COMPOUNDS AND METHODS FOR INHIBITING CATARACT FORMATION

Tp.πhn-iral Field

This invention relates generally to compounds which are useful for preventing or inhibiting cataract formation in warm-blooded animals and, more specifically, to compounds which function as anti-cataract agents and/or prodrugs to anti-cataract agents.

Background of the Invention

"Cataract" is the general term for any pathological condition in which the normal transparency of the ocular lens is substantially diminished. Although often regarded as an inevitable accompaniment of age, cataracts may develop at any time in life, even before birth. Risk factors for cataract formation include metabolic disorders (e.g., diabetes) , exposure to toxic agents in the environment (e.g., ultraviolet radiation, ionizing radiation) , drug side effects, and inherited traits. Due to the wide variety of causative agents and conditions, the pathogenesis of cataract has been the subject of much debate.

In contrast to cataract pathogenesis, the cellular structure of the lens is well characterized. The lens exhibits a high degree of regularity, consisting of fiber cells with hexagonal cross sections packed together to create a very regular parallel array of fiber cells which stretch from anterior to posterior pole. The lens fiber cells lose all intracellular organelles that could contribute to light scattering during the process of differentiation and the cytoplasmic protein concentration increases markedly.

Approximately 35%-60% of the total mass of the lens consists of structural proteins, the remainder being water. More than 90% of the total lens protein consists of alpha, beta and gamma crystallins, a group of structural proteins found at extremely high concentrations (in excess of 300 mg/ml) in the lens cell cytoplasm. The cytoplasmic concentration of the crystallins throughout the lens occurs along a continuous radial concentration gradient, in which the concentration is greatest in cells at the nucleus and decreases in peripheral cells of the lens cortex. The crystallin distribution determines the mean index of refraction and index gradient, which are in turn responsible for the special optical properties of the animal lens . An important optical property is lens transparency. In the normal lens, incident light is scattered in all directions by the macromolecular constituents of the lens. If the individual wavelets of the scattered light interfere destructively with one another, the lens is transparent. Destructive interference takes place in the normal lens because of the existence of short range order in the relative positions of the crystallins. If the uniformity of the protein concentration is sufficiently perturbed, a substantial fraction of the incident light is scattered in directions away from the forward direction. The scattering results in a distortion of the wave front of the transmitted light, and in opacity of the lens tissue. The opacity is responsible for the visual impairment in cataract disease. Cataracts are the leading cause of blindness in humans worldwide, and surgery remains the primary form of treatment. More than one million cataract extractions are performed annually in the United States, and it is estimated that 5-10 million individuals become visually disabled each year due to cataracts. Cataracts in animals also pose a significant veterinary problem. Accordingly,

significant effort is underway to identify compounds which can treat or inhibit cataract formation, thus avoiding or delaying the need for surgical lens replacement.

To date, significant progress has been made in the development of anti-cataract agents. Specifically, pantethine and pantetheine have now been found to be effective anti-cataract agents in animals, including humans (see U.S. Patent No. 5,091,421) . In addition, apparatus and methods now exist which can detect the very early stages of cataract formation (see U.S. Patent Nos. 4,957,113, 4,993,827 and 5,072,731) . Such early detection allows administration of anti-cataract agents to individuals in need thereof at a sufficiently early stage of cataract development to effectively inhibit or delay the subsequent visual impairment associated with cataract diseases.

While significant progress has been made in the development of anti-cataract agents, improvements in this field are desired, especially in the area of enhanced activity and improved delivery to the lens of the eye. The present invention fulfills these needs and provides further related advantages.

Summary of Invention Briefly stated, the present invention is directed to compounds and methods for preventing or inhibiting cataract formation in warm-blooded animals, including humans. The compounds of this invention function as anti-cataract agents and/or prodrugs to anti- cataract agents, including pantethine and pantetheine.

In one embodiment, a method is disclosed for inhibiting cataract formation in the lens of a warm¬ blooded animal by administering to the animal an effective amount of a compound of this invention. In general, the compounds of the present invention are derivatives of pantethine or pantetheine where one or more of the alcohol

groups have been modified, and/or are derivatives of pantetheine where the terminal sulfhydryl has been modified. In a preferred embodiment, the compounds are thioester or thiocarbamate derivatives of pantetheine. Other aspects of this invention will become apparent upon reference to the attached figures and the following detailed description.

Brief Description of the Drawings Figure 1 shows the HPLC elution of standard samples of both pantethine and pantetheine (Figure 1A) and pantethine alone (Figure IB) .

Figure 2A shows the HPLC elution of a representative compound of this invention (S-pivaloyl-D- pantetheine) after incubation with a corneal homogenate. Figure 2B shows that no pantetheine was detected when a corresponding sample of corneal homogentate was incubated without S-pivaloyl-D-pantetheine.

Figure 3A shows the HPLC elution of pantetheine after incubation of serum with S-pivaloyl-D-pantetheine.

Figure 3B shows that no pantethine was detected when a corresponding sample of serum was incubated without S- pivaloyl-D-pantetheine.

Figure 4 illustrates the enhanced corneal permeability S-pivaloyl-D-pantetheine (corneas #3 and #4) compared to pantethine (corneas #1 and #2) .

Figure 5 illustrates the steady state corneal penetration of S-pivaloyl-D-pantetheine.

Figure 6 illustrates the steady state corneal penetration of pantethine.

Detailed Description of the Invention

As mentioned above, the compounds and methods of this invention are useful for the prevention or inhibition of cataract formation in a warm-blooded animal, including a human. In one aspect, the compounds of this invention

serve as prodrugs to anti-cataract agents, including pantethine or pantetheine. As used herein, the term "prodrug" means a compound which, when administered to the animal, will be converted within the animal's body to a biologically active anti-cataract agent. Although not intending to be limited by the following theory, it is believed that pantethine and or pantetheine are biologically active forms of the prodrugs of this invention. In addition, the compounds of this invention may also possess utility as anti-cataract agents prior to conversion to pantethine or pantetheine. Thus, it is believed that the combined effect of the compounds (i.e., in vivo conversion to pantethine or pantetheine, as well as in vivo activity as anti-cataract agents) accounts for the effectiveness of the compounds of this invention.

The compounds of this invention may generally be represented by the following structures 1 and 2:

where X, Y and Z are the same or different, and are selected from hydrogen or a chemical group which is susceptible to enzymatic cleavage within the body of a patient, with the proviso that X, Y and Z of structure JL are not all hydrogen, and with the further proviso that X and Y of structure 2, are not all hydrogen (since the compounds of structure 2. are dimers, this proviso applies

when both X moieties and both Y moieties are hydrogen) .

Moreover, with regard to the compounds of structure 2, the individual X moieties may be the same or different, and the individual Y moieties may be the same or different. The compounds of the present invention may generally be described as derivatives of pantethine or pantetheine in which one or more of the alcohol groups have been modified

(i.e, X and/or Y modifications) , or as derivatives of panthethine were the terminal sulfhydryl has been modified (i.e., Z modifications) .

In one embodiment of the present invention, X, Y and Z are the same or different, and are selected from hydrogen and structures through £:

0 0 0 0

1 I I I I I I

1 1 . R — P — OR — S — OR

R ^^ N ^ I I

1

OR 0 R

2 4 £ £

For example, when X and/or Y is structure 2, esters of structures _L and 2 are formed, and when X and/or Y is structure __-, carbamates result. When X and/or Y is structure __. or £, phosphate esters or sulphonate esters are formed, respectively. Similarly, when Z is structure 2, thioesters of structure _ result, and when Z is structure 4., thiocarbamates are formed. When Z is structure 5_ or £, phosphate or sulfonate thioesters of structure 1 are formed, respectively.

In another embodiment, X and Y taken together form a single carbon bridge between the oxygen atoms as represented by structures through _L__:

1 £ 2 1£

Thus, cyclic acetals or ketals are achieved when X and Y taken together are represented by structure 2, and cyclic orthoesters are produced when X and Y taken together are selected from structures £, 1 or lfl. In yet a further embodiment, X and Y taken together may form a phosphate bridge having structure 11, or a sulfone bridge having structure 12:

0 OR o 11 12

Alternatively, X and Y taken together may form a cyclic carbonate having structure 12, or a cyclic oxalate having structure 14 :

π II w o o o

12 lit

In structures __. through 11 above, each R moiety may be the same or different chemical species, and are selected from hydrogen or saturated or unsaturated, branched or unbranched, substituted or unsubstituted C^ to C25 alkyl moieties, C3 to C25 cycloalkyl moieties, Cg to C25 aryl moieties, and combinations thereof. For example, C _] _ to C5 alkyl moieties include methyl, ethyl, propyl, isopropyl, butyl, sec-buty, tert-butyl, pentyl, isopentyl, sec-pentyl and neopentyl; C3 to Cg cycloalkyl moieties include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and Cg and C7 aryl moieties include phenyl, benzyl and tolyl . Substituted aryl moieties include substituted phenyl (such as p-acetylphenyl and p- acetimidylphenyl) and heteroaryl groups (such as furyl, thienyl and pyridyl) . Representative examples of suitable R moieties of this invention are listed in Table 1

Table 1 Representative R Moieties

^• CH —CH CH 2 3

CH(CH )CH CH C(CH ) CH 3 3 2 3 2 3

In another embodiment of this invention, Z is a sulfur-containing compound linked to the terminal sulfur atom of structure 1 by a disulfide linkage (i.e., -S-S-) . Suitable sulfur-containing compounds of this invention include the compounds disclosed in PCT Publication No. WO 93/06832 (hereby incorporated by reference in its

entirety) . Representative examples of such sulfur- containing compounds include (but are not limited to) the compounds listed in Table 2.

Table 2

Representative Z Moieties:

"Sul ur-Containing Compounds" cysteamine cysteine pantetheine aletheine phosphocysteamine N,N,dimethylcysteamine coenzyme A mercaptoethylgluconamide thiocholine dithiothreitol dithioerythritol aminopropanethiol aminobutanethiol aminopentaethiol glutathione

HOOCCH, NCH-> CH ₉ - SH H

HOOCCH ₂ N ( CH ₂ CH ₂ SH ) ₂

HOOC-CH-CH ₂ CH ₂ -NCH ₂ CH ₂ SH

CH,N(CH 2C ^** "H ^* - ^* 2.-S' H NH, Η

HOOCCHCH ₂ CH ₂ N ( CH ₂ CH ₂ SH ) ₂ :CH ₃ ) ₂ N(CH ₂ CH ₂ SH) ₂

NH,

H ₂ NCH ₂ CH ₂ NHCH ₂ CHSH

HN(CH ₂ CH ₂ SH) ₃

CH- _b NCH ₂ CH ₂ SH

OH, N0 ₂ , AcNH, , CN, CF ₃

a=l, 2, 3, 4

+ H ₂ N(CH ₂ CH ₂ SH) ₂

Physical data (i.e., proton NMR and melting point/TLC Rf values) of certain representative compounds of this invention are presented in the following Table 3.

Table 3 Proton NMR of Representative Compounds

A:

B

D:

E:

ft ppm in CDCl^: a b C d e f g

A t 3.74 d 1.03 d 0.97 S 4.1 m 7.0 t 2.72 t 3.6

B t 3.71 d 1.03 d 0.97 S 4.09 m 7.0 t 2.8 t 3.69

C t 3.8 d 0.99 d 0.96 S 4.1 m 7.1 t 2.6 t 3.51

D t 3.82 d 1.03 d 0.97 S 4.14 m 7.09 t 2.6 t 3.54

E m 3.3 2 1.1 s 1.1 S 3.9 m, br m t 6.8-7.1 3.3-3.5 2.5-2.6

F s 4.3 S 1.2 S 1.2 S 5.2 m, br m t 7.0-7.1 3.4-3.5 2.7-2.8

Melting Point and TLC of Representative Compounds

R Moiety Melting Point .°C) Rf ( LC)

—CH(CH )CH 69-71 3 3 0.7, B

—C(CH ) 3 3 0.3, A

CH C(CH ) CH 2 3 2 3 108-110 0.6, C

0.5, B

0 52-55 0.6, B

\\ //

70-71 0.7, C

TLC Solvent Systems:

A = 5% MeOH-CH ₂ Cl ₂ B = 10% MeOH-CH ₂ Cl ₂ C = 15% MeOH-CH ₂ Cl ₂ D = 20% MeOH-CH ₂ Cl ₂

In one aspect of this invention, the compounds have the ability to serve as prodrugs to biologically active anti-cataract agents, including pantethine and pantetheine. Although not intending to be limited by the following theory, the compounds of this invention are believed to undergo hydrolysis due to esterase activity within the body of the animal, including ocular tissues and fluids (such as the cornea, iris-ciliary complex, lens, aqueous humor and vitreous humor) , as well as upon contact with tissue and fluid of the blood, liver and kidney. The compounds of this invention may thus be assayed for their ability to convert to the biologically active form upon, for example, contact with corneal tissue and serum. Such assays may be accomplished by the procedures set forth below in Example 6. In that assay, corneal tissue and serum was collected from an animal, and contacted with the compound for a period of time. After precipitation of protein by addition of acid, the samples were centrifuged and the resulting solution analyzed by

HPLC with electrochemical and/or fluorescence detection. By such procedures, the ability of the compounds of this invention to be converted to the biologically active form may be determined. The compounds of this invention may be administered to an animal in any manner which results in the delivery of an effective amount of the compound to the lens of the eye. Suitable modes of administration of the compounds (and salts thereof) include topical, as well as oral, parenteral and other systemic forms of delivery.

Depending on the mode of administration, the compounds may be formulated in a variety of pharmaceutically acceptable forms, including liquid, semi-solid, solid and aerosol forms. Representative examples of such forms include liquids, ointments, tablets, pills, capsules, powders, suspensions and the like. One or more conventional

pharmaceutical carriers or excipients may also be present in addition to the compounds of this invention (or pharmaceutically acceptable salt thereof) .

The amount or dosage of compound administered to an animal will depend on a number of factors, including the animal's age and weight, the stage of cataract development and mode of administration. Dosage may be monitored for effectiveness in vivo by monitoring the initial stage of cataract by, for example, the apparatus and techniques disclosed in U.S. Patent Nos. 4,957,113, 4,993,827 and 5,072,731, followed by subsequent monitoring of the animal. For example, an effective dosage of the compounds of this invention may be in the range of 0.1 to 2,000 mg/kg/day if administered systemically (for a 70 kg human, this would amount to 7 mg to 14g/day) . The animal which is to be treated may also be an important consideration. For example, dosages may be higher in veterinary applications, such as for horses, dogs and cats, and may be more prolonged than might be possible or desirable with humans.

Preferably, the compounds of this invention are administered before the formation of detectable levels of high molecular weight protein aggregates in the lens cell cytoplasm, although it may also be applied up to and including the time that vision is noticeably impaired. It should also be applied as soon as possible after detection of any rise in phase separation temperature in the lens cell cytoplasm. The amount required for effective prophylaxis or treatment may be determined by measuring the decrease in phase separation temperature per mole. For in vivo application, it is necessary to decrease and maintain the phase separation temperature of the lens cell cytoplasm at less than body temperature while inhibiting the formation of high molecular weight aggregates. To this end, the compounds of this invention are particularly useful when administered prior to a cataract insult,

including (but not limited to) insults associated with both radiation and steroid therapy, as well as cataract surgery, such as vitrectomy.

As mentioned above, depending on the intended mode of administration, the compounds ^' used may be in the form of solid, semisolid, or liquid dosage forms, such as, for example, ointments, tablets, pills, capsules, powders, liquids, suspensions, or the like, preferably in unit dosage forms suitable for single administration of precise dosages. Such formulations will include a conventional pharmaceutical carrier or excipient in combination with the compound (or a pharmaceutically acceptable salt thereof) and may further include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc. More than one compound may be included in the formulation to achieve advantages not available from the separate administration of such compounds.

Parenteral administration of the compound is generally characterized by injection, either subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like. In addition, if desired, the formulations may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like.

For administration of a solid formulation, conventional nontoxic solid carriers may be used including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid formulations can, for example, be

prepared by dissolving or dispersing a compound of this invention in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to form a solution or suspension. If desired, the formulation may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate and triethanolamine oleate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art (see, e.g. ,

Remington's Pharmaceutical Scienc s, Mack Publishing Co.,

Easton, Penn. , 16th ed. , 1982) . The formulation to be administered will, in any event, contain a quantity of the compound(s) in an amount effective to prevent or inhibit the further development of cataract in the animal being tested.

For oral administration, a pharmaceutically acceptable nontoxic formulation is made by the incorporation of any of the normally employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. Such formulations include solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations, and the like, and may contain 10%- 95% active ingredient, preferably 25%-70%.

Another approach for parenteral administration employs the implantation of a slow-release or sustained- release system, such that a constant level of dosage is maintained (see. e.g. , U.S. Patent No. 3,710,795) . Alternatively, a contact lens delivery system may be employed.

Preparation of an ophthalmic solution requires careful consideration of such factors as isotonicity value, the need for buffering agents, the need for a

preservative and sterilization. Lacrimal fluid is isotonic with blood, having an isotonicity value corresponding to that of a 0.9% sodium chloride solution. Ideally, an ophthalmic solution should have this isotonicity value, but the eye can tolerate isotonicity values as low as that of a 0.6% sodium chloride solution and as high as that of a 3.0% sodium chloride solution without marked discomfort . Some ophthalmic solutions are necessarily hypertonic in order to enhance absorption and provide a concentration of the pharmaceutically active ingredient (s) strong enough to exert a prompt and effective action.

A boric acid vehicle, which is preferred in some ophthalmic preparations, has a pH slightly below 5.0. It may be prepared by dissolving 1.9 g of boric acid in sufficient water to make 100 mL of solution. A phosphate buffer system may also be employed, and adjusted for isotonicity provides a choice of pH ranging from 5.9 to 8.0. A pharmaceutical grade of methylcellulose (e.g., 1% if the viscosity is 25 centipoises, or 0.25% if 4000 centipoises) or other suitable thickening agents such as hydroxypropyl methylcellulose or polyvinyl alcohol occasionally are added to ophthalmic solutions to increase the viscosity and prolong contact of the compound with the tissue of the eye.

As mentioned above, the compounds of this invention have increased corneal permeability compared to their biologically active forms. Thus, in a preferred embodiment, the compounds are formulated for topical application to the animal's eye. The efficacy of the compounds of this invention to penetrate the cornea may be demonstrated with a Ussing Chamber in combination with a suitable analytical technique for the quantitation of pantethine or pantetheine. The Ussing Chamber contains two fluid-filled chambers which are separated by an excised cornea, and is described, for example, by

Schoenwald and Huang, J. Pharm. Sci. 22.:1266-1272, 1978 (hereby incorporated by reference) .

In this assay, the cornea is kept in a viable state with appropriate buffers perfused with both oxygen and carbon dioxide. A buffer solution containing a known concentration of compound (C ₀ ) is placed on the epithelial side of the cornea, and the concentration of the compound appearing of the endothelial side (Cn.) is assayed as a function of time (C-j_(t)) . After an initial time delay, the steady state rate of increase of the endothelial concentration (expressed as dC- (t)/dt) is calculated from the data. Using the known values of the total volume (V) , the endothelial chamber and the surface area (A) of the cornea which is exposed to either side of the chamber, the steady state penetration coefficient (K) may be approximated calculated by the following equation (1) :

V(dC _± (t)/dt)

KC

A

(1)

Equation (1) above assumes that C ₀ is essentially constant and that C^ is much smaller than C ₀ , conditions which were satisfied in this assay. A more detailed treatment of the corneal penetration of the compounds of this invention is set forth in Example 7. The ability of the compounds of the present invention to be converted in vivo to a biologically active form is demonstrated in Example 8. Specifically, the ability of pantethine to convert to pantetheine upon contact with ocular tissue is demonstrated. Based on the results of this experiment, other compounds having structure 1 (where Z is a sulfur-containing compound joined to the terminal sulfur atom via a disulfide bond) would be expected to undergo similar conversion to pantetheine.

The following disclosure is directed to the general synthesis of the compounds of this invention. As mentioned above, in one embodiment of this invention the prodrugs are esters where the primary or secondary alcohol of pantetheine or pantethine has been modified (e.g., X = -C(=0)R, Y = H) . Such ester compounds may be prepared by reaction of pantetheine or pantethine with a suitable carboxylic acid to form the corresponding ester. Suitable carboxylic acids include carboxylic acids themselves, or reactive derivatives of carboxylic acids, such as acid chlorides, mixed anhydride derivatives, or N- hydroxysuccinimide esters of carboxylic acid derivatives. Suitable carboxylic acids or their reactive derivatives are available from a variety of commercial sources. Under appropriate reaction conditions, treatment of pantetheine with a carboxylic acid or reactive derivative thereof yields a pantetheine ester according to structure 1 above where Y is hydrogen and X is -C(=0)R. Similarly, treatment of pantethine with a carboxylic acid or reactive derivative thereof yields a pantethine ester according to structure 2. where Y is hydrogen and X is -C(=0)R. For these compounds, R corresponds to the substituent of the carboxylic acid utilized in the esterification reaction. For example, treatment of pantetheine or pantethine with isobutyric acid chloride produces ester compounds where R is an isopropyl group. Similar treatment with carbamic acids and their reactive derivatives results in carbamate formation (e.g., where X = -C(=0)NHR and Y = H) .

Multiple ester-containing compounds of this invention may be made by esterification or carbamate formation. For example, the esters may be symmetric (i.e., X = Y = -C(=0)R) or asymmetric (i.e., X = -C(=0)R and Y = C(=0)R') . In addition, with respect to structure 2 _. , the X moieties and Y moieties may be the same or different (i.e., a compound of structure 2. has two X moieties and 2 Y moieties) . Thus, further asymmetry may

be imparted to the compounds by varying the individual X moieties, Y moieties, or both. Because esterification is a stepwise process, the above esters are readily achieved. Esterification initially results in the modification of the primary alcohol - that is, X = H is transformed into X -C(=0)R3. To achieve esterification of the second pantetheine or pantethine alcohol (i.e., a secondary alcohol) slightly more severe reaction conditions are necessary. Accordingly, esterification of the secondary alcohol with the same carboxylic acid derivative as utilized in esterification of the primary alcohol results in symmetric diesters, while esterification with a different carboxylic acid or reactive derivative produces the asymmetric esters. The symmetric and asymmetric dicarbamates may be prepared in the same manner using either the same or different carbamic acid derivatives, respectively. The ester-carbamate compounds may also be prepared by the same strategy to provide either X -C(=0)R and Y = -C(=0)NHR or X = -C(=0)NHR ₃ and Y = -C(=0)R, where R may differ from ester to carbamate.

In a further embodiment, the compounds of the present invention are cyclic derivatives in which both alcohol groups have been modified. In these cyclic derivatives, the 1,3-diol of pantetheine or pantethine may be modified with a reagent which effectively bridges the alcohol groups with one or more carbon atoms. Referring to structures _L and 2. above, X and Y taken together form the bridge between the alcohol groups. Preferred embodiments of these cyclic prodrugs include cyclic ketals, cyclic acetals, cyclic orthoesters, cyclic carbamate, and cyclic oxalates.

Cyclic ketal and acetal prodrugs may be prepared by condensation of pantetheine or pantethine with ketones or aldehydes, respectively. Condensation with a ketone (R-C(=0)-R) yields a prodrug where X and Y taken together form a single carbon bridge (see structure 2) • Such

cyclic ketal compounds may be prepared from any ketone, including cyclic and acyclic ketones. Condensation with cyclic ketones such as cyclopentanone or cyclohexanone provides a spiro-bridged prodrug, while condensation with an acyclic ketone, such as acetone, yields a compound of structure 2 where both R moieties are methyl . Suitable ketones include dialkyl ketones, such as acetone or methyl ethyl ketone; alkyl aryl ketones, such as acetophenone; and diaryl ketones, such as benzophenone. Cyclic acetal compounds may be prepared from the condensation of pantetheine or pantethine with a suitable aldehyde (e.g., R-CHO) . Suitable aldehydes include alkyl aldehydes, such as formaldehyde or ethanal; cycloalkyl aldehydes; and aryl aldehydes, such as benzaldehyde. Cyclic orthoesters are those compounds in which

X and Y taken together form a single carbon bridge, as illustrated in structures £ and J£ above. Treatment of pantetheine or pantethine with the appropriate orthoester reagent provides such cyclic compounds. For example, treatment of pantetheine or pantethine with trimethyl orthoformate yields a cyclic orthoester compound according to structures _L and 2. wherein X and Y taken together form structures JL or _£ (where R = H and OR = OCH3 ₎ . Similar treatment with trimethyl orthoacetate or trimethyl orthobenzoate provide cyclic orthoester prodrugs (where R methyl and phenyl, respectively, and OR is OCH3) .

Similarly, treatment of pantetheine or pantethine with other orthocarbonate reagents provide cyclic orthoesters in which X and Y taken together form a single carbon bridge of structure .]____. Reaction of pantetheine or pantethine with tetramethyl orthocarbonate provides such cyclic orthoester compounds where R is methyl .

The compounds of the present invention may also be pantetheine or pantethine cyclic carbonates. For these compounds, the carbon bridge is a single carbonyl group as

illustrated in structure 13.. Such cyclic carbonates may be prepared by treating pantetheine or pantethine with phosgene or its synthetic equivalent.

In addition, pantetheine or pantethine cyclic oxalates may also be formed. In this embodiment, the carbon bridge comprises two carbonyl groups as represented in structure ______ Such cyclic oxalates may be prepared by treatment of pantetheine or pantethine with oxalic acid or a suitable reactive derivative thereof. In a preferred embodiment of this invention, compounds of structure _L and 2 are synthesized according to the disclosure of U.S. Patent Application Serial No. (awaiting serial number), filed October 27, 1993, entitled "Compounds and Methods for Synthesizing Pantethine, Pantetheine and Derivatives Thereof" (which application is hereby incorporated by reference in its entirety) . In brief, the 1,3-diol functional group present in pantothenic acid as the site of chemical elaboration to produce ketal derivatives. Thus, pantothenic acid may serve as the starting point for the chemical synthesis of the compounds of this invention. The crystalline ketal intermediates may be subjected to recrystallization, as necessary, to afford ketals of high purity. Ketal purity may be ascertained by any one of a variety of techniques, including melting point determination, as well as spectrographic or chromatographic analysis. Hydrolysis of the ketal, if desired, regenerates the 1,3-diol functional group.

In this embodiment, the first synthesis step involves the ketalization of pantothenic acid. Sodium pantothenate (Aldrich Chemical Co., Milwaukee, I) is treated with acetone under acidic conditions to yield the acetone ketal of pantothenic aid, 1, 3-isopropylidene pantothenic acid. More specifically, a solution of sodium pantothenate in acetone may be treated with either a catalytic or stoichiometric amount of an acid, such as

sulfuric acid, and heated at reflux for several hours to effect conversion of the diol to the corresponding ketal. The ketal may be then be isolated via crystallization by dilution of the solution with a nonpolar solvent such as hexane. The crystallized ketal may be collected by filtration, washed with an appropriate solvent, and recrystallized as necessary to afford the purified ketal synthetic intermediate. The formation of the acetone ketal of pantothenic acid by the process described above may be referred to as direct ketalization.

Alternatively, pantothenic acid may be treated with a ketal of acetone such as 2,2-dimethoxypropane

(acetone dimethyl ketal) under acidic reaction conditions, to provide the acetone ketal of pantothenic acid by a process referred to as trans-ketalization. In this process, the acetone ketal is exchanged or transferred from the ketalizing reagent, 2,2-dimethoxypropane, to pantothenic acid. Specifically, a solution of sodium pantothenate in acetone may be treated with a single molar equivalent of sulfuric acid followed by treatment with 2, 2-dimethoxypropane. After heating the reaction mixture for several hours to complete ketal formation, the crude product may be isolated by an aqueous extractive process utilizing methylene chloride and water. The ketal thus obtained may be recrystallized from a suitable solvent system such as acetone-hexane (1:1) to provide highly pure 1, 3-isopropylidene-D-pantothenic acid. A representative experimental procedure for the formation of the acetone ketal of pantothenic acid is described in detail in Example 1. The acetone ketal of pantothenic acid, 1,3- isopropylidene-D-pantothenic acid, may be represented by structure 15 :

H C CH 3

15.

Other pantothenic acid ketals may be utilized which are derived from a variety of ketones . Suitable ketones are those which provide pantothenic acid ketals which are crystalline and capable of recrystallization to provide highly pure ketals. Preferred ketones include dialkyl ketones containing from four to eight carbons, such as methyl ethyl ketone (2-butanone) ; cyclic ketones containing from three to seven carbon atoms, such as cyclopentanone and cyclohexanone; alkyl aryl ketones, such as methyl phenyl ketone (acetophenone) ; and diaryl ketones, such as diphenyl ketone (benzophenone) . The pantothenic acid ketals derived from the above-mentioned ketones may be generally represented by structure 15 ' :

!____!

wherein R^ and R2 individually represent alkyl groups of a dialkyl ketone, alkyl and aryl groups of an alkyl aryl ketone, and aryl groups of a diaryl ketone as disclosed above, or wherein R]_ and R2 taken together represent the carbocyle of a cyclic ketone as disclosed above.

Ketal formation is accomplished under acidic conditions. Suitable acids for these conditions include organic acids and mineral acids . Organic acids include

carboxylic acids, ammonium salts, sulfinic acids, and sulfonic acids. Mineral acids include hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid. In a preferred embodiment, ketal formation utilizes sulfuric acid.

The coupling of either cystamine or cysteamine to the ketal of pantothenic acid is then performed. In this step, the amino group of either cystamine or cysteamine is coupled to the carboxy group of the pantothenic acid ketal to form an amide bond to produce the ketal of pantethine or pantetheine, respectively. This coupling reaction may optionally utilize a coupling agent to effect amide bond formation. Such coupling agents include those reagents which activate carboxylic acid groups toward nucleophilic substitution. Suitable reagents include thionyl chloride (which transforms carboxylic acids to acid chlorides) , various chloroformates (which convert carboxylic acids into reactive anhydrides) , and diimide reagents such as dicyclohexyl carbodiimide (DCC) and 1- (3- dimethylaminopropyl) -3-ethyl carbodiimide (EDC) (which convert carboxylic acids to active ester derivatives) . In the practice of this invention, a preferred coupling agent is carbonyl diimidazole (CDI) which converts carboxylic acids to carbonyl imidazoles.

Alternatively, the coupling of cystamine or cysteamine to the ketal of pantothenic acid may be accomplished by esterifying the ketal by refluxing in methanol with a catalytic amount of concentrated sulfuric acid. To this crude reaction mixture is added cysteamine (1 equivalent) or cysteamine (0.5 equivalents) , and refluxed for an additional period of time. The mixture is then evaporated to dryness, redissolved in methylene chloride and washed with dilute HCl, saturated NaHCθ3 and brine, and then dried (e.g., using MgSθ4) , filtered and evaporated to dryness.

More generally, the pantothenic acid ketal (i.e., structure 15 ' above) is coupled with cystamine to yield the pantethine ketal of structure _L__., or is coupled with cysteamine to yield the pantetheine ketal of structure 12- The coupling of cystamine or cysteamine to a representative pantothenic acid ketal (i.e., 1,3- isopropylidene-D-pantothenic acid) utilizing carbonyl diimidazole to produce the 1, 3-isopropylidenes of pantethine and pantetheine are described in detail in Examples 2 and 3, respectively. In general, these coupling reactions may be represented schematically as follows:

The purity of the ketals of pantethine and pantetheine (i.e., structures 1£ and 12) may be enhanced by recrystallization in a manner similar to that of the starting material, the pantothenic acid ketal.

The pantetheine ketal of structure 12 may be further esterified. In this method, the sulfhydryl group of the pantetheine ketal is esterified with an esterifying

agent to provide a pantetheine ketal thioester. As used herein, the term esterifying agent refers to any reactive acid derivative which is capable of reacting with a sulfhydryl group to form a thioester. A thioester may be thought of as resulting from the condensation of a sulfhydryl containing compound, a thiol, with an acid much in the same way that an ester results from the condensation of an alcohol with an acid. The pantetheine ketal thioesters produced by the methods of the present invention may be prepared from esterifying agents derived from carboxylic, carbamic, phosphoric, and sulfuric acid derivatives, and are represented by structure IS.:

ιa

wherein R^ and R2 are as described above, and R3 represents the residual portion of the esterifying agent.

The residual portion of the esterifying agent, R3, is the portion of the esterifying agent which corresponds to the acid from which the esterifying group is derived, less the -OH group of the acid. For example, when the esterifying agent is a carboxylic acid derivative, such as an acid chloride, the residual portion of the esterifying agent corresponds to the carboxylic acid (i.e., R-CO2H) from which the acid chloride is derived, less the OH group of the carboxylic acid (i.e., R3 in this case would be -C(=0)R) . Similarly, when the esterifying agent is a carbamic acid derivative, the residual portion of the esterifying agent corresponds to the carbamic acid (i.e., R2N-CO2H) less the OH group of the carboxylic acid (i.e., R3 is -C(=0)NR2) . When the

esterifying group is a phosphoric acid (H3PO4) or sulfuric acid (H2SO4) derivative, the residual portion of the esterifying group, R3, is -PO3H2 and -SO3H, respectively.

Esterifying agents derived from carboxylic acids include reactive carboxylic acid derivatives such as acid halides, carboxylic acid anhydrides, and reactive carboxylic ester derivatives, including p-nitrophenyl esters and N-hydroxysuccinimide ester. Suitable acid halide esterifying agents include acid chlorides such as acetyl chloride and benzyl chloride. The thioesters produced from carboxylic acid-derived esterifying agents by the methods of the present invention are represented by structure formula ________:

18a

wherein R^ and R2 as are as described above, and R represents the side chain of the carboxylic acid from which the esterifying agent is derived. Suitable R groups included hydrogen or saturated or unsaturated, branched or unbranched, substituted or unsubstituted C _] _ to C25 alkyl moieties, C3 to C25 cycloalkyl moieties, Cg to C25 aryl moieties, and combinations thereof. For example, C^ to C5 alkyl moieties include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec- pentyl and neopentyl; C3 to Cg cycloalkyl moieties include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and Cg and C7 aryl moieties include phenyl, benzyl and tolyl . Substituted aryl moieties include substituted phenyl (such as p-acetylphenyl and p-acetimidylphenyl) and heteroaryl groups (such as furyl, thienyl and pyridyl) . The

synthesis of a representative pantetheine ketal thioester, S-trimethylacetyl-1, 3-isopropylidene-D-pantetheine (i.e., structure 18a wherein R-|_ = R ₂ = CH ₃ and R = C(CH ₃ ) ₃ ) , by esterification utilizing trimethylacetyl chloride is described in detail in Example 4.

Closely related to carboxylic acid esterifying groups are esterifying groups derived from carbamic acid. These esterifying agents produce pantetheine ketal thioesters, which may also be referred to as pantetheine ketal thiocarbamates. The thioesters produced from carbamic acid-derived esterifying agents by the method of the present invention may be represented by formula 18b:

wherein R^, R2, and R are as described above.

The pantetheine ketal thioesters of the present invention derived from phosphoric and sulfuric acid derivatives may also be referred to as pantetheine ketal thiophosphonate and thiosulfonate esters, respectively. The thioesters produced from phosphoric and sulfuric acid- derived esterifying agents by the methods of the present invention are represented by formulas 18c and 18d _f respectively:

wherein R^ and R2 are as described above. The synthesis of structure 18c may be accomplished by reacting structure 17 with phosphoryl chloride. Similarly, structure 18d may be synthesized by reacting thiosulfate with the reaction product of structure 12 or its sulfenyl chloride.

Hydrolysis of the pantetheine ketal thioesters (i.e., structure IS.) yields the corresponding pantetheine thioesters. In this reaction step, hydrolysis of the ketals under aqueous acid conditions yields the corresponding pantetheine thioesters. The pantetheine thioesters produced by this method are represented by structure 12.:

12.

wherein R3 is as described above. The synthesis of a representative pantetheine thioester, S-trimethylacetyl-D- pantetheine (i.e., structure V where R3 = C (=0) C (CH ₃ ) 3) ,

by hydrolysis of the corresponding ketal under aqueous acidic conditions is described in detail in Example 5.

The compounds of this invention also include other 1,3-diol derivatives of pantethine and pantetheine. For example, in addition to ketones, 1,3-diols may be condensed with aldehydes (R-CHO) to provide acetals. (As disclosed above, this would be accomplished by forming an acetal of pantothenic acid, followed by addition of cystamine or cysteamine) . In addition, the 1,3-diols of pantothenic acid may be treated with orthoester reagents. Orthoester reagents are derived from the addition of two equivalents of an alcohol (R _a -OH) to an ester (R^-C(=0) -OR _a ) . Orthoester reagents are classified according to the ester and alcohols from which they are derived. Orthoester reagents derived from formate esters (R]-, is hydrogen) are referred to as orthoformates, and orthoester reagents derived from all other esters (R- _Q not hydrogen) are referred to as the originating ester. For example, orthoester derived from acetates (R- _Q is methyl) and benzoates (R^ is phenyl) are referred to as orthoacetates and orthobenzoates, respectively. Suitable orthoester reagents of this invention include orthoesters derived from esters and alcohols wherein Rj-, and R _a are as described above for R^_ (and R2) regarding the synthesis of ketals. Preferred orthoesters include trimethyl orthoformate, trimethyl orthoacetate, and trimethyl orthobenzoate.

Similarly, treatment of 1,3-diols of pantothenic acid with orthocarbonate reagents (C(0R _a ) ₄ ) provide cyclic orthocarbonate (wherein R^ and R2 are 0R _a ) . Suitable orthocarbonate reagents are derived from alcohols (R _a -0H) wherein R _a is as described above for the orthoesters.

The following examples are offered by way of illustration, not limitation.

EXAMPLES

Example 1 Synthesis of 1.3-Isopropylidene-D-Pantothenic Acid

(Pantothenic Acid Acetone Ketal)

To a solution of 24.1 g (0.10 mole) sodium D- pantothenic acid in 250 mL methanol was added 9.1 g (0.10 mole) concentrated sulfuric acid by dropwise addition followed by the addition of 50 mL acetone. The mixture was evaporated to dryness at 45°C under reduced pressure.

To the resulting syrup was added 150 mL dimethoxypropane and 50 mL acetone. After heating the solution for 10 hours at 65°C, the solution was concentrated under reduced pressure to yield the crude product as a thick slurry.

The slurry was partitioned in 250 mL methylene chloride and 250 mL saturated aqueous sodium chloride. The methylene chloride layer was separated and washed with 100 mL saturated aqueous sodium chloride and dried over anhydrous sodium sulfate. Filtration of the drying agent and concentration to dryness produced a white powder (85% yield) . Recrystallization from acetone-hexane (1:1 v/v) gave 1, 3-isopropylidene-D-pantothenic acid as a white crystalline solid, melting point 90-91°C (HPLC purity > 99%) .

The recrystallized product was characterized by: !H NMR: (see Table 3, compound D) Elemental Analysis:

Calculated for %C=55.58, %H=8.16, %N=5.40

Found:

%C=55.34, %H=8.14, %N=5.37

Example 2

Synthesis of 1.3-Isopropylidine-D-Pantethine

(Pantethine Acetone Ketal)

To a solution of 14.0 g (0.050 mole) 1,3- isopropylidene-D-pantothenic acid (prepared as described above in Example 1) in 140 mL tetrahydrofuran (dried over

4A molecular sieves) was added 0.10 g (0.050 mole) carbonyl diimidazole. The resulting solution was stirred at room temperature until the evolution of carbon dioxide gas had ceased (approximately 3 hours) . To the solution was added 11.25 g (0.050 mole) cystamine dihydrochloride and the resulting solution heated at reflux for 6 hours.

The solution was concentrated under reduced pressure and the resulting white solid was dissolved in 150 mL methylene chloride. The methylene chloride solution was washed sequentially with solutions of saturated aqueous sodium chloride, dilute hydrochloric acid, saturated sodium bicarbonate, again with saturated aqueous sodium chloride, and dried over anhydrous sodium sulfate. The solution was filtered, diluted with 150 mL hexanes, and allowed to stand overnight at room temperature. The crystallized product was collected by filtration and recrystallized from ether-hexane (1:1 v/v) to yield 11.81 g (0.020 mole, 80%) 1, 3-isopropylidene-D-panthethine as a white crystalline solid (melting point 117-118°C, HPLC purity > 99%) .

The recrystallized product was characterized by: ^J ^H NMR: (see Table 3, compound B) Elemental Analysis : Calculated for C28 ^H 52 ^N 4°8 ^S 2 ^:

%C=52.81, %H=8.23, %N=8.69 Found:

%C=53.11, %H=8.02, %N=8.69

Example 3 Synthesis of 1.3-Isopropγlidene-D-Pantetheine (Pantetheine Acetone Ketal) The synthesis of 1, 3-isopropylidene-D-pantetheine from D-pantothenic acid was as described above in Example 2 for the synthesis of 1,3-isopropylidene-D-pantethine, except that cysteamine hydrochloride was used instead of cystamine dihydrochloride (white powder, 87% yield, HPLC purity > 99%) . The recrystallized product was characterized by ¹ H NMR (see Table 3, compound A) .

Example 4

Synthesis of S-Trimethylacetyl-

_ .3-Tsopropvlidene-D-Pan ethei e To a solution of 2.76 g (0.0010 mole) 1,3- isopropylidene-D-pantetheine (prepared as described above in Example 3) in 20 mL methanol was added a solution of sodium methoxide (freshly prepared from 0.23 g sodium in 20 mL methanol) . The solution was concentrated to dryness under vacuum. The resulting powder was dissolved in 20 mL tetrahydrofuran (dried over 4A molecular sieves) and treated with 1.38 g (0.0110 mole) trimethylacetyl chloride (pivaloyl chloride) . The solution was stirred for 1 hour at 25°C and evaporated to dryness. The residue was dissolved in 100 mL methylene chloride and washed sequentially with solutions of saturated aqueous sodium chloride, dilute hydrochloric acid, saturated sodium bicarbonate, again with saturated aqueous sodium chloride, and dried over anhydrous magnesium sulfate. The solution was filtered and evaporated to dryness to yield S- trimethylacetyl-1, 3-isopropylidene-D-pantetheine as a clear, colorless oil (89% yield, HPLC purity > 99%) . The compound was characterized by ¹ H NMR (see Table 3, compound C) .

Example 5 Synthesis of S-Trimethγlacetyl-D-Pantetheine S-trimethylacetyl-D-pantetheine was prepared by hydrolysis of its corresponding acetone ketal. Specifically, a solution of 0.010 mole S-trimethylacetyl- 1, 3-isopropylidene-D-pantetheine (prepared as described above in Example 4) in 100 mL 80% aqueous acetic acid was heated for 6 hours at 65°C. The solution was concentrated to dryness under reduced pressure to provide a glassy solid. The solid was dissolved in 100 mL distilled water and washed with two 100 mL portions of methylene chloride. The aqueous layer was collected and freeze-dried to yield S-trimethylacetyl-D-pantethine as glassy material (93% yield, HPLC purity > 99%) .

Example 6

Conversion of S-Pivalovl Pantetheine to Pantetheine

Upon Contact with Corneal Tissue and Serum

This example illustrates the ability of corneal tissue and serum to convert a representative compound of this invention, S-pivaloyl pantetheine, to pantetheine.

Two rabbits were sacrificed and their eyeballs removed and placed on ice. Serum and plasma from the rabbits was also collected, and placed on dry ice. The corneas were then removed and homogenized by sonication in

0.9% NaCl solution, 20% w/v. Homogenate was added to vessels with and without the compound. Hydrolysis was carried out by maintaining the solution at 37°C for one

^' hour. Proteins were precipitated with acid and the homogenates centrifuged, yielding clear supernatants. The resulting supernatants were then analyzed by HPLC, employing either electrochemical or fluorescence detection techniques.

In this example, the HPLC included a Bioanalytical Systems PM-80 HPLC pump, LC-4C dual amperometric electrochemical detector, LC-44 thin layer

flow cell, and LC-26 vacuum degasser. The analytical column was a Bioanalytical Systems 3.2 x 100 mm, 3 μm, Phase II ODS cartridge column. An SGE 4.1 x 10 mm, 5 μm, ODS-Iguard column was employed. Mobile phase A was a 50 mM, pH 2.8 phosphate buffer. Mobile phase B was prepared by adding 100 mL of a 500 mM, pH 2.8 phosphate buffer to a 1 L volumetric flask, then adding 200 mL of acetonitrile and diluting to the mark with HPLC-grade water. The mobile phase was degassed and maintained under 2 psig of helium using a Kontes sparging manifold. The flow was set to 1.0 mL/minute. The mobile phase composition was changed linearly from 90%A:10%B to 10%A:90%B in 10 minutes, followed by holding at this composition for 10 minutes before equilibrating the system with 90%A:10%B.

A dual HG/Au electrode was prepared and equilibrated overnight in the system. A potential of - 1.250 V was applied to the upstream electrode and a potential of +0.250 was applied to the upstream electrode. The optimum potentials were determined by performing hydrodynamic voltammograms at the upstream and downstream electrode.

Figures 1A and IB display standard samples showing elution times of both pantetheine (Figures 1A and IB) and pantethine (Figure 1A) under the chromatographic conditions employed.

Figures 2A and 2B show results of HPLC analyses of corneal homogenates demonstrating hydrolysis of the S- pivaloyl pantetheine by the cornea tissue. Figure 2A shows the elution of S-pivaloyl pantetheine after incubation of corneal homogenates with S-pivaloyl pantetheine as described above. Figure 2B illustrates that no pantetheine was detected when a corresponding sample of corneal homogenate which was incubated without S-pivaloyl pantetheine present.

Figures 3A and 3B presents the results of HPLC analysis of control samples and of serum, demonstrating conversion of S-pivaloyl pantetheine to pantetheine in serum. Specifically, Figure 3A illustrates the elution of pantetheine after incubation of serum with S-pivaloyl pantetheine as described above. Figure 3B shows a corresponding sample of serum which was incubated without S-pivaloyl pantetheine, and no pantetheine was detected.

Example 7

Enhanced Corneal Penetration of S-Pivaloyl Pantetheine

This example illustrates the ability of a representative compound of this invention, S-pivaloyl pantetheine, to penetrate the cornea of an animal lens. For each compound tested (i.e., pantethine and

S-pivaloyl pantetheine) , a male New Zealand White rabbit

(weighing approximately 1.6-2.0 Kg) was sacrificed by injecting 0.3 mL phenobarbitol into the marginal ear vein.

The intact eyes, along with the lids and conjunctival sac, were enucleated in approximately 3 minutes for the first eye, and 8 minutes for the second eye. The exposed corneas of the enucleated eyes were carefully placed in a corneal holder, which maintained the corneal curvature and held the eye in place. Various tissues of the eyes were dissected, leaving the cornea, a small ring of scleral tissue, and the palpebral conjunctiva, which was tied to the corneal ring. The conjunctival and scleral tissue served as a gasket and permitted the cornea to be suspended within the corneal ring, which was then positioned between two large rings and placed in the center of the perfusion chamber. The chamber was put in a water bath to maintain the cornea and the perfusion solution at 37°C. Bicarbonated Ringer's solution was modified as described by Schoenwald and Huang (J. Pharm. Sci. 22:1266-1272, 1978) to preserve the integrity of the

tissue of the excised cornea over 6 hours and used through the perfusion study.

Within 30 minutes of death, the first cornea was mounted and clamped between two cylindrical compartments of the perfusion chamber. A measured volume (7.0 mL) of bicarbonated Ringer's solution was added first to the endothelial side (i.e., the "receiving solution") to prevent the cornea from buckling. An equal volume of solution containing either pantethine or the prodrug, S- pivaloyl pantetheine, was then added to the epithelial side (0 minutes) . Fluid was circulated inside each half- chamber by bubbling a mixture of O2-CO2 (95:5) through at a rate of 3-5 bubbles per second. This served not only to provide oxygen to the excised corneas, but also maintained the solution at a constant pH of about 7.7. Samples were withdrawn (0.5 mL) from the receiving chamber at 2, 15, 30, 60, 90, 120, 150, 180, 210 and 240 minutes after the compound was added to the epithelial side. After each sample was withdrawn, an equal volume (0.5 mL) of solution was immediately added to the receiving solution to maintain a constant volume. The first sample, withdrawn at 2 minutes, served as a control to detect leakage and rapid penetration. Sample solutions were transferred into autosampler vials for HPLC analysis immediately after finishing the collection of each sample.

The HPLC system included of the following components: Rainin solvent delivery module, Rainin Dynamax automatic sample injection (Model AI-1) , Rainin reversed phase C18 column, 5 μm particle diameter, pore size 100 Angstroms, 15 cm long with an I.D. of 4.6 mm, and a Rainin Dynamax ultraviolet absorbance detection (Model UV-D) detecting at a wavelength of 230 nm. Data was collected and processed with a Waters 860 System Data Station on a Digital Equipment MicroVax 3100 computer. The mobile phase consisted of methanol and water in the volumetric ratios 40:60 for pantethine and 60:40 for S-

pivaloyl pantetheine, and was delivered at a rate of 1.0 mL/minute from a reservoir. The column temperature was set to 25°C, injected sample volume was 20 μl, and run time was 10 minutes. After each permeation experiment, the corneas were trimmed of excess scleral tissue and conjunctiva, weighed, and dried in an oven overnight at 100°C. After each cornea was dried, it was reweighed so that the hydration level of the cornea could be determined. Each cornea had a hydration level of between 77-79%. A normal, undamaged cornea gives a hydration level of 76-80% following this same procedure, indicating the hydration levels in the test corneas were within satisfactory hydration limits. Four corneas were tested in the above manner, two corneas with pantethine (corneas #1 and #2) and two with S-pivaloyl pantetheine (corneas #3 and #4) . The initial concentration (C ₀ ) of pantethine for corneas #1 and #2 was 72 mM, and the initial concentration (C ₀ ) of S- pivaloyl pantethine for corneas #3 and #4 was 88 mM. After bathing the epithelial side of each cornea with concentration C ₀ of each compound, the concentration of pantethine and S-pivaloyl pantetheine appearing in the receiving chamber was measured. These result are expressed (mM as a function of time, Ci (t) ) in Table 4.

Table 4

Corneal Penetration of S-Pivaloyl Pantetheine

Compared to Pantethine

Ci(t) (mM)

Minutes Cornea #1 (ϋornea #2 Cornea #3 Cornea #4

0 0 0 0 0

2 0 0 0 0.013

10 0.0018 0.0004

15 0.002 0.014

30 0.028 0.0049 0.051 0.047

60 0.071 0.021 0.236 0.171

90 0.105 0.104 0.558 0.429

120 0.141 0.140 0.950 0.778

150 1.368 1.160

180 1.743 1.550

210 2.183 1.899

240 2.509 2.212

Figure 4 illustrates the data collected for corneas #1 through #4 of Table 4. More specifically, Figure 4 is a plot of the receiving chamber concentration of pantethine as a function of time (C-j_(t)) for the data collected from 0 to 120 minutes. This figure graphically illustrates the enhanced corneal permeability achieved in corneas #3 and #4 by S-pivaloyl pantetheine (i.e., the upper two lines) , compared to the corneal permeability of corneas #1 and #2 by pantethine (i.e., the lower two lines) .

By employing the equation (1) above (with V = 7 cm ³ , A = 0.8 cm ² , C ₀ = 72 mM and C ₀ = 88 mM) , the steady state penetration coefficient (K) may be calculated for both pantethine and S-pivaloyl pantetheine. Specifically, penetration coefficient for S-pivaloyl pantetheine was 2.0 x 10 ^~5 cm/seconds (see Figure 5) , and for pantethine was 2.6 x 10 ^~6 cm/seconds (see Figure 6) . Thus, K _s . _{pivaloyl p} an _t et _h eine ^was approximately 7 to 8 times greater than pan _t e _th ine' demonstrating that corneal permeability of representative compound of this invention was superior compared to that of pantethine.

Example 8 Conversion of Pantethine to Pantetheine

Upon Contact With Corneal Lens Tissue This example illustrates the conversion of the disulfide bond of pantethine to its sulfhydryl form,

pantetheine, upon contact with various lens tissue. In other words, this example illustrates the ability of compounds having structure 1 (where Z is a sulfur- containing compound joined to the terminal sulfur via a disulfide linkage) and structure 2, to be converted in vivo to the biologically active anti-cataract agent, pantetheine.

In this example, the following protein solutions were employed: (1) calf lens homogenate, (2) unfractionated alpha-, beta- and gamma-crystallin solutions, and (3) purified gamma-II crystallin and gamma- IV crystallin solutions. These solutions were prepared by homogenizing bovine calf lenses, separating the various constituents by gel filtration and ion exchange chromatography, and concentrating by ultrafiltration and/or centrifugation.

The protein solutions were then mixed with pantethine (7.5 mM) and incubated at 37°C for either 30 or 180 minutes. After incubation, proteins were precipitated by the addition of 2 parts methanol to 1 part reaction mixture, allowed to stand for 30 minutes, and centrifuged for 15 minutes (Beckman JA-20 rotor, 15,000 rpm, 4°C) . The supernatant from each tube was recentrifuged for 5 minutes and the final supernatant was placed into HPLC vials for analysis of pantethine and pantetheine concentrations.

The HPLC system included the following components: Rainin solvent delivery module, Rainin Dynamax automatic sample injector (Model AI-1) , Rainin reversed phase C18 column, 5 μm particle diameter, pore size 100 Angstroms, 15 cm x 4.6 mm (L x I.D.) , and Rainin Cynamax ultraviolet absorbance detector (Model UV-D) set to a wavelength of 230 nm. Data was collected and processed with a Waters 860 System Data Station on a Digital Equipment MicroVax 3100 computer. The mobile phase contained methanol and water in the volumetric ratios of 40:60, and was delivered at a

rated of 1.0 ml per minute from a reservoir. The column temperature was set to 25°C, the injected sample volume was 20 μL, and the run time was 10 minutes.

As controls, three types of samples were employed: (1) samples containing the relevant protein(s) but with no added pantethine, (2) samples containing pantethine but with no protein, and (3) samples containing water only. Each control was treated in identical fashion to the pantethine/protein(s) samples described above, including incubation, addition of methanol and centrifugation. No pantethine or pantetheine was detected in those sample which did not receive the addition of pantethine. Moreover, no pantetheine was detected in samples containing pantethine but no protein. The results of this experiment are summarized in

Table 5, and illustrate the ability of compounds of structure 1 (where Z is a sulfur-containing compound linked via a disulfide bridge to the terminal sulfur atom) and structure 2 to convert to a biologically active anti- cataract agent upon contact with ocular lens protein.

Table 5

Conversion of Pantethine to Pantetheine

Upon Contact With Ocular Lens Protein

Initial Resulting calf ns incubation Protein Pantethine Pantetheine

Protein Time Cone, Cone, Cone. Homogenate 30 min. 230 mg/ml 7.5 mM 6.6 mM

Homogenate 30 min. 182 mg/ml 7.5 mM 8.4 mM

Alpha- 30 min. 133 mg/ml 7.5 mM 1.2 mM Crystallin

Beta- 30 min. 124 mg/ml 7.5 mM 4.2 mM Crystallin

Gamma- 180 min. 4.8 mg/ml 7.5 mM 0.7 mM Crystallin

Gamma-II 180 min. 15.4 mg/ml 7.5 mM 1.8 mM Crystallin

Gamma-II 180 min. 30.7 mg/ml 7.5 mM 3.1 mM Crystallin

Gamma-IV 180 min. 9.3 mg/ml 7.5 mM 0.4 mM Crystallin

Gamma-IV 180 min. 18.5 mg/ml 7.5 mM 0.9 mM Crystallin

Example 9

Efficacy of S-Pivalovl Pantetheine

In Selenium-Induced Cataract

This example illustrates the efficacy of a representative compound of this invention, S-pivaloyl pantetheine, to prevent or inhibit cataract formation in the selenium-induced animal cataract model (pantethine was also used in this experiment for comparative purposes) . In this model, a mature cataract forms four or five days after a single subcutaneous injection of selenium, and has been described, for example, by Bunce and Hess, Exp. Eye Res. _____:505-514, 1981, and reviewed by Shearer et al. , Curr. Eye Res. ϋ:289-300, 1987 (which references are hereby incorporated by reference) .

Sodium selenite, dissolved in sterile saline to a concentration of 1.8 mg/ml, was injected subcutaneously into 14-day-old Spraque-Dawley rats such that each rat received a dose of 3.25 mg/kg of body weight. Rats treated with S-pivaloyl pantetheine or pantetheine were dosed one day prior to selenite injection, and on the day of selenite injection. On each day of dosing, four 10 μl drops of either 2% S-pivaloyl pantetheine or 40% pantethine were adinistered to each eye. On the day prior to selenite injection, the drops were administered over an

eight hour period, two in the morning and two in the afternoon. On the day of selenite injection, two drops were administered before injection, and two drops after injection. In this experiment, the rats were divided into three groups: selenite only group (control group) , selenium plus S-pivaloyl pantetheine treatment, and selenite plus pantethine treatment.

One day prior to selenite injection, gross opthalmic observations were performed to scale each eye from Stage 0 to Stage 6 as follows: Stage 0, completely clear lens; Stage 1, barely visible haziness at lens necleus; Stage 2, diffusely hazy nucleus; Stage 3, densely hazy nucleus; Stage 4, opaque (white) nucleus, small cataract; Stage 5, opaque nucleas, large cataract; and Stage 6, completely opaque lens.

At day 7 after selenite injection, the rats were again evaluated for cataract developement according to the above scal . The results of this experiment are are presented in Table 6 ("mature cataract" = stage 4-6; "slight catarct" = 1-3.5; and "clear lens" = stage 0) .

Table 6

Selenium Plus Selenium Selenium Plus 2% S-P valoyl Only 40% Pantethine Pantetheine

# of Eyes 16/20 3/20 8/18 With Mature

Cataract

# of Eyes 2/20 2/20 2/18 With Slight

Cataract

# of Eyes 2/20 15/20 8/18 With Clear

Lenses

Referring to the results presented in Table 6, most of the control animals (i.e., 16 out of 20) receiving only selenite developed mature cataract, while less than half of the animals receiving S-pivaloyl pantetheine developed mature cataract. More importantly, this protection against the onset of cataract was achieved with topical application of eye drops containing 2% of S- pivaloyl pantetheine. For comparison, the inhibitory effect of pantethine was achieved at a 40% concentration, 20 times the concentration of S-pivaloyl pantetheine. The ability of S-pivaloyl pantetheine to inhibit the onset of cataract associated with this cataract model is believed to be due, at least in part, to its enhanced corneal permeability compared to that of pantethine (as demonstrated above in Example 7) .

From the foregoing, it will be appreciated that, although specific embodiments of this invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.