Non-immunosuppressive Immunophilin Ligand Research Articles

The Neurotrophic Effects and Mechanism of Action for FK1706 in Neurorrhaphy Rat Models and SH-SY5Y Cells.

FK1706 is a novel non-immunosuppressive immunophilin ligand with neurotrophic activity and exerts its neurotrophic effect through NGF. The present study aimed to elaborate on the neurotrophic activity and the mechanism of action of FK1706 in end-to-side neurorrhaphy rats and SH-SY5Y cells. In the regenerating nerves of neurorrhaphy rats, FK1706 increased the thickness of myelin sheath and the level of nerve regeneration-related proteins. The mechanism of action of FK1706 on neurite regrowth was elucidated in vitro by incubating SH-SY5Y cells in different conditions (Control, NGF, FK1706, NGF + FK1706, NGF + FK1706 + geldanamycin). Under the conditions where NGF was used, the phosphorylation level of major proteins (Raf-1 and ERK) in the Ras/Raf/MAPK/ERK signaling pathway related to SH-SY5Y cell proliferation was significantly enhanced following the application of FK1706. The number of viable cells, cell viability and neurite length of SH-SY5Y cells was maximal when NGF and FK1706 were used simultaneously. The binding level of HSP90 and Raf-1 in FK1706 group was the highest. These results indicated that FK1706 could significantly promote nerve regeneration after neurorrhaphy. The putative mechanism of action stated that FK1706 could promote the binding of HSP90 and Raf-1, make Raf-1 continue to be activated, thereby affecting key proteins in the Ras/Raf/MAPK/ERK signaling pathway related to the neurotrophic effects of NGF to promote the proliferation and neurite regrowth of nerve cells.

Topical RT1640 treatment effectively reverses gray hair and stem cell loss in a mouse model of radiation-induced canities.

Gray hair is a visible sign of tissue degeneration during aging. Graying is attributed to dysfunction of melanocyte stem cells (McSCs) that results in depletion of their melanin-producing progeny. This non-lethal phenotype makes the hair follicle and its pigment system an attractive model for investigating mechanisms that contribute to tissue aging and therapeutic strategies to combat this process. One potential combination therapeutic is RT1640, which is comprised of two drugs that are known to stimulate hair growth (cyclosporine A [CsA] and minoxidil), along with RT175, a non-immunosuppressive immunophilin ligand that is implicated in tissue regeneration. Using the ionizing radiation-induced acute mouse model of hair graying, we demonstrate that RT1640, over CsA alone, promotes regeneration of the hair pigment system during and following treatment. In non-irradiated mice, RT1640 is also physiologically active and successfully speeds hair growth and expands the McSC pool. It appears that this effect relies on the combined activities of the three drugs within RT1640 to simultaneously activate hair growth and McSCs as RT175 alone was insufficient to induce hair cycling in vivo, yet sufficient to drive the upregulation of the melanogenic program in vitro. This study sets the stage for further investigation into RT1640 and its components in McSC biology and, ultimately, melanocyte hypopigmentary disorders associated with disease and aging.

FK1706, a novel non-immunosuppressive immunophilin ligand, modifies gene expression in the dorsal root ganglia during painful diabetic neuropathy

Objectives: FK1706, a non-immunosuppressive immunophilin ligand, potentiated nerve growth factor-induced neurite outgrowth, putatively mediated via FKBP-52 and the Ras/Raf/MAPK signaling pathway. It also improved mechanical allodynia accompanied by the recovery of intraepidermal nerve fiber density in a painful diabetic neuropathy in rats. The aim of this study was to demonstrate the gene expression profiling in dorsal root ganglion in streptozotocin-induced diabetic rats related to pain and anti-allodynia effects of FK1706 administration to elucidate the putative mechanisms of its neurotrophic activity in vivo. Here, we analyzed gene expression of the dorsal root ganglia using microarray together with behavioral measurement of mechanical allodynia in diabetic rats to try to capture the global fingerprint of changes in gene expression associated with FK1706 administration.Methods: The withdrawal threshold of streptozotocin-induced diabetic rats was measured by an electronic von Frey system. The gene expression of the ganglia from L4 to L6 obtained from streptozotocin-treated rats with or without chronic administration of FK1706 was analyzed using an Affymetrix GeneChip to extract interesting genes in the development of mechanical allodynia in diabetes and anti-allodynia effect of FK1706.Results: Daily oral administration of FK1706 improved mechanical allodynia without decreasing plasma glucose levels. From gene expression analysis, the expression of thioredoxin interacting protein gene was sustained to increased change, whereas those of collagen I alpha1, II alpha1 and IX alpha1 genes were decreased from 2 to 4 weeks after streptozotocin injection. While no changes occurred after 1 week of commencing of FK1706 administration (2 weeks after streptozotocin injection), changes in expression more than 1·5-fold were observed for genes such as Ckm, Actn3, Atp2a1, Bglap, Acta1, Myl1, Tnnc2, and Mylpf at 2 weeks of FK1706 administration (3 weeks after streptozotocin injection). The genes RGD1564519, Hbb, LOC689064, Arpc4 and S100a9 were upregulated in comparison with streptozotocin-injected control group at 3 weeks of FK1706 administration; on the other hand, those of Actn3, Atp2a1 were downregulated by FK1706.Discussion: FK1706 ameliorates mechanical allodynia with accompanying increases in gene expressions possibly related to neurite outgrowth, development, differentiation, and nociceptive sensitivity.

GM1485, a nonimmunosuppressive immunophilin ligand, promotes neurofunctional improvement and neural regeneration following stroke

Stroke is the leading cause of disability in the industrialized world, and the development of pharmacologic strategies to promote poststroke recovery is of paramount importance. GM1485, a nonimmunosuppressive immunophilin ligand, promotes regeneration of multiple cell types following injury. In the present study, we evaluated the effect of GM1485 treatment on functional recovery and neurogenesis following rat stroke. Transient cerebral ischemia was induced in rats receiving daily GM1485 (5 mg/kg) beginning 24 hr postischemia and continuing for a total of 6 weeks. Neurological function was evaluated over this period using a battery of neurobehavioral tests, and immunostaining for stem-cell markers was performed following animal sacrifice. An in vitro model of oxidative stress was also employed to evaluate the ability of GM1485 to mediate stem-cell-like induction and plasticity. GM1485-treated rats demonstrated improved neurological function as well as increased Sox2(+) cells in the ipsilateral SVZ and striatum relative to vehicle-treated rats. Additionally, GM1485-treated fibroblasts subjected to oxidative stress were reprogrammed to a stem-cell-like phenotype and were able to differentiate down a neuronal lineage. These data demonstrate that GM1485 administration improves neurological function and is consistent with an upregulation of endogenous neurogenesis following stroke in rats. Further experiments are necessary to characterize the molecular pathways involved in these processes.

Time-Dependent Inhibitory Effects of (1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-1,14-Dihydroxy-12-(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl-23,25-dimethoxy-13,19,21,27-tetramethyl-17-(2-oxopropyl)-11,28-dioxa-4-azatricyclo[22.3.1.04.9]octacos-18-ene-2,3,10,16-tetrone (FK1706), a Novel Nonimmunosuppressive Immunophilin Ligand, on CYP3A4/5 Activity in Humans in Vivo and in Vitro

We investigated the inhibitory effects of (1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-1, 14-dihydroxy-12-(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl-23,25-dimethoxy-13,19,21,27-tetramethyl-17-(2-oxopropyl)-11,28-dioxa-4-azatricyclo [22.3.1.0(4.9)]octacos-18-ene-2,3,10,16-tetrone (FK1706), a novel nonimmunosuppressive immunophilin ligand, on CYP3A4/5 in in vitro and in vivo settings. First, the inhibitory effects of FK1706 (preincubation dependence, inactivation rate estimation, and reversibility) were tested using human liver microsomes. Second, the effect of repeated oral doses of FK1706 (60 mg q.d. for 14 days) on the pharmacokinetics of midazolam (single oral 2-mg dose) was tested in healthy volunteers. Finally, pharmacokinetic modeling and simulation were performed. In vitro experiments showed that FK1706 inhibited CYP3A4/5 in a time-dependent and irreversible manner. The in vitro maximum inactivation rate constant (k(inact)) and concentration of inhibitor that gave half-maximal k(inact) (K(I)) were estimated to be 10.1 h(-1) and 2050 ng/ml, respectively. In the clinical study, FK1706 produced a 2-fold increase in the area under the time-concentration curve (AUC) of midazolam. A pharmacokinetic model developed for this study, which described the time course of concentrations of both FK1706 and midazolam and incorporated CYP3A4/5 inactivation in the liver and intestine, successfully predicted the change in the pharmacokinetics of midazolam using in vitro k(inact) and K(I) values (1.66- to 2.81-fold increases in AUC predicted) and estimated the in vivo inactivation rate to be 0.00404 to 0.0318 h(-1) x ml/ng. In conclusion, FK1706 weakly or moderately inhibited the activity of CYP3A4/5 in vitro and vivo at the tested dose. The model developed here would be helpful in predicting drug-drug interactions and in the design of dose regimens that avoid drug-drug interactions.

FK1706, a novel non-immunosuppressive immunophilin ligand, modifies the course of painful diabetic neuropathy

FK1706, a derivative of FK506, is a non-immunosuppressive immunophilin ligand with significant neurotrophic activity mediated via FKBP-52 and the RAS/RAF/MAPK signaling pathway. Here, we tested the effect of FK1706 on painful diabetic neuropathy in rat model of diabetes induced by streptozotocin (STZ). FK1706 ameliorated mechanical allodynia in this model at doses over 0.32 mg/kg, p.o., even if treatment was initiated after neuropathy was established, and did not affect plasma glucose levels. Furthermore, this improvement continued at least 4 weeks after the last administration. In morphological analysis, FK1706 treatment also restored intraepidermal nerve fiber density in footpad skin to almost normal levels. Gabapentin also improved mechanical allodynia in the same model, but efficacy disappeared the day after administration stopped. Allodynia responses were potentiated by co-administration of both compounds. Thus, FK1706 ameliorated painful diabetic neuropathy via a different mechanism from gabapentin and improved morphological outcomes, indicating that FK1706 improves painful diabetic neuropathy by modifying the underlying disease pathology.

Neuroimmunophilin Ligands Protect Cavernous Nerves after Crush Injury in the Rat: New Experimental Paradigms

ObjectivesWe investigated the effects of the orally bioavailable non-immunosuppressive immunophilin ligand GPI 1046 (GPI) on erectile function and cavernous nerve (CN) histology following unilateral or bilateral crush injury (UCI, BCI, respectively) of the CNs. MethodsAdult male Sprague-Dawley rats were administered GPI 15mg/kg intraperitoneally (ip) or 30mg/kg orally (po), FK506 1mg/kg, ip, or vehicle controls for each route of administration just prior to UCI or BCI and daily up to 7 d following injury. At day 1 or 7 of treatment, erectile function induced by CN electrical stimulation was measured, and electron microscopic analysis of the injured CN was performed. ResultsIntraperitoneal administration of GPI to rats with injured CN protected erectile function, in a fashion similar to the prototypic immunophilin ligand FK506, compared with vehicle-treated animals (93%±9% vs. 70%±5% vs. 45%±1%, p<0.01, respectively). Oral administration of GPI elicited the same level of significant protection from CN injury. GPI administered po at 30mg/kg/d, dosing either once daily or four times daily with 7.5mg/kg, provided nearly complete protection of erectile function. In a more severe BCI model, po administration of GPI maintained erectile function at 24h after CN injury. Ultrastructural analysis of injured CNs indicated that GPI administered at the time of CN injury prevents degeneration of about 83% of the unmyelinated axons at 7 d after CN injury. ConclusionsThe orally administered immunophilin ligand GPI neuroprotects CNs and maintains erectile function in rats under various conditions of CN crush injury.

The immunophilin ligand GPI1046 protects neurons from the lethal effects of the HIV‐1 proteins gp120 and Tat by modulating endoplasmic reticulum calcium load

The dysfunction and death of neuronal cells is thought to underlie the cognitive manifestations of human immunodeficiency virus (HIV)-associated neurological disorders. Although HIV-infected patients are living longer owing to the effectiveness of anti-retroviral therapies, the number of patients developing neurological disorders is on the rise. Thus, there is an escalating need for effective therapies to preserve cognitive function in HIV-infected patients. Using HIV-protein-induced neurotoxicity as a model system, we tested the effectiveness of a non-immunosuppressive immunophilin ligand to attenuate gp120 and Tat-induced modification of neuronal function. The immunophilin ligand GPI1046 attenuated endoplasmic reticulum (ER) calcium release induced by gp120 and Tat and protected neurons from the lethal effect of these neurotoxic HIV proteins. Both inositol 1,4,5 trisphosphate (IP(3)) and ryanodine-sensitive ER calcium release was attenuated by pre-incubation with GPI1046. Using the sarco/endoplasmic reticulum calcium pump inhibitor thapsigargin to release ER calcium, we determined that GPI1046 reduced the total ER calcium load. These findings suggest that non-immunosuppressive immunophilin ligands may be useful neuroprotective drugs in HIV dementia.

3-Normeridamycin: A Potent Non-Immunosuppressive Immunophilin Ligand is Neuroprotective in Dopaminergic Neurons

3-Normeridamycin (1), isolated from fermentation extracts of the soil actinomycete Streptomyces sp. LL-C31037, demonstrated potent neuroprotective activity. When challenged with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), known to induce parkinsonism, 1 restored functional dopamine uptake in a concentration-dependent manner, with an EC50 of 110 nM in dopaminergic neurons. The structure of 1 was determined via spectroscopic methods, and the immunosuppressive and immunophilin binding properties of the compound were also measured.

FK1706, a novel non-immunosuppressive immunophilin: neurotrophic activity and mechanism of action

Immunophilin ligands are neuroregenerative agents, characterized by binding to FK506 binding proteins (FKBPs), which stimulate recovery of neurons in a variety of injury paradigms. Here we report the discovery of a novel, non-immunosuppressive immunophilin ligand, FK1706. FK1706, a derivative of FK506, showed similarly high affinity for two FKBP subtypes, FKBP-12 and FKBP-52, but inhibited T-cell proliferation and interleukin-2 cytokine production with much lower potency and efficacy than FK506. FK1706 (0.1 to 10 nM) significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in SH-SY5Y cells, as did FK506. This neurite potentiation could be blocked by an anti-FKBP-52 antibody, as well as by specific pharmacological inhibitors of phospholipase C (PLC), phosphatidylinositol 3-kinase (PI3K), and the Ras/Raf/Mitogen-Activated Protein Kinase (MAPK) signaling pathway. FK1706 also potentiated NGF-induced MAPK activation, with a similar dose-dependency to that necessary for potentiating neurite outgrowth. Taken together, these data suggest that FK1706 is a non-immunosuppressive immunophilin ligand with significant neurotrophic effects, putatively mediated via FKBP-52 and the Ras/Raf/MAPK signaling pathway, and therefore that FK1706 may have therapeutic potential in a variety of neurological disorders.

Molecular Basis for the Neuroprotective Properties of FKBP-Binding Immunophilin Ligands

Non-immunosuppressive immunophilin ligands (NI-IPLs) are attracting attention as new candidate drugs for neuroprotection and / or neurorestoration, particularly, since they do not have the adverse effects of immunosuppressants. However, it is not yet enough to understand that NI-IPLs are useful drugs for treating neurological disorders. In particular, the molecular mechanism of NI-IPL activity in target cells in the brain remains obscure. In this review, we focused on the molecular basis of the neuroprotective properties of FK506 binding protein (FKBP)-binding IPLs. Our findings suggest that IPLs have neuroprotective effects mediated by multiple beneficial properties such as a GSH-activating effect, a NTF-activating effect, and an anti-apoptotic effect, but not by an immunosuppressive effect, both in cell cultures and in vivo. In particular, the GSH-activating effect and the NTF-activating effect of NI-IPLs may be essential to the expression of their neuroprotective properties. Thus, NI-IPLs might have a potentially beneficial effect by ameliorating neurological disorders, since they do not cause serious side effects such as immune deficiency. Keywords: neuroimmunophilins, non-immunosuppressive immunophilin ligand, glutathione, gdnf, bdnf, apoptosis

The non-immunosuppressive immunophilin ligand GPI-1046 potently stimulates regenerating axon growth from adult mouse dorsal root ganglia cultured in Matrigel

We used explant cultures of adult mouse dorsal root ganglia with spinal nerve attached growing in Matrigel to assess the effects of the non-immunosuppressive immunophilin ligand GPI-1046 [Snyder et al. (1998) TIPS 19, 21–26] on the growth rate of regenerating sensory axons and found a potent stimulation of axon growth. In these explant cultures, naked, unfasciculated axons emerge from the cut end of the spinal nerve and continue to grow in the Matrigel for up to eight days [Tonge et al. (1996) Neuroscience 73, 541–551]. Some axons are entirely smooth whilst others show prominent varicosities. Some of the former express the phosphorylated neurofilament epitope recognised by monoclonal antibody RT97, a marker for large calibre, myelinated axons, whilst the latter express calcitonin gene-related peptide, predominantly a marker for unmyelinated, and small diameter myelinated sensory axons. Many of the axons in these cultures also express the low-affinity neurotrophin receptor p75. GPI-1046 has been shown to have striking stimulatory effects on embryonic primary sensory axons growing in vitro and it was therefore of interest to see whether it could also enhance regenerating sensory axon growth from the adult ganglia in our cultures. GPI-1046 potently stimulated axon growth in our cultures in a dose-dependent manner. The stimulatory effect was not dependent on the class of sensory axon. These observations show that GPI-1046 is a potent stimulator of regenerating axons from adult, primary sensory neurones. The cellular site of action of GPI-1046 is unknown. To distinguish between a direct effect of the drug on neurones and an indirect effect we compared the effects of GPI-1046 on explant and dissociated cultures. In confirmation of previous results, we found that GPI-1046 potently stimulated axon outgrowth from explants of embryonic chick dorsal root ganglia. However, the drug was without effect on dissociated embryonic dorsal root ganglion neurones, suggesting that non-neuronal cells are important for axon growth stimulation.

A Novel Immunophilin Ligand: Distinct Branching Effects on Dopaminergic Neurons in Culture and Neurotrophic Actions after Oral Administration in an Animal Model of Parkinson's Disease

Protection or regeneration of the dopaminergic (DA) system would be of significant therapeutic value for Parkinson's disease. Immunophilin ligands, such as FK506, can produce neurotrophic effectsin vitroandin vivo,but their immunosuppressive effects make them unsuitable for neurological application. This study demonstrates that a novel, nonimmunosuppressive immunophilin ligand (V-10,367) increased the number of neurites extended by tyrosine hydroxylase positive (TH+) DA neurons in embryonic day 14 primary DA neuronal cultures. In contrast, the immunosuppressive immunophilin ligand FK506 increased the length of TH+ neurites. After oral administration in MPTP-treated mice, V-10,367 completely protected against MPTP-induced loss of striatal TH+ axonal density, while FK506 did not. These experiments demonstrate that nonimmunosuppressive immunophilin ligands specifically increase neurite branching in primary DA neuronal cultures and possess neurotrophic actionsin vivowith potential application to neurodegenerative disease.

Neurotrophic immunophilin ligands stimulate structural and functional recovery in neurodegenerative animal models.

Although immunosuppressant immunophilin ligands promote neurite outgrowth in vitro, their neurotrophic activities are clearly independent of their immunosuppressive activity. In the present report, a novel nonimmunosuppressive immunophilin ligand, GPI-1046 (3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate+ ++) is described. In vitro, GPI-1046 bound to FK506 binding protein-12 and elicited neurite outgrowth from sensory neuronal cultures with picomolar potency with maximal effects comparable to nerve growth factor. In vivo, GPI-1046 stimulated the regeneration of lesioned sciatic nerve axons and myelin levels. In the central nervous system, GPI-1046 promoted protection and/or sprouting of serotonin-containing nerve fibers in somatosensory cortex following parachloroamphetamine treatment. GPI-1046 also induced regenerative sprouting from spared nigrostriatal dopaminergic neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice or 6-hydroxydopamine (6-OHDA) toxicity in rats. The rotational abnormality in 6-OHDA treated rats was alleviated by GPI-1046. These neurotrophic actions in multiple models suggest therapeutic utility for GPI-1046 in neurodegenerative diseases.