EP0575556A1

EP0575556A1 - Palytoxin-related prodrugs and a method for their administration

Info

Publication number: EP0575556A1
Application number: EP92910620A
Authority: EP
Inventors: Gary S. Bignami; Paul G. Grothaus
Original assignee: Hawaii Biotechnology Group Inc
Current assignee: Hawaii Biotechnology Group Inc
Priority date: 1991-03-15
Filing date: 1992-03-10
Publication date: 1993-12-29
Also published as: AU1762392A; EP0575556A4; JPH06508114A; WO1992016203A1

Abstract

La palytoxine et ses analogues naturels sont produits sous la forme de promédicaments transformés en dérivés, clivables au moyen d'un catalyseur biologique. Ces promédicaments sont utiles dans des protocoles dans lesquels on peut cibler le catalyseur sur des cellules ou des tissus que l'on veut altérer ou détruire.Palytoxin and its natural analogues are produced in the form of prodrugs transformed into derivatives, cleavable by means of a biological catalyst. These prodrugs are useful in protocols in which the catalyst can be targeted at cells or tissues that are to be altered or destroyed.

Description

PALYTOXIN-RELATED PRODRUGS AND

A METHOD FOR THEIR ADMINISTRATION

Technical Field

The invention is directed to prodrugs of palytoxin and its related compounds and to administration of such prodrugs in concert with a targeted releasing agent. The administration protocol permits the targeted releasing agent to mediate localized liberation of the toxic drug.

Background Art

Palytoxin is an extremely toxic, nonprotein- aceous, relatively low molecular weight marine natural product isolated from tropical coelenterates of the genus Palythoa. The toxicity of the coral Palythoa toxica was first recognized by the ancient Hawaiians. Special warriors were designated to smear the exudate of the coral on spear tips prior to combat. In modern times, this legend permitted researchers at the University of Hawaii to rediscover the single set of tidepools containing Palvthoa toxica near Hana on the island of Maui Moore, R.E., et al., Oceanus (1982) 25(2. ;54-63. From the coral collected in these tidepools, highly purified toxin was isolated in 1971, and the chemical structure was reported by Moore, R.E., et al., J Am Chem Soc (1981) 103.2491. and independently by Uemura, D. , et al., Tet Let (1981) 22.-2781, who isolated the toxin from the more abundant species Palythoa tuberculosa. Additional toxic related compounds, including palytoxin carboxylic acid, homopalytoxin, bishomopalytoxin, neopalytoxin and deoxypalytoxin have also been identified (Wattenberg, E.V. , et al.. Cancer Res (1989) 49_:5837-5842, and Uemura, D., et al., Tetrahedron (1985) 41(6. :1007-1017) . Figure 1 shows the structures of these compounds. Additional minor* components have also occasionally been detected in Palvthoa species.

Palytoxin contains an amino group at position 115 shown in Figure 1, which araino group has been derivatized with carboxylic acids to obtain acylated palytoxin derivatives that have been shown to be 100- 1000 times less cytotoxic than native palytoxin . (Wattenberg, E.V., et al., Cancer Res (1989) 49;5837- 5842, and Ohizumi, Y. , et al., Pharmacol Exp Ther (1980) 214(1. :209-212) . Such acylation has been limited to derivatives for analysis and structural elucidation studies. Hirata, Y. , et al.. Pure & Appl C em (1979) .51:1875-1883, described the acetylation of palytoxin with p-nitrophenyl acetate to yield N-acetylpalytoxin. This compound was also described by Uemura, D. , et al. , Tet Let (1980) 1:4857-4860, MacFarlane, R.D. et al., J Am Chem Soc (1980) 102f2. :875-876. and Moore, R.E., Progress in the Chemistry of Organic Natural Products (1985) £8.:82-202. In addition, Moore, R.E., et al. , J Am Chem Soc (1980) 102.:7370-7372, and Moore (1985) supra. prepared N-(p-bromobenzoyl)palytoxin, and N-(p-bromo- benzoyl)bisho opalytoxin was prepared by Uemura et al. (supra) . Levine, L. , et al., Toxicon (1988) 26(12) :1115- 1121, describe the preparation of N-3^,-(4"-hydroxy-3¹¹- I-iodophenyl)propionylpalytoxin for use in a palytoxin radioimmunoassay, and Sachinvala, N.D., et al., unpublished results (1985), prepared N-(4^l-(N"-malei- midomethyl)cyclohexane-1-carboxyl)palytoxin, and N-(3*-(2"-pyridyldithio)propionyl)palytoxin as haptens for conjugation to protein in the production of palytoxin immunotoxins and immunogens.

In vitro assays have shown that the foregoing amino acylated derivatives are less toxic than the native molecule. Ohizumi, Y. , and Shibata, S., 3 Pharmacol Exp Ther (1986) 214.1) :209-212. showed that N-acetylpalytoxin was 100 times less active than palytoxin in inducing contractions of the isolated guinea pig vas deferens. Wattenberg, E.V., et al.. Cancer Res (1989) 49:5837- 5842, showed that N-(p-bromobenzoyl)palytoxin and

N-acetylpalytoxin were at least 100 times less effective in down-modulating epidermal growth factor binding to 3T3 cells, and that they were also less cytotoxic than native palytoxin. An additional cytotoxicity assay employing EL-4 murine T-lymphocytes (Hewetson, J.F., et al., FASEB J (1989) 3(4. :A1191) was shown by the inventors herein to give a differential in toxicity between palytoxin and N-acetylpalytoxin of up to 500-fold. Wattenberg et al. .supra, have suggested that the N-acylated palytoxins may be virtually inactive and that the remaining cytotoxicity results from traces of the contaminating palytoxin.

None of the N-acylated palytoxin derivatives prepared have been designed as substrates for enzymatic hydrolysis. In order to take advantage of technology which permits localized production of activity, the invention compounds are provided in forms which are susceptible to cleavage by biological catalysts.

Early approaches to targeted prodrug conversion proposed the use of localized endogenous amidases to effect the hydrolysis of amide prodrugs. In particular, cyclophosphoamide was designed to be activated by phosphamidase which was thought to be elevated in certain epithelial tumors (Gomori, G. , Proc Soc Exp Biol Med (1948) ϋ9_:407). Although cyclophosphamide is clinically useful, the original theory for its in situ cleavage turned out to be inaccurate, and difficulty was experienced generally in finding enzymes which convert prodrugs selectively in tumor tissue. More recent approaches have focused on selective delivery of prodrug- hydrolyzing enzymes to target tissues using enzyme- antibody conjugates (Senter, P.D., et al., PNAS (1988) 8Σ5:4842-4846; Senter, P.D., et al.. Cancer Res (1989) 49:5789-5792; Bagshawe, K.D. , Br J Cancer (1989) 60:275- 281; U.S. patent 4,975,278; Bagshawe, K.D. , et al., Br J Cancer (1988) .58.:700-703; Kerr, D.E., et al.. Cancer Immunol Im unother (1990) 11:202-206; PCT application WO88/07378, published 6 October 1988; EP 302,473, published 8 February 1989; and U.S. patent 4,975,278. For a general discussion of this approach, see Senter, P.D., et al., FASEB J (1990) 1:188-193.

According to this approach, an enzyme/prodrug pair is used to treat cancer by targeting the enzyme in the form of a conjugate to a monoclonal antibody to solid tumors and then delivering the prodrug which will be activated only at the tumor site. The administration of the prodrug is delayed until the enzyme conjugate has optimally localized in the tumor and cleared normal tissue and plasma. Enzyme/prodrug pairs which have been used include alkaline phosphatase/etoposide phosphate (Senter, P.D., et al., PNAS (1988) 85:4842-4846) and bacterial carboxypeptidase G2/p-N-bis-(2-chloroethyl)- aminobenzoyl glutamic acid (Bagshawe, K.D., et al., Br J Cancer (1988) 58.:700-703) , and penicillin V amidase/ doxorubicin amide (Kerr, D.E., et al. (1990) supra) .

Disclosure of the Invention

The present invention provides prodrugs of palytoxin-related compounds including novel amides of palytoxin and its naturally occurring analogs which are cleavable by penicillin amidase and thus useful as toxins in the above-described prodrug-activating system. These amides are the phenylacetyl, hydroxyphenylacetyl, phen- oxyacetyl, or hydroxyphenoxyacetyl amides. By utilizing prodrugs of the palytoxin family, targeted toxicity can be achieved with respect to any cells or tissues for which a targeting agent is available.

While, as indicated above, the invention is illustrated with reference to penicillin amidase- cleavable prodrugs, the invention includes compositions and methods using any palytoxin prodrug cleavable with a biological catalyst.

In one aspect, the invention is directed to pharmaceutical compositions containing the palytoxin- related prodrugs selected from the group consisting of palytoxin, palytoxin carboxylic acid, homopalytoxin, bishomopalytoxin, isopalytoxin, neopalytoxin, deoxy¬ palytoxin and their naturally occurring coexistent analogs, and to methods to destroy or impair the functioning of undesirable cells or tissue by ad inis- tering these prodrugs, typically in a protocol which includes administration of the relevant targeted biological catalyst which has the ability to release the palytoxin-related toxin from the prodrug.

In other aspects, the invention is directed to specific prodrugs of palytoxin and its analogs.

Preferred are amide prodrugs of penicillin amidase- cleavable forms which include the phenylacetyl, hydroxyphenylacetyl, phenoxyacetyl or hydroxyphenoxy¬ acetyl derivatives.

Brief Description of the Drawings

Figure 1 shows the structure of palytoxin and several naturally-occurring coexistent analogs. Figure 2 is a graphic representation of the cytotoxicity of the prodrug and palytoxin forms of the invention in the presence of penicillin amidase.

Figure 3 is a graphic representation of the cytotoxicity of prodrug, N-acetylpalytoxin and palytoxin in the presence of penicillin amidase.

Modes of Carrying Out the Invention

The invention is directed to palytoxin-related toxins which have been converted to prodrugs cleavable by specific biological catalysts which can be targeted to tissues. The invention compositions take advantage of the remarkable toxicity of palytoxin and its analogs, and the availability of functional groups on these toxins which permit derivatization to obtain prodrug forms.

Palytoxin and its analogs are isolated from native sources, and palytoxin is presently commercially available. The total synthesis of palytoxin carboxylic acid, which contains 64 σhiral carbons and thus 10 21 possible isomers, has recently been reported (Armstrong, R.W. , et al., J Am Chem Soc (1989) 111:7525-7530). Reviews of the chemistry, toxicology and pharmacology of palytoxin have been published by Haberman, E., Toxicon (1989) 27_£lϋ:1171-1187, and Hirata, Y. , et al., in Handbook of Natural Toxins. Vol 3, chapter 11: Chemistry and Pharmacology of Palytoxin, Tu, A.T. , ed. (1988) Marcel Dekker, Inc., New York, pp. 37-45. The structure of palytoxin and its analogs homopalytoxin, bishomopaly- toxin, neopalytoxin, deoxypalytoxin, isopalytoxin, and palytoxin carboxylic acid are shown in Figure 1. It is not unlikely that additional naturally occurring analogs coexisting in Palythoa species will also be found. Hence, as used herein, the named forms of palytoxin "and their naturally-occurring coexistent analogs" refers to the named compounds themselves and any related compounds of similar structure which occur in Palythoa species along with the named analogs.

Palytoxin is extremely toxic, having an D_5Q in a 24 hr intravenous dose in the range of 0.025- 0.45 μg/kg, depending on species. Administration by intramuscular, subcutaneous and intraperitoneal injection shows less toxicity, and intragastric administration is at least 200 times less effective than intravenous administration. With high doses of palytoxin, death occurs within minutes of administration and palytoxin is one of the most potent vasoconstrictors known, being about 100 times more potent than angiotensin-II. Effects shown in vivo include renal failure with shock and uremia, generalized hemorrhagic diathesis with necrotizing vasculitis and ischemia, congestive heart failure, hemorrhagic pneumonitis and susceptibility to infection. It is also reported to be a tumor promoter in a mouse skin model (Fujiki, H. , et al., "New Classes of Tumor Promotors: teleocidin, aplysiatoxin and palytoxin" Cellular Interaction by Environmental Tumor Promotors Fujiki, H., et al., eds., Japanese Scientific Society Press, Tokyo/VNU Science Press, Utrecht (1984) , pp. 37-45; Fujiki, H. , et al., Carcinogenesis (1986) 2:707) . n vitro demonstrations of toxicity have shown that the effects of palytoxin include semiselective monovalent cation pore formation in cell membranes, depolarization of excitable membranes, smooth muscle contractions, cardiac muscle contraction, erythrocyte hemolysis, norepinephrine release and histamine release, stimulated arachidonic acid metabolism, bone resorption, platelet aggregation and EGF receptor modulation.

Palytoxin and its analogs are uniquely suited for use as prodrugs .in a catalyst-mediated release system. For example, they are capable of forming amides which are stable absent the presence of an enzyme, and the prodrugs of the present invention are not cleavable by endogenous enzymes. As the molecular weight of the toxin is relatively low, tumor penetration through diffusion is relatively easy, and the prodrug form is greatly less potent than the toxic form. The toxic forms of these drugs are extremely potent.

Because palytoxin has a high affinity for the Na, K-ATPase on the surface of most cells (Bottinger, H. , et al., Biochi Biophys Acta (1986) 861:165-176) . once generated in the general vicinity of the tumor, the toxin is likely to bind all tumor cells regardless of whether a marker for the targeting agent is present. Furthermore, the potent vasoconstrictor activity of palytoxin is expected to reduce convective forces which prevent effective distribution of drugs through tumor tissue. The palytoxin prodrugs of the invention are designed to be cleavable to the effective palytoxins in the presence of an appropriate biological catalyst. By "biological catalyst" is meant a molecule compatible with biological systems that behaves in a manner with respect to affecting reaction rates analogous to that of an enzyme. The most commonly used biological catalysts are enzymes; however, other compatible molecules such as antibodies and RNA have been found to exhibit catalytic activity. These alternate forms are also included within the range of catalysts useful in the methods of the invention.

An illustrative embodiment of the invention includes novel palytoxin amides which are susceptible to cleavage by penicillin amidase, an enzyme not known to occur in mammalian tissue, thus making these prodrugs useful in a targeting protocol wherein the penicillin amidase is targeted to specific locations in a subject to be treated. Penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) is an 86 kd microbial enzyme which is commercially used to produce 6-aminopenicillanic acid from penicillin-G and penicillin-v (Lindsay, CD., et al., Eur J Biochem (1990) 192:133-141) . This enzyme has been used to activate amide prodrug derivatives of doxorubicin in vitro (Kerr, D.E., Cancer Immunol Immunother (1990) 11:202-206; Senter, P.D., FASEB J (1990) 4.:188-193). The coupling of this enzyme to monoclonal antibodies has been described by Kerr, D.E., et al., (supra), incorporated herein by reference. Briefly, the antibodies are provided with sulfhydryl groups by derivatization with 2-iminothiolane hydro- chloride and bound to the amidase which has been derivatized with the linker succinimidyl 4-(N-maleimi- domethyl)cyclohexane-l-carboxylate (SMCC) to provide the enzyme with a reactive maleimide group using standard coupling technology. Conjugates of penicillin amidase with any suitable targeting agent is useful in the therapeutic methods described herein.

Other biological catalysts which are suitable as cleavage agents for converting palytoxin-related prodrugs to the corresponding toxins include those appropriate for those derivatized forms of palytoxin which lose their toxic activity when thus derivatized. Candidate enzymes are found, for example, in PCT Application W088/07378, set forth above.

Synthesis of the Prodrugs Palytoxin is isolated from Palythoa tuberculosa according to the method of Moore, R.E., and Sheuer, P.J. , Science (1971) 172:495-498. Purity can be verified using standard analytical methods. In particular, the isolated drug should migrate as a single peak on HPLC using either a Zorbax ODS column (4.6 x 250 mm) at 40% acetonitrile/ 0.05 N acetic acid, 1 ml/min, with detection by UV absorbance at 263 nm or on a Showdex OHpak B804 column (8 x 500 mm) 10:1 0.02 N phosphate buffer, pH 4.6:ethanol, 1 ml/min, 263 nm. Acylated palytoxin is synthesized using standard acylation methods. These include the use of the activated form of the relevant carboxylic acids such as the acyl halides or use of condensing agents such as dii ide reagents, for example l-(3-dimethylaminopropyl)- 3-ethylcarbodiimide (EDCI) . If penicillin amidase is used as the biological catalyst, the carboxylic acid partner in the acylation reaction is chosen so as to render the resulting amide susceptible to cleavage with this enzyme and is normally selected from the group consisting of phenylacetic acid, hydroxyphenylacetic acid, phenoxyacetic acid, and hydroxyphenoxyacetic acid. In a typical preparation, the carboxylic acid in an inert polar aprotic solvent is converted to the activated hydroxysuccinimide ester by reaction with N- hydroxysuccinimide and EDCI. A tenfold excess of the NHS active ester is then used to treat the palytoxin in a nonaqueous, mildly basic solvent. The product amide is recovered using standard methods, and purified by chromatography. Cation exchange chromatography is particularly favored.

Alternate derivatives for the N-terminal amino group or other palytoxin functionality resulting in inactivation reversible by the action of other biological catalysts require selection of the appropriate substituent susceptible to the selected catalyst.

Various alternate standard modes of preparation of acyl amides or other derivatives can also be used. The resulting amides, can be separated from unreacted palytoxin using the HPLC methods set forth as criteria for purity of palytoxin isolates above, or by thin-layer chromatography. These procedures, conducted as analytical methods, may also be used in assessing the enzymatic hydrolysis of the prodrugs.

The purified and isolated prodrugs are then suitable for formulation into compositions for administration to subjects in treatments designed to impair or destroy unwanted cells or tissues.

Use and Administration The prodrugs of the invention are designed for administration in a protocol which generally employs a preliminary administration of a targeting agent^'-prodrug cleavage catalyst conjugate prior to administration of the prodrug itself. The conjugate is comprised of a targeting agent specifically designed to be reactive with markers at the targeted tissue, typically a tumor. Most typically, the targeting agent is an antibody or an immunologically-reacting fragment thereof such as an Fab, Fab", or F(ab*)₂ fragment which retains the immuno- reactivity of the complete immunoglobulin. A discussion of the nature of tumor-targeting antibodies is found in the above-referenced WO 88/07378 and EP 302473 applications. However, other targeting strategies can also be used, such as the use of ligands designed to bind receptor sites or other surface proteins on the target tissue. Various glycoprotein, carbohydrate, and other ligands are known in the art and the selection of the appropriate targeting agent depends on the nature of the cells or tissues to be destroyed. While tumor tissues are generally candidates for destruction, as set forth above, the strategy can be extended to other undesirable cells and tissues such as cells infected with virus, various benign growths, sites of inflammation, obstructions of vascular tissue, and the like. Suitable targeting agents for these cells and tissues will be understood by those of ordinary skill in the art.

The coupled biological catalyst is typically a conventional enzyme such as an amidase, but alternate forms such as catalytic antibodies or their functional fragments or nucleic acids may also be used.

The prodrugs of the invention are administered using conventional modes of administration and in formulations which are suitable for the selected mode. Such formulations are found, for example, in Remington's Pharmaceutical Sciences (latest edition) , Mack Publishing Co. , Easton, PA. The prodrugs of the invention can be ■injected using various carriers such as Hank's or Ringer's solution, and may be injected intravenously, intraperitoneally, intramuscularly, or subcutaneously, as dictated by the particular indication to be treated. The prodrugs may also be formulated into liposome or microcapsules for injection. In addition, the prodrugs may be formulated for implantation in slow release formulations, or may be administered in transdermal or transmucosal formulations as skin patches, nasal sprays, suppositories, and the like. Suitable formulations with penetrants, such as bile salts, fusidates, detergents, and the like are well known in the art. Oral adminis- tration as tablets, capsules, powders, syrups, and the like is also contemplated. In certain indications, where localized treatment is desirable, topical treatment in the form of pastes, salves, gels, or poultices may also be employed. In typical protocols, the targeting agent coupled to the biological catalyst effective to cleave the prodrug is first administered by any suitable route (analogous to those set forth above) which -permits systemic administration and homing to the target tissue. In this case, also, if localized treatment is possible. this may also be effected by direct injection into a tumor or other target or by topical treatment. If systemic administration is used, sufficient time is allowed to permit the targeting agent/catalyst conjugate to clear normal tissue and plasma and to home to the targeted cells or tissues. If the targeting agent/ catalyst couple is immunogenic, as has often been found to be the case in administration of immunotoxins, this side effect may be ameliorated by the use of immuno- suppressants such as cyclosporins.

The dosage levels and protocols are designed for the particular subject and indication as is generally practiced by medical and veterinary professionals. The dosage levels will depend on the mode of administration as well as the seriousness of the affliction and its nature. However, it is anticipated that dosage levels for the prodrug in the range of 0.1-500 μg/kg, preferably 1-50 μg/kg, of prodrug will be administered in most instances of systemic administration. Lower levels may be used in localized treatment.

The following examples are intended to illustrate but not limit the invention.

Example l Synthesis of N-C4'-hydroxyphenyl

Acetyl) alytoxin CNHPAP) Acylation of the amino group at position 115 as shown in Figure l is conducted as follows. To a solution of 4-hydroxyphenylacetic acid (107 mg, 700 μmol) in THF (7 ml) at ambient temperature under an argon atmosphere were added N-hydroxysuccinimide (81 mg, 700 μmol) and N,N-dicyclohexylcarbodiimide (144 mg, 700 μmol) . After stirring for one hr, the precipitated dicyclohexylurea was allowed to settle. Using a syringe, 70 μl of the supernatant (theoretically containing 7.0 μmol of the NHS active ester of 4-HPAcOH) was transferred to a second vial containing palytoxin (2.0 mg, 0.7 μmol) in dry pyridine (0.5 ml). After 1.5 hr at room temperature, TLC (E. Merck HPTLC NH₂ F₂₅₄ precoated #15647, 9:8:6 pyridine-water-n-pentanol; E. Merck HPTLC silica gel 60 F₂₅₄ precoated #13727, 7:6:7 pyridine-water-n- pentanol) indicated complete consumption of palytoxin. The solvent was evaporated jLn vacuo. The resulting residue was dissolved in water (2 ml) and washed with CH₂C1₂ (3 x 1 ml) . Following lyophilization of the aqueous portion, the product was purified by cation exchange chromatography (CM-Sephadex C-25, 0.02 M phosphate buffer, pH 4.5). The yield of NHPAP was estimated to be 0.87 mg (41%) by UV spectroscopy, using the reported extinction coefficient for palytoxin of 23,600 at 263 nm.

NHPAP is stable in storage for at least six months and remains penicillin amidase-cleavable.

Example 2

Synthesis of N-f4'-hvdroxyphenoxyacetyl)palytoxin (NHPOAP) The a ino group at position 115 as shown in Figure 1 is acylated using p-hydroxyphenoxyacetic acid (NHPOAP) as follows. To a solution of (4-hydroxy- phenoxy)acetic acid (118 mg, 700 μmol) in THF (7 ml) at ambient temperature under an argon atmosphere is added N-hydroxysuccinimide (81 mg, 700 μmol) and N,N-dicyclo- hexylcarbodiimide (144 mg, 700 μmol) . After stirring for one hr, the precipitated dicyclohexylurea is allowed to settle. Using a syringe, 70 μl of the supernatant (theoretically containing 7.0 μmol of the NHS active ester of (4-hydroxyphenoxy)acetic acid is transferred to a second vial containing palytoxin (2.0 mg, 0.7 μmol) in dry pyridine (0.5 ml). The reaction is monitored by TLC (E. Merck HPTLC NH₂ F₂₅₄ precoated #15647, 9:8:6 pyridine-water-n-pentanol; E. Merck HPTLC silica gel 60 F₂₅₄ precoated #13727, 7:6:7 pyridine-water-n- pentanol) . Following complete consumption of palytoxin, the solvent is evaporated in vacuo. The resulting residue will be dissolved in water (2 ml) and washed with CH₂C1₂ (3 x 1 ml) . Following lyophilization of the aqueous portion, the product is purified by cation exchange chromatography (CM-Sephadex C-25, 0.02 M phosphate buffer, pH 4.5). The yield is estimated by UV spectroscopy, using the reported extinction coefficient for palytoxin of 23,600 at 263 nm.

Example 3 Method for Thin-Laver Chromatography

Analysis of Palytoxin Prodrugs Palytoxin and palytoxin prodrugs are separated on silica gel 60 F₂c₄ (EM Science) thin-layer chromatog¬ raphy plates using a 7:6:7 mixture of pyridine:water:amyl alcohol. In this system, palytoxin migrates with an ^■ R - 0.51, while N-(4'-hydroxyphenyl acetyl)palytoxin migrates with an R_f - 0.69. The compounds are visualized by UV light (254 nm) or by treating the plate with p-anisaldehyde reagent (Recipe #22, "Dyeing Reagents for Thin Layer and Vapor Chromatography," E. Merck, Darmstadt, Germany, 1980) .

Example 4 Activation of NHPAP The EL-4-murine T-cell lymphoma line (ATCC

TIB39) was cultured in DMEM supplemented (Flow) medium with 10% calf serum, 1 mM L-glutamine and 2 mM sodium pyruvate. The cells were harvested, suspended in leucine-free Minimal Essential Medium supplemented with 10% (v/v) calf serum, 1 mM L-glutamine, and 2 mM sodium pyruvate, and plated at 105 cells/well in sterile,

96-well Costar microtiter plates. Each well was exposed to a predetermined concentration of NHPAP or palytoxin in the presence or absence of 0.3 μg/ml-33 μg/ml penicillin-G amidase (PGA) prepared from E . coli in a total volume of 0.15 ml. The specific activity of PGA used in the assay was 47 units/mg.

After 18 hr of incubation, the wells were treated with 0.05 μCi of [ 14C]-labeled leucme for 2 hr to determine viability. Viable cells were able to incorporate [ C]-leucine into cellular protein. The wells were then harvested onto glass fiber filters and processed for liquid scintillation counting. Percent survival in test wells was calculated as the mean cpm obtained from the replicate wells divided by mean cpm of replicate nontreated controls x 100. Controls treated only with enzyme and no drug or prodrug showed comparable cpm to untreated controls.

The results are shown in Figure 2 as a plot of % survival vs. picograms/ml NHPAP (squares) or palytoxin (circles) at various concentrations of PGA. As shown in the figure, when no enzyme was added, over 10,000 pg/ml of NHPAP could be added per ml without a decrease in survival; addition of palytoxin alone caused a decrease in survival to 20% of controls at 10 pg/ml. However, in the presence of as little as 3.3 μg/ml of PGA, NHPAP- treated cultures containing NHPAP showed dose response curves similar to those obtained with palytoxin alone. There was no effect on the curves obtained with palytoxin in the presence of enzyme.

In a similar experiment, EL-4 murine T-lymphoma cells (5 x 10 4 cells/well) were exposed to the indicated concentrations of palytoxin (PTX) , N-acetylpalytoxin (NACPTX) , or N-(4'-hydroxyphenoxyacetyl)palytoxin (NHPAP) , with or without PGA, for 2 h in a total volume of 0.1 ml leucine-free MEM containing 10% calf serum (MEM). The PGA was supplied as 3.3 μg/ml penicillin G amidase. Then 0.05 μCi [ C]-leucine was added in a volume of 0.05 ml MEM and the mixtures were incubated a further 2 h. Cells were then harvested onto glass fiber filters and processed for liquid scintillation counting. Viable cells incorporated [ C]-leucine into cellular protein.

The results are shown in Figure 3, where it is seen that NHPAP toxicity is increased significantly in the presence of PGA; PTX is toxic with or without PGA, and the toxicity of NACPTX is not increased in the presence of PGA.

Claims

Clai s

1. A pharmaceutical composition for the in vivo destruction of undesirable cells or tissue which comprises a prodrug of a toxin selected from the group consisting of palytoxin, palytoxin carboxylic acid, homopalytoxin, bishomopalytoxin, isopalytoxin, neopalytoxin, deoxypalytoxin, and their naturally- occurring coexistent analogs as active ingredient in admixture with a nontoxic, pharmaceutically acceptable excipient.

2. The composition of claim 1 which further contains an effective amount of a biological catalyst effective in releasing said toxin from the prodrug.

3. The composition of claim 2 wherein said biological catalyst is coupled to a target-specific reagent.

4. The composition of claim 3 wherein said target-specific reagent is an antibody or an immuno- logically reactive fragment thereof.

5. The composition of claim 2 wherein said biological catalyst is an enzyme.

6. The composition of claim 5 wherein said enzyme is penicillin amidase.

7. The composition of claim 2 wherein said biological catalyst is a catalytic antibody or functional fragment thereof.

8. A method to destroy or impair the functioning of undesirable cells or tissue in a subject, which method comprises administering to a subject in need of such treatment an effective amount of the composition of claim 1.

9. A method to destroy or impair the functioning of undesirable cells or tissue in a subject, which method comprises administering to a subject in need of such treatment an effective amount of the composition of claim 2.

10. A method to destroy or impair the functioning of undesirable cells or tissue in a subject, which method comprises administering to a subject in need of such treatment a composition comprising a conjugate of a biological catalyst effective in releasing a toxin selected from the group consisting of palytoxin, paly¬ toxin carboxylic acid, homopalytoxin, bishomopalytoxin, isopalytoxin, neopalytoxin, deoxypalytoxin, and their naturally-occurring coexistent analogs from a prodrug thereof, said biological catalyst being coupled to a targeting agent specifically reactive with said cells or tissue, allowing said conjugate to home to said cells or tissue, and administering to said subject an effective amount of said prodrug.

11. The method of claim 10 wherein the biological catalyst is an enzyme.

12. The method of claim 10 wherein the biological catalyst is a catalytic antibody or a functional fragment thereof.

13. An amide prodrug of a toxin selected from the group consisting of palytoxin, palytoxin carboxylic acid, homopalytoxin, bishomopalytoxin, isopalytoxin, neopalytoxin, deoxypalytoxin, and their naturally- occurring coexistent analogs which amide prodrug is the penicillin amidase-cleavable phenylacetyl, hydroxy¬ phenylacetyl, phenoxyacetyl or hydroxyphenoxyacetyl amide thereof.

14. The prodrug of claim 13 which is N-(4'-hydroxyphenylacetyl)palytoxin.

15. The prodrug of claim 13 which is N-(4*-hydroxyphenoxyacetyl)palytoxin.