Salvianolic acid B as a substrate and weak catechol-O-methyltransferase inhibitor in rats

Abstract

1.?The aim of this study was to investigate the biotransformation of salvianolic acid B (SAB) by catechol-O-methyltransferase (COMT) and its interaction with levodopa (l-DOPA) methylation in rats.

2.?The enzyme kinetics of SAB were studied after incubation with rat COMT. The in vivo SAB and 3-monomethyl-SAB (3-MMS) levels were determined after a single dose of tolcapone with or without SAB administration. For l-DOPA, the effect of SAB inhibition on l-DOPA methylation was studied in vitro. The l-DOPA and 3-O-methyldopa (3-OMD) levels were determined after single and multiple doses of SAB with or without l-DOPA administration.

3.?After incubation, we found that SAB was methylated mainly by rat liver and kidney COMT. Tolcapone strongly inhibited the formation of 3-MMS in vitro and in vivo, without any change in the plasma concentration of SAB. Moreover, tolcapone significantly increased the cumulative bile excretion of SAB from 3% to 40% in the rat. SAB inhibited the methylation of l-DOPA with an IC₅₀ value of 2.08?μM in vitro. In vivo, a single intravenous dose of SAB decreased the plasma concentration of 3-OMD, with no obvious effect on the pharmacokinetics of l-DOPA. Multiple doses of SAB given to rats also decreased the plasma concentration of 3-OMD, while SAB increased the plasma concentration of l-DOPA.     

Potential transbuccal delivery of l-DOPA methylester prodrug: stability in the environment of the oral cavity and ability to cross the mucosal tissue

Levodopa (l-DOPA) is the most effective pharmacologic agent in Parkinson’s disease and remains the “gold standard”. Nevertheless, in long-term treatments, dyskinesias and motor complications can emerge. In this work, the combined use of l-DOPA methylester hydrochloride prodrug (LDME) with transbuccal drug delivery was supposed as a good alternative method to optimize the bioavailability of l-DOPA, to maintain constant plasma levels and to decrease the drug unwanted effects. The effects of environmental pH on buccal delivery of LDME were evaluated ex vivo. The increase of pH value from 5.8 to 6.2 implies an improvement of drug permeation. Since the pH increase causes the raising of hydrolytic conversion of LDME to l-DOPA, the pH value 6.2 was considered as a good compromise between drug stability and permeation rate. It was found that during the passage through the biological tissue, LDME undergoes a primary conversion to l-DOPA catalyzed by membrane’s enzymes. Supplementation of delivery with Tween 80® produces substantial enhancement of LDME passage through the membrane. The drug could be loaded in the IntelliDrug mechatronic device, released close to the buccal mucosa, so achieving and maintaining constant therapeutic blood levels for extensive time.     

Effects of asimilobine on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05–0.2 μM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC₅₀ value of 0.13 μM. Asimilobine at 0.15 μM inhibited tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca^2 +  concentrations. In addition, asimilobine at 0.05–5.0 μM, but not 10 μM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 μM) associated with l-DOPA (20, 50, and 100 μM) for 24 h inhibited l-DOPA-induced increases in dopamine levels and enhanced l-DOPA-induced cell death when compared with l-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances l-DOPA-induced cytotoxicity in PC12 cells.     

Catalponol enhances dopamine biosynthesis and protects against l-DOPA-induced cytotoxicity in PC12 cells

The effects of catalponol (1) on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells were investigated. Catalponol at concentration ranges of 1–5 μM increased the intracellular levels of dopamine at 12–48 h. Catalponol at concentrations of up to 10 μM did not alter cell viability. Tyrosine hydroxylase (TH) activity was enhanced by 1 at 3 μM in a time-dependent manner, but aromatic l-amino acid decarboxylase activity was not. Catalponol also increased the intracellular levels of cyclic AMP and TH phosphorylation. In addition, catalponol at 3 μM associated with l-DOPA (20–50 μM) further enhanced the increases in dopamine levels induced by l-DOPA (50–100 μM) at 24 h. Catalponol at 2–5 μM inhibited l-DOPA (100–200 μM)-induced cytotoxicity at 48 h. These results suggest that 1 enhanced dopamine biosynthesis by inducing TH activity and protected against l-DOPA-induced cytotoxicity in PC12 cells, which was mediated by the increased levels of cyclic AMP.     

Levodopa delivery systems: advancements in delivery of the gold standard

Importance of the field: Despite the fact that Parkinson's disease (PD) was discovered almost 200 years ago, its treatment and management remain immense challenges because progressive loss of dopaminergic nigral neurons, motor complications experienced by the patients as the disease progresses and drawbacks of pharmacotherapeutic management still persist. Various therapeutic agents have been used in the management of PD, including levodopa (l-DOPA), selegiline, amantadine, bromocriptine, entacapone, pramipexole dihydrochloride and more recently istradefylline and rasagiline. Of all agents, l-DOPA although the oldest, remains the most effective. l-DOPA is easier to administer, better tolerated, less expensive and is required by almost all PD patients. However, l-DOPA's efficacy in advanced PD is significantly reduced due to metabolism, subsequent low bioavailability and irregular fluctuations in its plasma levels. Significant strides have been made to improve the delivery of l-DOPA in order to enhance its bioavailability and reduce plasma fluctuations as well as motor complications experienced by patients purportedly resulting from pulsatile stimulation of the striatal dopamine receptors.

Areas covered in this review: Drug delivery systems that have been instituted for the delivery of l-DOPA include immediate release formulations, liquid formulations, dispersible tablets, controlled release formulations, dual-release formulations, microspheres, infusion and transdermal delivery, among others. In this review, the l-DOPA-loaded drug delivery systems developed over the past three decades are elaborated.

What the reader will gain: The ultimate aim was to assess critically the attempts made thus far directed at improving l-DOPA absorption, bioavailability and maintenance of constant plasma concentrations, including the drug delivery technologies implicated.

Take home message: This review highlights the fact that neuropharmaceutics is at a precipice, which is expected to spur investigators to take that leap to enable the generation of innovative delivery systems for the effective management of PD.     

IPX066, a mixed immediate/sustained-release levodopa preparation for Parkinson’s disease

Introduction: l-DOPA has long been the ‘gold standard’ treatment for Parkinson’s disease (PD), but suffers from poor oral bioavailability and rapid pharmacokinetic elimination. A longer acting preparation has long been sought.

Areas covered: We conducted PubMed search for IPX066 and reviewed abstracts from meetings that included the topic of PD. IPX066 is a novel mixed immediate release (IR) and sustained-release levodopa preparation designed to prolong the clinical effect of a single dose. Pharmacokinetic studies demonstrate similar time to peak dose as regular IR l-DOPA, but a longer duration of time with > 50% of peak dose. This contrasts with available controlled release preparations that have a delay to onset. Clinic trials in fluctuating PD patients show that IPX066 provided more ‘on’ time despite fewer daily doses, compared to IR l-DOPA. As expected, it was also superior to placebo in early PD. However, it is not known whether it can achieve l-DOPA levels that are continuous enough to delay the onset of fluctuations when given early in the disease.

Expert opinion: Although not a radical advance in l-DOPA therapy, the drug will clearly have a role in more advanced patients taking multiple l-DOPA doses and may have a role as first-line therapy when starting l-DOPA.     

Inhibitory Effects of Plant Extracts on Tyrosinase,l-DOPA Oxidation,and Melanin Synthesis

For medical, pharmacological, and cosmetic reasons, the demand for effective and safe depigmentating agents has increased. In this study, 101 plant extracts (methanol or water extracts) were screened for their inhibitory activities against tyrosinase, (l–3, 4,-dihydroxyphenylalanine) l-DOPA oxidation, and melanin biosynthesis in B16 mouse melanoma cells. Of the extracts examined, 31 showed over 50% inhibition of mushroom tyrosinase at a concentration of 666 μg/Ml, and 11 inhibited l-DOPA auto-oxidation at this concentration. In particular, extracts of Broussonetia kazinoki var. humilis (leaves and stems), Broussonetia papyrifera (leaves and bark), Cornus officinalis (fruit), Rhus javanica (gallnut), and Pinus densiflora (leaves) inhibited both tyrosinase activity and l-DOPA oxidation in a concentration-dependent manner. Seventeen plant extracts that inhibited tyrosinase were further tested for their inhibitory effects on melanogenesis. In B16 mouse melanoma cells, extracts of Acorus gramineus, Capsella bursa-pastoris, Morus bombycis, Perilla frutescens var. crispa, Quercus dentate (bark), Rhus javanica (gallnut), Schizopepon bryoniaefolius, or Sophora flavescens markedly inhibited (>50%) melanin synthesis at 50 μg/Ml. These plants represent a potential source of novel whitening agents for ultraviolet (UV)-sensitive skin.     

Mavoglurant as a treatment for Parkinson’s disease

Introduction: A major unresolved issue in the Parkinson’s disease (PD) treatment is the development of l-DOPA-induced dyskinesias (LIDs) as a side effect of chronic l-DOPA administration. Currently, LIDs are managed in part by reducing the l-DOPA dose or by the administration of amantadine. However, this treatment is only partially effective. A potential strategy, currently under investigation, is the coadministration of metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators (NAMs) and l-DOPA; a treatment that results in the improvement of dyskinesia symptoms and that permits reductions in l-DOPA dosage frequency.

Areas covered: The authors examine the role of mGluR5 in the pathophysiology of PD and the potential use of mGluR5 NAM as an adjuvant therapy together with a primary treatment with l-DOPA. Specifically, the authors look at the mavoglurant therapy and the evidence presented through preclinical and clinical trials.

Expert opinion: Interaction between mGluR5 NAM and l-DOPA is an area of interest in PD research as concomitant treatment results in the improvement of LID symptoms in humans, thus enhancing the patient’s quality of life. However, few months ago, Novartis decided to discontinue clinical trials of mavoglurant for the treatment of LID, due to the lack of efficacy demonstrated in trials NCT01385592 and NCT01491529, although no safety concerns were involved in this decision. Nevertheless, the potential application of mGluR5 antagonists as neuroprotective agents must be considered and further studies are warranted to better investigate their potential.     

Structural features of a polysaccharide from Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao

A polysaccharide Astragalus polysaccharide (APS) was obtained from the boiling water extract of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. The results of gas chromatography (GC) indicated that APS consisted of l-rhamnose (l-Rha), d-xylose (d-Xyl), d-glucose (d-Glc), and d-galactose (d-Gal) in the molar ratio of 1:4:5:1.5. Its molecular weight was determined to be 3.01 × 10⁵ by high-performance gel filtration chromatography. The results of ¹H NMR and ¹³C NMR spectral analysis indicated that APS had a linear backbone mainly consisting of 1,3-linked β-d-Gal residues with insertion of β-Glc, 1,6-linked α-Gal, 1,5-linked β-Xyl, 1,4-linked β-Gal, β-d-Gal, 1,2-linked α-Rha, 1,2,4-linked α-Rha residues. HSQC spectrum indicated that C-2 and C-6 may link with H.     

Two new metabolites from the mangrove endophytic fungus No. 2524

Two new metabolites, the cyclo-(l-Phe-l-Leu¹-l-Leu²-l-Leu³-l-Ile) (1) and (3S,4R)-dihydroxy-(6S)-undecyl-α-pyranone (2) have been produced by the endophytic fungus no. 2524 isolated from a seed of mangrove Avicennia marina in Hong Kong. The structures have been elucidated by spectra including two-dimensional NMR, ESI tandem mass spectrometry and CD. These compounds show no activity toward human cancer cell lines Bel-7402, NCI-4460 and the normal human cell lines L-02.     

Phenolic acid glucosides from the seeds of Entada phaseoloides Merill

Phytochemical investigation of the defatted seeds of Entada phaseoloides Merill. (Mimosaceae) led to the isolation of three new phenolic acid glucosides, which were characterized as 2-hydroxy-5-methylbenzoyl-β-l-glucopyranoside (p-cresotyl glucoside, 1), 2-hydroxy-5-methylbenzoyl-β-l-glucopyranosyl (2 → 1)-β-l-glucopyranosyl (2 → 1)-β-l-glucopyranoside (p-cresotyl triglucoside, 2), and 2-hydroxybenzoyl-β-l-glucopyranosyl (2 → 1)-β-l-glucopyranosyl (2 → 1)-β-l-glucopyranosyl (2 → 1)-β-l-glucopyranoside (salicylic acid tetraglucoside, 5), along with sucrose and triglucoside. The structures of these phytoconstituents have been established on the basis of spectral data analysis and chemical reactions.     

Four new triterpenoid glycosides from the seed residue of Hippophae rhamnoides subsp. sinensis

Four new triterpenoid saponins (1–4) were isolated from the seed residue of Hippophae rhamnoides subsp. sinensis, named 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 3)]-[α-l-rhamnopyranosyl-(1 → 2)]-α-l-arabinopyranosyl-13-ene-19-one-28-oic acid 28-O-β-d-glucopyranosyl ester (1), 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 3)]-[α-l-rhamnopyranosyl-(1 → 2)]-α-l-arabinopyranosyl-13-ene-19-one-30-hydroxyolean-28-oic acid 28-O-β-d-glucopyranosyl ester (2), 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 3)]-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranosyl-13-ene-19-one-28-oic acid 28-O-β-d-glucopyranosyl ester (3), and 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl-(1 → 3)]-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranosyl-13-ene-19-one-30-hydroxyolean-28-oic acid 28-O-β-d-glucopyranosyl ester (4), and their structures were elucidated on the basis of spectroscopic and chemical methods.     

Antimycobacterial activity of cyclic dipeptides isolated from Bacillus sp. N strain associated with entomopathogenic nematode

Context: Tuberculosis (TB) is one of the leading causes of morbidity and mortality with a global mortality rate of two million deaths per year; one-third of the world’s population is infected with Mycobacterium tuberculosis.

Objective: The aim of this study was to determine the antimycobacterial activity of six diketopiperazines (DKPs) purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) sp.

Materials and methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration of DKPs were determined using the broth dilution method on Middlebrook 7H11 against M. tuberculosis H₃₇Rv. Time-kill assay was used to determine the rate of killing of M. tuberculosis H₃₇Rv by DKPs. The cytotoxicity of the DKPs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay against the VERO cell line.

Results: Out of six DKP-tested cyclo-(d-Pro-l-Leu), cyclo-(l-Pro-l-Met) and cyclo-(d-Pro-l-Phe) recorded antimycobacterial activity, the cyclo-(l-Pro-l-Met) showed the highest activity and MIC values of 4?μg/ml for M. tuberculosis H₃₇Rv. The MIC value for rifampicin was 0.06?μg/ml. Growth curve study by the MIC concentration of cyclic dipeptides recorded significant inhibition when compared with control. Time-kill curve showed maximum reduction of colony count was between 3 and 5 weeks. The DKPs are nontoxic to the VERO cell line up to 200?µg/ml. The antimycobacterial activity of cyclo-(d-Pro-l-Leu), cyclo-(l-Pro-l-Met) and cyclo-(d-Pro-l-Phe) is reported in this study for the first time.

Discussion and conclusion: In conclusion, the potency, low cytotoxicity and selectivity of these compounds make them valid lead compounds for treatment against TB.     

The administration of l-thyroxine as soft gel capsule or liquid solution

Introduction: Levothyroxine (l-T4) is the mainstay of treating hypothyroidism. The tablet is the traditional formulation of l-T4. Tablet l-T4 malabsorption results from either hindered gastric dissolution of the tablet or binding of l-T4 by sequestrants in the intestinal lumen.

Areas covered: This review provides an overview of the pharmacokinetics of l-T4 formulations available in the market: the tablet, the soft gel capsule and the oral solution. We review literature on the new formulations and anticipate the areas of future research.

Expert opinion: Failure of l-T4 treatment to reach target serum thyroid-stimulating hormone levels generally prompts the physicians to increase l-T4 daily dose. In vitro studies have shown that the soft gel capsule releases the active ingredient more consistently at varying pH than the tablet. In addition, in vivo studies have confirmed the in vitro data and have demonstrated that both the soft gel capsule and the liquid formulation are capable to solve tablet l-T4 malabsorption caused by certain drugs, bariatric surgery or coffee. These new formulations may be attractive also for patients who cannot/do not want to change their (improper) habits of l-T4 ingestion. Finally, the oral solution l-T4 could be suitable for patients who cannot swallow the solid formulations.     

New oleanane-type triterpenoid saponins isolated from the seeds of Celosia argentea

Three new oleanane-type triterpenoid saponins named celosins H (1), I (2), and J (3) were isolated from the seeds of Celosia argentea L. Their structures were characterized as 3-O-β-d-xylopyranosyl-(1 → 3)-β-d-glucuronopyranosyl-polygalagenin 28-O-β-d-glucopyranosyl ester, 3-O-β-d-glucuronopyranosyl-medicagenic acid 28-O-β-d-xylcopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-β-d-fucopyranosyl ester, and 3-O-β-d-glucuronopyranosyl-medicagenic acid 28-O-α-l-arabinopyranosyl-(1 → 3)-[β-d-xylcopyranosyl-(1 → 4)]-α-l-rhamnopyranosyl-(1 → 2)-β-d-fucopyranosyl ester by NMR, MS, and chemical evidences, respectively. In our opinion, celosins H–J could be used as chemical markers for the quality control of C. argentea seeds.     

l-3-n-Butylphthalide attenuates β-amyloid-induced toxicity in neuroblastoma SH-SY5Y cells through regulating mitochondrion-mediated apoptosis and MAPK signaling

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid-β protein (Aβ), the hallmark of AD, invokes a cascade of mitochondrial dysfunction and eventually leads to neuronal death. l-3-n-Butylphthalide (l-NBP) has shown the potent neuroprotective effects in stroke and AD animal models. The present study is to evaluate the neuroprotective effect of l-NBP on Aβ_25–35-induced neuronal injury and the possible mechanism in the human neuroblastoma SH-SY5Y cells. Our results showed that l-NBP significantly attenuated Aβ_25–35-induced cell death and reduced neuronal apoptosis. l-NBP significantly inhibited Aβ_25–35-induced mitochondrial dysfunction, including mitochondrial membrane potential reduction, and reactive oxygen species production. Furthermore, l-NBP could partially reverse the elevations of Aβ_25–35-induced active caspase-3, caspase-9, and cytochrome c expressions, and the downregulation of anti-apoptosis protein Bcl-2. Moreover, l-NBP markedly inhibited the activations of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway. These results demonstrated that l-NBP was capable of protecting neuronal cells from Aβ_25–35-induced toxicity through a mitochondrial-dependent apoptotic pathway. Thus, l-NBP shows promising candidate of multi-target neuronal protective agent for the treatment of AD.     

Metabolomic analysis on the toxicological effects of TiO2 nanoparticles in mouse fibroblast cells: from the perspective of perturbations in amino acid metabolism

Titanium dioxide nanoparticles (nano-TiO₂) have been widely applied in daily life and subsequent problem on the potential health risk are raised. Studies on the toxicity of nano-TiO₂ have shown that they could lead to toxic effects on human and environment. However, the mechanisms are still unclear. We investigated the change of amino acid levels in L929 cells after nano-TiO₂ exposure using gas chromatography with time-of-flight mass spectrometry (GC/TOFMS)-based metabolomics approach. Spectral profiles were subjected to multivariate statistics, namely, Principal Component Analysis (PCA), and Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA). Using MetaboAnalyst 2.0, it was found that 7 metabolic pathways (impact-value >0.10) among the regulated pathways were significantly perturbed. Twelve distinct amino acids are identified from these pathways, including l-α-alanine, β-alanine, glycine, l-aspartate, l-methionine, l-cysteine, glutamate, l-pyroglutamate, l-asparagine, l-glutamine, S-adenosylmethionine, and l-lysine. These results show that the disturbed amino acids played an important role in the nano-TiO₂-induced cytotoxicity. Along with earlier findings, we successfully used the metabolomics approaches to manifest nano-TiO₂ toxicity through triggering cellular oxidative stress, energy damage and the inhibition of DNA and RNA synthesis.     

Triterpene saponins from the leaves of Ilex kudingcha

Three new triterpene saponins, ilekudinosides T–V (1–3), along with six known saponins were isolated from the 70% ethanolic extract of the leaves of Ilex kudingcha. The new saponins were characterized as 3-O-β-d-glucopyranosyl-(1 → 3)-[α-l-rhamnopyranosyl-(1 → 2)]-α-l-arabinopyranosyl-3β,19α-dihydroxy-urs-12(13)-en-28,20β-lactone (1), 3-O-β-d-glucopyranosyl-(1 → 3)-[α-l-rhamnopyranosyl-(1 → 2)]-α-l-arabinopyranosyl-3β,19α-dihydroxy-12-ethoxy-urs-13(18)-ene-28,20β-lactone (2), 3-O-β-d-glucopyranosyl-(1 → 3)-[α-l-rhamnopyranosyl-(1 → 2)]-α-l-arabinopyranosyl-3β,19α-dihydroxy-11-oxo-urs-13(18)-ene-28,20β-lactone (3), respectively. The structures of compounds 1–3 were elucidated on the basis of the chemical and spectroscopic evidence, and the structures of known compounds were identified by comparison of their spectroscopic data with those reported in the literature.     

Phenylalanine monooxygenase and the sulfur oxygenation of S-carboxymethyl-l-cysteine in mice

1.?The extent of sulfoxidation of the drug, S-carboxymethyl-l-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-l-cysteine has been investigated.

2.?Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased C_max, AUC_{(0–180?min)} and AUC_{(0–∞?min)} values (15?- to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-l-cysteine (13?- to 22-fold lower).

3.?Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-l-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.     

Modification of translationally controlled tumor protein-derived protein transduction domain for improved intranasal delivery of insulin

Carrier peptides, termed protein transduction domains (PTDs), serve as provide promising vehicles for intranasal delivery of macromolecular drugs. A mutant PTD derived from human translationally controlled tumor protein (TCTP-PTD 13, MIIFRALISHKK) was reported to provide enhanced intranasal delivery of insulin. In this study, we tested whether its efficiency could be further improved by replacing amino acids in TCTP-PTD 13 or changing the amino acids in the carrier peptides from the l- to the d-form. We assessed the pharmacokinetics of PTD-mediated transmucosal delivery of insulin in normal rats and the activity of insulin in alloxan-induced diabetic rats. The safety/toxicity profile of the carrier peptides was evaluated based on the release of lactate dehydrogenase (LDH) in nasal wash fluid, body weight changes, and several biochemical parameters. Pharmacokinetic and pharmacodynamic studies showed that the l-form of a double substitution A6L, I8A (MIIFRLLASHKK), designated as l-TCTP-PTD 13M2 was the most effective carrier for intranasal insulin delivery. The relative bioavailability of insulin co-administered intranasally with l-TCTP-PTD 13M2 was 37.1% of the value obtained by the subcutaneous route, which was 1.68-fold higher than for insulin co-administered with l-TCTP-PTD 13. Moreover, co-administration of insulin plus l-TCTP-PTD 13M2 reduced blood glucose levels compared to levels in diabetic rats treated with insulin plus l-TCTP-PTD 13. There was no evidence of toxicity. These results suggest that the newly designed PTD is a useful carrier peptide for the intranasal delivery of drugs or biomolecules.