CN1163573A - Pharmaceutical compositions containing bile salt and buffer for increased bioavailability of active compound - Google Patents
Pharmaceutical compositions containing bile salt and buffer for increased bioavailability of active compound Download PDFInfo
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- CN1163573A CN1163573A CN 95194834 CN95194834A CN1163573A CN 1163573 A CN1163573 A CN 1163573A CN 95194834 CN95194834 CN 95194834 CN 95194834 A CN95194834 A CN 95194834A CN 1163573 A CN1163573 A CN 1163573A
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Abstract
Compositions containing a bile salt and a buffer such as a carbonate or bicarbonate salt which is adapted to buffer the gut to a pH of from 7.5 to 9 are capable of increasing the bioavailability of an active molecule whilst minimising the toxic side effects which are generally associated with bile salts.
Description
The present invention relates to a kind of pharmaceutical composition, particularly be difficult for by the protein of gastrointestinal absorption the Orally administered composition of peptide class and other bioactive molecule usually.
Many bioactive substances that are used for the treatment of or prevent multiple disease or disease have been used or have advised using in medical practice for many years.The most well known but be not that unique prescription biological activity protein is an insulin, it is used to control of diabetes.Other biological activity protein comprises somatomedin, interleukin and calcitonin, and the nonprotein bioactive substance comprises oligonucleotide and polysaccharide.
The easiest a kind of medication is oral.Thisly can use syrup, elixir, tablet, capsule, granule, the route of administration of powder or other any common dosage forms form is normally simple and directly, and often is minimum inconvenience and painful route of administration for the patient.
Yet unfortunately, when this class material of oral great majority, they extremely are difficult for being absorbed.At first, the preferred route of administering of protein drug and other bioactive substance is to make these materials by stomach, and stomach to be many materials comprise the unconformable environment of protein.Because the acidity of stomach, hydrolysis and proteolysis environment are aminoacid and oligopeptides with protein substance digestion effectively, carry out anabolism then, therefore, be difficult to the imagination, if adopt oral administration simply, can have in the multiple biological activity protein class material maybe can have several surviving and absorbed by health in small intestinal by stomach the time.
Adopt the enteric coating preparation can make its erosion that is not subjected to gastric acid environment, however, big relatively molecule such as peptide class and protein-based also being difficult in the small intestinal are absorbed by health.
As a result, concerning many diabeticss, numerous protein class medicine must be through parenterai administration, and promptly warp is commonly used subcutaneous, intramuscular or the administration of intravenous injection mode, thereby the patient will bear all inconvenience, shortcoming and the difficulty of bringing thus.
This is not isolated problem, because need be very general with the protein substance control disease.For example, there is a large amount of diabeticss in many in the world countries, and have many other diseases need use protein-based chemical compound or other macromole to treat.Osteoporosis is the another kind of disease that can treat with protein (using calcitonin in the case), and the albumen growth hormone can be used for treating dwarfism.
Therefore, need a kind of protein and other macromole class pharmaceutical preparation significantly, they can be oral, and the bioavailability of acceptable activity material is provided.
Known, when pharmaceutical active compounds during with some bile salts administration, their bioavailability increases.Ke Kaimi people such as (Kakemi) once discussed this problem; [" chemicals communication " (Chem.Pharm.Bull;) 18 (2) 275-280 (1970)], he thinks when multiple material during with taurocholate or glycocholate administration, can increase its bioavailability.Several agates of Ke people's such as (Kojima) further experiment [" chemicals communication " (Chem.Pharm.Bull), 25 (6) 1243-1248 (1977)] illustrates that sodium cholate can increase the absorption of multiple material, but also quickens the release of cell membrane part.
In addition, be as absorption enhancer in some other documents of prior art with bile salts, comprising GB-A-22444918, it tells us, when somatostatin during with chlolic acid derivatives such as chenocholic acid or ursodesoxycholic acid administration, the bioavailability of somatostatin can improve.
The absorption that it is believed that bile salts improvement bioactive substance is because they act on the cell membrane of epidermis cell.Cell membrane easier see through that become may be because the detergent action of bile salts has been removed fat from cell membrane, and makes them become easier to flow.A kind of theory thinks that cell membrane increases transparent performance makes active substance pass epidermis cell.In addition, because the Cytoplasm sodium that causes of permeability of cell membrane increase or the change of calcium level, may change the cytoskeletal structure of epidermis cell, this will cause the variation of tight conjunction integrity, thereby make active substance can pass intercellular gap.No matter which kind of mechanism as if all show, increase permeability of cell membrane absorption is increased.
Yet the pharmaceutical preparation that comprises bile salts always has problem because, when the bile salts that uses requirement when improving bioavailability, they have unacceptable high toxicity.Someone points out, the permeability increase of the epidermis cell that contacts with bile salts can increase ion and flow to or flow out cell.For making these cells keep survival, ion such as sodium in the cell, the concentration of potassium and calcium ion must remain in the relative narrow scope, and this scope may be that ion flow that the existence owing to bile salts causes increases and makes intracellular ion concentration exceed the scope of cell survival.Therefore, be that they always are considered to have unacceptable low therapeutic index with bile salts as the problem of absorption enhancer.The term that uses in whole description " therapeutic index " refers to following ratio:
Toxicity dose: strengthen absorbed dose
Therefore, can reduce bile salts toxicity and increase bile salts effectively is extremely useful at the therapeutic index of certain point, can be used to pharmaceutical composition and not have unacceptable toxic and side effects at this bile salts.
First aspect present invention provides a kind of medicine and/or veterinary composition, and said composition contains bioactive substance, bile acid or salt and a kind of with the buffer agent of enteral pH regulator to 7.5-9.
WO-A-9012583 discloses and has contained active ingredient, the pharmaceutical composition of the additional composition of bile salts and bile.Yet the document thinks that may have only additional composition is useful in this compositions, and this additional composition is additional bile salts and bile fat, particularly phospholipid.Though bicarbonate (the preferred pH regulator agent of the present invention) is a biliary component, does not mention bicarbonate in WO-A-9012583, clearly, bicarbonate is not look to as the useful composition that contains the compositions of bile salts and pharmaceutically active agents.Therefore, bicarbonate or other pH regulator agent acting on aspect effectively increasing the bile salts therapeutic index do not have open certainly in this existing document.
Should be appreciated that term " bile salts " and " bile acid " can exchange use in the present invention, because, no matter be whether salt or its corresponding acid exist, all will depend on the pH value of surrounding.Therefore, solid preparation of the present invention both can comprise bile salts, also can comprise bile acid.But if Pharmaceutical composition often is to use bile acid, then its pH wants to make bile acid be converted to the form of salt in solution.
When the present composition uses bile salts, preferably have suitable equilibrium ion and exist, as sodium or potassium.Can also use, for example, ammonium ion, but not as preferred ion.
Bile salts is the surfactant of natural generation.They are one group of chemical compounds that have based on common " skeleton " structure of cholestanic, have all found cholestanic in all mammals and high vegetable.Bile salts can be one, two or the trihydroxy thing; They comprise one 3 Alpha-hydroxy usually, and other hydroxyl, modal is at C
6, C
7Or C
12, both can (β) above the planes of molecules also can below (α).
Said bile salts is included in the polynary sterol of amphiphilic of the carboxyl that has conduct uncle pendant moiety in this compounds.Their prevailing examples come from the cholesterol metabolism in mammal, and are found in bile, and are present in whole intestinal with derivative form.
In this manual, this term also can be used for the synthetic analogues of natural generation bile salts, because they also show similar biological agent.Or be used for the microorganism derived molecules, as fusidinic acid and derivant thereof.
Bile salts both can be uncombined also can be bonded.Term " uncombined " refers to that wherein uncle's side chain has the bile salts of single carboxyl, and this carboxyl is in the end position and be not substituted.The example of non-binding bile salts comprises cholate, ursodeoxycholate, chenodesoxy cholate and dexycholate.Binds bile salts is that wherein uncle's side chain has a bile salts that replaces carboxyl.Substituent group is normally linked amino acid derivativges on the carboxyl of bile salts by nitrogen-atoms.The example of binds bile salts comprises taurocholate, glycocholate, taurodeoxycholate and sweet dexycholate.
The content of bile acid depends on selected concrete bile acid in single-dose preparations, and the bile acid dissolubility in the aqueous fluids and soluble end in intestinal.For chenodeoxy cholic acid and other acid of great majority, this content should be in the 10mg-1g scope, preferred 20-200mg, most preferably 30-100mg.For deoxycholic acid, consider that its activity is big slightly, so the maximum of this content generally is no more than 500mg.
The intestinal of many animals (especially people and other mammal) has natural adaptation to neutral following pH.The present composition contains the pH regulator of the intestinal buffer agent to pH7.5-9.This reagent both can rely on the chemical property of itself or pass through the control consumption, but normally relied on both " adaptively " to regulate pH value.Concerning intestinal, best pH is the scope that is adjusted to 7.8-8.3.
If the pH that can successfully use simple buffer agent to regulate intestinal is above-mentioned specified scope in the present invention, so preferably this pH regulator agent cushions the ability of intestinal pH to above-mentioned scope in addition.So just can obtain longer lasting drug effect, this needs in many occasions.And buffer agent has the ability of difference between bigger adaptation patient and the intestinal pH of patient own, and to the viability of any single patient viewing duration intestinal pH; Particularly, buffer agent can play the safety curtain effect, guarantees that the pH of patient's intestinal can not change in preparation administration process of the present invention beyond the safety limit basically.
Being used for the present invention, to regulate pH be carbonic acid and bicarbonate ion to two kinds of the optimum reagent of appropriate value, and they can use respectively, also can be used in combination.In the following discussion, mainly with the related principle of bicarbonate explanation, but same principle is applicable to other reagent that can produce similar effect to enteral pH.
Directly determine that with pH regulator necessary reagent dosage in the desired extent be very difficult, because enteral pH can change at 5-7, and wherein water content and the buffer capacity of itself are also changing, and its effect is to keep a low pH.Therefore, the suitable consumption that determine to control the reagent of pH just need be considered " worst case ".By observing the situation of change that potential pH regulator agent is added to pH in 50mMMES (morpholino ethyl sulfonic acid) solution of pH6.0, can obtain one and instruct consumption.Being used for concentration of the present invention is will be with pH regulator to 7.5-9; Preferred concentration is to be adjusted to 7.8-8.3.(because following will the explanation, the weight content of pH regulator agent represents it generally is the amount that produces desired concn in 10ml liquid).List the resulting pH value of preferred bicarbonate of the variable concentrations that is dispersed among distilled water or the MES below.Magnesium hydrogen salt concentration water MES (molal quantity)
0.002????????8.86????6.42
0.004????????8.67????6.58
0.008????????8.63????6.86
0.016????????8.57????7.14
0.031????????8.55????7.40
0.062????????8.49????7.68
0.125????????8.41????7.93
0.250????????8.30????8.14
0.500????????8.16????8.05
1.000????????8.02????7.96
Therefore, in most cases the minimum amount of bicarbonate of the present invention is about 0.045M, and it is about 7.5 at the pH that enteral produces.To the 10ml dispersion liquid, this is equivalent to the 40mg sodium bicarbonate.
(＜2M) pH is about 8.01 to the saturated aqueous solution of sodium bicarbonate.This shows that needn't consider the concentration of used preferred bicarbonate, pH can not reach more than 9.0.In some cases, the concentration of salt solution of bile acid itself has been enough to make pH to be raised to suitable level, although its effect does not have preferred bicarbonate strong.Therefore, though always do not need, also be possible as the pH regulator agent with bile salts itself or other bile salts.
The buffer capacity of compositions is big more, and the pH in the above-mentioned specified scope is long more in the time that enteral keeps, and then the effect time of the present invention also will be long more.
Be applicable to that two kinds in the optimized buffer agent of the present invention is carbonic acid and bicarbonate ion, they can use respectively, also can be used in combination.As mentioned above, the advantage of the present composition is that they can increase the therapeutic index of the bile salts in the compositions effectively.In conjunction with and non-binding bile salts all have this useful effect, but slightly different in the effect of carbonic acid or bicarbonate ion and the non-binding bile salts in the binds bile salts.
For binds bile salts, the existence of capacity bicarbonate or carbonate makes in cellular exposure effectively increases permeability of cell membrane during in the bile salts of specified rate, and does not influence cell viability.This means to reaching the consumption that the given value added of permeability can reduce bile salts, promptly when not increasing the bile salts side effect, increase the permeability of cell.
Non-binding bile salts under carbonate or the bicarbonate existence does not increase at cellular exposure cell permeability during bile salts, and on the contrary, the toxic action of pair cell reduces after being exposed to bile salts.This means that the dosage at bile salts under the situation that does not influence cell viability increases to some extent in the presence of bicarbonate radical or carbanion.
In a word, the consumption of pH regulator agent will depend on the administration situation of said composition to the patient when units dosage composition is dispersed in the appropriate amount of fluid (this liquid is present in whole enteral), promptly allow compositions be distributed in whole enteral.Though more accurate calculating can be with reference to above-mentioned MES buffer agent test, the concentration of pH regulator agent is at least about 0.01M.But for preferred carbonate and bicarbonate, the concentration of bicarbonate or carbonate is usually greater than 0.05M.Though can use higher concentration, to 1M, most preferred concentration is to be at least about 0.1M as height.
It is 30cm that the present composition may be distributed in enteral concrete length, may be about 10ml at the content liquid of the enteral of this segment length.But the 30cm that is arbitrarily chosen as suitable length should compress, and the present invention does not want the compositions that is distributed on this segment distance of small intestinal is limited.Reason is to want to allow the compositions that is distributed in enteral keep short or much longer time, and for example, the compositions that continues to discharge can be disperseed more lentamente, and the longer length that therefore can distribute in small intestinal.These compositionss are that those of ordinary skills are familiar with, and they can determine optimal types of compositions at an easy rate, to meet concrete treatment requirement.
If the compositions that adopts will be scattered in a segment distance long or short in the small intestinal, the consumption of water should correspondingly increase or reduce so, contains the units dosage composition that reaches prescribed concentration after the dispersion in the said water.
Being dispersed in the water with the unit dose of determining required bicarbonate minimum amount will be to give the dosage of patient's administration at any time.For liquid preparation, this dosage is that doctor or pharmacists pass through to calculate predetermined dosage.For solid preparation such as tablet or wafer, normally single tablet of unit dose or single capsule.But, also there is needs of patients to surpass the situation of an a slice or a capsules, for example, heavy dose of if desired active substance then can be divided into the bicarbonate of aequum two or more tablets or capsule in this case.
Obviously, because they can increase the dissolubility of bile acid, other beneficial effects of therefore preferred carbonate or bicarbonate ion also can improve.Generally, it is 6.8 or following corresponding acid that bile salts begins to be converted into pH, and should the water insoluble solution of acid.Because buffer agent has and the present composition is buffered to pH is about 7.5 or above effect, so lysed bile salts rather than insoluble bile acid will occur.When lysed bile salts was in solution, it can act on epithelial cell, and this is impossible when the solid acid form.The concentration of buffer agent is high more, and it is just fast more to obtain satisfied pH, and then bile acid or salt dissolve soon more; This will cause bile salts that higher local concentration is arranged in solution, and then cause the permeability of bigger efficient ground raising to bioactive substance.
Also a reason may be because the special beneficial effect of carbonate or bicarbonate ion is to be expressed in the such fact in enterocyte surface with the bicarbonate receptor in some way to interrelate.But, if the essence of this contact---its certain existence---not clear at present, and, under any circumstance, should emphasize that the correction of this theory or other situation all can not limit effectiveness of the present invention.The result who obtains with bicarbonate is normally unusual.
As mentioned above, can think that one of effect of bicarbonate radical or carbanion is to guarantee that bile salts can dissolve.Yet the existence of calcium ion has increased pH makes bile salts become common form, and, make insoluble bile acid begin from solution, to be precipitated out because pH value is low.PH value changes according to concrete bile salts, but wants to prevent the bile acid precipitation.For this reason, it is favourable often containing calcium ion chelator in the compositions.Said chelating agen comprises two or tricarboxylate (as citrate), edetate (EDTA), ethylene glycol bis (beta-amino ether) N, N, N ', N '-tetraacetate (EGTA) or phytate, and other polyphosphide.
When using citrate as calcium ion chelator, preferably it is the water-soluble form of energy.Therefore, although also can use ammonium citrate in some cases, optimal salt is sodium citrate or potassium.
Especially, term " bioactive substance " comprises the pharmaceutically active protein substance.This protein substance can be a true protein, or contains protein, for example, has not only contained protein but also has contained the glycoprotein of glycosyl.
This material both can be used as people's medicine or veterinary drug is used for the treatment of or prevent disease or its symptom, also can be used for face-lifting and diagnosis.The example that meets the present invention and can be used for the protein-based biological substance of oral or rectally preparation comprises protein or peptide hormone or releasing factor, and as insulin, no matter calcitonin and growth hormone are the human or animals, and is still semi-synthetic or complete synthesis; Or other biologically active peptide, as human interferon-alpha, and comprise IL-1, IL-2, IL-3, IL-4 and IL-5 are at interior interleukin.These protein and other proteinic analog and active fragment also can use.
Because be difficult in the pH value dissolving of the present invention's expection under the insulin normal condition, but the present invention can not only make insulin generation effect and make it act on finely, this point is extremely important.As if because the interaction of not expecting between insulin and the bile salts can only disperse insulin, wherein the effect of bile salts is to make it stable in bicarbonate or other high pH solution.
Bioactive substance also can be an oligonucleotide, as the antisense oligonucleotide and can be used for interfere RNA aspect viral infection or cancerous cell duplicate, or can be used for proofreading and correct the analog of other form aspect that is not suitable for proliferation of cells.Polysaccharide such as heparin also are applicable to the present invention, because polysaccharide is one or more protein, and the combining form of nucleic acid or polysaccharide.
The molecular weight of bioactive substance should preferably be no more than 20,000Da.Even this is because used the present composition that the permeability of cell is increased to some extent, but to reach also nothing the matter absolutely not of effective bioavailability with bioactive molecule greater than this amount.Therefore, generally speaking, bioactive molecule is more little, and its transmission is easy more.So preferred molecular weight is less than 10, the bioactive molecule of 000Da, most preferably molecular weight is less than 5,000Da's.
At present, the consumption of bioactive substance depends on its internal potential in essence.What all were necessary is still to show it under the situation that the capacity material exists to have required activity when oral.For medicine, dosage will be deferred to doctor or clinicist's guidance.
Other operable excipient comprises the lactose as filler, in order to the uniformity that guarantees compositions with help preparation; Ac-di-sol
TM(crosslinked hydroxyl methyl ether sodium cellulosate), a kind of extender that helps disintegration of tablet; And the polyvinyl pyrrolidone that in pelletization, is often used as binding agent.
Except liquid dosage form, also has solid dosage forms usually.Solid dosage forms is preferred, because rely on enteric coating can avoid active substance to be digested under one's belt at an easy rate, and, can guarantee more easily that active ingredient and bile salts complete sum arrive small intestinal simultaneously; This implements for liquid dosage form wants difficult ground many.If compositions is a solid dosage forms, for long-term storage, it should be done substantially.It can be a sheet, piece, powder, granule or particulate form, and can contain suitable, filler that those of ordinary skills know or binding agent.Powder, granule or microgranule can fill in capsule.As mentioned above, bile acid or bile salts may be used to solid preparation.
Liquid dosage form has above-mentioned deficiency.But, perhaps be syrup or the best administering mode of elixir with the enteric coating capsule for certain special requirement present composition also can be made into liquid form.
For rapidly or continue to discharge or with these two kinds of releasing pattern combinations, the present composition can be made into corresponding form.Said composition can also be made into to discharge immediately, postpones to discharge or pulse release type preparation.The amount of used buffer agent depends on that the formulation method of compositions disperses the needed time with it at enteral in the compositions.Therefore, the content of buffer agent is more much bigger than the consumption in the immediate release formulations usually in slow releasing preparation.
In a word, the technology can with medicament preparation field those of ordinary skill known of the present composition prepares by mixing various components simply.
The invention still further relates to the method for improving the active substance bioavailability, this method comprises the co-administered active substance to the patient, bile salts and enteral pH is adjusted to the buffer agent of 7.5-9.
Therefore, second aspect of the present invention provides bile salts and adjusts the purposes of buffer agent aspect certain preparation of preparation of enteral ph to 7.5-9, and said preparation is used to increase the bioavailability of uniting the bioactive substance of use with it.
The preferred feature of first aspect present invention has been done necessary explanation.
To further specify the present invention with embodiment and accompanying drawing below, wherein:
Fig. 1 represents that the bile salts viability afterwards that the Caco-2 cells in vitro is exposed in balanced salt or the bicarbonate solution compares, and the result represents with the maximum tolerated concentration mg/ml of bile salts;
Fig. 2 represent the Caco-2 cells in vitro be exposed to during the binds bile salts in HBSS or the bicarbonate solution and dimethyl diaminophenazine chloride permeability afterwards relatively, the result represents with the maximum tolerated concentration mg/ml of bile salts;
Fig. 3 represent the Caco-2 cells in vitro be exposed to during the non-binding bile salts in HBSS or the bicarbonate solution and dimethyl diaminophenazine chloride permeability afterwards relatively, the result represents with the maximum tolerated concentration mg/ml of bile salts;
The percentage ratio that dyestuff discharged after Fig. 4 curve representation cell dye was cultivated with the chenodesoxy cholate in the different buffer agents;
The percentage ratio that mitochondria activity reduced after Fig. 5 curve representation was cultivated with the chenodesoxy cholate in the different buffer agents;
Fig. 6 relates to embodiment 3, and its is represented to pH and bile salt concentration after pig injection (intrajejunally) administration the action effect of salmon calcitonin; And
Fig. 7 relates to embodiment 4, the degree that efficient improved when its expression oral insulin solid dosage forms was used in combination with bicarbonate.
The preparation of the used solution of embodiment
Prepared at concentrations dimethyl diaminophenazine chloride liquid storage with 0.5mg/ml in DMEM (Dulbecco ' s Modified Eagle Medium), then, use TCM (Tissue Cultrue Medium) or HBSS (Hanks Balanced Salt Solution) to be diluted to 1: 10 before use.
The HBSS of preparation contains following composition:
g/L?????mM
CaCl
2.2H
2O???0.19????1.26
KCl????????????0.40????5.37
KH
2PO
4???????0.06????0.44
MgCl
2.6H
2O???0.10????0.49
MgSO
4.7H
2O???0.10????0.41
NaCl???????????8.00????133.33
NaHCO
3????????0.35????4.17
Na
2HPO
4??????0.48????3.38
D-glucose 1.00 5.56
Phenol red 0.01.
Preparation 0.1M sodium bicarbonate distilled water solution (buffer solution).Prepare sodium acetate similarly, the 0.1M distilled water solution of sodium borate and sodium carbonate.They are all in an embodiment as buffer.
Prepare the testing liquid of containing various concentration bile saltss at HBSS or in a kind of 0.1M buffer.Test following bile salts: non-binding bile salts: sodium cholate, ursodesoxycholic acid sodium, chenodeoxy cholic acid sodium, NaTDC; Binds bile salts: sodium taurocholate, sodium glycocholate, sodium taurodeoxycholate, Sodium glycodeoxycholate..
To be the prepared at concentrations MTT liquid storage of 5mg/ml in distilled water, be diluted to 1: 10 with TCM before using.
Test described in the embodiment 1 and 2 is known test and meets British Standard (BS 5750).The Caco-2 cell is that those of ordinary skills generally believe only enterocyte external model.Embodiment 1 measures the preparation of the In vitro culture experimental cell of cytotoxicity and permeability of cell membrane
Adding concentration with branch ground such as 200 μ l in each hole of 96 hole microtiter plates is 1 * 10
5The Caco-2 cell suspending liquid of cell/ml.In outermost hole, add 200 μ l saline, with the payment evaporation.Contain 5% CO at 37 ℃
2Air in cell was cultivated 8 days in Iger medium (DMEM) tissue culture medium (TCM) (TCM) of high glucose Dulbecco modification, and add as required.Carry out the research of initial damage or recovery then with this cell.A. estimate the influence of toxicity to initial damage
The dimethyl diaminophenazine chloride dyestuff is absorbed by living cells.Having the cell of strong permeability film can lose dyestuff makes it to enter into culture medium and bathes.Therefore, after being exposed to substances, measure the permeability that the amount that remains in dyestuff in the cell can directly be estimated cell membrane.Thereby in the method, cell at first is colored, and cultivates with substances then, measures the degree that the cultivation of carrying out with substances causes that between exposure period dyestuff leaks from cell at last.Adopt 2 hour open-assembly time; The following method of this experimental basis is carried out.
From the cell monolayer in each hole, remove TCM, replace 200 μ l dyestuffs.With contain 5% CO of crowd's dish at 37 ℃
2Air in cultivated 2 hours, check level dyeing and normal morphology situation then.From each hole, remove dyestuff and replace the suitable buffer agent of 150 μ l.Be listed as the testing liquid of removing buffer agent the hole and replacing 150 μ l Cmaxs from the 2nd again.Add 150 μ l testing liquiies in the 3rd each hole of row, reach uniform mixing by aspirating repeatedly, wherein 150 μ l move on in the 4th row hole again.Each dish is repeated the diluent of this process with the substances that obtains another row dilution factor twice of every behavior.Have delegation to organize in contrast with HBSS in each dish, another row can be used as the buffer matched group if desired.
5% CO at 37 ℃
2Middle cultured cell 2 hours sways cultivation 20 minutes then from each hole suction dyestuff, and with each dish in containing the 50% ethanolysis absorption solution (100 μ l/ hole) of 1% glacial acetic acid.
In order to give the dyestuff that remains in the cell quantitative, in 550nm position measurement trap.Reading value and buffer matched group are relatively estimated the situation that dyestuff spills from cell.
Therefore, this test is used to be exposed to the measurement of permeability of cell membrane during the bile salts.B. the recovery after poisoning
The dimethyl diaminophenazine chloride dyestuff is absorbed by living cells but not survivaling cell is not colored.The cell dye content that the cell membrane permeability increases can reduce.In distinct methods, cell is at first cultivated with substances, cultivates with dyestuff then, measures the cultivation of carrying out with substances at last and cause that cell can not absorb/keep the degree of dimethyl diaminophenazine chloride after exposing.Adopt 2 hour open-assembly time and 3 hour recovery time in the optimum growh material, dyestuff is absorbed during this period.This is tested in order to following method:
From each hole, remove TCM and replace the suitable buffer of 150 μ l.
Be listed as the testing liquid of removing buffer the hole and replacing 150 μ l Cmaxs from the 2nd.
Add 150 μ l testing liquiies in the 3rd each hole of row, reach uniform mixing by aspirating repeatedly, wherein 150 μ l move on in the 4th row hole again.Each dish is repeated the diluent of this process with the substances that obtains another row dilution factor twice of every behavior.Have delegation to organize in contrast with HBSS in each dish, another row can be used as the buffer matched group if desired.5% CO at 37 ℃
2Middle cultured cell 2 hours.
From the cell monolayer in each hole, remove supernatant and replace 200 μ l dyestuffs.
With contain 5% CO of each dish at 37 ℃
2Air in cultured cell 3 hours.From each hole, aspirate dyestuff then, and each dish is swayed cultivation 20 minutes in containing the 50% ethanolysis absorption solution (100 μ l/ hole) of 1% glacial acetic acid.
In 550nm position measurement trap.Reading value and buffer matched group are relatively estimated the situation that dimethyl diaminophenazine chloride spills from cell behind the activation picked-up dyestuff.
Therefore, this test is used to be exposed to the measurement of inner cell membrane permeability between convalescent period behind the bile salts.
Bicarbonate and HBSS relatively list in table 1 and 2 and Fig. 2 and 3 to the action effect of the cell handled with bile salts.HBSS and bicarbonate, the action effect of acetate and borate buffer is relatively listed in Figure 4 and 5.
It should be noted that for HBSS, the ratio of each component is adjusted to and can makes the pH of solution reach 7.0-7.2 when they are dissolved in distilled water.In small intestinal, pH will be lower than this value.Comparatively speaking, as from before the table given can see, adopt bicarbonate can make pH approach 8.0, and, when described here each experiment finishes also certainly so.Embodiment 2 utilizes MTT dyeing to measure mitochondria activity and then measures the preparation that Cytotoxic In vitro culture is tested cell
Adding concentration with branch ground such as 200 μ l in each hole of 96 hole microtiter plates is 1 * 10
5The Caco-2 cell suspending liquid of cell/ml.In each dish outermost side opening, add 200 μ l saline, with the payment evaporation.Contain 5% CO at 37 ℃
2Air in cell was cultivated 8 days in high glucose DMEM tissue culture medium (TCM) (TCM), and add as required.The mitochondria activity test
MTT is a tetrazolium gold father-in-law salt, and it is an insoluble purple crystals of passing through the conversion formation of mitochondrial dehydrogenase in survivaling cell.Dead cell does not influence this conversion process.The available colorimetry of crystalline formation is carried out quantitative analysis, and is used for estimating mitochondria activity and cell survival.The MTT liquid storage, the preparation of testing liquid and buffer as mentioned above.
In the method, cell is at first cultivated with substances, cultivates with MTT then, measures the cultivation of carrying out with substances at last and has changed the active degree of cell mitochondrial.Adopt 2 hour open-assembly time and 3 hour recovery time, add dyestuff during this period.
From each hole, remove TCM and replace the suitable buffer agent of 150 μ l.
Be listed as the testing liquid of removing buffer agent the hole and replacing 150 μ l Cmaxs from the 2nd.
Add 150 μ l testing liquiies in the 3rd each hole of row, reach uniform mixing by aspirating repeatedly, wherein 150 μ l move on in the 4th row hole again.Each dish is repeated the diluent of this process with the substances that obtains another row dilution factor twice of every behavior.Have delegation to organize in contrast with HBSS (referring to embodiment 1) in each dish, another row can be used as the buffer agent matched group if desired.At 37 ℃ of 5% CO
2Middle cultured cell 2 hours.
From the cell monolayer in each hole, remove supernatant and replace 200 μ l MTT.
Each is coiled at 37 ℃ of 5% CO
2Middle cultured cell 3 hours detects by an unaided eye then to estimate the dye level in each hole.From each hole, aspirate dyestuff, and each dish is swayed cultivation 20 minutes in containing the anhydrous isopropyl alcohol desorption solution of 0.1M HCl (100 μ l/ hole).
Measure trap at the 550-650nm place.Reading value and buffer agent matched group are relatively estimated the variation of mitochondria activity.Painted the weakening of following matched group shows weakening of mitochondria activity, and to be that active substance is toxic in the culture medium demonstrate fully.The result
The Caco-2 cell of table 1-in the presence of bicarbonate or HBSS is at the Cmax (mg/mL) of external tolerant non-binding bile salts
| Bile salts | Test(ing) medium | Dimethyl diaminophenazine chloride (during) | Dimethyl diaminophenazine chloride (afterwards) | MTT viability (afterwards) |
| Cholate | The HBSS bicarbonate | ????0.313 ????0.625 | ????0.313 ????1.25 | ????0.313 ????2.5 |
| Ursodeoxycholate | The HBSS bicarbonate | ????1.25 ????0.313 | ????0.313 ????1.25 | ????0.625 ????1.25 |
| Chenodesoxy cholate | The HBSS bicarbonate | ????0.078 ????0.04 | ????0.03 ????0.125 | ????0.125 ????0.25 |
| Dexycholate | The HBSS bicarbonate | ????0.04 ????0.078 | ????0.01 ????0.04 | ????0.01 ????0.156 |
Table 2-in the presence of bicarbonate or HBSS the Caco-2 cell at the Cmax (mg/mL) of external tolerant binds bile salts
| Bile salts | Test(ing) medium | Dimethyl diaminophenazine chloride (during) | Dimethyl diaminophenazine chloride (afterwards) | MTT viability (afterwards) |
| Taurocholate | The HBSS bicarbonate | ????1.25 ????0.313 | ????5 ????5 | ????5 ????5 |
| Glycocholate | The HBSS bicarbonate | ????1.25 ????0.625 | ????2.5 ????2.5 | ????2.5 ????2.5 |
| Taurodeoxycholate | The HBSS bicarbonate | ????0.313 ????0.039 | ????0.313 ????0.313 | ????0.313 ????0.313 |
| Glycodeoxycholate | The HBSS bicarbonate | ????0.313 ????0.078 | ????0.313 ????0.313 | ????0.313 ????0.313 |
Table 1 and 2 expression embodiment 1 and 2 described experimental results.First has listed the bile salts in the test(ing) medium in the table, and itself lists in secondary series test(ing) medium.Test(ing) medium is HBSS or 0.1M sodium bicarbonate solution.
The result who is exposed to during the bile salts and afterwards dimethyl diaminophenazine chloride is measured provides the bile salts Cmax of representing with mg/mL, and wherein the cell permeability keeps uninfluenced (comparing with cultured cells in not having the culture medium of bile salts).Represent the Cmax of bile salts in the result of MTT string, wherein the viability of cell keeps uninfluenced after cultivating.
Table 1 has provided the experimental result of non-binding bile salts, and it is pointed out in all cases, it is bigger than the numerical value that applies by HBSS to apply bile salts by bicarbonate in dimethyl diaminophenazine chloride and MTT (after the exposure) two row, although little in the difference of dimethyl diaminophenazine chloride (between exposure period) string two numbers.This expression, the permeability of cell does not have big change when bicarbonate ion exists between with the bile salts culture period, still, because after cultivating in the cell toxic action appears, so need more bile salts.Therefore, this means when bicarbonate ion exists and toxicity acts on before the cell significantly, these cells can be exposed to more bile salts.Because can increase the consumption of bile salts, so the permeability of cell also can be increased under the sex situation of cell survival not being subjected to after cultivating.
Table 2 has provided the experimental result of binds bile salts, and it points out in all cases, and the numeral of dimethyl diaminophenazine chloride (between exposure period) row increases when using bile salts under the situation about existing at bicarbonate, and numerical value is constant in dimethyl diaminophenazine chloride and MTT (after the exposure) two row.This expression when cultivating with bile salts, has the permeability that bicarbonate exists needs less bile salts to increase cell than there not being bicarbonate to exist.
Listed result also represents, the influence that the permeability of cell and viability are not existed by bile salts after cultivating.Therefore, when bicarbonate ion exists, can use binds bile salts in the training period, under the situation that does not increase bile salt concentration, increase the permeability of cell, nor can oppositely influence cell survival.
Fig. 1 to Fig. 3 is table 1 and 2 listed results' a pictorial representation.Fig. 1 represents the action effect of bicarbonate to the viability of the aquicultural cell of usefulness bile salts, is the measurement result of MTT viability test.For binds bile salts, can see that the existence of bicarbonate does not influence the viability of cultivating cell afterwards with bile salts, and for non-binding bile salts, the concentration of bile salts obviously increases when bicarbonate exists, and does not influence their viability with the cell of such bile salts processing.
Fig. 2 is illustrated in measuring the cell survival of measuring by dimethyl diaminophenazine chloride between the binds bile salts culture period and afterwards.As can be seen, between exposure period, when the 0.1M bicarbonate exists, when being lower than HBSS and existing for the consumption that increases the needed bile salts of cell survival.But after exposing, the permeability of cell transfers to normally.This means that for the bile salts of specified rate, the cell permeability can obviously increase between exposure period.Yet, after exposing, with matched group (with bile salts with HBSS but not the bicarbonate cultured cells) viability of comparing cell keeps change.
Fig. 3 is similar to Fig. 2, is that non-binds bile salts exists the result under the situation.In this case, can see, bicarbonate does not very clearly influence the permeability of cell during being exposed to bile salts, but after exposing, and, increase at the Cmax that does not cause the bile salts that to use under the situation that the cell permeability increases, thereby the cell permeability obviously reduces when bicarbonate exists.
The curve representation of Fig. 4 is exposed to staining cell the percentage ratio that dyestuff discharges behind the bile salts of different pH value in dimethyl diaminophenazine chloride is measured, be example with the chenodesoxy cholate.This curve obtains quite clearly representing that the dyestuff from discharging the cell of 7.5 above culture medium culturings with pH wants much less, and this pair cell short-term and the long-term cultivation that recovers predictability can be better under the situation of rising pH.Equally, Fig. 5 is illustrated in that toxicity obviously reduces under the condition of rising pH, the figure shows the percentage ratio that mitochondria activity reduces after cultivating with the chenodesoxy cholate in the different buffer.
The preparation of pH7.0 culture medium is that the HBSS solution that will contain 3% (v/v) 1M HEPES (ethoxy propylidene ethyl sulfonic acid) transfers to pH7.0.The preparation of pH7.5 culture medium is to contain 50mMTRIZMA
TMHBSS solution transfer to pH7.5.The preparation of pH8.0 culture medium is to contain 50mM TRIZMA
TMHBSS solution transfer to pH8.0.The preparation of pH8.5 culture medium is that the HBSS solution that will contain 50mM Tricine transfers to pH8.5.This result also shows, is that 8.0 bicarbonate can be observed this phenomenon with 0.1M pH, but is that 7.5 HBSS does not then observe with pH only.
In the body that the following example is reported, can prove that the concentration of used bicarbonate has needed effect really in the enteral environment in the experiment.Embodiment 3 body internal efficiency researchs---salmon calcitonin (Salmon Calcitonin)
Earlier the pig that will be used to study (DABAI * DABAI/Landrace, male, 65kg) carry out preparatory operation after the anesthesia, after 10 days two electrodes are inserted dorsal aorta through central saphena tremulous pulse.The intubate of the about 1.6-2mm of internal diameter is inserted jejunum.Feed a little water arbitrarily for these pigs, one night of fasting, then every day twice of feeding sooner or later.Those days in treatment are forbidden the feeding in morning, after obtaining last blood sample.
Medicament salmon calcitonin (sCT) in intubate is instilled into jejunum, is washed with warm disinfectant then.Before and after the administration blood sampling repeatedly, each 8ml, and separated into two parts.A part allows to condense in room temperature, extracts serum and is stored in-20 ℃, for future use.Another part is transferred to the heparinization pipe, and adds 4000kiu (iu is an iu) aprotinin.Centrifugal decant blood plasma afterwards also is stored in-20 ℃.Measure serum calcium cancentration with colorimetry after the complexation of the responsive chelating agen arsenazo III of calcium.
Study with 9 pigs.Divide administration in 3 days, be separated by at least one day at every turn, in 3-Leg Intersection mode at random, therefore, all pigs are all accepted three treatments.Each therapeutic agent can only contain the 625iu sCT that is dissolved in the 2ml phosphate buffered saline (PBS), pH is 7.5, or use with 25mg chenodeoxy cholic acid sodium (_ Δ _), or use with 50mg chenodeoxy cholic acid sodium (_ ■ _), or use with 50mg chenodeoxy cholic acid sodium, pH is reduced to 6.5 (_ * _) with hydrochloric acid then.The results are shown in Fig. 6, represent with respect to the variation of calcitonin with plasma calcium concentration and time.This shows that bile salts is improving the concentration that efficient aspect the calcitonin intake depends on contained bile salts, and the pH value during administration.Embodiment 4 body internal efficiency research---insulins
With embodiment 3 used same procedure pig is carried out pretreatment.With the standard enzyme analytical method glucose of blood sample is analyzed.
Contain the solution (adding peptide to this solution when the pH5) of 200iu insulin with 2ml, or use the insulin solid preparation, and with or without sodium bicarbonate to animals administer.Each medicament contains the above-mentioned preparation of 300mg, and can be suspended in the 2ml phosphate buffered salt solution before use rapidly.The component of two kinds of preparations is listed in the table below with weight-weight form.No carbonic acid hydrogen salt (wt/wt) has bicarbonate (wt/wt) bovine insulin 2.4 bovine insulins 2.4 chenodeoxy cholic acids 16.7 chenodeoxy cholic acids 16.7 lactose 74.9 lactose 66.6Ac-di-sol 3.0 Ac-di-sol 3.0 polyvinyl pyrrolidone 3.0 polyvinyl pyrrolidone 3.0
Sodium bicarbonate 8.3pH＜7.0 pH＞7.5
The results are shown in Fig. 6, represent with respect to the variation of insulin with plasma glucose concentration and time.This shows that the bile acid chenodeoxy cholic acid obviously improves owing to the existence of sodium bicarbonate in the solid chemicals in the efficient that improves aspect the peptide intake of intestinal.
Claims (21)
1. a medicinal and/or veterinary composition comprises bioactive substance, bile acid or salt, and the buffer agent that enteral pH is transferred to 7.5-9.
2. the compositions of claim 1, wherein buffer agent transfers to 7.8-8.3 with enteral pH.
3. wherein there are the solubility equilibrium ion in claim 1 or 2 compositions.
4. the compositions of claim 1,2 or 3, wherein bile acid or salt are uncombined.
5. the compositions of claim 4, wherein bile acid or salt are chenodeoxy cholic acid or ursodesoxycholic acid or their salt.
6. the compositions of claim 1,2 or 3, wherein bile acid or salt are bonded.
7. arbitrary compositions among the claim 1-6, wherein the pH regulator agent is suitable for enteral pH is adjusted to described scope.
8. the compositions of claim 7, wherein buffer agent contains carbonate and/or bicarbonate ion.
9. the compositions of claim 8, wherein the concentration of bicarbonate radical or carbonate is at least about 0.1M.
10. claim 8 or 9 compositions, wherein containing is enough to make bicarbonate radical or carbonate concentration reach bicarbonate radical or the carbanion of 0.045M at least when units dosage composition being scattered in the 10ml water.
11. arbitrary compositions among the claim 1-10, wherein pH regulator agent itself contains bile acid or salt.
12. arbitrary compositions among the claim 1-11, comprising calcium ion chelator as two-or tri-carboxylic acids salt, for example, citrate, ethylenediaminetetraacetic acid (EDTA) or ethylene glycol bis (beta-amino ether)-N, N, N ', N '-tetraacethyl (EGTA), or polyphosphate such as phytate.
13. arbitrary compositions among the claim 1-12, wherein bioactive substance is protein such as insulin, calcitonin, growth hormone, interferon, interleukin or any active fragment in them.
14. arbitrary compositions among the claim 1-12, wherein bioactive substance is oligonucleotide (or its analog) or polysaccharide.
15. arbitrary compositions among the claim 1-14, wherein the molecular weight of bioactive substance is less than about 20,000Da.
16. the compositions of claim 15, wherein the molecular weight of bioactive substance is less than about 10,000Da.
17. arbitrary compositions among the claim 1-16 is made into the drying solid preparation.
18. the compositions of claim 17, it is tablet, pill, powder, granule or particulate form.
19. bile salts and the purposes that enteral pH is transferred to the buffer agent of 7.5-9 use buffer agent can increase the bioavailability of bioactive substance simultaneously.
In applying active substances, use bile salts jointly and enteral pH is transferred to the buffer agent of 7.5-9 20. a method of improving the active substance bioavailability, this method comprise to the patient.
21. one kind is improved the effectively method of therapeutic index of bile salts (to improve active substance as the permeability reinforcing agent relevant in the ability of enteral with it), this method comprises to the patient to be used bile salts jointly and enteral pH is transferred to the buffer agent of 7.5-9 in applying active substances.
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| CN115003293A (en) * | 2020-01-23 | 2022-09-02 | 爱克赛斯(英国)有限公司 | Cellular uptake |
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| CN115003293A (en) * | 2020-01-23 | 2022-09-02 | 爱克赛斯(英国)有限公司 | Cellular uptake |
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