EP1049473A1

EP1049473A1 - Pyrimidin 3-oxide compounds suitable for the treatment of pathologies of the skeletal muscle, in particular for the treatment of the hypokalemic paralysis

Info

Publication number: EP1049473A1
Application number: EP99904793A
Authority: EP
Inventors: Moreno Paolini; Domenico Tricarico
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-01-19
Filing date: 1999-01-18
Publication date: 2000-11-08
Also published as: ITMI980086A1; WO1999036072A1; IT1298155B1

Abstract

The present invention relates to pyrimidin 3-oxyde compounds particularly useful for the production of a medicament for the treatment of pathologies of the muscular skeletal apparatus. Among these compounds, the 6-[1-Piperidinyl]pyrimidin-2,4-diamin 3-oxide is particularly effective in the treatment of the familial primary hypokalemia (HOPP) and in the secondary forms induced by drugs, toxins, gastrointestinal K+ loss or by free K+ diet.

Description

PYRIMIDIN 3-OXIDE COMPOUNDS SUITABLE FOR THE TREATMENT OF PATHOLOGIES OF THE SKELETAL MUSCLE, IN PARTICULAR FOR THE TREATMENT OF THE HYPOKALEMIC PARALYSIS Technical Field

The present invention relates to pyrimidin 3-oxide compounds suitable for the treatment of pathologies of the skeletal muscle, in particular for the treatment of the hypokalemic paralysis.

In particular, the present invention regards new medicaments based on 3-oxide derivatives of the pyrimidine that can be useful in the field of the muscular-skeletal apparatus affections, such as the primary and secondary hypokalemic periodic paralysis. Background Art

Pyrimidin 3-oxide derivatives with antihypertensive properties are known from many years from U.S. patents No. 3.382.247 and 3.644.364.

Moreover, some derivatives of the pyrimidine have recently shown new therapeutic indications in the treatment of hair loss and of alopecia androgenetica.

Moreover, it is known that the 6-[l- Piperidinyl]pyrimidin-2,4-diamin 3-oxide shows the main therapeutic indication in the treatment of the malignant hypertension resistant to diazoxide, hydralazine, and other antihypertensive drugs. The 6- [ l-Piperidinyl]pyrimidin-2, 4- diamin 3-oxide causes, in vitro, a marked arteriolar vasodilatation at concentrations that range from 5 to 100 micromolar per liter. It has been proved that the drug vasodilates small arterioles in the gastrointestinal district, in the skeletal muscle and in the skin. Marked vasodilatation is observed also in the cardiac district but not in SNC. The drug is capable to relax in vitro arterioles precontracted with norepinephrine or with 20-30 mM of KC1. The effects of the 6-[ 1-Piperidinyl]pyrimidin- 2,4-diamin 3-oxide are extended also at renal level where the drug ameliorates the renal performance in particular in those patients in which the renal dysfunction is secondary to the hypertension.

It is known that the hypokalemic periodic paralyses are classified in two types: the primary paralysis and the secondary paralysis. These are associated to the lowering of the serum K⁺ concentration below 3.2 mEq/L. The incidence of the primary paralysis on human beings is 1/100000, whereas the secondary paralysis represents one of the most common phenomenon of toxicity of drugs and toxins, affecting a high percent and strongly variable number of patients treated with β-blockers and diuretics.

Although up to date familiarity in the subjects affected by the secondary paralysis was not demonstrated, in these subjects there is however a predisposition possibly linked to phenomena of the genetic polymorphism.

In the primary and secondary forms, the paralytic attacks are caused by a carbohydrate rich meal, glucose injection, rest after exercise, and emotional stress, conditions that release insulin from the pancreas, inducing paralysis. In the primary form high penetrance is observed in the male during the adolescent age, whereas the female are slightly affected. In these subjects, the hystophatological examination made on the muscle tissue shows the presence of vacuoles and morphological alterations of the sarcoplasmic reticulum. The paralytic attacks are separated by periods of prolonged muscle weakness associated to the lowering of the serum K concentration even if less pronounced. At the beginning, the lower limbs are affected, afterwards the upper limbs are affected too. In the most severe cases a complete flaccid paralysis of the lower and upper limbs, respiratory failure and cardiac arrhyt ias are observed. The electromyographic analysis does not show signs of spontaneous activity ( yotonia and muscle hyperexcitability) in the affected patients.

It is also known that the subcutaneous injection of a solution of insulin (4 U/L) and glucose (0.5 gr. ) in the patients affected by the primary paralysis and in the hypokalaemic rats induces flaccid paralysis in 3-4 hr from the injection. This represents a useful characteristic diagnostic provocative test currently used in clinics.

The treatment of the primary hypokalemia is currently limited at the use of drugs that restore the serum K levels such as the acetazolamide and, more rarely, spironolactone. The chronic treatment with these drugs entails the drawback of administering high concentration of drug for long periods of time. These therapies are therefore associated to the onset of side effects of certain severity. For example, the prolonged administration of acetazolamide causes, in a high percentage of treated patients, nephrotiliasis. Moreover, there are variants that do not respond to the drug. In some of these, the acetazolamide precipitates the paralysis.

Currently, another therapeutic intervention takes into account the administration of K salt. This therapeutic approach is however useful only in some patients being not effective in most of the cases. Further, the low management of this therapeutic approach causes frequent cases of hyperkalemia with critical consequences for the patients.

Moreover, in drug intoxications, β-blockers such as the propanolol are used. This compound is capable to restore the symptoms of the hypokalemia linked to an hyperactivity of the Na⁺/K⁺ ATPase pump, but entails the drawback of being contra-indicated in the asthmatic patients and in those with chronic respiratory failure.

The treatment of the intoxication by barium and chlorochine is based on the sole infusion of K salt that however causes hyperkalemia with severe clinical consequences. Currently, therefore, there are no commercially available drugs capable to significantly antagonise the effects of the primary and secondary hypokalemia. Disclosure of the Invention

An aim of the present invention is to provide new therapeutic uses for a class of well-known drugs. Another object of the present invention is to provide a new use for pyrimidin 3-oxide derivatives as pharmacological agents for the treatment of pathology of the muscular- skeletal apparatus.

Another object of the present invention is to provide for the ( 6- [1-Piperidinyl] pyrimidin 2,4-diamin 3-oxide) a new therapeutic use for the treatment of the primary and secondary hypokalemia paralyses.

On the basis of this aim, these objects and others that will become apparent hereinafter, it is provided, according to the present invention, the use of pyrimidin 3- oxide compounds of formula (I):

in which:

R_l is selected from the group consisting of C;_L-C4 alkyl, a -NHR group in which R is Ci-C-i alkyl, or H;

R2 is selected from the group consisting of H, Cι~C alkyl, a -NHR group in which R is as designated above;

R₃ is selected from the group consisting of H, halogen atom, nitro group, amino group, C_!-C alkyl optionally substituted by:

- a group OR5 in which R₅ is C₁-C₄ alkyl, a benzene ring optionally substituted by one or more Cχ-C-1 alkoxy groups;

R4 is selected from the group consisting of H, halogen atom, C₁-C₄ alkyl, heterocyclic moieties, a group -NXY in which:

X and Y are independently selected from the group consisting of C^Cg alkyl, Ci-Cg alkenyl, cicloalkyl of from 3 to 10 carbon atoms unsubstituted or alkyl substituted (preferably alkyl), phenylalkyl of from 7 to 12 carbon atoms, naphtylalkyl of from 11 to 14 carbon atoms, - said heterocyclic moieties are selected from the group consisting of aziridinyl, azetidinyl, pyrrolidinyl, piperidino, hexahydroazepinil, heptamethyleneimino, octamethyleneimino, morpholino, 4-alkylpiperazinyl, each of said heterocyclic moieties having optionally attached as substituents on carbon atoms thereof of from 0 to 3 alkyls, each of said alkyl having of from 1 to 8 carbon atoms; and their salts pharmaceutically acceptable, for the manufacture of a medicament for the treatment of a pathology of the muscular-skeletal apparatus.

Among these compounds, a compound is particularly preferred with formula (I) in which: R-l is NH2; R2 is NH2, R₃ is H;

R4 is a piperidinyl, referred to as 6-( l-piperidinyl)-2, 4-pyrimidin-diamine 3- oxide (minoxidil), or also as (6-[l-piperidinyl]pyrimidin- 2,4-diamin 3-oxide). According to a preferred embodiment of the present invention, the pyrimidin 3-oxide compounds of formula (I) are suitable and selective therapeutic agents in the treatment of the primary and secondary hypokalemic paralyses. in the present invention, the term pathologies of the skeletal muscle apparatus designates the disorders that affect the skeletal muscle, regardless of their nature. These pathologies include the primary hypokalemic paralysis of familial origin (HOPP), and the secondary caused by drugs, toxins or gastrointestinal K loss and free K diet. This term relates also to all the depolarising pathologies of the muscular-skeletal apparatus, such as myotonia and paramyotonia. High-frequency pathologies are also included, such as the mitochondria myophaty, that, to date, do not have a valid pharmacological approach.

This latter specific activity of the compounds of formula (I) can be ascribed to the presence of the receptors for these compounds on the mitochondrial membrane. It has been observed that their stimulation by agonists induces a consistent stimulation of the cellular respiration with a possible consequent benefit in the patients affected by mitochondrial myophathy.

Among the compounds of formula (I), it has been verified that the compound 6-( l-piperidinyl)-2, 4- pyrimidindiamin 3-oxide, generally termed Minoxidil, is particularly suitable in the production of a medicament for the treatment of the pathologies of the skeletal muscle, and in particular for the treatment of the primary and secondary hypokalemic periodic paralysis. An aspect of the present invention is to provide drugs or compositions with a therapeutically effective amount of a pyrimidin 3-oxide compound of formula (I) in association with a pharmaceutically acceptable carrier for the treatment of the disorders of the muscular skeletal apparatus.

Examples of compounds included in the present invention, are, in a not restricted sense:

4-chloride-l , 2-dihydro-l-hydroxy-2-imino-6-methylpyrimidine , 1 , 2-dihydro-l-hydroxy-2-imino-6-methyl- 4pyrrolidilpyrimidine, 6-aminomethyl-l , 2 , dihydro-l-hydroxy-2-imino-4-pyperidin- pyrimidine.

Further examples of suitable compounds of formula (I) are cited in US-3.644.364, or in US-3.382.247 or in EP 0 540 629 Bl and totally included in the present document.

The compounds/derivatives pyrimidin 3-oxide of formula (I) can also be used as corresponding salts with the addition of pharmaceutically acceptable acids such as chloridric acid, phosphoric, sulphoric, glycolic, salicylic, succinic, lactic, maleic acids, and with aminoacids.

The compounds of the formula (I) according to the present invention which are useful in the treatment of the pathology of the muscular skeletal apparatus, in particular in the primary and secondary hypokalemia, are administered to the patient in need of treatment, at a dosage ranging preferably between 18 and 150 μg/kg pro die. In particular, in an adult person of about 70 kg body weight, these dosages are equal to 1,3-10 mg/kg of body weight per day. More specific dosages can be used according to particular conditions of the patient, to the severity of the pathology, to the extension of the invalidity and to the activity of the compounds used. The determination of the optimal dosage for particular clinical situations regards specialists. For the preparation of a medicament or a pharmaceutical composition starting from compounds of the present invention, inert pharmaceutically acceptable carriers are used, both liquid or solid.

Medicaments or pharmaceutical compositions of the invention include powder, tablets, granular forms, capsule, cachet, solutions and suppository.

In formulations in solid form one or more physiologically acceptable substances can be used as solid carrier that act as a diluent, solubilizer, greasing, bonding, aggregating or dis-aggregating element.

As an example, in the pharmaceutical form of tablet the active ingredient of formula (I) is mixed with the vehicle or eccipient in proportion suitable for the solidity, in defined proportion according to the formulation of the pharmaceutical technique. In the powder formulation the carrier is a solid finely divided and mixed with the active ingredient of formula (I).

In the suppository form the active ingredient of formula (I) is in the dispersed form into a suitable eccipient such as a low melting wax, i.e. a mix of glycerol of fatty acids and cacao butter or in glycerine.

Suitable carriers in the preparation of drugs according to the invention include magnesium stearate, lactose, saccharose, magnesium carbonate, dextrin, starch, methylcellulose, pectin, carboxy-methyl-cellulose, wax, talc and other similar vehicles.

The liquid formulation includes solutions suitable for the parenteral or oral administration, or suspensions or emulsions suitable for the oral administration. Formulations or liquid preparations suitable for the parenteral administration are, for example, solutions of compounds of formula (I) in ethanol, water, propylene glycol and mixtures thereof.

Formulations or liquid preparations suitable for the oral administration may include one or more natural or synthetic viscous materials, such as gums, resins, carboxy- methyl-cellulose, and other suspended agents.

The pharmaceutical compositions of the invention can further include aromatic agents, colorants and substances commonly used in pharmaceutical preparations.

The use of the compounds of formula (I) advantageously entails the production of a medicament in single unit of dosage. In this form, the preparation is divided into unitary doses containing a suitable amount of the active ingredient. The form of unitary dosage can be a packaged preparation containing variable amounts of unit.

According to another aspect of the present invention the use of a pyrimidin 3-oxide compound of formula (I) in association with cilazapril for the manufacture of a medicament for the treatment of pathology of the muscular- skeletal apparatus is provided. In particular, this association is useful in the chronic or acute treatment of the muscular pathologies for the prevention of the possible enhancement of the ventricular mass that can be observed in patients treated for a long period of time (6 months-1 year) .

An association particularly useful in the treatment of the primary or secondary hypokalemia is the one between inoxidil and cilazapril. The use in the therapy of the association of these two active ingredients produces a particularly relevant potentiating activity.

The specific biological activity of the compounds of the present invention has been estimated by using the following examples and tests that are shown only as illustrative of the present invention and do not have to be intended in a restricted sense of the present invention, as defined in the appended claims.

EXAMPLE 1 Experiments on hypokalemic rats. A group of 35 male Wistar rats (Charles River, Milano) of 280+21 gr. was divided into two groups of 25 rats and 10 rats, respectively. The first group (hypokalemic rats) was fed with a K free diet (Mucedola S.r.l., Settimo Milanese, Province of Milano) of composition: casein 21.3%, saccharose 15%, solca floe 3%, multivitamins mixture 2%, mineral water free of K ion 3 %, D,L-methionine 15%, coline 0.25%, corn oil 5%, fat 5%, destrin 43.35%. The second group (normokalemic rats) was fed with a diet of the same composition as that of the first group of rats but enriched with K ion (0%). Both groups were fed with pellet of 30 gr./die and water ad libido. The ( 6- [1-Piperidinyl] pyrimidin 2,4-diamin 3-oxide) -chloride was dissolved in a sterile isotonic salt solution (0.9% NaCl) maintained at pH 7.4 with phosphate buffer and afterward used as placebo. After 23 days of K free diet, the body weight of the hypokalemic rats was reduced and was 340+37 gr. , whereas in the normokalemic rats it was 410+15 gr.. The serum K concentration, measured by flame spectrophotometry (Corning EEL 450 flame photometer) in the sera prepared from blood samples collected from the caudal vein, was significantly reduced (p<0.0001) in the hypokalemic animals

(significativity evaluated by student t-test). In these animals the serum K concentration was 2.5+0.2 mEq/L (n rats=25) whereas in the normokalemic rats it was 5.1+0.3 mEq/L (n rats=10). No significant change was observed in the serum Na levels between hypokalemic and normokalemic rats. The serum Na levels were 142+3 mEq/L (n rats=25) and 144+4 mEq/L (n rats=10) in the hypokalemic and normokalemic rats, respectively.

The hypokalemic rats were in turn divided into three groups two out of three composed by 10 rats and one by 15 rats. The first group (hypokalemic low dosage) was treated chronically orally per os by endogastric administration with a dose of ( 6- [ 1-Piperidinyl] pyrimidin 2,4-diamin 3-oxide)- chloride of 18 μg kg die for 10 days. The second group (hypokalemic high dosage) was chronically treated per os by endogastric administration with a dose of (6-[l- Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -chloride of 36 μg kg die for 10 days, the third group was treated with placebo and used as control. The residual 5 rats placebo were used for the evaluation of the acute effects of the (6- [l-Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -chloride. The normokalemic rats were divided in turn into two groups. The first group was treated for 10 days with the high dosage of the ( 6- [1-Piperidinyl] pyrimidin 2,4-diamin 3-oxide)- chloride, the second group was treated with placebo and was used as control. During the 10 days of the pharmacological treatment, the diet of the hypokalemic and normokalemic rats was not modified.

After 10 days of treatment with the low dosage of (6- [1-Piperidinyl] pyrimidin 2,4-diamin 3-oxide) -chloride, in the treated hypokalemic rats an increase in the body weight related to the dose was observed. This was of 375+28 gr. and 462+12 gr. for the dose of 18 μg kg^" die and 36 μg kg die , respectively. In these animals the serum K concentration was similar to that of the normokalemic rats being 3.3+0 mEq/L and 3+0.9 mEq/L for the 18 μg kg^" die and 36 μg kg die , respectively. No changes were observed in the serum Na level after treatment with (6-[l- Piperidinyl ]pyrimidin-2 , 4-diamin 3-oxide ) -chloride .

Test n'l: after 10 days of treatment, the insulin test was performed. The hypokalemic rats treated with (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -chloride and the rats treated with placebo were intraperitoneally injected with a physiological solution enriched with pig's/bovine insulin (4 U/100 gr. body weight) and glucose (0.5 gr. ) . This procedure causes flaccid paralysis in 3-4 hours from the injection in both the patients affected by the primary and the secondary forms of the paralysis.

It was observed that 100% of the hypokalemic rats treated with placebo were paralysed after 3-4 hours from the insulin and glucose injection. Whereas, the chronic treatment with the ( 6-[l-Piperidinyl]pyrimidin-2, 4-diamin 3- oxide) -chloride low dosage prevented the paralysis in 50% of the hypokalemic rats and in 80% of the hypokalemic rats treated with high dosage.

The normokalemic placebo rats did not show any kind of paralysis after injection of the insulin and glucose solution.

EXAMPLE 2

According to the flaccid paralysis observed following the acute administration of insulin and glucose (Test n°l), recordings of the membrane potentials of the skeletal muscle fibers performed in vitro by the two endocellular microelectrode techniques on the extensor digitorum longus muscle (EDL) excised from the hypokalemic paralysed rats (hypokalemic rats treated with placebo and fraction of hypokalemic rats that did not respond to the treatment with (6-[l-Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -chloride, (Test n°l) showed that in these animals the muscle fibers were depolarised, as shown in the tables 1 and 2 and following reported. The in vitro exposure of the EDL muscle of these animals to a Ringer low K solution further depolarised the muscle fibers (Table 1, 2). On the contrary, the muscle fibers of the hypokalemic not paralysed rats (fraction of hypokalemic rats that responded to the treatment with (6-[l-Piperidinyl]pyrimidin-2, 4-diamin 3- oxide) -chloride) were not depolarised when exposed to the Ringer low K solution as shown in the following Table 2.

According to the test n°l, the muscle fibers of the placebo normokalemic rats after injection with the insulin and glucose solution were markedly hyperpolarised when exposed to the Ringer low K solution (Table 1).

Table n°l Effects of an acute administration of insulin on membrane potentials of the fibers of the extensor digitorum longus muscle of hypokalemic and normokalemic rats

Experimental group Membrane potentials in Membrane potentials in Ringer normal Ringer solution low K⁺ solution ( V) <]5 )

Placebo normokalemic rats after the injection of -81.3+3.2 (n° fibers=45) -87.7+8.1 (n° fibers =52) insulin 4U/L+glucose 0.5 gr. 100% not paralysed

Placebo hypokalemic rats after the injection of -54.2+5.1* (n° fibers =76) -50.7+8.1* (n° fibers =90) insulin 4U/L+glucose 0.5 gr. 100% paralysed

The values are the mean+standard error of 10 individual experiments. *Values significantly different with respect to those of the normokalemic rats (p<0.001). The significativity was evaluated by student-t test.

Table n° 2 Effects of the chronic administration of ( 6-[l-Piperidinyl]pyrimidin-2, 4-diamin 3- oxide) -chloride on the paralysis induced by insulin and on membrane potentials of the fibers of the extensor digirorum longus muscle of the hypokalemic rats

Hypokalemic rats chronically Membrane potentials in Membrane potentials in treated with MINOXIDIL- normal Ringer Ringer low K⁺ chloride after the acute (mV) (mV) injection of insulin

50% paralysed -66+6.5 (n° fibers =96) -62.7+7.2 (n°fibers =100)

18 μg kg^_1die

50% not paralysed -77.3+2.9 * (n° fibers =20) -78.35+1.6* (n° fibers =20)

20% paralysed -63+6.1 (n° fibers =86) -57.7+8.1 (n° fibers =90)

36 μg kg die^"

80% not paralysed -84.4+3.2 * (n° fibers =40) -89.4+1.8* (n° fibers =43)

The values are the mean+standard error of 10 independent experiments. *Values significantly different with respect to those of the normokalemic rats (p<0.001). The significativity was evaluated by student-t test.

In another series of experiments, the (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide ) -sulphate, in vivo metabolite of the ( 6-[ l-Piperidinyl]pyrimidin-2 , 4-diamin 3- oxide) -chloride, was directly applied in vitro on EDL muscle of the placebo hypokalemic rats. It was observed that the drug at doses between 1 μM and 100 μM was capable to prevent the depolarisation of the muscle fibers induced by the Ringer low K solution enriched with insulin, as reported in the following Table 3.

Table 3 Effects of the acute administration of (6-[ l-Piperidinyl]pyrimidin-2, 4-diamin 3- oxide) -sulphate on membrane potentials of the fibers of the extensor digitorum longus muscle of hypokalemic rats preincubated "in vitro" with insulin.

Placebo hypokalemic rats Dose Membrane potentials (μM) in Ringer low K⁺

+ insulin (4U/L)

(mV)

Controls C

-53.1+2.1 (n° fibers=27)

( 6-[ 1-Piperidinyl ]pyrimidin- -57.4+2.1 * (n° fibers =34) 2,4-diamin 3-oxide) -sulphate

10 -66.1+5 * (n° fibers =33)

100 -87.2+4 * (n° fibers =31)

The values are the mean+standard error of 5 independent experiments. *Values significantly different with respect to those of the normokalemic rats (p<0.001 or less). The significativity was evaluated by student-t test.

EXAMPLE 3

In order to evaluate the effects of the (6-[l-

Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -sulphate on the

+ . + macroscopic K currents and on single ATP sensitive K channels of the muscle fibers of the hypokalemic rats, single fibers were isolated by enzymatic dissociation of the muscle biopsy (EDL) excised from the paralysed and not paralysed rats, and from the normokalemic rats. The muscles were then incubated with normal Ringer enriched with collagenase type

XI-S for 30 min., at 30° C, in a Dubnoff shaker, at 0.5 rpm, under 95% 02 and 5% CO2 atmosphere. Macroscopic currents

2 flowing through macropipettes (pipette area=5.2+0.9 μm ) and single channel current flowing through micropipettes (pipette

2 area=1.2+0.3 μm ) were recorded through the standard patch clamp technique in the membrane patches excised from the skeletal fibers (inside-out configuration). The recordings were performed at a voltage of -60 mV, at 22° C, in the presence of 150 mM of KC1 on both sides of the membrane patches under study, and in the presence or absence of MgADP

(50 μM) that it is known to induce the maximal current stimulation. The macroscopic current recordings were acquired by the Clampex software (AXON Instrument, U.S.A.) with a sampling rate of 5 kHz and filtered at 0.5 kHz by low bessel filters. The single channel recordings were acquired continuously at constant voltage by a video recorder at frequency of 98.4 kHz. Afterwards, these current traces were sampled back by Fetchex software (AXON Instrument, U.S.A.) at

20 kHz and filtered at 2 kHz for further analysis. The analysis of the macroscopic and microscopic traces was performed by Cla pfit, Fetchan and Pstat (AXON Instrument, U.S.A.) software. The membrane patches excised from the muscle fibers of hypokalemic rats that were paralysed (hypokalemic rats treated with placebo and fraction of hypokalemic rats that did not respond to the treatment with ( 6-[ l-Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -chloride Test n° 1), showed macroscopic K currents markedly reduced, due to the loss of the ATP dependent component, as shown in Figures 1 A, B, in which the macroscopic recordings of the ATP dependent K currents of placebo normokalemic rats (A) and of hypokalemic rats (B) are reported. The recordings were performed, on isolated membrane patches, at -60 mV (Vm) , at 20 °C, in the presence of 150 mM KCl on both sides of the membrane. The application of ATP on cytosolic face of the channels reduces the K currents indicating that these were sustained by ATP sensitive K channels.

In the placebo hypokalemic rats the macroscopic ATP sensitive K + current was -11.1+2.5 pA/μm2 (n° macropipettes/n° rats=98/10). In same membrane patches the

2 current was particularly reduced and was -1.4±2.1 pA/μ (n° macropipettes/n° rats=54/10). On the contrary, in the normokalemic placebo rats the macroscopic ATP sensitive K

2 current was high being -21.5+3 pA/μm (n° macropipettes/n" rats=170/10) . Single channel analysis revealed an abnormal reduction of the open probability (Popen) and of the number of functional ATP sensitive K channels (N) present in the patches under study, as is shown in the following table 4. In particular, two conductance levels (γ) of single K channel, which are blocked by ATP, were detected in the membrane patches of the hypokalemic rats (Table 4). These were due to fluctuations of the same channel between two different conductance states. Similar macroscopic and microscopic abnormalities were observed in the hypokalemic rats treated with ( 6-[ l-Piperidinyl]pyrimidin-2, 4-diamin 3-oxide ) -chloride that did not respond to the treatment (see Test 1). In contrast, only one conductance level was detected in the normokalemic placebo rats and in the rats treated with (6- [l-Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -chloride (Table n° 4). In the hypokalemic rats not paralysed that therefore responded to the treatment with low and high dosages of (6- [l-Piperidinyl]pyrimidin-2, 4-diamina 3-oxide) -chloride (see

+ Test 1), the macroscopic K currents were high due to the presence of the ATP sensitive component and were similar to those detected in the placebo normokalemic rats, as shown in the enclosed Figures 2 A, B in which the recordings of the macroscopic ATP sensitive K currents of the hypokalemic rats treated for 10 days with (6-[ l-Piperidinyl]pyrimidin-2,4- diamin 3-oxide) -chloride 36 μg kg -1 die-1 (A) and 18 μg kg-1

die (B) are reported. The recordings were performed, on isolated membrane patches, at -60 mV (Vm) at 20°C, in the presence of 150 mM KCl on both sides of the membrane. The application of ATP on the cytosolic face of the channels reduces the K currents indicating that these were sustained by ATP sensitive K channels.

2 In these animals the currents were -18.2+4 pA/μm (n°

2 macropipettes/n° rats=71/10) and -15.2+3 pA/μm (n° macropipettes/n° rats=51/10) in the hypokalemic rats treated with low and high dosages, respectively.

Also the single channel parameters were similar to those of the normokalemic rats (Table 4). Table 4 Effects of the chronic administration of ( 6-[ 1-Piperidinyl ]pyrimidin~2, 4-diamin 3-oxide)-chloride on the parameters of the ATP dependent K⁺ single channel of the fibers of the extensor digitorum longus muscle of the hypokalemic rats.

Experimental groups Y N/μm' open ( PS)

Placebo normokalemic rats 71+1.4 4+1 0.437+0.05

(n =26) (n =26) (n =10)

73 + 3 1 + 0 0.18+0.02

Placebo hypokalemic rats (n =28) (n=28) (n =3) paralysed

29+4 1 + 0 0.13+0.06

(n=7) (n=28) (n =3)

Hypokalemic rats treated with 18 μg kg -l 70+4 2 + 1 0.298+0.07 die^-1 of the drug (n=17) (n =17) (n=5) Not paralysed

Hypokalemic rats treated with 36 μg kg^" 71+5 4+1 0.334+0.02 die^-1 of the drug (n=ll) (n =11) (n=6)

Not paralysed

The values are the mean+standard error. From left to right are reported γ, the single channel conductance calculated in the range of membrane potentials between -10 mV e —70 mV; N, the number of functional channels normalised on the pipette area; Popen_# the open probability of the channel measured, at —60 mV, as ratio between the time spent by the channels in the open state over the total time of recording. The rats were treated with insulin and glucose (see Test n° 1) to induce paralysis.

No significant effect on membrane potentials (see Test n°2), and on the macro and microscopic currents was observed following the chronic treatment of the normokalemic rats with the high dose of (6-[ l-Piperidinyl]pyrimidin-2, 4-diamin 3- oxide) -chloride.

In another series of experiments, the (6-[l- Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -sulphate was applied in vitro directly on the membrane patches isolated from the muscle fibers of placebo hypokalemic rats. It was observed that the drug at the doses between 1 μM and 100 μM was capable to stimulate the macroscopic ATP dependent K currents without reaching the current values of the placebo normokalemic rats. Also some single channel parameters such as the Popen were similar to those of the placebo normokalemic rats. In contrast N was not modified by the (6- [l-Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -sulphate and was similar to those of the hypokalemic placebo rats as shown in the Figures 3 A, B, in which the typical continuous recordings of single ATP-sensitive K channels of placebo normokalemic rats (A) and placebo hypokalemic rats (B) are reported. The recordings were performed, on membrane patches, at -60 mV (Vm) , at 20 °C, in the presence of 150 mM KCl on both sides of the membrane. For convention, downward deflection of the current indicates channel openings. One conductance level is distinguishable in the trace of the normokalemic rats (A), whereas two conductance levels, 01 and 02, are present in the trace of the hypokalemic rat (B). The application of ATP at 16 sec (A) and 30 sec (B) of recordings on the cytosolic face of the channels inhibited the K channels indicating that these were ATP dependent. The application of ( 6-[ l-Piperidinyl]pyrimidin-2, 4-diamin 3- oxide)-sulphate induced a stimulation of the open probability of the channels of the normokalemic and hypokalemic rats. No significant effects were observed on the membrane patches of the normokalemic rats that already showed elevated macroscopic ATP-sensitive K currents. EXAMPLE 4

Test n° 4: In order to evaluate if a single dose of (6-[l-

Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -chloride prevents the paralysis in the hypokalemic rats, an acute administration of physiological solution enriched with (6-[l-

Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -chloride 150 μg kg

1 . -1 die to 5 hypokalemic rats was performed. After 90 min. from the administration (time needed for the hepatic metabolism to transform the (6-[l-Piperidinyl]pyrimidin-2, 4- dia in 3-oxide)-chloride to sulphate derivative, the active metabolite), these rats were treated with the usual injection of insulin and glucose. In 5 out of 5 rats no paralysis was observed in the first 4 hours from the administration. In these animals the serum K concentration at death was 3+0 mEq/L, values similar to those of the controls. EXAMPLE 5

Test n' 5: In order to evaluate if the chronic administration of (6-[l-Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -chloride was capable to induce, macroscopic alterations in the main organs, the weight of the dried organs was measured over the body weight of the rats at death. This type of experiment was performed on hypokalemic rats treated with placebo, on hypokalemic rats treated with ( 6- [ 1-Piperidinyl] pyrimidin- 2,4-diamin 3-oxide) -chloride at two different dosages, and on normokalemic rats treated with the high dose of the drug.

Table n° 5 shows a significant increase in the weight of the kidneys and lungs of the hypokalemic rats with respect to that of the normokalemic rats. No significant changes were observed in the weight of the other vital organs. The (6-[l- Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -chloride after chronic administration (10 days) at both tested dosages did not alter significantly the weight of the heart of the hypokalemic and normokalemic rats. This is particularly interesting, indeed the limit of the use of the drug as antihypertensive is the onset of cardiac hypertrophy that is manifested as a significant increase in the weight of the heart in the treated subjects. These toxic effects reported by other authors were observed at concentrations of 2 mg per day, that is the lower one used as anti-hypertensive for chronic administration for at least 6 weeks. Moreover, the same authors have reported that the acute treatment did not provoke any significant toxic effects. An interesting observation that has been made is that the chronic treatment with (6-[l-Piperidinyl]pyrimidin-2,4-diamin 3-oxide) -chloride was capable to prevent the significant increase in the weight of the lungs and kidneys of the hypokalemic rats with an unknown mechanism, as shown in the Table 5. Table n° 5 Effects of the chronic treatment with ( 6-[ l-Piperidinyl ]pyrimidin-2 , 4-diamin 3- oxide ) -chloride on the organ weight of the normokalemic and hypokalemic rats

Experimetal groups hear /body Kidney/ body Spleen/body Lung/ body EDL/ body Liver/body weight weight weight weight weight weight

Placebo normokalemic 0.0033+0. OOOl 0.003+0.0002 0.002+0.00Q3 0.005+0.0004 0.0005+0.0003 0.035+0.00 rats (n° rats =10) (n° rats =10) (n° rats =10) (n^* rats =10) (n° rats =10) (n° rats =1

(not paralysed)

Placebo hypokalemic 0.0031+0.0001 0.008+0.0001* 0.005+0.0001 0.02+0.005* 0.0005+0. OOOl 0.036+0.00 rats (n° rats =10) (n° rats =10) (n° rats =10) (n° rats =10) (n° rats =10) (n° rats =1

(Paralysed)

Hypokalemic rats (not paralysed) 0.0035+0.0003 0.005+0.0004 0.0025+0. OQl 0.008+0.0006 0.0005+0.0002 0.037—+0.00 treated with 18 μg kg~ (n° rats =5) (n° rats =5) (n° rats =5) (n° rats =5) (n° rats =5) (n° rats =5 die of the drug

Hypokalemic rats 0.003+0.0002 0.004+0.0006 0.003+0.0008 0. 008+0.0008 0.0005+0. OOOl 0.031+0.00 ( not paralysed) ( n ° rats =8 ) ( n ° rats =8 ) ( n ° rats =8 ) ( n ° rats =8 ) ( n ° rats =8 ) ( n ° rats =8 treated with 36 μg kg^" die^" of the drug

The values are the mean+standard error of the ratio between the organ-dried weight and the body weight of the animals. The rats were treated with insulin and glucose ( see Test n° 1 ) to induce the paralysis . *Values significantly different with respect to those of the normokalemic rats (p<0.001 or less ) . The signif icativity was evaluated by student-t test .

Experiments on human biopsy.

In line with the above-described experiments on the rodent, the following tests were performed on patients.

The effects of the ( 6-[ l-Piperidinyl]pyrimidin-2 , 4- diamin 3-oxide) -sulphate were evaluated in vitro on muscle biopsy of patients affected by primary hypokalemic periodic paralysis and in subjects that underwent orthopaedic surgery considered as controls. The two subjects affected by HOPP were an adolescent male of 18 year of age and an adult female of 40 year of age. Both patients showed the mutation R528H of the dihydropyridine receptor of the skeletal muscle that is one out of three of the most common found in the HOPP patients. The 18 year old patient was affected by frequent, 4-5 per week, severe attacks of hypokalemic paralysis having a minimum duration of 5 hours and a maximum duration of 48 hours. The paralysis usually nocturnal was observed after a period of physical stress, or following a carbohydrate rich meal . During the attacks the serum K⁺ concentration was £1.5 mEq/L. The attacks were then interrupted by periods of muscle weakness during which the serum K⁺ concentration was restored to 3-3.5 mEq/L. The electromyographic analysis did not show signs of myotonia. The family pedigree showed that the mother was affected.

The 40 year old woman showed a persistent muscle weakness that led to rare attacks, 1 per week generally nocturnal and of short duration (1-2 hours). During the attacks the serum K⁺ concentration was <2 mEq/L. The attacks were mainly provoked by a period of physical stress.

Both healthy subjects examined did not present pathology of certain importance, nor systemic infections or alterations of the blood parameters. EXAMPLE 6

Test n°6: As already observed for the hypokalemic rats, recordings of the membrane potentials of the skeletal muscle fibers performed in vitro by the two microelectrode technique on vastus lateralis muscles excised from two hypokalemic subjects have shown that in these biopsies the muscle fibers were slightly depolarised with respect to those of the healthy subjects (Table 6). The in vitro exposure of the muscles to a Ringer low K solution further depolarised the muscle fibers (Table 6). A further depolarisation was observed when the fibers were exposed to a Ringer low K⁺ solution enriched with insulin. On the contrary, the muscle fibers of the healthy subjects were hyperpolarised when exposed to Ringer low K⁺ solution and/or to insulin, as shown in the Table 6.

Table n' Membrane potentials of the fibers of the vastus lateralis muscle of healthy subjects and of hypokalemic patients

Experimental groups Membrane potentials Membrane potentials Membrane potentials in in normal Ringer in Ringer low K⁺ Ringer low K⁺+ insulin (m ) (mV) (mV)

Male healthy subject -80.3+3.6 -84.3+2.5 -89.3+4.1 of 17 year of age (n° fibers=31) (n° fibers =27) (n° fibers =38)

Female healthy subject -79.3+3.5 -86.3+3.5 -90.3+2.1 of 50 year of age (n° fibers=32) (n° fibers =21) (n° fibers =25)

Male patient affected -73.3+2.5 * -65.3+4.1* -52.7+3.2* by hypokalemic (n° fibers =27) (n° fibers =29) (n° fibers =23) periodic paralysis of 18 year of age

Female patient -75.3+1.5* -60.3+2.1* -50.7+8.1* affected by (n° fibers =32) (n° fibers =20) (n° fibers =15) hypokalemic periodic paralysis of 40 year of age

The values are the mean+standard error. *Values significantly different with respect to those of the healthy subjects (p<0.001 or less). The significativity was evaluated by student-t test. The muscles were incubated with insulin (4 U/L) for 30 in before recording.

The incubation for 30 min. of the muscle biopsies of the HOPP patients with ( 6- [ l-Piperidinyl]pyrimidin-2 , 4-diamin 3- oxide)-sulphate (1 μM and 100 μM) was capable to prevent in dose-dependent manner the characteristic depolarisation induced by Ringer low K⁺ solution and insulin restoring the membrane potentials of the muscle fibers, as shown in the following Table 7.

Table 7 Effects of the acute administration of ( 6- [ l-Piperidinyl]pyrimidin-2 , 4-diamin 3- oxide ) -sulphate on membrane potentials of the fibers of the vastus lateralis muscles of patients affected by hypokalemic periodic paralysis

Hypokalemic patients Dose (μM) Membrane potentials in Ringer low K⁺ + insulin (4U/L) (n>V)

Controls -51.3+1.1 (n° fibers=38)

( 6- [ 1-Piperidinyl ] pyrimidine- -56.4+3.1 * 2,4-diamin 3-oxide )-sulphate (n° fibers =39)

10 -68.1+4 * n° fibers =30)

100 -88.2+6 * (n° fibers =37)

The values are the mean±standard error of two independent experiments. *Values significantly different with respect to controls (p<0.001 or less).

No significant effects of the (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -sulphate was observed on the membrane potentials of the fibers of the vastus lateralis muscles of the healthy subjects. EXAMPLE 7 Test n° 7: In order to evaluate the effects of the (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -sulphate on the ATP dependent K currents and on the single ATP dependent K channels of the muscle fibers of the HOPP patients, single cells were obtained by enzymatic dissociation of the muscle biopsies of the vastus lateralis muscle with a Krebs solution enriched with collagenase type XI-S for 30 min., at 30° C, in a Dubnoff shaker incubator at 0.6 rpm under 95% 02 and 5% CO2 atmosphere. Macroscopic currents flowing through macropipettes (pipette area=5.01+04 μm 2 ) and single channel currents flowing through micropipettes (pipette area=l.1+0.4

2 μm ) were recorded by the standard patch clamp technique on membrane patches isolated from skeletal fibers. The macroscopic and microscopic recordings were performed as reported above (see Test n°3). As already reported for the hypokalemic rats, the membrane patches isolated form the muscle fibers of both 18 and 40 year old patients showed a macroscopic K current markedly reduced with respect to that recorded in the healthy subjects due to the loss of the ATP dependent component as shown in Figures 4 A, B, in which the recordings of the macroscopic ATP dependent K currents of the hypokalemic patients are reported. The recordings were performed, on isolated membrane patches, at -60 mV (Vm), at 20° C, in the presence of 150 M KCl on both sides of the membrane. The application of ATP on the cytosolic face of the channels reduced the K currents indicating that these were sustained by ATP sensitive K channels. The (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -sulphate, applied on the cytosolic face of the channels, induced a stimulation of the currents. A further evidence is shown in Figures 5 A, B in which the recordings of the macroscopic ATP dependent K currents of healthy subjects are reported. The recordings were performed, on isolated membrane patches, at -60 mV (V ) , at 20 °C in the presence of 150 mM KCl on both sides of the membrane. The application of ATP on the cytosolic face of the channels reduced the K currents indicating that these were sustained by ATP dependent K channels. The (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -sulphate, applied on the cytosolic face of the channels, induced a stimulation of the currents.

In the HOPP patients the ATP dependent K current was

2 2

-0.36+0.02 pA/μm (n° macropipette=8 ) and -0.16+0.036 pA/μm

(n° macropipette=13) in the 18 year old male and in the 40 year old female, respectively (Fig. 4 A, B). Whereas it was - 9.03+0 pA/μm² (n° macropipette=8 ) and -9.1+0.9 pA/μm² (n° macropipette=ll) in the control male subject of 18 years of age and in the female subject of 40 years of age, respectively (Fig. 5 A, B). In these muscle fibers, the single channel analysis showed an abnormal reduction of the Popen of the ATP dependent K channel and of the number of functional channels, as reported in the table 8.

As already observed in the rodent experiments, also in the human experiments at least two conductance levels of single ATP dependent K channels were observed, as shown in Figure 6 in which the continuous recordings of single ATP dependent K channels of a hypokalemic male patient of 18 year of age are reported. The recordings were performed, on isolated membrane patches, at -60 mV (Vm) at 20° C, in the presence of 150 mM KCl on both sides of the membrane. For convention, downward deflection in the current records indicates the channel openings. At least two conductance levels of different amplitude are represented in the trace reported. The application of ATP on the cytosolic face of the channels inhibited the K channels indicating that these were ATP dependent. The application of (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -sulphate induced a stimulation of the open probability of the channels of the patient. These levels were due to fluctuations of the same channel between two different conductance states. In contrast, only one level of conductance was observed in the healthy subjects as shown in Figure 7 in which typical continuous recordings of single ATP sensitive K channel of an healthy male subject of 18 year of age are reported. The recordings were performed, on isolated membrane patches, at - 60 mV, at 20 °C, in the presence of 150 mM KCl on both sides of the membrane. For convention, downward deflection in the current records indicates channel openings. At least three conductance levels of similar amplitude are present in the trace reported. The application of ATP on the cytosolic face of the channels inhibited the K channels indicating that these were ATP dependent. The application of (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -sulphate induced a stimulation of the open probability of the channels of the subject. The acute exposure of the membrane to (6-[l- Piperidinyl]pyrimidin-2, 4-diamin 3-oxide ) -sulphate induced a significant stimulation of the macroscopic ATP dependent K current in both healthy and HOPP patients (Fig. 4 A, B; 5 A, B). This effect was more marked in the 18 year old HOPP patient than in the 40 year old HOPP patient. At single channel level, after exposure of the membrane to the drug, an increase was observed of the open probability of the channels but not of the number of functional channels (Fig. 6; Tab 8). Moreover, in the presence of the drug only one conductance level was detected in the patients of amplitude similar to those of the healthy subjects (Table 8).

Table n° 8 Effects of the acute administration of ( 6-[ 1-P per α nyl ]pyr m-.dιr-2 , 4- diamm 3-oxιde) -sulphate on the parameters of ATP dependent K^* channels of fibers of vastus lateralis muscle of HOPP patients and healthy subjects

Experimental groups γ ( _PS ) N/μm" ^popen

Male healthy subject of 17 year of age 70+3 4+1 0.411^0.07

(n =6) (n =6) (n =4)

(6-[ l-Pιperιdιnyl]pyrιmιd ne-2,4-dιamιn 3-o de)- 70+2 4+2 0.699+0.08 sulphate 10 μM (n=3) (n=3) (n=3)

Female healthy subject of 50 year of age 71+5 4+1 0.334+0.02

(n =11) (n =11) (n =6)

( 6- [ 1-Pιperιdιnyl ] yr m d ne-2 , 4-dιamιn 73+3 4+2 0.639+0.05 3-oxιde) -sulphate 10 μM (n=4) (n=4) (n=3)

Male patient affected by hypokalemic periodic 72+2 3+2 0.06+0.002 paralisys of 18 year of age (n =11) (n =11) (n =5)

27+2 1+0 0.09+0.006 (n =5) (n =5) (n =3)

( 6- [ 1-Pιperιdιnyl ] pyrιmιd ne-2 , 4-dιamm 3-oxιde) -sulphate 10 μM 72+3 3+2 0.15+0.06 (n=3) (n=3) (n=3)

Female patient affected by hypokalemic periodic 71+1 2 + 1 0 . 07+0.002 paralisys of 40 year of age (n=4) ( n =10 ) ( n =4 )

26+2 2 +1 0.08+0.007

(n=3) (n =6) (n =3)

( 6- [ 1-Pιper idiny 1 ] pyrιmιdιn-2 , 4-d amm 3-oxιde) -sulphate 10 μM 73+4 3+1 0.17^0.03 (n=4) (n=4) (n=3)

The values are the mean+standard error. From left to right are represented v, single channel conductance calculated m the range of membrane potentials between -10 mV e -70 mV; N, number of functional channels normalised on the pipette area; ^popen, open probability of the channels measured at -60 mV as ratio between the time spent by the channels in the open state over the total time of recording. It has been verified that the ( 6-[ 1-Piperidinyl]pyrimidin-

2,4-diamin 3-oxide) -chloride chronically administrated for a period of 10 days at doses of 18 μg kg -1 die-1 and 36 μg kg-1

die was capable to prevent the characteristic paralysis induced by insulin in the hypokalemic rats in dose-dependent manner. Also the acute treatment with the drug under study at a dose of 150 μg kg die was effective in the prevention of the paralysis induced by insulin in the same animals.

Both the chronic and acute treatments with the (6-[l- Piperidinyl]pyrimidin-2 , 4-diamin 3-oxide) -chloride were capable to restore the serum K level of the hypokalemic rats without inducing episodes of hyperkalemia that is of certain importance in the clinical application.

According to the results obtained in vivo, it was observed that the drug was capable to repolarise the muscle fibers of the hypokalemic rats. This effect was observed in the biopsy of the hypokalemic not paralysed rats which therefore responded to the treatment, after direct application in vitro of the drug on the hypokalemic placebo rat muscles pre-incubated with insulin and Ringer low K solution. Similarly, after the chronic treatment with the drug under study, also the values of the macroscopic currents and the single channel parameters were similar to those observed in the normokalemic placebo rats used as controls. The direct application of the drug on the membrane patches was capable to restore the open probability and the single channel conductance, but was not capable to restore the number of functional channels present in the membrane. The efficacy of the chronic treatment was further confirmed by the fact that in the animals paralysed by insulin, and that therefore did not respond to the chronic treatment, the parameters recorded in vitro were similar to those of the placebo hypokalemic rats for both macroscopic and microscopic recordings.

The chronic treatment with ( 6- [ l-Piperidinyl]pyrimidin-2 , 4- dia in 3-oxide) -chloride did not produce alterations of the mass of the vital organs, such as for example the heart. Rather, beneficial effects of the drug treatment on lungs and kidney of the hypokalemic rats were observed. Indeed, in these animals the usual increase observed in the weight of the lungs and kidney was antagonised by the treatment with ( 6-[ 1-Piperidinyljpyrimidin- 2,4-diamin 3-oxide) -chloride. Additionally, the chronic treatment with (6-[l-Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) -chloride prevented the characteristic fall of the body weight of the hypokalemic rats.

According to the results obtained on the rodents, the acute application in vitro of ( 6-[ l-Piperidinyl]pyrimidin-2, 4-diamin 3- oxide) -sulphate on the muscle fibers of the HOPP patients repolarised in dose-dependent manner the fibers pre-incubated with insulin and Ringer low K . This observation is consistent with the fact that the drug was capable to stimulate the macroscopic currents sustained by the ATP sensitive K channels of the muscle fibers of these patients. As observed in the rats, the in vitro application of the drug on single channel was capable to restore the open probability and conductance, but not the number of functional channels present in the membrane. This indicates that in these HOPP patients, that carry a mutation

R528H of the dihydropyridine receptor, the number of functional

ATP sensitive K channels was irreversibly reduced. This observation is of a certain importance considering that currently although it is known that the familial hypokalemic periodic paralysis is linked to mutations of the dihydropyridine receptor there are no correlations between these mutations and the depolarisation of the membrane of the skeletal muscle fibers, the paralysis caused by insulin in the patients and the hypokalemia. In contrast, an alteration of the ATP sensitive K⁺ channel of the skeletal muscle appears to better explain most of the symptoms that are observed in the patients and in the hypokalemic rats. A drug as the ( 6-[ l-Piperidinyl]pyrimidin-2, 4-diamin 3-oxide) that is capable to selectively stimulate these channels that are altered in these types of pathologies will be particularly useful.

The disclosures in Italian Patent Application No. MI98A000086 from which this application claims priority are incorporated herein by reference.

Claims

CLAIMS 1. Use of a pyrimidin 3-oxide compound of formula ( I ]

in which: Ri is selected from the group consisting of C₁-C₄ alkyl, a - NHR group in which R is C₁-C₄ alkyl, or H; R2 is selected from the group consisting of H, Cχ-C alkyl, a -NHR group in which R is as designated above; R3 is selected from the group consisting of H, halogen atom, nitro group, amino group, Cι~C₄ alkyl optionally substituted by: - a group OR5 in which R5 is C]_-C₄ alkyl, a benzene ring optionally substituted by one or more C]_-C alkoxy groups; R4 is selected from the group consisting of H, halogen atom, C_!-C₄ alkyl, heterocyclic moieties, a group -NXY in which: - X and Y are independently selected from the group consisting of Ci-Cs alkyl, Cχ-C₈ alkenyl, cicloalkyl of from 3 to 10 carbon atoms unsubstituted or alkyl substituted (preferably C_1.-C₄ alkyl), phenylalkyl of from 7 to 12 carbon atoms, naphtylalkyl of from 11 to 14 carbon atoms, - said heterocyclic moieties are selected from the group consisting of aziridinyl, azetidinyl, pyrrolidinyl , piperidino, hexahydroazepinil, heptamethyleneimino, octamethyleneimino, morpholino, 4-alkylpiperazinyl, each of said heterocyclic moieties having optionally attached as substituents on carbon atoms thereof of from 0 to 3 alkyls each of said alkyl having of from 1 to 8 carbon atoms; and their salts pharmaceutically acceptable, for the manufacture of a medicament for the treatment of a pathology of the muscular-skeletal apparatus. 2. Use according to claim 1, in which: Ri is NH2; R2 is NH2, R3 is H; R4 is piperidinyl. 3. Use of a compound according to claim 1 or 2 for the manufacture of a medicament for the treatment of the primary or secondary hypokalemic paralysis. 4. Use according to one of the preceding claims 1-3, in which this medicament is in the form of tablet, capsule, solution suitable for the oral or parenteral administrations. 5. Use according to any one of preceding claims 1-4, in which the compound of formula (I) is in association with cilazapril. 6. Use of the compound of formula (I) according to claim 1, for the manufacture of a medicament for the treatment of the mitochondrial myophathy 7. Use according to claim 1 or 5 , in which the compound of formula (I) is 6-( 1-piperidinyl )-2, 4-pyrimidinediamine 3-oxide.