Herbal formulations

Abstract

A formulation comprising a variety of Chinese herbs is disclosed for the treatment of cancer and side effects associated with the treatment of cancer.

Claims

1. A method for reducing recurrence of bladder cancer, the method comprising administering to a subject in need thereof a herbal formulation comprising Poriae Cocos, Radix Paeoniae Lactiflorae, Ligustrum Lucidum, Ophiopogon Japonicus, Scutellaria Barbata, Taraxaci Mongolici Cum Radice Herba and Prunella Vulgaris.

2. A method for reducing recurrence of bladder cancer, the method comprising administering to a subject in need thereof a herbal formulation comprising Astragalus Membranaceus, Poriae Cocos, Radix Paeoniae Lactiflorae, Ligustrum Lucidum, Ophiopogon Japonicus, Oldenlandia Diffusa, Scutellaria Barbata, Taraxaci Mongolici Cum Radice Herba and Prunella Vulgaris.

3. The method according to claim 2, wherein the formulation further comprising at least one of Glehnia Littoralis, Citrus Reticulata, Radix Et Caulis Jixueteng, Atractylodes Macrocephala, Paeonia Obovata and Lycium Chinense.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

(2) FIG. 1 is a bar graph presenting the analysis of quality of life of the combined treatment according to the present embodiments.

(3) FIG. 2 is a bar representation showing the number of studied patients receiving growth factor drugs to enhance white blood or red blood count.

(4) FIG. 3 is a bar representation showing the number of studied patients in which a delay in chemotherapy was affected.

(5) FIG. 4 is a bar representation demonstrating the effect of a formulation of the invention on MDA-231 cells survival.

(6) FIG. 5 is a bar representation demonstrating the effect of a formulation of the invention on MDA-453 cells survival.

(7) FIG. 6 is a bar representation demonstrating the effect of a formulation of the invention on T47D cells survival.

(8) FIGS. 7A-C are light microscopy images depicting the effect of the formulation of the invention on breast cancer adenocarcinoma cell line (T47D). FIG. 7A shows an image of a control sample. FIG. 7B shows an image of cells treated with 3 mg/ml of a formulation of the invention; and FIG. 7C shows an image of cells treated with 50 mg/ml of a formulation of the invention, and

(9) FIGS. 8A-C are flow cytometry analysis of apoptosis of T47D carcinoma cell line: FIG. 8A shows analysis of a control sample; FIG. 8B shows an analysis of cells treated with 3 mg/ml of a formulation of the invention; and FIG. 8C shows the results of the analysis of cells treated with 50 mg/ml of a formulation of the invention.

DETAILED DESCRIPTION OF EMBODIMENTS

(10) The present invention, in most general terms, relates to uniquely designed herbal formulations and, more particularly, but not exclusively, to herbal formulations that are suitable as complementary therapy for cancer patients.

(11) It is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples, as these are provided only as non-limiting examples of the broad aspects of the invention. The invention is capable of other embodiments or of being practiced or carried out in various ways.

(12) As demonstrated herein, the formulations of the invention have been found to be highly efficacious when used in combination with chemotherapy, in reducing multiple side effects induced by the chemotherapy and in shielding the production of red blood cells, white blood cells and neutrophils from the toxic effects of chemotherapy.

(13) Chemotherapy-induced nausea and vomiting (CINV) is a common and serious problem encountered by many patients receiving anti-cancer therapy. Up to three fourths of all cancer patients experience chemotherapy-related emesis. CINV complicates or prevents administration of planned therapy and decreases quality of life. Although new agents, such as palonosetron and aprepitant, have improved outcomes, CINV is still a major concern for many patients. In older studies patients receiving chemotherapy reported nausea as the most severe symptom and vomiting as the fifth. Patients who do not receive preventive therapy have a 70% to 90% incidence of delayed emesis with high-risk agents (e.g., Adriamycine and Cyclophosphamide) and a 30% to 60% risk with moderate-risk agents (Taxol).

(14) In the studies disclosed herein, on subjects being treated according to methodologies of the invention, 90% of the patients suffered minor or no vomiting at all. Minor or no nausea has been noted in a very small number of patients, needing no supplementary conventional medication, except the dose in the treatment days.

(15) Febrile neutropenia (FN) is a severe clinical outcome that justifies the use of colony stimulating factor (CSFs) when the risk of FN is approximately 20% and no other equally effective regimen that does not require CSFs is available. Current evidence indicates that primary prophylaxis with a CSF results in a relative risk reduction of FN by approximately 50%. Significant reductions in documented infections have also been demonstrated. An analysis of five trials that have reported infection-related mortality failed to find a significant reduction with CSF support [summary odds ratio, 0.60 (0.30, 1.22); P=0.16]. However, the power of the combined analysis was insufficient to determine this outcome. An additional outcome demonstrated in these trials was the maintenance of dose intensity of the chemotherapeutic regimen through avoidance of dose reduction or delay secondary to neutropenia. The study reported significant reductions in the risk of FN from 37% to 20% (14 studies, n=3,091, relative risk reduction 46%, P<0.0001) and the risk of infection-related mortality from 3.3% to 1.7% (10 studies, n=2,468, relative risk reduction 48%, P=0.01).

(16) In studies described herein, employing methodologies of the invention, 80% of the patients treated with a formulation of the invention did not need to take growth factor drugs to enhance white blood or red blood count. In the majority of the cases blood production was maintained substantially stable throughout the chemotherapy.

(17) Cancer treatments may interfere with the patient's ability to taste, ingest, swallow or digest food. Drugs may cause nausea, diarrhea, and anorexia. Although many new agents have been developed to combat these side effects, patients still suffer greatly during treatment. One of the major side effects presenting a significant challenge to the cancer patient is alteration of taste, or dysgeusia.

(18) Chemotherapeutic drugs are secreted or excreted in tears, saliva, sweat, bile, or urine. Drugs in the saliva may markedly alter taste (dysgeusia), leading to food revulsion and avoidance. After early satiety, dysgeusia is the most common challenge in patients on chemotherapy.

(19) The two main types of dysgeusia are loss of taste acuity and distortion of taste. Loss of taste acuity comes from drug inhibition of a taste receptor function. Taste distortion results from drugs activating the taste receptor in an abnormally persistent fashion or preventing activation of the receptor

(20) The treatment of dysgeusia is typically very difficult. Treatment with agents such as zinc, folic acid, -lipoic acid, and vitamins of the B class may alleviate some of the metallic taste but are only mildly helpful. Zinc seems to work best with a sweet dysgeusia. Drugs such as antracyclines and taxanes may have the worst associated dysgeusia. These seem to be secreted in the saliva and after the first bite or chew often produce a horrible taste sensation. After drug cessation, the taste returns to normal over a period of 2 months.

(21) In studies described herein, employing methodologies of the invention, 85% of patients reported no or minor decrease in appetite and no patient reported a digestive dysfunction or dysgeusia.

(22) Fatigue was reported in 20% of 430 assessable patients receiving adjuvant chemotherapy (202 standard-doses, 228 high-doses) with at least a 3-year follow-up, without change over time or difference between treatment arms. Mean Hb levels were lower following high-dose chemotherapy. Only 5% of patients experienced fatigue and anemia. Mental health score was the strongest fatigue predictor at all assessment moments. Menopausal status had no effect on fatigue. Linear mixed effect models showed that the higher the Hb level (P=0.0006) and mental health score (P<0.0001), the less fatigue was experienced. Joint (P<0.0001) and muscle pain (P=0.0283) were associated with more fatigue.

(23) In conclusion of this study, within the patients being treated with a formulation of the invention, no patient reported severe fatigue.

(24) In conclusion, the results demonstrate that the herbal formulation treatment according to the present invention is safe and serves as an effective adjunctive therapy to ameliorate multiple side effects of chemotherapy and to enhance the immune system in cancer patients.

(25) An exemplary formula comprises the following herbal powder extracts: Astragalus Membranaceus (Huang Qi), Poriae Cocos (Fu Ling), Atractylodes Macrocephala (Bai zhu), Lycium Chinense (Gou Qi Zi), Ligustrum Lucidum (Niu Zhen Zhi), Paeonia Lactiflora (Bai Shao), Paeonia Obovata (Chi Shao), Citrus Reticulata (Chen Pi), Ophiopogon Japonicus (Mai men Dong), Milletia Reticulata (Ji Xue Teng), Oldenlandia Diffusa (Bai Hua She She cao), Scutellaria Barbata (Ban Zhi Lian), Prunella Vulgaris (Xia Ku Cao), Glehnia Littoralis (Bei Sha Shen), as described herein.

(26) Additional herbs that can be added into the formula include, for example, Radix Pseudostellariae Heterophyllae (Tai Zhi Shen), Radix Glycyrrhizae (Gan Cao), Massa Fermentata (Shen Qu) and Fructus Hordei Germinatus (Mai Ya).

(27) Reference is now made to the following examples, which together with the above description illustrate some embodiments of the invention in a non limiting fashion.

(28) Formulations of the Invention:

(29) The herbs used were encapsulated dried herbal extract powders. The herbal formula was prescribed in the dosage of 6 grams (2 grams3 times a day).

(30) The extracted herbal powders where imported under license in accordance with the regulations of the Israel Ministry of Health by ZEN Herb Company (Tel Aviv, Israel) and manufactured under GMP conditions. The placebo capsules where provided by the same company resembling the CHT capsules in taste, texture and appearance.

(31) A variety of formulations have been prepared and used. The following is a description of a group of formulations prepared according to the invention.

(32) TABLE-US-00001 TABLE 1 Formulation 1. Latin name Chinese name grms Wt % 1 Astragalus Membranaceus Huang QI 10 8.1 2 Poriae Cocos Fu Ling 6 4.9 3 Atractylodes Macrocephala Bai zhu 6 4.9 4 Lycium Chinense Gou Qi Zi 8 6.5 5 Ligustrum Lucidum Niu Zhen Zhi 10 8.1 6 Paeonia Lactiflora Bai Shao 8 6.5 7 Paeonia Obovata Chi Shao 6 4.9 8 Glehnia Littoralis Bei Sha Shen 8 6.5 9 Citrus Reticulata Chen Pi 6 4.9 10 Ophiopogon Japonicus Mai men Dong 8 6.5 11 Milletia Reticulata Ji Xue Teng 10 8.1 12 Oldenlandia Diffusa Bai Hua She She cao 15 12.2 13 Scutellaria Barbata Ban Zhi Lian 12 9.8 14 Prunella Vulgaris Xia Ku Cao 10 8.1

(33) As regards the formulations of the invention, additional herbs were used for particular individual symptoms; these include one or more of the following exemplary herbs: Radix Pseudostellariae Heterophyllae (Tai Zhi Shen), Radix Glycyrrhizae (Gan Cao), Massa Fermentata (Shen Qu) and Fructus Hordei Germinatus (Mai Ya), utilized in case of digestive disorders. Any other herb mentioned herein with reference to any one particular formulation may also be added to a formulation not indicated to comprise same.

(34) TABLE-US-00002 TABLE 2 Formulation 2. Latin name Chinese name Grms Wt % 1 Astragalus Membranaceus Huang Qi 20 12.4 2 Atractylodes Macrocephala Bai zhu 10 6.2 3 Redix Glycyrrhizae Uralensis Gan Cao 4 2.5 4 Poriae Cocos Fu Ling 8 5.0 5 Lycium Chinense Gou Qi Zi 12 7.5 6 Radix Paeoniae Lactiflorae Bai Shao 10 6.2 7 Paeonia Obovata Chi Shao 8 5.0 8 Curcumae, Tuber Yu Jin 10 6.2 9 Citrus Reticulata Chen Pi 6 3.7 10 Ligustrum Lucidum Niu Zhen Zhi 15 9.3 11 Oldenlandia Diffusa Bai Hua She She cao 15 9.3 12 Scutellaria Barbata Ban Zhi Lian 20 12.4 13 Prunella Vulgaris Xia Ku Cao 15 9.3 14 Anemarrhenae Asphodeloidis Zi Mu 8 5.0 Rhizoma

(35) TABLE-US-00003 TABLE 3 Formulation 3. Latin name Chinese name Grms Wt % 1 Atractylodes Macrocephala Bai zhu 10 5.8 2 Pseudostellaiae Heterophillae Tai Zi Shen 15 8.7 3 Poriae Cocos Fu Ling 12 7.0 4 Radix Paeoniae Lactiflorae Bai Shao 12 7.0 5 Radix Angelica Sinensis Dang Gui 15 8.7 6 Paeonia Obovata Chi Shao 8 4.7 7 Radix Et Caulis Jixueteng Ji Xue Tang 12 7.0 8 Cyperi Rotundi Rhizoma Xiang Fu 8 4.7 9 Ligustrum Lucidum Niu Zhen Zhi 10 5.8 10 Ophiopogon Japonicus Mai men Dong 10 5.8 11 Scutellaria Barbata Ban Zhi Lian 20 11.6 12 Radix Isatidis Seu Baphicacanthi Ban Lan Gen 15 8.7 13 Prunella Vulgaris Xia Ku Cao 15 8.7 14 Anemarrhenae Asphodeloidis Zi Mu 10 5.8 Rhizoma

(36) TABLE-US-00004 TABLE 4 Formulation 4. Latin name Chinese name Grms Wt % 1 Atractylodes Macrocephala Bai zhu 8 4.7 2 Ginseng Radix Ren Shen 20 11.8 3 Poriae Cocos Fu Ling 10 5.9 4 Lycium Chinense Gou Qi Zi 12 7.1 5 Radix Angelica Sinensis Dang Gui 15 8.9 6 Paeonia Obovata Chi Shao 8 4.7 7 Radix Salviae Miltiorrhizae Dan Shen 12 7.1 8 Radix Et Caulis Jixueteng Ji Xue Tang 10 5.9 9 Citrus Reticulata Chen Pi 6 3.6 10 Glehnia Littoralis Bei Sha Shen 8 4.7 11 Panacis Quinquefolii, Radix Xi Yang Shen 10 5.9 12 Flos Lonicerae Japonicae Jin Yin Hua 12 7.1 13 Pseudobulbus Shancigu Shan Ci Gu 18 10.7 14 Prunella Vulgaris Xia Ku Cao 20 11.8

(37) TABLE-US-00005 TABLE 5 Formulation 5. Latin name Chinese name Grms Wt % 1 Atractylodes Macrocephala Bai zhu 6 3.5 2 Radix Codonopsitis Pilosulae Dang Shen 12 6.9 3 Poriae Cocos Fu Ling 6 3.5 4 Lycium Chinense Gou Qi Zi 15 8.7 5 Radix Paeoniae Lactiflorae Bai Shao 12 6.9 6 Radix Salviae Miltiorrhizae Dan Shen 10 5.8 7 Radix Et Caulis Jixueteng Ji Xue Tang 15 8.7 8 Curcumae Tuber Yu Jin 18 10.4 9 Curcuma Zedoaria E-Zhu 15 8.7 10 Aucklandiae Lappae Radix Mu Xiang 12 6.9 11 Ligustrum Lucidum Niu Zhen Zhi 12 6.9 12 Smilacis Glabrae, Rhizoma Tu Fu Ling 15 8.7 13 Pseudobulbus Shancigu Shan Ci Gu 15 8.7 14 Anemarrhenae Asphodeloidis Zi Mu 10 5.8 Rhizoma

(38) TABLE-US-00006 TABLE 6 Formulation 6. Latin name Chinese name Grms Wt % 1 Astragalus Membranaceus Huang Qi 20 11.2 2 Atractylodes Macrocephala Bai zhu 12 6.7 3 Poriae Cocos Fu Ling 8 4.5 4 Radix Angelica Sinensis Dang Gui 12 6.7 5 Radix Salviae Miltiorrhizae Dan Shen 10 5.6 6 Radix Et Caulis Jixueteng Ji Xue Tang 10 5.6 7 Curcumae Tuber Yu Jin 12 6.7 8 Aucklandiae Lappae Radix Mu Xiang 8 4.5 9 Sangjisheng, Ramulus Sang Zi Sheng 10 5.6 10 Panacis Quinquefolii, Radix Xi Yang Shen 15 8.4 11 Radix Isatidis Seu Ban Lan Gen 18 10.1 Baphicacanthi 12 Flos Lonicerae Japonicae Jin Yin Hua 15 8.4 13 Prunella Vulgaris Xia Ku Cao 20 11.2 14 Anemarrhenae Asphodeloidis Zi Mu 8 4.5 Rhizoma

(39) TABLE-US-00007 TABLE 7 Formulation 7. Latin name Chinese name Grms Wt % 1 Pseudostellaiae Heterophillae Tai Zi Shen 15 8.5 2 Herba Lobeliae Chinensis Cum Ban Bian Lian 10 5.7 Radice 3 Rhizoma Alismatis Ze Xie 8 4.5 4 Rhemanniae Glutinosae Shu Di Huang 12 6.8 Conquitae Radix 5 Persicae Semen Tao Ren 12 6.8 6 Curcuma Zedoaria E-Zhu 15 8.5 7 Diospyri Kaki Calyx Xi Di 8 4.5 8 Radix Adenophorae Seu Sha Shen 12 6.8 Glehniae 9 Sophorae Subprostratae Radix Shan Dou Gen 15 8.5 10 Pseudobulbus Shancigu Shan Ci Gu 12 6.8 11 Rhizoma, Phragmitis Communis Lu Gen 15 8.5 12 Gardeniae Jasminoidis Fructus Zhi Zi 12 6.8 13 Nelumbinis Nuciferae Semen Lian Xin 12 6.8 14 Herba Lophatheri Gracilis Dan Zhu Ye 18 10.2

(40) TABLE-US-00008 TABLE 8 Formulation 8. Latin name Chinese name Grms Wt % 1 Radix Codonopsitis Pilosulae Dang Shen 12 6.9 2 Pseudostellaiae Heterophillae Tai Zi Shen 15 8.6 3 Coicis Lachryma-jobi Semen Yi Yi Ren 12 6.9 4 Semen Plantaginis Che Qian Zi 10 5.7 5 Arilus Euphoriae Longanae Long Yang Rao 8 4.6 6 Mori Albae Fructus Sang Shen 18 10.3 7 Paeonia Obovata Chi Shao 8 4.6 8 Persicae Semen Tao Ren 6 3.4 9 Cyperi Rotundi Rhizoma Xiang Fu 8 4.6 10 Plastrum, Testudinis Gui Ban 15 8.6 11 Panacis Quinquefolii, Radix Xi Yang Shen 15 8.6 12 Taraxaci Mongolici Cum Pu Gong Yin 18 10.3 Radice Herba 13 Sophorae Subprostratae Radix Shan Dou Gen 12 6.9 14 Prunella Vulgaris Xia Ku Cao 18 10.3

(41) TABLE-US-00009 TABLE 9 Formulation 9. Latin name Chinese name Grms Wt % 1. Radix Codonopsitis Pilosulae Dang Shen 20 11.6 2 Herba Lobeliae Chinensis Cum Ban Bian Lian 12 6.9 Radice 3 Fructus Kochia S copariae Di Fu Zi 10 5.8 4 Mori Albae Fructus Sang Shen 10 5.8 5 Flos Carthami Tinctorii Homg Hua 8 4.6 6 Persicae Semen Tao Ren 8 4.6 7 Diospyri Kaki Calyx Xi Di 12 6.9 8 Citri Aurantii Fructus Zhi Ke 10 5.8 9 Citri Aurantii Fructus Zhi Shi 10 5.8 Immaturus 10 Plastrum, Testudinis Gui Ban 18 10.4 11 Smilacis Glabrae, Rhizoma Tu Fu Ling 20 11.6 12 Pseudobulbus Shancigu Shan Ci Gu 15 8.7 13 Gardeniae Jasminoidis Fructus Zhi Z 10 5.8 14 Herba Lophatheri Gracilis Dan Zhu Ye 10 5.8
Study Design and Subjects:

(42) Breast cancer patients receiving formulation 1 of the invention in concurrent with chemotherapy, were reviewed over a period of 3 years. The patients aged 18-70, having localized tumors, exhibited Karnovsky performance status >80% and were candidates for adjuvant or neodajuvant chemotherapies with anthracyclines-based combination with or without taxanes at the Sorasky Oncology department Center. All patients had biopsy proven disease, were chemonaive and had normal liver and kidney function (up to X2 of ULN values) with initial blood count: Hb>10, WBC>3000, platelets>100,000. Thus, all patients included in this study were treated in parallel by conventional chemotherapy in oncology departments at academic conventional hospitals. The content of the herbal formulation was never made known to them or made public at the termination of the study.

(43) Treatment Applied:

(44) Chemotherapy applied to patients was adjuvant or neo-adjuvant standard protocol for breast cancer treatment, which, in the majority of the tested patients, included a combination of doxorubicin (Adriamycine), cyclophosphamide (Cytophosphan) and paclitaxel (Taxol) (AC+T) or doxorubicin (Adriamycine), cyclophosphamide (Cytophosphan) and 5-fluorouracil (5-FU) (CAF). The chemotherapy was applied according to a typical and well-known international chemotherapy protocols, as follows:

(45) 1. Four treatments of Adriamycine 60 mg/m.sup.2 and Cytophosphan 600 mg/m.sup.2, every 3 weeks for 4 cycles (AC protocol).

(46) 2. Four treatments of Adriamycine 60 mg/m.sup.2 and Cytophosphan 600 mg/m.sup.2, every 3 weeks for 4 cycles, followed by Taxol 80 mg/m.sup.2, every week for 12 weeks (AC+T protocol).

(47) 3. Three cycles of Cytophosphan 500 mg/m.sup.2, Adriamycine 50 mg/m.sup.2 and 5-FU 500 mg/m.sup.2, every 3 weeks for 6 cycles (CAF protocol).

(48) 4. Dose dense AC every 2 weeks followed by paclitaxel 175 mg/m.sup.2 every 2 weeks, with WBC stimulants (Neupogen/Neulastim) support (dd. AC+T protocol).

(49) Patients treated with one of first 2 regimens received Epirubicin 90 mg/m.sup.2 substituting Adriamycin at the discretion of attending physician.

(50) Following enrollment, patients were blindly randomized to receive either capsules containing a formulation according to the invention (typically formulation 1) or placebo capsules. Capsules containing formulation 1 or an equivalent according to the invention contained an effective amount of a homogeneous mixture of herbal extract powders. The extracted herbal powders where imported under license as described above.

Example 1

(51) The records from 20 consecutive breast cancer patients, having been treated with formulation 1 of the invention or an equivalent thereof, in parallel with conventional therapy, were analyzed for the study. Table 10 lists the baseline characteristics of these patients.

(52) TABLE-US-00010 TABLE 10 Baseline data on patients receiving adjuvant treatment in conjunction with chemotherapy. Values are numbers (percentages) of participants unless otherwise indicated. Baseline Data Patients Number 20 Mean age, years (SD) 52.64 (2.08) Body weight 65.47 (3.75) BRCA 1/2 4 (20%) Cancer + Stage IDC grade 2-3 16 (80%) ILC 2 (10%) DCIS 1 (5%) Metastatic cancer 1 (5%) Treatment protocols AC 5 (25%) AC + T 7 (35%) CAF 5 (25%) Other* 3 (15%) *Other protocols included: CEF + taxotere, CEF + taxol.

(53) Analysis of post-treatment scores, presented in FIG. 1, showed low scores of chemotherapy side-effects in the categories of vomiting (Avg=0.9), nausea (Avg=1.15), appetite (Avg=1.3), weakness (Avg=1.25), pain (Avg=0.7), bowel function (Avg=0.75), fatigue (Avg=1.3) and impaired daily function (Avg=1.25). As presented in FIG. 1, the majority of the patients treated with formulation 1 of the invention demonstrated reduced incidence of the various side effects.

(54) Since low scores of white blood cells (WBC), in particular, but sometimes also of red blood cells (RBC) is a common side effect of chemotherapy to breast cancer patients, the need for using growth factor drugs to enhance white blood or red blood count was also evaluated. As depicted in FIG. 2, the majority of patients did not require treatment with growth factor drugs; therefore, attesting to the ability of the formulation of the invention to effect maintenance and protection of blood cells production.

(55) The delay in chemotherapy treatment according to the clinical benefit questionnaires was also tested as means to understanding the effect of the formulation of the invention on the overall ability of a patient to undergo chemotherapy while being treated with a formulation of the invention. As shown in FIG. 3, out of 20 patients, only 3 patients reported delay in one treatment and one patient reported delay in two treatments. The majority of the patients were able to sustain the scheduled regimen and drug doses when treated also with the formulation of the invention, thereby increasing success for treatment.

(56) Importantly, no herbal-related adverse effects were reported at any time.

(57) The results of this study demonstrated that the use of the formulations described herein was effective in alleviating side effects of breast cancer patients receiving chemotherapy.

(58) In summary, the formulations of the invention have been shown to act as adjunctive treatment to ameliorate multiple side effects of chemotherapy, as follows:

(59) Vomiting and nausea: It is shown (FIG. 1) that 90% of the patients had minor or no vomiting at all. Minor or no nausea has been noted in very few patients, without any need for supplementary conventional medication (in addition to the applied chemotherapy).

(60) Febrile neutropenia (FN): There have been no reports of FN during the study.

(61) Use of growth factor drugs: Only four patients (20%) reported the need to take growth factor drugs to enhance white blood or red blood count.

(62) Alteration of taste (dysgeusia): In this study, 85% of patients reported no or minor decrease in appetite and no patient reported any significant digestive dysfunction or dysgeusia.

(63) Fatigue: The results demonstrate that none of the patients receiving the formulation of the invention reported severe fatigue.

(64) This study demonstrates that herbal formulation treatments according to the invention are safe and may serve as an effective adjunctive therapy to ameliorate multiple side effects of chemotherapy and to enhance the immune system in cancer patients.

Example 2

(65) This study aimed at assessing the efficacy, tolerability and side effects of the formulation of the invention as a complement to neoadjuvant and adjuvant chemotherapy treatment of breast cancer patients. The study shed light on the clinical importance and implications of the unique formulation of the invention described herein, as adjunctive treatment for ameliorating multiple side effects of chemotherapy in breast cancer treatment.

(66) Study methodology: The clinical study is a unicenter, randomized, double-blind placebo controlled clinical trial.

(67) Population: Breast cancer patients receiving neoadjuvant and adjuvant treatment of Tel Aviv Medical Center Oncology Department.

(68) Patients were treated for a minimum of 20 weeks, starting 2 weeks prior to chemotherapy, clinically evaluated every 6 weeks, up to 1 month post chemotherapy.

(69) The follow-up was according to the usual protocol in neoadjuvant and adjuvant chemotherapy.

(70) Imaging methods: Patients entering the trial were evaluated by imaging methods according to conventional protocol, as other patients treated with neoadjuvant and adjuvant chemotherapy. Patients receiving neoadjuvant chemotherapy in the trial had core biopsy histologically proven breast cancer. Clinical or sonographic suspicious auxiliary lymph nodes were further evaluated by FNA or core biopsy. For patients receiving neoadjuvant chemotherapy, metallic markers were introduced with ultrasonographic guidance, during the chemotherapeutic treatment. Post surgery patients were evaluated only by their pathological results from the tumor removed. The following methodologies were employed in the course of evaluation: 1. Mammography: Standard two views examinations of both breasts complemented by 90 lateral view and magnifications views. 2. Whole breast ultrasound with evaluation of the axillas. 3. Breast MRI of both breasts or PET-CT. Potentially more accurate of both previous methods in evaluation of the disease extension and in the evaluation of the contralateral breast for occult disease by other methods.

(71) Chemotherapy: Neoadjuvant, adjuvant and treatment standard chemotherapeutic protocols for breast cancer.

(72) Chemotherapy was administered according to standard adjuvant or neoadjuvant protocols for breast cancer treatment. Most of the patients received a combination of doxorubicin (Adriamycin), cyclophosphamide (Cytophosphan) and paclitaxel (Taxol) (AC+T) or doxorubicin (Adriamycin), cyclophosphamide (Cytophosphan) and 5-fluorouracil (5FU) (CAF), according to accepted international chemotherapy protocols.

(73) Herbal treatment: Formulation 1 of the invention. Each of the herbal components was in the form of an encapsulated dried herbal extract powder imported by the ZEN Herb Company and manufactured under GMP conditions.

(74) Patients were divided into two groups:

(75) Group A: received formulation 1 in a dosage of 6 grams (23 grams) per day.

(76) Group B: received placebo, resembling the herbal capsules in taste, texture and appearance, and consisting of flavored bred crams.

(77) Visit 1: 2 weeks prior to chemotherapy

(78) Patients meeting inclusion criteria received an information sheet, signed informed consent form and then evaluated by an oncologist and given the herbal formula. Next, patients were randomly divided into the 2 groups A and B, as above (a formulation group and a placebo group).

(79) Randomization was made in a manner that each group consisted of a similar number of patients of each malignancy.

(80) Visits 2-8: every 3 weeks (according to neo-adjuvant or adjuvant chemotherapy standard protocol)

(81) Visits included oncologist evaluation and physical examination according to standard protocol.

(82) Patients in the research were handled in a double blind-fashion. Each patient received a sealed container with a 21-day supply of pills, containing 315 pills: calculated as 5 pills 3 times a day for 21 days. During each follow up visit the patients received the next dosage. Instructions were to take the 5 capsules 30 min before meals, 3 times a day.

(83) Treatment was started 2 weeks prior to the first dose of chemotherapy and lasted to the last day of chemotherapy. Patients were seen by attending physicians at least every 6 weeks and had blood counts before each chemotherapy dose or when admitted for complication.

(84) Table 11 summarizes the demographic data for the study group which consisted of 65 breast cancer female patients with localized tumors. Thirty-four were randomized to receive formulation 1 and thirty-one to receive placebo. The median age was 52 years (range 24-68), about a third of the patients were treated in the neoadjuvant setting while the rest received adjuvant therapy. The use of dose-dense and taxane-based regimens was equally distributed among the study groups. Two patients in each group did not start the allocated protocol. Full compliance with treatment arm was assigned to 24 of 34 women allocated to formulation 1 and to 17 of those assigned to placebo.

(85) TABLE-US-00011 TABLE 11 Clinical and demographic characteristics at baseline. Formulation 1 Placebo P-value No of patients 34 31 Age median 47.58 52.16 range 24-67 28-68 Histology 0.32 Invasive Ductal (IDC) 20 24 Invasive Locular (ILC) 2 2 IDC + ILC 11 5 Other 1 Hormone receptors: ER, PR Negative 8 5 0.46 positive 26 26 Her-2 Negative 26 19 0.11 Intermediate 5 3 Positive 3 3 9 Protocol Compliance 0.39 Full compliance 24 17 Partial compliance 8 12 Not started 2 2

(86) Hematological toxicities associated with anemia (hemoglobin concentration of less than 10 gram %) and neutropenia and leucopenia (total WBC<3.010.sup.9/L or absolute neutrophile (ANC) count <100010.sup.9/L) were significantly reduced in patients treated with the formulation of the invention (Table 12). In sub-group analysis according to chemotherapy schedules, the protective effect against anemia was noticed only in groups receiving Adriamycin and Cytoxan every 3 weeks (hemoglobin concentration of less than 10 gram in 4% of patients treated with the formulation of the invention and 50% of placebo patients, p=0.003), while the protective effect against neutropenia was noticed only in the dose-dense group (ANC<1.510.sup.9/L in 33% of placebo patients and 0% of groups treated with the formulation of the invention, respectively). Neutropenic fever episodes occurred in 2 and 4 patients of the formulation-treated and placebo groups, respectively, non-significant difference (p=0.32). Sever infections occurred in 3 patients in the placebo group and none in the group treated with the formulation, non-significant difference (p=0.10)).

(87) TABLE-US-00012 TABLE 12 Hematological Toxicity by treatment group Grade 1 and Above grade 1 under (%) (Grade 2-4) (%) P value Anemia <10.0 g/dl >10.0 g/dl CHM >28(82%) 6(18%) 0.0081 Placebo 16(52) 15(48) Leukopenia <3.0 10.sup.9/L >3.0 CHM 28(82) 6(18) 0.0315 Placebo 18(58) 13(42) neutropenia <1.5 10.sup.9/L >1.5 CHM 25(73) 9(27) 0.063 Placebo 16(52) 15(48) Thrombocytopenia <75.0 10.sup.9/L >75.0 CHM 34(100) 0 0.258 Placebo 28(3) 2(7) Lymphopenia <0.8 >0.8 CHM 23(68) 11(32) 0.2139 Placebo 16(51) 15(49) Grade 2 and Above Grade 2 under (%) (%) P value Leukopenia <2.0 10.sup.9/L >2.0 CHM 33(97) 1(3) 0.0475 Placebo 18(58) 13(42) neutropenia <1.0 10.sup.9/L >1.0 CHM 30(88) 4(12) 0.0447 Placebo 21 (52) 10(32) Lymphopenia <0.5 10.sup.9/L >0.5 CHM 31(91) 3(9) 1.0 Placebo 28(90) 3(10)

Example 3

Cell Culture and Reagents

(88) The human Breast cell lines, MDA-231 and MDA-453, were obtained from the American Type Culture Collection (ATCC). Both cell lines were grown and maintained in Dulbecco's modified Eagle's medium (DMEM, Biological Industries, Beit HaEmek, Israel) supplemented with 10% fetal calf serum (FCS), 1% penicillin and 1% streptomycin (full medium) at 37 C., in an atmosphere of 95% oxygen and 5% CO.sub.2.

(89) The herb formulation is the formulation of the invention as described herein.

(90) Cell Viability Assay:

(91) Cells (2-510.sup.3/well) were seeded in 96-well plastic plates and incubated at 37 C. in full medium containing the tested formulation. After 48 and 72 hours, cell viability was assessed by the ability of metabolically active cells to reduce tetrazolium salt (XTT) to colored formazan compounds. The absorbance of the samples was measured with an ELISA reader (wavelength 450 nm, reference wavelength 630 nm). Each measurement was performed in triplicate. The data are mean values from three different experiments.

(92) The treatment was started after 24 h of cell culturing in order to determine the effect of the formulation of the invention on cell survival. This effect was calculated by comparing the density of the intact cells to the density of the treated cells. Cell density was determined by the XTT assay as follows. Cells (2-510.sup.3/well) were seeded in 96-well plastic plates and incubated at 37 C. in full medium containing the test drugs. The formulation of the invention was added in varying concentrations (1-100 mg/ml) to each of three replicate wells and incubated for 72 h. A freshly prepared mixture of XTT and an activation reagent (PMS) was added to each well (50 l). Following 2 h of incubation at 37 C., the plates were placed on a mechanical plate shaker of a computerized automatic micro-well plate spectrophotometer and shaken for 30 sec and the optical densities (ODs) of the dye were read at 450 nm. The measurements were repeated following 4 and 6 h of incubation. The time point of the assay at which there were optimal OD readings was chosen to count the cell number. When more than one time point fitted this criterion, the results for the different time points were normalized and averaged. The OD readings were previously shown to correlate well (r>0.97-0.99) with the number of cells/well.

(93) Light Microscopy.

(94) T47D breast adenocarcinoma cells were plated at a density of 510.sup.6 per 10-cm dish with different concentrations of the herbal formulation of the invention and after 24 h visualized by light microscopy 200.

(95) Flow Cytometry Analysis.

(96) T47D cells were plated at a density of 510.sup.6 per 10-cm dish with the test drugs at selected concentrations. The adherent and non-adherent cells were collected during exponential growth of the cells and counted. A total of 1-210.sup.6 cells were washed in phosphate-buffered saline (PBS) and the pellet was fixed in 3 ml ethanol for 1 h at 4 C. Before the analysis, the cells were pelleted and resuspended in 1 ml PBS and incubated for 30 min with 0.64 mg/ml RNAse at 37 C. They were stained with 45 propidium iodide (PI) for at least 1 h before analysis by flow cytometry. Data acquisition was performed on a FACScan and analyzed by CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, Calif., USA). All fluorescence and laser light scatter measurements were made with linear signal processing electronics. Data for at least 15,000 cells were collected for each data file. A standard protocol for cell cycle distribution and cell size was used. Necrotic cells were excluded by counting cells following staining with trypan blue before fixation. All experiments were done three times.

(97) Statistical Analysis.

(98) The results were calculated as meanSE. The difference between the intact and treated cells was evaluated by the one-way Student's T-test using an SPSS software package (SPSS Inc., Chicago, Ill., USA). Significance (p<0.05) was established by the post hoc Tukey's pairwise comparison.

(99) Effect of the Formulation of the Invention on Cell Survival.

(100) A dose-dependent inhibitory effect of the formulation of the invention on cells survival was found in MDA-231, MDA-453 and T47D human breast carcinoma cell lines (FIGS. 4, 5 and 6, respectively). IC50 was approximately similar (25 mg/ml) on all 3 carcinoma cell lines tested.

(101) Assays were also conducted with breast cancer adenocarcinoma cells (T47D). The results are presented in FIGS. 7A-C. As demonstrated, administration of the formula of the invention causes cell death of cancer cells and does not only reduce side effects associated with the treatment, thereby making chemotherapy treatments more effective and also having additional effect in reducing the recurrence of the treated disease.

(102) Induction of Apoptosis.

(103) The extent of apoptosis was assessed by flow cytometry analysis following 72 h of exposure of the cells to the different concentrations of the formulation of the invention. The formulation of the invention increased the percentage of cells with sub-diploid DNA content, the hallmark of apoptosis, in a dose-dependent manner in the T47D carcinoma cell line (FIGS. 8A-C).