Use of chymase inhibitors for the treatment of endometriosis, post operative fibrosis and diseases which are characterized by fibrosis formation

Abstract

The present application relates to the use of bicyclic-substituted uracil derivatives, alone or in combinations with other active ingredients for the treatment and/or prophylaxis of diseases, in particular for the treatment and/or prophylaxis of inflammatory and fibrotic disorders, for the treatment of endometriosis, of endometriosis-associated fibrosis, of adenomyosis and of pain associated with an endometriosis disorder and also of postoperative peritoneal fibrosis and adhesion formation.

Claims

1. A method for the treatment and/or prophylaxis of endometriosis, endometriosis-associated fibrosis, adenomyosis, and pain associated with an endometriosis disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of the formula (I) ##STR00016## in which R.sup.1 is hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.2 is a group of the formula ##STR00017##  where * is the point of attachment to the uracil nitrogen atom, A is —CH.sub.2—, —CH.sub.2—CH.sub.2—, —O—CH.sub.2-## or oxygen, in which ## is the point of attachment to the phenyl ring, m is a number 0, 1 or 2, R.sup.4 is halogen, difluoromethyl, trifluoromethyl, (C.sub.1-C.sub.4)-alkyl, difluoromethoxy, trifluoromethoxy or (C.sub.1-C.sub.4)-alkoxy, R.sup.5A is hydrogen or deuterium, R.sup.5B is hydrogen, deuterium or (C.sub.1-C.sub.4)-alkyl, R.sup.6 is hydrogen or fluorine, R.sup.7 is hydrogen or fluorine, R.sup.8 is halogen, difluoromethyl, trifluoromethyl, (C.sub.1-C.sub.4)-alkyl or nitro, R.sup.9 is hydrogen, halogen, difluoromethyl, trifluoromethyl, (C.sub.1-C.sub.4)-alkyl, nitro or (C.sub.1-C.sub.4)-alkylthio, R.sup.3 is a group of the formula ##STR00018## where # is the point of attachment to the uracil nitrogen atom, the ring Q is 5- to 7-membered heterocyclyl or 5- or 6-membered heteroaryl, in which 5- to 7-membered heterocyclyl and 5- or 6-membered heteroaryl may be substituted by 1 to 4 substituents independently selected from the group of halogen, difluoromethyl, trifluoromethyl, trideuteromethyl, (C.sub.1-C.sub.6)-alkyl, (C.sub.3-C.sub.7)-cycloalkyl, oxo, hydroxyl, (C.sub.1-C.sub.4)-alkylcarbonyl, (C.sub.1-C.sub.4)-alkoxycarbonyl, aminocarbonyl and (C.sub.1-C.sub.4)-alkyl sulfonyl, in which (C.sub.1-C.sub.6)-alkyl and (C.sub.3-C.sub.7)-cycloalkyl may in turn be substituted by 1 to 3 substituents independently selected from the group of halogen, cyano, trifluoromethyl, (C.sub.3-C.sub.7)-cycloalkyl, hydroxyl, (C.sub.1-C.sub.4)-alkoxy and 4- to 7-membered heterocyclyl,  and in which two (C.sub.1-C.sub.6)-alkyl radicals bonded to a carbon atom of 5- to 7-membered heterocyclyl and 5- or 6-membered heteroaryl, together with the carbon atom to which they are bonded, may form a 3- to 6-membered carbocycle, R.sup.24 is halogen, (C.sub.1-C.sub.4)-alkyl or (C.sub.1-C.sub.4)-alkoxy, n is a number 0, 1, 2 or 3, or a salt, solvate or solvate of a salt thereof.

2. The method according to claim 1, in which R.sup.1 is hydrogen, methyl or ethyl, R.sup.2 is a group of the formula ##STR00019## where * is the point of attachment to the uracil nitrogen atom, A is CH.sub.2—, —CH.sub.2—CH.sub.2—, —O—CH.sub.2-## or oxygen, in which ## is the point of attachment to the phenyl ring, R.sup.4A is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, R.sup.4B is hydrogen, fluorine, chlorine, trifluoromethyl or methyl, with the proviso that at least one of the R.sup.4A and R.sup.4B radicals is not hydrogen, R.sup.5A is hydrogen, R.sup.5B is hydrogen, R.sup.6 is hydrogen, R.sup.7 is hydrogen, R.sup.8 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R.sup.9 is fluorine, chlorine, difluoromethyl, trifluoromethyl or methyl, R.sup.3 is a group of the formula ##STR00020##  where # is the point of attachment to the uracil nitrogen atom, E.sup.1 is CR.sup.11 or N, in which R.sup.11 is hydrogen, (C.sub.1-C.sub.4)-alkyl, (C.sub.3-C.sub.7)-cycloalkyl or aminocarbonyl, E.sup.2 is CR.sup.12 or N, in which R.sup.12 is hydrogen, (C.sub.1-C.sub.4)-alkyl or (C.sub.3-C.sub.7)-cycloalkyl, E.sup.3 is NR.sup.14 or S, in which R.sup.14 is hydrogen, (C.sub.1-C.sub.4)-alkyl or (C.sub.3-C.sub.7)-cycloalkyl, G.sup.1 is C═O or SO.sub.2, G.sup.2 is CR.sup.16AR.sup.16B, NR.sup.17, O or S, in which R.sup.16A is hydrogen, fluorine, (C.sub.1-C.sub.4)-alkyl or hydroxy, R.sup.16B is hydrogen, fluorine, chlorine, (C.sub.1-C.sub.4)-alkyl or trifluoromethyl, or R.sup.16A and R.sup.16B together with the carbon atom to which they are bonded form a 3- to 6-membered carbocycle, R.sup.17 is hydrogen, (C.sub.1-C.sub.6)-alkyl, (C.sub.3-C.sub.7)-cycloalkyl or (C.sub.1-C.sub.4)-alkoxycarbonyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by 1 to 3 substituents independently selected from the group of fluorine, trifluoromethyl, cyano, (C.sub.3-C.sub.7)-cycloalkyl, hydroxyl, trifluoromethoxy, (C.sub.1-C.sub.4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, G.sup.3 is CR.sup.18AR.sup.18B, NR.sup.19, O or S, in which R.sup.18A is hydrogen, fluorine, (C.sub.1-C.sub.4)-alkyl or hydroxy, R.sup.18B is hydrogen, fluorine, chlorine, (C.sub.1-C.sub.4)-alkyl or trifluoromethyl, or R.sup.18A and R.sup.18B together with the carbon atom to which they are bonded form a 3- to 6-membered carbocycle, R.sup.19 is hydrogen, (C.sub.1-C.sub.6)-alkyl, (C.sub.3-C.sub.7)-cycloalkyl or (C.sub.1-C.sub.4)-alkoxycarbonyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by 1 to 3 substituents independently selected from the group of fluorine, trifluoromethyl, cyano, (C.sub.3-C.sub.7)-cycloalkyl, hydroxyl, trifluoromethoxy, (C.sub.1-C.sub.4)-alkoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, G.sup.4 is CH.sub.2, C═O or SO.sub.2, K.sup.1 is CH.sub.2 or O, K.sup.2 is CH.sub.2 or O, with the proviso that only one of the K.sup.1 and K.sup.2 groups is O, D.sup.1, D.sup.2, D.sup.3 and D.sup.4 are each independently CR.sup.23 or N, in which R.sup.23 is hydrogen, halogen, (C.sub.1-C.sub.6)-alkyl or (C.sub.3-C.sub.7)-cycloalkyl, with the proviso that not more than 2 of the D.sup.1, D.sup.2, D.sup.3 and D.sup.4 groups are N, R.sup.24 is fluorine or methyl, n is a number 0 or 1, R.sup.10 is (C.sub.1-C.sub.4)-alkyl or (C.sub.3-C.sub.7)-cycloalkyl, in which (C.sub.1-C.sub.4)-alkyl may be substituted by 1 or 2 substituents independently selected from the group of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R.sup.3 is hydrogen, (C.sub.1-C.sub.4)-alkyl or (C.sub.3-C.sub.7)-cycloalkyl, R.sup.15 is hydrogen, (C.sub.1-C.sub.6)-alkyl or (C.sub.3-C.sub.7)-cycloalkyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by 1 or 2 substituents independently selected from the group of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R.sup.20 is hydrogen, (C.sub.1-C.sub.6)-alkyl, (C.sub.3-C.sub.7)-cycloalkyl or (C.sub.1-C.sub.4)-alkylcarbonyl, in which (C.sub.1-C.sub.6)-alkyl may be substituted by 1 or 2 substituents independently selected from the group of fluorine, trifluoromethyl, cyclopropyl, cyclobutyl, hydroxyl, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R.sup.21 is hydrogen, (C.sub.1-C.sub.6)-alkyl, (C.sub.3-C.sub.7)-cycloalkyl or (C.sub.1-C.sub.4)-alkylsulfonyl, R.sup.22A is hydrogen or C.sub.1-C.sub.4-alkyl, R.sup.22B is hydrogen or C.sub.1-C.sub.4-alkyl, or R.sup.22A and R.sup.22B together with the carbon atom to which they are bonded form a carbonyl group.

3. The method according to claim 1, in which R.sup.1 is hydrogen, R.sup.2 is a group of the formula ##STR00021## where * is the point of attachment to the uracil nitrogen atom, A is —CH.sub.2—, R.sup.4A is chlorine or trifluoromethyl, R.sup.4B is hydrogen, R.sup.3 is a group of the formula ##STR00022## where # is the point of attachment to the uracil nitrogen atom, E.sup.1 is CR.sup.11, in which R.sup.11 is hydrogen, E.sup.2 is N, G.sup.1 is C═O, G.sup.2 is CR.sup.16AR.sup.16B, NR.sup.17, O or S, in which R.sup.16A is hydrogen, fluorine, methyl or hydroxy, R.sup.16B is hydrogen, fluorine, methyl or trifluoromethyl, or R.sup.16A and R.sup.16B together with the carbon atom to which they are bonded form a cyclopropyl ring, R.sup.17 is hydrogen, (C.sub.1-C.sub.4)-alkyl or (C.sub.3-C.sub.5)-cycloalkyl, in which (C.sub.1-C.sub.4)-alkyl may be substituted by 1 to 3 substituents independently selected from the group of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R.sup.24 is hydrogen or fluorine, R.sup.10 is (C.sub.1-C.sub.4)-alkyl, and R.sup.15 is hydrogen, methyl or ethyl, in which methyl and ethyl may be substituted by 1 substituent selected from the group of fluorine, trifluoromethyl and cyclopropyl.

4. The method according to claim 1, in which R.sup.1 is hydrogen, R.sup.2 is a group of the formula ##STR00023## where * is the point of attachment to the uracil nitrogen atom, R.sup.5A is hydrogen, R.sup.5B is hydrogen, R.sup.6 is hydrogen, R.sup.7 is hydrogen, R.sup.8 is fluorine, chlorine or trifluoromethyl, R.sup.9 is fluorine, chlorine, trifluoromethyl or methyl, R.sup.3 is a group of the formula ##STR00024## where # is the point of attachment to the uracil nitrogen atom, E.sup.1 is CR.sup.11, in which R.sup.11 is hydrogen, E.sup.2 is N, G.sup.1 is C═O, G.sup.2 is CR.sup.16AR.sup.16B, NR.sup.17, O or S, in which R.sup.16A is hydrogen, fluorine, methyl or hydroxy, R.sup.16B is hydrogen, fluorine, methyl or trifluoromethyl, or R.sup.16A and R.sup.16B together with the carbon atom to which they are bonded form a cyclopropyl ring, R.sup.17 is hydrogen, (C.sub.1-C.sub.4)-alkyl or (C.sub.3-C.sub.5)-cycloalkyl, in which (C.sub.1-C.sub.4)-alkyl may be substituted by 1 to 3 substituents independently selected from the group of fluorine, trifluoromethyl, cyano, cyclopropyl, cyclobutyl, hydroxyl, trifluoromethoxy, methoxy, ethoxy, azetidinyl, oxetanyl, tetrahydrofuranyl and pyrrolidinyl, R.sup.24 is hydrogen or fluorine, R.sup.10 is (C.sub.1-C.sub.4)-alkyl, and R.sup.15 is hydrogen, methyl or ethyl, in which methyl and ethyl may be substituted by 1 substituent selected from the group of fluorine, trifluoromethyl and cyclopropyl.

5. A method for the treatment and/or prophylaxis of chronic pain in the pelvic cavity which is not associated with endometriosis comprising administering to a patient in need thereof a therapeutically effective amount of the compound of formula (I) ##STR00025## in which R.sup.1 is hydrogen or (C.sub.1-C.sub.4)-alkyl, R.sup.2 is a group of the formula ##STR00026##  where * is the point of attachment to the uracil nitrogen atom, A is —CH.sub.2—, —CH.sub.2—CH.sub.2—, —O—CH.sub.2-## or oxygen, in which ## is the point of attachment to the phenyl ring, m is a number 0, 1 or 2, R.sup.4 is halogen, difluoromethyl, trifluoromethyl, (C.sub.1-C.sub.4)-alkyl, difluoromethoxy, trifluoromethoxy or (C.sub.1-C.sub.4)-alkoxy, R.sup.5A is hydrogen or deuterium, R.sup.5B is hydrogen, deuterium or (C.sub.1-C.sub.4)-alkyl, R.sup.6 is hydrogen or fluorine, R.sup.7 is hydrogen or fluorine, R.sup.8 is halogen, difluoromethyl, trifluoromethyl, (C.sub.1-C.sub.4)-alkyl or nitro, R.sup.9 is hydrogen, halogen, difluoromethyl, trifluoromethyl, (C.sub.1-C.sub.4)-alkyl, nitro or (C.sub.1-C.sub.4)-alkylthio, R.sup.3 is a group of the formula ##STR00027## where # is the point of attachment to the uracil nitrogen atom, the ring Q is 5- to 7-membered heterocyclyl or 5- or 6-membered heteroaryl, in which 5- to 7-membered heterocyclyl and 5- or 6-membered heteroaryl may be substituted by 1 to 4 substituents independently selected from the group of halogen, difluoromethyl, trifluoromethyl, trideuteromethyl, (C.sub.1-C.sub.6)-alkyl, (C.sub.3-C.sub.7)-cycloalkyl, oxo, hydroxyl, (C.sub.1-C.sub.4)-alkylcarbonyl, (C.sub.1-C.sub.4)-alkoxycarbonyl, aminocarbonyl and (C.sub.1-C.sub.4)-alkyl sulfonyl, in which (C.sub.1-C.sub.6)-alkyl and (C.sub.3-C.sub.7)-cycloalkyl may in turn be substituted by 1 to 3 substituents independently selected from the group of halogen, cyano, trifluoromethyl, (C.sub.3-C.sub.7)-cycloalkyl, hydroxyl, (C.sub.1-C.sub.4)-alkoxy and 4- to 7-membered heterocyclyl,  and in which two (C.sub.1-C.sub.6)-alkyl radicals bonded to a carbon atom of 5- to 7-membered heterocyclyl and 5- or 6-membered heteroaryl, together with the carbon atom to which they are bonded, may form a 3- to 6-membered carbocycle, R.sup.24 is halogen, (C.sub.1-C.sub.4)-alkyl or (C.sub.1-C.sub.4)-alkoxy, n is a number 0, 1, 2 or 3, or a salt solvate or solvate of a salt thereof.

Description

EXPERIMENTAL SECTION

(1) The synthesis of the compounds of the general formula (I) is described in WO2013/167495 A1.

(2) Assessment of the Pharmacological Activity

(3) The pharmacological activity of the compounds according to the invention on chymase is shown in the assays described in WO2013/167495 A1.

(4) For the sake of completeness, the results of the enzymatic chymase assay are listed here again.

(5) Enzymatic Chymase Assay (WO2013/167495)

(6) The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 μl of a 50-fold concentrated solution of test substance in DMSO, 24 μl of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 μl of substrate solution (final concentration 10 μM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) were combined in a white 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, Mannedorf, Switzerland).

(7) One test compound is tested on the same microtitre plate in 10 different concentrations from 30 μM to 1 nM in duplicate determination. The data are normalized (enzyme reaction without inhibitor=0% inhibition, all assay components without enzyme=100% inhibition) and IC50 values are calculated using in-house software. Compounds in the context of the invention which were tested in this assay inhibited chymase activity with an IC50 of less than 10 μM.

(8) IC50 values representative of the compounds of the invention are shown in Tables 1 and 2 below:

(9) TABLE-US-00001 TABLE 1 Example No. Hamster chymase 1 8 2 7 3 9 4 64 5 20 8 33 9 1500 10 1600 13 5 14 10 15 330 16 14 18 10 20 8 21 5 22 6 25 7 27 5 28 4 33 4 34 7 35 6 37 700 40 15 41 23 42 7 43 643 44 18 45 50 47 35 48 17 49 17 50 31 51 120 52 16 53 30 55 39 56 67 62 44 63 37 64 19 65 19 66 30 67 4 75 82 76 41 77 170 78 140 79 210 81 65 82 83 220 86 140 89 84 94 62 95 100 96 80 97 33 99 64 101 24 103 27 104 2 105 64 106 56 107 29 108 76 109 24 110 150 111 20 112 113 6 114 7 115 10 116 20 117 3 118 6 119 280 120 1025 121 3 122 2 123 4 124 7 125 6 126 10 127 34 128 7 129 450 130 350 131 4 132 2 133 465 134 2 135 4 136 2 137 4 138 4 139 2 140 1 141 2 142 1 143 2 144 2 145 2 146 1 147 2 148 4 149 2 150 5 151 2 152 19 153 4 154 4 155 5 156 12 157 6 158 10 159 92 160 32 161 53 162 58 163 28 164 34 165 40 166 62 167 91 168 49 169 370 170 20 171 17 172 27 173 110 174 44 175 8 176 29 177 30 178 16 179 10 180 7 181 4 182 4 183 10 184 170 185 140 186 23 187 4 188 4 189 3 190 140 191 16 192 5 193 8 194 13 195 4 196 6 197 10 198 54 199 8 200 4 201 7 202 4 203 20 204 39 205 3 206 3 207 4 209 13 211 20 213 18 214 20 215 26 216 183 217 1 218 4 219 5 220 6 221 10 222 12 223 3 224 2 225 4 226 3 227 2 228 14 229 4 230 170 231 21 232 6 233 470 234 270 235 9 236 5 238 45 239 490 240 67 241 2 242 40 243 6 244 2 245 67 246 1 247 1 248 2 249 200 250 37 251 420 252 190 253 1500 254 84 255 500 256 170 257 540 258 190 259 430 260 130 261 110 262 2100 263 38 264 31 265 2 266 59 267 16 268 18

(10) TABLE-US-00002 TABLE 2 Example No. Hamster chymase 269 14 270 6 271 23 272 11 273 1100 274 2 275 2300 276 4 277 2 278 5 279 4 280 250 281 1 282 88 283 40 284 11 285 42 286 37 287 4500 288 14 289 970 290 8 291 4 292 293 12 294 2 295 8 296 3 297 298 6 299 120 300 2 301 33 302 19 303 9
Rectovaginal Hamster Endometriosis-Fibrosis Model

(11) To investigate the effect of the test substances on fibrosis mediated by endometriosis or the adhesions produced thereby, female syrian hamsters (ca. 150 g) aged 3-4 months are used. The cycle of the animals is determined via a vaginal smear needle. Oestrus in the animals is defined by the flow of a pus-like secretion from the vagina. On the day after oestrus, the operation is carried out to induce rectovaginal endometriosis and generate the adhesions, fibroses and fistulae associated thereto. Rectovaginal endometriosis is induced in the animals as follows: one day after oestrus, the animals are anaesthetized with isoflurane and the abdominal wall is opened. Approximately ⅓ rd of the uterus is removed and, in the piece removed, the endometrium is separated from the myometrium. Punches with a diameter of 5 mm are taken from the endometrium and each of these is sutured with 3 stitches. Here, the peritoneum is sutured with the uterus in the rectovaginal pouch and between the bladder and uterus. Subsequently, the abdominal wall is closed off by placing a suture. Endometriosis is thus generated by the adhesions which form within 21 days.

(12) Starting on the day of the OP, the animals are treated daily with the test substance over a period of 21 days. The test substance is administered orally once daily at 2 ml/kg with ethanol, solutol, water (v/v/v=1/4/5) as vehicle and dosages of 3 mg/kg and 10 mg/kg. Disease severity is determined after sectioning the animals on day 21, based on adhesions and fistula development, in which the following scoring system is applied: 0=no adhesion, bladder freely moveable, no connection with the lesion, no fibrosis and adhesion, 1=slight adhesion, bladder not freely moveable, uterus moves slightly due to connection via the lesion, bladder slightly fused with the lesion and/or low adhesion of the lesion with the surrounding tissue, adhesion very fine and transparent, 2=moderate adhesion, bladder not freely moveable, uterus moves due to connection via the lesion, approximately half of the bladder fused with the lesion and/or adhesion of the lesion with the surrounding tissue, 3=severe adhesion, bladder not freely moveable, uterus moves vigorously due to connection via the lesion, bladder largely fused with the lesion and/or severe adhesion of the lesion with the surrounding tissue.

(13) FIG. 1 shows a significant reduction in the adhesions caused by fibrosis, which were caused by an induced endometriosis, by the test substance example compound 189.

(14) Hamster Adhesion Model

(15) To investigate the effect of the test substances on a postoperative fibrosis and/or postoperative adhesions and fistulae, female syrian hamsters (each ca. 150 g) aged 3-4 months are used. The cycle of the animals is determined via a vaginal smear needle. During oestrus, a pus-like secretion may flow out of the vagina. On the day of oestrus, the operation to generate adhesions is carried out. For this purpose, the animals are anaesthetized with isoflurane and the abdominal cavity is opened. Four ischaemic nodes are each placed laterally on the peritoneal wall by basal ligation of a fold at a diameter of ca. 5 mm in size using a 4-0 silk suture. The gap between the nodes is ca. 1 cm in each case. Subsequently, the abdominal cavity is closed off by placing a suture. Starting on the day of the OP, the animals are treated daily with the test substance for 7 days. The test substance is administered orally once daily at 2 ml/kg with ethanol, solutol, water (v/v/v=1/4/5) as vehicle and dosages of 1 mg/kg, 3 mg/kg and 10 mg/kg. On day 7, the animals are sacrificed by decapitation or under deep isoflurane anaesthesia and autopsy is subsequently carried out. The number of adhesions per ischaemic node is determined and also their size and condition before the tissue is taken for histological and molecular biological analysis. In addition, tissue is preserved from the surrounding peritoneum as control tissue. Disease severity is determined after sectioning based on adhesions and fistula development. The scoring is undertaken for each ischaemic node individually, in which the following scoring system is applied:

(16) 0=no adhesion is found at the ischaemic nodes,

(17) 1=one adhesion with a blood vessel is found at the nodes,

(18) 2=more than one adhesion is found at the nodes,

(19) 3=many adhesions are found at the nodes, but this is just visible.

(20) The average of the scores of all nodes per animal is then determined.