Takehiko Yajima

Mishandling of the Therapeutic Peptide Glucagon Generates Cytotoxic Amyloidogenic Fibrils

AbstractPurpose. Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application. Methods. Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concen- trations ranging from 1 mg/ml to 80 mg/ml and then aged at 37°C. Amyloidogenic properties were assessed by circular dichroism (CD), electron microscopy (EM), staining with β-sheet-specific dyes, and size-exclusion chromatography (SEC). Cytotoxic characteristics were determined concomitantly. Results. By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a β-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml. Conclusions. These results indicated that glucagon was self-associated by its β-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as β-amyloid (Aβ)1-42 and prion protein fragment (PrP)106-126 including conformational change to a β-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.

PACAP protects neuronal PC12 cells from the cytotoxicity of human prion protein fragment 106–126

Misfolding of the prion protein yields amyloidogenic isoforms, and it shows exacerbating neuronal damage in neurodegenerative disorders including prion diseases. Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) potently stimulate neuritogenesis and survival of neuronal cells in the central nervous system. Here, we tested these neuropeptides on neurotoxicity in PC12 cells induced by the prion protein fragment 106–126 [PrP (106–126)]. Concomitant application of neuropeptide with PrP(106–126) (5×10−5 M) inhibited the delayed death of neuron‐like PC12 cells. In particular, PACAP27 inhibited the neurotoxicity of PrP(106–126) at low concentrations (>10−15 M), characterized by the deactivation of PrP(106–126)‐stimulated caspase‐3. The neuroprotective effect of PACAP27 was antagonized by the selective PKA inhibitor, H89, or the MAP kinase inhibitor, U0126. These results suggest that PACAP27 attenuates PrP(106–126)‐induced delayed neurotoxicity in PC12 cells by activating both PKA and MAP kinases mediated by PAC1 receptor.

Bioactive analogues and drug delivery systems of vasoactive intestinal peptide (VIP) for the treatment of asthma/COPD

Vasoactive intestinal peptide (VIP) is one of the major peptide transmitters in the central and peripheral nervous systems, being involved in a wide range of biological functions. In an airway system where VIP-immunoreactive nerve fibers are present, VIP acts as neurotransmitter or neuromodulator of the inhibitory non-adrenergic and non-cholinergic airway nervous system and influences many aspects of pulmonary biology. A clinical application of VIP has been believed to offer potential benefits in the treatment of chronic inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD), however, its clinical application has been limited in the past for a number of reasons, including its extremely short plasma half-life after intravenous administration and difficulty in administration routes. The development of long-acting VIP analogues, in combination with appropriate drug delivery systems, may provide clinically useful agents for the treatment of asthma/COPD. In this review, development of efficacious VIP derivatives, drug delivery systems designed for VIPs and the potential application for asthma/COPD are discussed. We also include original data from our chemical modification experiments and formulation studies, which led to successful development of [R(15, 20, 21), L(17)]-VIP-GRR (IK312532), a potent VIP analogue, and a VIPs-based dry powder inhaler system.

Separation of metallothionein into isoforms by column switching on gel permeation and ion-exchange columns with high-performance liquid chromatography-atomic-absorption spectrophotometry.

Metallothionein in tissue supernatant was separated into isoforms by on-line switching from gel filtration to ion-exchange columns and the metals bound to the isoproteins were directly determined by using a flame atomic-absorption spectrophotometer as a specific detector for metals. Conditions for elution of the gel filtration column were established and Tris-HCl buffer solutions in low concentration were shown to be applicable to the column-switching experiment. The effects of pH, buffer and salt concentrations on the elution of metallothionein are discussed.

Development of a fluorescent chelating ligand for gallium ion having a quinazoline structure with two Schiff base moieties.

A novel chelating ligand, 2,4-[bis-(2,4-dihydroxybenzylidene)]-dihydrazinoquinazoline (DBHQ), was synthesized, and the fluorescence characteristics of its complex with metal ions were investigated. Thirty-five different metal ions were tested for the emission of fluorescence in the presence of DBHQ in aqueous solutions in a pH range of 3.0-10.5 (at a difference of 0.5 for each metal). It was observed that DBHQ fluoresces intensely at 470nm with an excitation wavelength of 405nm in the presence of Ga(3+) or Al(3+) in the pH range 3.0-4.0. The other metal ions did not show fluorescence with DBHQ. Although the presence of Cu(2+), Co(2+) and Fe(3+) decreased the fluorescence intensity of DBHQ-Ga(3+), the addition of a fluoride ion (NaF) recovered the fluorescence by masking the interfering ions. In addition, the fluoride ions were found to enhance the sensitive determination of Ga(3+) because the fluorescence intensity of DBHQ-Ga(3+) was further increased approximately 2.5-fold in the presence of F(-) (phi=0.658) from that in the absence of F(-) (phi=0.401). The fluoride ions also masked the Al(3+) ions, which emit fluorescence on chelation with DBHQ. Therefore, a selective and sensitive detection of Ga(3+) was achieved by using DBHQ in the presence of F(-). The detection limit of Ga(3+) was approximately 50nmolL(-1) (3.5ppb). The proposed method was applicable to determine Ga(3+) in river water.

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide attenuate glutamate-induced nNOS activation and cytotoxicity

Both vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in the central and peripheral nervous systems. Attention has been focused on these neuropeptides because among their numerous biological activities, they have been confirmed to show neuroprotective effects against ischemia and glutamate-induced cytotoxicity. It is well established that glutamate has excitatory effects on neuronal cells, and that excessive glutamate shows potent neurotoxicity, especially in neuronal nitric oxide synthase-containing neurons. Glutamate stimulates the production of nitric oxide (NO) in neurons, and the NO generated is tightly associated with the delayed death of neurons. We examined the effects of these neuropeptides on the glutamate-induced neural actions using PC12 cells, and we confirmed the important activities of PACAP/VIP on the production of NO as well as the delayed cell death stimulated by glutamate.

Pituitary adenylate cyclase activating polypeptide regulates the basal production of nitric oxide in PC12 cells.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon family, modulate neurotransmission via stimulation of protein kinases including cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the central and peripheral nervous systems. They are reported to co-exist with nitric oxide synthases (NOSs) and other neuropeptides within the nervous system and peripheral tissues. In the present study, we investigated the neuronal role of these peptides in NO production in PC12 cells. We showed that PACAP decreased NO production in a dose-dependent manner, and the activators of protein kinase A and C also inhibited the NO production in PC12 cells. RT-PCR experiments demonstrated that PC12 cells constitutively express the mRNAs for neuronal NOS and the PACAP-specific (PAC1) receptor, and we concluded that PACAP plays an important role in the regulation of nNOS activity through PAC1 receptor in PC12 cells.

Physicochemical and pharmacological characterization of novel vasoactive intestinal peptide derivatives with improved stability

Previously, [R(15,20,21), L(17)]-VIP-GRR (IK312532), a long-acting VIP derivative, was proposed as potential drug candidate for the treatment of asthma/COPD. The present work is aimed to elucidate solution-state stability of IK312532 and to develop further stabilized derivative with equipotent or higher biological functions. A stability study on IK312532 was carried out in solution state, and degradation mechanism was deduced by UPLC-MS and amino acid analyses. Three novel VIP derivatives were designed and chemically synthesized on the basis of stability data, being subjected to physicochemical and pharmacological characterization. Solution-state stability studies revealed the gradual degradation of IK312532, following pseudo-first-order kinetics. Chemical modification of IK312532, mainly position at 24, resulted in marked improvement of stability, although the chemical modification had no influence on the secondary structure, receptor binding, and activation of adenylate cyclase in rat lung cells. Novel derivatives also exhibited more potent neurite outgrowth in rat pheochromocytoma PC12 cells when compared to VIP and IK312532, possibly due to improved stability. Deamination of Asn at position 24 might be responsible for degradation of VIP derivative, and stability and chemical modification studies led us to the successful development of novel VIP derivatives with higher stability and biological functions.

Long-acting analogue of vasoactive intestinal peptide, [R15, 20, 21, L17]-VIP-GRR (IK312532), protects rat alveolar L2 cells from the cytotoxicity of cigarette smoke

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) act as neurotransmitters in numerous biological responses. We previously reported that the replacement of Lys by Arg, and Met by Leu in VIP (IK312532; [Arg15, 20, 21, Leu17]-VIP) resulted in a significant improvement in metabolic stability and biological activity. In the present study, we investigated the effect of VIP and its related peptides including long-acting VIP derivative (IK312532) and PACAP27 on the cytotoxicity of cigarette smoke extract (CSE), a causative factor of chronic obstructive pulmonary disease (COPD), in rat alveolar L2 cells. RT-PCR displayed the dominant expression of mRNA for the VIP-specific VPAC2 receptor in L2 cells, and VIP and the related peptides showed the specific binding activity and potent stimulation of adenylate cyclase. CSE at a concentration of 0.1% or higher induced significant apoptotic death of L2 cells. Interestingly, the addition of neuropeptides at a concentration of 10(-11) M or higher in L2 cells with CSE (0.25%) resulted in significant attenuation of cell death with the deactivation of CSE-evoked caspase-3 activity. IK312532 was much stable against the enzymatic digestion compared to VIP, and the protective effect of IK312532 was 1.6-fold higher than that of VIP. Taken together with our previous report showing that IK312532 has long-acting relaxant activity in the lung, IK312532 may be a potential candidate for drug treatment of asthma and COPD.

Simultaneous Analysis of Cocaalkaloids and Sugars in Illicit Cocaine Using Capillary Electrophoresis

Most illicit cocaine is adulterated with other substances such as sugars and polyhydric alcohols or local anesthestics. Various sugars have been detected in seized cocaine. Analysis of sugars, polyhydric alcohols, and cocaalkaloids yields helpful information that aids in identification of the sample seized as well as the possible route of sales. We analyzed illicit cocaine directly using capillary electrophoresis. As a result, we were able to separate and detect sugars and polyhydric alcohols and cocaalkaloids using a combination of Micelle electrokinetic chromatography (MEKC) and indirect UV detection.