[Conjugation to Branched Glycerol Oligomers, a Novel Strategy for Extremely Hydrophobic Agents].

Ascertaining the absorption, distribution, metabolism, and excretion (ADME) profile of drugs is one of the most crucial factors in the process of drug discovery. Since it is important to combine water solubility and cell permeability within the compound to achieve the desired ADME properties, an appropriate balance between lipophilicity and hydrophilicity is required. It is often necessary to facilitate hydrophilicity of very hydrophobic candidates, because quite lipophobic molecules are rarely hit as positive in molecular-targeted or cell-based screenings. For that purpose, it has been popular to conjugate hydrophobic molecules with polyethylene glycol (PEG). However, PEG is a polymer, and PEG-conjugated molecules are not uniform. Besides, the dosage should be much increased compared with the original molecule due to the increase in molecular weight. Therefore we have been developing alternative ways to endow hydrophobic compounds with extra hydrophilicity by conjugating with symmetrically branched glycerol oligomers. This technology is versatile and easily applicable to various hydrophobic compounds. Water-solubility of fenofibrate, one of the most hydrophobic medicines in clinical use, was facilitated by a factor of more than 2000, and its lipid-lowering effect in vivo improved more than ten-fold, by simply conjugating with branched glycerol trimer, for instance. Here we will briefly introduce the basic concepts and our successful experiences of applying branched glycerol oligomers including antitumor agents in terms of water-solubility, pharmacological effects, and pharmacokinetics, and merits and current issues will be discussed in this review.

Histamine H1 Receptor Gene Expression and Drug Action of Antihistamines.

The upregulation mechanism of histamine H1 receptor through the activation of protein kinase C-δ (PKCδ) and the receptor gene expression was discovered. Levels of histamine H1 receptor mRNA and IL-4 mRNA in nasal mucosa were elevated by the provocation of nasal hypersensitivity model rats. Pretreatment with antihistamines suppressed the elevation of mRNA levels. Scores of nasal symptoms were correlatively alleviated to the suppression level of mRNAs above. A correlation between scores of nasal symptoms and levels of histamine H1 receptor mRNA in the nasal mucosa was observed in patients with pollinosis. Both scores of nasal symptoms and the level of histamine H1 receptor mRNA were improved by prophylactic treatment of antihistamines. Similar to the antihistamines, pretreatment with antiallergic natural medicines showed alleviation of nasal symptoms with correlative suppression of gene expression in nasal hypersensitivity model rats through the suppression of PKCδ. Similar effects of antihistamines and antiallergic natural medicines support that histamine H1 receptor-mediated activation of histamine H1 receptor gene expression is an important signaling pathway for the symptoms of allergic diseases. Antihistamines with inverse agonist activity showed the suppression of constitutive histamine H1 receptor gene expression, suggesting the advantage of therapeutic effect.

8-Prenylnaringenin promotes recovery from immobilization-induced disuse muscle atrophy through activation of the Akt phosphorylation pathway in mice.

8-Prenylnaringenin (8-PN) is a prenylflavonoid that originates from hop extracts and is thought to help prevent disuse muscle atrophy. We hypothesized that 8-PN affects muscle plasticity by promoting muscle recovery under disuse muscle atrophy. To test the promoting effect of 8-PN on muscle recovery, we administered an 8-PN mixed diet to mice that had been immobilized with a cast to one leg for 14 days. Intake of the 8-PN mixed diet accelerated recovery from muscle atrophy, and prevented reductions in Akt phosphorylation. Studies on cell cultures of mouse myotubes in vitro demonstrated that 8-PN activated the PI3K/Akt/P70S6K1 pathway at physiological concentrations. A cell-culture study using an inhibitor of estrogen receptors and an in vivo experiment with ovariectomized mice suggested that the estrogenic activity of 8-PN contributed to recovery from disuse muscle atrophy through activation of an Akt phosphorylation pathway. These data strongly suggest that 8-PN is a naturally occurring compound that could be used as a nutritional supplement to aid recovery from disuse muscle atrophy.

Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin.

Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.

A novel benzofuran, 4-methoxybenzofuran-5-carboxamide, from Tephrosia purpurea suppressed histamine H1 receptor gene expression through a protein kinase C-δ-dependent signaling pathway.

Histamine H1 receptor (H1R) gene is upregulated in patients with allergic rhinitis (AR), and its expression level is strongly correlated with the severity of allergic symptoms. We previously reported isolation of the putative anti-allergic compound, 4-methoxybenzofuran-5-carboxamide (MBCA) from Tephrosia purpurea and its chemical synthesis (Shill et al., Bioorg Med Chem 2015;23:6869-6874). However, the mechanism underlying its anti-allergic activity remains to be elucidated. Here, we report the mechanism of MBCA on phorbol 12-myristate-13-acetate (PMA)- or histamine-induced upregulation of H1R gene expression in HeLa cells, and in vivo effects of MBCA were also determined in toluene-2,4-diisocyanate (TDI)-sensitized rats. MBCA suppressed PMA- and histamine-induced upregulation of H1R expression at both mRNA and protein levels and inhibited PMA-induced phosphorylation of PKCδ at Tyr(311) and subsequent translocation to the Golgi. Furthermore, MBCA ameliorated allergic symptoms and suppressed the elevation of H1R and helper T cell type 2 (Th2) cytokine mRNAs in TDI-sensitized rats. Data suggest that MBCA alleviates nasal symptoms in TDI-sensitized rats through the inhibition of H1R and Th2 cytokine gene expression. The mechanism of its H1R gene suppression underlies the inhibition of PKCδ activation.

The isolation and synthesis of a novel benzofuran compound from Tephrosia purpurea, and the synthesis of several related derivatives, which suppress histamine H1 receptor gene expression.

A novel naturally occurring compound with a benzofuran skeleton was isolated from a plant, Tephrosia purpurea collected in Bangladesh. The chemical synthesis of this compound confirmed its structure, and preliminary biological results showed its suppressive activity towards histamine H1 gene expression. One isomer and four derivatives were also synthesized, and their suppression activity was investigated. Although only small quantities of this compound can be isolated from its natural source, a 10 g scale synthesis was demonstrated by the newly developed method.

Maackiain is a novel antiallergic compound that suppresses transcriptional upregulation of the histamine H1 receptor and interleukin-4 genes.

Kujin contains antiallergic compounds that inhibit upregulation of histamine H1 receptor (H1R) and interleukin (IL)-4 gene expression. However, the underlying mechanism remains unknown. We sought to identify a Kujin-derived antiallergic compound and investigate its mechanism of action. The H1R and IL-4 mRNA levels were determined by real-time quantitative RT-PCR. To investigate the effects of maackiain in vivo, toluene-2,4-diisocyanate (TDI)-sensitized rats were used as a nasal hypersensitivity animal model. We identified (-)-maackiain as the responsible component. Synthetic maackiain showed stereoselectivity for the suppression of IL-4 gene expression but not for H1R gene expression, suggesting distinct target proteins for transcriptional signaling. (-)-Maackiain inhibited of PKCδ translocation to the Golgi and phosphorylation of Tyr(311) on PKCδ, which led to the suppression of H1R gene transcription. However, (-)-maackiain did not show any antioxidant activity or inhibition of PKCδ enzymatic activity per se. Pretreatment with maackiain alleviated nasal symptoms and suppressed TDI-induced upregulations of H1R and IL-4 gene expressions in TDI-sensitized rats. These data suggest that (-)-maackiain is a novel antiallergic compound that alleviates nasal symptoms in TDI-sensitized allergy model rats through the inhibition of H1R and IL-4 gene expression. The molecular mechanism underlying its suppressive effect for H1R gene expression is mediated by the inhibition of PKCδ activation.

Disruption of Heat Shock Protein 90 (Hsp90)-Protein Kinase Cδ (PKCδ) Interaction by (-)-Maackiain Suppresses Histamine H1 Receptor Gene Transcription in HeLa Cells.

The histamine H1 receptor (H1R) gene is an allergic disease sensitive gene, and its expression level is strongly correlated with the severity of allergic symptoms. (-)-Maackiain was identified as a Kujin-derived anti-allergic compound that suppresses the up-regulation of the H1R gene. However, the underlying mechanism of H1R gene suppression remains unknown. Here, we sought to identify a target protein of (-)-maackiain and investigate its mechanism of action. A fluorescence quenching assay and immunoblot analysis identified heat shock protein 90 (Hsp90) as a target protein of (-)-maackiain. A pull-down assay revealed that (-)-maackiain disrupted the interaction of Hsp90 with PKCδ, resulting in the suppression of phorbol 12-myristate 13-acetate (PMA)-induced up-regulation of H1R gene expression in HeLa cells. Additional Hsp90 inhibitors, including 17-(allylamino)-17-demethoxygeldanamycin, celastrol, and novobiocin also suppressed PMA-induced H1R gene up-regulation. 17-(Allylamino)-17-demethoxygeldanamycin inhibited PKCδ translocation to the Golgi and phosphorylation of Tyr(311) on PKCδ. These data suggest that (-)-maackiain is a novel Hsp90 pathway inhibitor. The underlying mechanism of the suppression of PMA-induced up-regulation of H1R gene expression by (-)-maackiain and Hsp90 inhibitors is the inhibition of PKCδ activation through the disruption of Hsp90-PKCδ interaction. Involvement of Hsp90 in H1R gene up-regulation suggests that suppression of the Hsp90 pathway could be a novel therapeutic strategy for allergic rhinitis.

N-myristoylated ubiquitin ligase Cbl-b inhibitor prevents on glucocorticoid-induced atrophy in mouse skeletal muscle.

A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120 µM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.

Prevention of aberrant protein aggregation by anchoring the molecular chaperone αB-crystallin to the endoplasmic reticulum.

The chaperone αB-crystallin (αBC) is a member of the small heat shock protein family and its point or truncated mutants cause the muscular disorder α-crystallinopathy. The illness is histologically characterized by accumulation of protein aggregates in muscle cells. Expression of the myopathy-causing R120G mutant of αBC, harboring an arginine-to-glycine mutation at position 120, results in aggregate formation. We demonstrated that tethering αBC to the endoplasmic reticulum (ER) membrane represses the protein aggregation mediated by the R120G mutant. ER-anchored αBC decreased the amount of the R120G mutant through autophagic proteolysis. In contrast, knockdown of ATG5, an E3 ligase essential for autophagy, in ER-anchored αBC-transfected cells restored the quantity of the R120G mutant. In this context, aggregate formation was still suppressed, indicating that ER-anchored αBC profoundly constrains aggregation competency of the R120G mutant separately from downregulating the abundance of the mutant. We have proposed that protein aggregation is prevented by manipulation of the ER microenvironment with αBC, and have shed light on a novel aspect of the ER as a therapeutic target.

Prenylation enhances quercetin uptake and reduces efflux in Caco-2 cells and enhances tissue accumulation in mice fed long-term.

Prenyl flavonoids are widely distributed in plant foods and have attracted appreciable attention in relation to their potential benefits for human health. Prenylation may enhance the biological functions of flavonoids by introducing hydrophobic properties in their basic structures. Previously, we found that 8-prenyl naringenin exerted a greater preventive effect on muscle atrophy than nonprenylated naringenin in a mouse model. Here, we aimed to estimate the effect of prenylation on the bioavailability of dietary quercetin (Q). The cellular uptake of 8-prenyl quercetin (PQ) and Q in Caco-2 cells and C2C12 myotube cells was examined. Prenylation significantly enhanced the cellular uptake by increasing the lipophilicity in both cell types. In Caco-2 cells, efflux of PQ to the basolateral side was <15% of that of Q, suggesting that prenylation attenuates transport from the intestine to the circulation. After intragastric administration of PQ or Q to mice or rats, the area under the concentration-time curve for PQ in plasma and lymph was 52.5% and 37.5% lower than that of Q, respectively. PQ and its O-methylated form (MePQ) accumulated at much higher amounts than Q and O-methylated Q in the liver (Q: 3400%; MePQ: 7570%) and kidney (Q: 385%; MePQ: 736%) of mice after 18 d of feeding. These data suggest that prenylation enhances the accumulation of Q in tissues during long-term feeding, even though prenylation per se lowers its intestinal absorption from the diet.

A novel prodrug strategy for extremely hydrophobic agents: conjugation to symmetrically branched glycerol trimer improves pharmacological and pharmacokinetic properties of fenofibrate.

Management of a lipophilic-hydrophilic balance is a key element in drug design to achieve desirable pharmacokinetic characters. Therefore we have created unique modular molecules, symmetrically branched oligoglycerols (BGL), as an alternative way to endow hydrophobic molecules with sufficient hydrophilicity. We have successfully demonstrated amelioration of the water solubility and thermal stability of several hydrophobic agents by covalent conjugation to BGL so far. However, it has not been clarified whether the molecular modification by BGL also improves the pharmacological and/or pharmacokinetic properties indeed. Recently, we synthesized a novel BGL-prodrug derivative of fenofibrate, which is an antihyperlipidemic agent and one of the most hydrophobic medicinal compounds currently used clinically, by conjugating fenofibric acid to symmetrically branched glycerol trimer (BGL003), the simplest BGL. We have previously demonstrated that the hydrophilicity and water solubility of fenofibrate are improved more than 2000 times just by conjugation to the BGL003. To verify our hypothesis that the prodrug strategy with BGL should improve pharmacological efficacy and pharmacokinetic properties of extremely hydrophobic agents such as fenofibrate by the rise in hydrophilicity, we evaluated the BGL003-prodrug derivative of fenofibrate (FF-BGL) using rodent models. Here we demonstrate that the lipid-lowering effects of fenofibrate are much potentiated by chemical conjugation to BGL003 without exhibiting significant toxicity. Plasma concentration of fenofibric acid, an active metabolite of fenofibrate, after single oral administration of FF-BGL was more than 3 times higher than that of fenofibrate, in accordance. In fasting rats, plasma concentration of fenofibric acid after fenofibrate administration was curtailed into less than half of that in ad libitum-fed rats, while FF-BGL showed about the same plasma level even in the starving rats. This is the first report showing that BGL-prodrug improves pharmacological and pharmacokinetic properties as well as hydrophilicity of highly hydrophobic compounds. Furthermore, prodrug strategy using BGL suggests the possibility of diminishing the food-drug interaction effects, which should be advantageous for promoting drug compliance. BGL will be a suitable prodrug strategy to ameliorate physical, pharmacological, and pharmacokinetic characteristics of extremely hydrophobic compounds.

Acute oral toxicity evaluation of symmetrically branched glycerol trimer in ddY mice.

Lipophilic-hydrophilic balance is a quite important determinant of pharmacokinetic properties of pharmaceuticals. Thus it is a key step to successfully manage lipophilic-hydrophilic balance in drug design. We have designed unique modular molecules, symmetrically branched oligoglycerols (BGL) as an alternative means to endow hydrophobic molecules with much hydrophilicity. We have succeeded in improving the water-solubility of several hydrophobic medicinal small molecules and thermal stability of artificial protein by covalent conjugation to BGL. We have also demonstrated that a representative BGL, symmetrically branched glycerol trimer (BGL003) does not exhibit significant cytotoxicity against human hepatocarcinoma HepG2 cells. However, there have been no reports suggesting whether BGL could be used in safety in vivo. Therefore, evaluation of acute oral toxicity of BGL003 in healthy mice was conducted. Here we demonstrate that an oral administration of BGL003 did not exhibit acute lethal toxicity up to 3,000 mg/kg. Body weight, food intake, blood glucose levels and weights of tissues were not affected by a short-term repetitive administration of increasing doses of BGL003. Biochemical indications related to hepatic disorders and tissue damage were unchanged, either. A single administration study revealed that 50% lethal dose of BGL003 should be more than 2,000 mg/kg. BGL003 will be safe and suitable approach to improve hydrophilicity of hydrophobic compounds.

Prevention of disuse muscle atrophy by dietary ingestion of 8-prenylnaringenin in denervated mice.

Flavonoids have attracted considerable attention in relation to their effects upon health. 8-Prenylnaringenin (8-PN) is found in the common hop (Humulus lupulus) and assumed to be responsible for the health impact of beer consumption. We wanted to clarify the effects of prenylation on the physiological functions of dietary flavonoids by comparing the effects of 8-PN with that of intact naringenin in the prevention of disuse muscle atrophy using a model of denervation in mice. Consumption of 8-PN (but not naringenin) prevented loss of weight in the gastrocnemius muscle further supported by the lack of induction of the protein content of a key ubiquitin ligase involved in muscle atrophy, atrogin-1, and by the activation of Akt phosphorylation. 8-PN content in the gastrocnemius muscle was tenfold higher than that of naringenin. These results suggested that, compared with naringenin, 8-PN was effectively concentrated into skeletal muscle to exert its preventive effects upon disuse muscle atrophy. It is likely that prenylation generates novel functions for 8-PN by enhancing its accumulation into muscle tissue through dietary intake.

Cytotoxicity evaluation of symmetrically branched glycerol trimer in human hepatocellular carcinoma HepG2 cells.

An appropriate balance between lipophilicity and hydrophilicity is necessary for pharmaceuticals to achieve fine Absorption, Distribution, Metabolism and Excretion (ADME) properties including absorption and distribution, in particular. We have designed and proposed symmetrically branched oligoglycerols (BGL) as an alternative approach to improve the lipophilic-hydrophilic balance. We have previously shown that stability in circulation and water-solubility of such molecules as proteins, liposomes and hydrophobic compounds are much improved by conjugation to BGL. Albeit these successful applications of BGL, little was known whether BGL could be used in safety. Thus we conducted evaluation of the cytotoxicity of a representative BGL, symmetrically branched glycerol trimer (BGL003) in the cultured cells to clarify its biological safeness. Here we demonstrate that water-solubility of an extremely hydrophobic agent, fenofibrate was more than 2,000-fold improved just by conjugated with BGL003. BGL003 did not exhibit any significant cytotoxicity in human hepatocarcinoma HepG2 cells. Thus BGL003 should be safe and suitable strategy to endow hydrophobic molecules with much hydrophilicity.

Synthesis of highly water-soluble fibrate derivatives via BGLation.

Three water-soluble fibrates (fenofibrate, bezafibrate and chlofibrate) conjugated with a symmetrically branched glyceryl trimer (BGL003) were synthesized, and an evaluation of the fenofibrate-BGL003 conjugate as a candidate for anti-hyperlipemia drug was carried out using rats. The water-solubility of the fenofibrate-BGL003 conjugate was several thousand times greater than that of the original fenofibrate. The lipid-lowering effects of the fenofibrate-BGL003 conjugate were as strong as those of the same grams of fenofibrate. The actual active species of fenofibrate, fenofibric acid, was detected in rats' blood, but neither the fenofibrate-BGL003 conjugate nor fenofibrate was detected, probably due to enzymatic hydrolysis of the ester bond. The plasma concentration of fenofibric acid derived from the fenofibrate-BGL003 conjugate was five times higher than that derived from fenofibrate 4h after administration.

Evaluation of the inhibitory effects of quercetin-related flavonoids and tea catechins on the monoamine oxidase-A reaction in mouse brain mitochondria.

Quercetin, a typical dietary flavonoid, is thought to exert antidepressant effects by inhibiting the monoamine oxidase-A (MAO-A) reaction, which is responsible for regulation of the metabolism of the neurotransmitter 5-hydroxytryptamine (5-HT) in the brain. This study compared the MAO-A inhibitory activity of quercetin with those of O-methylated quercetin (isorhamnetin, tamarixetin), luteolin, and green tea catechins ((-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin gallate) by measuring the formation of the oxidative deamination product of 5-HT, 5-hydroxyindole aldehyde (5-HIAL), in mouse brain mitochondria. Quercetin was inferior to luteolin in the inhibition of MAO-A activity, whereas isorhamnetin, tamarixetin, and tea catechins scarcely exerted inhibitory activity. Quercetin did not affect MAO-A activity in mouse intestinal mitochondria, indicating that it does not evoke side effects on the metabolism of dietary monoamines in the gut. These data suggest that quercetin is a weak (but safe) MAO-A inhibitor in the modulation of 5-HT levels in the brain.

Synthesis of paclitaxel-BGL conjugates.

Four kinds of symmetrically branched oligoglyceryl trimeric (BGL003)-paclitaxel conjugates and a corresponding heptameric (BGL007) conjugate were synthesized. Molecular weights of all the compounds were less than two times that of paclitaxel. The anti-tumor activity of the most water-soluble BGL003 conjugate was examined and found to be preserved in spite of the chemical modification that is displacement of the N3'-debenzoyl residue with the BGL003 succinyl residue.

Synthesis and evaluation of water-soluble resveratrol and piceatannol via BGLation.

Synthesis of four water-soluble resveratrol and piceatannol derivatives bearing symmetrically branched glyceryl trimer (BGL003) with a non-biocleavable linkage, and their biological evaluation as a mitochondrial fusion-inducing agent with cellular fat-reducing effect from cells, is described. The effect of Piceatannol-BGL003 conjugate was as high as that of original stilbenoids.