WO2003037309A2

WO2003037309A2 - USE OF 4α PHORBOL

Info

Publication number: WO2003037309A2
Application number: PCT/GB2002/004911
Authority: WO
Inventors: Anil Mehta
Original assignee: University Court Of The University Of Dundee
Priority date: 2001-10-30
Filing date: 2002-10-29
Publication date: 2003-05-08
Also published as: WO2003037309A3; GB0126000D0; AU2002349103A1

Abstract

The present invention relates to the use of 4α phorbol in maintaining or enhancing ciliary beat frequency such as in the treatment of subjects suffering from diseases associated with reduced or poor mucociliary clearance, such as cystic fibrosis, primary cilia dyskinesia, Young's syndrome or bronchiectasis.

Description

USE OF 4 PHORBOL

The present invention relates to the use of 4α phorbol in maintaining ciliary beat frequency.

In lung tissues, protein kinase C PKC regulates inter alia mucociliary clearance (MCC) , that process by which airway mucus is removed by the beating action of cilia [1,2]. This finding provides a quasi-real time bioassay of PKC activity in vitro because PKC reduces airway ciliary beat frequency (CBF) across species (for example, rabbit [4] ; sheep [5] ; dog [6] ; and human [7] ) .

It is an object of the present invention to provide an agent which maintains ciliary beat frequency. It is a further object to provide an agent which inhibits protein kinase C activity.

When phorbol esters such as phorbol-12-myristate-13- acetate (PMA) are used as PKC activators, it is accepted practice to apply 4 , 9 , 12β, 13α, 20-pentahydrotiglia-l, 6-diene-3-one(4 -phorbol; 4α-PHR; Isophorbol) as a negative control. The present invention is based in part on observations by the present inventors that 4α-PHR is not neutral in this regard, but has a potent activity on human cilia.

In a first aspect the present invention provides use of 4α phorbol; or a physiologically acceptable salt, ester or other physiologically functional derivative thereof; in the manufacture of a medicament for maintaining or enhancing ciliary beat frequency in an animal. Ciliary beat frequency may be enhanced with respect to a basal level, or the addition of 4α phorbol may be used to seek to maintain ciliary beat frequency. Maintaining ciliary beat frequency may be 50%, 60%, 70%, 80%, 90% or substantially 100% of the basal level of ciliary beat frequency observed, prior to the addition of an agent shown to reduce ciliary beat frequency.

The animal to which the medicament is applied is typically a mammal, such as a human, but may also include domestic and farm animals such as dogs, cats, horses, pigs, cattle, sheep, goats and the like.

In another aspect the present invention provides a pharmaceutical formulation comprising 4α phorbol or a physiologically acceptable salt, ester, or other physiologically functional derivatives thereof and a carrier therefor for use in maintaining ciliary beat frequency in an animal.

In a yet further aspect the present invention provides a method for maintaining ciliary beat frequency in vivo or in vitro which comprises administering to an animal or tissue or a cell thereof a ciliary beat frequency maintaining amount of 4 phorbol or a physiologically acceptable salt, ester or other physiologically functional derivative thereof.

In a yet further aspect the present invention provides use of 4α phorbol; or a physiologically acceptable salt, ester or other physiologically functional derivative thereof; in the manufacture of a medicament for inhibiting protein kinase C.

The medicament pharmaceutical formulation or method of treatment may be applied to subjects displaying reduced ciliary beat frequency. Such subjects may include subjects suffering from diseases associated with reduced or poor mucociliary clearance, such as cystic fibrosis, primary cilia dyskinesia, Young's syndrome or bronchiectasis. The medicament may also be added to counteract the effect of drugs, the side-effects of which are shown to be reduced ciliary beat frequency or decreased mucociliary clearance.

Examples of physiologically acceptable salts of 4α phorbol include acid addition salts formed with organic carboxylic acids such as acetic, lactic, tartaric, maleic, citric, pyruvic, oxalic, fumaric, oxaloacetic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulphonic, benezenesulfonic and p-tolunesulphonic acids and inorganic acids such as hydrochloric, sulphuric, phosphoric and sulphamic acids.

Physiologically functional derivatives of 4α phorbol are derivatives which can be converted in the body into the parent compound, or be active in their own right. Such physiologically functional derivatives may also be referred to as "pro-drugs" or "bioprecursors" .

It may be appreciated that 4α phorbol may exist in various stereoisomeric forms and 4α phorbol therefore includes all stereoisomeric forms and mixtures thereof, including enantio ers and racemic mixtures. The present invention includes within its scope the use of any such stereoisomeric form or mixture of stereoisomers, including the individual enantiomers of the compounds of formula (I) as well as wholly or partially racemic mixtures of such enantiomers.

4α phorbol or a physiologically acceptable salt, ester or other physiologically functional derivative thereof may be administered alone or in combination with other drugs as part of a therapeutic regimen for the maintenance or enhancement of ciliary beat frequency, or it may be administered as an adjunct to other forms of therapy.

The amount of a compound of 4 phorbol required for use in the maintenance or enhancement of ciliary beat frequency will depend inter alia on the route of administration, the age and weight of the patient and the nature and severity of the condition being treated and will ultimately be at the discretion of the attendant physician or veterinarian. In general, a suitable dose for administration to man is in the range of 0.1 to 100 mg. per kilogram bodyweight per day, for example from 1 g/kg. to 40 mg/kg. , per day particularly 5 to 15 mg/kg. per day. For administration by inhalation the dose may conveniently be in the range of 0.1 to 50 mg/kg/day, eg. 1 to 10 mg/kg/day.

It will be appreciated that for administration to neonates, lower doses may be required. For prophylactic treatment 4α phorbol or a physiologically acceptable salt, ester or other physiologically functional derivative thereof may also be given less frequently, eg. as a single dose on alternate days, once or twice per week or once or twice per month. The dosage for prophylactic treatment will depend inter alia on the frequency of administration, and, where a depot preparation or controlled release formulation is used the rate of release of the active ingredient. Thus for once- weekly administration a suitable prophylactic dose is in the range 1 to 100 mg/kg, eg. 5 to 50 mg/kg particularly 15 to 30 mg/kg.

For use according to the present invention 4 phorbol is preferably presented as a pharmaceutical formulation, comprising a compound of formula (I) or a physiologically acceptable salt, ester or other physiologically functional derivative thereof (hereinafter referred to as "active compound") together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic and/or prophylactic ingredients. The carrier (s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

An active compound may conveniently be presented as a pharmaceutical formulation in unit dosage form. Convenient unit dose formulation contains an active compound in an amount of from 25 g to 100 mg. Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual) , rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous) , nasal and pulmonary administration eg. by inhalation. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association an active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of an active compound. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tables may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed for example in a rice paper envelope. An active compound may also be formulated as dispersable granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged eg. in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.

Formulations for oral administration include controlled release dosage forms eg. tablets wherein an active compound is formulated in an appropriate release - controlling matrix, or is coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of an active compound with the softened or melted carrier (s) followed by chilling and shaping in moulds. S

Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of an active compound in aqueous or oleaginous vehicles. Injectable preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use. Alternatively, an active compound may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

An active compound may also be formulated as long- acting depot preparations, which may be administered by intramuscular injection or by implantation eg. subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.

Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing an active compound and desirably having a diameter in the range 0.5 to 7 microns are delivered into the bronchial tree of the recipient.

As one possibility such formulations are in the form of finely comminuted powders which may conveniently be presented either in a pierceable capsule, suitably of, for example, gelatin, for use in an inhalation device, or alternatively as a self-propelling formulation comprising an active compound, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent. Suitable liquid propellants include propane and the chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self- propelling formulations may also be employed wherein an active compound is dispensed in the form of droplets of solution or suspension.

Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.

As a further possibility an active compound may be in the form of a solution or suspension for use in an atomiser or nebuliser whereby an accelerated airstream or ultrasonic agitation is employed to produce a fine droplet mist for inhalation.

Formulations suitable for nasal administration include presentations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.

It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulations described above may include, as appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

Therapeutic formulations for veterinary use may conveniently be in either powder or liquid concentrate form. In accordance with standard veterinary formulation practice, conventional water soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. Thus particularly suitable powders of this invention comprise 50 to 100% w/w, and preferably 60 to 80% w/w of the active ingredient(s) , and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients. These powders may either be added to animal feedstuffs, for example by way of an intermediate premix, or diluted in animal drinking water. Liquid concentrates of this invention suitably contain a water-soluble compound of formula (I) or a salt thereof and may optionally include a veterinary acceptable water- miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the drinking water of animals.

The present invention will now be described further by way of Example only and with reference to the Figures which show:

Figure 1 shows ciliary beat frequency time-course profiles for cilia observed under zero flow conditions in PKC-modifying solutions compared to basal PKC activity at 32°C. Cilia were observe without fluid over time under normal (M199; closed circles, n=32) , activated PKC (InM PMA; open upward-pointing triangles, n=49) , and in InM of 4α-PHR, the control reagent for PMA (open squares, n=33) . Under 4α-PHR (control) conditions, the CBF was maintained at baseline levels for up to 90 minutes, whereas there was a steady decline in CBF in M199, especially beyond the first hour, and an even faster decline when PKC was activated. The differences between the CBF profiles in the different solutions were significant (p<0.0001, Student's paired t-test) ; and

Figure 2 shows ciliary beat frequency reaction to the presence of PMA and 4α-PHR at 32 °C. Cilia were observed at 32 °C under zero flow conditions for 30 minutes in InM PMA before a perfusion of fresh InM PMA was made at a rate of 0.125 ml min^-1 for 20 minutes (ie. activated PKC, open upward-pointing triangles; n=12) . In the subsequent experiment, observations were made in InM PMA for 30 minutes, before a perfusion of a mixture of InM 4α-PHR, a non-active phorbol, and InM PMA was made (closed diamonds, n=12) . In the final series, cilia were observed in a InM 4α-PHR solution, before the mixture of the 4α-PHR and PMA was perfused at the same slow rate over 20 minutes (open squares, n=12) . There was a significant difference between each profile reported here following the 20-minute perfusion (p<0.0001, Student's paired t-test) .

MATERIALS AND METHODS Materials

All reagents used were of analytical grade and included phorbol-12-myristate-12-acetate (PMA) , and dimethylsulphoxide (DMSO) , both of which were obtained from Sigma (Poole, UK); 4α, 9α, 12β, 13α, 20-pentahydrotiglia- l,6-diene-3-one(4α-phorbol; 4α-PHR; isophorbol) obtained from Calbiochem-Novabiochem (Nottingham, UK) , and medium 199 (M199) , obtained from ICN Biochemicals (Oxfordshire, UK) . The phorbol esters (PMA and 4α-PHR) were dissolved in DMSO first before being diluted to the desired concentrations in M199. Subject selection

Respiratory epithelium was obtained from normal volunteers or immediately after the induction of general anaesthesia from non-asthmatic, non-atopic patients undergoing routine operations unrelated to nasal disease (age range, 10 - 45 years) . These patients had had no respiratory infections for at least a month prior to the date of operation. Approval for the study was given by the Tayside Committee on Medical Research Ethics and informed written consent was obtained from the volunteers, patients and parents of minors in all cases.

Sample collection

This has been described in detail elsewhere [8]. Briefly, strips of ciliated epithelium were obtained with a cytology brush from the inferior turbinate. Cellular material adherent to the brush was dislodged by brisk agitation in Eppendorf tubes containing 1 ml of culture medium (Medium 199). The samples were kept on ice (4°C) until they were placed in the experimental chamber. All experiments were performed within 24 hours of sample collection [9] .

Measurement of ciliary beat frequency

This has been described in detail elsewhere [7, 10, 11] . Briefly, CBF was measured in a perfusion chamber at the normal physiological nasal temperature (32 ± 0.5°C) using our well characterised video-based, Hoffman contrast technique (Brian Reece Scientific, Newbury Berks) . The ambient temperature in the chamber was maintained using an air conditioner. This temperature was monitored continuously using a K-type thermocouple (51 k/J digital thermometer, John Fluke, Palatine, Illinois, USA) .

The Rose chamber loading and perfusion protocol has been described in detail elsewhere [11-13], Briefly, an aliquot of freshly collected strips of ciliated epithelial cells was transferred in M199 to a glass walled modified Rose chamber with an internal volume of 0.5 ml. The chamber was then connected to a syringe-operating non- peristaltic infusion pump (Medfusion Model 2010, Medex Medical Inc., Lancashire, UK) delivering 2.5 ml of the relevant test solution at a rate of 0.125 ml min^"1. This rate of infusion eliminated changes in CBF which would have been induced by higher perfusion rates as found in previous studies [7, 9, 14]. All readings were taken in the absence of flow. The post-perfusion CBF measurements were taken within 30 seconds of the termination of the flow of fluid. The pH of all solutions used was between 7.2 and 7.4, a range known not to adversely affect ciliary activity [15] .

Ciliated strips formed by a sheet of 10 or more cells attached to their basement membrane were selected at random and CBF recorded from the same point adjacent to the ciliated cell border.

Having chosen the ciliated strip of cells to be used in the experiment, the baseline CBF was established by taking readings every 5 minutes for 30 minutes, before the chamber was perfused using the infusion pump, and CBF readings continued (after the end of the perfusion) till the 120^th minute, for all experiments.

Experimental Protocols with test solutions

Baseline CBF profiles were established in the three test solutions (M199, 4α-PHR, and PMA) . For experiments where the reagents were changed, however, CBF readings were taken first for 30 minutes, either in PMA or in 4α-PHR, before the chamber was perfused with either InM PMA alone or a mixture of InM PMA and InM 4α-PHR according to each specific protocol.

In the first part of the study (see Figure 1) , CBF was recorded under zero flow conditions in M199, InM 4α-PHR, or InM PMA at 32°C. In the second part (see Figure 2) , CBF was recorded in InM PMA under zero flow conditions, for 30 minutes, before a perfusion of fresh InM PMA was made into the cell chamber at a rate of 0.125 ml min^"1 over 20 minutes and CBF readings continued after the cessation of the perfusion up to the 120^th minute. In the third group of experiments, HNE were observed in InM PMA for 30 minutes, before being perfused with a mixture of InM PMA and InM 4α- PHR at the same rate (see Figure 2) . In the final group of experiments, the cells were kept in InM 4o£-PHR, before being perfused with the mixture of InM PMA and InM 4α-PHR for twenty minutes as for the other series (see Figure 2) . Date analysis

As previously described [7], CBF was expressed as percentage of the first (ie. time 0) , baseline reading, for both time-courses, and after introducing the second reagent solution, in the latter part of the study. Significance of difference between reagent series was accepted at P<0.05, using Student's paired t-test.

Example 1: Time courses

Cilia observed without fluid-flow stress over time, under normal conditions in M199 (Figure 1, closed circles) , showed a steady decline in CBF from a baseline 100% (10.1±0.4 Hz, mean±SEM, n=32; range 5.8 - 12.9 Hz), to 60±7% of the baseline, by two hours. In contrast, in cells incubated with InM PMA, CBF fell rapidly from the baseline (9.9+0.4 Hz; range 6.5 - 14.1 Hz) to 29+5%, at the end of the same period (Figure 1, open triangles, n=49) . The difference between the decline in CBF in medium alone versus medium + InM PMA was highly significant (P<0.0001, Student's paired t-test). Unexpectedly, 1 nM of 4α-PHR, maintained CBF at baseline levels (9.0±0.4 Hz; range 6.0 - 11.5 Hz) up to 90 minutes. Thereafter, CBF declined to 75+6%, by two hours (Figure 1, open squares, n=33), but remained significantly above medium±PMA throughout (p<0.0001, Student's paired t-test). Example 2: Serial reagent perfusions

When cilia were pre-incubated for 30 minutes in InM PMA (baseline CBF 11.7±0.6 Hz; range 8.7 - 14.9 Hz), before re-perfusion with an equimolar solution of InM 4α-PHR and PMA, the CBF fell rapidly only up to the point when the 4α- PHR+PMA mixture was added. The arrest in decline of CBF (at 53±3% of the baseline) remained until the end of the experiment. In the reverse experimental protocol, cilia were pre-incubated in 4α-PHR first (baseline CBF 11.3±0.7 Hz; range 6.1 - 14.7 Hz), followed by the addition of the equimolar (InM) mixture of 4α-PHR and PMA. It was observed that the CBF did not decline significantly from baseline for the duration of the study (96+6% of the baseline by two hours) suggesting that the 4 -PHR negated the PMA effect. This was not an artefact because control experiments with cilia pre-incubated in PMA (baseline CBF 12.4+0.6 Hz; range 9.8 - 16.1 Hz), before re-perfusing with PMA, the CBF continued on its original path of descent reaching 20±4% of the baseline by two hours. There was a highly significant difference between the CBF profiles seen in the series where cilia were observed in PMA before PMA was re-perfused and those where 4α-PHR was added to the PMA in the second perfusion (P<0.0001, Student's paired t-test). The CBF profile obtained when cilia were observed in 4 -PHR before perfusing the mixture of 4α-PHR and PMA was also significantly different from the rest (P<0.0001, Student's paired t-test, in both cases) . To confirm that the PMA effect was mediated by PKC, we used a cell permeant and selective analogue peptide derived from the pseudosubstrate domain of PKC. This reagent inhibited the PMA effect as expected (data not shown) .

The present results demonstrate that where a cellular process is concerned, 4α-PHR is not neutral, but rather, inhibits PMA-mediated activation of PKC. The effect of this reagent to prevent the normal decline in CBF in the absence of PMA suggests that there exists a pathway that stabilises CBF ex vivo . This unexpected finding may explain why attempts by the present inventors to construct a conventional dose response failed. Thus, administration of a reduced concentration of 4α-PHR (lpM, two log orders below that of PMA) also prevented the expected PMA-induced decline in CBF. This suggests that 4α-PHR exerts its effects by a different route. Without wishing to be bound by theory, the simplest hypothesis is that other phorbol ester binding proteins such as the β-chiamerins [18] are involved. But, the high potency of the effect (pM) is also compatible with the recent observation that cells contain proteins with phorbol-binding domains that control G- protein activity [19, 20]. Thus cilia might possess moieties that specifically interact with non-PKC active phorbol esters at different sites from those responsible for interactions with PKC active phorbols. Another candidate for such an interaction is the recently described 3-phosphoinositide-dependent kinase-1 (PDK-1) , which activates membrane-bound PKC [21-23] by inducing autophosphorylation at two cardinally important sites in the Cl and C2 domains within the carboxy terminus of PKC. This enables the membrane-delimited enzyme to translocate into the cytosol where it remains inactive due to the binding of the autoinhibitory pseudosubstrate peptide. PMA interacts with this inactive species of PKC to effect the necessary conformational changes in the enzyme which result in the removal of the pseudosubstrate from the activation site, thus activating the enzyme.

REFERENCES

1. Wanner, A. (1977) . Clinical aspects of mucociliary transport. American Review of Respiratory Diseases 116, 73 - 125.

2. Sleigh, M. , Blake, J.R. & Liron, N. (1988). The propulsion of mucus by cilia. American Review of Respiratory Diseases 137, 726 - 741.

3. Satir, P., Barkalow, K. & Hamasaki, T. (1993). The control of ciliary beat frequency. Trends in Cell Biology

3. 409 - 412.

4. Kobayashi, K. , Tamaoki, J. , Sakai, N. , Chiyotani, A. and Takizawa, T. (1989). Lung 167, 277 - 284.

5. Salathe, M. , Pratt, M.M. and Wanner, A. (1993). J. Cell Sci. 106, 1211 - 1220.

6. Wong, L.B. and Yeates, D.B. (1992) . Am. J. Respir. Cell Mol. Biol. 7, 447 - 454.

7. Mwimbi, X.K. , Muimo, R. , Green, M. and Mehta, A. (2000). J. Aerosol Med. 13, 273 - 280.

8. Rutland, J. and Cole, P.J. (1980). Lancet 2, 564 - 565. 9. Di Benedetto, G. and Mehta, A. (1990). Euro. Resp. J. 3, 257S.

10. Rusznak, C, Devalia, J.L., Lozewicz, S. and Davies, R.J. (1994). Respir. Med. 88, 89 - 101.

11. Smith, R.P., Shellard, R. , Dhillon, D.P., Winter, J. and Mehta, A. (1996). J. Physiol. 496, 883 - 889.

12. Di Benedetto, G. , Magnus, C.J., Gray, P.A. and Mehta, A. (1991). J. Physiol. 439, 103 - 113.

13. Smith, R.P., Shellard, R. , Di Benedetto, G. , Magnus, C.J. and Mehta, A. (1996). Eur. Respir, J. 9, 86 - 92.

14. Di Benedetto, G. , Lopez-Vidriero, M.T. and Clarke, S.W. (1987) . Bull. Eur. Physiopathol. Respir. 23, 364S.

15. Luk, CM. and Dulfano, M.J. (1983) . Clin. Sci. (Colch) 64, 449 - 451.

16. Caloca, M.J., Garcia-Bermejo, M.L., Blumberg, P.M., Lewin, N.E., Kremmer, E. , Mischak, H., Wang, S., Nacro, K. , Bienfait, B., Marquez, V.E. and Kazanietz, M.G. (1999). Proc. Natl. Acad. Sci. USA. 96, 11854 - 11859.

17. Ron, D. and Mochly-Rosen, D. (1995). Proc. Natl. Acad. Sci. USA. 92, 492 - 496. 18. Souroujon, M. and Mochly-Rosen, D. (1998). Nat. Biotechnol. 16, 919- 924.

19. Chou, M.M., How, W. , Johnson, J. , Graham, L.K., Lee, M.H., Chen, C-S., Newton, A.C, Schauffhausen, B.S. and Toker, A. (1998). Curr. Biol. 8, 1069 - 1077.

20. Dutil, E.M., Toker, A. and Newton, A.C. (1998). Curr. Biol. 8, 1366- 1375.

21. Le Good, J.A. , Ziegler, W. H. , Parekh, D.B., Alessi, D.R., Cohen, P. and Parker, P.J. (1998). Science 281, 2042 - 2045.

Claims

1. Use of 4α phorbol; or a physiologically acceptable salt, ester or other physiologically functional derivative thereof; in the manufacture of a medicament for maintaining or enhancing ciliary beat frequency in an animal.

2. Use according to claim 1 wherein ciliary beat frequency is maintained at at least 50% of the basal level of ciliary beat frequency observed, prior to the addition of an agent shown to reduce ciliary beat frequency.

3. Use according to either of claims 1 or 2 wherein the medicament is intended to be applied to a mammal.

4. A pharmaceutical formulation comprising 4α phorbol or a physiologically acceptable salt, ester, or other physiologically functional derivatives thereof and a carrier therefor for use in maintaining ciliary beat frequency in an animal.

5. A method for maintaining ciliary beat frequency in vivo or in vitro which comprises administering to an animal or tissue or a cell thereof a ciliary beat frequency maintaining amount of 4 phorbol or a physiologically acceptable salt, ester or other physiologically functional derivative thereof.

6. Use of 4a phorbol; or a physiologically acceptable salt, ester or other physiologically functional derivative thereof; in the manufacture of a medicament for inhibiting protein kinase C

7. The use, formulation or method according to any preceding claim for treating subjects suffering from diseases associated with reduced or poor mucociliary clearance, such as cystic fibrosis, primary cilia dyskinesia, Young's syndrome or bronchiectasis.

8. The use, formulation or method according to any one of claims 1 to 6 for counteracting the effect of drugs, the side-effects of which are shown to be reduced ciliary beat frequency or decreased mucociliary clearance.

9. The use, formulation or method according to any preceding claim wherein said physiologically acceptable salts of 4α phorbol include acid addition salts formed with organic carboxylic acids such as acetic, lactic, tartaric, maleic, citric, pyruvic, oxalic, fumaric, oxaloacetic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulphonic, benezenesulfonic and p-tolunesulphonic acids and inorganic acids such as hydrochloric, sulphuric, phosphoric and sulphamic acids.

0. The use, formulation or method according to an preceding claim wherein said physiologically functional derivatives of 4 phorbol are derivatives which can be converted in the body into the parent compound.