阅读说明：本技术 腺苷受体激动剂及治疗膀胱泌尿功能障碍的药物 (Adenosine receptor agonist and medicine for treating bladder urinary dysfunction ) 是由 俞为群 于 2019-01-07 设计创作，主要内容包括：本发明公开了一种腺苷受体激动剂在制备治疗哺乳动物膀胱泌尿功能障碍的药物中的应用,可以有效的增加膀胱容量和减少排尿频率。(The invention discloses application of an adenosine receptor stimulant in preparing a medicine for treating urinary dysfunction of bladder of mammals, which can effectively increase bladder capacity and reduce urination frequency.)

1. An adenosine receptor agonist for use in the preparation of a medicament for the treatment of bladder urinary dysfunction, wherein the adenosine receptor agonist is a compound of formula (I):

2. the adenosine receptor agonist according to claim 1, wherein: the bladder is a mammalian bladder.

3. The adenosine receptor agonist according to claim 2, wherein: the mammal is a human, mouse, pig, cat or dog.

4. The adenosine receptor agonist according to claim 1, wherein: the adenosine receptor is its subunit A2 b.

5. The adenosine receptor agonist according to claim 1, wherein: the urinary bladder dysfunction includes frequent micturition, urgency, nocturia, odynuria, overactive bladder syndrome, urinary incontinence, interstitial cystitis, and drug cystitis.

6. A medicament for the treatment of bladder urinary dysfunction, characterized by: the medicament comprising an adenosine receptor agonist according to any one of claims 1 to 5.

Technical Field

The invention relates to the use of adenosine receptor agonists for the preparation of a medicament for the treatment of urinary bladder dysfunction, and a method for assessing the ability of adenosine receptor agonists to modulate urinary bladder dysfunction in a mammal.

Background

Bladder urinary dysfunction such as urinary frequency, urgency, nocturia, odynuria, overactive bladder syndrome, urinary incontinence, interstitial cystitis, drug cystitis (chloroaminoketone cystitis), and the like, affecting at least more than 50% of >40 years old people, current treatment options are limited, including fluid management, drug management, use of adult diapers, and surgical treatment, in terms of drugs, there are currently 1. first line anticholinergics, but current oral dosage forms have limited efficacy and great side effects (mostly abandoned last), 2. recently β 3 receptor agonists have been used for treatment, have some efficacy but great side effects, 3.FDA recently approved BOTOX for local injection treatment of bladder, by local paralysis of bladder nerve endings to treat overactive bladder.

Adenosine receptors can be divided into four subunits, a1, A2a, A2b, and A3. There have been no reports on how adenosine receptor signaling and its agonists modulate in vivo urinary bladder micturition function and thus treat urinary dysfunction as a potential clinical agent.

In addition, there is a great area of error in current research in the urinary field: the mechanical forces are equivalent to reducing the activity of the bladder and reducing the contractile force of the bladder smooth muscle. In fact, the reduction of the contraction force of the bladder smooth muscle may reduce the active degree of the bladder, but may also directly cause urinary retention or incomplete urination, thereby causing frequent increase of bladder urination and capacity reduction, and the bladder is over-active instead. Therefore, the regulation of the contractile force of the smooth muscle of the bladder by the drug in vitro cannot be directly used to speculate the in vivo regulation effect on the urinary function of the bladder. In addition, the bladder smooth muscle needs to slowly relax in the urine storage period under the condition of keeping a certain contractility, so that the in vitro inhibitory effect of the drug on the contractility of the bladder smooth muscle does not necessarily indicate the in vivo relaxation of the bladder in the urine storage period. Therefore, the regulation of bladder micturition function by drugs must be finally verified by in vivo experiments. Based on these erroneous findings, current research on urinary function mechanisms and drug evaluation are mostly focused on cell and tissue levels, and a comprehensive evaluation system is lacking. Thus severely hampering the study of corresponding drugs to modulate urinary function.

Disclosure of Invention

In view of the above-mentioned problems of the prior art, it is an object of the present invention to provide an adenosine receptor agonist which can be used in the preparation of a medicament for the treatment of urinary bladder dysfunction.

In order to achieve the purpose, the invention adopts the following technical scheme:

an adenosine receptor agonist for use in the preparation of a medicament for the treatment of bladder urinary dysfunction, wherein the adenosine receptor agonist is a compound of formula (I):

preferably, the bladder is a mammalian bladder.

Preferably, the mammal is a human, mouse, pig, cat or dog.

Preferably, the adenosine receptor is subunit A2b thereof.

Preferably, the bladder urinary dysfunction includes urinary frequency, urgency, nocturia, odynuria, overactive bladder syndrome, urinary incontinence, interstitial cystitis, and drug cystitis.

A method of evaluating an adenosine receptor agonist for modulating urinary bladder dysfunction in a mammalian bladder, the method comprising:

determining the regulation effect of adenosine receptor agonist on the contraction force of the bladder smooth muscle of the mammal by using the in vitro isolated smooth muscle;

knocking out mammalian adenosine receptor genes to observe changes in mammalian bladder function caused thereby;

normal mammals are injected directly subcutaneously with an adenosine receptor agonist to observe its modulating effect on bladder function under in vivo conditions,

the adenosine receptor agonist is a compound of formula (II):

preferably, the adenosine receptor is the subunit A2b gene thereof.

Preferably, the mammal is a human, mouse, pig, cat or dog.

Preferably, the bladder urinary dysfunction includes urinary frequency, urgency, nocturia, odynuria, overactive bladder syndrome, urinary incontinence, interstitial cystitis, and drug cystitis.

A medicament for the treatment of bladder urinary dysfunction, said medicament comprising an adenosine receptor agonist as described above.

The invention has the beneficial effects that:

by adopting the technical scheme, the invention can effectively increase the bladder capacity and reduce the urination frequency, thereby being an effective medicament for treating the urinary dysfunction.

Drawings

Figure 1 is experimental data for UroA002 selectively inhibiting purine-mediated contraction of bladder smooth muscle.

FIG. 2 is data from experiments in which administration of UroA002 injection decreased the number of urination times and increased the urine volume.

FIG. 3 is data from experiments in which UroA002 intragastric administration decreased the number of urination times and increased urine volume.

Detailed Description

The present invention includes a method of modulating contractility of smooth muscle of the bladder comprising injecting an effective dose of an adenosine receptor agonist, a novel A2b adenosine receptor agonist UroA002, into a mammal in need thereof.

The present invention also relates to a method of evaluating A2b adenosine receptor agonist for modulating urinary bladder dysfunction in a mammal:

(1) determining the effect of an A2b adenosine receptor agonist such as UroA002 on the regulation of the contractile force of mammalian bladder smooth muscle using smooth muscle ex vivo in vitro;

(2) a mammal such as a mouse is injected directly subcutaneously with the novel A2b adenosine receptor agonist UroA002 to observe its modulating effects on bladder function under in vivo conditions.

(3) A novel A2b adenosine receptor agonist UroA002 was gavaged in mammalian mice to observe its modulating effects on bladder function under in vivo conditions.

The mouse is taken as an experimental object, and the specific experimental process is as follows:

1. the novel A2b adenosine receptor agonist UroA002 selectively inhibits purinergic contractility of mouse bladder smooth muscle.

Purpose of the experiment: to test the effect of the novel A2b adenosine receptor agonist UroA002 on bladder smooth muscle contractility, we measured the response of the ionospheric bars to it using electromyography.

Experimental method the bladder smooth muscle of 12-16 week old male C57B L/6J mice was used for the experiment, the bladder smooth muscle after removal of the inner epidermis was divided into 4 strips of 5-7 mm long and 2-3 mm wide, one end of the strip was fixed on a rack by 6-0 silk thread and the other end was connected to a force sensor by 6-0 silk thread, the strip was located between two platinum electrodes on the rack and placed in a SI-MB 4 water bath system (World Precision Instruments, F L), the force signal of the strip was converted to a digital signal by a TBM 4M signal converter and observed in real time by Chart software amplified by Powerlab before the experiment started, the strip was equilibrated in a water bath for at least one hour, physiological saline solution (physiological saline solution, water bath 37 degrees) and continued to pass 95% oxygen and 5% carbon dioxide, we stimulated release of electrical fields (1, 2, 5, 10, 20, 50 z, 3 z) with different frequencies of electrical fields to stimulate release of neurotransmitter fibers (PSS, Grass, neurotransmitter, and nerve release at different frequencies.

The experimental results are as follows: under stimulation of electric fields of different frequencies, the bladder nerve fibers release mainly acetylcholine and ATP, thereby activating the cholinergic and purinic receptors of the bladder smooth muscle to cause contraction. As shown in fig. 1, the bladder smooth muscle induces significant contractile force under electrical field stimulation of different frequencies. Atropine can block the cholinergic force leaving the purine-induced contractile force. As shown in fig. 1, the new A2b adenosine receptor agonist UroA002 significantly inhibited the contractile force of bladder smooth muscle at nM level (EC50 ═ 3.2nM, and was able to almost completely inhibit the contractile force of bladder smooth muscle at-10 nM level), indicating that the A2b adenosine receptor agonist has excellent effect of inhibiting the contractile force of bladder smooth muscle.

In fig. 1, a, bladder smooth muscle strips induced contraction under 1, 2, 5, 10, 20, 50Hz electric field stimulation, atropine inhibited cholinergic forces while the remaining purine forces were inhibited by the novel A2b adenosine receptor agonist UroA002 in a concentration-dependent manner. B, curve fitting of UroA002 inhibitory concentration and contractile force.

2. Subcutaneous injection of the novel A2b adenosine receptor agonist UroA002 in mice significantly reduced the number of urination and increased the volume of urine per urination.

Purpose of the experiment: to verify the modulating effect of this novel A2b adenosine receptor agonist on bladder function in vivo, we injected UroA002 directly subcutaneously to observe the resulting bladder function change and its possible therapeutic effects.

The experimental method comprises the following steps: individual 12-16 week old mice were gently placed in standard AN75 polycarbonate cages. A28.5 cm long by 17.5 cm wide filter Paper (Blicks Cosmos Blotting Paper, Cat #:10422-. Within 4 hours of the experiment, the mice had no drinking water but were able to eat freely. The filter paper was collected after 4 hours. Blots of mouse urine on filter paper contain autofluorescent material that can be used for photographic imaging under ultraviolet (365nm) radiation (UVP Chromato-Vue C-75system, UVP, Upland, CA) (camera: EOSRebel T3). The imaged pictures were quantified and statistically analyzed using ImageJ software. By analyzing known volumes of the dot blots, we obtained 1mm²The urine of (2) is equal to 0.283. mu.l of urine. In the experimental group, mice were injected subcutaneously with 0.1-1mg/kg of UroA002 before the experiment, and in the control group, mice were injected subcutaneously with the same volume of solvent.

The experimental results are as follows: as shown in fig. 2, mouse urine blots can be clearly imaged under uv light for analysis. Along with the increase of the concentration of the UroA002, the frequency of urination of mice is obviously reduced, and the volume of urination per time is obviously increased, so that the research on the regulation effect of adenosine A2b receptors on the function of the bladder is proved, and the UroA002 is an effective medicament for treating urinary dysfunction.

In fig. 2, a and B are representative urine blot diagrams of the control group and the administration group; c and D are summaries of the data for the quantitative analysis.

3. The mouse gavage novel A2b adenosine receptor agonist UroA002 can significantly reduce the number of urination and increase the volume of urination per time.

Purpose of the experiment: to verify that the new A2b adenosine receptor agonist UroA002 also has a modulating effect on bladder function in vivo by the gavage route, we administered UroA002 by gavage to observe the resulting bladder function change and its possible therapeutic effects.

The experimental method comprises the following steps: individual 12-16 week old mice were gently placed in standard AN75 polycarbonate cages. A piece of 28.5 cm long by 17.5 cm wide filter Paper (Blicks Cosmos Blotting Paper, Cat #: 10422-. Within 4 hours of the experiment, the mice had no drinking water but were able to eat freely. The filter paper was collected after 4 hours. Blots of mouse urine on filter paper contain an autofluorescent material capable of being exposed to ultraviolet (365nm) radiation (UVP Chromato-Vue C-75system, UVP, Upland, CA)Can be used for photographic imaging (camera: EOSRebel T3). The imaged pictures were quantified and statistically analyzed using ImageJ software. By analyzing known volumes of the dot blots, we obtained 1mm²The urine of (2) is equal to 0.283. mu.l of urine. In the experimental group, mice were administered 0.2-2mg/kg UroA002 by gavage before the experiment, and in the control group, mice were administered the same volume of solvent by gavage.

The experimental results are as follows: as shown in fig. 3, mouse urine blots can be clearly imaged under uv light for analysis. Along with the increase of the concentration of the gastric lavage drug UroA002, the micturition frequency of mice is obviously reduced, and the micturition volume per time is obviously increased, so that the research on the regulation effect of adenosine A2b receptors on the bladder function is proved, and the UroA002 is an effective drug which can be used for treating urinary dysfunction through an oral route.

In fig. 3, a and B are representative urine blot diagrams of the control group and the administration group; c and D are summaries of the data for the quantitative analysis.

The above data indicate that the systemic study of the effect of adenosine A2b receptor in the regulation of bladder function, and the quantitative methodology of the novel adenosine A2b receptor agonist UroA002, are effective in increasing bladder capacity and reducing frequency of urination, and thus can be used as an effective drug for the treatment of urinary dysfunction.