Patent Application: US-18678805-A

Abstract:
in a method of measuring a relaxing effect on constricted human bronchi of a candidate substance a bronchus tissue preparation is mounted to a force transducer in a test apparatus . after conditioning the preparation is exposed for a contraction - effective dose of a known contraction - effective substance to make it assume a first tensioned state . the preparation is then exposed for a dose of the candidate substance to make it assume a second tensioned state . by comparing the contraction forces recorded for said tensioned states , a measure of the bronchorelaxing efficiency of the candidate substance is obtained . also disclosed is a candidate substance thus identified , its uses , and a corresponding bronchus tissue preparation .

Description:
dissection and mounting of lung tissue preparations . lung tissue was obtained from patients undergoing lobectomia or pulmectomia due to lung carcinoma . the tissue was placed in a dissection chamber continuously perfused with 10 ml min − 1 of a physiological saline solution ( pss ) at room temperature . an airway was identified in the cut part of the lobe , and a bronchus of 10 - 20 mm length and 1 - 2 mm diameter was obtained . the bronchus was cut into rings of a width of about 2 - 3 mm . each bronchial ring was cleaved to obtain an about rectangular oblong preparation , one end of which was tied to a small steel hook connected to a force transducer , while the other end of the preparation was attached to a fixed hook . this is followed by a period of adjustment , as described below . the preparation was mounted in an atmosphere containing 12 % of oxygen and 6 % of co 2 . experimental chamber . the experimental chamber has a a volume of 5 ml . it is perfused with pss at a rate of 3 ml min − 1 . two preparations are mounted in the chamber , and measurements on them are performed in parallel . for mechanical tensioning each force transducer ( ame 801 , sensonor a / s , horten , norway ) is connected to a micrometer screw . the substances to be tested , the reference substance ( capsazepine ), and transmitter ( ltd4 ) are injected upstream of the preparation ( s ). materials . pps ( physiological saline solution , in mm ): nacl , 117 ; kcl , 4 . 87 ; mgso 4 , 0 . 60 ; nahco 3 , 25 . 0 ; cacl 2 , 1 . 60 ; glucose , 5 . 23 . the solution is saturated with a mixture of 94 % oxygen and 6 % carbon dioxide , giving a ph of 7 . 40 ± 0 . 05 in the experimental chamber . all substances are prepared as stock solution dissolved in the vehicles ethanol or dmso . leukotriene d4 ( ltd4 ; keyman ltd . ): 10 μl of a 100 μm ethanol stock solution . capsazepine ( sigma aldrich ): 10 μl of a 0 . 1 m ethanol stock solution . substance to be tested : 10 - 100 μl of a 0 . 01 - 0 . 1 m ethanol or dmso stock solution . solution for establishing the passive tension level : calcium - free pss + 2 mm egta + 20 mm caffeine . to exclude effects by the test substance vehicle , ethanol or dmso , respectively , were added during the entire experiment except during the presence of test substance . an exemplary test is shown in fig1 in which capital letters indicate interference with the test system . the material for the preparation was a bronchus ( inner diameter about 1 mm ) from a male occasional smoker ( 41 yrs ) but with the epithelium intact . adjustment and stretch . after mounting as described above the preparation is allowed to adjust with a low passive tone in the experimental chamber . the composition of the gas is changed to 94 % ( v / v ) of oxygen . after a short adjustment period , pss with 10 nm ltd4 is added to the experimental chamber upstream of the preparation ( a ). the preparation is stretched repeatedly ( b ) until it exerts a contraction force of around 150 mg . when the contraction has levelled off , leukotriene - free solution is administered for 1 hour ( c ), resulting in a relaxation . a second injection of 10 nm ltd4 ( d ) makes the preparation return to the tensioned state . at the peak tension leukotriene - free solution is again administered ( e ). after a third injection of 10 nm ltd4 ( f ) the preparation returns to the tensioned state . at the peak , pss with 10 μm capsazepine ( g ) is added , resulting in a relaxation . after 1 h exposure to capsazepine , ltd4 is added , resulting in a contraction ( h ). in comparison with the control ltd4 contraction ( f ), a substantially weaker contraction is now observed ( h ). to obtain a measure of the test substance &# 39 ; s bronchorelaxing effect the test and control forces registered in the experiment are compared . in the present experiment a remaining contraction ( test force ) of about 55 % of that caused by the control force was registered . after allowing one hour for return to baseline conditions ( i ) 10 nm ltd4 is again injected ( j ) to determine the reversibility of the vr1 receptor inhibition . during steps c - f and i - j 10 μl ethanol per 100 ml pss is present to compensate for potential vehicle effects . the experiment is concluded by adding calcium - free solution with addition of 2 mm egta and 20 mm caffeine for 20 min to establish the passive tension level ( k ). a candidate can be tested for antagonist properties by substituting capsazepine for it in the test system . a measure for its blocking capacity is obtained by comparing the result (% blocking of contraction by ltd4 ) with that obtained with capsazepine . if the remaining contraction after exposure to a test substance is larger than after exposure to capsazepine , the test substance is less effective than capsazepine in regard to blocking properties . if , on the other hand , the remaining contraction after exposure to a test substance is smaller than after exposure to capsazepine , the test substance is more effective than capsazepine in regard to blocking properties . instead of capsazepine , any other suitable substance can be used as a standard for comparing blocking properties . a preparation is considered stable and thus fit for the evaluation of test substances if the difference in contraction between contractions d and f is less than 15 percent . it should be noted that , although ltd4 is normally used to contract the preparations , it is possible to use any other suitable transmitter or substance with a broncho - constrictive effect . examples of useful broncho - constrictive substances that may be used instead of ltd4 include cholinergic receptor agonists such as acetylcholine , charbacholine , metacholine and other m3 - agonists ; adenosine receptor agonists ; bombesin receptor agonists ; bradykinin receptor agonists ; cannabinoid receptor agonists ; chemokine receptor agonists ; cytokine receptor agonists ; dopamine receptor agonists ; glutamate receptor agonists ; glycine receptor agonists ; high concentrations of potassium chloride ; histamine receptor agonists such as histamine and other h1 - agonists ; leukotriene receptor agonists ; neuropeptide y receptor agonists such as neuropeptide y ; opioid receptor agonists such as fentanyl ; platelet activating factor receptor agonists such as platelet activating factor ( paf ); prostanoid receptor agonists such as prostaglandin f2 - alpha , other prostaglandins , tromboxane a2 ; tachykinin receptor agonists such as neurokinin a , neurokinin b , substance p . it is also possible to contract the preparation by stimulating it by applying an electrical field ( efs ). another possibility is to examine the relaxing effects by test substances on airway preparations displaying a spontaneous tension development , a so - called spontaneous tone . were tested for broncho - relaxing effect in the test system of example 1 . relevant sections of the recorded force v . time diagrams are shown in fig2 to 4 . ltd4 was used as a broncho - contracting agent . the three compounds were all found to exhibit a broncho - contracting effect . in comparison to capsazepine ( fig1 ) res 1 - 83 ( fig2 ) was found to be a substantially less effective , compound res 3 - 22 to be about equally effective , and compound res 5 - 21 to be substantially more effective in relaxing broncho - constriction . appendino g et al ., br j . pharmacol . 2003 ; 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