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She present invention relates to pharmaceutical compounds and, more particularly, to lipophllio pyrazin- a idβ prodrugs, which possess antimicrobial activity as ve l as compositions containing these prodrugs and methods of use.

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Microorganisms have adopted a wide variety of strategies which enable them to colonise a host. Among those organisms which are pathogenic those which are capable of causing or residing in the intracellular milieu are among those which are most difficult to treat. Such organisms are of a wide variety and include bacteria, protosoa, fungi, and viruses.

A typical representative of this group is Jfyoαbaσtarium aviun-ifltracβllulere complex (KAIC) . The Committee on

M, intrmcβllulΛrΛ Disease of the National Consensus Conference on Tuberculosis has reported in 1985 that in parts of the U.S., pulmonary MAZC disease is as common as tuberculosis (Ise an, et Λ1. _≠ Cheat, £2:1398, 1985). At the same time, an unprecedented increase in incidence of MAIC disease in the Acquired Immune Deficiency Syn- drome (AIDS) patient population has been reported. A

1985 study (published in March 1987) by the Cleveland Clinic Foundation noted that "slightly over one-half of the MAIC isolates were from patients with AIDS" (Woods, et al., Reviews of Infectious Dimeeice. _ \275,1987).

It has long been recognized that intracellular pathogens occupy a pharmacological sanctuary which often serves to protect the microorganisms from what might otherwise be an effective chemotherapeutic agent, studies have con irmed the concept that the efficacy of certain chemotherapeutic agents in treating pathogens which can •urvive through intracellular colonisation is related to the ability of the antibiotic to transit the host cell membrane. This problem is further complicated by the fact that drug* are normally designed to be as water soluble (hydrophilic) as possible in order to allow ready dissolution in an aqueous environment, unfortu¬ nately, such hydrophilic drugs are often incapable of adequately traversing the host cell membrane in order to exert the chemotherapeutic effect on the intracellular pathogen. On the other hand, while lipophilic (oil¬ like) drugs should be more likely to be absorbed into the intracellular domain of the host cell, their lipo¬ philic nature tends to make them insoluble such that they cannot be utilized intravenously and, as a conse- quence, must typically be administered in oral dosage form. Although the most convenient type of administra¬ tion, oral delivery of drugs is the least efficient with respect to the amount of drug reaching the target site ("bioavailability") which may be due to poor absorption through the gut, high metabolism (inactivation) in the liver, high systemic distribution into all body tissues, or a combination of some or all of the above.

In order to overcome the problems of drug delivery associated with intracellular parasites, there has been »uch interest in the use of targeted, or βitβ-dirβctβd, drug delivery systems such as liposomes. A number of 5 publications describe the combination of lipophllio anticancer prodrugs and liposomes, mainly to reduce toxicity, prolong the circulation half-life, or inhibit rapid metabolism of the anticancer drug (Schwendener, et al. _r Blochemica et Biophysica Research Communications _/ 10 12£:6β0, 1985; Rubaβ, et al.. International Journal of Cancer, 22:149, 1986; Superβaxo, et al , , Journal of Jficroencα sulation, £:1, 1988; Matsushita, et al . , Cancer Research, 41:2707, 1981; Hong, et al. , Journal of Medical Chemistry, 2&:171, 1985).

15 Pyrazinamide is a drug of moderate efficacy when admin¬ istered to patients suffering from pulmonary in ec¬ tions. However, because it is quite toxic, pyrazinamide therapy should only be utilized if other therapy fails. In the case of pulmonary M. avium-intraαellulare

20 infections, the organisms reside in pulmonary alveolar macrophages such that the organism is protected rom the drug which, in turn, allows the macrophages to act as a reservoir for chronic infection. Pyrazinamide is poten¬ tially more efficacious and less toxic when given in

25 encapsulated form, for example, in a liposoae, either directly to the lung via inhalation or systemically via intravenous injection. While inhalation is indicated for patients with an infection con ined to the lung, intravenous injection would be the route of choice for

30 patients suffering from disseminated infection, for

•xample, patients with AIDS who are suffering from Mycobacterium aviwα-intracellulare infection. Unfortu¬ nately, drugs which are highly water soluble, such as pyrazinamide, cannot be incorporated in liposomal or emulsion drug carriers in a stable fashion for prolonged periods of time. This is due to the fact that such drugs will diffuse out of the liposome into the water phase and, in essence, exist as though the carrier ere not present. This phenomenon of water soluble drug instability is known aβ "leakage".

As a consequence of the significant drawbacks of prior researoh, a strong need exists for new chemotherapeutic agents which are especially effective in the treatment of intracellular pathogenic infections. Especially useful would be a lipophllio antimicrobial drug which could be used in combination with a targeted delivery system.

fPHMARY OT MB MVBMTIOM

Recognizing the role that intracellular pathogens play in chronic infectious diseases and the severe therapeu¬ tic limitations of pyrazinamide, the inventors produced 5 and evaluated novel lipophilic prodrugs in an effort to develop a more effective chemotherapeutic agent. These e orts have culminated in the production and identifi¬ cation of new chemotherapeutic agents which are particu¬ larly effective against intracellular pathogens.

10 These prodrugs wore developed through the lipophilic derivatization of pyrazinamide which, unlike pyrazin¬ amide, can be stably incorporated into a targeted drug delivery system, such as a liposome. Surprisingly, these prodrugs are also capable of exerting an antimi-

15 crobial effect at much lower concentrations than seen with pyrazinamide.

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The present invention relates to novel lipophilic pro- drugs of pyrazinamide which are useful in the treatment

20 of diseases caused by intracellular pathogens. Because of their lipophilic nature, these prodrugs are particu¬ larly amenable to incorporation in a targeted drug delivery system, such as a liposome. The prodrugs comprise a core comprising pyrazinamide which is materi-

25 ally modified to have from about 13 to about 17 carbon atoms _*

Prodrugs consist of a parent drug and a second chemical moiety covalently linked to the parent drug which pro¬ vides certain desired modifications of the physicochemi- oal properties of the parent drug. Prodrugs can also be designed to protect drugs from rapid degradation, to direct drugs to specific areas in the body, or to make drugs more hydrophilic. A prodrug can be active in itself, or be inactive and become active following chemical or enzymatic cleavage and release of the origl- nal drug.

The term "effective amount" denotes both microbicidal (killing) or mlcrobistatic (inhibiting) activity of a compound or composition of the invention _*

The prodrugs of the invention are effective en intracβl- lular pathogens such as bacteria, viruses, fungi, and protozoans. These prodrugs are especially effective against bacteria of the genus Nyeobaeteriua.

The lipophilic prodrugs of the invention have the formula

wherein R, is selected from saturated or unsaturated straight or branched chain alkyl moieties having from about 13 to about 17 carbon atoms. Preferred compounds wherein K, is lauroyl, syristoyl, palaitoyl, and stear- oyl.

The lipophilic prodrugs of the invention can be prefera¬ bly synthesized by the reaction of pyrazinamide with an acid chloride in the presence of chloroform. Preferred are acid chlorides of the formula

,0

*< α

5 wherein R is from about 12 to about 16 carbon atoms.

Other functional groups, for example, other halogens or an anhydride, that yield the amide bond, can be substi¬ tuted for chlorine to form the lipophilic acylpyra- sina ide prodrug and are within the scope of the inven- 10 tion. such functional groups are known or readily diβcernablβ by those of skill in the art without undue experimenta ion.

R, and Sg are selected from saturated or unsaturated, straight or branch chain alkyl moieties _* Other R, and 15 K _f groups that function aquivalently to yield a lipo¬ philic aeylpyrazinamide are known to those of skill in the art and are within the scope of the invention.

The prodrugs of the invention are especially suited for use in targetable drug delivery systems such as synthet-

20 ie or natural polymers in the form of macromolecular complexes, nanocapsules, microspheres, or beads, and ipid-baβed systems including oil-in-water emulsions, micelles, mixed micelles, liposomes, and reβealed eryth- rocytes. These systems are known collectively as eol-

25 loidal drug delivery systems. Typically, such colloidal

particles containing the dispersed drugs are about 50 nm - 2 iM in diameter. The size of the colloidal particles allows them to be administered intravenously such as by injection, or as an aerosol. Materials used 5 in the preparation of colloidal systems are typically sterilizable via filter sterilization, nontoxic, and biodegradable, for example albumin, ethylcellulose, casein, gelatin, lecithin, phospholipids, and soybean oil. Polymeric colloidal systems are prepared by a 10 process similar to the coacervation of microencapsula- tion.

Most preferred as a targeted delivery system for the prodrugs of the invention are liposomes. When phospho¬ lipids are gently dispersed in aqueous media, they

15 swell, hydrate, and spontaneously form multilamellar concentric bilayer vesicles with layers of aqueous media separating the lipid bilayer. Such systems are usually referred to as multilamellar liposomes or multilamellar vesicles (M Vs) and have diameters ranging from about

20 lOOn to about 4um. When MLVs are sonicated, small unila ellar vesicles (SOVs) with diameters in the range of from about 20 to about 50 nm are formed, which con¬ tain an aqueous solution in the core of the SUV.

The composition of the liposome is usually a combination 25 of phospholipids, particularly high-phase-traneition- temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.

Examples of lipids useful in liposome production include 30 phosphatidyl compounds, such as phosphatidylglycerol.

5855

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phosphatidylcholine, phosphatidylserine, phosphatidyle- thanolaaine, sphingolipids, cerebroβideβ, and ganglio- sideβ. Particularly useful are diaoylphosphatidyl- glycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and are saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalnitoylphosphatidyloholine, and diβtearoylphosphatidylcholine.

In preparing liposomes containing the prodrugs of the invention, such variables as the efficiency of drug encapsulation, lability of the drug, homogeneity and size of the resulting population of liposomes, drug-to- lipid ratio, permeability instability of the prepara¬ tion, and pharmaceutical acceptability of the formula- tion should be considered. (Szeka, et al.. Annual Re¬ views of Biophysics and Bloengineering , £t467, 1980; Deamer, et al., in Liposomes, Marcel Dekker, New York, 1983, 27; Hope, et al. , Chem. Phys _* Lipids, A£:89, 1986) .

The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classi¬ fication is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle- specific. Mechanistic targeting can be further diβtin- guished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the retioulo-endo- thelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves the alteration of the liposome by coupling the

liposo e to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposomes themselves in order to achieve targeting to organs and cell types other than the naturally occurring sites of localisa¬ tion. Alternatively, liposomes may physically localize in capillary beds such as the lung or may be given by site-specific injection.

Another targeted delivery system which can be used with the prodrugs of the invention is resealed erythrocytes. When erythrocytes are suspended in a hypotonic medium, swelling occurs and the cell membrane ruptures. As a consequence, pores are formed with diameters of approxi¬ mately 200-500 A which allow equilibration of the intra- cellular and extracellular environment. If the ionic strengths of this surrounding media is then adjusted to isotonic conditions and the cells incubated at 37*C, the pores will close such that the erythrocyte reseals. This technique can be utilized with the prodrugs of the invention to entrap the prodrug inside the resealed erythrocyte. The resealed erythrocyte containing the prodrug can then be used for targeted delivery.

The surface of the targeted delivery system may be modified in a variety of ways. Non-lipid material nay be conjugated via a linking group to one or more hydro- phobic groups, for example, alkyl chains from about 12- 20 carbon atoms. In the case of a liposoaal targeted delivery system, lipid groups can be incorporated in to the lipid bylayer of the liposome in order to maintain the compound in stabile association with the liposomal

bilayer. Various linking groups can then be used for joining the lipid chains to the compound.

Whether a ligand or a receptor, the number of molecules bound to a liposome will vary with the size of a lipo- some, as well as the size of the molecule, the binding of affinity the molecule to the target cell receptor or ligand, as the case may be, and the like. In most instances, the bound molecules will be present on the liposome in from about 0.05 to about 2 mol%, preferably from about 0.1 to about 1 mol%, based on the percent of bound molecules to the total number of molecules in the outer membrane bilayer of the liposome.

In general, the compounds to be bound to the surface of the targeted delivery system will be ligandβ and recep- tors which will allow the targeted delivery system to actively "home in" on the desired tissue, A ligand may be any compound of interest which will specifically bind to another compound, referred to as a receptor, such that the ligand and receptor form and a homologous pair. The compounds bound to the service of the targeted delivery system may vary from small haptens of from about 125-200 molecular weight to much larger antigens with molecular weights of at least about 6000, but generally of less than 1 million molecular weight. Proteinaceous ligandβ and receptors are of particular interest _*

In general, the surface membrane proteins which bind to specific effector molecules are referred to as recep¬ tors. As presently used, however, most receptors will be antibodies. These antibodies may be monoclonal or

polyclonal and may be fragments thereof such as Fab, and F(ab') _t , which are capable of binding to an epitopio determinant.

The targeted delivery system containing the prodrug of 5 the invention may be administered in a variety of ways to a host, particularly a mammalian host, such as intra¬ venously, intramuscularly, subcutaneously, intraperi- toneally, intravascularly, topically, intraαavity, transdermally, intranasally, and by inhalation. The

10 concentration of the prodrug will vary upon the particu¬ lar application, the nature of the disease, the frequen¬ cy of administration, or the like. The targeted deliv¬ ery system-encapsulated prodrug may be provided in a formulation comprising other drugs as appropriate and an

15 aqueous physiologically acceptable medium, for example, saline, phosphate buffered saline, or the like.

The above disclosure generally describes the present invention. A further understanding can be obtained by reference to the following specific examples Which are 20 provided for purposes of illustration only and are not intended to be limiting unless otherwise specified.

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Pyrazinamide (I), acetic anhydride, caproyl chloride, capryl chloride, palmitoyl chloride, and silica gel (70- 5 230 mesh) were obtained from Sigma, St. Louis, MO. All other solvents were from Fisher, Springfield, NJ. All chemicals and solvents were of reagent grade and were used as received. N-Acetylpyrazinamide (II) was pre¬ pared according to the method of Kuβhner, et al . (Jotir-

10 naJ of the American Chemical Society, 21*3617, 1952) as illustrated in Scheme I. Purification in benzene was replaced with separation on a silica gel column. A mixture of 4 g (0.032 mol) of (I) and 20 ml (0.21 mol) of acetic anhydride was refluxed for 75 minutes at

15 approximately 140*C. After cooling to room temperature, a precipitate consisting of unreaoted (I) appeared. The precipitate wes filtered off and the solution evaporated to dryneβs under vacuum. The residue was dissolved in chloroform and loaded onto a silica gel column (21mm x

20 30 em) . The column was eluted with 250 ml chloroform, followed by a 80:1 (vol/vol) mixture of chloroform methanol. The fraction containing the pure product (II) was evaporated to drynesβ in vacuo at 70'C The yield was 2.5g (47.3%) of a colorless solid.

25 N-λcylpyrazinamides (lll)-(V) where prepared ae noted in Scheme 2. A mixture of 200 ml chloroform and a few drops (approximately 0.25 ml) of aoetylchloride were refluxed for about 30 minutes in a 250 ml three-necked round-bottom flask in order to remove moisture from the

30 chloroform and glass surfaces. Next, 4.3 g (0.035 mol) of (I) and amounts of either caproyl chloride, capryl

chloride and pal itoyl chloride corresponding to 0.03 mol each were added and the mixture refluxed for 24 hours. While refluxing, a total of 4.74 g (0.06 mol) of pyridine was added dropwise to the solution. Upon 5 completion of the reaction, the solution was cooled to room temperature and the needle-shaped crystals of unreacted (I) were removed by filtration. The solution was concentrated to approximately 20 ml by rotoevapor- ation and washed three times with 15 ml of cold St NaOK

10 solution, followed by repeated washing with water until the wash phase was neutral. The chloroform layer was dried with enhydrous sodium sulfate. The solution was loaded onto a silica gel column and eluted with chloro¬ form (III), chloroform/ethanol 9:1 (vol/vol) (IV), or

15 chloroform/ethanol 8:1 (vol/vol) (V), respectively. The yields of the individual reactions were 47.3% (II), 31% (III) , 25 % (IV), and 19% (V).

EP-HPU5 I

CHEMICAL ANALYSIS OF K-ACYLPYRAlimNIMS

20 The structural identification data including elemental analysis, 1H-NMR and mass spectra are summarized in Table 1. Elemental analysis was performed by Atlantic Microlabs, Inc., Atlanta, Gλ. Proton nuclear magnetic resonance spectra were recorded on a Varian EK390 NHR

25 spectrometer at 90 MHz. Chemical shifts were reported as parts per million (f) relative to the internal stan¬ dard tetra ethylsilane. Chemical ionization high reso¬ lution mass spectra were performed on a AEI MS30 mass spectrometer. Differential scanning calorimetry (DSC)

30 was performed on a Parkin-Elmer DSC4 calorimeter. The HPLC set-up consisted of a Rabbit-HP pump (Rainin)

equipped with a M190 injector, a Waters model 440 abβor- bance detector, set to 254 nm, and a Fisher Reαordall series 5000 recorder. Phosphate buffer (pH 3.4)/- acetonitril 95:5 (v/v) was used as mobile phase. An ODS 5 (Zorbax CDS) column (4.6mmx 15cm) was employed. The flowrate was set to l.m./min. Nicotinamide served as internal standard.

XfΛlΛ 1 STRUCTURAL IDENTIFICATION OF W-ACYLFYRASnmtlDM

10 . Mass

Compound Elemental analysis Η-NMR Spectra

%C,H,N Theoretical Spectral CU *

II C: 50.90 (50.91) 10.70, 9.26, 165.0 15 H: 4.31 (4.27) 8.93, 8.75,

N: 25.42 (25.44) 2.43

III C: 59.72 (59.71) 10.42, 9.26, 221.11 H: 6.83 (6.83) 8.86, 8.59,

N: 18.92 (18.99) 2.31, 1.25, 0.85

20 IV C: 64.86 (64.96) 10.30, 9.38, 277.18

H: 8.39 (8.36) 8.76, 8.56, 2.31, N: 15.05 (15.15) 1.73, 1.25, 0.86

V C: 69.75. (69.77) 10.50, 9.48, 361.27 H: 9.79 (9.76) 8.84, 8.62, 2.30, 25 N: 11.69 (11.62) 1.70, 1.25, 0.86

Yield, melting points and aqueous solubilities of the four products are shown in Table 2. Uncorrected melting points were determined on a Thomas Hoover capillary melting point apparatus.

TABLB 2 PHYfllCOCHBMICAL FSOPKM-IEB OT W-ACYLFYRASHIMrrnMB

Compound Yield (%) Melting Point (oC) Solubility (M)

*** micelle formation in aqueous solution

Rf-valueβ resulting from thin layer chromatography are shown in Table 3. Thin layer chromatography was per¬ formed on silica gel plates with fluorescent indicator (Eastman Kodak, Rochester, N.Y.) in chloroform (contain¬ ing 1.1% ethanol) as mobile phase, and UV detection at 254 nm.

T BU 3

« YM.VM Compound Rf Value

I 0.27

II 0.61

III 0.71

IV 0.74

V 0.77

KMffPLB 1

IN VITRO ANTIMICROBIAL ACTIVITY OF K-AOYLPTgaEIWlwrMsa

Five ml Middlebrook 7H9 broth in sterile 16X10Omm glass tubes with metal culture caps were inoculated with 70 ul 5 of MAIC (strain 8T48 4/89) stock suspension having

1.55X10 ⁷ bacilli/al and incubated in 5% CO, for 1 week in a humidified incubator. Drugs (I) - (V) were dis¬ solved in pure ethanol to give final concentrations of 0.1, 1.0, 10 and 100 ug/ml when added to the MAIC sus-

10 pension in 50 ul volumes. One untreated control and a control sample containing 50 ul ethanol without drug was included in the protocol. All samples were prepared in duplicate and incubated for 2 weeks with gentle agita¬ tion once e week. After this time, the samples were

15 diluted 1:1000 in Middlebrook 7H9 broth and plated on

Middlebrook 7H10 agar plates by placing ten 20ul drops/- plate such that the total volume per plate was 200 ul. The plates were incubated for 2 weeks under conditions as described above. Colony-forming units (CFU) were

20 counted on each side of the two halves of each duplicate plate. The results obtained are illustrated in Table 4 _*

25

As shown in Table 4, only compound V, N-palmitoyl pyra¬ zinamide showed a significant antimicrobial effect at 100 ul/ l. Concentrations of V at 0.1, 1.0, and 10.0 ug/ml, and compounds I-IV at all concentrations tested, showed no antimicrobial effect.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made without departing from the spirit or scope of the invention.