WO2015107196A1

WO2015107196A1 - Use of thalidomide or analogs thereof for preventing neurologic disorders induced by brain irradiation

Info

Publication number: WO2015107196A1
Application number: PCT/EP2015/050895
Authority: WO
Inventors: Franck LEBRIN; Carole SOUSSAIN; Jérémy THALGOTT
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Institut Curie; College de France
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Institut Curie; College de France
Priority date: 2014-01-20
Filing date: 2015-01-19
Publication date: 2015-07-23
Anticipated expiration: 2016-07-20

Abstract

The present invention relates to the use of thalidomide or analogs thereof for preventing neurologic disorders induced by brain irradiation.

Description

USE OF THALIDOMIDE OR ANALOGS THEREOF FOR PREVENTING NEUROLOGIC DISORDERS INDUCED BY BRAIN IRRADIATION

FIELD OF THE INVENTION

The present invention relates to the field of medicine, in particular oncology and radiotherapy.

BACKGROUND OF THE INVENTION

Treatment-induced central nervous system (CNS) toxicity remains a major cause of morbidity in patients with brain cancer and has become a crucial therapeutic issue.

Neurological side effects induced by radiotherapy are classified in three categories: acute (during radiation), early-delayed (up to 6 months post-irradiation) and late-delayed (more than 6 months post-irradiation).

Conventionally used low fractions (< 3 Gy) have substantially reduced the risk of acute side effects. In addition, steroids can help to lessen the severity of acute side effects.

Early-delayed complications include somnolence syndrome and transitory cognitive disturbances mainly affecting short-term memory and attention. A preventive role of steroid has been suggested. The severity of these early-delayed complications is not predictive of later, more severe, cognitive impairments.

Late-delayed side complications include focal cerebral and spinal cord radionecrosis although they are less likely to occur with more recent irradiation procedure. The most common and serious delayed complication is cognitive dysfunction related to radiation- induced leukoencephalopathy. In addition, some patients present a higher risk, especially risk associated with old age, diabetes, high blood pressure, combined radiochemotherapy treatment and presence of a mass. The course is unpredictable; most patients stabilize, some deteriorate slowly, and a few develop severe dementia and eventually die, especially in case of elderly patients. Then, several investigators even recommend to avoid cranial radiotherapy in patient older than 60 years with CNS lymphoma at a risk of a decreased tumor control.

Late side effects of radiotherapy are reported to cause early damage of blood vessels. It is becoming a major issue following recent improvement of therapeutic results in CNS and non central nervous system cancers. Despite the considerable burden of treatment-induced CNS toxicity on survival and quality of life of cancer patients, the mechanisms of neurotoxicity remain poorly understood and no therapeutic strategies are available. Experimental data on animal models consistently show a pattern of lesions that depends on both dose and time. The blood-brain barrier (BBB) and blood-spinal barrier might have a role in the pathophysiology of radiation-induced damage. After irradiation, there are several changes in the BBB that occur at different time points after exposure. The most consistently reported change is an acute, dose-dependent increase in permeability of the BBB, which recovers over a few weeks. This increase in permeability is followed, in secondary radio-necrosis, by a new wave of barrier disruption that is concomitant with parenchymal loss. Whilst the organization of the BBB is well described and includes endothelial cells, pericytes, neuron and glial cells, the cellular targets of irradiation and the mechanisms underlying radiation-induced BBB breakdown as well as the relation between early and late BBB dysfunctions remain unclear.

It is also important to note that the combination of radiotherapy and chemotherapy seems to have higher toxic effects, in particular more frequent and often more severe.

Accordingly, despite the considerable burden of treatment-induced central neurotoxicity on survival and quality of life of cancer patients, the mechanisms of neurotoxicity remain poorly understood and there is no prophylactic or curative therapeutic strategy available for CNS toxicity induced by radiotherapy or combined chemotherapy and radiotherapy.

SUMMARY OF THE INVENTION

The inventors have shown that pericytes are involved in brain response to radiation and that BBB dysfunctions are due to radiation-induced pericyte disengagement. Accordingly, it is hypothesized that it is a primary event preceding cognitive anomalies. The inventors have also demonstrated that thalidomide treatment may prevent radiation treatment induced CNS toxicity.

Therefore, the present invention relates to a compound selected from the group consisting of thalidomide, analogs thereof and pharmaceutically acceptable salts thereof for use for preventing in a patient a CNS complication/injury induced by central nervous system (CNS) irradiation. It also relates to the use of a compound selected from the group consisting of thalidomide, analogs thereof and pharmaceutically acceptable salts thereof for the preparation of a drug for preventing in a patient a CNS complication/injury induced by central nervous system (CNS) irradiation. It further relates to a method for preventing in a patient a CNS complication/injury induced by central nervous system (CNS) irradiation, wherein a therapeutically effective amount of a compound selected from the group consisting of thalidomide, analogs thereof and pharmaceutically acceptable salts thereof is administered to a patient submitted to CNS irradiation, thereby preventing or decreasing the CNS complication/injury induced by central nervous system (CNS) radiotherapy. More particularly, the CNS irradiation is a brain radiotherapy.

Optionall the compound may present one of the following formulae (I), (II) and (III)

(III)

wherein

X is C=0 or CH₂;

Ri is independently selected from -H, -Oi l, -CH₃, -CH;OZ (ethers ), -CH₂OCOZ (esters), -CH₂OCONZ (carbamates ), or -CH₂Z (alkyls), wherein Z is selected from II or - (CH₂)n-H, where n is 1 - 10;

R₂ is selected from -H, -NH₂, G -CK alkyl, -NH-NH₂ (hydrazine), -NH-OH (hydroxalamine), -NH-OR5, -N=N-R₅, -NH₂, -N(R₅)₂, -NHCOH, -NHCOCH3, pyrazoiidine, pyrazoline, tetrazole, imidazole, pyrazole, piprazine, o imidazoline, R5 being selected from the group consisting of pyrazoiidine, tetrazole, and pyrazoline; R3 is selected from -H, G-Cg alkyl. benzyl or halogen; and

R₄ is selected from -H, -NH₂, -NH-NH₂ (hydrazine), -NH-OH (hydroxalamine), -NH- OR₅, -N=N-R₅, -NH₂, -N(R₅)₂, -NHCOH, -NHCOCH3, pyrazoiidine, pyrazoline, tetrazole. imidazole, pyrazole, piprazine, or imidazoline. R5 being selected from the group consisting of pyrazoiidine, tetrazole, and pyrazoline.

Preferably, the compound is selected in the group consisting of thalidomide, lenalidomide, pomalidomide, CC-10015 (Celgene), CC-11006 (Celgene), 3-(5-amino-2-methyl-4-oxo- 4H-quinazolin-3-yl)-piperidine-2,6-dione (Celgene), and pharmaceutically acceptable salts thereof. More preferably, the compound is selected in the group consisting of thalidomide, lenalidomide, pomalidomide and pharmaceutically acceptable salts thereof. Still more preferably, the compound is selected in the group consisting of thalidomide, pomalidomide and pharmaceutically acceptable salts thereof. Even more preferably, the compound is pomalidomide or a pharmaceutically acceptable salt thereof.

Preferably, the CNS complication/injury is a late CNS complication/injury. More preferably, the late CNS complication/injury occurs at least six months after the beginning of the CNS irradiation treatment. Optionally, the CNS complication/injury is a progressive subcortical dementia, especially characterized by psychomotor slowing, executive and memory dysfunction, behavioral changes, gait ataxia, and/or incontinence.

Preferably, the patient has a brain tumor, either a benign brain tumor such as glioma, meningioma, schwannoma, pituitary adenoma, hemanglioblastoma, craniopharyngioma, or preferably a malignant brain tumor such as glioma, glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, ganglioglioma, mixed glioma, malignant nerve sheath tumor, or brain metastasis of solid tumor or primary CNS lymphoma.

In particular, the patient is at higher risk of developing a neurological disorder induced by central nervous system (CNS) radiotherapy. Accordingly, the patient may be selected among elderly patients, patients with cardiovascular risk, children, especially those of less than 5 years old, adolescents, patients suffering of diabetes or high blood pressure or patients having ApoE polymorphisms (e.g., the presence of the epsilon 4 allele of APOE). The compound can be administered before, simultaneously and/or after the radiotherapy. Preferably, the compound is to be administered during a period covering one, two, three or four weeks prior the radiotherapy and the radiotherapy duration, and optionally one, two, three or four weeks after the end of the radiotherapy. More specifically, the compound is to be administered during a period covering one or two weeks prior the radiotherapy and the radiotherapy duration, and optionally one or two weeks after the end of the radiotherapy. Preferably, the compound is to be administered during 5 to 9 weeks, more preferably 6 to 8 weeks. Optionally, the daily dose to be administered by oral route is comprised between 0.01 and 500 mg, preferably between 0.1 and 100 mg.

Optionally, the compound is used in combination with an additional therapeutic agent, in particular an antitumoral chemotherapy. DETAILED DESCRIPTION

The inventors have shown that, in vivo, CNS pericytes are an early target of irradiation. In an in vivo mice model of irradiation inducing late behavioral dysfunctions, they have shown that, early after irradiation, pericytes display phenotypic and molecular changes leading to a disruption of their interactions with endothelial cells. As a result, pericyte- mediated vascular constriction is altered. Increased blood brain barrier (BBB) permeability is concomitantly observed.

More importantly, the inventors have shown that thalidomide was proved effective to prevent the radiation-induced pericyte dysfunctions and the increased BBB permeability. The present results demonstrate a pivotal role of pericytes in the radiation-induced CNS toxicity amenable to pharmacological intervention in order to prevent neurological disorders induced by CNS radiotherapy.

Accordingly, the present invention relates to a compound selected from the group consisting of thalidomide, analogs thereof and pharmaceutically acceptable salts thereof for use for preventing in a patient a neurological disorder induced by central nervous system (CNS) irradiation, in particular radiotherapy.

It also relates to the use of a compound selected from the group consisting of thalidomide, analogs thereof and pharmaceutically acceptable salts thereof for the preparation of a medicament for preventing in a patient a neurological disorder induced by central nervous system (CNS) irradiation, in particular radiotherapy.

It further relates to a method for preventing in a patient a neurological disorder induced by central nervous system (CNS) irradiation, in particular radiotherapy, wherein a therapeutically effective amount of a compound selected from the group consisting of thalidomide, analogs thereof and pharmaceutically acceptable salts thereof to said patient submitted to CNS radiotherapy, thereby preventing or decreasing the neurological disorder induced by central nervous system (CNS) irradiation, in particular radiotherapy.

Thalidomide, analogs thereof, pharmaceutically acceptable salt thereof

The compounds used in the present invention are thalidomide, analogs thereof and pharmaceutically acceptable salts thereof.

The most known analogs are lenalidomide (also called CC-5013, 3-(4-amino-l-oxo-l,3- dihydro-isoindol-2-yl)-piperidine-2,6-dione) and pomalidomide (also called CC-4047, 4- (amino)-2-(2,6-dioxo-(3-piperidyl))-isoindoline-l,3-dione).

Thalidomide pomalidomide Lenalidomide

Therefore, in a preferred embodiment, the compound is selected from the group consisting of thalidomide, lenalidomide, pomalidomide and pharmaceutically acceptable salts thereof. In a most preferred embodiment, the compound is selected from the group consisting of thalidomide, pomalidomide and pharmaceutically acceptable salts thereof. In a very specific embodiment, the compound is pomalidomide or a pharmaceutically acceptable salt thereof.

However, several analogs of thalidomide have been developed and described. In particular, Celgene Corp is a company developing analogs of thalidomide.

Examples of analogs are disclosed in the following patent applications: WO03/014315, US 5,874,448, WO98/03502, WO98/54170, WO02/059106, WO94/20085, US 6,458,810. In particular, the compound may be also selected in the following lists: 3,6- dichlorothalidomine, 3,6-diaminothalidomine, 3,6-dihydrazinothalidomine, 5-(amino)-2- (2,6-dioxo-(3-piperidyl))-isoindoline-l,3-dione, 3-(5-amino-2-methyl-4-oxo-4H- quinazolin-3-yl)-piperidine-2,6-dione, CC-10015 (Celgene), CC-11006 (Celgene), ENMD- 0995 (S-3-Amino-phthalimido-glutarimide or S-3APG), CPS11, CPS45, CPS49, l-oxo-2- (2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline, l,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine- 3-yl)isoindoline, tetra substituted 2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindolines, 1-oxo and l,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines, l,3-dioxo-2-(3-methyl-2,6- dioxopiperidin-3-yl) 4-aminoisoindoline, preferably in the following list: 3,6- dichlorothalidomine, 3,6-diaminothalidomine, 3,6-dihydrazinothalidomine, 5-(amino)-2- (2,6-dioxo-(3-piperidyl))-isoindoline-l,3-dione, l,3-dioxo-2-(2,6-dioxo-3-fluoropiperidine- 3-yl)isoindoline, l,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines, l,3-dioxo-2-(3- methyl-2,6-dioxopiperidin-3-yl) 4-aminoisoindoline.

In an alternative embodiment, the preferred thalidomide analogs could be defined by the following one of the following formulae (I), (II) and (III)

(HI)

wherein

X is C=0 or CH₂;

Ri is independently selected from -H, -Oi l, -CH₃, -CH₂OZ ( ethers ), -CH₂OCOZ (esters), - CH₂OCONZ (carbamates ), or -CH₂Z (alkyls), wherein Z is selected from I I or - ( CH : ),,-! I, where n is 1 - 10;

R₂ is selected from -I I, -NH₂, Ci-C₈ alkyl, -NH-NH; (hydrazine), -NH-OH (hydroxalamine ), -NH-OR5, -N=N-R₅, -Nth, -N(R₅)₂, -NHCOH, -NHCOCH3, pyrazol idine, pyrazol ine, tetrazole, imidazole, pyrazole, piprazine. or imidazoline, R5 being selected from the group consisting o pyrazolidine, tetrazole, and pyrazol ine;

R3 is selected from -I I. Ci-Cg alkyl. benzyl or halogen; and

R t is selected from -H, -NH₂, -NH-NIL (hydrazine), -NH-OH ( hydroxalamine ), -NH-OR5, -N=N-R₅, -NH₂, -N(R₅)₂, -NHCOH, -NHCOCH3, pyrazolidine, pyrazoline, tetrazole, imidazole, pyrazole, piprazine, or imidazoline, R5 being selected from the group consisting of pyrazolidine, tetrazole, and pyrazoline.

In a preferred embodiment, Ri is -H.

In a preferred embodiment, R₂ is -H or -NH₂.

R x is preferably -I I, -F, -CI, -CM 3 or benzo. in particular -H. -F. or -CI 13. In a preferred embodiment. R is -I I.

In a preferred embodiment. R₄ is I I.

For instance, such compounds that can be used in the present invention are well-disclosed in WO2007/028047 (pages 27-42), the disclosure of which is incorporated herein by reference.

The pharmaceutically acceptable salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, maleic, methanesulfonic and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, and in Handbook of Pharmaceutical Salts: Properties, Selection, and Use edited by P. Heinrich Stahl and Camille G. Wermuth 2002. In a preferred embodiment, the salt is selected from the group consisting of maleate, chlorhydrate, bromhydrate, and methanesulfonate.

Thalidomide and its analogs, Lenalidomide and Pomalidomide, are used in the treatment of hematologic malignancies, especially multiple myeloma. Lenalidomide has also been developed for the treatment of myelodysplasia syndromes (MDS). It is undergoing clinical trial as a treatment for Hodgkin's lymphoma, non-Hodgkin's lymphoma, and chronic lymphocytic leukemia.

In the scientific literature, thalidomide and its analogs have been extensively studied in order to define their utility in cancer treatments, alone or in association with chemotherapy or radiotherapy. Chang et al (2004, Int J Radiat Oncol Biol Phys, 60, 353-357), Chang et al (2012, Int J Radiat Oncol Biol Phys, 82, 817-825), Hoang et al (2012, J Clin Oncoln 30, 616-622), Viani et al (2009, J Exp Clin Cancer Res, 28, 1), Anscher et al (2006 Int J Radiat Oncol Biol Phys, 66, 477-482) reported that the addition of thalidomide does not provide any therapeutic benefit, sometimes with increased toxicity. However, other authors reported therapeutic benefits: Molinari et al (2012, Radiat Res, 177, 59-68) on oral cancer in hamster models; Drappatz et al (2009, Int J Radiat Oncol Biol Phys, 73, 222-227) in preliminary study for defining appropriate lenalidomide dosage; Lee et al (2006, Oncol Rep, 15, 1162-1168) with the rat orthopic glioma model; Hsu et al (2006, Jpn J Clin Oncol, 36, 93-99) on advanced hepatocellular carcinoma; Ansiaux et al (2005, Clin Cancer res, 11, 743-750) on mice FSAII tumor model; Morabito et al (2004, Oncol Rep, 11, 93-95) on a group of 17 patients affected by recurrent glioblastoma; Fine et al (2000, J Clin Oncol, 18, 708-715) on a group of 36 patients and with an antitumor activity in a minority of patients with recurrent high-grade gliomas.

However, it has never been reported nor suggested that Thalidomide and its analogs could present a therapeutic advantage in order to prevent radiation induced neurocognitive disorders. On the opposite, in the clinical trial RTOG 0118 relating to randomized patients with multiple brain metastases treated by radiotherapy with or without thalidomide, the authors reported that the neurocognitive decline induced by radiotherapy is not significantly different when patients are treated with thalidomide (Corn et al, 2008, Int J Radiation Oncology Biol Phys, 1, 71-78). Moreover, two other clinical trials reported either an increased toxicity with no therapeutic benefit (Turner et al, 2007, J Neurooncol, 82, 95-101, Knisely et al, 2008, Int J Radiat Oncol Biol Phys, 71, 79-86) or a neurotoxic effect (Alexander et al, 2013, J Neurooncol, 111, 33-39) of thalidomide when used in association with radiotherapy for the treatment of brain tumors (i.e., brain stem gliomas and glioblastomas). In addition, the scientific literature always discloses treatments with thalidomide or analogs thereof in which the highest tolerable dosage is used and the treatment durations are long (i.e., several months). More specifically, in the above- mentioned clinical trials, thalidomide is given from the first day of radiotherapy up to several months after its end with a dose escalation up to 1,200 mg/day.

Therefore, the inventors surprisingly identified that thalidomide and its analogs could be used for preventing radiation induced toxicity, in particular for preventing neurological disorders induced by central nervous system (CNS) radiotherapy. More specifically, the inventors defined that thalidomide and its analogs can have advantageous effects for preventing radiation induced toxicity when used with short period of treatment, especially when compared with the very long period tested in the prior art (e.g., several months, more frequently one year or more). This short treatment period allows to obtain therapeutic benefits while avoiding the well-known possible side effects of thalidomide and its analogs. More preferably, the treatment period begins before CNS radiotherapy.

CNS complication/injury

In the context of the present invention, the CNS complication/injury affects in particular the cognitive functions. Cognitive functions can be impaired. Severe cases result in a subcortical dementia characterized by psychomotor slowing, executive and memory dysfunction, behavioral changes, gait ataxia, and/or incontinence. Lethargy, focal deficits, seizure can be associated. More particularly, the cognitive dysfunction is related to radiation-induced leukoencephalopathy. Pathological findings include vascular lesions such as stenosis, thrombosis, haemorrhage, fibrinoid vascular necrosis, and demyelination. However, the present invention may also concern spinal cord radiotherapy. In this context, the complication is rather myleopathy.

In a preferred embodiment, the CNS complication/injury concerns late complications, such as delayed cognitive impairment. Late CNS complication/injury occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 months after the beginning of the CNS irradiation treatment. Preferably, late CNS complication/injury occurs at least 3, 4, 5, 6, 7, 8, 9, 10 months after the beginning of the CNS irradiation treatment. More preferably, late CNS complication/injury occurs at least 5, 6, 7 months after the beginning of the CNS irradiation treatment. Even more preferably, late CNS complication/injury occurs at least 6 months after the beginning of the CNS irradiation treatment. Alternatively, late CNS complication/injury occurs at least 3 months after the beginning of the CNS irradiation treatment. Tumors and patients

The present invention relates to CNS or brain tumor, either a benign brain tumor such as glioma, meningioma, schwannoma, pituitary adenoma, hemanglioblastoma, craniopharyngioma, or preferably a malignant brain tumor such as glioma, glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, ganglioglioma, mixed glioma, malignant nerve sheath tumor, or brain metastasis of solid tumor or primary CNS lymphoma. Preferably, the brain tumor is a malignant one. In particular, it relates to CNS or brain tumors that can be treated by radiotherapy, alone or combined with chemotherapy. In a particular embodiment, the CNS or brain tumor is a primary CNS lymphoma.

The patient is a human being. He could be a child, an adolescent, an adult, even an elderly subject. By elderly subject is intended a person of 60 years old or more.

In a particular embodiment, the patient is a human being of 60 years old or older. Preferably, the patient is a human being of 65 years old or older, more preferably, a human being of 70 years old or older.

In a particular embodiment, the patient presents an increased risk of radiation-induced CNS complications/injuries. Accordingly, the patient can be selected in the group consisting of a child, in particular a child of less than 5 years old, an adolescent, a elderly subject, patients at cardiovascular risk, patients suffering of diabetes or high blood pressure and patients having APOE polymorphisms. Indeed, APOE polymorphisms have been described a factor increasing the risk of CNS complication/injury, in particular the presence of the epsilon 4 allele of APOE (Ahles el al. Psychooncology. 2003, 12(6):612- 9). The risk also depends on the volume of the brain tissue to be treated, the total irradiation dose, and the fractionation schedule.

In a particular embodiment, the CNS irradiation treatment is the first line of treatment of the patient. Preferably, the patient has not been previously treated by radiotherapy, in particular CNS irradiation treatment, by chemotherapy or by the combination of radiotherapy and chemotherapy.

Alternatively, the patient did not relapse since his former line of treatment and/or the patient's condition is stable for at least 1, 2, 3, 4, 5 or 6 months prior to the CNS irradiation treatment.

The patients can also be a non-human mammal such as pets, horses, or farm animal. Therefore, the veterinary use of the invention is also contemplated.

CNS irradiation, in particular radiotherapy

Irradiation includes therapeutic irradiation of the CNS and accidental irradiation.

By "accidental irradiation" is not intended to refer to natural irradiation. It refers to radiation accidents, namely accidents involving radioactive materials or ionizing radiation from artificial sources such as x-ray tubes and particle accelerators. For instance, accidents relating to operators of irradiation facility or radiological facility have been reported.

Therapeutic irradiation of the CNS, also referred to as CNS radiotherapy, includes but is not limited to γ-rays or X-rays radiotherapy, preferably X-rays radiotherapy. Therapeutic irradiation can be applied by external beam radiation therapy, by brachytherapy or systemic radioisotope therapy. Preferably, CNS radiotherapy is selected from the group X- rays radiotherapy applied by external beam radiation therapy.

In particular, the present invention relates to CNS radiotherapy. More specifically, it relates to brain radiotherapy. Alternatively, it may also relate to spinal cord radiotherapy. Generally, a total safe dose of irradiation, e.g. 40-60 Gy, is delivered to a field as restricted as possible. In addition, the total safe dose is applied as daily fractions in order to avoid radiation induced injury, usually 4-7 fractions a week, for instance fractions of less than 2 Gy, in particular of 1.8-2.0 Gy.

Several methods for delivering the radiotherapy are known and available to the one skilled in art, e.g. external beam radiation therapy (EBRT), Three-dimensional conformal radiation therapy (3D-CRT), Intensity modulated radiation therapy (IMRT), Conformal proton beam radiation therapy, or Stereotactic radiosurgery/stereotactic radiotherapy.

The radiotherapy can be used in combination with an antitumoral agent such as therapeutic antibody, bevacizumab, everolimus, rituximab, or with a chemotherapeutic agent. In a preferred embodiment, the chemotherapy is carried out before the radiotherapy, especially in case of primary CNS lymphoma. Generally, the chemotherapeutic agent is methotrexate. Alternatively, the chemotherapy can be carried out simultaneously with the radiotherapy, especially in case of glioblastoma. Generally, the chemotherapeutic agent is temozolomide.

Dosage and schedules

Thalidomide, the analogs thereof or the pharmaceutical salts thereof can be administered prior, simultaneously or concomitantly and/or after radiotherapy.

The duration of treatment with thalidomide, the analogs thereof or the pharmaceutical salts thereof is relatively short. Duration of less than 8, 7, 6, 5, 4, 3, 2 or 1 weeks is contemplated. For instance, duration comprised between 1 and 8 weeks is contemplated, preferably between 2 and 6 weeks, more preferably between 3 and 4 weeks. A single period or cycle of treatment is contemplated.

Thalidomide or analog or salt thereof is to be administered during a period covering one, two, three or four weeks prior the radiotherapy and the radiotherapy duration, and optionally one, two, three or four weeks after the end of the radiotherapy.

In a preferred embodiment, the treatment period may begin between one and two weeks before the beginning of the radiotherapy, more preferably about one week before.

In a preferred embodiment, the treatment period include the period of radiotherapy, especially when fractioned radiotherapy is chosen.

Preferably, the treatment is carried on after the end of radiotherapy, in particular during a period of between one and four weeks, in particular about 1, 2, 3, or 4 weeks. More preferably, the period of treatment after the radiotherapy lasts one or two weeks.

Thalidomide, the analogs thereof or the pharmaceutical salts thereof can be administered by any convenient route. For instance, it can be administered by oral route (in the form of discrete units as capsules, sachets, tablets or lozenges) or by systemic route, for instance subcutaneous, intravenous injection or infusion.

By "therapeutically effective amount" it is meant the quantity of the composition of the invention which prevents, removes or reduces the CNS complication/injury in mammals, including humans. It is understood that the administered dose may be adapted by those skilled in the art according to the patient, the pathology, the mode of administration, etc. A daily dose, by oral route, can vary and could be comprised between 0.01 and 500 mg, preferably between 0.01 and 100 mg or 0.1 and 100 mg, more preferably between 0.01 and 50 mg and even more preferably between 0.1 and 50 mg. Alternatively, a daily dose, by oral route, can vary and could be comprised between 0.01 and 40 mg, preferably between 0.01 and 25 mg, more preferably between 0.01 and 10 mg, and even more preferably between 0.1 and 10 mg. As illustration, by oral route, a daily dose of thalidomide can be comprised between 50 and 500 mg, preferably between 100 and 300 mg, still more preferably between 150 and 250 mg. Alternatively, by oral route, a daily dose of thalidomide can be comprised between 1 and 500 mg, between 1 and 200 mg, between 1 and 150 mg, between 1 and 50 mg and even more preferably between 1 and 25 mg. For lenalidomide, by oral route, a daily dose of lenalidomide can be comprised between 1 and 50 mg, preferably between 5 and 30 mg, more preferably between 0.1 and 30 mg, still more preferably between 5 and 30 mg, and even more preferably between 10 and 25 mg. For pomalidomide, by oral route, a daily dose of pomalidomide can be comprised between 0.01 and 10 mg or between 0.1 and 10 mg, preferably between 0.01 and 15 mg, more preferably between 0.1 and 10 mg, still more preferably between 1 and 7 mg, and even more preferably between 3 and 5 mg.

In a particular aspect of the invention, low doses can be used, in particular 80, 70, 60 or 50 % of the above mentioned daily dose.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Characterization of the radiation-induced model of toxicity on the central nervous system Irradiation was delivered to the whole brain excluding the eyes and the olfactory bulb at doses ranging from 10 to 40 Gy, to 5-6 8-weeks old mice for each dose level, a. Weight curves represent the median of weight variations from baseline for each dose level after irradiation, b. Survival curves in each group. Mice were sacrificed between 136 and 139 days after irradiation, right after the activity tests, c-d. Activity tested 4 months after increasing doses of irradiation. Results were obtained from 5-6 mice in each group, c. To perform the stand up test, mice were observed for 5 min and the observer counted every stand up position, d. To perform the traction test, mice were placed in the center on the horizontal part of a hanger and the observer recorded the time till the mice fell. Values are given as means + s.e.m. *P < 0.05, and **P < 0.01, results from unpaired t test. e-f. Radiation activates TGF-b signaling pathways. Quantification of tgfbl, Tgfbrl and N-cadherin mRNA expression levels in control retinas (n=10) and 9-day- irradiated retinas (n=8). Radiation activates Notch signaling pathways. Quantification of Hey-1, Hes-1 and Jagged-1 mRNA expression levels in control retinas (n=10) and 9-day-irradiated retinas (n=8). Values are given in median + interquartile range. *P < 0.05, **P < 0.01 and ***P < 0.001, results from Mann Whitney U test and denoted statistical significance.

Figure 2. Irradiation induces changes in pericytes phenotype, which is prevented by peri-irradiation treatment with thalidomide, (a-e) 12 old-week mice were euthanized 8 days after a single fraction of 20 Gy irradiation delivered to the head including the eyes. Thalidomide or vehicle was intraperitoneally injected at 150 mg/kg on day -7, -5, -2, +3 and +6 from irradiation, a. Confocal images of retinas from untreated, irradiated and irradiated with thalidomide NG2DsRed mice (red, pericytes) stained for isolectin B4 (blue, endothelial cells) and ccSMA (green, vascular smooth muscle cells), b. High magnifications reveal that pericytes are only loosely attached to the endothelium in irradiated NG2DsRed retinas compared to control retinas and that thalidomide prevents irradiation-mediated pericyte detachment, c. Pericyte density in NG2DsRed retinas from control mice (n= 10) and 8 day- irradiated mice (n= 10). 60 images were analyzed per mouse, d. Surface of pericyte somas in NG2DsRed retinas from control mice (n= 10), 8 day-irradiated mice (n= 10) and 8 day-irradiated mice treated with thalidomide at 150mg/kg body weight (n=10). e. Quantification of the roundness, as measured by Image J software (roundness = 4 x area/(n x sqr(major ,mice (n= 10) and 8 day-irradiated mice treated with thalidomide at 150mg/kg body weight (n=10). f. Quantification of rgs5, pdgfr, cspg4, desmin and acta 1 mRNA expression in NG2DsRed retinas from control mice (n= 10) and 8 day-irradiated mice (n=8). Results are given at means + s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001, ****P < 0.0001 results from unpaired t test and denoted statistical significance.

Figure 3: Measures of areas of soma pericytes and their roundness at different time points after irradiation. Measures were obtained from control mice (DO) and five days (D5), seven days (D7), eight days (D8) and nine days (D9) after irradiation (20 Gy single fraction) (n = 3 to 12 mice). *p < 0.05, **p < 0.01 and ***p < 0.001, results from unpaired t test a. Areas of soma pericytes were smaller in irradiated mice than in control mice from 5 to nine days after irradiation. No difference was noted between the different time points after irradiation, b. Roundness of soma pericyte was higher after irradiation than in control mice from 5 to nine days after irradiation. No difference was noted between the different time points after irradiation.

Figure 4: Pericyte-mediated vessel constriction (a) capillary with pericytes before, during and after stimulation. Erythrocytes are present within capillary; thin structures outside capillary are astrocyte endfeet. Pericyte-mediated constriction is intensity- dependent, (b) Confocal images of retinas from NG2DsRed (top left) and Cx40- EGFP mice (top right) stained with isolectin B4 (endothelial cells, blue) and a smooth muscle actin (perivascular cells, green) or with isolectin B4 (endothelial cells, blue) and a smooth muscle actin (perivascular cells, red) respectively, show arterio-venous separation. At Bottom left, higher magnification of an arterial vessel that shows regular coverage of smooth muscle cells with the cells being perpendicular to the direction of blood flow. At bottom right, higher magnification of an arterial capillary covered with pericytes that are either positively or negatively stained for a smooth muscle actin. Minimal intensity currents necessary to cause vessel constriction of the different vascular beds are indicated in the graph. Values are given as means + s.e.m.

Figure 5: Pericyte-mediated vessel constriction is impaired in irradiated mice, an effect reversed by pharmacological treatment with thalidomide, a-d. Pericyte-induced vessel constriction after electrical stimulation of arteriolar vessels and arteriolar capillaries. Images represent vessels before, during and after pericyte-induced constriction of control mice (a), 8 day-irradiated mice treated with vehicle (b), with thalidomide alone at 150mg/kg (c) or with thalidomide at 150mg/kg in combination with STI-571 at 50mg/kg (d). Graphs: Distribution of the proportion of arteriolar vessels (left graph) and arteriolar capillaries (right graph) that constricts according to the intensity used to stimulate pericyte contraction. An average of 15 pericytes were stimulated per retina and between 4 to 7 mice were used per condition, e. Percent of pericytes that is able to induce an arteriolar vessel constriction at 1.5 μΑ. f. Percent of pericytes that is able to induce an arteriolar capillary constriction at 2.0 μΑ. *P < 0.05, **P < 0.01 and ***P < 0.001, ****P < 0.0001, Chi- square test. 95 % CI

Figure 6: The radiation-induced blood-brain barrier permeability is prevented by Thalidomide. Mice were euthanized 8 days after irradiation. Thalidomide was administered as intraperitoneal injection (150 mg/kg body weight per injection on days -7, -5, -2, +3 and +6 from irradiation). Vehicle (DMSO) was administered on the same days than thalidomide in control and irradiated animals. Cadaverin Alexa-Fluor-555 was injected intravenously as a tracer of vasculature permeability, a. Confocal images of whole mount retina stained for isolectin B4 (endothelial cells, green) of control or irradiated mice treated with vehicle or with thalidomide and injected with cadaverin Alexa fluor-555 (red) 2h before being euthanized. Quantification of Alexa fluor-555 extravasation, b. High magnification images of retinas stained for Tuj l (neurons, green) show accumulation of cadaverin Alexa Fluor-555 in tuj 1 neurons after irradiation, c. Upper panel. Whole brains photographed after injection of cadaverin Alexa Fluor-555 in control mice or irradiated mice treated with vehicle or with thalidomide, c. Middle panel. 100 μιη thick brain coronal sections stained for PEC AM- 1 (endothelial cell, green) of control mice or irradiated mice treated with vehicle or with thalidomide and injected with cadaverin Alexa Fluor-555 (red) before being euthanized, c. Bottom panel. High magnification images of the brain coronal sections show that the tracer accumulates in perivascular space in irradiated mice. Number of mice used is between 2-4 per condition. Values are given as means + s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001, results from unpaired t test, 95 % CI.

EXAMPLES

Example 1: Thalidomide prevents radiation-induced CNS toxicity by inhibiting pericyte disengagement from endothelium

To study the effect of radiation on the CNS pericytes, the inventors set up an in vivo model of CNS irradiation in mice. Single fractions of irradiation delivered to the brain of adult mice, at doses ranging from 10 to 30 Gy, did not affect weight or survival of mice during 5 months follow up (Fig 1 a-b). Four months after irradiation, mice that had received at least 20 Gy exhibited a lower score on basic motor activity tests (stand up and traction tests) (Fig lc-d)). The inventors took advantage of the vasculature of the retina as a surrogate site of the CNS because the eye is anatomically an extension of the CNS, it is a well-described model for analyzing pericyte-endothelial cells interactions, and finally because both CNS and retina vasculatures are the most pericyte-covered organs. They tested increasing doses of irradiation delivered to the eyes ranging from 15 to 25 Gy and observed a change of the pericytes phenotype at the 20 Gy level. As expected, TGF-β and Notch signaling pathways were activated in isolated retinas. Indeed, N-cadherin RNA and the Notch target gene Hey 1 were over expressed in retina nine days after irradiation at the dose of 20 Gy (Fig le-f). The radiation dose of 20 Gy was thus selected for further experiments.

Pericytes mainly through their interactions with endothelial cells (ECs) play a pivotal role in the brain microvascular homeostasis including the maintenance of the BBB permeability and regulation of the cerebral microvessels blood flow. The inventors first investigated structural changes in pericytes of the retina occurring early after irradiation of mice and then tested the effects of thalidomide given before (days -7, -5, -2) and after (days +3 and +6) irradiation. The general architecture of the vasculature and the alpha-smooth muscle actin (cc-SMA) coverage of arteries were unchanged 8 days after irradiation with or without thalidomide (Fig 2a). However, significant morphological changes of arteriolar and arteriolar capillaries pericytes were observed after irradiation (Fig 2b). Whereas in control mice pericytes displayed flattened somas closely attached to the endothelial cell, after irradiation most of the pericytes adopted a round shape, offering less contact with the underlying endothelial cell. As a result, compared to control (Ctl), the area of pericytes somas was smaller after irradiation (RT) and their "roundness" increased. Both changes were prevented by thalidomide. Pericytes from irradiated mice treated with thalidomide were similar to pericytes from control mice. In Ctl, RT and RT + Thai, mean areas of pericytes somas and roundness were 12226, 10527, 11705 pixels and 0.39, 0.49, 0.41 respectively. No differences were observed in the measures of area and roundness of pericytes soma at different time points after irradiation (day 5, 7, 8 and 9) (Fig 3). Further experiments were therefore only performed on day 8 after irradiation.

To further characterize features of pericytes after irradiation, the inventors realized immunostaining for Ki67 and cleaved caspase 3 and found no signal of proliferation or apoptosis in pericytes. In accordance with these results, density of pericytes was stable after irradiation (Fig2c-e). At a molecular level, radiation-induced morphological changes in pericytes were associated with an increase of Platelet-derived growth factor receptor β (pdgfrb) and cspg4 Neuron-Glial 2 (NG2) chondroitin sulfate proteoglycan) mRNA expression, two markers of activated pericytes. Expressions of actal ccSMA), rgs5 and desmin, which were used as markers of different stages of pericyte maturation were not affected by irradiation (Fig 2 f).

Pericytes and ECs interacts in the neuro-vascular unit for the regulation of blood flow in arterioles and capillaries, in response to changes in neural activity. The contractile property of pericyte can be measured on ex vivo retina. When an electrical stimulation is applied with a pipette pressed on the pericyte soma, it induces a pericyte contraction that in turn mediates the underlying vessel constriction (Peppiatt et al, 2006, Nature, 443, 700-704), which requires a close interaction between pericyte and EC. The inventors hypothesized that the morphological changes observed in pericytes after irradiation would disrupt the pericyte-EC communication and impair the pericyte-mediated vessel constriction. To test this hypothesis, they next characterized the contractile properties of pericytes in control and irradiated mice in arteriolar and arteriolar capillaries sites. They previously showed that pericyte-mediated constriction was bed dependant (Figure 4). Briefly, three zones could be characterized. The arteriolar site was identified by positivity of the endothelial cell for the connexin 40 and a median diameter of pericytes of 39 pixels (range, 27-49). In the arteriolar site, most pericyte-mediated constriction responses occurred when an electric current up to 1.5 μΑ was applied on the pericyte. The alteriolar capillaries were identified by a positive reactivity with the cc-SMA immunostaining and a weaker positivity for the connexin 40, and a median diameter of arteriolar capillaries of 23 pixels (range, 11-39). In the arteriolar capillaries site, a 2 μΑ electric current applied on pericytes induced the contriction of most of the arteriolar capillaries. Venous capillaries, negative for con40 and CC-SMA immuno staining were unresponsive to electrical stimulation of pericytes.

In the present experiments, the inventors identified arteriolar (group 1) and arteriolar capillaries (group 2) by their diameters. Endothelial cell (EC)-pericyte interactions were impaired in both groups of vessels (Fig 5). Compared to controls, pericytes from irradiated mice required higher intensities of electrical current for inducing a constriction of the underlying vascular bed, both at the arteriolar (group 1) and arteriolar capillary levels (group 2) (fig 5a-b). Pericyte-mediated vessel constriction was preserved in irradiated mice treated with thalidomide (Fig 5c). Recruitment of pericytes to the vascular wall is under the control of the PDGF /PDGFR- pathway To test whether the protective properties of thalidomide on radiation-induced pericyte dysfunctions were mediated through the PDGF /PDGFR- pathway, the inventors co-administered thalidomide with STI-571 (imatinib or gleevec), a potent inhibitor of PDGF receptor tyrosine kinase activity. They observed that gleevec partially abolished the protective properties of thalidomide as measured by the intensity of electric current required for inducing a vessel constriction when applied on group 1 and group 2 pericytes (Fig 5d).

The proportions of pericytes from group 1 that induced a vessel constriction when stimulated by a current intensity below 1.5 μΑ were significantly different according to treatment. Proportion of contractile pericytes in control, irradiated (RT), irradiated mice treated with thalidomide (RT + Thai), irradiated mice treated with thalidomide and STI- 571 (RT + Thai + STI-571) were 83%, 9 %, 61 %, and 33 % respectively (p < 0.0001, Chi-square 95 % CI)). Same pattern was observed for pericytes from group 2 and for an electrical stimulation by a current intensity below 2μΑ. Proportions of contractile pericytes in control, RT, RT+ Thai and RT+Thal+STI-571 were 75%, 29 %, 64 %, and 54 % respectively (p < 0.0001, Chi-square 95 % CI), although the difference between RT + Thai and RT+Thal+STI-571 did not reach statistical significance (fig 5e-f). The inventors deemed these radiation-induced consequences on pericytes and pericytes-ECs interactions to result in in vivo deregulation of the arteriolar and capillary blood flow, which may contribute to late radiation-induced brain damages.

Increased BBB permeability is a hallmark of pericytes-deficient mice models and has been consistently observed after in vivo models of CNS irradiation. To address whether radiation-induced changes in the phenotype and contractile properties of pericytes translated in concomitant impaired vascular permeability, the inventors used cadaverine Alexa Fluor-555 as a fixable tracer (950 D). The fluorescent dye cadaverine Alexa Fluor- 555 significantly extravasated in the retina and in the brain parenchyma of irradiated mice (fig. 6a, b, d)) but not in control mice. Extravascular Cadaverine Alexa Fluor-555 localized mainly to neurons (fig 6c). No leakage of the tracer was observed when thalidomide was administered to irradiated mice.

Altogether, the present data demonstrate that CNS pericytes are an early target of irradiation, which results in disengagement of the endothelium from the surrounding mural cells. As a consequence, the contractile properties of pericytes involved in the control of the cerebral blood flow are impaired. Increased BBB permeability is temporally associated with these disorders. Mechanisms underlying the effects of irradiation on pericytes and pericytes-ECs disengagement need, however, to be further studied. A short treatment with thalidomide has proved effective in the present model to prevent radiation-induced vascular damage, suggesting that this drug could be used in CNS lymphoma and brain tumor patients.

Materials and Methods

. Animals

Experiments were done on adult mice (8 to 12 weeks old). The inventors took advantage of NG2DsRedBAC-transgenic mice, which express the red fluorescent protein (DsRed) under the control of the Cspg4 (encoding NG2) promoter Zhu, et al, 2008, Development, 135, 145-157). When fluorescence of pericytes was not necessary, experiments were done on C57/BL6 adult male. Groups of 3 to 10 mice were constituted for each experiment. Mice were bred under isolator condition and maintained in filtered-top microisolator cages. Food and water were provided ad libitum. The local welfare committee approved experimental protocols. Mice were killed by quick elongation.

2. Irradiation procedures

For irradiation of the head including the eyes, animals were immobilized in a containment device without anesthesia. The heads of adult mice were exposed to a X-ray generator Philips/YXYLON type MG325 (200 kV voltage, 21 mA intensity), the X-ray beam was collimated by a bloc of lead located at 50 cm from the source. Four mice were irradiated by run. A total dose of 20 Gy was delivered in a single fraction at a dose rate of 0.9 Gy/min. Dosimetry was performed on 4 mice. For behavioral and cognitive testings, irradiation was confined to the brain. Mice were anesthesized with inhalational isofluorane. Brains were exposed to a X-ray generator Xrad320 (PXI) (200 kV voltage, 20 mA intensity). Mice were individually irradiated. A total dose of 20 Gy was delivered in a single fraction at a dose rate of 0.97 Gy/min by two lateral beams. The irradiation field was a 2x2.5 cm² rectangular field. Dosimetry was performed on 4 mice.

Beams were similar between the two generators.

3. Treatment

Thalidomide ((+)-2-(2,6-Dioxo-3-piperidinyl)-lH-isoindole-l, 3(2H)-dione) was synthetized par Green Parma. Thalidomide was diluted in dimethylsulfoxyde (DMSO) (95 mg/ml). Mice received intraperitoneal injection of vehicle (DMSO) or thalidomide (150 mg/kg) on days -7, -5, -2, +3 and +6 from irradiation. STI-571 (Gleevec) was purchased from euromedex (#S2475) and diluted in water. Mice received intraperitoneal injection of 150 μΐ vehicle or gleevec (50 mg/kg) on the same days as thalidomide.

4. Antibodies and immunohistochemistry

For retina, the following primary antibodies were used: biotinylated Isolectin B4 (Invitrogen), rabbit anti-mouse Ki67 (cat # 66155, Abeam), rabbit anti-mouse cleaved caspase 3 (cat # 9664, Cell signaling), and FITC-anti alpha smooth muscle antibody (ccSMA) (#F3777, Sigma-aldrich). For brain, the following primary antibodies were used: Rat anti-mouse CD31 (cat # 553370 BD Bioscience), rabbit anti-mouse Glut 1 (cat # 07- 1401, Millipore), rat anti-mouse collagen IV (cat # 2150-1470, AbD Serotec). Secondary antibodies used were : Alexa fluor 488 Donkey anti-Rabbit IgG (1/250 ; A21206 ; Life technologie) ; Alexa fluor 555 Goat anti-Rat IgG (1/250 ;A21434 ; Life Technologie). Isolectin B4 was revealed with Cy2, Cy3 or Cy5 Streptavidin (1/100 ; GE Healthcare). 5. Morphometry and quantitative analysis

Specimen were analyzed with a confocal laser-scanning microscope inversed-SP5 (Leica). Confocal images of Immunohistochemistry results are presented as 3D reconstructions of z-stacks.

Image J software was used to measure the area of the pericytes soma, and their roundness was automatically calculated. Roundness formula as given by Image J software is "roundness" = 4 x area/(n x sqr(major axis). Density of pericytes in retina was calculated as the number of pericytes per field (magnification x 63) related to the vessel length measured by image J software.

6. Electrostimulation Whole retina were mounted and maintained in an extracellular solution (37 °C)(NaCl 140 mM; Glucose 25 mM; KC1 5.5 mM; CaC12 1.8 mM; MdC12 1 mM; Hepes 10 mM; ph = 7.3)). On whole mounted retinas, pericytes were patched with pipette filled with the same solution. Resistance of the pipette was measured before each experiment in order to define the input voltage (pulses 0.02 ms, 10 Hz for 5 s) to be applied so as to deliver a current intensity increasing from 1 to 7 μΑ. Pericytes were stimulated by applying voltage pulses 20 secondes after the patch. A total of 300 images was recorded in 2.5 minutes.

7. Quantitative PCR analysis

RNA was isolated from adult mice retinas using the RNeasy Mini Kit (74106, Qiagen). In vitro transcription reactions were performed from every RNA sample using Superscript III first-Strand Synthesis Kit for real-time (RT)-PCR (18080-051, Invitrogen) according to the manufacturer's instructions. The quantity of RNA extracted was assessed using a Biomate 3 Spectrophotometer. Real-time quantitative PCR (qPCR) reactions were performed in triplicate using the MylQTM real-time PCR system (Biorad). Each 25 μΐ reaction contained 5 ng cDNA, 12.5 μΐ iQTM SYBR® Green Supermix (170.8882, Biorad), 250 nM forward and reverse primers and nuclease free water. The following primers were used: for mouse PDGFR-β (QTOOl 13148); for PDGF-β, primer set (QT00266910); for Cspg4 (NG2), primer set (QT00120407) NG2; for cc-SMA, primer set (QT0088102); for RGS-5, primer set (QT00102592); for Desmine, primer set (QT00102333); for ALK5, primer set (QT100034117); for N-Cadherin, primer set (QT00148106); for Heyl, primer set (QT 00115094); for Hes, primer set (QT00313537); for Jag 1, primer set (QTOOl 15703); for VE-cadherin, primer set (QT00595840).

The data were normalized to a reference pool including mouse GAPDH and HPERT. Fold changes were calculated using the comparative CT method.

8. Intravenous injections ofBBB permeability tracer and detection of injected tracer. Lysine-fixable cadaverine conjugated to Alexa Fluor-555 (5 mg/ml in saline) (Invitrogen), was injected intravenously into the tail vein in adult (12 weeks) C57/B16 wt mice. Animals were perfused with HEPES buffer saline for whole brain examination, and with 4% paraformaldehyde for immunohistochemistry on brain sections and whole mounted retinas. Whole brains were examined under a fluorescent macroscope (Leica). Images were taken with the exposure time where the fluorescence signal deriving from the brain of irradiated (RT) animals was not saturated. Immunostainig of whole mounted retina and 100 μιη brain coronal sections were pictured with a confocal microscope (Leica). Brain sections visualizing cortex, corpus callosum and ventricules were selected. Dosage of Cadaverine Alexa Fluor-555 was 120 g/20 g for whole brain examination, and 500 g/20 g for immunohistochemistry. Quantification of Alexa Fluor-555 in retina was performed with image J software from confocal images that were all taken with the same photomultiplier gain.

9. Behavioural testing

For behavioural testings used for setting the irradiation model, groups of 5-6 animals were obtained for each dose of irradiation. Motor activity was scored after traction and stand up tests.

10. Statistical analysis

Statistical analyses were performed with Prism 4 software (GraphPad) using unpaired Mann Whitney test and Chi- square tests, p values < 0.05 were considered statistically significant. Results are given with 95 % CI.

Example 2: Social behavior of mice under Thalidomide treatment

The goal of this study is to analyze the social behavior of mice through a social interaction test. This test is based on a test designed by Cambon K et al (Neuroscience, 2010, 171 (3), 840-51).

In total, 80 mice are needed for this experiment, 40 are to be tested (4 groups of 10 mice) and 40 are used as interaction partners. The 4 groups are the followings: Group 1 without irradiation and thalidomide; Group 2 without irradiation but with thalidomide; Group 3 irradiated without thalidomide; Group 4 irradiated with thalidomide.

Before the study, the mice to be tested are isolated (individual cages) for 3 weeks. However, the interaction partner mice are raised in social cages.

Before the test, the mouse to be tested is introduced in a new and neutral environment (a Plexiglas cage) for 30 minutes. Then, a second mouse (the interaction partner), raised in social conditions, is introduced. The test lasts 8 minutes and begins when the mice are put in presence of each other. The second mouse has to be unknown from the first and to share the same genotype. The interactions between the 2 mice are filmed during 8 minutes.

The social behavior is analyzed manually and only the first 4 minutes are to be analyzed. The analysis is done by a behavioral specialist. The experimenter in charge of the experiments works in a blind manner (without knowing to which group the animals belong).

The following parameters are studied:

Total contact time. Number of pursuits.

Number of paw controls (the tested mouse put a fore paw on the back or the head of the other).

Number of bites and latency of the first attack (if any).

- Number of tail rattling.

Number of circling (around the second mouse).

Example 3: Spatial memory of mice under Thalidomide treatment

The goal of this study is to analyze the spatial working memory abilities of mice for a spontaneous alternation task in a T labyrinth. This test is based on a test designed by

Vandesquille M. et al (Psychopharmacology, 2011, 215, pp 709-720).

40 mice are needed for this experiment (4 groups of 10 mice, the same than Example 2).

Each mouse is tested for a week (7 trial a day) after a period of habituation to the dispositive. The period of habituation to the dispositive includes labyrinth habituation, opening and closing of the doors of the dispositive habituation. The Inter Trial Intervals

(ITI) time are variable but fixed: 5 sec, 30 sec or 120 sec.

The experimenter in charge of the experiments works in a blind manner (without knowing to which group the animals belong).

The following parameters are studied:

- Alternation percentage.

Choices delays.

Example 4: Long-term memory of mice under Thalidomide treatment

The goal of this study is to analyze the long-term memory abilities of mice in an object memory task. This test is based on a test designed by Wilkund A et al (Neuroreport, 2009, 20 (16), pp 1419-23).

40 mice are needed for this experiment (4 groups of 10 mice, the same than Example 2). Each mouse is subjected to a protocol including several successive conditions: dispositive habituation, being put in presence of 2 objects, being put in presence of a new object.

The mice to be tested have a period of dispositive habituation before testing. Then, they are subjected to the memory object test: 3 sessions of 5 minutes with 2 identical objects, then, 24 hours later, a session with a new object.

The experimenter in charge of the experiments works in a blind manner (without knowing to which group the animals belong). The following parameters are studied: - Contact time with objects.

Number of contacts with objects.

Claims

1- A compound selected from the group consisting of thalidomide, analogs thereof and pharmaceutically acceptable salts thereof for use for preventing in a patient a CNS complication/injury induced by central nervous system (CNS) irradiation.

2- The compound for use according to claim 1, wherein the CNS irradiation is a brain radiotherapy.

3- The compound for use according to anyone of claims 1-2, wherein the compound presents one of the followin formulae (I), (II) and (III)

(III)

wherein

X is C=0 or CH₂;

Ri is independently selected from -H, -OH, -CH3, -CH2OZ (ethers ), -CH2OCOZ (esters), -CH2OCONZ (carbamates ), or -CH₂Z (alkyls), wherein Z is selected from II or - (CH₂)n-H, where n is 1 - 10;

R: is selected from -H, -NH₂, O-Cs alkyl, -NH-NH2 (hydrazine), -NH-OH (hydroxalamine), -NH-OR5, -N=N-R₅, -NH₂, -N(R₅)₂, -NHCOH, -NHCOCH3, pyrazolidine, pyrazoline, tetrazole, imidazole, pyrazole, piprazine, or imidazoline, R5 being selected from the group consisting of pyrazol idine, tetrazole, and pyrazoline; R3 is selected from -H, C i -Cs alkvl, benzyl or halogen; and

R t is selected from -H. -Nl , -N H-NH2 (hydrazine), - N H-OH (hydroxalamine), -NH- OR5, -N=N-R₅, -NH2, -N(R5>2, -NHCOH, -NHCOCH3, pyrazolidine, pyrazoline, tetrazole, imidazole, pyrazole, piprazine, or imidazoline, R5 being selected from the group consisting of pyrazolidine, tetrazole, and pyrazoline.

4- The compound for use according to anyone of claims 1-3, wherein the compound is selected in the group consisting of thalidomide, lenalidomide, pomalidomide, CC-

10015 (Celgene), CC- 11006 (Celgene), 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3- yl)-piperidine-2,6-dione (Celgene), and pharmaceutically acceptable salts thereof.

5- The compound for use according to anyone of claims 1-4, wherein the compound is selected in the group consisting of thalidomide, pomalidomide and pharmaceutically acceptable salts thereof.

6- The compound for use according to anyone of claims 1-5, wherein the CNS complication/injury is a late CNS complication/injury.

7- The compound for use according to claim 5, wherein the late CNS complication/injury occurs at least six months after the beginning of the CNS irradiation treatment. 8- The compound for use according to anyone of claims 1-7, wherein the CNS complication/injury is a progressive subcortical dementia, especially characterized by psychomotor slowing, executive and memory dysfunction, behavioral changes, gait ataxia, and/or incontinence. 9- The compound for use according to anyone of claims 1-8, wherein the patient has a brain tumor, either a benign brain tumor such as glioma, meningioma, schwannoma, pituitary adenoma, hemanglioblastoma, craniopharyngioma, or preferably a malignant brain tumor such as glioma, glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, ganglioglioma, mixed glioma, malignant nerve sheath tumor, or brain metastasis of solid tumor or primary CNS lymphoma.

10- The compound for use according to anyone of claims 1-9, wherein the patient is selected among elderly patients, patients with cardiovascular risk, children, especially of less than 5 years old, adolescents, patients suffering of diabetes or high blood pressure, or patients having ApoE polymorphisms (e.g., the presence of the epsilon 4 allele of APOE).

1- The compound for use according to anyone of claims 1-10, wherein the compound > to be administered before, simultaneously and/or after the radiotherapy.

12- The compound for use according to claim 11, wherein the compound is to be administered during a period covering one, two, three or four weeks prior the radiotherapy and the radiotherapy duration, and optionally one, two, three or four weeks after the end of the radiotherapy.

13- The compound for use according to claim 11, wherein the compound is to be administered during a period covering one or two weeks prior the radiotherapy and the radiotherapy duration, and optionally one or two weeks after the end of the radiotherapy.

14- The compound for use according to anyone of claims 1-13, wherein the compound is to be administered during 5 to 9 weeks, preferably 6 to 8 weeks.

15- The compound for use according to anyone of claims 1-14, wherein the compound is used in combination with an antitumoral chemotherapy.

16- The compound for use according to anyone of claims 1-14, wherein the daily dose, by oral route, is comprised between 0.01 and 500 mg, preferably between 0.1 and 100 mg.