Patent 6,929,808

Assay-guided affinity fractionation and reverse phase high pressure liquid chromatography (HPLC) methodology to isolate, test and characterize the most active water-soluble ingredients within Cat's Claw, or Uncaria tomentos. These components appear to account for the majority of the amyloid or Aβ fibrillogenesis inhibitory activity. Individual fractions and/or compounds as isolated by HPLC are tested in relevant in vitro and/or animal models, and found to consistently demonstrate inhibition of amyloid or Aβ fibrillogenesis. Related extraction methods are disclosed.

Description of the Invention

TECHNICAL FIELD

The invention relates to the method of isolation and use of amyloid-inhibiting compounds derived from Uncaria tomentosa and related plants for the therapeutic intervention of Alzheimer's disease, type II diabetes, Parkinson's disease and other disorders involving amyloid accumulation; more particularly, it relates to methods of isolating amyloid-inhibiting compounds from Uncaria tomentosa and related plants, and to the use of those compounds.

BACKGROUND OF THE INVENTION

Alzheimer's disease is characterized by the accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein or Aβ, in a fibrillar form, existing as extracellular amyloid plaques and as amyloid within the walls of cerebral blood vessels. Fibrillar Aβ amyloid deposition in Alzheimer's disease is believed to be detrimental to the patient and eventually leads to toxicity and neuronal cell death, characteristic hallmarks of Alzheimer's disease. Accumulating evidence implicates amyloid as a major causative factor of Alzheimer's disease pathogenesis.

A variety of other human diseases also demonstrate amyloid deposition and usually involve systemic organs (i.e. organs or tissues lying outside the central nervous system), with the amyloid accumulation leading to organ dysfunction or failure. In Alzheimer's disease and "systemic" amyloid diseases, there is currently no cure or effective treatment, and the patient usually dies within 3 to 10 years from disease onset.

The amyloid diseases include, but are not limited to, the amyloid associated with Alzheimer's disease, Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (wherein the specific amyloid is referred to as beta-amyloid protein or Aβ), the amyloid associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever (wherein the specific amyloid is referred to as AA amyloid or inflammation-associated amyloidosis), the amyloid associated with multiple myeloma and other B-cell dyscrasias (wherein the specific amyloid is referred to as AL amyloid), the amyloid associated with type II diabetes (wherein the specific amyloid protein is referred to as amylin or islet amyloid polypeptide), the amyloid associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie (wherein the specific amyloid is referred to as PrP amyloid), the amyloid associated with long-term hemodialysis and carpal tunnel syndrome (wherein the specific amyloid is referred to as beta2-microglobulin amyloid), the amyloid associated with senile cardiac amyloid and Familial Amyloidotic Polyneuropathy (wherein the specific amyloid is referred to as transthyretin or prealbumin), and the amyloid associated with endocrine tumors such as medullary carcinoma of the thyroid (wherein the specific amyloid is referred to as variants of procalcitonin).

Discovery and identification of new compounds or agents as potential therapeutic agents to arrest amyloid formation, deposition, accumulation and/or persistence that occurs in Alzheimer's disease, Parkinson's disease and other amyloidoses are desperately sought.

DISCLOSURE OF THE INVENTION

Methods of isolation for the identification and purification of the potent amyloid inhibitory ingredients within Uncaria tomentosa and related plants are disclosed. Use of such extracts from the inner bark and root parts of Uncaria tomentosa and related plant materials are anticipated to benefit human patients with Alzheimer's disease, type II diabetes, Parkinson's disease and other amyloidoses, due to the previously unknown ability of these compounds to inhibit amyloid fibril formation, and cause disruption/ dissolution of pre-formed amyloid fibrils.

The present invention pertains to the surprising discovery that specific extraction methods (and compounds derived from such extraction methods) when applied to the inner bark and root parts of Uncaria tomentosa, otherwise known as Uña de Gato (or Cat's claw), leads to the purification of a group of compounds (the group referred to herein as PTI-777), and their individual components (such as "compound H") which act as impressive inhibitors of Alzheimer's disease beta-amyloid protein (Aβ) formation and growth.

Previously our studies led to the identification of a natural substance derived from the Amazon rain forest woody vine, Uncaria tomentosa, and referred to as PTI-00703. See for instance U.S. patent applications Ser. Nos. 09/079,829, 09/198,824, and 09/208,278, which describe the initial discovery of derivatives of Uncaria tomentosa and related plant material extracts as inhibitors of amyloidosis of Alzheimer's disease, type II diabetes and other amyloid disorders.

In the present application, we used assay-guided affinity fractionation and reverse phase high pressure liquid chromatography (HPLC) methodology to isolate, test and characterize the most active water-soluble ingredients within PTI-00703 (collectively referred to as PTI-777) that appear to account for the majority of the Aβ fibrillogenesis inhibitory activity. PTI-777 and its individual fractions and/or compounds as isolated by HPLC were tested in relevant in vitro and/or animal models, and found to consistently demonstrate inhibition of Aβ fibrillogenesis. The present invention describes extraction methods for the isolation of PTI-777 and its individual fractions and/or components.

Further purification and in vitro testing of each of the PTI-777 compounds, as well as initial structural characterization studies suggest that the Aβ inhibitor compounds derived from Uncaria tomentosa are small molecules (˜200-500 molecular weight) that belong to the general class of aromatic polyphenolic compounds. Two such compounds, chlorogenic acid (C₁₆H₁₈O₉; FW 354.31) and epicatechin (C₁₅H₁₄O₆; FW 290.27) were purified and identified by analytical techniques. In addition, data indicates that "compound H", the major compound within "fraction H" isolated from PTI-777 is a most potent inhibitor of Aβ amyloid fibrillogenesis.

In addition, PTI-777 has the ability to enter the brain as demonstrated by radiolabeling experiments, indicating that it has the potential to be very useful as a therapeutic agent for Alzheimer's disease, Parkinson's disease, and other central nervous system disorders involving deposition and accumulation of fibrillar proteins.

A primary object of the present invention is to establish new methods for the treatment of the amyloid diseases. In addition, the alpha-synuclein protein which forms fibrils, and is also Congo red and Thioflavin S positive, is found as part of Lewy bodies in the brains of patients with Parkinson's disease (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer, Berline pp.920-933, 1912; Pollanen et al, J. Neuropath. Exp. Neurol. 52:183-191, 1993; Spillantini et al, Proc. Natl. Acad. Sci. USA 95:6469-6473, 1998; Arai et al, Neurosc. Lett. 259:83-86, 1999). For purposes of this disclosure, Parkinson's disease, due to the fact that fibrils develop in the brains of patients with this disease (which are Congo red and Thioflavin S positive, and which also contain predominant beta-pleated sheet secondary structure), are regarded as a disease that also displays the characteristics of an amyloid-like disease.

Yet another object of the present invention is to use fraction "H" contained within Uncaria tomentosa and related plant materials for the treatment of amyloid formation, deposition, accumulation and/or persistence in Alzheimer's disease, type II diabetes, other amyloidoses and Parkinson's disease.

Yet another object of the present invention is to provide methods to isolate the active water-soluble amyloid inhibitory ingredients present within Uncaria tomentosa and related plant materials for use as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, amyloid protein-amyloid protein interactions, and/or cause a dissolution/disruption of pre-formed or pre-deposited amyloid fibrils in Alzheimer's disease, type II diabetes, other amyloidoses and Parkinson's disease.

Yet another object of the present invention is to provide compositions and methods involving administering to a subject a therapeutic dose of an Uncaria tomentosa and related plant material extract which inhibits amyloid deposition. Accordingly, the compositions and methods of the invention are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs. The compounds of the invention can be used therapeutically to treat amyloidosis or can be used prophylactically in a subject susceptible to amyloidosis. The methods of the invention are based, at least in part, in directly inhibiting amyloid fibril formation, inhibiting amyloid fibril growth, and/or causing dissolution/disruption of preformed amyloid fibrils.

Yet another object of the present invention is to provide pharmaceutical compositions for treating amyloidosis. The pharmaceutical compositions include a therapeutic compound of the invention in an amount effective to inhibit amyloid deposition and a pharmaceutically acceptable vehicle.

Yet another object of the present invention is the use of any and all synthetic compounds made similar to an Uncaria tomentosa and related plant material extract for use as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, amyloid protein-amyloid protein interactions, and/or cause a dissolution/ disruption of pre-formed or pre-deposited amyloid fibrils in Alzheimer's disease, type II diabetes, other amyloidoses and Parkinson's disease.

In a particular aspect of the invention there is a method of isolation to purify and identify the water-soluble amyloid inhibitory ingredients from Uncaria tomentosa and/or extracts thereof. In one such method, an extract prepared from commercially obtained pills, tablets, caplets, soft and hard gelatin capsules, lozenges, sachets, cachets, vegicaps, liquid drops, elixers, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositories, sterile injectable solutions, sterile packaged powders, bark bundles and/or bark powder, using the methods described in the present invention.

Another object of the present invention is to use the methods of extraction as described herein to provide an extract from Uncaria tomentosa and related plant materials for promoting mental alertness and for inhibiting the formation of brain amyloid deposits in a subject.

Yet another object of the present invention is to use the extract from Uncaria tomentosa and related plant materials for mental acuity; to promote mental alertness; to provide nutritional support for age or related cognitive or memory decline; to promote cognitive well being; to support brain function; to improve cognitive ability, mental performance or memory; to promote concentration and mental sharpness; to improve mental vitality; to promote greater mental clarity and alertness; to improve short term memory, for age associated cognitive or memory decline; to support normal brain function; to enhance learning or memory; to improve concentration; to enhance mental performance; to reduce mental decline; to reduce likelihood of age related brain disorders; to maintain good brain health; to reduce, eliminate, prevent, inhibit or disrupt/dissolve amyloid fibril or protein deposits, brain associated amyloid fibril deposits or brain associated amyloid protein deposits, amyloid fibril formation and growth or age associated amyloid fibril formation and growth, brain associated amyloid fibril formation and growth; to support healthy pancreatic function; to promote pancreatic function by helping to promote normal insulin function; to reduce, eliminate, prevent, inhibit or disrupt/dissolve amyloid fibril or protein deposits, and pancreas associated amyloid fibril formation and growth.

In particular the disclosure is directed to novel applications of assay guided fractionation leading to novel compounds and novel methods of use of those novel compounds, such as:

A method for isolating compounds that possess amyloid inhibitory activity from plant matter of the genus Uncaria having the following steps:

a) preparing a polar solvent extract (preferably a methanol extract) of Uncaria plant matter, where the polar solvent extraction is extraction with water, extraction with a polar alcohol or a water solution of a polar alcohol, extraction with a water solution of acetonitrile, or extraction with a water solution of another polar organic solvent such as triethanolamine, acetone, or the like, and running the extract through a first column that has a hydroxy group containing resin, a resin having hydrophobic characteristics but without any hydroxy groups, or a mixture of both;

b) eluting the first column with distilled water, followed by eluting with not more than 2-4 column bed volume washings with a dilute polar alcohol (such as methanol)/water solution having an alcohol/water ratio not greater than about 50/50, depending on which alcohol is used, and discarding any eluate, the object being to wash non-active material and fractions away, without appreciably eluting any active fractions (during particularization of any separation protocol-choice of solvent and concentration, volume of washings, flow rates and the like, appropriate analytical testing of putative discardable eluates is desirable, as will be appreciated by those skilled in the art, and such persons will know what tests to perform, such as for instance standard Thioflavin T testing to detect amyloid inhibiting substances);

c) eluting the first column with one or more column bed volume washings of a polar alcohol/water solution having an alcohol/water ratio somewhere at, or between, 50/50 and substantially pure alcohol, and collecting and drying the eluted volumes to a dried material. These volumes and their dried material contain the active amyloid inhibiting ingredients referred to in this disclosure as PTI-777.

It will be appreciated that in the drying step above, alternate conventional drying procedures may be substituted by those skilled in the art without departing from the scope of coverage, and in some cases, the drying step may be omitted

In the method of above, the column that comprises hydroxy containing resin, resin having hydrophobic characteristics but without any hydroxy, or a mixture of both, may advantageously be a column such as a C2 column, C4 column, C18 column, or the like, or Tris-acrylate column, LH-20 column, Affi-prep 10 gel column, or the like. Also the polar alcohol/water solution preferably has an alcohol/water ratio of 75/25 or higher, and more preferably is pure or nearly pure alcohol, and preferably methanol.

The plant matter of the genus Uncaria is preferably taken from one or more of the various Uncaria species such as tomentosa, attenuata, elliptica, guianensis, pteropoda, bernaysli, ferra DC, kawakamii, rhyncophylla, calophylla, gambir, and orientalis, and more preferably, Uncaria tomentosa. The Uncaria tomentosa plant matter is preferably taken from the inner bark and/or the root.

Optionally the isolation method set forth above is extended with the further steps:

d) applying an aqueous solution of the dried material from step (c) to a second column comprising a hydrophobic resin, the second column having been preparatorily equilibrated in a solvent comprising about 95% water/5% acetonitrile, referred to herein as solvent A, and then eluting the second column with more solvent A and discarding the eluate.

e) eluting the second column with a mixture of solvent A containing about 10-15%, and preferably about 12.5%, of a solvent comprising about 95% acetonitrile/5% water, referred to herein as solvent B, and collecting and drying the eluted volumes to a dried material.

"About" as applied to solvent percentage compositions and generally to other percentages expressed in this disclosure generally refers to +/-; about 2% points; thus 'about 95% water/5% acetonitrile', for example, can lie anywhere at or between 97% water/3% acetonitrile to 93% water/7% acetonitrile. In other instances, the words 'about' or 'substantially' are understood to mean a figure or amount somewhere close to the stated figure, varying from the stated figure or amount by as much as +/-;5%-20% of the stated figure or amount.

Optimally, TFA (typically about 0.1%) is added to the solvents indicated for acid stability and added efficacy in resin column work, as will be appreciated by those skilled in the art.

The isolation method above may be yet further advantageously enhanced by having a hydrophobic resin in the second column, and selecting a column from one of the many so called 'carbon columns', or carbon/hydrophobic columns, each preferably containing no hydroxy groups, such as for instance a C18 SPE, Varian Chroma..Zone™, or other HPLC columns, or the like.

The isolation method above may be yet further advantageously extended by having the following additional steps:

f) making one or more injections of a solution of the dried material of step (c) or the dried material of step (e) in a solvent such as water, water/dilute alcohol or a solution of solvent A comprising no more than 10% solvent B, into an HPLC instrument with a diode array uv/vis detector and graphic display and a reverse-phase column;

g) eluting the material through the HPLC column using a solvent gradient profile as follows: 10% solvent B for about the first 20 minutes from start of elution, 10 to 100% solvent B gradient for about minutes 20 to 30 from start of elution, and 100 to 10% solvent B gradient for about minutes 30 to 32 from start of elution, while observing the uv/vis detector graphic display during the elution gradient over time, and separating fractions of the eluate at elution times corresponding to times associated with the graphic display peaks.

Suitable reverse phase columns will occur to, and be well known by, those skilled in the art, and with minor adjustments to the protocol described above, may be interchanged for any columns set forth here. As discussed above, one of the many so called 'carbon columns', or carbon/hydrophobic columns, each preferably containing no hydroxy groups, such as for instance a C18 SPE, Varian Chroma..Zone™, or other HPLC columns, or the like, may be employed.

It should be noted that the preferred diode array detector may be advantageously substituted with alternate detectors such as a RI (refractive index) detector, a total ion detector, or the like, in order to monitor and record intensity peaks over time that correspond to elution fractions, as does the uv/vis detector preferred.

In a particular embodiment, the reverse-phase column has dimensions of about 2.2 cm×25 cm and contains about 95 ml of C18 reverse phase resin. The solution of the dried material is advantageously a solution of about 50 mg of the dried material of step (c) in about 1-2 ml of solvent A, and the step of injecting the solution of dried material into the HPLC may be repeated as required to load the column. An HPLC column solution gradient flow rate is preferably set to about 5 mls per minute, and the solvent gradient profile is preferably 10% solvent B for 0 to 20 minutes, followed by 10 to 100% solvent B gradient for minutes 20 to 30, and 100% to 10% solvent B gradient from minutes 30 to 31; such that fractions F though N of the eluate are collected at the following times: fraction G (13-14 minutes), fraction F (15-16 minutes), fraction H (17-20 minutes), fraction I (21 minutes), fraction J (22-23 minutes), fraction K1 (24 minutes), fraction K2 (25 minutes), fraction L (26-27 minutes), fraction M (27-28 minutes), and fraction N (28-29 minutes).

In another embodiment, the reverse-phase column has dimensions of 1.0 cm×25.0 cm and contains about 20 ml of C18 reverse phase resin. The solution of the dried material of step (c) is a solution of about 50 μg of the dried material in 50-100 μl of solvent A, wherein the step of injecting the solution into the HPLC is repeated multiple times, wherein a HPLC column solution gradient flow rate is set to about 1.5 mls per minute, and further wherein the solvent gradient profile is 10% solvent B for 0 to 20 minutes, followed by 10 to 100% solvent B gradient for minutes 20 to 30, and 100% to 10% solvent B gradient from minutes 30 to 31; such that fractions F though O of the eluate are collected at the following times: fraction G (12-13 minutes), fraction F (13-14 minutes), fraction H (15 minutes), fraction I (16 minutes), fraction J (18-19 minutes), fraction K1 (20 minutes), fraction K2 (21 minutes), fraction L (21-23 minutes), fraction M (23 minutes), fraction N (24 minutes), and fraction O (26-27 minutes).

Steps (f) and (g) of the isolation method set forth above may alternatively proceed as follows:

f) injecting a solution of 1 gram of the dried material of step (c) in 5-10 ml of solvent A into an HPLC instrument having a Varian model 320 uv/vis detector set at 230 nm with a graphic display, the HPLC further comprising a 4.14 cm×25 cm Varian Dynamax column further comprising 380 ml of C-18 reverse phase resin, the column fitted to a Varian Prostar 215 solvent delivery system, or the like.

g) eluting the HPLC column at a solution gradient flow rate of about 50 ml/mlnute, and further wherein the solvent gradient profile is with a solvent C/solvent D gradient (referred to in the art for HPLC solvent gradients as "A/B", but as C/D here to avoid confusions with other A/B gradients referred to herein as standards of protocol elsewhere in this disclosure) as follows: 0-4 minutes, 25% D; 4-11 minutes, 25-30% D gradient; 11-14 minutes, 30-90% D gradient; 14-17 minutes, 90% D; and 17-19 minutes, 90-25% D gradient, where C is water and D is methanol, such that fractions F through O of the eluate are separated at elution times corresponding to times associated with the graphic display peaks.

Those skilled in the art will appreciate, and readily accommodate, without undue experimentation, that adjusting flow rates and gradients for substitution of various A/B gradient setups, such as substituting water/methanol for water/acetonitrile, will be necessary and appropriate, because for instance methanol is more polar than acetonitrile, and thus more methanol (25%) is needed compared to acetonitrile (10%) in the discussions herein. Even so, specific percentages, times and flow rates will readily be selectable for various choices of solvents, all in accordance with the teachings disclosed herein.

Alternatively the preparation in step (a) of the extract of Uncaria may proceed as follows:

	1) adding 4000 ml of methanol to 1 kg of Uncaria tomentosa and mixing
	2) centrifuging the mixture at ×2,500 g using a centrifuge for 30 minutes and collecting the supernatant;
	3) extracting the insoluble material about 3 more times as steps a and b above;
	4) combining the supernatants and evaporating to a dried extract, or to at least about 500 ml volume, using a rotary evaporator at 50° C.;
	5) washing the dried extract, or the 500 ml volume, 4 times with 300 ml of petroleum ether, and discarding the ether layer;
	6) further evaporating any remaining methanol to dryness using a rotary evaporator at 50° C.;
	7) extracting the dried extract 5 times with 150 ml of distilled water, followed by centrifugation at 2,500×g for 30 minutes each time, and
	8) combining the supernatants and then lyophilizing using a freeze-dryer.

Further preparation of the extract of Uncaria from the resulting lyophilized extract can use the following additional steps:

9) dissolving the resulting lyophilized extract into about 500 ml of distilled water, and applying 50-100 ml portions to a 400 ml LH-20 column equilibrated with distilled water.

10) eluting the LH-20 column with 1,100 ml of distilled water (˜3 column volumes) and discarding the amber/yellow, non-active fractions;

11) eluting the LH-20 column with 1,100 ml of 100% methanol (˜3 column volumes) and collecting a set of active fractions and evaporating to dryness using a rotary evaporator at 50° C.

Alternatively the aqueous solution of a dried material from step (c) may be further prepared by the following steps:

1) dissolving the dried material in water at 80 mg/ml and applying 5 ml at a time to a disposable C18 SPE column (10 gram) equilibrated in a first solvent comprising about 95% water/5% acetonitrile/0.1% TFA;

2) washing with 3 column bed volumes of the first solvent and discarding the eluate.

3) eluting with 3 column bed volumes of the first solvent further comprising about 12.5% of a second solvent comprising about 95% acetonitrile/5% water/0.1% TFA, and

4) lyophilizing the corresponding fractions using a freeze-dryer.

It will be appreciated that the various drying and volume reducing methods disclosed are well known to those skilled in the art, and effective substitutes are also well known. Other drying methods, where at least one object is to avoid oxidation of the extracted material, such as nitrogen atmosphere, or vacuum drying will occur to those skilled in the art without departing from the scope of invention set forth herein.

Alternately the aqueous solution of a dried material from step (c) is further prepared by the following steps:

1) dissolving the lyophilized fractions at 5 grams in 20 ml water and applying 20 ml at a time to a Varian Chroma..Zone™apparatus

2) washing with 3 column bed volumes of a first solvent comprising about 95% water/5% acetonitrile/0.1% TFA and discarding the eluate;

3) eluting with 3 column bed volumes of the first solvent further comprising about 12.5% of a second solvent comprising about 95% acetonitrile/5% water/0.1% TFA, and

4) collecting and drying the next 3 column bed volumes of eluate.

Another, more particular, method for isolating water-soluble components from Uncaria tomentosa that possess amyloid inhibitory activity has the following steps:

a) adding 4000 ml of methanol to 1 kg of Uncaria tomentosa and mixing

b) centrifuging the mixture at ×2,500 g using a centrifuge for 30 minutes and collecting the supernatant, where it is understood that means for separating suspended matter from the liquid, such as overnight sedimentation by gravity or filtration may be substituted by those skilled in the art to separate suspended solids from solution,

c) extracting the insoluble material about 3 more times as steps a and b above;

d) combining the supernatants and evaporating to dryness (or until about 500 ml volume is reached) using a rotary evaporator at 50° C.,

e) taking the powdered extract (or about 500 ml volume), washing 4 times with 300 ml of petroleum ether, or other non-polar organic solvent, and discarding the ether (non-polar) layer,

f) evaporating the methanol to dryness using a rotary evaporator at 50° C.;

g) extracting the solid material 5 times with 150 ml of distilled water, followed by centrifugation at 2,500×g for 30 minutes each time;

h) combining the supernatants and then lyophilizing using a freeze-dryer;

i) dissolving the resulting lyophilized extract into about 500 ml of distilled water, and applying 50-100 ml portions to a 400 ml LH-20 column equilibrated with distilled water.

j) eluting the LH-20 column with 1,100 ml of distilled water (˜3 column volumes) and discarding the amber/yellow, non-active fractions;

k) eluting the LH-20 column with 1,100 ml of 100% methanol (˜3 column volumes) and collecting a set of active fractions and evaporating to dryness using a rotary evaporator at 50° C.;

l) dissolving the fractions of step k in water (80 mg/ml) and applying 5 ml at a time to a 10 gm disposable C18 SPE column equilibrated in solvent A (solvent A is 95% water/5% acetonitrile/0.1% TFA);

m) washing the column with 3 volumes of solvent A and discarding the eluate;

n) eluting the column with 3 volumes of solvent A containing 12.5% solvent B (solvent B is 95% acetonitrile/5% water/0.1% TFA) and lyophilizing the eluate;

o) taking 50 mg of the lyophilized eluate of step n and injecting multiple times into a Hewlett-Packard 1100 Series HPLC instrument with diode array detector, fitted with a 2.2 cm×25 cm Vydac 218TP1022 C18 reverse-phase column maintained at 25° C. and at a flow rate of 5 ml/min;

p) eluting the sample with the following solvent profile, 10% B for 0 to 20 minutes, 10-100% B gradient for minutes 20 to 30, and 100-10% B gradient for minutes 30-31, where B is 95% acetonitrile/5% water/0.1% TFA;

q) and separating and collecting the fractions into 11 major components defined as fraction G (13-14 minutes), fraction F (15-16 minutes), fraction H (17-20 minutes), fraction I (21 minutes), fraction J (22-23 minutes), fraction K1 (24 minutes), fraction K2 (25 minutes), fraction L (26-27 minutes), fraction M (27-28 minutes), and fraction N (28-29 minutes).

A novel composition further referred to herein as PTI-777 may thus be isolated according to any of the isolation processes set forth above. And other compositions further referred to herein as PTI-777 fractions, such as PTI-777 fraction G, PTI-777 fraction F, PTI-777 fraction H, PTI-777 fraction I, PTI-777 fraction J, PTI-777 fraction K₁, PTI-777 fraction K₂, PTI-777 fraction L, PTI-777 fraction M, PTI-777 fraction N, and PTI-777 fraction O, may also be isolated according to any of the processes set forth above that employ HPLC fractionation.

A further novel compound H may be isolated by a method having steps (a) through (c) as set forth above, and further having the steps:

d) applying an aqueous solution of the dried material from step (c) to a second column, LH-20 or the like, eluting the material from the column with successive column volumes of water/methanol mixtures containing 0.1% TFA, beginning with 25% methanol and increasing to 100% menthol in 25% increments, and collecting and combining the fractions;

e) separating, combining and drying a fraction to a dried material, referred to hereafter as compound H, by analytical HPLC, the fraction containing a peak occurring between 7-8 minutes from start of elution on a Dynamax 5μ C-18 column having dimensions of about 4.6 mm×25 cm, using an elution gradient of water for solvent A and methanol for solvent B, A and B each containing about 0.1% TFA, with detection at 280 nm, the gradient conditions being 0 to 9 min fro 25% to 36% B gradient, 3 to 10 min for 36 to 100% B gradient, 10 to 12 min for 100% B and 12 to 13 min for 100 to 25% B gradient, all at a flow rate of about 20 ml/min;

f) making one or more injections of a solution of the dried material of step (e) above in a solvent comprising water/methanol 80/20 containing about 0.1% TFA and applied at about 150 mg/run to a preparative HPLC Dynamax 5 μ C-18 column with dimensions of about 21.4 mm×25 cm, using substantially the same elution gradient as used in step (e) above, with detection at 280 and 300 nm, the gradient conditions being 0 to 3 min for 20% to 25% B gradient, 3 to 9 min for 25 to 45% B gradient, 9 to 10 min for 45 to 100% B gradient, 10 to 12 min for 100% B and 12 to 13 min for 100 to 25% B gradient, all at a flow rate of about 20 ml/min, the compound H fraction eluting between 7-8 minutes from start of elution, and;

g) repeating steps (e) and (f) above until the peak as seen on analytical HPLC in step (e) is relatively pure, thus ending, when appropriately dried, with substantially pure compound H.

Also disclosed is a method of treatment, prevention or management of an amyloidosis, or a disease related to alpha-synuclein, in a mammalian subject susceptible to, or afflicted by, the amyloidosis or alpha-synuclein disease. The method includes the step of administering to the subject a therapeutic amount of the composition produced in accordance with any of the methods set forth above, such as, in particular, PTI-777 and/or compound H., or any of fraction G, fraction F, fraction H, fraction I, fraction J, fraction K₁, fraction K₂, fraction L, fraction M, fraction N or fraction O.

This method may be efficaciously applied to any amyloidosis which has an associated amyloid, such as amyloidoses associated with Alzheimer's disease, Down's syndrome, hereditary cerebral hemorrhage with amyloidosis of the Dutch type, the amyloidosis associated with type II diabetes, the amyloidosis associated with chronic inflammation, various forms of malignancy and Familial Mediterranean Fever, the amyloidosis associated with multiple myeloma and other B-cell dyscrasias, the amyloidosis associated with the prion diseases including Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru and animal scrapie, the amyloidosis associated with long-term hemodialysis and carpal tunnel syndrome, the amyloidosis associated with endocrine tumors such as medullary carcinoma of the thyroid, and the alpha-synuclein associated diseases including Parkinson's disease and Lewy body disease, and in particular, Alzheimer's disease.

In this method the associated amyloid may be either beta-amyloid protein or Aβ, AA amyloid or inflammation-associated amyloid, AL amyloid, amylin or islet amyloid polypeptide, PrP amyloid, beta₂-microglobulin amyloid, transthyretin or prealbumin, or variants of procalcitonin.

Another method for the treatment, inhibition, prevention or management of amyloid fibril or alpha-synuclein fibril formation, deposition, accumulation, aggregation and/or persistence in a mammalian subject is disclosed, and the method includes the step of administering to the subject a therapeutic amount of any of the compositions isolated by any of the methods disclosed herein. Contemplated routes of administration of the method of treatment include oral administration, parenteral injection, intraperitoneal injection, intravenous injection, subcutaneous injection, or aerosol spray administration.

A novel pharmaceutical agent is disclosed that is comprised of a therapeutically effective amount of a material made according to any of the disclosed isolation processes, with the therapeutic amount of the material selected for efficacy in treating an amyloid disease in a patient.

Another pharmaceutical agent is disclosed that is comprised of a therapeutically effective amount of a chlorogenic acid and/or epicatechin, the compound and the therapeutic amount of the compound selected for efficacy in treating an amylold disease in a patient.

In either or both of the pharmaceutical agents disclosed above, the therapeutically effective amount of a material is a dosage in the range of from about 10 to 1,000 mg/kg of body weight of the patient, and more particularly from about 10 to 100 mg/kg of body weight of the parent. The pharmacological agent may also contain a pharmaceutically a cceptable carrier, diluent, or excipient. A therapeutically effective amount of the material is defined as an amount that has an amyloid inhibitory activity or efficacy greater than 50%, as compared to placebo, or no material at all.

Claim 1 of 2 Claims

1. A method for isolating amyloid inhibitory components from Uncaria tomentosa, the method comprising the steps:

a) adding 4000 ml of methanol to 1 kg of Uncaria tomentosa and mixing;

b) centrifuging the mixture at ×2,500 g using a centrifuge for 30 minutes and pouring off the supernatant from a remaining residue;

c) adding a second volume of methanol to the residue and repeating step b;

d) combining the supernatants from step b and c and evaporating until reduced in volume to about 4-5% of the volume of the supernatents using a rotary evaporator at 50° C.;

e) taking the reduced volume, washing 4 times with 300 ml of petroleum ether, and discarding the ether layer;

f) evaporating the methanol to dryness to form a solid material using a rotary evaporator at 50° C.;

g) extracting the solid material 5 times with 150 ml of distilled water, followed by centrifugation at 2,500×g for 30 minutes each time;

h) combining the supernatants from step g and then lypohilizing using a freeze-dryer;

i) dissolving the resulting lypohilized extract into about 500 ml of distilled water, and applying 50-100 ml portions of the dissolved extract to a 400 ml LH-20 column equilibrated

j) eluting the LH-20 column with ˜3 column volumes of distilled water and discarding the amber/yellow eluate;

k) eluting the LH-20 column with ˜3 column volumes of methanol, collecting the eluate and evaporating it to dryness using a rotary evaporator at 50° C;

l) dissolving the product of step k in water to a concentration of ˜80 mg/ml and applying 5 ml at a time to a 10 gm disposable C18 SPE column equilibrated in solvent A, where solvent A is 95% water/5% acetonitrile/0.1% TFA;

m) washing the column with 3 volumes of solvent A and discarding the eluate;

n) eluting the column with 3 volumes of solvent A containing 12.5% solvent B, where solvent B is 95% acentronitrile/5% water/0.1% TFA, and lyophilizing the eluate;

o) injecting 50 mg portions of the lyophilized eluate of step n into a Hewlett-Packard 1100 Series HPLC instrument with diode array detector, fitted with a 2.2 cm×25 cm Vydac 218TP1022 C18 reverse-phase column maintained at 25° C. and at a flow rate of 5 ml/min;

p) eluting the sample with the following solvent profile, 10% solvent B for minutes 0 to 20, 10-100% solvent B gradient for minutes 20 to 30, and 100-10% solvent B gradient for minutes 30-31; and

q) seperating and collecting at least one fraction selected from the following fractions: fraction G (˜13-14 minutes), fraction F (˜15-16 minutes), fraction H (˜17-20 minutes), fraction I (˜21 minutes), fraction J (˜22-23 minutes), fraction K1 (˜24 minutes), fraction K2 (˜25 minutes), fraction L (˜26-27 minutes), fraction M (˜27 -28 minutes), and fraction N (˜28-29 minutes).

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

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