Side-effects of analgesic kyotorphin derivatives: advantages over clinical opioid drugs.

The adverse side-effects associated with opioid administration restrain their use as analgesic drugs and call for new solutions to treat pain. Two kyotorphin derivatives, kyotorphin-amide (KTP-NH2) and ibuprofen-KTP-NH2 (IbKTP-NH2) are promising alternatives to opioids: they trigger analgesia via an indirect opioid mechanism and are highly effective in several pain models following systemic delivery. In vivo side-effects of KTP-NH2 and IbKTP-NH2 are, however, unknown and were evaluated in the present study using male adult Wistar rats. For comparison purposes, morphine and tramadol, two clinically relevant opioids, were also studied. Results showed that KTP-derivatives do not cause constipation after systemic administration, in contrast to morphine. Also, no alterations were observed in blood pressure or in food and water intake, which were only affected by tramadol. A reduction in micturition was detected after KTP-NH2 or tramadol administrations. A moderate locomotion decline was detected after IbKTP-NH2-treatment. The side-effect profile of KTP-NH2 and IbKTP-NH2 support the existence of opioid-based mechanisms in their analgesic actions. The conjugation of a strong analgesic activity with the absence of the major side-effects associated to opioids highlights the potential of both KTP-NH2 and IbKTP-NH2 as advantageous alternatives over current opioids.

Antimicrobial properties of analgesic kyotorphin peptides unraveled through atomic force microscopy.

Antimicrobial peptides (AMPs) are promising candidates as alternatives to conventional antibiotics for the treatment of resistant pathogens. In the last decades, new AMPs have been found from the cleavage of intact proteins with no antibacterial activity themselves. Bovine hemoglobin hydrolysis, for instance, results in AMPs and the minimal antimicrobial peptide sequence was defined as Tyr-Arg plus a positively charged amino acid residue. The Tyr-Arg dipeptide alone, known as kyotorphin (KTP), is an endogenous analgesic neuropeptide but has no antimicrobial activity itself. In previous studies new KTP derivatives combining C-terminal amidation and Ibuprofen (Ib) - KTP-NH(2), IbKTP, IbKTP-NH(2) - were designed in order to improve KTP brain targeting. Those modifications succeeded in enhancing peptide-cell membrane affinity towards fluid anionic lipids and higher analgesic activity after systemic injection resulted therefrom. Here, we investigated if this affinity for anionic lipid membranes also translates into antimicrobial activity because bacteria have anionic membranes. Atomic force microscopy revealed that KTP derivatives perturbed Staphylococcus aureus membrane structure by inducing membrane blebbing, disruption and lysis. In addition, these peptides bind to red blood cells but are non-hemolytic. From the KTP derivatives tested, amidated KTP proves to be the most active antibacterial agent. The combination of analgesia and antibacterial activities with absence of toxicity is highly appealing from the clinical point of view and broadens the therapeutic potential and application of kyotorphin peptides.

Chemical conjugation of the neuropeptide kyotorphin and ibuprofen enhances brain targeting and analgesia.

The pharmaceutical potential of natural analgesic peptides is mainly hampered by their inability to cross the blood-brain barrier, BBB. Increasing peptide-cell membrane affinity through drug design is a promising strategy to overcome this limitation. To address this challenge, we grafted ibuprofen (IBP), a nonsteroidal anti-inflammatory drug, to kyotorphin (l-Tyr-l-Arg, KTP), an analgesic neuropeptide unable to cross BBB. Two new KTP derivatives, IBP-KTP (IbKTP-OH) and IBP-KTP-amide (IbKTP-NH(2)), were synthesized and characterized for membrane interaction, analgesic activity and mechanism of action. Ibuprofen enhanced peptide-membrane interaction, endowing a specificity for anionic fluid bilayers. A direct correlation between anionic lipid affinity and analgesic effect was established, IbKTP-NH(2) being the most potent analgesic (from 25 µmol · kg(-1)). In vitro, IbKTP-NH(2) caused the biggest shift in the membrane surface charge of BBB endothelial cells, as quantified using zeta-potential dynamic light scattering. Our results suggest that IbKTP-NH(2) crosses the BBB and acts by activating both opioid dependent and independent pathways.

Translocating the blood-brain barrier using electrostatics.

Mammalian cell membranes regulate homeostasis, protein activity, and cell signaling. The charge at the membrane surface has been correlated with these key events. Although mammalian cells are known to be slightly anionic, quantitative information on the membrane charge and the importance of electrostatic interactions in pharmacokinetics and pharmacodynamics remain elusive. Recently, we reported for the first time that brain endothelial cells (EC) are more negatively charged than human umbilical cord cells, using zeta-potential measurements by dynamic light scattering. Here, we hypothesize that anionicity is a key feature of the blood-brain barrier (BBB) and contributes to select which compounds cross into the brain. For the sake of comparison, we also studied the membrane surface charge of blood components-red blood cells (RBC), platelets, and peripheral blood mononuclear cells (PBMC). To further quantitatively correlate the negative zeta-potential values with membrane charge density, model membranes with different percentages of anionic lipids were also evaluated. From all the cells tested, brain cell membranes are the most anionic and those having their lipids mostly exposed, which explains why lipophilic cationic compounds are more prone to cross the blood-brain barrier.

Drug-lipid interaction evaluation: why a 19th century solution?

The affinity of a drug candidate for a biological membrane (its lipophilicity) is closely related to the pharmacologically crucial events of absorption, biodistribution, metabolization and excretion. The evolution of knowledge of biological membranes during the past two decades contrasts with the rudimentary parameter most commonly used to assess lipophilicity: P(o/w), the octanol-water partition coefficient. P(o/w) is especially unrealistic when testing molecules that are polar or partially charged. By contrast, lipid vesicle-based methods determine the extent of the actual partition of a drug to a membrane much more accurately, and have the additional advantage of enabling the choice of the lipid composition considered most suitable to answer a specific biological or pharmaceutical question. In addition, some of these methods are appropriate for high throughput screening, thus shifting determinations of membrane partition to a more preliminary stage of drug development. This streamlines research and development, by saving the time and money that would be spent on unpromising leads.

Molecular interaction studies of peptides using steady-state fluorescence intensity. Static (de)quenching revisited.

Protein-protein interactions, as well as peptide-peptide and peptide-protein interactions are fields of study of growing importance as molecular-level detail is avidly pursued in drug design, metabolic regulation and molecular dynamics, among other classes of studies. In membranes, this issue is particularly relevant because lipid bilayers potentiate molecular interactions due to the high local concentration of peptides and other solutes.However, experimental techniques and methodologies to detect and quantify such interactions are not abundant. A reliable, fast and inexpensive alternative methodology is revisited in this work. Considering the interaction of two molecules, at least one of them being fluorescent, either intrinsically (e.g. Trp residues) or by grafting a specific probe, changes in their aggregation state may be reported, as long as the fluorophore is sensitive to local changes in polarity, conformation and/or exposure to the solvent. The interaction will probably lead to modifications in fluorescence intensity resulting in a decrease ('quenching') or enhancement ('dequenching'). Although the presented methodology is based on static quenching methodologies, the concept is extended from quenching to any kind of interference with the fluorophore. Equations for data analysis are shown and their applications are illustrated by calculating the binding constant for several data-sets.