CN1393266A - Slow-releasing hirudin medicine applied orally - Google Patents

Abstract

A slow-releasing hirudin medicine as the strongest thrombase suppressor is disclosed. Its advantages are long life of in body, high blood concentration up to 6-24, and relatively good biological utilization rate.

Description

Orally-taken hirudin delays (control) release dosage form

The design and the preparation technology of novel form released in oral slow (control) that this patent relates to a kind of hirudin.

Hirudin (Himdin) is a kind of small protein that is belonged to (Himdomedicinalis) Hirudo salivary gland secretion by Annelida Hirudinea doctor trematodiasis, be so far known to the strongest naturally occurring thrombin inhibitor.Since hirudin can be directly effectively anticoagulant enzymatic activity, it is considered to a kind of new drug that is used to prevent and treat cardiovascular and cerebrovascular disease that is expected to become.But natural hirudin source is limited and output very little, so utilize the synthetic lepirudin 023 ludon of genetic engineering to be paid attention to widely in recent years.So far, people have obtained the lepirudin 023 ludon of biologically active from escherichia coli and yeast expression system.

The single chain polypeptide that hirudin is made up of 65 or 66 amino acid residues is a kind of " family " that is made up of multiple shaped body.The homology of height between shaped body only has the difference of several amino acid residue, and mean molecule quantity is 7,000D.Amino acid sequence analysis shows, contains Arg in the hirudin molecule, Met, and Trp is rich in Glu, Asp, Gln, Asn.Its C end is rich in Glu, and Asp forms acidic ending; Tyr ₆₃By sulphation; There is Pro at the peptide middle part ₄₆-Lys ₄₇-Pro ₄₈The distinguished sequence fragment is difficult for by general protease hydrolysis, and with thrombin action in play a significant role.Lepirudin 023 ludon Tyr ₆₃Not by sulphation, the specific activity natural hirudin is low slightly.According to zoopery and clinical research report, hirudin has multiple biological function in vivo: it is anticoagulation efficiently, antithrombotic forms, and stops the thrombin activation and the platelet aggregation reaction of catalyzed by thrombin, the inductive various kinds of cell propagation of Trombin inhibiting.According to other anticoagulant contrast experiment, the result shows that the hirudin anticoagulant effect is better than other conventional anticoagulants far away, as heparin, aspirin, trisodium citrate etc.Hirudin and heparin compare, and anticoagulant effect hirudin is than the strong hundred times of heparin, and the consumption much less.

More hirudin class medicinal application has been arranged both at home and abroad in clinically to the treatment of cardiovascular and cerebrovascular disease.More domestic are only arranged is the Chinese patent medicine goods of main component with the hirudin, " Maixuekang " produced as the how safe drugmaker in Chongqing etc.They all are directly to be used as medicine with Hirudo to make gastrointestinal tract and directly release the oral formulations such as capsule of separating administering mode.There has been several families genetic engineering lepirudin 023 ludon class medicine to enter clinical or list marketing abroad, as hirudin of the hirudin of the Leprudin of the Hoest Marion company of Germany, U.S. HMR company, European Novartis company etc., but they are intramuscular injection or vein titration on dosage form design.

This patent has been invented the Orally-taken hirudin slow (control) that most of chronic hypercoagulability patient's long-term anticoagulant therapy are adapted and has been released novel formulation at hirudin some characteristics in clinical practice.Oral slow (control) release formulation is meant that oral drugs discharge by the slow non-constant speed of designing requirement (or constant speed) in gastrointestinal tract, medication every day number of times and the preparation that the response ordinary preparation reduces once more at least or administration time prolongs at interval to some extent.Ideal slow (control) release formulation should be like this: medicine reaches ideal valid density at site of action (receptor) rapidly, and keep this concentration reasonable time, then there are not drug distribution or drug level at other position of body only in the least concentration scope, in case reach therapeutic purposes, medicine is eliminated from site of action immediately.For reaching this purpose, fibrous root is according to the pharmacokinetics principle, and to the drug release rate of slow (control) release formulation, the dose ratio of rapid release and slow-released part is reasonably designed, calculates, and makes preparation have permanent, equilibrated blood drug level.

The technological principle of the related slow releasing preparation of this patent is mainly based on the principal agent dissolubility being reduced and the principal agent diffusion velocity being slowed down.1. reducing dissolution is the technology of cardinal principle.

1-1. under the prerequisite that keeps hirudin activity, be modified into little salt of dissolubility or fat.

1-2. under the prerequisite that keeps hirudin activity, hirudin and some macromolecular carrier are passed through covalent bonds, can obtain having the hirudin delivery system of slow-release function.

1-3. the surface area of medicine is relevant with the dissolution rate of medicine, the release that the surface area of increase drug particle can slow down medicine.

1-4. hirudin is contained in erodible skeleton, promptly makes slow releasing tablet with main matrix such as fat, wax, hydrogenated vegetable oil, stearic acid butyl esters.Hirudin is dissolved in or is suspended in these hot substrate, is ground into powder or granule after cold, is contained in the capsule or makes tablet, and wherein fatty acid ester is hydrolyzed complexity decision rate of release.

1-5. hirudin is contained in hydrophilic colloidal material.Promptly be skeleton, add diluent such as lactose, make tablet in gastrointestinal tract solution with the hydrophilic colloid, tablet imbibition gradually, hirudin is diffused into the surface gradually and is dissolved in the gastro-intestinal Fluid.Hydrophilic gel body and function methylcellulose commonly used, sodium carboxymethyl cellulose, hydroxypropyl starch, PVP, carbopol etc.2. reduce the technology that diffusion coefficient is a cardinal principle

2-1. coating.This is the main method of preparation slow releasing preparation, and the piller or the tablet that are about to hirudin block the material coating with coating.Coating retardance material can be divided into two classes: enteric material and blocker.

2-1-1. enteric material coating.Be coated on the hirudin principal agent surface with enteric material coatings such as cellulose acetate-phthalate (CAP), EudragitL, EudragitS, polyvinylether/maleic anhydride half fat, these materials can be resisted the effect of gastric acid and can dissolve in intestinal juice.

2-1-2. blocker coating.Be prepared into piller with hydrophobic polymer materials such as Lac and hirudin principal agent mixing granulation tabletting or with excipient, in coating pan, be sprinkled into the hirudin fine drug powder, become medicine clothing piller, and then use the blocker coating.

2-2. using slow (control) release formulation of microcapsule technology preparation is newer method.Microcapsule membrane is a semipermeable membrane, and in gastrointestinal tract, moisture can penetrate in the capsule, hirudin medicine in the pocket, form saturated solution, be spread in then in the outer Digestive system of capsule and absorbed by body, rate of release is determined by factors such as capsule thickness, micropore size, micropore flexibility.

2-3. be prepared into insoluble matrix tablet.Hirudin principal agent and insoluble plastics (non-toxic polyvinyl chloride, polyethylene, polyvinylacetate, polymethacrylates, silicone rubber etc.) mixing, add plastics the organic solvent solution moistening, mix thoroughly, be prepared into soft material, granulate.The flexibility influence in the dissolubility of medicine, the porosity of skeleton, aperture and hole discharges the speed of medicine.

2-4. as preparation is liquid dosage form, can add gelatin, PVP etc. increases solution viscosities with prolong drug action time.

2-5. as preparation is liquid dosage form, can be prepared into water/oil type Emulsion, and long-acting is arranged in vivo.

Example explanation I: the structure of yeast expression system:

Hirudin gene is used recurrence PCR method and is linked to be full-length gene order through chemosynthesis three segment DNAs, and sequence is with blood-eating hirudo salivary gland hirudin HV ₂Be the basis, 47 Asn sequences of coding changed into the sequence of coding Lys.Select for use yeast to finish codon, with the secreted expression carrier construction recombination plasmid, but through having obtained the Pichia barms of high-efficiency expression recombinant hirudin after repeatedly screening.Pichia yeast secretion type expression system efficiently expresses by the methanol induction hirudin, and after 72 hours fermentation, every liter of fermentation liquid can obtain lepirudin 023 ludon 1.5g.Can improve 10 times through the hirudin concentration in the juice after centrifugal, the ultrafiltration.Behind centrifugal exchange and reversed phase high efficiency liquid phase purification, hirudin purity is greater than 95%, and about 11000ATU/mg, yield is greater than 60% than vigor.Pyrogen content is less than 2EU/mg albumen.The purification hirudin is about 7000 dalton through its molecular weight of 8M carbamide SDS-PAGE electrophoretic determination, conforms to theoretical value; The polyacrylamide gel isoelectric focusing electrophoresis shows that its isoelectric point, IP is between 3.5～4.0, meets theoretical value; Terminal 15 amino acid sequence analysis of N-prove that its sequence is entirely true, amino acid profiles is analyzed, peptide figure, HPLC etc. analyze also consistent with design load, and the ratio vigor of its Trombin inhibiting is higher, all points out the structure of lepirudin 023 ludon and the correctness of physicochemical property.Example explanation II: the foundation of slow release measure: the hirudin dosage form of external (Hoechst company) is a freeze dried powder, intravenous drip.Little based on the hirudin molecular weight, can be by characteristics such as protease hydrolysiss in the intestinal, at the needs of most of chronic hypercoagulability patient's long-term anticoagulant therapy, this research is used hirudin effect convenient, that effectively also can prevent blood coagulation, thrombosis for a long time in order to reach, and has adopted unique peroral dosage form.At first, studied common oral enteric capsule formulation, carried out the rat interior animal experiment, find that the blood coagulation index has clear improvement, but required dosage has been bigger, this may be a short relatively cause of half-life in the hirudin body.For this reason, use the enteric coated capsule that slow release has the protection casing instead, can improve the utilization ratio of medicine greatly its release time about about 10-12 hour.Hirudin is coated on the hirudin principal agent surface with enteric material after lyophilizing, can resist the effect of gastric acid and is dissolved in intestinal juice.Example explanation III: zoopery result:

Zoopery according to the Ministry of Public Health anticoagulation medicine requires and domestic and international corresponding document, and we carry out the interior animal drug effect preliminary experiment of body (entrusting pharmacology teaching and research room of Huaxi Medical Univ to finish) of lepirudin 023 ludon.Oral lepirudin 023 ludon dosage is respectively 100 and 400ATU/kg, and the anticoagulant index of 7 days inner bloods is obviously improved after 3 days, and the venous thrombosis of administration treated animal significantly reduces, and drug effect and dosage positive correlation.Acute toxicity testing is experiment material with the kunming mice, is safe when finding oral dose for 10mg/kg.Experimental results show that its safety and effectiveness, oral novel lepirudin 023 ludon dosage form gastrointestinal availability is good, good stability, and the effect of anticoagulant thromboembolism preventing is obvious in the body of oral back.

Claims (9)

1. one kind is principal agent with natural hirudin or genetic engineering lepirudin 023 ludon, is aided with necessary adjuvant, through slow (control) release dosage form of orally-taken hirudin that slow (control) releases effect that has of slow (control) release formulation technology gained.

2. natural hirudin described in the clause 1 comprises that all can secrete the hirudin that is extracted the Hirudo of hirudin from the Annelida Hirudinea; This hirudin can be pure product, also can be to be the mixture of the certain purity of necessity or main component with this hirudin.

3. the lepirudin 023 ludon described in the clause 1 comprises the anomaly and the saltant of various hirudins, and its expressive host comprises antibacterial, yeast, mammal cell line, insect cell line and animals and plants.

4. necessary adjuvant comprises following aspect described in the clause 1:

4.1. oil infiltration agent.As glyceryl monostearate, castor oil hydrogenated, Dormant oils, polysiloxanes, dimethyl polysiloxane etc.

4.2. hydrophilic colloid.As cellulose derivative (CMC-Na, HPC, HPMC), PVP, arabic gum, sodium alginate, card wave spectrum etc.

4.3. coating blocker.As EC, HPMC, PVP, CAP, allyl resin.

(4.4.PVA polyvinyl alcohol).

(4.5.EVA ethylene-vinyl acetate copolymer).

4.6. polypropylene.

4.7. polylactic acid.

4.8. other polymer.As Polyethylene Glycol, poly-acetic acid, polyglycolic acid, polyamino acid etc.

4.9. other adjuvant commonly used.As sugar etc.

5. clause 1 described slow (control) release formulation technology comprises following aspect:

5.1. make the little complex of dissolubility;

5.2. generate insoluble chemical compound with macromolecular compound;

5.3. control drug particle size, it is long-pending to increase particle surface;

5.4. pharmaceutical pack is hidden in the erodible skeleton;

5.5. pharmaceutical pack is hidden in the hydrophilic colloid;

5.6. with coating blocker coating;

5.7. make microcapsule;

5.8. make insoluble matrix tablet;

5.9. increase viscosity reduces diffusion coefficient;

5.10. make Emulsion;

5.11. make osmotic pump controlled release tablet.

Oral slow (control) described in the clause 1 release effect and refer to that oral drugs non-lentamente on request constant speed in specified solvent discharges (or constant speed) that medication every day number of times reduces once more at least with relative ordinary preparation or the prolongation to some extent of administration time interval.

Slow (control) described in the clause 1 to release the action time scope be 6～24 hours.

8. oral slow (control) release dosage form comprises oral formulations such as tablet, capsule, granule, pellet, microspheres agent, microcapsule, micelle, water preparation, Emulsion, liposome, cyclodextrin inclusion compound described in the clause 1.

9. function is an anticoagulation in the concrete body of natural hirudin and lepirudin 023 ludon described in the clause 1, antithrombotic effect.