Novel compositions for the treatment of cancer

Abstract

The present invention is directed to novel compositions for treating cancer. The present invention relates to novel compositions comprising proteolytic enzymes and clarifying agents and methods of using these compositions to treat and / or prevent cancer.

Description

[0001] This application is a divisional application of the Chinese national phase patent application No. 201580061402.5, which was filed on May 11, 2017, after the international application No. PCT / US2015 / 058139, the international application date was October 29, 2015, and the invention title was "Novel Composition for Treating Cancer". Invention Field

[0002] This invention relates to novel compositions comprising proteolytic enzymes and clarifying agents, and methods of treating and / or preventing cancer using these compositions. Background Technology

[0003] Selected natural and synthetic digestive (proteolytic) enzymes were used to artificially interact with an elastic extracellular matrix model to observe whether proteins in the extracellular matrix could be broken down. Therefore, if the extracellular matrix could be broken down, organic compounds (clarifying agents) could be used to form ionic bonds with the products from matrix degradation, thereby initiating a cleavage process to remove them. Summary of the Invention

[0004] This article provides novel compositions comprising at least one proteolytic enzyme and a clarifying agent. Methods for treating cancer using these novel compositions are also provided.

[0005] In some embodiments disclosed herein, the mammal is a human. In some embodiments disclosed herein, the mammal is a dog, cat, horse, or bird.

[0006] In some embodiments, the compositions provided herein are administered orally. In other embodiments, the compositions provided herein are administered intravenously.

[0007] This document also provides articles comprising packaging material, the enzymes and clarifying agents described herein within the packaging material, and a label indicating that the composition is used to treat, prevent, or improve one or more symptoms of the treated disease or condition.

[0008] Other objects, features, and advantages of the methods and compositions described herein will become apparent from the following detailed description. However, it should be understood that the detailed descriptions and specific embodiments are given by way of illustration only when indicating particular implementations, as various changes and modifications within the spirit and scope of this disclosure will be readily apparent to those skilled in the art from this detailed description.

[0009] Incorporate by reference

[0010] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference. Attached Figure Description

[0011] This patent or application document includes at least one color drawing. Upon request and payment of the necessary fees, our firm will provide a copy of the published patent or application with color drawings.

[0012] Details of the invention can be gathered in part by studying the accompanying drawings, all of which are photographs taken 24 hours after processing at 200x magnification.

[0013] Figure 1 : Control photograph - In the control, sporangia were grown in deionized water for 24 hours without any other treatment. The presence of hatching was evident through a single slit running down the middle of the shell to create a partial fringing line and an opening for hatching. Complete hatched nauplii were also present, indicating that the nauplii were able to survive in this artificial environment (normal conditions require physiological saline / salt conditions). The shell showed no signs of any kind of degenerative pattern and remained consistently solid in color. The sporangia remained dark brown within their shells. This indicates that the sporangia could grow in deionized water without any factors affecting their growth, and that deionized water did not contribute to the degradation of the sporangium shell or extracellular matrix.

[0014] Figure 2 Kiwifruit. After treating sporangia with kiwifruit juice for 24 hours, the development of nauplii was observed inside the sporangia, as shown by the circular dots in the center of some sporangia. A fully hatched nauplii (pear-shaped) and a destroyed nauplii (curved mass) were present for comparison. The presence of a lighter-colored shell was observed, which differed from the control. Figure 1 The image shows a gradient of different dark brown sporangiocarps color before the initial treatment. Different reticulate or spongy degenerative patterns are observed, with the reticulate portions appearing darker on top of the lighter-colored overall sporangiocarps. Clear portions of the sporangiocarps are also present and can be observed floating in the medium, indicating that the main sporangiocarps have degenerated, leading to the later separation of the thinner portions. This differs from conventional hatching, which produces complete dark brown half-shells.

[0015] Figure 3 Kiwiberry. Some differences were observed 24 hours after treatment of these sporangia. Translucent fragments of the sporangium shell with some rounded edges were observed floating around the medium. This indicates that the sporangium shell was partially separated, which contrasts with the dark brown hatching half-shell. Figure 1 The differences are as follows. A degenerative pattern with dark brown spots is observed on one sporangium in the photograph with a translucent body. The sporangium is intact, while the shell is degenerating. This indicates that the structural integrity of the main sporangium is not affected. Other sporangia observed in the photograph remain a relatively solid dark brown color, and the degenerative pattern is not yet present.

[0016] Figure 4 Kiwiberry. Figure 3 The continuation, stemming from the same result but different parts. The transparent sporangial sheath separates from the main sporangium, which in turn... Figure 1 It appears to be a lighter brown hue. This transparent shell fragment with rounded edges indicates that it previously contained the sporangium on which it rested. The transparent shell is quite different from the shells that produce only a dark brown half-shell upon hatching of regular nauplius larvae. The sporangium left from the transparent fragment is also smaller in shape.

[0017] Figure 5 Kiwiberry. Figure 3 The continuation, from the same result, different parts. Complete nauplii can be observed hatching from the sporangium, indicating its viability under the current treatment conditions. On the same sporangium that produced the hatching nauplii, degenerative patterns on the shell can be observed while the hatching of the nauplii is taking place, without affecting the hatching process. The degenerative patterns here include spots of varying sizes missing from the dark brown shell of the circular sporangium, revealing a lighter-colored internal body. In the same photograph, as observed in the lower right, there is another sporangium that has not yet been affected by the treatment, still remaining a pure dark brown, without any species of degenerative patterns.

[0018] Figure 6 Banana. Different distinct observations were made 24 hours after treatment. Different cyst gradients were observed, ranging from dark brown to medium or light brown. Complete hatching nauplii were observed, indicating their ability to survive and thrive under the current treatment conditions. A cyst in the upper right corner of the photograph shows a type exhibiting "cap"-like detachment and degeneration, where the entire shell separates from the host cell. The host cell, lighter in color than the separated "cap" plate, was able to hatch at different fringes, unaffected by the degeneration process. Another cyst with a reticulate or spongy degeneration pattern was also present, featuring a black reticulate pattern on top of a lighter brown body.

[0019] Figure 7 Silica sol. Different degradation patterns were observed 24 hours after treatment. On one of the sporangia, a larger, cap-like fragment was observed rising from the sporangia. The fragment detached from the sporangia was dark brown, while the rest of the intact sporangia was light brown. Figure 1 As shown, the shape of the fragment separated from the sporangium differs from that of a normally hatching sporangium half-shell. Another sporangium was observed with a gradient, spongy degenerative pattern, where the spongy pattern is dark brown over the entire intact sporangium, which is a lighter, translucent brown. The sporangium is currently actively hatching, with clear fringes visible and nauplius hatching in the distance. Other sporangium cells are present in the photograph, varying in color from dark brown to medium brown.

[0020] Figure 8 Silica sol. Figure 7 The continuation, from the same result, different parts. Most of the sporangia observed in this photograph are... Figure 1 Those that are lighter in color are compared to the others. The complete hatching of the nauplius indicates that the sporangia can reproduce and survive under the current treatment conditions. The entire transparent shell containing the sporangia is observed, with a deterioration in activity and the absence of the black patches, thus revealing the lighter brown, translucent sporangia underneath. Another sporangia with a "cap"-like degenerate portion is observed when the sporangia hatch in the right half of the photograph. The remaining sporangia has a smooth, textured appearance, unlike the surrounding sporangia, and it receives a slight reflection of light.

[0021] Figure 9 Pineapple + ginger + albumin. After 24 hours, using the combined enzymes and clarifying agent, nauplii were able to hatch intact and survive under the current treatment conditions. No damaged nauplii were observed. The seemingly dark sporangiocarps also exhibited a faint light crack pattern observed relative to the medium-dark brown shell color, demonstrating degeneration during active hatching. This degeneration process did not affect the hatching or growth process.

[0022] Figure 10 Pineapple + ginger + albumin. Figure 9 The continuation, from the same results of the treatment, occurred in different parts. The entire sporangium cell shape was observed to be intact; however, the dark brown sheath was degenerating, thus exposing the entire sporangium, which was a lighter brown and translucent color. This occurred in several different sporangia, as seen in the photograph. Some remnants of the separated and degenerated dark brown sheath were also present, which is consistent with... Figure 1 The observed hatching half-shells were different. Compared with the few that remained pure dark brown sporangia, reticulate or spongy degenerative patterns and the separation and detachment of sporangium fragments were observed. Detailed Implementation

[0023] The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will readily be apparent to those skilled in the art, and the general principles set forth herein can be applied to other embodiments without departing from the spirit and scope of the invention. Therefore, it should be understood that the specification presented herein represents currently preferred embodiments of the invention and thus represents the subject matter of a broad consideration. It should also be understood that the scope of the invention fully encompasses other embodiments that may be apparent to those skilled in the art, and the scope of the invention is therefore not limited.

[0024] Selected natural and synthetic digestive (proteolytic) enzymes were used to artificially interact with an elastic extracellular matrix model to observe whether proteins in the extracellular matrix could be broken down. Therefore, if the extracellular matrix could be broken down, organic compounds (clarifying agents) could be used to form ionic bonds with the products from matrix degradation, thereby initiating a cleavage process to remove them.

[0025] Digestive enzymes are a naturally occurring feature in all living species—serving as mechanisms that selectively break down large or complex macromolecules into smaller, more useful molecules, such as nutrients that can be extracted for digestive reasons recognizable to the body. A range of digestive enzymes from natural and synthetic sources were tested on the extracellular matrix of Artemia salina. Artemia can remain dormant and relatively unharmed in their cyst state. Their extracellular matrix protects larvae, or nauplii, from hatching for more than twenty years. These different digestive enzymes were selected to study their artificial effects on the extracellular matrix of Artemia salina to observe whether the extracellular matrix could safely dissolve to expose intact nauplii undergoing mitosis.

[0026] A small cluster of Artemia dormant cysts was grown in vivo, with each experiment in petri dishes under different conditions and treatments serving as a model for studying the prevalent condition of malignant-like tumors before actual testing in humans. Controls were established for each experimental run, in which a cluster of Artemia was grown simply suspended in deionized water without any treatment of its growth factors. Individual treatments of UV exposure against the control tests were used to illustrate the role of UV treatment in affecting the extracellular matrix of the dormant cysts during growth. Each digestive enzyme was individually tested on Artemia for its individual performance. Dilutes of the digestive enzymes for each test were performed to find the optimal concentration for the enzyme's effect on the extracellular matrix surface area. Observations were made daily to record any significant changes. Enzyme selection was based on its known digestive properties, but enzymes were eliminated or diluted based on the state of the Artemia's extracellular matrix.

[0027] Diluted, naturally derived digestive enzymes were found to break down the extracellular matrix of Artemia more quickly and at lower concentrations, causing less damage to nauplius growth. A transparent membrane remained around the nauplius. Mitotic growth of the nauplius could be observed under a microscopic light source until they hatched. The key indicator of success was the dissolved extracellular matrix, in which the interior of the sporangium was observed to be a fully hatched, motile, growing Artemia. The synthetic origin of the digestive enzymes also indicated the dissolution of the extracellular matrix and the observation of unhatched nauplius. Organic compounds tested for their function against the extracellular matrix of Artemia showed a visible degradation pattern different from that of the digestive enzymes, leading to a smooth, gradual degradation of the extracellular matrix.

[0028] Therefore, it has been found that the artificial breakdown of the extracellular matrix by digestive enzymes can be carried out in combination with organic compounds to further break down portions of the extracellular matrix while maintaining the integrity and viability of the rest of the cell. Sustainable and naturally occurring digestive enzymes and organic compounds exist that can be used to dissolve these extracellular matrices in Artemia, thus these enzymes could serve as a feasible model for the safe dissolution of malignant tumors in humans through further human testing.

[0029] Some exemplary terms

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. Where multiple definitions exist for terms herein, those in this section shall prevail. When URLs or other such identifiers or addresses are referenced, it should be understood that such identifiers are subject to change, and specific information on the Internet may be subject to appropriate alteration, but equivalent information may be found by searching the Internet. References to such information demonstrate their availability and public dissemination.

[0031] It should be understood that the general description above and the detailed description below are exemplary and interpretive only, and do not limit any of the claimed subject matter. In this application, the singular is used to include the plural unless expressly stated otherwise. It must be noted that, unless the context clearly specifies otherwise, the singular forms “a / an” and “described” as used in the specification and appended claims include the plural indicator. In this application, unless otherwise specified, the use of “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “include,” “includes,” and “included” is non-limiting.

[0032] The chapter headings used herein are for organizational purposes only and should not be construed as limiting the subject matter. All references or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are expressly incorporated herein by reference in their entirety for any purpose.

[0033] It should be understood that the methods and compositions described herein are not limited to the specific methods, schemes, and reagents described herein, and are therefore subject to variation. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the methods and compositions described herein. Numerous variations, modifications, and substitutions will appear to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The appended claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of those claims, as well as their equivalents.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] As used herein, the term “acceptable” in relation to formulations, compositions or ingredients means that it has no lasting adverse effect on the overall health of the treated subject.

[0036] As used herein, improvement of symptoms of a particular disease, condition, or symptom by administration of a particular pharmaceutical composition means any reduction, delay in onset, slowing of progression, or shortening of duration of severity (whether permanent or temporary, continuous or transient) attributable to or related to the administration of the compound or composition.

[0037] As used herein, the terms “administer,” “administering,” “administration,” etc., refer to methods that can be used to deliver a composition to a desired site of biological action. These methods include, but are not limited to, oral administration, duodenal administration, parenteral administration (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, and / or infusion), local administration, and rectal administration. Those skilled in the art are familiar with administration techniques that can be used with the compositions and methods described herein. In some embodiments, the compositions described herein are administered orally.

[0038] As used herein, the term "co-administration" and similar terms refer to the administration of selected therapeutic agents to a single patient and are intended to include treatment regimens in which the agents are administered via the same or different routes of administration or at the same or different times. Co-administration includes simultaneous administration of individual compositions, administration of individual compositions at different times, or administration of a composition in which two agents are present.

[0039] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an administration of a sufficient amount of a medicine to provide adequate relief for one or more symptoms of a disease or condition being treated. The result may be a reduction and / or mitigation of the signs, symptoms, or cause of the disease, or any other desired alteration of the biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising an enzyme and a clarifying agent as disclosed herein required to provide a clinically significant reduction in the symptoms of a disease. The appropriate "effective" amount in any individual case may be determined using techniques such as dose escalation studies.

[0040] As used herein, the terms “enhance” or “enhancing” mean to increase or prolong the potency or duration of a desired effect. Therefore, in relation to enhancing the effect of a therapeutic agent, the term “enhancement” refers to the ability to increase or prolong the potency or duration of the effect of another therapeutic agent on the system. As used herein, “enhancing effective amount” refers to an amount sufficient to enhance the effect of another therapeutic agent on the desired system.

[0041] As used herein, the term "drug combination" refers to a product obtained by mixing or combining more than one active ingredient, and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that both the active ingredient and the adjuvant are administered to a patient simultaneously as a single entity or dose. The term "non-fixed combination" means that the active ingredient and the adjuvant are administered to a patient simultaneously, concurrently, or sequentially as separate entities without a specific time interval, wherein such administration provides an effective level of both compounds in the patient's body. The latter also applies to cocktail therapies, such as the administration of three or more active ingredients.

[0042] The terms “kit” and “product” are used as synonyms.

[0043] The terms "subject" or "patient" encompass both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees and other apes; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0044] As used herein, the terms “treat,” “treating,” or “treatment” include alleviating, reducing, or improving a disease or condition, symptoms; preventing additional symptoms; improving or preventing the underlying metabolic cause of symptoms; inhibiting a disease or condition, such as preventing its progression; relieving a disease or condition; causing a disease or condition to regress; alleviating symptoms caused by a disease or condition; or preventively and / or therapeutically stopping symptoms of a disease or condition. Furthermore, therapeutic benefits are achieved by eradicating or improving one or more physiological symptoms associated with an underlying condition so that improvement is observed in a patient, although the patient may still be troubled by the underlying condition. For preventative benefits, the composition may be administered to patients at risk of developing a specific disease or to patients reporting one or more physiological symptoms of a disease (even if a diagnosis of the disease may not yet have been made). Treatment or improvement of symptoms may be based on objective or subjective parameters; including the results of physical examination, functional (self) evaluation, and / or any form of visual evaluation.

[0045] The term "in vivo" refers to events that occur within the body of a subject.

[0046] cancer

[0047] In some embodiments, methods of treating cancer using the compositions disclosed herein are disclosed herein.

[0048] As used in this article, the term "cancer" refers to the abnormal growth of cells that tend to proliferate in an uncontrolled manner and, in some cases, metastasize (spread). Types of cancer include, but are not limited to, solid tumors (such as those of the bladder, intestines, brain, breast, endometrium, heart, kidneys, lungs, liver, uterus, lymphoid tissue (lymphoma), ovary, pancreas or other endocrine organs (thyroid), prostate, skin (melanoma or basal cell carcinoma)) or hematologic malignancies (such as leukemia and lymphoma) at any stage of disease, with or without metastasis.

[0049] Non-limiting examples of cancer include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumors, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brainstem glioma, brain tumors, brain and spinal cord tumors, breast cancer, bronchial tumors, Burkitt lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, desmoidoma, embryonal tumors, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, and the Ewing sarcoma family of tumors. Tumors, including ocular cancer, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gastrointestinal stromal cell tumors, germ cell tumors, gliomas, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (endocrine pancreas), Kaposi's sarcoma, renal cancer, and Langerhans cell histiocytosis. Histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, lymphoma, Waldenstrom macroglobulinemia. Macroglobulinemia, medulloblastoma, medullary epithelioma, melanoma, mesothelioma, oral cancer, chronic myeloid leukemia, myeloid leukemia, multiple myeloma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumors, low-grade potential ovarian tumors, pancreatic cancer, papillomatosis, thyroid carcinoma, penile cancer, pharyngeal cancer, moderately differentiated pineal parenchymal tumors, pineal cell tumors and supratentorial primitive neuroectodermal tumors, pituitary tumors, plasma cell tumors / multiple myeloma, pleural pulmonary germ cell tumors, primary central nervous system lymphoma, prostate cancer.Rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach / gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

[0050] In some embodiments, the compositions disclosed herein are used to treat ovarian cancer, prostate cancer, breast cancer, lung cancer, melanoma, head and neck cancer, colorectal cancer, thyroid cancer, glioblastoma, follicular lymphoma, renal cancer, Hodgkin's lymphoma, hepatocellular carcinoma, pancreatic cancer, or melanoma.

[0051] In some embodiments, the compositions disclosed herein are used to treat bone metastases.

[0052] In some embodiments, the compositions disclosed herein are used to treat oral cancer, prostate cancer, rectal cancer, non-small cell lung cancer, lip and oral cavity cancer, liver cancer, lung cancer, anal cancer, kidney cancer, vulvar cancer, breast cancer, oropharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, urethral cancer, small bowel cancer, bile duct cancer, bladder cancer, ovarian cancer, laryngeal cancer, hypopharyngeal cancer, gallbladder cancer, colon cancer, colorectal cancer, head and neck cancer, parathyroid cancer, penile cancer, vaginal cancer, thyroid cancer, pancreatic cancer, esophageal cancer, Hodgkin's lymphoma, leukemia-related diseases, mycosis fungoides, or myelodysplastic syndrome.

[0053] In some embodiments, the compositions disclosed herein are used to treat non-small cell lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, or head and neck cancer.

[0054] In some embodiments, the compositions disclosed herein are used to treat cancer, tumors, lesions, lymphomas, melanomas, gliomas, sarcomas, or germ cell tumors.

[0055] In some implementations, cancer is selected from the group consisting of: carcinoma, adenocarcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, adrenocortical carcinoma, well-differentiated carcinoma, squamous cell carcinoma, serous carcinoma, small cell carcinoma, invasive squamous cell carcinoma, large cell carcinoma, islet cell carcinoma, oat cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, verrucous carcinoma, renal cell carcinoma, papillary serous adenocarcinoma, Merkel cell carcinoma, hepatocellular carcinoma, soft tissue carcinoma, bronchial adenocarcinoma, capillary carcinoma, bartholin gland carcinoma, basal cell carcinoma, carcinosarcoma, papilloma / carcinoma, clear cell carcinoma, endometrioid adenocarcinoma, mesothelioma, metastatic carcinoma, mucoepidermoid carcinoma, cholangiocarcinoma, actinic keratosis, cystadenoma, and hepatic adenoma.

[0056] In some implementations, the tumor is selected from the group consisting of: astrocytic tumors, malignant mesotheliomas, ovarian germ cell tumors, supratentorial primitive neuroectodermal tumors, Wilms' tumors, pituitary tumors, gonadal extragerminal tumors, gastrinomas, germ cell tumors, gestational trophoblastic tumors, brain tumors, pineal and supratentorial primitive neuroectodermal tumors, pituitary tumors, somatostatin-secreting tumors, endodermal sinus tumors, carcinoid tumors, central brain astrocytomas, glucagonomas, hepatic adenomas, islet tumors, medullary epitheliomas, plasmacytomas, vipomas, and pheochromocytomas.

[0057] In some implementations, the tumor is selected from the group consisting of: intraepithelial neoplasia, multiple myeloma / plasma cell tumor, plasma cell tumor, intraepithelial squamous cell tumor, endometrial hyperplasia, focal nodular hyperplasia, hemangioendothelioma, lymphangioleiomyomatosis, and malignant thymoma.

[0058] In some implementations, lymphoma is selected from the group consisting of: nervous system lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, and Waldenström's macroglobulinemia.

[0059] In some implementations, melanoma is selected from the group consisting of: acral lentigines melanoma, superficial diffuse melanoma, uveal melanoma, malignant lentigines melanoma, melanoma, intraocular melanoma, adenocarcinoma nodular melanoma, and hemangioma.

[0060] In some implementations, the sarcoma is selected from the group consisting of: adenoma, adenosarcoma, chondrosarcoma, endometrial stromal sarcoma, Ewing's sarcoma, Kaposi's sarcoma, leiomyosarcoma, rhabdomyosarcoma, sarcoma, uterine sarcoma, osteosarcoma, and pseudosarcoma.

[0061] In some implementations, gliomas are selected from the group consisting of gliomas, brainstem gliomas, and hypothalamic and optic pathway gliomas.

[0062] In some implementations, the germ cell tumor is selected from the group consisting of: pulmonary germ cell tumor, pleural pulmonary germ cell tumor, retinoblastoma, neuroblastoma, ductoblastoma, glioma, and angioblastoma.

[0063] Exemplary enzymes

[0064] In the various embodiments provided herein, compositions comprising one or more natural and / or synthetic digestive (proteolytic) enzymes are used to break down the elastic extracellular matrix.

[0065] Non-limiting examples of proteolytic enzymes suitable for use in this invention include the cysteine ​​protease family, which can be found in papain and papain chymopapain from papaya fruit, fig protease from fig, kiwi protease from kiwifruit, and Chinese gooseberry / mango / banana. Specific examples of fresh proteolytic enzymes suitable for use in this invention are found in kiwifruit, kiwiberry, turmeric, papaya, banana peel, mango, turmeric + ginger, and fig. Enzymes that function like proteases, such as diarylheptane metabolites (curcumin from fresh turmeric), are also suitable. In specific embodiments, one or more enzymes are selected from bromelain, kiwi protease, ginger protease (GP), or zingibain.

[0066] Exemplary clarifying agent

[0067] Once proteolytic enzymes break down the extracellular matrix into byproducts, clarifying agents are used to remove those byproducts. Another characteristic of clarifying agents is their ability to remove byproducts by precipitating and / or binding proteins. Therefore, in the various embodiments provided herein, once the extracellular matrix has been broken down, organic compounds such as clarifying agents are used to form ionic bonds with the products from matrix degradation to carry out a cleavage process to remove them.

[0068] Non-limiting examples of clarifying agents suitable for use in the compositions described herein are carbon, gelatin, casein, albumin, PVPP, fish glue (isinglass / ichtyocolle), silica sol, chitosan, silica sol + chitosan, sodium alginate, diatomaceous earth, alginate, and bentonite. In a specific embodiment, the clarifying agent is albumin.

[0069] Exemplary pharmaceutical compositions

[0070] Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers (including excipients and adjuvants) that facilitate the processing of the active ingredient into a pharmaceutically usable formulation. The appropriate formulation depends on the chosen route of administration. Any well-known techniques, carriers, and excipients may be used when appropriate and as understood in the art. A summary of the pharmaceutical compositions described herein can be found, for example, in the following references: Remington: The Science and Practice of Pharmacy, 19th edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, HA and Lachman, L., editors, Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition (Lippincott Williams & Wilkins 1999), the entirety of which is incorporated herein by reference.

[0071] In some embodiments, the combination of enzymes and clarifying agents described herein is administered alone or in combination with pharmaceutically acceptable carriers, excipients, or diluents in the pharmaceutical composition. Administration of the compositions described herein can be achieved by any method of delivering the enzymes and clarifying agents to the site of action. These methods include, but are not limited to, administration via enteral routes (including oral, gastric or duodenal feeding tubes, rectal suppositories, and rectal enemas), parenteral routes (injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrasheathal, intravascular, intravenous, intravitreal, supradural, and subcutaneous), inhalation, percutaneous, transmucosal, sublingual, buccal, and topical (including epidermal, dermal, enema, eye drops, ear drops, intranasal, and vaginal) administration, but the most suitable route may depend on, for example, the recipient's condition and illness. By way of example only, the compositions described herein can be applied topically to areas requiring treatment, such as by local infusion during surgery, topical application such as creams or ointments, injections, catheters, or implants. Application can also be achieved by direct injection at the site of the diseased tissue or organ.

[0072] This document provides pharmaceutical compositions comprising the enzymes and clarifying agents described herein, as well as pharmaceutically acceptable diluents, excipients, and / or carriers. Furthermore, the compositions described herein can be mixed with other active ingredients, such as in combination therapies.

[0073] As used herein, a pharmaceutical composition refers to a mixture of one or more enzymes and clarifying agents described herein with other chemical components (such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients). The pharmaceutical composition facilitates the administration of these components to a living organism. In practicing the treatments or methods of use provided herein, a therapeutically effective amount of the enzymes and clarifying agents described herein is administered in the pharmaceutical composition to a mammal suffering from the disease or symptom to be treated. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the enzymes and clarifying agents used, and other factors. Components in the compositions provided herein can be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0074] Orally administered pharmaceutical compositions include tablets, sublingual tablets, chewable / soluble tablets, push-fit capsules made from gelatin, and sealed soft capsules made from gelatin and plasticizers (such as glycerin or sorbitol). Tablets can be made by compression or molding, optionally using one or more excipients. Compressed tablets are prepared by compression in a suitable machine of an active ingredient in a free-flowing form (such as powder or granules), optionally mixed with a binder, inert diluent or lubricant, surfactant or dispersant. Molded tablets are prepared by molding in a suitable machine a mixture of powdered compounds moistened with an inert liquid diluent. In some embodiments, tablets are coated or scored and formulated to provide a slow or controlled release of the active ingredient therein. All formulations for oral administration should be in a dose suitable for said administration. Push-fit capsules may contain an active ingredient mixed with a filler (such as lactose), a binder (such as starch), and / or a lubricant (such as talc or magnesium stearate), and optionally a stabilizer. In soft capsules, enzymes and clarifying agents may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In some embodiments, stabilizers are added. The tablet core has a suitable coating. For this purpose, a concentrated sugar solution may be used, optionally containing gum arabic, talc, polyvinylpyrrolidone, carbomer gum, polyethylene glycol, and / or titanium dioxide, lacquer solution, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablet or tablet coating for the identification or characterization of different dosages.

[0075] In some embodiments, the pharmaceutical composition is formulated for parenteral administration via injection (e.g., by bolus or continuous infusion). Formulations for injection may be presented in unit dosage forms with added preservatives, for example, in ampoules or in multi-dose containers. The composition may be in the form of suspensions, solutions, or emulsions in oily or aqueous media, and may contain formulations such as suspending agents, stabilizers, and / or dispersants. The composition may be presented in single-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in powder form or in a lyophilized (freeze-dried) state, requiring only immediate addition of a sterile liquid carrier, such as physiological saline or sterile pyrogen-free water, prior to use. Immediate-use solutions and suspensions may be prepared from sterile powders, granules, and tablets of the previously described types.

[0076] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous (oil-based) sterile injectable solutions of the compositions, which may contain antioxidants, buffers, antibacterial agents, and solutes to make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Suitable lipophilic solvents or mediators include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or reagents that increase the solubility of enzymes and clarifying agents to allow the preparation of high-concentration solutions.

[0077] Pharmaceutical compositions can also be formulated as reservoir-type formulations. Such long-acting formulations can be administered via implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, enzymes and clarifying agents can be formulated with suitable polymeric or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or formulated as sparingly soluble derivatives, such as sparingly soluble salts.

[0078] For administration by inhalation, the pharmaceutical composition is conveniently delivered from an inhaler, a nebulizer-pressurized pack, or other convenient means of delivering aerosol spray. The pressurized pack may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a measured amount. Alternatively, for administration by inhalation or inhalation, the pharmaceutical formulation may be in the form of a dry powder composition, such as a mixture of enzymes and clarifying agents with a suitable powder matrix (e.g., lactose or starch). This powder composition may be presented in unit dosage forms, for example, in capsules, cartridges, gelatin, or blister packs, from which the powder can be administered by means of an inhaler or inhaler.

[0079] The compositions described herein are applicable topically and can be formulated into a variety of topical compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, or ointments. Such pharmaceutical compositions may contain solubilizers, stabilizers, tonicants, buffers, and preservatives known in the art.

[0080] Formulations suitable for transdermal administration of the compositions described herein may use transdermal delivery devices and transdermal delivery patches, and may be lipophilic emulsions or buffered aqueous solutions dissolved and / or dispersed in a polymer or binder. Such patches may be configured for continuous, pulsed, or on-demand delivery of the pharmaceutical agent. Furthermore, transdermal delivery of the compounds described herein may be achieved via iontophoresis patches, etc. Additionally, transdermal patches may provide controlled delivery of the compositions described herein. The absorption rate may be slowed by using a rate-controlled membrane or by trapping enzymes and clarifying agents within a polymer matrix or gel. Conversely, absorption enhancers may be used to increase absorption. Absorption enhancers or carriers may comprise absorbable, pharmaceutically acceptable solvents to aid penetration through the skin. For example, a transdermal device may be in the form of a bandage comprising a back member, a reservoir containing the compound (optionally) and a carrier, an optional rate-controlled barrier (to deliver the compound to the host's skin at a controlled and predetermined rate over an extended period of time), and a device for securing the device to the skin.

[0081] It should be understood that, in addition to the ingredients specifically mentioned above, the compounds and compositions described herein may contain other pharmaceutical agents conventional in the art in relation to the types of formulations discussed, such as flavoring agents for oral administration.

[0082] Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers (including excipients and adjuvants) that facilitate the processing of the active compound into a pharmaceutically usable formulation. The appropriate formulation depends on the chosen route of administration. Any well-known techniques, carriers, and excipients may be used when appropriate and as understood in the art. Pharmaceutical compositions comprising the compounds described herein can be manufactured in a conventional manner, such as (by way of example only) through conventional mixing, dissolving, granulation, tablet preparation, grinding, emulsification, encapsulation, embedding, or tableting methods.

[0083] Aqueous suspensions may also contain one or more polymers as suspending agents. Useful polymers include water-soluble polymers, such as cellulose polymers, for example hydroxypropyl methylcellulose; and water-insoluble polymers, such as cross-linked carboxyl-containing polymers. Useful compositions may also contain mucosal adhesion polymers selected from, for example, carboxymethyl cellulose, carbomer (acrylic polymer), poly(methyl methacrylate), polyacrylamide, polycarbofil, acrylic / butyl acrylate copolymer, sodium alginate, and dextran. Oral suspensions / liquids may be in powder form until the user adds water. In various embodiments, the pharmaceutical composition is an oral syrup or an oral g / mL solution.

[0084] The composition may also contain a solubilizer to facilitate the dissolution of the enzymes and / or clarifying agents described herein. The term "solventizer" generally includes agents that cause the formation of a micelle solution or a true solution of the pharmaceutical agent. Certain acceptable nonionic surfactants (e.g., polysorbate 80) may be used as solubilizers, as may eye-acceptable glycols, polyethylene glycols (e.g., polyethylene glycol 400), and glycol ethers.

[0085] The composition may also contain one or more pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethyl)aminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are contained in amounts necessary to maintain the pH of the composition within an acceptable range.

[0086] Other compositions may also contain one or more preservatives to inhibit microbial activity. Suitable preservatives are known in the art and include, but are not limited to, mercury-containing substances such as merfen and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, hexadecyltrimethylammonium bromide, and hexadecylpyridinium chloride.

[0087] Other compositions may contain one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octylphenyl polyol 10 and octylphenyl polyol 40.

[0088] Other compositions may contain one or more antioxidants to enhance chemical stability when needed. Suitable antioxidants include (by way of example only) ascorbic acid and sodium metabisulfite.

[0089] In some embodiments, the compositions provided herein comprise a preservative. Non-limiting examples of preservatives suitable for use in this invention include parabens (alkyl esters of parabens, such as methylparaben and propylparaben), benzoic acid, sorbic acid, benzyl alcohol, phenoxyethanol, chlorocresol, benzalkonium chloride, centrimide, benzyl chloride, chlorhexidine, chlorobutanol, methylparaben, phenethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, propylparaben, and thimerosal.

[0090] The effective amount of the composition can vary based on a variety of factors, including but not limited to the physiological characteristics of the subject, the nature of the condition to be treated, and the route and / or method of administration. Advantageously, the methods described herein allow for reductions in therapeutic indications, side effects, dose levels, dose frequencies, duration of treatment, tolerability, and / or other factors.

[0091] Exemplary administration methods and treatment regimens

[0092] The compositions described herein can be used to prepare medicaments for the treatment or prevention of specific diseases or symptoms. Furthermore, methods for treating any of the diseases or symptoms described herein in a subject requiring such treatment involve administering a pharmaceutical composition containing at least one enzyme and a clarifying agent to the subject in a therapeutically effective amount.

[0093] Compositions containing the compounds described herein may be administered for preventative and / or therapeutic purposes. In therapeutic use, the composition is administered to a patient who already suffers from a disease or symptom in an amount sufficient to cure or at least partially stop the symptoms of said disease or symptom. The effective amount for such use will depend on the severity and duration of the disease or symptom, prior therapy, the patient's health status, weight and response to the drug, and the judgment of the attending physician. It is believed that determining such an effective therapeutic amount through routine experiments (including, but not limited to, dose-escalation clinical trials) is clearly within the capabilities of those skilled in the art.

[0094] In prophylactic applications, compositions containing the compounds described herein are administered to patients who are susceptible to a particular disease, condition, or symptom, or who are otherwise at risk of a particular disease, condition, or symptom. This amount is defined as a “preventative effective amount or dose.” In this application, the precise amount also depends on the patient’s health condition, weight, etc. It is believed that determining such a preventative effective amount through routine experiments (e.g., dose-escalation clinical trials) is clearly within the capabilities of those skilled in the art. When used in patients, the effective amount for this purpose will depend on the severity and duration of the disease, condition, or symptom, prior therapy, the patient’s health condition and response to the drug, and the judgment of the attending physician.

[0095] If the patient’s condition does not improve, the compound described herein may be administered for a prolonged period of time, including throughout the patient’s life, at the physician’s discretion, in order to improve or otherwise control or limit the symptoms of the patient’s disease or condition.

[0096] If the patient's condition does improve, the compound described herein may be administered continuously at the physician's discretion; alternatively, the dosage of the compound described herein may be temporarily reduced or temporarily postponed for a period of time (i.e., a "withdrawal period"). The length of the withdrawal period may vary between 2 days and 1 year, and includes, by way of example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dose reduction during the withdrawal period can be 10% to 100%, including, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100%.

[0097] Once the patient's condition improves, a maintenance dose is administered as needed. Subsequently, the dose or frequency, or both, may be reduced to a level that maintains the improvement in the disease, condition, or symptom, depending on the symptoms. However, in the event of any recurrence of symptoms, the patient may require long-term intermittent treatment.

[0098] The amount of a given agent corresponding to this quantity will vary depending on factors such as the specific compound, the disease condition and its severity, and the identity of the subject or host requiring treatment (e.g., age, weight, sex, etc.), but can be routinely determined in a manner known in the art based on the specific circumstances surrounding the case, including, for example, the specific composition administered, the route of administration, the condition being treated, and the subject or host being treated. The required dose can be conveniently presented as a single dose or as a fractionated dose administered simultaneously (or over a short period of time) or at appropriate intervals (e.g., sub-dose twice, three, four, or more times daily).

[0099] The pharmaceutical compositions described herein may be presented in unit dosage forms suitable for precise dosing, for single administration. In a unit dosage form, the formulation is divided into unit doses containing appropriate amounts of one or more compounds. The unit dose may be in the form of a package containing discrete amounts of the formulation. Non-limiting examples include packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions may be packaged in single-dose, non-resealable containers. Alternatively, multi-dose, resealable containers may be used, in which case the composition typically contains a preservative. By way of example only, formulations for parenteral injection may be presented in unit dosage forms, including but not limited to ampoules, or in multi-dose containers with added preservatives.

[0100] Treatment regimens depend on the individual and the treatment sought. In some embodiments, the composition is applied once daily, twice daily, three times daily, four times daily, five times daily, or six times daily. In alternative embodiments, the composition is applied hourly, every 2 hours, every 3 hours, every 4 hours, or every 5 hours over a course of one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, or one month.

[0101] In various embodiments, the composition is administered over a period of one day, two days, three days, four days, five days, six days, seven days, or daily for a period of one week, two weeks, three weeks, or four weeks. In other embodiments, the composition is administered over a period of one day for a period of one month, two months, three months, four months, five months, or six months. In a specific embodiment, treatment lasts for 5 to 7 days, but may be continued as needed.

[0102] In various embodiments, the composition is stored at room temperature to maintain the activity of the enzymes and clarifying agents.

[0103] When administering the composition to adolescents, the dosage is based on the standards shown in Table 1A below:

[0104] Table 1A

[0105] Cancer staging dose* 0 to I Low II to III medium* IV high*

[0106] When administering the composition to adults, the dosage is based on the standards shown in Table 1B below:

[0107] Table 1B

[0108] Cancer staging dose* 0 to I Low to medium* II to III Medium to High* IV high

[0109] *The dosages in Tables 1A and 1B are described in Examples 38 and 39 below. Low doses can be injected intravenously, but moderate and high doses must be administered by infusion (intravenous infusion) over several hours.

[0110] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided by way of illustration and are not intended to limit the invention unless otherwise stated.

[0111] Example

[0112] Example 1: Pineapple from theoretical values

[0113] Dilute 3 drops of freshly ground pineapple juice (from the mortar and pestle) into 12.0 mL of H2O using a 5 mL graduated pipette.

[0114] • 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0115] • 5 mL graduated pipette = 25 drops / mL

[0116] • 25 drops / mL = 0.04mL / drop

[0117] Using x volumes / drops of pineapple juice as the volume of pineapple juice used for dilution = total volume of pineapple juice used for dilution. (3) x (0.04 mL) = 0.12 mL of pineapple juice solute. Using the percentage solution equation (vol / vol %):

[0118]

[0119] Fresh pineapple juice stock solution = 1% in 12.0 mL of H2O. Then add 1 drop of pineapple stock solution to 4.0 mL of H2O for the experiment. Use the dilution formula C1V1 = C2V2:

[0120]

[0121] Theoretical result: 0.0001% final pineapple juice concentration.

[0122] Example 2: Pineapple Experimental Values

[0123] Dilute 3 drops of freshly ground pineapple juice (from the mortar and pestle) into 13.2 mL of H2O using a 5 mL graduated pipette.

[0124] • 13.2 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.4 mL

[0125] • 5 mL volumetric pipette = 22.7 drops / mL

[0126] • 22.7 drops / mL = 0.044mL / drop

[0127] Using x volumes / drops of pineapple juice as the volume of pineapple juice used for dilution = total volume of pineapple juice used for dilution. (3) x (0.044 mL) = 0.132 mL of pineapple juice solute. Using the percentage solution equation (vol / vol %):

[0128]

[0129] Fresh pineapple juice stock solution = 1% in 13.2 mL of H2O. Then add 1 drop of pineapple stock solution to 4.4 mL of H2O for the experiment. Use the dilution formula C1V1 = C2V2:

[0130]

[0131] Experimental results: 0.0001% final pineapple juice concentration.

[0132] Example 3: Ginger root from theoretical value

[0133] Dilute 3 drops of freshly ground ginger root (from mortar and pestle) into 12.0 mL of H2O using a 5 mL graduated pipette.

[0134] • 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0135] • 5 mL graduated pipette = 25 drops / mL

[0136] • 25 drops / mL = 0.04mL / drop

[0137] Using x volumes / drops of ginger juice = total volume of ginger root juice used for dilution. (3) x (0.04 mL) = 0.12 mL of ginger juice solute. Using the percentage solution equation (vol / vol %):

[0138]

[0139] Fresh ginger root extract stock solution = 1% in 12.0 mL of H2O. Then add 1 drop of ginger extract stock solution to 4.0 mL of H2O for the experiment. Use the dilution formula C1V1 = C2V2:

[0140]

[0141] Theoretical result: 0.0001% final ginger root juice concentration was used in the experiment.

[0142] Example 4: Experimental values ​​of ginger

[0143] Dilute 3 drops of freshly ground ginger root juice (from the mortar and pestle) into 13.2 mL of H2O using a 5 mL graduated pipette.

[0144] • 13.2 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.4 mL

[0145] • 5 mL volumetric pipette = 22.7 drops / mL

[0146] • 22.7 drops / mL = 0.044 mL / drop

[0147] The volume of ginger root juice used per drop = the total volume of ginger juice used for dilution. (3) x (0.044 mL) = 0.132 mL of ginger root juice solute. Using the percentage solution equation (vol / vol %):

[0148]

[0149] Fresh ginger root extract was added to 13.2 mL of H2O at a concentration of 1%. Then, 1 drop of the ginger extract was added to 4.4 mL of H2O for the experiment. The dilution formula C1V1 = C2V2 was used.

[0150]

[0151] Experimental results: 0.0001% final ginger root juice concentration.

[0152] Example 5: Protein from theoretical values

[0153] Dilute 2 drops of freshly ground pineapple juice (from the mortar and pestle) into 12.0 mL of H2O using a 5 mL graduated pipette.

[0154] • 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0155] • 5 mL graduated pipette = 25 drops / mL

[0156] • 25 drops / mL = 0.04mL / drop

[0157] The volume of protein used per drop = the total volume of protein used for dilution. (2) x (0.04 mL) = 0.8 mL of protein solute. Using the percentage solution equation (vol / vol %):

[0158]

[0159] Fresh protein stock solution = 0.67% in 12.0 mL of H2O.

[0160] Then add 1 drop of protein stock solution to 4.0 mL of H2O for the experiment. Use the dilution formula C1V1 = C2V2:

[0161]

[0162] Theoretical result: 0.000067% final protein concentration.

[0163] Example 6: Protein experimental values

[0164] Dilute 2 drops of fresh protein (from the mortar and pestle) into 13.2 mL of H2O using a 5 mL graduated pipette.

[0165] • 13.2 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.4 mL

[0166] • 5 mL volumetric pipette = 22.7 drops / mL

[0167] • 22.7 drops / mL = 0.044 mL / drop

[0168] Using x volumes / drops of pineapple droplets = Total volume of protein used for dilution. (2) x (0.044 mL) = 0.088 mL of protein solute. Using the percentage solution equation (vol / vol %):

[0169]

[0170] Fresh protein stock solution = 0.67% in 13.2 mL of H2O. Then add 1 drop of protein solution to 4.4 mL of H2O for the experiment. Use the dilution formula C1V1 = C2V2:

[0171]

[0172] Experimental result: 0.000067% final protein concentration.

[0173] Example 7: Experimental values ​​of kiwi juice

[0174] Dilute 2 drops of freshly ground kiwi juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0175] • 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0176] • 3 mL graduated pipette = 25 drops / mL

[0177] • 25 drops / mL = 0.04mL / drop

[0178] Using x volumes / drops of kiwi juice as the liquid volume = the total volume of kiwi juice used for dilution. (2) x(0.04mL) = 0.08 mL of kiwi juice solute. Using the percentage solution equation (vol / vol %):

[0179]

[0180] Example 8: Exemplary Fresh Kiwi Juice Stock Solution – Concentration #2 (Medium)

[0181] *Testing of fresh kiwi juice stock before dilution

[0182] Dilute 5 drops of freshly ground kiwi juice (from the mortar and pestle) into 16.0 mL of H2O using a 3 mL graduated pipette.

[0183] • 16.0 mL = 4 times (4x) the amount needed for 100 drops (100 tests) in the experiment. 100 drops = 4.0 mL

[0184] • 3 mL graduated pipette = 25 drops / mL

[0185] • 25 drops / mL = 0.04mL / drop

[0186] Using x volumes / drops of kiwi juice as the volume of liquid droplets = Total volume of kiwi juice used for dilution. (5) x(0.04mL) = 0.2 mL of kiwi juice solute. [Use percentage solution equation (vol / vol %)]

[0187]

[0188] Fresh kiwi juice stock solution = 1.25% in 16.0 mL of H2O. Then add 1 drop of kiwi juice stock solution to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0189]

[0190] The final kiwi juice concentration of .000125% was used in the experiment.

[0191] Example 9: Kiwi juice experimental values ​​– Concentration #1 (Strong)

[0192] Dilute 2 drops of freshly ground kiwiberry juice (with skin) from the mortar and pestle into 4.0 mL of H2O using a 3 mL graduated pipette.

[0193] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0194] - 3 mL volumetric pipette = 25 drops / mL

[0195] - 25 drops / mL = 0.04mL / drop

[0196] Using x volumes / drops of kiwifruit juice as the volume of kiwifruit juice used for dilution = total volume of kiwifruit juice used for dilution. (2) x(0.04mL) = 0.08 mL of kiwifruit juice solute. [Use percentage solution equation (vol / vol %)]

[0197]

[0198] Example 10: Exemplary Fresh Kiwi Juice Stock – Concentration #2 (Medium)

[0199] *Testing of fresh kiwifruit juice stock before dilution.

[0200] Diluted kiwiberries: Dilute 3 drops of freshly ground kiwiberry juice (with skin) from the mortar and pestle into 8.0 mL of H2O using a 3 mL graduated pipette.

[0201] • 8.0 mL = twice the amount needed for 100 drops (100 tests) in the experiment (2x). 100 drops = 4.0 mL

[0202] • 3 mL graduated pipette = 25 drops / mL

[0203] • 25 drops / mL = 0.04mL / drop

[0204] Using x volumes / drops of kiwifruit juice as the number of drops = the total volume of kiwifruit juice used for dilution. (3) x(0.04mL) = 0.12 mL of kiwifruit juice solute [using percentage solution equation (vol / vol %)]

[0205]

[0206] Fresh kiwifruit juice stock solution = 1.5% in 8.0 mL of H2O

[0207] Then add 1 drop of kiwiberry stock solution to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0208]

[0209] .00015% final kiwi juice concentration was used in the experiment.

[0210] Example 11: Mango Juice Experimental Values ​​– Concentration #1 (Strong)

[0211] Dilute 2 drops of freshly ground mango juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0212] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0213] - 3 mL volumetric pipette = 25 drops / mL

[0214] - 25 drops / mL = 0.04mL / drop

[0215] The volume of mango juice used for dilution is calculated using x volumes / drops of mango.

[0216] (2) x (0.04 mL) = 0.08 mL mango juice solute. [Use percentage solution equation (vol / vol %)]

[0217]

[0218] Example 12: Exemplary Fresh Mango Juice Stock Solution – Concentration #2 (Medium)

[0219] *Test the fresh mango juice stock before dilution.

[0220] Diluted mango Dilute 3 drops of freshly ground mango juice (from the mortar and pestle) into 8.0 mL of H2O using a 3 mL graduated pipette.

[0221] • 8.0 mL = twice the amount needed for 100 drops (100 tests) in the experiment (2x). 100 drops = 4.0 mL

[0222] • 3 mL graduated pipette = 25 drops / mL

[0223] • 25 drops / mL = 0.04mL / drop

[0224] The volume of mango juice used per drop = the total volume of mango juice used for dilution. (3) x (0.04 mL) = 0.12 mL of mango juice solute. [Use percentage solution equation (vol / vol %)]

[0225]

[0226] Fresh mango juice stock solution = 1.5% in 8.0 mL of H2O.

[0227] Then add 1 drop of mango stock solution to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0228]

[0229] The final mango juice concentration of 0.00015% was used in the experiment.

[0230] Example 13: Banana Juice Experimental Values ​​– Concentration #1 (Strong)

[0231] Dilute 2 drops of freshly grated banana (with peel) juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0232] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0233] - 3 mL volumetric pipette = 25 drops / mL

[0234] - 25 drops / mL = 0.04mL / drop

[0235] The volume of banana juice used per drop = the total volume of banana juice used for dilution. (2) x (0.04 mL) = 0.08 mL of banana juice solute. [Use percentage solution equation (vol / vol %)]

[0236]

[0237] Example 14: Exemplary Fresh Banana Juice Stock Solution – Concentration #2 (Medium)

[0238] *Test the fresh banana juice stock before dilution.

[0239] Diluted bananas: Dilute 4 drops of freshly grated banana (with peel) juice (from the mortar and pestle) into 8.0 mL of H2O using a 3 mL graduated pipette.

[0240] • 8.0 mL = twice the amount needed for 100 drops (100 tests) in the experiment (2x). 100 drops = 4.0 mL

[0241] • 3 mL graduated pipette = 25 drops / mL

[0242] • 25 drops / mL = 0.04mL / drop

[0243] The volume of banana juice used per drop = the total volume of banana juice used for dilution. (4) x (0.04 mL) = 0.16 mL of banana juice solute [using the percentage solution equation (vol / vol %)].

[0244]

[0245] Fresh banana juice stock solution = 2% in 8.0 mL of H2O. Then add 1 drop of banana stock solution to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0246]

[0247] 0.0002% final banana juice concentration used in the experiment

[0248] Example 15: Curcuma Experimental Values ​​– Concentration #1 (Medium)

[0249] Dilute 2 drops of freshly ground turmeric juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0250] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0251] - 3 mL volumetric pipette = 25 drops / mL

[0252] - 25 drops / mL = 0.04mL / drop

[0253] Using x volumes / drops of turmeric droplets = Total volume of turmeric juice used for dilution. (2) x (0.04 mL) = 0.08 mL of turmeric juice solute [using percentage solution equation (vol / vol %)]

[0254]

[0255] Example 16: Exemplary Turmeric Stock Solution – Concentration #2 (Strong)

[0256] *Testing of fresh turmeric juice reservoirs

[0257] Dilute 3 drops of freshly ground turmeric juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0258] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0259] - 3 mL volumetric pipette = 25 drops / mL

[0260] - 25 drops / mL = 0.04mL / drop

[0261] Using x volumes / drops of turmeric droplets = Total volume of turmeric juice used for dilution. (3) x (0.04 mL) = 0.12 mL of turmeric juice solute [using percentage solution equation (vol / vol %)]

[0262]

[0263] Example 17: Experimental values ​​of fresh papaya juice – Concentration #1 (Very strong)

[0264] *Testing of fresh papaya juice stock before dilution

[0265] Dilute 1 drop of freshly ground papaya juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0266] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0267] - 3 mL volumetric pipette = 25 drops / mL

[0268] - 25 drops / mL = 0.04mL / drop

[0269] The volume of papaya juice used per drop = the total volume of papaya juice used for dilution. (1) x (0.04 mL) = 0.04 mL of papaya juice solute [using the percentage solution equation (vol / vol %)].

[0270]

[0271] Example 18: Experimental values ​​of fresh papaya juice – Concentration #2 (Strong)

[0272] *Testing of fresh papaya juice stock before dilution

[0273] Dilute 2 drops of freshly ground papaya juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0274] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0275] - 3 mL volumetric pipette = 25 drops / mL

[0276] - 25 drops / mL = 0.04mL / drop

[0277] Using x volumes / drops of papaya juice as the number of drops = the total volume of papaya juice used for dilution. (2) x (0.04 mL) = 0.08 mL of papaya juice solute [using the percentage solution equation (vol / vol %)].

[0278]

[0279] Example 19: Experimental values ​​of fresh papaya juice – Concentration #3 (medium)

[0280] Diluted papaya: Dilute 3 drops of freshly ground papaya juice (from the mortar and pestle) into 12.0 mL of H2O using a 3 mL graduated pipette.

[0281] • 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0282] • 3 mL graduated pipette = 25 drops / mL

[0283] • 25 drops / mL = 0.04mL / drop

[0284] Using x volumes / drops of papaya juice as the number of drops = Total volume of papaya juice used for dilution. (3) x (0.04 mL) = 0.12 mL banana juice solute [using percentage solution equation (vol / vol %)]

[0285]

[0286] Fresh papaya juice stock solution = 1% in 12.0 mL of H2O. Then add 1 drop of papaya stock solution to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0287]

[0288] .0001% final papaya juice concentration used in experiments

[0289] Example 20: Experimental values ​​of fresh papaya juice – Concentration #4 (slow-acting)

[0290] Diluted papaya Dilute 4 drops of freshly ground papaya juice (from the mortar and pestle) into 12.0 mL of H2O using a 3 mL graduated pipette.

[0291] • 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0292] • 3 mL graduated pipette = 25 drops / mL

[0293] • 25 drops / mL = 0.04mL / drop

[0294] Using x volumes / drops of papaya juice as the number of drops = Total volume of papaya juice used for dilution. (4) x (0.04 mL) = 0.16 mL of papaya juice solute [using percentage solution equation (vol / vol %)]

[0295]

[0296] Fresh papaya juice stock solution = 1.3% in 12.0 mL of H2O. Then add 1 drop of papaya stock solution to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0297]

[0298] .00013% final papaya juice concentration used in experiments

[0299] Example 21: Exemplary combination enzyme – ginger + turmeric experimental value – concentration #1 (very strong)

[0300] *Testing of fresh ginger and turmeric juice stock solutions prior to dilution.

[0301] Dilute 1 drop each of freshly ground ginger and turmeric juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0302] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0303] - 3 mL volumetric pipette = 25 drops / mL

[0304] - 25 drops / mL = 0.04mL / drop

[0305] Using x volumes / drops of (ginger + turmeric) of liquid drops = total volume of ginger and turmeric juice used for dilution. (2) x (0.04 mL) = 0.08 mL of ginger and turmeric juice solute [using percentage solution equation (vol / vol %)]

[0306]

[0307] Fresh ginger and turmeric juice stock solution = 2% in 4.0 mL H2O (* 1% fresh ginger + 1% fresh turmeric = 2%)

[0308] Example 22: Exemplary combination enzyme – ginger + turmeric experimental value – concentration #2 (medium)

[0309] Diluted ginger + undiluted turmeric: Dilute 3 drops of freshly ground ginger juice (from the mortar and pestle) into 12.0 mL of H2O using a 3 mL graduated pipette.

[0310] • 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0311] • 3 mL graduated pipette = 25 drops / mL

[0312] • 25 drops / mL = 0.04mL / drop

[0313] The volume of ginger juice used per drop = the total volume of ginger juice used for dilution. (3) x (0.04 mL) = 0.12 mL of ginger juice solute [using the percentage solution equation (vol / vol %)].

[0314]

[0315] Fresh ginger juice stock solution = 1% in 12.0 mL of H2O. Then add 1 drop of ginger stock solution to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0316]

[0317] .0001% final ginger juice concentration used in the experiment

[0318] Dilute 1 drop of freshly ground turmeric juice (from the mortar and pestle) into 4.0 mL of H2O using a 3 mL graduated pipette.

[0319] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0320] - 3 mL volumetric pipette = 25 drops / mL

[0321] - 25 drops / mL = 0.04mL / drop

[0322] The volume of turmeric used per drop = the total volume of turmeric juice used for dilution. (1) x (0.04 mL) = 0.04 mL of turmeric juice solute [using the percentage solution equation (vol / vol %)].

[0323]

[0324] Fresh turmeric juice stock solution = 1% in 4.0 mL of H2O.

[0325] Final ginger + turmeric juice concentration = 1% turmeric + 0.0001% ginger = 1.001% combined ginger + turmeric juice in 4.0 mL H2O

[0326] Example 23: Fish glue clarifying agent experimental values ​​– Concentration #1 (rapid-medium to slightly strong)

[0327] Dilute 2 drops of fish glue in 4.0 mL of H2O using a 3 mL graduated pipette.

[0328] - 100 drops (100 doses) for experiments. 100 drops = 4.0 mL

[0329] - 3 mL volumetric pipette = 25 drops / mL

[0330] - 25 drops / mL = 0.04mL / drop

[0331] The volume of fish glue used per drop = the total volume of fish glue used for dilution. (2) x (0.04 mL) = 0.08 mL of fish glue solute [using the percentage solution equation (vol / vol %)].

[0332]

[0333] Fish gelatin stock solution = 2% in 4.0 mL H2O for experiments

[0334] Example 24: Experimental values ​​of silica sol clarifying agent – ​​Concentration #1 (slow)

[0335] Dilute 2 drops of silica sol into 4.0 mL of H2O using a 3 mL graduated pipette.

[0336] - 100 drops (100 doses) for experiments. 100 drops = 4.0 mL

[0337] - 3 mL volumetric pipette = 25 drops / mL

[0338] - 25 drops / mL = 0.04mL / drop

[0339] The volume of silica sol used per drop = the total volume of silica sol used for dilution. (2) x (0.04 mL) = 0.08 mL of silica sol solute [using the percentage solution equation (vol / vol %)].

[0340]

[0341] Silica sol stock solution = 2% in 4.0 mL H2O for experiments

[0342] Example 25: Chitosan Clarifying Agent Experimental Values ​​– Concentration #1 (Slow)

[0343] Dilute 1 drop of chitosan to 4.0 mL of H2O using a 3 mL graduated pipette.

[0344] - 100 drops (100 doses) for experiments. 100 drops = 4.0 mL

[0345] - 3 mL volumetric pipette = 25 drops / mL

[0346] - 25 drops / mL = 0.04mL / drop

[0347] Using x volumes / drops of chitosan droplets = Total volume of chitosan used for dilution. (1) x (0.04 mL) = 0.04 mL chitosan solute [using percentage solution equation (vol / vol %)]

[0348]

[0349] Chitosan stock solution = 1% in 4.0 mL H2O for experiments

[0350] Example 26: Chitosan Clarifying Agent Experimental Values ​​– Concentration #3 (Moderately Rapid)

[0351] Diluted chitosan (based on package instructions) According to the packaging instructions: 2 fl oz chitosan needs to be diluted in 30 mL of warm H2O.

[0352]

[0353] x = 23.65 mL of chitosan is needed for the experiment to produce 12 mL of H2O.

[0354] Dilute 591.25 drops of chitosan into 12.0 mL of H2O using a 3 mL graduated pipette.

[0355] - 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0356] - 3 mL volumetric pipette = 25 drops / mL

[0357] - 25 drops / mL = 0.04mL / drop

[0358] Using x volumes / drops of chitosan droplets = Total volume of chitosan used for dilution. (591.25) x (0.04 mL) = 23.65 mL chitosan solute [using percentage solution equation (vol / vol %)]

[0359]

[0360] Dilute the chitosan stock solution to 197% in 12.0 mL of H2O for the experiment. Then add 1 drop of the diluted chitosan stock solution to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0361]

[0362] The final chitosan concentration of 0.0194% was used in the experiment.

[0363] Example 27: Experimental values ​​of combined silica sol + chitosan clarifying agent – ​​Concentration #1 (rapid)

[0364] Dilute one drop each of undiluted silica sol and chitosan into 4.0 mL of H2O using a 3 mL graduated pipette.

[0365] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0366] - 3 mL volumetric pipette = 25 drops / mL

[0367] - 25 drops / mL = 0.04mL / drop

[0368] Using x volumes / drops of silica sol and chitosan droplets = Total volume of silica sol and chitosan used for dilution. (2) x (0.04 mL) = 0.08 mL of silica sol and chitosan solute. [Use percentage solution equation (vol / vol%)]

[0369]

[0370] Example 28: Combined silica sol + chitosan reservoir – Concentration #2 (slow)

[0371] Dilute one drop of undiluted silica sol into 4.0 mL of H2O using a 3 mL graduated pipette.

[0372] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0373] - 3 mL volumetric pipette = 25 drops / mL

[0374] - 25 drops / mL = 0.04mL / drop

[0375] The volume of silica sol used per drop = the total volume of silica sol used for dilution. (1) x (0.04 mL) = 0.04 mL of silica sol solute [using the percentage solution equation (vol / vol %)].

[0376]

[0377] The silica sol stock solution was prepared at 1% in 4.0 mL of H2O for the experiment. Then, 1 drop of chitosan-diluted stock solution was added to 4.0 mL of H2O for the experiment. [Use the dilution formula C1V1 = C2V2]

[0378]

[0379] The final chitosan concentration of 0.0194% was used in the experiment.

[0380] The final silica sol + chitosan concentration was 1.0194% in 4.0 mL of H2O for the experiment.

[0381] Example 29: Experimental values ​​of sodium alginate clarifying agent – ​​Concentration #1 (rapid)

[0382] 1:100 dilution. Dilute 0.12 g of undiluted sodium alginate to 12.0 mL of H2O using a 3 mL graduated pipette.

[0383] - 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0384] - 3 mL volumetric pipette = 25 drops / mL

[0385] - 25 drops / mL = 0.04mL / drop

[0386] According to the package instructions, the solution concentration is 2 grams of sodium alginate per 200 mL of H₂O. [Use percentage solution equations (weight / vol%)]

[0387]

[0388] Sodium alginate stock solution = 1% in 12.0 mL of H₂O for experiments.

[0389] Example 30: Experimental values ​​of sodium alginate clarifying agent – ​​Concentration #2 (slow)

[0390] 1:50 dilution. Dilute 0.24 g of undiluted sodium alginate to 12.0 mL of H2O using a 3 mL graduated pipette.

[0391] - 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0392] - 3 mL volumetric pipette = 25 drops / mL

[0393] - 25 drops / mL = 0.04mL / drop

[0394] According to the package instructions, the solution concentration is 2 grams of sodium alginate per 200 mL of H₂O. [Use percentage solution equations (weight / vol%)]

[0395]

[0396] Sodium alginate stock solution = 2% in 12.0 mL H2O for experiments

[0397] Example 31: Bentonite Clarifying Agent Experimental Values ​​– Concentration #1 (Slow)

[0398] Bentonite must be rehydrated before the experiment. Boil 2 teaspoons of bentonite with ½ cup of deionized H₂O at 60°C, then let stand at room temperature for 4 hours. Ratio: 1 tablespoon / gallon.

[0399] Dilute 1 drop of rehydrated bentonite in 4.0 mL of H2O using a 3 mL graduated pipette.

[0400] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0401] - 3 mL volumetric pipette = 25 drops / mL

[0402] - 25 drops / mL = 0.04mL / drop

[0403] The volume of bentonite used per drop = the total volume of bentonite used for dilution. (1) x (0.04 mL) = 0.04 mL of bentonite solute [using the percentage solution equation (vol / vol %)].

[0404]

[0405] Bentonite stock solution = 1% in 4.0 mL H2O for experiments

[0406] Example 32: Bentonite Clarifying Agent Experimental Values ​​– Concentration #2 (Medium)

[0407] Bentonite must be rehydrated before the experiment. Boil 2 teaspoons of bentonite with ½ cup of deionized H₂O at 60°C, then let stand at room temperature for 4 hours. Ratio: 2 tablespoons / gallon.

[0408] Dilute 2 drops of rehydrated bentonite into 4.0 mL of H2O using a 3 mL graduated pipette.

[0409] - 100 drops (100 doses) for the experiment. 100 drops = 4.0 mL

[0410] - 3 mL volumetric pipette = 25 drops / mL

[0411] - 25 drops / mL = 0.04mL / drop

[0412] The volume of bentonite used per drop = the total volume of bentonite used for dilution. (2) x (0.04 mL) = 0.08 mL of bentonite solute [using the percentage solution equation (vol / vol %)].

[0413]

[0414] Bentonite stock solution = 2% in 4.0 mL H2O for experiments

[0415] Example 33: Diatomaceous earth clarifying agent experimental values ​​– Concentration #1 (slow)

[0416] 1:100 dilution. Dilute 0.101 g of undiluted diatomaceous earth to 12.0 mL of H2O using a 3 mL graduated pipette.

[0417] - 12.0 mL = 3 times (3x) the amount needed for 100 drops (100 portions) in the experiment. 100 drops = 4.0 mL

[0418] - 3 mL volumetric pipette = 25 drops / mL

[0419] - 25 drops / mL = 0.04mL / drop

[0420] According to the package instructions = 1 teaspoon / 1 cup H2O [using percentage solution equation (weight / vol %)]

[0421]

[0422] Sodium alginate stock solution = 0.8% in 12.0 mL H₂O for experiments.

[0423] Example 34: Exemplary solution from theoretical values

[0424] The reservoir solution of the composition is prepared by combining the components described in the following table:

[0425] Components concentration volume Pineapple (Example 1) 0.0001% <![CDATA[4.0 mL H2O]]> Ginger (Example 3) 0.0001% <![CDATA[4.0 mL H2O]]> Protein (Example 5) 0.000067% <![CDATA[4.0 mL H2O]]> total 0.000267% <![CDATA[12.0 mL H2O]]>

[0426] Dilute the stock solution described in the table according to the following formula: Concentration x (Final Volume / Stock Volume) = Required Volume from Stock

[0427] 250 mL IV bag: 0.000267% x (250 mL / 12.0 mL) = 0.0267 x (250 mL / 12.0 mL) = 0.5563 mL of reservoir. Therefore, a 250 mL IV bag contains 0.56 mL of reservoir and 249.44 mL of IV-specific injection water.

[0428] 500 mL IV bag: 0.000267 % x (500 mL / 12.0 mL) = 0.0267 x (500 mL / 12.0 mL) = 1.113 mL of reservoir. Therefore, a 500 mL IV bag contains 1.113 mL of reservoir and 498.9 mL of IV-specific injection water.

[0429] Example 35: Exemplary 13.2 mL H2O stock solution from experimental values

[0430] The reservoir solution of the composition is prepared by combining the components described in the following table:

[0431] Components concentration volume Pineapple (Example 2) 0.0001% <![CDATA[4.4 mL H2O]]> Ginger (Example 4) 0.0001% <![CDATA[4.4 mL H2O]]> Protein (Example 6) 0.000067% <![CDATA[4.4 mL H2O]]> total 0.000267% <![CDATA[13.2 mL H2O]]>

[0432] Dilute the stock solution described in the table according to the following formula: Concentration x (Final Volume / Stock Volume) = Required Volume from Stock.

[0433] 250 mL IV bag: 0.000267% x (250 mL / 13.2 mL) = 0.0267 x (250 mL / 13.2 mL) = 0.5056 mL of reservoir. Therefore, a 250 mL IV bag contains 0.51 mL of reservoir and 249.49 mL of IV-specific injection water.

[0434] 500 mL IV bag: 0.000267% x (500 mL / 13.2 mL) = 0.0267 x (500 mL / 13.2 mL) = 1.011 mL of reservoir. Therefore, a 500 mL IV bag contains 1.0 mL of reservoir and 499.00 mL of IV-specific injection water.

[0435] Example 36: Exemplary solution from a 250 mL IV bag of experimental data

[0436] Prepare a 250 mL IV bag from the reservoir using the following formula: 0.000267% x (250 mL / 13.2 mL) = 0.0267 x (250 mL / 13.2 mL) = 0.5056 mL of reservoir.

[0437] Example 37: Exemplary solution from a 500 mL IV bag of experimental data

[0438] Prepare a 500 mL IV bag from the reservoir using the following formula: 0.00267 % x (500 mL / 13.2 mL) = 0.0267 x (500 mL / 13.2 mL) = 1.011 mL of reservoir.

[0439] 500 mL IV bag = 1.0 mL stock + 499.00 mL IV-specific injection water.

[0440] Example 38: Exemplary 250 mL IV bag dose from experimental values

[0441] Lower dose: Prepare a 250 mL lower-dose IV bag from the reservoir using the following formula: 0.25 mL reservoir + 249.75 mL IV-specific injection water (.000134% concentration, ½ of the medium dose).

[0442] Moderate dose: Prepare a 250 mL intermediate-dose IV bag from the reservoir using the following formula: 0.51 mL reservoir + 249.49 mL IV-specific injection water (.000267% concentration).

[0443] Higher dose: Prepare a 250 mL higher-dose IV bag from the reservoir using the following formula: 0.76 mL reservoir + 249.24 mL IV-specific injection water (.0004005% concentration, 1.5x the medium dose).

[0444] Example 39: Exemplary 500 mL IV bag dose from experimental values

[0445] lower dose Prepare a 500 mL lower-dose IV bag from the reservoir using the following formula: 0.51 mL reservoir + 499.49 mL IV-specific injection water (0.000134% concentration, ½ of the medium dose).

[0446] moderate dose Prepare a 500 mL intermediate-dose IV bag from the reservoir using the following formula: 1.0 mL reservoir + 499.00 mL IV-specific injection water (.000267% concentration).

[0447] higher dose Prepare a 500 mL higher-dose IV bag from the reservoir using the following formula: 1.5 mL reservoir + 498.5 mL IV-specific injection water (0.0004005% concentration, 1.5x the medium dose).

[0448] Example 40: Exemplary veterinary intramuscular injection solution of 5 mL.

[0449] The veterinary injection solution was prepared using the stock solution described in Example 8 according to the following formula: 0.000267% x (5 mL / 13.2 mL) = 0.0267 x (5 mL / 13.2 mL) = 0.01 mL stock solution.

[0450] Prepare a 5 mL standard dose injection (0.000267% concentration) using 0.1 mL of stock solution + 4.99 mL of IM-specific injection water.

[0451] Example 41: Exemplary veterinary intramuscular injection solution of 10 mL.

[0452] The veterinary injection solution was prepared using the stock solution described in Example 8 according to the following formula: 0.000267% x (10 mL / 13.2 mL) = 0.0267 x (10 mL / 13.2 mL) = 0.02 mL stock solution.

[0453] Prepare a 10 mL standard dose injection using 0.2 mL of stock solution + 9.8 mL of IM-specific water for injection. Prepare a 10 mL higher dose injection (0.0004005% concentration, 1.5x the standard dose) using 0.3 mL of stock solution + 9.7 mL of IM-specific water for injection.

[0454] Example 42: Treatment of Stage III Colon Cancer

[0455] A 65-year-old Vietnamese-American woman was diagnosed with stage II-III colon cancer. In September 2012, she was diagnosed with stage II cancer and underwent surgery to remove the malignant portion of her colon. Post-operative evaluation confirmed progression to stage III cancer.

[0456] The replacement therapy began in January 2013. This treatment consisted of: daily juice of ½ a fresh pineapple and 2 cm slices of fresh ginger root (to prepare approximately 12 ounces), along with one soft-boiled egg. The juice and egg were consumed daily for three months; two months before radiation and chemotherapy, and one month during radiation and chemotherapy.

[0457] From March to April 2013, the patient received radiation and chemotherapy. A total of 20 radiation sessions and 4 chemotherapy sessions were administered. In April 2013, the patient was discharged based on a clean bill of health. No side effects were observed during the radiation and chemotherapy treatment.

[0458] In April 2013, specialists issued COVID-free health certificates and the alternative treatment was modified to twice a week for preventative purposes only.

[0459] Example 43: Treatment of Stage III Uterine Cancer

[0460] A 47-year-old Vietnamese woman was diagnosed with stage III uterine cancer. She underwent her first surgery in 2013 to remove a 1.6 kg tumor, and a second surgery in February 2014 to remove another recurrent tumor growing in the same area.

[0461] From March to April 2014, a replacement therapy was administered once daily, consisting of 1 / 3 of a fresh pineapple, 3-4 cm slices of freshly squeezed ginger, and 1 raw egg white, prepared to make approximately 12 ounces of cup. Perirectal bleeding was only observed on the first day after the replacement therapy was started. This treatment lasted for one month. Follow-up in April 2014 showed no disease-free health certificates, and no residual cancer cells were identified.

[0462] Example 44: Treatment of Stage 0 Breast Cancer

[0463] A 60-year-old Vietnamese-American woman was diagnosed with stage II-III colon cancer. In September 2012, she was also diagnosed with breast cancer. Surgery was performed to remove non-benign tumors or dense areas of breast tissue. In November 2013, a mammogram showed several dense patches of breast tissue and evidence of hyperplastic cell accumulation. A biopsy was recommended for the presence of hyperplastic cells indicating a malignant tumor.

[0464] Prepare approximately 16-ounce cups of the alternative treatment by applying once daily, consisting of ¼ fresh pineapple + 2-3 cm of freshly juiced ginger + 1 raw egg, once a day for only two months.

[0465] Prior to replacement therapy, the patient had experienced daily fever for 10-12+ years. There were no cold or flu symptoms, but the fever occurred daily, and the only way to control it was by taking Tylenol or Ibuprofen daily. In addition, during this period, the patient had a constant daily blood pressure of at least 170-180 (systolic) / 95-100 (diastolic). Three different blood pressure medications were used to control the blood pressure to within the normal range. One week after replacement therapy began, the patient's fever subsided, and the blood pressure returned to normal.

[0466] A biopsy was performed after the patient's symptoms subsided, and no malignant tumor was detected in the dense breast tissue. The patient has remained cancer-free and asymptomatic since June 2014.

[0467] Example 45: Treatment of Stage IV Lung Cancer

[0468] A 73-year-old Vietnamese-American man was diagnosed with stage IV lung cancer. In December 2012, his prognosis was estimated at four months to live.

[0469] Beginning in February 2013, a daily intake of 2-3 cups of replacement therapy (each 12-ounce cup containing ¼ of a fresh pineapple, 2-4 cm of fresh ginger, and 1 raw egg white) was continued for 3 months. The initial tumor size in the lung had a radius of 2.5 cm. After 3 months of this treatment, tumor restaging tests showed that the tumor radius had decreased to 1.8 cm.

[0470] The patient progressed from stage IV to stage III while receiving chemotherapy infusions. At this point in the treatment, only chemotherapy pills were administered, with no further infusions. The patient was able to operate more independently, with virtually no side effects from the chemotherapy infusions.

[0471] Ultimately, the patient discontinued treatment and passed away (returning to stage IV, chemotherapy, and side-effect medications), but the patient did extend their life by up to 14 months from the original 4-month prognosis, for a total of 18 months.

[0472] Example 46: Treatment of Stage III-IV Lung Cancer

[0473] A 63-year-old Vietnamese-American woman was diagnosed with stage III-IV lung cancer. In 2013, she received chemotherapy pills, and the cancer progressed from stage I to stage III. She is currently undergoing chemotherapy and is in stage IV.

[0474] From July to October 2013, the patient received a replacement therapy three times daily, consisting of ¼ oz fresh pineapple, 3-4 cm slices of juiced fresh ginger root, and one raw egg white per 12 oz cup. Prior to the replacement therapy, the patient was bedridden, had pale skin with itchy lumps, a lemon-sized hole in the neck, severe weight loss, and was emaciated. Three months of this regimen resulted in a return to "normal" physical condition; weight gain, pink skin, the absence of itchy lumps and lesions, and the ability to move independently again with normal energy levels.

[0475] Due to the monotony of treatment, the patient discontinued alternative therapy after October 2013. Cancer recurrence indicates that this discontinuation led to the progression of the cancer from stage III to stage IV.

[0476] Example 47: Treatment of Stage III-IV Colon Cancer

[0477] A 68-year-old Vietnamese-American woman developed stage III-IV colon cancer. In May 2014, she underwent surgery to remove the malignant portion of her colon during the stage III cancer phase. The surgery subsequently led to progression to stage IV cancer.

[0478] Since April 2014, the patient has been taking a 12-ounce cup of alternative therapy made from ¼ of a fresh pineapple, 2-3 cm slices of juiced fresh ginger, and 1 raw egg white, twice daily. The treatment is ongoing.

[0479] Example 48: Treatment of Stage IV Breast Cancer + Lymphoma + Bone Cancer

[0480] A 64-year-old Russian-American woman was diagnosed with stage IV breast cancer, lymphoma, and bone cancer. She had been undergoing radiation and chemotherapy concurrently since being diagnosed with stage IV breast cancer that had spread to the lymph nodes and bone tissue in April 2013.

[0481] The alternative therapy program is underway and began in May 2013. The program consists of juice from one whole pineapple plus a 6-7 cm slice of ginger every two days, and one fresh egg white to each cup before consumption. The 12-ounce composition is consumed twice a day.

[0482] Example 49: Treatment of Stage IV Prostate Cancer + Lymphoma

[0483] A 71-year-old American male patient was diagnosed with stage IV prostate and lymphoma. He had previously undergone surgery on his bladder to remove the cancerous portion, but the cancer has now recurred.

[0484] Prepare two 12-ounce cups using a replacement therapy consisting of 4-5 cm slices of juiced fresh ginger and half a pineapple. Consume one cup for breakfast and refrigerate the other for the evening. For each cup, add a single raw egg white and shake well. Patients should consume the replacement therapy daily for at least two weeks.

[0485] Prior to replacement therapy, the patient was diagnosed with stage IV cancer, indicating that the cancer was spreading to parts of the bladder, colon, and lymph nodes. After two weeks of replacement therapy, a PET scan showed that the cancer was now confined to the pelvic region. The patient is able to move independently daily and has a normal appetite. Treatment is ongoing.

[0486] Example 50: Exemplary reagents / enzymes tested and results

[0487] Saline solution: Observation of ionic effects successfully induced the degradation of the extracellular matrix in patches such as clarifying agents.

[0488] White vinegar (acetic acid): was chosen as a disinfectant with low toxicity. However, for effectiveness, successful experiments removing the extracellular matrix require more complex dilutions than those using proteolytic enzymes, without damaging the cells.

[0489] 10% liquid bleach (sodium hypochlorite): Selected because of its properties as a disinfectant / bleach that is strong enough to successfully remove blood, but is toxic and removes the extracellular matrix at a very low rate.

[0490] Calcium hypochlorite: Selected for its disinfectant properties and as a bleaching agent, unlike sodium hypochlorite, it is safe for use in drinking water. It successfully removed the extracellular matrix in experiments, however, at a much slower rate than proteolytic enzymes.

[0491] Pineapple (bromelain + kiwifruit protease): Successful, but dilution is needed to achieve safe efficacy without damaging whole cells. Once optimal concentrations are reached, observable differences in the extracellular matrix are slowly observed over time.

[0492] Powdered bromelain (meat tenderizer): An alternative to fresh pineapple, but unsuccessful because it does not achieve the desired effect of removing the extracellular matrix, but only damages the cells.

[0493] Ginger root (GP / zingibain): An alternative to fresh pineapple was chosen due to its proteolytic properties. It successfully removed the extracellular matrix at a rate similar to that of fresh pineapple, but required dilution.

[0494] Turmeric: Selected for its properties of healing wounds and reducing scars.

[0495] 3% H2O2 (hydrogen peroxide): Selected for its disinfecting and bleaching properties on surfaces and wounds. It cannot successfully remove the extracellular matrix because it has no effect on it.

[0496] UV: Selected as a UV (and therapeutic) display that can be applied to cells - removing the shell and embryo before hatching.

[0497] Albumin: Although it is a protein in itself, it is chosen for its known wine-clarifying properties to remove impurities while remaining non-toxic to wine. Success in experiments requires dilution and very small ratios for use to avoid exceeding its intended application.

[0498] 70% ethanol: chosen because of its disinfectant properties, but unsuccessful, completely damaging cells.

[0499] Destaining agent: Selected for its dye removal properties in microbial staining tests while maintaining the integrity of the cells under study. However, successful removal of the extracellular matrix requires dilution to slow down the rate of extracellular matrix degradation.

[0500] Acidic alcohol (acetone): chosen for its cleaning and peeling properties, it has proven unsuccessful because the cells are severely damaged.

[0501] Powdered urea: Selected for its use in the kidneys to remove nitrogenous waste. It successfully removes the extracellular matrix, but requires vigorous dilution to achieve its effectiveness.

[0502] Ammonia: Selected for its cleaning properties and similar to urea, but more mobile than urea. Successful, but firmly diluted.

[0503] Sodium bicarbonate (baking soda): Selected for its cleaning and fermentation properties; it has low toxicity because it can be consumed. It effectively removes the extracellular matrix, however, at a very slow rate.

[0504] Purigen: Used for its ability to remove impurities from water and nitrogenous organic waste from aquariums. Since it is safe to use in the presence of fish, it will have similar effectiveness to proteolytic enzymes in the presence of brine shrimp.

Claims

1. A liquid composition comprising two or more proteolytic enzymes selected from the group consisting of: bromelain, kiwifruit protease, ginger protease, papain, diarylheptane metabolites, fig protease, and a clarifying agent, wherein the clarifying agent is selected from the group consisting of: fish glue (swim bladder glue), gelatin, silica sol, chitosan, silica sol + chitosan, sodium alginate, bentonite, diatomaceous earth, and albumin.

2. The composition of claim 1, wherein the clarifying agent is fish glue (swim bladder glue), gelatin, silica sol, chitosan, silica sol + chitosan, sodium alginate, bentonite, albumin, or diatomaceous earth.

3. The composition of claim 1, wherein the composition comprises at least two proteolytic enzymes of bromelain, kiwifruit protease, or ginger protease.

4. The composition of claim 1, wherein the clarifying agent is albumin.

5. The composition of claim 1, wherein the composition is in the form of an intravenous dosage form.

6. The composition of claim 1, wherein the composition is in an oral dosage form.

7. A liquid composition comprising two or more proteolytic enzymes selected from the group consisting of: bromelain, kiwifruit protease, ginger protease, papain, diarylheptane metabolites, fig protease; a clarifying agent, wherein the clarifying agent is fish glue (swim bladder glue), gelatin, silica sol, chitosan, silica sol + chitosan, sodium alginate, bentonite, diatomaceous earth, or albumin; and a pharmaceutically acceptable carrier, including excipients and adjuvants.

8. The composition of claim 7, wherein the composition is in the form of an intravenous dosage form.

9. The composition of claim 7, wherein the composition is in the form of an oral dosage form.

10. Use of the composition of any one of claims 1-9 in the preparation of a medicament for treating and / or preventing cancer in a subject, wherein the composition comprises a therapeutically effective amount of two or more proteolytic enzymes and a clarifying agent.

11. The use as claimed in claim 10, wherein the subject comprises a mammal.

12. The use as claimed in claim 10, wherein the subject is a human.

13. The use as claimed in claim 10, wherein the subject is a dog, cat, horse, or bird.

14. The use of claim 10, wherein the cancer is selected from the group consisting of: solid tumors with or without metastasis and hematologic malignancies.

15. The use of claim 10, wherein the cancer is selected from the group consisting of: solid tumors of the bladder, intestine, brain, breast, endometrium, heart, kidney, lung, liver, uterus, lymphatic tissue, ovary, pancreas or other endocrine organs, prostate, and skin; and leukemia and lymphoma.

16. The use of claim 10, wherein the cancer is selected from the group consisting of: ovarian cancer, prostate cancer, breast cancer, lung cancer, melanoma, head and neck cancer, colorectal cancer, thyroid cancer, glioblastoma, follicular lymphoma, renal cancer, Hodgkin's lymphoma, hepatocellular carcinoma, pancreatic cancer, and melanoma.

17. The use of claim 10, wherein the cancer includes bone metastases.

18. The use of claim 10, wherein the cancer is selected from the group consisting of: oral cancer, prostate cancer, rectal cancer, non-small cell lung cancer, lip and oral cavity cancer, liver cancer, lung cancer, anal cancer, kidney cancer, vulvar cancer, breast cancer, oropharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, urethral cancer, small intestine cancer, bile duct cancer, bladder cancer, ovarian cancer, laryngeal cancer, hypopharyngeal cancer, gallbladder cancer, colon cancer, colorectal cancer, head and neck cancer, parathyroid cancer, penile cancer, vaginal cancer, thyroid cancer, pancreatic cancer, esophageal cancer, Hodgkin's lymphoma, leukemia-related diseases, mycosis fungoides, and myelodysplastic syndrome.

19. The use of claim 10, wherein the cancer is selected from the group consisting of: non-small cell lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, and head and neck cancer.

20. The use of claim 10, wherein the cancer is a leukemia selected from the group consisting of: acute lymphoblastic leukemia, acute myeloid leukemia, myeloblastic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

21. The use of claim 10, wherein the cancer is a lymphoma selected from the group consisting of: nervous system lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, and Waldenström's macroglobulinemia.

22. The use of claim 10, wherein the cancer is a melanoma selected from the group consisting of: acral lentigines melanoma, superficial diffuse melanoma, uveal melanoma, malignant lentigines melanoma, intraocular melanoma, adenocarcinoma nodular melanoma, and hemangioma.

23. The use of claim 10, wherein the cancer is a sarcoma selected from the group consisting of: adenoma, adenosarcoma, chondrosarcoma, endometrial stromal sarcoma, Ewing's sarcoma, Kaposi's sarcoma, leiomyosarcoma, rhabdomyosarcoma, uterine sarcoma, osteosarcoma, and pseudosarcoma.

24. The use of claim 10, wherein the cancer is a glioma selected from the group consisting of: brainstem gliomas and hypothalamic and optic pathway gliomas.

25. The use of claim 10, wherein the cancer is a germ cell tumor selected from the group consisting of: pulmonary germ cell tumor, pleural pulmonary germ cell tumor, retinoblastoma, neuroblastoma, ductoblastoma, glioma, and angioblastoma.

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