WO1995006649A1

WO1995006649A1 - Macrolides as antagonists of macrophilin binding immunosuppressants

Info

Publication number: WO1995006649A1
Application number: PCT/EP1994/002894
Authority: WO
Inventors: Theodor Fehr; Jean-Jacques Sanglier
Original assignee: Sandoz Erfindungen Verwaltungs GmbH; Sandoz AG
Current assignee: Novartis Pharma GmbH Austria; Sandoz AG
Priority date: 1993-09-01
Filing date: 1994-08-31
Publication date: 1995-03-09
Anticipated expiration: 1996-03-01
Also published as: GB9318144D0; AU7692594A

Abstract

The invention provides macrolides of formula (I) wherein R1 is H, alkyl, aryl, or acyl; R2 is H, or -OR5, such that R5 is H, alkyl, aryl, or acyl; R3 is -CH2- or -CH2CH2-; and R4 is H, alkyl, acyl, or aryl; preferably wherein 'alkyl' refers to C1-6 alkyl; 'aryl' refers to an aromatic hydrocarbon radical having one or two aromatic rings; and 'acyl' is alkylcarbonyl or arylcarbonyl. Pharmaceutical compositions, pharmaceutical uses, assays, methods for producing the macrolides, and a novel producer strain are also provided.

Description

MACROLIDES AS ANTAGONISTS OF MACROPHILIN BINDING IMMUNOSUPPRESSANTS

This invention relates to certain novel macrolides which are antagonists of macrophilin binding immunosuppressants, such as FK506 and rapamycin.

Rapamycin (sirolimus) is a macrolide antibiotic that is produced by Streptomyces hvgroscopicus. The structure of rapamycin is given, e.g., in McAlpine et al. (1991) J. Antibiotics 44: 688 and Schreiber et al. (1991) J. Am. Chem. Soc. 113: 7433. Rapamycin is an extremely potent immunosuppressant and also has anti-tumor and anti-fungal activity. However the utility of rapamycin as a pharmaceutical is restricted by its low and variable bioavailability and its high toxicity.

FK506 (tacrolimus) is a macrolide antibiotic that is produced by Streptomvces tsukubaensis No 9993. The structure of FK506 is given, e.g., in Tanaka et al. (1987) /. Am. Chem. Soc. 109: 5031. FK506 also is a potent immunosuppressant. FK506, however, is also toxic, particularly to the nervous system.

FK506 and rapamycin are believed to have somewhat different mechanisms of inducing immunosuppression; however, both bind strongly to macrophilins and suppress lymphocyte proliferation.

This invention provides novel macrolides having a novel profile of biological activity. Although the compounds bind strongly to macrophilins and inhibit adhesion molecule expression and epithelial cell proliferation, they do not suppress lymphocyte proliferation. These novel macrolides are useful as pharmaceuticals, e.g., as inhibitors of adhesion molecule expression, as antidotes to macrophilin binding immunosuppressants of the FK506 or rapamycin type, as steroid potentiators, and as Mip inhibitors, as well as in diagnostic and screening assays. The novel macrolides are of formula I:

Formula I

wherein

R, is H, alkyl, aryl, or acyl;

R₂ is H, or -OR₅, such that R₅ is H, alkyl, aryl, or acyl;

R₃ is -CH₂- or -CH₂CH ; and

R₄ is H, alkyl, acyl, or aryl.

Preferably, "alkyl" refers to C,.₆ alkyl, e.g., methyl or ethyl; "aryl" refers to an aromatic hydrocarbon radical having one or two aromatic rings, e.g., phenyl or benzyl; and "acyl" refers to alkylcarbonyl or arylcarbonyl (preferably a physiologically hydrolysable and acceptable acyl; that is, a residue which is cleavable under physiological conditions to yield an acid which is itself tolerated at the doses to be administered), e.g., acetyl, benzoyl, or salicyl. In a preferred embodiment, the macrolides of the invention are those of formula Ia:

Formula Ia

wherein R, is H or -CH₃; R₂ is H or -OH; and R₃ is -CH₂- or -CH₂CH₂-.

Especially preferred compounds of formula Ia include the following:

Compound A, wherein R, is H, R₂ is H, and R₃ is -CH₂CH₂-; Compound B, wherein R, is H, R₂ is -OH, and R₃ is -CH₂CH₂-; Compound C, wherein R, is -CH₃, R₂ is -OH, and R₃ is -CH₂CH₂-; Compound D, wherein R, is H, R₂ is H, and R₃ is -CH₂-.

The macrolides of formula I (the Macrolides) are shown to inhibit adhesion molecule expression. Cellular adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin, are expressed on the surface of endothelial cells and keratinocytes in response to pro- inflammmatory mediators including TNFα, IFNγ, ELI and LPS. Corresponding counter- ligands, e.g. LFA-1, VLA-4 and SLE", are expressed on the surfaces of circulating blood cells. Transendothelial migration of leucocytes during inflammatory processes as well as extravascular cell-cell interactions are regulated as a result of the interactions between these adhesion molecules and their counter-ligands. Consequently inhibitors of adhesion molecule expression offer potential for the treatment of many disease states. However, no suitable low molecular weight inhibitors of adhesion molecule expression are currently available.

The Macrolides are shown to inhibit VCAM, ICAM and ELAM expression in vitro at low micromolar or submicromolar levels. This activity can be confirmed in vivo; for example, a topical solution containing 1.2% of Compound B shows 39% inhibition of oxazolone induced allergic contact dermatitis compared to controls.

Inhibition of adhesion molecule expression makes the Macrolides useful as pharmaceuticals in a variety of applications. For example, the compounds are useful for the treatment or prophylaxis of disease processes which involve expression of cellular adhesion molecules. These disease processes include many acquired and inherited diseases/disorders where leucocyte trafficking plays a prominent role in the pathogenic process, most notably acute and chronic inflammation (e.g. allergy, asthma, psoriasis, reperfusion injury, rheumatoid arthritis and septic shock) and autoimmune states (e.g. multiple sclerosis). Other indications for the compounds of the invention include tumour metastasis (e.g. melanoma, osteocarcinoma) and allograft/xenograft rejection, since it is known that inhibition of vascular adhesion molecules can greatly improve the prognosis of these processes. Also the compounds have therapeutic potential in hyperproliferative skin diseases (e.g. psoriasis) as well as various malignancies in view of their inhibitory activity at low micromolar concentrations when tested for 72 hours in a keratinocyte-based proliferation assay.

The Macrolides are also potent macrophilin binders. The macrophilin binding activity of the Macrolides can be shown using the macrophilin binding assay (MBA), which measures the ability of the Macrolides to compete with a macrophilin binding immunosuppressant for binding to a macrophilin. FK506, rapamycin, and immunosuppressive derivatives of these drugs are known to bind in vivo to macrophilin- 12 (also known as FK506 binding protein or FKBP-12), so the ability of the macrolide to compete with FK506 or rapamycin for binding to macrophilin- 12 can be measured. In this assay, FK506 is coupled to bovine serum albumin (BSA) and is then used to coat microtiter wells. Biotinylated recombinant human macrophilin- 12 (biot-MAP) is allowed to bind in the presence or absence of the Macrolide to the immobilized FK506. After washing (to remove macrophilin- 12 which is not specifically bound to the immobilized FK506), the amount of bound biot-MAP is assessed by incubation with a streptavidin-alkaline phosphatase conjugate, followed by washing and subsequent addition of p-nitrophenyl phosphate as a substrate. The read-out is the OD at 405nm. Any binding of the Macrolide to the biot-MAP results in a decrease in the amount of biot-MAP that is bound to the FK506 and thus in a decrease in the OD 405. Repeating the test at various concentrations of the macrolide allows the determination of the concentration resulting in 50% inhibition of the biot-MAP binding to the immobilized FK506 (IC₅₀). The Macrolides, e.g., Compounds A, B, C, and D, have an IC₅₀ of less than 10 nM in this assay, comparable to FK506 or rapamycin.

That the Macrolides have a substantially different mechanism of action from FK506 or rapamycin can be shown in standard in vitro assays in comparison to FK506 and rapamycin. FK506, for example, is known to be a potent inhibitor of IL-2 transcription, as can be shown in an IL-2 reporter gene assay. Rapamycin, although not active in the IL-2 reporter gene assay, strongly inhibits IL-6 dependent T-cell proliferation. Both compounds are very potent inhibitors of the mixed lymphocyte reaction. The Macrolides, by contrast, are inactive in all of these assays.

Because the Macrolides bind strongly to macrophilin without suppressing lymphocyte proliferation, they can be used in the treatment of overdoses of macrophilin-binding immunosuppressants, such as FK506 and rapamycin. The Macrolides are particularly potent inhibitors of rapamycin activity. Compounds B and C, for example, completely abrogate the biological activity of rapamycin at approximately equimolar concentrations. To abrogate completely the biological activity of FK506, approximately a 100-fold molar excess is desirable.

The macrophilin binding activity of the Macrolides also makes them useful in enhancing or potentiating the action of corticosteroids. Combined treatment with the compounds of the invention and a corticosteroid, such as dexamethasone, results in greatly enhanced steroidal activity. This can be shown, e.g., in the murine mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene assay, e.g., as described in Ning, et al., J. Biol. Chem. (1993) 268: 6073. This synergistic effect allows reduced doses of corticosteroids, thereby reducing the risk of side effects in some cases.

Additionally, the Macrolides bind to and block a variety of Mip (macrophage infectivity potentiator) and Mip-like factors, which are structurally similar to macrophilin. Mip and Mip-like factors are virulence factors produced by a wide variety of pathogens, including those of the genera Chlamidia. e.g., Chlamidia trachomatis: Neisseria. e.g., Neisseria meningitidis; and Legionella. e.g., Legionella pneumophilia: and also by the obligately parasitic members of the order Rickettsiales. These factors play a critical role in the establishment of intracellular infection. The efficacy of the Macrolides in reducing the infectivity of pathogens which produce Mip or Mip-like factors can be shown by comparing infectivity of the pathogens in cells culture in the presence and absence of the macrolides, e.g., using the methods described in Lundemose, et al., Mol. Microbiol. (1993) 7: 777.

Accordingly this invention also provides the macrolides of formula I, e.g., of formula Ia, e.g., Compound A, B, C, or D, for use as a pharmaceutical, and pharmaceutical compositions comprising any of them, optionally in combination or association with a pharmaceutically acceptable diluent or carrier, e.g., for use in any of the following indications:

-5 ppressing adhesion molecule expression or

-treatment or prevention of any of the indications set forth above, e.g.,

-treating or preventing acute or chronic inflammation (e.g. allergy, asthma, psoriasis, reperfusion injury, rheumatoid arthritis and septic shock)

-treating or preventing autoimmune autoimmune diseases (e.g. multiple sclerosis),

-treating or preventing tumour metastasis (e.g. melanoma, osteocarcinoma),

-treating or preventing allograft xenograft rejection, optionally in combination with known immunosuppressants, e.g., cyclosporin or FK506, and/or corticosteroids,

-treating or preventing hyperproliferative skin diseases (e.g. psoriasis) as well as various malignancies,

-treating or preventing dermatitis,

-treating overdoses of a macrophilin binding immunosuppressant, e.g., FK506 or rapamycin,

-potentiating the action of steroids, e.g., for treating or preventing inflammation or for one of the other uses known for corticosteroids,

-treating or preventing infectious diseases, e.g., prophylaxis or treatment of infections or infectuous diseases caused by organisms producing Mip or Mip-like factors (including organisms of the genera Chlamidia. e.g., Chlamidia trachomatis: Neisseria. e.g., Neisseria meningitidis: and Legionella. e.g., Legionella pneumophilia: and also the obligately parasitic members of the order Rickettsiales), optionally in combination with one or more other anti- infective agents.

The invention also provides the use of a Macrolide in the manufacture of a medicament for use in any of these indications. The invention also provides a method of treatment or prophylaxis of any of these indications, said method comprising administering a pharmaceutically effective amount of a Macrolide to a subject, e.g., a larger mammal, e.g., man, in need of such treatment.

The Macrolides may be produced synthetically, e.g., by total synthesis using a procedure analogous to that described for rapamycin by Nicolaou, et al., J. Am. Chem. Soc. (1993) 115: 4419, or by fermentation as described below, or by a combination of synthetic and biosynthetic means, e.g., by isolating fermentation products and further chemically modifying them.

Preferably, the Macrolides are produced by fermentation using a microorganism of the genus Micromonospora. preferably the novel species of the genus Micromonospora designated A92-306401; for example, the strain which has been deposited by the applicant under the Budapest Convention on July 30, 1993 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1 B, D-38124 Braunschweig, Germany under accession number DSM 8429. Compounds A, B, C, and D may be obtained by cultivating the microorganism in an appropriate culture medium and then isolating the macrolide, e.g., by chromatography.

Compounds A, B, C, and D are then optionally further modified to give other compounds of formula 1. Alkylation or arylation of hydroxy groups, to yield macrolides of formula I wherein R„ R₄, and/or R₅ is/are selected from alkyl or aryl, is performed, e.g., as described for rapamycin in WO 94/09010 or as described for FK506 in WO 92/20688, or for example by reacting Compound A, B, C, or D with an organic radical attached to a leaving group (e.g., RX where R is the organic radical, e.g., an alkyl, allyl, or benzyl moiety, which is desired as the O-substituent, and X is the leaving group, e.g., CCl₃C(NH)O or CF₃SO₃) under suitable reaction conditions, e.g., in the presence of an acid like trifluoromethanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid or their respective pyridinium or substituted pyridinium salts when X is CCl₃C(NH)O or in the presence of a base like pyridine, a substituted pyridine, diisopropylethylamine or pentamethylpiperidine when X is CF₃SO₃. Acylation to form macrolides of formula I wherein R_ls R₄, and/or R₅ is/are acyl is accomplished by esterification of the hydroxy group using conventional means, e.g., as described in US 4 316 885 for rapamycin, or e.g., reacting Compound A, B, C, or D with an anhydride, e.g., acetic anhydride, or acid halide, e.g., alkylcarbonyl halide, in the presence of an acid binding agent, e.g. a base, e.g., pyridine, under suitable reaction conditions.

The invention therefore provides a process for the production of a macrolide, e.g., a macrolide of formula 1, e.g., of formula la, comprising cultivating a microorganism of the genus Micromonospora. e.g., of the species Micromonospora sp.A92-306401, e.g., of the deposited strain DSM 8429 in an appropriate culture medium and isolating the macrolide. An appropriate culture medium is a medium containing suitable sources of nitrogen, carbon, and trace minerals. Preferably the sources of carbon in the culture medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, and dextrin. Preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cottonseed meal, soybean meal, casein hydrolysates, soybean hydrolysates, yeast hydrolysates, and the like and inorganic and organic nitrogen-containing compounds such as ammonium salts, urea, amino acids and the like. Conventional fermentation agents and trace materials may also be added.

The invention also includes macrolides made using this fermentation process, including macrolides which are not within the scope of formula I.

The invention also includes the novel macrolide-producing Micromonospora described herein.

The appropriate dosages of the macrolides will vary depending on the nature of the condition to be treated, but in general however satisfactory results are obtained on administration orally at dosages on the order of from 0.05 to lOmg/kg/day, preferably 0.1 to 7.5 mg/kg/day, more preferably 0.1 to 2 mg/kg/day, administered once or, in divided doses, 2 to 4 times per day. On administration parenterally, for example by i.v. drip or infusion, dosages on the order of from 0.01 to 5 mg/kg/day, preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1 to 1.0 mg/kg day can be used. Suitable daily dosages for patients are thus on the order of from 2.5 to 500 mg p.o., preferably 5 to 250 mg p.o., more preferably 5 to 100 mg p.o., or on the order of from 0.5 to 250 mg i.v., preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50 mg i.v..

Dosaging may also be arranged in a patient specific manner to provide pre-determined trough blood levels, as determined by the RIA technique. Thus patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml; analogously to methods of dosaging currently employed for Ciclosporin immunosuppressive therapy.

The Macrolides may be administered by any conventional route, in particular enterally, parenterally, or topically. Suitable enterally administered forms are solutions for drinking, tablets or capsules. Suitable parenteral forms are injectable solutions or suspensions. Suitable unit dosage forms for oral administration may comprise from 1 to 50 mg of the Macrolides; usually 1 to 10 mg. Pharmaceutical compositions for topical administration include ointments or creams, e.g., containing 0.1-10%, preferably about 1-2% of Macrolide together with a topical earner or in a topical formulation.

In another embodiment, the invention provides for Macrolides to be used as a screening tool to determine the presence of macrophilin-binding immunosuppressants in broths. This can be done using standard competitive assays based on the FK506 antagonistic properties of the Macrolide. Preferably a Macrolide is immobilized in microtiter wells and then allowed to bind in the presence of a test broth to labelled macrophilin- 12. EXAMPLE I - Characterization and cultivation of producer strain

a. Description of the strain

Micromonospora sp.A92-306401 was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg lb, D-3300 Braunschweig, Germany on July 30, 1993 under the terms of the Budapest Treaty, and has been assigned the accession number DSM 8429. The strain DSM 8429 can be assigned to the genus Micromonospora as a novel species, designated A92-306401, using standard classification systems, e.g., as described in Bergey's Manual (1989) and The Prokaryotes (1992).

The cell walls of this microorganism contain meso-diaminopimelic acid. The fatty acids are iso- and anteiso-branched straight and unsaturated and characteristically 18:0 (stearic acid). The sugar spectrum is composed of ribose, arabinose, xylose, mannose, galactose, glucose , meso-inositol and an unknown sugar which is not madurose (retention time 16.5 using the method of Saddler et al.) The mycelium is formed of thin hyphae and does not break down in fragments. No aerial mycelium is formed on the traditionally used media. Spores are in most of the cases rare, non-motile and singular. The strain grows on various organic and inorganic media. The substrate mycelium grows as hyphae and is generally orange to salmon. The ability of A92-306401 to grow on usual biological media, its carbon utilization, and its physiological characteristics are presenting in the following tables.

Table 1 : Growth on various biological media

Culture medium Culture characteristics

yeast extract/ growth: good malt agar substrate mycelium: orange-ocher soluble pigment: none

oatmeal growth: good substrate mycelium: dark ocher soluble pigment: none

glucose-asparagine growth: good substrate mycelium: salmon soluble pigment: none

Inorganic salts/ growth: spare starch agar substrate mycelium: ocher soluble pigment: none

Sucrose/ growth: sparse nitrate agar substrate mycelium: weak salmon soluble pigment: none

Glycerol/ growth: good asparagine agar substrate mycelium: orange soluble pigment: none

Nutrient agar growth: sparse substrate mycelium: salmon soluble pigment: none

Table 2 : Carbon utilization good: arabinose, sucrose, xylose, rhamnose, m-inositol medium: glucose, mannose, fructose negative: cellulose, raffinose

Table 3: Physiological characteristics

nitrate reduction: positive but limited starch hydrolysis: negative tyrosine degradation: positive but limited milk peptonization: positive melanin formation: negative on peptone-yeast-iron-agar; positive on tyrosine agar gelatine liquefaction: negative growth temperatures: 13-37°c. No growth at 45°C. ^ily sparse at 13°C. pH-range: 5 to 9, but onl_^ sparse growth at pH 5

NaCl resistance: up to 2%.

As with any microorganism, the strain A92-396401 can be mutated or modified into different forms by conventional techniques, e.g., by UV radiation or by treatment with a chemical mutagen such as N-methyl-N'-nitro-nitrosoguanidine. Recombinant clones can be obtained by protoplast fusion. It is intended that all such mutants or recombinants or modified forms, capable of producing the Macrolides and related compounds in a quantity at least as great as described in the example be within the scope of this application.

b. Culture conditions

The producing strain may be cultured at suitable temperatures on various culture media using appropriate nutrients and mineral substances, as aerobic or immersion cultures. The fermentation media should contain a utilizable source of carbon, sources of nitrogen and mineral salts including trace elements, all of which can be added in the form of well defined products or as complex mixtures, as are found in biological products of various origins.

i) Agar starting culture

Agar slant cultures of the strain DSM 8429 are grown for 10 to 14 days at 27°C on the following agar medium:

Glucose lO.Og

Soluble starch 20.0g

Yeast extract 5.0g

(Gistex, Gist Braocades)

NZ-Amine,Type A (Sheffield) 5.0g

Calcium carbonate l.Og

Agar (Bacto) 15.0g

Demineralized water to 1000ml

The medium is adjusted to pH 6.6-6.8 with NaOH/H₂SO₄, then sterilized for 20 min. at 121°C. The cultures can be stored at -25°-70°C. Suspension in glycerol-peptone can be stored under liquid nitrogen.

ii) Preculture

Ten starting cultures are suspended in 100 ml of a 0.9% salt solution. Five 2 Liter-Erlenmeyer flasks each containing 1 liter of preculture medium are inoculated each with 20 ml of this suspension. The composition of the preculture medium is as follows:

Glucose (technical) 7.50g Yeast extract (BBL) 1.35g

Malt extract liquid (Wander) 7.50g

Starch soluble 7.50g

NZ-Amine,Type A (Sheffield) 2.50g

Soya protein (Siber Hegner) 2.50g

L-asparagine l.OOg

Glycerin 7.50g

KH₂PO₄ 0.25g

K₂HPO₄ 0.50g

MgSO₄ ^• 7H2O 0.10g

CaCO₃ 0.050g

NaCl 0.050g

Trace element solution A 1ml

Agar (Bacto) ig

Demineralised water to 1000ml

The medium is adjusted to pH 6.8-7.2 with NaOH/H₂SO₄ and sterilized for 20m at 121°C.

The composition of the trace element solution A is as follows:

FeSO₄ 7H₂O 5.0g

ZnSO₄ ^• 7H₂O 4.0g

MnCl₂ 4H₂O 2.0g

CuSO₄ 5H₂O 0.2g

CoCl₂ ^• 6H₂O 2.0g

H,BO₃ O.lg

KI 0.05g

H₂SO₄ (95%) 1ml Demineralized water to 1000 ml

The precultures are fermented for 96 hr. at 27 °c on a rotary shaker at 200 rpm with an eccentricity of 50mm.

iii) Intermediate culture

One 60 liter bioreactor containing 50 liters of preculture medium is inoculated with 5 liters of the preculture and fermented for 72 hr. at 27°C. The fermenter is rotated at 150 rpm. during the first 48 hours and thereafter at 200 rpm. Air is introduced at a rate of 0.5 liter per minute per liter medium.

iv) Main culture

One 600 liter fermentation vessel containing 500 liters of the main culture medium is inoculated with 50 liters of intermediate culture. The composition of the main culture medium is as follows:

Mannitol 20.0g

Yeast Extract (BBL) l.Og

Pharmatone 20.0g

KH₂PO₄ O.lg

MgSO₄ ^• 7H₂O 0.05g

CaCl₂ 6H₂O 0.02g

Trace element solution A 1ml

Agar (Bacto) ig demineralised water to 1000ml. The pH is adjusted to 6.5 with KOH/H₂SO₄. The medium is sterilized for 20 min at 121 °C. The main culture is incubated for five days at 24°C. The fermentation vessel is rotated at 60 rpm during the 24 first hours and herafter at 80 rpm. Air is introduced at 0.5 liter per minute per liter medium during the 24 first hours and thereafter at 0.8 liter per minute per liter medium.

EXAMPLE II - Isolation and purification of compounds

The whole broth of a 700 liter fermentation is stirred twice for 2 hours with 700 liters of ethyl acetate in a Dispax apparatus and thereafter the two phases are separated in a separator. The combined ethyl acetate phases are evaporated to dryness under reduced pressure. The crude extract is then defatted by separation with 3x 101 of methanol/water 9:1 and 3x 101 of hexane. The methanol/water phases are combined and the methanol is distilled off under reduced pressure. The residue is extracted twice with 5 liter ethyl acetate. Evaporation to dryness under reduced pressure gives ca. 250 g defatted extract.

The extract is dissolved in 1 liter of ethyl acetate and 600 g of silica gel are added and evaporated to dryness under reduced pressure in a rotary evaporator. This impregnated silica gel is added to a column of 1 kg silica gel and the components of the extract are separated by chromatography using methyl-tertiary -butyl-ether (MTBE)/hexane 1 :1 as eluent. The polarity of the eluent is increased by using MTBE/hexane 3:1, MTBE, MTBE + 5% methanol, MTBE+ 10% methanol and methanol. Fractions of 2 liters are collected and analyzed by HPLC and MBA (macrophilin binding assay). Fractions 3 through 11 showing good MBA activity are combined and kept in 500 ml methanol solution. A precipitate of inactive material is formed which is removed by filtration. The filtrate is added to a column of 1 kg Sephadex LH20 and chromatography is started collecting fractions of 800 ml. Fractions 3 and 4 are taken together and again chromatographed on a column of 1.25 kg Sephadex LH20 in methanol solution. Active fractions 2 to 6 from this separation are taken for further chromatographic separation on a column of 3 kg Lichroprep RP18 (Merck) in methanol water 8:2. The most active fractions are combined and separated on a column of 300 g Silicagel H with a gradient hexane/acetone 8:2 to 1:9. Fractions 3 and 5 are the most active ones in the MBA test showing that more than one compound is responsible for the biological activity. Fraction 3 is again chromatographed on a 3 kg column filled with Lichroprep RP18 in methanol/water 8:2. Fractions 3 to 6 from this run are dissolved in 1 ml methanol to give crystals of macrolide Compound C. The mother liquor is further separated on 20 g silica gel with methylenchloride/methanol/water 92:7.5:0.5. With the aid of HPLC, crystals of pure macrolide Compound D can be collected. The fractions 8 to 11 of the reversed phase chromatography are dissolved in 5 ml methanol, resulting in crystallization of Compound A. Fraction 5 is taken for another chromatography run on 3 kg Lichroprep RP18 with methanol/water 8:2. HPLC analysis reveals that fraction 6 and 7 comprise compound B.

Compound A molecular formula C₃₇H₅₇NO₉ (659.87)

UV (MeOH) absorption: 222 nm (extinction 4.19)

IR (KBr crystal) spectrum is given in Figure 1

Mass spectrum is given in Figure 2

Compound B molecular formula C₃₇H₅₇NO₁₀ (675.87)

UV (MeOH) absorption: 222 (extinction 4.07)

IR (KBr crystal) spectrum is given in Figure 3

Mass spectrum is given in Figure 4

Compound C molecular formula C₃₈H₅₉NO₁₀ (689.89)

UV (MeOH) absorption: 223 nm (extinction 4.08 ) IR (KBr crystal) spectrum is given in Figure 5 Mass spectrum is given in Figure 6

Compound D molecular formula C₃₆H₅₅NO₉ (645.84)

UV (MeOH) absorption: 223 (extinction 4.07)

IR (KBr crystal) spectrum is given in Figure 7

Mass spectrum is given in Figure 8

EXAMPLE HI - Inhibition of adhesion molecule expression and cell proliferation

a. ICAM-1 Cell ELISA

The ICAM-1 cell Elisa used to determine inhibition of ICAM-1 expression is substantially as described by Winiski and Foster (1992, J. Invest. Dermatol., 99, 48-52). HaCaT cells (a spontaneously-transformed, non-tumorigenic human keratinocyte cell line with highly preserved phenotypic differentiation characteristics of normal keratinocytes (Boukamp et al., 1988 J. Cell Biol. 106. 761-771)) are seeded in 96 well microtiter plates (2xl0⁴ cell_ well in culture medium: DMEM with 5% FCS, 100 U/ml Penicillin, lOOμg/ml Streptomycin, 2mM Glutamine, 1 mM Na Pyruvate), grown to confluency, and then incubated in fresh test medium (as for culture medium but with 0.5% FCS instead of 5%) with or without IFN-γ/TNF-α stimulation medium (test medium + 1000 U/ml IFN-γ / 3ng/ml TNF-α) both in the presence and absence of compound A or compound B for ca. 24 hrs.. The medium is then washed away and the cell monolayers are fixed with 1% parafomraldehyde. The monolayers are incubated with saturating amounts of primary (mouse anti-ICAM-1 monoclonal) and secondary (goat anti-mouse peroxidase conjugated) antibodies. The subsequent peroxidase reaction uses 3-amino-9-ethylcarbazole (AEC) as substrate and generates an insoluble, colored product, which is easily measured in a standard microtiter plate reader. After the AEC reaction to detect ICAM-1 is completed, the HaCaT monolayers, are rinsed with PBS (200 μL), the PBS is poured off from the plates which are then patted dry on top of a paper towel to remove excess liquid. The bottom surfaces of the microtitre plates are gently wiped with a moist facial tissue and then again with a dry facial tissue and absorbance read at 492nm. Before the monolayers can dry out 0.1 ml of 0.1% crystal violet solution in PBS (passed first through a 0.2 μm filter) is added to each well. The plates are then incubated at room temperature for 10 minutes, washed thoroughly 5x with PBS, excess fluid removed as described above and their absorbance read again at 492nm before the monolayers are able to dry out. Subtraction of optical densities before and after staining gives values due to crystal violet staining and is hence related to the amounts of cell monolayer present in the wells. These values are used to correct the AEC values.

Compounds tested were active in this assay, e.g., Compound B has an IC50 of ca. 5 μM in this assay.

b. VCAM-1 (or E-selctin) cell ELISA

The assay employs non-hazardous, non-radioactive, commercially available reagents. It is based on a 96-well cell Elisa method using the human microvascular endothelial cell line HMEC-1. Cells are pretreated for four hours with the test compounds/broth extracts, stimulated for the next 16-18 hours with a cytokine "cocktail" (TNFα/INFγ), then parafomaldehy de-fixed for subsequent evaluation of VCAM-1 or E-selectin expression by an indirect immunoperoxidase staining technique. Cytotoxic effects are determined by counting the relative number of cells (Giemsa nuclear stain) after exposure to the test substances, in comparison to the control wells (solvent and media only). Compounds are scored positive if they exhibit >50% VCAM-1 or E-selectin inhibition with <25% cell loss.

The VCAM-1 /E-selectin assay utilizes an immortalized (SV-40 virus large T antigen) human microvascular endothelial cell line (HMEC-1; Ades et al., Jrl Invest Dermatol 99: 683-690, 1992). HMEC-1 cells constitutively express low levels of ICAM-1 which are upregulated by inflammatory mediators. However, they only express VCAM-1 or E-selectin following cytokine stimulation. Dose-response and time-course experiments are performed to determine the optimal conditions for inducing VCAM-1 and E-selectin expression.

HMEC-1 cells are grown in T-75 flasks (Nunc) under standard conditions (37°C, 5% CO₂) with 1.5 x 10⁶ cells/ml culture medium (CM = Endothelial Cell Basal Medium [EBM; Clonetics] supplemented with 10% FCS, 10 ng/ml human EGF (Boehringer), lμg/ml hydrocortisone (Sigma # 0888), 2.2 g/1 NaHCO₃, 15 mM Hepes, 0.11 g/1 sodium pyruvate, 4 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin). After mild trypsinization (0.25% trypsin + 0.1% EDTA for 8 min) and resuspension, the cells are reseeded every 2-3 days at a 1 :3 splitting ratio.

96 well flat-bottom microtiter plates are precoated with bovine fibronectin (FN; Sigma # Fl 141) and then seeded with 2 x 10⁴ cells/well in 200 μl of EBM growth medium and incubated overnight. The following day the culture medium (CM) is initially replaced with 200 μl/well of EBM assay medium (CM supplemented with 5% FCS instead of 10%) and subsequently replaced with 180 μl of medium containing either (1) appropriate concentrations of compound A or compound B, (2) corresponding concentrations of solvent/methanol- extracted medium, or (3) EBM assay medium alone and incubated for 4 hr at 37° C. Each 96-well assay is performed with duplicate wells. The cells are then stimulated by adding 20 μl of concentrated cytokine solution (1500 U/ml INFγ + 2000 U/ml TNFα) and incubated for 16-18 hr at 37° C.

The cell monolayer is then washed with 1 % paraformaldehyde in EBM medium, fixed in 2% parafomaldehyde for 15 min at room temperature (RT) and rinsed several times with PBS. The PBS is removed from the cells, and the monolayer is incubated for 30 min in PBS containing 10% normal goat serum (NGS). The NGS solution is replaced with 100 μl/well of the anti-VCAM or anti-ELAM monoclonal antibody (mAb; 1:500 dilution, both from Genzyme) in PBS containing 5% NGS) and incubated overnight at 4° C. The mAb solution is then removed and the cells rinsed several times with PBS, followed by incubation with PBS containing 10% NGS for 30-60 min at RT. The NGS solution is removed and lOOμl of horseradish peroxidase-conjugated goat F(Ab')₂ anti-mouse IgG antibody (Tago; 1:500 dilution in PBS containing 5% NGS) is added and the plates incubated for 1 hr at RT. The secondary antibody is then removed and the cells rinsed in PBS, which is then replaced with 150 μl/well of a freshly-prepared and filtered AEC solution (3-amino-9ethyl-carbazole; Sigma) and the plates incubated for 45-60 min at RT. The peroxidase substrate is removed and the cells rinsed in PBS. AEC absorbance values are read on a microtiter plate reader at 550 nm and corrected for "blank" or reference values at 690 nm.

The cells are destained by replacing the PBS with 95% ethanol for 20 min (two 10 min changes) with control by microscopic evaluation. The cells are then rinsed in distilled water (Aquadest) and the monolayer covered with a 33% Giemsa solution in Aquadest for 5 min at RT. The wells are then washed with Aquadest and air dry for at least 15 min. Microscopic evaluation is used to check that only the nuclei are stained, with essentailly no cytoplasmic staining. Giemsa absorbance values are read on a microtiter plate reader at 550 nm and corrected for "blank" values (rows without cells) at 690 nm.

The AEC values for constitutive VCAM-1 or E-selectin expression (unstimulated control wells) are essentially equal to those of an isotype-matched control mAb and represent the background stain. In every 96-well plate, the mean constitutive value is subtracted from the mean AEC value for each cytokine-stimulated group (EBM and solvent controls, as well as test substance), resulting in a number which represents inducible VCAM-1 or E-selectin Cell adhesion molecule (CAM) expression (referred to as AEC-CAM). Each AEC-CAM value is then divided by the corresponding mean Giemsa value, resulting in a number which estimates relative levels of VCAM-1 or E-selectin expression for a given cell density, based on the number of nuclei (referred to as AEC: Giemsa ratio).

AEC (stimulated) - AEC (unstimulated) = AEC-CAM

AEC-CAM / Giemsa = AEC:Giemsa ratio

Therefore "actual" VCAM-1 or E-selectin IC50 values are determined by comparing the AEC:Giemsa values for a test substance with those of the stimulated control (EBM, solvent). These values are then analyzed relative to the IC50 values for Giemsa alone. Strict criteria determine whether the VCAM or E-selectin inhibition versus cytotoxicity (Giemsa) profile indicates a "real" hit which should be pursued.

Compounds of the invention tested, e.g., Compound A and B, show inhibition of VCAM expression in this assay with an IC₅₀ of less than 2 μM.

Compounds of the invention tested in an ELAM expression ELISA using swine endothelial cells were active in suppressing ELAM expression as well; Compound B, for example, had an IC50 of 0.6 μM.

c. HaCaT cell proliferation assay

HaCaT cells are cultivated in DMEM (Gibco # 074-02100) supplemented with 2.2 g/1 NaHCO₃, 0.1 1 g/1 sodium pyruvate, 15 mM Hepes, 5% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 μg/ml), and glutamine (to increase the final concentration by 4 mM). For the proliferation assay, cells are detached by trypsinization, suspended in fresh medium, and seeded into 96-well microtiter plates at a final density of 4000 cells/0.2 ml/well. After 24 hours (day 0) the medium is replaced with fresh medium containing graded concentrations of test compound. After 3 days of incubation at 37°C/5%CO₂, the extent of cellular proliferation in comparison to solvent controls is measured by a colorimetric assay that measures relative cell mass using the dye sulforhodamine B (Skehan et al, 1990, J. Natl. Cancer Inst. £2, 1107-1112). The "starting cell number" is determined by measuring the relative cell mass on day 0. The results are expressed as % Inhibition = 100 - % control absorbance (where solvent control = 100%) and represent the average ± standard deviation of three measurements. A dose-response curve is plotted semi-logarithmically and the concentration required for half-maximal inhibition (IC₅₀) is determined by linear interpolation. Maximal inhibition without net loss of cells is represented by the "starting cell number" and is usually between 90-98%. Compounds of the invention tested in this assay, e.g., Compound B, are active with an IC_J0 of ca. 2 μM.

Claims

1. A macrolide of formula I:

Formula I

wherein

R, is H, alkyl, aryl, or acyl;

R₂ is H, or -OR₅, such that R₅ is H, alkyl, aryl, or acyl;

R₃ is -CH₂- or -CH₂CH₂-; and

R₄ is H, alkyl, acyl, or aryl; wherein "alkyl" refers to C__₆ alkyl; "aryl" refers to an aromatic hydrocarbon radical having one or two aromatic rings; and "acyl" is alkylcarbonyl or arylcarbonyl.

2. A macrolide according to claim 1 of formula Ia

Formula Ia

wherein R, is H or -CH₃; R₂ is H or -OH; and R₃ is -CH₂- or -CH₂CH₂-

3. A macrolide according to claim 2 wherein a) R, is H, R₂ is H, and R₃ is -CH₂CH₂-; b) R, is H, R₂ is -OH, and R₃ is -CH₂CH₂-; c) R, is CH₃, R₂ is -OH, and R₃ is -CH₂CH₂-; or d) R, is H, R₂ is H, and R₃ is -CH₂-.

4. A process for the production of a macrolide comprising cultivating a producing strain of the genus Micromonospora in an appropriate culture medium and isolating the macrolide.

5. A process according to claim 4 wherein the producing strain is the deposited strain DSM 8429.

6. A macrolide produced according to the process of claim 4 or 5.

7. A macrolide according to any one of claims 1, 2, 3, or f use as a pharmaceutical.

8. A pharmaceutical composition comprising a macrolide a.» ording to any one of claims 1, 2, 3 or 6 together with a pharmaceutically acceptable diluent or carrier.

9. Use of a macrolide according to any one of claims 1, 2, 3, or 6 in the manufacture of a medicament for:

(i) suppressing adhesion molecule expression or

(ii) treatment or prevention of any of the indications set forth above, e.g.,

(iii) treating or preventing acute or chrome inflammation (e.g. allergy, asthma, psoriasis, reperfusion injury, rheumatoid arthritis and septic shock) (iv) treating or preventing autoimmune autoimmune diseases (e.g. multiple sclerosis), (v) treating or preventing tumour metastasis (e.g. melanoma, osteocarcinoma), (vi) treating or preventing allograft/xenograft rejection, (vii) treating or preventing hyperproliferative skin diseases (e.g. psoriasis), (viii) treating or preventing dermatitis, (ix) treating overdoses of a macrophilin binding immunosuppressant, e.g., FK506 or rapamycin; (x) potentiating the action of steroids, (xi) treating or preventing infectious diseases, e.g., prophylaxis or treatment of infections or infectuous diseases caused by organisms producing Mip or Mip-like tors.

10. A biologically pure culture of DSM 8429.