DE102004011988A1 - Cyclosporin derivatives and pharmaceutical compositions containing them - Google Patents

Abstract

The present invention relates to novel cyclic peptides of the cyclosporin series, whose ability to inhibit the protein phosphatase calcineurin can be switched and thereby act immunosuppressive. Unexpectedly, it has been found that by modifying the hydroxyl group of a [D-Ser] .8.-cyclosporin by means of suitable activatable substituents, it is possible to eliminate the immunosuppressive effect of such cyclosporin derivatives. Since therapeutically used doses of cyclosporin A have many undesirable side effects, the value of the present invention is that the immunosuppressive activity of the provided cyclosporin derivatives can be switched off. It is also possible to use the non-immunosuppressive cyclosporin derivative in a mixture with the parent substance or exclusively therapeutically. In the case of therapeutic use of switchable cyclosporin derivatives according to the invention, their effect during the therapy can be influenced.

Description

background the invention

pathobiochemical changes of the immune system are a large group of autoimmune diseases and chronic inflammation underlying, such as the following diseases .: Systemic lupus erythematosus (SLE), Chronic Rheumatoid Arthritis, Type 1 Diabetes, Chronic inflammatory Intestinal diseases, biliary cirrhosis, uveitis, multiple sclerosis and other diseases such as Crohn's disease, ulcerative colitis, bullous pemphigoid, Sarcosidosis, Psoriasis, Atopic Dermatitis, Ichthyosis or Graves Opthalmopathy. Although the underlying pathogenesis respectively may be different, is the common biochemical feature these diseases the occurrence of autoantibodies or autoaggressive Lymphocytes, which cause inflammation and finally destruction lead the affected cells. In the same way it can after bone marrow or organ transplantation for the formation of antibodies, or against the graft-targeted immune cells come what ultimately leads to graft rejection. Like many publications (e.g., Altschuh D. Journal of Molecular Recognition 15 (5): 277-285, 2002; Griffiths B. and Emery P. Lupus. 10 (3): 165-170, 2001; Nasonov EL. et al. Terapevticheskii Arkhiv. 72 (5): 67-71, 2000; Winkler M. Biodrugs. 14 (3): 185-193, 2000) and patents (e.g., US 491488; US 5948755) graft rejections therapeutically by administering cyclosporins. Another relatively well-researched indication of certain cyclosporins, such as. Cyclosporin A (CsA) has the observed inhibitory effect against Human Immunodeficiency Virus (HIV) (such as: Zavyalov V. P. et al. APMIS. 103 (6): 401-415, 1995). Investigations on corticosteroid-dependent asthma patients show that here the lung function can be improved by CsA as a therapeutic agent (e.g., Powell N. et al., Journal of Allergy & Clinical Immunology, 108 (6): 915-917, 2001; Eckstein J.W. and Fung J. Expert Opinion on Investigational Drugs. 12 (4): 647-653, 2003). The best known side effect of CsA is its nephrotoxicity and beyond Side effects such as abnormal liver function, hirsutism, gum hypertrophy, Tremor, hyperesthesia and gastrointestinal disorders and Increase in incidence for different tumors (e.g., Herman M. Journal of Laboratory & Clinical Medicine 137 (1): 14-20, 2001; Landewe R.B.M. et al. Nature Medicine 5 (7): 714, 1999).

cyclosporine are a family of immunosuppressant compounds which are by fermentation of various fungi, e.g. Tolypocladium inflatum to win (Lexikon der Biochemie, Spektrumverlag, Germany). Generally speaking, the class of cyclosporins can be called cyclic peptides with 11 amino acids (formula I).

The compound cyclosporin A (CsA) R ^{1 is} methyl (4R) -4 - [(E) -2-butenyl] -4-methyl-L-threonine, R ^{2 is} α-aminobutyric acid, R ^{3 is} N- Methyl glycine, R ⁴ , R ⁶ , R ⁹ and R ¹⁰ for N-methyl-L-leucine, R ⁵ for L-valine, R ¹ for L-alanine, R ⁸ for D-alanine and R ¹¹ for N- methyl-L-valine.

The biological activity of CsA is manifold. CsA, for example, inhibits the activity of the cyclophilin-type enzyme peptidyl-prolyl cis / trans isomerase (PPlase), as shown in Example 1a. These proteins, also referred to as folding helper enzymes or peptidyl-prolyl cis / trans isomerases, hereinafter referred to as PPlases, are prepared according to the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology ("Enzyme Nomenclature", Academic Press, 1992 ) under EC number 5.2.1.8. These enzymes can catalyze the cis / trans isomerization of peptide bonds in peptides and proteins (Rail-Fischer C. et al., Biological Chemistry, 383 (12): 1865-1873, 2002; Tradler T. et al., FEBS Letters. 2): 184-190, 1997; Ivery MTG Medicinal Research Reviews 20 (6): 452-484, 2000. It has been demonstrated in numerous publications that this catalysis performs a switch function in both protein folding and cellular signal transduction Of the various members of these PPlases, at least 12 genetically distinct cyclophilins are encoded in the human genome, the interaction with cyclophilin 18 (Cyp18) is believed to be of particular importance for immunosuppression, with the binary CsA / Cyp18 complex inhibiting the protein phosphatase calcineurin (Liu et al .: Cell 66 (1991) 807), as also summarized in Example 1b The inhibition of this phosphatase in lymphocytes ultimately leads, as a result of the e show numerous publications (eg: Ho et al .: Clin. Immune. and Immunopathology 80 (1996) 40), for immunosuppression. The determination of this effect is shown in Example 2. The interaction of CsA to Cyp18 occurs via residues 9, 10, 11, 1, 2 of CsA, which are also referred to as "binding domain." In contrast, calcineurin binds to CsA via residues 4, 5, 6, 7, 8 of CsA designated as "effector domain". Another pharmacologically important effect of CsA is the sensitization of cancer cells to chemotherapeutics, such as vincistin (VCR) or daunorubicin (DNR). For example, a phenomenon known as "multi drug resistance" (MDR) has long been reported in the literature (eg, Duraj J. et al, Anticancer Research, 22 (6A): 3425-8, 2002, Chorvath B. et al., International Journal of Cancer 72 (5): 916-7, 1997, Li G. et al., Chinese Journal of Oncology, 24 (4): 370-4, 2002, Suarez L. et al., Haematologica, 86 (12): 1287-95, 2001) that tumor cells can become resistant to initially effective therapeutics.This undesirable resistance can be favorably influenced by therapeutic administration of CsA has been demonstrated in several studies, including: Yamamoto M et al., Anti-Cancer Drugs. 6 (4): 570-7, 1995, Slater L. et al., Proc., Assoc. Cancer Res 27 (1982) 392, Pallis M. et al., Leukemia & Lymphoma, 43 (6): 1221-8, 2002; Jachez B. and Loor F. Anti-Cancer Drugs 4 (6): 605-15, 1993; Malingre MM et al., British Journal of Cancer. 84 (1): 42-7, 2001.

Maybe over one Inhibition of allergen-induced proliferation and IL-5 production special white Blood cells (peripheral mononuclear Blood Cells (PBMC)) causes CsA to improve lung function in asthmatics (Powell N. et al., Journal of Allergy & Clinical Immunology. 108 (6): 915-917, 2001). Also, the protection of the lungs by CsA-gifts against hypoxic damage is used therapeutically, for example: Matthew et al. American Journal of Physiology - lung Cellular & Molecular Physiology. 20 (5): L786-L795, 1999). Such effects could be found in different studies on larger patient populations be secured (so for example: Lock S.H. et al American Journal of Respiratory & Critical Care Medicine. 153 (2): 509-514, 1996).

A Another important pharmacological effect of CsA is its strong Inhibitory activity against retroviruses, especially against the human immunodeficiency virus (HIV). This effect was proven by different authors become, so for example: Franke EK. and Luban J. Virology. 222 (1): 279-82, 1996; Drewe J. et al. Biochemical Pharmacology. 57 (10): 1147-52, 1999; Tour L. et al. Journal of the American Academy of Dermatology. 37 (3 Pt 1): 501-2, 199; Sapphire AC et al. Immunologic Research. 21 (2-3): 211-7, 2000 or has been deposited in patents, e.g., WO97 / 04005; US6270957; US5948755. While CsA as an immunosuppressant in a wide variety of formulations currently being used worldwide, its use is for treatment the MDR or HIV infections due to the high levels needed Doses and associated side effects not possible. Indeed Some work suggests that non-immunosuppressive Cyclosporins, although still inhibitors of PPlase activity, both used to treat HIV infections (Bartz et al., 1995), Proc. Natl. Acad Sci USA 92: 5381, Rosenwirth et al. (1994) Antimicrobial Agents and Chemotherapy 38: 1763; US6270957) as well as the MDR (Lopez et al., Leukemia Research 27 (5): 413-423, 2003; Atadja P. et al. Cancer & Metastasis Reviews 17 (2): 163-168, 1998; Naito M. Tsuruo T. Cancer Chemotherapy & Pharmacology. 40 (Suppl S): S 20-S 24, 1997) are suitable, without for CsA observed to show side effects.

As Numerous examination findings prove (Pong K. and Zaleska MM. Current Drug Targets - Cns & Neurological Disorders. 2 (6): 349-56, 2003; Gold BG. and Villafranca JE. Current Topics in Medicinal Chemistry. 3 (12): 1368-75, 2003; Udina E. and Navarro X. Neurologia. 17 (4): 200-13, 2002 ) CsA also has neuroprotective or regenerative properties in various neurological indications, e.g. in Alzheimer's, Amyotrophic lateral sclerosis, or Parkinson's.

Farther In particular, the antimicrobial (High KP. Transplantation. 57 (12): 1689-700, 1994) and anti-parasitic (e.g., Bell A. et al., General Pharmacology 27 (6): 963-71, 1996; Chappell LH et al. Parasitology. 105 Suppl: S25-40, 1992) Properties in of animal and human medicine.

Non-immunosuppressive cyclosporine

In order to selectively utilize the differential effects of CsA either as an inhibitor of PPlase activity of cyclophilins or the immunosuppressive property leading to inhibition of calcineurin activity, numerous changes have been made to cyclic peptides of the cyclosporin series. In particular, changes to the "effector domain" have made it possible to create cyclosporin derivatives which are non-immunosuppressive and no longer inhibit calcineurin. [Allgemein Allgemein] Generally, non-immunosuppressive cyclic peptides of the cyclosporin series are claimed in, inter alia, US4914188, EP484281 or WO9704005 Substances such as (gamma-hydroxy-N-methylleucine) -cyclosporin, melle-4-cyclosporin or McVal-4-cyclosporin and McAla-6 or McAbu-6-cyclosporin have virtually no immunosuppressive activity, although these substances are the PPlase Inhibit activity of Cyp18, they no longer have a nephrotoxic effect (US4914188) .Non-surprisingly, these non-immunosuppressants with simultaneous administration of immunosuppressive CsA to relieve its nephrotoxic property without impairing its immunosuppressive properties, as described in the patent US4914188 and in the literature, such as Lazarova T et al .: J.Med.Chem. 46 (3003). 674 is listed. A possible compensation mechanism of these non-immunosuppressant compounds is shown in Example 2.

The The object underlying the present application is the provision new cyclosporin derivatives immunosuppressive by inhibition of Protein phosphatase calcineurin act, but specifically manipulated can lose this property while retaining PPlase inhibition.

A Another object of the invention is the provision of pharmaceutical Compositions for the therapeutic use of the provided cyclosporin derivatives are suitable in medicine, as well as aids, with their Help the properties of the provided cyclosporin derivatives targeted in desired Way changed can be.

A Another object of the invention is the provision of methods with which the cyclosporin derivatives of the invention can be produced.

The solution These objects are the objects of the invention described in the claims.

According to the above-mentioned concept of an "effector domain", chemical modifications of the R ⁸ -position (N-methyl-glycine) still belonging to this domain should significantly influence the inhibition constants of the protein phosphatase calcineurin and thus the immunosuppressive properties of CsA surprisingly, even large volume changes, such as the introduction of a (nitroveratryl) oxycarbonyl radical, only marginally affect the inhibition of calcineurin (Compound 11, Table 1). Table 1. Cyp 18 and CaN inhibition of various CsA derivatives

Also the introduction strongly charged groups, such as the introduction of different phosphoryl-D-serines (Compound 3, 4, and 5), or the introduction of an O-methyl or O-carboxymethyl-D-serine (Compound 2 or 6) have little influence on the calcineurin inhibition. The same applies the extensive changes, which are listed in the structures of compounds 7 to 11 of Table 1. All the more surprising was that under the chosen one Test conditions for the compound Cs12 and its analogues not shown in the table, by feeder of suitable energy, such as: UV light of 330 nm (example 3,4) or by the action of an enzyme (Example 5) or by modification of the pH (Example 6) from a compound Cs10 or their Analogs can be produced, no inhibition of protein phosphatase Calzineurin was more detectable. This will be surprising with this Discovery of two different cyclosporin derivatives (Cs10 and Cs12) provided:

Cs10:

Compounds which correspond to the structure of Cs10 and which can significantly inactivate the PPlase activity of Cyp18, which is understood here to mean significantly an IC ₅₀ value of less than 10 μM, which furthermore possess the property of significantly inhibiting calcineurin IC ₅₀ value of less than 10 μM, and which have the property to be converted by suitable activation into a compound which corresponds to the structure of Cs12 or its analogues in Table 1, wherein the activation in photolabile groups by means of suitable light sources occurs in pH-sensitive groups by a sufficient pH change and catalytically sensitive groups by the effect the corresponding enzyme.

Cs12:

Compounds obtained from the structure Cs10 (Table 1) or its analogues by appropriate activation and corresponding to the structure Cs12 or its analogues, or produced by chemical synthesis and corresponding to the structure Cs12 or its analogues, all of which have the property have to significantly inactivate the PPlase activity of Cyp18, which is understood to mean significantly an IC ₅₀ value of less than 10 μM, and these substances furthermore have the property that they do not significantly inhibit calcineurin here by not significantly an IC ₅₀ value of greater than 10 μM is understood.

worin
R¹ für Methyl(4R)-4-[(E)-2-butenyl)-4-methyl-L-threonin (MeBMT) oder Dihydro-McBMT steht;
R² für α-Aminobuttersäure (Abu) oder ein an einer oder mehreren Positionen der Seitenkette fluoriertes Derivat dieser Aminosäure steht; wie z.B.

worin X₁ bis X₅ unabhängig voneinander für H oder F stehen, mit der Maßgabe, daß mindestens eins der X₁, X₂, X₃, X₄ and X₅ für F steht;
R³ für N-Methyl-glycin (Sar), N-Methyl-D-alanin oder ein fluoriertes Derivat davon steht;
R⁴, R⁶, R⁹ and R¹⁰ unabhängig voneinander für N-Methyl-L-leucin (MeLeu) oder ein fluoriertes Derivat davon stehen;
R⁵ für L-Valin (Val) oder ein fluoriertes Derivat davon steht; R⁷ für L-Ala oder ein fluoriertes Derivat davon steht, und
R¹¹ für L-MeVal oder ein fluoriertes Derivat davon steht.As shown in the examples (7, 8, 9), the substances of the type Cs10 and Cs12 and their analogs can be prepared in a variety of ways in larger quantities. The analogs A10 and A12 listed below correspond in their properties with respect to the inhibition with respect to Cyp18 or calcineurin to the substances Cs10 and Cs12 and may be as follows:

wherein
R ^{1 is} methyl (4R) -4 - [(E) -2-butenyl) -4-methyl-L-threonine (MeBMT) or dihydro-McBMT;
R ^{2 is} α-aminobutyric acid (Abu) or a derivative of this amino acid fluorinated at one or more positions on the side chain; such as

wherein X ₁ to X ₅ independently represent H or F, with the proviso that at least one of X ₁ , X ₂ , X ₃ , X ₄ and X _{5 is} F;
R ^{3 is} N-methyl-glycine (Sar), N-methyl-D-alanine or a fluorinated derivative thereof;
R ⁴ , R ⁶ , R ⁹ and R ¹⁰ independently represent N-methyl-L-leucine (MeLeu) or a fluorinated derivative thereof;
R ^{5 is} L-valine (Val) or a fluorinated derivative thereof; R ^{7 is} L-Ala or a fluorinated derivative thereof, and
R ^{11 is} L-MeVal or a fluorinated derivative thereof.

Another embodiment is cyclosporin analogs of formula (I) wherein R ^{1 is} McBmt or dihydro-MeBmt;
R ^{2 is} Abu;
R ^{3 is} Sar or D-MeAla;
R ⁴ , R ⁶ , R ⁹ and R ^{10 may} independently represent McLeu or a fluorinated analog;
R ^{5 is} Val or a fluorinated analog;
R ^{7 is} Ala; and
R ^{11 represents} McVal or a fluorinated analog.

Another embodiment is cyclosporin analogs of formula (I) wherein R ^{1 is} McBmt;
R ^{2 is} Abu;
R ^{3 is} sar;
R ⁴ , R ⁶ , R ⁹ and R ^{10 represent} McLeu;
R ^{5 is} Val;
R ^{1 is} Ala; and
R ¹¹ stands for McVal.

• (a)
  
  wobei X für O oder S steht, Y für H oder Methyl steht, m einen Wert zwischen 0 und 5 hat, n eine ganze Zahl zwischen 3 und 10 ist, Z für N-R¹²R¹³ oder NHC(O)OR¹⁴ steht, wobei R¹² für H, Methyl, Ethyl, n-Propyl, iso-Propyl oder eine photolysierbare Gruppe steht und R¹³ für H oder einen C1-C8-Alkylrest steht, und R¹⁴ für eine säure-/basenlabile oder eine enzymatisch spaltbare Gruppe steht; oder
• (b)
  
  wobei X für O, S oder CH₂ steht, Y für H oder Methyl steht, m einen Wert zwischen 0 und 5 hat, n eine ganze Zahl zwischen 3 und 10 ist, Z für N-R¹²R¹³ oder NHC(O)OR¹⁴ steht, wobei R¹² für H, Methyl, Ethyl, n-Propyl, iso-Propyl oder eine photolysierbare Gruppe steht, und R¹³ für H oder einen C1-C8-Alkylrest steht, und R¹⁴ für eine säure-/basenlabile oder eine enzymatisch spaltbare Gruppe steht.

In all the compounds listed above, R ⁸ stands for

• (A)
  
  wherein X is O or S, Y is H or methyl, m has a value between 0 and 5, n is an integer between 3 and 10, Z is NR ¹² R ¹³ or NHC (O) OR ¹⁴ , where R ^{12 is} H, methyl, ethyl, n-propyl, iso-propyl or a photolysable group and R ^{13 is} H or a C ¹ -C ⁸ -alkyl radical, and R ^{14 is} an acid / base labile or an enzymatically cleavable group ; or
• (B)
  
  where X is O, S or CH ₂ , Y is H or methyl, m has a value between 0 and 5, n is an integer between 3 and 10, Z is NR ¹² R ¹³ or NHC (O) OR ¹⁴ wherein R ^{12 is} H, methyl, ethyl, n-propyl, iso-propyl or a photolysable group, and R ^{13 is} H or a C ¹ -C ⁸ alkyl radical, and R ^{14 is} an acid / base labile or a enzymatically cleavable group.

The photolysable group may be an optionally substituted nitroaryl group. Examples of the optionally substituted nitroaryl group are 2-nitroveratryloxycarbonyl (NVOC), α-carboxy-2-nitrobenzyl (CNB), 1- (2-nitrophenyl) ethyl (NPE), 1- (4,5-dimethoxy-2-nitrophenyl) ethyl (DMNPE) or 5-carboxymethoxy-2-nitrobenzyl (CMNB). The acid / base labile or enzymatically cleavable group can be, for example, methyl, ethyl, methoxymethyl (MOM), -CH ₂ CH ₂ F, methylthiomethyl (MTM), β-glucuronide, D-glucopyranosyl, β-D-galactopyranosyl, tetra-O- Acetyl-D-glucopyranosyl, or tetra-O-acetyl-β-D-galactopyranosyl.

Preferred examples of compounds according to the invention are the [O- (NVOCNH (CH ₂ ) ₅ NHC (O) CH ₂ -) D-Ser] ⁸ CsA or the [O- (NH ₂ (CH ₂ ) ₅ NHC (O) CH ₂ - ) D-Ser] ⁸ CsA.

The A12 compounds of the invention may be prepared by conventional methods using [D-Ser] ⁸ or [D-Cys] ⁸ -CsA (where D-Ser or D-Cys may also be replaced by other suitable amino acids), CPG- Bromoacetate and a compound of the formula NH ₂ (CH ₂ ) _n NH-APG, where n is an integer between 3 and 10, APG is an amino-protecting group and CPG is a carboxyl-protecting group.

Likewise, the A10 type compounds of the present invention can be replaced by conventional methods using [D-Ser] ⁸ or [D-Cys] ⁸ CsA (where D-Ser or D-Cys may also be replaced by other suitable amino acids) CPG Bromoacetate and a compound selected from the group comprising NVOC, CNB, NPE, DMNPE and CMNB, where n is an integer between 3 and 10, APG is an amino-protecting group and CPG is a carboxyl-protecting group.

When Amino or carboxyl protecting groups can be used as customary protecting groups as they are known to those skilled in the art, e.g. from N. Sewald, H.-D. Jakubke, Peptides: Chemistry and Biology, published by Wiley-VCH. Examples of amino protecting groups are carbobenzoxy, Boc, Fmoc, Bpoc, NPS, triphenylmethyl or tosyl. examples for Carboxy protecting groups are methyl, ethyl, tert. Butyl, benzyl, nitrobenzyl or methoxybenzyl. Preference is given to tert. Butyl as carboxy-protecting group and Boc as amino protecting group.

Examples for the Preparation of the compounds of the invention are given in Examples 7, 8 and 9.

specificity change

• a) Photoschaltung: Durch Licht schaltbare chemische Gruppierungen sind seit längerem bekannt und Gegenstand zahlreicher Patentschriften so z.B. in US6392089, US6242258, US6160024, US5952311, US5767288, US6057096, US5786198, US5780287, EP1208079 oder US5503721 bzw. sind in Übersichtsarbeiten, wie z.B.: Pirrung, M.C. et al. J.Org.Chem 60 (1995) 1116; Gurney A.M. et al. Physiological Reviews 67 (1987) 583; Pillai V.N.R. Reviewspp 1–27 (1980); Cameron J.F. et al. J.Chem.Soc. Chem. Commun 923 (1995); Pirrung M.C. et al. J.Org.Chem 59 (1994) 3890; McCray J.A. et al. Annu.Rev.Biophys.Chem. 18 (1989) 239; Cummings R.T. et al. Tetrahedron Letters 29 (1988) 65; Mendel D. et al. J.Am. Chem. Soc. 113 (1991) 2578 oder Sheehan J.C. et al. J. Am. Chem. Soc 93 (1971) 7222 beispielgebend dargestellt. Ebenso wurden zahlreiche Verfahren dokumentiert, mit deren Hilfe die gewünschte Aktivierung durchgeführt werden kann, so z.B. in US6461567, US6524447, US6397102, US6433343, US5459322, US6258319, US5854967, US5683661 oder US5503721. So wird zum Beispiel zur Umwandlung von Substanz Cs10 in Substanz Cs12 vorzugsweise UV-Licht mit einer Wellenlänge von etwa 330 nm benutzt. Wahl der Lichtquelle und Dauer der Bestrahlung der Substanz um die gewünschte Umwandlung von A10 nach A12 zu erreichen, hängen naturgemäß neben der verwendeten photolabilen Gruppe und Stärke der Lichtquelle von weiteren Faktoren ab. Befindet sich A10 z.B. in Körperflüssigkeiten, wie z.B. Blut, werden Teile der Lichtenergie auch von absorbierenden Blutbestandteilen absorbiert werden. Beim Bestrahlen von Hautbereichen, sind wiederum andere störende Effekte, wie z.B. durch unterschiedliche Pigmentierung zu erwarten. Eine optimale Aktivierung ist aber leicht an der Bildungsgeschwindigkeit von A12 zu erkennen wie dies in Beispiel 3 dargestellt ist.
• b) Enzymatische Schaltung: Die gezielte Einführung von Substratanaloga in eine als Elternsubstanz bezeichnete Verbindung, aus der dann durch Wirkung unterschiedlicher Enzyme eine gewünschte Tochtersubstanz entsteht, ist dem Fachmann bekannt und in der Literatur umfassend dokumentiert (z.B.: Gum A.W. et al. Chem.Eur.J. 6 (2000) 3714; deBont D.B.A. et al. BioorganicMedicinal Chemistry 5 (1997) 405; Lazarova T. et al J.Med.Chem. 46 (2003) 674). So kann z.B. durch Einführung einer Estergruppierung die Aktivität von Hydrolasen/Esterasen, durch Einführung einer geeigneten sensitiven Peptidbindung die Aktivität von Proteasen oder bei Verwendung von Glucose- oder Galactosederivaten die Aktivität von Glycosidasen genutzt werden, um A10 in A12 zu verwandeln. Beispiel 5 zeigt die gezielte Hydrolyse einer solchen Muttersubstanz A10 am Beispiel einer Estergruppierung.
• c) pH-Schaltung: Die gezielte Einführung von Substratanaloga in eine als Elternsubstanz bezeichnete Verbindung, aus der dann durch Säure- oder Basenkatalyse eine gewünschte Tochtersubstanz entsteht, ist dem Fachmann bekannt und in der Literatur umfassend dokumentiert, wie z.B. in „Catalysis in Chemistry and Enzymology" von William P. Jencks. (1987). Geeignet für diese nichtenzymatische Katalyse sind chemische Bindungen, welche gegenüber Säuren – oder Basen, im einfachsten Fall gegenüber Protonen oder Hydroxylionen, empfindlich sind. Charakteristisches Kennzeichen solcher chemischer Bindungen ist, daß die Hydrolysegeschwindigkeit in einem direkten Zusammenhang zur Konzentration der katalytisch wirksamen Ionen steht. Beispiel 6 zeigt die Umwandlung eines Substanzanalogen der Verbindung A10 in die Verbindung A12 mittels erhöhter Hydroxylionenkonzentration.

The appropriate method for changing the specificity of a type A10 substance to an A12 type substance is determined by the activatable grouping. Such activatable groups and their suitable activation methods are known to the person skilled in the art and have been described in detail several times. Depending on the intended method, one or more daughter substances can be produced selectively by influencing these chemical functionalities from a parent substance, wherein the daughter substance according to the invention has a specificity changed with respect to the protein phosphatase calcineurin.

• a) Photo Switching: Light-switchable chemical groupings have been known for some time and are the subject of numerous patents such as in US6392089, US6242258, US6160024, US5952311, US5767288, US6057096, US5786198, US5780287, EP1208079 or US5503721 or are in reviews, such as: Pirrung, MC et al. J. Org. Chem 60 (1995) 1116; Gurney AM et al. Physiological Reviews 67 (1987) 583; Pillai VNR Review 1-27 (1980); Cameron JF et al. J. Chem. Chem. Commun 923 (1995); Pirrung MC et al. J. Org. Chem 59 (1994) 3890; McCray JA et al. Annu.Rev.Biophys.Chem. 18 (1989) 239; Cummings RT et al. Tetrahedron Letters 29 (1988) 65; Mendel D. et al. J. Am Chem. Soc. 113 (1991) 2578 or Sheehan JC et al. J. Am. Chem. Soc. 93 (1971) 7222 by way of example. Numerous methods have also been documented by means of which the desired activation can be carried out, for example in US6461567, US6524447, US6397102, US6433343, US5459322, US6258319, US5854967, US5683661 or US5503721. For example, for the conversion of Substance Cs10 in Substance Cs12, UV light having a wavelength of approximately 330 nm is preferably used. Choice of the light source and duration of irradiation of the substance in order to achieve the desired conversion of A10 to A12, of course, depending on the photolabile group used and strength of the light source depend on other factors. For example, if A10 is in body fluids such as blood, some of the light energy will also be absorbed by absorbing blood components. When irradiating skin areas, in turn, other disturbing effects, such as expected by different pigmentation. However, optimal activation is easily recognized by the rate of formation of A12 as shown in Example 3.
• b) Enzymatic Circuit: The targeted introduction of substrate analogues into a compound designated as the parent substance, from which a desired daughter substance is then produced by the action of different enzymes, is known to the person skilled in the art and comprehensively documented in the literature (for example: Gum AW et al., Chem J. 6 (2000) 3714; deBont DBA et al., Bioorganic Medicine Chemistry 5 (1997) 405; Lazarova T. et al J. Med. Chem. 46 (2003) 674). Thus, for example, by introducing an ester grouping, the activity of hydrolases / esterases, the introduction of a suitable sensitive peptide bond, the activity of proteases or, if glucose or galactose derivatives are used, the activity of glycosidases can be used to convert A10 into A12. Example 5 shows the specific hydrolysis of such a parent substance A10 using the example of an ester grouping.
• c) pH-switching: The targeted introduction of substrate analogues into a compound designated as the parent substance, from which a desired daughter substance is then formed by acid or base catalysis, is known to the person skilled in the art and comprehensively documented in the literature, as described, for example, in "Catalysis in Chemistry and Enzymology "by William P. Jencks (1987). Suitable for this nonenzymatic catalysis are chemical bonds which are sensitive to acids or bases, in the simplest case to protons or hydroxyl ions is a direct relationship to the concentration of the catalytically active ions Example 6 shows the conversion of a substance analog of the compound A10 into the compound A12 by means of increased hydroxyl ion concentration.

Place of activation:

In addition to the application for scientific purposes, where the activation and conversion of the substance A10 to A12 occurs while changing the specificity in a cuvette, a main area of application for medical purposes is to be seen. Here it may be advantageous to administer the substance A10 externally, nasally, orally, intracranially, parenterally, by means of a drip, catheter or similar aids, in order subsequently to locally activate the immunosuppressive property by converting the compound A10 into A12 in the desired tissue. To pick up blood cells. Irradiation can be carried out over the skin over a large area, as is customary, for example, for the treatment of elevated bilirubin (US Pat. No. 3,877,437, US Pat. No. 6,664,715, US Pat. No. 6,045,575) or by means of catheters, as described, for example, in US Pat. No. 5,503,721 or US Pat. However, it may also be advantageous to temporarily subject extracorporeal body fluids, cell material or entire organs to such an activation that only the substance constituents of the substance A10 present in these body fluids, cells or organs are converted into A12. Methods to temporarily extracorporeal liquid The clinical physician is well-known. Thus, for example, by means of plasmapheresis (eg US6099734) largely cell-free body fluid can be obtained. Or it can be made continuously available extracorporeally by means of the widely used dialysis techniques (eg: US5277820). Both methods have in common that body fluid is spent extracorporeally, here an activation is accessible and then fed back to the natural body fluid flow. But it can also be a discontinuous procedure of advantage. Thus, body fluids, organs, tissues or cells, such as bone marrow, can be harvested to make them or only components of these body fluids, organs, tissues or cells accessible for activation to eliminate the immunosuppressive properties of Cs10 by conversion into Cs12. Subsequently, these body fluids, organs, tissues or cells or components of these body fluids, organs, tissues or cells can be re-administered to another person using techniques known to those skilled in the art either from the person from whom the material was taken or, if beneficial that the benefits obtained by converting A10 to A12 will have a therapeutic benefit to the recipient.

Administration of the substances

substances which are summarized under A10 and A12, either each as a single substance or as a mixture in therapeutic applications and should continue to be called an effective agent. It can It may be beneficial to use A12 from A10 during drug activation therapy manufacture. In cases, where the immunosuppressive effect of A10 compounds is entirely undesirable, it may also be beneficial to forego the substance activation and to administer therapeutically A12 compounds. The Administerments can self be made orally, topically or parenterally in formulations. The formulations can additionally conventional non-toxic pharmaceutically acceptable carriers, Adjuvants and solvents contain. Under the term parenteral subcutaneous, intravenous, intramuscular, intracranial or intrasternal injections or infusion techniques Understood.

The Pharmaceutical compositions may contain the active ingredient (s) or optionally with A10 their precursor in one for the oral Use suitable form, e.g. as aerosol, tablets, Pastilles, candies, aqueous or oily suspensions, Powder or grains, Emulsions, hard or soft capsules, in syrup or elixirs.

aerosols, wherein aerosol is a dispersion of solid or liquid particles in gases, can be manufacture methods known in the art, in particular by Provision of patient-to-use vessels, which may contain the therapeutic and optionally other substances that Therapeutically applicable aerosol immediately before use is produced. Technical solutions, in particular for the production of aerosols, due to their physical condition of an effective droplet size well absorbed by the lungs can be are in big Scope has been described and applicable, such as: US06136295, US05985309, US06447753, US06447752, US06436443, US06399102, US06338809, USRE037053, US05855913. Applicable aerosols for the therapeutic use of cyclosporins are also multiple the subject matter of patents (such as: US06241969, US05958378, US Pat. US06413547, US05693336, US05683714).

compositions the for are intended for oral use, can be known by those skilled in the art Establish methods. Such compositions may contain other substances, which enable a pharmaceutically elegant production and continue for cause positive acceptance in the patient, e.g. one or several substances selected from the group of sweeteners, flavorings or the Dyes selected can be.

tablets, which the active agent as an admixture with non-toxic pharmaceutical contain acceptable substances, can be determined by a person skilled in the art produce known methods. As excipients or as for the production of tablets important resources can different substances are used. Thus, as (a) inert substance e.g. Calcium carbonate, lactose, calcium phosphate or sodium phosphate; as (b) granulating agent e.g. Starch or alginic acid; when (c) proppant e.g. Gelatin, starch or gum arabic, and as (d) dispersing agent e.g. magnesium stearate, stearic acid or talc. The tablets themselves may be unprotected or provided with an outer protective cover be to delay the residence time in the digestive tract and thus a longer time To achieve effect. Materials that influence the residence time, are e.g. Glycerol monostearate or glyceryl distearate. By means of known Techniques (e.g. US4256108, US4160452, US4265874) can also be used Make tablets that temporally control the active agent or based on the digestive tract in defined areas release the tablet.

In some cases It may be beneficial to use formulations for oral use in the Form of gelatin capsules in which the active agent with one or more several other inert substances mixed. Such substances can e.g. Calcium carbonate, calcium phosphate or kaolin. The gelatine capsules can but also, in addition to the active agent, water or oils, e.g. Nut oil, olive oil or too paraffin oil contain.

Aqueous suspensions usually contain the active agent in mixtures with admixtures, which are necessary for their preparation are such as (a) suspending agents such as sodium carboxymethyl cellulose, Methylcellulose, hydroxypropylmethylcellulose, sodium algenate, polyvinylpyrrolidone, gum tragacanth and gum arabic; (b) dispersing or wetting agent Fabrics, like natural occurring phosphatides such as e.g. lecithin; (c) condensation products an alkylene oxide with a fatty acid, e.g. polyoxyethylene; (d) condensation products of an ethylene oxide with a long chain aliphatic alcohol, e.g. Heptadekaethyleneoxycetanol; (E) Condensation products of an ethylene oxide with a partial ester, that of a fatty acid and a hexitol, e.g. Polyoxyethylene sorbitol monooleate, or (f) Condensation products of an ethylene oxide with a partial ester, which of a fatty acid and a hexitol anhydride, e.g. Polyoxyethylensorbitnamonooleat is formed.

The aqueous suspensions can also one or more preservatives, e.g. Ethyl or N-propyl-p-hydroxybenzoate; one or more dyes, one or more flavorings and one or more flavorings such as. Contain sucrose or saccharin.

Oily suspensions can by putting the active agent in a vegetable oil or mixtures these oils, such as. arachis, Olive oil, Sesame oil, Nut oil or else in a mineral oil, such as. liquid Paraffin, to be obtained. The oily Suspension may be a thickening agent, such as e.g. Beeswax, paraffin or cetyl alcohol. additionally can Sweetener, Flavorings or dyes may be added to a good acceptance reach the patient. To extend the shelf life of Composition can Antioxidants such as e.g. Ascorbic acid added. As well can the addition of dispersing powders of different particle size for the production the aqueous solution advantageous be. These accessories allow one relatively homogeneous and stable mixture together with all others mentioned above advantageous additives.

The Pharmaceutical composition may also be in the form of an oil-in-water emulsion. there can the oily Phase a vegetable oil, such as. olive oil or arachis oil, or a mineral oil, such as. liquid Paraffin or a mixture of these oils contain. Emulsionsvermittler- or stabilizing additives can to be there: (1) of course occurring gums, e.g. Gum arabic or gum tragacanth, (2) Naturally occurring phosphatides, e.g. from soybean or lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides have been derived, such as Sorbitol monooleate, (4) condensation products the above mentioned Partial ester with ethylene oxide, e.g. Polyoxyethylene monooleate. The emulsions themselves can again additives, like sweeteners, Flavorings or dyes included.

syrups and elixirs can with sweeteners, such as. Glycerol, propylene glycol, sorbitol or sucrose be. These formulations can additionally other substances, such as a demulsifier, an emulsifier, a preservative, a flavoring agent containing a dye or more of these additives.

The pharmaceutical composition can also take the form of a sterile injectable aqueous or oily suspension are located. Such suspensions can be prepared by methods known to those skilled in the art by using dispersing, emulsifying or wetting Additions, as listed above were, manufacture. The sterile injectable composition can also in the form of a sterile injectable solution or suspension with a non-toxic parenteral-compatible solvent or diluents, such as. 1,3-butanediol consist. The acceptable additives include, in particular, water, Ringer's solution and isotonic sodium chloride solution. Also sterile non-volatile Generally used oils may be used as a solvent or as an additive to the suspension be used, including synthetic Mono or diglycerides. additionally can fatty acids, such as. Oleic acid in these be included in injectable preparations.

An active agent of the formula 1 can also be present in a suppository which is customary for rectal or intravaginal use. Such suppositories customary for rectal use can be prepared by methods known to the person skilled in the art and using known substances and additives. In this case, the active agent with a conventional, non-irritating excipient, which is characterized in that it is solid at room temperature and liquid at body temperature and therefore melts in the rectum and so the active agent can release, mixed. Common excipients for making rectal suppository are z. As cocoa butter and various polyethylene glycols.

For external Applications, can e.g. Creams, ointments, foams, Lotions or suspensions containing the active agent used become.

The Amount of active agent associated with one or more of the above listed support materials together is going to be one of the above Making application forms can be very different. The Quantity hangs depending on the type of application as well as the application target. So can e.g. a formulation for oral intake of 5 mg up to 1 g of the containing active agent, wherein the additives listed above between 5 and 95% of the total amount. It can also be partial be necessary, considerable use smaller amounts of the active agent or quantities, which exceed 1 g. Preferably, dosage forms may between 25 and 500 mg of the active agent. It lies however in the nature of medical applications, that specific dose levels of the active Agent depends both on the type of agent itself but also on the individual Properties of the patient to be treated, e.g. Age, body weight, general health, sex, diet, time and nature of Administration, rate of elimination, eventual Combination with other drugs, the type of therapy and the specific Illness.

The Figures show:

1

1 shows the dependence of calcineurin activity on the concentration of Cyp18 / inhibitor complex. The experimental procedure is described in Example 1.

2

2 describes the competition of calcineurin inhibition by CsA and is explained in more detail in Example 2.

3

3 shows the influence of photoswitching and different concentrations of cyclosporin derivatives on transfected stimulated Jurkat cells using a luciferase assay. The experimental procedure is described in Examples 4 and 5.

4

4 shows the photoconversion of Cs10 in Cs12 by means of HPLC separations and is explained in more detail in Example 4.

5

5a describes the influence of Cs12 on the proliferation of stimulated blood cells compared to CsA. 5b shows the influence of Cs12 on cytokine production in stimulated blood cells compared to CsA. The experimental procedure is described in Examples 3, 5, 11 and 12.

The explain the following examples the present invention, without this to the examples described limit.

Example 1: enzyme activity assays

• a) Der PPlase-aktivitätsassay wurde nach Fischer et al. Biomed. Biochim Acta 43 (1984) 1101 durchgeführt, mit Suc-AFPF-NHNp als Substrat und alpha-Chymotrypsin (Merck, Darmstdt) als Hilfsprotease in 30 mM HEPES-Puffer bei pH 7,8 und 7°C. Nach 10 min Vorinkubation einer gewünschten Konzentration des Inhibitors mit 2.5 nM Cyp18, wurde die Reaktion durch aufeinanderfolgende Zugabe von Substrat und Chymotrypsin gestartet und bei 390 nm mit einem Spektralphotometer (HP 8452) die Absorption gemessen. Die IC₅₀-Werte konnten durch Auftragen der nach einer Geschwindigkeitsgleichung 1. Ordnung berechneten Geschwindigkeitskonstanten gegen die Inhibitorkonzentration berechnet werden.
• b) Die Aktivität der Proteinphosphatase Calzineurin wurde nach Baumgraß et al. J.Biol.Chem. 276 (2002) 47914 mittels biotinyliertem RII-Peptid bestimmt. Dazu wurde das RII-Peptid mit PKA und gamma ³³P-ATP phosphoryliert und nachfolgend mittels einer 1 ml RP-C2 Extraktionssäule (Amchro, Sulzbach) bis zur chromatografischen Einheitlichkeit (HPLC, 0 zu 100 Azetonitril-Gradient) gereinigt. Das so hergestellte Peptid (PRII) wurde im „Scintillation Proximity"-Assay verwendet. Im einzelnen wurden zusammen mit geeigneten Konzentrationen an Inhibitor eine Mischung aus Calmodulin (50 nM) und Calzineurin (1.32 nM) in Assay-Puffer (40 mM Tris/HCl, pH 7.5, 100 mM NaCl, 6 mM MgCl₂, 0.5 mM DTT, 1 mM CaCl₂, 0.1 mg/ml BSA) für 30 min bei 30°C inkubiert. Danach wurde PRII bei einer Endkonzentraion von 100 nM bei einem Endvolumen von 100 μl für weitere 30 min bei 30°C inkubiert. Danach wurden 90 μl abgenommen und in kommerziellen mit Streptavidin belegten Titerplatten (ScintiStrip) inkubiert. Nach 20 min Inkubation bei 22°C wurden die Platten gewaschen und das nichtumgesetzte PRII mittels MicroBeta-Counter (Wallac, Turku, Finnland) bestimmt. 1 zeigt eine typische Abhängigkeit der CaN-Aktivität von der Inhibitorkonzentration.

For the classification of the CsA derivatives, the inhibition of the PPlase activity of the commercially available human PPlase Cyp18 (Sigma: C3805) and the phosphatase activity of the commercially available calcineurin (Sigma: C1907) was used.

• a) The PPlase activity assay was performed according to Fischer et al. Biomed. Biochim Acta 43 (1984) 1101, with Suc-AFPF-NHNp as substrate and alpha-chymotrypsin (Merck, Darmstdt) as auxiliary protease in 30 mM HEPES buffer at pH 7.8 and 7 ° C. After preincubation of a desired concentration of the inhibitor with 2.5 nM Cyp18 for 10 min, the reaction was started by successive addition of substrate and chymotrypsin and the absorption was measured at 390 nm using a spectrophotometer (HP 8452). The IC ₅₀ values could be calculated by plotting the rate constants calculated from a first-order rate equation versus the inhibitor concentration.
• b) The activity of the protein phosphatase calcineurin was determined according to Baumgraß et al. J. Biol. 276 (2002) 47914 using biotinylated RII peptide. For this purpose, the RII peptide was phosphorylated with PKA and gamma ³³ P-ATP and subsequently purified by means of a 1 ml RP-C2 extraction column (Amchro, Sulzbach) until chromatographic homogeneity (HPLC, 0 to 100 acetonitrile gradient). The peptide thus prepared (PRII) was used in the Scintillation Proximity Assay Specifically, together with appropriate concentrations of inhibitor, a mixture of calmodulin (50 nM) and calcineurin (1.32 nM) in assay buffer (40 mM Tris / HCl , pH 7.5, 100 mM NaCl, 6 mM MgCl ₂ , 0.5 mM DTT, 1 mM CaCl ₂ , 0.1 mg / ml BSA) for 30 min at 30 ° C. Thereafter, PRII was incubated at a final concentration of 100 nM at a final volume of 100 μl was incubated for a further 30 min at 30 ° C. Thereafter, 90 μl were removed and incubated in commercial streptavidin-coated titer plates (ScintiStrip) .After incubation for 20 min at 22 ° C., the plates were washed and the unreacted PRII was incubated by means of MicroBeta counter (Fig. Wallac, Turku, Finland). 1 shows a typical dependence of CaN activity on inhibitor concentration.

Example 2: Competition CaN inhibition

The phosphatase activity of calcineurin can be inhibited by the Cyp18 / CsA complex. Thus, the administration of 1 μM of this complex leads to the almost complete inhibition of a CaN concentration of 1.32 nM, as shown in 1 (Example 1) is shown. Since only the Cyp18 / CsA complex can inhibit CaN, but not the Cyp18 / Cs12 complex, the inhibition of CaN activity by CsA can be competed depending on the concentration of Cyp18. So shows 2a at a concentration of 100 μM Cyp18 only at higher CsA concentrations a CaN inhibition, while such an effect is no longer observed when increasing the Cyp18 concentration to 500 nM.

Example 3: Imunsuppression

T cell proliferation: To determine the effect of the inhibitors on T cell proliferation, 2 x 10 ⁶ cells (peripheral blood mononuclear cells, PBMC) per ml were labeled with 5- (6-) carboxyfluoresoresin diacetate succinimidyl ester and anti-CD3 plus anti -CD28 antibodies in the presence of suitable inhibitor concentrations (inhibitor stock solution: 1 mg / ml DMSO) at a final DMSO concentration of 0.5% for 5 days at 37 ° C and then subjected to flow cytometry (BD FACScalibur).

Cytokine production: To this end, 2x10 ⁶ cells (PBMC) with the desired inhibitor concentration were preincubated at 37 ° C for 10 min. Subsequently, the cells were incubated with 10 ng / ml PMA and 1 μl of ionomycin (Sigma, Steinheim) for 2 hours and after addition of brefeldin A (5 μg / ml) for a further 3 hours. After fixing the cells with 2% paraformaldehyde for 20 min, permeabilizing with a solution of 0.5% saponin, and 1% FCS in PBS, the cytokine concentration could be determined by flow cytometry and anticytokine PE-conjugated antibodies (BD Biosciences). This procedure for detecting cytokine production was comprehensively described by Wittmann, B. et al. 1999 in Blood (Vol. 94, pages 1717-1726).

Luciferase Reporter Genassay: In order to visualize the influence of desired inhibitor concentrations and the photo-switching, Jurkat cells transfected with NFAT-luc reporter plasmid (Strategene, The Netherlands) were produced by electroporation (Amaxa, Cologne). After incubation of these cells for 20 min and a further incubation with or without irradiation at 366 nm for 30 min, the cells were stimulated by addition of 10 ng / ml PMA and 1 ug / ml ionomycin and incubated for a further 5 h. Thereafter, the concentration of luciferase was determined by means of commercial luciferase assay (Promega, Mannheim). The internal control standard used was the expression of β-galactosidase cotransfected cells. The results obtained are in the 5a and 5b summarized, which are explained below:
5a Jurkat cells are stimulated with PMA / ionomycin and then exposed to various cyclosporin derivatives. CsA and Cs10 lead to complete inhibition of stimulation in a concentration-dependent manner. If Cs11 is irradiated analogously to Example 3 and then added to the stimulated cells, the inhibition effect is no longer observed in a concentration-dependent manner as for the chemically synthesized substance Cs12.
5b In contrast to experiment 5a, the irradiation of the Jurkat cells is now carried out after application of Cs10. It clearly shows that here too the inhibition can be abolished by the irradiation.

Example 4: Photo switching (I)

The photoconversion of Cs10 into Cs12 is readily accomplished by RP-HPLC separation and subsequent mass spectrometry, as exemplified herein. For this purpose, light with a hand lamp (Dr. Gröbel UV Electronics GmbH, 2-5062, 254/366 nm) for 10, 20, 30 or 60 min to an ethanolic sample of Cs10 (10 mg / ml) at 23 ° C in directed to a cuvette. Immediately after irradiation, the sample is separated by HPLC. The 4 shows a typical running behavior of the substance irradiated at different times compared to the untreated sample. The prepared mass spectrograms of the separated samples corresponded to compound Cs10 (before irradiation) and Cs12 (after 60 min irradiation).

Example 5: Photo switching (II)

• a) Die Zugabe von PMA/Ionomycin führt zur NFAT Reportergenaktivierung
• b) Die zusätzliche Gabe zu (a) von Cs10 hemmt diese Aktivierung dosisabhängig
• c) Die zusätzliche Gabe zu (a) von mit Licht bestrahltem Cs10 hemmt diese Aktivierung nur in hohen Dosen
• d) Die Bestrahlung der Zellen nach Applikation von Cs10 führt zu ähnlichen Effekten wie die Applikation von Substanz Cs12

The NFAT reporter gene assay listed in Example 3 is well suited to visualize the effect of activation of the substance Cs10 to Cs12 on the basis of the NFAT translocation. As in 5 as outlined above, Jurkat cells transfected with reporter plasmid were incubated with Cs10 at 20 to 100 nM for 20 min, then irradiated with a light source (366 nm) for 30 min, and then stimulated with PMA / ionomycin for 5 h measured the luciferase activity. In order to control the observed effects, radiation was dispensed with or the pure substance Cs12 was used in comparison to Cs10. The evaluation of in 5a summarized results can be summarized as follows:

• a) The addition of PMA / ionomycin leads to NFAT reporter gene activation
• b) The additional administration to (a) of Cs10 inhibits this activation in a dose-dependent manner
• c) The addition of (a) light-irradiated Cs10 inhibits this activation only in high doses
• d) The irradiation of the cells after application of Cs10 leads to similar effects as the application of substance Cs12

Example 6: Activation by enzymatic cleavage

By preparing a cyclosporin derivative at the 8-position (corresponding to Formula 1) the substituent -CH ₂ -O-CH ₂ C (O) NH (CH ₂ ) ₂ NHC (O) OC ₂ H _{5 is introduced} according to an analog of the A10 type this compound is now accepted by esterases (EC 3.1.1) as a substrate. After hydrolysis of the ethyl ester, spontaneous CO ₂ cleavage occurs and an A12 analogue forms, which here corresponds to a cyclosporin derivative having a -CH ₂ -O-CH ₂ C (O) NH (CH ₂ ) ₂ NH ₂ substituent. The esterase used was a commercial porcine liver commercial carboxylesterase (Sigma, E3019) under the following reaction conditions: 100 μg of CsA ⁸ -CH ₂ -O-CH ₂ C (O) NH (CH ₂ ) ₂ NHC (O) OC ₂ H ₅ are added in Dissolved 100 μl of DMSO and then added to 2 ml of 25 mM Tris buffer (pH 8) containing 30 units of esterase and allowed to stand overnight at room temperature (about 23 ° C). The separation of the reaction mixture by means of HPLC shows 4 corresponding reaction conversion with almost complete formation of the substance CsA ⁸ -CH ₂ -O-CH ₂ C (O) NH (CH ₂ ) ₂ NH ₂ which corresponds to the analog A12. The conversion of the substance by enzymatic hydrolysis can be prevented by adding appropriate amount of esterase inhibitor, such as the human esterase inhibitor C-1 (Sigma, E0518).

Example 7: Activation by change of the pH

By preparing a cyclosporin derivative wherein in the 8-position (corresponding to formula 1) the substituent -CH ₂ -O-CH ₂ C (O) NH (CH ₂ ) ₃ NHC (O) OCH ₃ corresponding to an analog of type A10 was introduced , this compound becomes sensitive to catalysis by acids or bases. After hydrolysis of the methyl ester, there is spontaneous CO ₂ cleavage and the formation of an A12 analog, which here corresponds to a cyclosporin derivative with a substituent -CH ₂ -O-CH ₂ C (O) NH (CH ₂ ) ₃ NH ₂ . The following reaction condition was used: 200 μg of CsA ⁸ -CH ₂ -O-CH ₂ C (O) NH (CH ₂ ) ₃ NHC (O) OCH ₃ are dissolved in 150 μl of DMSO and then added to 2 ml of 50 mM glycine buffer (pH 13 ) and allowed to stand overnight at room temperature (about 23 ° C). The separation of the reaction mixture by HPLC shows one of 4 corresponding reaction conversion with almost complete formation of the substance CsA ⁸ -CH ₂ -O-CH ₂ C (O) NH (CH ₂ ) ₃ NH ₂ which corresponds to the analog A12.

Example 8: Synthesis of [O-carboxymethyl D-Ser] ⁸ CsA (Cs6)

A solution of 60 mg of [D-Ser] ⁸ CsA, tert-butyl bromoacetate (20 mg) and 5 mg of benzyltriethylammonium chloride in 1 ml of CH ₂ Cl ₂ was stirred for 2 hours at 23 ° C with 2 ml of 30% NaOH aqueous solution. Thereafter, the solution was diluted with 10 ml of water and extracted twice with ether. Thereafter, the organic phase was dried with Na ₂ SO ₄ . Then, without further separation steps, 30 ml of methanolic KON solution (60 mM) were added thereto and the mixture was stirred for a further three hours. After adding an equivalent amount of acetic acid, most of the acetic acid and methanol were removed by rotary evaporation. Thereafter, 200 ml of ethyl acetate were added and the mixture was washed with water. After separation of the organic phase and drying with Na ₂ SO ₄ , the desired compound was separated by RP HPLC (94%). The MALDI mass spectrum (m / z) of 1276.8 [MH] ⁺ agreed very well with the calculated cal. 1275.7.

Example 9: Synthesis of [O- (NH ₂ (CH ₂ ) ₅ NHC (O) -CH ₂ -) D-Ser] ⁸ CsA (Cs12)

One part (100 mg) of Cs6 (Example 8), 3 parts of NH ₂ (CH ₂ ) ₅ NNBoc, 4 parts of PyBop and 8 parts of DIPEA are added to 5 ml of CH ₂ Cl ₂ and stirred at 23 ° C overnight , Thereafter, 40 ml of ethyl acetate are added and the organic phase washed successively with 5% NaHSO ₄ , 5% NaHCO ₃ and saturated aqueous NaCl solution, and then dried with Na ₂ SO ₄ . After removal of the solvent by means of a vacuum evaporator, the product (Cs9) could be separated off by means of C8 HPLC (96%). The MALDI mass spectrum (m / z) of 1461.3 [MH] ⁺ agreed very well with the calculated one of 1460.2. To remove the Boc protecting group, Cs9 was added with 5 ml of ZnCl ₂ / ether in ether under inert gas (N ₂ ) for 3 hours. It was then precipitated by the addition of 0.1 ml of water and 15 ml of acetonitrile and then filtered. After removal of the organic solvent by means of a vacuum evaporator, Cs12 could be obtained by means of C8 HPLC (86%). The product was analyzed by ¹ H NMR: δ: 9.50 (m), 8.42 (m), 8.08 (m), 7.56 (m), 6.84 (m), 5.62 (s), 5.52 (s), 5.40 (m), 5.28 (m), 5.19 (s), 4.60 (m), 4.10 (m), 3.58-3.40 (m), 3.14 (m), 3.03 (m), 2.84 (m), 2.75 (m), 2.70 (s ), 1.81 (m), 1.72 (m), 1.59 (m), 1.18-1.40 (m), 0.70-1.15 (m), and MALDI mass spectrometry (m / z) are determined to be 1361.3 [MH] ⁺ and corresponded thereto good of the calculated mass of 1360.1

Example 10: Synthesis of [O- (NVOCNH (CH ₂ ) ₅ NHC (O) CH ₂ -) D-Ser] ⁸ CsA (Cs10)

One part (30 mg) of Cs12, 2 parts of NVOC-Cl, 4 parts of DIPEA are added to 2 ml of DMF and stirred at 23 ° C overnight. Subsequently, 40 ml of ethyl acetate were added and the organic phase washed successively with 5% NaHSO ₄ , 5% NaHCO ₃ and saturated aqueous sodium chloride solution and subsequently dried with Na ₂ SO ₄ . After narrowing with a rotary evaporator, the product was separated by C8 HPLC (90%). The MALDI mass spectrum (m / z) of 1601.0 [MH] ⁺ agreed very well with the calculated cal. 1599.6.

Example 11: Influence of Cs12 on proliferation

Cs12 is synthesized either chemically according to Example 8 or prepared by irradiation from Cs10 (Example 3). Blood cells (PBMCs) are labeled with CFSE and stimulated with anti-CD3 / anti-CD28 as indicated in Example 2 (T-cell proliferation). After incubation with DMSO (as a control), Cs12 (1 μM) or CsA (50 nM) for 5 days, the labeling is measured as a function of the cell number by means of FACS. In 5a the result is shown. The percentages entered correspond to the concentration of proliferating cells. In contrast to CsA, Cs12 has no influence on the proliferation of these cells.

Example 12: Influence of Cs12 on cytokine production

Cs12 is synthesized either chemically according to Example 8 or prepared by irradiation from Cs10 (Example 4). A FACS analysis and stimulation procedure with PMA / ionomycin, including the necessary administration of brefeldin during the stimulation process of PBMC, was carried out largely analogously to the procedure given in Example 2 (T cell proliferation) and is based essentially on the processants customary in the literature ( such as Wittmann, B. et al., Blood 94 (1999) 1717). The effectors are added to the PBM cells at 37 ° C 10 min prior to the stimulation described above. In 5b is the result of the effect shown. Percentages entered represent the percentage of cells that have interleukin 2 ( 5b above) or IFN-gamma ( 5b below). In contrast to CsA, Cs12 has no influence on cytokine production and corresponds in its effect to the control, which contained only the solvent of the active ingredients (DMSO).

Claims (32)

Cyclosporin analog of general formula (I)

wherein R ^{1 is} N-methyl- (4R) -4 - [(E) -2-butenyl] -4-methyl-L-threonine (MeBMT) or dihydro-MeBMT; R ^{2 is} α-aminobutyric acid (Abu) or a derivative of this amino acid fluorinated at one or more positions on the side chain; R ^{3 is} N-methyl-D-alanine (D-McAla) or N-methyl-glycine (sarcosine (Sar)) or a fluorinated derivative thereof; R ⁴ , R ⁶ , R ⁹ and R ¹⁰ independently represent N-methyl-L-leucine (L-McLeu) or a fluorinated derivative thereof; R ^{5 is} L-valine (Val) or a fluorinated derivative thereof; R ^{1 is} L-alanine (Ala) or a fluorinated derivative thereof; R ^{11 is} N-methyl-L-valine (L-McVal) or a fluorinated derivative thereof; and R ⁸ is (a)

wherein X is O or S, Y is H or methyl, m has a value between 0 and 5, n is an integer between 3 and 10, Z is NR ¹² R ¹³ or NHC (O) OR ¹⁴ , where R ^{12 is} H, methyl, ethyl, n-propyl, iso-propyl or a photolysable group and R ^{13 is} H or a C ¹ -C ⁸ -alkyl radical, and R ^{14 is} an acid / base labile or an enzymatically cleavable group ; or (b)

where X is O, S or CH ₂ , Y is H or methyl, m has a value between 0 and 5, n is an integer between 3 and 10, Z is NR ¹² R ¹³ or NHC (O) OR ¹⁴ wherein R ^{12 is} H, methyl, ethyl, n-propyl, iso-propyl or a photolysable group, and R ^{13 is} H or a C ¹ -C ⁸ alkyl radical, and R ^{14 is} an acid / base labile or a enzymatically cleavable group. A cyclosporin analog according to claim 1, wherein the photolysable Group is an optionally substituted nitroaryl group. A cyclosporin analog according to claim 2 wherein the optionally substituted nitroaryl group 2-nitroveratryloxycarbonyl (NVOC), α-carboxy-2-nitrobenzyl (CNB), 1- (2-nitrophenyl) ethyl (NPE), 1- (4,5-dimethoxy-2-nitrophenyl) ethyl (DMNPE) or 5-carboxymethoxy-2-nitrobenzyl (CMNB) is. A cyclosporin analog according to claim 1 wherein R ^{12 is} H. A cyclosporin analog according to claim 1 wherein the acid / base labile or enzymatically cleavable group is methyl, ethyl, methoxymethyl (MOM), -CH ₂ CH ₂ F, methylthiomethyl (MTM), β-glucuronide, D-glucopyranosyl, β-D-galactopyranosyl , tetra-O-acetyl-D-glucopyranosyl, or tetra-O-acetyl-β-D-galactopyranosyl. A cyclosporin analog according to any one of claims 1 to 5, wherein R ^{2 is} Abu; R ^{3 is} Sar or D-MeAla; and R ^{7 is} Ala. A cyclosporin analog according to any one of claims 1 to 6, wherein R ^{1 is} McBmt; R ^{3 is} sar; R ⁴ , R ⁶ , R ⁹ and R ^{10 represent} L-MeLeu; R ^{5 is} L-Val; and R ^{11 stands} for L-MeVal. Cyclosporin analog according to one of claims 1 to 3 or 5 to 7, which inhibits cyclophilin and calcineurin. The cyclosporin analog of claim 8, wherein the peptidyl-prolyl isomerase activity is inhibited by cyclophilin becomes. A cyclosporin analog according to claim 8 or 9 wherein the protein phosphatase activity of calcineurin is inhibited. A cyclosporin analog according to any one of claims 8 to 10, wherein the IC ₅₀ value for the inhibition of cyclophilin is less than 10 μM, and the IC ₅₀ value for the inhibition of calcineurin is less than 10 μM. Cyclosporin analog according to one of claims 8 to 11, wherein the calcineurin-inhibiting effect by photolysis, addition of an enzyme or change of the pH can be reduced. A cyclosporin analog according to claim 12 which is [O- (NVOCNH (CH ₂ ) ₅ NHC (O) CH ₂ -) D-Ser] ⁸ CsA. A cyclosporin analog according to claim 4 which is cyclophilin, but not calcineurin inhibits. A cyclosporin analog according to claim 14, wherein the Peptidyl-prolyl isomerase activity inhibited by cyclophilin becomes. The cyclosporin analog of claim 14 or 15, wherein the IC ₅₀ value for the inhibition of cyclophilin is less than 10 μM, and the IC ₅₀ value for the inhibition of calcineurin is greater than 10 μM. A cyclosporin analog according to claim 16 which is [O- (NH ₂ (CH ₂ ) ₅ NHC (O) CH ₂ -) D-Ser] ⁸ CsA. A process for the preparation of the cyclosporin analog according to any one of claims 4 or 14 to 16 using [D-Ser] ⁸ or [D-Cys] ⁸ cyclosporin A, CPG bromoacetate and a compound of the formula NH ₂ (CH ₂ ) _n NH-APG, where n is an integer between 3 and 10, APG is an amino-protecting group and CPG is a carboxyl-protecting group. A process for preparing the cyclosporin analog according to any of claims 1 to 3 or 6 to 13 using [D-Ser] ⁸ or [D-Cys] ⁸ cyclosporin A, CPG bromoacetate, a compound of the formula NH ₂ ( CH ₂ ) _n NH-APG, where n is an integer between 3 and 10, APG is an amino-protecting group and CPG is a carboxyl-protecting group, and a compound selected from the group comprising NVOC, CNB, NPE, DMNPE and CMNB. A method according to any one of claims 18 or 19, wherein the carboxyl-protecting group tert-butyl and the amino protecting group is Boc. A method of cleaving the residue R ^{12 of} the cyclosporin analog according to any one of claims 1 to 3 or 6 to 13, wherein the cyclosporin analog is irradiated with ultraviolet light. A method for cleaving the C (O) OR ¹⁴ group of the cyclosporin analog according to any one of claims 1 to 3 or 5 to 13, wherein the cyclosporin analog is added with an enzyme, an acid or a base. Medicament containing a cyclosporin analog according to one of the claims 1 to 17. A pharmaceutical composition containing a cyclosporin analog according to any one of claims 1 to 3 or 5 to 13, wherein the action of the drug can be altered by photolysis, addition of an enzyme or change in pH. The pharmaceutical composition of claim 24, wherein the effect changed before or after administration can be. Use of the cyclosporin analogue after one the claims 1 to 3 or 5 to 13 for the preparation of a medicament for immunosuppression, and / or for the treatment of autoimmune diseases, chronic inflammation, HIV infection, multi-drug resistance (MDR) and / or asthma and / or to prevent rejection of grafts, to sensitize cancer cells for chemotherapy and / or to protect the lung from hypoxic damage, Neuroprotection or neuroregeneration, for the treatment of bacterial or parasitic Diseases of humans and animals. Use according to claim 26, wherein the immunosuppressive Effect and / or side effects of the drug by photolysis, Adding an enzyme or change of the pH can be reduced. Use according to claim 27, wherein the photolysis, Adding an enzyme or change the pH before or after the administration of the drug can be done. Use of the cyclosporin analogues after one the claims 4 or 14 to 17 for the manufacture of a medicament for the treatment immunosuppression, and / or for the treatment of autoimmune diseases, chronic inflammation, HIV infection, multi-drug resistance (MDR) and / or asthma and / or Prevention of rejection of grafts, to sensitize cancer cells for chemotherapy and / or to protect the lung from hypoxic damage, Neuroprotection or neuroregeneration, for the treatment of bacterial or parasitic Diseases of humans and animals. Use according to claims 26 and 29, wherein the autoimmune diseases and chronic inflammation selected are from the group comprising Systemic Lupus Erythematosus (SLE), Chronic Rheumatoid Arthritis, Type 1 Diabetes, Chronic Inflammatory Intestinal disease, biliary cirrhosis, uveitis, multiple sclerosis, morbus Crohn, ulcerative colitis, bullous Pemphigoid, sarcosidosis, psoriasis, atopic dermatitis, ichthyosis or Graves Opthalmopathy. Cyclosporin analog according to claim 12 or method according to claim 22 or use according to claim 27 or 28, wherein the enzyme is selected is from the group comprising hydrolases, esterases, proteases, peptidases and glycosidases. Use according to claim 27, 28 or 31, wherein the Side effect of the drug is selected from the group comprising nephrotoxicity abnormal liver function, hirsutism, gum hypertrophy, tremor, hyperesthesia, gastrointestinal disorders and Increase in incidence for different tumors.