Small peptidic compounds useful for the treatment of glaucoma

Small peptidic compounds containing a small and branched chained amino acid residue, pharmaceutical compositions containing at least one such compound active against glaucoma and intraocular hypertension and a method for treating glaucoma and intraocular hypertension.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to small peptidic compounds, pharmaceutical 
preparations containing such compounds and a method for treating glaucoma. 
2. Description of the Prior Art 
Glaucoma is a very common eye disease affecting millions of people in the 
later stages of their life. Glaucoma is characterized by abnormally high 
intraocular pressure and, if untreated, damage to the optic nerves which 
may cause narrowing of the visual field, and eventually irreversible 
blindness. 
The intraocular pressure is determined by the rates of inflow and outflow, 
i.e. the dynamics of the aqueous humour. The aqueous humour enters into 
the posterior chamber of the eye, and then flows through the pupil to the 
anterior chamber, from where it eventually leaves the eye through the 
trabecular meshwork. 
The aqueous humour supplies nutrients to the lens and cornea, and its 
proper supply is thus of the utmost importance for maintaining healthy 
eyes. 
Any disturbance of aqueous humour dynamics by either excess inflow, or 
reduced outflow, results in an increase in the intraocular pressure above 
the normal value (for adults) of 17-20 mm Hg, i.e. the eye becomes 
hypertensive. A prolonged hypertensive state will result in nerve damage 
and blindness. Detailed descriptions on glaucoma can be found in "An 
Outline of Ophthalmology", by R. L. Coakes, and P. J. Holmer Sellars, 
published by Wright, Bristol (1985), cf. pp. 54/57, and in the series: 
Current Topics in Eye Research", edited by J. A. Zadunaisky and K. Davson, 
Academic Press. 
All known antiglaucoma drugs on the market lower the intraocular pressure, 
either by decreasing formation of aqueous humour, or by increasing the 
outflow, i.e. the elimination of aqueous humour from the eye. Glaucoma 
drugs are thus all hypotensive agents. 
The most common class of antiglaucoma agents are adrenergic antagonists; 
many of them are .beta.-blockers (the most widely used of this type is 
timolol), adrenergic agonists, dopaminergic agents, cholinergic agents 
(the most widely used of this type is pilocarpine), or several other 
classes of compounds. For detailed overviews, see for example Annual 
Reports in Medicinal Chemistry, Vol. 20, chapter 9: "Antiglaucoma Agents", 
by M. F. Sugrue and R. L. Smith (1985, Academic Press), and the text: "The 
Pharmacological Basis of Therapeutics" by A. Goodman and L. Gilmans. 
Thus one of the characteristics of glaucoma theory is the fact that an 
enormous variety of chemical structural types can be used to reduce 
excessively high intraocular pressure. 
None of the currently used drugs is fully satisfactory. There are serious 
side effects affecting the heart, the kidneys, the lungs and/or the 
libido. Some of the side effects are, especially in the case of carbonic 
anhydrase inhibitors, .alpha.-adrenergic antagonists and .beta.-adrenergic 
antagonists, directly implicated with the different modes of action, while 
others are not. Furthermore, there are problems of metabolic stability 
which necessitates several applications of eye drops per day. Great 
efforts are therefore made to develop new antiglaucoma agents which would 
be free of the above constraints. Recently, an entirely new chemical 
structural type of compounds, namely peptides and peptide derivatives, was 
described as having antiglaucoma activity, i.e. as hypotensive agents. 
Examples are carboxyalkyl dipeptides (European Patent No. 0088350) and the 
atrial natriuretic factor, a long peptide of 29 amino acids in length 
(Fortschritte der Ophthalmologie, Volume 89, pp. 89/91 (1989)). 
U.S. Pat. No. 4,634,698 describes ophthalmological pharmaceutical 
compositions comprising carboxyalkyl dipeptides Joined through a 
sulfonamido group to a benzothiadiazinyl sulfonylphenyl moiety and to a 
method for using said composition in the treatment of glaucoma. The 
compositions contain as active agent cyclic, proline-type amino acids, 
which differ substantially from the compounds according to the .invention. 
Besides the peptide moiety being different from the one in the compounds 
claimed in the present invention the known compounds further obligatorily 
contain sulfonamido groups. The sulfonamido group is also present in the 
older antiglaucoma drug acetazolamide which is a carbonic anhydrase 
inhibitor. 
Danish Patent Application No. 1315/85, which has lapsed, discloses a 
process for treatment of glaucoma and/or intraocular hypertension by using 
ACE inhibitors. The ACE inhibitors mentioned were said to be useful also 
for lowering high blood pressure of different genesis. However, the 
proposed ACE inhibitors are not of the type proposed in the present 
invention since they as one of the two amino acids contain one in which 
the .alpha.-amino group and the side chain together obligatorily form an 
at least C4 heterocyclic ring system. Further it is not rendered possible 
that the compounds have the claimed effect. 
Furthermore, hydrolysates of milk proteins were also described as having 
antiglaucoma activity (WO 86/04217 and EP 210204). The peptide 
compositions described therein are not well defined chemical compounds as 
are the compounds of the present invention, rather they are mixtures which 
resulted from the hydrolysis of milk proteins. 
The applicants' previous patent application No. PCT/DK90/00322, filed on 
Dec. 7, 1990, concerns peptide derivatives of the formula 
EQU R.sub.1 --A--B--C--D--E--R.sub.2 I 
wherein 
A is absent or is a non-hydrophobic, uncharged amino acid or a derivative 
thereof, 
B is absent or is an uncharged amino acid or an uncharged N-methylated 
amino acid, 
C is an uncharged amino acid or an uncharged N-methylated amino acid, 
D is an uncharged amino acid with a non-hydrophilic or absent side chain, 
E is cysteine or a cysteine homologue, the sulfhydryl group being free or 
substituted, 
R.sub.2 is optionally substituted NH.sub.2, optionally substituted OH, 
--O-glycosyl, an L- or D-.alpha.-amino acid, or R.sub.2 is absent. 
These compounds are active with glaucoma and intraocular hypertension. 
Preferred compounds are H-Asn-Gly-Gly-Val-Cys(Acm)-NH.sub.2 (SEQ ID NO.1) 
and H-Asn-Leu-Gly-Val-Cys(Acm)-NH.sub.2 (SEQ ID NO.2). One of the 
compounds has been tested on human beings and has proved itself suitable 
against glaucoma and intraocular hypertension by topical application, 
while no side effect was found on blood pressure or heart rate. The 
absence of these cardiovascular effects of this compound has also been 
demonstrated by i.v. administration in rats. 
It has now surprisingly been found that smaller entities of such a parent 
compound of dipeptidal structure may in themselves be active core 
structures. As another such class of minimal structures which are 
significantly different (see the concurrently filed DK patent application 
No. 0531/91) has been identified, both the mode of action, metabolism and 
possible side effect profiles may be envisaged to be different. Thus the 
separation of these new core structures from the parent structure may 
constitute a significant advantage as a base for design of optimal 
pharmaceutical preparations targeted specifically towards different forms 
of glaucoma, treatment profiles and patient groups, while further reducing 
risk of side effects. 
The present invention relates to compounds with dipeptidal structure, and 
derivatives thereof, which lower the intraocular pressure, IOP, in 
relevant animal models. 
SUMMARY OF THE INVENTION 
The compounds of the invention are of the general formula 
##STR1## 
or an .omega.-amino-.alpha.-carboxy cyclic form thereof, or a compound 
transformed into or releasing any of the above basic structures under 
physiological conditions in humans, 
wherein 
R.sub.1 is H, or 
##STR2## 
where R is H, straight, branched alkyl or cycloalkyl up to C.sub.20, 
optionally containing double bonds and/or substituted with halogen, nitro, 
amino, sulfo, phospho or carboxyl, or aralkyl or aryl optionally mono- or 
polysubstituted with halogen, hydroxy, nitro, amino, sulfo, phospho, 
carboxy or alkyl, or R or R.sub.1 is glycosyl, nucleosyl, or R.sub.1 is an 
L- or D-.alpha. amino acid or a peptide moiety of 2 to 8 residues, 
connected by bonds of type [P], 
F is 
##STR3## 
or absent wherein R.sub.1 ' is as defined for R.sub.1 and is absent, when 
the compound is an N--C-cyclic form, 
a is 0, 1 or 2, 
C.sub.x and C.sub.y are tetrahedral carbon atoms (SP3 hybridized) 
independently having R or S configuration or C.sub.x is achiral, 
C.sub.z is a triplanar carbon atom (SP2 hybridized) and D is absent or 
C.sub.z is a tetrahedral carbon atom and D is H.sub.2, 
A is H or CH.sub.3, 
B is with respect to C.sub.y .alpha.- or .beta.-branched C.sub.3 -C.sub.7 
alkyl or C.sub.4 -C.sub.7 cycloalkyl, or phenyl or benzyl, 
[P] is a peptide bond CO--NH, substituted forms thereof, e.g. CO--NR.sub.6, 
wherein R.sub.6 is C.sub.1 -C.sub.3 alkyl, or isosteres thereof such as 
CH.sub.2 --NH, CH.sub.2 --S, CO--CH.sub.2, 
retroinverse forms thereof, such as NH--CO, 
R.sub.2 is H, OH, 
##STR4## 
NH--NH.sub.2, NHOH or NR.sub.3 R.sub.4, wherein R.sub.3 and R.sub.4 are 
independently H, straight or branched alkyl or cycloalkyl, aralkyl or aryl 
optionally mono- or polysubstituted with halogen, carboxy, sulfo, phospho, 
amino or nitro, 
OR.sub.5, where R.sub.5 is H, straight or branched alkyl or cycloalkyl, 
aralkyl or aryl, optionally substituted as defined for R.sub.3 and 
R.sub.4, 
O-glycosyl, or 
an L- or D-.alpha.-amino acid or a peptide moiety of 2 to 8 residues, 
or R.sub.2 is absent when the compound is an N--C cyclic form, 
b is 1, 2, 3 or 4, 
and R.sub.1 and R.sub.2 together comprise no more than 10 amino acid 
residues, 
and wherein hydrogen atoms may be replaced by fluorine, 
or a derivative or salt thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Preferred compounds of the invention are of the general formula 
EQU R.sub.1 --(C[P]G).sub.b --R.sub.2 I' 
wherein C is Gly, .beta.-Ala, Ala or GABA, G is Val, Ile, tert.Leu, Leu, 
Tyr, Phe or cyclohexylalanin, in L-configuration, and [P], b, R.sub.1 and 
R.sub.2 are as defined in claim 1, except that R.sub.2 cannot be Cys or 
Cys-homologues, or a derivative or salt thereof. 
Especially preferred compounds contain the sequence C'-Val wherein C' is 
.beta.-Ala or Gly. 
Examples of active compounds are 
EQU H-Gly-Gly-Val-OEt, (SEQ ID NO.3) 
EQU H-Asn-Gly-Gly-Val-NH.sub.2, (SEQ ID NO.4) 
EQU H-Asn-Leu-Gly-Val-NH.sub.2, (SEQ ID NO.5) 
EQU H-Asn-Leu-Gly-Tyr-NH.sub.2, (SEQ ID NO.6) 
EQU H-Gly-Val-Tyr-NH.sub.2, (SEQ ID NO.7) 
EQU Ac-Gly-Gly-Val-NH.sub.2 (SEQ ID NO. 8) 
and 
EQU H-Gly-Val-OBzl (SEQ ID NO.9) 
EQU H-.beta.-Ala-Val-OBzl (SEQ ID NO.10) 
and derivatives or salts thereof. 
A number of small peptides which contain some of the basic substructures 
belonging to the class of compounds defined in the present invention are 
known, see e.g. WO 87/03485, WO 90/14358, EP 0133225, EP 0174245, EP 
0188629, EP 89485, EP 0278787, EP 0410372, DE 3200273, DE 3412445, DE 
3544375, GB 1420909, Patent Abstract of Japan, 11, 133, Patent Abstract of 
Japan, 8, 251, and Tetrahedron Letters, 29, (1988): 13, pp. 1565-1568. 
None of the compounds disclosed are said to have antiglaucoma effect. 
Especially effective peptides for treating glaucoma and intraocular 
hypertension are 
EQU H-Gly-Val-Benzylester (SEQ ID NO.9) 
and 
EQU H-.beta.-Ala-Val-Benzylester (SEQ ID NO.10) 
These novel peptides and their structurally related active derivatives are 
described in claim 1. 
Further, the invention relates to a pharmaceutical composition containing a 
compound according to the invention in an amount effective to treat 
glaucoma or intraocular hypertension and a pharmaceutically acceptable 
diluent or excipient. 
Additionally, the invention relates to a method for treating glaucoma or 
intraocular hypertension, comprising administering to a mammal an 
effective antiglaucoma or intraocular pressure lowering amount of a 
peptide derivative according to the invention. 
The peptide derivatives of this invention are preferably used in topically 
applicable aqueous isotonic and sterile solutions or in sterile solutions 
or dispersions in an oil as used for the topical treatment of the eye. A 
typical oil for ocular treatment is sterile castor oil. These topical 
solutions or dispersions contain 0.01-10%, in particular 0.1-5%, 
preferably 0.25-1% (percent by weight) of at least one of the peptide 
derivatives of this invention. The normal dosage of these solutions is 1 
to 5 drops administered to the conjunctival sac of the eye. This dosage is 
normally administered 2 to 6 times per day. [20 drops of a DAB-9 dropper 
(Tropfenzahler gemass "Deutsches Arzneibuch 9") will give about 1 ml]. 
In the present invention the term amino acid is to be understood to not 
only cover the 20 natural amino acids, but also to embrace amino acid 
replacements and substituents as recognized in the art. 
The term alkyl is to be understood to cover all saturated hydrocarbons as 
exemplified in e.g. IU. As examples are mentioned methyl, ethyl, 
n-propyl, isopropyl, n-butyl, isobutyl and tert.-butyl for C.sub.1 
-C.sub.4 alkyl. In similar way the term aryl is as defined in e.g. IU, 
and halogen means chlor, brom, iod or fluor. 
The term peptide is to be understood to embrace peptide bond replacements 
and/or peptide mimics, i.e. pseudopeptides, as recognized in the art (see 
for example: Proceedings of the 20th European Peptide Symposium, eat. G. 
Jung, E. Bayer, pp. 289-336, and references therein), as well as salts and 
pharmaceutical preparations and/or formulations which render the bioactive 
peptide(s) particularly suitable for topical application as drops, or for 
oral delivery. Such salts, formulations, amino acid replacements and 
pseudopeptide structures may be necessary and desirable to enhance the 
stability, formulation, deliverability, or to improve the economy of 
production, and they are acceptable, provided they do not negatively 
affect the required biological activity of the peptide as a hypotensive 
agent suitable for lowering of elevated intraocular pressure and glaucoma. 
The actual pharmacological activity effects are envisaged as mediated 
through binding of the structurally active center(s) of the molecules to 
one or more hitherto unestablished and perhaps unknown receptors in the 
eye. Thus, so far no receptor displacement, in vivo or in vitro, assays 
performed on the compound HAsnLeuGlyValCys(Acm)NH.sub.2 (SEQ ID NO.2), a 
potent compound according to PCT DK90/00322, has been able to demonstrate 
any .alpha.-adrenergic agonistic or .beta.-adrenergic antagonistic 
effects, cholinergic effects or carbonic anhydrase inhibitory effects. 
The pharmacological efficacy, potency and duration of effect may be 
modulated through additional structural features, such as chain 
elongation, optical isomerism, the substitution of peptide bond isosters, 
or substitution with one or more groups, which in case of susceptibility 
to enzymatic or spontaneous chemical conversion under the pharmacological 
conditions may also constitute prodrug forms. Different additives and 
vehicles may also affect pharmacokinetic and therapeutic effects. 
The modulation may in some cases lead to significant improvement of 
performance because of enhanced stability, eye penetration, transport to 
the receptor, or controlled release. An example of the-use of amino acid 
and N-terminal substitutions to enhance stability is given in "Enzyme 
resistant immunomodulatory peptides" U.S. Pat. No. 4,505,583 (1985), 
Goldstein, G. et al. An example of peptide prodrugs is mentioned in Int. 
J. of Pharmaceutics 52, p. 255 (1989), Bundgaard, H. An example of the use 
of additives is given in "Evaluation of mucoadhesive polymers in ocular 
drug delivery. 1. Viscous solutions", Pharmaceuticals Res. 8, p. 1039 
(1991), Davies, N. M. et al. 
Apart from substitutions, three particular forms of peptide mimetic and/or 
analogue structures of particular relevance when designing bioactive 
peptides, which have to bind to a receptor while risking the degradation 
by proteinases and peptidases in the blood and elsewhere, may be mentioned 
specifically, illustrated by the following examples: Firstly, the 
inversion of backbone chiral centers leading to D-amino acid residue 
structures may, particularly at the N-terminus, lead to enhanced stability 
for proteolytic degradation while not impairing activity. An example is 
given in the paper "Tritiated D-Ala.sup.1 -Peptide T Binding", Smith, C. 
S. et al, Drug Development Res. 15, pp. 371-379 (1988). Secondly, 
stability and sometimes also receptor binding may be enhanced by forming 
cyclic analogues. An example of this is given in "Conformationally 
restricted thymopentin-like compounds", U.S. Pat. No. 4,547,489 (1985), 
Goldstein, G. et al. Finally, the introduction of ketomethylene, 
methylsulfide or retroinverse bonds to replace peptide bonds, i.e. the 
interchange of the CO and NH moieties may both greatly enhance stability 
and potency. An example of the latter type is given in the paper 
"Biologically active retroinverso analogues of thymopentin", Sisto A. et 
al in Rivier, J. E. and Marshall, G. R. (eds.) "Peptides, Chemistry, 
Structure and Biology", Escom, Leiden (1990), p. 722-773. 
A more closely related example of modulation of effect by structural 
modification not related directly to receptor binding is taken from 
PCT/DK90/00322 in which the pentapeptide HAsnLeuGlyValCys(Acm)NH.sub.2 
(SEQ ID NO.2) was shown both to penetrate the sclera of the eye and to be 
a potent pressure lowering agent. It further contains two activity 
centers, one according to the present application and one according to the 
concurrently filed DK patent application No. 0531/91 together forming the 
tripeptide moiety-GlyValCys(Acm)NH.sub.2 (SEQ ID NO.11). However, when the 
corresponding particular N-.alpha.-unprotected tripeptide 
HGlyValCys(Acm)NH.sub.2 (SEQ ID NO.11) was tested in the stress induced 
rabbit model for antagonizing effect, this was found to be significantly 
lower than expected. However, merely acetylating the tripepride to 
AcGlyValCys(Acm)NH.sub.2 (SEQ ID NO.12) partially restored activity. 
Parallel studies, e.g. on HAsnValCys(Acm)NH.sub.2 (SEQ ID NO.13) and 
HGlyValOBzl (SEQ ID NO.9), have shown that the lower efficacy is not due 
to the free amino terminus per se, since these had a good efficacy. 
Without wanting to be committed to one particular theory, it may be 
speculated that the overall amphiphilicity of the molecule by acetylation 
in case of HGlyValCys(Acm)NH.sub.2 (SEQ ID NO.11) is made more favourable, 
thus improving penetration and transport through the eye and/or to the 
receptor. Some enhanced enzymatic stability may also be envisaged from the 
acetylation. 
The peptides of the invention can be synthesized by various methods which 
are known in principle, namely by chemical coupling methods (cf. Wunsch, 
E.: "Methoden der organischen Chemie", Volume 15, Band 1+2, Synthese yon 
Peptiden, Thieme Verlag, Stuttgart (1974), and Barrany, G.; Merrifield, R. 
B.: "The Peptides", eds. E. Gross, J. Meienhofer., Volume 2, Chapter 1, 
pp. 1-284, Academic Press (1980)), or by enzymatic coupling methods (cf. 
Widmer, F., Johansen, J. T., Carlsberg Res. Commun., Volume 44, pp. 37-46 
(1979), and Kullmann, W.: "Enzymatic Peptide Synthesis", CRC Press Inc., 
Boca Raton, Fla. (1987), and Widmer, F., Johansen, J. T. in "Synthetic 
Peptides in Biology and Medicine", eds., Alitalo, K., Partanen, P., 
Vatieri, A., pp. 79-86, Elsevier, Amsterdam (1985)), or by a combination 
of chemical and enzymatic methods if this is advantageous for the process 
design and economy. 
The peptide derivatives of the invention can be produced by the above 
listed general synthetic methods, or by an advantageous combination 
thereof. 
The described peptides which constitute this invention can be used for the 
treatment of glaucoma in pharmaceutical preparations, possibly in 
combination with pharmaceutical carriers and delivery systems and/or other 
useful and pharmaceutically acceptable additives. 
It was shown in an animal experiment where the intraocular pressure, IOP, 
in the rabbit eye was experimentally raised above the normal level, that 
the compounds of the invention were able to achieve a lowering of the 
intraocular pressure in a similar way as when timolol was applied. Timolol 
is commonly used to treat glaucoma, but, however, being a .beta.-blocker, 
it has serious side effects on the heart, lungs and/or sexual functions. 
It is anticipated that with the compounds according to the invention, many 
of these and other side effects can be avoided. Indeed, a particular 
pentapeptide according to PCT/DK90/00322 containing a characteristic 
structure of the compounds according to the invention, 
HAsnLeuGlYValCys(Acm)NH.sub.2 (SEQ ID NO.2), has been especially 
thoroughly examined for side effects, especially blood pressure and heart 
rate effects, toxicity and mutagenicity as well as local irritant or 
anaesthetic effects in a variety of animal and microbial models. 
The animal model on which the IOP lowering effect of the antiglaucoma 
compound(s) was first established, is a clinically relevant model which 
was developed in the laboratory of one of the inventors who has positively 
shown in this model the pressure lowering effect of many .beta.-blockers 
(such as timolol) and adrenergic agonists, and thus has demonstrated the 
clinical relevance of the model on known and putative glaucoma drugs. 
The main feature of this clinical model is a stress induced elevation of 
the IOP in the rabbit eye above the initial and normal value. The stress 
is exerted, i.e. applied, in the form of measuring the pressure (at 12 
hour intervals) with the help of a SHIOTZ-Tonometer, which is loaded with 
7.5 grams. The pressure first begins to rise after 5 measurements, i.e. 
after 21/2 days, and reaches a maximum after 10 measurements, i.e. after 5 
days. 
Known antiglaucoma drugs lower the intraocular pressure when they are 
applied after the intraocular pressure (IOP) has clearly been established, 
in spite of the fact that the trauma, i.e. the measuring of the pressure, 
continued during the treatment. 
If the treatment with the antiglaucoma drugs is started simultaneously with 
the traumatization, i.e. the exertion of stress by measuring of the 
pressure at the start of the animal experiment, the active antiglaucoma 
drugs antagonize the development of an elevated IOP above the initial and 
normal value, while the inactive compounds will not antagonize, and thus 
result in an elevated pressure. The relevance of this model has been 
demonstrated in many experiments with clinically used antiglaucoma drugs. 
Detailed description of the model is found in: Stainbach, T., Dissertation, 
Universitats-Augenklinik Hamburg-Eppendorf, 1986: "Adrenergica und neue 
Peptide bei Augeninnendruck: Beziehung zum Prostaglandin im Kammerwasser 
yon Kaninchen". 
The IOP activity of the compounds of the present invention has likewise 
been demonstrated on this model as shown in the examples. These peptidic 
compounds are thus likely candidates for the treatment of glaucoma. 
The peptide compositions described in the above doctoral thesis are as 
mentioned not well defined chemical compounds as are the peptide 
derivatives of this invention, rather they are mixtures which resulted 
from the hydrolysis of milk proteins. These peptides and their various 
activities, among which is antiglaucoma, are described in the European 
Patent No. 210 204 by one of the present inventors. 
The findings of IOP lowering effects in the stress induced rabbit model 
have been confirmed and further studied by using another elevated eye 
pressure rabbit model. In this model, the widely applied water load model, 
elevation of the intraocular pressure is achieved by injecting a large 
volume of sterile water intraperitoneally into the rabbits. Following 
onset of eye drop treatment in one eye while the other eye is treated with 
saline placebo, the intraocular pressure of both eyes is then measured at 
various intervals and the .pressure difference between the eyes is taken 
as an expresion of the pharmaceutical effect. In this model pilocarpine, a 
well-known pressure lowering cholinergic agent, was shown to have a 
pressure lowering effect. 
The advantage of the compounds of the invention is their defined chemical 
nature, Which allows for proper registration and, if deemed desirable, for 
logic and systematic structural modification to produce analogues of even 
better properties than the ones invented and claimed now. 
Furthermore, the compounds according to the invention are of low molecular 
weight (.ltoreq.800), and thus topically applicable, unlike the atrial 
natriuretic factor described in Fortschritte der Ophthalmologie, Volume 
86, p. 89-91 (1989), which has a molecular weight of .about.3000, and 
needs to be administered by injection to achieve an antiglaucoma effect. 
Moreover, the atrial natriuretic factor is a cardiovascular hormone and 
thus not suited to be used for treatment of glaucoma over prolonged 
periods of time. Finally, both the peptidic protein hydrolysate mixtures 
(which are not necessarily strictly peptidic in chemical structural terms) 
and the atrial natriuretic factor are of a size which may give rise to an 
immune response followed by the production of antibodies. Such a response 
is unlikely to occur with the low molecular weight compounds according to 
the invention. 
The mechanism, or mechanisms, by way of which the peptides according to the 
invention work, is so far not known in detail and may be of hitherto 
unknown types or related to some known mechanisms. With the apparent lack 
of .beta.-blocking effects, .alpha.-agonistic effects, cholinergic effects 
and inhibitory effects on the enzyme carbonic anhydrase other effects on 
aqueous humour outflow could be working. Some indications of mechanisms of 
the latter type have been found in in vitro studies. Thus, an in vitro 
study conducted at an early stage demonstrated that the parent compound 
HAsnGlyGlyValCys(Acm)NH.sub.2 (SEQ ID NO.1) induced a marked and 
significant decrease of uptake of glycosamines in cultured bovine 
trabecular meshwork cells. From this decrease in the synthesis of 
glucosamineglycanes of importance in the outflow resistance was inferred. 
The invention is now further explained and documented by way of examples. 
PHARMACOLOGICAL EXAMPLES 
Antagonizing of the Intraocular Pressure in the stressed Rabbit's Eyes 
Model 
The compound lowers the experimentally increased IOP in the rabbit animal 
model, or it antagonizes, i.e. prevents the increase in pressure when it 
is applied simultaneously with the treatment which inflicts the increase 
in the pressure. 
The compound was a freeze-dried powder, and was applied to the rabbit eye 
as a powder, or as drops, dissolved in 0.9% NaCl aqueous solution. 
Negative control was 0.9% NaCl solution in water. 
Water Load Model Effects in Rabbit's. Eyes 
The studies utilized a "water load" animal model. 
Thirty minutes before drug solution instillation, rabbits were injected 
intraperitonally with 60 ml/kg of sterile distilled water for injection 
(30.degree. C.) spiked with an antibiotic mixture (Sigma P9032). 
At time zero, 50 .mu.l of a drug solution was instilled to one eye and an 
equal volume of a saline solution was instilled to the other eye. The IOP 
in each eye was monitored at the time points indicated. The change in IOP 
at each time point is computed by subtracting the IOP in the dosed eye 
from that in the undosed-eye. 
Plots of this data were made showing the IOP versus time including standard 
deviation. From these plots were assessed the maximal IOP effect, the time 
to reach this and the time for returning to a zero or insignificant level 
of IOP lowering effect. These figures were taken as a measure of potency 
and duration of effect. 
General note for pharmacological examples 1-6 
Peptides were tested for the intraocular pressure lowering or antagonizing 
effects in the water load model described above or the stress induced 
antagonizing model respectively, in groups of four to ten rabbits, as 
described below. The tests were performed on homogeneous groups of 
randomly sexed rabbits, weight 2.5-3.0 kg, but of different breeds in 
various laboratories several places in the world. Thus, in some cases 
intergroup variations were found in the absolute starting pressure of the 
rabbit's eyes. 
In the case of the water load model structure, each rabbit served as its 
own reference control for the duration of the experiment, and in the case 
of the stress induced antagonistic model, each group of rabbits served as 
reference control, at the beginning and end of 10 stress units. Usually, 
the peptides were dissolved in plain isotonic saline, but in one case in 
the waterload model, a TRIS-buffer at physiological pH was included. 
In both cases a negative saline control group showed no effect on the 
pressure. The relevant TRIS-buffer control group showed also no effect in 
the waterload model, while a 2.6% solution of the known miotic glaucoma 
drug, pilocarpine, gave a similar response to some of the preferred 
compounds as illustrated in the drawing of FIG. 4. The compounds listed in 
the tables were then classified as active on the following criterion: In 
the water load model one drop of a 1% solution in one eye resulted in a 
significant pressure lowering effect corresponding to the control treated 
eye, which was at maximum at least 1 mm Hg within 1 hour and with a 
lowering effect duration of at least 90 minutes for the group on average. 
In the stress induced antagonizing model, the pressure increase following 
10 stress units for the treated group on average was found to be smaller 
than 2 mm Hg and to be significantly less compared to untreated controls, 
which normally gave 8 to 18 mm Hg. 
The following examples are further explained by means of the drawing in 
which 
FIG. 1 shows the change in the intraocular pressure, .DELTA.IOP, in mm Hg 
as a function of the time in minutes for the compound Peptide 3, 1%, 
AcGlyGlyValNH.sub.2 (SEQ ID NO.8), 
FIG. 2 shows the change in the intraocular pressure, .DELTA.IOP, in mm Hg 
as a function of the time in minutes for the compound Peptide 1.4, 1%, 
HGlyValOBzl (SEQ ID NO.9), 
FIG. 3 shows the change in the intraocular pressure, .DELTA.IOP, in mm Hg 
as a function of the time in minutes for the compound Peptide No. 109, 1%, 
H-.beta.-Ala-Val-OBzl (SEQ ID NO.10), and 
FIG. 4 shows the change in the intraocular pressure, .DELTA.IOP, in mm Hg 
as a function of the time in minutes for the positive reference control 
2,6% pilocarpine. 
EXAMPLE 1 
Antagonizing effect of tri- and tetrapeptides containing the sequence 
Gly-Gly-Val with various N- and C-substitutions on the stress induced 
intraocular pressure in the rabbit's eye, following 10 stress units. 
The peptides were applied topically as a 1% solution in 0.9% aqueous NaCl 
in aliquots of 60 .mu.l 3 times daily over a period of 5 days. 
______________________________________ 
Peptide Antagonizing Effect 
______________________________________ 
H-Gly-Gly-Val-OEt Active 
H-Asn-Gly-Gly-Val-NH.sub.2 (SEQ ID NO. 9) 
Active 
______________________________________ 
EXAMPLE 2 
Antagonizing effect of tetrapeptides containing the sequence 
Asn-Leu-Gly-X-NH.sub.2, where X is Val or Tyr, on the stress induced IOP 
in the rabbit's eye, following 10 stress units. 
The peptide was applied topically as a 1% solution in 0.9% aqueous NaCl in 
aliquots of 60 .mu.l 3 times daily over a period of 5 days. 
______________________________________ 
Peptide Antagonizing Effect 
______________________________________ 
H-Asn-Leu-Gly-Val-NH.sub.2 (SEQ ID NO. 5) 
Active 
H-Asn-Leu-Gly-Tyr-NH.sub.2 (SEQ ID NO. 6) 
Active 
______________________________________ 
EXAMPLE 3 
Antagonizing effect of tripeptides containing the sequence Gly-Val on the 
stress induced intraocular pressure in the rabbit's eye, following 10 
stress units. 
The peptides were applied topically as a 1% solution in 0.9% aqueous NaCl 
in aliquots of 60 .mu.l 3 times daily over a period of 5 days. 
______________________________________ 
Peptide Antagonizing Effect 
______________________________________ 
H-Gly-Val-Tyr-NH.sub.2 (SEQ ID NO. 7) 
Active 
Ac-Gly-Gly-Val-NH.sub.2 (SEQ ID NO. 8) 
Active 
______________________________________ 
EXAMPLE 4 
Pressure lowering effect of tripeptides containing the sequence Gly-Val on 
water load induced hypertension in the rabbit's eye by single dose 
treatment. 
50 .mu.l of a 1% solution of the peptides in 0.9% aqueous saline were 
applied in one eye and 50 .mu.l of 0.9% aqueous saline in the other eye 30 
minutes after the intraperitonal water loading and IOP were measured in 
both eyes for 2 hours and the difference calculated. 
______________________________________ 
Pressure 
Peptide Lowering Effect 
______________________________________ 
H-Gly-Val-Tyr-NH.sub.2 (SEQ ID NO. 7) 
Active 
Ac-Gly-Gly-Val-NH.sub.2 (SEQ ID NO. 8) 
Active 
______________________________________ 
The time curve for the pressure lowering effect in the waterload model for 
AcGlyGlyValNH.sub.2 is given in FIG. 1, where the peptide has the 
designation No. 3. 
EXAMPLE 5 
Antagonizing effect on the stress induced intraocular pressure and pressure 
lowering effect in the experimentally hypertensive rabbit's eye of the 
dipeptide ester Glycyl-L-Valine-Benzylester, H-Gly-Val-OBzl (SEQ ID NO.9). 
The peptide was applied topically as a 1% solution in 0.9% aqueous NaCl and 
tested in the two models as described in Examples 1 and 4. 
______________________________________ 
Antagonizing Effect 
Pressure Lowering Effect 
______________________________________ 
Active Active 
______________________________________ 
The time curve for pressure lowering effect in the waterload model for this 
compound is given in FIG. 2, where the peptide has the designation No. 
1.4. 
EXAMPLE 6 
Pressure lowering effect of a dipeptide containing the sequence 
.beta.Ala-Val on water load induced hypertension in the rabbit's eye by 
single dose treatment, the dipeptide ester .beta.-alanyl L-valine 
Benzylester, H-.beta.-Ala-Val-OBzl (SEQ ID NO.10). 
50 .mu.l of 1% solution of the peptides in 0.9% isotonic saline containing 
TRIS-buffer pH 7.4 was applied in one eye and 50 .mu.l of 0.9% isotonic 
saline containing TRIS-buffer. pH 7.4 in the other eye 30 minutes after 
the interperitonal water loading and intraocular pressure was measured in 
both eyes for 2 hours and the difference calculated. 
______________________________________ 
Peptide Pressure Lowering Effect 
______________________________________ 
H-.beta.-Ala-Val-OBzl (SEQ ID NO. 10) 
Active 
______________________________________ 
The time curve for the pressure lowering effect in the waterload model for 
this peptide is given in FIG. 3, where the peptide has the designation No. 
109. 
Synthesis of compounds according to the invention 
The abbreviations used in this description for amino acids and protecting 
groups are in agreement with the IU-IUB standard rules for 
nomenclature. 
In addition; and in particular, the following abbreviations are used: 
______________________________________ 
HONSu : N-hydroxysuccinimide 
DCC : Dicyclohexylcarbodiimide 
Boc : tert.-butyloxycarbonyl 
OBzl : Benzylester 
DMF : Dimethylformamide 
DCU : Dicyclohexylurea 
TEA : Triethylacetic acid 
EtOAc : Ethylacetate 
OEt : Ethylester 
Ac : Acetyl 
______________________________________ 
EXAMPLE 7 
Synthesis of G1]-Val-OBzl (SEQ ID NO.9) 
Boc-Gly-Val-OBzl (SEQ ID NO.14) 
4.4 g (25 mmoles) Boc-Gly-OH and 3.0 g (26.3 mmoles) HONSu were dissolved 
in 50 ml DMF and cooled to 0.degree. C. in an icebath. 5.6 g (27.5 mmoles) 
DCC dissolved in 50 ml cold DMF was then added and the mixture was stirred 
cold for 5 hours. 10.4 g (27.5 mmoles) of Val-OBzl paratosylate were then 
added, dissolved in 50 ml DMF and 3.8 ml (27.5 mmoles) TEA. The mixture 
was stirred at room temperature overnight, and further for one day, 
following addition of further 2.0 ml TEA. The mixture was then filtered, 
evaporated to dryness and dissolved in EtOAc and extracted with aqueous 
NaHCO.sub.3 followed by extraction with 10% citric acid, dried and again 
taken to dryness under reduced pressure to give an oil. Yield 10.0 g 
(92%). 
HCl, Gly-Val-OBzl (SEQ ID NO.9) 
To 9.0 g (20.6 mmoles) oily Boc-Gly-Val-OBzl (SEQ ID NO.14) were added 250 
ml 2.6M HCl in EtOAc. The mixture was stirred for 80 min. and taken to 
dryness Under reduced pressure. Following repeated additions of EtoAc and 
evaporation to dryness under reduced pressure HCl, Gly-Val-OBzl (SEQ ID 
NO.9) was isolated as a white powder, Yield 5.6 g (79%). 
EXAMPLE 8 
Synthesis of Ac-Gly-Gly-Val-NH.sub.2 (SEQ ID NO.8) 
1.75 g (10 mmoles) Ac-Gly-Gly-OH and 1.73 g (15 mmoles) HONSu were 
dissolved in 25 ml acetonitrile and cooled to 0.degree. C. 2.46 g DCC (12 
mmoles) were then added, the mixture allowed to warm to room temperature 
and stirred for 2 hours until complete formation of the active ester. 
After filtering off the DCU, 1.16 g of Val-NH.sub.2 free base (10 mmoles) 
in 25 ml H.sub.2 O at pH 8 were added slowly and pH maintained above 7. At 
completion of the reaction the acetonitrile was removed under vacuum and a 
small residue filtered off from the aqueous phase. The product was then 
purified by reverse phase HPLC using water/ethanol/acetic acid buffers. 
Yield 0.8 g (34%). Purity by HPLC: &gt;95% at 220 nm. 
.sup.13 C-NMR proved correct structure by assignment of all carbon atoms. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 14 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note="Cys(Acm)-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AsnGlyGlyValCys 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note="Cys(Acm)-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AsnLeuGlyValCys 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..3 
(D) OTHER INFORMATION: /note="Val-OEt" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GlyG lyVal 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..4 
(D) OTHER INFORMATION: /note= "Val-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AsnGlyGlyVal 
1 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..4 
(D) OTHER INFORMATION: /note="Val-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
AsnLeuGlyVal 
1 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..4 
(D) OTHER INFORMATION: /note="Tyr-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
AsnLeuGlyTyr 
1 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..3 
(D) OTHER INFORMATION: /note="Tyr-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GlyValTyr 
1 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..3 
(D) OTHER INFORMATION: /note="Ac-Gly Gly Val-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GlyGlyVal 
1 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..2 
(D) OTHER INFORMATION: /note="Where X is Benzylester 
(OBzl), Val- X" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GlyVal 
1 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..2 
(D) OTHER INFORMATION: /note="Where X is Benzylester 
(OBzl) and Z- Ala is beta-alanine, Z-Ala Val-X" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AlaVal 
1 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..3 
(D) OTHER INFORMATION: /note="Cys(Acm)-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GlyValCys 
1 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..3 
(D) OTHER INFORMATION: /note="Ac-Gly Val Cys(Acm)-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GlyValCys 
1 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION: 1..3 
(D) OTHER INFORMATION: /note="Cys(Acm)-NH2" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
Asn ValCys 
1 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..2 
(D) OTHER INFORMATION: /note="Boc-Gly Val-Obzl" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GlyVal 
1