The present invention relates to the use of protease inhibitors,
especially cysteine protease inhibitors.
Trichomonas gallinae is a flagellated protozoan parasite
which infects a variety of birds all over the world. Avian
trichomonosis , caused by T . gallinae, has been reported from
several continents as a major disease for numerous avian species
of the orders Columbif ormes , Falconif ormes and Psittacif omes .
They may infect other avian species as well, like Galliformes
and Passerif ormers . The domestic pigeon (Columba livia) is the
primary host of this flagellate which has been considered re
sponsible for the worldwide spread of T . gallinae. Furthermore,
serious losses among wild birds, in particular wild finches, due
to T . gallinae were reported recently. T . gallinae colonizes
mainly in the upper digestive tract of the birds, where, it can
cause granulomatous lesions that occlude the oesophagus lumen,
leading to the death of birds as a result of severe starvation.
The parasite produces a variety of pathological changes depend
ing on the virulence of the strain and the species of bird in
fected. Earlier studies with T . gallinae have demonstrated a
wide spectrum of virulence among different T . gallinae strains.
Accordingly, some T . gallinae strains do not cause clinical
signs and can induce certain immunity against a highly virulent
one. Virulent strains are able to produce a systemic infection
in its host and affect mainly the liver and lung beside the oro
pharynx. Pathological changes following experimental infection
of a pigeon with a virulent strain of T . gallinae were described
in detail in the prior art.
Applying molecular technologies genetic differences between
T . gallinae strains could be demonstrated in different studies,
for example it could be shown that T . gallinae clones 5895-C1/06
and 8855-C3/06 are members of a particular clade (T. gallinae
like isolates) belonging to two different subgroups. In contrast
clone 231-C1/07 was grouped in a different clade (Trichomonas
tenax like isolates) , based on phylogenetic analyses of the two
ribosomal RNA internal transcribed spacers (ITS1 and ITS2) and
the 5 .8S rRNA gene .
In order to assess the virulence of T . gallinae in vivo a
suitable system is needed premising a simple working protocol
and the protection of animals. Embryonated chicken eggs have
been one of the most common substrate for isolation, propagation
and characterization of different avian viruses as well as for
the production of viral vaccines. Additionally, embryonated eggs
have been used to investigate the virulence of various bacteria
and fungi .
WO 97/05867 A l discloses the treatment of Trichomonas in
fections with ditiocarb or disulfiram. WO 00/63350 A2 D2 dis
closes a cysteine protease and/ or a nucleic acid encoding a
cysteine protease as a vaccine against neurocysticerocosis ; also
WO 98/44943 A l discloses a Leishmania vaccine comprising a cys
teine protease and/ or a nucleic acid encoding a cysteine prote
ase. WO 2008/043180 A l discloses immunostimulatory compositions
comprising recombinant BCG Mycobacterium expressing a mammalian
cysteine protease. WO 2010/000398 A l discloses the use of nifurtimox
for treating diseases caused by Trichomonadida . Iwalewa et
al . (J. Ethnopharm. 117 (2008), 507-511) report about antiprotozoan
activities of Harungana madagascariensis stem bark ex
tract on trichomonads and malaria. Amin et al . (PLoS ONE 7
(2012), e37417) disclose that cysteine peptidases that are se
creted by Trichomonas gallinae are involved in the cytopathogenic
effects on a permanent chicken liver cell culture.
Recently, peptidases are described as synonyms for proteas
es, proteinases and peptide hydrolases which are the best known
enzymes which catabolize proteins and polypeptides through
cleavage of peptide bonds. Cysteine peptidases have been report
ed as essential factors in the biology and the pathogenesis of
various parasitic diseases. In this context it was reported that
cysteine peptidases are involved in degradation of extracellular
matrix components such as fibronectin and laminin. Previously,
it was demonstrated that liver abscesses produced by Entamoeba
histolytica in severe combined immunodef icient (SCID) mice were
greatly reduced by preincubation of the trophozoites with the
cysteine peptidase inhibitor namely L-3-carboxyl-2 ,3-transepoxysuccinyl-
leucylamido (4-guanidino) butane (E-64). Moreover,
application of a specific inhibitor to major Trypanosoma cruzi
cysteine peptidases (cruzain) was able to protect the infected
mice from lethal infection.
Recently, cathepsin L-like cysteine peptidases were identi
fied as secreted virulence products in a cell-free filtrate of
axenic T . gallinae clonal culture. Additionally, it could also
be shown that certain peptidase inhibitors are capable to reduce
the cytopathogenic effects of T . gallinae in a permanent chicken
liver (LMH) cell line. However, evidence that peptidase inhibi
tors could significantly decrease the protozoal peptidases of T .
gallinae activity in vivo is still lacking.
It is therefore an object of the present invention to pro
vide suitable means for combatting infections with T . gallinae
in birds.
Therefore the present invention provides protease inhibi
tors for use in the treatment of a Trichomonas gallinae infec
tion of an animal, selected from the orders Galliformes, Passeriformes,
Psittacif ormes , Columbif ormes and Falconif ormes .
With the present invention it is demonstrated that the pro
gression of avian trichomonosis is linked with the presence of
cysteine peptidases which can be neutralized by co-application
of peptidase inhibitors without toxicity to the host. Such pep
tidase inhibitors had no direct cytotoxic effect on the viabil
ity of the trichomonads . This indicates that the reduction of
pathogenicity of T . gallinae in the presence of peptidase inhib
itors is due to the inhibition of cysteine peptidase activity
rather than a direct effect on the trichomonad cells. The pre
sent investigations also show the importance of cysteine pepti
dases as drug targets as a strategy for chemotherapy.
The present invention is based on the surprising fact that
the protease inhibitors are successful in preventing or reducing
the pathogenic effects of T . gallinae after oral administration
to the animals. Although proteases have been suggested for com
batting parasitic diseases for more than 2 0 years (e.g. Rosen
thal et al., J . Clin. Invest. 82 (1988), 1560-1566), no success
ful application of this concept has yet made its way to applied
medicine, neither in humans nor in animals. This might also be
due to the different nature and protease strategy such pathogens
have with respect to their host.
The fact that the strategy of the present invention can be
successfully applied for combatting T . gallinae caused patho
genicity stands in contrast to recently published results that
the haemolytic activity of T . gallinae does not correspond with
clinical virulence (Gerhold et al ., Vet. Parasitol. 160 (2009),
221-224). The successful administration of protease inhibitors
according to the present invention shows that those results do
not necessarily exclude combatting clinical virulence of T . gal
linae, although the results according to the present invention
could not have been predicted on the basis of e.g. Gerhold et
al .. For example, it was also surprising that T . gallinae, in
contrast to the closely related Tetratrichomonas gallinarum (Ma
lik et al., PLoS ONE 6(2011): e20774) indeed induces distinctive
cytopathogenic effects in tissue cultures.
Preferably, the protease inhibitors according to the pre
sent invention are protease inhibitors selected from cysteine
protease inhibitors and serine protease inhibitors, especially
cysteine protease inhibitors.
Proteases are enzymes that degrade polypeptides.
Cysteine proteases have a common catalytic mechanism that
involves a nucleophilic cysteine thiol in a catalytic dyad. The
first step is deprotonation of a thiol in the enzyme's active
site by an adjacent amino acid with a basic side chain, usually
a histidine residue. The next step is nucleophilic attack by the
deprotonated cysteine's anionic sulfur on the substrate carbonyl
carbon. In this step, a fragment of the substrate is released
with an amine terminus, the histidine residue in the protease is
restored to its deprotonated form, and a thioester intermediate
linking the new carboxy-terminus of the substrate to the cyste
ine thiol is formed. Therefore they are also sometimes referred
to as thiol proteases. The thioester bond is subsequently hydrolysed
to generate a carboxylic acid moiety on the remaining sub
strate fragment, while regenerating the free enzyme.
Serine proteases are enzymes that cleave peptide bonds in
proteins, in which serine serves as the nucleophilic amino acid
at the active site. Serine proteases fall into two broad catego
ries based on their structure: chymotrypsin-like (trypsin-like)
or subtilisin-like . The main player in the catalytic mechanism
in the serine proteases is the catalytic triad. The triad is lo
cated in the active site of the enzyme, where catalysis occurs,
and is preserved in all serine protease enzymes. The triad is a
coordinated structure consisting of three essential amino acids:
histidine, serine ("serine protease") and aspartic acid. Located
very near one another near the heart of the enzyme, these three
key amino acids each play an essential role in the cleaving
ability of the proteases. In the event of catalysis, an ordered
mechanism occurs in which several intermediates are generated.
The catalysis of the peptide cleavage can be seen as a Ping-Pong
catalysis, in which a substrate binds (in this case, the poly
peptide being cleaved) , a product is released (the N-terminus
"half" of the peptide) , another substrate binds (in this case,
water) , and another product is released (the C-terminus "half"
of the peptide) .
Inhibitors to proteases are known to the person skilled in
the art. In fact, numerous protease inhibitors with specificity
to e.g. serine proteases or cysteine proteases are available.
The terms "serine proteases" or "cysteine proteases" and
"protease inhibitors", especially "serine protease inhibitors"
and "cysteine protease inhibitors" are therefore accepted prod
uct classes in the present field. The compounds showing the de
sired serine or cysteine protease functionality (or the inhibi
tion of this specific enzymatic activity) are part of the common
general knowledge and this functionality is accepted in the pri
or art to have a clear technical meaning. The functionality can
be verified using tests or procedures adequately specified in
the prior art and widely known to the skilled person. The nature
of a compound being a serine or cysteine protease inhibitor is
therefore known to a person skilled in the art (see also the
prior art cited herein) or can easily be defined by standard en
zymatic inhibition assays (e.g. the assays for testing cysteine
or serine inhibiting function disclosed in the prior art docu
ments as cited herein) . Such tests can therefore be performed as
routing methods and do not require undue experimentation. A s al
so disclosed herein, the present invention unambiguously shows
that this functional activity is essential for the solution of
the technical problem underlying the present invention, regard
less of the structure of the compounds.
It is known to a person skilled in the art that not only
the term "protease inhibitors" as such is directed to a clearly
defined class of compounds, this holds specifically true for
"cysteine protease inhibitors" as well as for "serine protease
inhibitors" .
According to a preferred embodiment of the present inven
tion, the protease inhibitors are cysteine protease inhibitors
selected from the group consisting of E-64 (trans-Epoxysuccinyl-
L-leucylamido (4-guanidino) butane) , L-trans-3-Carboxyoxiran-2-
carbonyl-L-leucylagmatine, N- (trans-Epoxysuccinyl) -L-leucine 4-
guanidinobutylamide) , TLCK (tosyl-L-lysine chloromethyl ketone) ,
TPCK (tosyl-L-phenylalanine chloromethyl ketone) , or mixtures
thereof .
A group of preferred cysteine protease inhibitors is the
group of cathepsin L inhibitors, such as thiochromanone thiosemicarbazone
analogs 6-bromo-TST, acetyl-leu-leu-norleucinol ,
NPI-8343, NPI-8344, NPI-2349, NPI-2019, NPI-3485 and NPI-3469,
epoxysuccinyl acid derivatives containing aziridine-2 ,3-
dicarboxylic acid as the electrophilic alpha-amino acid, inhibi
tors as disclosed in JP-00987265 A , FEBS Lett. 458 (1999), 6-10,
EP 0 0611 756 A , and Antibiotics 51 (1998), 629-634; 3-(N-(l-(N-
(4-aminobutyl) N- (3-aminopropyl ) carbamoyl) 2- (4-hydroxyphenyl)
ethyl) carbamoyl) oxirane 2-carboxylic acid, WF-14685A and WF-
14865B. Examples are also given in Fig. 9 .
Cysteine protease inhibitors and various suitable tests and
procedures for easily observing such cysteine protease inhibit
ing function are also (among many other documents) disclosed in
WO 99/53039 Al, WO 02/076939 A2, WO 2003/097664 A2, WO
2005/003150 A2, WO 2006/091610 A2, WO 2007/012180 Al, WO
2007/041775 Al, WO 2009/067797 Al, WO 2010/025314 A2 (especially
paragraphs [0056] to [0063]), WO 2010/033658 A2 (especially par
agraphs [0036] to [0045]), WO 2010/054042 A2, WO 94/06280 Al, WO
95/23229 A l and Rosenthal et al ., J.Clin. Invest 82 (1988),
1560-1566) . Cysteine protease inhibitors, including their func
tionality and mechanistic properties are also reviewed by Otto
et al. (Chem. Rev. 97 (1997), 133-171, especially 147-164), e.g.
peptidyl-aldehydes , such as leupeptins, chymostatins , antipain,
elastinal and b-MARI (b-microbial alkaline protease inhibitor);
peptidyl-semicarbazones , such as Z-Arg-Ile-Phe-Sc, Z-Ile-Phe-Sc,
Z-Phe-Gly-Sc and Z-Gly-Phe-Gly-Sc; peptidyl-methyl ketones and -
trif luormethyl ketones, such as Z-Phe-Ala-CH 3, Z-Phe-Ala-CF 3,
Ac-Phe-Gly-CH 3, Z-Phe-CH 3, Bz-Phe-CHF 2, Bz-Phe-CF 3, Z-Val-Phe-CF 3
and Z-Val-D-Phe-CF 3; peptidyl-a-keto acids, - - eto esters, -aketo
amides and -diketones, such as Bz-Phe-COOMe, Z-Phe-COOEt,
Z-Phe-COCH 3, Z-Val-Phe-COOMe, Z-Val-Phe-COCH 3, Z-Phe-Gly-COOH,
Z-Phe-Gly-COOCH 3, Ac-Phe-Gly-COOCH 3, Ac-Phe-Gly-COCH 3, Z-Phe-Gly-
CONHEt, Z-Phe-Gly-CO-LeuOMe, Z-Phe-Gly-COOn-But , Z-Gly-Phe-Gly-
COOn-But, Z-Phe-Gly-C0 2CH2C02Et, Z-Phe-Gly-C0 2 (CH2)3C02Me, Z-Leu-
Phe-CONH, Z-Leu-a-aminodimethylacetic acid-CONH 2, Z-Leu-Phe-
CONHR, Z-Leu-a-aminodimethylacetic acid-CONHR, Z-Leu-Phe-CONEt 2,
Z-Leu-a-aminodimethylacetic acid -CONR 2, Z-Leu-Phe-COOH, Z-Leua-
aminodimethylacetic acid-COOH, Z-Leu-Phe-COOEt , Z-Leu-aaminodimethylacetic
acid-COOR, Z-Leu-nLeu-COOEt , Z-Leu-Met-
COOEt, Z-Leu-a-aminodimethylacetic acid-CO-NHEt-S0 2Et, (CH3)2NCO-
Leu-a-aminodimethylacetic acid-CONHEt and morpholino-CO-Leua-
aminodimethylacetic acid-CONHEte ; peptidyl-nitriles , such as
Z-NH-CH (CH2OH) -CN, Ac-NH-CH 2-CN, Bz-NH-CH 2-CN, Ac-Phe-NH-CH 2-CN,
CH3-0-CO-Phe-NH-CH 2-CN, Ac-Phe-NH-CH (i-But) -CN, Gly-NH-CH (CH2PH) -
CN, Ac-NH-CH 2-CSNH 2, Bz-NH-CH2-CSNH 2 and CH3-0-CO-Phe-NHCH 2-
CSNH 2; peptidyl-halomethyl ketones, such as TLCK, Z-Phe-Phe-
CH2C1, Z-Phe-Phe-CH 2F , Z-Phe-Ala-CH 2C1, Z-Phe-Ala-CH 2F , Z-Leu-
Tyr-CH 2F , Z-Ala-Phe-CH 2F , Z-Leu-Leu-Tyr-CH 2F , Z-Tyr-Ala-CH 2F , ZLeu-
Leu-Phe-CH 2C1, Pro-Phe-Arg-CH 2Cl, Leu-Leu-Phe-CH 2C1, Leu-Leu-
Lys-CH 2C1, Ala-Phe-Lys-CH 2C1, Ala-Phe-Lys-CH 2F , Z-Leu-Gly-CH 2C1,
Z-Leu-Gly-CH 2Br, Z-Leu-Ala-CH 2C1 and Z-Leu-Phe-CH 2C1; peptidyldiazomethanes,
such as Z-Phe-Ala-CHN 2, Z-Phe-Thr (OBzl )-CHN 2 ZAla-
Ala-Pro-CHN 2, Z-Gly-Pro-CHN 2, Z-Lys-CHN 2, Z-Ala-Phe-Ala-CHN 2,
Z-Pyrod-Glu-CHN 2, Pyro-CHN 2, Z-Leu-Val-Gly-CHN 2, Z-Phe-Tyr (O-t-
But)-CHN 2, Ser (OBzl) -CHN 2, Gly-Phe-CHN 2, Z-Leu-Leu-Tyr-CHN 2 and
Z-Tyr (I)-Ala-CHN 2; pept idylacyloxymethyl ketones, such as Z-Phe-
Ala-CH 2-0-CO- (2, 6- (CF3)2)-Ph, Z-Phe-Ala-CH 2-0-CO- (2,5- (CF3)2)-Ph,
Z-Phe-Ala-CH 2-0-CO- (2, 6-Me 2-4-COOMe) -Ph, Z-Phe-Ala-CH 2-0-CO-4-
N02-Ph, Z-Phe-Ala-CH 2-0-CO- (2, , 6-Me 3)-Ph, Z-Phe-Ala-CH 2-0-COCMe
3, Z-Phe-Ala-CH 2-0-C 6F5, Z-Phe-Lys-CH 2-0-CO- (2 ,6- (CF3)2)-Ph, ZPhe-
Lys-CH 2-0-CO- (2,4, 6-Me 3)-Ph, Z-Phe-Ser (OBzl) -CH2-0-CO- (2, 6-
(CF3)2)-Ph, Z-Phe-Cys (SBzl) -CH2-0-CO- (2, 6- (CF3)2)-Ph, Z-Leu-Leu-
CH2-0-CO- (2, 6- (CF3)2)-Ph, Z-Val-Phe-CH 2-0-CO- (2, 6- (CF3)2)-Ph, ZLeu-
Leu-Phe-CH 2-0-CO- (2, 6- (CF3)2)-Ph, Z-Asp-CH 2-0-CO- (2, 6-Cl 2-
Ph) , Z-Val-Asp-CH 2-0-CO- (2, 6-Cl 2-Ph) , Z-Val-Ala-Asp-CH 2-0-CO-
(2,6-Cl 2-Ph) and Z-Glu-CH 2-0-CO- (2 ,6-Cl 2-Ph) ; peptidylmethlysulf
onium salts, such as (CH3)3-S+, Z-Phe-CH 2-S+- (Me) 2, ZPhe-
CH 2-S+- (Me) (Bzl) , Z-Phe-CH 2-S+- (Et )2 Z-Phe-Ala-CH 2-S+- (Me) 2,
Z-Phe-Lys-CH 2-S - (Me) 2, Z-Phe-Lys-CH 2-S - (Me) (Bzl) , Z-Lys-CH 2-S -
(Me) 2, Ala-Lys-Lys-CH 2-S+- (Me) 2 , Ala-Lys-Arg-CH 2-S+- (Me) 2, Ala-
Arg-Lys-CH 2-S+- (Me) 2, Ala-Arg-Arg-CH 2-S+- (Me) 2, Bz-Phe-Arg-CH 2-S+-
(Me) 2 and Z-Leu-Leu-Phe-CH 2-S+- (Me) 2 eposuccinyl peptides, such
as E-64, E-64 D , Ep 459 (E-64a; HO-Eps-Leu-NH- (CH2) -NH 2), Ep 460
(E-64aZ; HO-Eps-Leu-NH- (CH2)4-NH-Z) , Ep 459-Ac, Ep 479 (HO-Eps-
Leu-NH- (CH2)7-NH2 ) , Ep 174 (HO-Eps-Leu) , Eps (Eps=2 (S),3 (S)-
transepoxysuccinate) LeuOBzlb, Ep 47 LL, (E-64c) LD, DL, DD, Ep
420 (Bzl-DL-Eps-Ile-TyrOMe) , Ep 429 (E-64b; HO-Eps-Leu-Leu) ,
EpsLeuProOBzl, EtO-EpsLeuProOBzl , i-ButNH-EpsLeuProOBzl ,
EpsLeuPro, Et-EpsLeuPro, i-ButNH-EpsLeuPro, EpsPheOBzl, EpsArgOBzl,
EpsIleOBzl, EpsPheNBzl, EpsLeuNBzl, E-64, E-64c, E-64d,
CA-074 (nPr-NH-Eps-Ile-Pro) , CA-028 (HO-Eps-Ile-Pro) , CA-030
(Et-OEps-Ile-Pro) , CA-030-OMe, EtO-Eps-Pro-Pro, EtO-Eps-Thr-Ile,
EtO-Eps-Ile-Ala, EtO-Eps-Gly-Pro, HO-Eps-Leu-OEt , HO-Eps-Leu-
OBzl, Bzl-O-Eps-Leu-OEt, Ho-Eps-Ile-OBzl, HO-Eps-Phe-OEt , HOEps-
Orn (Z) -OEt, HO-Eps-Arg (N02)-OMe, HO-Eps-Arg-OMe, HO-Eps-Arg
and HO-Eps-Lys (Z)-OBzl ; unsaturated derivatives using the M i
chael system, such as DC-11, Fum (Fum=EtOOCCH=CH-CO trans) -Phe-
OBzl Fum-Ile-NH-nBu, Fum-Ile-Pro-OBzl, N-ethylmaleimide, N -
butylmaleimide, N-hexylmaleimide, N-decylmaleimide, Ac-Phe-NHCH2-
CH=CH-COOMe, Ac-Phe-NH-CH2-CH=CH, Gly-NH-CH (Bzl )-X
(X=CH=CHCOOMe trans), Gly-NH-CH (Bzl )-Y (Y= CH=CHS0 2Me trans),
Ac-NH-CH (Bzl) -X, Ac-NH-CH (Bzl )-Y, NH2-CH (Bzl) -X, NH2-CH (Bzl) -Y
and Mu (morpholine urea) -Phe-Hphe (homophenylalanine)
VsPh (vinylsulf onyl )benzene CH=CHS0 2Phe ) ; disulfides, azapeptides,
azobenzenes, O-acylhydroxamates , lysosomotropic bases,
such as ammonium chloride, methylamine, tributylamine, nigericin,
gramicidin and chloroquine ; calmodulin antagonists, aziridines
and thiiranes, 1 , 3 ,2-dioxathiolane dioxide derivatives,
Ac-Gly-Phe-Nle-OH, Ac-Gly-Phe-Nle-OS0 2-CH 3, aspartyl R-[(lphenyl-
3- (trif luoromethyl) pyrazol-5-yl] -oxy] methyl ketones and
aspartyl R- [(diphenylphosphinyl )oxy ]-methyl ketones.
A further group of preferred cysteine protease inhibitors
is the group 1-Naphthalenesulf onyl-Ile-Trp-CHO (1), or
[(3S) -3- [[(2S) -4-me thy 1-2- (10H-phenothiazine-2-carbonylamino) pen
tanoyl] amino] oxolan-2-yl] (BN 82270 (and BN 82204)) (2), Z-Phe-
Tyr (OtBu) -COCHO · H20 (3), tert-butyl N - [(2S) -1- [2- [2- (2-
ethylanilino) -2- oxoethyl] sulf anylcarbonylhydrazinyl ]-3- (1Hindol-
3-yl) -l-oxopropan-2-yl ]carbamate and N_-[(S)-2-
tertbutoxycarbonylamino-3- (lH-indol-3-yl) -propionyl] -hydrazine
carboxylic acid 2- (3, 4-dihydro-2H-quinolin-l-yl) -2-oxo-ethyl es
ter (4), N-Acetyl-L-leucyl-L-leucyl-L-methional (5), (2S,3S)-
oxirane-2 ,3-dicarboxylic acid 2- [((S) -l-benzylcarbamoyl-2-
phenyl-ethyl )-amide] 3-{ [2- (4-hydroxy-phenyl) -ethyl] -amide} (6) ,
[N- (4-Biphenylacetyl) -S-methylcysteine- (D) -Arg-Phe- b-
phenethylamide (7), 1-Naphthalenesulf onyl-Ile-Trp-CHO (8), ZPhe-
Tyr (t-Bu) -diazomethylketone (9) and acetyl-leu-leunorleucinol
(10) . This group is characterised by a strong inhib
itory capacity on cysteine proteases (IC5o = 1.9 mM (1); calpain
and cell death inhibition in C 6 glial cells (IC50) of 13.34 and
15.5 mM (2); Ki = 600, exhibits over 360-fold greater selectivi
ty for cathepsin L compared to cathepsin B (Ki = 214 nM) (3) ;
IC50 of 1.0 nM and 0,4 nM (4); very potent inhibitor of cathep
sin L (K = 0.6 nM) and the strongest inhibitor of cathepsin B
(K = 100 nM) (5) ; inhibits rat liver cathepsin L with IC 50 val
ues of 1.9 nM (6); inhibitor of human recombinant cathepsin-L
(Ki = 19 nM) (7); inhibitor of cathepsin L (IC50 = 1.9 nM) (8);
about 10.000-fold more effective against cathepsin L than ca
thepsin S (9) ).
Besides the inhibitors mentioned above, the present inven
tion can also apply structural alternatives (variants) of the
substances mentioned above, e.g. derivates, conjugates and iso
mers of such substances which preserve the general structure and
the general inhibitory capacity of the molecule, but slightly
deviates in structure. For example, (1) and suitable alterna
tives for (1) are disclosed in WO 1996/016079 A2 and EP 0 611
756 A2 (especially the compounds of and claims 1 and 14 of the
EP 0 611 756 B l document); (2) and suitable alternatives for (2)
(BN 82270 and BN 82204) are disclosed in Auvin et al ., Bioorg.
Med. Chem. Lett. 14 (2004), 3825-3828; (3) and suitable alterna
tives for (3) are disclosed in Lynas et al ., Bioorg. Med. Chem.
Lett. 10 (2000), 1771-1773; (4) and suitable alternatives for
(4) are disclosed in WO 2009/136997 A2) .
Administration amounts are typically dependent on the indi
vidual inhibitory capacity of each inhibitor and its toxicity.
Preferred administration dosages for the inhibitors are in the
mg range, such as 1 to 1000 mg/kg bodyweight /day, preferably 5
to 500 mg/kg bodyweight /day, especially 10 to 100 mg/kg bodyweight
/day .
Cysteine protease inhibitors have been specifically effec
tive in the present invention, especially for combatting T . gallinae
pathogenicity in preferred avian species. This has been
confirmed within the course of the present invention by using
chicken embryos, as a disease model. Accordingly, it is pre
ferred to use the protease inhibitors according to the present
invention in the treatment of a T . gallinae infection of an an
imal, selected from a species belonging to the orders Galliformes,
Columbif ormes , Psittacif ormes or Passerif ormes , espe
cially Columba livia, Melopsittacus undulates, Serinus canaria
forma domestica, or Falco spp .. |
