Second basic pKa: An overlooked parameter in predicting phospholipidosis-inducing potential of diamines.

In this paper, we present the phospholipidosis-inducing potential (PLIP) of forty fragment-sized diamines derived from N-benzyl-4-(methylamino)piperidine and discuss the relationship between their PLIP and the physicochemical properties. Our results demonstrate that the previously reported methods are not suitable for predicting the PLIP of fragment-sized diamines; the second basic pKa can distinguish PLIP-positive diamines from PLIP-negative diamines more accurately than ClogP or most basic pKa. To the best of our knowledge, this is the first report describing the relationship between PLIP and second basic pKa.

Fibroblast growth factor receptor modulators employing diamines with reduced phospholipidosis-inducing potential.

SUN13837 (1), a fibroblast growth factor receptor modulator, has been an attractive candidate for treating neurodegenerative diseases. However, one of its metabolites, N-benzyl-4-(methylamino)piperidine (BMP), turned out to possess phospholipidosis-inducing potential (PLIP) in vitro. To obtain SUN13837 analogs with reduced phospholipidosis risk, we replaced BMP with other diamines possessing low PLIP. Our effort led to the discovery of compound 6 with increased efficacy. Further structural modifications to reduce hydrogen bond donors afforded 17 with improved brain exposure. Oral administration of 17 at 1 mg/kg once daily for 10 days showed enhanced recovery of coordinated movement in a rat acute stroke model, suggesting that it is a promising follow-up compound for 1 with reduced risk of phospholipidosis.

Discovery of 1,2,3-triazole-based fibroblast growth factor receptor modulators.

To avoid production of a phospholipidosis-inducing metabolite, we replaced the amide structure of SUN13837 (1) with a 1,2,3-triazole. The resulting 1,2,3-triazole analog of 1 (compound 2) displayed greater neuroprotective activity than 1. Structural modification of 2 yielded compound 10, which showed improved neuroprotective activity and negligible mechanism-based inactivation against CYP3A4. In addition, installation of a methyl group at the 5-position of 1,2,3-triazole of 10 significantly boosted the neuroprotective activity. These 1,2,3-triazole derivatives displayed reduced phospholipidosis risk, sufficient systemic exposure, and high central nervous system penetration, and therefore may be potentially useful agents for the treatment of neurodegenerative diseases.

Introduction of antioxidant-loaded liposomes into endothelial cell surfaces through DNA hybridization.

Ischemia-reperfusion damage is a problem in organ transplantation. Reactive oxygen species are produced in cells by blood-mediated reactions at the time of blood reperfusion. In this study, we developed a method to immobilize and internalize antioxidants in endothelial cells, using vitamin E-loaded liposomes. The liposomes loaded with vitamin E and human umbilical vein endothelial cells (HUVECs) were modified with poly(ethylene glycol)-phospholipid conjugates carrying 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of complementary deoxythymidylic acid (oligo(dT)20), respectively. The liposomes were effectively immobilized on HUVECs through DNA hybridization between oligo(dA)20 and oligo(dT)20. The liposomes loaded with vitamin E were gradually internalized into HUVECs. Then, the cells were treated with antimycin A to induce oxidative stress. We found the amount of reactive oxygen species was greatly reduced in HUVECs carrying vitamin E-loaded liposomes.

Synthetic molecules that protect cells from anoikis and their use in cell transplantation.

One of the major problems encountered in cell transplantation is the low level of survival of transplanted cells due to detachment-induced apoptosis, called anoikis. The present study reports on the chemical synthesis and biological evaluation of water-soluble molecules that protect suspended cells from anoikis. The synthetic molecules bind to and induce clusters of integrins and heparan-sulfate-bound syndecans, two classes of receptors that are important for extracellular matrix-mediated cell survival. Molecular biological analysis indicates that such molecules prolong the survival of suspended NIH3T3 cells, at least in part, by promoting clustering of syndecan-4 and integrin ß1 on the cell surface, leading to the activation of small GTPase Rac-1 and Akt. In vivo experiments using animal disease models demonstrated the ability of the molecules to improve cell engraftment. The cluster-inducing molecules may provide a starting point for the design of new synthetic tools for cell-based therapy.

Small-molecule-induced clustering of heparan sulfate promotes cell adhesion.

Adhesamine is an organic small molecule that promotes adhesion and growth of cultured human cells by binding selectively to heparan sulfate on the cell surface. The present study combined chemical, physicochemical, and cell biological experiments, using adhesamine and its analogues, to examine the mechanism by which this dumbbell-shaped, non-peptidic molecule induces physiologically relevant cell adhesion. The results suggest that multiple adhesamine molecules cooperatively bind to heparan sulfate and induce its assembly, promoting clustering of heparan sulfate-bound syndecan-4 on the cell surface. A pilot study showed that adhesamine improved the viability and attachment of transplanted cells in mice. Further studies of adhesamine and other small molecules could lead to the design of assembly-inducing molecules for use in cell biology and cell therapy.

Synthesis and biological evaluation of truncated α-galactosylceramide derivatives focusing on cytokine induction profile.

A series of truncated analogs of α-galactosylceramide with altered ceramide moiety was prepared, and evaluated for Th2-biased response in the context of IL-4/IFN-γ ratio. Phytosphingosine-modified analogs including cyclic, aromatic and ethereal compounds as well as the C-glycoside analog of OCH (2) with their cytokine inducing profile are disclosed.

Islet surface modification with urokinase through DNA hybridization.

Transplantation of islets of Langerhans (islets) has been proposed as a safe, effective approach to treating patients with insulin-dependent diabetes mellitus (type I diabetes). It has been reported, however, that many islets are lost in the early phase after intraportal transplantation by instant blood coagulation-mediated inflammatory reactions. In this study, DNA hybridization was applied to conjugate the fibrinolytic enzyme urokinase on the islet surface. We synthesized amphiphilic polymers, PEG-lipids carrying oligo(dT)(20) (oligo(dT)(20)-PEG-lipid; PEG MW = 5000) and urokinase (UK) carrying oligo(dA)(20). The oligo(dT)(20)-PEG-lipid was spontaneously incorporated into the cell membrane through interactions between the hydrophobic parts of the PEG-lipids and the lipid bilayer, and UK was conjugated on the cell surface through DNA hybridization between oligo(dT)(20) on the cell and complementary oligo(dA)(20) on the UK. The activity of UK was maintained on the islet surface. The surface modification with UK did not influence islet morphology or islet ability to secrete insulin in response to changes in glucose concentration. No practical volume increase was observed with our method, indicating that islet graft loss could be suppressed at the early stage of intraportal islet transplantation.

Systemic treatment with a novel basic fibroblast growth factor mimic small-molecule compound boosts functional recovery after spinal cord injury.

Neurotrophic factors have been regarded having promising potentials for neuronal protection and regeneration, and thus promoting beneficial effects of kinesiological functions. They can be suspected to play important roles in cell/tissue grafting for various neural diseases. The clinical applications of such trophic factors to the central nervous system (CNS), however, have caused problematic side effects on account of the distinctive bioactive properties. In the course of developing synthetic compounds reflecting beneficial properties of basic fibroblast growth factor (bFGF), we conducted screening candidates that stimulate to trigger the intracellular tyrosine phosphorylation of FGF receptor and lead to the subsequent intracellular signaling in neurons. A small synthetic molecule SUN13837 was characterized by mimicking the beneficial properties of bFGF, which have been known as its specific activities when applied to CNS. What is more remarkable is that SUN13837 is eliminated the bioactivity to induce cell proliferation of non-neuronal somatic cells. On the bases of studies of pharmacology, behavior, physiology and histology, the present study reports that SUN13837 is characterized as a promising synthetic compound for treatment of devastating damages onto the rat spinal cord.

Coaggregates of Regulatory T Cells and Islet Cells Allow Long-term Graft Survival in Liver Without Immunosuppression.

BACKGROUND: Transplantation of islets of Langerhans (islets) has been investigated in the clinic to treat patients with insulin-dependent diabetes mellitus. Islet grafts have been maintained by administering immunosuppressive drugs, which can lead to complications in the long term. Alternatives to immunosuppressive therapy are eagerly desired. In this study, we examined the transplantation of coaggregates of CD4CD25 regulatory T (Treg) cells. METHODS: Coaggregates of Treg cells from C57BL/6 mice and islet cells from BALB/c mice were prepared on agarose hydrogel with small round-bottomed wells. Four hundred coaggregates were transplanted into the livers of streptozotocin-induced diabetic C57BL/6 mice without systemic immunosuppression. RESULTS: The Treg cells and islet cells were distributed randomly in the coaggregates. When 400 coaggregates were transplanted into 9 C57BL/6 mice via the portal vein, 6 of the 9 recipients demonstrated blood glucose less than 250 mg/dL for more than 100 days. A number of insulin-positive cells were observed in the livers at 120 days after transplantation. CONCLUSIONS: The Treg cells and islet cells were distributed randomly in the coaggregates. After intraportal transplantation of the coaggregates, Treg cells in the aggregates enabled the long-term survival of allogeneic islet cell grafts in the liver without the use of immunosuppressive drugs.

Cell patterning on polylactic acid through surface-tethered oligonucleotides.

Polylactic acid (PLA) is a candidate material to prepare scaffolds for 3-D tissue regeneration. However, cells do not adhere or proliferate well on the surface of PLA because it is hydrophobic. We report a simple and rapid method for inducing cell adhesion to PLA through DNA hybridization. Single-stranded DNA (ssDNA) conjugated to poly(ethylene glycol) (PEG) and to a terminal phospholipid (ssDNA-PEG-lipid) was used for cell surface modification. Through DNA hybridization, modified cells were able to attach to PLA surfaces modified with complementary sequence (ssDNA'). Different cell types can be attached to PLA fibers and films in a spatially controlled manner by using ssDNAs with different sequences. In addition, they proliferate well in a culture medium supplemented with fetal bovine serum. The coexisting modes of cell adhesion through DNA hybridization and natural cytoskeletal adhesion machinery revealed no serious effects on cell growth. The combination of a 3-D scaffold made of PLA and cell immobilization on the PLA scaffold through DNA hybridization will be useful for the preparation of 3-D tissue and organs.

Sequence-specific nuclease-mediated release of cells tethered by oligonucleotide phospholipids.

Single-stranded oligonucleotide-conjugated lipids (ssDNA-PEG-lipids) that associate with the cell membrane confer to the cell an artificial adhesive capability via sequence-specific hybridization to complementary oligonucleotides, forming bonds of double stranded oligonucleotides (dsDNA). Such artificial tethers permit surface patterning of cells or controlled formation of cellular aggregates. However, the hybridization responsible for tethering cells to surfaces or to other cells is not trivially reversed under physiological conditions. In this study, we approach the unbinding of tethered cells by cleaving dsDNA bonds with restriction endonuclease BamHI or digesting bonds with the nonspecific nuclease Benzonase. The procedure was applied to CCRF-CEM cells bearing dsDNA suspended in isolation, cells tethered to glass substrates, and cells aggregated heterotypically with other ssDNA-bearing cells. Cells liberated from surfaces with BamHI could be flushed from flow chambers and viably recovered while the majority of cells not bearing enzyme recognition sequences were retained on the surface, and DNA-tethered cells could be nonspecifically recovered viably from surfaces after Benzonase treatment. Heterotypic aggregates of cells joined by recognition sequence DNA could be dispersed with 10 min exposure to BamHI while undispersed cells heterotypically aggregated with a control sequence remained. Likewise, 10 min exposure to Benzonase was sufficient to disperse aggregates independently of sequence. The potential to undo artificially engineered DNA-mediated adhesion offers new possibilities in the controlled arrangement of cells relative to other cells and in the study of membrane biophysics.

SUN11602-induced hyperexpression of calbindin D-28k is pivotal for the survival of hippocampal neurons under neurotoxic conditions.

Basic fibroblast growth factor (FGF-2/bFGF) possesses neuroprotective activity and promotes cell proliferation. In this study, the novel synthetic compound 4-({4-[[(4-amino-2,3,5,6-tetramethylanilino)acetyl](methyl)amino]-1-piperidinyl}methyl)benzamide (SUN11602) exhibited neuroprotective activities similar to those of FGF-2 without promoting cell proliferation. In primary cultures of hippocampal neurons, stimulation with SUN11602 or FGF-2 increased calbindin D-28k (CalB) gene expression and prevented glutamate-induced neuronal death. These effects were abolished by pretreatment with PD166866 (FGF receptor 1 [FGFR1] tyrosine kinase-specific inhibitor). This indicated that FGFR1 activation and increased CalB expression were involved in SUN11602-mediated neuroprotection. However, receptor-binding assays revealed that unlike FGF-2, SUN11602 did not alter the binding of (125)I-labeled FGF-2 to FGFR1. To investigate the possible proliferative activity of SUN11602, we utilized BHK21 and SKN cells expressing endogenous FGFR1. FGF-2 promoted cell proliferation whereas SUN11602 did not. In in vivo studies, wild-type (WT) and CalB-deficient (CalB(-/-)) mice were injected with aggregated Aß1-40 and ibotenate (NMDA receptor agonist) to severely damage the hippocampal tissue. Treatment with SUN11602 (orally) or FGF-2 (intraparenchymally) at the midpoint of Aß1-40 and ibotenate injections prevented the hippocampal damage in WT mice, however this effect was abolished in CalB(-/-) mice. Thus, SUN11602 exerted protective effects on hippocampal neurons through activation of FGFR1 and increased CalB expression. Moreover, the neuroprotective effects of SUN11602 depended upon the various biological activities of FGF-2.

Transplantation of co-aggregates of Sertoli cells and islet cells into liver without immunosuppression.

BACKGROUND: Transplantation of islets of Langerhans (islets) was used to treat insulin-dependent diabetes mellitus. However, islet grafts must be maintained by administration of immunosuppressive drugs, which can lead to complications in the long term. An approach that avoids immunosuppressive drug use is desirable. METHODS: Co-aggregates of Sertoli cells and islet cells from BALB/c mice that were prepared by the hanging drop method were transplanted into C57BL/6 mouse liver through the portal vein as in human clinical islet transplantation. RESULTS: The core part of the aggregates contained mainly Sertoli cells, and these cells were surrounded by islet cells. The co-aggregates retained the functions of both Sertoli and islet cells. When 800 co-aggregates were transplanted into seven C57BL/6 mice via the portal vein, six of seven recipient mice demonstrated quasi-normoglycemia for more than 100 days. CONCLUSIONS: The hanging drop method is suitable for preparing aggregates of Sertoli and islet cells for transplantation. Notably, transplantation of these allogeneic co-aggregates into mice with chemically induced diabetes via the portal vein resulted in long-term graft survival without systemic immunosuppression.

SUN11602 has basic fibroblast growth factor-like activity and attenuates neuronal damage and cognitive deficits in a rat model of Alzheimer's disease induced by amyloid ß and excitatory amino acids.

Basic fibroblast growth factor (bFGF/FGF-2) is known to possess neuroprotective and neurite outgrowth activity properties. In this study, the effects of a novel synthetic compound that mimics the neuroprotective properties of bFGF - SUN11602 - were examined in vitro and in vivo. SUN11602 promoted neurite outgrowth of primarily cultured rat hippocampal neurons. For the in vivo study, an Alzheimer's disease (AD) model with severe damage to the hippocampal tissue was constructed by injecting the hippocampi of rats with aggregated Aß1-40, followed 48 h later by an injection of ibotenate [an agonist for N-methyl-d-aspartate (NMDA) receptor]. Oral administration of SUN11602 at the midpoint of Aß1-40 and ibotenate injections attenuated short-term memory impairment in the Y-maze test, as well as spatial learning deficits in the water maze task. In addition, the SUN11602 treatment inhibited the increase of peripheral-type benzodiazepine-binding sites (PTBBS), which are a marker for gliosis. A negative correlation was found between PTBBS numbers and learning capacity in the water maze task. These results suggest that SUN111602 improved memory and learning deficits in the hippocampally lesioned rats by preventing neuronal death and/or promotion of neurite outgrowth. Taken together, these results indicate that SUN11602, a bFGF-like compound with neuroprotective and neurite outgrowth activity, may be beneficial for the treatment of progressive neurodegenerative diseases such as AD.

Immobilization of Sertoli cells on islets of Langerhans.

Sertoli cells play a crucial role in creating the immunoprivileged environment of the testis. We examined the survival of islets of Langerhans after co-transplantation with Sertoli cells. Sertoli cells near islets should protect the graft from rejection. In this study, conjugates of single stranded oligonucleotides, poly(ethylene glycol) and phospholipids (ssDNA-PEG-DPPE) were used to immobilize Sertoli cells on islets. The 20-mer of deoxyadenylic acid (oligo(dA)20) and 20-mer of deoxythymidylic acid (oligo(dT)20) were presented as ssDNAs on the surfaces of Sertoli cells and islets, respectively, through the hydrophobic interaction between a lipid unit of the conjugates and the cell membrane. The Sertoli cells were immobilized on the islets through hybridization between oligo(dA)20 and oligo(dT)20. When Sertoli cell-immobilized islets were infused into the liver of mice through the portal vein, the Sertoli cells remained around the islets.

SUN11602, a novel aniline compound, mimics the neuroprotective mechanisms of basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) offers some measure of protection against excitotoxic neuronal injuries by upregulating the expression of the calcium-binding protein calbindin-D28k (Calb). The newly synthesized small molecule 4-({4-[[(4-amino-2,3,5,6-tetramethylanilino)acetyl](methyl)amino]-1-piperidinyl}methyl)benzamide (SUN11602) mimics the neuroprotective effects of bFGF, and thus, we examined how SUN11602 exerts its actions on neurons in toxic conditions of glutamate. In primary cultures of rat cerebrocortical neurons, SUN11602 and bFGF prevented glutamate-induced neuronal death. This neuroprotection, which occurred in association with the augmented phosphorylation of the bFGF receptor-1 (FGFR-1) and the extracellular signal-regulated kinase-1/2 (ERK-1/2), was abolished by pretreatment with PD166866 (a FGFR-1 tyrosine kinase-specific inhibitor) and PD98059 (a mitogen-activated protein kinase [MAPK]/[ERK-1/2] kinase [MEK] inhibitor). In addition, SUN11602 and bFGF increased the levels of CALB1 gene expression in cerebrocortical neurons. Whether this neuroprotection was linked to Calb was investigated with primary cultures of cerebrocortical neurons from homozygous knockout (Calb(-/-)) and wild-type (WT) mice. In WT mice, SUN11602 and bFGF increased the levels of newly synthesized Calb in cerebrocortical neurons and suppressed the glutamate-induced rise in intracellular Ca(2+). This Ca(2+)-capturing ability of Calb allowed the neurons to survive severe toxic conditions of glutamate. In contrast, Calb levels remained unchanged in Calb(-/-) mice after exposure to SUN11602 or bFGF, and due to a loss of function of the gene, these neurons were no longer resistant to toxic conditions of glutamate. These findings indicated that SUN11602 activated a number of cellular molecules (FGFR-1, MEK/ERK intermediates, and Calb) and consequently contributed to intracellular Ca(2+) homeostasis as observed in the case of bFGF.

Minimum structure requirement of immunomodulatory glycolipids for predominant Th2 cytokine induction and the discovery of non-linear phytosphingosine analogs.

Analogs of immunomodulatory glycolipid OCH (2) were prepared and minimum structure requirement to exhibit equivalent profiles was disclosed. Analogs bearing non-linear hydrocarbon chain in the phytosphingosine moiety (18, 19) were shown for the first time to possess comparable cytokine inducing profile to 2. Molecular modeling of 2/hCD1d complex based on the crystal structure of alpha-GalCer (1)/hCD1d complex is also described.