ION PAIRS OF HYPERICIN COMPOUNDS HAVING ANTIVIRAL
ACTIVITY
FIELD OF THE INVENTION
The present invention relates to ion pairs of hypericin and their use to inhibit the growth of viruses.
BACKGROUND TO THE INVENTION Hypericin, a constituent of plants of the genus
Hypericum, has been obtained in pure form from plants (Brockman, et al., Ann. 553:1 (1942)), and has also been totally synthesized, (Brockman, et al, Chem. Ber. 90:2302- 2310 (1957) and Brockman, et al, Chem. Ber. 90:2480-2491 (1957)).
The Merck Index. 11th Edition, 4799, 1989, reports that hypericin has the following structure:
It further reports that it gives solvated blue-black needles from pyridine + methanolic HC1 dec. 320°; is freely soluble in pyridine and other organic bases yielding cherry-red solutions with red fluorescence; is almost insoluble in most other organic solvents; is solu¬ ble in alkaline aqueous solutions; and is red in solutions below pH 11.5 and is green with red fluorescence in solu¬ tions above pH 11.5.
Hypericin, both of plant origin and synthetic, has been found to be a potent inhibitor of a wide spectrum of DNA and RNA containing viruses, and particularly of retroviruses, such as Human Immunodeficiency Virus (HIV) ,
the presumed causative agent of AIDS and other condi¬ tions.
U.S. Patent No. 4,898,891 issued February 6, 1990, discloses antiviral pharmaceutical compositions containing hypericin, pseudohypericin or pharmaceutically acceptable salts thereof and methods for using these compositions to treat viral infections.
Meruelo et al, Proc. Na l. Acad. Sci. USA, 85:5230- 5234 (1988), and U.S. patent 5,047,435 reported antiretroviral activity of hypericin and pseudohypericin in vitroand i vivo . The authors also reported that these aromatic polycyclic diones also were able to inhibit HIV from infecting individual cells.
Halm, Bulletin of Pharmacy. 33:217-218 (1978) and Nozaki, JP 063254, published Oct. 29, 1984, disclose the extraction and use of an antiviral agent from Hypericum sp.
U.S. Patent No. 5,149,718, issued September 22, 1992, describes compositions and methods for inactivating viruses and retroviruses present in blood, other body fluids and, more generally, biological fluids. The compo¬ sitions include the antiviral compounds hypericin, pseudohypericin, derivatives, analogs, isomers, homologs, salts and mixtures thereof. PCT patent publications WO 89/09055 and 89/09056 disclose compositions for treating retroviral infections with hypericin, pseudohypericin, salts and mixtures with nucleoside analogues, such as AZT.
However, it has been noted that different prepa- rations of hypericin, obtained from plants of the genus Hypericum or prepared synthetically, varied in their biological activity. Also the hypericin preparations differed in some of their physical properties, including solubility in organic solvents and formation of disper- sions in water. This variation apparently resulted from the way hypericin was extracted and purified. Therefore, the need arose to find compounds possessing a hypericin
moiety which would give consistent antiviral and antiretroviral activity. There is also a need to obtain compounds possessing a hypericin or related moiety with increased anti-viral activity, and physical properties more suitable for biological function.
SUMMARY OF THE INVENTION The present invention relates to novel compounds possessing a hypericin moiety consisting of ion pairs having anti- viral activity. These ion pairs, consisting of negatively charged hypericin ions bound to cationic species, either organic or inorganic, are useful in the treatment of various diseases caused by viruses and par¬ ticularly retroviruses in humans and animals and are also useful in neutralizing the infectivity of viruses in vitro . The ion pairs can be prepared by first treating the hypericin with acid to yield free hypericin. Free hypericin is then dispersed in an organic or inorganic base to form an ion pair. The hypericin ion pair may be used for the prophylactic or therapeutic treatment of an individual exposed to or suspected of being infected with a virus. The hypericin ion pair may also be used for inhibiting the growth of a virus in vi tro or disinfecting fluids and materials.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph plotting the percent inhibi¬ tion of Friend virus induced splenomegaly in mice as a function of the administered amount of hypericin-sodium ion pair and hypericin-lysine ion pair.
Fig. 2 is a graph plotting the percent inhibi¬ tion of Friend virus induced splenomegaly in mice as a function of the administered amount of hypericin-sodium ion pair and hypericin-Tris ion pair.
Fig. 3 is a graph plotting the percent inhibi¬ tion of Friend virus induced splenomegaly in mice as a
function of the administered amount of hypericin-sodium ion pair and hypericin-ammonium ion pair.
Fig. 4 is a graph plotting the antiretroviral activity of various hypericin ion pairs by monitoring the direct inactivation of murine radiation leukemia virus as measured by the inhibition of virus-particle derived reverse transcriptase activity.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS It has now been discovered that the form of hypericin which has heretofore been obtained by isolation from plants contains a certain amount of sodium ions. The usual way to isolate hypericin from plant material is by extraction with polar solvents such as ethanol, methanol, acetone and the like, followed by chromatography on silica gel. As obtained, this form of hypericin has sodium ions originating from silica gel which invariably contains sodium ions. The relative quantities of sodium ions in the pure hypericin obtained by chromatography on silica gel may vary, depending on the type of the silica gel used, the solvent applied in the elution procedure, and the rate of the elution. It has also been discovered that the hypericin prepared by chemical synthesis contains sodium ions, since generally its purification also employs chromatography on silica gel.
The alternative way of obtaining hypericin in the pure form described in the above-cited Merck Index and in the literature is crystallization or precipitation from pyridine solutions in the absence or in the presence of hydrogen chloride. These crystals are very slightly soluble in organic solvents, and practically insoluble in water. These may be converted to a soluble form of hypericin also by chromatography on silica gel.
The present inventors have discovered by means of X-ray diffraction that such crystals of hypericin precipitated from pyridine solutions contain two molecules of pyridine and one of water. According to the diffrac-
tion pattern, one of the hydrogen atoms of the two hydroxyl groups which are adjacent to one another at the right side of the structure shown as (I) hereinabove, is included between the two pyridine molecules. This indi- cates that these crystals of hypericin are composed of negatively charged hypericin and positively charged pyridinium moiety.
The significance of this discovery is that it shows that hypericin is already ionized at slightly alka- line or lower pH. This discovery was corroborated by the measurements of UV-visible absorption spectra, indicating that the pKa value of hypericin is ca. 2.5, and that the first ionization of the above mentioned hydroxy group occurs at that pH. The same spectral measurements showed us that the second pKa value of hypericin is ca. 11 demon¬ strating that at above this pH value two hydroxy groups ionize. Thus, under conditions above pH 2.5, only one of the hydroxy groups is capable of ionization forming an ion pair with a base, while at above pH 11 both hydroxy groups ionize forming a salt with two cations.
It is believed that some of the difference in the physical properties, including solubility, and the variable biological activities which have been noted with varying batches of hypericin, whether isolated from plants or chemically synthesized, were due to inconsistent amounts of sodium, bound to the hypericin molecule or to lack of sodium at all. In order to differentiate from the form of hypericin in which both hydroxy groups are ionized and the form in which only a single hydroxy group is ionized, the former will hereinafter be referred to as a salt and the latter as an ion pair.
To reduce the problem of variability of solubil¬ ity and biological activity between different batches of hypericin, the compound is first acidified to form free hypericin (I) in which none of the hydroxy groups are ionized and any pyridine or sodium paired to the molecule during work-up is removed. Free hypericin is acidic and
has different IR, UV-visible and fluorescent spectra characteristics than are obtained from the starting form of hypericin.
Once the free hypericin is formed, it is reacted with an organic or inorganic base at a pH below about 11.5 to result in a compound which consists of a negatively charged hypericin moiety bound to an organic or inorganic cationic species as a hypericin ion pair. Hypericin ion pairs differ from hypericin salts, the green colored material described above, by their solubility in organic polar solvents, their color and their very low electric conductance. Thus, the ion pairs behave in all respects as organic compounds and can be defined as such and not as salts.
By first acidifying to free hypericin before reacting with a predetermined quantity and pH of base, a compound having a structure of improved predictability and reproducibility is formed with improved predictability and reproducibility with respect to the properties of solubil¬ ity and biological activity.
The hypericin ion pairs of the present invention have the general formula:
wherein either X is a monovalent cation and n is 1, or X is a divalent cation and n is 2, X not being a sodium or pyridinium cation.
Thus, the present invention comprehends the use of any organic or inorganic base, with the exception of NaOH and pyridine, as the cation in the ion pair as long as the pH is not such as to create the divalent salt of
hypericin. Preferred inorganic cationic species include the alkali and alkaline earth metal cations, such as potassium, lithium, calcium and magnesium, as well as ammonium. Preferred organic cations are protonated nitro- gen base moieties of the general formula NR1R2R3R4 where R1 -R* are the same or different and may be hydrogen; alkyl, alkenyl or cycloalkyl optionally substituted by one or more of hydroxy, alkoxy, phenoxy, amino or carboxy groups; or aryl, aralkyl or alkaryl groups such as phenyl, phenylalkyl and phenyl substituted by alkyl, alkoxy, hydroxy or halogen; or alkyl interrupted by one or more nitrogen or oxygen atoms or by nitrogen atoms substituted by alkyl or by alkyl interrupted .by one or more nitrogen or oxygen atoms. By appropriate selection of R1 -R , the cation may be any of the basic amino acids.
Two of the R groups may, together with the nitrogen atom to which they are attached, form a pyrrolidine, piperidine, morpholine, piperazine or an N- alkyl substituted piperazine ring, or three of the R groups form together with the nitrogen atom to which they are attached an alkyl-substituted pyridine ring.
The various ion pairs of the present invention have varying solubility in water depending on the cation which is chosen. The preferred organic cationic species are those of the general formula, NHR1R2R3 in which R1 -R3 are all ethyl or 2-hydroxyethyl; R1 and R2 are hydrogen and R3 is octyl, 2-hydroxyethyl, 2-aminoethyl, 5-amino-5- carboxypentyl (lysine) or 1,3-dihydroxy-2-hydroxymethyl-2- propyl; R1 is hydrogen and R2 and R3 are 2-hydroxyethyl or 2-hydroxypropy1; or R1 is hydrogen, R2 is methyl and R3 is 1-desoxy-1-sorbityl.
Compounds wherein X is an inorganic monovalent or divalent cation may be produced by addition of the free hypericin to a water solution or suspension of the corre¬ sponding inorganic base such as alkali carbonate, bicar¬ bonate, hydroxide, phosphate, etc. For example, compounds
wherein X is ammonium, lithium or potassium cations may be prepared by addition of free hypericin to a water solution of ammonium hydroxide, lithium hydroxide or potassium hydroxide, respectively, preferably using one equivalent, followed by lyophilization of the resulting solution to dryness. When X is an inorganic divalent cation, for example calcium or magnesium cation, the ion pairs may be prepared by addition of free hypericin to a water solution of calcium hydroxide or magnesium hydroxide, respectively, preferably using half an equivalent, followed by lyophilization of the resulting solution to dryness.
The hypericin ion pairs wherein X is an organic cation derived from a nitrogen base bound to hydrogen of the structure NHXR2R3 may be prepared by treating free hypericin dissolved in an organic polar solvent with the respective nitrogen base, preferably using one equivalent. Alternatively, a suspension of the free hypericin in water may be treated with the nitrogen base.
For example, the hypericin ion pair wherein X is NHR1R2R3, where R =H, R2=CH3 and R3 =CHZ (CHOH) hCHZOH, may be prepared by addition of free hypericin to a water solution of N-methyl glucamine, followed by lyophilization of the resulting solution. In another example wherein R1=R2=R3 =CH2CH2OH, the hypericin ion pair may be prepared by addition of free hypericin to an ethanol solution of triethanolamine, followed by evaporation to dryness.
The hypericin ion pairs, wherein X is a quaternary ammonium cation of the structure N"1" (R1 ,R2 ,R3 ,R may be prepared by treating a hypericin ion-pair, preferably containing inorganic monovalent cationic species which may include sodium, potassium, ammonium or lithium cations dissolved in an organic polar solvent or water, with the respective quaternary ammonium salt. For example, the hypericin ion pair, wherein X is ST (R1 ,R2 ,R3 ,R* ) where R1=R2=R3 = * =CH3 may be prepared
by addition of hypericin ammonium ion-pair to a water solution of tetramethylammonium chloride, followed by lyophilization of the resulting solution. The resulting dry material is then heated in vacuum. In another example the hypericin ion pair wherein X is
NT (R1 ,R2 ,R3 ,R* ) , where R1 =R2 =R3 =CH3 and R = (CH2 ) __ 5 may be prepared by addition of hypericin sodium ion-pair to cetyltrimethylammonium bromide in water, followed by lyophilization of the resulting solution. The resulting dry material is then extracted with an organic solvent, preferably one in which sodium bromide is insoluble, like toluene, acetonitrile, etc. Evaporation of the solvent to dryness results in the respective quaternary ammonium ion pair. It has been observed that quaternary hypericin ion pairs behave in all aspects as organic compounds and cannot be defined as salts.
The hypericin ion pairs of the present invention are characterized by their solubility in organic polar solvents. Some of these derivatives can be dispersed in water, forming high molecular weight, loosely associated polymeric structures containing occluded water which can be decomposed back to the monomeric hypericin by addition of organic solvents.
The dispersability of hypericin ion-pairs in water has a unique influence on their bioavailability. In water dispersions, hypericin ion-pairs are in an aggregate state, but inside cellular or viral membranes they exist in a monomeric form. Thus, the bioabsorption of the hypericin ion-pairs necessitates dissociation of the aggregates into monomeric molecules. This slow down in the bioabsorption effect will provide slow-release of active substances. It is to be anticipated that in such cases a constant level of the active substances in the body will be kept for comparatively long periods. Some ion pairs exhibit slower bioabsorption than others. Liposomes are a model for all membranes. High solubility in liposomes is indicative that the compound
enters the cell rapidly and thus more frequent doses have to be administered, while low solubility indicates that the compound enters the cell slowly. Thus, amounts of the compound remain in the organism and act as slow release agents, thus necessitating less frequent administration of doses.
Solubility of hypericin ion-pairs in liposomes is given in Table 1„ A solution of liposomes was prepared from lecithin (10%) „ This solution was mixed with an equal volume of water containing various hypericin ion- pairs. The maximum binding capacity of the liposomes for the following hypericin ion-pairs was established.
Table 1
Ion-Pair Compound Solubility (mg/ml of
5% lecithin solution)
Hypericin-sodium 0.06 -lithium 0.06
-lysine 0.02
-ethylene diamine 0.01
The ability of hypericin-lysine and hypericin- ethylene diamine ion-pair to act as slow release vehicles for the target organs is shown by their comparative low solubility in liposomes (as compared with the solubility of sodium and lithium ion-pairs) . However, all ion-pairs would be expected to be less soluble than salts which are fully dissolved in monomeric form.
Because the hypericin-sodium ion pair conven¬ tionally obtained from isolation or synthesis has antiviral, including antiretroviral, activity, it is expected that all of the various ion pairs which can be made in accordance with the present invention will also have some degree of antiviral and antiretroviral activity. Of course, the specific activity with respect to specific viruses or retroviruses would be expected to vary from compound to compound, and optimum ion pair cations for use with any given virus or retrovirus can be determined
empirically. Antiviral activity against Friend virus has been measured using ion pairs of hypericin with sodium, lysine, ethylenediamine, diethanolamine, N- methylglucamine, triethanolamine, Tris and ammonium. The hypericin ion pairs of the present invention can be advantageously used as prophylactic antiviral agents, for treatment of virally-infected mammals or for inactivating viruses and retroviruses present in biologi¬ cal fluid as in U.S. Patent No. 5,149,718. The effective- ness of hypericin and pseudohypericin for antiviral effects has been shown in U.S. Patent 4,898,891. The hypericin ion pairs of the invention can be combined for antiviral therapy with nucleoside analogs such as azidothymidine (AZT) or dideoxyinosine (DDI) , acyclovir or other reverse transcriptase inhibitors when treating retroviral infections. For other viral infections, the hypericin ion pairs of the invention may be used with other antiviral agents such as adamantine, rifampicin, or with vaccines or antibody preparations. "In conjunction" includes successive administration, co-administration, substantially contemporaneous administration of different preparations or alternating administration of nucleoside analog therapy and hypericin ion pairs.
The hypericin ion pairs may be used in conjunc- tion with other biologically effecting substances. Other known antibiotics may be used in conjunction with hypericin ion pairs even if that antibiotic does not have any effect on viral growth per se. For example, when being administered to an immunosuppressed individual, it may be desirable to include one or more antibacterial or antifungal agents in combination to prevent opportunistic infections. Furthermore, hypericin ion pair containing compositions may include one or more immunomodulating chemicals. Biological materials and transplants may also contain hypericin ion pairs as part of the therapy.
The hypericin ion pairs of the present inven¬ tion, in their water-dispersed or water-solubilized forms
have a wide spectrum of effectiveness in inhibiting vi¬ ruses and retroviruses. Non-limiting examples of the viruses which are inhibited by the compounds of the pres¬ ent invention are cytomegalovirus, Herpes Simplex Virus (HSV) , influenza virus, Vesicular Stomatitis Virus (VSV) , Hepatitis B virus, papilloma virus and retroviruses, such as HIV, HTLV I, HTLV II and feline leukemia virus.
Effective inhibition of a given virus may be achieved by using one of the compounds of the present invention or a combination of two or more of such com¬ pounds. Moreover, a single hypericin ion pair may consti¬ tute the sole active ingredient of the compositions of the present invention or may be employed in conjunction with other antiviral agents acting by any means. When treating mammals suffering from infections caused by viruses according to the present invention, the determination of the most effective compound or mixture of compounds and the concentration of effective treatment of the particular virus or retrovirus responsible for the infection, can be ascertained by routine experimentation using suitable experimental models well-known in the art.
When employed to treat AIDS, viremia, or sepsis, the hypericin ion pairs of the present invention may be administered orally, topically or parenterally, and pref- erably intravenously at dosages broadly ranging between about 0.0001 micrograms and about 100,000 micrograms per kilogram (kg) of body weight of the treated mammal per treatment. More preferably, the dosage range may between about 0.1 and about 50,000 micrograms per kg body weight of the treated mammal per treatment.
The hypericin ion pairs of the present invention may also be used on various surfaces to inactivate or inhibit viruses in vitro. Among the surfaces to be treated include surgical equipment, prosthetic devices, interior and/or exterior surfaces of gloves, condoms, body fluid handling devices such as needles, syringes, cathe-
ters, etc. The compositions of the invention may be used as antiseptics broadly as well to disinfect organs, flu¬ ids, skin, table tops, containers, equipment, etc. A thin film of the composition may remain on the surface. The present invention also provides pharmaceuti¬ cal compositions and formulations for treating viral infections. The hypericin ion pairs of the present inven¬ tion can be incorporated in conventional solid or liquid pharmaceutical formulations in any concentration desired. For example, tablets, capsules, caplets, injectable or orally administrable solutions may be used for treating mammals that are afflicted with viral infections. The pharmaceutical formulations of the invention comprise an effective amount of the hypericin ion pair of the present invention as the active ingredients. Other active or inert ingredients may be added.
For example, a parenteral therapeutic composi¬ tion may comprise a sterile isotonic saline solution containing between 0.001 micrograms of the hypericin ion pair of the present invention. It will be appreciated that the unit content of active ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount provided that the effective amount can be reached by administration of a plurality of doses.
Each formulation according to the present inven¬ tion may additionally comprise inert constituents in¬ cluding pharmaceutically-acceptable carriers, diluents, fillers, salts, and other materials well-known in the art, the selection of which depends upon the dosage form uti¬ lized, the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field and the properties of such additives. Examples of carriers and diluents include carbohydrates and lipids including, without limitation, phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine,
triglycerides, tocopherol, retinoic acid, cyclodextrins and their derivatives.
Hypericin ion pairs of the present invention may additionally be incorporated into transdermal delivery systems or liposomes, the latter for use as specific drug carriers. Such liposomes may also comprise other active agents, e.g., specific anti-HIV antibodies directed against viral proteins expressed by virally infected cells such as HIV gpl20, pl20, gp41, p 41 and p24 to act as specific targeting agents.
Topical use of the hypericin ion pairs in a suitable carrier may also be used. This may be of partic¬ ular importance in viral rashes, herpes simplex vesicles (type I or type II) , shingles, etc. While the present specification specifically refers to hypericin as the compound which is formed into an ion pair, it should be understood to those of ordinary skill in the art that this ion pair technology is also applicable to other polycyclic aromatic diones which have antiviral and antiretroviral activity and which have two reactive hydroxyl groups in their free acid form as does hypericin. For example, pseudohypericin has the same structure as hypericin except that one of the two methyl groups shown on the right-hand side of formula (I) is replaced by CH20H. Other modifications to the substituents of hypericin may also be made on moieties other than the hydroxy groups on which the ion pair is formed, so long as such modifications do not affect the solubility properties of the ion pairs and do not substan- tially affect the antiviral and antiretroviral properties of the compounds themselves. These compounds will be referred to herein as hypericin analogs and derivatives. For example, the methyl groups may be replaced by other alkyl, alkylamino, alkoxy, and alkylaminoalkyl groups. These two groups may be combined to form a six or seven membered ring. The remaining four hydroxy groups on the hypericin molecule (other than those involved in the ion
pair reaction) may be eliminated (H) or substituted with an ether group (OR) or an amino group (NRR) in which R may be hydrogen or alkyl. Preferably, not all of these OH groups are eliminated in the same compound. The remaining four positions on the ring structure which are not substi¬ tuted may be substituted with OH or alkyl. All of these compounds are expected to form ion pairs in the same manner as discussed above for hypericin and are intended to be included within the scope of the present invention. The hypericin used in the following examples was prepared according to the methods in U.S. Patent No. 4,898,891. Pharmaceutical preparations described in this patent may also be used with hypericin ion pairs of the present invention.
EXAMPLE 1 - Free Hypericin
Free hypericin was prepared by mixing a solution of hypericin (lg) in acetone (150 mL) and treating it with hydrochloric acid solution 7% (5 mL) . The resulting brown precipitate was filtered and washed with water until it became neutral. The precipitate was collected and dried in high vacuum at 70°C.
IR spectrum (KBr) showed adsorption bands at 3400, 1625, 1598, 1473, 1426, 1399, 1369, 1330, 1300, 1224, 1188', 1153, 1116, 1090, 844, 806, 794, 673 cm-1.
UV visible absorption spectrum (EtOH) λmax 572, 531, 461 nm
NMR (DMSO) δ 2.69, 6.51, 7.35, 14.04, 14.67ppm (all singlets)
EXAMPLE 2 - Hypericin-Sodium Ion Pair
Hypericin sodium ion pair was prepared by slowly adding powdered free hypericin (0.25g), obtained in Exam¬ ple 1, to a stirred solution of acetone (50 ml) and 5% aqueous sodium bicarbonate (168 ml, 2mM) . After about 0.5 hours, the resulting solution was filtered and the fil-
trate evaporated to dryness. The ion pair was further dried in high vacuum at 70°C
IR spectrum (KBr) showed adsorption bands at 3400, 1615, 1590, 1559, 1500, 1465, 1422, 1353, 1386, 1337, 1260, 1187, 1114, 843, 805, 685, 604, 567, 538, 527 cm" 1 UV visible absorption spectrum (EtOH) λmax 589, 545, 470, 382, 325nm
NMR (DMSO) 18.40, 14.7, 14.1, 7.4, 6.5, 2.7ppm (all singlets)
EXAMPLE 3 - Hypericin Potassium Ion Pair
Hypericin potassium ion pair was prepared by slowly adding powered free hypericin (0.25g) , obtained in Example 1, to a stirred solution of acetone (50 ml) and 2% aqueous potassium hydroxide (5.6 ml, 2mM) . After about
0.5 hours, the resulting solution was filtered and the filtrate evaporated to dryness. The ion pair was further dried in high vacuum at 70°C. IR spectrum (KBr) showed adsorption bands at 3400, 1618,
1586, 1557, 1499, 1458, 1386, 1368, 1333, 1299, 1183,
1114, 851, 804 cm" 1
UV visible absorption spectrum (EtOH) xm a x 590, 547, 509, 474 nm
NMR (DMSO) δ 18.26, 14.63, 1401, 7.29, 6.41, 2.63 ppm (all singlets)
EXAMPLE 4 - Hypericin Lithium Ion Pair Hypericin lithium ion pair was prepared by slowly adding powdered free hypericin (0.25g) , obtained in Example 1, to a stirred solution of acetone (50 ml) and 0.3 ml of 3% aqueous lithium hydroxide monohydrate (2mM) . After about 0.5 hours, the resulting solution was filtered and the filtrate evaporated to dryness. The ion pair was further dried under high vacuum at 70°C. IR spectrum (KBr) showed adsorption bands at 3400, 1590, 1558, 1501,
1465, 1422, 1393, 1367, 1336, 1288, 1260, 1188, 1113, 843, 666, 625, 603, 567, 528 cm"1
UV visible absorption spectrum (EtOH) λmax 589, 545, 470, 382, 325 nm.
NMR (DMSO) δ 18.3, 14.65, 14.02, 7.31, 6.43, 2.64 (all singlets)
EXAMPLE 5 - Hypericin Aπmonium Ion Pair Hypericin ammonium ion pair was prepared by slowly adding powdered free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50ml) and 0.32 ml of 5% ammonium bicarbonate (2 mM) . After about 0.5 hours, the resulting solution was filtered and the filtrate evaporated to dryness. The ion pair was further dried under high vacuum at 70°C.
IR spectrum (KBr) showed adsorption bands at 3400, 3200, 1620, 1585, 1556, 1499, 1461, 1418, 1365, 1334, 1260, 1183, 1114, 893, 850, 831, 804, 671, 624 cm"1 UV visible absorption spectrum: λmax 590, 547, 509, 474 nm
NMR (DMSO) δ 14.65, 14.02, 7.15, 6.44, 2.60 ppm (all singlets)
EXAMPLE 6 - Hypericin Calcium Ion Pair
Hypericin calcium ion pair was prepared by slowly adding free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50 ml) and 1 ml at 2% aqueous calcium hydroxide. After about 0.5 hours, the resulting solution was filtered and the filtrate evapo¬ rated to dryness. The ion pair was further dried under high vacuum at 70°C.
IR spectrum (KBr) showed adsorption bands at 3500, 1620, 1585, 1563, 1490, 1463, 1418, 1386, 1334, 1260, 1180, 1116, 845, 806, 677, 625, 604, 571 cm-1
UV visible absorption spectrum: (EtOH) λma3C 590, 547,
509, 474 nm NMR (DMSO) δ 18.37, 14.70, 14.06, 7.38, 6.51, 2.70 ppm (all singlets)
EXAMPLE 7 - Hypericin Magnesium Ion Pair Hypericin magnesium ion pair was prepared by slowly adding powdered free hypericin (0.25g), obtained in Example 1, to a stirred solution of acetone (50 ml) and 7 mL of magnesium hydroxide pentahydrate in 2% aqueous basic magnesium carbonate. After about 0.5 hours, the resulting solution was filtered and the filtrate evaporated to dryness. The ion pair was further dried under high vacuum at 70°C.
IR spectrum (KBr) showed adsorption bands at 3500, 1620, 1585, 1563, 1490, 1463, 1418, 1386, 1334, 1260, 1180, 1116, 845, 806, 677, 625, 604, 571 cm"1
UV visible absorption spectrum: (EtOH) λmax 590, 547,
509, 474 nm
NMR (DMSO) 18.16, 14.70, 13.95, 7.20, 6.34, 2.51 ppm (all singlets)
EXAMPLE 8 - Hypericin Triethylaππonium Ion Pair
Hypericin triethylammonium ion pair was prepared by mixing a solution of triethylamine (0.05g) in acetone (50mL) , treating it with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room tempera¬ ture for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
IR spectrum (KBr) showed absorption bands at 3500, 1590, 1554, 1499, 1421, 1392, 1367, 1338, 1292, 1185, 841 cm"1 UV visible absorption spectrum (EtOH): λmax 590, 547, 509, 474 nm
NMR (DMSO) δ: 18.34, (s) , 14.67 (s) , 7.33 (s) , 6.45 (s) , 3.35 (m) , 3.05 (q,J=7Hz) , 2.60 (s) , 1.17 (t,J=7Hz)
EXAMPLE 9 - Hypericin Octyla-mmonium Ion Pair
Hypericin octylammonium ion pair was prepared by mixing a solution of octylamine (0.065g) in acetone (50
mL) , treating it with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room tempera¬ ture for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness. UV visible absorption spectrum: λmax 590, 547, 509, 474 nm
NMR (DMSO) 18.37 (s) , 7.39 (s) , 5.09 (bs) , 3.45 (bs) , 2.68 (s) ppm
EXAMPLE 10 - Hypericin 2-hydroxyethylaιπmonium Ion Pair
Hypericin 2-hydroxyethylammonium ion pair was prepared by mixing ethanolamine (0.03g) in acetone (50 mL) , treating it with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room tempera- ture for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
IR spectrum (KBr) showed absorption bands at 1617, 1530, 1552, 1464, 1419, 1395, 1383, 1364, 1310, 1261, 1187, 1115, 883, 829, 644, β,' ' 605, 570 cm'1 UV visible absorption spectrum: λmax 590, 547, 509, 474 nm
NMR (DMSO) δ: 7.45 (s) , 6.58 (s) , 2.79 (Z, J=6Hz) , 3.52, (Z, J=6Hz) , 2.74 (s)
EXAMPLE 11 - 2-amino-ethyl mmonium Ion Pair
Hypericin 2-amino-ethylammonium ion pair was prepared by mixing ethylenediamine (0.037g) in acetone (50 mL) , treating it with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room tempera- ture for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
IR spectrum (KBr) showed adsorption bands at 3500, 1580, 1554, 1490, 1445, 1390, 1368, 1292, 1258, 1185, 1115, 940, 847, 807, 622 cm"1 UV visible absorption spectrum: λmax 590, 547, 509, 474 nm NMR (DMSO) δ 7.44 (s) , 6.58 (s) , 2.74 (s) , 1.01 (m) ppm
EXAMPLE 12 - Bis- (2-hydroxyethyl)ammonium Ion Pair
Hypericin bis- (2-hydroxyethyl)ammonium ion pair was prepared by mixing diethanolamine (0.05g) in acetone (50 mL) , treating with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room tempera¬ ture for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness. IR spectrum (KBr) showed adsorption bands at 3450, 1590, 1556, 1551, 1421, 1392, 1367, 1336, 1290, 1258, 1114, 1066, 840, 624 cm'1
UV visible absorption spectrum : (EtOH) λmax 589, 547, 509, 473 nm NMR (DMSO) δ 7.39 (s) , 6.52 (s) , 3.57 (t, J=4.4Hz), 2.86 (t, J=4.4Hz), 2.7 (s) ppm
EXAMPLE 13 - Hypericin Bis- (2-hydroxypropane)ammonium Ion Pair
Hypericin bis- (2-hydroxypropane) ammonium ion pair was prepared by mixing diisopropanolamine (0.05g) in acetone (50 mL) , treating with free hypericin powder (0.25g), obtained in Example 1, and then stirring at room temperature for 30 minutes. The resulting dark red solu¬ tion was evaporated in vacuum to dryness. IR spectrum (KBr) showed adsorption bands at 3450, 1589, 1554, 1464, 1420, 1394, 1368, 1336, 1291, 1259, 1118, 1114, 804, 623 cm"1
UV visible absorption spectrum: λraax 589, 547, 509, 473 nm
NMR (DMSO) δ 18.4 (s) , 7.4 (s) , 6.5 (s) , 3.3 (m) , 2.7 (s) , 1.16 (d, J=6Hz)
EXAMPLE 14 - Tris- (2-hydroxyethyl)ammonium Ion Pair
Hypericin tris- (2-hydroxyethyl)ammonium ion pair was prepared by mixing triethanolamine (0.075g) in acetone (50 mL) , treating with free hypericin powder (0.25g),
obtained in Example 1, and stirring at room temperature for 30 minutes. The resulting dark red solution was evaporated in vacuum to dryness.
IR spectrum (KBr) showed adsorption bands at 3350, 1591, 1558, 1500, 1487, 1465, 1423, 1407, 1336, 1291, 1259, 1187, 1117, 1097, 1094, 1065, 1031, 917, 841, 825 cm"1 UV visible absorption spectrum (EtOH): λmax 590, 547,
509, 473 nm
NMR (DMSO) 18.3 (s) , 14.64 (s) , 14.02 (s) , 7.28 (s) , 6.41 (s) , 3.71 (t,=5Hz). 3.22 (t,J=5Hz), 2.63 (s)
EXAMPLE 15 - Hypericin Lysinium Ion Pair
Hypericin lysinium ion pair was prepared by mixing lysine (0.08g) in water (20 mL) , treating with free hypericin powder (0.25g), obtained in Example 1, and stirring at room temperature overnight. The resulting solution was lyophilized until dry.
IR spectrum (KBr) showed adsorption bands at 3500, 3100, 3000, 1618, 1585, 1502, 1482, 1419, 1386, 1335, 1259, 1182, 1114, 839, 804, 675, 666 cm"1
UV visible absorption spectrum (EtOH): λmax 589, 547,
510, 474, 383, 328 nm
NMR (DMSO) δ 18.4 (s) , 14.07 (s) , 7.43 (s) , 6.77 (s) , 2.81
(m) , 2.73 (s) , 1.54 (m) ppm
EXAMPLE 16 - Hypericin N-methylgluca-πmonium Ion Pair Hypericin N-methylglucammonium ion pair was prepared by mixing N-methylglucamine (0.2g) in water (20 mL) , treating with free hypericin powder (0.25g), obtained in Example 1, and stirring at room temperature overnight.
The resulting solution was lyophilized until dry.
IR spectrum (KBr) showed adsorption bands at 1590, 1557,
1502, 1464, 1421, 1393, 1336, 1259, 1187, 1113, 1084,
1031, 841, 624, 603, 526 cm"1 UV visible absorption spectrum: (EtOH) λmax 589, 547,
510, 474, 383, 328 nm
NMR (DMSO) δ 18 . 4 (s ) , 7 . 43 ( s ) , 6 . 57 ( s ) , 3 . 31 (m) , 2 . 73 ( s ) ppm
EXAMPLE 17 - Hypericin Tris Ion Pair Hypericin Tris ion pair was prepared by mixing
2-amino-2-hydroxymethyl-l,3-propanediol Tris (0.06g) in water (20 mL) , treating with free hypericin powder (0.25g), obtained in Example 1, and stirring at room temperature overnight. The resulting solution was lyophilized until dry.
IR spectrum (KBr) showed adsorption bands at 3500, 1589, 1557, 1463, 1421, 1389, 1367, 1335, 1259, 1185, 1114, 1057, 843, 624, 603, cm"1 UV visible absorption spectrum: (EtOH) λmax 589, 547, 510, 474, 383, 328 nm
NMR (DMSO) 18.36 (s) , 7.4 (s) , 5.09 (m) , 3.45 (m) , 2.68 (s) ppm
EXAMPLE 18: Hypericin- etramethylaπiπonium ion pair. A solution of tetramethylammonium chloride
(0.2g) in water (50ml) was treated with hypericin-ammonium ion pair (0.5g) . The dark red solution obtained was lyophilized, and the resulting dry powder was heated under vacuum at 80°, resulting in the title compound.
EXAMPLE 19: Hypericin-cetyltrimethylammonium ion pair.
A solution of cetyltrimethylammonium bromide (0.5g) in water (100ml) was treated with hypericin-sodium ion pair (0.5g) . The dark red solution obtained was lyophilized, and the resulting dry powder was dissolved in acetonitrile (100ml) . Evaporation to dryness resulted in the title compound.
EXAMPLE 20 - Solubility of Hypericin Ion Pairs The concentration of water dispersions of vari¬ ous selected hypericin compounds were measured. The
following data was found: Compound Solubility (mg/ml)
Hypericin-sodium 1-3.5 Hypericin-lysine 11 Hypericin-ethylenediamine 0.13 Hypericin-N-methylglucamine 9.1 Hypericin-triethanolamine 4.7 Free Hypericin 0.05 Hypericin-pyridine 0.05
EXAMPLE 21: The Antiretroviral Activity of Hypericin- Lysine Ion Pair Against Murine Friend Virus in BALB/c mice.
Mice infected with Friend Virus develop a virus- induced erythroleukemia which results in death of 100% of the animals within 25-45 days. An early manifestation of the infection is a large increase in spleen size (splenomegaly) . The spleens undergo a 4-8-fold enlarge¬ ment in size within 10 days after infection with the virus. Inhibition of splenomegaly can, therefore, be used to assay and to quantitate the antiviral activity of various agents in vivo . In this experiment, mice (in groups of 3) were infected with Friend virus and hypericin-lysine ion pair was then administered intrave¬ nously, within one hour of the infection, in a single dose of 1, 10, 50 and 150 micrograms/mouse. 0.5 ml solution of the compound in PBS was administered. Standard hypericin (obtained by a process which includes chromatography on silica gel and thus is a sodium ion pair) , solubilized in 0.5% aqueous benzyl alcohol and administered at the same concentrations, was used as a control, along with normal control mice infected with PBS. All mice were sacrificed after 10 days and analyzed for spleen weights. The re¬ sults, shown in Fig. 1, show that both hypericin-lysine and hypericin-sodium inhibited splenomegaly of Friend
Virus infected mice by approximately 70-80% at dose ranges of 10-50 micrograms/mouse.
EXAMPLE 22 - The Antiretroviral Activity of Hypericin-Tris Ion Pair Against Murine Friend Virus in
BALB/c Mice
An experiment was conducted as described in example 21 but using hypericin-Tris ion pair. The results are shown in Fig. 2.
EXAMPLE 23 - The Antiretroviral Activity of Hypericin-
Aπmonium Ion Pair Against Murine Friend Virus in BALB/c Mice
An experiment was conducted as described in example 21 but using hypericin-ammonium (Hy-NH,, + ) ion pair. The results are shown in Fig. 3.
EXAMPLE 24 - Determination of the Antiretroviral Activity of Various Hypericin Ion Pairs by Monitoring the Direct Inactivation of Murine Radiation Leukemia Virus as Measured by the Inhibition of Virus-Particle Derived Reverse Transcriptase Activity
Virus particles released into the growth medium of the AQR lymphoblastoid cell line which is infected with and producing RadLV, were exposed to hypericin- ethylenediamine (eda) , hypericin-triethanalamine (tea) , hypericin-diisopropanolamine (dpa) and hypericin-n- methylglucamine (nmg) ion pairs. In addition, two batches of hypericin sodium (bv and pf#4) and a sodium titrated batch (Na) were tested. The virus and compounds were incubate on ice for a period of 30 minutes after which time the virus was precipitated by ultracentrifugation at
40,000 RPM for 1 hour in a Beckman ultracentrifuge and analyzed for Reverse-transcriptase according to Stephenson et al. (Stephenson, H.R. , Reynolds, R.K. and Aaronson,
S.A., Virology. 48:749-756, 1972). The results shown in
Fig. 4 are expressed as count per minute of 3H-thymidine incorporation. They show significant inhibition of virus-
particle derived reverse transcriptase with eda being the least efficacious. See Fig. 4.
Example 25 - In Vitro Assay of Hypericin and Two Ion Pairs thereof against Murine Cytomegalσvirus
(MCMV)
The antiviral activity of the test compounds was assayed at final concentrations of 32, 10, 3.2, 1.0, 0.32 and 0.1 μg/ml. Stock solutions were prepared by dis- solving a preweighed amount of test compound in dimethylsulfoxide (DMSO) , then bringing up the volume with medium so that the final stock concentration was 1 mg/ml. Stock solutions, and all dilutions made from the stock solutions, were prepared in Earles MEM medium with 2% fetal calf serum (2% MM) .
Antiviral activity was evaluated in mouse mamma¬ ry tumor fibroblasts (C127L) grown in 24-well tissue culture plates. Four wells were used for each concentra¬ tion of each test article. After aspirating the medium from the tissue culture plates, three of the wells re¬ ceived 0.5 ml of virus inoculum; the fourth well received 0.5 ml of 2% MM. The plates were incubated for 1 hr at 37°C in 5% C02 with continuous agitation. After incuba¬ tion, the virus inoculum was removed and an agar overlay added. A separate overlay was made for each concentration of test compound. Each overlay consisted of 3 ml of test compound (prepared as a 2x solution) plus 3 ml of agar overlay. After mixing, 1 ml of the combination overlay was added to each of the four wells, the fourth well being the test compound toxicity control. Cell control and virus control wells were included on each plate. Acyclovir, the positive antiviral control, and a virus titration were run simultaneously with the test. The plates were incubated at 37°C in 5% C02 for six days, after which time the overlay was removed and the plates were fixed and stained with 1% crystal violet. The
plaques were read using an inverted microscope and the number of plaques in each well was recorded.
The average plaque forming units (PFU) per ml and percent reduction results from this in vitro assay are shown in Table 2, with the plaque counts for individual wells shown in Table 3. A test compound concentration resulting in a reduction of 50% or more in the number of plaques as compared to the virus control (<44 PFU/ml) is an indication of good antiviral activity. Hypericin was toxic at both the 32 and 10 μg/ml concentrations, but showed a 51% plaque reduction at 3.2 μg/ml, with no reduc¬ tion at any of the other concentrations tested. Both hypericin ion pairs were toxic at 32 μg/ml and moderately toxic at the 10 μg/ml concentration. The N- methylglucamine derivative, even though moderately toxic at 10 μg/ml, showed an 88% reduction in plaques, with no reduction in plaques at any of the other concentrations. The lysine derivative showed a 100% reduction in plaques at the 10 μg/ml concentration. Only 25% of the cell sheet was still attached when the assay was read, but these cells were normal and there were no plaques present. None of the other concentrations tested showed any signs of reduction. Acyclovir, the positive antiviral control, showed complete protection at all of the concentrations tested.
TABLE 2 - Hurine CytoaegaloYirus Plaque Assay
Concentration in /tg/ml
32 10 3.2 0.32 0.1
Test Compound PFU/mla %b PFU/ml PFU/ml PFU/ml PFU/ml PFU/ml
Hypericin TOXIC TOXIC 43 si- ios 117 124
Hypericin TOXIC 11c 88" 126 91 117 107 N-methylglucamine ion pair 1
Hypericin TOXIC 0C 100 111 95 106 125 lysine ion pair
Acyclovir 100 100 100 100
a Average nu-rber of plaque-forming units in three wells of cells.
" Percent reduction in plaque number. c Both derivatives moderately toxic. Cells treated with glucamine derivative showed moderate toxicity with few plaques. Cells treated with lysine derivative had approximately 25% normal cell sheet remaining with no plaques present.
" Samples showing 50% or greater reduction in plaques; the mean and standard deviation for thirty untreated, virus control wells was 87 ± 15.3 PFU/ml.
TABLE 3 - Hurine Cytfegatovirus Plague Assay
Concentration in μg/ml
32 10 3.2 1 0.32 0.1
Test
CompoL-nd PFU/mla PFU/ml PFU/ml PFU/ml PFU/ml PFU/ml
Hypericin TOXIC TOXIC 42, 44, 44 98, 116. 102 126, 118, 106 130, 136, 106
Hypericin TOXIC 6, 10, 16 144, 114, 120 98, 84, 90 124, 126, 100 80, 128, 114
N-methylglucamine ion pair to
00
Hypericin TOXIC 0, 0, 0 106, 140, 88 100, 90, 90 106, 120, 92 110, 136, 128 lysine ion pair
Acyclovir 0, 0, 0 0, 0, 0 0, 0, 0 0, 0, 0
Results are shown for the individ-ial assay wells at each drug concentration. The mean and standard deviation for thirty untreated, virus control wells was 87 ± 15.3 PFU/ml.
In conclusion, in the in vitro assay, hypericin was toxic at the 32 and 10 μg/ml concentrations, but showed a 51% reduction of plaques at the 3.2 μg/ml concen¬ tration. Both hypericin derivatives were toxic at the 32 μg/ml concentration, and, even though both were moderately toxic at the 10 μg/ml concentration, the N-methylglucamine derivative showed a plaque reduction of 88% and the lysine derivative showed a plaque reduction of 100%.
Example 26 - In Vivo Assay of Hypericin and Two Ion Pairs thereof against MCMV
For the in vivo evaluation of the three test articles against MCMV, dosing concentrations of 50, 25, and 10 mg/kg body weight (mpk) were used. Virus antibody- free four week old female CD1 mice were obtained from Charles River Breeding Laboratories (Portage, Michigan) . The animals were randomly housed five per cage after being examined grossly for general activity and alertness. The mice were quarantined for twelve days prior to dosing and were provided with food and water ad libitum throughout the quarantine period and the study. Three days before dosing began, the animals were weighed and assigned to groups with dosages being determined on a group weight basis, with each group consisting of ten mice. The Groups were assigned as follows:
Group 1 Hypericin - 10 mpk
Group 2 Hypericin - 25 mpk
Group 3 Hypericin - 50 mpk
Group 4 Hypericin N-methylglucamine ion pair - 10 mpk Group 5 Hypericin N-methylglucamine ion pair - 25 mpk
Group 6 Hypericin N-methylglucamine ion pair - 50 mpk
Group 7 Hypericin lysine ion pair - 10 mpk
Group 8 Hypericin lysine ion pair - 25 mpk
Group 9 Hypericin lysine ion pair - 50 mpk Group 10 Acyclovir - 25 mpk
Group 11 Ara-A - 25 mpk
Group 12 Vehicle Control
All dosing solutions were prepared at concentra- tions such that the desired dosage of compound could be
administered in a volume of 0.2 ml. For the 50 mpk dosing solutions, the three test compounds were dissolved in sterile water at concentrations of 5.75 mg/ml for hypericin, 5.75 mg/ml for the N-methylglucamine deriva- tive, and 5.5 mg/ml for the lysine derivative. The 25 and 10 mpk dosing solutions were prepared by dilution with sterile water from the 50 mpk concentration. The positive control compounds, Acyclovir and adenine-9-B-D-arabinoside (Ara-A) , were dissolved in 0.25% methyl cellulose. Dosing solutions were prepared fresh daily.
Mice were administered the test articles in a volume of 0.2 ml by intraperitoneal injection once each day for eight consecutive days. The vehicle control group re¬ ceived daily injections of 0.2 ml of sterile water. On the second day, approximately five hours after receiving the second dosing of test compound, all mice were adminis¬ tered a 0.2 ml challenge dose of MCMV by intraperitoneal injection. Mice were observed for mortality for twenty- one days after virus challenge. Percent mortality and mean survival times (MST) were calculated for each group. The results for this experiment are summarized in Table 4. In this experiment we observed a mortality of 60% in the vehicle control group, with a MST of 11.9 days. Hypericin, and both of its derivatives, showed statist!- cally significant increases in the mean survival times for treated animals when compared to the vehicle control. Treatment with hypericin at the 25 and 50 mpk concentra¬ tions reduced mortality to 10% and 0%, respectively, while the mean survival times increased to 19.7 and 21 days. The survival of animals treated with hypericin at 10 mpk was not significantly different from the vehicle control group. The N-methylglucamine ion pair derivative showed reduced mortality and an increased MST at all three con¬ centrations. The 10, 25, and 50 mpk concentrations re- suited in mean survival times of 18, 21, and 21 days, with
mortalities of 20%, 0%, and 0%, respectively. The lysine ion pair derivative reduced mortality at the 25 and 50 mpk concentrations to 20% and 0%, respectively, with increased MST to 18 and 21 days. Survival of the 10 mpk group was not significantly different from that of the vehicle control group. For the positive control compounds, Acyclovir treatment at 25 mpk resulted in a MST of 19.4 days with a mortality of 10%, while Ara- A at 25 mpk produced a MST of 21 days with a mortality of 0%.
TABLE 4
Survival Time and Mortality of Mice Treated by the Intraperitoneal Route and Challenged with Murine Cytomegalovirus
a Mice were dosed once daily for eight consecutive days. Virus challenge occurred five hours after the second daily dosage.
D MST, mean survival time in days. c Numbers of dead mice/total challenged.
* The difference in MST is statistically significant (p < 0.05) by the Dunnetts analysis of variance.
In conclusion, in the .in vivo assay, hypericin, at 25 and 50 mpk, the N-methylglucamine ion pair deriva¬ tive, at 10, 25 and 50 mpk, and the lysine ion pair deriv- ative, at 25 and 50 mpk, significantly increased mean survival times and reduced mortality, when compared to animals treated with the dosing vehicle only.
The foregoing description of the specific em¬ bodiments reveal the general nature of the invention so that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.
All references and copending patent applications mentioned in this application are incorporated by refer¬ ence.