236
`
`Fingolimod Mitsubishi Pharma/Novartis
`Francis J Dumont
`
`Address
`421 Dogwood Avenue
`Egg Harbor Township
`NJ 08234
`USA
`Email: Francisj_dumont@yahoo.com
`
`IDrugs 2005 8(3):236-253
`© The Thomson Corporation ISSN 1369-7056
`
`Mitsubishi Pharma Corp and Novartis AG are developing
`fingolimod, an orally active
`immunosuppressant affecting
`lymphocyte re-circulation,
`for
`the potential prevention of
`transplant rejection and the treatment of autoimmune diseases,
`including multiple sclerosis. Fingolimod is a synthetic sphingosine
`analog that becomes phosphorylated in vivo and acts as a
`sphingosine-1-phosphate receptor agonist.
`
`Introduction
`Contemporary immunosuppressive therapies are largely
`unsatisfactory, which is in part due to the low therapeutic
`index of the two current mainstay immunosuppressants
`cyclosporin A (CsA) and tacrolimus. These drugs potently
`block lymphokine production by inhibiting calcineurin
`function during T-cell activation, but exert serious
`mechanism-based
`toxicity
`[371251],
`[402151],
`[505878].
`Significant adverse side effects also limit the utility of other
`drugs that suppress lymphocyte activation or proliferation
`at different
`levels,
`such as
`sirolimus, everolimus,
`leflunomide and mycophenolate mofetil (MMF) [371251],
`[483827]. The latter agents are useful in multi-drug regimens
`mitigating their own toxicity and that of calcineurin
`inhibitors or corticosteroids. However, there is a pressing
`need for immunosuppressants with novel modes of action
`and improved safety to provide for better prophylaxis of
`transplant rejection and more effective treatment of chronic
`autoimmune/inflammatory diseases (such as rheumatoid
`arthritis and multiple sclerosis (MS) [505878].
`
`researchers at
`(FTY-720), discovered by
`Fingolimod
`Yoshitomi Pharmaceutical Industries Ltd (now Mitsubishi
`Pharma Corp) [176944], [225279], may go some way toward
`fulfilling this need. While still not entirely elucidated, the
`mechanism of action of fingolimod appears to be quite
`unique since it reflects an alteration of the trafficking of
`lymphocytes rather than of their activation or proliferation
`[371332],
`[558907],
`[558908]. Numerous studies have
`demonstrated the ability of fingolimod to prolong allograft
`survival in rodents, dogs and non-human primates and to
`act synergistically with
`inhibitors of calcineurin or
`proliferation, without exerting major toxic effects [558907].
`The immunosuppressive activity of fingolimod was also
`established
`in various rodent models of autoimmune
`diseases
`[558907]. These encouraging preclinical data
`prompted the development of fingolimod, by Mitsubishi
`and Novartis AG, for potential use in transplant rejection
`and autoimmune diseases [522816], [538366]. Over recent
`years, phase I and II clinical trials in renal transplantation
`have provided preliminary evidence that fingolimod is well
`tolerated
`and
`efficacious when
`administered
`in
`
`
`
`Originator Mitsubishi Pharma Corp
`
`Licensee Novartis AG
`
`Status Phase III Clinical
`
`Indications Autoimmune disease, Cancer, Inflammatory
`bowel disease, Insulin-dependent diabetes, Multiple
`sclerosis, Myocarditis, Transplant rejection
`
`Actions Apoptosis inducer, Anticancer,
`Immunosuppressant, Lymphocyte trafficking modulator,
`Sphingosine kinase substrate, Sphingosine-1-phosphate
`receptor agonist
`
`Technology Oral formulation
`
`Synonym FTY-720
`Registry Nos: 162359-56-0, 162359-55-9
`
`
`
`
`
`OH
`
`OH
`
`NH2
`
`CH3
`
`
`
`
`combination with CsA [558910], [558912]. The preclinical
`and clinical studies described below were all carried out
`using oral delivery, unless otherwise specified.
`
`Synthesis and SAR
`Fingolimod is a synthetic sphingosine analog initially
`generated by the chemical modification of myriocin (ISP-1),
`a natural product from the ascomycete Isaria sinclairii
`[371535]. Myriocin, first described as an antifungal antibiotic
`in 1972 [371531], was re-discovered more than 20 years later
`as an immunosuppressive metabolite. Although potently
`immunosuppressive
`in vivo, myriocin
`caused
`fatal
`gastrointestinal toxicity [371535], and various synthetic
`derivatives were thus tested to identify a safer compound.
`Simplified
`2-alkyl-2-amino-1,3-propanediol
`structures
`demonstrated
`reduced
`toxicity while
`retaining
`immunosuppressive activity [176999], [186975], [225279],
`[226420]. Insertion of a phenyl ring into the alkyl side chain
`led to the development of fingolimod, which exhibited
`improved immunosuppressive activity and safety [176944],
`[225279]. Synthesis of additional analogs demonstrated that
`while the length of the hydrophobic alkyl chain is not
`critical, the position of the phenyl ring is highly important
`for activity, with the optimum length between the phenyl
`ring and the quaternary carbon being two carbon atoms
`[371349],
`[377090]. None of
`these analogs proved
`pharmacologically superior to fingolimod [371349], [377090].
`Of
`the
`two
`hydroxymethyl
`groups present
`in
`the hydrophilic portion of
`fingolimod, only
`the
`pro-S hydroxymethyl group appeared essential
`for
`immunosuppressive activity [371349], [377090]. Moreover,
`only the R-enantiomer configuration at the chiral carbon of a
`fingolimod analog was
`immunosuppressive
`[371349],
`[377090]. Procedures for the synthesis of fingolimod and its
`
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`
`

`

`Fingolimod Dumont 237
`
`chiral analogs and corresponding phosphates have been
`described [378422], [477725], [530769], [558913], [558914],
`[559658], [579915].
`
`Preclinical Development
`Although fingolimod was initially reported to potently
`inhibit mouse T-cell proliferation in mixed-lymphocyte
`cultures [176944], this was not confirmed in subsequent
`studies [558965]. At concentrations up to 1 µM, fingolimod
`did not substantially affect either proliferation or interleukin
`(IL)-2 production of antigen- or mitogen-stimulated rat
`[371352], [371354] or human T-cells [371356], nor did it
`inhibit IL-2-driven T-cell growth [371352]. In this respect, the
`action of fingolimod clearly differed from CsA and
`tacrolimus (which inhibit IL-2 production) [371338] and
`sirolimus (which
`inhibits IL-2-dependent proliferation)
`[371358]. Sub-micromolar concentrations of fingolimod
`nevertheless exerted a synergistic effect with CsA and
`sirolimus in suppressing T-cell proliferation in vitro [371356].
`At higher concentrations (> 4 to 5 µM), fingolimod alone
`induced apoptosis of mature T-cells [371360], [371362],
`thymocytes [371363] and non-lymphoid cells [371363],
`[371364], [558969], [558970]. Fingolimod also promoted
`apoptosis of
`lymphocytes
`[371365]
`and
`enhanced
`superantigen-mediated T-cell deletion in vivo [371368], but,
`as discussed below, it is highly improbable that such effects
`contribute to the mechanism of immunosuppressive action
`of fingolimod.
`
`Despite its weak immunosuppressive activity in vitro,
`fingolimod proved to be a potent immunosuppressant in
`rodent models of graft rejection. Administration of the
`compound at > 0.1 mg/kg dose-dependently prolonged the
`survival of skin [212189], [242515], heart [212190] and liver
`[371356] allografts in rats. Fingolimod was efficacious when
`administered at 0.5 or 1 mg/kg for 2 to 5 weeks in rats with
`small bowel allotransplant known to elicit a strong rejection
`response [371371], [558982], [558983]. At higher doses
`(3 mg/kg),
`fingolimod significantly augmented
`limb
`[371369] and
`joint [371370] allograft survival in rats.
`Immunosuppression with fingolimod also protected corneal
`allograft from rejection [558984], [558985], and promoted
`long-term pancreatic islet allograft survival and function
`[371506], [477727], [477833] in mice and rats. In cardiac
`transplantation models, fingolimod not only prolonged the
`survival of the allograft [371372], but also reduced the
`development of graft atherosclerosis associated with chronic
`rejection [371372], [558986]. In these studies, fingolimod
`treatment was usually
`initiated
`the day before
`transplantation and continued for several weeks thereafter.
`In some instances fingolimod was also effective when
`administered only for 2 days, either from the time of
`transplantation of heart [371352] or liver [371377] allograft,
`or before transplantation of kidney allograft [371378].
`Moreover, fingolimod prolonged liver allograft survival in
`rats if given at 5 mg/kg only on days 3 and 4 post-
`transplantation [371377]. This dose also reversed ongoing
`acute rejection of cardiac allograft if administered on day 3
`to 7 post-transplantation in mice [558987]. Fingolimod
`treatment (0.5 mg/kg/day), delayed to 20 weeks after
`transplantation, ameliorated chronic allograft nephropathy
`induced by CsA (1.5 mg/kg/day) in a renal transplantation
`model [558988]. However, fingolimod failed to prolong skin
`
`allograft survival in rats when administered from day 4
`post-transplantation
`[378421], and overall,
`fingolimod
`proved more potent if given before transplantation, rather
`than post-operatively only. Importantly, fingolimod was
`also active in transplantation models in larger species. At a
`dose of 5 mg/kg, fingolimod, administered for only 2 days
`prior to transplantation, delayed the rejection of renal
`allograft in dogs [371377]. Chronic treatment at lower doses
`also prolonged the survival of renal and liver allografts in
`dogs [233473], [371379], [371380], [584577]. Furthermore,
`once-daily administration of fingolimod at a dose of
`3 mg/kg/day, initiated at least 2 days before transplantation
`and continued thereafter, extended renal allograft survival
`by 33 to 85 days in cynomolgus monkeys [477762].
`
`In addition to benefits derived from lone administration,
`fingolimod displayed
`strong
`synergy with
`other
`immunosuppressive agents
`in various
`transplantation
`models. This was first demonstrated for skin allograft in
`rats, and cardiac allograft in rats and dogs, in which low
`doses of fingolimod potentiated the effect of sub-therapeutic
`doses of CsA [212189], [212190], [212191]. Subsequent
`studies replicated this observation in rat models of skin,
`cardiac, small bowel or liver transplantation [371356],
`[371390], [371391], [371394], [477824] and in mouse models
`of
`cardiac
`transplantation
`[371372],
`[477825]. The
`combination of fingolimod with CsA proved highly effective
`in rat models of small bowel transplantation, where it
`prevented graft rejection and graft-versus-host reaction
`[371371], as well as cardiac transplantation, where it
`abrogated chronic rejection [558986]. Fingolimod plus CsA
`treatment prevented graft vessel disease in a rat carotid
`artery transplantation model [477821], [477831]. Moreover,
`fingolimod (1 or 3 mg/kg) administered every day or every
`other day in combination with CsA (15 mg/kg) inhibited the
`rejection of porcine islet xenografts in rats, while treatment
`with either drug alone was ineffective [558992]. Synergistic
`effects between
`fingolimod and CsA were
`further
`documented in canine models of kidney [371377], [371392],
`[371396] and liver [371380] allotransplantation. Similarly,
`fingolimod (0.1 to 0.3 mg/kg/day) given intravenously or
`orally synergized with sub-therapeutic doses of CsA (10 to
`30 mg/kg/day) to markedly prolong renal allograft survival
`in cynomolgus monkeys [371394], [477762]. Rejection-free
`graft survival was extended to between 32 and 101 days
`with this low-dose, combined-treatment regimen [477762].
`Fingolimod was also demonstrated to synergize with low
`doses of tacrolimus in preventing rejection of skin [371398],
`heart [371398], [371401] and liver [371403] allografts in rats.
`Furthermore, the combination of fingolimod (5 mg/kg) with
`tacrolimus (1 mg/kg) significantly improved survival of
`rat-to-hamster skin xenografts [371404]. However, in a liver
`transplantation model
`in dogs,
`the combination of
`fingolimod (0.1 mg/kg) with tacrolimus (0.5 mg/kg) was
`less effective than tacrolimus alone and caused mortality
`from
`infectious
`complication
`due
`to
`over-
`immunosuppression [371380]. This suggested that careful
`dose adjustment would be needed if fingolimod and
`tacrolimus were to be used together in clinical regimens.
`Fingolimod was further demonstrated to exert synergistic
`effects when administered with immunosuppressants other
`than calcineurin inhibitors. For example, the combination of
`fingolimod with sirolimus or everolimus resulted in potent
`
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`
`

`

`[559040],
`[477724],
`(EAE)
`[477832], encephalomyelitis
`[559041], [559042] and type 1 diabetes [371388], [371411],
`[371433],
`[492753],
`[559045].
`Chronic
`fingolimod
`administration prevented the spontaneous development of
`autoimmune diabetes in NOD mice [559046], [559047] and
`slowed the progression of systemic lupus erythematosus-
`like syndrome in MRL-lpr/lpr mice [559048]. It must be
`noted that fingolimod treatment generally needed to be
`initiated before, or at the time of disease induction, to be
`effective in these models. However, the pathology of EAU
`could still be significantly reduced when the drug was
`administered after disease onset [371444], and in the case of
`thyroiditis induced by neonatal thymectomy and irradiation,
`fingolimod significantly reversed ongoing autoimmune
`disease [371438]. Furthermore, administration of fingolimod
`(3 mg/kg/day ip) to SJL mice with established relapsing-
`remitting EAE, a chronic disease
`that mimics
`the
`predominant form of human MS, resulted in a rapid and
`sustained improvement in the clinical status of the mice,
`which was maintained as long as dosing was continued
`[559042]. Similarly, in IL-10 gene knockout mice, a model of
`inflammatory bowel disease,
`fingolimod significantly
`decreased the severity of colitis when administered for
`4 weeks after disease onset [559051]. While such data
`suggest a role for fingolimod monotherapy in the treatment
`of autoimmunity,
`it appears probable
`that, as
`for
`transplantation, the drug may be of greater utility when
`combined with other immunosuppressive agents. This
`possibility has not been explored in the studies published so
`far, but is suggested in patent application WO-2004028521
`(described below).
`
` A
`
` striking feature of the in vivo action of fingolimod,
`invariably observed in the aforementioned studies, was the
`induction (at immunosuppressive doses) of a marked
`decrease in the number of peripheral blood lymphocytes
`(PBLs). For example, a single-dose administration of
`fingolimod (0.1 mg/kg) in rats reduced PBL counts by
`> 90% between 3 and 24 h, with a return to baseline level
`within a week [371354], [396003]. In baboons or cynomolgus
`monkeys receiving fingolimod (0.1 or 0.3 mg/kg/day),
`peripheral lymphopenia occurred as soon as 4 h after
`treatment, reaching 60 to 80% by 24 to 48 h [371447],
`[417322]. This effect was somewhat more rapid and
`pronounced on T-cells than B-cells, with CD4+ cells being
`more greatly reduced than CD8+ cells [371354], [371447],
`[417322]. In cynomolgus monkeys that were chronically
`treated with fingolimod, PBL counts decreased to ~ 30 and
`14% of pretreatment values at doses of 0.03 and 3.0
`mg/kg/day, respectively and only ~ 4% of peripheral CD4+
`T-cells were refractory to depletion by the drug, compared
`with ~ 30% for CD8+ T-cells [477762]. As a correlate of this
`peripheral lymphopenia, fingolimod reduced the infiltration
`of allografts by T-cells [371483], [371489], [558982], [558985],
`especially
`if
`the drug was administered before
`this
`infiltration occurred [378421]. Significantly diminished T-cell
`infiltration of autoimmune disease target organs was
`similarly documented along with PBL depletion in animal
`models of autoimmunity,
`following
`treatment with
`fingolimod [371388], [559040], [559047]. Interestingly, in the
`transplantation studies, the few T-cells present in the grafts
`of fingolimod-treated animals expressed IL-2 and interferon
`(IFN)γ mRNA [371483], whereas these cytokines were
`
`238 IDrugs 2005 Vol 8 No 3
`
`suppression of allograft rejection in rats [371356], [396003],
`mice [477828] and monkeys [477762], [558993]. In the latter
`model,
`the
`triple-daily
`combination of
`fingolimod
`(0.1 mg/kg)/CsA (10 mg/kg)/everolimus (0.25 mg/kg)
`resulted in further increased graft survival (from 47 to > 100
`days) compared with either drug given alone or in double
`combination [477762]. The co-administration of a low, non-
`toxic dose of mycophenolate sodium (10 mg/kg/day) with
`low doses of fingolimod (0.03 or 0.1 mg/kg/day) was also
`synergistic and prolonged heart allograft survival in rats
`[477726], [477728]. Moreover, fingolimod synergized with
`the blockade of CD28-mediated T-cell co-stimulation by
`CTLA-4-immunoglobulin (Ig) to prevent cardiac allograft
`rejection [559003] or obliterative bronchiolitis in tracheal
`transplantation [559005].
`
`Fingolimod facilitated the induction of tolerance to allografts
`in experimental systems involving the administration of
`allochimeric class I major histocompatibility complex (MHC)
`antigen [371409], or intrathymic injection of donor splenic
`cells [371505], in rats. In contrast, fingolimod prevented
`tolerance induction by donor-specific blood transfusion in
`intestinal transplantation [396001], [559007] or by an anti-
`CD4 mAb in a rat kidney transplantation model [559008].
`However, establishment of transplant tolerance was not
`influenced by fingolimod co-treatment in other models
`[559007], [559009].
`
`Several studies revealed that fingolimod may help alleviate
`grafted organ damage due to ischemia-reperfusion (IR)
`injury, a significant problem in clinical transplantation. This
`was observed in rat models involving cold preservation of
`kidney graft in which fingolimod treatment of recipients,
`either
`immediatedly (1 mg/kg
`iv) [559019] or 24 h
`(0.5 mg/kg po) [559026] prior to reperfusion ameliorated the
`morphological and
`functional consequences of post-
`transplant IR injury. A protective role of fingolimod
`(1 mg/kg iv) was also suggested in renal IR injury models in
`mice [409000], [559032]. Similarly, fingolimod pretreatment
`diminished the biochemical and histological manifestations
`of tissue injury in rat models of warm hepatic IR [477817],
`[559036], although an earlier study reported that such a
`treatment may aggravate IR-induced liver injury [371381].
`More recently, in a rat model of segmental hepatic ischemia,
`fingolimod (1 mg/kg iv) prevented hepatocyte apoptosis
`and decreased the acute phase inflammatory response in
`both normal and cirrhotic livers when administered 20 min
`before ischemia and 10 min before reperfusion [584578].
`
`Fingolimod inhibited various other T-cell-mediated immune
`responses in rodents, in addition to transplant rejection.
`These
`included graft-versus-host
`reactions
`[212192],
`[474285],
`contact
`allergy
`[477810],
`delayed-type
`hypersensitivity [371388], acute viral myocarditis [559576]
`and airway inflammation induced by adoptive transfer of
`Th1 or Th2 cells [530768]. Fingolimod prevented the
`spontaneous development of dermatitis in NC/Nga mice, a
`model for human atopic dermatitis [559575]. Importantly,
`fingolimod proved efficacious at suppressing several
`experimentally induced autoimmune diseases in mice or
`rats,
`including myocarditis
`[371443],
`experimental
`autoimmune uveoretinitis
`(EAU)
`[371444],
`thyroiditis
`[371438], collagen- and adjuvant-induced arthritis [371538],
`
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`
`

`

`Fingolimod Dumont 239
`
`[559497], [559499], which are known to play prominent roles
`in lymphocyte homing to secondary lymphoid organs
`[559052]. Although other chemokine receptors, such as
`CCR2 and CXCR4, may participate in the action of
`fingolimod [431191], [559502], [559577], further observations
`indicated
`that
`fingolimod-induced
`lymphocyte
`sequestration resulted from an inhibition of lymphocyte
`emigration from LN and PP rather than from an enhanced
`attraction to these organs [558908]. In this respect, it is worth
`noting that lymphocyte re-circulation to the blood requires
`lymphocytes to enter the thoracic duct lymph (TDL) after
`their transit through secondary lymphoid organs [371457],
`[559052]. Interestingly, fingolimod treatment decreased
`lymphocyte counts in the TDL to a greater extent than in the
`blood
`[371354]. Histological analyses of LN
`from
`fingolimod-treated mice revealed an accumulation of
`lymphocytes on the abluminal side of the lymphatic
`endothelium, along with an emptying of lymphatic sinuses,
`indicating that lymphocyte egress into lymph was blocked
`[558965], [559503]. This may result in an inhibition of both
`the re-circulation of naïve T-cells and the release of antigen-
`activated T-cells from the draining lymph node to lymph
`and to the blood compartment [558965]. In addition,
`fingolimod inhibited the passage of mature T-cells from the
`thymus into blood [371477], [559504].
`
` A
`
` breakthrough in understanding the pharmacological
`effects of fingolimod came from the discovery that the drug
`is rapidly phosphorylated in vivo. Furthermore, the resulting
`phosphorylated
`fingolimod
`(fingolimod-P)
`inhibited
`lymphocyte re-circulation and acted as a potent agonist on
`several members of the sphingosine-1-phosphate (S1P)
`receptor family, namely S1P1, S1P3, S1P4 and S1P5 [477724],
`[559503]. Upon binding their natural sphingolipid ligands,
`such cell-surface G protein-coupled receptors have been
`reported to elicit a variety of responses in diverse cell types
`[559505], [559507]. S1P1 receptors, which are expressed on
`lymphocytes and endothelial cells [559508], appear to be the
`most
`important S1P
`receptors with
`regard
`to
`the
`immunosuppressive action of fingolimod. This was first
`suggested by structure-activity analyses of semi-selective
`S1P receptor agonists [559509], [559510]. Moreover, a potent
`S1P1-selective agonist, structurally unrelated to S1P and
`fingolimod-P,
`induced peripheral
`lymphopenia
`by
`preventing the entry of lymphocytes into lymph, in a
`manner similar to fingolimod [559511]. A role for S1P1 in
`lymphocyte trafficking was further demonstrated by the
`observation that mice
`lacking this receptor on their
`lymphocytes exhibited an almost complete absence of
`T-cells, and severe deficiency of B-cells, in their blood
`[559513]. Moreover, S1P1-negative T-
`and B-cells,
`transferred to a normal host, accumulated in secondary
`lymphoid organs from which they failed to exit [559513]. A
`study conducted with knockout mice revealed that selective
`deletion of S1P1 in T-cells produced a block in the egress of
`mature T-cells from the thymus into the periphery [559515].
`Most importantly, exposure of normal lymphocytes to
`fingolimod-P in vivo or in vitro downregulated expression of
`S1P1 through internalization and rapid degradation, thereby
`inducing an S1P1-negative phenotype [559513], [559520].
`This implied that fingolimod-P behaves as a partial S1P1
`agonist in lymphocytes, rather than a full agonist as
`originally thought [477724], [559503]. Therefore, expression
`
`tacrolimus-treated
`in CsA- or
`markedly suppressed
`recipients [371403], [371483]. The combination of fingolimod
`with either CsA or tacrolimus abrogated both the T-cell
`infiltration and cytokine mRNA expression in the graft
`[371403], [371483], which may account for the synergism in
`the graft protection mentioned above. Furthermore, studies
`with fingolimod analogs revealed that their ability to cause
`lymphopenia correlated well with
`their efficacy
`in
`promoting rat skin allograft survival [377090]. There was
`also a close correlation between the degree of circulating
`lymphocyte depletion and heart allograft survival in rats
`treated with low doses of fingolimod (0.01 to 0.1 mg/kg) in
`conjunction with everolimus [396003]. This further suggests
`that lymphopenia and the associated reduction of graft
`infiltrating
`lymphocytes play a crucial role
`in
`the
`immunosuppressive effect of fingolimod.
`
`It was initially proposed that the lymphopenia caused by
`fingolimod reflected apoptotic cell death [371360], [371452],
`[371507]. However,
`the
`blood
`concentrations
`of
`immunosuppressive doses of fingolimod proved to be
`> 2-fold lower than those required to induce apoptosis
`[371354]. In addition, apoptotic cells could not be detected in
`the PBL of baboons following treatment with low doses of
`fingolimod [371447]. Similarly, apoptosis rates were not
`increased in the PBL of patients receiving fingolimod
`[438642]. In mice, dye-labeled lymphocytes that had been
`depleted from the blood after fingolimod treatment (0.3
`mg/kg) reappeared after cessation of treatment [371454],
`and there was no evidence for deletion of antiviral memory
`cells by the drug [371388]. Moreover, an S-enantiomer
`analog of fingolimod, which did not induce lymphopenia,
`proved
`as
`potent
`as
`the
`lymphopenia-inducing
`R-enantiomer analog in causing lymphocyte apoptosis in
`vitro [477818]. Therefore, lymphocyte apoptosis is unlikely to
`play a significant role in fingolimod-induced lymphopenia.
`
`An alternative and more plausible explanation for the
`lymphopenic effect of fingolimod was provided by the
`finding that, concomitant with a reduction of PBL numbers,
`the drug increased lymphocyte numbers in the peripheral
`and mesenteric lymph nodes (LN) and in Peyer's patches
`(PP), but not in spleen [371354], [371455], [371473]. This
`suggested
`that
`fingolimod altered
`the
`trafficking of
`lymphocytes such that they became sequestered in LN and
`PP. One possibility could be that fingolimod accelerated the
`homing of lymphocytes to these tissues, a process known to
`involve both
`specialized
`adhesion molecules
`and
`chemokines [371457], [559052]. While antibodies directed
`against lymphocyte homing molecules such as CD62L,
`CD49d and CD11a interfered with fingolimod-induced
`lymphocyte sequestration, expression of these molecules
`was not altered by the drug [371354], [371462]. Furthermore,
`evidence was obtained
`that
`fingolimod
`can
`act
`independently of CD62L [559481]. An augmentation of
`lymphocyte responses to homing chemokines was also
`postulated
`to mediate fingolimod-induced
`lymphocyte
`sequestration
`in LN and PP [371332], [477822]. This
`hypothesis was based on the observation that nanomolar
`concentrations of the drug stimulated T-cell chemotaxis to
`certain chemokines in vitro [559484]. However, fingolimod
`proved capable of producing lymphocyte sequestration in
`mice lacking the chemokine receptors CCR7 and CXCR5
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`in rats, monkeys and humans, giving rise to the biologically
`active
`fingolimod-P metabolite
`[477724],
`[559503].
`Phosphorylation of fingolimod also occurred
`in cells
`incubated with the compound [559503], and the MDR-1
`multidrug transporter appeared to mediate the efflux of
`fingolimod-P from cells [559545]. Moreover, fingolimod-P
`was
`detected
`in
`plasma
`following
`intravenous
`administration of fingolimod in rats, and of the two chiral
`forms of a fingolimod analog, the R-enantiomer was readily
`phosphorylated, whereas the S-enantiomer exhibited only
`trace phosphorylation in rat blood [477724], [559503], which
`was
`consistent with
`the R-enantiomer being
`the
`immunosuppressive isomer [377090], [477818]. Following
`oral administration of fingolimod, the blood level of
`fingolimod-P exceeded that of the parent compound by up
`to 4-fold [477724]. Fingolimod was phosphorylated in vitro
`by both sphingosine kinase type 1 (SPHK1) and type 2
`(SPHK2), but SPHK2 was more effective, suggesting that it
`may be the relevant enzyme in vivo [530765], [559546],
`[585265]. While phosphorylation of fingolimod appeared
`reversible in vivo [477724], the compound was irreversibly
`metabolized by hepatic oxidation
`to carboxylic acid
`derivatives devoid of immunosuppressive activity that were
`excreted in urine and feces [371344], [413804]. A study using
`human liver microsomes suggested that CYP4F3, or another
`closely related form of P450 enzyme, was the primary
`catalyst of fingolimod oxidation, which resulted in the
`formation of two metabolic products [371490]. Since none of
`the major drug-metabolizing P450 enzymes appear to be
`involved in this metabolism, interactions of fingolimod with
`potential co-medications such as CsA,
`tacrolimus or
`sirolimus are unlikely. This is consistent with earlier
`findings in dogs [371377], and with data from human
`studies that demonstrated the lack of pharmacokinetic or
`pharmacodynamic cross-interference between fingolimod
`and CsA [559547], [559548]. However, a potentiation of
`fingolimod exposure by CsA co-administration was noted in
`cynomolgus monkeys [417322].
`
` A
`
` pharmacokinetic study in baboons revealed that when
`administered as a single oral dose, fingolimod (0.3 mg/kg)
`displayed a Cmax of 2.16 ng/ml, an AUC(0-48 h) of 77.9 ng.h/ml
`and a t1/2 of 36 ± 12 h. Upon repeated dosing at 0.03 mg/kg,
`the compound accumulated over time to reach a stable
`blood concentration (0.72 ng/ml) by days 7 to 9 [371447]. In
`cynomolgus monkeys treated with fingolimod as a single
`dose either orally (0.1 or 1 mg/kg) or intravenously
`(0.1 mg/kg),
`a
`linear
`three-compartment model
`characterized the time course of fingolimod concentrations
`with a t1/2 of ~ 31 h, a Cl of ~ 0.53 l/h/kg and a
`bioavailability of ~ 38% [448400]. This long terminal t1/2 most
`likely reflected extensive tissue distribution and binding of
`the compound, particularly as the steady state Vd of the
`drug was also notably large (Vdss = 15 l/kg) [448400].
`
`The pharmacokinetics of fingolimod (0.25 to 3.5 mg) were
`analyzed following the administration of single doses in
`renal transplant patients (n = 20) [477737]. Fingolimod
`displayed a prolonged absorption phase with a Tmax of 12 to
`24 h and an elimination t1/2 ranging from 89 to 157 h, which
`was dose-independent. The Cmax (0.16 to 2.8 ng/ml) and
`AUC (28 to 434 ng.h/ml) were proportional to the dose,
`with low interpatient variability. Moreover, fingolimod
`
`240 IDrugs 2005 Vol 8 No 3
`
`of functional S1P1 by lymphocytes appears to be required
`for their egress from the thymus and secondary lymphoid
`organs. The egress-blocking effects of fingolimod and
`consequent depletion of T- and B-cells from the circulation
`may thus result from the inactivation of this receptor by the
`fingolimod-P metabolite. In addition, the downregulation of
`S1P1 by fingolimod on marginal zone B-cells, a unique
`subset of sessile B-cells with a partially activated phenotype,
`was shown to cause their rapid relocalization to lymphoid
`follicles [559540]. This indicated that the action of the drug
`may extend beyond a blockade of lymphocyte egress and
`affect lymphoid tissue compartmentalization. Fingolimod
`was further demonstrated to suppress the antibody response
`to a T-dependent antigen by inhibiting the formation of
`germinal centers in peripheral lymphoid tissues of mice
`[559543].
`
`Evidence was obtained that fingolimod may also alter the
`barrier function of the vascular endothelium. Fingolimod-P
`(10 nM) induced calcium mobilization and MAP kinase
`activation, and promoted survival and adherens junction
`assembly in endothelial cells in vitro [515624], [530766],
`[559544]. Consistent with these observations in vitro, the
`enhanced vascular permeability
`for macromolecules
`induced by vascular endothelial growth factor (VEGF) in
`vivo was inhibited by fingolimod treatment (10 µg) in mice
`[530766]. Whether such an effect contributes
`to
`the
`aforementioned ability of the drug to attenuate graft vessel
`disease [477831] and IR injury [559036] remains to be
`investigated.
`
`Fingolimod was also demonstrated to exert antitumor
`effects, albeit through still poorly defined mechanisms. At
`micromolar concentrations in vitro, the drug inhibited
`proliferation and promoted the apoptosis of various human
`or mouse tumor cell lines, including glioma [558969],
`bladder cancer [558970] breast cancer [452318], [585263],
`prostate cancer [585260], [585264], hepatoma [514900],
`[515624], [585261] and myeloma [574214], while affecting
`normal cell counterparts to a lesser extent. Tumor cell death
`induced by fingolimod may be due to activation of
`pro-apoptotic signaling pathways [585263], [585264], and/or
`inhibition of anti-apoptotic pathways,
`such as
`the
`phosphoinositide 3-kinase/Akt pathway [585261], but there
`is no evidence that this could be mediated via S1P receptors.
`An intriguing possibility, which remains to be confirmed, is
`that high concentrations of
`fingolimod may
`inhibit
`sphingosine ki