Treatment of Solid Cancerous Tumors by Oral Administration of a Halogenated Xanthene
The present invention contemplates a method of treating a solid cancerous tumor in a mammalian subject by the oral administration of a solid cancerous tumor-effective amount of a halogenated xanthene (HX), the lactone thereof, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof (collectively an HX compound) to that mammalian subject, and liquid and solid pharmaceutical compositions for administering the HX compound. In addition, a method of inhibiting the growth of a GI tract carcinoma of a mammalian subject by the oral administration of a solid cancerous tumor-effective amount of an HX compound, as well as solid and liquid pharmaceutical compositions therefor.
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The present invention contemplates methods and compositions for treating solid cancerous tumors by oral treatment with a halogenated xanthene.
BACKGROUND ARTCancers are classified in two ways: by the type of tissue in which the cancer originates (histological type) and by primary site, or the location in the body where the cancer first developed. The following discussion is directed towards cancer classification based on histological type.
The international standard for the classification and nomenclature of histology is the International Classification of Diseases for Oncology, Third Edition (ICD-O-3), World Health Organization, Geneva, Switzerland. From a histological standpoint there are hundreds of different cancers, which are grouped into six major categories: Carcinoma, Sarcoma, Myeloma, Leukemia, Lymphoma, and Mixed Types.
Those six groups are also further subdivided into solid tumors and blood cancers. The solid tumors include Carcinoma, Sarcoma, Lymphoma, and Mixed Types, whereas Myeloma and Leukemias are blood cancers. This invention is directed towards solid tumors.
Sarcomas are a type of cancer that begins in the bones or in the soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissue. Different types of sarcoma are based on where the cancer forms.
Lymphoma is a cancer that begins in the cells of the immune system. There are two basic categories of lymphomas. One kind is Hodgkin lymphoma, which is marked by the presence of a type of cell called the Reed-Sternberg cell. The other category is non-Hodgkin lymphomas, which includes a large, diverse group of cancers of immune system cells. Non-Hodgkin lymphomas can be further divided into cancers that have an indolent (slow-growing) course and those that have an aggressive (fast-growing) course.
Mixed Type cancer is a rare type of cancer that is made up of at least two different types of germ cell tumors (tumors that begin in cells that form sperm or eggs). These can include choriocarcinoma, embryonal carcinoma, yolk sac tumor, teratoma, and seminoma. Mixed germ cell tumors occur most often in the ovary or testicle, but they may also occur in the chest, abdomen, or brain.
Carcinomas, malignancies of epithelial tissue, account for 80 to 90 percent of all solid tumor cancer cases. The word “carcinoma” refers to a malignant neoplasm of epithelial origin or cancer of the internal or external lining of the body.
Epithelial tissue is found throughout the body. It is present in the skin, as well as in the covering and lining of organs and internal passageways, such as the gastrointestinal (GI) tract. Most carcinomas affect organs or glands capable of secretion, such as the breasts, which produce milk, or the lungs, which secrete mucus, or the colon or the prostate or the bladder.
Carcinomas are divided into two major subtypes: adenocarcinomas, which develop in an organ or gland, and squamous cell carcinoma, which originates in the squamous epithelium. Adenocarcinomas generally occur in mucus membranes and are first seen as a thickened plaque-like white mucosa. They often spread easily through the soft tissue where they occur. Squamous cell carcinomas occur in many areas of the body.
Of the carcinomas, those of the gastrointestinal tract are of particular interest to the present invention. The gastrointestinal tract is comprised of the organs that food and liquids travel through when they are swallowed, digested, absorbed, and leave the body as feces. These organs include the mouth, pharynx (throat), esophagus, stomach, small intestine, large intestine, rectum, and anus. The GI tract is also called the alimentary tract and the digestive tract.
Switching from histological nomenclature types, while maintaining carcinoma, each of the above-named organs can have an organ-named carcinoma to which it is more usually referred, such as esophageal cancer, stomach cancer, colon cancer and the like. Organs such as the pancreas, the gall bladder, and the appendix that are or can sometimes be deemed to be part of the gastrointestinal tract are not included herein. Although those organs are connected to the route from mouth to anus, little of what goes into the mouth goes into those three organs. Rather, the flow is largely one way, from each of the organs into the path to the anus.
Starting at the mouth, there are several carcinomas of the mouth (oral cavity) and surrounding areas. Mouth cancer is one of several types of cancers grouped in a category called head and neck cancers. Mouth cancer and other head and neck cancers are often treated similarly.
Mouth carcinomas most commonly begin in the flat, thin cells (squamous cells) that line the lips and the inside of the mouth. Most oral cancers are squamous cell carcinomas.
Carcinomas of the nose and throat are also relatively common. Such cancers can arise in the nasopharynx (upper throat), oropharynx (middle throat), and hypopharynx (lower throat). These typically arise from squamous cells that line the mouth and throat.
The most common esophageal tumor in the proximal (upper) two thirds of the esophagus is squamous cell carcinoma; adenocarcinoma is the most common in the distal (lower) one third. Most adenocarcinomas arise in Barrett's esophagus, which results from chronic gastroesophageal reflux disease and reflux esophagitis. In Barrett esophagus, a metaplastic, columnar, glandular, intestine-like mucosa with brush border and goblet cells replaces the normal stratified squamous epithelium of the distal esophagus during the healing phase of acute esophagitis when healing takes place in the continued presence of stomach acid.
Stomach (gastric) cancers tend to develop slowly over many years. Before a true cancer develops, pre-cancerous changes often occur in the inner lining (mucosa) of the stomach. These early changes rarely cause symptoms, so they often go undetected.
Cancers starting in different sections of the stomach can cause different symptoms and tend to have different outcomes. The cancer's location can also affect treatment options. For example, cancers that start at or grow into the gastroesophageal (GE) junction are usually staged and treated the same as cancers of the esophagus.
Most cancers of the stomach (about 90% to 95%) are adenocarcinomas. These cancers develop from. the gland cells in the innermost lining of the stomach (the mucosa).
There are two main. types of stomach adenocarcinomas. The intestinal type tends to have a slightly better prognosis. The cancer cells are more likely to have certain gene changes that might allow for treatment with. targeted drug therapy. The diffuse type tends to grow and spread more quickly. It is less common than the intestinal type, and it tends to be harder to treat.
The types of cancer found in the small intestine are adenocarcinoma, sarcoma, carcinoid tumors, gastrointestinal stromal tumor, and lymphoma. Adenocarcinoma starts in glandular cells in the lining of the small intestine and is the most common type of small intestine cancer. Most of these tumors occur in the part of the small intestine near the stomach.
Colorectal cancer is the third most common cancer in both men and women in the U.S., with an incidence of approximately 150,000 new cases annually, and the second most common cause of cancer mortality, resulting in nearly 53,000 deaths [Cancer Facts & Figures 2021, American Cancer Society, Atlanta, Ga., 2021]. Although incidence has been declining by approximately 1% per year between 2013 and 2107, and mortality has declined approximately 2% per year between 2014 and 2018, incidence in adults younger than 50 years has increased at double the rate of the general population, foreshadowing a potential reversal of progress in mortality driven by younger adults. Each of these statistics highlights a compelling need for further improvement in detection, treatment and prevention.
More than 90 percent of colorectal malignancies develop from adenomatous polyps into adenocarcinomas. These can be defined as well demarcated masses of epithelial dysplasia, with uncontrolled crypt cell division. Although several lines of evidence indicate that carcinomas usually originate from pre-existing adenomas, this does not imply that all polyps undergo malignant changes, and does not exclude “de novo” carcinogenesis [Ponz et al., Digest Liver Dis 33(4):372-388 (May 23001)]. The incidence and epidemiology, etiology, pathogenesis, and screening recommendations are common to both colon cancer and rectal cancer.
Clinical studies demonstrating that certain non-steroidal anti-inflammatory drugs (NSAIDs) can inhibit colorectal carcinogenesis and may be chemopreventive [Chan et al., Ann. Intern. Med., 140:157-166 (2004] offer hope that these trends may improve. Additional preclinical and early clinical studies have suggested that the benefits of NSAIDs may extend to COX-2 inhibitors via antiangiogenic and proapoptotic effects [Gasparini et al., Lancet Oncol., 4: 605-615 (2003); Chen et al., Cancer Sci., 94:253-258 (2003); Rahme et al., Gastroenterology, 125: 404-412 (2003); Evans et al., Am. J. Clin. Oncol., 26:S62-65 (2003); and Rao et al., Curr. Cancer Drug Targets, 4: 29-42 (2004)]. Unfortunately, the most effective NSAID doses may be well above those typically used for long-term therapy, suggesting potential risk with prophylactic use [Chan et al., Ann. Intern. Med., 140:157-166 (2004); Rao et al., Curr. Cancer Drug Targets, 4: 29-42 (2004); Mamdani et al., Lancet, 363:1751-1756 (2004); Mukherjee et al., J Amer Med Assoc, 286:954-959 (2001); and Fitzgerald, N. Engl. J. Med., 351:1709-1711 (2004)] and emphasizing the need for further clinical development and drug discovery.
Anal cancer is an uncommon type of cancer that occurs in the anal canal. The anal canal is a short tube (about 3 to 5 cm) at the end of the rectum to the anal verge through which feces leave the body. The anal verge is where the anal canal connects to the outside skin at the anus. This skin around the anal verge is called the perianal skin.
At the anal verge, the squamous cells of the lower anal canal merge with the skin just outside the anus. This skin around the anal verge is called the perianal skin and is also made up of squamous cells, but it also contains sweat glands and hair follicles, which are not found in the lining of the lower anal canal. The inner lining of the anal canal is the mucosa. Most anal cancers start from cells in the mucosa, and about 90 percent of the anal cancers in the United States are squamous cell carcinomas.
Rose Bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein; RB) is a non-toxic small molecule with an extensive history of safe use in humans spanning a period of almost 90 years. Assessments of new therapeutic uses of RB for dermatology and oncology are available in the literature [Wachter et al., Lasers Surg. Med., 32:101-110 (2003); Wachter et al., “Functional Imaging of Photosensitizers Using Multiphoton Microscopy”, in: A. Periasamy and P. T. So, P. T. (Eds), Multiphoton Microscopy in the Biomedical Sciences II. SPIE, Bellingham, Wash., vol. 4620, pp. 143-147 (2002); and Wachter et al., (2002) “Imaging Photosensitizer Distribution and Pharmacology Using Multiphoton Microscopy”, in: D. L. Farkas and R. C. Leif, R. C. (Eds) Optical Diagnostics of Living Cells V. SPIE, Bellingham, Wash., vol. 4622, pp. 112-118 (2002)]. A thorough review of long-term toxicology data has also been published [Ito et al., J Natl Cancer I, 77:277-281 (1986)].
Reviewing these publications, we noticed a striking but apparently unrecognized trend: continuous oral feeding of RB (at a concentration of 0.1-0.5% in drinking water) to B6C3F1 mice led to substantially greater survival rates at 82 weeks age relative to untreated controls (i.e., 94% vs. 52% for male controls, 90% vs. 64% for female controls). Because this mouse strain exhibits a marked tendency for spontaneous neoplasia, particularly liver adenoma and carcinoma [Haseman et al., Toxicol. Pathol., 26:428-441 (1998)], these data suggested to us that continuous administration of RB might yield a preventive, therapeutic, chemopreventive or chemotherapeutic effect against some tumors.
BRIEF SUMMARY OF THE INVENTIONThe present invention contemplates a method of treating a solid cancerous tumor in a mammalian subject by the oral administration of an effective amount of a halogenated xanthene (HX), the lactone thereof, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof to the mammalian subject. Preferably, that HX, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof is rose bengal disodium. Specifically contemplated pharmaceutically acceptable salts, C1-C4 alkyl and aromatic esters are discussed in detail hereinafter. A contemplated halogenated xanthene, its pharmaceutically acceptable salt, and C1-C4 alkyl or aromatic ester thereof are collectively grouped hereinafter under the term “HX compound”, for efficiency of expression.
One aspect of the treatment of solid cancerous tumors is that the treated tumors are carcinomas, sarcomas, lymphomas, and mixed types tumors. In a preferred aspect, the treated solid tumors are carcinomas. In a further preferred aspect, the carcinoma is present in the gastrointestinal (GI) tract.
Exposure of solid tumor tissue can occur through direct interaction (contact) of an administered HX compound within the gastrointestinal (GI) tract and/or through absorption of administered HX compound from the GI tract into the bloodstream and resultant distribution and delivery via the bloodstream of a portion of the administered HX to tumor sites in the GI tract or elsewhere in the body beyond the GI tract.
An exemplary aspect that has arisen as part of our study of the herein-contemplated oral treatment solid tumors is that oral administration of a contemplated HX compound to a mammalian subject can inhibit the formation of a carcinoma of the gastrointestinal tract (GI) such as a colorectal carcinoma, as well as treat an already-formed colorectal carcinoma.
A preferred HX compound is rose bengal, its disodium (RB disodium) salt, or rose bengal lactone. The method is typically repeated several times, particularly when inhibiting the formation of a gastrointestinal tract carcinoma is intended.
In a preferred aspect, the HX compound is dissolved or dispersed in an aqueous diluent when administered to a mammalian subject. It is more preferred that the aqueous diluent is free of tonicity agents except for those sugars and/or buffering agents present as flavorants.
In another preferred aspect, the HX compound is within a solid diluent matrix as part of a solid pharmaceutical composition when administered to the mammalian subject. Preferably, the composition releases the HX compound at the pH value of the portion of the GI tract in which the carcinoma is located or where systemic absorption is optimized. Illustrative carcinoma locations are in the stomach, the small intestine or the colon. Illustrative locations for release for optimized systemic absorption are the duodenum and the small intestine.
In one aspect, the solid pharmaceutical composition is a tablet. In another aspect, the solid pharmaceutical composition is a capsule that contains a plurality of coated particles that contain the HX compound. Preferably, the coating of those coated particles dissolves or disintegrates at a preselected pH value to release the HX compound.
It is preferred in each embodiment and aspect of the invention above that the HX compound active ingredient rose bengal (RB), which once in the body can be present as a salt or ester. Disodium rose bengal (RB disodium) is particularly useful because of its solubility in aqueous media. Further, rose bengal in the lactone form is also preferred because it can be prepared in an extremely pure form and converts to the absorbable salt form at intraluminal pH values great that about pH 5.
In the drawings forming a part of this disclosure:
The present invention contemplates a method of treating a solid cancerous tumor in a mammalian subject by the oral administration of an effective amount of a halogenated xanthene (HX), the lactone thereof, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof (HX compound) to that mammalian subject.
That any cancer, let alone a solid cancerous tumor such as a solid cancerous tumor of the GI tract like colorectal cancer, could be affected in any way by an orally administered HX compound was quite unexpected because of low HX compound bioavailability, first pass losses of the drug, and also because of the relatively short half-time (about 30 minutes) previously reported for HX compounds such as rose bengal (RB) in other contexts. It was thus unexpected that oral administration of rose bengal disodium, an illustrative HX compound in a pharmaceutically acceptable salt form, could slow the progress of colorectal tumor development in animals specially bred to develop colorectal tumors in the absence of any treatment. It was even more unexpected that an orally-delivered contemplated HX compound could prevent formation of a colorectal cancerous tumor in those specially bred animals.
According to eNews published by The Jackson Laboratory (Apr. 8, 2015), the purveyor of study animals used, one hundred percent (100%) of the (C57BL/6J-Min/+ (heterozygous) mice (ApcMin) raised on a high fat diet develop in excess of 30 adenomas throughout the intestinal tract and most die by 120 days of age [Moser et al., Science 247:322-324 (1990)]. The data of
Assessment of the change in tumour burden is an important feature of the clinical evaluation of cancer therapeutics: tumour shrinkage (objective response), tumor stasis (disease control), and disease progression are useful endpoints in clinical trials. Criteria for assessment of a change in tumor burden were published by Eisenhauer et al., Eur J Cancer 45:228-247 (2009). Those revised guidelines are referred to as Revised RECIST guideline (version 1.1) and are set out below:
4.3.1. Evaluation of Target Lesions
Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm.
Partial Response (PR): At least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.
Progressive Disease (PD): At least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression).
Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
Using the above criteria, the HX compound-containing composition is administered at least until stable disease is achieved (disease control). More preferably, that administration is continued until a partial response is achieved. Most preferably, treatment continues until a complete response is obtained.
The administration is thus preferably repeated a plurality of times and in some instances, for the remainder of the mammalian subject's lifetime where prevention is contemplated. Treatment can be given once or more often each day or less frequently as ordered by a treating physician.
A mammalian subject having a having a solid cancerous tumor in need of treatment to which a pharmaceutical composition containing an HX compound such as disodium RB is administered can be a primate such as a human; an ape such as a chimpanzee or gorilla; a monkey such as a cynomolgus monkey or a macaque; a laboratory animal such as a rat, mouse, or rabbit; a companion animal such as a dog, cat, horse; or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama, or the like.
In one aspect, the invention contemplates treating mammalian subject with a solid tumor cancer by orally administering to that mammalian subject a carcinoma-treating effective amount of a halogenated xanthene (HX), the lactone thereof, a pharmaceutically acceptable salt, a C1-C4 alkyl or aromatic ester thereof, or collectively “HX compound.”
A particular aspect of the above aspect contemplates a method of treating or inhibiting the formation of a gastrointestinal tract carcinoma of a mammalian subject. That method comprises orally administering to that mammalian subject, in whose GI tract (a) a carcinoma is to be inhibited from forming or (b) a carcinoma is present to be treated, a carcinoma-formation-inhibiting or carcinoma-treating effective amount of a halogenated xanthene (HX), the lactone thereof, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof.
Oral Administration of Halogenated XanthenesA solid cancerous tumor-treating effective amount of a halogenated xanthene, the lactone thereof, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof can be orally administered in solid or liquid form. The halogenated xanthene is preferably rose bengal, and more preferably a pharmaceutically acceptable salt of rose bengal such as the disodium or dipotassium salt.
A contemplated HX compound includes rose bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodo-fluorescein; RB) that is particularly preferred, erythrosin B, phloxine B, 4,5,6,7-tetrabromo-2′,4′,5′,7′-tetra-iodofluorescein, 2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein, 4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein, 2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein, 4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein, 2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein, 4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein, 4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein, 4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, and 4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein.
The reader is directed to Berge, J. Pharm. Sci. 1977 68(1):1-19 for lists of commonly used pharmaceutically acceptable acids and bases that form pharmaceutically acceptable salts with pharmaceutical compounds, such as the above halogenated xanthenes. Illustrative cations include alkali metals such as sodium, potassium, as well as ammonium and alkaline earth salts such as magnesium and calcium. The disodium salt of rose bengal is particularly preferred.
A C1-C4 alkyl ester of one of the above halogenated xanthene compounds can also be used, with the C2; i.e., ethyl ester, being preferred. In vitro studies using each of RB, ethyl-Red 3 (erythrosine ethyl ester; 2′,4′,5′,7′-tetraiodo-fluorescein ethyl ester), 4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein and ethyl-Phloxine B (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetrabromo-fluorescein ethyl ester) exhibited similar anti-tumor activities against CCL-142 renal adenocarcinoma.
A contemplated aromatic ester is formed by a reaction between an HX compound and an aromatic alcohol having a 5- or 6-membered aromatic ring, or a 5,6- or 6,6-fused aromatic ring system that contains 0, 1 or 2 hetero ring atoms that are independently nitrogen, oxygen or sulfur. When an aromatic ester is used, it is preferably a benzyl, phenyl or a 2-, 3-, or 4-pyridyl (pyridyl) ester, other aromatic single and fused ring-containing esters are contemplated as discussed hereinafter. It is to be understood that although a benzyl ester is often considered to be an “aralkyl ester”, for the purposes of this invention, a benzyl ester is deemed an aromatic ester.
Illustrative examples of such aromatic alcohol ester portions are shown and named below, where O is an oxygen atom and line-O indicates the ring-oxygen can be from any available carbon of the ring and the O-line crossed by a wavy line indicates that the depicted alkoxy group is a portion of another molecule, the esterified HX molecule.
Rose bengal is a preferred HX compound and its disodium salt, rose bengal disodium, is the most preferred. A structural formula of rose bengal disodium is shown below:
Further details of the medicinal use of a pharmaceutical composition containing an above-noted HX compound are described in U.S. Pat. Nos. 5,998,597, 6,331,286, 6,493,570, 7,390,688, 7,648,695, 8,974,363, 9,107,887, 9,808,524, 9,839,688, 10,130,658 and 10,471,144, whose disclosures are incorporated by reference herein in their entireties.
Dosing—FIG. 3Upon exposure of tumor cells to an HX compound, irreversible accumulation of the HX compound occurs in tumor lysosomes, precipitating tumor autolysis once a sufficient concentration is achieved to destabilize lysosomal integrity [Wachter et al., SPIE 4620:143-147 (2002)]. This suggests that this immunogenic mechanism of cell death can be elicited over a range of exposure conditions based on a concentration·time function, where cytotoxicity is proportional to the product of these two parameters (i.e., cytotoxicity=f([HX]·t)).
For example, when RB is administered in vivo by intralesional injection to a range of solid tumors (e.g., melanoma, hepatocellular carcinoma, breast carcinoma) acute tumor cytotoxicity is evident within approximately 30 minutes for intratumoral RB concentrations of approximately 25-50 mg/g tumor tissue (25-50 mM) [Thompson et al, Melanoma Res 18:405-411 (2008)].
Swift et al. [Oncotargets Ther 12:1293-1307 (2019)] demonstrated cytotoxicity of treatment-refractory pediatric solid tumors (neuroblastoma and neuroepithelioma) upon in vitro exposure to RB for 96 hours at concentrations of approximately 50-100 μM. Additionally, Swift et al., J Clin Oncol 36:Suppl; abstr 10557 (2018), showed cytotoxicity in additional treatment-refractory pediatric solid tumors (Ewing sarcoma, osteosarcoma and rhabdomyosarcoma) under equivalent exposure.
Extended exposure to RB in the context of continuous oral feeding has been shown to prevent formation of colon cancer (prophylactic activity) and to arrest colon cancer (therapeutic activity) in the murine ApcMin colorectal tumor model. For therapeutic use, symptomatic mice receiving RB ad libitum in drinking water at a concentration of 1 mg/mL had an approximate 38% increase in mean survival relative to untreated mice (12.3±0.5 weeks vs 9.8±0.8 weeks). Assuming a daily drinking water consumption rate of approximately 2 mL/10 g body weight, this corresponds to consumption of approximately 2 mg RB/10 g (200 mg/kg). Bioavailability of RB disodium administered in aqueous solution via the oral route appears to be limited based on mass balance studies conducted by the inventors, and can be estimated at 0.1-1%, corresponding to a daily systemic exposure of 0.2-2 mg/kg. Presuming this amount is distributed through the bloodstream, and that blood volume comprises approximately 10% of body weight, this equates to an estimated concentration of 2-20 μM RB in the blood.
Plotting these data confirm that the hypothesized relationship (i.e., cytotoxicity=f([HX]·t)) is supported by experimental results, as illustrated in
More importantly, this functional relationship allows prediction of dose level and schedule appropriate to achieve either an anti-tumor prophylactic outcome or an anti-tumor therapeutic outcome upon systemic administration. For extended treatment schedules equivalent to that investigated with the ApcMin model low micromolar concentrations (i.e., about 10 μM) of circulating HX compound are sufficient to achieve lysosomal accumulation and tumor cell destruction over a period of approximately 3 months, whereas micromolar to submicromolar concentrations (i.e., about 1 μM) are sufficient to achieve tumor cell destruction over a period of approximately 12 months.
The ApcMin data herein show that a simple formulation of the disodium salt of RB is sufficient to deliver a therapeutically active level of RB; however, this may be less than ideally efficient as to bioavailability. Determining a suitable formulation to achieve efficient liberation and absorption of an orally delivered HX compound is thus a matter of standard pharmaceutical development familiar to those of skill in the art, where the properties of the formulation can be varied to achieve desired bioavailability by control of liberation (disintegration, disaggregation and dissolution) at an appropriate point within the GI tract so as to maximize absorption of the dissolved HX compound into the bloodstream.
Formulary optimization can be guided by standard pharmacokinetic study of absorption such that dose level and formulation are adjusted to achieve the necessary systemic exposure on the desired dose schedule (e.g., about 100 μM in the bloodstream for short duration exposure on the order of several days, about 1 to about 10 μM for intermediate duration exposure on the order of several months, to about <1 μM or lower for long-term exposure on the order of a year or more).
The dibasic salt forms of the HX compounds exist in solution having a pH greater than approximately 5, whereas at pH <5 the HX compounds spontaneously convert to their lactone form. Because the dibasic salt forms are highly soluble in aqueous media, whereas the lactone forms are insoluble in aqueous media, the former exhibit higher bioavailability in the GI tract compared to the latter. Thus, optimizing formulation to properly compensate for the pH value of the GI tract is perhaps the most important parameter affecting bioavailability. For example, in the stomach, where pH <4, dissolved HX compound is rapidly converted to the insoluble lactone form. Once in the lactone form, the HX compounds exhibit hysteresis and hinderance to saponification back to the absorbable salt form, delaying or inhibiting downstream bioavailability.
However, the intraluminal pH value rapidly increases from highly acidic in the stomach to around pH 6 in the duodenum, and further increases in the small intestine from pH 6 to about pH 7.4 in the terminal ileum; pH drops to 5.7 in the caecum before gradually increasing to pH 6.7 in the rectum. [pubmed.ncbi.nlm.nih.gov/10421978/.] Thus, by applying standard means in the art of pharmaceutical formulation to achieve intestinal liberation, where the favorable pH values facilitate HX compound liberation in a dissolvable, absorbable dibasic salt form, bioavailability can be optimized.
For a 12-month treatment regimen, these data indicate that a target concentration of approximately 1 μM (1 mg/L) should be achieved in the bloodstream. For a 70 kg adult human, where blood volume comprises approximately 10% of body weight (i.e., about 7 L), this implies absorption of 7 mg HX compound/day. If bioavailability is limited to 1% of administered HX compound, then 700 mg HX compound per os (PO) would be required daily to achieve this target blood level.
However, by optimizing absorption to 50% of administered dose, the necessary PO dose is reduced to approximately 15 mg daily. For a shorter treatment regimen (i.e., 3 months), these data indicate that a target blood concentration of approximately 10 μM (10 mg/L) should be achieved. Presuming 1% bioavailability, then 7 g HX compound PO would be required daily, whereas at 50% bioavailability, the necessary dose is reduced to approximately 150 mg daily.
In one aspect of the invention, a contemplated HX compound for oral administration is typically used dissolved or dispersed in a sterile aqueous pharmaceutical composition. Sterile tap water or sterile water from another source can be used.
An HX compound is typically present in a contemplated aqueous pharmaceutical composition at about 0.1 to about 20% (w/v). More preferably, that concentration is about 0.2 to about 10% (w/v), most preferably, the concentration is about 0.2 to about 5% (w/v). Thus, for example, the above dose of 150 mg daily could readily be achieved by use of 3 mL of a 5% (w/v) aqueous solution.
The bioavailability of various HX compounds such as disodium rose bengal has not been well characterized. Studies commissioned by one of the assignees concluded that bioavailability of disodium rose bengal is less than (<) 1% based on radiolabel studies where 14C-RB in aqueous solution was given orally to mice. In the stomach, with a pH value <4, an HX compound is likely to be in the lactone form. Conversion to lactone in the stomach does not destroy the HX compound, but such conversion into the lactone form can present a kinetic and/or thermodynamic barrier to reconversion to the soluble salt form necessary for absorption in the intestines.
The study discussed hereinafter in Apcmin mice showed that those mice consumed 4 mg/mL ad libitum in drinking water arrested onset of disease. Those Apcmin mice are understood to have thereby consumed 8 mg/10 g/day=800 mg/kg/day. That amount is consistent with toxicology data showing such a dose is tolerated. Thus, Ito et al., J Natl Cancer I, 77:277-281 (1986) studying rose bengal as a food coloring (Food Red No. 105) found that rose bengal fed ad libitum continuously for 2 years at a dose of 970 mg/kg/day to C57BL6N mice was well tolerated. The previously used intravenous (IV) liver diagnostic delivered 112 mg of rose bengal as a bolus; for a standard 60 kg adult human, which equates to 1.9 mg/kg; this has not reported to yield morbidity.
General Characteristics of Contemplated Pharmaceutical CompositionsIt is preferred that the composition be free of tonicity agents (or tonicity-adjusting agents) such as sugars like mannitol and dextrose, C3-C6 polyhydroxy compounds such as propylene glycol, glycerol and sorbitol, isotonic salts such as sodium or potassium chloride, and/or buffering agents other than those such as citric acid, malic acid, acetic acid and other food acids and their salts that can be provided for flavor and mild buffering (less than 5 mmol of buffering agent). The stomach and lower GI tract are well adapted to provide the proper tonicity to materials flowing through such that further salts and/or buffers are not needed. One or more pharmaceutically acceptable taste-masking agents or flavorants as are well-known can be present at up to about 5% by weight to enhance the potability of the composition.
Liquid Pharmaceutical CompositionsFor treating a solid cancerous tumor in the GI tract or elsewhere in the body, the aqueous liquid pharmaceutical composition containing a solid cancerous tumor-treating amount of an HX compound can be administered by drinking or by gavage. Where the tumor to be treated is in the buccal cavity, that aqueous liquid pharmaceutical composition can be gargled and maintained within the buccal cavity and then spit out. For tumors such as nasopharyngeal carcinomas, a neti pot can be used to administer the liquid to the nasal cavity and eliminated as directed for a neti pot device.
The amount of HX compound delivered by a solid medicament composition is substantially the same as that from an aqueous composition. This is particularly the case where HX compound-release occurs at the duodenum or beyond toward the rectum and a pH value-sensitive release coating is utilized for the solid medicament at the intraluminal pH value of the duodenum or intestines so that uncontrolled formation of the lactone form of the HX compound in the stomach can be avoided.
It is preferred that the pH value of a pharmaceutically acceptable aqueous diluent be about 5 to about 9, to yield maximum solubility of the HX compound in an aqueous vehicle and assure compatibility with biological tissue. A particularly preferred pH value is about 5 to about 8, and more preferably between about 6 to about 7.5. At these pH values, the halogenated xanthenes typically remain in dibasic form, rather than the lactone that forms at low pH values.
An HX compound such as rose bengal is dibasic, having pKa values of 2.52 and 1.81. pKa value determinations for several contemplated halogenated xanthenes can be found in Batsitela et al., Spectrochim Acta Part A 79(5):889-897 (September 2011).
Although liquid pharmaceutical compositions for oral delivery of an HX compound such as rose bengal disodium can be particularly useful for treating or preventing a solid cancerous tumor of the mouth, larynx and esophagus, that use can be unsightly because the HX compound typically stains any tissues contacted. As a result, a solid form of an HX compound-containing pharmaceutical composition is also contemplated as another aspect of the invention.
Solid Pharmaceutical CompositionsIt is further contemplated that the HX compound such as RB or disodium RB, or a HX compound lactone such as RB lactone, be administered in a solid pharmaceutical composition for oral administration that is enterically-coated to pass through the stomach and release the HX compound relatively close to the site of the cancer (closer than the mouth) so that there will be a lesser amount of wasted HX compound bound to tissues dorsal to the site of the tumor and less likely visible tissue staining. The HX compound is typically dissolved in or dispersed in or on a solid diluent matrix.
There are several factors at play in the dissolution of an orally administered solid pharmaceutical product in a mammalian body. Among those factors are residence time of the medicament at different locations along the GI tract, particle size, solubility of the individual components of the medicament in the bodily fluids likely to be encountered from mouth to anus, the order in which various coating layers, when present, are applied to the medicament, as well as the pH value at which a particular coating layer is soluble.
For example, the highly acidic gastric environment (pH 1.5-2 in the fasted state; pH 3-6 in the fed state) rises rapidly to about pH 6 in the duodenum and increases along the small intestine to pH 7.4 at the terminal ileum. The pH value in the cecum drops just below pH 6 and again rises in the colon reaching pH 6.7 at the rectum [Hua, Front Pharmacol 11:Article 524 (April 2020)]. Observation of solutions of disodium RB mixed into a water solution having the pH value of the human stomach revealed rapid clouding of the admixture and clumping of the previously soluble disodium RB, presumably into the lactone form.
Gastric transit can range from 0 to 2 hours in the fasted state and can be prolonged up to 6 hours in the fed state. In general, the transit time in the small intestine is considered relatively constant at around 3 to 4 hours, but can range from 2 to 6 hours in healthy individuals. Colonic transit times can be highly variable, with ranges from 6 to 70 hours reported [Hua, Front Pharmacol 11:Article 524 (April 2020)].
One approach useful for predictable release of a medicament to a particular location in the GI tract relies upon pH-specific coatings and matrices that are dissolve or disintegrate at preselected GI tract pH values such as those noted previously. Particularly preferred for release in or near the colon, neutral or slightly alkaline pH values are utilized to release the drug in the distal part of the small intestine or in the colon.
The table below shows some examples of pH-dependent polymer coatings that have been used for the purpose of colonic targeting (local treatment) either alone or in combination, including some methacrylic resins (commercially available from Evonik Industries, AG, Essen, Germany as Eudragit®), and hydroxypropyl methylcellulose (HPMC; available from DuPont, Wilmington, Del. as Methocel™; and Ashland, Inc., as Benecel™, Wilmington, Del.) derivatives. In addition to triggering release at a specific pH value range, the enteric coating can protect the incorporated active agent against the harsh GI tract environment (e.g., gastric juice, bile acid, and microbial degradation) and can create an extended and delayed drug release profile to enhance therapeutic efficiency.
The Table below lists several commercially available enteric coating polymers and the “published pH release” value from their manufacturer. The “published pH release” values are not absolute for all compositions or environments, and pH values for dissolution or disintegration stated herein are based on those published values.
For the local treatment of colonic cancers, colon-targeted drug delivery systems have been actively pursued because conventional non-targeted therapy can have undesirable side-effects and low efficacy due to the systemic absorption of drug before reaching the target site. Liu et al., Eur. J. Pharm. Biopharm. 74:311-315 (2010), adopted dual coating approach by using the alkaline aqueous solution of Eudragit® S with buffering agents for inner layer and the organic solution of Eudragit® S for outer layer, accelerating the drug dissolution at pH values greater than 7. Subsequently, Varum et al., Eur. J. Pharm. Biopharm. 84:573-577(2013), evaluated in vivo performance of this dual coated system in humans, demonstrating more consistent disintegration of dual coated tablets mainly in the lower intestinal tract.
Hashem et al., Br. J. Pharm. Res. 3:420-434 (2013), developed microspheres combining time-and pH-dependent systems for colonic delivery of prednisolone. By using a combination of Eudragit® S and ethyl cellulose, they achieved greater colonic drug delivery while preventing premature drug release in the upper intestine.
Eudracol® is another example of a multi-unit technology providing targeted drug delivery to the colon, with delayed and uniform drug release. This system is based on coating the pellet with Eudragit® RL/RS and Eudragit® FS 30D, providing colon-specific drug release in a pH-and time-dependent manner [Patel, Expert Opin. Drug Deliv. 8:1247-1258 (2011)].
One composition that targets the small intestine comprises a diluent matrix of sugar/sucrose beads coated with particulate rose bengal (RB) that is coated with one or a plurality of layers of a (meth)acrylate copolymer that is composed of about 60 to about 95% by weight free radical polymerized C1-C4-alkyl esters of acrylic or methacrylic acid and about 5 to about 40% by weight (meth)acrylate monomers with an acidic group in the alkyl radical.
Particularly suitable (meth)acrylate copolymers include about 10 to about 30% by weight methyl methacrylate, about 50 to about 70% by weight methyl acrylate and about 5 to about 15% by weight methacrylic acid (Eudragit® FS type). Similarly suitable, are (meth)acrylate copolymers of about 20 to about 40% by weight methacrylic acid and about 80 to about 60% by weight methyl methacrylate (Eudragit® S type). Use of the word “(meth) acrylate” is used to mean that either or both of acrylate and methacrylate monomers can be used.
These coating polymers permit little if any HX compound release prior to the particles leaving the stomach. The pH value of the fluid within the duodenum typically is about 6 and rises to about 7.4 toward the ileum. Thus, if the cancerous tumor is closer to the stomach, a coating polymer with a greater amount of free carboxylic acid groups is utilized, whereas if the tumor is further toward the ileum, a polymer with a lesser of amount of acid groups can be utilized.
A usual tablet or lozenge can be prepared by admixture of lactose (20%) and active ingredient (80%; HX compound) mixed in a high-speed mixer (DIOSNA type P10, Osnabruck, Germany). An aqueous solution containing the excipient polyvinylpyrrolidone (PVP) such as povidone (Sigma-Aldrich International GmbH, Buchs, CH) is added in small amounts until a homogeneous composition is obtained. The moist powder mixture is screened. Tablets are subsequently made therefrom as is well-known, and dried.
The resulting tablets or lozenges are thereafter preferably coated with a protective polymer film, often using fluidized bed equipment. Film-forming polymers are normally mixed with plasticizers and release agents by a suitable process. The film formers can in this case be in the form of a solution or suspension. The excipients for the film formation can likewise be dissolved or suspended. Organic or aqueous solvents or dispersants can be used. Stabilizers can be used in addition to stabilize the dispersion (for example: Tween® 80 or other suitable emulsifiers or stabilizers).
Examples of release agents are glycerol monostearate or other suitable fatty acid derivatives, silicic acid derivatives or talc. Examples of plasticizers are propylene glycol, phthalates, polyethylene glycols, sebacates or citrates, and other substances mentioned above and in the literature.
Another preferred type of medicament is a water-soluble capsule or blister containing a plurality of particles of an HX compound such as rose bengal disodium or rose bengal lactone that is covered with one or more layers of polymeric resin that release the HX compound quickly upon dissolution or disintegration of the capsule in water or body fluid. Capsules are typically made of gelatin and are often referred to as gelcaps. Gelatin is an animal product. Vegetarian capsules are often made of hydroxypropyl methyl cellulose (HPMC).
In some embodiments, the HX compound is directly layered with one or more coats of the polymer to form particles that are generally spherical in shape. Such particles are often referred to as beads. In a preferred aspect, particles (beads) are sized so as that about 90 percent by weight pass through a 20 mesh sieve (opening=850 μm) screen and about 90 percent by weight are retained on an 80 mesh sieve (opening=180 μm) screen.
Exemplary pH value-sensitive coating polymeric resins are discussed above. Exemplary pH value-insensitive coating polymeric resins are discussed above. The pH value-sensitivity of coating polymeric resins is to be understood in terms of physiologically present pH values along the GI tract such as those discussed above.
In other embodiments, small pellets such as sugar/starch seeds, non-pareils or prills, which are small, generally spherically-shaped cores, are coated with one or a plurality of layers of the HX compound and one or more layers of polymeric coating. Illustrative sugar/starch cores are sugar spheres NF that pass through an about 40 mesh sieve (425 mm opening) screen to an about 50 mesh sieve (300 mm opening) screen, that contain not less than 62.5 percent and not more than 91.5 percent sucrose, calculated on the dry basis, the remainder consisting primarily of starch. (USP NF 1995 2313).
In an illustrative example, a 100 kilogram (kg) quantity of disodium rose bengal, a 7.1 kg quantity of cross-linked carboxymethyl cellulose (preferably croscarmellose sodium NF), and an 11.9 kg quantity of starch NF, are each divided in half, and the three constituents are blended together to form two identical batches. Each of the batches is milled through an 80 mesh screen using a mill such as a Fitzpatrick Mill. The two milled batches are then blended to form a mixture, which is tested for composition in accordance with accepted quality assurance testing methods that are well-known by those skilled in the art.
The disodium rose bengal mixture is subsequently divided into three equal parts, with a first part remaining whole, and second and third parts each divided into lots of 50 percent, 30 percent and 20 percent. A 25.6 kg quantity of 40-50 mesh sugar/starch seeds, (e.g., sugar spheres NF) is placed in a stainless steel coating pan. An 80 liter (L) quantity of 5 percent povidone/IPA solution is prepared for spraying onto the particles.
The coating pan is started with the sugar spheres, onto which is sprayed an application (approximately 0.173 kg per application) of the povidone-alcohol solution, and onto which is sifted an application (approximately 0.32 kg) of the disodium rose bengal mixture from the first part (that part that remained whole). Sifting is done using a standard sifter. The spraying and sifting steps are continued until the first part of the mixture has been applied to the sugar spheres to form a batch of partially coated spheres.
The partially coated spheres are then divided into two equal lots, each lot being placed in a coating pan. Separately for each of the two lots, spraying of the povidone/IPA solution and sifting of the disodium rose bengal mixture as divided into the 50 percent lots continues until the 50 percent lots have been applied to the spheres. Following application of the 50 percent lots, the spheres can be screened using a 25 mesh screen if necessary.
The spraying of the povidone/IPA solution and sifting of the disodium rose bengal mixture as divided into the 30 percent lots commences and continues until the 30 percent lots have been applied to the spheres. The coated spheres can be rescreened using a 25 mesh screen.
Spraying of the povidone/IPA solution and sifting of the disodium rose bengal mixture continues using the mixture as divided into the 20 percent lots until the 20 percent lots have been applied to the spheres. At this point in the process, the entire quantity of the disodium rose bengal mixture has been applied to the spheres, and about 50 kg of the 5 percent povidone/IPA solution has been applied to the spheres.
A 7.5 percent povidone/IPA solution is prepared and applied to the spheres as a sealant. The sealed spheres are tumble dried for about one hour, weighed, and placed in an oven at about 122° F. (50° C.) for 24 hours. After drying, the spheres are screened through a 20 mesh screen and a 38 mesh screen to form the immediate (quick or fast as compared to delayed) release particles.
The above-discussed HX compound-containing spheres or their capsule (or blister) can also be coated with a pH value-sensitive enteric coating polymer as discussed previously so that once released in the GI tract, the spheres do not provide their active ingredient, HX compound, to their surroundings unless the pH value is that of a desired GI tract location.
Another way to control the location of HX compound release is to further coat the spheres (HX-coated particles) discussed above, with a dissolution-controlling coat of polymeric resin applied to the surface of the spheres such that the release of the HX compound from the spheres is controlled and released over a 6-10 hour period. The materials used for this purpose can be, but are not limited to, ethylcellulose, hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, hydroxyethylcellulose, nitrocellulose, carboxymethyl-cellulose, as well as copolymers of ethacrylic acid and methacrylic acid (Eudragit®), or any other acrylic acid derivative (Carbopol®, etc.) can be used.
In addition, an enteric coating material can also be employed, either singularly, or in combination to the above non-pH-sensitive coatings. These materials include, but are not limited to, hydroxypropylmethylcellulose phthalate and the phthalate esters of all the cellulose ethers. In addition, phthalate esters of the acrylic acid derivatives (Eudragit®), or cellulose acetate phthalate.
These coating materials can be employed in coating the surfaces in an amount of about 1.0% (W/W) to about 25% (W/W). Preferably, these coating materials are present at about 8.0 to about 12.0 percent (W/W).
Excipients
Excipients customary in pharmacy can be employed in a manner known per se in the production of the drug form. These excipients can be present in the core or in the coating agent.
Polymers
Polymeric materials used as adhesives in helping to adhere an HX compound to a sugar prill or sphere is deemed to be an excipient where coating layers of an HX compound are employed. Illustrative of such polymers are polyvinyl pyrrolidone and polyvinyl alcohol as are other water-soluble, pharmaceutically acceptable film-forming polymers such as hydroxypropyl cellulose.
Dryers (Non-Stick Agents)
Dryers have the following properties: they have large specific surface areas, are chemically inert, are free-flowing and comprise fine particles. Because of these properties, they reduce the tack of polymers containing polar comonomers as functional groups. Examples of dryers are: alumina, magnesium oxide, kaolin, talc, fumed silica, barium sulphate and cellulose.
Disintegrants
Disintegrants are added to oral solid dosage forms to aid in their disaggregation. Disintegrant are formulated to cause a rapid break-up of solids dosage forms on contacting moisture. Disintegration is typically viewed as the first step in the dissolution process. Illustrative disintegrants include sodium croscarmellose, an internally cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone (crospovidone) and sodium starch glycolate.
Release Agents
Examples of release agents are: esters of fatty acids or fatty amides, aliphatic, long-chain carboxylic acids, fatty alcohols and their esters, montan waxes or paraffin waxes and metal soaps; particular mention should be made of glycerol monostearate, stearyl alcohol, glycerol behenic acid ester, cetyl alcohol, palmitic acid, carnauba wax, beeswax, and the like. The usual proportionate amounts are in the range from 0.05% by weight to 5, preferably 0.1 to 3, % by weight based on the copolymer.
Other Excipients Customary in Pharmacy
Mention should be made here of, for example, stabilizers, colorants, antioxidants, wetting agents, pigments, gloss agents. They are typically used as processing aids and are intended to ensure a reliable and reproducible production process and good long-term storage stability. Further excipients customary in pharmacy may be present in amounts from 0.001% by weight to 10% by weight, preferably 0.1 to 10% by weight, based on the polymer coating.
Plasticizers
Substances suitable as plasticizers ordinarily have a molecular weight between 100 and 20,000 and comprise one or more hydrophilic groups in the molecule, e.g. hydroxyl, ester or amino groups. Citrates, phthalates, sebacates, castor oil are suitable. Examples of further suitable plasticizers are alkyl citrates, glycerol esters, alkyl phthalates, alkyl sebacates, sucrose esters, sorbitan esters, dibutyl sebacate and polyethylene glycols 4000 to 20 000. Preferred plasticizers are tributyl citrate, triethyl citrate, acetyl triethyl citrate, dibutyl sebacate and diethyl sebacate. The amounts used are between 1 and 35, preferably 2 to 10, % by weight, based on the (meth)acrylate copolymer.
Optimizing Systemic BioavailabilityAlthough the solid pharmaceutical compositions described herein have been discussed in the context of optimizing direct delivery to diseased tissue in the GI tract, these approaches for controlling site of delivery and kinetics of delivery are equally applicable to optimizing bioavailability for systemic uptake based on controlling site of delivery and kinetics.
Combination TreatmentA second therapeutic agent useful for combination treatment with an HX compound in an oncology indication is an immune checkpoint inhibitor, which can also be viewed as a special systemic anti-cancer medication. An immune checkpoint inhibitor is a drug that binds to and blocks certain checkpoint proteins made by immune system cells such as T cells and some cancer cells. When not blocked, those proteins inhibit immune responses, helping keep immune responses in check and keeping T cells from killing cancer cells. Blocking those immune checkpoint proteins releases the “brakes” on the immune system permitting T cells to become activated and kill cancer cells.
Such an immune checkpoint inhibitor can function additively or synergistically with the HX compound via their respective actions on the adaptive immune system, where the HX compound serves to activate antitumor function of the adaptive immune system through downstream signaling initiated by cancer cell death debris and STING activation, whereas the immune checkpoint inhibitor serves to function on the same adaptive immune system components through inhibition of down-regulation of such components.
A useful immune checkpoint inhibitor is preferably a human or humanized monoclonal antibody or binding portion thereof whose administration blocks the action of those self-recognizing T cells. That blockage permits the immune system to recognize the cancer cells as foreign and assist in eliminating those cancer cells from the body.
Illustrative immune checkpoint inhibitors include the anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) monoclonal antibodies ipilimumab and tremelimumab that are designed to counter down-regulation of the immune system by blocking CTLA-4 activity and thus augment T-cell response against cancer. Similarly, monoclonal antibodies such as pidilizumab, nivolumab, and pembrolizumab bind to PD-1 (programmed death 1) receptor to counter down-regulation of the immune system and augment T-cell responses to cancerous cells. Three antibodies that target the immune checkpoint protein ligand for the PD-1 receptor (PD-L1) are atezolizumab, avelumab and durvalumab. Initial work with antibodies to the PD-1 receptor ligands, PD-L1 and PD-L2, such as BMS-936559 and MEDI4736 (durvalumab) to PD-L1, also indicate inhibition of down-regulation of the immune system and an augmented T-cell response against cancer.
Another group of antibodies with checkpoint inhibitor-like activity immunoreact with the cell surface receptor OX40 (CD134) to stimulate proliferation of memory and effector T-lymphocytes, and thereby stimulate a T-cell-mediated immune response against cancerous cells. Exemplary such humanized anti-OX40 monoclonal antibodies include those presently referred to in the literature as gsk3174998 (IgG1), pogalizumab (MOXR0916), tavolixizumab (MED10562), and the human anti-OX40 IgG2 antibody designated PF-04518600 (PF-8600). Because the activities of anti-OX40 antibodies are so similar to those shown by the above immune checkpoint inhibitors, the anti-OX40 monoclonal antibodies are deemed to be immune checkpoint inhibitors for the purposes of this invention.
Intact monoclonal antibodies, as well their paratope-containing portions (binding site-containing portions) such as Fab, Fab′, F(ab′)2 and Fv regions, as well as single-stranded peptide antibody binding sequences can be useful as immune checkpoint protein inhibitors.
An immune checkpoint inhibitor is administered as directed on their individual labels. Intact checkpoint inhibiting monoclonal antibodies have half-lives in a human body of about one to three weeks [e.g., Yervoy® (ipilimumab) terminal t1/2=15.4 days; package insert December 2013; Keytruda® (pembrolizumab) terminal t1/2=23 days; package insert March 2017, Tecentriq (atezolizumab) terminal t1/2=27 days; package insert May 2020, and single-stranded oligo or polypeptides tend to have shorter half-lives in vivo.
Because an HX compound first anti-cancer agent such as RB disodium is administered orally, and the second anti-cancer agent such as a check point inhibitor is administered parenterally, typically by IV injection, the two medicaments are in separate compositions. It is preferred to administer both types of anticancer agent within minutes to about 8 hours of each other. More preferably, both are administered within less than one hour of the other.
As used herein, “administration” is used to mean the beginning of a treatment regimen. Thus, swallowing a liquid, tablet or other per os dosage form is the beginning of a treatment regimen, as is the time at which an IV flow is begun.
Although it is preferred that both anticancer agents be administered as discussed above, where the second anticancer agent is an immune checkpoint inhibitor such as a monoclonal antibody, the HX compound and the second anticancer agent immune checkpoint inhibitor can be administered at the same time or one before the other, with the second anticancer agent immune checkpoint inhibitor administration commencing up to about one month prior to or after administering the HX compound. More preferably, the two anticancer agents are administered at about the same time or with the second anticancer agent immune checkpoint inhibitor being administered within a few days before or after the HX compound.
Studies
The chemopreventive and chemotherapeutic effects of RB dissolved in sterilized tap water (autoclave-sterilized and then passed through a 0.2 μm filter) as a pharmaceutically acceptable carrier or diluent, as an illustrative HX compound, were assessed using the ApcMin mouse model. This model has an autosomal dominant mutation of chromosome 18 at the murine adenomatosis polyposis coli (Apc) gene that can lead to spontaneous multiple intestinal neoplasia (Min) [Su et al., Science, 256:668-670 (1992)]. This gene is homologous with the human Apc gene, making the model a valuable surrogate for both sporadic and inherited forms of human colorectal cancer (adenocarcinoma).
ApcMin mice were treated with orally-administered (ad libitum) aqueous rose bengal disodium vs. untreated (sterilized tap water) control female mice (n=8 for both groups). Treatment began at approximately 5 weeks of age (prior to onset of symptoms) with treatment mice orally receiving 4 mg/mL RB in sterilized tap water and controls receiving sterilized tap water with no additive. The treatment group mice remained asymptomatic (to 30 weeks of age), whereas controls began exhibiting symptoms at 16 weeks. Mean survival of the control group=19.6±0.6 weeks (SE); mean survival of the treatment group mice not defined. These data are shown in
The eight treated ApcMin mice were returned to sterilized tap water for 6 weeks and developed intestinal bleeding. The 8 mice were then randomized and divided into two groups of four mice. One group was treated with 1 mg/mL RB disodium in water orally-administered vs. untreated control mice (n=4 for both groups) treated only with tap water. Treatment began upon onset of symptoms. Mean survival of the control group=8.9±0.8 weeks whereas survival of the RB disodium treatment group=12.3±0.5 weeks. These data are shown in
Control mice in the chemoprevention phase of the study had a mean survival time of 19.6±0.6 weeks of age (range 16.4 to 20.4 weeks). In contrast, all mice undergoing continuous treatment with oral RB disodium survived the duration of this phase of the study (to 30 weeks of age). These results are illustrated in
Control mice in the subsequent chemotherapeutic phase of the study had a mean survival time of 8.9±0.8 weeks (range 8.1 to 11.3 weeks) following disease onset. In contrast, mice undergoing continuous treatment with oral RB disodium exhibited a mean survival of 12.3±0.6 weeks (range 11.3 to 13.4 weeks). These results are illustrated in
In the chemoprevention phase of the study all control group mice were positive for fecal occult blood (FEOB) by approximately 100 days of age, whereas none of the RB treatment group mice exhibited positive FEOB results (FEOB positive). Mice in the control group exhibited stable body weight after approximately 100 days of age; in contrast, mice in the treatment group continued to gain body weight throughout the study.
The first unequivocal morbidity was observed when three control group mice appeared ill at approximately 115 days of age; one severely ill mouse in this group was euthanized at this point (Table A, below). Over the next several weeks the remainder of the control group mice became ill and were euthanized or found dead. At no point during this phase of the study (25 weeks total duration, to 30 weeks of age) did any mice receiving RB appear ill nor were any of these mice lost.
Table A, below, provides a summary of morbidity and mortality for chemoprevention study. The subject animals' ages in days (d) are shown. Mean body weight and standard deviation are shown for control and treatment group mice during study. The column labeled “Sym” represents the number of symptomatic animals (i.e., exhibiting anemia or wasting) in each group at the indicated age, whereas “Dec” represents cumulative number of deceased animals; “Sur” is the number of surviving mice. By 100 days of age, all control group mice were FEOB positive, whereas none of the treatment group mice exhibited positive results. RB disodium treatment began at approximately 5 weeks of age (prior to onset of symptoms).
At the outset of the chemotherapeutic phase of the study 7 mice (of a combined total of 8) were FEOB positive. Three mice in the control group were found dead at 8.1 weeks, and the final mouse in this group was euthanized at 11.3 weeks. Treatment group mice were euthanized upon onset of severe illness after 11.3, 11.9, 12.6 and 13.4 weeks of treatment.
Retrospective AnalysisFor comparative purposes, survival data reported in a long-term oral feeding study of 0.125 or 0.5% RB disodium in drinking water to B6C3F1 mice were analyzed using the Fisher exact test [original data presented in Ito et al., J Natl Cancer I, 77:277-281 (1986)]. The B6C3F1 mouse is a hybrid strain that is produced as a cross between a male C3H mouse and a female C57BL/6 mouse. The B6C3F1 mouse exhibits a marked tendency for spontaneous neoplasia, particularly liver adenoma and carcinoma [Haseman et al., Toxicol Pathol 26:428-441 (1998)], and have been used for many years in murine carcinogenicity evaluations performed for the National Toxicity Program (NTP) bioassay program. These studies have generated a very large historical control database for background incidences of spontaneous tumors in this mouse mode.
Survival following 82 weeks of continuous feeding with 0.5% RB showed statistically significant increases in survival for treatment group mice relative to the control groups (P<0.001 for males, at 94% vs 52% survival; P=0.004 for females, 90% vs 64%). Similar analyses of data for 0.125% RB showed a statistically significant increase in survival for male mice (P<0.001, 92% vs 52% survival) but not for female mice (P=0.4, 74% vs 64%). Published survival percentages at the end of 2-year tests and weight-gain patterns are often used as controls to guide investigators using these same animals in long-term studies [Cameron et al., Fundam Appl Toxicol 5(3):526-538 (June 1985)].
DiscussionThe ApcMin model represents a particularly severe test of chemopreventive or chemotherapeutic properties of a drug because these mice have an autosomal dominant defect of the Apc gene that continuously promotes abnormal cell proliferation and transformation into cancer. Heterozygous mice with an induced defect in the Apc tumor suppressor gene are highly susceptible to spontaneous intestinal adenoma formation (typically developing an excess of 30 intestinal adenomas) with a life expectancy of approximately 120 days (i.e., 17 weeks). These mice generally become anemic and eventually suffer wasting as lesions proliferate. There is no possibility of cure in the model, making it particularly challenging relative to the task of combating isolated, spontaneous tumors.
Chemopreventive EffectsSurvival of naive control ApcMin mice in the chemopreventive phase of this study (i.e., mean survival time 19.6±0.6 weeks of age) was consistent with life expectancy. Because all ApcMin mice undergoing continuous treatment with oral RB disodium survived the duration of this phase of the study (to 30 weeks of age;
These differences in outcome are reinforced by the morbidity data, wherein control group mice exhibited onset of intestinal bleeding within 14 weeks of age and obvious clinical illness beginning at approximately 16 weeks of age. In contrast, none of the treatment group mice exhibited clinical symptoms throughout the duration of the study.
Trends in weight gain reflect disease onset in the control group, diverging from those of the treatment group concurrently with onset of positive FEOB status. Continued increases in body weight of treatment group animals further demonstrates preservation of overall health among these animals. Taken together with the survival data, these results demonstrate that RB disodium treatment successfully suppressed onset of disease and its symptoms, including intestinal bleeding and subnormal growth.
This apparent chemopreventive effect is consistent with that evident upon retrospective analysis of data from long-term oral feeding studies of B6C3F1 mice [Ito et al., J Natl Cancer I, 77:277-281 (1986)], where reported survival data following nearly 2 years of continuous feeding with 0.5% RB showed statistically significant increases in survival for treatment group mice. While a lesser effect was apparent at lower dose (i.e., 0.125% RB), both males and females exhibited increased survival relative to controls. This suggests the existence of a chemopreventive effect in these mice. However, except for thyroid tumors, no significant trends in tumor incidence were noted in this notoriously tumor-prone model [Haseman et al., Toxicol. Pathol., 26:428-441 (1998)], so it is impossible to definitively ascertain an underlying basis for these differences in survival.
Although prolonged oral administration of RB disodium has been associated with colloid goiter in B6C3F1 mice through apparent inhibition of conversion of 3,5,3′-triiodothyronine (T3) to thyroxine (T4) and potential inhibition of thyroid peroxidase [Kurebayashi et al., J. Toxicol. Sci., 13:61-70 (1988)], administration of RB disodium to male Wistar rats was not associated with thyroid tumorigenesis or abnormal hormonal levels [Hiasa et al., Jpn. J. Cancer Res., 79:314-319 (1988); and Kanno et al., Toxicology, 99:107-111 (1995)]. Thus, the increased incidence in thyroid adenomas observed by Ito et al. [J Natl Cancer I, 77:277-281 (1986)] is heuristic, but may represent a unique feature of the B6C3F1 model rather than a significant thyroid risk in other species.
Chemotherapeutic EffectsDisease progression was qualitatively similar in treatment and control group animals when treatment was begun following onset of symptomatic disease.
Nonetheless, survival of treatment group mice was significantly enhanced over that of control mice (comprising an approximate 38% increase in mean survival). Although the magnitude of this effect is not as dramatic as that observed for chemoprevention, such a response suggests a common anti-neoplastic mechanism underlying both phenomena. In addition, the dose of RB administered was one-fourth that administered in the prevention study.
MechanismAdditional studies of the interaction of RB with several tumor lines provide possible insight into the mechanism underlying the chemopreventive and chemotherapeutic effects of RB in the ApcMin model. For instance, murine hepatocellular carcinoma cells [Wachter et al., “Functional Imaging of Photosensitizers Using Multiphoton Microscopy”, in: A. Periasamy and P. T. So, P. T. (Eds), Multiphoton Microscopy in the Biomedical Sciences II. SPIE, Bellingham, Wash., vol. 4620, pp. 143-147 (2002); and Wachter et al., (2002) “Imaging Photosensitizer Distribution and Pharmacology Using Multiphoton Microscopy”, in: D. L. Farkas and R. C. Leif, R. C. (Eds) Optical Diagnostics of Living Cells V. SPIE, Bellingham, Wash., vol. 4622, pp. 112-118 (2002)] exhibit highly selective uptake of RB (both in vitro and upon systemic delivery using tumor homografts), ultimately leading to irreversible accumulation of the amphoteric molecule in acidic lysosomes. Additional tissue culture studies by the inventors have shown similar lysosomal accumulation in human breast carcinoma (HTB-133) and multidrug resistant small cell lung carcinoma (H69AR) cell lines.
In each case, this accumulation leads to autolysis of these cells upon RB-mediated lysosomal rupture. In contrast, exclusion of RB from normal cells appears to prevent similar intracellular accumulation and cytotoxicity.
It is believed that a similar process may lead to the chemopreventive effects noted in the present study by selectively destroying nascent dysplasias or neoplasias in the intestinal tract upon accumulation of RB disodium in the lysosomes of those tissues. This appears particularly likely considering the dual exposure of such lesions to circulating RB in the blood stream as well as to high levels of intact RB continuously excreted by the liver and delivered topically to aberrant tissues in the intestines.
ConclusionsBased on the positive results of this study, systemic RB disodium appears to exhibit substantial chemopreventive and chemotherapeutic utility against a carcinoma of the GI tract, colorectal cancer. Given the long and extensive history of safe use of RB disodium as an intravenous liver diagnostic [Delprat et al., Arch. Intern. Med., 34:533-541 (1924)], as a topical ophthalmic diagnostic [Sjogren, Acta Ophthalmol. (suppl. 2), 11:1-151 (1933)], and as a food dye [Tanaka et al., Jpn. J. Hyg., 30:574-578 (1975); and Sako et al., Tox. Appl. Pharma., 54: 285-292 (1980)], this novel use is expected to be safe in addition to being effective.
Materials and MethodsColorectal Cancer
Rose BengalA solution for administration was prepared by dissolving 1 mg/mL (0.1% w/v) or 4 mg/mL (0.4% w/v) rose bengal disodium (Sigma-Aldrich, St. Louis, Mo.) in autoclave-sterilized tap water followed by filter sterilization (0.2 μm).
AnimalsSixteen female ApcMin mice approximately 4 weeks of age (C57BL/6J-Ap-min/J, Jackson Laboratory, Bar Harbor, Me.) were used for the study. Animal housing and care were based on standards established by the Association for Assessment and Accreditation of Laboratory Animal Care, International and guidelines set forth in the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. All animals were housed in micro-isolator cages and fed a standard radiation sterilized diet (7912, Harlan Teklad, Madison, Wis., a nutrient enriched grain diet consisting of 19% protein, 5% fat and 5% fiber).
Group AssignmentsMice were randomly divided into two treatment groups (n=8 per group) for assessment of chemoprevention. Treatment was begun immediately following acclimatization (at approximately 5 weeks of age), well prior to the age typical of onset of symptomatic illness (i.e., anemia characteristic of polyps or wasting indicative of neoplasia).
Treatment group animals (average weight, 16.0±1.1 g at outset) were permitted to drink RB solution (4 μg/mL) continuously ad libitum; control group animals (16.8±0.7 g) were provided sterilized tap water ad libitum.
At the conclusion of the chemopreventive phase of the study, surviving mice from the RB treatment group were returned to tap water for a period of 6 weeks to permit disease onset; at this point 7 of 8 mice became definitely symptomatic for intestinal neoplasia, exhibiting positive FEOB results. These mice were re-randomization into treatment and control groups (n=4 per group) for assessment of chemotherapeutic potential. Treatment group animals received 1 mg/mL RB solution continuously ad libitum, whereas control animals were provided sterilized tap water ad libitum. During both phases of the study, RB and tap water supplies were changed weekly.
Clinical ObservationMice were observed daily for behavioral or physical changes (i.e., languid behavior, anemia or wasting) indicating onset of serious illness. Mice exhibiting symptoms consistent with onset of severe disease were immediately euthanized by carbon dioxide inhalation. At periodic intervals fecal occult blood was determined for all mice using a commercial test kit (Hemoccult Fecal Occult Blood Test, Beckman Coulter, Inc., Fullerton, Calif.). Immediately prior to FEOB assay, treatment group mice were provided autoclaved tap water for 3 days in order to minimize potential interference of RB (which is bright red in color and excreted unmetabolized in feces) with the colorimetric FEOB test; RB treatment was resumed for these animals immediately following stool sampling.
Statistical AnalysisSurvival of mice was compared between the treatment and control groups. Data were analyzed using the log-rank test to determine the statistical significance of any difference in survival experienced between treatment and control groups. Retrospective analysis of literature data was performed using the Fisher exact test. SigmaStat 3.0 (Systat Software, Inc., Point Richmond, Calif.) was used for all statistical analysis.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Each of the patents, patent applications and articles cited herein is incorporated by reference.
Claims
1. A pharmaceutical composition for oral delivery that comprises a solid tumor treating-effective amount of a halogenated xanthene (HX), the lactone thereof, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof (HX compound) contained within an enterically-coated solid diluent matrix, said enteric coating dissolving or disintegrating at a physiological pH value of 5 or greater.
2. The pharmaceutical composition according to claim 1, wherein said enterically-coated solid diluent matrix is in the form of a tablet, lozenge, or a plurality of generally spherical sugar prills.
3. The pharmaceutical composition according to claim 1, wherein said HX compound is dissolved in or dispersed in or on a solid diluent matrix.
4. The pharmaceutical composition according to claim 3, wherein solid diluent matrix is comprised of sugar spheres coated with one or a plurality of layers of said HX compound.
5. The pharmaceutical composition according to claim 1, wherein said aromatic ester is formed by a reaction between the halogenated xanthene and an aromatic alcohol having a 5- or 6-membered aromatic ring, or a 5,6- or 6,6-fused aromatic ring system that contains 0, 1 or 2 hetero ring atoms that are independently nitrogen, oxygen or sulfur.
6. The pharmaceutical composition according to claim 5, wherein said aromatic alcohol is selected from the group consisting of one or more of benzyl, phenyl, pyridyl, thienyl, furyl, oxazolyl, thiazolyl, naphthyl, quinolyl, quioxalinyl, benzofuranyl, benzo[b]thienyl and benzoxazinyl alcohols.
7. The pharmaceutical composition according to claim 1, wherein said HX compound is rose bengal disodium or rose bengal lactone.
8. The pharmaceutical composition according to claim 1, wherein said enteric coating dissolves or disintegrates at a published pH value of 5.5 to 7.0.
9. A method of treating a solid cancerous tumor in a mammalian subject that comprises orally administering an effective amount of a halogenated xanthene (HX), a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof (HX compound) to that mammalian subject.
10. The method according to claim 9, wherein said administration is repeated a plurality of times at least until the solid cancerous tumor has been reduced in volume by at least 30 percent.
11. The method according to claim 9, wherein said aromatic ester is formed by a reaction between said HX and an aromatic alcohol that is selected from the group consisting of one or more of benzyl, phenyl, pyridyl, thienyl, furyl, oxazolyl, thiazolyl, naphthyl, quinolyl, quioxalinyl, benzofuranyl, benzo[b]thienyl, and benzoxazinyl alcohols.
12. The method according to claim 9, wherein said HX compound is administered as part of an aqueous pharmaceutical composition.
13. The method according to claim 9, wherein said HX compound is administered as part of a solid pharmaceutical composition.
14. The method according to claim 13, wherein said solid pharmaceutical composition is in the form of a tablet or a plurality of generally spherical prills.
15. The method according to claim 13, wherein said solid pharmaceutical composition is coated with a polymeric film that dissolves or disperses at a pH value above that of the stomach.
16. The method according to claim 9, wherein said HX compound is rose bengal disodium or rose bengal lactone.
17. A method of treating or inhibiting the formation of a carcinoma of the gastrointestinal tract in a mammalian subject that comprises orally administering to said mammalian subject a carcinoma formation-inhibiting or carcinoma-treating effective amount of a halogenated xanthene (HX), a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof to said mammalian subject.
18. The method according to claim 17, wherein said HX is rose bengal disodium.
19. The method according to claim 17, wherein said aromatic ester is formed by a reaction between said HX and an aromatic alcohol having a 5- or 6-membered aromatic ring, or a 5,6- or 6,6-fused aromatic ring system that contains 0, 1 or 2 hetero ring atoms that are independently nitrogen, oxygen or sulfur.
20. The method according to claim 19, wherein said aromatic alcohol is selected from the group consisting of one or more of benzyl, phenyl, pyridyl, thienyl, furyl, oxazolyl, thiazolyl, naphthyl, quinolyl, quioxalinyl, benzofuranyl, benzo[b]thienyl, and benzoxazinyl alcohols.
21. The method according to claim 17, wherein said administration is repeated.
22. The method according to claim 17, wherein said administration treats a carcinoma of the gastrointestinal tract of said mammalian subject.
23. The method according to claim 17, wherein said HX, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof is dissolved or dispersed in an aqueous diluent when administered to said mammalian subject.
24. The method according to claim 17, wherein said aqueous diluent is free of tonicity agents except for those sugars and/or buffering agents present as flavorants.
25. The method according to claim 17, wherein said HX, a pharmaceutically acceptable salt, or a C1-C4 alkyl ester or aromatic thereof is present as part of a solid medicament when administered to said mammalian subject.
26. The method according to claim 17, wherein said administration treats a carcinoma of the gastrointestinal tract of said mammalian subject and said solid medicament releases said HX, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof at the pH value of the portion of the GI tract in which said carcinoma is located.
27. The method according to claim 26, wherein said carcinoma is in the stomach.
28. The method according to claim 26, wherein said carcinoma is in the small intestine.
29. The method according to claim 26, wherein said carcinoma is in in the colon.
30. The method according to claim 26, wherein solid medicament is a tablet.
31. The method according to claim 26, wherein said HX compound is disodium rose bengal or rose bengal lactone.
32. The method according to claim 17, wherein said solid medicament is a capsule that contains a plurality of coated particles that contain said HX, the lactone thereof, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof.
33. The method according to claim 32, wherein the coating of said coated particles dissolves or disintegrates at a preselected pH value to release said HX, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof.
34. The method according to claim 33, wherein said HX, the lactone thereof, a pharmaceutically acceptable salt, or a C1-C4 alkyl or aromatic ester thereof is disodium rose bengal or rose bengal lactone.
Type: Application
Filed: Mar 26, 2021
Publication Date: Sep 30, 2021
Applicants: Provectus Pharmatech, Inc. (Knoxville, TN), Governors of the University of Calgary (Calgary)
Inventors: Eric A. WACHTER (Oak Ridge, TN), Dominic RODRIGUES (Knoxville, TN), Edward V. PERSHING (Knoxville, TN), Bruce HOROWITZ (Knoxville, TN), Timothy C. SCOTT (Knoxville, TN), H. Craig DEES (Knoxville, TN), Aru NARENDRAN (Calgary)
Application Number: 17/214,590
