Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity.

AIM: The research was aimed to analyze a level of triglycerides in blood serum as a possible new marker of toxicity, particularly in patients with excess body weight, receiving cisplatin. MATERIALS AND METHODS: Study involved 20 oncological patients with stage III lung cancer, who received palliative treatment with cisplatin. High-performance liquid chromatography was used for quantitative determination of pure cisplatin in urine and blood samples. Cisplatin concentration of the test samples was determined based on the data obtained from the calibration graph. RESULTS: Quantitative determination of pure cisplatin is quite complicated. The elimination half-time for one of the groups was observed higher almost by half than for other patients. Higher dose of cisplatin showed a significant association with increase in triglyceride levels. We found a close correlation between body mass index and triglyceride changes during chemotherapy (p = 0.001; r = 0.67). The results indicate that a higher body mass index gives higher fluctuations of triglyceride levels in blood serum. Analyses of correlation between level of triglycerides and elimination half-time show that by an increase in the level of triglycerides in the blood serum cisplatin elimination half-time is prolonged (R2 Linear = 0.596). Cisplatin concentration in urine is higher and elimination takes longer time at elevated levels of triglycerides, where close correlation between fraction of excreted substance in urine and concentration parameters was seen (p < 0.01). Also good correlation for body mass index with fraction of excreted substance in urine and concentration parameters was observed (p < 0.05). CONCLUSION: Clearance of cisplatin, which was determined by the chromatographic method, is reduced in individuals with increased adipose tissue mass. Research data suggest that overweight affects cisplatin elimination from the body. The greater body fat mass can contribute to a greater rise of triglyceride level in blood serum. Triglycerides in blood plasma may serve as an additional indicator of higher cisplatin toxicity as a cardiotoxicity marker.

Determinants of mental health stigma among pharmacy students in Australia, Belgium, Estonia, Finland, India and Latvia.

BACKGROUND: Healthcare professionals commonly exhibit negative attitudes toward people with mental disorders. Few international studies have sought to investigate the determinants of stigma. OBJECTIVE: To conduct an international comparison of pharmacy students' stigma towards people with schizophrenia, and to determine whether stigma is consistently associated with stereotypical attributes of people with schizophrenia. METHOD: Students (n = 649) at eight universities in Australia, Belgium, India, Finland, Estonia and Latvia completed a seven-item Social Distance Scale (SDS) and six items related to stereotypical attributes of people with schizophrenia. RESULTS: Mean SDS scores were 19.65 (+/- 3.97) in Australia, 19.61 (+/- 2.92) in Belgium, 18.75 (+/- 3.57) in India, 18.05 (+/- 3.12) in Finland, and 20.90 (+/- 4.04) in Estonia and Latvia. Unpredictability was most strongly associated with having a high social distance in Australia (beta = -1.285), the perception that people will never recover in India (beta = - 0.881), dangerousness in Finland (beta = -1.473) and the perception of being difficult to talk to in Estonia and Latvia (beta = -2.076). Unpredictability was associated with lower social distance in Belgium (beta = 0.839). CONCLUSION: The extent to which students held stigmatizing attitudes was similar in each country, however, the determinants of stigma were different. Pharmacy education may need to be tailored to address the determinants of stigma in each country.

EPR investigation of in vivo inhibitory effect of guanidine compounds on nitric oxide production in rat tissues.

The aim of the present study was to evaluate in vivo effects on NO production of pharmacologically widely used, commercially available NOS inhibitors, structurally related to guanidine. We compared the NO inhibitory potency and selectivity of L-NAME, aminoguanidine and guanabenz in tissues of normal and LPS-stimulated rats using ex vivo EPR measurements of the NO radical in its complex with dithiocarbamate-Fe(II). The tissues studied were the brain cortex, kidney, liver, heart and testis. Differential inhibitory effects were seen for L-NAME, aminoguanidine and guanabenz when applied during basal or LPS-stimulated conditions. Aminoguanidine exerted inhibition of NO only after stimulation with LPS. Guanabenz had little effect on NO in liver, kidney, testis and heart under normal conditions, while it reduced the basal NO in brain cortex. After stimulation with LPS guanabenz afforded a partial inhibition of the NO formation in all tissues studied. L-NAME was a potent inhibitor of NO synthesis in all tested tissues, both during basal and LPS stimulated conditions. Our results show that compounds containing a guanidine moiety might possess different NOS inhibitory profiles in vivo.

MC(1) receptors are constitutively expressed on leucocyte subpopulations with antigen presenting and cytotoxic functions.

The expression of melanocortin MC(1) receptors on human peripheral lymphocyte subsets was analysed by flow cytometry using rabbit antibodies selective for the human MC(1) receptor and a panel of monoclonal antibodies against lymphocyte differentiation markers. The MC(1) receptor was found to be constitutively expressed on monocytes/macrophages, B-lymphocytes, natural killer (NK) cells and a subset of cytotoxic T-cells. Interestingly T-helper cells appeared to be essentially devoid of MC(1) receptors. The results were confirmed by RT-PCR which indicated strong expression of MC(1) receptor mRNA in CD14(+), CD19(+) and CD56(+) cells. However, only a faint RT-PCR signal was seen in CD3(+) cells, in line with the immuno-staining results that indicated that only part of the CD3(+) cells (i.e. some of the CD8(+) cells) expressed the MC(1) receptor. The MC(1) receptors' constitutive expression on immune cells with antigen-presenting and cytotoxic functions implies important roles for the melanocortic system in the modulation of immune responses.

Detection of regions in the MC1 receptor of importance for the selectivity of the MC1 receptor super-selective MS04/MS05 peptides.

We have investigated the ability of our earlier identified MS04-MS05 MSH-peptide analogues to bind to chimeric MC1-MC3 receptors. While the MS04 and MS05 peptides bind with nanomolar and sub-nanomolar affinities to the wild type MC1 receptor, they bind only with micromolar affinities for the wild type MC3 receptor, thus being the hitherto most MC1 receptor selective ligands. Upon exchanging portions involving transmembrane regions TM1, TM2-3, and TM6-7 of the MC1 receptor with corresponding portions of the MC3 receptor both of these peptides showed major losses of affinities. By contrast exchanges involving TM4-5 did not appreciably affect the affinity of either MS04 or MS05. Our data suggest that the binding pocket for the MS04-MS05 MSH-peptides is located between TM1-3 and TM6-7 of the melanocortin receptors.

PLS modeling of chimeric MS04/MSH-peptide and MC1/MC3-receptor interactions reveals a novel method for the analysis of ligand-receptor interactions.

A novel method has been developed for the analysis of ligand-receptor interactions. The method utilizes binding data generated from the analysis of chimeric proteins with chimeric peptides. To each chimeric part of the peptide and receptor are assigned descriptors, thus creating a matrix of X descriptors. These descriptors are then correlated with the experimentally determined interaction binding affinities for each chimeric receptor/peptide pair by use of partial least-squares projection to latent structures (PLS). The method was applied to analyze the interactions of chimeric MSH-peptides with wild-type MC1 and MC3 receptors, and MC1/MC3 receptor chimeras (in total 40 peptide-receptor combinations). Two types of PLS models could be created, one that revealed the relationships between receptor and peptide structure and peptide binding pK(i) values (i.e., affinity) (R2 and Q2 being 0.71 and 0.62, respectively), and another that revealed the relationships between peptide and receptor structure and peptide-receptor selectivity (R2 and Q2 being 0.64 and 0.57, respectively). After addition of cross-terms these models improved significantly; the R2 and Q2 being 0.93 and 0.75 for affinity, and 0.92 and 0.72 for selectivity, respectively. The analysis shows that the high affinity of the MSH-peptides is primarily achieved by interactions of the peptides' C-terminal amino acids with TM2 and TM3 of the receptor, and, to a lesser extent, by the interaction of the N-terminus with TM1, TM2 and TM3 of the receptor. However, in contrast, the MC1 receptor selectivity is primarily determined by an interaction of the peptides' N-termini with TM2/3 of the receptor. Moreover, the cross-terms of the PLS model revealed the existence of a strong interaction between TM6/7 and TM2/3 of the receptors.

Design of new small cyclic melanocortin receptor-binding peptides using molecular modelling: Role of the His residue in the melanocortin peptide core.

The conserved core of melanocyte stimulating hormones (MSH), His-Phe-Arg-Trp, was probed by comparing a cyclic pentapeptide containing His-DPhe-Arg-Trp, with three structurally similar cyclic peptides, that lacked the His residue. All three peptides bound to the MC(1), MC(3), MC(4) and MC(5) receptors with similar affinities. Molecular modelling indicated that the 3D structure of the DPhe-Arg-Trp of all three peptides were closely similar. The data indicate that the His residue of the small rigid cyclic MSH core peptides does not participate in binding with the melanocortin receptors.

Effects of melanocortin peptides on lipopolysaccharide/interferon-gamma-induced NF-kappaB DNA binding and nitric oxide production in macrophage-like RAW 264.7 cells: evidence for dual mechanisms of action.

The pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) mediates broad anti-inflammatory and immunomodulatory effects, which include inhibition of the production and release of proinflammatory cytokines and nitric oxide (NO) from macrophages. We investigated the effects of alpha-MSH, alpha-MSH(1-10), and alpha-MSH(11-13) on NO production and nuclear factor-kappaB (NF-kappaB) translocation in RAW 264.7 macrophages. After stimulation of the cells with bacterial lipopolysaccharide/interferon-gamma (LPS/IFN-gamma), all three peptides inhibited NO production with an order of potency alpha-MSH > or = alpha-MSH(11-13) > alpha-MSH(1-10). All three MSH peptides inhibited NF-kappaB nuclear translocation with the maximal effect of alpha-MSH and alpha-MSH(11-13) being seen in the range 1 nM-1 microM, and that of alpha-MSH(1-10) at 1 microM. By use of (125)I-(Nle(4),D-Phe(7))alpha-MSH(NDP-MSH) radioligand binding, MC(1) receptor-binding sites were demonstrated on RAW 264.7 cells. alpha-MSH and alpha-MSH(1-10) competed with the (125)I-NDP-MSH binding at these MC(1) receptor-binding sites, but alpha-MSH(11-13) even in concentrations up to 1 mM did not. Moreover, alpha-MSH and alpha-MSH(1-10) caused powerful stimulation of cyclic 3',5'-adenosine monophosphate (cAMP) in the RAW 264.7 cell, whereas alpha-MSH(11-13) was ineffective. Forskolin stimulated cAMP and inhibited NO production to the same extent as alpha-MSH and alpha-MSH(1-10), but did not modify the translocation of NF-kappaB. Whereas the protein kinase A inhibitor H89 did not modify the effect of alpha-MSH on NF-kappaB translocation, H89 caused a partial inhibition of the inhibitory effect of alpha-MSH, alpha-MSH(1-10), alpha-MSH(11-13), and forskolin on NO production. In addition alpha-MSH, alpha-MSH(1-10), alpha-MSH(11-13), and forskolin also inhibited the activity of an NF-kappaB-dependent luciferase reporter and these effects were partially counteracted by H89. We suggest that melanocortin peptides act via dual mechanisms of action: one cAMP-independent and causing inhibition of NF-kappaB translocation and the other dependent on MC(1) receptor/cAMP activation.

New aspects on the melanocortins and their receptors.

Knowledge of melanocortins and their receptors has increased tremendously over the last few years. The cloning of five melanocortin receptors, and the discovery of two endogenous antagonists for these receptors, agouti and agouti-related peptide, have sparked intense interest in the field. Here we give a comprehensive review of the pharmacology, physiology and molecular biology of the melanocortins and their receptors. In particular, we review the roles of the melanocortins in the immune system, behaviour, feeding, the cardiovascular system and melanoma. Moreover, evidence is discussed suggesting that while many of the actions of the melanocortins are mediated via melanocortin receptors, some appear to be mediated via mechanisms distinct from melanocortin receptors.

New highly specific agonistic peptides for human melanocortin MC(1) receptor.

A peptide with very high specificity for the human melanocortin MC(1) receptor identified by phage display was used as a lead for the design of new peptides. Two new peptides, MS05 and MS09, were synthesized and found to bind with sub-nanomolar affinities to the MC(1) receptor. Both these peptides showed strong agonistic activity at the MC(1) receptor. The MS05 was the most MC(1) receptor selective as it showed virtually no binding affinity for the MC(4) and MC(5) receptors and only micromolar affinity for the MC(3) receptor. The selectivity and potency of the new peptides make them potent tools for studies of MC(1) receptors, as well as novel potential candidate drugs for the treatment of inflammatory conditions.

Behavioural responses of gamma-MSH peptides administered into the rat ventral tegmental area.

The behavioural effects induced by alpha-, gamma1- and gamma2-MSH peptides (0.3 and 3 nmole per rat) injected into the left ventral tegmental area (VTA) of rats were compared. alpha- and gamma1-MSH caused grooming of comparable magnitude, and also additional vertical activity (rearing). By contrast gamma2-MSH caused a moderate but stable catalepsy, and practically no grooming. Moreover, intra-VTA pre-treatment with gamma2-MSH, 15 min prior to intra-VTA gamma1-MSH, markedly attenuated both the gamma1-induced grooming and vertical activities. The differences in the behavioural response of the MSH peptides indicate that they act differentially on MC receptors in the VTA.

Thyrotropin releasing hormone (TRH) selectively binds and activates the melanocortin 1 receptor.

We tested the endogenous tripeptide TRH (thyrotropin releasing hormone) ability to bind to MC (melanocortin) receptor subtypes. We discovered that TRH binds to the human MCI receptor expressed in COS cells and to murine B16 melanoma cells with 5790+/-1010 nM and 6370+/-1260 nM Ki's, respectively. TRH did not bind to the human MC3, MC4 or MC5 receptor subtypes. Moreover, TRH also stimulated cAMP production in murine B16 melanoma cells reaching the same maximum level of cAMP as found for alpha-MSH. However, several analogues of TRH, including TRH-OH, TRH-Gly-NH2 and other analogues, where each of the three amino acid residues in TRH had been exchanged by a related residue, did not bind to any of the MC receptors tested in this study. C(alpha) atoms of molecular models of TRH and the core of a MSH peptide were aligned with r.m.s. of 0.01 A. Moreover, TRH could be docked into a binding pocket of a molecular model of the MC1 receptor at only a little higher energy than a short cyclic MSH peptide. The data indicate a similarity in the mode of TRH and MSH activation of the MCI receptor.

Long term orexigenic effect of a novel melanocortin 4 receptor selective antagonist.

1. We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors. 2. One of the substances HS028 (cyclic [AcCys11, dichloro-D-phenylalanine14, Cys18, Asp-NH2(22)]-beta-MSH11-22) showed high affinity (Ki of 0.95nM) and high (80 fold) MC4 receptor selectivity over the MC3 receptor. HS028 thus shows both higher affinity and higher selectivity for the MC4 receptor compared to the earlier first described MC4 receptor selective substance HS014. 3. HS028 antagonised a alpha-MSH induced increase in cyclic AMP production in transfected cells expressing the MC3 and MC4 receptors, whereas it seemed to be a partial agonist for the MC1 and MC5 receptors. 4. Chronic intracerebroventricularly (i.c.v.) administration of HS028 by osmotic minipumps significantly increased both food intake and body weight in a dose dependent manner without tachyphylaxis for a period of 7 days. 5. This is the first report demonstrating that an MC4 receptor antagonist can increase food intake and body weight during chronic administration providing further evidence that the MC4 receptor is an important mediator of long term weight homeostasis.

Further pharmacological characterization of the selective melanocortin 4 receptor antagonist HS014: comparison with SHU9119.

SHU9119 and HS014 are cyclic MSH analogues which are widely used to elucidate the physiology behind the various effects of the MSH peptides and their receptors. We carefully compared the potency of SHU9119 and HS014 in cells expressing the MC receptor clones. We found that both the peptides are partial agonists for the MC1 and MC5 receptors while they are potent antagonists for the MC3 and MC4 receptors. In agreement with earlier binding data, we found that SHU9119 has equal potency for the MC3 and MC4 receptor whereas HS014 has at least 10-fold higher potency for the MC4 receptor than the MC3 receptor in cAMP assay. Moreover, we synthesized analogues of HS014 where the C-terminal was truncated. We found that this C-terminal fragment of HS014, in particular the Lys(14), has a major influence on the affinity for the MC4 receptor without any particular influence on the affinity for the other MC receptors.

Discovery of a novel superpotent and selective melanocortin-4 receptor antagonist (HS024): evaluation in vitro and in vivo.

Several novel cyclic MSH analogs were synthesized, and their binding properties were tested on cells transiently expressing the human melanocortin-1 (MC1), MC3, MC4, and MC5 receptors. We discovered a novel substance (HS024) that showed about 20-fold selectivity and very high affinity (Ki = 0.29 nM) for the MC4 receptor. HS024 (cyclic [AcCys3,Nle4,Arg5,D-Nal7,Cys-NH2(11)]alpha-MSH-(3-11)) has a 29-membered atom ring structure that includes an Arg in position 5. HS024 was found to antagonize an alphaMSH-induced cAMP response in cells expressing the human MC1, MC3, MC4, and MC5 receptor DNAs. HS024 also caused a dose-dependent increase in food intake, with a maximum response (4-fold increase) at a 1-nmol dose injected intracerebroventricularly in free feeding rats. We also tested SHU9119, a previously described nonselective MC receptor antagonist, and found HS024 and SHU9119 to have similar potencies for increasing food intake, although SHU9119 appeared to induce more serious side-effects. HS024 increased the food intake of free feeding rats to levels comparable to those in food-deprived rats, indicating that blockade of the MC4 receptor is a highly effective way to increase feeding. Moreover, we tested the effects of intracerebroventricular injections of HS024 in elevated plus-maze and open-field experiments on rats. In these tests, HS024 did not appear to affect emotionality or locomotor activity, suggesting that the MC4 receptor does not mediate the anxiogenic-like and locomotor effects related to the melanocortic peptides.

Evidence indicating that the extracellular loops of the mouse MC5 receptor do not participate in ligand binding.

The mMC5 receptor was cloned from a genomic library, mutated in the extracellular loops (EL's), expressed and tested for binding to melanocyte stimulating hormone (MSH) peptides. The EL's show low amino acid homology within the MC receptor family. Two mutants of the mMC5 receptor were created in order to investigate the participation of these regions in ligand binding. The EL1 and EL3 were separately altered by multiple mutagenesis so that their amino acid sequences became identical with the hMC1 receptor. The mutants were expressed in COS cells and found to bind peptide ligands in the same fashion as the wild type mMC5 receptor clone. The results indicate that the amino acids that were mutated in the mMC5 receptor do not participate in binding of MSH peptides. Comparison of the wild type mMC5 receptor with the hMC5 receptor showed that it has the same potency order for the MSH peptides but considerably higher affinity than the hMC5 receptor.

Chimeric melanocortin MC1 and MC3 receptors: identification of domains participating in binding of melanocyte-stimulating hormone peptides.

The melanocortin receptors MC1 and MC3 are G protein-coupled receptors that have substantial structural similarities and bind melanocyte peptides but with different affinity profiles. We constructed a series of chimeric MC1/MC3 receptors to identify the epitopes that determine their selectivities for natural melanocyte peptides and synthetic analogues. The chimeric constructs were made by a polymerase chain reaction that used identical regions in or just outside transmembranes (TM) 1, 4, and 6 and divided the receptors into four segments. Saturation and competition studies on the expressed chimeric proteins indicate that TM1, TM2, TM3, and TM7 are involved in the subtype-specific binding of melanocyte peptides to these receptors. The results support the hypothesis that TM4 and TM5 may not contribute to the ligand-binding specificity of the MC receptors. This is the first report to describe the subtype-specific hormone-binding domains of the melanocortin receptor family.

Selective properties of C- and N-terminals and core residues of the melanocyte-stimulating hormone on binding to the human melanocortin receptor subtypes.

We synthesised nine analogues of [Nle4,D-Phe7]alpha-MSH (melanocyte-stimulating hormone) (NDP) where (1) the N- or C-terminals were deleted or exchanged by those of beta- or gamma-MSH and (2) the core residues His6, Phe7, Arg8 and Trp9 were individually substituted by Glu6, beta-(2-naphthyl)-D-alanine (D-Nal7), Lys8 and His9, respectively. We tested these analogues in ligand binding assays with cells transiently expressing the human melanocortin MC1, MC3, MC4 and MC5 receptors. The results show that the N-terminal segment (Ser1-Tyr2-Ser3) of NDP was not important for binding to melanocortin MC1 and MC4 receptors whereas it affects binding to melanocortin MC3 and MC5 receptors. The C-terminal segment (Gly10-Lys11-Pro12-Val13) of NDP was clearly important for binding to all the four melanocortin receptor subtypes. The data indicate that the low affinity of gamma-MSH for the melanocortin MC4 receptor is due to its C-terminal (Asp10)-Arg11-Phe12). Substitution of D-Phe7 by D-Nal7 increased the affinity for the melanocortin MC4 receptor but not for the other melanocortin receptor subtypes. The other core residue substitutions lowered the affinity in a differentiated manner for each of the melanocortin receptors. These results are valuable for the molecular modelling and design of selective drugs for the melanocortin receptors.

Discovery of novel melanocortin4 receptor selective MSH analogues.

1. We synthesized a novel series of cyclic melanocyte stimulating hormone (MSH) analogues and tested their binding properties on cells transiently expressing the human melanocortin1 (MC1), MC3, MC4 and MC5 receptors. 2. We discovered that compounds with 26 membered rings of [Cys4,D-Nal7,Cys11]alpha-MSH(4-11) displayed specific MC4 receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys4D-Nal7Cys11]alpha-MSH(4-11) analogues are distinct from all other known MSH analogues, particularly as they bind the MC4 receptor with high and the MC1 receptor with low relative affinities. 3. HS964 and HS014 have 12 and 17 fold MC4/MC3 receptor selectivity, respectively, which is much higher than for the previously described cyclic lactam and [Cys4,Cys10]alpha-MSH analogues SHU9119 and HS9510. 4. HS964 is the first substance showing higher affinity for the MC5 receptor than the MC1 receptor. 5. HS014, which was the most potent and selective MC4 receptor ligand (Ki 3.2 nM, which is approximately 300 fold higher affinity than for alpha-MSH), was also demonstrated to antagonize alpha-MSH stimulation of cyclic AMP in MC4 receptor transfected cells. 6. We found that a compound with a 29 membered ring of [Cys3,Nle10,D-Nal7,Cys11]alpha-MSH(3-11) (HS010) had the highest affinity for the MC3 receptor. 7. This is the first study to describe ligands that are truly MC4 selective and a ligand having a high affinity for the MC3 receptor. The novel compounds may be of use in clarifying the physiological roles of the MC3, MC4 and MC5 receptors.

Evaluation of behavioural effects of neural melanocortin receptor antagonists injected ICV and in VTA in rats.

The natural melanocortic peptides are known to exert a variety of effects after central administration. Recently, we discovered the first potent and selective substances for the MC4 receptor, i.e. HS964 and HS014. We found HS964 to be an antagonist for the MC1, MC3, MC4 and MC5 receptors in vitro. HS014 is an antagonist for the MC3 and MC4 receptors and a partial antagonist for the MC1 and MC5 receptors. We injected alpha-MSH and these substances, both intracerebroventricular (ICV) and in the ventral tegmental area (VTA) in rats and scored several behavioural effects. The results show that alpha-MSH caused intensive grooming which was antagonized by pre-treatment of both HS014 and HS964. The data give further support to the hypothesis that it is the MC4 receptor which mediates grooming in rodents. The grooming effects of alpha-MSH were more pronounced after intra-VTA administration compared to the ICV administration. Both alpha-MSH, HS014 and HS964 caused an increase in vertical activity of the rats after intra-VTA administration but not after ICV administration. Horizontal activity was virtually not affected by the administration of the peptides. The data indicate that the neural MC3 and MC4 receptors are not likely to be an important mediators of locomotor activity in rats.