Reduction in intraocular pressure induced by colchicine and related compounds

European Journal of Pharmacology, 77 (1982) 17-24 Elsevier/North-Holland Biomedical Press

17

R E D U C T I O N IN INTRAOCULAR P R E S S U R E INDUCED BY C O L C H I C I N E AND RELATED COMPOUNDS R I C H A R D N. W I L L I A M S and P A R I M A L B H A T T A C H E R J E E

Department of Pharmacology, We//come Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, England Received 10 March 1981, revised MS received 27 August 1981, accepted 28 September 1981

R.N. W I L L I A M S and P. B H A T T A C H E R J E E , Reduction in intraoeular pressure induced by colchicine and related compounds, European J. Pharmacol. 77 (1982) 17-24.. The effects of various microtubule inhibitors on the intraocular pressure (IOP) of albino rabbits were investigated. The compounds produced a marked fall in the IOP which was more prolonged after intravitreal injection than after topical administration. Tolerance to this response developed although no cross tolerance between colchicine and vinblastine was apparent. The drugs produced no effect on the pupil diameters and fluorescein angiography revealed no leakage of dye from the vessels of the iris. Lumicolchicine and trimethylcolchicinic acid were without effect on the lOP. The iris-ciliary processes contained more [3H]colchicine after intravitreal injection than after topical administration, and most was associated with the ciliary processes, the site of formation of aqueous humour. Further colchicine and vinblastine were bound within the tubulin containing fraction of this tissue. We suggest that colchicine and related compounds reduce the IOP via mechanisms of microtubule disruption which may partially inhibit the formation of aqueous h u m o u r by the ciliary processes. Aqueous h u m o u r formation Ciliary processes

Microtubule inhibitors

Colchicine

1. Introduction

The intraocular pressure (lOP) is normally maintained between a balanced control of aqueous humour formation in the ciliary processes and drainage of aqueous humour through the trabecular plexus and episcleral veins. An imbalance in one or both of these systems can lead to either a rise or fall in the lOP, the former producing the pathological condition of glaucoma which, if not checked, causes serious visual impairment. Recently Bhattacherjee and Eakins (1978) reported the lowering of IOP in rabbits by colchicine and vinblastine, plant alkaloids known to disrupt microtubules. These compounds are known to inhibit both fast and slow axoplasmic transport (Paulson and McClure, 1975), prevent the assembly of microtubules (Owellen et al., 1972) and arrest cell division in metaphase (Taylor, 1973), effects attributed to the drugs binding specifically to tubulin, the protein sub-unit of the microtubule. In this study we have extended the observations

Intraocular pressure

Tubulin

of Bhattacherjee and Eakins (1978) in an attempt to explain the mechanism of action of microtubule inhibitors on the lOP. We have investigated the effects of several microtubule inhibitors and their inactive analogues, himicolchicine and trimethylcolchicinic acid (TMCA), on the lOP in the albino rabbit. In addition we compared the distribution and binding of [3H]colchicine and vinblastine in various ocular tissues 24 h after intravitreal injection and topical administration.

2. Materials and methods

Adult New Zealand Albino rabbits (2-3 kg) of either sex were used in all the experiments.

2.1. l O P effects 2.1.1. Topical administration One eye of each rabbit was treated with the required concentration of drug solution (20 ~tl)

0014-2999/82/0000-0000/$02.75 © 1982 Elsevier/North-Holland Biomedical Press

18

mmHg +0.5

0

1

2

3

4

5

6

7

8

9 (days)

A

>, ¢1

~-1.0

J....IJ /

Y

i

a.

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...........

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Fig. I. Effects of colchicine • (5.0 #g) and vinblastine © ( 1,0 #g) on the intraocular pressure (mean -+ S.E.M.) after sub-acute topical administration (once/24 h). Tolerance to the IOP response developed over a period of several days.

mmHg A

COLCHICINE(EtR) COLCHICINE(5tJg)+VINBLASTINE (21Jg)

1.0

0 o-1.0

~ -2.0 0. -3.0 O Z -4.0 iii

o -5.0 Z ,< -Irj

2

4

6

8

10

12

14

16

18

20

22

24

TIME (days) Fig. 2. The effect of vinblastine on the intraocular pressure after tolerance to colchicine had developed. After tolerance to the IOP response to colchicine developed, vinblastine was administered topicaUy (once/24 h). Vinblastine still produced a marked faU in IOP either in the presence or absence (not shown) of colchicine, indicating that cross tolerance to these two compounds had not developed. However tolerance to vinblastine also developed after 5 - 6 days of administration. Vertical bars indicate the standard error of mean values determined from 5 observations.

19 once daily for periods of a few days up to two weeks. The contralateral eyes received equal volumes of vehicle. The l O P was measured using an Alcon applanation pneumatonometer immediately before and 24h after each administration of drug. During the period of treatment corneal sensitivity and pupiliary light reflexes were tested, pupil diameter measured and any vascular reactions assessed. If and when an intense vascular reaction appeared the treatment was stopped. The condition of the vessels in the iris was determined by fluorescein angiography as previously described (Edwards, 1975).

2.1.2. Intravitreal injection Freshly prepared solutions of the alkaloids were injected (20 #1) into the vitreous body using a 30 gauge needle attached to polythene tubing which had previously been calibrated to contain a fixed volume per unit length. Contralateral eyes were injected with the same volume of vehicle. The lOPs were measured as above until they returned to pre-injection values. 2.2. Preparations of drug solutions Colchicine, demecolcine, vinblastine, podophyllotoxin and TMCA were purchased from Sigma. Colchicine and TMCA were dissolved in 50 mM phosphate buffer pH 7.4. Vinblastine, demecolcine and podophyllotoxin were dissolved in absolute alcohol and then diluted with phosphate buffer to give the required concentration of drug, the final concentration of alcohol being no greater than 5 %. Lumicolchicine was prepared by irradiating a colchicine solution (25 m g / m l in absolute alcohol) with ultraviolet light directly from a mercury lamp until the spectrum showed no absorbance at 350 nm (Wilson and Friedkin, 1967). This solution was diluted with phosphate buffer so that the final concentration of alcohol was 4%. Drug solutions for intravitreal administration were prepared as above except that sterile isotonic saline was used in place of phosphate buffer.

2.3. Distribution of [3H]colchicine in ocular tissues Tritiated colchicine (Radiochemical Centre, Amersham, England) of specific activity 285

G B q / m M o l (7.7 C i / m M o l ) was used in all the experiments. The original solvent, absolute alcohol, was evaporated under a stream of nitrogen and the residue redissolved in phosphate buffer for topical administration or sterile saline for intravitreal injection.

2.3.1. Topical administration Tritiated colchicine (5 #Ci) was instilled onto the rabbit conjunctiva in volumes of 20 #1. After 24 h the animals were killed by an overdose of sodium pentobarbitone injected into the marginal ear vein. The lower conjunctiva was removed, the aqueous humour aspirated and the eye then enucleated, before the cornea, lens and iris were dissected free, washed in saline, blotted dry and weighed. The tissues were dissolved in Soluene 350 (Packard) and the radioactivity in each determined by standard liquid scintillation counting techniques. As colchicine is known to undergo very little metabolism (Wallace, 1974) no attempt was made to isolate the source of tritium label. 2.3.2. lntravitreal injection Tritiated colchicine (5/xCi) was injected in vols of 20/~1 into the vitreous body and 24 h later the animals were killed and the ocular fluids and tissues removed as described above. 2.4. Sub-cellular distribution and binding of colchicine and vinblastine Tritiated colchicine (5/~Ci) was injected into the vitreous body and 24h later the animals were killed, iris ciliary processes from 4 eyes pooled and homogenized in 5 ml of 0.25 M sucrose solution. The subcellular fractions were prepared by centrifugation according to the method of Berman (1971) and the activity in each determined. Similar fractions were prepared after intravitreal injections of [ 3H]vinblastine (5 #Ci). The percentage of 'bound' drug in each fraction was determined using a method modified from that of Wilson (1975). An aliquot of each subcellular fraction was shaken (10-20 sec) with a mixture of dextran (3%; mol. wt. 40000) and charcoal (1 : 5, w/w). This mixture was centrifuged for 5 rain and the activity in the supernatant determined as

2O

above. The dextran charcoal mixture adsorbs over 90% of the unbound drug leaving the protein bound complex in the supernatant.

3. Results

tolerance to colchicine and vinblastine did not occur. After tolerance to colchicine developed, administration of vinblastine (2/~g) still produced a fall in the IOP (fig. 2) although the onset was delayed (72 h). However, tolerance to vinblastine also developed after a period of a few days.

3.1. Topical administration

3.2. Intravitreal administration

All the compounds which disrupt the organisation of microtubules, with the exception of demecolcine, produced a dose-dependent fall in the intraocular pressure (table 1) which was slow in onset ( > 4 h) but prolonged ( > 24 h). The drugs produced mild dose-dependent vascular reactions, namely dilation of episcleral and limbal vessels and sometimes hyperaemia of the iris, vinblastine producing the more severe reactions. Fluorescein angiography revealed no leakage of dye from the ocular blood vessels into the anterior chamber and no difference between the pupil diameters of the test or control eyes was observed. Repeated topical administration (once/24 h) of microtubule inhibitors resulted in tolerance to the lOP response (fig. 1), the lOP returning to normal levels even in the presence of the drug. During this period of repeated treatment, corneal sensitivity decreased after 4-5 days but returned to normal after treatment was stopped. There was little or no effect on the pupillary light reflex during treatment. Cross-

As with topical administration all the compounds with an effect on the assembly of microtubules produced a fall in IOP (table 1). However, in contrast to its effects after topical administration, demecolcine produced quite a marked fall in the IOP, the difference possibly relating to the permeability characteristic of the drug. The fall in the IOP was greater than that produced after topical administration, this being especially so for vinblastine where 5 #g produced as much as twice the fall observed after topical administration. Furthermore, the fall in IOP after intravitreal injection was prolonged, lasting for several days, the actual duration depending on the particular drug and concentration used (fig. 3). The maximum fall in IOP produced by the drugs after intravitreal injection only occurred 48-72h after administration. In contrast to effects after topical administration corneal sensitivity was seldom depressed although inhibition of the pupillary light reflex accompanied the IOP response, except after de-

TABLE 1 The effects of microtubule inhibitors on the intraocular pressure 24 h after topical administration and intravitreal injection. The intraocular pressures (mean -+S.E.M. (n)) were measured using an Alcon applanation tonometer immediately before and 24 h after drug administration. The drug solution was administered in constant volumes of 20 p,1 to one eye of each animal, the contralateral eye receiving vehicle only. Microtubule inhibitor

Colchicine

Vinblastine

Podophyllotoxin Demecolcine

Topical administration

Intravitreal injection

Dose (~t g)

Change in IOP (mmHg) Test-control eye

Dose (~ g)

20 l0 5 5 2 1 20 20

- 5.14 + 1.05 (7) - 5 . 0 -+0.0 (4) --2.2 --+0.58 (5) --4.3 -+0.49 (5) --2.4 --+0.66 (5) -- 1.8 -+0.58 (5) - 2.8 -4-0.88 (3) 0.0 (3)

20 l0 0.5 5.0 2.5

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5.0 10

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Change in IOP (mmHg) Test-control eye (2) (2) (4) (2) (2)

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~ -12.0 Fig. 3. The duration of the reduction in intraocular pressure (mean ±S.E.M.) produced after a single intravitreal injection of podophyllotoxin • (5/Lg), demecolcine C) (10/~g), vinblastine • (5/tg) and colchicine A (10 Fg). The intraocular pressure was measured before and once every 24 h after intravitreal injection until the pressure returned to preinjection values.

mecolcine where no inhibition of the reflex was observed. All the drugs induced marked vascular reactions after intravitreal injection, demecolcine being the least potent in this respect. As noted after topical administration, fluorescein angiograp h y revealed that there was no increased leakage of fluorescein from the ocular blood vessels with respect to the contralateral eye, even during the most intense vascular reactions. Lumicolchicine and T M C A produced no effect on the l O P when administered either topically (lumicolchicine 20 /~g; T M C A 10/~g) for several days or after a single intravitreal injection (lumicolchicine 20/xg; T M C A 5 t~g).

3.3. Distribution of [ 3H]colchicine in ocular tissues Ocular absorption of colchicine was widespread, all tissues being permeated to some degree 2 4 h after topical or intravitreal administration. As might be expected, the anterior structures of the eye e.g. cornea and conjunctiva contained the highest activity after topical a d m i n i s t r a t i o n whereas the more posterior structures, the iris ciliary processes, retina and vitreous b o d y contained considerably higher concentrations of drugs after intravitreal administration. A comparison of the distribution of [3 H]colchicine 24 h after topical

and intravitreal administration showed the difference in tissue concentrations after the two routes of administration (fig. 4). Further, when the iris was separated from the ciliary processes, most

1000 CPM mg'l 100

1°°

i

1.0 0.1 CORNEA CON-

LENS

IRIS

RETINA

JUNCTIVA Fig. 4. Comparison of the distribution of [3H]colchicine in ocular tissues 24 h after topical [] or intravitreal [] administration. The iris-ciliary process and retina were the only tissues

containing a higher concentration of drug after intravitreal injection, the route of administration producing the more pronounced fall in IOP (mean ± S.E.M., n=3).

22 10000

5000

TOTAL

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Fig. 5. Sub-cellular distribution and binding of [3H]colchicine and [3H]vinblastine in the iris-ciliary processes 24 h after intravitreal injection of the drugs. The iris-ciliary process homogenate (H) was separated into nuclear (N), mitochondrial (M), microsomal (Mic) and soluble (S) fractions and the total drug present in each determined []. The percentage of bound drug in each fraction t~ was determined by adsorbing free drug on charcoal, The soluble fraction contained the greatest amount of drug and only in this fraction were the drugs found in a bound form (mean ±S.E.M., n=3).

(79.7%---3.3) of the total drug present was recovered from the latter tissue.

3. 4. Sub-cellular distribution of [ 31-1]colchicine and vinblastine Twenty four hours after intravitreal injection of [3H]colchicine or vinblastine most of the radioactivity in the iris ciliary processes was associated with the 100000 × g soluble fraction which contains tubulin and microtubule fragments (fig. 5). Significantly only this fraction contained colchicine and vinblastine in a bound form (colchicine 15.7 ± 3.9; vinblastine 62.2% ± 25.7), presumably reflecting a tubulin-drug complex.

4. Discussion

Most effects of colchicine and related compounds are caused specifically by disrupting microtubule assembly and the evidence presented in

this study suggest a similar mechanism of action of these drugs in reducing the intraocular pressure. Several compounds known to disrupt microtubules produced this fall in lOP whereas lumicolchicine and TMCA were without effect. Colchicine, demecolcine, podophyllotoxin and vinblastine, although of differing chemical structures, share the common pharmacological property of causing microtubule disruption in a number of cellular systems and an identical response to these drugs has been taken as direct evidence of an effect on microtubules (Wolff and Williams, 1973; Zweig and Chignell, 1973). Since all these compounds produced a fall in lOP, it seems that microtubules are involved in this response which, being slow in onset, was characteristic of the binding kinetics of these drugs with tubulin (Paulson and McClure, 1975). The structural dissimilarity between the microtubule inhibitors is also reflected by the receptors on the tubulin molecule where colchicine and vinblastine have separate and independent binding sites (Wilson, 1975). Indeed this may offer an explanation for cross-tolerance not developing between colchicine and vinblastine after repeated topical administration. However the actual mechanism of tolerance to the lOP response is more difficult to explain but an increased rate of tubulin turnover may be responsible as the synthesis of this protein is increased several fold by a wide variety of factors (Pipeleers et al., 1977). Indirect evidence that colchicine and vinblastine act on microtubules to reduce the lOP was provided by our findings that both drugs were bound, presumably to tubulin, in the microtubule-containing fraction of the irisciliary processes. A number of reports have cautioned against explaining all the actions of colchicine and vinblastine on the basis of microtubule disruption as these compounds exhibit other properties which appear to be unrelated to microtubules. Lumicolchicine and TMCA, both chemically similar to colchicine, do not bind to tubulin but possess other properties of colchicine (Mizel and Wilson, 1972; Zweig and Chignell, 1973) and have been widely used as controls to determine whether microtubules are involved in a given pharmacological response. In this study lumicolchicine and TMCA

23

had no effect on the IOP, further evidence of rnicrotubule involvement in the lOP response. Most drugs used clinically to reduce the lOP in glaucoma affect the autonomic nervous system pre- or postsynaptically. The possibility that the formation or release of autonomic neurotransmitters may be altered by the microtubule inhibitots exists since there are several reports which show inhibition of axoplasmic transport in sympathetic and parasympathetic nerve fibres (Dahlstrbm et al., 1975), a phenomenon which also accounts for the development of supersensitivity to noradrenaline and acetylcholine in the cat eye following intravitreal injection of high doses (200300/xg) of colchicine (Hahnenberger, 1976). However, Bhattacherjee and Eakins (1978) reported an absence of supersensitivity to noradrenaline in colchicine treated eyes. Further, Cheney et al. (1973) were unable to show any changes in the adrenergic innervation of the rat iris after large intravenous doses of colchicine and vinblastine. Therefore it seems unlikely that the lOP lowering effect of microtubule inhibitors is due to an interaction with the adrenergic nervous system in the eye. However the finding that corneal sensitivity was reduced 3-5 days after topical administration may indicate an inhibition of axoplasmic transport in the sensory nerves of the cornea as none of these compounds have local anaesthetic properties. Recently colchicine and related compounds have been shown to inhibit a number of secretory processes through microtubular disruption. Thus colchicine inhibits the secretion of milk from mammary epithelial cells (Nickerson et al., 1980), the transport of water across epithelial bladder cells (Taylor et al., 1973) and the release of various hormones (Temple et al., 1972). As the epithelial cells of the ciliary processes are known to be involved in the secretion of aqueous humour (Cole, 1977) and since [3H]colchicine was found in relatively high concentrations in a bound and free state in this tissue, it is possible that the microtubule inhibitors may lower the lOP, at least in part, by inhibiting the secretion of aqueous humour. In conclusion the microtubule inhibitors cause a marked fall in intraocular pressure by an action which appears to be unrelated to the autonomic nervous system. A possible mechanism of action

appears to be disruption of microtubules in the ciliary processes resulting in inhibition of aqueous humour formation.

Acknowledgement Part of this work was done at the Institute of Ophthalmology under a grant from the Medical Research Council (U.K.).

References Berman, E.R., 1971, Acid hydrolases of the retinal epithelium, Invest. Ophthalmol. 10, 64. Bhattacherjee, P. and K.E. Eakins, 1978, The intraocular pressure lowering effect of colchicine, Exp. Eye Res. 27, 649. Cheney, D.L., I. Hanin, R. Massarelli, M. Trabucchi and E. Costa, 1973, Vinblastine and vincristine: a study of their action on tissue concentrations of epinephrine, norepinephrine and acetylcholine, Neuropharmacology 12, 233. Cole, D.F., 1977, Secretion of the aqueous humour, Exp. Eye Res. Suppl. 25, 161. Dahlstr~m, A., P.O. Heiwall, J. Haggendal and N.R. Saunders, 1975, Effect of antimitotic drugs on the intra-axonal transport of neurotransmitters in rat adrenergic and cholinergic nerves, Ann. N.Y. Acad. Sci. 253, 5017. Edwards, J., 1975, A new apparatus for fluorescein angiogra. phy of the anterior segment of the eye, J. Physiol. 246, 39P. Hahnenberger, R.W., 1976, Influence of intraocular colchicine and vinblastine on the cat iris, Acta Physiol. Scand. 98, 425. Mizel, S.B. and L. Wilson, 1972, Nucleoside transport in mammalian cells. Inhibition by colchicine, Biochemistry I 1, 2573. Nickerson, S.C., J.J. Smith and T.W. Keenan, 1980, Role of microtubules in milk secretion: action of colchicine on microtubules and oxocytosis of secretory vesicles in rat mammary epithelial cells, Cell Tissue Res. 207, 361. Owellen, R.J., A.H. Owens and D.W. Donegan, 1972, The binding of vincristine, vinblastine and colchicine to tubulin, Biochem. Biophys. Res. Comm. 47, 685. Pipeleers, D.G., M.A. Pipeleers and D.M. Kipris, 1977, Physiological regulation of total tubulin and polymerised tubulin in tissues, J. Cell Biol. 74, 351. Paulson, J.C. and W.O. McClure, 1975, Microtubules and axoplasmic transport. Inhibition of transport by podophyllotoxin: an interaction with microtubule protein, J. Cell Biol. 67, 461. Paulson, J.C. and W.O. McClure, 1975, Inhibition of axoplasmic transport by colchicine, podophyllotoxin and vinblastine: an effect on microtubules, Ann. N.Y. Acad. Sci. 253, 517. Taylor, A., M. Mamelak, E. Reaven and R. Marly, 1973, Vasopressin: possible role of microtubules and microfilaments in its action, Science 181,347.

24 Taylor, E.W., 1973, Macromolecule assembly inhibitors and their action on the cell cycle, in: Drugs and the Cell Cycle, eds. A.M. Zimmerman, G.M. Padilla and I.L. Cameron, (Academic Press, London) p. 11. Temple, R., J.A. Williams, J.F. Wilber and J. Wolff, 1972, Colchicine and hormone secretion, Biochem. Biophys. Res. Commun. 46, 1455. Wallace, S.L., 1974, Colchicine semin, Arthritis. Rheum. 3,369. Wilson, L., 1975, Microtubules as drug receptors: pharmacological properties of microtubule protein, Ann. N.Y. Acad. Sci. 253, 213.

Wilson, L. and M. Friedkin, 1967, The biochemical events ot mitosis. II. The in vivo and in vitro binding of colchicine in grasshopper embryoes and its possible relation to inhibition of mitosis, Biochemistry 6 (10), 3126. Wolff, J. and J.A. Williams, 1973, The role of microtubules and microfilaments in thyroid secretion, Recent Prog. Hormone Res. 29, 229. Zweig, M.H. and C.F. Chignell, 1973, Interaction of some colchicine analogues, vinblastine and podophyllotoxin with rat brain microtubule protein, Biochem. Pharmacol. 22, 2141.