WO2019140429A1 - Treating chemotherapy associated cognitive impairment - Google Patents

Abstract

A therapeutic method for treating Chemotherapy Associated Cognitive Impairment. The method comprises administering a therapeutically-effective amount of a mammalian liver extract, the extract being characterized by being heat stable, insoluble in acetone and soluble in water, peptide or peptide fragment.

Description

TREATING CHEMOTHERAPY ASSOCIATED COGNITIVE IMPAIRMENT

SPECIFICATION

BACKGROUND OF THE DISCLOSURE

1. Field of the Disclosure

[0001] The present disclosure is directed to a method of treating Chemotherapy Associated Cognitive Impairment using a mammalian liver extract that is efficacious in treating this condition.

2. Description of the Related Art

[0002] Mammalian liver extract has been used for the treatment of a wide range of infectious and noninfectious dermatologic conditions, including ache vulgaris, Journal Invest Dermatology, 2:205-218 (1939); first and second degree burns, Mississippi Valley Medical Journey, 76: 199 (1954); sunburn, Clinical Medicine, 3 :245 (1956); poison ivy dermatitis, Clin. Med., 3 :425 (1956) and Herpes zoster, Southern Medical Journal, 50: 1524 (1957). The active principle and mechanism have not been described. Although some medical practitioners have used liver extract for the treatment of dermatologic conditions, it is not regarded as an antiviral or immune modulator agent even for skin therapy.

[0003] Mammalian liver extract has been reported to have bradykinin potentiating activity. Tewksbury et al., Arch. Blochem. Biophys. (U.S.), 112, 453 (1965); Tewksbury, Archives Int'l. de Pharmacodynamie et de Therapie, 173, 426 (1968); Tewksbury, Dissertation Abstracts International-Part II, Vol. 25/04, p. 2214 (1964). Further, one commercially-available liver extract (sold under the trademark KUTAPRESSIN by Kremers-Urban Co., Milwaukee, Wisconsin) exerts its action, according to product literature, only with respect to tissues that have been injured and when inflammation and edema are present.

[0004] One commercially available mammalian liver abstract useful for purposes of the present disclosure is sold under the trademark KUTAPRESSIN by Kremers-Urban Co., of Milwaukee, Wis. According to product literature, this extract exerts its action only with respect to tissues that have been injured, and particularly when inflammation and edema are present.

[0005] U.S. Pat. No. 5,055,296, filed by certain inventors common to the present application further discloses use of a particular heat stable, acetone-insoluble, water-soluble mammalian liver extract, designated as "KU 10,001" that was shown to be effective in the treatment of mammals infected with a non-dermatological virus and, in particular, chronic fatigue syndrome. The disclosure of this patent as to such extracts are incorporated herein by reference. This patent also discloses preliminary results of this extract as a protector of MT-2 cells using an in vitro test culture, when exposed to HIV-l .

[0006] U.S. Pat. No. 5,284,664, filed by certain inventors common to the present application, disclose a heat stable, acetone-insoluble, water-soluble mammalian liver extract, that is said to be effective in the treatment of symptoms of Alzheimer's Disease. The liver extract was also partially sequenced, and that sequence listing is incorporated herein by reference.

[0007] U.S. Pat. No. 5,316,775, filed by certain inventors common to the present application, discloses the same mammalian liver extract and demonstrates it to be effective in the treatment of Hepatitis B infections. [0008] U.S. Pat. No. 5,334,395, filed by certain inventors common to the present application, discloses use of the same mammalian liver extract and demonstrates it to be effective in the treatment of Epstein-Barr viral infections.

[0009] U.S. Pat. No. 4,254,103 discloses a method of extracting Hepatoprotector Factor (HF) from bovine liver to be used in the treatment of cirrhosis of the liver and viral hepatitis.

[0010] U.S. Pat. No. 4,883,660 discloses gel bases for use in pharmaceutical compositions comprising a glycol solvent, and ethoxylated fatty alcohols or ethoxylated behenyl alcohol. The composition is suitable for topical, systemic and oral administration of pharmaceutical agents.

[0011] U.S. Pat. No. 5,492,937 discloses a composition which is a liquid at or below room temperature, and forms a highly viscous gel at body temperature. This composition is comprised of a cellulose ether, a surfactant, and other optional additives. It may be used for oral or local administration of a pharmaceutical to the skin, mucous membrane, eye, or body cavity.

[0012] U.S. Pat. No. 5,589, 192 discloses a gel formulation for use with local anesthetics. A support of non-woven fabric with an impervious backing sheet is coated with a copolymer containing a local anesthetic. A polymer/drug matrix is formed, and an aqueous fluid is used to coat the surface of the matrix, converting the matrix into a gel with good percutaneous absorption.

[0013] U.S. Pat. No. 5,595,760 discloses a composition which includes a soluble, gelable salt of a peptide, and a pharmaceutical carrier. The composition forms a gel upon interacting with body fluids after injection, and the peptides are released continuously over a period of at least three days. [0014] U.S. Pat. No. 6, 156,349, filed by certain inventors common to the present application, discloses use of the same mammalian liver extract and demonstrates it to be effective in the treatment of Human Immunodeficiency Virus (HIV) when administered in suppository form.

[0015] U.S. Pat. No. 6,350,472 filed by certain inventors common to the present application, discloses use of the same mammalian liver extract and demonstrates it to be effective in the treatment of Human Immunodeficiency Virus (HIV) when administered as a transdermal gel.

[0016] Against this background, the inventors have endeavored to discover a method to treat Chemotherapy Associated Cognitive Impairment, using a heat stable, acetone-insoluble, water- soluble mammalian liver extract.

[0017] An illness referred to as Chemotherapy Associated Cognitive Impairment (CACI) has been associated with a range of drugs used in the treatment of cancer referred to as chemotherapy drugs. CACI may be caused by the chemotherapy drugs or other conditions related to cancer and its treatment. CACI symptoms often start during or just after treatment, though they may persist or even get worse after treatment is over. Patients suffering from CACI demonstrate numerous symptoms including fatigue, short term memory problems, and cognitive problem. The cognitive problems may include being unusually disorganized, confusion, difficulty concentrating, difficulty finding the right word, difficulty learning new skills, difficulty multitasking, a feeling of mental fogginess, shortened attention span, and taking longer than usual to complete routine tasks. To date, there is no known effective treatment for CACI.

BRIEF SUMMARY OF THE DISCLOSURE [0018] In aspects, the present disclosure is related to methods for treatment of CACI in human patients, specifically using a mammalian liver extract.

[0019] One embodiment according to the present disclosure includes a method of treating Chemotherapy Associated Cognitive Impairment (CACI) which comprises administering to a person suffering from CACI a therapeutically-effective amount of a mammalian liver extract, the extract being characterized by being heat stable, insoluble in acetone and soluble in water. In some embodiments, the liver extract may be contained in a pharmaceutically-acceptable carrier at a concentration of about 2.5% by weight solids. In some embodiments, the liver extract may be contained in a pharmaceutically-acceptable carrier at a concentration of about 2.5% by weight solids. In some embodiments, the liver extract is contained in water.

[0020] Examples of the more important features of the disclosure have been summarized rather broadly in order that the detailed description thereof that follows may be better understood and in order that the contributions they represent to the art may be appreciated. There are, of course, additional features of the disclosure that will be described hereinafter and which will form the subject of the claims appended hereto.

[0021] While the inventions disclosed herein are susceptible to various modifications and alternative forms, only a few specific embodiments are described herein. The detailed descriptions of these specific embodiments are not intended to limit the breadth or scope of the inventive concepts or the appended claims in any manner. Rather, the detailed written descriptions are provided to illustrate the inventive concepts to a person of ordinary skill in the art, and to enable such persons to make and use one or more of the inventive concepts.

DETAILED DESCRIPTION OF THE DISCLOSURE [0022] In aspects, the present disclosure is related to a method for treating CACI in humans. Specifically, the present disclosure is related to administering a mammalian liver extract to a human patient. The present invention is susceptible to embodiments of different forms. There are shown in the drawings, and herein will be described in detail, specific embodiments with the understanding that the present invention is to be considered an exemplification of the principles and is not intended to limit the present invention to that illustrated and described herein.

[0023] One or more illustrative embodiments incorporating the invention disclosed herein are presented below. Not all features of an actual implementation are described or shown in this application for the sake of clarity. It is understood that in the development of an actual embodiment incorporating the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related and other constraints, which vary by implementation from time to time. While a developer's efforts might be complex and time consuming, such efforts would be, nevertheless, a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0024] Accordingly, it is an object of the present disclosure to provide a method of treating CACI through the administration of a mammalian liver extract to a human patient.

[0025] While the compositions and methods are described in terms of "comprising" various components or steps (interpreted as meaning "including, but not limited to"), the compositions and methods can also "consist essentially of or "consist of the various components and steps, such terminology should be interpreted as defining essentially closed-member groups.

[0026] The portion of mammalian liver extract that has been discovered to be effective in treating CACI is the fraction which is heat stable, insoluble in acetone and soluble in water. The liver extract prepared according to the disclosure herein is free from fatty acids, and vitamins, and specifically is free from vitamin B-12, a vitamin naturally occurring in liver. Work in progress suggests polysaccharides may be present in KUTAPRESSIN (Kremers-Urban Co.) in the form of proteoglycans and/or glycoproteins. The same liver extract has been used heretofore in treating skin conditions and Epstein-Barr virus infections.

Preparation of the Liver Extract

[0027] The liver extract employed in the present disclosure is prepared by separating a fraction from mammalian livers, preferably porcine liver. The starting material may be a liver preparation as described in Pharmacopeia of the United States, Vol. 15, p. 379 (which describes a boiled liver extract suitable for parenteral use), in National Formulary, Vol. XII, p. 222 (which describes an aqueous solution of the thermostable fraction of mammalian liver) or in National Formulary, Vol. XI, p. 192-94 (which describes several thermostable liver preparations). Alternatively, the starting material may be fresh liver, frozen liver or a commercially-available liver preparation.

[0028] An acetone-insoluble fraction is separated from the starting material. This may be accomplished by admixing a large excess of acetone with the starting material which results in an acetone-insoluble fraction that is separated from the acetone. The treatment with acetone may be repeated. The acetone-insoluble fraction, after being separated from the acetone, is dissolved in water. Any remaining acetone is removed by, for example, distillation. The material effective in treating Epstein-Bart virus infection is contained in the water solution.

[0029] Alternatively, and preferably, before the acetone extraction, the starting material is dissolved in water with phenol. The solution is incubated at room temperature and after incubation, the solution is clarified by filtration, and the solution is passed over a cation exchange resin. The resulting resin-treated solution is then concentrated by evaporation, diluted with water, and centrifuged. The acetone-insoluble fraction is then separated from the supernatant by adding a large excess of acetone and further processed as described above.

[0030] The acetone-insoluble fraction may be further purified to remove the color pigments by treatment with activated charcoal. For example, the acetone-insoluble fraction may be dissolved in water and contacted with ammonia-activated charcoal.

[0031] A pharmaceutically-acceptable preservative usually is added to the water solution. For instance, phenol at from about 0.05 to about 1%, preferably about 0.5% may be added.

[0032] The liver extract useful in the present disclosure may be prepared according to, but is not limited to, the following examples.

Example 1 Preparation of Liver Extract

[0033] Liver Fraction I, described in National Formulary XI, page 193, was dissolved in water to a concentration of 16% by weight. Phenol was added to a final concentration of 1%. The solution was mixed and incubated for seven days at room temperature. It was then clarified by filtration, and diluted to 8% solids by weight in water.

[0034] This aqueous solution was then passed three times through a cation exchange resin (sulfonated polystyrene). The resin-treated solution was clarified by filtration and concentrated to 40% total solids by weight by evaporation under vacuum at 65-70 degrees C. Cold water (5-10 degrees C) was added (five volumes of water to seven volumes of liver solution) with mixing. The resultant solution was then centrifuged and the supernatant collected (Sharples-type centrifuge at 1 liter per minute). Phenol was added to a final concentration 0.5-1%. [0035] The solution was adjusted to pH 6.0-7.0, with HC1 or NaOH as necessary, clarified by filtration, and heated to 40 degrees C. Then acetone was added (20-30 liters acetone per liter liver solution). The acetone-precipitable material was allowed to settle and most of the acetone was decanted off. The remaining suspension was incubated overnight at room temperature, after which the suspension was diluted to 10 liters with water, and the acetone was removed by distillation. Phenol and water were then added to give a final preparation containing 0.5% phenol and greater than 25 mg total solids per ml (herein designated "KU 10,000").

[0036] KU 10,000 was adjusted to pH 6.0-7.0 with HC1 or NaOH, as necessary and diluted to 25 mg total solids per ml. with water (i.e., 2.5% by weight solids). The solution was then sterile filtered in suitable vials for use. This final solution is referred to herein as "KU 10,001 ".

Example II— Physically Active Polypeptide Separation

[0037] A large excess of acetone " 1800 ml" was added to eight vials KU 10,001 prepared according to Example I, 20 ml/vial, total of 160 ml, and left to stand at room temperature for four hours. After the precipitate settle down at the bottom of the beaker, the clear acetone layer decanted and the remaining suspension centrifuged for five minutes at 3000 RPM. The pellet then dissolved in 160 ml of water and freeze dried to produce about 4.0 g of dry powder consists of 0.3 mg protein/l mg of dry powder weight. These samples were designated KU 10,172, KU 10,185, KU 10,211, KU 10,244 and KU 10,275.

[0038] One gram of the dry powder was taken in 7 ml of 50 mM phosphate buffer, pH 7.5 and passed through a 100 x 2.5 centimeter column packed with Sephadex G50 suitable for use as a molecular sieve that exclude (does not retard) molecules with a molecular weight greater than 30,000 or BIOGEL plO which exclude molecules with a molecular weight greater than 20,000. The column was equilibrated with 50 mM phosphate buffer before use at flow rate of 36 ml/hr. The column was eluted with 50 mM phosphate buffer pH 7.5. Seven ml fractions were collected, and read at A280. Fractions were tested for angiotensin converting enzyme inhibition (using Furylacryloylphenylalanylglycylglycine as substrate) as described by Bush, Henry and Slasarchyk; J. of Antibiotics 37(4), 330 (1984). All fractions eluted before angiotensin converting enzyme inhibition were pooled according to the following table.

Loaded KU# KU # Pool # Tube #

10,244 10.245 1 22-30

10.246 2 31-50

10, 185 10.190 1 14-16

10.191 2 18-20

10.192 3 21-23

10.193 4 26-29

10,275 10.275-1 1 35-46

10.275-P 2 47-58

10.275-IP 3 59-68

[0039] All pooled samples were concentrated, dialyzed in cellulose dialysis tubing with a molecular weight cut off of 1,000 and lyophilized.

Example III— Physically Active Polypeptide Separation

[0040] KU 10,172 prepared according to Example II was fractionated on reverse phase C. sub.18 prep column, eluted with buffer A: 20 mM ammonium acetate pH 7.0, B: 80% acetonitrite in buffer A, gradient run at 214 nm, programmed zero to 80% B in 80 min. at 8.4 ml/min. Fractions collected 8.4 ml/test tube. All tubes were analyzed by analytical C₁₈ reverse phase column and size exclusion high pressure liquid chromatography column Tsk 125 and pooled to twelve fractions based on its retention times. Eight fractions KU 10,201 to KU 10,208 were tested for anti-viral activity and showed a significant cell protection activity.

Example IV— Further Purification of Physically Active Polypeptide

[0041] KU 10,203 and KU 10,207 prepared according to Example III were further purified on reverse phase Ci₈ prep column, eluted with buffer A: 20 mM ammonium actuate pH 7.0, B: 80% acetonitrile in buffer A, gradient run at 214 nm, programmed: Zero to 80% B in 80 min, at 8.4 ml/min. Fractions collected 8.4 ml test tube. All tubes were analyzed by analytical Ci₈ reverse phase column and size exclusion high pressure liquid chromatography column (Tsk 125) and pooled according to its retention times to produce KU 10,214 and KU 10,215. The cDNA from rat liver for the KU 10,214 and KU 10,215 fractions that were active in the bioassay was isolated and cloned using the polymerase chain reaction technique. The desired sequence to be amplified was that of the gene in pig liver cells that encodes the peptides in the KU 10,214 and KU 10,215 fractions.

Active Fraction Peptide Amino Terminal Amino Acid Sequencing

[0042] Ten amino acids of amino terminal sequence of KU 10,214 and KU 10,215 were determined by Edman degradation using an Applied Biosystems model 477A automated peptide sequencer with attached High Pressure Liquid Chromatography model 120A on-line phenyl isothiocyanate analyzer found to be (Ala or Val or lie) Glu (His or Pro) Gly (Tyr or Met or Thr) His Gly Pro His Gly. More specifically, [0043] KU 10,214 has the amino acid sequence: (Ala or Val or Ile) - (Glu or Gln) - (His or Pro or Arg) - Gly - Thr - His - X - Pro - His - Gly

[0044] KU 10,215 has the amino acid sequence: (Ala or Val or Ile) - (Glu or Gln) - (His or Pro) - Gly - (Tyr or Met) - His - Gly - X - His - Gly - X - X - Gly - X - Gln

[0045] Due to the similarity of both sequences, we proposed a sequence to be used for our Polymerase Chain Reaction (PCR) work using the following deca peptide sequence. Ala - Glu - His - Gly - Tyr - His - Gly - Pro - His - Gly.

Polymerase Chain Reaction Primer Design

The oligonucleotide primer 5'CATGGICCICATGGI3' [the letter“I” indicates Inosine] was designed based on the five amino acid sequence (HGPHG) region sequence that was common to both the KU 10,214 and KU 10,215 fractions active in the bioassay. This primer corresponds to the indicated amino acids regardless of codon usage except for His. Codon bias analysis for all pig gene sequences found in the databank Genbank 66 showed His codon at CAT to be used about 2.5 times as often as His codon CAC. This and the rarity of CG dinucleotides in peptide coding regions of mammalian genomes (Sambrook, I, Fritsch, E. F. and Maniatis, T. (1989) in "Molecular Cloning: A Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) dictated use of CAT and not CAC for His in this sequence producing a primer sequence of 5'CATGGICCICATGGI 3'. This primer was prepared by conventional techniques using an Applied Biosystems DNA synthesizer.

RNA Isolation [0046] Total RNA (2.5 mg) was isolated from 1.5 grams of fresh liver tissue from a female pig by rapid homogenation in guanidine thiocyanate followed by extraction with phenol (Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem 162, 156-159). After two successive precipitations with isopropanol, the purified RNA was dissolved in water. The RNA was then subjected to poly(U) Sepharose chromatography, (Jacobson, A. (1987) Meth. Enzymology 152, 254-261), which yielded 21 pg of Poly(A)+RNA from 1 mg of total RNA. cDNA Synthesis and Intermediate Polymerase Chain Reaction Amplification

[0047] Double stranded cDNA was prepared from the Poly(A)+RNA by a modification of the method of Gubler and Hoffman (Gene 25, 283 (1983)). Five pg of Poly(A)+RNA was used with an oligo(dT)-Hind III primer and AMV reverse transcriptase to synthesize the first strand, and second strand conversion was accomplished using RNase H and E. Coli DNA polymerase I. The yield was 30% first strand conversion and 100% second strand conversion. The double stranded cDNA was extracted with phenol and precipitated with ethanol by conventional methods. After treatment with T4 DNA polymerase to flush the ends, the cDNA was ligated with UNIAMP adaptors (Clontech Labs Inc. 800-662-CLON) under conditions described by the manufacturer. Following ligation, 3 pi of a 1 : 10 diluation of the cDNA was amplified by polymerase chain reaction in the Perkin Elmer Cetus GENEAMP System using a single UNIAMP primer (Clontech) under conditions described by the manufacturer. A sample of the reaction products was analyzed by agarose gel electrophoresis in the presence of ethidium bromide. The results showed a distribution of cDNA products that closely matched the pattern of unamplified cDNA. The remainder of the reaction products were purified by extraction with phenol and SEPHAROSE (Pharmacia Co.) CL-4B chromatography. These intermediate amplification and purification steps produced a sufficient amount of pure cDNA free of extraneous sequences which could have interfered with subsequent procedures.

Polymerase Chain Reaction Amplification with the Specific Primer

[0048] The amplified cDNA was precipitated with ethanol, collected by centrifugation and dissolved in 20 pi water. A 1 mΐ sample was used for polymerase chain reaction amplification with the specific primer described above in combination with oligo (dT)-Hind III primer. Conditions for polymerase chain reaction were 30 cycles at 94 degrees C for 1 minute, and 72 degrees C for 2 minutes per cycle, with a final extension at 72 degrees C for 5 minutes. A fraction of the reaction products were analyzed by agarose gel electrophoresis in the presence of ethidium bromide. The results showed 3 major DNA species in the range of 200 bp (base pairs), 400 bp and 500 bp. Control reactions in which the primers were omitted produced no detectable products.

Cloning of the Polymerase Chain Reaction Products

[0049] The remainder of the polymerase chain reaction products was treated with T4 DNA polymerase to flush the ends, and then purified by phenol extraction and ethanol precipitation. The DNA was ligated with ECOR I linkers under standard conditions. Following digestion with ECO RI and Hind III and removal of small molecules by SEPHAROSE (Pharmacia Co.) CL-4B chromatography, the prepared DNA was ligated with ECO RI/Hind III EXLOX vector arms, packaged in vitro, and plated on E. Coli by standard methods. The resulting library contained 3xl0⁵ independent clones and was amplified to a titer of 3xl0¹⁰ pfp/ml.

Verification of cloning

[0050] The library was plated at a density corresponding to approximately 1,000 plaques per 82 mm plate. Plaque lifts were prepared and hybridized with random-primer labeled DNA probes by conventional methods. Using a probe from DNA amplified with the specific primer described above, virtually every plaque produced a positive hybridization signal. This indicated the library contained the desired inserts of polymerase chain reaction products.

DNA sequencing

[0051] Six randomly chosen plaques were converted to plasmid subclones for DNA sequence analysis. Restriction enzyme analysis showed an insert of approximately 500 bp in four of these isolates. Plasmid DNA was prepared and sequenced directly using T7 DNA polymerase (promega) and chain-terminating dideoxynucleotides (Mierendorf, R. C. and Pfeffer, D. (1987) Meth. Enzymol. 152, 556-562).

DNA Sequencing

[0052] Sequencing experiments determined 96 base pairs at the 5' end and 110 pairs at the 3' of the cDNA insert, which appeared to be identical in the clones sequenced. [0053] The 5' 96 base pairs encoded a 32 amino acid polypeptide (Sequence Id. No. 1) whose first four amino acids GPHG corresponded to those of the primer HPGHG. (Sequence Id. No. 2) The 110 pairs at the 3' end (Sequence Id. No. 3) had a TAA or ATG stop codon in all three reading frames, so the C-terminal of the polypeptide is L (encoded by 5'CTA3'), (Sequence Id. No. 4 which has 8 amino acids and is encoded by (Sequence Id. No. 5) and/or (Sequence Id. No. 6) which has 21 amino acids and is encoded by Sequence Id. No. 7. As such the polypeptides are characterized by the Sequence Id. No. 1 at the 5' end and Sequence Id. Nos. 4 and 6.

Example V Physical and Chemical Tests on Physiologically Active Polypeptide

[0054] Thus, the physiologically-active polypeptide may be characterized by its physical and chemical properties. The active polypeptide is insoluble in acetone, and soluble in water. It has a molecular weight as determined by molecular sieve chromatography experiments to be about 5,000-40,000.

ADMINISTRATION OF POLYPEPTIDES

[0055] The polypeptides useful in the present disclosure may administered via injection, for example, intramuscular injection. However, other forms of administration are contemplated, which may include orally, such as pill or liquid form, transdermally, such as a cream or patch applied to the skin, and in suppository form. The polypeptides may be employed in the form of pharmaceutically-acceptable salts of the components, such as the alkali metal salts. The pharmaceutically-acceptable amides, lower alkyl esters, protected derivatives, other derivatives and analogues of the components of the polypeptides are also contemplated.

[0056] Although, as indicated, the polypeptides may be used as a water solution, it may also be utilized in association with other pharmaceutical carriers, for example, in saline solution. In any case, since the polypeptide is preferably administered by injection, it is contemplated that the extract will be contained in a water base carrier. A preferred product is a polypeptide water solution containing about 2.5% by weight of polypeptide. More generally, the polypeptide ranges from 5 pg to 500 pg per ml of carrier.

ADMINISTRATION OF LIVER EXTRACT

[0057] The acetone-insoluble liver extract useful in the present disclosure may be administered by injection, for example, intramuscular injection. However, other forms of administration are contemplated.

[0058] The liver extract may be employed in the form of pharmaceutically-acceptable salts of the components, such as the alkali metal salts. The pharmaceutically-acceptable amides, lower alkyl esters, protected derivatives, other derivatives and analogues of the components of the liver extract are also contemplated.

[0059] Although, as indicated, the liver extract may be used as a water solution, it may also be utilized in association with other pharmaceutical carriers, for example, in saline solution. In any case, since the liver extract is preferably administered by injection, it is contemplated that the extract will be contained in a water base carrier. A preferred product is a water solution containing about 2.5% by weight of liver extract solids.

[0060] Dosages may vary depending upon the condition of the patient. Generally, however, it has been found that the administration of 2 cc. of KU 10,001 prepared as described in Example 1 intramuscularly 2 cc will produce beneficial results in as little as about 3 days. [0061] In another embodiment, the acetone-insoluble liver extract useful in the present disclosure may be administered by transdermally. A transdermal delivery system has been discovered to be a particularly effective method of delivering a mammalian liver extract, such as KUTAPRESSIN, when further concentrated according to the present disclosure. Such a transdermal delivery system allows the concentrated liver extract to be absorbed into the blood in concentrations apparently adequate for the treatment of CACI.

Preparation of a Transdermal Colloidal Dispersion Delivery System for Kutapressin

[0062] The transdermal colloidal dispersion employed in the present disclosure is one of the preferred methods for administering the liver extract.

[0063] A soy lecithin gel is prepared from 10 grams of soy lecithin, to which 11.7 ml of isopropyl palmitate and 0.2 g of sorbic acid are added. The gel is then allowed to meld for 24 hours. A 20% poly(oxypropylene)-poly(oxy ethylene) copolymer gel, such as Pluronic 127 (BASF), is prepared by adding 0.2 g of potassium sorbate and 100 ml of distilled water to 20 g of Pluronic 127. This gel is also allowed to meld for 24 hours.

[0064] Dessicated liver extract is used to prepare the gel formulation. One way in which this may be accomplished is described below by dessicating 3 vials (60 ml) of commercially available KUTAPRESSIN. A Nalgene dessicating system is set up using calcium sulfate as the dessicating absorbant. Two evaporating dishes containing the KUTAPRESSIN are placed in the apparatus and a vacuum is applied via a pump for 7 minutes. The dessicating absorbant must be changed every 24 hours, and the vacuum pressure must be reapplied after each change of absorbant to restore the vacuum. After 72 hours, the liquid will be absorbed, leaving only active ingredient. [0065] Six ml of preserved water and 4 drops of phenol are added to the KUTAPRESSIN, and it is allowed to solubilize. This is the resolubilized liver extract preparation. The liquid is placed in a 30 ml syringe attached to another 30 ml syringe by a Luer Lock adapter. Seven ml of soy lecithin gel is added to the solubilized KUTAPRESSIN in the syringe. The liquids are transferred between the two syringes until an emulsion forms. With the syringes still attached and the emulsified liquid in one syringe, 17 ml of 20% Pluronic 127 is added to the empty syringe. The Pluronic 127 is then transferred to the KUTAPRESSIN emulsion. This results in a colloidal dispersion. The colloidal dispersion is then transferred between the syringes about 20 times to further emulsify the mixture. This final colloidal dispersion volume is about 30 ml, and it contains KUTAPRESSIN at a concentration of about 50 mg/ml.

[0066] It is also anticipated that a transdermal delivery system comprised of lecithin and other penetration enhancers; vehicles other than lecithin which alter the molecular environment of the epidermis; an adhesive patch containing a drug reservoir, with or without a rate controlling membrane, covered by an occlusive backing; iontophoresis; and phonophoresis may be used to deliver the liver extracts of the present disclosure.

Administration of Liver Extract

[0067] An acetone-insoluble liver extract useful in the present disclosure preferably is administered percutaneously, for example, using a transdermal colloidal dispersion delivery system in the form of a patch applied to the skin, or by applying the colloidal dispersion directly to the skin. However, other forms of administration are contemplated.

[0068] The liver extract may be employed in the form of pharmaceutically acceptable salts of the components, such as alkali metal salts. The pharmaceutically acceptable amides, lower alkyl esters, protected derivatives, other derivatives and analogs of the components of the liver extract are also contemplated.

[0069] While a transdermal colloidal dispersion formulation is preferred, other pharmaceutical carriers, for example, a saline solution, could be employed. The liver extract preferably is administered percutaneously while contained in a colloidal dispersion. A preferred product is a colloidal dispersion comprised of any of a variety of transdermal delivery vehicles and penetration enhancers containing KUTAPRESSIN which has been concentrated to a level of about 50 mg/ml. Iontophoretic and phonophoretic methods of introducing KUTAPRESSIN transdermally are also contemplated.

[0070] Dosages may vary depending upon the condition of the patient. Generally, however, it has been found that the administration of 400 mg of KUTAPRESSIN per day will produce beneficial results in as little as about 4 weeks.

EXAMPLE VI Transdermal application

[0071] A patient suffering from CACI may receive a periodic dose of KUTAPRESSIN percutaneously. The dosage amount may vary based on the concentration of the drug, the characteristics of the patient, and the condition of the patient.

[0072] Although the disclosure has been described primarily in connection with special and preferred embodiments, it will be understood that it is capable of modification without departing from the scope of the disclosure. The following claims are intended to cover all variations, uses, or adaptations of the disclosure, following, in general, the principles thereof and including such departures from the present disclosure as come within known or customary practice in the field to which the disclosure pertains, or as are obvious to persons skilled in the field. [0073] While embodiments in the present disclosure have been described in some detail, according to the preferred embodiments illustrated above, it is not meant to be limiting to modifications such as would be obvious to those skilled in the art.

[0074] The foregoing disclosure and description of the disclosure are illustrative and explanatory thereof, and various changes in the details of the illustrated apparatus and system, and the construction and the method of operation may be made without departing from the spirit of the disclosure.

Claims

CLAIMS What is claimed is

1. A method of treating Chemotherapy Associated Cognitive Impairment which comprises administering to a person suffering from Chemotherapy Associated Cognitive

Impairment a therapeutically-effective amount of a mammalian liver extract, the extract being characterized by being heat stable, insoluble in acetone and soluble in water.

2. The method of claim 1 wherein the liver extract is contained in a pharmaceutically- acceptable carrier at a concentration of about 2.5% by weight solids.

3. The method of claim 2 wherein the liver extract is contained in a pharmaceutically - acceptable carrier at a concentration of about 2.5% by weight solids.

4. The method of claim 3 wherein the liver extract is contained in water.