US20110046091A1 - Composition and methods used during anti-hiv treatment - Google Patents

Composition and methods used during anti-hiv treatment Download PDF

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US20110046091A1
US20110046091A1 US12/811,474 US81147408A US2011046091A1 US 20110046091 A1 US20110046091 A1 US 20110046091A1 US 81147408 A US81147408 A US 81147408A US 2011046091 A1 US2011046091 A1 US 2011046091A1
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acid
hiv
ionic form
inhibitor
cells
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Pierre Cau
Patrice Bourgeois
Vincent Bonniol
Nicolas Levy
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Aix Marseille Universite
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61K31/365—Lactones
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    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/665—Phosphorus compounds having oxygen as a ring hetero atom, e.g. fosfomycin

Definitions

  • the invention relates to an anti-HIV composition and processes for treating an HIV-infected patient.
  • This invention is, for example, very useful in the treatment of side effects caused by certain anti-HIV treatments, for example premature aging and lipodystrophy, which can be caused by protease inhibitors or reverse-transcriptase inhibitors.
  • references between parentheses refer to the list of references at the end of the examples.
  • the references between parentheses with the author's name and the date also refer to this list of references.
  • the nucleus of eukaryotic cells is delimited by a porous double membrane, the nuclear envelope, which controls molecular exchanges between the two nuclear and cytoplasmic compartments.
  • This envelope partially isolates the contents of the nucleus, i.e. the genetic material and all of the enzymatic machinery needed for the functions of the nuclear genome.
  • the nuclear envelope consists of two concentric membranes, the outer membrane, continuous with the endoplasmic reticulum, and the inner membrane. The latter is bordered on its inner face by a dense fibrillated mesh called the nuclear lamina. It is a protein network composed essentially of polymers of lamins and associated proteins. In the vertebrae, two subclasses of lamins are distinguished: lamins of type A (lamins A and C) and type B (lamins B1, B2 and B3), all of which are involved in the development of the lamina. The latter is held in place by the association with other proteins, attached to the inner membrane of the nuclear envelope (cf. Gruenbaum & al. 2005 (19)).
  • Lamins are filament-shaped proteins belonging to the family of intermediate filaments (type V), all of which have a common structure: a short globular N-terminal (head) segment separated from another globular C-terminal (tail) segment by a long central domain organized in a plurality of alpha helices (rod domain).
  • the globular tail contains in particular a nuclear localization signal (NLS) enabling the addressing to the nucleus after synthesis.
  • the central domain enables two parallel lamin molecules to be associated and organized in filaments by a “head-to-tail” association of dimers. This structure gives them very resistant mechanical properties.
  • Lamin C is directly synthesized in its mature form.
  • the precursor of lamin A and lamins B is terminated by a characteristic CaaX motif (C is a cysteine, a is an amino acid with an uncharged aliphatic chain and X is any amino acid, in this case a methionine; cf. Levy & Cau 2003 (29)).
  • the C-terminal CaaX motif enables the attachment of a fatty acid (in general a C15 fatty acid, farnesyl) owing to a farnesyl-transferase.
  • This prenylation (the farnesyl motif is derived from a C5-based aliphatic unit called isoprene) enables the prelamins to be inserted in the membrane of the endoplasmic reticulum after their synthesis in cytosol. They are subjected to the action of an endoprotease itself inserted into the envelope membrane of the reticulum and of which the active site is cytosolic.
  • prelamin A The specific endoprotease of prelamin A is Face1 (or ZMPSTE24, Zinc Metallo-Protease, an STE24 yeast homolog), while Face2 (or Rce1, ras-converting enzyme) is specific to prelamins B. These enzymes catalyze the hydrolysis of the peptide bond between the cysteine and the next (aliphatic) amino acid, shortening the prelamins of 3 amino acids. The carboxyl end of the farnesylated cysteine is then recognized by an isoprenylcysteine-carboxymethyl transferase (ICMT), which attaches a methyl group thereto by esterification.
  • ICMT isoprenylcysteine-carboxymethyl transferase
  • prenylation we mean the attachment to the thiol group of a cysteine, either by a farnesyl chain with 15 carbon atoms, thus farnesylation, or by a geranyl-geranyl chain of 20 carbon atoms, thus geranyl-geranylation (Reid & al. 2004 (39)), or by any other isoprene derivative.
  • Farnesylation catalyzed by farnesyl-transferase (FTase), which recognizes the C-terminal consensus sequence (CaaX), preferably binds a farnesyl group to the cysteine residue of the motif.
  • Geranyl-geranylation is the attachment by Geranyl-geranyl-transferase (GGTase) of a Geranyl-geranyl group on the cysteine residue of the motif.
  • GTTase Geranyl-geranyl-transferase
  • fatty acids are produced by biosynthesis, which on the basis of hydroxymethyl-glutaryl-Coenzyme A, is used by the cells to produce in particular cholesterol, steroids, the heme of hemoglobin and ubiquinones (Hampton & al. 1996 (20)).
  • the family of prenylated proteins comprises around 300 members in the human genome, of which the majority can be identified by the C-terminal motif CaaX (Reid & al. 2004 (39)).
  • the proteins of the Ras, Rho and Rab families (Leung & al. 2006 (28)), certain proteins ensuring an import function to the mitochondria (HDJ2), and certain mitotic proteins (CENPE, CENPF) are in particular prenylated (Winter-Vann & Casey 2005 (51)).
  • X is a serine, a methionine, a cysteine, an alanine or a glutamate
  • the isoprenoid preferably grafted is farnesyl.
  • the LMNA gene located at 1q21.2-q21.3 (Wyder & al. 1996 (52)), gives lamins A and C by alternative splicing.
  • the LMNA gene is composed of 12 exons.
  • the start of the exon 1 codes the globular N-terminal end common to lamins A and C; the end of exon 1 and up to the start of exon 7 code the central helical part; finally, the other exons code the globular C-terminal end (Levy & Cau 2003 (29)).
  • the gene codes for 4 products spliced differently, the 2 main ones being the lamins C and the prelamin A (Lin & Worman 1993 (31)).
  • the differential production of lamins A and C is done by using an alternative splicing site at the level of the exon 10 of the pre-messenger, so that the lamin C is coded by exons 1 to 10 and lamin A is coded by exons 1 to 9, the first 90 base pairs of exon 10, and exons 11 and 12 (lamin Aspecific).
  • the prelamin A and lamin C peptides are identical at the level of the first 566 amino acids, while the C-terminal ends of the lamins C and the prelamin A then contain, respectively, 6 and 98 specific amino acids.
  • the lamins of type B include three different proteins (Shelto & al. 1981 (43)): lamins B1, B2 (the two isoforms best represented) and B3.
  • the LMNB1 gene is located at 5q23.3-q31.1 and comprises 11 exons coding the lamin B1 (Lin & Worman 1995 (30)).
  • the LMNB2 gene is localized at 19p13.3 and codes for lamins B2 and B3 by an alternative splicing mechanism (Biamonti & al. 1992 (2)).
  • Lamins B are constitutively expressed in all of the cells from the first stages of development, while lamins of type A are generally absent from the embryonic stem cells (Stewart et al. 1987 (45)) and are expressed in all of the differentiated somatic cells. Their expression is subject to regulations according to the tissue and the life course (Duque & al. 2006 (9)). It appears that their expression is not necessary, since mice in which the expression of lamin A was specifically blocked, but which still express lamin C and other lamins, do not have an apparent phenotype (Fong & al. 2006 (14)).
  • Lamins interact with a very large number of protein partners having a wide variety of functions; they are consequently involved in a large number of nuclear processes, including DNA replication and repair, control of transcription and splicing, and organization of the chromatin structure (cf. Shumaker & al. 2003 (44), Zastrow & al. 2004 (54), Hutchison & al. 2004 (26), Gruenbaum & al. 2005 (19)).
  • the alterations of the structure of the lamina are at the origin of numerous human hereditary pathologies. They are due to mutations of genes coding the lamins, or other proteins of the lamina. These pathologies have been grouped together under the generic term laminopathies (Broers & al. 2006 (5), Mattout & al.
  • RNAi RNA-interference
  • murine model Vergnes & al. 2004 (50)
  • lamins of type B are essential for cell development and integrity.
  • a lamin B1 deficiency causes perinatal lethality in mice.
  • nuclei of the embryonic fibroblasts of the same LMNB1-deficient mice show remarkable alterations in nuclear morphology, similar to those observed in patients with LANA gene mutations.
  • lamins B are necessary for the formation of the division spindle during mitosis, which tends to prove that they have a dynamic and multifaceted role over the course of the cell cycle, and that their role is not restricted to maintaining the nucleus architecture (Tsai & al. 2006 (48)).
  • a recent article demonstrates the structural function of lamins B: cells artificially deprived of lamins B1 have a “floating” nucleus in the cell, which turns around (Liu & al. 2007 (45)).
  • the functional redundancy existing between the two lamins B1 and B2 is undoubtedly also a direct reflection of their importance, exerting a strong selection pressure and masking the effect of any potential mutations in the sequence of the corresponding genes.
  • the functional alterations of lamins A/C, due to mutations of the LMNA gene, are the origin of at least 15 disorders including a wide range of pathologies in a clinical spectrum ranging from mild forms, affecting a single tissue in isolation, to systemic forms that are lethal in the perinatal period.
  • a number of mutations of the LMNA gene notably modify the assembly of proteins in the nuclear envelope and disrupt its functioning. In the cells of various tissues, the morphology of the nuclei is altered: they often have bulges that extrude genetic material into the cytoplasm (Goldman & al. 2004 (18)).
  • lamins B The proteins normally associated with the nuclear envelope, lamins B, certain nuclear pore proteins and LAP2 proteins, are absent from the edge these bulges. These morphological abnormalities are followed by functional alterations, and end by causing cell death. Among all of the pathologies included under the term laminopathies, only those associated with the abnormal accumulation of a prenylated protein form are concerned by this invention.
  • Hutchinson-Gilford or Progeria syndrome (De Sandre-Giovannoli & al. 2003 (7), (Eriksson & al. 2003 (11)), and restrictive dermopathy (Navarro & al. 2004 (36)).
  • Hutchinson-Gilford or Progeria syndrome (De Sandre-Giovannoli & al. 2003 (7), (Eriksson & al. 2003 (11)), and restrictive dermopathy (Navarro & al. 2004 (36)).
  • the physiopathological cause is an accumulation and persistence of immature farnesylated prelamin in the cells of patients.
  • Restrictive dermopathy lethal around the natal period, is characterized by clinical signs that are almost always the consequence of a cutaneous deficit that restricts in utero movements. This pathology is very rare.
  • the skin is rigid and taut, and gives way in places, causing, for example, tears at the armpits or the neck.
  • the eyelashes, the eyebrows and the skin down are absent or very sparse. Hydramnios is often present, and the decrease in fetal movements is observed from the 6 th month of pregnancy.
  • the radiography shows contractures of all joints, congenital convex pes valgus, thin, dysplasic and bi-partite clavicles, thin ribs, long tubular arm bones and demineralization of the skull.
  • the mutation considered responsible located at exon 11 of the LMNA gene, activates a cryptic splicing site of the pre-mRNA, leading to an mRNA with 150 nucleotides deleted (De Sandre-Giovannoli & al. 2003 (7), Eriksson & al. 2003 (11)).
  • This deleted mRNA involves an abnormal prelamin A, progerin, which cannot mature into a normal lamin A: the absence of 50 amino acids of the exon 11 comprising the protease recognition site blocks the 2 nd cleaving of the progerin, of which the C-terminal end preserves its farnesyl group.
  • the second study shows that the treatment of cells of HGPS patients by morpholino (antisense oligonucleotides) targeting the cryptic splicing site eradicates the mutant phenotype (Scaffidi & Misteli 2005 (43)).
  • progerin is also synthesized by a subpopulation of dermal fibroblasts and keratinocytes, cells in which it accumulates with age. Progerin could therefore be a marker of skin aging (McClintock et al. 2007 (34)).
  • the prior art describes two therapeutic approaches to improving the cell phenotype caused by the pathological production of progerin.
  • the first of these solutions is very simply to prevent the use by the spliceosome of this cryptic splicing site in exon 11, by “masking” it by a treatment with an antisense oligonucleotides (Scaffidi & Misteli 2005 (41)), or with a retrovirus producing an siRNA (Huang & al. 2005 (25)).
  • the results are promising in vitro, but this is “gene” therapy, and the development of a drug based on this approach is extremely long and complicated, with all of the inconveniences associated with OAS vectorization in order to obtain an in vivo effect.
  • the second solution consists of inhibiting farnesyl-transferase, the enzyme that catalyzes the transfer of the farnesyl group to the prelamins from farnesyl-pyrophosphate.
  • FTI farnesyl-transferase
  • HGPS Progeria
  • FTIs are specific to only one of the protein prenylation pathways, and cannot be envisaged as general post-translational prenylation inhibitors.
  • mevalonate-kinase is lethal during infancy (homozygotic mutation loss-of-function of the gene coding for this enzyme, syndrome reported by Hoffmann & al. 2003 (24)).
  • HIV-infected patients subjected to an antiretroviral treatment show clinical and biological signs of accelerated aging comparable to that observed in patients with a genetic progeroid syndrome.
  • OMIM 277700 mutated helicase in Werner syndrome
  • OMIM 277700 a premature aging syndrome associated with a predisposition to cancer and atherosclerosis
  • OMIM 277700 recruits cell protein cofactors essential for the transactivation of the LTR of HIV-1 and to the replication of the virus.
  • the slightest availability of helicase in infected cells could lead to aging and immunosuppression (Sharma et al., 2007).
  • Another example is modifications of the transport of cholesterol in macrophages.
  • the viral protein Nef inhibits permeases of the ABC family responsible for cholesterol efflux (Bukrinsky and Sviridov, 2006; Mujawar et al., 2006; Wang and Rader, 2007).
  • the accumulation of cholesterol in macrophages transforms them into foam cells involved in the formation of atheromatous plaques in the vascular walls (Pennings et al., 2006).
  • Antiretroviral drugs also inhibit the cholesterol efflux of macrophages and contribute to the formation of atheromatous plaque (Azzam et al., 2006; Dressman et al., 2003; Wang et al., 2007).
  • OMIM 176670 Hutchinson-Gilford Progeria
  • OMIM 248370 acromandibular dysplasia
  • OMIM 275210 restrictive dermopathy associated with mutations in the LMNA gene coding for lamins A and C or with mutations in the ZMPSTE24 protease (FACE1) responsible for cleavage of prelamin A during its maturation into lamin A:
  • the three main components of the mitochondrial theory of aging are i/the increase in production of reactive oxygen species (ROS) by complexes I and II of the respiratory chain, ii/progressive dysfunctioning thereof, and iii/accumulation of mitochondrial DNA lesions (Balaban et al., 2005; Meissner, 2007; Wallace, 2005).
  • ROS reactive oxygen species
  • the mitochondrial DNA is more sensitive than nuclear DNA to the effects of ROS, which target the DNA polymerase gamma (Graziewicz et al., 2002; Richter et al., 1988).
  • aging is accompanied by a decrease in mitochondrial functions, including those of the respiratory chain (Short et al., 2005; Trifunovic et al., 2005), and an increase in deletions in the mitochondrial DNA, very specifically observed in neurodegenerative diseases (Bender et al., 2006; Kraytsberg et al., 2006).
  • a mitochondria-to-nucleus signaling mechanism which is not yet understood, causes the protein p32 to become involved, in particular during aging or in ROS-induced pathologies (Jiang et al., 1999; Storz, 2006).
  • This protein coded by the nuclear genome, then imported into the mitochondrial matrix, is re-exported to the nucleoplasm (Brokstad et al., 2001) where it controls transcription (Chattopadhyay et al., 2004) and splicing (Petersen-Mahrt et al., 1999) of mRNA. It interacts with the lamin B receptor, localized the nucleoplasmic face of the nuclear envelope (Mylonis et al., 2004; Simos and Georgatos, 1994).
  • the nucleus-to-mitochondria signaling mechanism involves the around 1500 proteins coded by the nuclear genome, which are synthesized in the cytosol, then imported into the mitochondria (Calvo et al., 2006; Truscott et al., 2003).
  • Protein p53 is involved in accelerated aging in a murine model (Tyner et al., 2002) and participates in deregulations in the context of the “nuclear” theory (see below).
  • Protein p53 controls mitochondrial respiration (Matoba et al., 2006), and exerts pro- and anti-oxidant effects owing to the control of nuclear gene transcription (Bensaad and Vousden, 2005; Sablina et al., 2005). It maintains the stability of the mitochondrial DNA by interacting with DNA polymerase gamma (Achanta et al., 2005).
  • mitochondrial fission and fusion mechanisms involving a plurality of GTPases of the outer mitochondrial membranes (Drpl, mitofusins, etc.) and inner mitochondrial membranes (OPA1) and a plurality of associated proteins (Fis1, etc.) modulate the cell aging process: the elongation of the mitochondrial network by permanent fusion induces a cell aging process with a decrease in the difference in potential between the two faces of the inner membrane, and increase in the production of ROS and DNA lesions.
  • These phenotype modifications are neutralized by fragmentation of the mitochondrial network (Lee et al., 2007).
  • Lamins are proteins forming intermediate filaments localized exclusively in the nucleoplasm.
  • the LMNA gene codes primarily for lamins A and C, produced by alternative splicing. Two other genes code for lamins B1 and B2.
  • Lamins A and B are synthesized in the cytosol in the form of a precursor that is subject to a plurality of maturation steps.
  • the 3 proteins have, at their C-terminal end, the CaaX box (a cysteine, two aliphatic amino acids, one indifferent amino acid).
  • This CaaX sequence enables the farnesylation of lamins A and B (and around 300 other proteins of the human genome).
  • the farnesyl residue is synthesized in an intermediate step of the cholesterol synthetic pathway (appended FIG. 7 ).
  • a cytosolic enzyme, farnesyl-transferase binds the farnesyl residue to the cysteine.
  • prelamins A and B are then cleaved by FACE1 (ZMPSTE24) or FACE2 (Rce1), respectively, and lose their last three C-terminal amino acids aaX.
  • FACE1 ZMPSTE24
  • FACE2 FACE2
  • ICMT second enzyme
  • lamins B ends with this step.
  • These proteins glide in the plane of the envelope membrane of the ER, then of the cytosolic face of the nuclear envelope, pass through the nuclear pore and are localized in the nucleoplasmic layer of the nuclear envelope, where they become involved in the formation of the nuclear lamina and where they interact with a plurality of proteins, including the lamin B receptor.
  • the anchoring to the envelope results in the absence of lamins B in the nuclear matrix, inside the nucleoplasm and at a distance from the envelope.
  • the farnesylated and carboxymethylated prelamin A on the same C-terminal cysteine is subjected to a final maturation step by proteolytic cleavage of the last 15 amino acids by FACE1 (and perhaps by Rce1).
  • the lamin A loses its anchoring to the ER, becomes soluble, and is imported into the nucleoplasm (like lamin C) through the nuclear pore, like all of the soluble proteins, owing to its nuclear localization signal, which recruits the necessary importation complex (appended FIG. 8 ).
  • lamin A (and lamin C) is localized in the nuclear lamina, under the nuclear envelope, where it interacts with numerous proteins inserted in the nucleoplasmic layer of the envelope.
  • Lamins A and C (unlike lamins B) are also dispersed in the rest of the nucleoplasm where they become involved in the formation of the nuclear matrix or nucleoskeleton (appended FIG. 6 ).
  • the nuclear matrix controls the functioning of the nuclear genome: replication, DNA repair, RNA transcription, mRNA splicing, maturation of other RNA, etc., and its constituents, including lamins A and C, interact with a large number of proteins imported into the nucleoplasm (p53, SREBP, etc.) as shown by the abnormalities observed in genetic laminopathies (Broers et al., 2006; Vlcek et al., 2001; Vlcek and Foisner, 2006).
  • OMIM 248370 Hutchinson-Gilford progeria
  • OMIM 176670 Hutchinson-Gilford progeria
  • OMIM 275210 restrictive dermopathy
  • the mutation of the LMNA gene causes a disappearance of the site of recognition by FACE1 of prelamin A: the protein is not cleaved, and therefore preserves its farnesyl residue, and is imported into the nucleoplasm by gliding along the envelope membranes like lamins B (Pan et al., 2007).
  • the farnesylated prelamin A remains anchored in the nucleoplasmic face of the nuclear envelope where it disrupts the organization of the lamina and the interactions between the lamina and the membrane proteins of the envelope, which involves characteristic nuclear deformations (Goldman et al., 2004).
  • the rest of the nuclear matrix is free of lamin A, with multiple functional consequences, for example the activation of pathways downstream of p53 (Varela et al., 2005), abnormalities of mitosis (Cao et al., 2007; Dechat et al., 2007), and of DNA repair (Liu et al., 2005; Liu et al., 2006; Liu et al., 2007b), etc., leading to cell aging (Kudlow et al., 2007).
  • the analysis of the transcriptome of cells of progeria patients has shown abnormalities in the expression of genes involved in atherosclerosis (Csoka et al., 2004).
  • the mutation of the LMNA gene responsible for progeria unmasks a cryptic splicing site that leads to the synthesis of an mRNA deleted of 150 nucleotides coding for progerin, a prelamin A deleted of 50 amino acids and which preserves its farnesyl group.
  • This same protein is produced during physiological aging, in the absence of any mutation of the LMNA gene, due to an error, associated with age, of the splicing machinery (Scaffidi and Misteli, 2006).
  • Progerin is accumulated in dermal fibroblasts localized under the basal lamina, as well as in the keratinocytes. It could be a marker of cutaneous aging (McClintock et al., 2007).
  • the nuclear modifications (bulges of the nucleoplasm into the cytosol, breaching of the envelope, nuclear contour irregularities, etc.) observed in the cells of patients with progeria are also similar to the transient modifications induced by the viral protein Vpr, which facilitates the entrance, by transient breaching of the nuclear envelope and the lamina, of the pre-integration complex of the AIDS virus, which complex is too large to be imported into the nucleoplasm via the nuclear pores (de Noronha et al., 2001). For a recent review of these nuclear importation mechanisms (Suzuki and Craigie, 2007).
  • genetic laminopathy therefore covers the pathologies resulting from mutations in the genes coding for the lamins, very specifically LMNA coding for lamins A and C, but also those caused by mutations of associated proteins, including those participating in lamin maturation.
  • the abnormalities induced by PIs at the level of the nuclear matrix therefore represent an acquired and iatrogenic laminopathy. Whether they are genetic or acquired, these abnormalities in the nuclear matrix result in genomic instability and cell aging (Mehta et al., 2007; Oberdoerffer and Sinclair, 2007).
  • telomere aging is controlled by a “clock” measuring the length of telomeres, which are shortened in each mitosis, a phenomenon that can be counterbalanced by the activity of the catalytic telomerase subunit (Gilson and Geli, 2007; Stewart and Weinberg, 2006).
  • telomere length was measured in particular in mononucleated blood cells.
  • telomerase activity was performed on cells of patients treated with antiretrovirals, with variable results, in particular depending on the T-lymphocyte subtype analyzed (Effros, 2000).
  • telomere activity inhibits telomerase activity (Olivero, 2007; Yamaguchi et al., 2001).
  • telomere shortening Abnormalities in lamin A cause a reduction in telomerase activity, resulting in accelerated telomere shortening. These effects are observed in the cells of patients with progeria, whether or not the cells had been transfected to overexpress the catalytic telomerase subunit (Wallis et al., 2004). Comparable results were obtained in fibroblasts of healthy subjects overexpressing, after transfection, lamin A, normal, mutated or deleted, as in progeria (Huang et al., 2008). The nuclear matrix and the proteins of the nuclear envelope associated with it at the level of the lamina therefore directly or indirectly control telomerase activity (Pandita et al., 2007).
  • Certain Transcription Regulation Factors in Particular p53 and NF- K B, are Involved in Aging or regulation thereof.
  • the protein p53 is also involved in the abnormalities induced by laminopathies (Varela et al., 2005), in the functioning of helicase, the mutation of which is responsible for Werner syndrome (Brosh et al., 2001; Sommers et al., 2005), telomere regulation (Wynford-Thomas, 1996), and control of mitochondrial respiration (Matoba et al., 2006) independently of the role of p53 in the activation of apoptosis by its interaction with apoptogenic molecules of the outer membrane (Manfredi, 2003; Mihara et al., 2003; Moll et al., 2005). p53 also regulates the “insulin-IGF1-klotho” metabolic pathway (see below).
  • NF- K B is also involved in many metabolic pathways. Its inactivation by RNA interference in fibroblasts in culture protects them from aging (Hardy et al., 2005). Similarly, the blockage that can be induced by the expression of NF- K B in mouse epidermal cells slows their aging via variations in the expression of numerous genes analyzed by microarray (Adler et al., 2007).
  • Caloric restriction increases the lifespan of rodents (Borone and Guarente, 2005). It activates deacetylase sirtuin, which targets histones (with remodeling of the chromatin) (Vijg and Suh, 2006), numerous other nuclear proteins (transcription factors including p53, proteins involved in DNA repair, etc.) as well as cytosolic (tubulin) and mitochondrial proteins (Guarente and Picard, 2005; North and Verdin, 2004; Porcu and Chiarugi, 2005).
  • Caloric restriction causes biogenesis of mitochondria via the expression of endothelial NO-synthase, eNOS (Nisoli and Carruba, 2006; Nisoli et al., 2005), resulting in an increase in ROS production (Gredilla and Barja, 2005).
  • eNOS endothelial NO-synthase
  • ROS ROS production
  • Caloric restriction also inhibits the signaling pathways regulated by the klotho hormone (Kurosu et al., 2005; Unger, 2006), of which mutations are accompanied by premature aging in the mouse (Kuro-o et al., 1997).
  • Klotho is a regulator of the response induced by the FGF-receptor (Kurosu et al., 2006).
  • the mutations of klotho and FGF-23 induce the same premature aging phenotype, which is known to be caused by hypervitaminosis D and which is corrected by the deletion of a gene increasing the effect of vitamin D (Razzaque and Lanske, 2006).
  • Klotho also blocks the response of Wnt receptors (Liu et al., 2007a), which shows the diversity of the metabolic pathways involved in aging. Finally, insulin stimulates, via phosphatidyl-inositol 3 kinase, the cleavage of the transmembrane precursor of klotho by metalloproteases of the ADAM family and the release of klotho into the extracellular medium (Chen et al., 2007).
  • the protein p53 is also involved in the regulation of these metabolic pathways (Campisi, 2004; de Oliveira, 2006; Kim et al., 2007; Schmid et al., 2007).
  • the protein p66 shc represents one of the communication signals between the plasma membrane receptors for insulin and for IGF1, the nucleus and the mitochondria (Martin and Friedman, 2004).
  • KO p66 shc ⁇ / ⁇ mice show high resistance to stress and an increase in their lifespan (Migliaccio et al., 1999).
  • P66 shc is a target of p53, localized in the mitochondria of which it controls the metabolism and ROS production (Migliaccio et al., 2006; Nemoto et al., 2006; Orsini et al., 2004; Trinei et al., 2002).
  • a deletion of p66 shc inhibits aging of cardiac stem cells and prevents cardiac accidents, two phenomena induced by diabetes (Rota et al., 2006).
  • P66 shc is also a redox enzyme. In response to cell stress, p66 shc is imported into the mitochondria and is localized in the intermembrane space where it synthesizes H 2 O 2 (i.e.
  • H 2 O 2 may represent an intracellular signal for lifespan regulation (Giorio et al., 2007).
  • the inventors also observed that the association of a hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and a farnesyl-pyrophosphate synthase inhibitor has an additive effect in the restoration of the normal phenotype in fibroblasts of patients with Progeria.
  • the effect of the association is clearly superior to the effect of one or the other of the inhibitors used individually (Varela et al. 2008 (54 bis)).
  • the use of the association on cells of patients with Progeria leads to an inhibition in the prenylation of proteins, and therefore the appearance of non-farnesylated prelamin A and the improvement of nuclear symptoms and the partial correction of anomalies of DNA repartition.
  • Antiretroviral treatments target a plurality of metabolic pathways and a plurality of cell compartments, the cytosol, the nucleoplasm and the mitochondria.
  • lamin A causes deleterious consequences at the level of a number of nuclear metabolic pathways (DNA replication and repair, gene transcription, telomere shortening, etc.), and also have deleterious consequences in other cell compartments, including the mitochondria.
  • the general dysfunction of the cell causes accelerated aging and a reduced lifespan thereof.
  • FIG. 9 schematically shows the aging theory developed by the present inventors and based on abnormalities in the maturation of prelamin A and their functional consequences.
  • the objective is therefore to analyze the mechanisms by which these two types of antiretroviral treatment cause accelerated aging in patients infected with the AIDS virus and to verify that these mechanisms are comparable to those causing accelerated aging in patients with genetic laminopathy.
  • This composition can be intended, for example, for treating situations, pathological or not, associated with the accumulation and/or persistence in the cells of prenylated proteins.
  • this composition is useful in the treatment of an HIV-infected patient.
  • This invention also relates to a composition according to the invention in which the anti-HIV agent is an antiretroviral agent or a mixture of antiretroviral agents.
  • This invention also relates to a composition according to the invention in which the anti-HIV agent is a protease inhibitor or a reverse transcriptase inhibitor.
  • This invention also relates to a composition according to the invention in which the anti-HIV agent is a protease inhibitor chosen from the group including fosamprenavir, lopinavir, ritonavir, amprenavir, atazanavir and indinavir.
  • the anti-HIV agent is a protease inhibitor chosen from the group including fosamprenavir, lopinavir, ritonavir, amprenavir, atazanavir and indinavir.
  • This invention also relates to a composition according to the invention in which the anti-HIV agent is a reverse transcriptase inhibitor chosen from the group including zidovudine, lamivudine, didanosine and epzicom.
  • the anti-HIV agent is a reverse transcriptase inhibitor chosen from the group including zidovudine, lamivudine, didanosine and epzicom.
  • the anti-HIV agent can also be an association of one or more antiproteases of the virus and/or one or more reverse transcriptase inhibitors of the virus and/or one or more inhibitors of the entrance of the virus into the cells and/or one or more integrase inhibitors and/or any other treatment having an antiviral effect, in particular any treatment recognized by the national and/or international regulatory institutions and the scientific community.
  • This invention also relates to a composition according to the invention in which compounds are used that are both hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors and farnesyl-pyrophosphate synthase inhibitors.
  • HMG-CoA hydroxymethylglutaryl-coenzyme A
  • composition according to the invention is intended to treat an HIV-infected patient developing side effects associated with the accumulation and/or persistence of progerin in the cells; even more specifically, the treatment of situations associated with the accumulation and/or persistence, in the cells, of farnesylated prelamin A, whether or not it is truncated or modified.
  • any farnesyl-pyrophosphate synthase inhibitor or one of its physiologically acceptable salts can be used in the preparation of the composition according to the invention.
  • the physiologically acceptable salts can be, for example, salts formed with hydrochloric, hydrobromic, nitric, sulfuric or phosphoric acid, carboxylic acids such as, for example, acetic, formic, propionic, benzoic, maleic, fumaric, succinic, tartric, citric, oxalic, glyoxylic, aspartic acid, sulfonic alkanes acid such as sulfonic methane or ethane acids, and arylsulfonic alkanes such as sulfonic benzene or paratoluene acid.
  • carboxylic acids such as, for example, acetic, formic, propionic, benzoic, maleic, fumaric, succinic, tartric, citric, oxalic, glyoxylic, aspartic acid, sulfonic alkanes acid such as sulfonic methane or ethane acids, and arylsulfonic alkan
  • the farnesyl-pyrophosphate synthase inhibitor can be one of the members of the family of polyphosphonates, particularly aminobiphosphonates (NBP), or one of its physiologically acceptable salts.
  • NBP aminobiphosphonates
  • Polyphosphonates are synthetic molecules commonly used in the treatment of osteoporosis and bone regeneration.
  • phosphonate applies to molecules very similar to phosphate:
  • BP biphosphonates
  • a simple biphosphonate would be equivalent to ADP, with the 2 phosphate groups (O 3 P ⁇ ) being replaced by the biphosphonate group.
  • the central carbon unlike the oxygen of the phosphates, can again be involved in 2 bonds, and it is the nature of the groups grafted onto this carbon that constitutes the specificity of the biphosphonates.
  • lateral chains comprise an amine function (NH), or more generally one or more nitrogen atoms, we refer to amino-biphosphonate, or NBP.
  • aminobiphosphonate farnesyl-pyrophosphate synthase inhibitor
  • aminobiphosphonate farnesyl-pyrophosphate synthase inhibitor
  • zoledronic acid also called zolendronic acid
  • zoledronate also called zolendronate
  • any HMG-CoA reductase inhibitor, or one of its physiologically acceptable salts can be used in the preparation of the composition.
  • the HMG-CoA reductase inhibitor can be a molecule of the statin family, whether it is liposoluble or hydrosoluble, or one of its physiologically acceptable salts.
  • Statins have been identified in fungi. They have an inhibiting activity on HMG-CoA reductase, a key enzyme in the biosynthesis of cholesterol and steroids, which catalyze the reduction of hydroxymethylglutarate coupled to Coenzyme A in mevalonic acid (mevalonate in solution). This inhibition is ensured by their structural similarity with the hydroxymethylglutarate skeleton.
  • the metabolic pathway involved is admittedly that of cholesterol biosynthesis, but it is also that of the synthesis of prenyl groups, polymers of the 5 isoprene carbon base unit used to modify around 300 proteins in the cells and attach a lipophilic tail, enabling in particular their anchoring in the membranes.
  • the main polyprenes are geranyl (C10), farnesyl (C15) and geranyl-geranyl (C20). All of the statins are globally hepatoselective, but all do not have the same mode of entry into the cells. Indeed, pravastatin and rosuvastatin are both hydrophilic, and therefore hydrosoluble, unlike all of the others, which are lipophilic, and therefore can diffuse freely through the plasma membranes (lipid bilayers), which undoubtedly explains their higher toxicity.
  • the hydrosoluble statins need a specific transporter in order to enter the cell, Organic Anion Transporter 3, or OAT3, or SLC22A8 (Takedaa & al. 2004 (46)).
  • a statin can be chosen from atorvastatin, simvastatin, pravastatin, rivastatin, mevastatin (or compactin), fluindostatin, velostatin, fluvastatin, dalvastatin, cerivastatin, pentostatin, rosuvastatin, lovastatin, pitavastatin, or one of their physiologically acceptable salts.
  • Lovastatin, pravastatin and simvastatin are molecules derived from fungal metabolites, while the others (atorvastatin, cerivastatin, fluvastatin, pitavastatin and rosuvastatin) are entirely synthetic.
  • pravastatin, a semi-natural, hydrosoluble statin is used.
  • the composition can be intended both for the treatment of an HIV-infected patient and the treatment of side effects of the anti-HIV treatment, for example skin aging, body or head hair loss, osteoporosis and lipodystrophy.
  • the farnesyl-pyrophosphate synthase inhibitor and the HMG-CoA reductase inhibitor are advantageously present in the composition at physiologically effective doses.
  • the amounts to be administered can be adapted according to the patient, the pathology, the mode of administration, and so on. It is understood that repeated uses are possible, optionally in combination with other active ingredients or any carrier.
  • the daily dose of inhibitors will be the minimum dose needed to obtain the desired effect.
  • the hydroxymethyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, the farnesyl-pyrophosphate synthase inhibitor, and the anti-HIV agent can be used in the composition, in a mixture with one or more inert, i.e. physiologically inactive and non-toxic, excipients or carriers. It is possible to cite, for example, the ingredients normally used in drugs intended to treat HIV-infected patients and accompanying the anti-HIV agent.
  • composition of this invention can also include at least one other active ingredient, particularly another therapeutically active ingredient, for example for a simultaneous or separate use or a use spread out over time according to the galenic formulation used.
  • This other ingredient can be, for example, an active ingredient used, for example, in the treatment of opportunistic diseases that can develop in an HIV-infected patient.
  • the composition of this invention is a composition that can be used both to treat an HIV-infected patient and to prevent and/or treat skin disorders caused by the use of the anti-HIV agent.
  • This composition can be used as part of a multitherapy for an HIV-infected patient.
  • the anti-HIV agent can be a single agent or multiple agents (a mixture of a plurality of anti-HIV agents). In the case of multitherapy (for example, di-, tri- or quadritherapy), the mixture of inhibitors can accompany one or more of the anti-HIV agents.
  • the anti-HIV agent can also be an association of one or more antiproteases of the virus and/or one or more reverse transcriptase inhibitors of the virus and/or one or more inhibitors of the entry of the virus into the cells and/or one or more integrase inhibitors and/or any other treatment having an antiviral effect, in particular any treatment recognized by the national and/or international regulatory institutions and the scientific community.
  • This invention therefore also relates to a process for treating an HIV-infected patient, including the administration of a composition according to the invention.
  • the composition of the invention is as defined above.
  • the administration can be performed according to any of the routes known to a person skilled in the art for administering an anti-HIV composition. It can be, for example, an oral administration or an injection.
  • the administered dose of the composition of the invention depends upon the patient's needs and is also determined by taking into account what is physiologically acceptable by the patient.
  • the quantity of inhibitors in the composition of this invention can be such that it enables, as an example, the administration of a dose of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor of 0.01 to 2 mg/kg of body weight and a dose of farnesyl-pyrophosphate synthase inhibitor of 0.01 to 40 mg/kg of body weight.
  • HMG-CoA hydroxymethylglutaryl-coenzyme A
  • the ratio of farnesyl-pyrophosphate synthase inhibitor to hydroxymethyl glutaryl coenzyme A reductase inhibitor, or one of their physiologically acceptable salts can be between 0.01 and 0.2, and preferably 0.05 to 0.35.
  • the quantity of anti-HIV agent in the composition of this invention is conventionally determined according to the current knowledge of a person skilled in the art in the treatment of HIV-infected patients. It can be chosen, for example, from those conventionally used in HIV-infected patients.
  • anti-HIV agent concentrations for each of the anti-HIV agent examples described in this document and each of the mixtures or associations of anti-HIV agents described in this document are provided in the VIDAL Dictionary (registered trademark), for example Edition 2007. The concentrations indicated in this Dictionary are also those authorized for humans.
  • This invention also relates to a process for treating side effects of premature aging and/or lipodystrophy caused in a patient undergoing an anti-HIV treatment, which process includes the administration of a mixture including at least one hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and at least one farnesyl-pyrophosphate synthase inhibitor.
  • HMG-CoA hydroxymethylglutaryl-coenzyme A
  • the conditions, quantities and routes of administration can be as described in this document, for example above.
  • the anti-HIV agent is, for example, an anti-HIV agent or an association as defined above.
  • This invention relates in general to an application of the mixture of inhibitors mentioned above as an adjuvant for treatments having an iatrogenic effect, for example premature aging, including, for example, the application of an “adjuvant to the anti-HIV therapies comprising at least one anti-protease” in particular causing this iatrogenic effect.
  • an adjuvant to the anti-HIV therapies comprising at least one anti-protease” in particular causing this iatrogenic effect.
  • This invention also relates to a process for treating an HIV-infected patient including, in any order, the following steps:
  • said mixture and said anti-HIV agent can be co-administered. This involves the process of the invention defined above.
  • the anti-HIV agent can be as defined above.
  • At least one of the administrations can be performed orally or by injection.
  • the two administrations can be performed in the same way or differently.
  • each compounds of the composition may be administered concomitantly with the other compounds (for example in a single composition or in two compositions or in three compositions, each of these compositions including one or several of the above mentioned compound, the mode of administration of each compounds or composition(s) may be identical or different), or independently from one another, for example successively, for example independent administration of a hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, independent administration of at least one farnesyl-pyrophosphate synthase inhibitor and independent administration of an anti-HIV agent, these administrations are performed on the same patient, concomitantly or successively or alternatively, in the above mentioned order or another order.
  • HMG-CoA hydroxymethylglutaryl-coenzyme A
  • composition or co-administration may be performed independently from each other or in a combined manner (composition or co-administration), by an identical or different mode of administration (injection, ingestion, topical application, etc.), one or several times daily, for one or several successive, or not, days.
  • the administration of said mixture of inhibitors can be performed, for example, with a dose of the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor of 0.01 to 2 mg/kg of body weight and with a dose of the farnesyl-pyrophosphate synthase inhibitor of 0.01 to 40 mg/kg of body weight.
  • HMG-CoA hydroxymethylglutaryl-coenzyme A
  • the administration of the anti-HIV agent can be performed as indicated above.
  • dosages are described in the following table for the implementation of one or the other of the processes of the invention defined above.
  • Examples of amounts of inhibitors and anti-HIV agent in a composition according to this invention can be derived from this table.
  • the dosages concerning the anti-HIV agent that can be used to implement this invention may be known to a person skilled in the art. They can be, for example the dosages described in the VIDAL Dictionary (registered trademark), for example in Edition 2007. For each of the examples of anti-HIV agent described above and each of the mixtures or associations of anti-HIV agents described above, one will also find, in the VIDAL Dictionary (registered trademark), for example Edition 2007, dosages that can be used to implement this invention.
  • FIG. 1 shows the results obtained with a Western Blot on “normal” control fibroblasts treated with increasing doses of a hydrosoluble statin (pravastatin P, 20 to 100 ⁇ M), and an aminobiphosphonate (NBP, zoledronate Z, 20 to 100 ⁇ M) (tracks A to I, respectively P20/Z20, P20/Z60, P20/Z100, P60/Z20, P60/Z60, P60/Z100, P100/Z20, P100/Z60, P100/Z100).
  • Track J is a positive test for the presence of prelamin A (fibroblasts of DR patients), and track K is the negative test, treated with the solvent alone (PBS).
  • FIG. 2 shows the results obtained at effective doses of each of the products.
  • FIG. 3 shows the superior effect obtained with the administration of the two products together.
  • FIG. 4 shows the action of the association of the two products on aged cells.
  • FIG. 5 shows the cell multiplication of fibroblasts by a measurement of the mitotic index as a function of cell culture conditions.
  • the mitotic index corresponds to the ratio between the number of nuclei marked (entering the division process) with respect to the number of total nuclei of the field observed as a function of each treatment.
  • FIG. 6 Schematic representation of the mitochondrial theory of aging.
  • the mitochondrial targets of the antiretroviral treatments are identical to the mitochondrial targets of the antiretroviral treatments.
  • NRTI nucleosides inhibitors of inverse transcriptase
  • PI protease inhibitors
  • mtDNA mitochondrial DNA
  • ROS Reactive oxygen species
  • FIG. 7 Schematic representation of the biosynthetic pathway of isoprenoids and inhibitors thereof.
  • FIG. 8 Schematic representation of the post-translational maturation of prelamin A, its nuclear importation and its localization in the nucleoplasm.
  • FIG. 9 Schematic representation of the theory of aging based on abnormalities of lamins and their functional consequences.
  • PI viral protease inhibitors
  • FIG. 11 Mass spectrometry analysis of proteins extracted from the nuclear envelope of untreated cells (a), cells of progeria patients treated with FTI (2.5 ⁇ M, b) or treated with the pravastatin+zoledronate mix (1 ⁇ M each, c).
  • FIG. 12 Mass spectrometry analysis of proteins extracted from the nuclear envelope of untreated fibroblasts (a) or treated with the pravastatin+zoledronate mix (1 ⁇ M each, b) from Zmpste24 ⁇ / ⁇ mice.
  • FIG. 13 Lamin A (control cells) and prelamin A (Zmpste24 ⁇ / ⁇ mice cells) were analysed by mass spectrometry (MALDI-ToF).
  • MALDI-ToF mass spectrometry
  • FIG. 14 representations of results of different experiments demonstrating the synergic effect of the pravastatin+zoledronate combination on the accumulation of prelamin A in control cells and progeria patients:
  • FIG. 15 representation of the results of the treatment with pravastatin+zoledronate showing the correction of the nuclear morphology and the induction of a partial relocating of the isoforms of the A/C lamin and of the B1 lamin of the nuclear lamina in the nucleoplasm, in the progeria patients cells.
  • FIG. 16 representation of the effect of the pravastatin and of the zoledronate whether or not associated on the nuclear morphology of cells of Zmpste24 ⁇ / ⁇ mice (a) and of control mice (b) in culture, in the presence of farnesol, of geranylgeraniol or of the two molecules.
  • FIG. 17 Effect of the pravastatin+zoledronate treatment on the anomalies of the double-strand break repair (DSB) of the DNA in the cells of progeria patients.
  • Immunodetection of the foci of the H2AX phosphorylated histone detected 24 h after irradiation, foci corresponding to the unrepaired double-strand breaks (images at the top).
  • Nuclear marking of the DAPI images at the bottom).
  • Curves at bottom change in the number of foci of H2AX phosphorylated histone according to time after irradiation in the control cells (solid square) and the progeria cells (empty circle) incubated with the PBS or treated with pravastatin+zoledronate.
  • Each curve represents the mean ⁇ standard error of the mean of at least 3 experiments.
  • FIG. 18 Effect of the pravastatin+zoledronate treatment on the progeroid phenotype of Zmpste24 ⁇ / ⁇ mice:
  • the panel at the bottom represents the relative bone volume and the number of bone trabecules in Zmpste24 ⁇ / ⁇ non-treated and treated mice.
  • (f) Relative expression of the target genes of the p53 in the liver and the heart of Zmpste24, Zmpste24 ⁇ / ⁇ and Zmpste24 ⁇ / ⁇ treated mice, analysed by quantitative RT-PCR. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001. The error bars represent the mean ⁇ standard error of the mean.
  • FIG. 19 Effect of the pravastatin alone or of the zoledronate on the lifespan of Zmpste24 ⁇ / ⁇ mice: Kaplan-Meier curves pravastatin only (a) and zoledronate only (b) on Zmpste24 ⁇ / ⁇ treated (empty diamond) and non-treated (solid circles) mice.
  • the cell lines are either control fibroblasts AG16409 from the Coriell Institute or fibroblasts from biopsies of patients with restrictive dermopathy. They are cultivated at 37° C. under 5% CO 2 in room P2.
  • the usual complete culture medium is:
  • the harvesting of cells is performed by trypsinization as follows (protocol for a large flask, 75 cm 2 , BD Falcon):
  • the cells obtained are either frozen for later use or are subcultured from this washed pellet.
  • the pravastatin solution (hydrosoluble statin) used is prepared as follows:
  • pravastatin Sigma Aldrich, P4498
  • the final concentrations tested were 500 nM, 1, 5, 50 and 100 ⁇ M, obtained by diluting the stock solution in the complete medium.
  • NBP zoledronate solution
  • a (1-hydroxy-2-imidazol-1-yl-1-phosphono-ethyl) phosphonic acid stock solution (0.8 mg/ml, Novartis) is adjusted to a concentration of 2 mM.
  • the final concentrations tested were 500 nM, 1, 5, 50 and 100 ⁇ M, obtained by diluting the stock solution in the complete medium.
  • the cells are washed with PBS 1 ⁇ and placed in the complete medium substituted with the treatment.
  • the cells are incubated for the treatment time (6 to 72 hours, sequentially or simultaneously) in the incubator at 37° C.
  • the cells are trypsinized (protocol above) and the pellet obtained is stored at ⁇ 80° C. until protein extraction.
  • the cell pellet is placed in 300 ⁇ l of lysis buffer
  • the cells are exposed to sonication 2 times for 30 seconds (Brandson Sonifier Cell Disruptor B15).
  • the cell debris is centrifuged for 10 minutes at 10,000 rpm at 4° C.
  • the protein supernatant is preserved at ⁇ 80° C. until its use.
  • the protein assay is performed upon thawing.
  • An 8% acrylamide gel is conventionally used to detect the various forms of lamins A/C.
  • a concentration gel is poured over the separation gel.
  • the protein concentration of the samples is assayed, and fractions are adjusted to 50 ⁇ g per tube in the lysis buffer in a qsf 15 ⁇ l.
  • the samples are denatured by heating for 5 minutes at 95° C. and deposited in wells.
  • the transfer membrane (Hybon P, Amersham Bioscineces) is prepared by soaking in ethanol, followed by a 5-minute bath in sterile water, and 10 minutes in the transfer buffer:
  • the gel is humidified for 20 minutes in the transfer buffer, then the sandwich is assembled (Miniprotean system, Biorad).
  • the transfer generally takes place overnight, in a cold chamber, at 10 volts.
  • the membrane is rinsed in PBS 1 ⁇ , preserved in a humid environment, and used as is for detection.
  • the membrane is incubated for 1 hour at room temperature in a saturation solution:
  • the primary antibody is diluted in the saturation buffer (details and dilution, see immunolabeling below).
  • the membrane is incubated with the primary antibodies for 1 hour at room temperature while stirring.
  • the secondary antibody (system coupled to peroxidase, Jackson Immunoresearch) is diluted to 1/10000 in the saturation buffer.
  • the membrane is incubated with this solution for 30 to 45 minutes at room temperature while stirring.
  • the detection is performed with the ECL Plus Western Blotting System kit of Amersham Bioscience, according to the manufacturer's instructions.
  • the membrane After detection, the membrane is exposed on a Biomax MR film (Kodak), for the amount of time necessary to have a satisfactory signal.
  • a Biomax MR film Kodak
  • a cell culture is trypsinized, and the cells are counted on a Neubauer cytometer.
  • Labtech culture wells (Nunc, ref. 177399) are seeded in an amount of 5 ⁇ 10 4 cells per well.
  • the complete culture medium is supplemented by the treatment(s) (statin, NBP, both), and the cells are cultivated for the ad hoc time.
  • the culture medium is then suctioned, and the wells are disassembled.
  • the cells are fixed in a paraformaldehyde 4% solution (in PBS) for 10 minutes at room temperature.
  • the cells are dehydrated by a series of 3 minute baths in solutions with increasing ethanol percentages (70, 90, 100%, with the last bath being repeated).
  • the slides are stored at ⁇ 80° C. until use.
  • the cells After thawing, the cells are incubated for 5 minutes at room temperature in a humidity chamber in 50 ⁇ l of permeabilization solution:
  • the primary antibody is diluted to 1/100 in 50 ⁇ l of incubation solution and placed in contact with the cells for 1 hour at room temperature in a humidity chamber.
  • the primary antibodies used are of two types:
  • the incubation with the secondary antibody takes place for 1 hour in 50 ml of incubation solution at room temperature in a humidity chamber.
  • the secondary antibodies are of two types:
  • a final rinsing is performed for 5 minutes in a solution of Tween 20 at 0.1% in PBS.
  • the cells are immersed in one drop of VectaShield (Vector), covered with an object-cover slide and observed under a fluorescence microscope (Leica DMR, Leica Microsystems), equipped with a coolSNAP camera (Princeton).
  • Normal control fibroblasts were treated with a hydrosoluble statin (pravastatin P, 20 to 100 ⁇ M), and with an aminobiphosphonate (NBP zoledronate Z, 20 to 100 ⁇ M) in association (Tracks A to I, respectively P20/Z20, P20/Z60, P20/Z100, P60/Z20, P60/Z60, P60/Z100, P100/Z20, P100/Z60, P100/Z100).
  • pravastatin P 20 to 100 ⁇ M
  • NBP zoledronate Z 20 to 100 ⁇ M
  • the Western blot shows the “appearance” of a band corresponding to the size of the non-mature prelamin A (non-truncated) as a function of the increase in concentration of the two molecules, confirming that farnesylation is necessary for maturation of lamin A.
  • This result shows that the blockage of the farnesyl-PP synthesis at 2 points of the metabolic pathway is more effective than a blockage at only one point on the inhibition of the farnesylation of prelamin A, at least ex vivo.
  • pravastatin 100 ⁇ M for 12 hours, Zoledronate 20 ⁇ M for 6 hours shows better efficacy, since the prelamin A level produced in healthy cells treated (estimated at 35%) is much higher in association than if the molecules are added alone (respectively 25% and 15%). Moreover, the rate of deformed nuclei (sign of toxicity on healthy cells) is minimal (below 10%), and below what it is on cells treated with pravastatin alone (around 12%).
  • HGPS cells As a function of the number of “passages” (number of cell subcultures), therefore the age of the cells, the proportion of abnormal nuclei increases. This characteristic is typical of non-treated HGPS cells. If HGPS cells are treated with Pravastatin 1 ⁇ M for 24 hours and Zoledronate 10M for 12 hours, this proportion is maintained, and even decreases slightly (less than 40% by comparison with more than 80% in cells treated with a placebo).
  • the pravastatin/zoledronate association is effective at doses for which almost no effect is observed when the molecules are administered separately.
  • a composition according to the invention including a hydrosoluble hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and a farnesyl-pyrophosphate synthase inhibitor on the rate of cell division (mitotic index) of fibroblasts was measured.
  • HMG-CoA hydrosoluble hydroxymethylglutaryl-coenzyme A
  • farnesyl-pyrophosphate synthase inhibitor a farnesyl-pyrophosphate synthase inhibitor
  • fibroblasts two batches of fibroblasts, aged fibroblasts (batch no. 9052) and young fibroblasts (batch no. 7080) were placed in culture in a RPMI (Invitrogen) medium containing 10% fetal bovine serum without antibiotics for 24 hours after trypsinization of the dishes provided.
  • RPMI Invitrogen
  • the various active agents were added at a final concentration of 1 ⁇ M, each for 24 hours (a 1000 dilution of a stock solution in water for compounds A1, A2 and B1, or in Ethanol 100% for compound B2, was produced).
  • the mitotic index was evaluated by incorporating Bromodeoxyuridine (BrdU) over 45 minutes after 24 hours of incubation of the cells with one of the active agent combinations. An immunohistochemical detection showed the cells in the DNA synthesis phase (pre-division cell). Staining of the nuclei (genetic material) was performed by incorporating diamino-phenylindole (DAPI).
  • DAPI diamino-phenylindole
  • the mitotic index corresponds to the ratio of the number of nuclei having incorporated BrdU over the number of nuclei having incorporated DAPI.
  • An average index is statistically calculated with a standard deviation of between 0.005 and 0.061.
  • a bilateral Student test enabled the statistical significance of the results obtained to be measured.
  • the young fibroblasts showed a division capacity superior to that of the aged fibroblasts.
  • the division capacity of aged fibroblasts was below 5%, while the division capacity of young fibroblasts was 15.6%.
  • the difference in division capacity between the aged non-treated fibroblasts and the young non-treated fibroblasts was therefore equal to 3.
  • the number of aged fibroblasts without any treatment in the DNA synthesis phase was extremely low: less than 5% (see FIG. 5 , column 1).
  • the mitotic index was also not very high for the young fibroblasts: on the order of 15% (see FIG. 5 , column 6).
  • the Zmpste24 ⁇ / ⁇ KO mice used here are those described in the cited article of Varela & al., 2005 (49).
  • Evidence of efficacy of the association of the two molecules (pravastatin and zoledronate) was reported in collaboration with a Spanish laboratory (C. Lopez-Otin). The efficacy is obtained at combined doses that do not have an effect when the products are used separately, demonstrating an additive effect.
  • the two molecules (Zoledronic acid (Zometa (registered trademark)) 100 ⁇ g/kg/day and Pravastatin 100 mg/kg/day) were diluted in PBS 1 ⁇ and injected intraperitoneally, on a daily basis, in 1-month-old mice until their death.
  • the controls are wild mice of the same range, treated with PBS 1 ⁇ alone.
  • the survival of the treated mice was significantly improved, and was maximal for females, with an average lifespan increase of around 80%.
  • the clinical symptoms of the disease were all considerably reduced with respect to the individuals treated with PBS alone.
  • an effect was observed of the treatment on the skin and the regrowth of fur on these mice with respect to the mice treated with PBS, which showed large bald areas.
  • the tests are performed on skin from a donor around 60 years of age.
  • a preparation of 21 explants of human skin is produced and kept alive in a BEM medium (BIO-EC's Explants Medium).
  • the explants are distributed into three batches of six explants and one batch CO of three explants, as follows:
  • the treatment is performed on a different day, the first day (D0), 2 hours after preparation of the explants, then D+1 day, D+2 days, D+4 days, D+6 days, D+8 days and D10+ days.
  • the products are applied to the explants as follows:
  • the treatment is performed by topical application of the composition of the invention.
  • the composition is then distributed over the entire surface of the explant, using a spatula.
  • Half of the culture medium is renewed every two days and the explants are kept alive at 37° C. in a humid atmosphere enriched with 5% CO 2 .
  • the three explants of batch CO are obtained.
  • D+6 days and D+11 days three explants of each batch are obtained.
  • the samples are cut in two, and one half is fixed in formaldehyde while the other half is frozen at ⁇ 80° C., according to the BIO-EC procedure “P006-PPEH”.
  • the immunolabeling of cells in mitosis is performed on frozen cross-section cuts with the anti-KI 67 polyclonal antibody (Novo Castra) detected in DAB.
  • the positive cells are counted over the entire epidermal length and averages are returned for the number of cells labeled per cm.
  • the immunolabeling of collagen I will be performed on frozen cross-section cuts with the anti-collagen I polyclonal antibody detected in FITC.
  • the nuclei are counterstained with propidium iodide.
  • the immunolabeling of collagen III is performed on frozen cross-section cuts with the anti-collagen III polyclonal antibody detected in DAB.
  • the nuclei are counterstained with Masson's hemalun.
  • the immunolabeling of collagen IV is performed on frozen cross-section cuts with the anti-collagen IV polyclonal antibody (Cliniscience) detected in FITC.
  • the nuclei are counterstained with propidium iodide.
  • the immunolabeling of collagen VII is performed on frozen cross-section cuts with the anti-collagen VII monoclonal antibody detected in FITC.
  • the nuclei are counterstained with propidium iodide.
  • Endothelial cells can be viewed by immunolabeling of PECAM-1, performed on frozen cross-section cuts with the anti-PECAM-1 monoclonal antibody detected in Fast-red.
  • This example uses the same combinations of active agents used in example 2 above. These various combinations of active agents are used in vitro in order to evaluate their effect on physiological parameters involved in skin aging.
  • the combinations used in this example are A1B1, A1B2, A2B1, A2B2, respectively. These four combinations are tested at a plurality of concentrations, and the experiments are performed in triplicate (representing at least 36 experimental points).
  • the concentrations of the four combinations are proposed by the applicant, and, therefore in vitro cytotoxicity is not envisaged at this stage of the study (unless as a simple test).
  • the experimentation is performed on cell cultures of a fibroblast line as shown in example 1. This test is also applied to keratinocytes cultures. The following parameters are examined for the four combinations of active agents at the concentrations indicated.
  • the measurement of the mitotic index is performed after exposure of the cells to the active agents in a single time.
  • the index is evaluated by an image analysis count of the cell nuclei having incorporated a thymidine analog rendered fluorescent, over the total number of nuclei.
  • a plurality of fields are analyzed.
  • the photos are archived for iconography.
  • the remodeling of the extracellular matrix induced by the fibroblasts exposed to the active agents is evaluated by incorporating these cells into the collagen lattices and by quantifying their capacity to retract these lattices.
  • the retracted surface evaluation gives a remodeling index.
  • the photos are archived for iconography.
  • the measurement of DNA genome repair is performed after irradiation of the cells at a UV-B dose mimicking sun exposure conditions.
  • the objective is first to evaluate the effect of the active agents during DNA repair monitored over a period of 3 times.
  • the quantification is performed by cyclobutane pyrimidine dimer image detection and analysis by UVB irradiation using an immunohistochemical technique.
  • the photos are archived for iconography.
  • the patients of group A needing, during their follow-up, to start an antiretroviral treatment have an additional evaluation when starting the treatment and are analyzed starting on that date with the patients of group B.
  • the patients of group B needing an antiretroviral treatment modification during the studies have a clinical and paraclinical evaluation identical to those provided in the context of the follow-up when there is a change in treatment.
  • the follow-up data on these patients is taken into account to calculate the incidence of disruptions of nuclear and mitochondrial functions observed in patients exposed to antiretrovirals.
  • the study duration is 36 months.
  • the patients of group A needing to start an antiretroviral treatment have a clinical and paraclinical evaluation identical to that provided in the context of the follow-up when starting the treatment.
  • the follow-up data on these patients is analyzed from this date in the group of patients treated (group B).
  • a Treatment Associated a Statin and an Aminobiphosphonate Increases the Lifespan of a Mouse Model Reproducing a Human Syndrome of Premature Aging
  • mice The production of Zmpste24 ⁇ / ⁇ and Lmna ⁇ / ⁇ mice has been described (Pendas and al. 2002 (38); Sullivan and al., 1999 (285).
  • the computerised bone microtomography of the mice was carried out using the micro-CT SkyScan 1172 system (SkyScan—trademark)). All of the experiments on mice are governed by the rules set down by the Animal Experiment Committee of the University of Oviedo (Spain).
  • the pravastatin (100 mg/kg/day) and the zoledronate (100 mg/kg/day) diluted in PBS are administered to the mice every day.
  • the mice receiving the pravastatin-zoledronate treatment or the control mice receiving only PBS do not show any apparent damage or stress.
  • the dermal fibroblasts of a control subject (GM00038) and of patients afflicted with progeria and carriers of the mutation G608G (AG11498 and AG01972) were obtained from the Coriell Cell Repository.
  • the HeLa cells come from the American Type Culture Collection.
  • the cells are cultivated in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% antibiotic-antimycotic (Gibco).
  • the fibroblasts come from tails of 12-day-old mice (Varela and al., 2005).
  • the concentration and the duration of treatment with the statin and the aminobiphosphonate are indicated in the legende of the figures.
  • the fibroblasts are cultivated in Lab Tek chambers (Nalgen Nunc International), washed in the PBS, then fixed in paraformaldhyde 4% for 15 min.
  • the cells are dehydrated in ethanol baths in increasing concentration and are permeabilised 5 min at 25° C. in PBS containing Triton X-100 (0.5%), 5% serum (goat or rabbit).
  • the slides are pre-incubated at 25° C. in PBS for 5 min.
  • the dilution of the primary antibodies is 1/100 for the goat polyclonal anti-prelamin A antibody (Sc-6214, Santa Cruz Biotechnologies), 1/40 for the anti-lamin A/C antibody (4A7 provided by G.
  • the dilution of the secondary antibodies is as follows: 1/100 for the IgG of donkey anti-mouse coupled with FITC (Jackson ImmunoResearch), 1/400 for the IgG of donkey anti-goat coupled with the Alexa 488, 1/800 for the IgG of donkey anti-goat coupled with the Alexa 568 (Molecular Probes) and 1/100 for the IgG of donkey anti-rabbit coupled with tetramethylrhodamine isothiocyanate (Sigma).
  • the cells are then washed, the nuclei coloured for 15 min at 25° C. with DAPI (100 ng/ml, Sigma-Aldrich), finally washed 3 times with PBS containing 0.5% of Tween 20.
  • the slides are mounted in Vectashield (Vector). Digital images are recorded with a Leica DMR microscope equipped with a CoolSnap camera or with a Leica TCS SP5 confocal microscope. The nuclei are observed in the cells after marking of the lamin A/C. More than 100 nuclei were analysed in the control fibroblasts for each of the treatments. The number of nuclei of cells of patients afflicted with progeria analysed is 250 (passage 8), 198 (passage 13) and 150 (passage 20).
  • the cells of progeria patients and the 1BR3 control cells are irradiated with an Philips X device.
  • the X ray is produced by an tungsten anode subjected to a voltage of 200 kV under an intensity of 20 mA with a copper filter of 0.1 mm in diameter.
  • the dose rate is 1.243 Gy/min.
  • the H2AX phosphorylated histone is detected with antibodies that specifically recognise the phosphorylated serine 139 (Upstate Biotechnology-Euromedex, Mundolsheim, France) at the dilution 1/800 and of the anti-mouse antibodies conjugated with the FITC (1/100, Sigma-Aldrich).
  • Nt N0 (1/1+ ⁇ t) ⁇ , where ⁇ and ⁇ are adjustable parameters and Nt and N0 are the number of DSB in time t and in time 0.
  • the cells are homogenised in the following medium: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 50 mM NaF, 1 mM dithiothreitol, 2 mg/ml pepstatin A, in the presence of protease inhibitors (Complete inhibitor cocktail, Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktails I and II, Sigma).
  • the proteins are transferred onto nitrocellulose membranes blocked with 5% of delipidated milk powder, using the TBS-T buffer (20 mM Tris pH 7.4, 150 mM NaCl and 0.05% Tween-20), and incubated 1 h with either a specific anti-lamin A/C antibody (4A7, 1/500), or a specific anti-lamin A (C20, Santa Cruz Biotechnology, 1/250) or an anti-beta actin (A5441, Sigma, 1/5000).
  • the antibodies are diluted in TBS-T containing 3% of delipidated milk powder.
  • the blots are then incubated with an antibody coupled with the peroxidase (goat anti-mouse or anti-rabbit, Jackson ImmunoResearch) in TBS-T containing 1.5% of delipidated milk powder, then washed, finally revealed via chemiluminescence (Immobilon Western chemiluminescent HRP substrate, Millipore—trademark).
  • an antibody coupled with the peroxidase goat anti-mouse or anti-rabbit, Jackson ImmunoResearch
  • TBS-T 1.5% of delipidated milk powder
  • the fibroblasts of control and Zmpste24 ⁇ / ⁇ mice as well as the cells lymphoblastoids of progeria patients have been homogenised, the nuclei isolated via ultracentrifugation and the nuclear proteins obtained as described in Blobel and Potter, V. R. Nuclei from rat liver: isolation method that combines purity with high yield, Science 154, 1662-1665, 1966.
  • the proteins of the nuclear lamina were separated via SDS-PAGE electrophoresis, and the strips containing the lamin A, the prelamin A and the progerin were excised.
  • the fragments of the gel were washed twice with 180 ml of a ammonium bicarbonate/acetonitrile mixture (70/30, 25 mM), dried (15 min, 90° C.) and digested (1 h, 60° C.) with trypsin (12 ng/ml, Promega) in the ammonium bicarbonate 25 mM.
  • 1 ml of CHCA a-cyano-4-hydroxycinnamic acid, Waters
  • HeLa cells were cultivated in the presence of farnesyl transferase inhibitors (FTI-277, Sigma-Aldrich) and/or geranyl-geranyl transferase type I (GGTI-2147) at the concentrations indicated. Only the combination of the two inhibitors allows for a sufficient build-up of not prenylated prelamine A in the cells with regard to the effect of each inhibitors solely used.
  • FTI-277 farnesyl transferase inhibitors
  • GGTI-2147 geranyl-geranyl transferase type I
  • FIG. 10 is a photography of an obtained Western blot, displaying the detection of the A/C lamine in the HeLa cells treated by the farnesyl transferase inhibitors and/or the type I geranyl-geranyl transferase.
  • LA lamine A
  • LC lamine C
  • Pre prelamin A.
  • the farnesyl transferase (FTI) inhibitor induces the compensatory geranylation of the progerin (in the cells of patients afflicted with progeria) and in the Zmpste24 ⁇ / ⁇ mouse fibroblasts
  • the mass spectrometry analysis indicates, as expected, the presence of tryptic peptides of the farnesyled and carboxymethylated in Zmpste24 mouse cells but not in control mice cells. These results are shown in FIG. 13 a .
  • the farnesyled peptide lacks the 3 SIM residues which shows that the Zmpste24 is not indispensable for the first clivage during the maturing of the prelamine A.
  • the FTI-treated mice cells there was observed a reduction in the quantity of farnesyled prelamine A.
  • the geranyl-geranylised peptides there was no peptide derived from the prelamine A detectable in the cells of the non-treated Zmpste24 ⁇ / ⁇ mice. But there was detected a peptide which mass is compatible with a geranyl-geranylated fragment of the prelamine A, after FTI treatment.
  • the results are shown in FIG. 13 b.
  • the mass spectrometry analysis shows that the association of pravastatin (statin) zoledronate (aminobiophosphate) stimulates the appearance of a peptide which corresponds to the C-terminal extremity not prenylated of the progerin, a peptide not detectable in the FTI-treated cells; while neither farnesylated peptides nor the geranyl-geranylated peptides are detected any more, as shows on FIG. 11 c .
  • the statin+aminobiophosphate treatment inhibits the prenylation of the progerin. The same was observed with the prelamine A, as indicated in FIG. 12 .
  • FIG. 13 legend: the lamine A (control cells) and the prelamine A (Zmpste ⁇ / ⁇ mouse cells) have been analysed by a mass spectrometry analysis (MALDI-ToF).
  • a, b portions of the spectrum corresponding to the farnesylated tryptic peptides (a) and the geranylgeranylated peptides (b).
  • Each of the peack is marked by the theorical mass (between parentheses) of the peptide from the trypsin digestion of lamin A, or the prelamin A. The number of the residues is indicated in blue.
  • the sequence of the peptides and their masses are indicated in red.
  • FIG. 11 legend: mass spectrometry analysis of the proteins extracted from the nuclear envelop of the untreated cells (a), of the cells of the progeria patients treated with FTI (2.5 ⁇ M, b) or treated with the mixture of pravastatin+zoledronate (1 ⁇ M apiece, c).
  • the portion of the spectra corresponding to the non-modified proteins, the farnesylated proteins and the geranylgeranylated proteins is indicated on the top, in the center and at the bottom of the figure.
  • Each peak corresponds to the tryptic peptide of the progerin and is marked with the measured monoisotopic mass in experiment and with the theoretical mass (between parentheses).
  • the number of the amino acid residues is indicated in blue.
  • the progerin is predominantly farnesylated (F) and carboxymethylated (Cm) in the unaffected cells (a, centre panel); while, with the effect of the FTI, this peak is considerable reduced, and the progerin appears geranylgeranylated and phosphoryl (b, lower panel).
  • F farnesylated
  • Cm carboxymethylated
  • the unmodified progerin is the predominant form.
  • FIG. 12 legend: a mass spectrometry analysis of the proteins extracted from the nuclear envelop of the untreated fibroblasts (a) or treated with a mixture of pravastatin+zoledronate (1 ⁇ M apiece, b) stemming from Zmpste24 ⁇ / ⁇ mice.
  • the portion of the spectra corresponding to the unmodified proteins, the farnesylated proteins and the geranylgeranylated proteins, is indicated on the top, in the center and on the bottom of the figure.
  • Each peak corresponds to a tryptic peptide of the progerin and is marked with a monoisotopic mass measured the experiment and with the theoritical mass (in parentheses).
  • the number of the amino acid residues is indicated in blue.
  • the sequence of the peptides and their mass is indicated in red.
  • the prelamine A is predominantly farnesylated (F) and carboxymethylated (Cm) in the untreated cells (a, centre panel), while the unmodified or geranylgeranylated forms are not detected.
  • F farnesylated
  • Cm carboxymethylated
  • the prenylated peptides are no longer detectable, and the non-modified form of the prelamine A is predominant (b, upper panel).
  • the treatment with pravastatin+zoledronate corrects the nuclear anomalies of the progeria patients cells and Zmpste24 ⁇ / ⁇ mice cells being in culture; and partially restore the DNA lesion repair mechanisms induced by X-rays ( FIGS. 14 , 15 , 16 and 17 ).
  • the pravastatin+zoledronate treatment causes prelamin A to appear in the nucleus of control cells ( FIG. 14 a ), as in the cell nucleus of progeria patients cells, but with a significant improvement in nuclear morphology in the latter cells ( FIG. 14 b ).
  • Quantitative analysis shows an increase in nuclear anomalies in the cells of progeria patients as the number of passes increases; this number of anomalies decreases under the effects of the pravastatin+zoledronate treatment ( FIG. 14 c .).
  • the cells of progeria patients When observed under a confocal microscope, the cells of progeria patients contain aggregates of lamin A/C and deep invaginations of the nucleoplasmic surface of the nuclear envelope in the nucleoplasm (nuclear reticulum), marked by anti-calreticulin ( FIG. 15 a - f ). These lamin A/C aggregates are absent from the cells of control subjects ( FIG. 14 j - l ) and disappear from the cells of progeria patients under the effects of the pravastatin+zoledronate treatment ( FIG. 14 g - i ). The location of the lamin B1, a farnesylated component of the nuclear lamina, is modified under the effects of the treatment, confirming that the treatment blocks the prenylation of lamins.
  • the pravastatin+zoledronate treatment causes a reduction in the number of phosphorylated histone H2AX foci; these foci are directly correlated with the number of unrepaired DNA double-strand breaks ( FIG. 17 ).
  • the in vitro data gathered shows that the pravastatin+zoledronate combination partially inhibits farnesylation and geranylgeranylation and causes the expected changes of location within the lamina and redistribution within the nucleoplasm of non prenylated prelamin A and progerin in the Zmpste24 ⁇ / ⁇ cells and in progeria patients.
  • the decreased quantity of farnesylated progerin within the lamina and its relocation to the nucleoplasm explains the beneficial effects of the treatment on the cells of progeria patients.
  • the pravastatin+zoledronate treatment corrects the nuclear morphology and leads to a partial relocation of lamin A/C and lamin B1 isoforms from the nuclear lamina into the nucleoplasm, in the cells of progeria patients.
  • the farnesyl pyrophosphate and geranylgeranyl pyrophosphate precursors nullify the effect of the pravastatin+zoledronate treatment in Zmpste24 ⁇ / ⁇ mouse cells in culture.
  • the pravastatin+zoledronate treatment partially corrects the anomalies in the repair of DNA double-strand breaks (DSBs) in the cells of progeria patients.
  • Control fibroblasts and fibroblasts from progeria patients were incubated with the pravastatin+zoledronate mixture (1 ⁇ M each) or with PBS and were irradiated with X rays (2 Gy). Immunodetection of phosphorylated histone H2AX foci detected 24 hours after irradiation, where the foci correspond to unrepaired double-strand breaks (top images). Nuclear marking with DAPI (bottom images). Bottom graphs: Variation in the number of phosphorylated histone H2AX foci over time after irradiation in control cells (shaded square) and progeria cells (unshaded circle) incubated with PBS or treated with pravastatin+zoledronate. Each curve shows the mean ⁇ standard error of the mean from at least 3 experiments.
  • the Zmpste24 ⁇ / ⁇ mice and the control mice were treated on a daily basis with pravastatin and zoledronate or a combination of both drugs, at a dose that had previously been shown to be non-toxic in mice.
  • neither of the drugs in isolation, pravastatin or zoledronate increases the lifespan of Zmpste24 ⁇ / ⁇ mice ( FIG. 19 ).
  • the combination of both drugs significantly improves the progeroid phenotype of Zmpste24 ⁇ / ⁇ mice: the treatment leads to improved weight gain, increases the quantity of subcutaneous fat, reduces the scale of kyphosis and alopecia and increases the lifespan.
  • the survival time increased from 101 to 179 days and the maximum survival time increased from 151 to 222 days (P ⁇ 0.001, FIG. 18 c ). It should be noted that all phenotype signs corrected by the treatment in mice are also characteristic of progeria in humans.
  • the combined treatment corrects the decrease in bone density, which is one of the characteristics of Zmpste24 ⁇ / ⁇ mice and of patients with progeria or a related progeroid syndrome.
  • Computerised bone microtomography shows an increase in bone mineralisation and an increase in the thickness of the tibial cortex in the treated mice ( FIG. 18 d ).
  • the pravastatin+zoledronate treatment has no effect on mice devoid of lamin A/C.
  • progeroid syndromes including Hutchinson-Gilford progeria
  • Hutchinson-Gilford progeria are caused by the accumulation on the nuclear envelope of a farnesylated form of prelamin A deleted (progerin) or not deleted.
  • the progerin is also produced during the course of physiological aging.
  • FTI farnesyl-transferase inhibitors
  • the inventors show here that prelamin A and progerin undergo an alternative prenylation via the geranyl-geranyl-transferase when the farnesyl-transferase is inhibited, which could explain the low degree of efficacy of FTI in improving the phenotype of murin models of these progeroid syndromes.
  • statin and an aminobiphosphonate effectively inhibit the farnesylation as well as the geranyl-geranylation of the prelamin A and of the progerin, significantly improves the phenotype of aging of mice in which has been inactivated the gene coding the metalloprotease Zmpste24 involved in the maturation of the prelamin A.
  • the improvement of the phenotype includes that of the growth curve, weight, lipodystrophy, hair loss and bone anomalies.
  • the pravastatin+aminobiphosphonate treatment is being applied in Marseille for upcoming 3 years on children afflicted with progeria within the framework of a European therapeutic test (15 children) placed under the responsibility of Nicolas Levy, financed by the Ministry of Health (PHRC 2008) and the French Association against Myopathies (AFM) and which has received authorisation from the AFSSAPS and the South Mediterranean CCP.

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US11542309B2 (en) 2019-07-31 2023-01-03 Salk Institute For Biological Studies Fibroblast growth factor 1 (FGF1) mutant proteins that selectively activate FGFR1B to reduce blood glucose

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AU2008353145B2 (en) 2013-09-12
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US9545412B2 (en) 2017-01-17
EP2234620A2 (fr) 2010-10-06
US20150342970A1 (en) 2015-12-03
JP5570999B2 (ja) 2014-08-13
DK2234620T3 (en) 2016-06-06
CN103372215A (zh) 2013-10-30
SG10201408158SA (en) 2015-03-30
JP5622713B2 (ja) 2014-11-12
SG187420A1 (en) 2013-02-28
CN101990432A (zh) 2011-03-23
JP2011508764A (ja) 2011-03-17
BRPI0821962A2 (pt) 2019-05-07
CN102698276B (zh) 2014-12-10
CN101990432B (zh) 2016-01-20
PL2234620T3 (pl) 2016-09-30
WO2009115652A3 (fr) 2010-01-21
ES2573641T3 (es) 2016-06-09
JP2012062322A (ja) 2012-03-29
CN102698276A (zh) 2012-10-03
WO2009115652A2 (fr) 2009-09-24
CA2711569C (en) 2017-08-01
CN103372215B (zh) 2016-03-09
AU2008353145A1 (en) 2009-09-24
PT2234620E (pt) 2016-06-20

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