OF MEDICINAL HERBS AND COMPOSITIONS THEREOF

Related Application

This -application claims the benefit of priority of United States provisional appl ication serial number US62/574,440 of identical title filed October 19, 2017, the entire contents of which are incorporated by reference herein.

Field of the invention

The presented invention relates to certain medicinal herbs and their extracts which have been found to inhibit the androge receptor and are useful in the treatment of disease states and conditions which result from hyperactivity of the androgen receptor.

Pharmaceutical compositions based upo these herbs and/or extracts obtained therefrom represent an additional aspect of the invention. Methods of treating a variety of disease states which are a result of hyperactivity of the androgen receptor as otherwise disclosed herein represent an additional aspect of the invention.

Background of the invention

The prostate is a gland i the male reproductive system. Most prostate cancers grow slowly; however, some grow more rapidly. The cancer cells may spread from the prostate to other parts of the body, particularly the bones and lymph nodes. Initially, prostate cancer may exhibit no symptoms. Symptoms arising at a later stage of development may include difficulty urinating, blood in the urine, or pain in the pelvis, back or when urinating. Other !ate symptoms may include fatigue due to low levels of red blood ceils.

No single gene is believed to be responsible for prostate cancer; many different .genes have been implicated. Mutations in BRCAl and BRCA2, important risk factors for ovarian cancer and breast cancer in women, have also been implicated in prostate cancer. Other linked genes inc lude the Hereditary Prostate cancer gene S (HPC1), the androgen receptor, and the vitamin D receptor. TMPRSS2-ETS gene family fusion, specifically TMPRSS2- ERG or TMPRSS2-ETV i 4 promotes cancer cell growth.

The prostate glands require male hormones, known as androgens, to work properly. Androgens include testosterone, which is made in the testes; deiiydroepimidrosterone, made in the adrenal glands; and di hydro testosterone, which is converted from testosterone within the prostate itself. Androgens are also responsible for secondary sex characteristics such as facial hair and increased muscle mass. The androgen receptor (AR), also known as NR3C4 (nuclear receptor subfamily 3, group C, member 4), is a type of nuclear receptor that is activated by binding either of th androgenic hormones, testosterone, or dihydrotestosterone in the cytoplasm and then translocating into the nucleus. The main .function of the androgen receptor is as a DMA-binding transcription factor that regulates gene expression; however, the androgen receptor has other functions as well. The androgen receptor helps prostate cancer cells to survive and its inhibition is a target for many anti cancer research studies; so far, no: ^' composition, .has been found that is e.i feetive in inhibiting the androgen receptor in humans.

Prostate cancer, alopecia, hepatocellular carcinoma, and acne vulgaris are a few examples of the myriad of diseases linked to androgen receptor signaling. These diseases have a. significant impact on human health; for example. The American Cancer Society estimates that in 2009, prostate cancer will cause 27,360 deaths and 192.280 new cases will be diagnosed. In fact, one man in si will get prostate cancer in his lifetime and one in thirty- five will die from this disease. Androgen receptor inhibitors are the primary treatment option for androgen-related diseases. Current inhibitors prevent ligand binding to the androgen receptor, but these treatments can result in acquired resistance and serious side effects. Due to the limitations of current treatment options, alternative anti androgen therapies are urgently needed.

Brief .Description of the invention

The present invention recognizes that certain herbs, which have a long history of use by humans, may be able to inhibit the androgen receptor. Those herbs can be used singly or in combination with others, or as a iead for isolation of active chemicals for inhibition of the androgen receptor. Those herbs may be useful for cancer prevention particularly prostate hyperplasia or prostate cancer and other diseases caused by hyperactivity of the androgen receptor.

To address this potential, the inventors studied the effects of herbal water extracts from over 250 formulations, across 25 signaling pathways, using primary hiciferase reporter assay and other enzymatic reactions. Eleven (1 1) herbs showed androgen receptor inhibition. The present invention therefore relates to extracts obtained front the group of 11 (eleven) herbs consisting of Aloe barbadensis (Aloe vbera) ( ^' :F3 ^' Rheum palmatum

L . ( ^{" '} ;B6), Stephania tetrandra (St |¾ 3 :C4), Phellodendrem ch nense Sehneid.

(H¾:DS), Euphorbia humifusa (¾I|¥:S6), Eclipta prostrate (S¥ ^> ¾:i2) _J Apocyaum Sanguisorba officinalis L.

(¾ i :E5), Camellia sinensis var.assamica (Mast.) itaraura Chaag (puer tea, 'tf¾:PE}, Punica and mixtures thereof, including mix tures of two, three, four , five, six , seven, eight, nine, ten and eleven of the aforementioned herbs and/or their extracts). Such herbs are inhibitors of the androgen receptor and accordingly are effective in

pharmaceutical compositions or nutritive supplements to treat, inhibit, prevent, reduce the incidence of, ameliorate and/or resolve any of a number of disease states or conditions resulting from androgen receptor hyperactivity. These diseases and/or conditions include for example, prostate hyperplasia, prostate cancer, including castration resistant prostate cancer, drug resistant prostate cancer, especially including drug resistant cancers associated with AR- Vs (androgen receptor splice variants), bicalutamide and/or enzalutamide resistant prostate cancer, hepatocellular cancer, hair loss and/or the growth of hair, especially in the scalp and in other regions of the body where hair growth is desirable, pattern hair loss (androgeaetic alopecia.) caused by high levels of DHT, acne, seborrhea, hirsutism (excessive body hair), hidradenitis suppurativa, paraphilias, precocious puberty in boys and polycystic ovary syndrome in women, among others.

One or more the above herbs and/or extracts, preferably a solvent extract such as an aqueous (water or a mixture of water and at least one Ci~C% alcohol) or Ci-C? alcohol

(preferably methanoSic or ethanolic more preferably ethanolic) extract pursuant to the present invention, can be used alone or in combination with a pharmaceutically acceptable carrier, additive or excipient to treat, inhibit, prevent, reduce the incidence of, ameliorate and/or resolve a number of disease s tates or c onditions inc luding, for example, prostate hyperplasia, prostate cancer and other diseases caused by hyperactivity of the androgen receptor, in certain embodiments, the composition can cause a redaction in hair loss and/or the growth of hair, especially in the scalp and in other regions of the body where hair growth is desirable. In certam embodiments, die composition can be used to treat castration resistant prostate cancer, drug resistant prostate cancer including drug resistant cancers associated with AR-Vs (androgen receptor splice variants) arid or over -expression of androgen receptors. In an embodiments, the composition can be used to treat bicahitamide and/or enzalutamide resistant prostate cancer and hepatocellular cancer, in additional embodiments, the composition can he used to treat prostatic hyperplasia, pattern hair loss (androgenetic alopecia) caused by high level of DHT, acne, seborrhea, hirsutism (excessive body hair), hidradenitis suppurativa, paraphilias, precocious puberty in boys and polycystic ovary syndrome in women.

The present invention also relates to the discovery that a compound according to the group consisting of aloe-emodin, emodln, chrysophanol, rhein, sennoside- A., sennoside-C _* sennoside-D, gallic acid, epigallocatechin, gallocatechin, quercetirt, kearnpferoL

epigallocatechin gallate, polyphenol fraction of Camellia assamica (PE1 ) and mixtures thereof preferably, aloe-emodin, gallic acid, epigallocatechin, galloeateehm, epigallocatechin gallate, quercetin, keampferol, and polyphenol .fraction of Camellia assamica (PE l ) and mixtures thereof may be used alone or in combination with a pharmaceutically acceptable carrier, additive or excipient to treat, inhibit, prevent, reduce the incidence of, ameliorate and ^' or resolve a number of disease states or conditions including, for example, prostate hyperplasia, prostate cancer, and other diseases caused by hyperactivity/overactivity of the androgen receptor. In certain embodiments, the compound or mixtures of compounds may be used in the reduction in hair loss and/or the growth of hair, especially in the scalp and in other regions of the body where hair growth is desirable. In certain embodiments, the compound or mixture of compounds can be used to treat, inhibit, reduce the incidence or likelihood of, ameliorate and/or resolve castration resistant prostate cancer, drag resistant prostate cancer including drag resistant cancers associated with Ail- Vs (androgen receptor splice variants) and or over expression of androgen receptors. In an embodiment, the compounds or mixtures of compounds can be used to treat enzalutamide resistant prostate cancer and/or

hepatocellular cancer. In additional embodiments, the compound or mixture of compounds can be used to treat prostatic hyperplasia, pattern hair loss (androgenetic alopecia) caused by high level of DHT, acne, seborrhea, hirsutism (excessive body hair), hidradenitis suppurativa, paraphilias, precocious puberty in boys and polycystic ovary syndrome in women. Of course, herbs which contain one or more of these compounds may be used in place of these compounds for the biological effects these compounds exhibit. The above herbs and/or their extracts may be ^' used alone or in .further combination with at least one additional compound selected from die group consisting of aloe-emodin, gallic acid, epigaHoca.tec.hin (EGC), gallocatechin (GC), epigallocatechin gallate (EGCG), quercetin and keampferoL Each of these agents or herbs which contain these compounds may be used alone or in combination wit each other for the treatment of one or more of the disease states and/or conditions disclosed herein.

Since these herbs and or compounds listed above can inhibit the growth of22RVt, which are resistant to biealiitaniide and enzalutamide, these herbs and/or compounds may be developed to target biealytan de and/or enza taniide resistant prostat cancer.

Brief Description of the Dra wings

Figures 1 A-I are graphs showing the effects of selected herbal water extracts on androgen receptor mediated transcription activity of 22RV1 AR-fcciferase reporter cells in condition with dihydrotestosterone (DHT), The selected herbal extracts were nine (9); Aloe barbadensis (jS#:F3), Rheum palmatum L. (^]¾:B6) _; Stephania tetrandra(¾!¾ d.:C4), Phellodendro chinense Schneid.( ^' f $ή :D8), Euphorbia humifusa(ftl¾$:S6), Eclipta prostrata(ll ¾:12), A. venetum L. (^¾ ^* :F ), Portukca oleracea L.i ¾ l:F5), Sanguisorba officinalis L. (ifefifrES).

Figure 2 shows the effect of different batches of PE tea (puer tea) water. on the androgen receptor .mediated. transcription activity of 22RV1 AR-lactferase reporter cells m condition with DHT.

Figures 3A-D are graphs showing the effect of herbal water extracts on the niRNA expression of androgen receptor target genes: kalHkrein KLK (A, C) and prostate specific antigen/PSA (B, D) of 22RVT. in condition with and without DOT, qRT~PCR was used to determine the relative tnRNA expression where beta-actin was used as internal control. Aloe vera(ll ^: if:F3), Rheum palmatum L. ( ^" ivjf ;B6), Stephania tetrandra(R i¾? 3 :C4),

Phellodendron chinense Schneidiff fB:D8), Euphorbia huniifusai lli #kS<5), Eclipta prostrata(H ^ 1:12), A. venetum L. (ϋ¾ ^* ^:Ρ ), Po tulaca oleracea L.( ¾ii £:F5} _? Sanguisorba officinalis L. (¾¾|:E5), Camellia assamica (Mast) Chang (puer tea, ll¾¾l: PE). I» figure 3E and 3F, water extract prepared -using instant dissol ving powder of puer tea (PE-P) and water extract prepared using raw tea material of puer tea were compared for their effect on KLK and PSA mRNA expression of 22RV.1 cells.

Figure 4A shows the effect of ^' herbal water extracts on androgen receptor protein expression of 22EV.1 m conditions with and without DHT. Rabbit monoclonal antibody was used to detect androgen receptor protein expression following 24h treatment, β-actin was used as loading control. Aloe vera (It ^' ll :F3), Rheum palmatum L. (?i ¾:B6} _; Stephama tetrandra(¾.i^ :C4) _f Phellodendron eliraense Schneid.($t¾:D8) ₅ Euphorbia

A. venetum L. ( I¾ ^' J :F1 ), Portulaca oleracea L.{-¾¾.¾:F5), Sanguisorba officinalis t. (¾ϋ :Ε·5) ₅ Camellia assaniica (Mast) Chang (puer tea, PE). }& Figure 4B _; water extract prepared using instan dissolving po wder of puer tea (PE-P) a»d water extract prep ared using raw tea materia l of puer tea were compared for their effect on AR protein expression of 22RVI cells.

Figures S an B show the elect of herbal water extracts on androgen receptor mRNA expression of 22RV1 in conditions without DHT (A,C) and with DHT (B,C). qRT- PCR was used to quantify the AR mRNA expression, β-aetin was used for normalization. Aloe vera ( il :F3), Rheum palmatum L. (^;ft:B6), Stephania teu¾ndra(¾¾j¾ B:C4), Phellodendron chmense Schneid.(it 0:D8), Euphorbia ht iifusa(¾_l¾^ :S6), Eclipta prostrata(S ^¾:I2) _; A. venetum L. ,$I $£:F1), Portulaca oleracea L.(--¾¾ ^' $ :F5), Sanguisorba officinalis L, (%. ;E5) Camellia assamica (Mast) Chang (puer tea,

PE). In Figure 5C, water extract prepared using instant dissolving powder of puer tea (PE-P) and water extract prepared using raw tea material of puer tea were compared for their effect on AR mRNA expression of 22RV1 cells.

Figure 6 shows the effect of herbs water extracts on the interaction between AR protek and DNA (with androgen binding site). Androgen receptor -DNA interaction pull down assay was performed by mixing biotin label DNA 5 ^* ~

gtaattgeAGAACAgcaAGTGCTagctctc-3 ^'> (with androgen binding site) and nuclear lysis with androgen receptor protein (extracted from 22RV1 cells pre-stimulated with DHT 25nM for overnight). Sireptavidin-Dynabead was used to trap down the DMA- AR complex under magnetic condition. -Western blotting was used to delect the amount of pail down AR protein. Inhibition between DNA and androgen receptor binding will reduce the amount of AR protein pui! down. Aloe vera (¾¾".F3), Stephania ietrandra(¾!¾ ^" 3;C4) _S Euphorbia EcHpta tosttata( ¥- 1:12), A. venetum L. (H¾f |ft:Fl), Portulaca oleracea L.(¾¾$ :F5), Sanguisorba officinalis L. ( v||i:E5) _? Camellia assamica (Mast) Chang (piier tea, If PE).

Figure 4, Table 1 shows the possible target of the following herbs on AR signaling pathway. Aloe vera (S ^" # ^' : 3), Rheum palmaturn L. C H ;B6) _S Stephania

tetrandra(¾!¾i 3:C4), Phellodendron chinense Schnetd.( ^' ^-¾:D8), Euphorbia

EcHpta rostrata( § ¾:I2}, A. venetum L. Portulaca oleracea L.( ~|¾ S:F5), Sanguisorba officinalis L. (¾I¾¾:E5). Figure 6B, Table 2 shows the cytotoxicity of herbal water extracts for 22 V1 cells and DuI 5 cells with DHT. Aloe vera (1S¾".F3) _; Rheum palmaturn L. (λΊ :Β6), Stephania tetrandra(¾ S:C4), Phellodendron chinense Schneid.(¾ 0:D8} ₅ Euphorbia hun i&sa(¾lil£:S6} ₅ Eciipt prostrata(S 5l:i2} _} A. venetum L. (Il- ^: f i)#:Fl), Portulaca oleracea L.(%1¾¾F5),, Sanguisorba officinalis L, (j&flf ;E5), Camellia assamica (Mast) Chang (puer tea, #¾f^: PE), Punica granatum water extract ((Q¾:PG, pomegranate).

Figures 7A-L are graphs showing the effects of herbal water extract on the growth of DU145 and 22RV1 xenograft in vivo. Curves shown significant inhibition on the growth of xenograft were label with * (P«X0S). Aloe barbadensis (S #:F3), Euphorbia humifusa

officinalis L, {¾H:E5).

Figures 8A-H shows the effect o S6, F5 _S PE I water extract and Enzaluiamide on the growth of LMCaP xenograft (A-C) and Dul45 (D~F) in nude .mice. Significant inhibition on the growth of xenograft were label with * (P<0.05) t-test at day 14. (G. H) Effects of different treatment on the L 2 and PSA. mRNA of LNCaP tumor where significant inhibition was labeled with * (P 0.05). Figure 9 shows the inhibitory activity .-of the fractions of F3 obtained from preparatve HPLC. A. Inhibitory activit of the fractions of F3 obtained from preparative HPLC using luciferase .reporter assay in which SOOug/m! (equivalent dose to F3 crude water extract) was used for all fractions. B. Different dose of fraction 35 and 32 were tested for their AR inhibition acti vity using luciferase reporter assay.

Figure 10 shows the LC-MS for fraction 31 and 32 from fractionation of F3 using preparative HPLC. insert figure showed that the equivalent amount of aloe-emodin was compared to crude water extract of F3 using luciferase report assay.

Figure 11 shows the effect of aloe-emodin derivatives on the activity of AR using luciferase reporter assay.

Figure 12 shows the LC-MS for water elution aad 10% ethanol elution (Ei %) of S6 water extract from solid phase extraction CIS column, inserted figure showed that water elution and 10% ethanol elution (E10%) had relative stronger inhibitory effect on AR than other elution using luciferase report assay.

Figures 13A-F show the effect of gallic acid on AR activity. (A) the equivalent amount of gallic acid was compared to crude water extract of S6 using luciferase report assay. (B, C, D) .Effect of gallic acid on K.L 2, PSA and AR mRN expression of 22RV1 cells with or without DHT. (E, F) Arrii-tumor effect of gallic acid on 22 V1 tumor and Dul45 tumor of nude mice.

Figures I4A-C. (A) shows the effect of Fl (Ap cynnm venetum) and Luo-bu- ma(LBM) tea from China on AR activity using luciferase reporter assay , (B, C) show the effect of Fi (Apocynum venetum) and Luo-hu-ma(LBM) tea from China on KLK2 and PSA mRNA expression using qPCR assay.

Figures 15A-C show the _. anti-AR activity of ractions of Fl using solid phase extraction column and list of active chemicals. (A) Anti-AR activity of fractions of Fl water extract using sold phase extraction CI 8 column. Water extract of Fl passed through CIS column and then elufion with different concentration of ethanoL Fractions of Fl (Lmg/rnl equivalent to Fl v crude water extarct) were tested for their anti-AR activity using luciferase reporter assay. (B) Anti-AR. activity of detectable chemicals in different f action of Fl. (C) Dose response of different purified eompunds of F l on AR. ac vty using hiciferase reporter assay.

Figures 16A-C. (A) shows Polyphenols (PP) (extract from PE) obtained from obtained from Gaoligongshan co.ltd showed ami- androgen receptor using 22RV1 hiciferase reporter ceil assay. (B, C) show the anti-AR activity of different tractions of PE! or PP (polyphenol. ^' fraction).

Figure 17 shows the LC-MS profiles for 30% ethanol. ehjtion of Fl and

PP(polyphenoi).

Figure 18 shows the effect of epkateehiii, catechin, ga!locaiechm (GC),

Epigalioca echin (EGC) and epiga!locatechin gal late (EGCG) on AR acitivty which is determined, using iucif rase report assay.

Figure 19 shows the LC-MS profile for determination of gallic acid in PE using low molecular weight scan setting.

Figure 20 shows, that fraction 9 exhibited the strongest anti-andrpgen receptor activity from preparative B PLC fractions.

Figure 21 shows that fraction 9 from preparati e HPLC fraction contains gallic acid. Inset, shows gallic acid activity displacing DHT at androgen receptors in 22RV1 cells.

Figure 22 shows the cytotoxicity of certain herbal extracts of Fl, IBM tea and LBM mixed tea samples for 22RV1 cell and Dul45 cells with or without DHT.

Figure 23 shows the anii-androgen activity of different teas in 22RV I luciterase report cell assay. PB-P was from. Yunnan iasiydee pure biological tea group co.ltd. and PE-1 , PE-2, PE-B was obtained from Gaoligongshan co.ltd. Each of the samples showed anti- androgen receptor activity, although there was a slight difference in potency exhibited. Figure 24 shows die relative luciferase activity of components of ΡΕΪ extract isolated using HPLC.

Figure 25 shows the LC-MS from the preparative HPLC for PE Tea grade I and that fraction 13 corresponded to 5-gajloylquinic acid being the active component in mat fraction.

Figure 26 shows the LC-MS from t he preparative HPLC for PE Tea grade 1 and that fraction 21 corresponded to ga!loyl-beta ^» ghicose being the active component in that fraction.

Figure 27 shows the LC-MS from the preparative HPLC for PE Tea grade 1 and that fraction 32 corresponded to epicalechin gaiiate (ECG) being the active component in that fraction.

Figure 28 shows the LC-MS from the preparative HPLC for PE Tea grade 1 and that fraction 33 also corresponded to epicatechin gaiiate (ECG) being the active component in that fraction.

Figure 29 shows the LC-MS from the preparative HPLC for PE Te grade 1 and that fraction 42 also correspoaded to epigalloeatec m gaiiate (EGCG) being the active component ^' in that traction.

Figure 36 shows the cytotoxicity ofvarioBs herbs (left axis) according to the present invention in a number of cell lines (top axis).

Figures 31 -E shows the effect of Punka ^■ granatum water extract (PG, or commonly called pomegranate) on androgen receptor activity of 22RV1 cells. ^' (A) PG water extract showed inhibitory effect on AR mediated transcription of 22RV1 cells in luciferase report assay. (B and C) PG water extract inhibited PSA and KL 2 mRNA expression of 22RVI in present or absent of DHT conditions in qRT-PCR assays. (D ) PG water extract inhibited AR and AR- V protein of 22RVI cells in western blot assay . (E) PG water extract down regulated AR. mRNA of 22R.V1 in qRT-PCR assay. Detailed Description of the Invention

The following definitions ate used to describe the present invention. In instances where a term, is not specifically defined, the definition to be used is that which one of ordinary skill in the art would use to define that term within the context of that term's use.

The term "patient" or "subject" is used to describe an animal, including a

domesticated animal such as a dog, cat, cow, horse, sheep, goat or other similarly

domesticated animal, especially a human patient in need, who receives medical attention, care, or treatment of the present in vention.

The term "effective" is used to describe an amount of a component, extract, material or solvent which is used to produce an intended effect m amount consistent with the effect desired and may vary with the effect desired or which occurs.

The term "extract" is used to describe an aqueous or C Cs alcoholic (preferably, methanoiic or ethanolic) extract of one or more of the following herbs selected from the group consisting of Aloe barbadensis (¾ ^; li;F3), Rheum palmatum L. (λ;¾ ^' :Β6), Stephania tetrandra (tXi¾ fi:C4), Phellodendron chinense Schneid. ( ¾tt:D8), Euphorbia humifusa (i&f®¾S6), Eclipta prostrata (:1?-¾:I2), A. venetnm L. (li¾Jtl:FI), Portulaca o!eracea. L, (•¾S ¾:F5), and Sanguisorba officinalis L. (Ι¾ !;Ε5}, Camellia sinensis var.assamica (Mast.) itamura (¾¾:P£), Punka granatum. (¾ f:PG) _; and mixtures thereof (including mixtures of extracts from 2, 3, 4, 5, 6, ?:, 8 or 9 herbs),

Extracts of the present invention are prepared, by exposing one or more of the herbs which are described above to an effective amount of a solvent, preferably an aqueous or Cj- Cj alcoholic (preferably, ethanolic) solvent, preferably heated (including boiling) for a period of time effec tive to extract medicinal components of the herbs into the solvent (for a period from a few minutes, to several boors to several day or more). Extracts, of solvents may b prepared using standard methods readily available in the art and may include the preferred methods of preparation as otherwise described herein. The term "aqueous" is used to describe a solvent which comprises water in an amoun Preferably, extracts are provided using water or water/alcohol, preferably, water/ethanol. Aqueous solvents used to provide extracts preferably comprise at least about

50% by volume water within this mixture and often water and another alcohol such as ethanol, isopropanol or methanol, among others. In preferred aspects, the solvent is heated (preferably boiled). The use of water or a water/ethanol mixture is preferred. It is noted that other solvents may also be used to provide extracts according to the present invention ("extraction sol vent"), but the use of an aqueous alcohol, especially aqueous ethanol (wherein water preferably comprises at least about 5% up to about 95+%) is preferred. Water, ethanol, isopropanol, methanol., propanol and buianoS and mixtures.. thereof, are generally used as solvents to provide extracts according to the invention.

The term "methanolic", "methanol ic solvent", "ethanolic" or "ethanolic solvent" is used to describe a solvent which comprises methanol or ethanol in amounts greater man 50% by volume. As noted, the term "methanol" or "ethanol ic" may overlap with the term

"aqueous" as otherwise defined herein.

The term "solid extract" is used to describe an extract of one or more of the herbs as otherwise disclosed herein which has been dried, dehydrated, lyophilized or otherwise solidified to avoid the composition containing appreciable quantities of solvent.

The term "androgen receptor" is used to describe or denominate a protein comple typically occurring in cells, that binds to male hormones, known as androgens including testosterone. dehydroepjandiOsterone, and dihydrotestosterone. More specifically, androgen receptor (A ), also known as R3C4 (nuclear receptor subfamily 3, group C, member 4), is a type of nuclear receptor that is activated by binding either of the androgenic hormones, testosterone, or dihydrotestosterone in the cytoplasm and then translocating into the nucleus.

Hyperactivity of the androgen receptor (All) is a key factor of carcinogenesis in prostate tissue and many other diseases including hepatocellular carcinoma, and acne vulgaris.

The term "prostate cancer" is used to describe a disease in which cancer develops in the prostate, a gland in the male reproductive system. It. occurs when cells of the prostate imitate and begin to multiply uncontrollably. These cells may metastasize (metastatic prostate cancer) from the prostate to virtually an other part of the body , particularly the bones and lymph nodes, but the kidney, bladder and even the brain, among other tissues. Prostate cancer may cause pain, difficulty in urinating, problems during sexual intercourse, erectile dysfunction. Other symptoms can potentially develop during later stages of the disease.

Rates of detection of prostate cancers vary widely across the world, with South and East Asia detecting less frequently than k Europe, and especially the United States, Prostate cancer develops most frequently in men over the age of fifty and is one of the most prevalent types of cancer in men. However, many inert who develop prostate cancer never have symptoms, undergo no therapy, and eventually die of other causes. This is because cancer of the prostate is, in most cases, slow-growing, and because most of those affected are over the age of 60. Hence, they often die of causes unrelated to the prostate cancer. Many factors, including genetics and diet, have been implicated in the development of prostate cancer. The presence of prostate cancer may be indicated by symptoms, physical examination, prostate specific antigen (PSA), or biopsy. There is concern about the accuracy of the PSA test and its usefulness in screening. Suspected prostate cancer is typically confirmed by taking a biopsy of the prostate and examining it under a microscope. Further tests, such as CT scans and bone scans, may be performed to determine whether prostate cancer has spread.

Treatment options for prostate cancer with intent to cure are primarily surgery and radiation therapy. Other treatments such as hormonal therapy, chemotherapy, proton therapy, cryosurgery, high intensity focused ultrasound (HIFU) also exist depending on the clinical scenario and desired outcome.

The age and underlying health of the man, the extent of metastasis, appearance under the microscope, and response of the cancer to initial treatment are important in determining the outcome of the disease . The decision whether or not to treat localized prostate cancer (a tumor that is contained within the prostate) with curative intent is a patient trade-off between the expected beneficial and harmful effects in terms of patient survival and quality of life.

An important part of evaluating prostate cancer is determining the stage, or how far the cancer has spread. Knowing the stage helps define prognosis and is useful when selecting therapies. The most common system is the four-stage TNM system (abbreviated from

riimor/Nodes/ etastases). Its components include the size of the tumor, the number of involved lymph .nodes, and. the presence of any other metastases.

The most important distinction made by any staging system is whether or not the cancer is still confined to the prostate or is metastatic, to the TNM system, clinical Tl and T2 cancers are found only in the prostate, while T3 and T4 cancers have spread elsewhere and metastasized into other tissue. Several tests can be used to look for evidence of spread. These include computed tomography to evaluate spread within the pelvis, bone scans to look for spread to the bones, and endorectal coil magnetic resonance imaging to closely evaluate the prostatic capsule and the seminal vesicles. Bone scans often reveal osteoblastic

appearance due to increased bone density in the areas of hone meiasiasis - opposite to what is found in many other cancers that metastasize. Computed tomography (CT) and magnetic resonance imaging (MR!) currently do not add any significant information in the assessment of possible lymph node metastases in patients with prostate cancer according to a metaanalysis.

Prostate cancer is relatively easy to treat if found early. After a prostate biopsy, a pathologist looks at the samples under a microscope. If cancer is present, the pathologist reports the grade of the tumor. The grade tells how much the tumor tissue differs from normal prostate tissue and suggests how fast the tumor is likely to grow. The Gleaso system is used to grade prostate tumors from ^' 2 to 10, where a Gleason score of .10 indicates the most abnormalities. The pathologist assigns a number from 1 to 5 for the most common pattern observed under the microscope, then does the same for the second most common pattern. The sum of these two numbers is the Gleason score. The Whitrnore-Jewett stage is another method sometimes used. Proper grading of the tumor is critical, since the grade of the tumor is one of the major factors used to determine the treatment recommendation.

Early prostate cancer usually causes no symptoms. Often it is diagnosed during the workup for an elevated PSA noticed during a routine checkup. Sometimes, however, prostate cancer does cause symptoms, often si milar to those of diseases such as benign prostatic hypertrophy. These include frequent urination, increased urination at night, difficult starting and maintaining a steady stream of urine, blood in the urine, and painful urination. Prostate cancer is associated with urinary dysfunction as the prostate gland surrounds the prostatic ^• urethra. Changes within the gland therefore directly affect urinary .function. Because the vas deferens deposits seminal fluid into the prostatic urethra, and secretions from the prostate gland itself are included in semen content, prostate cancer may also cause problems wit sexual function and performance, such as difficulty achieving erectio or painful ejaculation.

Advanced prostate cancer can spread to other parts of the body and this ma cause additional symptoms. The most common symptom is bone pain, often in the vertebrae (bones of the spine), pelvis or ribs. Spread of cancer into other bones such as the femur is usually to the proximal part of the bone. Prostate cancer in the spine can also compress the spinal cord, causing leg weakness and urinary nd fecal, incontinence.

The specific causes of prostate cancer remain unknown. A man's risk of developing prostate cancer is related to his age. genetics, race, diet, lifestyle, medications, and other factors. The primary risk factor is age. Prostate cancer is uncommon in men less than 45, but becomes more common with advancing age. The average age at tire time of diagnosis is 70. However, many men never know they have prostate cancer.

A man's genetic background contributes to his risk of developing prostate cancer. This is suggested by an increased inc idence of prostate cancer found in certain racial groups, in identical twins of men with prostate cancer, and in men with certain genes. Men who have brother or father wi th prostate cancer have twice the usual risk of developing prostate cancel ^' . Studies of twins i Scandinavia suggest that forty percent of prostate cancer risk, can be explained by inherited factors. However, no single gene is responsible for prostate cancer; many different genes have been implicated. Two genes (BRCA i and BRCAI) that are important risk factors for ovarian cancer and breast cancer in women have also been

implicated in prostate cancer.

Dietary amounts of certain foods, vitamins, and minerals can contribute to prostate cancer risk. Dietary factors that may increase prostate cancer risk include low intake of vitamin E, the mineral selenium, green tea and vitamin D. A large study has implicated dairy, specifically low-fat milk and other dairy products to which vitamin A palmitate has been added. This form of synthetic vitamin A has been linked to prostate cancer because it reacts with zinc and protein to form an unabsorbable complex. Prostate cancer has also been linked to the inclusion of bovine somatotropi hormone in certain dairy products. There are also some links between prostate cancer and medications, medical

procedures, and medical conditions. Daily use of anti-inflammatory medicines such as aspirin, ibuprofen, or naproxen may decrease prostate cancer risk. Use of the cholesterol- lowering dregs known as die statins may also decrease prostate cancer risk, infection or inflammation of the prostate (prostatitis) ay increase the chance for prostate cancer, and infection with the sexually transmitted infections chlamydia, gonorrhea, or syphilis seems to increase risk. Obesity and elevated blood levels of testosterone may increase the risk for prostate cancer.

Prostate cancer is classified as an adenocarcinoma, or glandular cancer, that begins when normal semen-secreting prostate gland cells mutate into cancer cells. The region of prostate gland where the adenocarcinoma is most common is the peripheral zone. Initially, small clumps of cancer cells remain confined to otherwise normal prostate glands, a condition known as carcinoma in situ or prostatic intraepithelial neoplasia (PIN). Although there is no proof that PIN is a cancer precursor, it is closely associated with cancer. Over time these cancer cells begin to multiply and spread to the surrounding prostate tissue (the stroma) forming a tumor. Eventually, the tumor may grow large enough to invade nearby organs such as the seminal vesicles or the rectum, or the tumor celts may develop the ability to travel in the bloodstream and lymphatic system. Prostate cancer is considered a malignant tumor because- it is a mass of cells which can invade other parts of the body. This invasion of other organs is called metastasis. Prostate cancer most commonly metastasizes to the bones, lymph nodes, rectum, and bladder.

In prostate cancer, the regular glands of the normal prostate are replaced by irregular glands and clumps of cells. When a man has symptoms of prostate cancer, or a screening test indicates an increased risk for cancer, more invasive evaluatio is offered. The only test which can fully confirm the diagnosis of prostate cancer is a biopsy, the removal of small pieces of the prostate for microscopic examination. However, prior to a biopsy, several other tools may be used to gather more information about the prostate and the urinary tract .

Cystoscopy shows the urinary tract from inside the bladder, using a thin, flexible camera tube inserted down the urethra. Transrectal ultrasonography creates a picture of the prostate using sound waves from a probe in the rectum. After biopsy, the tissue samples are then examined under microscope to determine whether cancer ceils are present, and to evaluate the microscopic features (or Gleason score) of any cancer found, hi addition, tissue samples may be stained for the presence of PSA and other tumor markers in order to determine the origin of raaiigant cells that have metastasized. A number of other potential approaches for diagnosis of prostate cancer are ongoing such as early prostate cancer antigen-2 (EPCA-2), and prostasome analysis. in addition to therapy using the compounds according to the present invention, therapy (including prophylactic therapy) for prostate cancer supports roles in reducing prostate cancer for dietary - -selenium; vitamin E, iyeopene, so foods, vitamin I , gree tea, omega- 3 fatty acids and phytoestrogens. The selective estrogen receptor modulator drug toremifene has shown promise in early trials. Two medications which block the conversion of testosterone to dihydrotestosterone (and reduce the tendency toward cell growth), finasteride and dutasteride, are shown to be useful The phytochen icals indo!e~3~earhinol and diindolylmethane, found in cruciferous vegetables (calif ower and broccholi), have favorable antiandtogenie and im m e modulating properties. Prostate cancer risk is decreased in a vegetarian diet.

Treatment for prostate cancer may involve active sirrveillance. surgery (prosiatecom or orchiectomy), radiation therapy including brachytherapy (prostate brachytherapy) and external beam radiation as well as hormonal therapy. There are several forms of hormonal, therapy whic include the following, each of which may be combined wit or used in combination with compounds and/or compositions according to the present invention.

* Antiandrogens such as fiutami e, bicaiutamide, nHutamtde, and cyproterone acetate which directly block the actions of testosterone and DHT within prostate cancer cells.

* Medications such as ketoconazote and aminogiutethimide which block the production of adrenal androgens such as DHEA. These medications are generally used only in combination with other methods that can block the 95% of androgens made by the testicles. These combined methods are called total androgen blockade (TAB), which can also be achieved using antiandrogens.

_* Gn H modulators, including agonists and antagonists. GnRH antagonists suppress the production of LH directly, while GnRH agonists suppress LB through the process of do wnregulation after an initial stimulation effect. Abarelix is an example of a GnRH antagonist, while the GnRH agonists include leuproii.de, goserelin, triptorelm, and buserelin.

« The use of abiraterone acetate can be used to reduce PSA levels and tumor sizes in aggressive end-stage prostate cancer for as high as 70% of patients. Sorafenib may also be used to treat metastatic prostate cancer.

Each teatment described above has disadvantages which limit its use in certain circumstances, GnRH agonists eventually cause the same side effects as orchiectom but may cause worse symptoms at the beginning of treatment. When GnRH agonists are first used, testosterone surges can lead to increased bone pain from metastatic cancer, so antiandrogens. or a ^' barelix are often added to blunt these side effects. Estrogens are not commonly used because they increase the risk, for cardiovascular disease and blood clots. The antiandrogetis do not generally cause impotence and usually cause less loss of hone and muscle mass.

etoconazole can cause liver damage with prolonged use, and aminoglutethimide can cause skin rashes.

Palliative care for advanced stage prostate cancer focuses on extending fife and relieving the symptoms of metastati c disease. As noted above, abiraterone acetate shows some promise in treating advance stage prostate cancer as does sorafenib. Chemotherapy may be offered to slow disease progression and postpone symptoms. The most commonly used regimen combines the ehemoiherapeutic drug doce axel with corticosteroid such as prednisone. Bisphosphonates such as zo!edrooie acid have been shown to delay skeletal complications such as fractures or the need for radiatio therapy i patients with hormone- refractory metastatic prostate cancer, Alpharadin may be used to target bone metastasis. The phase II iesimg shows prolonged patient survival times, reduced pain and improved quality of life.

Bone pain due to metastatic disease is treated with opioid pain relievers such as morphine and oxycodone. External beam radiation therapy directed at bone metastases may provide pain relief. I njections of certain radioisotopes, such as strontiom-89, phosphorus-32, or samarium-153, also target bone metastases and may help relieve pain.

As an alternative to active surveillance or definitive treatments, alternative therapies may also be used for the management of prostate cancer. PSA has been shown to be lowered in men with apparent localized prostate cancer using a vegan diet (fish, allowed), regular exercise, and stress reduction. Many other single agents have been shown to reduce PSA, slow PSA. doubling times, or have similar effects on secondary markers in men with localized cancer in short term trials, such as pomegranate juice or genistein, an isoflavone found in various legumes.

Manifestations or secondary conditions or effects of metastatic and advanced prostate cancer may include anemia, bone marrow suppression, weight loss, pathologic fractures, spinal cord compression, pain, hematuria, ureteral and or bladder outlet obstruction, urinary retention, chronic renal failure, urinar incontinence, and symptoms related to bony or soft- tissue metastases, among others.

Additional prostate drugs which can be used in combination with the compounds and or compositions according to the present invention include, for example, the enlarged prostate drugs/agents, as well as eulexin, flutamide, goserelin, leuprolide, Supron, niiandron, mlutamide, zoladex and mixtures thereof. Enlarged prostate drugs/agents as above, include for example, anibenyl, ambophen, amgenal, airosept, bromanyl, bromodiphenhydramine- codeine, bron otuss-eodeine, Cardura, chiorphemramme-hydrocodone, ciclopirox,

elotriniazole-betamethasone, do!sed, dutasteride, finasteride, tlomax, gecH, hexaSol, lamisil, lanased, loprox, Sotrisone, metheaam e, niethen-bella-meth Bl-phen sal meth-hyos-atrp- biue-BA-phsal, MHF-A, mybanU, prosed/DS, Ro-Sed, S~T Forts, iamsniosin, erbinafine, trae, tussionex; ty-mefhate, urarnine, nratin, nretron, uridon, uro-ves, instat, usept and mixtures thereof

The present invention relates to herbal compositions and/or herb extract compositions, especially including solid extracts or extracts which are based preferably at least in part on aqueous, or Cj-Cs alcoholic (preferably eihanolic) solvents of herbs selected from the group consisting of Aloe barbadensis (It W :F3), Rheum paimatum L . ( t | ^' :B6), Stephania tetrandra (¾ !¾ 5:C4), Phellodendron chinense Schnetd. (¾tt:D8), Euphorbia humifus

(Jfef$ ^j $i:S6), Eciipta prostrata (Ρ- ·¾:12),, A. venetum L. (li#\!f£:F I.), Portulaca oleracea L. ( ¾ £ i"iS:F5), and Sanguisorha officinalis L. (¾ _' |. Έ5), Camellia sinensis var.assamica (Mast.) Kitamura ( # :PE), Punica granatum. (-¾¾:PG) and mixtures thereof. Further aspects of the invention relate to compositions which comprise an effective amount of an herb extract ^' liquid, semi-solid or solid form, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient These compositions may be used to prevent, treat, ameliorate, or reduce the incidence of various disease states or conditions which arise from androgen receptor (AR) hyperactivity, comprising administering an effective amount of an extract as otherwise described herein to a patient in need thereof Disease states or conditions which may be -treated incl ude, for example, prostate hyperplasia, prostate cancer, including castration resistant prostate cancer, drug resistant prostate cancer, especially including dmg resistant cancers associated with AR- Vs (androgen receptor splice variants), bicalutaraide and/or enzalutamide resistant prostate cancer, hepatocellular cancer, hair loss and/or the growth of hair, especially in the scalp: and in. other regions of the body where hair growth is desirable, pattern hair loss (androgenetie alopecia) caused by high level ofDHT, acne, seborrhea, hirsutism (excessive body hair), hidradenitis suppurativa, paraphilias, precocious pubert in boys aad polycystic ovary syndrome in women, among others.

Pharmaceutical compositions according to the present invention comprise an effective amount of one or more compounds according to the present invention optionally in combination with a pharmaceutically acceptable additive, carrier or excipient.

I another aspect, the present inventio is directed to the use of one or more herbal extracts according to the present in vention in a phannaceuticail acceptable carrier, additi ve or excipient at a suitable dose ranging from about 0.05 to about 100 mg/kg of body weight per day, preferably within the range of about 0.1 to 50 mg kg day, most preferably in the range of i to 20 mg/kg/day. The desired dose may conveniently be presented in a single dose or as di vided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day.

Ideally, the active ingredient should be administered to achieve etTecti ve peak plasma concentrations of the active compound preferably within the range of from about 0,05 to about 5 uM. This may be achieved, for example, by oral or other route of administration administration as otherwise described herein. Oral dosages, where applicable, will depend o the bioa vailability of the compounds from the GI tract, as well as the pharmacokinetics of the compounds to be administered. While it is possible that for use in therapy, a compound of the invention may be administered as the raw chemical, it is preferable to present the active ingredient as a pharraaceuticaS formulation, presented in combination with, a

pharmaceutically acceptable carrier, excipieat or additive.

Pharmaceutical formulations include those suitable for oral, rectal, nasal, topical (including buccal and sab-lingual), or parenteral (including intramuscular., sub-cutaneous and intravenous) administration. Compositions according to the present invention may also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form. -of, for example, aqueous or oily suspensions,

solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), o preservatives. When desired, the above described formulations may be adapted to provide sustained release characteristics of the active mgredient(s) in the composition using standard methods well-known in the art.

In the pharmaceutical aspect according to the present invention, the compound(s) according to the present invention is formulated preferably in admixture with a

pharmaceutically acceptable carrier. In general, it is preferable to administer the

pharmaceutical composition orally, but certain formulations may be preferably administered parenterall and in particular, in intravenous o intramuscular dosage form, as well as via other parenteral routes, such as transdermal, buccal, subcutaneous, suppository or other route, including via inhalation, including intranasally. Oral dosage forms are preferably

administered in tablet or capsule (preferably, hard or soft gelatin) form or as solutions and or suspensions. Intravenous and intramuscular formulations are preferably administered in. steri le saline. Of course, one of ordinary skill in the art may modify the formulations within the teachings of the speci fication to provide numerous formuiations for a particular route of administratiofl without rendering the compositions of the present invention unstable or compromising their therapeutic activity.

In particiuar, the modification of the present compounds to render them more soluble m water or other vehicle, for example, may be easily accomplished by minor modifications (such as salt formulation, etc.) which are well within the ordinary skill in the art. It is also well within the routineer's skill to modify the route of administration and dosage regimen of a particular compound or composition in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect to the patient.

Formulations containing the compounds of the Invention mm take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, capsules, powders, sustained-release formulations, solutions, suspensions, emulsions, suppositories, creams, ointments, lotions, aerosols or the like, preferabl in unit dosage forms suitable for simple administration of precise dosages.

The compositions typically include a conventional pharmaceutical carrier, additive or excipient and may additionally include other medicinal agents, carriers, and the like.

Preferably, the composition will be about 0.05% to about 75-80% by weight of an extract or extracts of the invention, with the remainder consisting of suitable pharmaceutical additives, carriers and or exciptents. For oral administration, such exeipients include pharmaceutical grades of manmtol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.

Liquid compositions can be prepared by dissolving or dispersing the extracts in liquid, semi-solid or solid form (often about 0.5% to about 20%), and optional -pharmaceutical additives, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanoi, to form a solution or suspension. For use in oral liquid preparation, the composition may be prepared as a solution, suspension, emulsion, or swop, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.

When the composition is employed in the form of solid preparations for oral administration, the preparations may be tablets, granules, powders, capsules or the like. In a tablet formulation, the composition is typically formulated with additives, e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl eellu- lose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations.

An injectable composition for parenteral administration mil typically contain the compound in a suitable i.v. solution, such as sterile physiological salt solution. The composition may also be formulated as a suspension in a lipid or phospholipid, in a liposomal suspension, or in an aqueous emulsion.

The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulati on and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance

bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

Methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see "Remington's Pharmaceutical Sciences" (17th Ed., Mack Pub. Co, 1 85). The person of ordinary skill will take advantage of favorable

pharmacokinetic parameters of the pro-drug forms of the present invention, where applicable, in delivering the present compounds to a patient suffering from a viral infection to maximize the intended effect of the compound.

The pharmaceutical compositions according to the invention may also contain other active ingredients in the treatment of any one or more of the disease states or conditions which are treated with herbal extracts according to the present invention. Effective amounts or concentrations of each of the active compounds of these herbal extracts may be included within the pharmaceutical compositions according to the present invention.

The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. These include especially, for example, one or more of aloe-emodin, emodin, chrysophano!, rheio, sennoside- A, sennoside-C, seunoside-D, gallic acid, epigallocatechin (EGC), gal!ocatechin (GC), quercetin, keampferol, epigallocatechin gallate (EPCG) and mixtures thereof. When one or more of the compounds according to the present invention is used in combination with a second tiierapeuiic agent active the dose of each compound may be either the same as or differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art.

In method aspects according to the present invention, one or more pharmaceutical compositions according to the present invention ma be administered to a patient in the treatment or prevention of any disease state or condition previously mentioned. An effective amount of an herbal extract as otherwise described herein is administered to a patient exhibiting symptoms of a disease state or condition as otherwise described herein in order to treat the symptoms of the disease states and/or conditi ons and reduce or eli minate the likelihood that the disease state or condition will deteriorate.

Pharmaceutical compositions according to the present invention comprise an effective amount of one or more of the extracts in liquid, semi-liquid or solid form, otherwise described herein, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient, and foxther optionally i combination with at least one additional agent useful in treating a disease state or condition which is related to or .modulated through androgen receptor (AR) protein. In this aspect of the invention, multiple compounds may be advantageously formulated to be coadministered for the prophylactic and/or therapeutic treatment of any one or more of the disease states or conditions described hereinabove.

The individual components of such combinations as described above may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. When one or more of the extracts according to the present invention is used in combination with a second therapeutic agent active the dose of each may be either the same as or differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. Essence of the Invention General Method

Eleven (1.1) herbs, including Aloe barbadensis (Aloe Vera) (ll lf .FS), Rheum patmatum L. (λ'; ^' ;Β6), Step ama tetrandra (¾!¾J5-"C4), Phelkxiendron chinense Schneid. (¾tt:D8), Euphorbia hu ifusa S6), EcHpta prostrata (¾^¾:I2) _f A. venetum L.

( i ^' -fiJ .ftrFl), Portulaca oleracea L, (.S¾@¾;F5), Sanguisorba officinalis L, (¾#tf:E5), Camellia sinensis var.assamka (Mast.) Kitaraura (i |:PE) and Punka granatuni.

(¾ ^' :PG were found to have in ibitor : effect o androgen receptor and could be used for the prevention or treatment of prostate hyperpiasia or prostate cancer as well as diseases caused by hyperactivity of androgen receptor.

These 11 herbs, either individually or in any combination could be used for the treatment of both A -positlve and AR-negative prostate cancers as well as other type of cancers.

Among the eleven (1 1 ) herbs, A ioe-emodin of Aloe barbadensis and gallic acid of Euphorbia huraifusa were found to play key role in inhibiting androgen receptor activity. Herbs or herbal formulations contain aloe-emodin and gallic acid (Table 3, beloxv) will have poten tial for the preventioo or treatment of prostate hyperplasia or prostate cancer as well as diseases caused by over activity of androgen receptor. In addition to aloe-emodin and gallic- acid, epigallocateehra (EGC), gal!ocatechin (GC), quercetin, keampferoi, epigalSocatechio gallate (EGCG) or the polyphenol fraction of Camellia assamica also exhibit inhibitory effect on androgen receptor activity and each alone or i any combination may be used to reduce the likelihood, inhibit and/or treat prostate hyperplasia, prostate cancer or other diseases caused by over activity of androgen receptor.

Table 3. List of ga!iic add containing herbs.

PSatycarya stxobilacea Sieb. et Zmc,

Sapium sebiferum (L)Roxb..

Puuiea graaaiom L,

Acaciagmbica WiUd.

mm m Rhus eoriaria Linn.

Rosa cMneasis cq.

puer tea

Mr Gaiia Chinerssis

Stcd in Aizoom L.

arnur maple

iit!l f Phylia hus erabiica L.

Angelica sinensis

m Paeoma iacfiflora Pali. P.veitchii Lynch

Oldenlandia cantonensis How grape seed

Loquat

Paeonia!aciiflora

Piiheceliobium eiypearia Benth.

hyperictim aseyron

Frucfus Chebuiae

«-b tsing!ing bergenia rhizome

-fi hull of water chestnut

m®. Querctts moagolicus

Geranium wi!fordii Maxim

«« Herba Ardisiae iaponicae

goava leaf

¾w Saxifrage

Ciioerospofidias axi Haris

Qing ^' iorigyi

««»]· Juglans mandshwrica leaves

Wild Rose Hip

aropelopsis j aponica

E ryaie ferox Salisb.

- broadieaf paris

msM Euphorbia firucalh L,

Chinese mugwort leaf

« Polygonum bistorta L.

t* green tea.

J ¾ Garden Burse

«¥ Polygonum capttatum

Phyllaoth s armaria L,

^: Trapa manshurica

Melastoma dodecandrom lour.

Calyx kaki

Humifose Euphorbia

Canariura album Raeusch.

Geranium strietipe J Geranium. earoSinianurn L.

an Red Common Stonecrop Herb

{ Copperieaf

[ Chinese gall Beihua

[ Bau mia aurea

Epigallocatechin, galiocatechin, quercettn and keampferol of Fl .showed inhibition cm DFJT induced androgen receptor activity of 22Rv l cells. These chemicals could be used for the prevention or treatment of prostate hyperplasia or prostate cancer as well as diseases caused by over activity of androgen receptor.

Results

Based on a comprehensive database (STAR) evolved from our own research - studying the effects of herbal water extracts from over 250 formulations, across 25 signaling pathways, using primary iuciferase reporter assay and other enzymatic reactions. The

inventors selected 9 herbs: Aloe vera (j:¾ :F3), Rheum palmatum L. (;> ^! vff :B6), Stephanie tetrandra(¾.i¾ ^' G:C4), Phellodendron chinense Schneid.(. l¾:D8), Euphorbia

oteraeea L.(¾ ¾M-'F5), Sangisisorha officinalis L. (j¾Jtf.E5) which shown ami -androgen receptor (AR) activity in the ueiferase receptor assay (Figure I), hi addition, Camellia

assamica (Mast) Chang (puer tea, PE) and Punica granatum Eff^PG,

pomegranate) were shown to have inhibitory activity on androgen receptor mediated

transcription activity of 22RV.1 AR-luciferase reporter cells in condition wit DHT (Figure 2 and 31 ). To further validat their effect on androgen receptor target genes (PSA, L 2) mRNA expression. All these herbs could inhibit DHT induced PSA and KL 2 mRNA

expression (Figure 3 and 31 ).

Different herbs may .have different, mechanism of actions against AR signaling. F3 and

F5 could dow regulate both AR protein and mRNA (Figure 4 and 5). B6, C4 and D8 could only down regulate AR protein but had weak effect on AR mRNA (Figure 4 and S), S6, 12,

FI, E5 and PE had no effect on AR protein and mRNA (Figure 4 and 5) and they may

directly act on the AR. A DNA pull down assa was used to access if herbal water can inhibit the interaction between Androgen receptor and DNA. Androgen receptor -DMA interaction pull down assay- was performed by mixing biotin label DNA 5 '-gtaattgcAGA ACAgcaA.GTGCTagctctc-3 ' (with androgen binding site) and nuclear ly sis with androgen receptor protein (extracted from 22RV1. cells pre-stiniulated with DHT 25n for overnight). Streptavidin-Dynabead was used to trap down the DNA-AR complex under magnetic condition. Western blotting wa -used to detect the amount of pull down AR protein. Inhibition between DNA and androgen receptor binding will reduce the amount of AR protein pull down. As shown in Figure 6, F3 and C4 did not inhibit DNA-AR protein interaction while FL S6, E5, F5, 12, PE1 could inhibit DNA- AR protein interaction with different potency. Possible mechanism actions of these herbs are listed on table 1.

9 herbs showed cytotoxic effect on two prostate cancer cells: 1. 22RV1 cells which express expresses the full-length AR. and the constitutively active, truncated AR, which is responsible for bicatutamide and enzaiutamide resistance. 22RV1 cell growth is partially dependent on androgen and its growth can be reduced by down regulating both AR isofortns. 2. Dal 45 cells which do not express AR protein, and its growth is androgen independent. As shown in Table 2, F3, B6, C4, S6, 12, Fl, show stronger effect on inhibiting the growth of 22RV1 cells than DuI45 cells . These herbs may have advantage for targeting prostate cancer cells with AR protein expression. However. DS and E5 were more toxic towards Dul45 cells than 22R.VI. ^' This suggests that D8 and E5 may have other target sites, which is independent on AR, for inhibiting the growth of Dnl45. Overall, all these herbs may have potential for treatment of prostate cancer with or without expressing A protein.

As shown in Figure 7, water extract of ES (Ig/kg BID), Fl(!g/1 g BI D), F3{ Ig/kg BID), 12(1 g/kg BID), S6 (!g/kg BID) and Gallic acid (23nig/kg BID; equivalent dose to S6 I /kg: BID) were feed orally to mouse implanted with 22RVl(AR+ve) and DU ! 45(AR-ve) xenografts. FL F3 _S 32, S6 showed significant inhibition on the DU 145 tumor growth while E5, Fl, F3, S6, showed significant inhibition on the 22RV1 tumor growth (Figure 7). B5 may have higher selectively to inhibit prostate cancer with AR protein in vivo. 12 may have higher selectively to inhibit prostate cancer without AR protein expression in vivo. hi another animal experiment, we compared the anti-prostate tumor growth effect of S6, F5, and PE1 (each SOOmg/kg bid) to eazaiotaraide { !On g kg qd) (targeting on AR) on LNCaF (androgen dependent) xenograft tumor and Du145 xenograft tumor in nude mice for two weeks. As predicted tirai enzalutamide effectivel inhibited the growth of LNCaF tumors bin not Dai 45 tumors (Fig 8 A~F), S6, F5 and PE1 showed significant inhibition on the growth of LNCaP (P<0.05) but S6, F5 and PE! have no significant inhibition on the growth of Dal 45 tumors (Fig 8.A-F). q T-PCR results further supported that S6, F5, FBI could down regulate the AR target genes, KL 2 or PSA of LNCaP tumor (Fig 8G, 8 B), In

conclusion, S6, F5, PE1 appears to have selectivity on inhibiting the growth of androgen dependent tumor thought targeting AR.

Further experiments were conducted pursuant to the present invention. The resul ts of these experiments are presented in Figures 20-30 and described herein.

As shown in Figure 20, preparative HPLC was conducted to fractiona!ize S6 water extract. To that end, ! Oul of S6 ^" water extract was subjected to HPLC with a CIS preparative column running with an increasing gradient of acetonitrile. Fraction 9 (star) from the preparative HPLC fractions evidenced the strongest anti-androgen receptor activity in AR luciferase report assay. Fraction 9 was coilected and then subjected to LC-MS analysis using negative mode scanning from MW160 to MW ^' 800. Fraction 9 (red) contained gallic acid (MW170), The small insert panel depicted in Figure 21 evidenced that pure gallic acid was active (IC50 was about 28uM) against AR activity of 22RY 3 ceils in the luciferase report assay.

Several tea extracts were tested for cytotoxicit against 22RV1 and Dul45 cells. The results are presented in Figure 22. In particular, the cytotoxicity of each of Fl (Apocynura venetum) and the teas labelled as Chinese "Luo-hu-ma" (LBM tea) (which is sometimes Apocynum venetum or "Apocynum pictum" in China) and LBM mix tea which contains the leaf, flower and green tea was tested. All of these teas exhibited cytotoxicity on 22RV 1 cells and Dul45 ceils (which don't express androgen receptor) as shown, Fi showed stronger cytotoxicity tha LBM tea or LBM mix. tea. Methylene blue was used to stain the total ceils following 3 -day treatment with the water extracts.

Figure 23 shows a comparison of polyphenol extracts (PE), PE-powder from Yunnan taslydee pure biological tea group co.ltd. or PE-1 , PE~2, PE-B from Gaoligongshan Co. Ltd was tested for their anti-androgen receptor activity using 22RV 1 luciferase reporter cell assay. The results are shown in Figure 23, with all extracts exhibiting activity and PE-1. exhibiting the strongest activity.

A preparative HPLC was conducted to fractionaiize a further PEI water extract. I mi of S6 (iOOmg/rnl) was subjected to HPLC using a CI S preparative column miming with an increasing gradient of acetooitri!e. Fractions 13, 21. 32 (star) from the preparative HPLC fractions bad relative stronger anti-androgen receptor activity in AR hjciferase report assay. These fractions were subjected to further analysis. This is show in Figure 24. HPLC analysis of fraction 13 (red) of PEI from preparative HPLC and PEI crude water extract (Figure 25) showed that S-Qalloylquinic acid could be found in identifiable quantities in fractio 13, evidencing that this compound was most likely responsible for the activity exhibited by the fraction, HPLC analysis for f action 21 (red) of PEI from the preparative HPLC of PEI crude water extract showed that 1 -Ga!loyl-beta-glucose could be found in identifiable quantities in fraction 21 and was likely responsible for the activity of fraction 21. See Figure 26. HPLC analysis of f action 31 (red) of the PEI water extract from the preparative HPLC evidenced identifiable quantities of epigal!ocatechm (EGC) as the likely active component. This is shown in Figure 27. HPLC analysis of fraction 33 (red) of PEI from the preparative HPLC also evidenced identifiable quantities of EGC as the likely active component. This is show in Figure 28. HPLC analysis of fraction 43 (red) and 44 (green) of PEI extract from preparative HPLC showed that fraction 43 had identifiable quantities of EGC and fraction 44 had identifiable quantities of epigaUocatechia gailate (EGCG). Results indicated that fraction 44 contained EGCG in identifiable quantities. The fraction also appeared to contain kaempferol-3-0~R-L-rhaninopyranosyi (lf6)~p-D~gafactopyraiioside, quercetin~3~0~p~D- g!ucopyranoside or gallic acid-3-O-(60-O-gailoyl)-p-D-glucoside in appreciable quantities. This is indicated in Figure 29,

Various extracts according to the present invention were tested for cytotoxicity of different on different cancer cell lines, for example. 22RVT, HepG2, KB, B- MDR(rnultidrug resistant). KB-300(CPT1 resistant) as indicated in Figure 30. The average ICs _f j from three independent experiments is presented in the figure. methylene blue assay was used to determine the cell growth following tire treatment of different herb water extracts for 3 days. Potential active chemicals)

Aloe barbadensis (F3):Prepa.raiive 11PLC was. used to firae lionize water ex tract of F3. As shown in figure 9 A and 9B, fraction 31 and 32 showed similar inhibitory effect on. AR action. LC-MS result indicated that fraction 31 and 32 contained similar amount of Aloe- eniodln MW271 and compound with MW6S6.1 which could be Elgomea-dimet. Standard Aloe-emodm was purchased and was confirmed to have anti-AR activity using !iiciferase reporter assay (Figure 10). The equivalent amount of aloe-emodin was compared to crude water extract of F3 using lucif erase report assay (Figure 10, insert figure). Result indicated that aloe-emodin is key ingredient of F3 for inhibiting AR activity. However; Aloe-emodm are relative weaker titan the whole herbal mixture of F3, This suggests that other chemicals in F3 may be also active against AR.

Different similar structure to aloe-emodin were selected to tested for thei inhibitory effect on AR using Inciferase reporter assay. Result indicated that in addition to aloe-emodin, emodin and sennoside A, but no chrysophanol, sennoside C or sennoside D, also activity in against AR (Figure 11)

Euphorbia humifusa .($6): Solid phase extraction column was used to fractionate S6 water extract. Water and 10% ethanol elution had relative stronger inhibitory effect on AR using luciierase reporter assay (Figure 12 insert). LC-MS detected gallic acid in water eiutton and 10% ethanol elation. Equi valent dose of gallic acid (as compare to S6) were shown to have inhibitory effect of androgen receptor mediated transcriptional activity (Figure 1.3 A). Gallic acid was shown to hav inhibitory effect on KLK2 and PSA but no AR (Figure OB, 13C, 13D). These result indicated that they are key ingredient of S6 for inhibiting AR activity. However, gallic acid are relative weaker than the whole herbal mixture of S6. This suggests that other chemicals in S6 may be also active against A or other chemicals may enhance the action of gallic acid against AR. Furthermore , gallic acid exhibit in vi vo anti-tumor activity in against the growth of 22RV1 and Disl45 tumor in nude mice (Figure 13E and 13F).

A, venetttm L« (! ftn&FI ): Figure 14, F.1 (Apocynum venetum) and other tea label with Chinese tto~bu-ma:LBM:" which could be. Apocynum venetum or "Apocynum pictum" in China were compared for their anti-A activity using Inctferase reporter assay.. Some LBM tea may also contain its leaf, its flower and green tea. All these teas had anti-androgen receptor activity, but PI had higher ant -androgen receptor than the others (LBM, LBM mix tea and LBM mix tea-Nile). FL LBM and LBM tea mix showed inhibition on KLK2 and PSA mRNA expression (Figure 14B, 14C).

Solid phase extraction CIS column was used to fractionate Fl water extract (Figure I SA). 10%, 20%, 40% ethanol elution were found to have relative high anti-AR activity using lueiferase reporter assay. LC-MS detect Catechin/Epicatecliin, Epigallocatechin or gal!ocatechra, caffiec, Chiorogenic acid. Isoquercetin. Hyperoside. Astragalk, Trifo!in, Aeetylated hyperoside Or Acetylated Isoquercetin, qaereetin, eampierol io those fractions (Figure 158). EpigaSlocatechm, gallocateehin, quercetin and Keampferol were shown to have different potency on inhibiting A using lueiferase reporter assay (Figure 1 SC).

Camellia assaniica (Mast) Chang (puer tea, ¾¾^: PE)

Polyphenols (PP) (extract from PE) obtained from obtained from Gaoligongshan eo.ltd showed anti -androgen receptor using 22RV1 lueiferase reporter cell assay (Figure 16 A). When PEI or PP water extract passing though C18 solid phase extraction column, 30% elution (arrow) showed the most potent anti-androgen receptor activity us using 22RVI lueiferase reporter cell assay (Fig 16B ₅ 16C).

Since the potency of PEi and PP is about 5: 1 , 1ml of lOOmg/ml PEI or lml of 20mg ml PP were chosen as the input for solide phase extraction. LC_ S showed that 30% ethanol elution of PEi or PP, some mass peaks (black) showed very similar intensity and. some peaks (highlight as green) bad different intensity. The common peaks should be more related to the anti-androgen receptor activity (Figure 17) . But we don't exclude if the green highlighted compounds are also active.

Five pure compunds, epicatechin, catecMn, gallocateehin (GC), Epigallocatechin (EGC) and epigallocatechin galSate EGCG which exist in F I were tested for their anti- androgen receptor activity in present of DHT !OnM using lueiferase reporter assay. GC, EGC, EGCG were found to have anti-androgen receptor activity. EGC showed highest potency against AR (Figure 18). When LC-MS scanning parameter was set to have low molecular weight scan. Gallic acid could be detected in PE (Figure 1 ). Fo PE tea 1C50 125ug ml contains about 4.8oM Gallic acid which may partially play a role in inhibiting AR. Pumca grawatum or common name pomegranate (¾¾¾ :PG) extract was found to have inhibitory effect on DHT induced androgen receptor activity of 22RV1 cells (Figure 31. A). PG also inhibited DHT induced KLK2 and PSA mRNA expression of 22RVI cells (Figure 31 B and C). PG reduced the basal level of androgen receptor protein and DHT induced androgen receptor protein of 22EV I cells (Figure 3 ID). In addition, PG showed inhibitio on AR mRN A expression (Figure 31 E). a. Novelty and major advantages:

L The 1 1 herbs including Aloe harhadensis Rheum palmatum

L. (Λ ft:B6), Stephama tetrandra (¾ |¾ fi :C4), Phellodendron chinense Schneid.

(H¾:D8), Euphorbia humifusa (¾IS¾S6), Eclipta prostrata (11^11:12}, A. venetum L. (S¾1#:F1), Portulaca oleracea L, (^¾¾¾:F5), Sanguisorba officinalis L. (¾H:E5) _r or Cameliia sinensis var.assamica (Mast) Kitamura (¾f$ :PE)- Pu ica granatura (¾ ^" ϋ:ΡΟ) and/or extracts of these herbs and/or their acti ve components (, which can suppress AR. receptor activity, can be used alone or in combination for the prevention or treatment of prostate hyperplasi or prostate cancer as well as diseases caused by hyperactivity of androgen receptor.

2, Herbs or the formulation containing those herbs listed above in combination with other chemicals such as Aioe-emodin, Gallic acid, Epigallocatechin, gallocatechin, epigaliocatechin gallate, quercetin and Keampferol which could be useful for targeting disease or symptoms caused by hyperactivity of androgen receptor.

3, Some of those herbs can be developed as h igh -end food supplements with scientific evidence for prostate cancer prevention. For example, Fl is commonly used as an herbal tea for controlling the blood pressure in China. F5 is commonly used as vegetable in many places including Europe, the middle east, Asia, and Mexico. PE is a tea which commonly be used. These herbs are considered safe to consume.

4, Aioe-emodin, Gallic acid, Epigaliocatechin, gallocatechm, epigaliocatechin gaSlate, quercetin and Keampferoi containing herbs or vegetable (table 3) could be developed for the prevention or treatment of prostate hyperplasia or prostate cancer as well as diseases caused by overactivity of androgen receptor. 5. Since these ^' herbs can inhibit the growth of 22RV1 cells which resistance to hicalutamide and eixzalutaniide, these herbs or their acti ve ingredient can be developed to target bicalutamide and enzaiutamtde resistant prostate cancer.

Experimen tal Proced u res

The goal of this study by the present inventors was to investigate wha effects, if any, herbal medicines have on androgen receptor activity, then to delve further to isolate the specific effects these herbs have on the androgen receptor activity and identify active compounds in these herbs responsible for that activity. This was done in three parts, the first of whic h was an initial dose-response screening of about 250 herbal medicines for androgen receptor activit using luciferase reporter cells (22RV1 cells transfected with PSA promoter luciferase reporter). RT- PCR for KLK2 and PSA. which are target genes of androgen reporter, was used to confirm the action of those herbal extracts.

Materials and Methods

PSA luciferase reporter cells - 22RV1 prostate cancer cells were used in the screening study. 22RVI cell lines were stably transfected with PSA promoter-PGL4,2 luciferase reporter. 25nM DHT was used to induce androgen receptor for 24k

Luciferase Screening -22RV1 androgen reporter cells were treated with herbal extracts a 30, 100 300, and 1000 pg/ml for 24h. in a 37°C-CQ2 incubator with or without 25nM DHT which was used androgen receptor activity. Cells were Sysed using luciferas lysis buffer after which Luciferase buffer with luciferin was added to generate luminescence. Luminescence was recorded using a luminescence microplate reader.

Real Time QuantiUuive PCR (RT-qPCR) ofNRF2 and downstream genes RNA was extracted from herb treated cells using the Roche High Pure RNA isolation kit. cDNA was then generated from NA samples using Bio-rad iScript Advanced cDNA synthesis kit for RT-qPCR. qPCR was performed using human MRF2, HOI, NQGl and j¾- ctio primer (as shown in the table below) and iTaq™ Universal SYBR<S> Green Supermix in CFX PCR machine (Bio-rad). Relative mRNA expression was calculated based on the change of the threshold cycle relati ve to the internal control, β-actm, using a standard curve generated by purified PC products.

Table 4: Primer Sequences for T-qPCR

Western Blot Protocol - Total cell lysis was prepared using 2x SDS sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM DTT, and 0.05% brompherioi blue) and sonicated for 10s to shear DNA. Cell nuclei were isolated using Tris buffer saline with 0.4% NP40, Cell extracts were then eleetrophoresed through 10% SDS-polyacry!amide gels and transferred to 0.2um niuoce ^' llulose membranes (Bio-Rad Laboratories, Hercules, CA) with a Mini protein II ixansferring apparatus (Bio-Rad). The membranes were blocked and probed in TBS-T buffer (1.x TBS buffer, 0.2% Tween 20) containing 5% non-fat milk. Monoclonal rabbit aiiti~AR (1 :5000) _; was used to detect androgen recptor (Abeam #133273) and a. monoclonal actio antibody dilated 1 :2500 (Sigma, St. Louis, MO) was used to detect β-aetm as the iniemai control to confirm equal protein loading. The membranes were then incubated with horseradish peroxidase-conjugated anti-moose IgG and anti-rabbit IgG (1 : 5,000;

Sigma). Enhanced chemi!iiminescence reagents (Perkin-Elmer Life Science Products, Boston, MA) were used to visualize the immunoreactive bands and the densities of protein bands were scanned using and analyzed using ImageJ software from the NIIL Standardized preparation of herbal extracts

An often expressed concern of holistic medicinal treatments is a lack of

standardization when preparing herbal reagents for testing, in order to maintain consistency between preparations for direct quantitative comparisons in functional assays, a standard aqneoas extraction protocol was designed. Extracts were prepared by heating dried, powdered herbs (0.5 g) in ultra-pure water (10 ml, > 5 megaO resistance) at 85*C for 30 minutes. The aqueous fraction was then filter sterilized and stored at 4°C in the dark until use. Adherence to this standard protocol not only permits accurate quantitative comparisons between herbal extracts, but also affords the opportunity to compare different harvests of the same teas to account for seasonal variations. In addition, after freeze-drying and

reconstituting the most active tea, described, later, consistent values were obtained in bioactivity screens, suggesting that the active ingredients in this tea are stable and that this method is a reliable method for standardizing teas.