KPV

Inflammation-triggered self-immolative conjugates enable oral peptide delivery by overcoming gastrointestinal barriers.

Juan Cheng, Peng Wu, Chenwen Li +5 more

Oral delivery of peptide therapeutics remains challenging due to gastrointestinal (GI) degradation and poor intestinal absorption. Here, we propose a self-immolative peptide prodrug conjugate (SIPPC) platform for inflammation-targeted oral delivery, integrating a hydrophilic polyethylene glycol segment, a reactive oxygen species (ROS)-responsive hydrophobic self-immolative module, and a hydrolyzable scaffold, which collectively enable spontaneous assembly into micelle-like nanoparticles. Using three anti-inflammatory peptides (KPV, Ac-QAW, and IRW), we demonstrated that the engineered conjugates exhibit remarkable GI stability, efficient mucus penetration, and ROS-responsive release at inflamed sites. In colitis mice, the KPV-based conjugate (proKPV) achieved a 3.8-fold greater colonic accumulation than free KPV, with enhanced efficacy even at a 20-fold lower dose. Beyond therapeutic effects in the colitis model, oral proKPV substantially accumulated in inflamed lungs and exhibited potent anti-inflammatory efficacy in mice with acute lung injury. Ac-QAW and IRW-based conjugates exhibited comparable benefits, underscoring SIPPC as a transformative paradigm for oral peptide therapeutics, offering substantial promise for clinical translation in inflammatory disorders.

Exploring the Role of Tripeptides in Wound Healing and Skin Regeneration: A Comprehensive Review.

Siti Balqis Adnan, Manira Maarof, Mh Busra Fauzi +1 more

Wound healing is a complex and dynamic process that requires the coordination of cellular, molecular, and physiological events to restore tissue integrity. Despite notable advances in treatment strategies, optimizing healing outcomes, particularly in chronic wounds, remains a major challenge. Emerging evidence highlights the therapeutic promise of peptides, especially tripeptides, in accelerating tissue repair through diverse mechanisms. These short peptides regulate key processes such as cell migration, proliferation, and differentiation, while also modulating inflammation, promoting angiogenesis, and facilitating extracellular matrix (ECM) remodeling. This review, covering studies published between 2016 and 2025, explores the role of tripeptides in enhancing wound repair, emphasizing their biological functions, mechanisms of action, and therapeutic applications. Recent findings demonstrate that tripeptides can stimulate fibroblast migration, enhance collagen deposition, and support angiogenesis. In addition, they exhibit antimicrobial and anti-inflammatory properties, making them valuable candidates for both acute and chronic wound management. GHK-based formulations, including nanoparticle conjugates, hydrogels, and clinical derivatives such as TriHex and TriHex 2.0, enhance fibroblast migration, ECM remodeling, collagen and elastin synthesis, and wound closure while providing antimicrobial activity. KdPT mitigates hyperglycemia-induced oxidative stress and restores keratinocyte function, whereas KPV-loaded hydrogels reduce inflammation, promote tissue regeneration, and combat MRSA infections. Additionally, lipotripeptides (DICAMs) inhibit and disrupt bacterial biofilms, and GPE supports neuroprotection and regeneration through ERK and PI3K/Akt signaling activation. Beyond wound repair, this review also discusses comparative physicochemical properties and wound healing applications of tripeptides versus larger peptides, factors influencing their performance, strategies for combination with biomaterial scaffolds, and emerging applications in fields such as cancer and cosmetics. Collectively, tripeptides represent a promising class of multifunctional bioactive molecules in wound care, offering novel avenues for targeted tissue regeneration. Future research should focus on improving their stability, bioavailability, and delivery systems to fully harness their clinical potential in regenerative medicine.

A nanoparticle platform for combined mucosal healing and immunomodulation in inflammatory bowel disease treatment.

Valentina Marotti, Yining Xu, Cécilia Bohns Michalowski +11 more

Current treatments for inflammatory bowel disease (IBD) treatment consist of anti-inflammatory products. In this study, we sought to induce the physiological secretion of glucagon-like peptide 2, a peptide with intestinal growth-promoting activity, via nanoparticles while simultaneously providing with immunomodulation by tailoring the nanoparticle surface. To this end, we developed hybrid lipid hyaluronate-KPV conjugated nanoparticles loaded with teduglutide for combination therapy in IBD. The nanocarriers induced (or did not induce) immunosuppression depending on the presence (or absence) of a hyaluronan-KPV functionalization. This strategy holds promise as a nanoparticle platform for combined mucosal healing and immunomodulation in IBD treatment.

Critical role of PepT1 in promoting colitis-associated cancer and therapeutic benefits of the anti-inflammatory PepT1-mediated tripeptide KPV in a murine model.

Emilie Viennois, Sarah A Ingersoll, Saravanan Ayyadurai +7 more

The human intestinal peptide transporter 1, hPepT1, is expressed in the small intestine at low levels in the healthy colon and upregulated during inflammatory bowel disease. hPepT1 plays a role in mouse colitis and human studies have demonstrated that chronic intestinal inflammation leads to colorectal cancer (colitis-associated cancer; CAC). Hence, we assessed here the role of PepT1 in CAC.

KPV and RAPA Self-Assembled into Carrier-Free Nanodrugs for Vascular Calcification Therapy.

Li Zhang, Dongze Li, Yierpani Aierken +7 more

Cardiovascular disease (CVD) is a leading cause of death globally, and vascular calcification (VC) is an important independent risk factor for predicting CVD. Currently, there are no established therapeutic strategies for the treatment of VC. Although recognized combination therapies of nanomedicines can provide effective strategies for disease treatment, the clinical application of nanomedicines is limited because of their complex preparation processes, low drug loading rates, and unpredictable safety risks. Thus, developing a simple, efficient, and safe nanodrug to simultaneously regulate inflammation and autophagy may be a promising strategy for treating VC. Herein, an anti-inflammatory peptide (lysine-proline-valine peptides, KPV) and the autophagy activator rapamycin (RAPA) are self-assembled to form new carrier-free spherical nanoparticles (NPs), which shows good stability and biosafety. In vivo and in vitro, KPV-RAPA NPs significantly inhibit VC in mice compared to the other treatment groups. Mechanistically, KPV-RAPA NPs inhibit inflammatory responses and activated autophagy. Therefore, this study indicates that the new carrier-free KPV-RAPA NPs have great potential as therapeutic agents for VC combination therapy, which can promote the development of nanodrugs for VC.

PepT1-targeted nanodrug based on co-assembly of anti-inflammatory peptide and immunosuppressant for combined treatment of acute and chronic DSS-induced ColitiS.

Daifang Zhang, Longqi Jiang, Fengxu Yu +4 more

Inflammatory bowel disease (IBD), as a chronic and recurrent inflammatory bowel diseases with limited therapeutic outcomes, is characterized by immune disorders and intestinal barrier dysfunction. Currently, the most medications used to cure IBD in clinic just temporarily induce and maintain remission with poor response rates and limited outcomes. Therefore, it is urgently necessary to develop an appropriate therapeutic candidate with preferable efficacy and less adverse reaction for curing IBD.

Skin-adaptive film dressing with smart-release of growth factors accelerated diabetic wound healing.

Yingzheng Zhao, Lantian Huang, Gaolong Lin +7 more

The general treatment of diabetic wound was use of wound dressings to absorb excess exudate. However, traditional wound dressings neither mimic the skin-like properties nor easily be withdrawn from the wound. Herein, the skin-adaptive three-layered films (AGB) dressing has been designed by alternatively depositing phenylboronic acid-grafted γ-PGA (PBA-PGA) and polyvinyl alcohol (PVA). The thickness of AGB film was only 479 μm and its flexibility was obviously strengthen by the boronic ester cross-linking. Besides, the dry AGB film was conveniently adhered to the fresh wound, where its adhesive force reached to 1267 ± 330 mN. Moreover, the adhered AGB film was easily peeled without any second damage after hydration. An anti-inflammatory tripeptide (KPV) and epidermal growth factor (EGF) as biologic factors were respectively encapsulated in the bottom layer and the middle-top two layers of AGB film. KPV was firstly released within 3 day and EGF was subsequently released in a glucose-responsive manner. AGB film containing KPV and EGF (K-E-AGB) could significantly improve the repair rate of full-thickness skin wound on diabetic mice. The mechanism of wound healing was associated with inflammatory inhibition, angiogenesis and collagen deposition. Collectively, skin-adaptive film may be a promising dressing as delivery of biologic factors for the chronic wound.

Mitigating the toxicity of palmitoylated analogue of α-melanocyte stimulating hormone(11-13) by conjugation with gold nanoparticle: characterisation and antibacterial efficacy against methicillin sensitive and resistant Staphylococccus aureus.

Sayani Mitra, Aftab Hossain Mondal, Kasturi Mukhopadhyay

In an attempt to develop potent and non-toxic antimicrobial agent, the palmitoylated analogue of α-melanocyte stimulating hormone(11-13), Pal-α-MSH(11-13) was conjugated with gold nanoparticles (GNPs) for the first time and the efficacy of derived complex was investigated against two strains of Staphylococccus aureus. The GNPs were synthesized using tri-sodium citrate as reductant and Pal-α-MSH(11-13) was conjugated thereafter. The particles were characterised by UV-vis spectroscopy, transmission electron microscopy, dynamic light scattering, fourier transform infrared spectroscopy etc. Conjugation occurred via electrostatic interaction between anionic GNPs and cationic Pal-α-MSH(11-13). The zeta potential of GNP-Pal-α-MSH(11-13) was - 26.91, indicating its stability. The antibacterial activity was determined by minimal inhibitory concentration (MIC) and killing kinetics assay, whereas, inhibition of biofilm formation was studied by determining the biofilm biomass by crystal violet dye binding method, viability of biofilm-embedded cells by counting CFUs and metabolic activity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The toxicity was analysed by hemolysis assay against murine RBCs and cytotoxicity against 3T3 fibroblasts. The MIC was 18 µM for GNP-Pal-α-MSH(11-13) and 12 µM for Pal-α-MSH(11-13). The killing kinetics and biofilm inhibition studies indicated the comparable efficacy of peptide before and after nano-conjugation. Importantly, the conjugation resulted in diminished toxicity, as evidenced by 0.29 ± 0.03% hemolysis and 100% viable fibroblasts at 72 µM compared to the Pal-α-MSH(11-13), showing 74.99 ± 1.59% hemolysis and 59.39 ± 1.06% viable fibroblasts. The nano-fabrication drastically reduced the peptide toxicity without compromising its antibacterial efficacy. The anionicity of the conjugate may be responsible for non-toxicity that makes them suitable for pharmaceutical applications.

Self-Cross-Linked Hydrogel of Cysteamine-Grafted γ-Polyglutamic Acid Stabilized Tripeptide KPV for Alleviating TNBS-Induced Ulcerative Colitis in Rats.

Jie Sun, Pengpeng Xue, Jiayi Liu +7 more

KPV (Lys-Pro-Val), which is a tripeptide derived from α-MSH (α-melanocyte-stimulating hormone), has an anti-inflammatory effect on colitis. However, KPV solution is very unstable when rectally administered, compromising its therapeutic efficacy. Herein, cysteamine-grafted γ-polyglutamic acid (SH-PGA) was synthesized by conjugating cysteamine with the carboxyl groups of γ-PGA. The synthesized SH-PGA has the thiol grafting amount of 4.5 ± 0.3 mmol/g. Without the use of the cross-linker, the SH-PGA hydrogel with 4% of the polymer was formed by self-cross-linking of thiol groups. Moreover, the formation of the SH-PGA hydrogel was not affected by KPV. The KPV/SH-PGA hydrogel presented higher elastic modulus (G') than the corresponding viscous modulus (G″) at 0.01-10 Hz, exhibiting good mechanical stability. The KPV/SH-PGA hydrogel presented a shear-thinning behavior, which was helpful for rectal administration. Only 30% of KPV was released from the KPV/SH-PGA hydrogel within 20 min, followed by a sustained-release behavior. Importantly, the stability of KPV in the SH-PGA hydrogel was obviously enhanced, which was presented by detecting its anti-inflammatory activity and promoting cell migration potential after 2 h of exposure to 37 °C. The enhanced therapeutic effect of the KPV/SH-PGA hydrogel on colitis was confirmed on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ulcerative colitis rats. The colitis symptoms including body weight loss and the disease activity index score were obviously attenuated by rectally administering the KPV/SH-PGA hydrogel. Besides, the KPV/SH-PGA hydrogel treatment prevented the colon shortening of TNBS-infused rats and decreased the colonic myeloperoxidase level. The morphology of the colon including the epithelial barrier, crypt, and intact goblet cells was recovered after KPV/SH-PGA hydrogel treatment. Besides, the KPV/SH-PGA hydrogel decreased the expression of proinflammatory cytokines such as tumor necrosis factor α and interleukin 6. Collectively, the KPV/SH-PGA hydrogel may provide a promising strategy for the treatment of ulcerative colitis.

Novel anti-inflammatory and chondroprotective effects of the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride and human melanocortin MC3 receptor agonist PG-990 on lipopolysaccharide activated chondrocytes.

Vedia C Can, Ian C Locke, Magdalena K Kaneva +5 more

Human melanocortin MC1 and MC3 receptors expressed on C-20/A4 chondrocytes exhibit chondroprotective and anti-inflammatory effects when activated by melanocortin peptides. Nearly 9 million people in the UK suffer from osteoarthritis, and bacterial infections play a role in its development. Here, we evaluate the effect of a panel of melanocortin peptides with different selectivity for human melanocortin MC1 (α-MSH, BMS-470539 dihydrochloride) and MC3 ([DTrp8]-γ-MSH, PG-990) receptors and C-terminal peptide α-MSH11-13(KPV), on inhibiting LPS-induced chondrocyte death, pro-inflammatory mediators and induction of anti-inflammatory proteins. C-20/A4 chondrocytes were treated with a panel of melanocortin peptides prophylactically and therapeutically in presence of LPS (0.1 μg/ml). The chondroprotective properties of these peptides determined by cell viability assay, RT-PCR, ELISA for detection of changes in inflammatory markers (IL-6, IL-8 and MMP-1, -3 and -13) and western blotting for expression of the anti-inflammatory protein heme-oxygenase-1. C-20/A4 expressed human melanocortin MC1 and MC3 receptors and melanocortin peptides elevated cAMP. LPS stimulation caused a reduction in C-20/A4 viability, attenuated by the human melanocortin MC1 receptor agonist BMS-470539 dihydrochloride, and MC3 receptor agonists PG-990 and [DTrp8]-γ-MSH. Prophylactic and therapeutic regimes of [DTrp8]-γ-MSH significantly inhibited LPS-induced modulation of cartilage-damaging IL-6, IL-8, MMPs -1,-3 and -13 mediators both prophylactically and therapeutically, whilst human melanocortin MC1 and MC3 receptor agonists promoted an increase in HO-1 production. In the presence of LPS, activation of human melanocortin MC1 and MC3 receptors provided potent chondroprotection, upregulation of anti-inflammatory proteins and downregulation of inflammatory and proteolytic mediators involved in cartilage degradation, suggesting a new avenue for osteoarthritis treatment.

Are melanocortin peptides future therapeutics for cutaneous wound healing?

Markus Böhm, Thomas Luger

Cutaneous wound healing is a complex process divided into different phases, that is an inflammatory, proliferative and remodelling phase. During these phases, a variety of resident skin cell types but also cells of the immune system orchestrate the healing process. In the last year, it has been shown that the majority of cutaneous cell types express the melanocortin 1 receptor (MC1R) that binds α-melanocyte-stimulating hormone (α-MSH) with high affinity and elicits pleiotropic biological effects, for example modulation of inflammation and immune responses, cytoprotection, antioxidative defense and collagen turnover. Truncated α-MSH peptides such as Lys-Pro-Val (KPV) as well as derivatives like Lys-d-Pro-Thr (KdPT), the latter containing the amino acid sequence 193-195 of interleukin-1β, have been found to possess anti-inflammatory effects but to lack the pigment-inducing activity of α-MSH. We propose here that such peptides are promising future candidates for the treatment of cutaneous wounds and skin ulcers. Experimental approaches in silico, in vitro, ex vivo and in animal models are outlined. This is followed by an unbiased discussion of the pro and contra arguments of such peptides as future candidates for the therapeutic management of cutaneous wounds and a review of the so-far available data on melanocortin peptides and derivatives in wound healing.

A PepT1 mediated medicinal nano-system for targeted delivery of cyclosporine A to alleviate acute severe ulcerative colitis.

Ya Wu, Minghui Sun, Dan Wang +7 more

To effectively alleviate acute severe ulcerative colitis (ASUC), we developed a colon-specific delivery system-PLGA-KPV/MMT/CS multifunctional medicinal nanoparticles loaded with cyclosporine A (CyA). The lysine-proline-valine (KPV) tripeptide, which possesses anti-inflammatory properties and high affinity to peptide transporter 1 (PepT1), can target therapy-related cells (colonic epithelial cells and macrophages) via overexpression of PepT1. Montmorillonite (MMT)/chitosan (CS) coating can reduce CyA leakage in the upper gastrointestinal tract (GIT) and enhance nanoparticle adhesion to the inflamed colon. The bio-distribution demonstrated that nanoparticles can specifically accumulate in the inflamed tissues and can be retained for up to 36 h. After being treated with the CyA-PLGA-KPV/MMT/CS nanoparticles (PKMCN), the mice with DSS-induced ulcerative colitis exhibited significant improvements in body weight, colon length, and disease activity index. Moreover, biochemistry and immunohistochemical analysis showed that the PKMCN treatment group performed as well as the healthy group. Intriguingly, PKMCN without CyA also presented marked therapeutic effects. Our results suggested that PKMCN could be a promising drug delivery system for ASUC therapy by targeting inflamed cells, prolonging curative time, and mitigating colitis.

Structural modification of the tripeptide KPV by reductive "glycoalkylation" of the lysine residue.

Abigael C Songok, Pradip Panta, William T Doerrler +2 more

Peptides that exhibit enzymatic or hormonal activities are regulatory factors and desirable therapeutic drugs because of their high target specificity and minimal side effects. Unfortunately, these drugs are susceptible to enzymatic degradation, leading to their rapid elimination and thereby demanding frequent dosage. Structurally modified forms of some peptide drugs have shown enhanced pharmacokinetics, improving their oral bioavailability. Here, we discuss a novel glycomimetic approach to modify lysine residues in peptides. In a model system, the ε-amine of Ts-Lys-OMe was reductively alkylated with a glucose derivative to afford a dihydroxylated piperidine in place of the amine. A similar modification was applied to H-KPV-NH2, a tripeptide derived from the α-melanocyte stimulating hormone (α-MSH) reported to have antimicrobial and anti-inflammatory properties. Antimicrobial assays, under a variety of conditions, showed no activity for Ac-KPV-NH2 or the α- or ε-glycoalkylated analogs. Glycoalkylated peptides did, however, show stability toward proteolytic enzymes.

Peptide Receptor-Targeted Fluorescent Probe: Visualization and Discrimination between Chronic and Acute Ulcerative Colitis.

Meiying Zeng, Andong Shao, Hui Li +7 more

The inflammatory activity of ulcerative colitis plays an important role in the medical treatment. However, accurate and real-time monitoring of the colitis activity with noninvasive bioimaging method is still challenging, especially in distinguishing between chronic and acute colitis. As a good receptor, the oligopeptide transporter (PepT1) is overexpressed in the colonic epithelial cells of chronic ulcerative colitis, which can deliver tripeptide KPV (Lys-Pro-Val, the C-terminal sequence of α-MSH) into cytosol in the intestine. Herein, we report a PepT1 peptide receptor-targeted fluorescent probe, dicyanomethylene-4H-pyran (DCM)-KPV, with the strategy of conjugating the KPV into the DCM chromophore. The diagnostic fluorescent probe bestows a specific receptor-targeted interaction with PepT1 through the KPV moiety, possessing several beneficial characteristics, such as efficient long emission, low photobleaching, negligible cytotoxicity, and high cytocompatibility in living cells. We build the overexpressed PepT1 on the cytomembrane of ulcerative colitis model Caco-2 cell as the efficient receptor to accumulate the targeted tripeptide KPV in the cytoplasm and nucleus. With the co-localization of DCM-KPV and the DNA-specific fluorophore, DAPI, the specifically long emission from chromophore DCM and efficient receptor-targeted peptide KPV, the fluorescent probe of DCM-KPV makes a breakthrough to the direct noninvasive observation of the accumulation in colon inflammation regions via intestinal mucosa, even successfully distinguishing the chronic, acute ulcerative colitis and normal groups. Compared with the traditional unenhanced magnetic resonance imaging and hematoxylin and eosin (H&E) staining, we make full use of exploiting the specific target-receptor interaction between the tripeptide unit, KPV, and the oligopeptide transporter, PepT1, for sensing selectivity. The desirable diagnostic ability of DCM-KPV can guarantee the real-time tracking and visualization of the role of intracellular KPV on ulcerative colitis, which provides an alternative to replace the time-consuming and tissue sampling-invasive H&E staining diagnosis.

Orally Targeted Delivery of Tripeptide KPV via Hyaluronic Acid-Functionalized Nanoparticles Efficiently Alleviates Ulcerative Colitis.

Bo Xiao, Zhigang Xu, Emilie Viennois +6 more

Overcoming adverse effects and selectively delivering drug to target cells are two major challenges in the treatment of ulcerative colitis (UC). Lysine-proline-valine (KPV), a naturally occurring tripeptide, has been shown to attenuate the inflammatory responses of colonic cells. Here, we loaded KPV into hyaluronic acid (HA)-functionalized polymeric nanoparticles (NPs). The resultant HA-KPV-NPs had a desirable particle size (∼272.3 nm) and a slightly negative zeta potential (∼-5.3 mV). These NPs successfully mediated the targeted delivery of KPV to key UC therapy-related cells (colonic epithelial cells and macrophages). In addition, these KPV-loaded NPs appear to be nontoxic and biocompatible with intestinal cells. Intriguingly, we found that HA-KPV-NPs exert combined effects against UC by both accelerating mucosal healing and alleviating inflammation. Oral administration of HA-KPV-NPs encapsulated in a hydrogel (chitosan/alginate) exhibited a much stronger capacity to prevent mucosa damage and downregulate TNF-α, thus they showed a much better therapeutic efficacy against UC in a mouse model, compared with a KPV-NP/hydrogel system. These results collectively demonstrate that our HA-KPV-NP/hydrogel system has the capacity to release HA-KPV-NPs in the colonic lumen and that these NPs subsequently penetrate into colitis tissues and enable KPV to be internalized into target cells, thereby alleviating UC.

Transdermal Iontophoretic Delivery of Lysine-Proline-Valine (KPV) Peptide Across Microporated Human Skin.

Kasturi Pawar, Chandra S Kolli, Vijaya K Rangari +1 more

Lysine-proline-valine (KPV) is a C-terminal peptide fragment of α-melanocyte stimulating hormone with potent anti-inflammatory properties. Present study investigates various transdermal enhancement strategies such as iontophoresis (ITP), microneedles (MN), and their combination (ITP + MN) on KPV delivery across dermatomed human skin. KPV attains a positive charge at pH less than 7.0, thus anodal ITP was used. The influence of current strength, KPV concentration, and duration of current application on the KPV delivery was investigated. At defined ITP parameters, the influence of MN on KPV delivery (ITP + MN) across skin was also determined. KPV permeation was less than detectable levels (limit of detection, 0.01 μg/mL) by simple passive diffusion. However, KPV permeation was increased to 4.4 μg/cm2/h by MN treatment. Furthermore, ITP and ITP + MN increased the permeation rate by 8 and 35 fold, respectively, as compared to MN alone. The skin retention levels of KPV by MN, ITP, and ITP + MN were increased by 5, 10, and 10 fold, respectively, as compared to passive diffusion. Confocal studies indicate that fluorescein isothiocyanate-labeled KPV migrated through the stratum corneum, along the microchannels and into the lower epidermal tissue because the fluorescence was observed beyond the depth of 100 μm.

Stability-indicating HPLC assay for lysine-proline-valine (KPV) in aqueous solutions and skin homogenates.

Kasturi R Pawar, Vanisree Mulabagal, Forrest Smith +3 more

A simple, sensitive and stability-indicating high-performance liquid chromatographic (HPLC) assay method was developed and validated for a bioactive peptide, lysine-proline-valine (KPV) in aqueous solutions and skin homogenates. Chromatographic separation was achieved on a reversed phase Phenomenex C18 column (4.6 × 250 mm, packed with 5 µm silica particles) with a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B). The proposed HPLC method was validated with respect to accuracy, precision, linearity, repeatability, limit of detection (LOD) and limit of quantitation (LOQ). The calibration curve was linear with a correlation coefficient (r) of 0.9999. Relative standard deviation values of accuracy and precision experiments were <2. The LOD and LOQ of KPV were 0.01 and 0.25 µg/mL, respectively. Under stress conditions (acid, alkali and hydrogen peroxide) KPV yielded lys-pro-diketopiperazine as major degradation product, which was identified by flow injection MS analysis. The developed HPLC method was found to be efficient in separating the active peptide from its degradation products generated under various stress conditions. Also, the validated method was able to separate KPV from other peaks arising from endogenous components of the skin homogenate.

Alpha-melanocyte stimulating hormone: an emerging anti-inflammatory antimicrobial peptide.

Madhuri Singh, Kasturi Mukhopadhyay

The alpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide belonging to the melanocortin family. It is well known for its anti-inflammatory and antipyretic effects and shares several characteristics with antimicrobial peptides (AMPs). There have been some recent reports about the direct antimicrobial activity of α-MSH against various microbes belonging to both fungal and bacterial pathogens. Similar to α-MSH's anti-inflammatory properties, its C-terminal residues also exhibit antimicrobial activity parallel to that of the entire peptide. This review is focused on the current findings regarding the direct antimicrobial potential and immunomodulatory mechanism of α-MSH and its C-terminal fragments, with particular emphasis on the prospects of α-MSH based peptides as a strong anti-infective agent.

Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors.

Ramesh Alleti, Josef Vagner, Dilani Chathurika Dehigaspitiya +10 more

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.

Single administration of tripeptide α-MSH(11-13) attenuates brain damage by reduced inflammation and apoptosis after experimental traumatic brain injury in mice.

Eva-Verena Schaible, Arne Steinsträßer, Antje Jahn-Eimermacher +7 more

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11-13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11-13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11-13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11-13) injected mice. The expression of the inflammatory markers TNF-α and IL-1β as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11-13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury.

The synthetic melanocortin (CKPV)2 exerts anti-fungal and anti-inflammatory effects against Candida albicans vaginitis via inducing macrophage M2 polarization.

Hai-xia Ji, Yu-lian Zou, Jing-jing Duan +14 more

In this study, we examined anti-fungal and anti-inflammatory effects of the synthetic melanocortin peptide (Ac-Cys-Lys-Pro-Val-NH2)2 or (CKPV)2 against Candida albicans vaginitis. Our in vitro results showed that (CKPV)2 dose-dependently inhibited Candida albicans colonies formation. In a rat Candida albicans vaginitis model, (CKPV)2 significantly inhibited vaginal Candida albicans survival and macrophages sub-epithelial mucosa infiltration. For mechanisms study, we observed that (CKPV)2 inhibited macrophages phagocytosis of Candida albicans. Meanwhile, (CKPV)2 administration inhibited macrophage pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) release, while increasing the arginase activity and anti-inflammatory cytokine IL-10 production, suggesting macrophages M1 to M2 polarization. Cyclic AMP (cAMP) production was also induced by (CKPV)2 administration in macrophages. These above effects on macrophages by (CKPV)2 were almost reversed by melanocortin receptor-1(MC1R) siRNA knockdown, indicating the requirement of MC1R in the process. Altogether, our results suggest that (CKPV)2 exerted anti-fungal and anti-inflammatory activities against Candida albicans vaginitis probably through inducing macrophages M1 to M2 polarization and MC1R activation.

Copper-62 labeled ReCCMSH peptide analogs for melanoma PET imaging.

Xiuli Zhang, Zhiwei Yue, Bao-Yuan Lu +7 more

High-specific activity radiolabeled melanocortin peptide preparations are necessary for optimal melanoma imaging due to the relatively low number of melanocortin-1 receptors (MC1-Rs) per tumor cell. In this study, a one-step synthesis of 62Cu-labeled MC1-R targeting peptide Re(Arg11)CCMSH was developed, which yielded high specific activity radiolabeled peptide preparations that required no post-labeling purification. DOTA and NOTA conjugated Re(Arg11)CCMSH peptides were synthesized and examined for 62Cu radiolabeling and cell binding properties. Biodistribution and PET imaging studies were performed to assess the in vivo tumor targeting and imaging characteristics of the optimal radiolabeled peptide. Melanoma cell binding affinities for NOTA-, NOTA-GGG-, and NOTA-GSG- conjugated Re(Arg11)CCMSH were determined to be 1.3×10-9 M, 1.9×10-9 M and 6.0×10-9 M. The 62Cu radiolabeling efficiencies of DOTA- and NOTA- conjugated Re(Arg11)CCMSH analogs were 30% and > 98% after 2 min at 24° C, while 0.5 μg of NOTA-GGG-peptide could be labeled to > 95% with a maximum specific activity of 138 Ci/μmol. Tumor uptake of 62Cu- NOTA-GGG-Re(Arg11)CCMSH in B16/F1 melanoma bearing mice was 4.65±0.48% ID/g and 9.43±2.69% ID/g at 20 and 40 min post injection and was visualized by PET imaging. High specific activity 62Cu-NOTA-GGG-Re(Arg11)CCMSH was prepared in a one-step procedure at 24°C in 6 min. 62Cu-NOTA-GGG-Re(Arg11)CCMSH exhibited MC1-R selective binding and rapid tumor uptake in B16/F1 melanoma bearing mice that was confirmed by PET imaging studies. High specific activity 62Cu from a 62Zn/62Cu generator coupled with simple one step radiolabeling procedures makes 62Cu an attractive radionuclide for PET imaging of low-density receptor targets.

Inhibition of cellular and systemic inflammation cues in human bronchial epithelial cells by melanocortin-related peptides: mechanism of KPV action and a role for MC3R agonists.

Stephen C Land

Chemokine signaling from airway epithelium regulates macrophage recruitment to the lung in inflammatory diseases such as asthma. This study investigates the mechanism by which the α-melanocyte stimulating hormone-derived tripeptide, KPV, and the agonist of the dominant melanocortin receptor in airway epithelium (MC3R), γ-melanocyte stimulating hormone (γ-MSH), suppress inflammation in immortalised human bronchial airway epithelium.

Terminal signal: anti-inflammatory effects of α-melanocyte-stimulating hormone related peptides beyond the pharmacophore.

Thomas Brzoska, Markus Böhm, Andreas Lügering +2 more

During the last two decades a significant number of investigations has established the fact that α-Melanocyte-stimulating hormone (α-MSH) is a potent anti-inflammatory mediator. The anti-inflammatory effects of α-MSH can be elicited via melanocortin receptors (MC-Rs) broadly expressed in a number of tissues ranging from the central nervous system to cells of the immune system and on resident somatic cells of peripheral tissues. α-MSH affects various pathways regulating inflammatory responses such as NF-κB activation, expression of adhesion molecules, inflammatory cytokines, chemokine receptors, T-cell proliferation and activity and inflammatory cell migration. In vivo α-MSH has been shown to be anti-inflammatory as well in animal models of fever, irritant and allergic contact dermatitis, cutaneous vasculitis, fibrosis, in ocular, gastrointestinal, brain and allergic airway inflammation and arthritis. A broad range of effects of α-MSH exerted beyond the field of inflammation, its pigmentory capacity being only the most visible aspect, has been one of the major impediments limiting the use of α-MSH in human inflammatory disorders. Interestingly KPV, C-terminal tripeptide of α-MSH, which lacks the entire sequence motif required for binding to any of the known MC-Rs, retains almost all of the anti-inflammatory capacity of the full hormone, but in its activities display a lack of any pigmentory action. While the exact signaling mechanism utilized by KPV and related peptides currently is unknown it has been demonstrated already that significant similarities between anti-inflammatory signaling of α-MSH and those short peptides exist. These α-MSH related tripeptides thus may be useful alternatives for anti-inflammatory peptide therapy. KdPT, a derivative of KPV corresponding to IL-1β(193-195), currently is emerging as another tripeptide with potent anti-inflammatory effects. A more limited spectrum of biologic activities, potentially advantageous physicochemical, pharmacokinetic and pharmacodynamic properties as well as the expectation of low costs for pharmaceutical production make these agents interesting candidates for the treatment of immune-mediated inflammatory skin and bowel diseases, allergic asthma and arthritis.

C-terminal amino acids of alpha-melanocyte-stimulating hormone are requisite for its antibacterial activity against Staphylococcus aureus.

Madhuri Singh, Kasturi Mukhopadhyay

Alpha-melanocyte-stimulating hormone (α-MSH) is an endogenous neuropeptide that is known for its anti-inflammatory and antipyretic activities. We recently demonstrated that α-MSH possesses staphylocidal activity and causes bacterial membrane damage. To understand the role of its amino acid sequences in the staphylocidal mechanism, in the present study we investigated the antimicrobial activities of different fragments of α-MSH, i.e., α-MSH(6-13), α-MSH(11-13), and α-MSH(1-5), and compared them with that of the entire peptide. Our results showed that peptides containing the C-terminal region of α-MSH, namely, α-MSH(6-13) and α-MSH(11-13), efficiently killed >90% of both methicillin-sensitive and -resistant Staphylococcus aureus cells in the micromolar range and ∼50% of these cells in the nanomolar range; their efficiency was comparable to that of the entire α-MSH, whereas the peptide containing the N-terminal region, α-MSH(1-5), was found to be ineffective against S. aureus. The antimicrobial activity of α-MSH and its C-terminal fragments was not affected by the presence of NaCl or even divalent cations such as Ca2+ and Mg2+. Similar to the case for the parent peptide, α-MSH(6-13) and α-MSH(11-13) also depolarized and permeabilized Staphylococcus cells (∼70 to 80% of the cells were depolarized and lysed after 2 h of peptide exposure at micromolar concentrations). Furthermore, scanning and transmission electron microscopy showed remarkable morphological and ultrastructural changes on S. aureus cell surface due to exposure to α-MSH-based peptides. Thus, our observations indicate that C-terminal fragments of α-MSH retain the antimicrobial activity of entire peptide and that their mechanism of action is similar to that of full-length peptide. These observations are important and are critical in the rational design of α-MSH-based therapeutics with optimal efficacy.

A retro-inverso α-melanocyte stimulating hormone analog with MC1R-binding selectivity.

Timothy Weeden, Jim Stefano, Su Duan +6 more

α-melanocyte stimulating hormone (α-MSH) is a tridecapeptide fragment of pro-opiomelanocortin (POMC) with broad effects on appetite, skin pigmentation, hormonal regulation, and potential roles in both inflammation and autoimmunity. The use of this peptide as an anti-inflammatory agent is limited by its low selectivity between the melanocortin receptors, susceptibility to proteolytic degradation, and rapid clearance from circulation. A retro-inverso (RI) sequence of α-MSH was characterized for receptor activity and resistance to protease. This peptide demonstrated surprisingly high selectivity for binding the melanocortin receptor 1 (MC1R). However, RI-α-MSH exhibited a diminished binding affinity for MC1R compared to α-MSH. Mapping of the residues critical for agonist activity, receptor binding, and selectivity by alanine scanning, identified the same critical core tetrapeptide required for the native peptide. Modest improvements in affinity were obtained by conservative changes employing non-natural amino acids and substitution of the C-terminal sequence with a portion of a MC1R ligand peptide previously identified by phage display. Recombination of these elements yielded a peptide with an identical K(i) as α-MSH at MC1R and a lower EC(50) in Mel-624 melanoma cells. A number of other structural modifications of the RI peptide were found to differ in effect from those reported for the L-form α-MSH, suggesting a significantly altered interaction with the MC1R.

Drug-loaded nanoparticles targeted to the colon with polysaccharide hydrogel reduce colitis in a mouse model.

Hamed Laroui, Guillaume Dalmasso, Hang Thi Thu Nguyen +3 more

One of the challenges to treating inflammatory bowel disease (IBD) is to target the site of inflammation. We engineered nanoparticles (NPs) to deliver an anti-inflammatory tripeptide Lys-Pro-Val (KPV) to the colon and assessed its therapeutic efficacy in a mouse model of colitis.

Prolylcarboxypeptidase regulates food intake by inactivating alpha-MSH in rodents.

Nicholas Wallingford, Bertrand Perroud, Qian Gao +12 more

The anorexigenic neuromodulator alpha-melanocyte-stimulating hormone (alpha-MSH; referred to here as alpha-MSH1-13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of alpha-MSH1-13 maturation and inactivation is incompletely understood. Here we have provided insight into alpha-MSH1-13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of alpha-MSH1-13, producing alpha-MSH1-12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where alpha-MSH1-13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet-induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active alpha-MSH1-13 levels.

Melanoma imaging using (111)In-, (86)Y- and (68)Ga-labeled CHX-A''-Re(Arg11)CCMSH.

Lihui Wei, Xiuli Zhang, Fabio Gallazzi +8 more

A novel alpha-melanocyte-stimulating hormone peptide analog CHX-A''-Re(Arg(11))CCMSH, which targeted the melanocortin-1 receptor (MC1-R) overexpressed on melanoma cells, was investigated for its biodistribution and tumor imaging properties.