Method for suppression of fear sensation

FIELD: organic chemistry, medicine, pharmacy, pharmacotherapy.

SUBSTANCE: method involves administration in mammal the effective dose of 6-hydroxy-8-[4[4-(2-pyrimidinyl)piperazinyl]butyl]-8-azaspiro[4,5]-7,9-dione or its pharmaceutically acceptable salt of acid addition or its hydrate. Method expands arsenal of medicinal agents used for suppression of fear sensation.

EFFECT: valuable properties of agent.

3 tbl, 6 dwg, 4 ex

 

The object of this invention is a method of suppressing feelings of fear by treatment anxioliticeski effective amount of 6-hydroxy-8-[4-[4-(2-pyrimidinyl)-piperazinil]-butyl]-8-azaspiro[4.5]vs.-7.9bn-dione. This compound was first described Jajoo, et al., Drug Metab. and Disposition. 17/6, pp.634-640, 1989, as one of several metabolites used in the clinic anxiolytic drug buspirone. The structure of this metabolite was confirmed by comparison with the obtained synthetically authentic sample of the compound. This metabolite is indicated BMY 28674.

Original drug, buspirone, has the following structure:

Buspirone, the chemical name is 8-[4-[4-(2-pyrimidinyl)-1-piperazinil]-butyl]-8-azaspiro(4,5)-decane-7,9-dione is a pharmaceutically active compound that has been found effective in the treatment of anxiety (fear) and depression. It is considered that buspirone has an effect using the serotonin receptor 1A (5-HT1A). However, buspirone has a high degree of primary metabolism and, in General, only about 4% of a therapeutic dose of buspirone gets into the bloodstream nematerializiranih form after oral administration (Mayol et al., Clin. Pharmacol. Ther.. 37, R, 1985). Also observed large differences in the absorption of buspirone between different people. It was demonstrated owano by measuring the maximum concentration of drug substance in plasma of patients up to 10-fold differences (Gammans, et al., American J. Med., 80, Suppl. 3B, pp.41-51, 1986).

Synthesis of buspirone and related analogues and their psychotropic properties described by Wu, et al., U.S. patent 3717634. Buspirone hydrochloride as a new anti-fobia agent for the treatment of patients suffering from neurosis described Casten, et al.. U.S. Patent 4182763.

BMY 28674 previously experienced on anti-fobia properties using laboratory methods specifically designed to detect anti-fobia properties azapirone compounds, such as buspirone, gepirone and structural analogues. When these tests have BMY 28674 not there has been a noticeable anti-fobia activity. For this connection was never described the biological activity of any type. Indeed, except for 1-pyrimidinylpiperazine (1-RR), no noticeable anti-fobia activity has not been described for any of the known metabolites of buspirone (see Gammans, et al., JAMA. (March, 1986), Vol. 80, Supp. 3B, pp.43-44). So, I think that oral doses of buspirone for the treatment of fear (phobias) is optimized when it is carried out in such a way as to maximize the concentration of the medicinal substance, not modified in the spending of metabolites.

In U.S. Patent 5431922 described the drug buspirone prolonged action as providing the advantage with a dose of medicinal substance, to the to levels of unchanged buspirone in the blood is increased, while the levels of metabolites decreased if the measure ratio of levels of buspirone and 1-PP-metabolite in plasma. However, these drugs were not provided no data on their effectiveness, not specified, whether they are produced industrially.

In U.S. Patent 5633009 described and claimed transdermal patch for delivery of buspirone. As expected, transdermal delivery of reliably ensured higher levels of buspirone in blood (AUC) at a significantly reduced content of metabolites in the measurement relative to 1-PP. A typical patch was designed to deliver 60 mg of buspirone in 24 hours. Unexpected clinical trials have shown that anti-fobia no different from placebo.

Later in U.S. Patent 6008222 was patented an improved method of oral administration of buspirone in which the bioavailability of unchanged buspirone increases, and the formation of metabolites is reduced. The described method included co-administration of buspirone drug substance nefazodone, an inhibitor of cytochrome RA (CYP3A4). After preliminary evaluation of clinical data was not planned any further improvement of a pharmaceutical product on the basis of combinations of these drugs.

In short, although it was found that BMY 28674 is one of the few h is human metabolites, resulting from oral administration of anxiolytic drugs, buspirone, prior to the present invention, any biological activity were not associated with the connection. In particular, no anxiolytic activity was not detected in previous tests. Recommendations for dosage for buspirone was done in accordance with the assumption that the inhibition of metabolism of buspirone (metabolism) will cause a stronger anti-fobia reaction. The clinical observation that a certain percentage of patients suffering from phobias (sick with fear, anxiety), did not feel relief when taking buspirone, attributed to the fact that these "not responding" patients not achieving adequate levels of original drug. The second clinical observation regarding seven to ten days latent period before the onset anxiolytic effect is attributed due to the necessity of changing the dynamics of the receptor site in result of chronic administration of buspirone. An unexpected discovery anxiolytic actions of BMY 28674 suggests other explanations for these observations.

Brief description of drawings

Figure 1. The effect of BMY 28674 on induced isolation and ultrasonic motor activity of young rats (the rats).

Figure 2. Effect of buspirone on the Indus the new insulation and ultrasonic motor activity of rats.

Figure 3. Concentrations of buspirone in human blood after oral administration of buspirone.

Figure 4. The concentration of 1-PP in human blood after oral administration of buspirone.

Figure 5. The concentration of BMY 28674 in human blood after oral administration of buspirone.

6. In vitro studies of metabolism: metabolism of buspirone in human liver microsomes with the formation of BMY 28674.

Conclusions and a detailed description of the invention

The inventors have found that 6-hydroxy-8-[4-[4-(2-pyrimidinyl)-piperazinil]-butyl]-8-azaspiro[4.5]vs.-7.9bn-dione (I) is effective as an agent for the treatment of fear, and he is called in this description of BMY 28674. The compound has the following structural formula:

and, as I believe, is the active metabolite of buspirone.

As a result of preliminary preclinical (small-scale) tests clinically applicable anxiolytic agent buspirone and several of his alleged and actual metabolites prevailing point of view that the anxiolytic effect is caused mainly by the buspirone at low (if it exists) the contribution of metabolites of buspirone. For example, systemic injection (I.V or intragastric) some alleged metabolites to rats gives weak, or does not, in General, inhibition of excitation is of Aronov dorsal (dorsal) of the weld. On the contrary, the buspirone inhibits the excitation of neurons in the dorsal suture (see VanderMaelen, et al., Eur. J. Pharmacol., 129, pp.123-30, 1986). Although one metabolite, structural fragment of 1-(2-pyrimidinyl)piperazine, also known as 1-RR

showed weak inhibition of excitation of the dorsal suture, and some anti-fobia activity in some other pre-clinical testing (see U.S. Patent 4409223), he also showed entries properties in other behavioral experiments (see Cervo, et al., Life Sciences, 43, pp.2095-2102, 1988; Martin, Psychopharmacology, 104, pp.275-278, 1991). Possible biological effect of 1-PP is mediated by alpha 2-adrenergic mechanism, as 1-RR does not show binding to 5-HT1A receptor. Currently, the clinical effect of 1-PP unclear.

If you take the General position that the active anxiolytic agent is itself buspirone, at the present time, instructions for dosage are given in accordance with the maximize in blood suffering from phobias patients levels of unchanged buspirone. In patients who have Engibarov metabolism of buspirone, which is associated either with the levels of hepatic enzyme activity of the patient, or with the intake of substances that have an inhibitory effect on hepatic metabolism, in particular CYP3A4; patients were advised to reduce the quantity of buspi is it. Interestingly, it was not established specific correlation side effects with higher levels of buspirone in the blood. With the opening of this active metabolite (invention) nature of these indications, dosage should be changed with regard to conditions conducive to the preferred enzymatic formation of BMY 28674. In fact, the dose of buspirone should enhance, not diminish, in patients with inhibited metabolism of buspirone.

Another common hypothesis is related to the fact that buspirone does not reduce feelings of anxiety, a certain percentage of patients. This lack of effect was attributed due to the insufficient level of unchanged buspirone in the blood "areagirls" patients, even if the fact that the levels of buspirone in the blood of all patients is very low. Scientific confirmation of this explanation failure in the treatment of missing. An alternative explanation is evident from the opening of the active metabolite of BMY 28674 and its anxiolytic action. More believable bad treatment of some patients is explained on the basis of the ratio of the anxiolytic effectiveness and level of BMY 28674 in the blood. "Nerealiausi" are those patients who have when exchanging the transformation of buspirone in BMY 28674 not sufficient to achieve an effective level of BMY 28674. This explanation corresponds wide razpro the levels of buspirone in the blood, observe how the same patient and for different patients following oral administration. This variability may result from certain fluctuations, levels of activity metabolism in human liver during the day. However, since the levels of buspirone in the blood are very low, the differences between the levels of buspirone in the blood is usually small compared with the differences between the levels of the more common metabolites in the blood.

Similarly, the latent period before the onset anxiolytic actions after starting treatment with buspirone may include the time required for accumulation of the metabolite, as well as for "preregulation" dynamics of the receptor site. Typically, the dependence of the anxiolytic actions from the appearance of the metabolite BMY 28674 well correlated with clinical observations on patients who are experiencing feelings of fear, receiving oral buspirone.

Based on the fact that the intact buspirone provides a useful anti-fobia activity observed in the clinic, was developed delivery system in the form of a transdermal patch (see U.S. Patent 5633009). It was assumed that the transdermal patch with buspirone is the best tool for the medical treatment of withdrawal fear, because the transdermal delivery system of a drug has minimal metabolism of buspirone, thereby providing the tsya greater number of the parent drug at much lower levels of metabolites. Unexpectedly, when applied in the clinic transdermal patch with buspirone observed low anxiolytic activity or no observed. This unexpected result led to the reassessment of metabolites of buspirone and to the discovery of a strong anti-fobia actions BMY 28674.

The diagram below metabolism (Scheme 1) buspirone taken from Jajoo, et al., Xenobiotica. 1990, Vol. 20, No. 8, pp.779-786, "In vitro metabolism of the antianxiety drug buspirone as a predictor of its metabolism in vivo". ("In vitro metabolism of medicinal substance against fear of buspirone as a forecast of its metabolism in vivo").

Scheme 1

The scheme of the metabolism of buspirone in microsomes and hepatocytes of rat liver

New work began with an assessment of receptor binding of metabolites of buspirone. Accordingly, in vitro activity of buspirone (Bu; MJ 9022) and its metabolite 1-PP (BMY 13653), 3'-Oh-buspirone (BMY 14295), 5-Oh-buspirone (BMY 14131) and 6'-Oh-buspirone (BMY 28674) was evaluated for its activity against human 5-HT1A receptor. The results of these experiments are presented in Table 1.

Table 1
ConnectionIC50[nm]STDEVToivalueN
8-OH-DPAT (standard)2,50,918
Buspirone (MJ 9022)3018158
6-HE-buspirone (BMY 28674)11485577
5-Oh-buspirone (BMY 14131)9281764647
3-HE-buspirone (BMY 14295)6524023267
1-PP (BMY 13653)>1000--3

As you can see in the Table 1 summary of new in vitro effect of MJ 9022 (buspirone) and its metabolites BMY 13653 (1-PP), BMY 14131 (5-Oh-buspirone), BMY 14295 (3-Oh-buspirone) and BMY 28674 (6-Oh-buspirone) on human 1A serotonin receptor (5-HT1A). Buspirone exhibits high affinity to human 5-HT1A-receptor (Ki=15 nm). BMY 28674 has a binding affinity approaching the affinity of buspirone (Ki=57 nm). Other verifiable metabolites have a relatively weak affinity for the human 5-HT1A-receptor compared with buspirone.

BMY 28674, apparently, is the active metabolite of buspirone. Not only the second most frequently occurring metabolite formed during the research exchange from human urine (5-hydroxy-PP is the most common)

but, more importantly, Uro is no BMY 28674 in human blood is about 40 times higher what levels of buspirone in the blood, and several times higher than the levels of 1-PP in the blood. Also of importance is the fact that the skeletal structure of buspirone remains intact in BMY 28674. In addition, the data binding to 5-HT1A receptor indicate that BMY 28674 is closer to the buspirone binding affinity, in contrast to other metabolites, which exhibit only a weak interaction between 5-HT1A site. Currently, the 5-HT1A receptor is understood as a serotonergic receptor, is closely involved in the regulation of fear. The focus of this study focused on metabolites of buspirone, which retain the skeletal structure of buspirone and contain in the molecule one hydrophilic hydroxy-group. The presence of more than one hydrophilic hydroxy-group, apparently, reduces distribution and transport of such polyhydroxylated products of metabolism in the CNS area of the body, thus making unlikely the necessary receptor interaction in targeted areas.

Earlier in-house testing of functional properties of BMY 28674 used in vivo test, which is a modification of the conflict-reaction Vogel (Vogel), a simple robust conflict-methods for testing suppress the fear agents (substances against fear) (see Vogel, et al., Psychopharmacologia. (Berl.) 21, pp.1-7, 1971). However, BMY 28674 does not give the reaction suppressing the fear waste Vogel. Earlier in BMY 28674 not noticed any useful anti-fobia activity.

Ultrasonic vibrations emitted by pups of rats after they were separated from their mothers and litter and were subjected to various environmental effects (e.g., low temperature), as it turns out, are a sensitive method to assess the potential anxiolytic and entries compounds (Winslow and Insel, 1991, Psychopharmacologia. 105:513-520). Psychoactive compounds, prospective anxiety, suppress the frequency of the ultrasonic signals, while the signals increase under the action of medicinal substances with entries properties. More importantly, caused by isolation ultrasonic changes, apparently, are the most sensitive for detecting anxiolytic properties in a wide range of classes of drugs, such as benzodiazepines, 5-HT inhibitors re-absorption, agonists at 5-HT1A, as well as NMDA antagonists. In this study, 6-gidroksilirovanii metabolite of buspirone, BMY 28674 having affinity to human 5-HT1A receptor (Ki=57 nm), evaluate the potential anxiolytic activity in young rats aged 9-11 days, which are taken from mothers, separated from the litter and placed in cold (18-20° (C) a die in order to "identify" stress ultras ukovich signals (Figure 1). Figure 2 shows the results obtained in this test for buspirone.

The introduction of BMY 28674 (0.03 to 1 mg/kg, sc; Figure 1) for 30 min before the test gives a dose-dependent suppression of ultrasonic signal cub rats on the cold plate [F(4,45)=19,27, p=0.0001]. Dose of BMY 28674, predictable reducing the number of signals by 50% (ID50), is 0.13 mg/kg, Motor activity decreases significantly after the introduction of BMY 28674 [F(4,45)=5,85, p=0.007]. However, the dose ID50(0,41 mg/kg) BMY 28674, which is estimated to reduce locomotor activity, about 3 times higher than the dose ID50(0.13 mg/kg) to suppress ultrasonic signals, suggesting that, like buspirone anxiolytic properties of BMY 28674 appear at lower doses.

The introduction of buspirone (0.03 to 1 mg/kg, sc; 2) for 30 min before testing gives a dose-dependent suppression of ultrasonic signal cub rats on the cold plate [F(4,42)=15,44, p=0.0001]. Projected dose of buspirone, reducing the number of signals by 50% (ID50), was 0.10 mg/kg Locomotor activity was also decreased [F(4,42)=4,343, p=0.005] at doses approximately 5 times higher than doses that suppress ultrasonic signals.

The results obtained demonstrate that, like buspirone, a metabolite of BMY 28674 shows anksioliticescuu activity on the ultrasonic signal model of fear arising from the separation is consistent cub rats. Anxiolytic activity due BMY 28674, occurs at doses significantly lower than the dose required to suppress motor activity. In short, the above in vitro and in vivo tests show positive results soothing test for buspirone and BMY 28674; however, the levels of concentrations of buspirone in the blood after oral administration of the small man. Prior to this work no information regarding the concentrations of BMY 28674 in clinical blood was not.

Conducted pharmacokinetic studies in humans and they gave amazing results, further confirming the role of BMY 28674 as the active acid metabolite.

Subjects the subjects (n=13) were given oral buspirone for 25 days with a total dose of 10-60 mg the dose divided into five times and give a 5-day intervals, with increasing visit bid dose in each interval. Pharmacokinetic measurements were carried out on the 5th day of each interval, and these data were used to estimate the pharmacokinetics of buspirone, 1-PP and BMY 28674. Regimen person shown in Table 2.

Table 2
The interval, in scheme applicationBuspirone BID dose (mg)Dimension RK (study Day)
1 55
27,510
31515
42020
53025

These multiple-dose oral accepted buspirone in five different doses, as it was discovered, are safe and generally well tolerated in healthy adult patients participating in the 25-day study.

Figure 3, 4 and 5 shows the average concentrations in the blood of buspirone, 1-PP and BMY 28674, respectively, in 12 hours after the dose on the last day of each interval. Levels of buspirone (Figure 3), in General, very low (about 1-2 ng/ml at higher doses) and fall to less than 1 ng/ml two hours after the dose. In contrast, the levels of 1-PP (Figure 4) and BMY 28674 (Figure 5) is much higher and prolonged (highlight) the action is comparable with buspirone. The concentration of BMY 28674 several times higher than the concentration of 1-PP, and about 30-40 times higher than concentrations of buspirone.

To date, studies show that after oral administration of buspirone metabolite levels BMY 28674 in the blood actually (mostly) is comparable with the observed minor levels of buspirone in the blood. Although it has been shown that buspirone exhibits anxiolytic properties in IP is ytheme models such as the young rats USV presented in this description, low blood concentrations observed in humans, lead to the conclusion that it is a common metabolite of BMY 28674 mediates observed clinically calming effect. Prior to the present evaluation of metabolites of buspirone relative prevalence of BMY 28674 in humans after oral administration of buspirone was not known.

Although it is obvious that to get a good effect, you can enter the BMY 28674, oral administration of his predecessor, buspirone made in certain circumstances, may also be a superior way of introduction BMY 28674 in the body (system) restless person. To determine this status, were conducted in vitro experiments on the metabolism with the aim of obtaining metabolism of buspirone in human liver microsomes (HLM). Choose numerous drugs HLM on the basis of their CYP3A4-enzymatic activity. Drugs HLM get from Gentest Corporation (Woburn, MA: Catalog NN NO, NO, NO and H112), characterize them using standard techniques. In addition to determining the loss of the parent compound during the incubations also determine the levels of metabolite BMY 28674.

In the first stage of experiments to determine the correlation depicted in Fig.6, between the specific activity of CYP3A4 in the HLM preparations and metabolism of buspirone and products metab the lita. 14With-buspirone with the same concentration (10 μm) thermostatic with five HLM drugs from the liver of five donors. Within 15 minutes, incubated with 0.5 mg/ml microsome protein and use NADPH-generating system. Levels14C-buspirone and BMY 28674 in incubated samples determined by reversed-phase HPLC, applying for detecting radioactivity online, and by comparison with authentic standards.14With recovered quantitatively. Figure 6 shows the ratio of the concentrations of BMY 28674 and buspirone compared to CYP3A4 specific activity measured in HLM-liver drugs from five donors (average of two different definitions). These results show that the ratio of BMY 28674 to buspirone increases as CYP3A4-specific activity in HLM increases. The change in the ratio with increasing CYP3A4 activity is marked as rising levels of BMY 28674, and lower concentrations of buspirone (results not shown).

The second series of incubation is carried out with the aim of identifying a potential inhibitor of CYP3A4, changing the ratio of BMY 28674 to buspirone at HLM-incubations. The same concentration of14C-buspirone (10 μm) incubated with United HLM (merged by combining equal volumes of the liver above five donors). Ketoconazole, a well-described inhibitor of CYP3A4, add to cu the dummy samples with different concentrations. All other conditions of incubation, and analysis of samples described above. In the absence of ketoconazole after 15-minute incubation of buspirone from United HLM attitude BMY 28674 to buspirone is 0.42 (table 3). No effect on the metabolism of buspirone is not observed at concentrations of ketoconazole down and 0.125 μm. With 0.25 μm of ketoconazole against the 6'-hydroxypyrene to buspirone decreases to 0.32. At higher concentrations of 1.25 μm and 2.5 μm, the ratio of BMY 28674 to buspirone drops further to 0.06 and 0.01, respectively. These results show that when ketoconazole is incubated together with buspirone in United HLM, the ratio of BMY 28674 and buspirone decreases with increasing concentrations CYP3A4 inhibitor, ketoconazole. The change in the ratio with increasing concentrations of ketoconazole is different as lower levels of BMY 28674, and increasing concentrations of unchanged buspirone.

Table 3

Inhibition of metabolism of buspirone with ketoconazole
[ketoconazole][buspirone][BMY 28674][BMY 28674/ buspirone]
(µm)Relative CPM(Ratio)
02005484000,42
0,0251864578840,42
0,1251964882010,42
0,252195770520,32
1,253611721110,06
2,5437125760,01

Therefore, some fragment (these in vitro experiments show dependence as metabolism of buspirone and appearance of the metabolite BMY 28674 on the activity of CYP3A4 in human liver.

The purpose of this invention is to provide improved (improved) way of detecting anxiolytic response in patients experiencing anxiety. This goal is achieved by providing anxiolytic levels of BMY 28674 in the blood of patients experiencing anxiety. The most obvious way of achieving this goal is the systematic use of sick of the BMY 28674. Therefore, one aspect of the present invention relates to a method for removal of the alarm condition in a mammal in need of such treatment, by using a system of effective sedative dose of BMY 28674.

Effective dose should, as a rule, to establish the minimum concentration (CMIN) BMY 28674 in the blood, which comprise at least 1-2 ng/ml is Usually the time of measurement levels CMIN is after 12 the Asses after taking a dose, i.e. immediately before the next visit bid-dose. BMY 28674 you can enter in a variety of ways, including, without limitation, oral, sublingual, transnasal or parenteral, such as intramuscular, intravenous, subcutaneous, etc.

In order therapy BMY 28674 you can give one of these methods in the form of a preparation containing an effective anxiolytic amount of BMY 28674, or one of its pharmaceutically acceptable salts accession acid or hydrate in a pharmaceutically acceptable carrier. Pharmaceutical compositions which provide from 5 to 50 mg of the active ingredient on the standard dose are preferred and can be usually prepared in the form of aqueous solutions and aqueous or oil suspensions. BMY 28674 can also be given orally if you cook it in the form of oral dosage forms such as tablet, pellet, capsule, syrup, elixir, an aqueous solution or suspension.

Pharmaceutically acceptable salt accession acids to BMY 28674 are also considered applicable as anxiolytic agents. By definition, such salts are salts in which the anion does not contribute a significant contribution to the toxicity or pharmacological activity of the basic form of BMY 28674.

Salt accession acids are produced either by the reaction of BMY 28674 with an organic or inorganic acid, predpochtitel is about, when contacting in solution, or by any standard method, described in detail in the literature and are available to any practice specialist in a given field of technology. Examples of applicable organic acids are carboxylic acids such as maleic acid, acetic acid, tartaric acid, propionic acid, fumaric acid, setinova acid, succinic acid, pamula acid and the like; applicable inorganic acids are kaleidotrope acid, such as Hcl, NVG, HI; sulfuric acid, phosphoric acid, etc.

Preferred oral compositions are compositions in the form of tablets or capsules, and in addition BMY 28674 they may contain conventional excipients such as binding agents (e.g. syrup, Arabian gum, gelatin, sorbitol, tragakant or polyvinylpyrrolidone), fillers (e.g. lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine), lubricants (e.g. magnesium stearate, talc, polyethylene glycol or silica), substances that contribute to crushing (e.g., starch) and surface-active agents (e.g. sodium lauryl sulphate). A solution or suspension of BMY 28674 with conventional pharmaceutical carriers used for parenteral compositions, such as aqueous solution for intravenous injection or an oily suspension for intramuscular inj the work. Such compositions having the desired clarity, stability and adaptability for parenteral use, receive, dissolving 0.1 to 10 wt.% active ingredient (BMY 28674 or its pharmaceutically acceptable salt accession acid or its hydrate in water or in media containing polyhydric aliphatic alcohol, such as glycerine, propylene glycol or polyethylene glycols, or mixtures thereof. The glycols containing a mixture of non-volatile, normally liquid polyethylene glycols, soluble in both water and organic liquids and having a molecular weight of about 200-1500.

BMY 28674 can be synthesized by methods well described in the chemical literature and well-known specialist in the field of synthetic organic chemistry. One way to get uses buspirone as an initial matter, this method is depicted in Scheme 2.

Scheme 2

Getting BMY 28674

This method of obtaining serves as a useful example and illustrates a convenient synthesis of BMY 28674.

You must also take into account that BMY 28674 you can get enzyme (liver microsomes human or rat) transformation of buspirone in vitro (see Jajoo, et al., Xenobiotica. 1990, Vol. 20, No. 8, pp.779-786).

System introduction you can also implement the second method, which checks for an effective anxiolytic in own BMY 28674 in the blood, namely orally administered precursor of BMY 28674. Such proletarienne form should be entered in the quantity that results in an effective anxiolytic effects, preventing harmful or unwanted side effects. That is, systemic administration of BMY 28674 can be done by introducing mammal oral predecessor or Palekastro the form of BMY 28674, for example, buspirone.

However, this method of system introduction BMY 28674 improved in comparison with the known standard method of oral administration of buspirone and differs from it. The prodrug buspirone is used in an improved method for making anxiolysis according to this invention. Therefore, another aspect of the present invention is aimed at weakening (withdrawal) anxiety in a mammal using an improved method of oral administration of buspirone. Improvement (improvement) includes oral administration of buspirone in such a way that it is preferable metabolic production of BMY 28674, thereby anxioliticeski effective amount of BMY 28674 the patient. This contradicts the currently accepted methods of introduction, which is aimed at maximizing levels of unchanged buspirone in the blood. As an example: earlier in the dosage indicated that if buspirone is administered in the us is the conditions, conducive to the inhibition of its metabolism, i.e. when the result is higher levels of unchanged buspirone and lower levels of the metabolite, the dose of buspirone should be lowered. An improved method is the opposite of the previous method of oral administration of buspirone. Instead of lowering the dose of buspirone, when its metabolism is inhibited, dose of buspirone should be increased in order to achieve suitable levels of BMY 28674. There is no specific correlation between serious side effects and receiving high amounts of buspirone.

Summarizing, we can say that the second aspect of the present invention relates to an improved method of reducing unwanted anxiety in a mammal using oral administration of buspirone or its pharmaceutically acceptable salt accession acid in a way that favors the metabolic production of BMY 28674 the mammal. Generally, you should take this amount of buspirone (dose), which will result in a minimum concentration (CMIN) BMY 28674 in the blood of 1-2 ng/ml Definition CMIN patients usually do 12 hours after a dose of medicinal substance and immediately before the next dose.

An example of one of the factors that affect the metabolic formation of BMY 28674, is the impact of the look at oral doses of buspirone. According to the package insert BUSPART(oral tablets buspirone Hcl) suggested that when taking buspirone with food increases the concentration of unchanged buspirone in the plasma.

The second example modification of dosing is offered in to the package insert BUSPARTis the recommendation to lower the dose of buspirone, if it is given in combination with an inhibitor of CYP3A4.

From the point of view of the improved method of reducing unwanted anxiety with oral administration of buspirone on this improved method according to this invention, it is recommended exactly the opposite of what is suggested in the above examples. Instead the dose of medicinal substance during a meal it is recommended to take it for about two hours or more before meals or two hours or more after eating. Similarly, in the case of weak action of cytochrome P450 4 (CYP3A4) dose of buspirone should enhance, not diminish, as recommended by leaflet-to the package insert BUSPART.

Other changes in use of the drug, proposed to improve anxiolytic method system the introduction of BMY 28674 using oral administration of buspirone include the following:

- Buspirone be administered orally in accordance with fluorescent peak CYP3A4 activity in a mammal.

- Stun the IC receiving concomitant medication or food, which inhibit the activity of CYP3A4.

- Increase the dose of buspirone for adjustments reduced CYP3A4 activity, if the discontinuation of concomitant (simultaneous) a drug that inhibits cytochrome activity, from a medical point of view is inappropriate.

In General, oral administration of buspirone patient in a state of fear (anxiety) is modified in such a way and to a degree in accordance with the useful medical practice to the metabolic formation of BMY 28674 was preferred. In accord with a reliable practice preferably BMY 28674 or predecessor in such concentrations that cause anxiolytic effect by preventing harmful or unwanted side effects.

Description of specific options inventions

The connection, which is the essence of this invention, and the method thereof can be better understood in the light of the following examples which are given only to illustrate but not to limit the scope and volume.

EXAMPLE 1

Getting BMY 28674 (I)

A. Di-4-nitrobenzenediazonium(III)

Di-4-nitrobenzenediazonium get using modified literature methods (F.Strain, et al., SoC., 1950. 72, 1254). So, for ice to a solution of 4-nitrobenzylamine (10,11 g, 4.7 mmol) in acetone (20 ml) EXT the keys dropwise within 30 minutes, cooled with ice, the mixture of 30% H 2O2(2.7 ml, 24 mmol) and 2,35 N NaOH (20 ml, 47 mmol). The mixture is vigorously stirred for 15 minutes and then filtered, and the residue on the filter is washed with water and then hexane. The resulting crude solid is dissolved in methylene chloride, the solution dried (Na2SO4and then it was diluted with an equal volume of hexane. The concentration of this solution at 20°in a rotary evaporator gives a crystalline residue, which was filtered, washed with hexane and dried in vacuum to obtain compound III (6,82 g, 74%) as a pale yellow microcrystalline substances, so pl. 104°C (decomp.).

It was found that di-4-nitrobenzenediazonium relatively stable, decomposed So when melting with a weak greenhouse gas. For comparison: dibenzyldithiocarbamate (Cf. M.P. Gore, J.C. Vederas, J.Org.Chem., 1986, 51, 3700) decomposes with sudden energetic ejection of matter from the capillary to determine TPL

Century 6-(4-Nitrobenzenesulfonyl)-8-[4-[4-(2-pyrimidinyl)-piperazinil]-butyl]-8-azaspiro[4.5]-7.9-dione (II)

To a solution of 8-[4-[4-(2-pyrimidinyl)-piperazinil]-butyl]-8-azaspiro[4.5]vs.-7.9bn-dione (buspirone: 10 g, 26 mmol) in dry THF (250 ml) was added LiN(Me3Si)2(28,5 ml of 1 M solution in THF) at -78°C and stirred for 3 hours, then added dropwise over 1 hour a solution of di-4-nitrobenzenesulfonate (11.2 g) in dry THF (150 ml). Premesis the tion at -78° With continued for 1 hour.

Remove the cooling bath and the reaction solution is poured into a mixture of N2O and EtOAc. The organic phase is separated and washed with water and then brine. The organic phase is dried and then evaporated to obtain a viscous oil. Flash chromatography of this oil by elution from a column of silica gel with a mixture of MeCN-EtOAc (1:2) gives the crude product, which was washed with acetone to remove unreacted buspirone, and get 6,23 g of a white solid (46%), the product (II).

C. 6-Hydroxy-8-[4-[4-(2-pyrimidinyl)-piperazinil]-butyl-8-azaspiro [4.5]vs.-7.9bn-dione (I; BMY 28674)

A mixture of II (4.0 g, 6,9 mmol) and 10% Pd/C (about 1 g) in Meon (100 ml) hydronaut in the vibrator when Parra 276-310 kPa for 1 hour. Gidrirovannoe the mixture is filtered through a layer of zeolite, which is then washed with EtOAc. The filtrate is evaporated to a resin, which is purified flash chromatography, using a column with silica gel and elwira EtOAc, obtaining 0,41 g off-white solid (I).

Analysis. Calculated for C21H31N5About3: C, 62,82; N, 7,78; N, 17,44.

Found: C, 62,84; N, 7,81; N, 13,33.

EXAMPLE 2

Analysis of the 5-HT1A-receptor binding

Membrane binding is prepared using human 5-HT1A-receptor expressing NECK cells. Cells collect and destroy using a homogenizer of the downs. Cells are centrifuged at 18000xg 10 minutes and the precipitate of dreams is suspended in the buffer for analysis, frozen in liquid nitrogen and stored at -80°until (days) analysis.

Well take a total of 30 μg of protein. The analysis is performed in 96-well tablets with deep holes. Buffer for analysis represents 50 mm HEPES, containing 2.5 mm MgCl2and 2 mm EGTA. The preparation of the membrane incubated at 25°C for 60 minutes with 0.1 to 1000 nm of the test compound and 1 nm 3H-8-OH-DPAT. 10 mm Serotonin serves as a blocking agent to determine nonspecific binding. The reaction is stopped by adding 1 ml of a cooled ice 50 mm HEPES-buffer, and quickly filtered through the harvester cells Brendel (Brandel), using filters Whatman (Whatman) GF/B. Precipitation on the filters consider using a liquid scintillation counter, LKB Trilux. The values of the IC50determine, using a nonlinear regression method in Excel.

EXAMPLE 3. The analysis of the ultrasonic signals induced by isolation of baby rats

Pup Harlan Sprague-Dawley (males and females) are placed in a cell made of polycarbonate with the uterus prior to 9-11 days old. 30 minutes prior to the testing of rats taken from the uterus, placed in a new cage with his little litter, bring to the laboratory and placed under the lamp (light)to maintain body temperature at 37°C. Then calves weigh, mark and return to the label up behavior assessment (behaviorally assessment). The test is carried out in the region by the traditional chamber made of plexiglass, in which there is a metal plate supported 18-20°With grid 5×5 cm, placed on the plate. For reception of the ultrasonic signals at a height of 10 cm above the plate hung the microphone. Ultrasonic signals are recorded by a system Noldus UltraVox, providing analysis of the frequency and duration of the signals in (online) online. The number of cells, which include calf, also summarize by visual assessment. Calves that do not emit at least 60 signals per 5-minute period before the test, exclude from pharmacological evaluation. Immediately after collection of baseline data pups injected carrier or drug subcutaneously in the scruff of the neck (cervical spine) and return to the brothers on the label. Thirty minutes cubs again tested using the respective characteristics (ultrasonic signals and intersections of the lattice cells) to assess the effects of medicinal substances. Unless otherwise specified, each calf is used only once. Deviation from the baseline (change) and change in percent compared to the baseline frequency of the ultrasonic signals and intersections of the lattice analyze one-way ANOVA method. For an accurate assessment of the effects of medicinal substances perform Bonferroni/Dunn post hoc comparisons with the media in kacestvennaja. To assess dose (milligrams per kilogram) each predicted agonist, induced inhibitory insulation ultrasonic signals by 50% (ID50)using log-probit analysis. All comparisons are conducted with an experimental error rate (α) type I of 0.05.

The dose of each drug can be placed in an arbitrary (irregular) order multiple pomatum. BMY 28674 and buspirone dissolved in physiological solution (0.9% NaCl, media). All injections do subcutaneously in a volume of 10 ml/kg Dose of the drug is related to weight salt.

EXAMPLE 4. The study of metabolism in vitro: the Transformation of buspirone in BMY 28674

- Choose 5 donor HLM preparations on the basis of their CYP3A4 cytochrome P450 enzyme activity.

-14With buspirone (10 μm) incubated with five drugs HLM liver five donors (15 minutes with 0.5 mg/ml microsome protein).

Levels14C-buspirone and BMY 28674 in the incubation samples determined by reversed-phase HPLC, using real-time detection of radioactivity and comparison with authentic standards.

In a second series of incubations carried out to determine the possibilities of ketoconazole (CYP3A4 inhibitor) to change the ratio of BMY 28674 to BUSPARIwhen the HLM incubations.

-14With buspirone (10 μm) incubated with United HLM (15 minutes with 0.5 mg/ml microsome protein).

The method of suppressing the feeling of fear is a mammal, which consists in the introduction to the mammal 6-hydroxy-8-[4-[4-(2-pyrimidinyl)-piperazinil]-butyl]-8-azaspiro[4.5]vs.-7.9bn-dione or its pharmaceutically acceptable salt accession acid or its hydrate.



 

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