Dual signaling of Wamide myoinhibitory peptides through a peptide-gated channel and a GPCR in Platynereis.

Neuropeptides commonly signal by metabotropic GPCRs. In some mollusks and cnidarians, RFamide neuropeptides mediate fast ionotropic signaling by peptide-gated ion channels that belong to the DEG/ENaC family. Here we describe a neuropeptide system with a dual mode of signaling by both a peptide-gated ion channel and a GPCR. We identified and characterized a peptide-gated channel in the marine annelid Platynereis dumerilii that is specifically activated by Wamide myoinhibitory peptides derived from the same proneuropeptide. The myoinhibitory peptide-gated ion channel (MGIC) belongs to the DEG/ENaC family and is paralogous to RFamide-gated ion channels. Platynereis myoinhibitory peptides also activate a previously described GPCR, MAG. We measured the potency of all Wamides on both MGIC and MAG and identified peptides that preferentially activate one or the other receptor. Analysis of a single-cell transcriptome resource indicates that MGIC and MAG signal in distinct target neurons. The identification of a Wamide-gated ion channel suggests that peptide-gated channels are more diverse and widespread in animals than previously appreciated. The possibility of neuropeptide signaling by both ionotropic and metabotropic receptors to different target cells in the same organism highlights an additional level of complexity in peptidergic signaling networks.-Schmidt, A., Bauknecht, P., Williams, E. A., Augustinowski, K., Gründer, S., Jékely, G. Dual signaling of Wamide myoinhibitory peptides through a peptide-gated channel and a GPCR in Platynereis.

A PEGylated analog of short-length Neuromedin U with potent anorectic and anti-obesity effects.

Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.

Manufacturing of a Secretoneurin Drug Delivery System with Self-Assembled Protamine Nanoparticles by Titration.

Since therapeutic peptides and oligonucleotides are gathering interests as active pharmaceutical ingredients (APIs), nanoparticulate drug delivery systems are becoming of great importance. Thereby, the possibility to design drug delivery systems according to the therapeutic needs of APIs enhances clinical implementation. Over the last years, the focus of our group was laid on protamine-oligonucleotide-nanoparticles (so called proticles), however, the possibility to modify the size, zeta potential or loading efficiencies was limited. Therefore, at the present study we integrated a stepwise addition of protamine (titration) into the formation process of proticles loaded with the angiogenic neuropeptide secretoneurin (SN). A particle size around 130 nm was determined when proticles were assembled by the commonly used protamine addition at once. Through application of the protamine titration process it was possible to modify and adjust the particle size between approx. 120 and 1200 nm (dependent on mass ratio) without influencing the SN loading capacity. Dynamic light scattering pointed out that the difference in particle size was most probably the result of a secondary aggregation. Initially-formed particles of early stages in the titration process aggregated towards bigger assemblies. Atomic-force-microscopy images also revealed differences in morphology along with different particle size. In contrast, the SN loading was only influenced by the applied mass ratio, where a slight saturation effect was observable. Up to 65% of deployed SN could be imbedded into the proticle matrix. An in-vivo biodistribution study (i.m.) showed a retarded distribution of SN from the site of injection after the application of a SN-proticle formulation. Further, it was demonstrated that SN loaded proticles can be successfully freeze-dried and resuspended afterwards. To conclude, the integration of the protamine titration process offers new possibilities for the formulation of proticles in order to address key parameters of drug delivery systems as size, API loading or modified drug release.

Phase IA Clinical Trial Evaluating the Tolerability, Pharmacokinetics, and Analgesic Efficacy of an Intrathecally Administered Neurotensin A Analogue in Central Neuropathic Pain Following Spinal Cord Injury.

We evaluated CGX-1160 in a Phase Ia clinical trial to determine the safety of escalating doses in patients with central neuropathic pain following spinal cord injury (SCI). Our secondary objective was to detect a trend toward analgesic efficacy. Four subjects received 3 consecutive escalating doses of CGX-1160 starting at 25 µg/h over 6 hours until 2 consecutive subjects experienced any adverse effect; 2 of the 4 subjects received 2 sequences of 3 consecutive dose escalations. Maximum tolerated dose was defined by the development of diarrhea (900 µg/h over 6 hours). Cerebrospinal fluid (CSF) and blood were collected for pharmacokinetic (PK) evaluation. The CSF concentration-versus-time data fit to a biexponential PK model, showing a rapid redistribution phase followed by a significantly slower terminal elimination phase. Incorporating an effect site delay into the model improved the fit to the data (concentration producing 50% of the maximum effect [C50 ], 58.7 ug/mL at the site of drug effect). Maximal reduction from the baseline pain intensity was 63%. In summary, CGX-1160 was generally well tolerated when administered intrathecally at doses up to 1000 µg/h. Peak analgesic effect occurred after the peak intrathecal concentration, indicating the presence of an effect site compartment to the PK model to represent the concentration and effect profiles for this unique compound.

Distribution in Rats Internal Organs of Intraperitoneally Given 125I-Labeled Heptapeptide [2-8]-Leucopyrokinin ([2-8]-LPK), a Truncated Analog of Insect Neuropeptide Leucopyrokinin.

BACKGROUND: It was previously found that synthetic, insect-derived octapeptide leucopyrokinin (LPK) applied directly into the lateral brain ventricle induced a significant antinociceptive effect in rats. Its synthetic truncated analog heptapeptide [2-8]-leucopyrokinin displayed a stronger antinociceptive effect in comparison to native LPK. Moreover it was previously found a high accumulation of these both 125I-labeled peptides in adrenals, as well as in hypothalamus and in hippocampus of rats brain. OBJECTIVES: The aim of the present study was to assess the distribution of 125I-labeled [2-8]-leucopyrokinin in rats' internal organs an in several parts of the brain after peripheral - intraperitoneal (i.p.) administration. MATERIAL AND METHODS: The study was performed on male Wistar rats. A synthetic [2-8]-leucopyrokinin ([2-8]-LPK) was iodinated with Na125I. On the day of experiment a solution of 125I-[2-8]-LPK was i.p. injected and the next after 1 and 24 h animals were sacrificed by decapitation. Radioactivity levels in samples of parts of the brain and of internal organs were determined by counter Gamma Auto Count. RESULTS: A uniform, low accumulation 125I-[2-8]-LPK was found in evaluated samples of the brain and in internal organs. CONCLUSIONS: The results of the present study indicate a weak penetration into the brain and internal organs of intraperitoneally applied 125I-[2-8]-LPK in rats and correspond with previously determined weak biological effects of i.p. injected LPK and [2-8]-LPK.

[Pharmacokinetic and pharmacodynamic synergism between neuropeptides and lithium in the neurotrophic and neuroprotective action of cerebrolysin].

OBJECTIVE: To study the synergism between neuropeptides and lithium ions. MATERIAL AND METHODS: An experimental model of stroke (chronic bilateral occlusion of the common carotid arteries in rats), neuronal culture studies, histomorphological analyses, determination of micronutrient profile of brain substrates were used. RESULTS: A complex of experimental studies revealed that the effect of cerebrolysin is influenced by the synergism between lithium ions and the neuropeptide contentof this drug. Pharmacokinetic synergism promotes the accumulation of lithium in brain tissues during cerebrolysin treatment. The existence of the pharmacokinetic synergism is evident from the potentiation of neuroprotective effects of the drug under the action of lithium ions established in the model of stroke. CONCLUSION: Lithium ions potentiate neuroprotective effects of cerebrolysin.

Development of neuropeptide analogs capable of traversing the integument: A case study using diapause hormone analogs in Helicoverpa zea.

Diapause hormone and its analogs terminate pupal diapause in Helicoverpa zea when injected, but if such agents are to be used as effective diapause disruptors it will be essential to develop simple techniques for administering active compounds that can exert their effect by penetrating the insect epidermis. In the current study, we used two molecules previously shown to have high diapause-terminating activity as lead molecules to rationally design and synthesize new amphiphilic compounds with modified hydrophobic components. An assay for diapause termination identified 13 active compounds with EC50's ranging from 0.9 to 46.0 pmol per pupa. Three compounds, Decyl-1963, Dodecyl-1967, and Heptyl-1965, selected from the 13 compounds most active in breaking diapause following injection, also successfully prevented newly-formed pupae from entering diapause when applied topically. These compounds feature straight-chain, aliphatic hydrocarbons from 7 to 12 carbons in length; DH analogs with either a short-chain length of 4 or an aromatic phenethyl group failed to act topically. Compared to a high diapause incidence of 80-90% in controls, diapause incidence in pupae receiving a 10 nmole topical application of Decyl-1963, Dodecyl-1967, or Heptyl-1965 dropped to 30-45%. Decyl-1963 and Dodecyl-1967 also remained effective when topically applied at the 1 nmole level. These results suggest the feasibility of developing DH agonists that can be applied topically and suggest the identity of new lead molecules for development of additional topically-active DH analogs. The ability to penetrate the insect epidermis and/or midgut lining is critical if such agents are to be considered for future use as pest management tools.

Chronic delivery of α-melanocyte-stimulating hormone in rat hypothalamus using albumin-alginate microparticles: effects on food intake and body weight.

Chronic delivery of neuropeptides in the brain is a useful experimental approach to study their long-term effects on various biological parameters. In this work, we tested albumin-alginate microparticles, as a potential delivery system, to study if continuous release in the hypothalamus of α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, may result in a long-term decrease in food intake and body weight. The 2-week release of α-MSH from peptide-loaded particles was confirmed by an in vitro assay. Then, daily food intake and body weight were studied for 18 days in rats injected bilaterally into the paraventricular hypothalamic nucleus with particles loaded or not with α-MSH. A decrease in body weight gain, persisting throughout the study, was found in rats injected with α-MSH-charged particles as compared with rats receiving non-charged particles and with rats injected with the same dose of α-MSH in solution. Food intake was significantly decreased for 3 days in rats receiving α-MSH-loaded particles and it was not followed by the feeding rebound effect which appears after food restriction. The presence of α-MSH-loaded particles in the hypothalamus was confirmed by immunohistochemistry. In conclusion, our study validates albumin-alginate microparticles as a new carrier system for long-term delivery of neuropeptides in the brain and demonstrates that chronic delivery of α-MSH in the hypothalamus results in a prolonged suppression of food intake and a decrease of body weight gain in rats.

The role for IGF-1-derived small neuropeptides as a therapeutic target for neurological disorders.

INTRODUCTION: Exogenous IGF-1 protects the brain from ischemic injury and improves function. However, its clinical application to neurological disorders is limited by its large molecular size, poor central uptake and mitogenic potential. AREAS COVERED: In this review, the authors have discussed the efficacy, pharmacokinetics and mechanisms of IGF-1 derivatives on protecting acute brain injury, preventing memory impairment and improving recovery from neurological degenerative conditions evaluated in various animal models. We have included natural metabolites of IGF-1, glycine-proline-glutamate (GPE), cleaved from N-terminal IGF-1 and cyclic glycine-proline (cGP) as well as the structural analogues of GPE and cGP, glycine-2-methyl-proline-glutamate and cyclo-l-glycyl-l-2-allylproline, respectively. In addition, the regulatory role for cGP in bioavailability of IGF-1 has also been discussed. EXPERT OPINION: These small neuropeptides provide effective neuroprotection by offering an improved pharmacokinetic profile and more practical route of administration compared with IGF-1 administration. Developing modified neuropeptides to overcome the limitations of their endogenous counterparts represents a novel strategy of pharmaceutical discovery for neurological disorders. The mechanism of action may involve a regulation of IGF-1 bioavailability.

The proton permeability of self-assembled polymersomes and their neuroprotection by enhancing a neuroprotective peptide across the blood-brain barrier after modification with lactoferrin.

Biotherapeutics such as peptides possess strong potential for the treatment of intractable neurological disorders. However, because of their low stability and the impermeability of the blood-brain barrier (BBB), biotherapeutics are difficult to transport into brain parenchyma via intravenous injection. Herein, we present a novel poly(ethylene glycol)-poly(d,l-lactic-co-glycolic acid) polymersome-based nanomedicine with self-assembled bilayers, which was functionalized with lactoferrin (Lf-POS) to facilitate the transport of a neuroprotective peptide into the brain. The apparent diffusion coefficient (D*) of H(+) through the polymersome membrane was 5.659 × 10(-26) cm(2) s(-1), while that of liposomes was 1.017 × 10(-24) cm(2) s(-1). The stability of the polymersome membrane was much higher than that of liposomes. The uptake of polymersomes by mouse brain capillary endothelial cells proved that the optimal density of lactoferrin was 101 molecules per polymersome. Fluorescence imaging indicated that Lf101-POS was effectively transferred into the brain. In pharmacokinetics, compared with transferrin-modified polymersomes and cationic bovine serum albumin-modified polymersomes, Lf-POS obtained the greatest BBB permeability surface area and percentage of injected dose per gram (%ID per g). Furthermore, Lf-POS holding S14G-humanin protected against learning and memory impairment induced by amyloid-ß25-35 in rats. Western blotting revealed that the nanomedicine provided neuroprotection against over-expression of apoptotic proteins exhibiting neurofibrillary tangle pathology in neurons. The results indicated that polymersomes can be exploited as a promising non-invasive nanomedicine capable of mediating peptide therapeutic delivery and controlling the release of drugs to the central nervous system.

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Evaluation of neuropeptide loaded trimethyl chitosan nanoparticles for nose to brain delivery.

Leucine-enkephalin (Leu-Enk) is a neurotransmitter or neuromodulator in pain transmission. Due to non-addictive opioid analgesic activity of this peptide, it might have great potential in pain management. Leu-Enk loaded N-trimethyl chitosan (TMC) nanoparticles were prepared and evaluated as a brain delivery vehicle via nasal route. TMC biopolymer was synthesized and analyzed by (1)H NMR spectroscopy. TMC nanoparticles were prepared by ionic gelation method. Mean peptide encapsulation efficiency and loading capacity were 78.28±3.8% and 14±1.3%, respectively. Mean particle size, polydispersity index and zeta potential were found to be 443±23 nm, 0.317±0.17 and +15±2 mV respectively for optimized formulations. Apparent permeability coefficient (Papp) of Leu-Enk released from nanoparticles across the porcine nasal mucosa was determined to be 7.45±0.30×10(-6) cm s(-1). Permeability of Leu-Enk released from nanoparticles was 35 fold improved from the nasal mucosa as compared to Leu-Enk solution. Fluorescent microscopy of brain sections of mice showed higher accumulation of fluorescent marker NBD-F labelled Leu-Enk, when administered nasally by TMC nanoparticles, while low brain uptake of marker solution was observed. Furthermore, enhancement in brain uptake resulted into significant improvement in the observed antinociceptive effect of Leu-Enk as evidenced by hot plate and acetic acid induced writhing assay.

[Influence of membranes on alaptide permeation from hydrogels]. / Vplyv membrán na permeáciu alaptidu z hydrogélov.

The paper evaluates the liberation of alaptide from gels through various types of permeable membranes. The gels were prepared on the basis of three different polymers (3% chitosan; 2.5% hydroxypropyl cellulose; 3% hydroxyethyl cellulose) in different concentrations with additions of humectants (5 %; 15% propylene glycol and 10% glycerol) and the preserving agent, 0.3% Sepicide HBR with 1% alaptide, and finally without the drug. The permeation of the drug from gels into the acceptor solution was evaluated with the use of the following membranes: the hydrophilic membrane from Chemosvit, the chicken skin, the stripped snakeskin, and the wall of the small intestine. The measurements showed that the highest percentage of the drug penetrated through the small intestine, a smaller percentage through the chicken skin, and the smallest amount through the snakeskin. Rheological properties of the prepared hydrogels were evaluated as well. The pseudoplastic flow was only confirmed for the hydrogel prepared on the basis of hydroxypropyl cellulose. An utterly opposite situation was with the hydrogels prepared on the basis of chitosan and hydroxyethyl cellulose. They showed a significant thixotropic character and the degree of thixotropy increased with time. Based on the results of the pH measurement, the samples prepared on the basis of chitosan and hydroxypropyl cellulose have been shown to be inconvenient because they reached a lower pH and had a potential of causing skin irritation. The hydroxyethyl cellulose hydrogel matched the physiological values of skin pH even after 14 days since its preparation.

Bioanalytical determination of unstable endogenous small peptides: RFRP3 and its metabolites in rat blood.

BACKGROUND: Targeting the gonadotropin-releasing hormone pathway for the treatment of endometriosis leads to an interest in monitoring for endogenous modulators of this pathway (RFRP3 and kisspeptin) as baseline controls for treatment development. RESULTS: Stabilization of RFRP3 was shown to be extremely difficult in a highly enzymatically active matrix, such as rat blood. Sample denaturing with solvent at collection was necessary due to enzyme inhibition being unsuccessful at stabilization leading to difficulties in sample processing. Monitoring multiple fragments formed in blood can aid in profiling these peptides once in-source conversion is controlled. CONCLUSION: generic high-sensitivity LC-MS/MS assay was developed for RFRP3 and the fragments formed from it in whole blood. Use of 2D chromatography circumvents concentration and retention issues related to small fragments with a normal flow setup, making a more open-access approach feasible.

Insulin-like growth factor -1 (IGF-1) derived neuropeptides, a novel strategy for the development of pharmaceuticals for managing ischemic brain injury.

Insulin-Like Growth Factor-1 (IGF-1) is neuroprotective and improves long-term function after brain injury. However, its clinical application to neurological disorders is limited by its large molecular size, poor central uptake, and mitogenic potential. Glycine-proline-glutamate (GPE) is naturally cleaved from the IGF-1 N-terminal and is also neuroprotective after ischemic injury, thus providing a potential novel strategy of drug discovery for management of neurological disorders. GPE is not enzymatically stable, thus intravenous infusion of GPE becomes necessary for stable and potent neuroprotection. The broad effective dose range and treatment window of 3-7 h after the lesion suggest its potential for treating acute brain injuries. The neuroprotective action of GPE is not age selective, is not dependent on cerebral reperfusion, plasma glucose concentrations, and core body temperature. G-2mPE, a GPE analogue designed to be more resistant to enzymatic activity, has a prolonged plasma half-life and is more potent in neuroprotection. Neuroprotection by GPE and its analogue may be involved in modulation of inflammation, promotion of astrocytosis, inhibition of apoptosis, and in vascular remodeling. Small neuropeptides have advantages over growth factors in the treatment of brain injury, and modified neuropeptides, designed to overcome the limitations of their endogenous counterparts, represent a novel strategy of pharmaceutical discovery for neurological disorders.

Insertion of a lysosomal enzyme cleavage site into the sequence of a radiolabeled neuropeptide influences cell trafficking in vitro and in vivo.

Radiolabeled neuropeptides are widely explored for targeting tumours for either imaging or radiotherapeutic purposes. After binding to their receptors, these peptides are rapidly internalized into lysosomes, where they are degraded by proteolytic enzymes, such as cathepsins. The aim of this study was to investigate the effect of the inclusion of specific cleavage sites for cathepsin B into the peptide sequence. The cleavage site, GFLG, together with a series of dipeptides for pharmacokinetic modification of radiometabolites, were, therefore, inserted into a peptide that binds to the gastrin/CCK2 receptor. The receptor binding of the peptides was explored in AR42J cells, rates of internalization, and externalization of the radionuclide were measured and the nature of the radiometabolites explored. The effects of the modifications on biodistribution in tumor-bearing mice was explored by high-resolution single-photon emission computed tomography imaging. Differences in rates of externalization from tumor cells in vitro and in the rates of washout from tumor and kidney in vivo were observed. These results indicate that insertion of an enzymatic cleavage site, such as that for cathepsin B, into a neuropeptide appears to have an influence on the intracellular processing, which results in a change in the rate of egress of radioactivity from target and nontarget tissues.

Bioavailability of backbone cyclic PK/PBAN neuropeptide antagonists--inhibition of sex pheromone biosynthesis elicited by the natural mechanism in Heliothis peltigera females.

The bioavailability (i.e. ability to penetrate the insect cuticle, to reach the target organ and to exert bioactivity) of two backbone cyclic (BBC) pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) antagonistic peptides was tested by applying them topically to Heliothis peltigera females and monitoring the resulting inhibition of sex pheromone production elicited by the natural (endogenous) mechanism during scotophase. Peptides were applied at various time points before the onset of scotophase, in aqueous or organic solvents, and pheromone content was examined at the 5th or 6th hour of scotophase. Both peptides penetrated the cuticle very efficiently and inhibited sex pheromone biosynthesis elicited by the natural mechanism for up to 8 or 9 h after application. The degree of inhibition differed between solvents: those applied in double-distilled water (DDW) were more active than those applied in dimethylsulfoxide (inhibition by 53-73% and 15-38%, respectively, for BBC-25, and 46-67% and 36-40%, respectively for BBC-28). Peptides applied in dimethylsulfoxide and hexane exhibited slightly more persistent inhibitory activity than those applied in DDW. The solvents themselves did not affect sex pheromone production. Multiple applications (at -2, 0, +2 and +4 h) resulted in almost complete (87%) inhibition of sex pheromone biosynthesis, compared with 52% inhibition following a single application. The present study is the first demonstration of the ability of topically applied PK/PBAN antagonists to inhibit sex pheromone biosynthesis elicited by the natural mechanism in female moths, and provides important information on the bioavailability of BBC peptides and the mechanism responsible for sex pheromone production in these insects.

Delivery of neuropeptides from the periphery to the brain: studies with enkephalin.

Many peptides with the potential of therapeutic action for brain disorders are not in clinical use because they are unable to cross the blood-brain barrier (BBB) following peripheral administration. We have developed two potential strategies for the delivery of peptides to the brain and demonstrated their feasibility with enkephalins. In the first approach, designated induced reversible lipophilization, Leu/Met Enkephalins were converted to 9-fluorenylmethoxycarbonyl (Fmoc) derived lipophilic prodrug analogues, which undergo slow, spontaneous hydrolysis under physiological conditions, generating the native agonists. In contrast to Enkephalin, Fmoc-Met-Enkephalin was found to facilitate an analgesic effect following intraperitoneal administration in mice. Fmoc-Leu-Enkephalin was not analgesic. In the second approach, Enkephalin was linked to BBB transport vectors through an Fmoc based linker spacer, forming conjugates that slowly release Enkephalin under physiological conditions. A pronounced antinociceptive response was thus obtained following intraperitoneal administration of either cationized-human serum albumin-Fmoc-Enkephalin or polyethylene glycol(5)-Fmoc-Enkephalin. Derivatives of Enkephalin covalently linked to the same BBB-transport vectors through a stable (nonreversible) chemical bond were not analgesic. In summary, we have demonstrated that lipophilicity can be conferred to hydrophilic peptides to a degree permitting the permeation of the BBB by passive diffusion, without the drawback of agonist inactivation, which is often caused by irreversible derivatization. Similarly, in the second strategy, the conjugation to BBB-permeable vectors overcomes the obstacle of peptide inactivation by releasing the active form in the central nervous system.

Bioavailability of beta-amino acid and C-terminally derived PK/PBAN analogs.

The ability of linear beta-amino acid substituted peptides (PK-betaA-1: Ac-YFT[beta(3)P]RLa; PK-betaA-2: Ac-Y[beta(3)homoF]TPRLa; PK-betaA-3: Ac-Y[beta(3)F]TPRLa; PK-betaA-4: Ac-[beta(3)F]FT[beta(3)P]RLa) and unsubstituted analogs (Ac-YFTPRLa and YFTPRLa) of the pyrokinin(PK)/pheromone biosynthesis-activating neuropeptide (PBAN) family to penetrate the insect cuticle and exert biological activity (i.e., stimulate sex pheromone biosynthesis), was tested by topical application on Heliothis peltigera moths. The present results clearly indicate that small linear synthetic peptides can penetrate the cuticle very efficiently by contact application and activate their target organ. The time responses of the peptides applied in DDW and DMSO were tested and the activities of topically applied and injected peptides were compared. The results clearly indicate that PK-betaA-4 and PK-betaA-3 exhibited high bioavailability (ability to penetrate through the cuticle and exertion of bioactivity) with the latter showing longer persistence in both solvents than any other analog in the study; indicative that incorporation of a beta-amino acid at the Phe(2) position can enhance longevity in topical PK/PBAN analogs. PK-betaA-4 was significantly more active in DMSO than in DDW, and significantly more active than the parent peptide LPK in DMSO. PK-betaA-1 and PK-betaA-2 exhibited negligible activity. Interestingly, Ac-YFTPRLa was highly potent in both solvents; its activity in DDW did not differ from that of PK-betaA-4 and PK-betaA-3, and was higher than that of LPK. Even the unacylated peptide YFTPRLa was active in both solvents, at a similar level to LPK. Topically applied PK-betaA-4 and Ac-YFTPRLa exhibited significantly higher activity than the injected peptides. PK-betaA-3 and YFTPRLa were equally potent in both routes of administration.

Bioavailability of insect neuropeptides: the PK/PBAN family as a case study.

The ability of unmodified linear peptides to penetrate the insect cuticle and exert bioactivity (e.g., stimulation of sex pheromone biosynthesis) was tested by topical application onto Heliothis peltigera moths of four insect neuropeptides (Nps) of the pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) family: Helicoverpa zea PBAN (Hez-PBAN), Pseudaletia (Mythimna) separata pheromonotropin (PT), Leucophaea maderae PK (LPK) and Locusta migratoria myotropin (Lom-MT-II). The time kinetic of the peptides applied in double distilled water (DDW) or dimethylsulfoxide (DMSO) was tested and the activities of topically applied and injected peptides were compared. The results clearly indicated that all four peptides were highly potent but with differing activities in the two solvents: PBAN was most active in water, and PT in DMSO. The activity of PBAN in DDW lasted up to 8h post-application and its activity in this solvent showed a faster onset and a longer persistence than in DMSO. LPK and MT differed less in their kinetics between the two solvents. Topically applied PBAN at 1 nmol exhibited an equivalent or even significantly higher potency than the injected peptide at several different times post-treatment. Similar results were obtained with topically applied and injected LPK. The present results add important information on the bioavailability of unmodified linear peptides in moths, clearly indicate that linear hydrophilic peptides can penetrate the cuticle by contact application in aqueous solutions and in organic solvents very efficiently, reach their target organ and activate it.