populations are in addition described in terms of Time to first relapse (TFQR variable) in Kaplan-Meier plots.

3. RESULTS

3.1 DESCRIPTION OF DATA TO ANALYZE

3.1.1. Efficacy candidate markers

The four efficacy candidate markers tested in this study are three SNPs previously identified as markers of response to Rebif: rs2229207 (in gene IFNAR2), rs2071543 (in gene PSMB8), rs469304 (in gene MX1 ) (see WO 2010/010057); and the presence/absence of the HLA- DRB1*15 allele (susceptibility marker of Multiple Sclerosis). Descriptive statistics are summarized in Table 5. 3.1.2. Whole genome scan markers

Seven hundred thirty five (735) samples have been genotyped with Affymetrix 5.0 chip technology. Out of the 500,578 SNPs present on the chip, 91 ,958 have been filtered out because of low frequency (MAF<5%, including 5,447 non-polymorphic SNPs) and 75,593 have been filtered out because of missing data rate greater than 5%, resulting in 333,027 SNPs kept for analysis.

3.1.3. ADME candidate markers

A subset of 124 samples from the three treatment groups (44 high-dose, 42 low-dose, 38 placebo) were genotyped for a selection of 1 ,145 ADME candidate markers, using three different technologies: Affymetrix® DMET™ v2.0 array (1 , 120 markers), TaqMan® (22 markers), Affymetrix® Genome-Wide SNP array 5.0 (3 markers) and sequencing (2 markers). Two markers were investigated using 2 technologies.

For each treatment group, about 8% of markers (89, 91 and 98 markers in high-dose, low-dose and placebo groups, respectively) were filtered out as exceeding the accepted rate of missing data (5%).

Among remaining markers, those showing a minor allele frequency (MAF) below 5% amounted to about 69% in each group (738 markers in high-dose, 725 in low-dose and 712 in placebo groups, including respectively 622, 593, and 620 markers not showing any polymorphism). Filtering out these markers resulted in respectively 318, 329, and 335 markers qualified for further association analysis.

Table 5 - Descriptive statistics of efficacy candidate markers.

(*): number differences are due to missing genotype data.

3.2. DATA ANALYSIS

3.2.1. Primary end point: relapse rate

3.2.1.1. Whole BM/PGx population

The distribution per treatment group of the imputed number of relapses between Study Day 1 and Week 96 is plotted in Figure 3 The average numbers of relapses are 0.21 , 0.28, and 0.55 in the High Dose (n=256), Low Dose (n=247) and Placebo (n=239) subgroups respectively. Compared with Placebo, the effect sizes (reduction of the number of relapses over 96 weeks of treatment) are 61 % and 49% in High Dose and Low Dose subgroups respectively. When High and Low Dose groups are merged, the average number of relapses is 0.25 (effect size = 55%).

3.2.1.2. Stratification analyses - Whole genome scan

This analysis has been run on 333,027 SNPs genotyped on 735 patients of the BM/PGx population with Affymetrix 5.0 technology. Filtering criteria described in methods (Bonferroni corrected p-value <5%, power > 90%, etc.) have been applied and resulted in the selection of: - 248,865 markers (out of 4 x 333,027 = 1 ,332, 108) in the "High dose vs. Placebo" contrast.

1 , 138 markers in the "Low dose vs. Placebo" contrast. The high proportion of selected markers in the "High dose vs. Placebo" comparison can be explained by the important difference observed in terms of number of relapses between those two treatment groups in the whole BM/PGx population (without any stratification: average relapse rate 0.21 in High Dose group vs. 0.55 in Placebo). As a consequence, any large enough couple of sampled sub-populations from High Dose and Placebo subgroups is likely to still display a significant difference in the number of relapses. Therefore, we judged the "High Dose vs. Placebo" contrast as only slightly informative in terms of stratification analyses and we decided to focus on 422 markers commonly selected in the "Low dose vs. Placebo" and "High dose vs. Placebo" contrasts. For those markers, the effect size (reduction of the number of relapse after 96 weeks of treatment compared to Placebo) is negatively correlated with marker frequency.

3.2.1.3. Stratification analyses - Candidate markers

The same statistical approach has been applied on the following 4 candidate markers:

- Three markers (rs2229207 in IFNAR2 gene, rs2071543 in PSMB8 gene, and rs469304 in MX1 gene) previously indentified in BM/PGx studies as efficacy stratification markers of response to Rebif. (see WO 2010/010057).

Presence of the HLA-DRB1*15 allele, associated with susceptibility to Multiple Sclerosis.

Stratification candidate markers with an uncorrected p-value smaller than 5% are listed in Table 6. Effect sizes in stratified population based on those significant markers vary in the range of 48% - 57% on average, which is comparable to effect sizes observed on the whole BM/PGx population (55% in merged treated groups). Therefore we conclude that none of the 4 candidate markers is associated with Cladribine efficacy in terms of reduction of number of relapses. As examples, results obtained with HLA-DRB1*15 presence and absence are displayed in Figure 4 and Figure 5.

3.2.2. Secondary endpoints

3.2.2.1. Null relapse

In the whole BM/PGx population, the relapse-free proportions are 82%, 81 % and 62% in respectively High Dose, Low Dose and Placebo subgroups. 3.2.2.1.1. Whole genome scan

Before multiple-testing correction, 28,504 markers have been found associated with relapse- free status at 96 weeks in merged treated subgroups (uncorrected exact Fisher p-value < 5%) that are not associated in Placebo subgroup (uncorrected exact Fisher p-value > 5%). Two markers withstand the Bonferroni correction at 5%: rs5971598 (corrected p-value = 0.014) and rs17338758 (corrected p-value = 0.030). No additional marker is selected with FDR lower than 20%. Visual inspection of the QQ plot (Figure 6) shown that the third ranked marker, rs6628643, might be of interest as well (uncorrected p-value = 3.7e-6, estimated FDR for the 3 SNP selection < 40%).

Those 3 SNPs are located in a 40 kb region of the DMD gene on chromosome X. The rs5971598 is located in intron 49 of DMD and rs17338758 and rs6628643 in intron 50. Summary statistics are listed in Table 7. DMD is not located in the pseudo-autosomal region (PAR) of the chromosome X. Therefore males carry only one allele for those markers. We note hereinafter AA and BB the two possible alleles for a A B SNP on males' X chromosome, which allows the comparison with AA and BB homozygote genotypes on females' X chromosome. The three SNPs are in Linkage Disequilibrium: r² \s 0.41 between rs5971598 and rs17338758, 0.42 between rs17338758 and rs6628643, and weaker (0.13) between rs5971598 and rs6628643.

The C-C-T haplotype of the rs5971598 - rs17338758 - rs6628643 SNP triplet is associated with relapse-free status (chi-square p-value = 3.06E-06, see Table 8). The Odds Ratio (OR) of the C-C-T haplotype is 2.15 [ 1.36 - 3.42 ], which is less strong than the signal obtained with rs5971598 alone: OR for the CC genotype = 3.68 [ 2.08 - 6.52 ], OR for the presence of the C allele (CC or CT genotypes) = 2.56 [ 1.62 - 4.05 ]. Therefore we focus the detailed analysis on the rs5971598 SNP below.

Association of rs5971598 with relapse status is illustrated in Figure 7. The relapse-free status proportions are 59%, 78%, 87% in the rs5971598-CC, -CT and -TT treated subpopulations respectively, compared to 81 % in the whole treated population and 62% in the Placebo arm. The stratification effect is comparable in High and Low Dose subgroups: 60% - 79% - 87% and 58% - 78% - 87% respectively Figure 8 ). In the whole treated BM/PGx population, males' relapse-free status is smaller than females' (75% vs. 84%). This trend impacts the stratification effect of rs5971598 by sex: the relapse-free status proportions are 70%, 78%, 90% in the rs5971598-CC, -CT and -TT treated subpopulations of women, and 53% and 82% in the rs5971598-CC and -TT treated subpopulations of men (Figure 9). Because rs5971598 is located on the non-pseudo-autosomal region of the X chromosome, there is no CT heterozygote in males. Extreme stratification effects are observed in CC males (only 53% relapse-free) and in TT females (90% relapse-free). The relapse-free differences in stratified subpopulations are not due to a cofounding effect of region (Figure 10). Figure 1 1 and Figure 12 display additional graphical representations of the association of rs5971598 with the relapse- free status in treated subgroup.

For this interesting marker, the stratification effect in terms of reduction of the number of relapses (primary endpoint) was also investigated. In the rs5971598-CC subpopulation, the average numbers of relapses are 0.53 and 0.58 in the High Dose and Low Dose subgroups respectively, which corresponds to -23% and -16% decrease compared to Placebo (0.69 relapse on average). Hence, the rs5971598-CC subpopulation responds similarly to the whole (i.e. not stratified) Placebo subgroup (on average 0.55 relapse). In contrast, patients with the TT genotype respond better: on average 0.14 and 0.16 relapse in the High Dose and Low Dose subgroups respectively, which corresponds to -74% and -70% decrease compared to Placebo. Investigated separately, the heterozygote-stratified subpopulation shows an intermediary response (Figure 13). Summary statistics are listed in Table 9. This stratification effect is also illustrated in terms of time to first relapse in Figure 14, Figure 15 and Figure 16.

From the results obtained with the different rs5971598 subpopulations, the capacity of an individual subject to respond to cladribine treatment can also be determined. This quantitative data is presented in Table 14 below, showing the likelihood that an individual Clad ri bine-treated patient of a given rs5971598 genotype, will have zero or 1 , 2, 3, 4, 5 or 6 relapses compared to a patient of the same genotype who is receiving placebo.

3.2.2.1.2. Candidate markers

The p-values of association of candidate markers with relapse-free status are listed in Table 10. No marker has a p-value smaller than 5%.

3.2.2.2. Disability progression

Disability progression is defined as a sustained change in EDSS score of at least one point, or at least 1 .5 point is baseline EDSS score was 0, observed over a period of at least three months. In the whole BM/PGx population, the disability progression-free proportions are 87%, 87% and 81 % in respectively High Dose, Low Dose and Placebo subgroups. 3.2.2.2.1 Whole genome scan

Before multiple-testing correction, 29,134 markers have been found associated with disability progression-free status at 96 weeks in merged treated subgroups (uncorrected exact Fisher p- value < 5%) that are not associated in Placebo subgroup (uncorrected exact Fisher p-value > 5%). rs5971598, the SNP associated with relapse-free status in DMD gene (see above), is moderately associated with progression-free status: p-value = 0.02 (Figure 17).

3.2.2.3. MRI activity

MRI activity is defined as the apparition of a new Gd-enhancing T1 lesion, a T2 lesion, or a Combined Unique lesion. In the whole BM/PGx population, the MRI activity-free proportions are 61 %, 58% and 23% in respectively High Dose, Low Dose and Placebo subgroups.

3.2.2.3.1 Whole genome scan

Before multiple-testing correction, 32,063 markers have been found associated with MRI activity-free status at Week 96 in merged treated subgroups (uncorrected exact Fisher p-value < 5%) that are not associated in Placebo subgroup (uncorrected exact Fisher p-value > 5%). None withstands the Bonferroni correction at 5%. rs5971598, the SNP associated with relapse-free status in DMD gene (see above), is not statistically associated with MRI activity-free status: p-value = 0.12. However, patients without MRI activity are less frequent in the rs5971598-CC treated subpopulation (51 %) than in the whole BM/PGx treated population (60%) or rs5971598-CT and rs5971598-TT subpopulations (61 %) (see Figure 18).

3.2.2.4. No disability progression and no relapse

3.2.2.4.1. Whole genome scan

In the whole BM/PGx population, the proportions of patients who have shown both no disability progression (as defined in 0) and no relapse are 75%, 73% and 54% in respectively High Dose, Low Dose and Placebo subgroups. Before multiple-testing correction, 29,443 markers have been found associated with no disability and no relapse at 96 weeks in merged treated subgroups (uncorrected exact Fisher p-value < 5%) that are not associated in Placebo subgroup (uncorrected exact Fisher p-value > 5%). None withstands the Bonferroni correction at 5%. Thirteen SNPs are selected below a 20% FDR threshold.

Table 1 1 :

- Of these, three SNPs, rs593531 1 , rs5935310 and rs7892480, are located in intron 1 of FRMPD4 (FERM and PDZ domain containing 4) on chromosome X (Table 1 1 ). They are located in a 1.1 kb region and are in Linkage Disequilibrium: /^ is 0.96 between rs593531 1 and rs7892480, 0.79 between rs593531 1 and rs5935310, and 0.76 between rs5935310 and rs7892480. Association of rs593531 1 with absence of progression and relapse is illustrated in Figure 19.

- Further, amongst these thirteen SNPs, two SNPs, rs17338758 and rs5971598 (Figure 20), are the two SNPs in DMD intron previously found associated with the null relapse only (see 0 above) (Table 1 1 ). Those SNPs withstand the Bonferroni correction for association with null relapse only but not with combined null relapse and no progression. They are located also on the chromosome X.

Table 6 - Stratification candidate markers associated with significant reduction in number of relapses in treated groups vs. Placebo.

Marker p-value Frequency effect Effect lowCI95% Effect highCI95%

BM/PGx

pop

rs2229207 TT 1.98E-06 77% 55% 37% 67%

rs2071543 G 2.61 E-06 99% 51 % 34% 63%

rs2229207 T 7.96E-06 98% 49% 31 % 62%

rs469304 G 9.15E-06 70% 57% 38% 71 %

rs2071543 GG 3.25E-05 81 % 48% 29% 62%

rs469304 A 0.000497 76% 45% 23% 60%

drb1_15 Y 0.000636 46% 52% 27% 68%

drb1_15 N 0.001498 54% 48% 22% 66% Table 7 - Markers associated with relapse-free status in treated subgroups.

SNP Ge Genotype Genotype p-value FDR Position Freq. Relap no counts in counts in (Bonfer- on chr. in se ty patients relapse- roni) X treate -free pes with free d prop. relapses patients group

s

rs5971598 TT 38 255 4.6E-08 1.4% 31 ,753,712 60% 87%

CT 31 1 1 1 (1.4%) 27% 78% cc 25 36 13% 59% rs17338758 TT 59 331 9.5E-08 1.5% 31 ,734,584 79% 85%

CT 20 57 (3.0%) 15% 74% cc 15 14 6% 48% rs6628643 cc 47 278 3.7E-06 38.6% 31 ,715,948 65% 86%

CT 27 94 (100.0%) 24% 78%

TT 20 30 1 1 % 60%

Table 8 - Association of rs5971598-rs17338758-rs6628643 haplotypes with relapse-free status in treated subgroups.

Table 9 - Summary of rs5971598 stratification effect in terms of primary endpoint. rs5971598 Reduction Reduction Relapse- Relapse- Relapse- Marker

(women/men) #relapses #relapses free free free frequency

Low dose High dose status status status

Low dose High dose Placebo

Whole -48% -59% 80% 82% 62% N.A.

BM/PGx

population

CC/C -16% -23% 58% 60% 58% 13%

CT/- -15% -43% 78% 79% 72% 27%

TT T -70% -74% 87% 87% 60% 60% Table 10 - Association of candidate markers with relapse-free status.

Marker Association p-value

Polymorphism Genotype High Dose Low Dose Placebo rs2071543 TT 1 0.58 0.53

GG 0.69 0.22 0.48 rs2229207 TT 0.72 0.84 0.15

CC 0.64 1 0.56 rs469304 GG 0.58 0.85 0.14

AA 0.21 0.38 0.45

HLA-DRB1*15 Presence 0.42 1 0.35

Absence 0.63 0.87 0.35

Table 11 - Markers associated with progression-free and relapse-free status in treated subgroups.

SNP Ge Genotype Genotype p-value FDR ChromoFreq. MRI no counts in counts in (Bonfer- some in activi ty patients progressio roni) Gene treate ty pes with n and Position d -free progress- relapse-free group prop. sion or patients s

relapse

TT 62 234 X 60% 79%

CT 33 102 7.1 E-07 FRMPD4 27% 76% rs593531 1 CC 33 31 (0.22) 1 1 % 12,287,884 13% 48%

TT 87 303 X 79% 78%

CT 24 53 3.9E-06 DMD 16% 69% rs17338758 CC 17 12 (1.00) 18% 31 ,734,584 6% 41 %

AA 67 252 X 64% 79%

AG 35 86 7.8E-06 FRMPD4 24% 71 % rs5935310 GG 26 30 (1.00) 22% 12,286,979 12% 54%

CC 67 253 X 65% 79%

CT 35 84 7.8E-06 FRMPD4 24% 71 % rs7892480 TT 26 31 (1.00) 20% 12,286,796 1 1 % 54%

TT 60 233 X 59% 80%

CT 40 102 8.4E-06 DMD 29% 72%

Rs5971598 TT 28 33 (1.00) 20% 31 ,753,712 12% 54% 4. CONCLUSIONS OF EXAMPLE 2

The aim of the Genetics and the Gene expression analyses in this trial were to carry out exploratory research to identify efficacy stratification markers, safety stratification markers and markers of the Mode of Action (MoA).

Efficacy

Efficacy to treatment with Cladribine was clinically measured using the relapse rate (absence or number of relapses), the time to first relapse, the EDSS score progression and the MRI activity. In this study, seven hundred thirty five (735) patients (63% of the patients completing treatment) were genotyped using the Affymetrix 500K GeneChip arrays as well as efficacy stratification candidate markers for response to Rebif. A subgroup of 124 patients was genotyped using the Affymetrix DMET2.0 chip.

A statistical analysis was performed in order to compare differences in genetic markers frequencies among subgroups of patients as defined based in the clinical outcomes. Statistical results were classified according to their p-value (<0.05) after correction for multiple testing (Bonferroni and <FDR) and filtering for minor allelic frequency (<5%) and missing data (>5%).

The Chr. X-DMD-SNP-5971598 marker has been identified as associated with both the absence of relapse and a reduced number of relapses. This marker is linked to the gender and the TT (female) or T (male) genotype predicts the good response to the Cladribine treatment. In addition, the most present genotype (TT/T) was associated with the best response, whereas heterozygotes presented an intermediate response and the less present genotype (CC/C) a response similar to patients in the placebo arm, suggesting a dose effect of the genotype (Table 13). Furthermore, this SNP is moderately associated with a reduced disease progression as measured by the EDSS score and a trend could be seen between presence of the TT or T genotype and the absence of MRI activities (Table 12). Therefore, this marker is a good candidate for efficacy stratification. Interestingly, this SNP is part of a C-C-T haplotype (Chr. X- DMD-SNP-5974598, X-DMD-SNP-17338758 and X-DMD-SNP-6628643) which is also found associated with Cladribine efficacy (Table 12). The DMD gene is a huge gene located on chromosome X and under extensive investigation. The neuronal DMD protein is thought to participate to the signal transduction in synapses through anchorage of receptors within the membrane.

In contrast, efficacy candidate markers to Rebif response were not associated with the Cladribine response. Table 12 - Efficacy analysis: p-values observed per marker for each clinical endpoint analyzed., (b) markers withstanding the Bonferronni correction at 5% and presenting a FDR <20%, (c) markers presenting a FDR <20% only n.d. not done, n.s., not significant

Table 13 - Efficacy analysis: frequency of the genotypes per marker.

Major Homozygote Heterozygote Minor Homozygote

Genotype Count Frequency Genotype Count Frequency Genotype Count Frequenc rs5971598 TT 439 60% CT 196 27% CC 97 13% rs593531 1 TT 437 60% CT 204 28% CC 92 13%

Table 14 : Relative likelihood of a cladribine-treated individual having x number of relapses compared to likelihood that a subject of the same genotype receiving placebo would have x relapses, according to rs5971598 genotype

Number of relapses

rs5971598

Treatment genotype 0 1 2 3 4 6

High dose CC 0% +9% -7% -2% 0% 0% at least one C +5% +6% -7% -4% 0% 0%

CT +5% +6% -7% -5% 0% 0% at least one T +21% -11% -7% -3% 0% -1%

TT +27% -17% -7% -2% 0% -1%

Total +19% -9% -7% -3% 0% 0%

Low dose CC + 1% +9% -11% + 1% 0% 0% at least one C +6% -1% -4% -2% + 1% 0%

CT +7% -4% -1% -3% +2% 0% at least one T +21% -14% -5% -2% + 1% -1%

TT +27% -18% -7% -1% 0% -1%

Total +18% -11% -6% -1% 0% 0%

High+Low

CC 0% +9% -9% 0% 0% 0% at least one C +6% +2% -6% -3% + 1% 0%

CT +6% 0% -3% -4% + 1% 0% at least one T +21% -13% -6% -2% 0% -1%

TT +27% -18% -7% -2% 0% -1%

Total +18% -10% -6% -2% 0% 0%

* "High + Low dose" corresponds to combined figures from high and low dose groups.

REFERENCES

1. Chitnis T. The role of CD4 T cells in the pathogenesis of multiple sclerosis. Int Rev

Neurobiol 2007;79:43-72

2. Franciotta D, Salvetti M, Lolli F et al. B cells and multiple sclerosis. Lancet Neurol

2008;7:852-858

3. Kleinschnitz C, Meuth SG, Kieseier BC et al. Immunotherapeutic approaches in MS: update on pathophysiology and emerging agents or strategies 2006. Endocr Metab Immune Disord Drug Targets 2007;7:35-63

4. Cohen BA, Rieckmann P. Emerging oral therapies for multiple sclerosis. Int J Clin Pract 2007;61 : 1922-1930

5. Beutler E. Cladribine (2-chlorodeoxyadenosine). Lancet 1992;340:952-956

6. Guarnaccia JB, Rinder H, Smith B. Preferential depletion of lymphocyte subpopulations by cladribine in a phase III clinical trial in multiple sclerosis. Poster presented at the World Congress on Treatment and Research in Multiple Sclerosis, Montreal, Canada, 17-20 September 2008 (Abstract P55).

7. Leist T, Weissert R. The mechanism of action of cladribine and its implications for oral therapy in multiple sclerosis (Meeting Abstract, Annual Meeting of the Consortium of Multiple Sclerosis Centers, Atlanta, 27-30 May 2009). Int J MS Care 2009;ln press:

8. Rice GP, Filippi M, Comi G. Cladribine and progressive MS: clinical and MRI outcomes of a multicenter controlled trial. Cladribine MRI Study Group. Neurology 2000;54:1 145-1 155

9. Bartosik-Psujek H, Belniak E, Mitosek-Szewczyk K et al. lnterleukin-8 and RANTES levels in patients with relapsing-remitting multiple sclerosis (RR-MS) treated with cladribine. Acta Neurol Scand 2004; 109:390-392

10. Janiec K, Wajgt A, Kondera-Anasz Z. Effect of immunosuppressive cladribine treatment on serum leucocytes system in two-year clinical trial in patients with chronic progressive multiple sclerosis. Med Sci Monit 2001 ;7:93-98

1 1. Kopadze T, Dobert M, Leussink VI et al. Cladribine impedes in vitro migration of

mononuclear cells: a possible implication for treating multiple sclerosis. Eur J Neurol 2009;16:409-412

12. Laugel B, Challier J, Siegfried C et al. Cladribine exerts a modulatory effect on T-cell activation (Meeting Abstract; ECTRIMS 2008) (P80). Mult Scler 2008;14:S52-S52

13. Niezgoda A, Losy J, Mehta PD. Effect of cladribine treatment on beta-2 microglobulin and soluble intercellular adhesion molecule 1 (ICAM-1 ) in patients with multiple sclerosis. Folia Morphol (Warsz ) 2001 ;60:225-228 McDonald Wl, Compston A, Edan G et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001 ;50: 121-127

Fazekas F, Barkhof F, Filippi M et al. The contribution of magnetic resonance imaging to the diagnosis of multiple sclerosis. Neurology 1999;53:448-456

Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 1983;33: 1444-1452

Goodin D. The return of natalizumab: weighing benefit against risk. Lancet Neurol 2006;5:375-377

Mikol DD, Barkhof F, Chang P et al. Comparison of subcutaneous interferon beta-1a with glatiramer acetate in patients with relapsing multiple sclerosis (the REbif vs Glatiramer Acetate in Relapsing MS Disease [REGARD] study): a multicentre, randomised, parallel, open-label trial. Lancet Neurol 2008;7:903-914

O'Connor P, Arnason B, Comi C et al. Interferon beta-1 b 500mcg, interferon beta-1 b 250 meg and glatiramer acetate: primary outcomes of the Betaferon®/Betaseron® Efficacy Yielding Outcomes of a New Dose study. Program and abstracts of the American Academy of Neurology 60th Annual Meeting; April 12-19, 2008; Chicago, Illinois LBS 004 2009;

Curado MP, Edwards B, Shin HR et al. Cancer incidence in five continents. [IX] GLOBOCAN. The GLOBOCAN 2002 database. http://www- dep.iarc.fr/qlobocan/database.htm

Breslow NE, Day NE. Statistical methods in cancer research. Vol II: The design and analyses of cohort studies. No.82

Claims

1. Method for determining the capacity of a subject suffering from Relapsing-Remitting Multiple Sclerosis (RRMS) to respond to treatment with Cladribine, comprising determining the polymorphism genotype of the subject in at least one SNP located on chromosome X.

2. Method according to claim 1 wherein the at least one SNP is located in the Duchenne Muscular Dystrophy (DMD) gene.

3. Method according to Claim 2 wherein the at least one SNP comprises rs5971598.

4. Method according to claim 3 wherein the presence of at least one T allele in the subject's genotype for rs5971598 is predictive of a positive Cladribine response.

5. Method according to claim 4 wherein the subject is female and the presence of two T alleles in the subject's genotype for rs5971598 is predictive of a positive Cladribine response.

6. Method according to any one of claims 1 to 5 wherein the capacity of the subject to respond to treatment with Cladribine is the capacity to respond to orally administered Cladribine.

7. Method according to claim 3 wherein the at least one SNP further comprises rs17338758 and / or rs6628643.

8. Cladribine for use in treating Relapsing-Remitting Multiple Sclerosis (RRMS) in a subject, wherein

a) the subject has undergone an analysis of genotype for rs5971598 resulting in confirmation of a genotype of TT for a female subject and T for a male subject, and

b) Cladribine is to be administered orally as a treatment regimen comprising the sequential steps :

9. Cladribine for use according to claim 8 wherein the induction period (i) has a duration of up to 4 months.

10. Cladribine for use according to claim 8 or 9 wherein the cumulative dose reached at the end of the induction period is about 1.75 mg/kg.

1 1. Cladribine for use according to any one of claim 8 to 10 wherein the first Cladribin-free period (ii) has a duration of up to 10 months.

12. Cladribine for use according to any one of claims 8 to 1 1 wherein the maintenance period (iii) has a duration of up to 4 months.

13. Cladribine for use according to any one of claims 8 to 12 wherein the cumulative dose reached at the end of the maintenance period is about 1.75 mg/kg.

14. Cladribine for use according to any one of claims 8 to 14 wherein the second Cladribine-free period (iv) has a duration of up to 10 months.

15. Cladribine for use according to any one of claims 9 to 14 wherein the periods (i), (ii), (iii) and (iv) together have a total duration of about 2 years.

16. Cladribine for use according to any one of claims 9 to 15, wherein the subject does not receive combination therapy with beta interferon at any time during periods (i), (ii), (iii) or (iv).

17. Cladribine for use according to any one of claims 9 to 16 wherein the subject is non-responsive to beta-interferon and / or is beta-interferon naive.

18. Cladribine for use in treating Relapsing-Remitting Multiple Sclerosis (RRMS) in a subject, wherein

a) the subject has undergone an analysis of genotype for rs5971598 resulting in confirmation of a genotype having at least one C allele, and

i) an induction period during which Cladribine is administered such that the total dose reached at the end of the induction period is between 3.0 and 4.5 mg/kg, followed by ii) a first cladribine-free period in which no Cladribine is administered, iii) a maintenance period during which Cladribine is administered such that the total dose reached at the end of the maintenance period is between 1.5 and 2.25 mg/kg, and iv) a second cladribine-free period in which no Cladribine is administered.

19. Cladribine for use according to claim 17 wherein the induction period (i) has a duration of up to 4 months.

20. Cladribine for use according to claim 17 or 18 wherein the cumulative dose reached at the end of the induction period is about 3.5 mg/kg.

21. Cladribine for use according to any one of claim 17 to 19 wherein the first Cladribin-free period (ii) has a duration of up to 10 months.

22. Cladribine for use according to any one of claims 17 to 20 wherein the maintenance period (iii) has a duration of up to 4 months.

23. Cladribine for use according to any one of claims 17 to 21 wherein the cumulative dose reached at the end of the maintenance period is about 1.75 mg/kg.

24. Cladribine for use according to any one of claims 17 to 22 wherein the second Cladribine-free period (iv) has a duration of up to 10 months.

25. Cladribine for use according to any one of claims 17 to 23 wherein the periods (i), (ii), (iii) and (iv) together have a total duration of about 2 years.

26. Cladribine for use according to any one of claims 17 to 24 wherein the subject is female and has a CC genotype or a CT genotype for rs5971598.

27. Cladribine for use according to any one of claims 17 to 25 wherein the subject is male and has a C genotype for rs5971598.

28. Cladribine for use according to any one of claims 17 to 26 wherein Cladribine is to be administered in combination with beta interferon.

29. Cladribine for use according to claim 27 wherein the beta interferon is to be administered during the induction period (i) and / or the maintenance period (iii).

30. Kit for the adaptation of the treatment of Relapsing-Remitting Multiple Sclerosis (RRMS) to a subject's response profile to Cladribine, comprising :

a pharmaceutical composition comprising cladribine for oral administration,

means for determining the polymorphism genotype of the subject in at least one SNP located on chromosome X,

31. Kit according to claim 30 wherein the at least one SNP comprises rs5971598.

32. Kit according to claim 31 or 32 wherein the means for determining the polymorphism genotype comprises means for nucleic acid amplification, sequencing and / or DNA scan chip technology.

33. Use of at least one SNP located on chromosome X, preferably within the Duchenne Muscular Dystrophy (DMD) gene, as a marker for Cladribine efficacy in subjects suffering from Relapsing- Remitting Multiple Sclerosis (RRMS).

34. Use according to claim 33 wherein the at least one SNP is selected from rs5971598, rs17338758, rs6628643 or a combination thereof.

35. Use according to claim 33 or 34 wherein the at least one SNP is selected from rs5935310, rs7892480, rs593531 1 or a combination thereof.