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.

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.

Melanotan II injection resulting in systemic toxicity and rhabdomyolysis.

INTRODUCTION: Melanotan products are currently purchased over the Internet and are designed to induce melanogenesis to create sunless tanning as well are used as sexual stimulants. We report a novel case of systemic toxicity with sympathomimetic excess and rhabdomyolysis after use of Melanotan II. CASE REPORT: A 39 year-old Caucasian male injected subcutaneously 6 mg of Melanotan II purchased over the Internet in an attempt to darken his skin during wintertime. This dose was six times the recommended starting dose per the patient. In the emergency department two hours post injection, he complained of diffuse body aches, sweating, and a sensation of anxiety. Vital signs included BP 151/85 mmHg, HR 130 bpm that peaked at 146 bpm, and temperature of 97.8°F. Physical exam demonstrated a restless and anxious appearing male with mydriasis, diaphoresis, tachycardia, and diffuse muscle tremors. Pertinent laboratory values were creatinine 2.25 mg/dL, CPK 1760 IU/L, troponin 0.23 ng/mL, WBC 19.1 k/µL. Urinalysis demonstrated 3 + blood with red cell casts but 0-2 RBC/hpf. Qualitative urine drug screen was negative for metabolites of cocaine and amphetamines but positive for opiates. The patient received benzodiazepines for agitation and anxiety and had improvement in his symptoms. He was admitted to the ICU and during hospitalization his CPK elevated to 17773 IU/L 12 hours later. He continued to receive intravenous fluids with sodium bicarbonate for rhabdomyolysis and his CPK decreased to 2622 IU/L with improvement of creatinine to 1.23 mg/dL upon discharge from the ICU after 3 days. The substance, which he injected, was analyzed via mass spectrometry and was confirmed to be Melanotan II when compared with an industry purchased standard sample. DISCUSSION: Melanotan products are purchased via the Internet and have three main formulations (Melanotan I, Melanotan II, and bremelanotide). Melanotan I increases melanogenesis and eumelanin content to produce sunless tanning. Melanotan II also increases skin pigmentation but also produces spontaneous penile erections and sexual stimulation. Bremelanotide is a variation of Melanotan II that is specifically designed for sexual stimulation. This unique case highlights the potential of systemic toxicity with sympathomimetic excess, rhabdomyolysis, and renal dysfunction from Melanotan II use. CONCLUSION: Melanotan II use resulted in systemic toxicity including apparent sympathomimetic symptoms, rhabdomyolysis, and renal dysfunction.

[Subacute toxicity study of combination of adrenocorticotropine 1-13 and met-enkephalin]. / Studija subakutne toksicnosti kombinacije adrenokortikotropnog hormona 1-13 i metenkefalina.

PURPOSE: Combination FAR4 consists from two peptide components: met-enkephalin and alpha adrenocorticotropine 1-13 (ACTH 1-13) named before as alpha-melanocyte-stimulating hormone-like (alpha-MSH-like). Met-enkephalin and alpha-MSH exhibited cytoprotective effects individually and statistically significant additive effect was registered when both peptides were applied in combination on the model of ethanol induced gastric lesions in rats. We performed subacute toxicity study with subcutaneous application. WORK METHOD: Wistar rats were randomized in 3 test groups (treatments) consisted of 10 male and to female rats and one control group consisted of 20 male and 20 female rats. One daily dose was applied 3 days a week. Three dose ranges as multiplication of expected maximal human therapeutic dose (10 mg of met-enkephalin and 2 mg of alpha-ACTH 1-13) were estimated: equivalent dose, dose that was 5 times higher and 10 times higher dose. Animals were treated during 4 weeks with 10-days long observation period without the treatment after. After the planned scarification at the end of study, necropsy with histopathology examination was performed. RESULTS: No lethality, toxic signs or histopathological changes were observed during the subacute toxicity testing. Variation of laboratory animals body mass was observed through six terms of body mass deternimation. Increase in body mass was noted in all test and control groups. Statistical analysis with Kruskal Wallis single variance test showed statisticaly significant difference in the number of respirations between the groups of ma-. les for the first measurement (p = 0.040332) and second measurement (p = 0.016852), but multiple comparation with control group showed statisticaly significant difference. Afterthe planned scarification at the end of the study, necropsy did not reveal changes in macroscopic structure of organs and tissues. Histopathology examination was performed on the samples of liver, kidneys, lungs, heart, brain, spleen and thymus and no pathological changes were noted, while microscopic structure of tissues was perserved. The changes regarding postmortem organ mass as percentual ratio towards total group mass were not noted nor for males, nor for females. DISCUSSION: Study was conducted following the rules of the Guide for the Care and Use of Laboratory Animals made by the U.S. National Institutes of Health. Methodologicaly our study complys with rutine design of thistipe of studies. Subacute toxicity studies usually last for fourweeks and the way of test substance application to laboratory animals should comply to future way of application in human use. In our study no lethality was registered and low toxicity

Receptor-mediated cytotoxicity of alpha-MSH fragments containing melphalan in a human melanoma cell line.

Four alpha-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep 1 and 2), the latter being the 4-10 sequence known to be the main alpha-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize alpha-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep 1 and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled alpha-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicity in terms of IC50 values, Pep 1 being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep 1 and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an alpha-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep 1 to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with alpha-MSH central fragments in human melanoma cells due to the presence of alpha-MSH-specific receptors. This mechanism appeared to be both peptide- and cell-type-specific.

Administration of melanotropic peptides during gestation in the rodent.

A potent analogue of alpha-MSH (alpha-melanocyte stimulating hormone, S-alpha-melanotropin), [Nle4,D-Phe7] alpha-MSH, induces darkening of follicular melanocytes when injected or applied topically to the skin of mice [1]. This analogue also results in increased pigmentation when injected subcutaneously (s.c.) in humans. Toxicological studies have been performed on rodent models with administration topically or by intraperitoneal (i.p.) injection. No toxicity was observed and no pathological or significant biochemical changes were found. However there has been some controversy in the literature revolving around whether or not alpha-MSH is trophic for fetal growth and whether the hormone affects fetal adrenal development. These questions have been addressed in this study. All previous studies on the possible reproductive function of alpha-MSH have involved use of the natural hormone only. This is first to demonstrate the effects of the more potent analogue. The rat was used as the animal model to determine if the potent analogue of alpha-MSH affects events in gestation and embryonic fetal development and to determine if the analogue was a developmental toxicant. This study also examines the effect of a melanotropic peptide delivered directly to the conceptus in utero during organogenesis. No changes were found in the parameters examined (sex ratio, weight, morphology or histology, etc.) between treated and control fetuses. There was no evidence of premature parturition or pigmentation changes in the fetuses. The work reported in this study is of relevance if such a melanotropic peptide is to be used in women of childbearing age to induce pigmentation of the skin. Although the present results cannot necessarily be extrapolated to humans, indications are that, in rodents at least, [Nle4,D-Phe7] alpha-MSH and natural alpha-MSH have no adverse effects when administered during gestation and fetal development.

Toxicologic studies of a superpotent alpha-melanotropin, [Nle4, D-Phe7]alpha-MSH.

A toxicology study was performed in mice given a superpotent alpha melanocyte stimulating hormone (MSH) analog. This 13 amino acid derivative, [Nle4, D-Phe7]alpha-MSH or NDP-MSH, is a melanotropin which is very slowly biodegraded in vivo and is active at 1/1,000 the concentration of natural alpha-MSH. Mice were administered up to 2 mg/kg of the analog daily and weekly over 4 or 12 weeks by both topical application (in 90% DMSO) or by IP injections (in physiologic saline). At the end of this period, no toxic effects were observed in various organs, on hematologic indices, or on weight gain. A slight increase in triglyceride and platelet levels were noted in mice given the analog weekly for 12 weeks. There was no evidence of an effect on behavior nor ACTH-like endocrine actions such as elevated serum cortisol levels. Transdermal drug delivery studies performed in vitro showed reproducible diffusion of the NDP-MSH analog through full-thickness mouse skin. Approximately 0.002% to 0.05% of a 10(-4) M preparation was transdermally delivered using a DMSO/water solution or a PEG/alcohol cream base, respectively. This superpotent analog is now entering a Phase I clinical trial with possible therapeutic applications for the treatment of hypomelanotic disorders such as vitiligo and for pharmacologic tanning without the need for sunlight exposure.

Genetic assembly and selective toxicity of diphtheria-toxin-related polypeptide hormone fusion proteins.

The reports of Miyanohara et al. (1986) and Murphy et al. (1986) were the first to describe the genetic construction, expression, and receptor-specific selective toxicity of a chimaeric toxin. In the present report, we have extended these earlier observations and have shown that the fusion of a modified gene encoding IL-2 to a truncated diphtheria toxin gene also results in the expression of a biologically active chimaeric IL-2 toxin. In both instances we have used receptor-binding-domain substitution and have genetically coupled those portions of the diphtheria toxin structural gene that encode the ADP-ribosyl transferase activity of fragment A and lipid-associating domains of fragment B to modified genes which encode either the polypeptide hormone alpha-MSH or the T-cell growth factor IL-2. The chimaeric toxins expressed from these gene fusions have been shown to be selectively targeted to those eukaryotic cells that carry specific surface receptors for the ligand compounds of the hybrid. For example, in the case of the IL-2 toxin, it is clear that the selective action of this hybrid protein is based upon both its diphtheria-toxin and IL-2-related components. Following binding to the IL-2R on activated and/or malignant T-cell, IL-2 toxin is internalized by receptor-mediated endocytosis. Upon acidification of the endosome, diphtheria toxin fragment B portions of the chimaeric toxin facilitate the delivery of fragment A to the cytosol where it catalyses the ADP ribosylation of EF-2. The assembly of chimaeric toxins at the level of the gene offers several advantages over chemical linkage. Since chemical linkage of the toxophore and ligand components of the conjugate toxins requires activation of the epsilon-amino moiety of lysine residues with reagents that will allow for subsequent disulphide linkage, the precise site of coupling is generally not known. In addition, there has been considerable concern over the lability of the disulphide bond between the toxophore and ligand components in vivo due to the action of disulphide reductases. The assembly of chimaeric toxins at the level of the gene allows for precise linkage of the toxophore and ligand components. Since the linkage between the toxophore and ligand is a peptide bond, the chimaeric toxin should be stable in vivo. In addition, the genetic construction of chimaeric toxins also allows for further protein engineering through site-directed mutagenesis.(ABSTRACT TRUNCATED AT 400 WORDS)