JP2003502270A - 21-Hydroxy-6,19-oxideprogesterone (21OH-6OP) and its use as an agent to treat glucocorticoid excess - Google Patents

Abstract

  (57) [Summary] The sterically highly bent steroid 21-hydroxy-6,19-oxideprogesterone (21OH-6OP) has a substantial loss of mineralocorticoid or glucocorticoid properties, as well as affinity for MR or PR. It is a selective anti-glucocorticoid that is used to treat diseases involving glucocorticoid excess in the body, such as Cushing's syndrome and depression.

Description

Detailed Description of the Invention

The present invention provides for the treatment or prevention of diseases associated with glucocorticoid excess,
Relating to using pure anti-glucocorticoid. In particular, the invention relates to the use of 21-hydroxy-6,19-oxide progesterone (21OH-6OP) for the treatment of Cushing's syndrome or depression.

Cushing's syndrome or pituitary basophilia is a disease caused by an increase in cortisol secretion from the adrenal cortex. Hyperfunction of the adrenal cortex may be ACTH-dependent or ACTH-independent, for example, by increased cortisol due to adenomas or carcinomas of the adrenal cortex. Administration of exogenous cortisol or a synthetic analogue related thereto in an amount exceeding the physiological level suppresses adrenal cortical function and causes symptoms similar to ACTH-independent hyperactivity. ACTH-dependent hyperfunction of the adrenal cortex is due to hypersecretion of ACTH from the pituitary gland, non-pituitary tumors, such as ACTH secretion from small cell carcinoma of the lung (ectopic ACTH).
Syndrome) or by administration of exogenous ACTH. The term Cushing's syndrome is used for clinical symptoms caused by an excess of cortisol, regardless of its cause.However, hyperadrenocorticism caused by excess ACTH of the pituitary is also called Cushing's disease and has a specific physiological value. It means abnormal condition. Patients with Cushing's disease may have pituitary basophilic or refractory adenomas. Microadenomas are usually imaged by CT scan, preferably by MRI scan, using high resolution techniques utilizing gadolinium. However, even with such a method, it is difficult to find some small adenomas. Histological abnormalities of the pituitary may not be found despite clear evidence of overproduction of ACTH.

[0003] As used herein, Cushing's syndrome refers to a clinical condition caused by an excess of cortisol, regardless of its cause. This condition may also be iatrogenically caused by ACTH injection or direct administration of cortisol or its synthetic analogs. The synthetic analogs are, for example, prednisone, prednisolone, dexamethasone, etc., and are widely used for various diseases including allergic, asthmatic, inflammatory or immunological ones. Cushing's syndrome also includes adrenal tumors that secrete corticoids, ectopic ACTH
Secretions and Cushing's disease are also included.

[0004] The clinical symptoms include a "moon face" that is red and has a rounded shape. Body obesity is seen, with marked fatty bumps (“buffalo aneurysms”) on the clavicle and dorsal neck. The extremities and fingers are usually fairly thin. Skeletal muscles are reduced and weakened. The skin becomes thin and atrophied, which leads to poor wound healing and easy bruising. Purple lines may be seen on the abdomen. Hypertension, renal stones, osteoporosis, glucose intolerance, reduced resistance to infection, and mental disorders are also common. In children, characteristically, linear growth stops. Women usually have irregular menstruation. In men, along with cortisol, increased androgen also presents hirsutism, transient baldness, and other signs of virilization.

Up to now, the use of anti-glucocorticoids (eg, mifepristone) in clinical therapy has been limited to cases in which surgery is not possible due to non-pituitary Cushing's syndrome. When using mifepristone in this indication, very high doses (20-800 mg per day) are required to be effective.

The glucocorticoid receptor (GR) was cloned in 1985 (1) and is a protein of a closely related intracellular receptor that functions as a transcription factor (2) activated by a ligand. Belong to the super family. Steroid ligands that can activate GR and elicit a biological response have a slight twist of the steroid nucleus at the A / B-ring junction for optimal glucocorticoid (GC) activity (3 ,Four) .
On the other hand, mineralocorticoid (MC) agonists require an overall flat conformation due to Na-retaining activity (5). The mineralocorticoid receptor (MR) was cloned by Arriza et al. In 1987 (6) and showed high homology with GR. Due to such fairly high homology, it was not surprising that natural steroids, even synthetic steroids, show cross-reactivity between GR and MR. The progesterone receptor (PR) is the third member of the above closely related steroid receptor superfamily. Its natural ligand, progesterone, is also associated with MR
It shows cross-reactivity with both GR.

Since a systematic strategy was used to increase activity and reduce cross-reactivity and adverse side effects, higher efficacy and efficacy in the field of new antihormonal agents, especially antiestrogens and antiandrogens The development of selective agents has made significant progress.

The development of selective anticorticoids is more limited. Reference may be made to therapeutic spironolactone family steroids. Those inhibitors may belong to the rapidly separating anti-MC group (7). Another synthetic steroid, ZK91587, described as an anti-MC, specifically binds to renal MR (8) and hippocampal type I MR (9) but not hippocampal type II MR. Therefore, it would be useful as a tool in investigating MR function in tissues with both receptor systems.

[0009] Long-term, ongoing research on GC antagonists ultimately led to the early 1980s,
The development of the 11β-aminophenyl substituted 19-nor steroid RU38486 was successful (10). Its name was later shortened to RU486. Soon, however, this compound was also found to have potent antiprogestin activity. Therefore, it was used as a contraceptive. Avoiding cross-reactivity between GR and PR is an unmet goal for pharmacologists.

The present inventor has found that the synthetic steroid 21-hydroxy-6,19-oxide progesterone (2
We have found that 1OH-6OP) (I) is a selective anti-glucocorticoid that cannot cross-react with uterine PR or renal MR.

[Chemical 1] This new structure was developed as a result of a systematic study of MC properties in a series of 21-deoxysteroids, among which the highly curved structure
-6,19-Oxidoprogesterone (6OP-) (Fig. 1) does not show any Na ⁺ retention property.

Preliminary studies have also shown that this steroid lacks GC properties. The 21-hydroxy group was introduced into the 6,19-oxide pregnane skeleton, thereby imparting anti-GC properties to the skeleton structure.

Therefore, the pure anti-glucocorticoids of the present invention are used for the treatment of diseases associated with glucocorticoid excess. The anti-glucocorticoid in that case substantially lacks mineralocorticoid or glucocorticoid properties, as well as MR
Alternatively, those lacking affinity for PR are strongly required. In particular, the anti-glucocorticoid of the present invention is used for treating Cushing's syndrome and depression associated with excess glucocorticoid in the body. The present invention therefore provides a 21-hydroxy-6,19-oxide progesterone of formula I for use as a drug.

Another subject of the invention is the use of 21-hydroxy-6,19-oxide progesterone of formula I in the manufacture of a medicament for treating and preventing diseases associated with glucocorticoid excess. Preferably, the compound of formula I is used in the manufacture of a medicament for treating Cushing's syndrome, iatrogenic hypercorticoids, or depression. The compounds of formula I can be formulated with one or more suitable carriers according to the composition of conventional steroids.

The invention will be described with reference to the following examples. Example 1 Materials and Methods Reagents: Aldosterone (ALDO), corticosterone (B), progesterone (P ₄ ) and dexamethasone (DXM) were purchased from Sigma Chemical Co. (St. Louis, MO).
6,19-Oxidoprogesterone (6OP) and its 21-hydroxy derivative (21OH-6OP)
(Formula I) was synthesized according to (12) and (13), respectively. ( ³ H)-Corticosterone (SA
. = 80Ci / mmol), ( 3 H) -ALDO (SA = 59Ci / mmol), (3 H) -P 4 (SA = 90Ci / mmol), and
( ³ H) -ZK91587 (SA = 86.5 Ci / mmol) was purchased from New England Nuclear (Boston, MA). RU28362 was obtained from Roussel / Uclaf (Romainville, France). All reagents used were of analytical grade.

Binding test: Adrenalectomized male Sprague-Dawley rats were sacrificed by blood bleeding by cardiac puncture. A large amount of cold 0.9% NaCl solution was injected into the aorta until the organs were completely white. The thymus was used as a raw material for GR and the kidney as a raw material for MR. The homogenization and incubation conditions for each were as described in (14) and (15). In summary, cold TEGM buffer pH 7.4 (0.1 M Tris-HCl, 10 mM EDTA, 10 mM β-mercaproethanol, 20 mM Na ₂ MoO ₄ , 25% glycerol, 0.1 mM PMSF, 2.0 TIU / ml aprotinin and 1 mg% leupeptin. ) Was homogenized and centrifuged at 37,000 rpm for 30 minutes at 0 ° C. The supernatant after centrifugation was called "cytoplasm". The cytoplasm of the thymus or kidney, in the presence of 5nM ⁽³ H) -B or ⁽³ H) -ALDO, and steroids incubated with unlabeled stepwise increased amount. The binding of ( ³ H) -P ₄ to uterine PR was described by Jordan et al.
Tested using immature female rats according to. (16).

1.0 μM RU28362 was added to the kidney cytoplasm to suppress the cross-reactivity of ( ³ H) -ALDO with GR. 1.0 μM cortisol (F) was added to the uterine cytoplasm to suppress the cross-reactivity of ( ³ H) -P ₄ with GR and transcortin (CBG). Relative binding affinity (RBA) with partially purified CBG was determined by steroid antagonism of ( ³ H) -B binding according to (14).

Biopsy: Using adrenalectomized male Sprague-Dawley rats (180-220 g), glycogen accumulation in the liver was measured as follows: Steroid was added as an ethanol / propylene glycol / 0.9% NaCl solution (3 : 3: 34) and injected intramuscularly the night before at 100 μg / 100 g body weight. On the morning of the experimental day, the same dose was again injected intraperitoneally. After 3 hours, the rat was killed by cervical amputation and the liver immediately removed. Krisma
Glycogen was purified and quantified according to method (17) of n. The Na ⁺ retention property was measured according to (5). Tyrosine aminotransferase (TAT)
Induction with rat isolated hepatocytes according to the method of y et al. (18). Granner and To
Its TAT activity was determined according to mkins (19).

Results FIG. 2 shows the binding properties of 21OH-6OP to GR (panel A) derived from rat thymus, CBG partially purified from rat plasma (panel B), and MR (panel C) derived from rat kidney. It is a thing. For comparison, the displacement curve with 6,19-oxide progesterone is also shown.

The steroid exhibits a moderate RBA (125 nM) for GR and a low RBA (1.27 μM) for CBG (Kd values in parentheses). It does not effectively compete with ( ³ H) -ALDO for MR, even when added in 1000-fold excess. 21 functional hydroxyl groups
Introduced at the position, the binding property of 6OP to GR was increased 40-fold and the binding to CBG was increased 100-fold. With this introduction, the 6,19-oxide pregnane skeleton and
Coupling with MR is not improved (panel C).

FIG. 3 shows the biological properties of the in vitro and in vivo methodologies. Compared to ADX control, DXM or B increased hepatic glycogen accumulation by 3 to
4-fold, and 5-fold increase in TAT activity. Despite the increase in RBA against GR,
The hydroxylated derivative at position 21 was unable to elicit a GC response. In fact
As shown in Figures 3A and 3B, neither 6OP nor 21OH-6OP evokes a GC response. 21OH-6OP lacks most of the MC properties compared to 6OP, which already has a weak Na ⁺ retention capacity (Fig. 3C).
). Similar to others with weaker Na ⁺ retention, 6OP and 21OH-6OP show a parabolic dose-response curve at higher doses, with maximum retention of 21OH-6OP and 6OP at 40% and 50%, respectively. But then loses activity at higher doses (see also 51).

To investigate the binding of ALDO to renal MR without antagonizing the relatively more abundant GR, GR was suppressed by the specific ligand RU28362 (7). This avoided the cross-reaction of ALDO with GR, and hence the Scatchard plot with two slopes.

Addition of 1.0 μM RU28862 makes the plot a single straight line corresponding to the type I site, as for the selective ligand ZK91587 for MR. The calculated Kd for the type I site under various conditions was 0.4-1 nM, which is at least 8-fold higher than the affinity for the type II site (Table 1). When the novel synthetic steroid 21OH-6OP of the present invention is added to the incubation solution containing ( ³ H) -ALDO,
A single binding site was again found with kinetic parameters comparable to MR. This means that 2.5 μM of 21OH-6OP can suppress the cross reaction of ( ³ H) -ALDO with GR. From the Hill coefficient (n _g ) calculated under each condition, it can be seen that there is no allosteric effect (Table 1).

Consistent with these binding properties, an antagonistic effect of 21OH-6OP on GC was observed. In fact, it can be seen from FIG. 4 that the novel pregnane steroids of the present invention antagonize TAT induction by corticosterone and dexamethasone. The basal TAT activity of hepatocytes, called control in the figure, was measured by adding a solvent (final concentration of 0.2% ethanol) to the culture medium. Both 6OP and 21OH-6OP are TA
Although not promoting T activity (hatched bars), 100 nM B (white bars) and 100 nM DXM (black bars) stimulate enzyme activity 5-6 fold. 2.5 μM 21OH-6OP suppresses 80% of TAT induction by incubation with B or DXM.

[0024]   FIG. 5 shows that 21OH-6OP is a negative control of 17β estradiol (E ₂ ) Bound to uterine receptors as weakly as³H) -P_FourShows to replace
It In contrast, unlabeled P_FourWas able to effectively replace the tracer.

From the above data, 21-hydroxy-6,19-oxide progesterone may also be a useful tool for studying steroid receptor interactions in systems where one or more receptors co-exist. I understand. Its RBA for GR is indicated by the average affinity of ( ³ H) in the thymic cytoplasm (125 nM). Despite these moderate affinities, 21OH-6OP showed no interactive activity on both MR and PR. In the kidney cytoplasm where not only GR and MR coexist but also CBG derived from two sources (inevitable plasma contamination (22,23) and renal biosynthesis (24)) is present in the kidney cytoplasm. Was also able to block the type II receptor. From Figure 2B, 2.5 μM 21OH-6
It was pointed out that OP could simultaneously block almost 60% of the binding sites of CBG. Figure 2
Showed only 75% tracer substitution, instead of the expected 95% saturation according to mass action, probably due to the two origins of CBG acting as antagonists to GR (22,23). It depends.

The induction of TAT activity evoked by B and DXM in hepatocytes was significantly inhibited by 21OH-6OP. This means that this new steroid is a potent GC antagonist. The oxidopregnan steroids of the present invention are PR
It is a potent selective GR blocker because it does not cross-react with (Figure 5). Since most of the available anti-progestins and anti-glucocorticoids cross-react with other receptors (7), to treat diseases such as Cushing's syndrome and depression, the glucocorticoid excess characteristic of those diseases is treated. A selective, pure anti-glucocorticoid is required to antagonize the antagonism, and therefore a pure anti-GC steroid of formula I has long been sought.

Table 1: Dissociation constants, apparent number of sites, and Hill coefficient at the type I receptor in the presence of RU28362 and 21OH-6OP. ─────────────────────────────────── Tracer Receptor type additive Kd Q n _H (nM) (fmol / mg) ─────────────────────────────────── ( ³ H) -ZK91587 I-0.42 98 0.91 ( ³ H) -ALDO I-0.44 104 0.97 ( ³ H) -ALDO I 10 μM RU28362 0.72 124.4 0.90 ( ³ H) -ALDO I 2.5 μM 21OH-6OP 0.80 110 0.88 ( ³ H) -ALDO II-64.4 2900 0.86 ─── ──────────────────────────────── (I): Type I part (II): Type II part

[Brief description of drawings]

FIG. 1: The most stable structure of 21-hydroxy-6,19-oxide progesterone (21OH-6OP): steric optimality by AMPAC5.0 (Semichem) according to the semi-experimental method of AMI. Optimized.

Figure 2: Binding characteristics of 21OH-6OP: using following binding proteins, 12 h at 0 ° C., and 5nM ⁽³ H) -B, and unlabeled steroids amount is increased stepwise Made a replacement for:
Panel A: Cytoplasm of thymus (6.0 mg protein / ml); Panel B: Partial purification (Experiment 1,
Materials and methods) CBG (1 mg protein / ml). Panel C, in the presence of 1.0 μM RU28362 to suppress the cross-reaction with GR, showing the antagonism of unlabeled steroid and 5nM ⁽³ H) -ALDO for kidney cytosol (10mg protein / ml).

FIG. 3: Biological properties of 21OH-6OP: A) In vivo glycogen storage: As in Example 1, male rats were injected with 100 μg steroid per 100 g body weight. Glycogen was purified from liver and quantified with Krisman reagent (17). Controls were injected with solvent. The results are expressed as mean ± SEM (n = 6). *: Significant difference from control
It shows P <0.02. B) In vitro TAT induction: Rat hepatocytes were incubated in HAM-F12 medium for 5 hours in the presence of steroids and TAT activity was measured. The results are expressed as the ratio of the enzyme activity to the control incubated in the presence of the solvent (mean ± SEM, n = 4). *: Significant difference P <0.01 from control is shown. C) Sodium-retaining activity: Adrenalectomized rats were treated with the indicated dose of steroids. The results are expressed as the ratio of the sodium exclusion rate in steroid-injected rats to the exclusion rate in vehicle-injected rats. In this figure,
A representative experimental result when 5 rats are used for each dose is shown.

FIG. 4: Antagonistic effect of 21OH-6OP on TAT induction: in hepatocytes, 2.
In the presence of 5 μM 6OP (+ 6OP) or 21OH-6OP (+ 21OH-6OP), or in their absence (control), 100nM DXM (black bar) or 100nM B (white bar)
Induced TAT activity. The shaded bars represent the case of incubation in the presence of solvent instead of agonist. Only at the incubations marked *, ie stimulation with DXM and B (control and + 6OP), TAT activity shows a significant difference (P <0.01) compared to basal activity.

FIG. 5: Non-cross-reactivity of 21OH-6OP with progesterone receptor: Jordan and Dix (1
Accordance 6), in TEGM, in the presence of unlabeled antagonists of various amounts, at 12 hours 0 ° C., it was binding experiments ⁽³ H) -P ₄ on uterine cytosol.

[Table 1]

[Table 2]

[Table 3]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SZ, UG, ZW), EA (AM , AZ, BY, KG, KZ, MD, RU, TJ, TM) , AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, D K, EE, ES, FI, GB, GE, GH, GM, HR , HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, L V, MD, MG, MK, MN, MW, MX, NO, NZ , PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, U S, UZ, VN, YU, ZW F-term (reference) 4C086 AA01 AA02 AA03 ZA12 ZC08                 4C091 AA01 BB05 CC01 DD01 EE07                       FF01 GG01 HH01 JJ01 KK01                       LL01 MM03 NN01 PA05 PB02                       QQ07 QQ17

Claims (6)

[Claims] 1. A 21-hydroxy-6,19-oxidoprogesterone of formula I for use as a drug. 2. Use of 21-hydroxy-6,19-oxide progesterone of formula I in the manufacture of a medicament for treating or preventing a disease associated with glucocorticoid excess. 3. The use according to claim 2, wherein the disease is Cushing's syndrome. 4. Use according to claim 2, wherein the disease is iatrogenic hypercorticoid. 5. Use according to claim 2, wherein the disease is depression. 6. A 21-hydroxy-6,19-oxide progesterone of formula I and 1
A pharmaceutical composition comprising one or more types of pharmaceutically suitable carriers.