CN1721431A - Novel alpha-melanocyte stimulating hormone analogue and use thereof - Google Patents
Novel alpha-melanocyte stimulating hormone analogue and use thereof Download PDFInfo
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- CN1721431A CN1721431A CN 200510026927 CN200510026927A CN1721431A CN 1721431 A CN1721431 A CN 1721431A CN 200510026927 CN200510026927 CN 200510026927 CN 200510026927 A CN200510026927 A CN 200510026927A CN 1721431 A CN1721431 A CN 1721431A
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Abstract
The present invention relates to medicine technology, and is especially one new type of alpha-melanophore stimulin analog in the chemical structural expression of Ac-Ser-Ser-Ile-Nle-Gln-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2. Extracorporeal cell experiment and animal experiment proves its excellent antiphlogistic effect, so that the alpha-melanophore stimulin analog may be used in preparing antiphlogistic medicine.
Description
Technical field
The present invention relates to medical technical field, be the analogue of alpha-melanocyte stimulating hormone and use it for the preparation antiphlogiston purposes.
Background technology
(α-Melanocyte-stimulating hormone, α-MSH) derive from proopiomelanocortin (POMC) are a kind of straight chain neuroendocrine immunomodulatory peptides that is made of 13 amino acid to alpha-melanocyte stimulating hormone.Its chemical structural formula is Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-N H
2Alpha-melanocyte stimulating hormone plays a role by casting skin matter hormone receptor (MC1-R, MC3-R, MC4-R, MC5-R).Wherein the main transmitting inflammation of MC1-R and MC5-R suppresses and immunoregulation effect, and MC3-R and MC4-R mainly participate in diet, body weight and energy metabolism equilibrated and regulate.Aspect anti-inflammatory, α-MSH not only has downward modulation effect (Rajora N to the generation and the effect of multiple inflammatory mediator, Boccoli G, Burns D, et al.Alpha-MSH modulates local and circulating tumor necrosis factor-alpha inexperimental brain inflammation.J Neurosci, 1997; 17 (6): 2181-2186), and can raise generation (the Bhardwaj RS of anti-inflammatory factors such as IL-10, Schwartz A, Becher E, et al.Pro-opiomelanocortin-derived peptides induce IL-10production in human monocytes.J.Immunol, 1996; 156:2517-2521).External and our research in the past all shows; α-MSH is to experimental lethality endotoxemia, adult respiratory distress syndrome (acute respiratory distress syndrome; ARDS) significant provide protection is arranged; can obviously reduce the inflammatory reaction of internal organs such as lung, the heart, liver, kidney, brain, reduce mortality ratio.In endotoxin shock patient body, serum α-MSH density loss, and convalescent patient α-MSH concentration rises.So far find no cumulative toxicity, with glucocorticosteroid its obvious superiority (Catania A is arranged relatively, Cutuli M, Garofalo L, et al.Plasma concentrations and anti-L-cytokineeffects of alpha-melanocyte stimulating hormone in septic patients.Crit Care Med.2000; 28 (5): 1403-1407).α-MSH can be easily enters body through approach such as vein, subcutaneous, abdominal cavity, suctions, and clinical use is convenient in consumption accurately control easily.But regulate polypeptide as small molecules in the body, also there is shortcoming in α-MSH itself, and it has higher selectivity to MC1-R and MC3-R, and is very low to the avidity of MC4-R and MC5-R.[Nle
4, D-Phe
7] α-MSH (NDP-MSH) is one of ideal alpha-MSH analogue comparatively, have advantages such as efficient, that acting duration is long, the proteolytic ferment resistivity is strong, stability is high, solubility is good, be used for the I clinical trial phase, but its selectivity to acceptor is not high, and tangible pigmentation is arranged.
Summary of the invention
The invention provides a kind ofly has highly selective, alpha-melanocyte stimulating hormone analogue that antiphlogistic effects is good to MC1-R and MC5-R.The present invention carries out the 3D molecular simulation under the support of InsightII software, α-MSH structure is carried out following modification: 2Ser replace 2Tyr, and 3Ile replaces 3Ser, and 4Nle replaces 4Met, and 5Gln replaces 5Glu, and 7DPhe replaces 7Phe.Sequence is Ac-Ser-Ser-Ile-Nle-Gln-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH
2Test in external body, prove its antiphlogistic effects apparently higher than α-MSH, on many evaluation indexes (pathological manifestations of IL-1 β, liver and lung), effect is better than NDP-MSH.Therefore can be used for preparing anti-inflammatory drug.
Description of drawings
The secretion level graphic representation of Fig. 1 THP-1 cell TNF-α after different treatment
The time-survivor curve figure of endotoxin shock mouse after Fig. 2 different treatment
TNF-alpha levels histogram in the endotoxin shock mice serum after Fig. 3 different treatment
Fig. 4 not homopolypeptide handles the horizontal histogram of IL-1 β in the endotoxin shock mice serum of back
Fig. 5 not homopolypeptide handles the horizontal histogram of NO in the endotoxin shock mice serum of back
Embodiment
Now in conjunction with the embodiments, the present invention is described in detail.
The preparation of novel alpha-melanocyte stimulating hormone analogue of the present invention entrusts the biochemical company limited of gill to finish synthetic.
The experiment of embodiment 1 cell in vitro anti-inflammatory
Recovery person monocytic cell strain THP-1 cell (available from ATCC) is cultivated in 37 ℃ with RPMI1640 complete culture solution (including 10% foetal calf serum).Inoculation 5 * 10
5Cells/well adds 100nM PMA (stroking ripple fat) to 24 hole tissue culturing plates, changes liquid after 3 days.Add 10ng/ml LPS (lipopolysaccharides) stimulation and add 50nM α-MSH (available from Sigma company), NDP-MSH (available from Sigma company), novel alpha-melanocyte stimulating hormone analogue simultaneously to corresponding hole, each sample is established 3 multiple holes.Collect supernatant after 12,24,36,48 hours, measure TNF-α.The results are shown in Figure 1, as seen from Figure 1, after LPS stimulated, the TNF-α secretory volume of THP-1 cell obviously increased, and reaches more than the 3000pg/ml.α-MSH, NDP-MSH suppress the secretion of TNF-effectively.α-MSH can reduce TNF-alpha levels 30%, and novel alpha-melanocyte stimulating hormone analogue can reduce TNF-alpha levels 40-50% (P＜0.01), and wherein the effect of novel alpha-melanocyte stimulating hormone analogue and NDP-MSH does not have significant difference (P＞0.05).
The mensuration of embodiment 2 avidity
Before the experiment 48h with the HEK-293 cell of stably express hMCRs (human melanocortin receptor) with 5 * 10
4/ hole density is cultivated in 24 orifice plates, when growing to the 90-95% individual layer, removes substratum, with lavation buffer solution (50mM Tris/HCl, 100mM NaCl, 50mM KCl, 2mM CaCl
2, pH7.2) washing is 2 times.If control group is selected the HEK-293 cell of not expressing MCRs for use, in every hole, add binding buffer liquid 0.25m1 (50mM Tris/HCl, 100mM NaCl, 50mM KCl, 2mM CaCl
2,, 5%Hanks ' balanced salt solution, 0.5%BSA, pH7.2).Add final concentration and be 40pmol/L [
125I]-NDP-MSH, the unmarked part of adding different concns in every hole, each concentration is done 3 hole replicate(determination)s.In non-specific group, add 10
-5Mol/L does not add unmarked part in summary is set up jointly, put 37 ℃ and hatch 2h, places 15min for 0 ℃, with the binding buffer liquid washing of 10 ℃ of 0.5ml 2 times, with 0.5ml 0.05M NaOH from flat board peptic cell to final volume 1ml.γ-counter is surveyed its radioactivity.Data are imported computer, adopt GraphPad Prism to analyze IC
50Value.According to formula K
i=IC
50/ { 1+ ([L]/K
D) calculating K
iValue, data mean ± standard error (X ± SE) expression.The results are shown in Table 1, is MC1-R＞MC5-R＞MC4-R＞MC3-R by the visible novel alpha-melanocyte stimulating hormone analogue of the present invention of table 1 to the selectivity order of acceptor, and MC1-R and MC5-R are had higher selectivity.Especially MC5-R being shown stronger avidity, is 4000 times of α-MSH, 4 times of NDP-MSH.
The K of table 1 α-MSH and analogue thereof
iValue (mol/L)
| Part | ?MC1-R | ?MC3-R | ?MC4-R | ?MC5-R |
| α-MSH | ?0.255±0.067 | ?38.756±4.780 | ?1080±130 | ?8270±740 |
| NDP-MSH | ?0.096±0.008 | ?0.578±0.023 | ?5.760±0.680 | ?8.347±0.720 |
| Novel alpha-melanocyte stimulating hormone analogue | ?0.159±0.040 | ?35.430±6.743 | ?19.293±2.780 | ?2.230±0.670 |
Embodiment 3 novel alpha-melanocyte stimulating hormone analogues are to the provide protection of endotoxin shock mouse
According to the method for Galanos, with PBS damping fluid dissolving LPS and D-Gal (D-galactose glycosides), every mouse peritoneal is injected 1 μ g LPS and 20mg D-Gal, sets up the endotoxin shock mouse model.BALB/c mouse is divided into 5 groups (A～E group), 10 every group at random.The A group: the PBS abdominal injection is organized in contrast; B group: 1 μ g LPS and 20mg D-Gal abdominal injection; C group: abdominal injection α-MSH in the 30min behind 1 μ g LPS and the 20mg D-Gal abdominal injection, 2.5mg/kg.D group: abdominal injection NDP-MSH in the 30min behind 1 μ g LPS and the 20mg D-Gal abdominal injection, 2.5mg/kg.E group: press 2.5mg/kg abdominal injection novel alpha-melanocyte stimulating hormone analogue behind 1 μ g LPS and the 20mg D-Gal abdominal injection in the 30min.Take mouse orbit blood in time to peak, collect supernatant and measure TNF-α, IL-1 β, NO respectively.Observe the existence situation, do the conventional organization pathologic finding.The result shows: novel alpha-melanocyte stimulating hormone analogue of the present invention as shown in Figure 2 prolongs the endotoxin shock mouse survival time, and median survival time is 32h, prolongs 8h than α-MSH.According to statistical test, C group, D group, E group have been compared notable difference (P＜0.01) with the survival rate of B group.No significant difference (P＞0.05) between C group, D group and the E group.Give as shown in Figure 3 behind the LPS that the TNF-alpha levels increases to more than the 2000pg/ml in the mice serum, the TNF-alpha levels of each treatment group (C group～E group) all reduces (P＜0.01).Wherein the mouse TNF-alpha levels of handling through NDP-MSH supports most, C group and E group no significant difference (P＞0.05).As shown in Figure 4, IL-1 β level rising in the serum behind the abdominal injection LPS, the IL-1 β level minimum (P＜0.01) of E group mouse, D group and E group variant (P＜0.05).Serum NO raises significantly to more than the 50 μ Mol/L behind the abdominal injection LPS as shown in Figure 5, and each treatment group (C group～E group) all can reduce NO level (P＜0.01).The E group can reduce NO 69%, to 20 μ mol/L.Three groups of effects of statistical study are (P＞0.05) quite.As seen pathological study gives mouse cardiac muscle fibre array disorder behind the LPS 6h, a matter blood vessel and telangiectasis hyperemia; Liver cell muddiness, hydropic degeneration, spotty necrosis, sinus hepaticus and central vein extravasated blood; Pulmonary interstitial edema and massive inflammatory cells infiltrated; The snius lienis dilatation and congestion, splenic cords broadening, the hyperplasia of splenic cords endolymph is obvious; The pathological change of kidney and brain is not obvious.Give behind the LPS 30min abdominal injection α-MSH, NDP-MSH, novel alpha-melanocyte stimulating hormone analogue respectively, the pathological lesion of each main organs with only give the LPS group relatively, 6h does not still have obvious improvement, improves gradually behind the 24h.The 48h light microscopic shows down, C group cardiac muscle fibre marshalling, and a matter blood vessel still has hyperemia, and a matter congestion of blood vessel subtracts; The liver lobule structure exists, vacuolar degeneration of hepatic cell, sinus hepaticus extravasated blood; Snius lienis hyperemia alleviates, and splenic cords endolymph cell still has hyperplasia; The alveolus wall structure is normal, alveolus wall and between the matter blood vessel slightly expand, hyperemia.The pathological lesion of D group improves than the C group, and matter does not have obvious hyperemia between cardiac muscle; Liver cell is vacuolar degeneration still, and sinus hepaticus extravasated blood alleviates; Snius lienis hyperemia alleviates; The interstitial lung inflammatory cell infiltration alleviates, and blood vessel does not have obvious dilatation and congestion.E group cardiac muscle fibre is arranged more neat, and a matter congestion of blood vessel alleviates; The liver lobule structural integrity, liver cell does not have sex change, and sinus hepaticus does not have obvious extravasated blood, and is normal substantially; Snius lienis does not have obvious hyperemia; The alveolus wall structure is normal.
In sum, novel alpha-melanocyte stimulating hormone analogue of the present invention is a kind of agonist that MC1-R and MC5-R is had highly selective, by reinforcement the avidity of MC5R and the biological effect of MC5R mediation are worked in coordination with the performance anti-inflammatory action, and the selectivity of MC1-R is not so good as NDP-MSH.When therefore giving for a long time as if chronic inflammatory diseases, novel alpha-melanocyte stimulating hormone analogue of the present invention then has its superiority than the pigmentation of NDP-MSH.Novel alpha-melanocyte stimulating hormone analogue also has better therapeutic effect and application prospect to inflammation such as organ-graft refection, the autoimmune disorder etc. of immunne response mediation.Therefore, novel alpha-melanocyte stimulating hormone analogue of the present invention can be used for preparing anti-inflammatory drug.
Claims (2)
1. novel alpha-melanocyte stimulating hormone analogue, its chemical structure is as follows:
Ac-Ser-Ser-Ile-Nle-Gln-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH
2。
2. the application of the described alpha-melanocyte stimulating hormone analogue of claim 1 in the preparation anti-inflammatory drug.
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