THERAPEUTIC COMPOUNDS, COMPOSITIONS AND USES THEREOF

Abstract: The invention relates to the therapeutic use of vasodilators and calcium antagonists in combination in the treatment of deficiencies in cerebral blood supply. The vasodilator and calcium antagonist show a selectivity of site of action and are effective in increasing cerebral blood supply without adversely affecting other physiological parameters such as blood pressure and heart rate.

Description: THERAPEUTIC COMPOUNDS. COMPOSITIONS AMP USES THEREOF Field of the Invention This invention relates to the use of vasodilators and calcium antagonists in combination in treatment of deficiencies in cerebral blood supply and to pharmaceutical compositions containing them. In particular it relates to the use of CGRP and nimodipine in treatment of deficiences in cerebral blood supply and to pharmaceutical compositions containing them. Background to the Invention Calcium is an integral component of cell function. Systemic and coronary arteries and many other tissues in the body including cerebral tissue are influenced by the movement of calcium across cell membranes. For example, the contractile processes of cardiac and vascular smooth muscle depend upon the movement of extracellular calcium ions into these cells through specific ion channels in the cell membranes. Calcium channel blocking agents also referred to as slow channel blockers or calcium antagonists share the ability to inhibit the movement of calcium ions across the cell membrane. Calcitonin gene-related peptide is a product of the calcitonin gene expression system. Alternative processing of RNA transcribed from the calcitonin gene leads to the production in neuronal tissue of CGRP, a 37 amino acid peptide. CGRP has been discovered in a number of species including rats, chickens and humans. The CGRPs are a very closely related group of compounds differing from each other by no more than a few amino acids. To date CGRP has been described as being principally of use in the treatment of hypertension owing to its properties on the cardiovascular system where it has been found to cause vasodilatation and to lower blood pressure. CGRP has also been postulated to play a role in calcium regulation and gastric acid secretion. The cerebral blood supply may become deficient for a number of reasons including, for example, cerebrovascular spasm. Cerebrovascular spasm of the carotid artery is the body's natural response to cerebral bleeding, cutting off the blood supply to the brain. Cerebral haemorrhage such as subarachnoid haemorrhage has many causes including the rupture of weakened blood vessels within the brain and mechanical damage to the head (trauma). Unless the blood supply and hence oxygen supply to the brain is restored the cerebrovascular spasm normally leads to brain damage and sometimes to death. The blood supply to the brain is also deficient in cases of stroke due either to infarction, i.e. occlusion of the cerebral blood vessel, or to rupturing of the blood vessel resulting in cerebral haemorrhage. The cerebral blood supply is also interrupted during a migraine attack and by restoring cerebral blood supply it is therefore possible to alleviate the symptoms of the migraine attack. We have now found that a combination of a calcium antagonist and a vasodilator is useful in the treatment of deficiencies in the cerebral blood supply in human subjects. Summary of the Invention Accordingly in a first aspect the invention provides a vasodilator and a calcium antagonist for use in combination in the treatment of a deficiency in cerebrovascular blood supply. In order to be useful in the treatment of deficiencies in cerebral blood supply it is essential that the therapeutic agent is able to selectively affect the cerebrovascular bed, such that the blood supply is increased at the desired site. A further essential requirement is that the blood supply should be increased without substantially lowering the blood pressure. It is also desirable that the therapeutic agent achieves the desired effect without significantly raising the heart rate. Current therapeutic strategies to reverse constriction of cerebral blood vessels are unsatisfactory because they also result in lowering of blood pressure, so exacerbating the original problem. Additionally some of the therapies currently available are associated with an unwanted increase in heart rate. Hitherto, therefore, there has been a real need for an effective treatment for restoring cerebral blood flow and/or reversing cerebral vasospasm which does not have the side effects associated with current therapies. We have found that it is possible to achieve both the required selectivity of site of action and the desired increase in cerebral blood supply without substantially affecting blood pressure or significantly raising the heart rate by administration in combination of an appropriate amount of a calcium antagonist and a vasodilator. The use of a combination of the calcium antagonist nimodipine and the vasodilator CGRP results in a totally .unexpected synergistic effect. The use of CGRP in treatment of deficiences in cerebrovascular blood supply as described in published International Patent Application No. W089/03686, is commonly associated with a certain increase in heart rate which it would be desirable to reduce or eliminate. We have surprisingly found that use of a combination of CGRP and nimodipine appears to reduce or eliminate the CGRP-related increase in heart rate. The expectation following standard pharmacological principles was that the CGRP effect on heart rate would be dominant and that use of CGRP in combination with nimodipine would still result in an increase in heart rate. Additionally the use of CGRP in combination with nimodipine results in an improved blood flow to the internal carotid artery. The blood flow reaching a level greater than that seen when either agent is used singly and which is greater than the sum of the individual levels. SUBSTITUTE SHEET This means that it is possible to achieve the desired therapeutic effect in the absence of unwanted side effects by using a combination of CGRP and a calcium antagonist where each of the active ingredients is administered at a lower dose e.g. at a ten fold lower dose, than would be required to achieve the same effect if either compound were administered alone. The use of a combination of a calcium antagonist and a vasodilator surprisingly does not produce any unwanted additive effect. For example, both CGRP and nimodipine will exert hypotensive effects if administered at a sufficiently high dose and it would have been predicted that the use of a combination of both would result in an additive hypotensive effect with the effect on blood pressure being observed at a dose at which use of either compound alone would not affect blood pressure. No such effect is, however, found. There are many classes of compounds which have calcium antagonist activity such as for example phenylalkylamines and derivatives thereof, e.g. verapamil; benzothiazepines and derivatives thereof, e.g. diltiazem; diphenylalkylamines and derivatives thereof, e.g. prenylamine; cinnamyl; diphenylmethyl piperazines and derivatives thereof e.g. cinnarizine and flunarizine and dihydropyridine derivatives as disclosed in British Patent No. 1173862, German Patent No, DE 2407115 and US Patent No. 3485847. The use of dihydropyridine derivatives such as nifedipine (l,4-dihydrc—2,6-dimethyl-4-(2-nitrophenyl)-3,5, pyridinedicarboxylic acid dimethyl ester), nicardipine (l,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid methyl 2-fmethyl(phenylmethyl)amino] ethyl ester) and nimodipine (1,4-dihydro-2, 6-dimethyl-4-(3-nitroρhenyl-3,5-pyridine- dicarboxylic acid- isopropyl-(2-methoxy-ethyl)-ester) is preferred. We have found that nimodipine is especially useful and the use of nimodipine in combination with a vasodilator is especially preferred. SUBSTITUTESHEET As used herein the term vasodilator denotes those compounds which produce vasodilatation and which do not directly block calcium channels and which when in combination with a calcium antagonist, preferentially act on the cerebrovascular bed to differentially increase cerebral blood supply without substantially affecting blood pressure. The vasodilator for use according to the invention is preferably one which preferentially acts on the cerebrovascular bed to differentially increase cerebral blood supply without substantially affecting blood pressure both in isolation and in combination with a calcium antagonist. Examples of such vasodilators showing the desired profile of activity include CGRP and as used herein the term CGRP, in addition to naturally occurring CGRPs, includes also biologically active fragments, analogues and derivatives thereof which have the characteristic cerebrovascular blood supply affecting properties of CGRP; i.e. which preferentially act on the cerebrovascular bed to differentially increase cerebral blood supply without substantially affecting blood pressure such as for example those described in International Patent Application No. PCT/GB 89/01249 filed 20th October 1989. The CGRPs, fragments, analogues and derivatives may be naturally occurring or may be produced chemically e.g. by chemical modification, cleavage, or synthesis or they may be produced by employing recombinant DNA techniques. The fragments, analogues and derivatives may include non-peptide compounds as well as peptide compounds. The CGRP may comprise an animal CGRP, e.g. rat or chicken CGRP, though is preferably human calcitonin gene-related peptide (hCGRP). Human calcitonin gene-related peptide exists in at least two forms known as alpha hCGRP, for instance as described in US Patent No. 4549986, and beta hCGRP, for instance as described in published European Patent Application No. EP 188400A. As used herein, the term 'hCGRP' is used to denote α- and β- hCGRP. The use of α- hCGRP is especially preferred. SUBSTITUTESHEET The use of a combination of CGRP and nimodipine according to the invention is especially preferred. A combination of a vasodilator and a calcium antagonist is particularly useful in the treatment of deficiencies in cerebrovascular blood supply in human subjects such as are associated with subarachnoid haemorrhage, stroke, trauma and migraine. According to a second aspect of the invention we provide a method of treatment of a human subject suffering from a deficiency in cerebrovascular blood supply which comprises administering an effective amount of a calcium antagonist and a vasodilator to the subject. Typically the amount of the vasodilator and calcium antagonist used is an amount which in combination is effective to differentially increase cerebrovascular blood supply without substantially affecting blood pressure or significantly raising the heart rate. In a third aspect the invention provides a pharmaceutical composition comprising a vasodilator and a calcium antagonist. In a fourth aspect the invention provides a pharmaceutical composition in unit dosage form, each unit dose comprising an amount of a vasodilator and a calcium antagonist which acts to differentially increase cerebrovascular blood supply without substantially affecting blood pressure or significantly raising the heart rate in combination with a pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutical composition according to the fourth aspect of the invention preferably contains 0.01 to 1620yg CGRP, preferably 0.08μg to 1260yg CGRP, more preferably from 5 to 1260yg CGRP and most preferably from 5 to 900yg CGRP; preferably 0.07 to 84mg of nimodipine, more preferably 0.35 to 4.2mg nimodipine and most preferably 0.7 to 3.0mg nimodipine. SUBSTITUTE SHEET In a fifth aspect the invention provides a process for the production of a pharmaceutical composition according to the fourth aspect of the invention comprising bringing into association with a pharmaceutically acceptable carrier, excipient or diluent a vasodilator and a calcium antagonist. In one embodiment of the fifth aspect of the invention, the invention provides a process for the production of a pharmaceutical composition in unit dosage form comprising bringing into association with a pharmaceutically acceptable carrier, excipient or diluent, aliquot amounts of a vasodilator and a calcium antagonist sufficient to differentially increase cerebrovascular blood supply without substantially affecting blood pressure or significantly raising the heart rate to provide unit doses. In a sixth aspect the invention provides the use of a vasodilator in combination with a calcium antagonist for the manufacture of a medicament for the treatment of a deficiency in cerebrovascular blood supply. In a seventh aspect the invention provides a drug for therapy of deficiencies in cerebral blood supply comprising a vasodilator in combination with a calcium antagonist as active ingredients. In an eighth aspect, the invention provides a cerebral blood supply improver comprising a vasodilator in combination with a calcium antagonist. In a ninth aspect, the invention provides a method for the treatment of deficiencies in cerebral blood supply which comprises administering to a patient a vasodilator in combination with a calcium antagonist. SUBSTITUTESHEET It will be appreciated that there are a number of alternative ways of administering the vasodilator and calcium antagonist according to the method of invention. For example, the vasodilator and calcium antagonist may be administered sequentially in any order and with any time period in between each administration. Alternatively the vasodilator and calcium antagonist may be co-administered. As used herein the term co-administration covers the situation where the vasodilator and calcium antagonist are formulated together and administered in one pharmaceutical preparation, and also where the vasodilator and calcium antagonist are formulated separately and administered simultaneously. The vasodilator and calcium antagonist may be formulated differently, for example, one may be formulated for oral administration and the other for intravenous administration. Preferably the vasodilator will be formulated for intravenous administration, and the calcium antagonist for oral administration, and most preferably both the vasodilator and the calcium antagonist will be formulated for intravenous administration. Pharmaceutical compositions for use according to the present invention may be formulated in conventional manner, optionally with one or more physiologically acceptable carriers, diluents or excipients. The vasodilator and calcium antagonists for use according to the present invention may be formulated for oral, buccal, parenteral or rectal administration or in a form suitable for nasal administration or administration by inhalation or insufflation. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g. lactose, micro- SUBSTIT crystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium glycollate); or wetting agents (e.g. sodium lauryl sulphate) . The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents; emulsifying agents; non-aqueous vehicles; and preservatives. The preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. The vasodilator and calcium antagonist may be formulated for parenteral administration by injection e.g. by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use. The vasodilator and calcium antagonist may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides. SUBSTITUTESHEET In addition to the formulations described previously, the vasodilator and calcium antagonist may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or by intramuscular injection. For nasal administration or administration by inhalation the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurised packs or a nebuliser, with the use of a suitable propellent, e.g. dichlorodi-fluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack or dispenser device may be accompanied by instructions for administration. In a preferred embodiment of the first to ninth aspects of the invention the calcium antagonist is preferably nimodipine and the vasodilator is preferably CGRP, more preferably human CGRP and most preferably human α-CGRP The dose at which the combination of a calcium antagonist and vasodilator will be administered to man will be such that the cerebral blood supply is differentially increased and blood pressure is not substantially affected and there is no significant increase in heart rate. The precise dose of the vasodilator and the calcium antagonist trill depend upon the route of administration, the potency of the vasodilator and calcium antagonist and the body weight and pathology of the patient. The important factor is believed to be the concentration of the vasodilator and calcium antagonist which is present at the target vascular bed. On an individual patient basis the dose of the vasodilator and calcium antagonist which should be administered to cause the desired effect may be determined by administering a low dose of vasodilator in combination with a calcium SUBSTITUTE SHEET antagonist for 10 to 20 minutes and then increasing the dose every 10 to 20 minutes until the desired effect is seen. A vasodilator such as CGRP may be administered to an average 70kg man by IV infusion at doses in the range 0.01 to 32ng/kg/min, preferably in the range 0.06 to 24ng/kg/min, more preferably in the range 2 to 24ng/kg/min, and most preferably in the range 2 to 16ng/kg/min. For example, α- CGRP and β-CGRP may be administered to an average 70kg man by IV infusion at doses in the range 2ng/kg/min to 16ng/kg/ min over a time period of 20 minutes. In some cases it may be desirable to infuse the patient with CGRP for longer time periods e.g. for up to or greater than 1 hour e.g. for 12 hours, or for more than 24 hours. A calcium antagonist such as nimodipine may be administered to an average 70kg man by IV infusion at doses in the range 1-lOOyg/kg/h, preferably in the range 5-50μg/kg/h and most preferably in the range 10-35yg/kg/h. For oral administration a calcium antagonist may be administered to an average 70kg man at doses in the range 0.05 to 20mg/kg preferably in the range 0.5 to 8mg/kg. The calcium antagonist and non-calcium dependent vasodilator may be adminstered in combination as often as is required. For example, they may be administered more than once per day, e.g. 2 to 6 times per day. The duration of treatment may be for example from 20 minutes to 24 hours and the period of treatment may continue for, for example, up to 21 days. A calcium antagonist for use in the present invention may be obtained using standard chemical procedures. For example, nimodipine may be produced as described in British Patent No. 1173862; nicardipine as described in German Patent No. DE 2407115 and nifedipine as described in US Patent No. 3485847. CGRP for use in the present invention may be obtained using recombinant DNA technology as described in British Patent No. 2141430B and published European Patent Application No. EP-A-188400. Alternatively the CGRP may be produced by chemical synthesis using conventional techniques well known in the art, see for example published European Patent Application No. EP-A-188400. CGRP in combination with a calcium antagonist such as nimodipine is particularly useful in the treatment of deficiencies in cerebral blood supply since it shows the desired profile of activity i.e. it selectively affects the cerebrovascular bed without substantially affecting blood pressure or significantly increasing heart rate. The vasodilator and calcium antagonist may be tested for their ability to differentially increase cerebrovascular blood supply in animals and humans using the Doppler technique as described in the examples hereinafter. The effect of the vasodilator and calcium antagonist on blood pressure and heart rate may be measured using conventional techniques. Brief Description of Drawings The differential increase in cerebrovascular blood supply affected by CGRP and nimodipine has been demonstrated in rats using the Doppler technique as described below and, with reference to the accompanying Figures in which: Figure 1 shows a graph of the effect in rats of a 30 minute individual infusion of different concentrations of CGRP and nimodipine on heart rate, mean arterial pressure, and blood flow and blood velocity to the internal carotid artery where: SUBSTITUTE SHEET O.Oβnmol CGRP/h o-o O.όnmol CGRP/h _ _ 60nmol nimodipine/h D-α 600nmol nimodipine/h Figure 2 shows a graph of the effect in rats of a 30 minute combined infusion of 0.06nmol/h CGRP and 60nmol/h nimodipine on heart rate, mean arterial pressure and blood flow and blood velocity to the internal carotid artery. Figure 3 shows a graphical representation of the cardiovascular responses to human α-CGRP and/or nimodipine in conscious Long Evans rats (n=8). Left-hand panels: responses to a 30 min infusion of human α-CGRP ( __-• , O.Oόn ol h "" ), or nimodipine (Ar-A , δOn ol h~ ) or human α-CGRP (O.Oδnmol h~ ) plus nimodipine (60nmol h~ ) (O—O ). Values are mean + S.E.M. Right hand panels: responses to a 30 min infusion of human α-CGRP ( • e , O.βnmol h~ ), or nimodipine (fr-* , 600nmol h~ ) or human α-CGRP (O.όnmol h~ ) plus nimodipine (δOOnmol h ) (O—O ). Values are mean + s.e. . (n=8); Decription of Specific Embodiments Example 1 This series of experiments was designed to determine the effect on cerebral blood flow and blood pressure of administration of different doses of CGRP and nimodipine individually and in combination. SUBSTITUTE SHEET Male long Evans rats (350-400g) were used (n=8) Under sodium methohexitone anaesthesia (60mg/kg IP) the left external carotid artery was tied off (410 silk suture) and a miniature pulsed Doppler probe was sutured around the left common carotid artery (to monitor internal carotid flow). 7-10 days after probe implantation, animals were re-anaesthetised (sodium methohexitone, 40 mg/kg IP) and catheters were implanted in the abdominal aorta (via the caudal artery) for BP recording, and in the right jugular vein for drug/peptide administration. Experiments were run 1 and 2 days after catheter implantation with animals fully conscious and freely moving. Day 1: Nimodipine vehicle Nimodipine (60nmol/h) Nimodipine (600nmol/h) Day 2: CGRP (0.06nmol/h) CGRP (0.6nmol/h) CGRP (0.06nmol/h) plus Nimodipine (60nmol/h) Infusions were given for 30 min at 0.3ml/h At least 60 min was allowed between each infusion Nimodipine vehicle consisted of: 969g polyethylene glycol 400 lOOg distilled water 60g glycerine All nimodipine solutions were made up in a dark room under yellow filters (approx. 550nm). SUBSTITUTE SHEET Syringes and catheters used for administering nimodipine were protected from light by black shielding. CGRP (human α) was dissolved in saline containing 1% bovine serum albumin. The results are shown in Table 1, Figure 1 and Figure 2. Administration of CGRP 0.06nmol/h and nimodipine 60nmol/h in combination resulted in an increase in cerebral blood flow which was greater than that seen on administration of either compound alone and which was greater than the additive effect at these doses. This was achieved in the absence of an additive hypotensive effect. SUBSTITUTE SHEET TABLE 1 - 16 - CGEP O.Oβnmol/h TIME 10 20 30 40 50 60 (min) Δ HE +37(19) +33(14) +30(12) +25(13) +12(15) + 7(9) (b/ain) Δ HAP 0(1) - 2(1) - 1(1) - 1(2) + 1(2) 0(1) (__mHε) Δ CDS +10(3) +14(4) + 8(5) -10(2) - 4(6) - 2(3) (*) Δ CVE - 9(2) -13(4) - 6(5) +11(4) + 8(6) + 4(4) (*) CGEP 0.6 nmol/h TIME 10 20 30 40 50 60 (atin) Δ HE +92(17) +99(14) +91(13) +14(7) - 9(9) - 6(14) (b/ain) Δ MAP -15(1) -14(2) -15(2) + 1(2) + 3(3) + 1(2) (nαHg) Δ CDS + .5(9) +54(7) +48(6) + 7(4) + 2(6) + 8(5) (*) Δ CVE -.0(3) -43(2) -42(2) - 4(6) + 4(9) - 5(6) (1) NIMODIPINE 60naol/h TIME 10 20 30 40 50 60 (ain) Δ HE - 6(9) - 1(9) -14(14) -18(9) + 2(10) + 3(10) (b/ain) Δ MAP ♦ 2(2) + 2(3) + 3(4) - 1(3) + 4(2) + 3(2) (aαHs) Δ CDS +16(4) + 6(4) + 4(4) + 2(4) - 1(3) - 2(4) (t) Δ CVE -11(4) - 3(5) + 1(6) - 2(6) + 5(3) + 6(3) ( ) NIMODIPINE 600nmol/h TIME 10 20 30 40 50 60 (ain) Δ HR +34(16) +29(18) +27(15) +14(12) + 6(6) + 5(7) (b/ain) Δ MAP -19(2) -17(4) -19(1) - 9(2) - 6(2) - 5(2) (α__Hs) Δ CDS +32(8) +32(8) +40(10) ♦33(10) +29(14) +29(9) (%) Δ CVE -37(4) -36(4) -40(4) -29(5) -22(8) -23(7) (*) CGRP 0.06nmol/h + KIMODIPINE βOnmol/b TIME 10 20 30 40 50 60 (ain) Δ HE +13(8) + 4(9) +14(15) + 1(11) -13(8) +13(15) (b/ain) Δ MAP - 3(3) - 1(3) - 4(3) + 3(2) + 3(1) + 3(1) (tamHs) Δ CDS +30(5) +27(5) +40(10) +15(5) +14(5) +10(7) (1) A CVE -25(3) -22(3) -30(4) -10(4) - 8(4) - 4(5) <« Yalwes are a*an (ιaa) n - 8 CDS * internal earotid doppltr shift (flow) CVS - inttrnal earotid vascular resistance SUBSTITUTE SHEET Example 2 This series of experiments was designed to look at the effects of CGRP and nimodipine on the internal carotid blood vessel, on regional circulation, and on physiological parameters such as heart rate and blo pressure. All studies were carried out on male, Long Evans rats (350-450g). Animals were anaesthetized (sodium methohexitone, 60mg kg- i.p., supplemented as required) and had one of the following procedures carri out:- a) implantation of a pulsed Doppler probe (Haywood et al. , 1981) around the left common carotid artery after the external carotid arter on the same side had been ligated. This arrangement permitted assessm of flow through the patent internal carotid artery. b) implantation of pulsed Doppler probes around the left renal and superior mesenteric arteries and the distal abdominal aorta below the level of the ileocaecal artery (to monitor flow to the hindquarters). Following 7-14 days recovery animals were briefly re-anaesthetized (βodium methohexitone, 40mg kg~ ) for the implantation of intravascula catheters for drug or peptide administrations (jugular vein) and the recording of instantaneous heart rate (HR) and blood pressures (BP) fr the abdominal aorta (via the caudal artery). The experiments began on the next day and ran over 3 days. The protocols involved continuous recordings from conscious unrestrain animals of mean BP, HR and Doppler shifts from the implanted probes. Percentage changes in regional blood flows and vascular resistances we calculated as described elsewhere (Gardiner et al.. (1989), Am. J. Physiol. 256 R332-338). SUBSTITUTE SHEET The following protocols were run:- Animals with internal carotid probes 1. Effects of human α-CGRP Human α-CGRP (0.06 or O.βnmol h~ ) was infused for 30 min. Measurements were made before, during, and for 30 min after infusion. 2. Effects of nimodipine vehicle Nimodipine vehicle (see below) was infused for 30 min. Measurements were made before, during, and for 30 min after infusion. 3. Effects of nimodipine Nimodipine (60 or δOOnmol h~ ) was infused for 30 min. Measurements were made before, during and for 30 min after infusion. 4. Effects of human α-CGRP plus nimodipine Human α-CGRP and nimodipine (0.06 and 60nmol h~ , respectively, or 0.6 and βOOnmol h~ , respectively) were infused concurrently for 30 min. Measurements were made before, during and for 30 min after infusi Animals with renal, mesenteric and hindquarters probes Protocols 1) - 4) above were run also in animals (n=8) with renal, mesenteric and hindquarters probes. Drugs and Peptides Human α-CGRP (Celltech Ltd) was dissolved in isotonic saline containing IX bovine serum albumin. Nimodipine (pure substance, Bayer, U.K.) was dissolved in a mixture of PEG400, sterile water and glycerine BP in t ratio 9.69: 1.00 : 0.6 (by weight). Nimodipine solutions were made u a dark room under filtered light (wave length > 500nm) and all catheters and syringes were masked to avoid exposure of the solutions ambient lighting; the infusion rate was 0.4ml h~ . Statistics All data were subjected to non-parametric analysis of variance (Friedman's test) with Wilcoxon's rank sum test, or the Mann-Whitney test as appropriate. Results Animals with internal carotid probes 1. Effects of human α-CGRP Infusion of human α-CGRP at O.Oόnmol h~ caused an increase in HR and a modest internal carotid hyperaemia, accompanied by a fall in vascul resistance (shown in Figure 3 and Table 2). Human α-CGRP at O.όnmol h~ caused tachycardia, hypotension and marked hyperaemic vasodilation in the internal carotid vascular bed (shown in Figure 3 and Table 2). 2. Effects of nimodipine vehicle Infusion of nimodipine vehicle was associated with a significant (P<0.05) fall in HR (-23+9 beats min at 30 min) and a slight rise in mean BP (+5+2mmHg at 30 min; P<0.05), but no change in internal carotid haemodynamics. SUBSTITUTE SHEET 3. Effects of nimodipine Nimodipine infused at όOnmol h~ caused a transient increase in internal carotid flow and fall in vascular resistance (Figure 3) . The higher dose of nimodipine (όOOnmol h ) caused hypotension and substantial hyperaemic vasodilatation in the internal carotid vascular bed. These effects persisted after the infusion was stopped, unlike those of human α-CGRP, (Figure 3). 4. Effects of human α-CGRP plus nimodipine Infusion of human α-CGRP (O.Oόnmol h~ ) together with nimodipine (όOnmol h~ ) caused significantly (P<0.05) greater increases in internal carotid flow and falls in vascular resistance than seen with either compound alone (Figure 3 and Tables 2, 3 and 4). However, there were no significant changes in mean BP or HR (Figure 3). Concurrent administration of human α-CGRP (O.όnmol h~ ) and nimodipine (όOOnmol h~ ) did not enhance significantly the vasodilatation seen with either compound alone (Figure 3) . After infusion of human α-CGRP the vasodilatation did not persist whereas following infusion of nimodipine and human α-CGRP there was a persistent vasodilatation (Figure 3). However, this effect was no greater than that seen with nimodipine alone (Figure 3). Animals with renal, mesenteric and hindquarters probes 1. Effects of human α-CGRP Infusion of human α-CGRP caused dose-dependent tachycardia, hypotension and reductions in renal and mesenteric flows, accompanied by increases hindquarters flow (Table 2), although during infusion only the changes hindquarters resistance were significant (Table 2). Following infusion of the higher dose there was a significant increase in mesenteric vascular resistance (Table 2). SUBSTITUTE SHEET 2. Effects of nimodipine vehicle Infusion of nimodipine vehicle was associated with an overall bradycardia, but this was not significant at any specific time point. There were no changes in mean BP or regional blood flows. 3. Effects of nimodipine Infusion of nimodipine at όOnmol h~ had no significant effects. However, at an infusion rate of όOOnmol h~ nimodipine caused reductions in mean BP and renal flow accompanied by increases in HR an hindquarters flow, with no change in mesenteric flow (Table 3). Durin infusion there were falls in mesenteric and hindquarters resistances b a tendency for renal vascular resistance to rise. Indeed, after infus a slight further increase in renal vascular resistance made the overal change significant (see Table 3). When assessed from areas under curves all the cardiovascular effects o nimodipine (όOOnmol h~ ) were significantly (P<0.05) different from those of human α-CGRP (O.όnmol h~ ). 4. Effects of human α-CGRP plus nimodipine During infusion of human α-CGRP (O.Oόnmol h~ ) together with nimodipine (όOnmol h~ ) there was no significant tachycardia or change in renal or mesenteric haemodynamics, in contrast to the tachycardia a fall in mesenteric blood flow seen with human α-CGRP alone. The hindquarters vasodilatation persisted throughout the infusion of human α-CGRP and nimodipine unlike the transient effect seen with human α-CGRP alone; in the former case there was also a delayed hindquarters vaβoconstriction following infusion. SUBSTITUTE SHEET Concurrent infusion of human α-CGRP (O.όnmol h~ ) and nimodipine (ό.OOnmol h~ ) had significantly (P<0.05) greater effects than human α-CGRP alone on all cardiovascular variables except HR and mesenteric flow. However, administration of human α-CGRP and nimodipine together did not enhance significantly the hypotension or mesenteric or hindquarters vasodilatations seen with nimodipine alone (Tables 3 and 4 Nonetheless, combined administration of human α-CGRP and nimodipine caused a significantly greater reduction in renal blood flow and enhanc renal vasoconstriction than seen with nimodipine or human α-CGRP alone (Tables 3 and 4). Conclusion The results therefore show that low doses of human α-CGRP and nimodipine exerted synergistic internal carotid vasodilator effects without compromising other regional circulation or causing systemic hypotension. Co-administration of high doses of human α-CGRP and nimodipine did not cause enhanced internal carotid vasodilatation compared to either compound alone or compared to the effects of concurrent administration of low doses of human α-CGRP and nimodipine. SUBSTITUTE SHEET Table 2 Cardlovasoular changes during and after 30 ain lInnffuusslions of huaan a-OGRP (O.OCπaol and O.Cπaol h~ ) in oonaclous. Long Evana rata (n-l; ■ean (a. «.■.)). Huaan «-C0HP O.O naol Huaan .-COUP 0.6n__ol tT l 4 TIIC (ain) 10 20 30 40 50 to 10 20 30 40 50 60 70 A llaart rata +1«(«) ♦31(H)* ♦25(7)* +11(1) -4(7) +1(1) +121(17)* +143(13»* +1S-(1«)» +3»(1») +10(11) +3(5) +11(6) (beata ain ) A Mean BP -4(2) -3(1) -1(2) -1(2) -2(3) 0(4) -1«(1) ♦ -16(2)* -17(3)* 0(2) +3(2) +1(2) -1(2) (aallg) A Ranal f ou -1(1) -5(3) -3(4) -2(4) -3(3) -3(5) -12(3)* -12(3)* -14(3)* -2(3) 0(4) 0(6) -5(5) (%> A Hβsβntβrlo flow -7(3) -10(3)* -•(J>» -4(2) -6(3) -10(2)• -17(4 )• -22(4)* -20(5)* -14(2)* -9(5) -4(4) 0(6) (%> A llindquartara flow +11(11) +5(4) 4<(f) ♦0(4) -1(7) -3(5) +24(5)* +26(5)* +31(»>* +9(7) +2(6) +6(4) 0(5) A Ranal reslstanαa -3(4) +3(4) ♦2(4) ♦3(4) ♦1(5) ♦4( ) -5(4) -4(2) -2(4) +3(4) +4(4) +2(5) +7(_) (*) ω A Meaantarlα +5(6) +9(5) +7(4) ♦4(4) ♦5(5) +11(5) ♦ 1(6) +10(6) +-<_) +16(4)* +13(5)* +6(4) +1(4) raalatanos (%) A Hindquarters -10(5)* -7(3) -6(5) +1(4) +2(7) +5«) -33(3)* -32(4)* -35(3)* -«(«) +2(4) -4(5) +2(7) raalstanoa • P<0.03 varaua baseline (Frledaan's test) - 24 - Table 3. cardiovascular changes during and after a 30 ain infusion of Niaodipine (600naol h ) in conscious. Long Evans rats (n « 8; aean ( s.e.a.) ) . Kiaodinine 600nmol b J TIKS (min) 10 20 30 40 50 60 70 A Beart rate +78(11)* +89(10)* +81(8)* +53(13)* +39(14)* +34(15) +28(15) (beats ain " ) A Mean S? -27(3)* -27(3)* -30(3)* -22(3)* -15(2)* -13(3)* -11(2)* (aaHg) A ?.enal ow -27(4)* -32(4)* -34(5)* -30(4)* -23(5)* -15(4) -11(4) ( .) A Mesenteric flew +7(4) -1(4) +1(4) +3(5) +2(5) +11(4)* +7(4) ( .) A Hindquarters flσw +52(9)* +52(10)* +59(10)* +43(10)* +27(9)* +27(8)* +18(5)* (*) A ?.enal resistance +4(5) +12(5) +13(5) +15(4)* +13(5) +6(5) +2(3) (%) A Mesenteric -29(5)* -23(4)* -28(3)* -21(4)* -15(4)* -20(4)* -15(4)* resistance (%) A Hindquarters -50(3)* -49(4)* -53(3)* -42(4)* -30(5)* -28(5)* -23(4)* resistance • P<0.05 versus baseline (Frieda&n's test) Table 4. Cardlovasoular changes during and after 30 ain nfusion of huaan a-CORP + Nlaodlplnβ n oonaclous. Long Evans rats (n-l ; aean (s. e.a. M . Huaan o-CORP (0.06nagl h ) Huaan .-CCRP (0.6naol h_ + H laodlplns (60naol h ) + Wlaodlolne (600naol h " TIM (ain) 10 20 30 40 50 60 10 20 30 40. 50 60 70 • P<0.05 varaua baseline (Frladaan'a test)

Claims: Claims 1. A vasodilator and a calcium antagonist for use in combination in the treatment of a deficiency in cerebrovascular blood supply. 2. Calcitonin gene-related peptide and nimodipine for use in combination in the treatment of a deficiency in cerebrovascular blood supply. 3. A method of treatment of a human subject suffering from a deficiency in cerebrovascular blood supply which comprises administerin an effective amount of a calcium antagonist and a vasodilator to the subject. 4. A pharmaceutical composition comprising a vasodilator and a calci antagonist. 5. A pharmaceutical composition in unit dosage form each unit dose comprising an amount of a vasodilator and a calcium antagonist which ac to differentially increase cerebrovascular blood supply without substantially affecting blood pressure or significantly raising the hea rate in combination with a pharmaceutically acceptable carrier, excipie on diluent. 6. A process for the production of a pharmaceutical composition according to claim 5 comprising bringing into association with a pharmaceutically acceptable carrier excipient or diluent a vasodilator and a calcium antagonist. SUBSTITUTE SHEET 7. The use of a vasodilator in combination with a calcium antagonis for the manufacture of a medicament for the treatment of a deficiency cerebrovascular blood supply. 8. A drug for therapy of deficiencies in cerebral blood supply comprising a vasodilator in combination with a calcium antagonist as active ingredients. 9. A cerebral blood supply improver comprising a vasodilator in combination with a calcium antagonist. 10. A method for the treatment of deficiencies in cerebral blood su with comprises administering to a patient a vasodilator in combination with a calcium antagonsit. SUBSTITUTE SHEET

Inventors: BENNETT TERENCE, GARDINER SHEILA MARGARET, NEY URSULA MARY, BENNETT, TERENCE, GARDINER, SHEILA, MARGARET, NEY, URSULA, MARY
Applicants: CELLTECH LTD, CELLTECH LIMITED
Application year: 1990
Main classification: A61K37
IPC codes: A61K38, A61K45, A61P3, A61P9