Biological properties of a new fluorescent biphalin fragment analogue

Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH<--X, where X=Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X=DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both mu and delta opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation.     

Neuroprotective Potential of Biphalin,Multireceptor Opioid Peptide,Against Excitotoxic Injury in Hippocampal Organotypic Culture

Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. Findings: (1) 0.025–0.1 μM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge.     

Brain and Spinal Cord Distribution of Biphalin: Correlation with Opioid Receptor Density and Mechanism of CNS Entry

Abstract: Biphalin [(Tyr-d -Ala-Gly-Phe-NH)₂] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [¹²⁵I-Tyr¹]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [¹²⁵I-Tyr¹]-biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of δ- and μ-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [¹²⁵I-Tyr¹]biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [¹²⁵I-Tyr¹]biphalin was decreased with naloxone, d -Phe-Cys-Tyr-d -Trp-Arg-Thr-Pen-Thr-NH₂, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to δ- and μ-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [¹²⁵I-Tyr¹]biphalin, which could be described by Michaelis-Menten kinetics with a K_m of 2.6 ± 4.8 µM, V_max of 14.6 ± 2.89 pmol^?1·min^?1·g^?1, and K_d of 0.568 ± 0.157 µl·min^?1·g^?1. Brain entry of [¹²⁵I-Tyr¹]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and l -phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.     

The opioid peptide analogue biphalin induces less physical dependence than morphine

We compared the physical dependence liability of biphalin, a dimeric enkephalin analogue that possesses high antinociceptive activity, with that of morphine in equipotent intravenous doses. Naloxone challenge produced severe withdrawal signs after a 5-day infusion of morphine but only minor withdrawal signs after a 5-day biphalin infusion. In a cross-dependence study, biphalin did not suppress body weight loss after morphine withdrawal, but successfully suppressed weight loss after pentazocine withdrawal. These data support consideration of biphalin as a new analgesic with a novel pharmacological profile and minimum dependence liability.     

Biological activity of fragments and analogues of the potent dimeric opioid peptide, biphalin.

The synthesis and biological activity of two fragments of the very potent opioid peptide biphalin, showed that Tyr-D-Ala-Gly-Phe-NH-NH<-Phe is the minimal fragment necessary to express equal affinities and the same biological activity profile as the parent biphalin. The replacement of N'-Phe with other L- or D- lipophilic amino acids showed the possibility of modification of receptor efficacy of the analogues.     

Interaction of enkephalin peptides with anionic model membranes

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen²,D-Pen⁵]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu,delta, and kappa opioid receptors

In an effort to improve biphalin’s potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1–5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki?=?0.27, 0.46, and 0.87?nM; EC₅₀?=?3.47, 1.45, and 13.5?nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.     

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1β (IL-1β). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Crystal structure of biphalin sulfate: a multireceptor opioid peptide.

Biphalin is a dimeric opioid peptide, composed of two tetrapeptides connected 'tail-to-tail', that exhibits a high affinity for all three opioid receptor types (i.e. mu, delta and kappa). This study presents the X-ray crystal structure of biphalin sulfate and compares it to other opioids that interact with the same biological targets. Both halves of the molecule have a folded backbone conformation but differ significantly from one another. Residues 1-4 in biphalin, which compare well with the delta selective opioid peptide DADLE, fold into a random coil. Residues 5-8, which can be fit to the mu selective peptide D-TIPP-NH2, exhibit a fairly normal type III' beta bend. Biphalin also exhibits structural similarities with two naltrexone analogs, naltrexonazine and norbinaltorphamine, that are specific to mu and kappa receptor sites.     

A molecular dynamics study on opioid activities of biphalin molecule

Molecular dynamics simulations of the biphalin molecule, (Tyr-D-Ala-Gly-Phe-NH)₂, and the active tetrapeptide hydrazide, Tyr-D-Ala-Gly-Phe-NH-NH₂ were performed to investigate the cause of the increased μ and δ receptor binding affinities of the former over the latter. The simulation results demonstrate that the acylation of the two equal tetrapeptide fragments of biphalin produces the constrained hydrazide bridges C₄^a - C₄￠- N₉ - N₁₀ {\hbox{C}}_4^{\alpha } - {{\hbox{C}}_4}\prime - {{\hbox{N}}_9} - {{\hbox{N}}_{{10}}} and N₉ - N₁₀ - C₅￠- C₅^a {{\hbox{N}}_9} - {{\hbox{N}}_{{10}}} - {{\hbox{C}}_5}\prime - {\hbox{C}}_5^{\alpha } , which in turn increase the opportunity of conformations for binding to μ or δ receptors. Meanwhile, the connection of the two active tetrapeptide fragments of biphalin also results in the constrained side chain torsion angle χ² at one of the two residues Phe. This constrained side chain torsion angle not only significantly increases the δ receptor binding affinity but also makes most of the δ receptor binding conformations of biphalin bind to the δ receptor through the fragment containing the mentioned residue Phe.     

Synthesis and biological activity of the first cyclic biphalin analogues

Biphalin is a linear octapeptide with strong opioid activity. Its structure is based on two identical sequences derived from enkephalins joined C-terminal to C-terminal by an hydrazide bridge (Tyr-D-Ala-Gly-Phe-NH-NH<--Phe<--Gly<--D-Ala<--Tyr). In this study we present the design, synthesis, and biological evaluation of the first cyclic biphalin analogues. d-Alanine residues in positions 2, 2' of the parent peptide were replaced by d- and l-cysteine and an intramolecular disulfide bond between the cysteine thiol groups was introduced. We obtained two cyclic analogues with quite different biological profiles.     

Characterization and analysis of biphalin: an opioid peptide with a palindromic sequence.

Among the many opioid peptides developed to date as nonaddictive analgesics, biphalin has exhibited extraordinary high potency and many other desirable characteristics. Biphalin is an octapeptide consisting of two monomers of a modified enkephalin, attached via a hydrazine bridge, and with the amino acids assembled in a palindromic sequence. Its structure is (Tyr-D-Ala-Gly-Phe-NH-)-2. However, this unique peptide, like any other synthetic peptide, needs strict quality control because of certain drawbacks associated with peptide synthesis. This paper discusses our approaches to characterizing and analyzing biphalin. Many techniques were used, including elemental analysis, amino acid analysis, amino acid sequence analysis (AASA), mass spectrometry (MS), 1H-NMR, 1H-correlated spectroscopy (COSY)-NMR, high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Electrospray ionization (ESI) mass spectrometry, which included both ESI-MS and ESI-MS/MS, was performed to confirm the full sequence because AASA results alone verified only the monomer sequence, and not the full sequence. Although the 1H-NMR results led to a preliminary assignment of many protons, the 1H COSY-NMR results allowed for unequivocal assignment of almost all protons. Peptide purity was determined using two techniques, reversed-phase HPLC and CE. The counter-ion of the peptide, trifluoroacetic acid, was determined by CE, using an indirect detection method developed previously in our laboratory. This paper illustrates successful application of nonconventional techniques to characterize and analyze a structurally modified peptide, biphalin, when standard techniques for peptide analysis are inadequate.     

New potent biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4' positions and non-hydrazine linkers

We report the synthesis and the biological evaluation of two new analogues of the potent dimeric opioid peptide biphalin. The performed modification is based on the replacement of two key structural elements of the native biphalin, namely: the hydrazine bridge which joins the two palindromic moieties and the phenylalanine residues at the 4,4′ positions of the backbone. The new analogues 9 and 10 contain 1,2-phenylenediamine and piperazine, respectively, in place of the hydrazidic linker and p-fluoro-l-phenylalanine residues at 4 and 4′ positions. Binding values are: K_\texti^m = 0.51 \textnM K_{\text{i}}^{\mu } = 0.51\,{\text{nM}} and K_\texti^d = 12.8 \textnM K_{\text{i}}^{\delta } = 12.8\,{\text{nM}} for compound 9, K_\texti^m = 0.09 \textnM K_{\text{i}}^{\mu } = 0.09\,{\text{nM}} and K_\texti^d = 0.11 \textnM K_{\text{i}}^{\delta } = 0.11\,{\text{nM}} for analogue 10.     

不同程度的尾损伤对镇海林蛙(Rana zhenhaiensis)蝌蚪游泳速度的影响

无尾两栖类蝌蚪的尾巴通过产生强大的游泳速度在反捕食中起到了重要的作用。以镇海林蛙(Rana zhenhaiensis)蝌蚪为实验动物来评估断尾的运动代价。以74尾具有完整尾蝌蚪作为实验组,通过截去不同尾长片段,人为分成轻微尾损伤组(30%)和严重尾损伤组(30%)并测定两组蝌蚪在断尾前后的游泳速度。以16尾完整尾蝌蚪作为对照组在实验组断尾前后同时进行游泳速度的测定。实验结果显示断尾影响蝌蚪的游泳速度,但仅在尾损伤程度达到尾长的30%以上时才产生不利的影响。这表明轻微尾损伤并不对镇海林蛙蝌蚪的游泳速度产生严重影响。在断尾前后实验组蝌蚪的游泳速度均与尾长呈正相关。在相同尾长状态下,尾损伤蝌蚪的相对游泳速度明显快于完整尾蝌蚪。因此,尾损伤的镇海林蛙蝌蚪有可能通过改变尾和身体的摆动频次等方式在断尾后对游泳速度进行了一定的补偿。尾损伤在野外频繁发生于蝌蚪的尾远端,据此推测镇海林蛙蝌蚪在自然条件下的尾损伤并不会产生严重运动代价。     

More tail lesions among undocked than tail docked pigs in a conventional herd

The vast majority of piglets reared in the European Union (EU) and worldwide is tail docked to reduce the risk of being tail bitten, even though EU animal welfare legislation bans routine tail docking. Many conventional herds experience low levels of tail biting among tail docked pigs, however it is not known, what the prevalence would have been had the pigs not been tail docked. The aim of this study was to compare the prevalence of tail lesions between docked and undocked pigs in a conventional piggery in Denmark with very low prevalence of tail biting among tail docked pigs. The study included 1922 DanAvl Duroc×(Landrace×Large White) female and castrated male pigs (962 docked and 960 undocked). Docked and undocked pigs were housed under the same conditions in the same room but in separate pens with 20 (±0.03) pigs/pen. Pigs had ad libitum access to commercial diets in a feed dispenser. Manipulable material in the form of chopped straw was provided daily on the floor (~10 g/pig per day), and each pen had two vertically placed soft wood boards. From weaning to slaughter, tail wounds (injury severity and freshness) were scored every 2^nd week. No clinical signs of injured tails were observed within the tail docked group, whereas 23.0% of the undocked pigs got a tail lesion. On average, 4.0% of the pigs with undocked tails had a tail lesion on tail inspection days. More pens with tail lesions were observed among pigs weighing 30 to 60 kg (34.3%; P<0.05) than in pens with pigs weighing 7 to 30 kg (13.0%) and 60 to 90 kg (12.8%). Removal of pigs to a hospital pen was more likely in undocked pens (P<0.05, 47.7% undocked pens and 22.9% docked pens). Finally, abattoir meat inspection data revealed more tail biting remarks in undocked pigs (P<0.001). In conclusion, this study suggests that housing pigs with intact tails in conventional herds with very low prevalence of tail biting among tail docked pigs, will increase the prevalence of pigs with tail lesions considerably, and pig producers will need more hospital pens. Abattoir data indicate that tail biting remarks from meat inspection data severely underestimate on-farm prevalence of tail lesions.     

Which is the most preventive measure against tail damage in finisher pigs: tail docking,straw provision or lowered stocking density?

One challenge of intensive pig production is tail damage caused by tail biting, and farmers often decrease the prevalence of tail damage through tail docking. However, tail docking is not an optimal preventive measure against tail damage and thus, it would be preferable to replace it. The aim of the current study was to investigate the relative effect of three possible preventive measures against tail damage. The study included 112 pens with 1624 finisher pigs divided between four batches. Pens were randomly assigned to one level of each of three treatments: (1) tail-docked (n=60 pens) v. undocked (n=52 pens), (2) 150 g of straw provided per pig per day on the solid floor (n=56 pens) v. no straw provided (n=56 pens), (3) stocking density of 1.21 m²/pig (11 pig/pen; n=56 pens) v. 0.73 m²/pig (18 pigs/pen; n=56 pens). Tail damage was recorded three times per week throughout the finisher period by scoring the tail of each individual pig. A pen was recorded as a tail damage pen and no longer included in the study if at least one pig in a pen had a bleeding tail wound; thus, only the first incidence of tail damage on pen level was recorded. Data were analysed by a Cox regression for survival analysis assuming proportional hazards. Results are presented as hazards, and a higher hazard means that a pen has a higher risk of tail damage and of it happening earlier in the finisher period. Pens with undocked pigs had a 4.32-fold higher hazard of tail damage compared with pens with docked pigs (P<0.001). Pens with no straw provided had a 2.22-fold higher hazard of tail damage compared with pens with straw provided (P<0.01). No interactions was seen between the treatments, but the effect of tail docking was higher than the effect of straw provision (P<0.001). Stocking density did not have a significant effect on the hazard of tail damage (hazard rate ratios (HRR)=1.67; P=0.064). However, a combination of straw provision and lowered stocking density showed a similar hazard of tail damage as seen with only tail docking (HRR=1.58; P=0.39). In conclusion, tail docking and straw provision were preventive measures against tail damage, and tail docking reduced the risk more than straw provision. A combination of other preventive measures is necessary to reduce the risk of tail damage in undocked pigs to the same level as in docked pigs.     

Structure of the receptor-binding protein of bacteriophage det7: a podoviral tail spike in a myovirus

A new Salmonella enterica phage, Det7, was isolated from sewage and shown by electron microscopy to belong to the Myoviridae morphogroup of bacteriophages. Det7 contains a 75-kDa protein with 50% overall sequence identity to the tail spike endorhamnosidase of podovirus P22. Adsorption of myoviruses to their bacterial hosts is normally mediated by long and short tail fibers attached to a contractile tail, whereas podoviruses do not contain fibers but attach to host cells through stubby tail spikes attached to a very short, noncontractile tail. The amino-terminal 150 residues of the Det7 protein lack homology to the P22 tail spike and are probably responsible for binding to the base plate of the myoviral tail. Det7 tail spike lacking this putative particle-binding domain was purified from Escherichia coli, and well-diffracting crystals of the protein were obtained. The structure, determined by molecular replacement and refined at a 1.6-Å resolution, is very similar to that of bacteriophage P22 tail spike. Fluorescence titrations with an octasaccharide suggest Det7 tail spike to bind its receptor lipopolysaccharide somewhat less tightly than the P22 tail spike. The Det7 tail spike is even more resistant to thermal unfolding than the already exceptionally stable homologue from P22. Folding and assembly of both trimeric proteins are equally temperature sensitive and equally slow. Despite the close structural, biochemical, and sequence similarities between both proteins, the Det7 tail spike lacks both carboxy-terminal cysteines previously proposed to form a transient disulfide during P22 tail spike assembly. Our data suggest receptor-binding module exchange between podoviruses and myoviruses in the course of bacteriophage evolution.     

Nonlethal injury caused by an invasive alien predator and its consequences for an anuran tadpole

Nonlethal tail injury resulting from unsuccessful predation attempts is common in anuran larvae and can potentially induce significant fitness costs in terms of survival and growth. We tested the hypotheses that the alien red swamp crayfish, Procambarus clarkii, is an important inducer of tail injury in tadpoles of the Iberian spadefoot toad, Pelobates cultripes, and that tail damage can have important consequences for the tadpoles’ life history and morphology. This was investigated by first estimating frequencies of caudal injury in P. cultripes tadpoles in temporary ponds, with and without crayfish. Secondly, we performed a laboratory experiment in which four levels of tail injury frequency were combined with two levels of food availability.The frequency of tadpoles with damaged tails was higher in ponds with crayfish and the presence of this predator was the strongest predictor of tail injury frequency in a pond. Induced tail loss decreased larval survivorship and affected tail morphology, with injured tadpoles developing deeper tail muscles and shallower tail fins. The magnitude of these effects depended on injury frequency, as well as on food availability. The results suggest that P. clarkii is inflicting tail injuries at much higher levels than those occurring before its introduction; these injuries affect tadpole morphology and may induce delayed fitness costs.     

Tail development and regeneration throughout the life cycle of the four-toed salamander Hemidactylium scutatum

The salamander tail displays different functions and morphologies in the aquatic and terrestrial stages of species with complex life cycles. During metamorphosis the function of the tail changes; the larval tail functions in aquatic locomotion while the juvenile and adult tail exhibits tail autotomy and fat storage functions. Because tail injury is common in the aquatic environment, we hypothesized that mechanisms have evolved to prevent altered larval tail morphology from affecting normal juvenile tail morphology. The hypothesis that injury to the larval tail would not affect juvenile tail morphology was investigated by comparing tail development and regeneration in Hemidactylium scutatum (Caudata: Plethodontidae). The experimental design included larvae with uninjured tails and with cut tails to simulate natural predation. The morphological variables analyzed to compare normally developing and regenerating tails were 1) tail length, 2) number of caudal vertebrae, and 3) vertebral centrum length. Control and experimental groups do not differ in time to metamorphosis or snout-vent length. Tails of experimental individuals are shorter than controls, yet they display a significantly higher rate of tail growth and less resorption of tail tissue. Anterior to the site of tail injury, caudal vertebrae in juveniles display greater average centrum lengths. Results suggest that regenerative mechanisms are functioning not only to produce structures, but also to influence growth of existing structures. Further investigation of juvenile and adult stages as well as comparative analyses of tail morphology in salamanders with complex life cycles will enhance our understanding of amphibian development and of the evolution of amphibian life cycles. J Morphol 233:15–29, 1997. © 1997 Wiley-Liss, Inc.