WO2013127196A1 - 环状核心序列与生物素或穿膜肽相连而成的促黑激素类似物 - Google Patents
环状核心序列与生物素或穿膜肽相连而成的促黑激素类似物 Download PDFInfo
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- WO2013127196A1 WO2013127196A1 PCT/CN2012/084555 CN2012084555W WO2013127196A1 WO 2013127196 A1 WO2013127196 A1 WO 2013127196A1 CN 2012084555 W CN2012084555 W CN 2012084555W WO 2013127196 A1 WO2013127196 A1 WO 2013127196A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- C07K14/68—Melanocyte-stimulating hormone [MSH]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- A61K38/34—Melanocyte stimulating hormone [MSH], e.g. alpha- or beta-melanotropin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
Definitions
- the present invention relates to a melatonin analog which is a cyclic core sequence linked to biotin or a transmembrane peptide, and a preparation method and application thereof.
- the first-line drug for treating male sexual dysfunction is a phosphodiesterase inhibitor such as sildenafil, but such drugs are ineffective for female patients, and some patients report a gradual disappearance of response.
- a phosphodiesterase inhibitor such as sildenafil
- drugs for the treatment of obesity such as sibutramine and orlistat.
- peptide drugs for the treatment of hypopigmentation PT-141 and ⁇ - ⁇ are two kinds of peptide drugs currently under development for men and women with sexual dysfunction, obesity, and hypopigmentation.
- a-MSH is a linear tripeptide derived from pro-opiomelanocortin (POMC).
- POMC pro-opiomelanocortin
- ⁇ -MSH is a linear tridecapeptide with an amide structure at the C-terminus and an N-terminal acetylation. Its primary structure is as follows: Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp- Gly-Lys-Pro-Val-NH
- CPP Cellular penetrating peptide
- HIV-1 transactivator can translocate into the cell membrane and nucleus across the membrane. It has also been found that one of the HIV-1 prions is rich in basic amino acid fragments, which are closely related to protein conduction function and are called protein transduction domains. Further studies revealed that the sequence consisting of residues 49-57 of the ⁇ protein of the full length of 86 amino acid residues fully functions as a protein and is cytotoxic. These findings provide a promising future for the development of an efficient and safe vector that actively mediates the passage of polypeptide macromolecules across biofilm barriers.
- Biotin also known as vitamin ⁇ , is one of the essential vitamins in the human body. It has a promoting effect on growth and has a certain effect on white hair and baldness. White hair is one of the symptoms of hyperpigmentation. It has a strong therapeutic effect on melatonin. It combines the core sequence of melatonin with biotin creatively, which can promote the blackening of hair while promoting it. Growth, which is an innovation of the invention in the field of medicine or cosmetics.
- a melatonin-promoting analog having a polypeptide structure including all enantiomers, diastereomers, stereoisomers, etc.) as follows: XZ-Arg-c (Asp-Dab-D-Phe-Arg-Trp- Lys)-Y and its non-physiologically toxic salts, wherein
- a physiologically toxic salt is a salt which retains the expected physiological activity of the parent compound without causing any unintended toxic side effects, and may specifically be a hydrochloride, a hydrobromide, a hydroiodide, a sulfate, a phosphate, Nitrate, acetate, trifluoroacetate, oxalate, tartrate, succinate, malate, benzoate, alginate, methanesulfonate, naphthalenesulfonate, potassium salt, A sodium salt, a lithium salt, a zinc salt, a copper salt, a barium salt or a calcium salt.
- the melatonin analog has a polypeptide structure of the following six kinds, and the therapeutic effect is particularly good:
- Biotin-Arg-c (Asp-Dab-D-Phe-Arg-Trp-Lys)-NH 2 and its non-physiologically toxic salts,
- Biotin-Arg-c (Asp-Dab-D-Phe-Arg-Trp-Lys)-OH and its non-physiological salts,
- the non-physiologically toxic salt is preferably an acetate.
- the amino acid residues in the above melatonin-like analogs are (L) except for D-Phe.
- the method for preparing the melatonin-promoting analog uses a solid phase synthesis method, and specifically includes the following steps: First, preparing a functional unit of a non-side chain protecting group that is not cleaved with a solid phase resin on a solid phase resin; Amino acid sequence, then biotinylating the N-terminus of the functional unit amino acid sequence on a solid phase resin, or continuing to sequentially sequence the amino acid residues in the loading unit amino acid sequence from the C-terminus to the N-terminus, respectively The terminal is ligated, purified, and dried to form a peptide after removing the N-terminal protecting group of the last amino acid residue; the N-terminal biotinylated solid phase resin containing the functional unit amino acid sequence is also subjected to cleavage, purification, and drying. a step of forming a peptide; wherein, the functional unit amino acid sequence is: Arg-c (Asp-Dab-D-Phe-Arg-T
- the loading unit amino acid sequence is: Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg or Arg-Arg-Arg.
- the administration mode of the melatonin analog is preferably mucosal administration, dermal administration or injection administration.
- the drug contains at least one of the polypeptide components having the following six structures and a pharmaceutically acceptable carrier or excipient -
- Biotin-Arg-c (Asp-Dab-D-Phe-Arg-Trp-Lys)-NH 2 ,
- the pharmaceutically acceptable carrier or excipient may be physiological saline containing a phosphate buffer.
- the MC-Rs refer only to the MC-1 receptor, the MC-3 receptor, the MC-4 receptor or the MC-5 receptor.
- the 47th to 60th residues of the HIV-1 TAT protein are composed of fourteen amino acid residues, and the primary structure is:
- the present invention binds a polypeptide sequence of a melatonin analog having MC-Rs agonistic activity to a different polypeptide fragment consisting of different residues in the 47th to 60th residues of biotin or HIV-1 TAT protein, A melatonin analog that can be inhaled through the mucosa or skin is designed.
- the melatonin analog has the activity of an MC-Rs agonist and can be used for the treatment of sexual dysfunction, obesity or hyperpigmentation in men and women.
- the polypeptide provided by the present invention is composed of a functional unit and two parts of biotin or a load unit, and the functional unit and the biotin or the load unit are linked by an amide bond.
- c Asp-Dab-D-Phe-Arg-Trp-Lys
- Dab-D-Phe-Arg-Trp is its core sequence.
- the N-terminal Arg is a linking amino acid.
- This functional unit consists of a heptapeptide. Structurally, the side chains are connected in a ring shape, and the fat solubility is increased, which is more favorable for transmucosal and skin, and the sequence is similar to but different from other melatonin receptor agonists.
- polypeptide 1 The functional unit N-terminus and biotin are linked to form two polypeptide structures, namely, polypeptide 1 and polypeptide 2, and the structural formula is as follows:
- Biotin-Arg-c (Asp-Dab-D-Phe-Arg-Trp-Lys)-NH 2 (multi-month too 1);
- Biotin-Arg-c (Asp-Dab-D-Phe-Arg-Trp-Lys)-OH (polypeptide 2).
- the load unit consists of different amino acid fragments in fragments 47 to 60 of the HIV-1 TAT protein.
- the load unit 1 consists of the 55th to 57th amino acid fragments of the HIV-1 TAT protein and contains three amino acid residues, and its structure is: Arg-Arg-Arg-OH.
- the load unit 2 consists of the 48th to 57th amino acid fragments of the HIV-1 TAT protein, and contains ten amino acid residues, and its structure is: Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg- Arg-OH.
- the functional unit N-terminus is linked to the load units 1, 2, respectively, to form four polypeptide structures, namely polypeptides 3, 4, 5 and 6, the structural formula is as follows: Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH 2 (poly) 3 too 3 );
- Biotin is biotinyl
- c is a cyclic peptide
- Dab is 2,4 diaminobutyric acid
- Gly is glycine
- Arg is arginine
- Lys is lysine
- Gin is glutamate
- D-Phe is dextrophenylalanine
- Trp is tryptophan
- TFA is trifluoroacetic acid
- HBTU is benzotriazole-1-tetramethylhexafluorophosphate
- NMM is N-methylmorpholine.
- the present invention tests after different load units and functional units are linked by peptide bonds.
- the experimental results show that the peptide formed by the 10-peptide loading unit and the functional unit is the most easy to penetrate different mucous membranes (including oral mucosa, gastric mucosa, nasal mucosa); the peptide formed by the tripeptide loading unit and the functional unit is the easiest to wear. Through the skin.
- the load unit provided by the present invention can carry the functional unit provided by the present invention through the mucosal and capillary walls and penetrate the blood-brain barrier into the cerebral cortex.
- the polypeptide formed by the binding unit or biotin provided by the present invention and the functional unit provided by the present invention can be easily absorbed through the digestive tract mucosa and the respiratory mucosa at a relatively small dose, thereby allowing oral and mucosal administration. It is possible that this is difficult for other peptide drugs. Also, since the load unit of the present invention can carry a functional unit through the skin, it is possible to subject the polypeptide to subcutaneous administration.
- the present invention respectively provides a tripeptide vector (Arg-Arg-Arg-OH ), decapeptide carrier (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH), -j ⁇ -peptide carrier (Tyr-Gly-Arg-Lys-Lys-Arg-Arg -Gln-Arg-Arg-Arg-OH) is coupled with green fluorescent protein and applied to the oral mucosa, nasal mucosa, gastric mucosa and skin of rats respectively. Blood is taken 15 minutes after administration and the rats are killed. Oral mucosa, nasal mucosa, gastric mucosa and skin specimens, the depth of penetration was observed by histochemical methods, and the content of green fluorescent protein in blood was also detected.
- decapeptide carriers are the easiest to penetrate different mucosa, while tripeptide carriers are the easiest to penetrate the skin.
- peptides 5 and 6 can be induced by penile erection in male rats and courtship behavior in female rats by oral mucosal administration, or by administering polypeptides 1, 2 via injection, or by transdermal administration of peptides 3 and 4. .
- 17 had obvious penile erections and had courtship behavior.
- the duration of an erectile response is 30 minutes to 2 hours with an average of 60 minutes.
- Ten female rats showed strong courtship behavior.
- In the control group only normal saline was administered orally, and 20 male rats and 10 female rats did not show significant erection and courtship behavior.
- polypeptides 3, 4, 5, 6 can be stimulated by dermal administration or by administering polypeptides 1, 2 by injection.
- the skin blackness was much higher than that of the control group; the control group was given only saline, and no skin color was deepened.
- the polypeptide of the present invention activates the melatonin MC-1 receptor and acts by promoting the melatonin MC-1 receptor to promote skin pigmentation like natural neurotransmitters.
- A is a physiological saline control group bullfrog
- B is a polypeptide 1 test group bullfrog.
- Step 1 Accurately weigh 10 g (0.36 mmol/g) of Fmoc-Lys(Mmt)-Linker Amide AM Resin into a 200 ml reaction flask, and add 100 ml of DMF swelling resin for 30 minutes, drain, and then The resin was washed three times with DMF, 50 ml each time, once every 2 minutes, drained, and 50 ml of deprotecting reagent (20% piperidine/DMF, ie piperidine/DMF mixture containing 20% by volume of piperidine) was added. The reaction was shaken for 30 minutes, the deprotection solution was withdrawn, the resin was washed three times with DMF, and dried, and a little ninhydrin was detected and positive.
- Step 2 sequentially adding 50 ml of DMF, three times the molar amount of Fmoc-Trp-OH, three times the molar amount of HBTU, three times the molar amount of NMM, shaking reaction for 30 minutes, taking a little resin for ninhydrin Detection, negative, withdraw the reaction solution, and then wash the resin three times with DMF shaking, about one minute each time, drain, add 50 ml of deprotection solution to the resin, shake the reaction for 30 minutes, take a little resin for ninhydrin detection, Positive, the deprotection solution was withdrawn, and the resin was washed three times with DMF shaking and drained.
- Step 3 According to the method of Step 2, the protected amino acids Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Asp (0-2- Phipr)-OH, Fmoc-Arg(Pbf)-OH is attached to the upper resin, and the N-terminal Fmoc-Arg (Pbf) has no deprotection step to form a polypeptide resin.
- the structure is:
- Step 4 the dried upper step polypeptide resin is added to the reaction bottle, and then the prepared 400 ml _ -
- TFA/DCM reaction solution ie 1% by volume of TFA in TFA/DCM mixture
- the reaction was carried out at 25 °C for 1 hour. After the reaction was completed, the resin was washed three times with DCM, and the resin was washed 4 times with DMF shaking.
- NMM/DMF reaction solution ie, 5% by volume of NMM in NMM/DMF mixture
- NMM/DMF mixture was washed three times, drained, and tested by ninhydrin.
- Step 5 The peptide resin containing the amino acid sequence of the functional unit is obtained by removing the Fmoc protecting group on the N-terminal Arg by the deprotection method of the above step, and the structure is:
- the functional unit amino acid sequence polypeptide resin obtained in Example 1 was linked to biotin according to the procedure of Step 2 in Example 1, and the connection time was 2 hours, and no deprotection step was carried out to obtain a polypeptide resin.
- the structure was:
- the upper step resin was cleaved with trifluoroacetic acid to obtain a crude peptide, and the crude peptide was purified by high performance liquid chromatography (column C-18, eluent eluted with acetonitrile/water/1% TFA, reversed phase). , freeze-dried to obtain polypeptide 1, the structure is:
- the functional unit amino acid sequence polypeptide resin prepared in Example 1 was subjected to the steps of Step 2 in Example 1 to sequentially protect the protected amino acids Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf). -OH is linked thereto to obtain a polypeptide resin having the structure: Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-c(Asp-Dab(Boc)-D-Phe-Arg(Pbf)- Trp-Lys)-Linker Amide AM Resin.
- the upper step resin was cleaved with trifluoroacetic acid to obtain a crude peptide, and the crude peptide was purified by high performance liquid chromatography (column C-18, eluent eluted with acetonitrile/water/1% TFA, reversed phase). , lyophilized to obtain polypeptide 3, the structure is:
- the functional unit amino acid sequence polypeptide resin prepared in Example 1 was subjected to the steps of Step 2 in Example 1 to sequentially protect the protected amino acids Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf).
- the upper step resin was cleaved with trifluoroacetic acid to obtain a crude peptide, and the crude peptide was purified by high performance liquid chromatography (column C-18, eluent eluted with acetonitrile/water/1% TFA, reversed phase). , lyophilized to obtain polypeptide 5, the structure is:
- Step 1 Accurately weigh 10 g (0.73 mmol/g) of Fmoc-Lys(Mmt)-Wang Resin into a 200 ml reaction flask, and add 100 ml of DMF swelling resin for 30 minutes, drain, and then use DMF. Rinse the resin three times, 50 ml each time, once every 2 minutes, drain, add 50 ml of deprotection reagent (20% piperidine / DMF, ie piperidine / DMF mixture containing 20% by volume of piperidine) After 30 minutes, the deprotection solution was withdrawn, the resin was washed three times with DMF, drained, and a little ninhydrin was detected and positive.
- Step 2 sequentially adding 50 ml of DMF to the reaction flask, three times the molar amount of Fmoc-Trp-OH, three times the molar amount of HBTU, three times the molar amount of NMM, shaking reaction for 30 minutes, taking a little resin for ninhydrin Detection, negative, withdraw the reaction solution, and then wash the resin three times with DMF shaking, about one minute each time, drain, add 50 ml of deprotection solution to the resin, shake the reaction for 30 minutes, take a little resin for ninhydrin detection, Positive, the deprotection solution was withdrawn, and the resin was washed three times with DMF shaking and drained.
- Step 3 according to the method of step 2, the protected amino acids Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Boc)-OH, Fmoc-Asp (0-2- Phipr)-OH, Fmoc-Arg(Pbf)-OH is attached to the upper resin, and the N-terminal Fmoc-Arg (Pbf) has no deprotection step to form a polypeptide resin.
- the structure is:
- Step 4 the dried upper step polypeptide resin is added to the reaction flask, and then the prepared 400 ml of 1 TFA/DCM reaction solution (ie, 1% by volume of TFA in the TFA/DCM mixture) is added to the reaction.
- 1 TFA/DCM reaction solution ie, 1% by volume of TFA in the TFA/DCM mixture
- the reaction was carried out at 25 °C for 1 hour.
- the resin was washed three times with DCM, and the resin was washed 4 times with DMF, and then 5 NMM/DMF reaction solution (that is, the NMM/DMF mixture contained 5% by volume of NMM).
- the resin was washed three times, drained, and the ninhydrin test was positive.
- polypeptide 5 of Example 4 can induce penile erection.
- polypeptides of the invention act through the central MC-3 and MC-4 receptors.
- SHU-9119 selectively blocks MC-3 and MC-4 receptors, and in our experiments, SHU-9119 (dose 2 ng/g body weight) was injected while applying the polypeptide of the present invention. It was found that the polypeptide of the present invention (dose of 12.5, 25, 50, and 100 ng/g body weight) was unable to induce an erection, and in 20 rats, only at 100 ng/g body weight, 2 cases were still observed. Mild erection. This demonstrates that the polypeptides of the invention act through the central MC-3 and MC-4 receptors.
- the unit is Nakheike, and 0 is the saline control. The same below.
- injection administration of the present invention 2 can induce pubic erection:
- the polypeptide of the present invention is a polypeptide which can be absorbed into the bloodstream through mucosa and muscle.
- the animal experiment proves that it has the function of causing male penile erection, and can be used for treating male erectile dysfunction.
- the polypeptide of the invention acts through the central MC-3 and MC-4 receptors
- SHU-9119 selectively blocks MC-3 and MC-4 receptors.
- SHU-9119 dose 2 ng/g body weight
- the polypeptide of the invention dose of 12.5, 25, 50, and 100 ng/g body weight
- was unable to induce significant courtship behavior and in 10 rats, only at 100 ng/g body weight, 2 cases were still observed to be light Degree courtship behavior.
- the polypeptide of the present invention is a polypeptide which can be absorbed into the bloodstream through mucosa and muscle.
- the animal experiment proves that it has the function of improving female sexual desire and can be used for treating female sexual desire.
- mice Male and female Kunming mice, weighing about 30 grams, were divided into several groups of 6 animals each. Cage, SPF-serving environment, simulating natural daylight and dark night light, plenty of food and water, giving mice plenty of time to adapt to the environment before the experiment.
- the polypeptide of the invention acts through the central MC-3 and MC-4 receptors
- SHU-9119 selectively blocks MC-3 and MC-4 receptors.
- SHU-9119 dose 2 ng/g body weight
- the polypeptide of the invention at a dose of 100 ng/g body weight
- Muscles of the present invention can inhibit the appetite of mice.
- the polypeptide of the present invention is a polypeptide which can be absorbed into the bloodstream through mucosa and muscle, and the animal experiment proves that it has an appetite suppressing effect and can be used for the treatment of obesity.
- polypeptide 1 of Example 2 promotes melanin
- the polypeptide of the present invention is a polypeptide which can be absorbed into the bloodstream through muscles and skin. It has been confirmed by animal experiments to promote melanin, which can be used for hypopigmentation and anti-ultraviolet radiation therapy.
- the hair of the rat abdomen was removed with a depilatory agent, 10 males and 10 females, and the polypeptide of the present invention (polypeptide 3 of Example 3) was physiologically used.
- the saline solution was formulated into a 50 mg/ml solution, and the exposed skin of the abdomen was applied three times a day for 8 hours.
- the penile erection of the male rats and the estrus of the female rats were observed after 2 hours of application (effects and peptides). 5 similar); After 7 days of continuous administration, it can be seen that the skin of the rat was blackened at the abdominal administration site.
- transdermal administration of the polypeptide of the present invention 3 induces penile erection in male rats, and estrus in females.
- the polypeptide of the present invention is a polypeptide which can be absorbed into the bloodstream through the skin, and the animal experiment proves that it has the functions of promoting erection and libido, promoting melanin, and acting on MC-1, MC-3, MC- 4 receptors.
- the recommended dose is 0.1 mg per kilogram of body weight.
- the recommended dosage form is a subcutaneous or oily agent, or a mucosal inhaler.
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Abstract
提供了环状核心序列与生物素或穿膜肽相连而成的促黑激素类似物及其制备方法和应用,所述促黑激素类似物由功能单位与生物素或穿膜肽相连而成,功能单位结构为Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys),穿膜肽结构为Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg或者Arg-Arg-Arg。该促黑激素类似物可透过粘膜或皮肤吸入,用于男女性功能障碍、肥胖症、色素沉着不足症的治疗。
Description
环状核心序列与生物素或穿膜肽相连而成的促黑激素类似物 技术领域
[0001] 本发明涉及一种环状核心序列与生物素或穿膜肽相连而成的促黑激素类似物及其制 备方法和应用。
背景技术
[0002] 随着生活节奏的加快, 工作压力的加大, 遗传因素的影响, 男女性功能障碍、 肥胖 症、 色素沉着不足症都日益凸显出来, 严重影响人们的生活质量。 其中, 肥胖症是众所周知 的导致动脉硬化、 高血压、 心脏病、 糖尿病等常见疾病的因素之一, 而男女性功能障碍和色 素沉着不足症导致的性生活不和谐和皮肤癌等疾病也严重影响到患者的身心健康和家庭幸 福。
[0003] 目前, 治疗男性性功能障碍的一线药物为西地那非等磷酸二脂酶抑制剂, 但此类药 物对女性患者无效, 而且部分患者报道响应逐渐消失。 治疗肥胖症的药物也只有少数几个, 如西布曲明和奥利司他。 治疗色素沉着不足症的多肽药物也少之又少。 PT-141和 ΜΤ-Π是两 种目前正在研发中的对男女性功能障碍、 肥胖症、 色素沉着不足症有一定疗效的多肽类药 物。
[0004] 由于生物细胞膜的阻挡性, 多肽等大分子药物很难由细胞膜直接进入体内, 故目前 使用的多肽类药物大多采用注射给药的给药方式, 而男女性功能障碍、 肥胖症、 色素沉着不 足症的治疗是长期的, 如能研发出能经粘膜或皮肤给药的多肽类药物则可免除患者长期注射 给药的不便和痛苦。 因此, 研发出一种能经粘膜或皮肤给药的用于治疗男女性功能障碍、 肥 胖症、 色素沉着不足症的多肽类药物是必须和当务之急的, 其具有广阔的市场前景。
[0005] a -MSH是一种源于前阿黑皮素 (POMC) 的线性十三肽。 早在 20世纪 50年代, 人 们就发现对狗、 猴、 猫以及兔子等中枢给予 α -MSH 后能引起其性兴奋和食欲抑制, 其作用 机理是通过作用于促黑激素受体亚型 MC-Rs中 MC-3、 MC-4受体而产生性行为和食欲抑制 调节效应。 后来, 人们还发现对牛蛙等动物给予 α -MSH后, 其皮肤颜色会加深, 其机制是 通过作用于促黑激素受体亚型 MC-Rs 中的 MC-1 受体而产生皮肤色素调节效应。 研究表 明, MC-Rs 激动剂在治疗男女性功能障碍、 肥胖症、 色素沉着不足症等方面有潜在的应用 价值。
[0006] α -MSH是一个线性十三肽, C端含有酰胺结构, N端乙酰化, 其一级结构如下: Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH
[0007] 人们后来研究发现: α -MSH 的最小活性序列是 His-Phe-Arg-Trp, 围绕着此核心序
列, 人们设计合成了一系列此类化合物, 包括线性肽和环肽, 在治疗男女性功能障碍、 肥胖 症、 色素沉着不足症等方面有一定的疗效。 其中, PT-141、 ΜΤ-Π便是其中的两种。
[0008] 由于生物细胞膜的屏障, 多肽等大分子不能直接进入细胞, 这给许多疾病的治疗带 来了困难。 细胞穿膜肽 (CPP) 是一类能携带大分子物质进入细胞的短肽, 其穿膜能力不依 赖经典的胞吞作用, 也有研究者称这类短肽为蛋白传导域 (PTD ) 或特洛伊木马肽。
[0009] 人们发现和证实, 人免疫缺损病毒 HIV-1 的反式激活蛋白 ΤΑΤ能跨膜转移到细胞膜 和细胞核内。 人们还发现 HIV-1 的 ΤΑΤ蛋白中的一个富含碱性氨基酸片段, 此带正电荷的 多肽片段与蛋白传导功能密切相关, 称之为蛋白传导域。 进一步研究发现, 全长 86 个氨基 酸残基的 ΤΑΤ蛋白中的第 49一 57残基组成的序列即可完全行使蛋白传导的功能, 并且无细 胞毒性。 这些发现为研制一种高效、 安全的载体, 主动介导多肽类大分子跨越生物膜屏障提 供了广阔的前景。
[0010] 生物素又称维生素 Η, 是人体必需维生素之一, 有促进生长的作用, 对白发、 秃发有 一定疗效。 白发症是色素沉着不足症之一, 促黑激素对其有很强治疗作用, 将促黑激素环状 核心序列与生物素创造性的结合在一起, 在使毛发变黑的同时又能促进其生长, 这是本发明 在医药或化妆品领域的一种创新。
发明内容
[0011] 本发明的目的在于提供一种促黑激素类似物及其制备方法和应用。
[0012] 为实现上述目的, 本发明采取了如下技术方案:
一种促黑激素类似物, 其多肽结构 (包括所有的对映体、 非对映体、 立体异构体等) 如下: X-Z-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-Y和其无生理毒性的盐, 其中,
X=生物素酰基或无,
Z=Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg或 Arg-Arg-Arg或无,
Y=NH2或 OH,
c=环肽,
当 X=生物素酰基时, Z=无;
当 Z=Gly-Arg-Lys-Lys-Arg-Arg-Gln- Arg-Arg-Arg或 Arg-Arg-Arg时, X=无;
X、 Z不能同时为无;
无生理毒性的盐是指可保留母体化合物预期生理活性而不会产生任何意料之外毒副作用的 盐, 具体可以为盐酸盐、 氢溴酸盐、 氢碘酸盐、 硫酸盐、 磷酸盐、 硝酸盐、 乙酸盐、 三氟乙 酸盐、 草酸盐、 酒石酸盐、 琥珀酸盐、 苹果酸盐、 苯甲酸盐、 海藻酸盐、 甲磺酸盐、 萘磺酸 盐、 钾盐、 钠盐、 锂盐、 锌盐、 铜盐、 钡盐或钙盐。
" '
[0013] 所述的促黑激素类似物, 其多肽结构为下述六种时, 疗效尤其好:
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2和其无生理毒性的盐、
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH和其无生理毒性的盐、
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2和其无生理毒性的盐、
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH和其无生理毒性的盐、
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2禾卩其无生 理毒性的盐或
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH 禾口其无生 理毒性的盐; 其中, Biotin=D-(+)生物素酰基, Dab= (L) 2,4 二氨基丁酸残基, D-Phe=右旋 苯丙氨酸残基 (结构式如下)。
[0014] 所述无生理毒性的盐优选为乙酸盐。 上述促黑激素类似物中氨基酸残基除 D-Phe 外, 其余皆为 (L) 型。
[0015] 所述促黑激素类似物的制备方法, 采用固相合成法, 具体包括以下步骤: 先在固相 树脂上制备出未与固相树脂裂解的、 未脱侧链保护基的功能单位氨基酸序列, 然后在固相树 脂上将功能单位氨基酸序列的 N 末端进行生物素酰化, 或者继续将负载单位氨基酸序列中 的氨基酸残基依次从 C端到 N端分别与功能单位氨基酸序列的 N末端连接, 在脱除最后一 个氨基酸残基的 N 端保护基后经裂解、 纯化、 干燥成肽; N 端生物素酰化的包含功能单位 氨基酸序列的固相树脂也要经裂解、 纯化、 干燥成肽的步骤; 其中, 功能单位氨基酸序列 为: Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys);
负载单位氨基酸序列为: Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg或 Arg-Arg-Arg。
[0016] 所述促黑激素类似物在制备用于治疗男性勃起功能障碍、 女性性欲低下、 肥胖症、 色素沉着不足症药物中的应用。
[0017] 所述促黑激素类似物的给药方式优选为粘膜给药、 皮肤给药或注射给药。
[0018] 所述药物中含有具有下述六种结构的多肽成分中的至少一种以及可药用载体或赋形 剂-
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2、
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH>
替换页 (细则第 26条)
_ -
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2 g¾
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OHo
[0019] 其中: 可药用载体或赋形剂可以为含有磷酸缓冲液的生理盐水。
[0020] 所述促黑激素类似物在制备促黑激素 MC-Rs激动剂中的应用。
[0021] 所述 MC-Rs仅指 MC-1受体、 MC-3受体、 MC-4受体或 MC-5受体。
[0022] HIV-1 TAT蛋白的第 47〜60残基片段由十四个氨基酸残基构成, 其一级结构为:
Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-OHo
[0023] 本发明将具有 MC-Rs激动活性的促黑激素类似物的多肽序列与生物素或 HIV-1 TAT 蛋白的第 47〜60 残基片段中的不同残基组成的不同多肽片段结合, 设计出一种能经粘膜或 皮肤吸入的促黑激素类似物, 该促黑激素类似物具有 MC-Rs 激动剂的活性, 可用于男女性 功能障碍、 肥胖症或色素沉着不足症的治疗。
[0024] 本发明提供的多肽由功能单位和生物素或负载单位两部分构成, 其功能单位与生物 素或负载单位之间由酰胺键连接。
[0025] 功能单位的结构是: Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-Y (Y=NH2或 OH), 其环状结 构由 Lactam 交联桥连接。 c(Asp-Dab-D-Phe-Arg-Trp-Lys)为其环状核心序列。 Dab-D-Phe- Arg-Trp为其核心序列。 N末端 Arg为连接性氨基酸。 该功能单位由一个七肽构成。 从结构 上看, 其侧链连接成环状, 脂溶性增加, 更有利于透过粘膜、 皮肤, 其序列与其他促黑激素 受体激动剂相似但不相同。
[0026] 将功能单位 N端和生物素相连接构成 2种多肽结构, 即多肽 1和多肽 2, 结构式如 下:
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2 (多月太 1 );
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH (多肽 2)。
[0027] 负载单位由 HIV-1 TAT蛋白的第 47〜60片段中的不同氨基酸片段组成。
[0028] 负载单位 1由 HIV-1 TAT蛋白的第 55〜57氨基酸片段组成, 含三个氨基酸残基, 其 结构为: Arg-Arg-Arg-OH。
[0029] 负载单位 2由 HIV-1 TAT蛋白的第 48〜57氨基酸片段组成, 含十个氨基酸残基, 其 结构为: Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH。
[0030] 将功能单位 N端分别与负载单位 1、 2连接组成 4种多肽结构, 即多肽 3、 4、 5和 6, 结构式如下:
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2 (多] 3太 3 );
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH (多肽 4);
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2 ( 多 肽
5);
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH ( 多 肽
6)。
[0031] 本发明中常见英文縮写的含义为:
Biotin为生物素酰基
c为环肽
Dab为 2,4二氨基丁酸
Gly为甘氨酸
Arg为精氨酸
Lys为赖氨酸
Gin为谷氨酰胺酸
Asp为天冬氨酸
D-Phe为右旋苯丙氨酸
Trp为色氨酸
DMF为二甲基甲酰胺
DCM为二氯甲烷
TFA为三氟乙酸
HBTU为苯并三唑 -1-四甲基六氟磷酸酯
NMM为 N-甲基吗啡啉。
[0032] 本发明将不同的负载单位与功能单位以肽键相连后进行试验。 经实验结果证明: 十 肽负载单位与功能单位相连后形成的多肽最容易穿透不同粘膜 (包括口腔粘膜、 胃粘膜、 鼻 粘膜); 三肽负载单位与功能单位相连后形成的多肽最容易穿透皮肤。 实验证明, 本发明提 供的负载单位可携带本发明提供的功能单位穿透粘膜和毛细血管壁, 并可穿透血脑屏障进入 大脑皮质。 将本发明提供的负载单位或生物素与本发明提供的功能单位连接后形成的多肽作 为药物, 在相对很小剂量即可很容易经消化道粘膜和呼吸道粘膜吸收, 从而使口服和粘膜给 药成为可能, 这是其他多肽类药物很难做到的。 同样, 由于本发明负载单位可携带功能单位 穿透皮肤, 使多肽经皮外给药成为可能。
[0033] 为观察不同的多肽载体对不同粘膜的穿透性, 本发明分别将三肽载体 (Arg-Arg-Arg-
OH )、 十肽载体 (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH )、 -j ^一肽载体 (Tyr-Gly- Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-OH) 与绿色荧光蛋白偶联在一起, 分别应用到大鼠的 口腔粘膜、 鼻粘膜、 胃粘膜及皮肤, 用药后 15 分钟取血, 并杀死大鼠取口腔粘膜、 鼻粘 膜、 胃粘膜及皮肤标本, 用组织化学方法观察穿透深度, 同时检测血中绿色荧光蛋白的含
[0034] 实验证明, 十肽载体最容易穿透不同粘膜, 而三肽载体最容易穿透皮肤。
[0035] 不同载体对不同粘膜的穿透性
口腔粘膜 胃粘膜 鼻粘膜 皮肤
三肽载体 ++ + ++ ++++
十肽载体 ++++ ++ ++++ ++
^一肽载体 +++ +++ +++ ++
动物实验证明, 多肽 5、 6经口腔粘膜给药, 或多肽 1、 2经注射给药, 或多肽 3、 4经皮肤 给药, 都可诱发雄性大鼠的阴茎勃起及雌性大鼠的求偶行为。 在 20只雄性大鼠中, 有 17只 有明显阴茎勃起, 并有求偶行为。 有勃起反应持续时间为 30分钟到 2小时, 平均 60分钟。 10只雌性大鼠 8只出现强烈求偶行为。 对照组只口服生理盐水, 20只雄性大鼠和 10雌性大 鼠均未出现明显勃起和求偶行为。 体内试验证明, 在给予本发明多肽的同时, 同时给予促黑 激素 MC-3、 MC-4受体阻滞剂 SHU-9119后, 各受试大鼠均未出现明显勃起和求偶行为。 这 说明本发明多肽可激活中枢促黑激素 MC-3、 MC-4受体, 并且是通过中枢促黑激素 MC-3、 MC-4 受体起作用的, 其作用类似于天然神经递质。 由于其作用靶点是中枢神经系统勃起中 心, 除生理作用外还有心理正反馈作用, 可同时用于心理性和器质性男性勃起功能障碍的治 疗。
[0036] 动物实验同时提示, 在有外界性刺激条件下, 本发明多肽能唤醒性欲低下女性的性 欲, 并增加阴道壁血流。 据统计性欲低下约发生于 30%的女性, 目前尚无有效药物对其进行 治疗。 我们在动物实验中发现, 本发明多肽可选择性刺激性欲低下雌性大鼠的求偶行为, 同 时发现, 阴道内温度升高, 说明阴道血流增加, 这些证据表明黑色素能神经系统不仅调节男 性阴茎勃起和性欲, 而且也调节女性的性欲, 是目前所知的唯一明确调节女性性欲的系统, 也成为治疗女性性欲低下的希望。
[0037] 动物实验证明, 多肽 5、 6 经口腔粘膜给药或多肽 1、 2 经注射给药, 都可抑制小鼠 的觅食行为, 从而减轻小鼠的体重。 在 6只小鼠中, 均有明显体重下降, 与对照组相比平均 下降 5%; 对照组只口服生理盐水, 各 6只小鼠未见明显体重下降。 体内试验证明, 在给予 本发明多肽的同时, 同时给予促黑激素 MC-3、 MC-4受体阻滞剂 SHU9119后, 受试小鼠未
见明显体重下降。 这说明本发明多肽能激活促黑激素 MC-3、 MC-4 受体, 并是通过促黑激 素 MC-3、 MC-4受体起作用的, 其作用类似于天然神经递质。
[0038] 动物实验证明, 多肽 3、 4、 5、 6 经皮肤给药或多肽 1、 2 经注射给药, 都可刺激黑 色素的生成。 在受试牛蛙或大鼠中, 与对照组相比, 皮肤黑度提高许多; 对照组只给予生理 盐水, 未见皮肤颜色加深。 这说明本发明多肽可激活促黑激素 MC-1受体, 并是通过促黑激 素 MC-1受体发挥作用, 象天然神经递质一样起到促进皮肤色素生成作用。
[0039] 经大量动物实验表明, 在试验狗体内长期大剂量给药, 未发现明显低血压, 也未发 现明显的肝脏及肾脏损害。
附图说明
[0040] 图 1为实施例 12的牛蛙试验结果, 其中, A为生理盐水对照组牛蛙; B为多肽 1试 验组牛蛙。
具体实施方式
[0041] 以下实施例仅用于解释本发明, 但不能由此限制本发明。
[0042] 实施例 1
在氨基(NH2) 型固相树脂上制备功能单位氨基酸序列
步骤 1、 精确称取 10克 (0.36毫摩尔 /克) 的 Fmoc-Lys(Mmt)-Linker Amide AM Resin加入 200毫升的反应瓶中, 并加入 100毫升 DMF溶涨树脂 30分钟, 抽干, 再用 DMF振荡洗涤 树脂三次, 每次 50毫升, 2分钟一次, 抽干, 加 50毫升脱保护试剂 (20%哌啶 /DMF, 即哌 啶/ DMF混合液中含有 20%体积百分数的哌啶) 振荡反应 30分钟, 抽出脱保护液, 用 DMF 洗涤树脂三次, 抽干, 取少许茚三酮检测, 呈阳性。
[0043] 步骤 2、 依次向反应瓶中加入 50毫升 DMF, 三倍摩尔量 Fmoc-Trp-OH, 三倍摩尔量 HBTU、 三倍摩尔量 NMM, 振荡反应 30分钟, 取少许树脂做茚三酮检测, 呈阴性, 抽出反 应液, 再用 DMF振荡洗涤树脂三次, 每次大约一分钟, 抽干, 加 50 毫升脱保护液于树脂 中, 振荡反应 30 分钟, 取少许树脂做茚三酮检测, 呈阳性, 抽出脱保护液, 用 DMF振荡 洗涤树脂三次, 抽干。
[0044] 步骤 3、 按步骤 2 的操作方法依次将保护氨基酸 Fmoc-Arg(Pbf)-OH、 Fmoc-D-Phe- OH、 Fmoc-Dab(Boc)-OH、 Fmoc-Asp(0-2-Phipr)-OH、 Fmoc- Arg(Pbf)-OH 连接到上步树脂 上, N末端 Fmoc- Arg(Pbf)无脱保护步骤, 构成多肽树脂, 结构为:
Fmoc-Arg(Pbf)-Asp(0-2-Phipr)-Dab(Boc)-D-Phe-Arg(Pbf)-Trp-Lys(Mmt)-Linker Amide AM Resin。
[0045] 步骤 4、 将已干燥的上步多肽树脂加入反应瓶中, 再将已配制的 400 毫升
_ -
1 TFA/DCM反应液 (即 TFA/DCM混合液中含有 1%体积百分数的 TFA) 加入反应瓶中,
25度下反应 1小时, 反应结束后, 用 DCM洗涤树脂三次, DMF振荡洗涤树脂 4次, 再用
5 NMM/DMF反应液 (即 NMM/DMF混合液中含有 5%体积百分数的 NMM) 洗涤树脂三 次, 抽干, 茚三酮检测呈阳性。 依次向反应瓶中加入 50毫升 DMF、 3.8克 HBTU、 1.1毫升
NMM, 振荡反应 1 小时, 取少许树脂做茚三酮检测, 呈阴性, 抽出反应液, 再用 DMF振 荡洗涤树脂三次, 甲醇洗涤树脂三次, 干燥, 得到如下结构肽树脂:
Fmoc- Arg(Pbf)-c(Asp- Dab(Boc)-D-Phe-Arg(Pbf)-Trp-Lys)- Linker Amide AM Resin
步骤 5、 将上步多肽树脂按步骤 2中的脱保护方法脱除 N端 Arg上的 Fmoc保护基后制得含 功能单位氨基酸序列的肽树脂, 结构为:
Arg(Pbf)-c(Asp- Dab(Boc)-D-Phe-Arg(Pbf)-Trp-Lys)- Linker Amide AM Resin。
[0046] 实施例 2
多肽 1的合成
将实施例 1制得的功能单位氨基酸序列多肽树脂按实施例 1中步骤 2的操作将生物素与其连 接, 连接时间为 2小时, 无脱保护步骤, 得到多肽树脂, 结构为:
Biotin-Arg(Pbf)-c(Asp- Dab(Boc)-D-Phe-Arg(Pbf)-Trp-Lys)- Linker Amide AM Resin。
[0047] 将上步树脂用三氟乙酸裂解得粗肽, 粗肽用高效液相色谱纯化 (色谱柱用 C-18, 洗 脱液用乙腈 /水 /1%TFA, 反相洗脱) 后, 冻干制得多肽 1, 结构为:
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH
[0048] 实施例 3
多肽 3的合成
将实施例 1制得的功能单位氨基酸序列多肽树脂按实施例 1中步骤 2的操作依次将保护氨基 酸 Fmoc-Arg(Pbf)-OH、 Fmoc-Arg(Pbf)-OH、 Fmoc-Arg(Pbf)-OH 与其连接, 得到多肽树脂, 结构为: Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-c(Asp-Dab(Boc)-D-Phe-Arg(Pbf)-Trp-Lys)-Linker Amide AM Resin。
[0049] 将上步树脂用三氟乙酸裂解得粗肽, 粗肽用高效液相色谱纯化 (色谱柱用 C-18, 洗 脱液用乙腈 /水 /1%TFA, 反相洗脱) 后, 冻干制得多肽 3, 结构为:
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH
[0050] 实施例 4
多肽 5的合成
将实施例 1制得的功能单位氨基酸序列多肽树脂按实施例 1中步骤 2的操作依次将保护氨基 酸 Fmoc-Arg(Pbf)-OH、 Fmoc-Arg(Pbf)-OH、 Fmoc-Arg(Pbf)-OH、 Fmoc-Gln(Trt)-OH、 Fmoc-
Arg(Pbf)-OH、 Fmoc-Arg(Pbf)-OH、 Fmoc-Lys(Boc)-OH、 Fmoc-Lys(Boc)-OH、 Fmoc-Arg(Pbf)- OH、 Fmoc-Gly-OH与其连接, 得到多肽树脂, 结构为:
Gly-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)- Arg(Pbf)-c(Asp- Dab(Boc)-D-Phe-Arg(Pbf)-Trp-Lys)- Linker Amide AM Resin。
[0051] 将上步树脂用三氟乙酸裂解得粗肽, 粗肽用高效液相色谱纯化 (色谱柱用 C-18, 洗 脱液用乙腈 /水 /1%TFA, 反相洗脱) 后, 冻干制得多肽 5, 结构为:
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp- Dab-D-Phe- Arg-Trp-Lys)-NH2
[0052] 实施例 5
在羟基 (OH)型固相树脂上制备功能单位氨基酸序列
步骤 1、 精确称取 10克 (0.73毫摩尔 /克) 的 Fmoc-Lys(Mmt)-Wang Resin加入 200毫升的反 应瓶中, 并加入 100毫升 DMF溶涨树脂 30分钟, 抽干, 再用 DMF振荡洗涤树脂三次, 每 次 50毫升, 2分钟一次, 抽干, 加 50毫升脱保护试剂 (20%哌啶 /DMF, 即哌啶 /DMF混合 液中含有 20%体积百分数的哌啶) 振荡反应 30分钟, 抽出脱保护液, 用 DMF洗涤树脂三 次, 抽干, 取少许茚三酮检测, 呈阳性。
[0053] 步骤 2、 依次向反应瓶中加入 50毫升 DMF, 三倍摩尔量 Fmoc-Trp-OH, 三倍摩尔量 HBTU、 三倍摩尔量 NMM, 振荡反应 30分钟, 取少许树脂做茚三酮检测, 呈阴性, 抽出反 应液, 再用 DMF振荡洗涤树脂三次, 每次大约一分钟, 抽干, 加 50 毫升脱保护液于树脂 中, 振荡反应 30 分钟, 取少许树脂做茚三酮检测, 呈阳性, 抽出脱保护液, 用 DMF振荡 洗涤树脂三次, 抽干。
[0054] 步骤 3、 按步骤 2 的操作方法依次将保护氨基酸 Fmoc-Arg(Pbf)-OH、 Fmoc-D-Phe- OH、 Fmoc-Dab(Boc)-OH、 Fmoc-Asp(0-2-Phipr)-OH、 Fmoc- Arg(Pbf)-OH 连接到上步树脂 上, N末端 Fmoc- Arg(Pbf)无脱保护步骤, 构成多肽树脂, 结构为:
Fmoc- Arg(Pbf)-Asp(0-2-Phipr)-Dab(Boc)-D-Phe-Arg(Pbf)-Trp-Lys(Mmt)-Wang Resin。
[0055] 步骤 4、 将已干燥的上步多肽树脂加入反应瓶中, 再将已配制的 400 毫升 1 TFA/DCM反应液 (即 TFA/DCM混合液中含有 1%体积百分数的 TFA) 加入反应瓶中, 25度下反应 1小时, 反应结束后, 用 DCM洗涤树脂三次, DMF振荡洗涤树脂 4次, 再用 5 NMM/DMF反应液 (即 NMM/DMF混合液中含有 5%体积百分数的 NMM) 洗涤树脂三 次, 抽干, 茚三酮检测呈阳性, 依次向反应瓶中加入 50毫升 DMF、 3.8克 HBTU、 1.1毫升 NMM, 振荡反应 1 小时, 取少许树脂做茚三酮检测, 呈阴性, 抽出反应液, 再用 DMF振 荡洗涤树脂三次, 甲醇洗涤树脂三次, 干燥, 得到如下结构肽树脂:
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[0063] 实施例 9
实验动物
成年雄性及雌性 SD大鼠, 重约 225克到 250克。 单个笼养, SPF伺养环境, 模拟自然白昼 及黑夜光线, 充足食物和水, 实验前给大鼠充足时间以适应环境, 所有行为学研究都固定时 间完成 (14一 18点)。
[0064] 实验方法及结果
口腔滴注本发明多肽 (实施例 4的多肽 5 ) 可诱发阴茎勃起。
[0065] 实验发现经口腔滴注本发明多肽可诱发阴茎勃起。 口腔滴注不同剂量本发明多肽 5 (25, 50, 100和 200纳克 /每克体重) 后, 动物实验证明, 经口腔粘膜给药, 功能单位氨基 酸序列与负载单位氨基酸序列相连的多肽可诱发大鼠阴茎勃起, 在剂量为 100 纳克 /每克体 重时, 20只大鼠中, 有 17只有阴茎勃起, 11只有强烈阴茎勃起, 并有求偶行为, 有勃起反 应持续时间为 30 分钟到 2 小时, 平均 60 分钟 (见表 1 )。 本实验设对照组, 为生理盐水 组, 各 20只大鼠中, 未见明显阴茎勃起。
[0066] 本发明多肽是通过中枢的 MC-3和 MC-4受体起作用。
[0067] SHU-9119可选择性的阻滞 MC-3和 MC-4受体, 在我们的实验中应用本发明多肽的 同时, 注射 SHU-9119 (剂量 2纳克 /每克体重), 结果发现, 本发明多肽 (剂量为 12.5,25,50, 和 100纳克 /每克体重) 无法诱导勃起, 在 20只大鼠中, 只有在 100纳克 /每克体重时, 2例 仍观察到轻度勃起。 说明本发明多肽是通过中枢的 MC-3和 MC-4受体起作用。
注: 表中剤 单位为纳克海克 重, 0表示生理盐水对照, 下同。
[0068] 采用同样的剂量和施用方法, 用多肽 6代替多肽 5取得近似的实验结果 (见表 2)。
[0070] 结论: 本发明多肽是一种可经粘膜、 肌肉吸收进入血流的多肽, 动物实验证实其具 有引发男性阴茎勃起的功能, 可用于治疗男性勃起功能障碍。
[0071] 实施例 10
实验动物
成年雄性及雌性 SD大鼠, 重约 225克到 250克。 单个笼养, SPF伺养环境, 模拟自然白昼 及黑夜光线, 充足食物和水, 实验前给大鼠充足时间以适应环境, 所有行为学研究都固定时 间完成 (14一 18点)。
[0072] 实验方法及结果
口腔滴注本发明多肽 (实施例 4的多肽 5) 可诱发雌性大鼠求偶行为
实验证明本发明多肽可使雌性大鼠发生求偶行为的改变。 实验雌性大鼠在麻醉后行双侧卵巢 切除术后, 成为性欲低下型大鼠。 实验中发现, 口腔滴注本发明多肽 5 ( 100 纳克 /每克体 重) 后, 雌性大鼠进入雄性大鼠所在的上层的频率由平均 3次增加为 11 次, 首次进入上层 的时间由平均 11分钟减少为 3分钟。 说明, 口腔滴注本发明多肽 5可刺激性欲低下的雌性 大鼠的求偶行为。 对阴道温度改变的观察中发现, 阴道内温度升高, 说明阴道血流增加。 对 照生理盐水无以上作用。
[0073] 本发明多肽是通过中枢的 MC-3和 MC-4受体起作用
SHU-9119可选择性的阻滞 MC-3和 MC-4受体, 在我们的实验中应用本发明多肽的同时, 注射 SHU-9119 (剂量 2纳克 /每克体重), 结果发现, 本发明多肽 (剂量为 12.5,25,50,和 100 纳克 /每克体重) 无法诱导明显求偶行为, 在 10只大鼠中, 只有在 100纳克 /每克体重时, 2 例仍观察到轻度求偶行为。 说明本发明多肽是通过中枢的 MC-3和 MC-4受体起作用。
[0074] 采用同样的剂量、 同样的施用方法, 用多肽 6代替多肽 5取得近似的实验结果。
[0075] 采用同样的剂量、 注射给药的方式, 用多肽 1或多肽 2也取得近似的实验结果。
[0076] 结论: 本发明多肽是一种可经粘膜、 肌肉吸收进入血流的多肽, 动物实验证实其具 有提高女性性欲的功能, 可用于治疗女性性欲低下。
[0077] 实施例 11
实验动物
成年雄性及雌性昆明种小鼠, 体重 30克左右, 分成若干组, 每组 6只。 笼养, SPF伺养环 境, 模拟自然白昼及黑夜光线, 充足食物和水, 实验前给小鼠充足时间以适应环境。
[0078] 实验方法及结果
肌肉注射本发明多肽 (实施例 2的多肽 1 ) 可抑制食欲
实验发现经肌肉注射本发明多肽可抑制食欲。 肌肉注射不同剂量本发明多肽 1 ( 100纳克 /每 克体重) 后, 动物实验证明, 经肌肉注射给药, 功能单位氨基酸序列与生物素相连的多肽可 抑制小鼠食欲, 在剂量为 100纳克 /每克体重时, 6只大鼠中平均体重比对照组下降 5%。 本 实验设对照组, 为生理盐水组, 6只小鼠中, 未见明显体重下降, 结果见表 5。
[0079] 本发明多肽是通过中枢的 MC-3和 MC-4受体起作用
SHU-9119可选择性的阻滞 MC-3和 MC-4受体, 在我们的实验中应用本发明多肽的同时, 注射 SHU-9119 (剂量 2纳克 /每克体重), 结果发现, 本发明多肽 (剂量为 100纳克 /每克体 重) 无法抑制食欲, 说明本发明多肽是通过中枢的 MC-3和 MC-4受体起作用。
[0080] 用同样的剂量、 同样的施用方法用多肽 2 代替多肽 1 取得近似的实验结果 (见表 6)。
表 6、 肌肉往射本发明多服 可抑制小鼠食欲。
[0081] 用同样的剂量、 口腔滴注的方式用多肽 5 或多肽 6 也取得近似的实验结果 (见表 7 和 8)。
, - 表 7、 口腔滴注本发明多胜 5可抑制小鼠食欲。
[0082] 结论: 本发明多肽是一种可经粘膜、 肌肉吸收进入血流的多肽, 动物实验证实其具 有抑制食欲的作用, 可用于肥胖症的治疗。
[0083] 实施例 12
实验动物: 牛蛙。 将牛蛙在自然光线下照射一周, 使其肤色均匀。
[0084] 实验方法及结果
皮下注射本发明多肽 (实施例 2的多肽 1 ) 可促生黑色素
实验发现经皮下注射本发明多肽可促生黑色素。 皮下注射不同剂量本发明多肽 1 (25, 50, 100和 200纳克 /每克体重) 后, 动物实验证明, 经皮下注射给药, 功能单位氨基酸序列与生 物素相连的多肽可促生黑色素, 在剂量为 100 纳克 /每克体重时, 牛蛙可见皮肤颜色加深。 对照组只注射生理盐水, 其牛蛙中, 未见明显肤色变化 (见附图 1 )。 说明本发明多肽是通 过 MC-1受体起作用的。
[0085] 用同样的剂量、 同样的施用方法用多肽 2代替多肽 1取得近似的实验结果。
[0086] 用 50毫克 /毫升溶液的剂量、 皮肤涂抹的方式, 用多肽 5或多肽 6进行试验, 也取得 近似的实验结果。
[0087] 结论: 本发明多肽是一种可经肌肉、 皮肤吸收进入血流的多肽, 动物实验证实其具 有促生黑色素的作用, 可用于色素沉着不足和抗紫外线辐射治疗。
[0088] 实施例 13
实验动物
成年雄性及雌性 SD大鼠, 重约 225克到 250克。 单个笼养, SPF伺养环境, 模拟自然白昼 及黑夜光线, 充足食物和水, 实验前给大鼠充足时间以适应环境, 所有行为学研究都固定时 间完成 (14一 18点)。
[0089] 实验方法及结果
将大鼠腹部的毛用脱毛剂脱除, 雌雄各 10只, 将本发明多肽 (实施例 3 的多肽 3 ) 用生理
盐水配成 50毫克 /毫升的溶液, 每日三次涂抹腹部裸露皮肤处, 每次间隔 8小时, 涂药 2小 时后可观察到雄性大鼠的阴茎勃起现象和雌性大鼠发情现象 (效果和多肽 5相近); 经 7天 的连续给药, 可见大鼠腹部给药处皮肤变黑。
[0090] 表 经皮给药本发明多肽 3可诱发雄鼠阴茎勃起, 雌鼠发情
剂量 50毫克 /毫升的溶液
雄鼠勃起总数 9/10 0/10
雌鼠发情总数 7/10 0/10
注: 0表示生理盐水对照。
[0091] 用多肽 4代替多肽 3后效果相似。
[0092] 表 经皮给药本发明多肽 4可诱发雄鼠阴茎勃起, 雌鼠发情
剂量 50毫克 /毫升的溶液
雄鼠勃起总数 8/10 0/10
雌鼠发情总数 6/10 0/10
注: 0表示生理盐水对照。
[0093] 结论: 本发明多肽是一种可经皮肤吸收进入血流的多肽, 动物实验证实其具有促进 勃起和性欲、 促生黑色素的作用, 其作用于 MC-1、 MC-3、 MC-4受体。
[0094] 实施例 14
初步大动物实验: 在对实验狗皮下大剂量 (200 微克 /每千克体重) 皮下注射本发明多肽 (实施例 3的多肽 5) 的实验。 注射后未见明显血压波动, 肝肾功能指标正常, 反应症状包 括食欲降低, 其中 2次注射后发生呕吐。 在用药后半年内观察无明显长期副作用。
[0095] 根据动物实验, 建议剂量为每公斤体重 0.1毫克, 建议剂型为水剂或油剂舌下含服, 也可制成粘膜吸入剂。
Claims
1. 一种促黑激素类似物, 其特征在于, 其多肽结构如下:
X-Z-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-Y和其无生理毒性的盐, 其中,
X=生物素酰基或无,
Z=Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg或 Arg-Arg-Arg或无,
Y=NH2或 OH,
c=环肽,
当 X=生物素酰基时, Z=无;
当 Z=Gly-Arg-Lys-Lys-Arg-Arg-Gln- Arg-Arg-Arg或 Arg-Arg-Arg时, X=无;
X、 Z不能同时为无;
无生理毒性的盐为盐酸盐、 氢溴酸盐、 氢碘酸盐、 硫酸盐、 磷酸盐、 硝酸盐、 乙酸盐、 三氟 乙酸盐、 草酸盐、 酒石酸盐、 琥珀酸盐、 苹果酸盐、 苯甲酸盐、 海藻酸盐、 甲磺酸盐、 萘磺 酸盐、 钾盐、 钠盐、 锂盐、 锌盐、 铜盐、 钡盐或钙盐。
2. 如权利要求 1所述的促黑激素类似物, 其特征在于, 其多肽结构为:
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2和其无生理毒性的盐、
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH和其无生理毒性的盐、
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)- NH2和其无生理毒性的盐、
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH和其无生理毒性的盐、
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2禾口其无生 理毒性的盐或
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH 禾口其无生 理毒性的盐; 其中,
Biotin=D-(+)生物素酰基,
Dab= ( L) 2,4二氨基丁酸残基,
D-Phe=右旋苯丙氨酸残基。
3. 如权利要求 2所述的促黑激素类似物, 其特征在于, 所述无生理毒性的盐为乙酸盐。
4. 权利要求 1 或 2所述促黑激素类似物的制备方法, 其特征在于, 包括以下步骤: 先在固 相树脂上制备出未与固相树脂裂解的、 未脱侧链保护基的功能单位氨基酸序列, 然后在固相 树脂上将功能单位氨基酸序列的 N 末端进行生物素酰化、 或者继续将负载单位氨基酸序列 中的氨基酸残基依次从 C端到 N端分别与功能单位氨基酸序列的 N末端连接, 在脱除最后 一个氨基酸残基的 N端保护基后经裂解、 纯化、 干燥成肽; N 端生物素酰化的包含功能单 位氨基酸序列的固相树脂也要经裂解、 纯化、 干燥成肽的步骤; 其中, WO 2013/127196 +π χ, , + - - PCT/CN2012/084555
权 利 要 求 书
功能单位氨基酸序列为: Arg-c(Asp-Dab-D-Phe- Arg-Trp-Lys);
负载单位氨基酸序列为: Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg或 Arg-Arg-Arg。
5. 权利要求 1 或 2 所述促黑激素类似物在制备用于治疗男性勃起功能障碍、 女性性欲低 下、 肥胖症或色素沉着不足症药物中的应用。
6. 权利要求 5 所述促黑激素类似物在制备用于治疗男性勃起功能障碍、 女性性欲低下、 肥 胖症或色素沉着不足症药物中的应用, 其特征在于, 促黑激素类似物的给药方式为粘膜给 药、 皮肤给药或注射给药。
7. 权利要求 5 所述促黑激素类似物在制备用于治疗男性勃起功能障碍、 女性性欲低下、 肥 胖症或色素沉着不足症药物中的应用, 其特征在于, 所述药物中含有具有下述结构的多肽成 分中的至少一种以及可药用载体或赋形剂:
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2
Biotin-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2
Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH
Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH2 g¾ Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Arg-c(Asp-Dab-D-Phe-Arg-Trp-Lys)-OHo
8. 权利要求 1或 2所述促黑激素类似物在制备促黑激素 MC-Rs激动剂中的应用。
9. 权利要求 8 所述促黑激素类似物在制备促黑激素 MC-Rs 激动剂中的应用, 其特征在 于 , 所述 MC-Rs指 MC-1受体、 MC-3受体、 MC-4受体或 MC-5受体。
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