# Patent

## Title
Methods and compositions for controlled delivery of phytochemical agents

## Abstract

The presently-disclosed subject matter provides compositions and methods for treating a cancer by providing a composition comprising a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent, a combination of phytochemical agents, or a phytochemical agent and a chemotherapeutic agent. Further, provided is a device for uterine cervical insertion for local delivery.

## Background
RELATED APPLICATIONS
This application claims priority from U.S. Provisional Application Ser. No. 61/035,153, filed Mar. 10, 2008, and U.S. application Ser. No. 61/096,108, filed Sep. 11, 2008, each of which are incorporated herein by this reference.


GOVERNMENT INTEREST
Subject matter described herein was made with U.S. Government support under Grant Numbers CA-90892, CA-118114, and CA-125152 awarded by the National Institutes of Health. The government has certain rights in the described subject matter.


TECHNICAL FIELD
The presently-disclosed subject matter relates to methods and compositions for the controlled delivery of phytochemical agents. In particular, the presently-disclosed subject matter relates to methods and compositions for treating cancer wherein a phytochemical agent is incorporated into and released in a controlled manner from a biocompatible and biodegradable polymeric matrix.
BACKGROUND
Despite recent advances in cancer detection and treatment strategies, cancer continues to contribute significantly to human mortality and remains second only to heart disease as the leading cause of death in the United States. In 2008 there were an estimated 565,650 deaths from cancer and each year about 1,437,180 new cases of cancer are diagnosed. Among those, lung cancer, breast cancer, and cervical cancer are among the most significant. For example, lung cancer remains the leading cause of cancer death in men and women, resulting in more than 215,000 new cases a year and over 161,000 deaths each year. Similarly, breast cancer and cervical cancer continue to be significant causes of cancer death in women with estimates of over 40,000 and over 3,500 women dying each year from breast and cervical cancer, respectively.
Overall, the survival rate for cancer has risen in recent years, but designing effective treatment strategies for cancer patients still represents a major challenge. In this regard, it has become increasingly clear that the problem of cancer mortality cannot be solved by treatment alone, but must also be approached from a prophylactic standpoint. Intervention with compounds of natural origin, such as phytochemical agents, is considered one such effective means to not only treat cancer, but also to prophylactically address cancer development. However, many of the same problems that are encountered with the administration of traditional chemotherapeutic agents are also encountered with these compounds of natural origin.
Typically, chemotherapeutic agents are administered orally or intravenously in bolus doses to elicit a response. Oral delivery of the chemotherapeutic agents often presents a bioavailability problem, as a large part of the agents is destroyed in the gut and the remainder is, at least in part, detoxified during first pass in the liver. Systemic delivery overcomes this hurdle, but this route of administration often leads to undesirable dose spikes. Moreover, a major limitation of both oral and intravenous delivery of traditional chemotherapeutic agents is that high doses of the agents are usually required in order to achieve an effective tissue or plasma concentration, but the high doses generally result in undesirable toxicity.
Accordingly, there remains a need in the art for methods and compositions for the delivery of compounds of natural origin in controlled low doses over extended periods of time. In particular, there remains a need in the art for methods and compositions for delivering low doses of phytochemical agents in a sustained manner such that phytochemical agents can effectively be used to address cancer mortality from both a therapeutic and prophylactic standpoint.
SUMMARY
The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.
This Summary describes several embodiments of the presently-disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
In some embodiments of the presently-disclosed subject matter, a composition is provided that comprises a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent. In some embodiments, the composition is capable of releasing a controlled low dose of the phytochemical agent over a time period. In some embodiments, the time period is at least about 18 months.
In some embodiments of the presently-disclosed subject matter, the biocompatible polymeric matrix comprises a polymer that can be selected from polycaprolactone, cyclodextrin, F68, polyethylene glycol, and combinations thereof. In some embodiments, the biocompatible polymeric matrix is comprised of polycaprolactone in combination with a second polymer that, in some embodiments, can be selected from cyclodextrin, F68, and polyethylene glycol. In some embodiments, the polycaprolactone is combined with the second polymer in a ratio of about 4:1 or about 9:1 to form the biocompatible polymeric matrix. In some embodiments, the biocompatible polymeric matrix comprises polycaprolactone and F68 in a ratio of about 4:1. In some embodiments, the biocompatible matrix is biodegradable.
With regard to the phytochemical agents that are incorporated into the biocompatible polymeric matrices of the presently-disclosed subject matter, in some embodiments, the phytochemical agent comprises about 2% to about 50% of the weight of the polymer or polymers forming the biocompatible polymeric matrix. In some embodiments, the phytochemical agent is selected from curcumin, green tea polyphenols, punicalagin, diindolylmethane, oltipraz, tocotrienol, tocopherol, plumbagin, cyanidin, delphinidin, lycopene, lupeol, curcurbitacin-B, Withaferin A, indole-3-carbinol, genestein, equol, resveratrol, co-enzyme Q-10, ellagic acid, petunidin, malvidin, peonidin, fennel extract, and combinations thereof.
In some embodiments, the phytochemical agent incorporated into the biocompatible polymeric matrix is a combination of one or more individual phytochemical agents. In some embodiments, the phytochemical agent is a combination of: oltipraz and curcumin; curcumin, ellagic acid, co-enzyme Q-10, and lycopene; curcumin, green tea polyphenols, diindolylmethane, and punicalagin; delphinidin, petunidin, malvidin, peonidin, and cyanidin; or punicalagin and delphinidin. In some embodiments, each phytochemical agent can be incorporated into the same biocompatible polymeric matrix or, in some embodiments, each phytochemical agent one or more additional biocompatible polymeric matrices such that the one or more additional biocompatible polymeric matrices each incorporate a single phytochemical agent. In some embodiments, a second biocompatible matrix is provided that incorporates at least one phytochemical agent.
In some embodiments, the compositions of the presently-disclosed subject matter can further comprise an effective amount of an anti-inflammatory agent or an effective amount of a chemotherapeutic agent. In some embodiments, the anti-inflammatory agents and the chemotherapeutic agents are incorporated into the same biocompatible polymeric matrix that also incorporates a phytochemical agent. In some embodiments, the anti-inflammatory agents and the chemotherapeutic agents are incorporated into a second biocompatible polymeric matrix.
Further provided, in some embodiments of the presently-disclosed subject matter, are methods for treating a cancer. In some embodiments, a method for treating a cancer is provided that comprises administering to a subject in need thereof an effective amount of a composition comprising a biocompatible polymeric matrix incorporating a phytochemical agent, where the phytochemical agent is incorporated into and released over a time period from the biocompatible polymeric matrix at a controlled low dose of the phytochemical agent. In some embodiments, the time period is at least about 18 months.
In some embodiments, a method of treating a cancer is provided that further comprises administering an effective amount of an anti-inflammatory agent or a chemotherapeutic agent that is incorporated into and released over a time period from the biocompatible polymeric matrix or a second biocompatible polymeric matrix.
In some embodiments of the presently-disclosed methods for treating a cancer, the cancer is selected from breast cancer, lung cancer, or cervical cancer. In some embodiments, the subject is at an increased risk of developing a primary cancer, which, in some embodiments, is breast cancer, lung cancer, or cervical cancer. In some embodiments, the subject is at an increased risk of developing a secondary cancer.
In some embodiments, a method for treating a cancer is provided wherein a phytochemical agent is administered to a subject by subcutaneous implantation of a biocompatible matrix incorporating a phytochemical agent. In some embodiments, the phytochemical agent is administered by implantation of a biocompatible polymeric matrix incorporating that phytochemical agent at a site distant from the site of the cancer. In some embodiments, the implantation occurs at a site of a cancer or at a site suspected of developing a cancer.
Still further provided, in some embodiments of the presently-disclosed subject matter, is a device for insertion into a cervical canal. In some embodiments, the device comprises: a substantially cylindrical shaft having a proximal end and a distal end, and defining a canal that extends from the proximal end to the distal end; and, a cap attached to the proximal end of the cylindrical shaft that includes a central opening in registry with the canal defined by the shaft. In some embodiments, the device is comprised of a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent in accordance with the presently-disclosed subject matter.
In some embodiments, a device is provided where the cylindrical shaft is suitably adapted for engaging the walls of a cervical canal and where a bottom surface of the cap is suitably adapted for engaging an external orifice of a uterus. In some embodiments, a device is provided where the canal defined by the cylindrical shaft allows for the passage of bodily fluids from the uterus.
In some embodiments of the device for cervical insertion, the bottom surface of the cap has a concave shape such that the bottom surface of the cap suitably engages an external orifice of the uterus. In some embodiments, a device for cervical insertion is provided where the distal end of the cylindrical shaft is tapered. In some embodiments, a device for cervical insertion is provided where the cylindrical shaft is about 19 to about 25 mm in length and about 9 to 11 mm in diameter; the cap is about 20 to about 25 mm in diameter; and, the canal is about 4 mm to about 5 mm in diameter.
Advantages of the presently-disclosed subject matter will become evident to those of ordinary skill in the art after a study of the description, Figures, and non-limiting Examples in this document.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a picture of exemplary compositions of the presently-disclosed subject matter, and including an image of a biocompatible polymeric matrix without the incorporation of a phytochemical agent (Sham) and images of biocompatible polymeric matrices incorporating various phytochemical agents including curcumin, ellagic acid, oltipraz, and green tea polyphenols (GTPs).
FIG. 2 includes graphs depicting the release of curcumin from a biocompatible polymeric matrix in vitro. FIG. 2A is a graph depicting the daily release of curcumin from a biocompatible polymeric matrix where the amount (μg) of curcumin released per day (y-axis) and the percentage of curcumin released from the matrix per day (y-axis) are plotted against a release period measured in days (x-axis). FIG. 2B is a graph depicting the cumulative release of curcumin from a biocompatible polymeric matrix where the total amount (μg) of curcumin released from the matrix (y-axis) and the total percentage of curcumin released from the matrix (y-axis) are plotted against a release period measured in days (x-axis).
FIG. 3 includes graphs depicting the release of green tea polyphenols (GTPs) from a biocompatible polymeric matrix in vitro. FIG. 3A is a graph depicting the daily release of GTPs from a biocompatible polymeric matrix where the amount (μg) of GTPs released per day (y-axis) and the percentage of GTPs released from the matrix per day (y-axis) are plotted against a release period measured in days (x-axis). FIG. 3B is a graph depicting the cumulative release of GTPs from a biocompatible polymeric matrix where the total amount (μg) of GTPs released from the matrix (y-axis) and the total percentage of GTPs released from the matrix (y-axis) are plotted against a release period measured in days (x-axis).
FIG. 4 includes graphs depicting the release of diindolylmethane (DIM) from a biocompatible polymeric matrix in vitro. FIG. 4A is a graph depicting the daily release of DIM from a biocompatible polymeric matrix where the amount (μg) of DIM released per day (y-axis) and the percentage of DIM released from the matrix per day (y-axis) are plotted against a release period measured in days (x-axis). FIG. 4B is a graph depicting the cumulative release of DIM from a biocompatible polymeric matrix where the total amount (μg) of DIM released from the matrix (y-axis) and the total percentage of DIM released from the matrix (y-axis) are plotted against a release period measured in days (x-axis).
FIG. 5 includes graphs depicting the release of punicalagins from a biocompatible polymeric matrix in vitro. FIG. 5A is a graph depicting the daily release of punicalagins from a biocompatible polymeric matrix where the amount (μg) of punicalagins released per day (y-axis) and the percentage of punicalagins released from the matrix per day (y-axis) are plotted against a release period measured in days (x-axis). FIG. 5B is a graph depicting the cumulative release of punicalagins from a biocompatible polymeric matrix where the total amount (μg) of punicalagins released from the matrix (y-axis) and the total percentage of punicalagins released from the matrix (y-axis) are plotted against a release period measured in days (x-axis).
FIG. 6 includes graphs depicting the release of oltipraz from a biocompatible polymeric matrix in vitro. FIG. 6A is a graph depicting the daily release of oltipraz from a biocompatible polymeric matrix where the amount (μg) of oltipraz released per day (y-axis) is plotted against a release period measured in days (x-axis). FIG. 6B is a graph depicting the cumulative release of oltipraz from a biocompatible polymeric matrix where the total amount (μg) of oltipraz released from the matrix (y-axis) is plotted against a release period measured in days (x-axis).
FIG. 7 includes graphs depicting the release of lycopene from a biocompatible polymeric matrix in vitro. FIG. 7A is a graph depicting the daily release of lycopene from a biocompatible polymeric matrix where the amount (μg) of lycopene released per day (y-axis) is plotted against a release period measured in days (x-axis). FIG. 7B is a graph depicting the cumulative release of lycopene from a biocompatible polymeric matrix where the total amount (μg) of lycopene released from the matrix (y-axis) is plotted against a release period measured in days (x-axis).
FIG. 8 includes graphs depicting the release of resveratrol from a biocompatible polymeric matrix in vitro. FIG. 8A is a graph depicting the daily release of resveratrol from a biocompatible polymeric matrix where the amount (μg) of resveratrol released per day (y-axis) is plotted against a release period measured in days (x-axis). FIG. 8B is a graph depicting the cumulative release of resveratrol from a biocompatible polymeric matrix where the total amount (μg) of resveratrol released from the matrix (y-axis) is plotted against a release period measured in days (x-axis).
FIG. 9 includes graphs depicting the effect of incorporating various loads of curcumin into a biocompatible polymeric matrix in vitro, including incorporating 2% w/w of curcumin (♦), 5% w/w of curcumin (), and 20% w/w of curcumin (▴) into separate biocompatible polymeric matrices. FIG. 9A is a graph depicting the daily release of curcumin from each of the biocompatible polymeric matrices where the amount (μg) of curcumin released per day (y-axis) from each biocompatible polymeric matrix is plotted against a release period measured in days (x-axis). FIG. 9B is a graph depicting the cumulative release of curcumin from each biocompatible polymeric matrix where the total amount (μg) of curcumin released from each matrix (y-axis) is plotted against a release period measured in days (x-axis).
FIG. 10 includes graphs depicting the effect of incorporating various loads of GTPs into a biocompatible polymeric matrix in vitro, including incorporating 1% w/w of GTPs (♦), 2.5% w/w of GTPs (), 5% w/w of GTPs (▴), and 10% w/w of GTPs () into a separate biocompatible polymeric matrices comprised of polycaprolactone, MW 65K polymers and polycaprolactone, MW 15K polymers that were combined in a 1:4 ratio. FIG. 10A is a graph depicting the daily release of GTPs from each of the biocompatible polymeric matrices where the amount (μg) of GTPs released per day (y-axis) from each biocompatible polymeric matrix is plotted against a release period measured in days (x-axis). FIG. 10B is a graph depicting the cumulative release of GTPs from each biocompatible polymeric matrix where the total amount (μg) of GTPs released from each matrix (y-axis) is plotted against a release period measured in days (x-axis).
FIG. 11 is a graph depicting the short-term in vivo release of curcumin from a biocompatible polymeric matrix where the total amount (mg) of curcumin released (y-axis) and the total percentage of curcumin released (y-axis) from the matrix are plotted against a release period measured in days (x-axis).
FIG. 12 includes graphs depicting the levels of curcumin (ng/g) in lung tissue (FIG. 12A) and liver tissue (FIG. 12B) of rats (y-axis) at different time intervals (x-axis), where the rats were treated with a composition comprising a biocompatible matrix incorporating 20% w/w curcumin.
FIG. 13 includes graphs depicting the long-term in vivo release of curcumin from a biocompatible polymeric matrix. FIG. 13A is a graph depicting the daily release of curcumin from a biocompatible polymeric matrix where the total amount (mg) of curcumin released per week (y-axis) and the total percentage of curcumin released per week (y-axis) from the matrix are plotted against a treatment period measured in weeks (x-axis). FIG. 13B is a graph depicting the cumulative release of curcumin from a biocompatible polymeric matrix where the total amount (mg) of curcumin released (y-axis) and the total percentage of curcumin released (y-axis) are plotted against a treatment period measured in weeks (x-axis).
FIG. 14 includes graphs depicting the long-term in vivo release of GTPs from a biocompatible polymeric matrix. FIG. 14A is a graph depicting the daily release of GTPs from a biocompatible polymeric matrix where the total amount (mg) of GTPs released per week (y-axis) and the total percentage of GTPs released per week (y-axis) from the matrix are plotted against a treatment period measured in weeks (x-axis). FIG. 14B is a graph depicting the cumulative release of GTPs from a biocompatible polymeric matrix where the total amount (mg) of GTPs released (y-axis) and the total percentage of GTPs released (y-axis) are plotted against a treatment period measured in weeks (x-axis).
FIG. 15 includes graphs depicting the long-term in vivo release of DIM from a biocompatible polymeric matrix. FIG. 15A is a graph depicting the daily release of DIM from a biocompatible polymeric matrix where the total amount (mg) of DIM released per week (y-axis) and the total percentage of DIM released per week (y-axis) from the matrix are plotted against a treatment period measured in weeks (x-axis). FIG. 15B is a graph depicting the cumulative release of DIM from a biocompatible polymeric matrix where the total amount (mg) of DIM released (y-axis) and the total percentage of DIM released (y-axis) are plotted against a treatment period measured in weeks (x-axis).
FIG. 16 includes graphs depicting the long-term in vivo release of punicalagins from a biocompatible polymeric matrix. FIG. 16A is a graph depicting the daily release of punicalagins from a biocompatible polymeric matrix where the total amount (mg) of punicalagins released per week (y-axis) and the total percentage of punicalagins released per week (y-axis) from the matrix are plotted against a treatment period measured in weeks (x-axis). FIG. 16B is a graph depicting the cumulative release of punicalagins from a biocompatible polymeric matrix where the total amount (mg) of punicalagins released (y-axis) and the total percentage of punicalagins released (y-axis) are plotted against a treatment period measured in weeks (x-axis).
FIG. 17 is a graph depicting the effect of curcumin, administered by subcutaneous implantation of a biocompatible polymeric matrix incorporating 20% w/w curcumin or by diet, on the formation of DNA adducts in liver and lung tissue of rats treated with a single bolus dose of benzo[a]pyrene.
FIG. 18 is a graph depicting the effect of oltipraz, administered by subcutaneous implantation of a biocompatible polymeric matrix incorporating 10% w/w oltipraz, on the formation of DNA adducts in liver tissue of rats treated with a bolus dose of dibenzo[a,1]pyrene.
FIG. 19 is a graph depicting the effect of GTPs, administered by subcutaneous implantation of a biocompatible polymeric matrix incorporating 10% w/w GTPs, on the formation of DNA adducts in liver tissue of rats treated with a bolus dose of dibenzo[a,1]pyrene.
FIG. 20 is a graph depicting the effect of curcumin, administered by subcutaneous implantation of a 1 cm biocompatible polymeric matrix incorporating 20% w/w curcumin, on the formation of DNA adducts in liver and lung tissue of rats treated with a sustained low dose of benzo[a]pyrene.
FIG. 21 is a graph depicting the effect of GTPs, administered by subcutaneous implantation of two 2-cm biocompatible polymeric matrices incorporating 10% w/w GTPs, on the formation of DNA adducts in liver tissue of rats treated with a sustained low dose of benzo[a]pyrene.
FIG. 22 includes graphs depicting the effect of GTPs, administered by subcutaneous implantation of two 2-cm biocompatible polymeric matrices incorporating 10% w/w GTPs, on expression of various cytochrome P450 (CYP) mRNA in liver tissue of rats treated with a sustained low dose of benzo[a]pyrene. FIG. 22A is a graph depicting the effect of GTPs on the expression of CYP 1A1. FIG. 22B is a graph depicting the effect of GTPs on the expression of CYP 1B1.
FIG. 23 includes graphs depicting the modulation of 17β-estradiol-induced mammary tumor volume (FIG. 23A) and mammary tumor multiplicity (FIG. 23B) by silastic tubing implants incorporating either ellagic acid, lycopene, or curcumin.
FIG. 24 includes graphs depicting the inhibition of 17β-estradiol-induced mammary tumor multiplicity (FIG. 24A) and mammary tumor volume (FIG. 24B) by ellagic acid, administered by a silastic tubing implant incorporating ellagic acid (slow release) or by dietary supplementation with ellagic acid.
FIG. 25 is a graph depicting the effect of a biocompatible silastic tubing implant incorporating fennel extract on 17β-estradiol-induced mammary cell proliferation as measured by cytological evaluation of histological sections for cellular markers of proliferation, including proliferating cell nuclear antigen (PCNA) and the Ki-67 protein.
FIG. 26 includes graphs depicting the inhibition of 17β-estradiol-induced mammary tumor volume (FIG. 26A) and mammary tumor multiplicity (FIG. 26B) by the combined administration of phytochemical agents incorporated into biocompatible silastic tubing implants and implanted subcutaneously (A: sham implant; B: one implant each of oltipraz and curcumin; and, C: one implant each of curcumin, ellagic acid, coenzyme-Q 10, and lycopene), where the numbers on the bars represent percent reduction.
FIG. 27 is a graph depicting the effect of combined administration of curcumin, GTPs, DIM, and punicalagin implants, comprised of a biocompatible polymeric matrix, on mammary cell proliferation in rats treated with 17β-estradiol, where the percentage of PCNA positive cells is plotted against treatment groups at different time intervals.
FIG. 28 is a graph depicting the synergistic activity of various phytochemical agents in inhibiting growth of H1299 human lung cancer cells, where the various phytochemical agents (y-axis) are plotted against the half maximal inhibitory concentration (IC50) for each agent (x-axis).
FIG. 29 is a graph depicting the synergistic activity of various phytochemical agents in inducing apoptosis in H1299 human lung cancer cells where the various phytochemical agents (y-axis) are plotted against the percentage of cells undergoing apoptosis (x-axis).
FIG. 30 is a side view of a device for insertion into a cervical canal made in accordance with the presently-disclosed subject matter.
FIG. 31 is a side view of a device for insertion into a cervical canal made in accordance with the presently-disclosed subject matter and depicting exemplary dimensions of features of the device.
FIG. 32 includes perspective views (FIGS. 32A, 32B, 32C, and 32D) of an exemplary device for cervical insertion.
FIG. 33 includes schematic diagrams depicting the placement of an exemplary device for cervical insertion. FIG. 33A is a schematic diagram depicting a front view of an exemplary device and showing the device engaging the walls of a cervical canal and the external orifice of a uterus. FIG. 33B is a schematic diagram depicting a side view of the device and showing the device engaging the walls of a cervical canal and the external orifice of a uterus.
FIG. 34 includes graphs depicting the release of curcumin from an exemplary device for cervical insertion in vitro. FIG. 34A is a graph depicting the daily release of curcumin from an exemplary device for cervical insertion where the amount (μg) of curcumin released per day (y-axis) and the percentage of curcumin released from the device per day (y-axis) are plotted against a release period measured in days (x-axis). FIG. 34B is a graph depicting the cumulative release of curcumin from an exemplary device for cervical insertion where the total amount (μg) of curcumin released from the device (y-axis) and the total percentage of curcumin released from the device (y-axis) are plotted against a release period measured in days (x-axis).



DESCRIPTION OF EXEMPLARY EMBODIMENTS
The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter.
Unless defined otherwise, 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 presently-disclosed subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are now described.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
The benefits of phytochemicals as therapeutic agents has been demonstrated in a variety of different physiological contexts and pathological conditions. Recently, epidemiological and experimental data has indicated that many phytochemical agents in fruits and vegetables are capable of lowering cancer risk (Steinmetz & Potter, 1991). β-carotene, for instance has been shown to have anti-oxidant properties and was demonstrated to inhibit carcinogenesis by preventing DNA damage (Sun, 1990), besides having immunomodulatory effects in animal models (Bendich, 1989). Similarly, previous studies have suggested a role for vitamin E in promoting immune function (Weitberg, et al., 1997) and have shown that pre-menopausal women with a family history of breast cancer who consumed a high quantity of vitamin E demonstrated a 43% reduction in breast cancer incidence (Zong, et al., 1999).
While phytochemical agents are known to have many health-promoting properties, including reducing cancer mortality, their application as an effective agent for the prophylaxis and treatment of cancer has been limited by the delivery of these agents orally in a capsule, tablet, or powdered format. In this regard, the biggest drawback of phytochemical agents in the prophylaxis and treatment of cancer is that oral delivery of these agents results in limited bioavailability. Similar to traditional chemotherapeutic agents, when phytochemical agents are administered orally, a large part of the agent is destroyed in the gut and a large portion of the remaining phytochemical agent is detoxified during first pass through the liver. As such, a high oral dose of the phytochemical agent is required to achieve a reasonable therapeutic effect. Systemic delivery overcomes this hurdle, but again, like traditional chemotherapeutic agents, systemic delivery can lead to undesirable dose spikes and potential toxicity. To that end, the presently-disclosed subject matter provides new compositions, and methods of using the same, for administering phytochemical agents in a controlled low dose over a period of time.
In some embodiments of the presently-disclosed subject matter, a composition is provided that comprises a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent. In some embodiments, the composition is capable of releasing a controlled low dose of the phytochemical agent over a time period. In some embodiments, the time period is at least about 18 months.
The term “biocompatible” is used herein to refer to a composition that is substantially non-toxic in the in vivo environment of its intended use, and that is not substantially rejected by the subject's physiological system (i.e., is non-antigenic). As will be recognized by those of ordinary skill in the art, the biocompatibility of a particular composition can be gauged by the composition's toxicity, infectivity, pyrogenicity, irritation potential, reactivity, hemolytic activity, carcinogenicity, and/or immunogenicity. When introduced into a majority of subjects, a biocompatible composition will not cause an undesirably adverse, long-lived, or escalating biological reaction or response, and is distinguished from a mild, transient inflammation, which typically accompanies surgery or implantation of foreign objects into a living organism.
A biocompatible polymeric matrix of the presently-disclosed subject matter can be fabricated from a variety of polymeric materials. Generally, polymeric materials are broadly classified as synthetic, natural, or blends thereof, and within these broad classes, the polymeric materials can be further classified as biodegradable or biostable. Biodegradable polymers degrade in vivo as a function of chemical composition, method of manufacture, and composition structure. Biostable polymers, on the other hand, remain intact in vivo for extended periods of time ranging from several years to more and include polymers such as ethylene-vinyl acetate copolymers, polyurethanes, polyacrylonitriles, and certain polyphosphazenes.
In some embodiments of the presently-disclosed subject matter, a biocompatible polymeric matrix is provided that is biodegradable. To provide a biocompatible polymeric matrix that is biodegradable, both synthetic and natural polymers can be used, with synthetic polymers being preferred due to a more uniform and reproducible degradation, as well as other physical properties. Examples of synthetic biodegradable polymers capable of being used in accordance with the presently-disclosed subject matter include, but are not limited to, polycaprolactone, polyanhydrides, polyhydroxyacids such as polylactic acid, polyglycolic acids and copolymers thereof, polyesters, polyamides, polyorthoesters, and certain polyphazenes. Examples of naturally-occurring biodegradable polymers capable of being used in accordance with the presently-disclosed subject matter include, but are not limited to, proteins and polysaccharides such as collagen, hylauronic acid, albumin, and gelatin.
In some embodiments, a biocompatible polymeric matrix is provided that is comprised of a polymers selected from polycaprolactone, cyclodextrin, F68, polyethylene glycol, and combinations thereof. In some embodiments, the biocompatible polymeric matrix comprises polycaprolactone in combination with a second polymer. In some embodiments, the polycaprolactone and the second polymer are combined in a ratio of about 4:1 or about 9:1. In some embodiments, the second polymer is selected from cyclodextrin, F68, and polyethylene glycol. In some embodiments, the biocompatible polymeric matrix comprises polycaprolactone and F68 in a ratio of about 4:1.
A composition of the presently-disclosed subject matter can be formulated by dissolving one or more polymers in an appropriate solvent to initiate the formation of the biocompatible polymeric matrix. One or more phytochemical agents are then dissolved in either the same solvent as the one or more polymers or another appropriate solvent depending on the solubility of the particular phytochemical agent. After mixing the polymer solution and the solution of phytochemical agents, the solvents can then be evaporated under reduced pressure to produce a composition where the phytochemical agents are embedded or entrapped within a polymeric matrix.
As discussed herein above, previously phytochemical agents generally have been delivered orally through the diet of a subject. Indeed, almost all published animal studies to date have used a dietary route for administering phytochemical agents, with the dosages of the phytochemical agents ranging from a 10-1000 mg/kg per day in a given diet. When these dose ranges are extrapolated for use in clinical studies, however, the dosage range becomes alarmingly high and thus imposes a risk of toxicity to the subject. By incorporating the phytochemical agents into a biocompatible polymeric matrix, such as those disclosed herein, the inventors of the presently-disclosed subject matter have discovered a composition that effectively releases phytochemical agents at levels that are significantly lower relative to levels provided by a dietary route (i.e., oral delivery) and furthers provides constant, steady-state dosages of the phytochemical agents over a time period, or in other words, provides a “controlled low dose” of the phytochemical agents. Incorporation of a predetermined-amount of a phytochemical agent into a biocompatible polymeric matrix of the presently-disclosed subject matter allows for similar amounts of the phytochemical agent to be released each day as the agent diffuses from the matrix and the biocompatible polymeric matrix degrades in a uniform manner. As such, the presently-disclosed compositions are capable of lowering the effective dose delivered to a given subject, if desired, to thereby minimize toxicity while also delivering the controlled dose at a constant, steady-state level over extended time periods to increase bioavailability and to elicit a biological response.
In some embodiments, the controlled low dose of the phytochemical agent is released over a time period of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 months from the time of administration. In some embodiments, a bolus dose of the phytochemical agent can be released from the biocompatible polymeric matrix over a time period of about 1 to about 2 weeks from the time of administration, and the controlled low dose of the phytochemical agent is released over a time period beginning at about 2 to about 3 weeks after administration and extending to about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, or about 24 months from the time of administration. In some embodiments, a composition of the presently-disclosed subject matter is provided that releases a controlled low dose of the phytochemical agent over a time period of at least about 18 months.
As used herein, the term “phytochemical agent” refers to a non-nutritive plant-derived compound, or an analog thereof, that is capable of “treating” a cancer, as defined herein below. Examples of phytochemical agents include, but are not limited to compounds such as monophenols; flavonoids, such as flavonols, flavanones, flavones, flavan-3-ols, anthocyanins, anthocyanidins, isoflavones, dihydroflavonols, chalcones, and coumestans; phenolic acids; hydroxycinnamic acids; lignans; tyrosol esters; stillbenoids; hydrolysable tannins; carotenoids, such as carotenes and xanthophylls; monoterpenes; saponins; lipids, such as phytosterols, tocopherols, and omega-3,6,9 fatty acids; diterpenes; triterpinoids; betalains, such as betacyanins and betaxanthins; dithiolthiones; thiosulphonates; indoles; and glucosinolates. As another example of a phytochemical agent disclosed herein, the phytochemical agent can be an analog of a plant-derived compound, such as oltipraz, which is an analog of 1,2-dithiol-3-thione, a compound that is found in many cruciferous vegetables. Table 1 provides a list of specific phytochemical agents that are exemplary of the broader classes of phytochemical agents described herein above.







TABLE 1





List of Exemplary Phytochemical Agents.

















PHENOLIC COMPOUNDS



Monophenols









Apiole



Carnosol



Carvacrol



Dillapiole



Rosemarinol









Phenolic acids









Ellagic acid



Gallic acid



Salicylic acid



Tannic acid



Vanillin



Capsaicin



Curcumin



Plumbagin









Hydroxycinnamic acids









Caffeic acid



Chlorogenic acid



Cinnamic acid



Ferulic acid



Coumarin









Lignans (phytoestrogens)









Silymarin



Matairesinol



Secoisolariciresinol



Pinoresinol



Lariciresinol









Tyrosol esters









Tyrosol



Hydroxytyrosol



Oleocanthal



Oleuropein









Stilbenoids









Resveratrol



Pterostilbene



Piceatannol









Hydrolyzable Tannins









Punicalagins









Flavonoids (polyphenols)









Flavonols









Quercetin



Gingerol



Kaempferol



Myricetin



Rutin



Isorhamnetin









Flavanones









Hesperidin



Naringenin



Silybin



Eriodictyol









Flavones









Apigenin



Tangeritin



Luteolin









Flavan-3-ols









Catechins



Gallocatechin



Epicatechin



Epigallocatechin



Epigallocatechin gallate



Epicatechin-gallate



Theaflavin



Theaflavin-gallate



Theaflavin-digallate



Thearubigins









Anthocyanins &



Anthocyanidins









Pelargonidin



Peonidin



Cyanidin



Delphinidin



Malvidin



Petunidin









Isoflavones (phytoestrogens)









Daidzein



Genistein



Equol



Glycitein









Dihydroflavonols



Chalcones



Coumestans









Coumestrol









TERPENES



Carotenoids (tetraterpenoids)









Carotenes









α-Carotene



β-Carotene



γ-Carotene



δ-Carotene



Tocotrienols



Tocopherols



Lycopene



Neurosporene



Phytofluene



Phytoene









Xanthophylls









Canthaxanthin



Cryptoxanthin



Zeaxanthin



Astaxanthin



Lutein



Rubixanthin









Monoterpenes









Limonene



Perillyl alcohol









Saponins



Lipids









Phytosterols









Campesterol



β-Sitosterol



γ-Sitosterol



Stigmasterol









Tocopherols



ω-3,6,9 fatty acids









γ-linolenic acid









Diterpene









Withaferins









Triterpenoid









Oleanolic acid



Ursolic acid



Betulinic acid



Moronic acid



Curcurbitacins



Lupeol









BETALAINS



Betalains









Betacyanins









Betanin



Isobetanin



Probetanin



Neobetanin









Betaxanthins









Indicaxanthin



Vulgaxanthin









ORGANOSULFIDES



Dithiolthiones









Sulphoraphane









Thiosulphonates









Allyl methyl trisulfide



Dialyl sulfide









INDOLES,



GLUCOSINOLATES



Indole-3-carbinol



sulforaphone



3,3′-Diindolylmethane



Sinigrin



Allicin



Alliin



Allyl isothiocyanate



Piperine









In some embodiments, the phytochemical agent is selected from curcumin, green tea polyphenols, punicalagin, diindolylmethane, oltipraz, tocotrienol, tocopherol, plumbagin, cyanidin, delphinidin, lycopene, lupeol, curcurbitacin-B, Withaferin A, indole-3-carbinol, genestein, equol, resveratrol, co-enzyme Q-10, ellagic acid, petunidin, malvidin, peonidin, fennel extract, and combinations thereof. In some embodiments, the phytochemical agent is a combination of oltipraz and curcumin; curcumin, ellagic acid, co-enzyme Q-10, and lycopene; curcumin, green tea polyphenols, diindolylmethane, and punicalagin; delphinidin, petunidin, malvidin, peonidin, and cyanidin; or punicalagin and delphinidin.
In some embodiments of the presently-disclosed subject matter, one or more phytochemical agents are incorporated into and a released from a single biocompatible polymeric matrix. In some embodiments, a second biocompatible polymeric matrix can be provided that incorporates at least one phytochemical agent such that multiple biocompatible polymeric matrices are provided, wherein each biocompatible polymeric matrix incorporates at least one phytochemical agent.
In some embodiments, a composition is provided that comprises a biocompatible matrix comprising polycaprolactone and F68 in a ratio of about 4:1 that incorporates an effective amount of curcumin such that the composition releases a controlled low dose of curcumin over a time period.
As described above, various phytochemical agents can be incorporated into and released from the biocompatible polymeric matrices of the presently-disclosed subject matter. In some embodiments, the phytochemical agent comprises about 2% to about 50% of the weight of the one or more polymers comprising the biocompatible polymeric matrix. In some embodiments, the phytochemical agents comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 18%, about 19%, about 20%, about 21%, about 22%, about 25%, about 30%, about 35%, about 40%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% of the weight of the one or more polymers. In some embodiments, the phytochemical agents are added to between about 2% to about 20% of the polymer weight. As will be recognized by those of ordinary skill in the art, the optimum amount of each phytochemical agent that is incorporated into a biocompatible polymeric matrix can vary depending on the particular phytochemical agents used and the desired dosage to be achieved. Determination and adjustment of the amount of a phytochemical agent to be used in a particular composition or application, as well as when and how to make such adjustments, can be ascertained using only routine experimentation.
Any phytochemical agent of the presently-disclosed subject matter can be provided in the form of a pharmaceutically acceptable salt or solvate. A salt can be formed using a suitable acid and/or a suitable base. Suitable acids that are capable of forming salts with the phytochemical agents of the presently disclosed subject matter include inorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, or the like. Suitable bases capable of forming salts with the phytochemical agents of the presently disclosed subject matter include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropyl amine, methyl amine, dimethyl amine, and the like), and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine, and the like).
As used herein, the term “solvate” refers to a complex or aggregate formed by one or more molecules of a solute, e.g. a phytochemical agent or a pharmaceutically-acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute and solvent. Representative solvents include, but are not limited to, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate. As such, the term “pharmaceutically-acceptable salt or solvate thereof” is intended to include all permutations of salts and solvates, such as a solvate of a pharmaceutically-acceptable salt of a phytochemical agent.
In some embodiments, a composition is provided that further comprises an effective amount of a chemotherapeutic agent that is incorporated into and released from a biocompatible polymeric matrix that also incorporates an effective amount of a phytochemical agent or from a second biocompatible polymeric matrix. Examples of such chemotherapeutic agents include, but are not limited to, platinum coordination compounds such as cisplatin, carboplatin or oxalyplatin; taxane compounds, such as paclitaxel or docetaxel; topoisomerase I inhibitors such as camptothecin compounds for example irinotecan or topotecan; topoisomerase II inhibitors such as anti-tumor podophyllotoxin derivatives for example etoposide or teniposide; anti-tumor vinca alkaloids for example vinblastine, vincristine or vinorelbine; anti-tumor nucleoside derivatives for example 5-fluorouracil, gemcitabine or capecitabine; alkylating agents, such as nitrogen mustard or nitrosourea for example cyclophosphamide, chlorambucil, carmustine or lomustine; anti-tumor anthracycline derivatives for example daunorubicin, doxorubicin, idarubicin or mitoxantrone; HER2 antibodies for example trastuzumab; estrogen receptor antagonists or selective estrogen receptor modulators for example tamoxifen, toremifene, droloxifene, faslodex or raloxifene; aromatase inhibitors, such as exemestane, anastrozole, letrazole and vorozole; differentiating agents such as retinoids, vitamin D and retinoic acid metabolism blocking agents (RAMBA) for example accutane; DNA methyl transferase inhibitors for example azacytidine; kinase inhibitors for example flavoperidol, imatinib mesylate or gefitinib; farnesyltransferase inhibitors; HDAC inhibitors; other inhibitors of the ubiquitin-proteasome pathway for example VELCADE®; or YONDELIS®.
In some embodiments, a composition is provided that further comprises an effective amount of a anti-inflammatory agent that is incorporated into and released from a biocompatible polymeric matrix that also incorporates an effective amount of a phytochemical agents or from a second biocompatible polymeric matrix. Example of anti-inflammatory agents that can be used in accordance with the methods and compositions of the presently-disclosed subject matter include, but are not limited to, non-steroidal anti-inflammatory agents (NSAIDS), such as aspirin, diclofenac, indomethacin, sulindac, ketoprofen, flurbiprofen, ibuprofen, naproxen, piroxicam, tenoxicam, tolmetin, ketorolac, oxaprosin, mefenamic acid, fenoprofen, nambumetone, acetaminophen, and combinations thereof, COX-2 inhibitors, such as nimesulide, flosulid, celecoxib, rofecoxib, parecoxib sodium, valdecoxib, etoricoxib, etodolac, meloxicam, and combinations thereof, glucocorticoids, such as hydrocortisone, cortisone, prednisone, prednisolone, methylprednisolone, meprednisone, triamcinolone, paramethasone, fluprednisolone, betamethasone, dexamethasone, fludrocortisone, desoxycorticosterone, rapamycin; or others or analogues of these agents or combinations thereof.
Further provided, in some embodiments of the presently-disclosed subject matter are methods for treating a cancer. In some embodiments, a method for treating a cancer is provided that comprises administering to a subject in need thereof an effective amount of a composition of the presently-disclosed subject matter comprising a biocompatible polymeric matrix incorporating a phytochemical agent, wherein the phytochemical agent is incorporated into and released over a time period from the biocompatible polymeric matrix at a controlled low dose of the phytochemical agent.
As used herein, the terms “treating” or “treatment” relate to any treatment of a cancer including, but are not limited to, therapeutic treatment and prophylactic treatment of a cancer. With regard to therapeutic treatment of a cancer, the terms “treating” or “treatment” include, but are not limited to, inhibiting the progression of a cancer, arresting the development of a cancer, reducing the severity of a cancer, ameliorating or relieving one or more symptoms associated with a cancer, and causing a regression or a cancer or one or more symptoms associated with a cancer.
As noted herein above, the terms “treating” or “treatment,” as used herein, further include the prophylactic treatment of a cancer including, but not limited to, any action that occurs before the development of a cancer. It is understood that the degree of prophylaxis need not be absolute (e.g. the complete prophylaxis of a cancer such that the subject does not develop a cancer at all), and that intermediate levels of prophylaxis, such as increasing the time required for at least one symptom resulting from a cancer to develop, reducing the severity or spread of a cancer in a subject, or reducing the time that at least one adverse health effect of a cancer is present within a subject, are all examples of prophylactic treatment of a cancer.
As further non-limiting examples of the treatment of a cancer by a composition described herein, treating a cancer can include, but is not limited to, killing cancer cells, inhibiting the development of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the available blood supply to a tumor or cancer cells, promoting an immune response against a tumor or cancer cells, reducing or inhibiting the initiation or progression of a cancer, increasing the lifespan of a subject with a cancer, or inhibiting or reducing the formation of DNA adducts by chemical carcinogens.
In some embodiments of the presently-disclosed subject matter, a method for treating a cancer is provided wherein the treating comprises inhibiting or reducing the formation of DNA adducts. The formation of DNA adducts (i.e. carcinogens covalently bound to DNA) is widely considered a prerequisite for the initiation and progression of cancer development. Many carcinogens are known to induce the formation of DNA adducts (Hemminki, 1995) and the presence of DNA adducts in humans has been strongly correlated with an increased risk for cancer development (Santella, 1997). For example, human studies have shown a higher accumulation of tissue DNA adducts in cigarette smokers than in non-smokers or individuals who have never smoked, indicating that DNA adduct formation is a viable target for the treatment of cancer.
As used herein, the term “cancer” refers to all types of cancer, neoplasm, or malignant tumors found in subjects, including leukemias, carcinomas, and sarcomas. Examples of cancers include, but are not limited to, cancer of the brain, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, prostate, sarcoma, stomach, uterus, skin, esophagus, pancreas, and medulloblastoma.
In some embodiments of the presently-disclosed subject matter, the cancer is selected from the group consisting of breast cancer, lung cancer, and cervical cancer. As used herein, the term “breast cancer” is meant to refer to any cancer of the breast and includes, but is not limited to, mammary carcinoma, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, inflammatory breast cancer, and hormone-dependent and hormone-independent tumors of the breast. The term “cervical cancer” is meant to refer to any cancer of the cervix and includes, but is not limited to, squamous cell carcinomas, adenocarcinomas, and adenosquamous carcinomas, or mixed carcinomas. The term “lung cancer” is meant to refer to any cancer of the lung and includes non-small cell lung carcinomas and small cell lung carcinomas. Examples of non-small cell lung carcinomas include, but are not limited to, squamous cell lung carcinomas, adenocarcinomas, bronchioalveolar carcinomas, adenosquamous carcinomas, papillary adenocarcinomas, mucoepidermoid carcinomas, adenoid cystic carcinomas, large cell carcinomas, and giant cell and spindle cell carcinomas.
In some embodiments of the presently-disclosed methods of treating a cancer, the subject is at an increased risk of developing a primary cancer. In some embodiments, the primary cancer is selected from the group consisting of breast cancer, lung cancer, and cervical cancer.
The phrase “increased risk” is used herein to refer to those subjects whose likelihood of developing a cancer in their lifetime is increased, as compared to a normal subject. Such subjects may be identified by factors that include, but are not limited to: a genetic pre-disposition to certain cancers; life style choices such as tobacco smoking, tobacco chewing, or dietary habits; medical treatments such as immunosuppression; or exposure to carcinogenic agents in the environment, such as viruses, chemicals, medications, and radiation, or age-related factors, such as hormone levels. For example, subjects at an increased risk of developing breast cancer may be identified by factors such as age, gender, early age of menarche, late age of menopause, nulliparity, use of oral contraceptives, prolonged estrogen replacement therapy, breast density, or mutations in two known breast-cancer related genes, BRCA1 and BRCA2. As another example, subjects at risk of developing cervical cancer may be identified by factors such as human papilloma virus (HPV) infection, life style choices such as tobacco smoking or dietary habits, human immunodeficiency virus (HIV) infection, use of oral contraceptives, multiple pregnancies, hormonal therapies, genetic predispositions to cervical cancer, and cervical dysplasia.
As used herein, the term “primary cancer” is meant to refer to an original tumor or cancer cell in a subject. Such primary cancers are usually named for the part of the body in which the primary cancer originates. Furthermore, a “secondary cancer” is used herein to refer to a cancer which has spread, or metastasized, from an initial site (i.e. a primary cancer site) to another site in the body of a subject, a cancer which represents a residual primary cancer, or a cancer that has originated from treatment with an antineoplastic agent(s) or radiation or both. In this regard, the term “secondary cancer” is thus not limited to any one particular type of cancer, including the type of primary cancer from which it derived. In some embodiments of the presently-disclosed subject matter, a method of preventing or treating a cancer is further provided where the subject is at risk of developing a secondary cancer.
As used herein, the term “subject” includes both human and animal subjects. Thus, veterinary therapeutic uses are provided in accordance with the presently disclosed subject matter. As such, the presently-disclosed subject matter provides for the treatment of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and/or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and/or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.
For administration of a composition as disclosed herein, conventional methods of extrapolating human dosage based on doses administered to a murine animal model can be carried out using the conversion factor for converting the mouse dosage to human dosage: Dose Human per kg=Dose Mouse per kg×12 (Freireich, et al., (1966) Cancer Chemother. Rep. 50:219-244). Drug doses can also be given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and excretionary functions. Moreover, body surface area can be used as a common denominator for drug dosage in adults and children as well as in different animal species as described by Freireich, et al. (Cancer Chemother. Rep. 1996; 50:219-244). Briefly, to express a mg/kg dose in any given species as the equivalent mg/sq m dose, multiply the dose by the appropriate kg factor. In an adult human, 100 mg/kg is equivalent to 100 mg/kg×37 kg/sq m=3700 mg/m2.
In some embodiments of the presently-disclosed subject matter, the phytochemical agent is administered to a subject by subcutaneous implantation of the biocompatible polymeric matrix incorporating the phytochemical agent. In some embodiments, the phytochemical agent is administered to a subject by implantation of a composition of the presently-disclosed subject matter at a site distant from the site of the cancer. For example, a composition of the presently-disclosed subject matter can be implanted subcutaneously in the arm of a subject such that the phytochemical agents, upon their release into systemic circulation, are capable of treating a breast, cervical, or lung cancer, which is located at a site distant from the site of the implantation of the presently-disclosed composition.
In some embodiments, the phytochemical agent is administered to a subject by implantation of the biochemical polymeric matrix incorporating the phytochemical agent at a site of a cancer or at a site suspected of developing a cancer. As an exemplary method of administering a composition of the presently-disclosed subject matter at the site of a cancer, the biocompatible polymeric composition can be implanted at the site of a tumor, either following surgical removal or resection of the tumor, or can be implanted into the surrounding tissue. As another example of administering a composition of the presently-disclosed subject matter at a site of cancer, the composition need not be implanted internally within the body of a subject, but can be implanted into an external orifice of the subject where a cancer is present. For example, a composition of the presently-disclosed subject matter can be implanted into the cervix of a subject by insertion of the biocompatible polymeric matrix incorporating the phytochemical agent into the cervical canal of a subject. Similarly, if a subject is suffering from cervical dysplasia, a biocompatible matrix incorporating a phytochemical agent can be administered to a site suspected of developing a cancer by the inserting the composition into the cervical canal of the subject such that an effective amount of the phytochemical agent is administered to the cervix of the subject.
The term “effective amount” is used herein to refer to an amount of the composition (e.g., a composition comprising a phytochemical agent that is incorporated into and released from a biocompatible polymeric composition) sufficient to produce a measurable biological response (e.g., inhibition of the development of tumor cells or a reduction in the number of tumor cells). Actual dosage levels of active ingredients in a therapeutic composition of the presently-disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject and/or application. The selected dosage level will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. Preferably, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.
For additional guidance regarding formulation and dose, see U.S. Pat. Nos. 5,326,902 and 5,234,933; PCT International Publication No. WO 93/25521; Berkow, et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, N.J.; Goodman, et al., (2006) Goodman & Gilman's the Pharmacological Basis of Therapeutics, 11th ed. McGraw-Hill Health Professions Division, New York; Ebadi. (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca Raton, Fla.; Katzung, (2007) Basic & Clinical Pharmacology, 10th ed. Lange Medical Books/McGraw-Hill Medical Pub. Division, New York; Remington, et al., (1990) Remington's Pharmaceutical Sciences, 18th ed. Mack Pub. Co., Easton, Pa.; Speight, et al., (1997) Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland/Philadelphia; and Duch, et al., (1998) Toxicol. Lett. 100-101:255-263.
Still further provided, in some embodiments of the presently-disclosed subject matter, is a device for insertion into a cervical canal. Referring to FIG. 30, in some embodiments of presently-disclosed subject matter, a device 10 for insertion into a cervical canal includes: a substantially cylindrical shaft 20 having a proximal end 22 and a distal end 24, and defining a canal 26 that extends from the proximal end 22 to the distal end 24; and, a cap 30 attached to the proximal end 22 of the cylindrical shaft 20, said cap including a central opening 32 in registry with the canal defined by the shaft.
Referring now to FIG. 33, an exemplary device 10 can be inserted into the cervix of a subject to “treat” a cancer, as defined herein. In some embodiments, a device 10 for insertion into a cervical canal is provided wherein the cylindrical shaft 20 is suitably adapted for engaging the walls of a cervical canal and the bottom surface 34 of the cap 30 is suitably adapted for engaging an external orifice of the uterus. In some embodiments, the bottom surface 34 of the cap 30 has a concave shape such that the bottom surface 34 of the cap 30 suitably engages the external orifice of the uterus. In some embodiments, a device 10 for insertion into a cervical canal is provided wherein the distal end 24 of the shaft 20 is tapered such that the device 10 is easily inserted into the cervix of a subject. In some embodiments, the canal 26 defined by the shaft 20 allows for the passage of bodily fluids from the uterus.
In some embodiments, the device is designed and fabricated such that the device contacts a transformation zone of a cervix of a subject. As used herein, the term “transformation zone” refers to a region of a cervix that separates the ectocervical region, which is comprised of squamous cells, from the endocervical region, which is comprised of columnar cells. In younger subjects, the transformation zone is typically found on the outside of the external orifice of the uterus. However, as a subject ages, the transformation zone decreases in size and reaches the cervical canal. As such, in some embodiments of the presently-disclosed subject matter, a device for cervical insertion is provided that can be utilized to treat cervical cancer in female subjects of all ages by maintaining contact with the transformation zone.
Referring now to FIG. 31, it is contemplated that an exemplary device for insertion into a cervical canal can be adapted for suitable insertion into the cervix of a human subject. In this regard, in some embodiments of the presently-disclosed device for insertion in a cervical canal, a device is provided wherein the cylindrical shaft is about 19 to about 25 mm in length and about 9 to about 11 mm in diameter, the cap is about 20 to 25 mm in diameter, and the canal is about 4 mm to about 5 mm in diameter.
As shown in FIG. 32A-D, an exemplary device of the presently-disclosed subject matter can be comprised of a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent, as described herein. For example, an exemplary device can be fabricated by extruding a dissolved mixture of one or more polymers and one or more phytochemical agents through a suitable mold and dried to provide a suitably shaped device. Further shaping of the device can additionally be performed during the drying process. As will be evident to those of ordinary skill in the art, different shaped molds can be provided such that other devices can be fabricated or adapted to provide suitably shaped or sized devices for insertion or implantation at other anatomical locations.
In some embodiments of the device for insertion into a cervical canal, the phytochemical agent is selected from curcumin, green tea polyphenols, punicalagin, diindolylmethane, oltipraz, tocotrienol, tocopherol, plumbagin, cyanidin, delphinidin, lycopene, lupeol, curcurbitacin-B, Withaferin A, indole-3-carbinol, genestein, equol, resveratrol, co-enzyme Q-10, ellagic acid, petunidin, malvidin, peonidin, fennel extract, and combinations thereof. In some embodiments, the phytochemical agent comprises about 2% to about 50% of the polymer weight.
In some embodiments, a device for cervical insertion is provided where the biocompatible polymeric matrix is comprised of polymers selected from polycaprolactone, cyclodextrin, F68, polyethylene glycol, and combinations thereof. In some embodiments, the biocompatible polymeric matrix comprises polycaprolactone in combination with a second polymer that, in certain embodiments, can be selected from cyclodextrin, F68, and polyethylene glycol. In some embodiments the polycaprolactone and the second polymer are combined in a ratio of about 4:1 or about 9:1. In some embodiments, the biocompatible matrix comprises polycaprolactone and F68 in a ratio of about 4:1 and, in some embodiments, the phytochemical agent comprises curcumin. In some embodiments, the biocompatible polymeric matrix is biodegradable.
The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. Some of the following examples are prophetic, notwithstanding the numerical values, results and/or data referred to and contained in the examples. Additionally, the following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.
EXAMPLES
Example 1
Formulation of Compositions
Compositions were prepared by first dissolving two polymers in an appropriate solvent: e.g. a water-insoluble, biodegradable, and biocompatible polycaprolactone (MW 65,000) and a water-soluble cyclodextrin. Polycaprolactone and cyclodextrin (in a 4:1 or 9:1 ratio) were dissolved in 2-5 volumes of dichloromethane (g/ml). Some preparations comprising polycaprolactone as a first polymer included either F68 or polyethylene glycol instead of cyclodextrin. Desired phytochemical agent(s), approximately 2% to 20% or more of the polymer weight, were then dissolved either in the same solvent as the polymer or another appropriate solvent (e.g., ethanol, acetone, tetrahydrofuran), depending upon the phytochemical agent's solubility. Typically, the amounts of phytochemicals incorporated into a biocompatible polymeric matrix ranged from less than 2 mg to as much as 20 mg of phytochemical agent per 100 mg of polymer. Depending on the particular phytochemical agent employed, however, it was possible to incorporate as much as 50 mg or more of the phytochemical agent per 100 mg of polymer.
After the phytochemical agent and the polymers were dissolved in appropriate solvents, the polymer solution and the phytochemical agent solution were mixed in a 100-200 ml glass beaker, and the solvents were evaporated under reduced pressure. The resulting composition was a polymeric material incorporating a phytochemical agent in a matrix, which was then cut into small pieces and filled in a syringe attached to a silastic tubing (4-6 cm long and 2.2 mm diameter). This entire assembly was then placed at 70-80° C. for several minutes to facilitate the extrusion of a molten polymeric material/phytochemical agent into the silastic tubing. After an appropriate amount of time had passed, the molten polymeric material/phytochemical agent was extruded into the silastic tubing and a few minutes after cooling at room temperature, the solid compositions were removed from the tubing and cut to produce solid, cylindrical compositions that were approximately 1 mm to 2.2 mm in diameter and approximately 1 cm to 3 cm in length. Exemplary compositions comprising a biocompatible polymeric matrix by itself (sham) or incorporating curcumin, ellagic acid, oltipraz, or green tea polyphenols (GTPs) are shown in FIG. 1.
Example 2
In Vitro Release of Phytochemical Agents
To determine daily release of the phytochemical agents in vitro, compositions, which were approximately 1 cm in length and approximately 1 mm to 2.2 mm in diameter, comprising a biocompatible polymeric matrix incorporating a desired phytochemical agent (20% w/w) were placed in a release medium (5-20 ml phosphate-buffered-saline (pH 7.4) containing 10% bovine serum) at 37° C. with constant agitation in a water bath to simulate an in vivo scenario. The medium was replaced daily and the amount of phytochemical agents released from the compositions per day was measured spectrophotometrically against a standard curve and expressed as micrograms per day.
FIGS. 2 to 8 show the amount of exemplary phytochemical agents released per day and the total amount of phytochemical agents released from a biocompatible polymeric matrix of the presently-disclosed subject matter over a particular time period, including data for curcumin (FIGS. 2A and 2B), GTPs (FIGS. 3A and 3B), diindolylmethane (DIM, FIGS. 4A and 4B), punicalagins (FIGS. 5A and 5B), oltipraz (FIGS. 6A and 6B), lycopene (FIGS. 7A and 7B) and resveratrol (FIGS. 8A and 8B). From this in vitro data, it can be seen that for a typical phytochemical agent approximately 4% to 10% of the phytochemical agent is released during the first week, followed by 2% to 5% per week for the following 2 to 3 weeks, and then after 3 weeks approximately 0.2 to 0.5% per week for periods up to several months. Without wishing to be bound by any particular theory, it is believed that the amount of phytochemical agents released from each biocompatible polymeric matrix is dependent on the lipophilicity of the phytochemical agent, with a higher initial release for highly water-soluble compounds (e.g. green tea polyphenols) and a somewhat lower release for more lipophilic agents (e.g. curcumin).
Example 3
Effect of Phytochemical Agent Load on In Vitro Release of Phytochemicals from a Biocompatible Polymeric Matrix
To determine the effect of incorporating various amounts of phytochemical agents into a biocompatible polymeric matrix on the in vitro release of the agents, 2% w/w, 5% w/w, and 20% w/w of curcumin were incorporated into separate biocompatible polymeric matrices and placed in a release medium (5-20 ml phosphate-buffered-saline (pH 7.4) containing 10% bovine serum) at 37° C. with constant agitation in a water bath to simulate an in vivo scenario. The medium was replaced daily and the amount of curcumin released from each composition per day was measured spectrophotometrically against a standard curve and expressed as μg/day.
As shown in FIG. 9, the amount of curcumin released from the composition per day (FIG. 9A) and the total amount of curcumin released from the composition (FIG. 9B) varied proportionally with the amount of curcumin incorporated into each biocompatible polymeric matrix. Similar to the results described in Example 2, a greater amount of curcumin was released during the first week after placing the composition into the medium. Following the first week, however, amounts of curcumin released from each composition approached steady-state levels where a similar amount of phytochemical agent was released from each composition per day, indicating that increasing the amount of phytochemical agent that is incorporated into each matrix is an effective way to increase the bioavailability of the phytochemical agent without adversely effecting the release of the agent from the matrix.
A similar experiment was also performed to examine the effect of incorporating various amounts of GTPs into a biocompatible polymeric matrix on the in vitro release of the agents. Briefly, 1% w/w, 2.5% w/w, 5% w/w, and 10% w/w GTPs were incorporated into a biocompatible polymeric matrix, comprised of polycaprolactone, MW 65K and polycaprolactone, MW 15K combined in a 1:4 ratio, and placed in a release medium (phosphate-buffered-saline (pH 7.4) containing 10% bovine serum) at 37° C. with constant agitation in a water bath. As shown in FIG. 10, the amount of GTPs released from the composition per day (FIG. 10A) and the total amount of GTPs released from the composition (FIG. 10B) varied proportionally with the amount of GTPs incorporated into each biocompatible polymeric matrix, again indicating that the amount of phytochemical agent incorporated into each matrix is an effective way to increase the bioavailability of the phytochemical agent without adversely effecting the release of the agent from the matrix.
Example 4
Short Term In Vivo Release of Curcumin from a Biocompatible Polymeric Matrix
To measure the average daily release of a phytochemical in vivo over a short time period in whole animals, two 2-cm biocompatible polymeric matrices incorporating 20% w/w of curcumin were implanted subcutaneously in the back of Sprague-Dawley rats. At predetermined time intervals, the animals were euthanized and the compositions were removed from the animals. The residual curcumin remaining in each matrix was then measured by solvent extraction and spectrophotometry. The total amount released at each time interval was determined by subtracting the unreleased amount of curcumin remaining in the matrix from the initial amount implanted into the rats, and this amount was divided by the initial amount implanted into the rats to determine the total percentage of curcumin released from the matrix at each time interval. As shown in FIG. 11, the total amount of curcumin (mg) released from the matrix and the total percentage of curcumin released from the matrix over the 40 day treatment period increased at each time interval indicating that a phytochemical agent can be effectively released from a biocompatible polymeric matrix in vivo and that this release is similar to the release kinetics observed for a phytochemical agent in vitro.
To determine the levels of curcumin released systemically into the rats treated with a biocompatible polymeric matrix incorporating 20% w/w curcumin, lung and liver tissues from each rat were removed after euthanizing the animals at 4, 7, 13, and 21 days after subcutaneous implantation of the compositions. Curcumin levels were measured in 0.5 g of tissue, either pooled from three rats randomly (lung) or analyzed from individual rats (liver), by solvent extraction and high-performance liquid chromatography (HPLC) in conjunction with a fluorescence detector and were expressed as nanograms (ng) of curcumin per gram (g) of tissue. As shown in FIG. 12A, curcumin was present at 4 days in lung tissue of treated rats at levels of approximately 15 ng/g. However, after 7 days these levels dropped to approximately 5 ng/g and this lower level of curcumin was maintained in the lung tissue at 13 days and 21 days post-implantation with only slight variation, indicating that a controlled low dose of curcumin can be sustained in tissue of subjects administered a biocompatible polymeric matrix incorporating a phytochemical agent by subcutaneous implantation. Similarly, in the liver tissue (FIG. 12B), curcumin was present at a level of approximately 40 ng/g at 4 days. The levels of curcumin in liver tissue dropped to about 5 ng/g at 7 days and then the levels remained constant at about 2.5 ng/g during the next 30 days.
Example 5
Long Term In Vivo Release of Phytochemicals from a Biocompatible Polymeric Matrix
To measure the release of a phytochemical agent from a biocompatible polymeric matrix in vivo over an extended time period, four 2-cm implants, one each of curcumin (20% w/w), GTPs (20% w/w), DIM (20% w/w), and punicalagins (20% w/w) were implanted simultaneously into the back of female ACI rats. At predetermined time intervals, the animals were euthanized and the compositions were removed from the animals. The residual phytochemicals remaining in each matrix were then measured by solvent extraction and spectrophotometry. The total amount released at each time interval was determined by subtracting the unreleased amount of the phytochemical remaining in each matrix from the initial amount implanted into the rats, and this amount was divided by the initial amount implanted into the rats to determine the total percentage of the phytochemical released from the matrix at each time interval.
FIGS. 13 to 16 show the amount of exemplary phytochemical agents released per day and the total amount of phytochemical agents released from a biocompatible polymeric matrix of the presently-disclosed subject matter in vivo over an extended time period of 18 weeks, including data for the long-term release of curcumin (FIGS. 13A and 13B), GTPs (FIGS. 14A and 14B), diindolylmethane (DIM, FIGS. 15A and 15B), and punicalagins (FIGS. 16A and 16B). As shown in FIGS. 13 to 16, a greater amount of each phytochemical was initially released from the biocompatible polymeric matrix in the first few weeks following implantation. However, after approximately five weeks post-implantation, the phytochemical agents were released at consistently lower levels indicating that a controlled low dose of the phytochemical agents can be sustainably released from a biocompatible polymeric matrix incorporating a phytochemical reagent following subcutaneous implantation of the compositions. This greater initial release of the phytochemical agent, followed by a sustained lower release, is consistent with the in vitro data depicted in FIGS. 2 to 5.
Example 6
Effect of Chemotherapeutic Compositions on Inhibition of Tissue DNA Adducts Treated with a Single Bolus Dose of a Carcinogen
Intervention with phytochemical agents is a useful strategy to inhibit or reduce cancer development. However, phytochemical agents must generally be administered in the diet at high doses in order to achieve effective plasma concentration and increased bioavailability. To reduce the effective dose of phytochemical agents compared to a dietary route, increase the bioavailability and minimize the toxicity of these phytochemical agents, the ability of a biocompatible matrix incorporating a phytochemical agent to inhibit the formation of DNA adducts after a single bolus dose of a carcinogen was determined. Briefly, female Sprague-Dawley rats were pretreated with curcumin either by diet (400 ppm, w/w) or by subcutaneous implantation of a 1 cm polycaprolactone (PCL) biocompatible polymeric matrix incorporating 20 mg of curcumin. Two weeks later, animals were challenged with a single intraperitoneal injection of benzo[a]pyrene (B[a]P) (50 mg/kg in dimethyl sulfoxide). The animals were then euthanized at predetermined time intervals, and liver and lung tissue samples were collected and analyzed for the formation of DNA adducts (FIG. 17). Analysis of the lung and liver DNA adducts by 32P-postlabeling (Gupta R C, 1996) showed that dietary curcumin inhibited the B[a]P-induced DNA adducts by 45% and 83% in the lung and liver (65±53 and 74±110 adducts/109 nucleotides), respectively compared with sham treatment (117±35 and 432±118 adducts/109 nucleotides). The adduct inhibition also occurred in animals that received curcumin via a biocompatible polymeric matrix, except that the degree of inhibition was 1.6 to 1.7 fold (17% and 48%, respectively) lower than the dietary administration. The total amount of curcumin administered via the polymeric matrix (less than 2 mg) was substantially lower than the amount administered by diet (about 120 mg), indicating a reduction of about 60-fold.
The lesser effectiveness of the curcumin administered via the biocompatible polymeric matrix was believed to stem from the low dose of curcumin, delivered by the implant, (approximately 80 μg/rat/day) that was available to combat against the bolus dose of B[a]P. To test this hypothesis, animals pretreated with curcumin implants for two weeks were further treated with a sustained, low dose of B[a]P incorporated into a polycaprolactone biocompatible polymeric matrix (1 cm incorporating 10 mg B[a]P). Analysis of tissue DNA adducts by 32P-postlabeling 30 days post B[a]P treatment showed 65% and 83% inhibition of adducts in the lung and liver (9±5 and 4±0.7 adducts/109 nucleotides), respectively compared with sham treatment (26±5 and 23±12 adducts/109 nucleotides). The average dose of curcumin over the duration of the study was approximately 110 μg/rat/day based on the amount left in the implants recovered from animals.
To determine the ability of further phytochemical agents incorporated into a biocompatible matrix to inhibit the formation of DNA adducts after a single bolus dose of a carcinogen, similar experiments were performed with biocompatible matrices incorporating either oltipraz or GTPs. Briefly, 10% w/w of oltipraz or GTPs was incorporated into a 2 cm polycaprolactone (MW 65K): cyclodextrin (combined in a 9:1 ratio) biocompatible polymeric matrix and subcutaneously implanted into female Sprague-Dawley rats. Eleven days later, the animals were treated with a bolus dose of dibenzo[a,1]pyrene. The animals were then euthanized 5 days after the carcinogen treatment and liver samples were collected. Analysis of DNA adducts in the liver of these rats by 32P-postlabeling revealed that treatment with both oltipraz (FIG. 18) and GTPs (FIG. 19) reduced the amount of B[a]P-induced DNA adducts when these phytochemicals were administered via their incorporation into a biocompatible polymeric implant.
Example 7
Effect of Chemotherapeutic Compositions on Inhibition of Tissue DNA Adducts in Subjects Treated with a Sustained Low Dose of a Carcinogen
Unlike bolus dose animal studies, humans are generally exposed to steady, low doses of carcinogens via the diet, smoking, occupation, etc. To determine the ability of the presently-disclosed compositions to inhibit tissue DNA adducts in subjects treated with sustained low carcinogen doses, biocompatible polymeric matrices comprised of polycaprolactone and incorporating benzo[a]pyrene (B[a]P) were first prepared by dissolving both B[a]P and polycaprolactone in an appropriate solvent, melting the polymer following removal of the solvent, and extruding the molten material through a mold.
To compare the effect of a bolus dose versus sustained low doses, female Sprague-Dawley rats were then treated with B[a]P either by a single intraperitoneal injection (10 mg in 500 μl sunflower oil) or by a subcutaneous PCL implant (1 cm containing 10 mg B[a]P). One group received sham treatment. Animals were euthanized after 6, 15, and 30 days, and different tissues were collected. Analysis of lung, liver and mammary DNA by 32P-postlabeling showed two distinct adducts. Total DNA adduction (in adducts/109 nucleotides) in the lung, liver and mammary tissues by bolus-dose regimen occurred after 6 days (117±35, 432±118, and 104±5, respectively), followed by a gradual decline after 15 days (88±13, 279±36 and 50 ±15, respectively) and 30 days (60±10, 156±28 and 53±8 respectively). In contrast, the B[a]P-PCL implant resulted initially in low adduct level in the lung (13±3) but the levels increased progressively after 15 days (23±12) and 30 days (26±5), indicating that animals were exposed to B[a]P continuously. Adducts were also detected in the liver (11±2, 21±6 and 23±4, respectively) and mammary tissue (16±2, 18±5, 23±5, respectively) and increased steadily with time. Measurement of the residual B[a]P in implants recovered from the animals showed that <1 mg of total B[a]P was released over 30 days. This release was consistent with an average daily release of 26±9 μg B[a]P when implants were stirred at 37° C. in phosphate-buffered-saline, pH 7.4 containing 10% serum. Notably, the sustained, low-doses of B[a]P resulted in a similar degree of DNA adduct accumulation in all of the three tissues analyzed as compared with the bolus-dose, which resulted in higher adduct accumulation in the liver. This data shows that the polymer-based delivery system is an effective means to study sustained, low-dose carcinogenesis.
Following the establishment of the presently-disclosed compositions as an effective means to study sustained, low-dose carcinogenesis, the ability of the presently-disclosed compositions to inhibit tissue DNA adducts in subjects treated with sustained low carcinogen doses was determined by examining the effect of curcumin administered via a biocompatible polymeric matrix on the inhibition of tissue DNA adduct formation induced by a sustained low dose of B[a]P. Briefly, one group of Sprague-Dawley rats received a 1 cm biocompatible polymeric matrix incorporating 20% w/w of curcumin, while a second group of rats received a sham biocompatible polymeric matrix that did not incorporate an agent. Both groups were maintained on a control diet. Two weeks later, the two groups of animals received a 1 cm biocompatible polymeric matrix incorporating B[a]P (10% carcinogen load) subcutaneously. The animals were then euthanized after 30 days and tissue DNA adducts were analyzed by a 32P-postlabeling assay. Analysis of DNA adducts in liver and lung tissue showed that curcumin administered via a biocompatible polymeric matrix inhibited the B[a]P-induced DNA adducts in both the liver and lung, as compared with the animals receiving a sham implant (FIG. 20).
A similar experiment was performed to determine the ability of GTPs administered via a biocompatible polymeric matrix to inhibit tissue DNA adducts in subjects treated with sustained low carcinogen doses. Briefly, one group of Sprague-Dawley rats subcutaneously received two 2-cm biocompatible polymeric matrices incorporating 10% w/w of GTPs, while a second group of rats received a sham biocompatible polymeric matrix that did not incorporate a phytochemical agent. At the same time the animals were receiving the biocompatible polymeric matrices incorporating GTPs, both groups of animals also received a 1 cm biocompatible polymeric matrix incorporating B[a]P (10% carcinogen load) subcutaneously. The animals were then euthanized 6 days after implantation of the matrices and tissue DNA adducts were analyzed by a 32P-postlabeling assay. Similar to the experiments described above with curcumin, analysis of DNA adducts in liver tissue of these animals showed that GTPs administered via a biocompatible polymeric matrix inhibited the formation of B[a]P-induced DNA adducts, as compared with the animals receiving a sham implant (FIG. 21).
Example 8
Effect of Green Tea Polyphenols on Cytochrome P450 Expression
Cytochrome P450 proteins are a diverse family of hemoproteins and are widely regarded as an important enzyme in the metabolism and formation of carcinogens. To determine the effect of GTPs administered via subcutaneous implantation of a biocompatible polymeric matrix incorporating the GTPs on cytochrome P450 1A1 and 1B1 mRNA expression, liver tissue was collected from Sprague-Dawley rats that subcutaneously received a 1 cm biocompatible polymeric matrix incorporating B[a]P (10% carcinogen load) and either a 1 cm biocompatible polymeric matrix incorporating 10% w/w of GTPs or a sham biocompatible polymeric matrix that did not incorporate a phytochemical agent, as described in Example 7. Analysis of the fold change in mRNA expression revealed that CYP 1A1 expression was not significantly affected by the administration of GTPs via a biocompatible polymeric matrix (FIG. 22A), but revealed that CYP 1B1 expression was reduced by the administration of the GTPs (FIG. 22B). Taken together, these results indicate that by reducing cytochrome P450 mRNA levels, the administration of a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent can be an effective means to lower the production of DNA-reactive metabolites of B[a]P and thus lower DNA adduct levels.
Example 9
Effect of Individual Phytochemical Agents on Mammary Carcinogenesis
Cancer chemoprevention approaches have been used to manipulate tumorigenesis and reduce the transformation of normal/pre-cancerous cells to malignancy. Many compounds that have worked in cell culture studies though have failed in animal models and clinical trials due to lack of bioavailability. Additionally, the high doses required to elicit biological response can be toxic. The following experiments were designed to test the hypothesis that the effective dose of phytochemical agents, administered systemically via implantation of a silastic implant incorporating the agents, can be reduced substantially compared with the traditional dietary route and were also designed to determine the effect of individual phytochemical agents on mammary carcinogenesis. The silastic implants used in the experiments described below were fabricated by filling silastic tubing with a desired agent. Although the silastic implants are not biodegradable like compositions disclosed herein comprising biocompatible polymeric matrices, the silastic implants provide proof of concept that a reduced dose of phytochemical agents administered systemically can be effective in inhibiting mammary carcinogenesis.
Initially, an experiment was performed by subcutaneously implanting individual phytochemical agents using a large number of animals per group (n=17-19). Briefly, four groups of ACI rats received either a biocompatible silastic implant with no phytochemical agent or a 3 cm biocompatible silastic implant incorporating either ellagic acid, lycopene, or curcumin (27 mg phytochemical agent per implant). Two weeks later, all groups received 3 cm biocompatible silastic implants incorporating 27 mg of 17β-estradiol. Six months later, after all animals had developed mammary tumors, the animals were euthanized and the mammary tumors were counted and their size measured. Tumor incidence or multiplicity, as measured by the number of mammary tumors, was not significantly reduced by any of the phytochemical agents (FIG. 23B). However, 30-55% reductions were found in the tumor burden with all test agents (FIG. 23A) as measured by tumor volume, with ellagic acid resulting in the most significant reduction in tumor burden (55%). The foregoing results thus support the effectiveness of ellagic acid, a significant component in certain berries, as an exemplary phytochemical compound that can be administered via a biocompatible silastic implant to inhibit or reduce estrogen-induced ACI rat mammary carcinogenicity.
To further evaluate the effectiveness of ellagic acid in estrogen-induced mammary carcinogenicity and to determine whether the effective dose of ellagic acid administered systemically via a biocompatible silastic implant can be reduced substantially compared with the traditional dietary route, ellagic acid was administered either in the diet (400 ppm) or by subcutaneous implantation of a biocompatible silastic implant incorporating an effective amount of ellagic acid (27 mg/implant, 4 implants). Two weeks later, all animals were treated with 17β-estradiol (9 mg) by a subcutaneous implantation of a biocompatible silastic implant incorporating the 17β-estradiol. All groups received diet and water ad libitum. The body weight and diet consumption were recorded weekly. The animals were palpated for mammary tumors, and were euthanized when the largest tumor reached >1 cm. The first tumor in the control group appeared after nearly 84 days. However, the first tumor appearance with ellagic acid implants or ellagic acid-supplemented diet was somewhat delayed by 2 and 3 weeks, respectively. The control group reached 100% tumor incidence after 26 wks, but only 75% or 69% of the animals developed palpable tumors in the groups that received ellagic acid by implants or diet, respectively. Ellagic acid also reduced the tumor burden (FIG. 24B) with both the implant (slow release, 454±105 mm3) and dietary (269±61 mm3) routes, as compared with the control group (787±170 mm3). Similar reductions were also observed in the tumor multiplicity (4.00±0.63 and 4.80±0.4 tumors/rat with implant and diet, respectively versus 6.77±0.87 in the control group) (FIG. 24A).
The total amount of ellagic acid administered during the entire duration of the study by diet was 800 mg/rat while the subcutaneous implantation of the biocompatible silastic implant incorporating ellagic acid delivered only 1.48±0.87 mg per implant, as determined by HPLC measurement of the material left in implants recovered after termination. Thus, with 4 implants per animal, an approximately 135-fold reduction of the ellagic acid dose produced similar biological responses when delivered by the slow release system. This approach thus opens new avenues for phytochemical agents whose use has been discontinued for therapeutic purposes, either due to lack of bioavailability or toxicity due to high doses.
To further determine the effects of phytochemical agents on mammary cell proliferation, an additional in vivo experiment was performed. Briefly, six week-old female ACI rats were divided in five groups (six per group). Animals received either purified (AIN-93M) control diet (Groups A-C) or diet supplemented with 2.5% cumin (Group D) or 2.5% fennel (Group E). Group C received implants of aqueous and non-aqueous extracts of fennel. The extracts were prepared from 1 g each of fennel. Concentrated aqueous and non-aqueous extracts were incorporated respectively in two 3-cm biocompatible silastic implants, and, thus, each animal received four implants, equivalent to 2 g fennel. Ten days later, all but Group A received a 1.2 cm implant of 17β-estradiol (9 mg) incorporated into a biocompatible silastic implant; Group A received sham implants. Experimental diets were continued until termination of the experiment. Diet and water were provided ad libitum. Animals were weighed and diet consumption recorded frequently. All animals were euthanized 20 days after estrogen treatment, blood and mammary and other organs were collected, and the tissues were weighed.
The sham-treated rat mammary tissues showed normal morphology. All rats treated with estrogen showed mild to moderate marked lobular hyperplasia with occasional dilate ducts filled with secretions. However, groups receiving intervention were less cellular than control group. Slides stained for Proliferating Cell Nuclear Antigen (PCNA) and Ki-67, cellular markers of proliferation, were scored by two experienced cytotechnologists to account for inter-observer variability and averages of the two were considered in interpretation of the data.
Analysis of PCNA staining in mammary tissues from rats without estrogen treatment showed essentially the same percentage of cells with moderate staining (41%) as with estrogen treatment, with (37-41%) or without (38%) phytochemical intervention. However, as shown in FIG. 25, the percentage of cells with intense staining for PCNA increased significantly with estrogen treatment, from 8% to 27%. This increase was significantly offset by intervention with the phytochemicals, with the percentage of cells with intense staining being decreased by 22% with dietary cumin (p=0.28), 40% (p=0.03) with dietary fennel, and 43% (p=0.005) with fennel extract implants. This conclusion was seconded by analysis of Ki-67, another marker for cell proliferation. These data show cumin and fennel phytochemicals have anti-proliferative activity and demonstrates that these phytochemicals administered by a slow-release system elicited the same efficacy as dietary route.
Example 10
Inhibition of Mammary Cell Carcinogenesis by Combined Administration of Phytochemical Agents
To determine the effect of utilizing a combination of phytochemical agents in inhibiting mammary cell carcinogenesis, female ACI rats were divided into Groups A-C (8 animals/group). Group A subcutaneously received sham implants (e.g., biocompatible silastic implants without a phytochemical agent) while groups B and C received implants of 3 cm biocompatible silastic implants incorporating various phytochemical agents. Group B received one implant each of oltipraz and curcumin, and Group C received one implant each of curcumin, ellagic acid, co-enzyme-Q10, and lycopene. Two weeks later, all groups received implants of 17β-estradiol implants comprised of a biocompatible silastic implant incorporating the 17β-estradiol. Animals received standard rodent chow diet and water ad libitum. Six months later, animals were euthanized and mammary tumors were counted and their sizes were measured with a caliper. Comparison of the groups showed no difference in the tumor incidence, but the tumor multiplicity (FIG. 26B) in the groups receiving phytochemical agents, Group B (3.4±1.3; p=0.01) and Group C (6.3±3.8; p=0.34), was lower as compared with the control group, Group A (8.25±3.4). The tumor volume (in mm3/rat) was also reduced by both the combination of phytochemical agents (FIG. 26A), from 2,283±2,061 (Group A) to 447±429 (p=0.08) (Group B) and 124±76 (p=0.03) (Group C). Although the reductions in tumor burden in Group B and tumor multiplicity in Group C were insignificant, presumably due to the small sample size, the data indicate that the slow-release composition is effective in eliciting a biological response and in inhibiting mammary cell carcinogenesis.
Example 11
Inhibition of Mammary Cell Proliferation by Combined Administration of Phytochemical Agents
To determine the effect of administering a combination of phytochemical agents in inhibiting mammary cell proliferation, four 2-cm biocompatible polymeric matrices, one each incorporating curcumin (20% w/w), GTPs (20% w/w), DIM (20% w/w), and punicalagin (20% w/w) were implanted subcutaneously in the back of female ACI rats. Two weeks later, the animals received either an empty biocompatible polymeric matrix or a biocompatible polymeric matrix incorporating 17β-estradiol (9 mg/1.2 cm implant).
Three weeks later, animals were euthanized and mammary cell proliferation was measured by immunohistochemistry by staining for the cell proliferation marker PCNA. As shown in FIG. 27, analysis of the results showed that at three weeks post-implantation, the estrogen-treated animals that were administered the combination of phytochemical agents exhibited a significantly lower percentage of PCNA-positive cells, as compared to the estrogen treated animals that were not administered a phytochemical agent. These data thus indicate that a combination of phytochemical agents administered via the polymeric matrix is highly effective in inhibiting mammary cell proliferation.
Example 12
Synergistic Activity of Phytochemical Agents in Treating Lung Cancer
Increased cellular proliferation is an integral part of the cancer phenotype and agents that have the potential to slow the growth of these phenotypes are often considered prophylactic (Dorai T & Aggarwal BB, 2004). To determine whether phytochemical agents administered in combination would display synergistic activity as compared with phytochemical agents administered individually, human lung cancer cells (H1299) were incubated with vehicle only, with phytochemical agents individually, or with a combination of phytochemical agents. Following incubation for 1-3 days, the cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The resultant formazan product was solubilized in dimethyl sulfoxide and the absorbance was measured at 540 nm to determine the extent of cellular proliferation. As shown in FIG. 28, incubation of the H1299 cells with a mixture of delphinidin, petunidin, malvidin, peonidin, and cyanidin inhibited the growth of H1299 cells at a much lower concentration as compared to phytochemical agents administered individually. These data thus indicate that a combination of phytochemical agents can act synergistically in inhibiting the growth of lung cancer cells.
The effect of phytochemical agents on cell growth and proliferation can be explained by their ability to alter the expression of cell-cycle related proteins leading to the induction of cell cycle arrest and apoptosis. Inhibition of cell proliferation and/or induction of apoptosis are highly correlated with the activation of a variety of intracellular signaling pathways leading to the arrest of the cell cycle in a particular phase (Yun J M, et al., 2009). To further examine the synergistic activity of combinations of phytochemical agents in treating lung cancer, H1299 cells were incubated for 48 hours with a vehicle (dimethyl sulfoxide) only, with individual phytochemical agents, or with a combination of phytochemical agents. Following harvesting of the cells, the cell pellet was washed with phosphate-buffered saline and fixed in 70% ethanol. To measure the percentage of cells undergoing apoptosis, the cells were then treated with RNase A and propridium iodide, and analyzed using a FACS-420 Flow Cytometry Analyzer. The treatment of cells with growth-suppressive concentrations of phytochemicals resulted in significant accumulation of cells in G2/M and G0/G1 phases of the cell cycle. An increase in the fraction of cells with sub-G0/G1 DNA content subsequent to the treatment of the cells with phytochemical agents was also observed in the analysis of cell cycle distribution and this sub-G0/G1 fraction was considered the apoptotic fraction. As shown in FIG. 29, incubation of the cells with a mixture of delphinidin, cyanidin, malvidin, peonidin, and petunidin, or a mixture of punicalagins and delphinidin resulted in a marked increase in the number of H1299 cells undergoing apoptosis, thus indicating that the a combination of phytochemical agents can act synergistically in inducing apoptosis in lung cancer cells.
Example 13
Formulation and Use of Device for Cervical Insertion
A device for cervical insertion (FIG. 30) was made of biodegradable polymers (polycaprolactone: F68 in a 4:1 ratio) incorporating a desired phytochemical agent (see, e.g., FIGS. 32A-32D showing perspective view of an exemplary device for cervical insertion comprising a biocompatible polymeric matrix incorporating curcumin). The device was capable of incorporating a phytochemical agent load of 0.5% to as much as 20% or more and was prepared by extruding a molten polymer formulation through a plastic mold made of a polypropylene transfer pipet, a round spoon and a 10-ml plastic syringe. The cut-piece of the pipet mold was narrow on one end and broad on the other end. The narrow part of this cut-pipet was attached to the syringe, while the broader part was held against the spoon to allow the molten material to pass through the pipet mold onto the spoon. Once the pipet mold was filled, the molten material started to collect in the spoon. The assembly was then detached from syringe and the material on the spoon was flattened manually to provide a round shape. After drying, a 3-5 mm hole was drilled lengthwise through the device. Following this fabrication protocol, the typical device weighed approximately 2.5 to 4 g, depending upon the size of the mold.
The device for cervical insertion is capable of delivering phytochemical agents to uterine cervix locally. As shown in FIGS. 33A and 33B, the device can be inserted into a cervix such that the shaft of the device engages the walls of the cervical canal and the bottom surface of the cap engages the external orifice of the uterus. As such, the device is capable of delivering an effective amount of a phytochemical agent locally to a cervix or the device can also be used to deliver phytochemical agents via systemic delivery to other organs like the uterus and ovary or to an organ more distant from the site of insertion such as the lungs.
An exemplary device for cervical insertion is relatively non-invasive and can include a tapered distal end to allow for easy insertion into a cervix as well as a canal through the device to allow for the passage of normal bodily fluids from the uterus. As shown in FIG. 31, an exemplary device for cervical insertion can be fabricated such that the cylindrical shaft is about 19 to about 25 mm in length and about 9 to about 11 mm in diameter, the cap is about 20 to about 25 mm in diameter, and the canal is about 4 mm to about 5 mm in diameter. Without wishing to be bound by any particular theory, it is believed that by fabricating the device in the foregoing dimensions, the device correlates to the dimensions of the human anatomy that it engages such that the device can engage the surrounding tissue and effectively deliver a phytochemical agent.
Example 14
In Vitro Release of Curcumin from an Exemplary Device for Cervical Insertion
To determine daily release of a phytochemical agent from a device for cervical insertion in vitro, a cervical insertion device comprising a biocompatible polymeric matrix incorporating curcumin was placed in a release medium (phosphate-buffered-saline (pH 7.4) containing 10% bovine serum) at 37° C. with constant agitation in a water bath to simulate an in vivo scenario. The medium was replaced daily and the amount of curcumin released from the compositions per day was measured spectrophotometrically against a standard curve and expressed as micrograms per day.
FIGS. 34A and 34B show the amount of curcumin released per day and the total amount of curcumin released from an exemplary device for cervical insertion over a release period of 18 days. This data, which is similar to the data described herein for the in vitro and in vivo release of phytochemical agents, shows that over the initial five days following the placement of the device into the release medium a greater amount of curcumin is released from the device. However, after approximately 5 days in the release medium, the amount of curcumin being released decreased and approached lower steady-state levels indicating that an exemplary device for cervical insertion can effectively be used to release a controlled low dose of a phytochemical agent over a time period.
Example 15
Efficacy of Compositions in Inhibiting Growth of Human Cancer Cells in a Nude Mouse Model
To determine whether phytochemical agents inhibit tumor growth in a dose dependent manner and to determine whether the effective dose of the phytochemical agents is substantially lower than doses delivered by traditional oral administration, the ability of the presently-described compositions to inhibit growth of human lung, breast, and cervical cancer cells in a dose dependent manner is examined using a nude-mouse model. Briefly, nude mice are injected with 1-10 million tumor cells (lung, breast or cervical tumor cells) subcutaneously on the dorsal flank in 50-100 μl of PBS or growth media according to established protocols (Morton C L & Houghton P J, 2007). The mice also receive a 2 cm biocompatible polymeric matrix incorporating 2-20% (w/w) of a phytochemical agent as a subcutaneous implant. Tumors start appearing at the site of implantation after approximately 2-4 weeks. The tumor growth is then monitored twice a week and is measured using a digital caliper. Once the tumor has reached approximately 1.5 cm in diameter in the control group, all of the animals are euthanized and the tumor tissue is isolated for examination of biochemical and molecular parameters. Different phytochemical agents are examined for their antitumor efficacy and the efficacy of combinations of different phytochemical agents are also examined by administering the combinations as a single implant or in multiple implants. Analysis of tumor tissue also reveals the mechanism by which the phytochemical agents are able to exert their effects. As compared with animals receiving sham implants, a reduction in tumor volume is observed in mice receiving biocompatible polymeric implants incorporating an effective amount of a phytochemical agent, indicating that a composition and method that includes or makes use of a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent is useful for treating breast, lung, and cervical cancer.
Example 16
Efficacy of Compositions used in Combination with Chemotherapeutic Agents and Anti-inflammatory Agents
Phytochemical agents are often used in adjuvant therapy with conventional chemotherapeutic drugs to enhance their therapeutic efficacy and are recommended in adjuvant therapy with selective Cox-2 inhibitors (like celecoxib) for adenomatous polyposis. Selective Cox-2 inhibitors often leave other critical carcinogenic pathways unaltered, however, and phytochemicals can augment their activity by inhibiting such alternative pathways involved in carcinogenesis. Previous studies have found that silibinin sensitizes the hormone refractory DU145 prostate carcinoma cells to cisplatin and carboplatin-induced cell growth inhibition and apoptotic death, and can be combined to enhance the therapeutic efficacy of these compounds. Moreover, certain chemotherapeutics like paclitaxel induce expression of antiapoptotic (XIAP, IAP-1, IAP-2, Bcl-2, and Bcl-xL), proliferative (cyclooxygenase 2, c-Myc, and cyclin D1) and metastatic proteins (vascular endothelial growth factor, matrix metalloproteinase-9, and intercellular adhesion molecule-1) which can be inhibited by addition of phytochemical agents like curcumin. Additionally, certain agents like linalool moderately inhibit cell proliferation, but are known to exert synergistic effects with chemotherapeutics like doxorubicin by potentiating the cytotoxicity and proapoptotic effects of doxorubicin. Previous studies have shown that linalool increases doxorubicin accumulation and decrease Bcl-xL levels which results in its higher cytotoxicity for malignant cells. Phytochemical agents like licochalcone and licorice were also found to inhibit cisplatin induced hepatotoxicity and nephrotoxicity. In this regard, repeated oral administration of licochalcone prior to cisplatin has been found to exert a preventive effect by blunting the cisplatin induced increase in serum nitric oxide and tissue lipid peroxidation levels and be increasing the glutathione levels in the tissues. (Lee C K, et al., 2008).
To determine whether the presently-described biocompatible polymeric matrix can effectively be used in adjuvant therapy to administer a phytochemical agent with a chemotherapeutic agent and/or an anti-inflammatory agent, either in the same implant or in separate implants, and to mitigate the toxicity, increase the efficacy, or both of chemotherapeutic agents, a nude mouse model is utilized. Briefly, nude mice are injected with 1-10 million tumor cells (lung, breast or cervical tumor cells) subcutaneously on the dorsal flank in 50-100 μl of PBS or growth media according to established protocols (Morton C L & Houghton P J, 2007). When the tumors start appearing at the site of injection (approximately 2-4 weeks), the mice then receive a 2 cm biocompatible polymeric matrix incorporating 2-20% (w/w) of a phytochemical agent and a chemotherapeutic agent and/or anti-inflammatory agents as subcutaneous implant(s). The biocompatible polymeric matrix incorporating the chemotherapeutic agent and/or anti-inflammatory agents is prepared by the same method that is used to prepare the biocompatible polymeric matrices incorporating an effective amount of a phytochemical agent, which is described herein above in Example 1. The tumor growth is monitored twice a week and is measured using a digital caliper. The tumor is allowed to reach approximately 1.5 cm in diameter in the control group. At this point, all the animals are euthanized and the tumor tissue is isolated for the examination of biochemical and molecular parameters. Analysis of the results reveals that a lower dose of the chemotherapeutic agent is required to inhibit tumor growth when adjuvant therapy using natural phytochemical agent is used, indicating that a composition and method that includes or makes use of a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent and a chemotherapeutic agent is useful for treating lung, cervical, and breast cancer, and that by incorporating a chemotherapeutic agent in a biocompatible matrix a lower dose of the chemotherapeutic agent can be used to treat cancer. Furthermore, when an anti-inflammatory agent is also incorporated into a biocompatible polymeric matrix, and is administered in conjunction with a biocompatible polymeric matrix incorporating a phytochemical agent and a chemotherapeutic agent, a reduction in the local inflammatory reaction to the chemotherapeutic agent is observed, indicating that the inclusion of an anti-inflammatory agent is useful for overcoming any local inflammatory reaction to the chemotherapeutic agent.
Example 17
Efficacy of Chemotherapeutic Compositions in Inhibiting Growth of Chemically Induced Breast or Lung Tumors
To determine whether breast and lung tumors induced by selected carcinogens can be inhibited in the host environment by the administration of a biocompatible polymeric implant incorporating an effective amount of a phytochemical agent, and to determine whether the requisite effective dose of the phytochemical agents can be lowered as compared with the traditional oral route, a rodent model is used.
To determine the effect of a biocompatible polymeric matrix incorporating a phytochemical agent on inhibiting chemically-induced lung tumors, female A/J mice (4-5 weeks old) receive either a sham implant or a biocompatible polymeric matrix implant incorporating 2-20% (w/w) of one or more phytochemical agents. Two weeks after receiving the implants, the mice receive 11.65 mg/kg body weight of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by gavage three times a week for 10 weeks. Six mice are then euthanatized at 5-weeks after the first carcinogen treatment and 1-week after the last carcinogen treatment (11 weeks) for intermediate biomarker analysis. Six weeks after the last carcinogen treatment (16 weeks), all mice are euthanatized, lung tissue is removed, surface adenomas are counted, and tissue is serially sectioned to count internal tumors. Part of the lung is also snap frozen for biomarker analysis. A reduction in the tumor multiplicity, both on the surface of the lung and internally, and a reduction in the conversion of adenomas to carcinomas is observed in the group receiving a biocompatible polymeric matrix incorporating one or more phytochemical agents, indicating that the presently-disclosed compositions are useful for treating chemically-induced lung tumors.
To determine the effect of a biocompatible polymeric matrix incorporating a phytochemical agent on inhibiting chemically-induced breast tumors, a first group of female Sprague-Dawley rats are injected intra-peritoneally with 50 mg of 1-methyl-1-nitrosourea (MNU) per kg body weight at 21 days of age. The first palpable tumor, histologically classified as an adenocarcinoma, starts appearing around 30 days post-carcinogen administration and at the end of 35 days post MNU administration, the cumulative incidence of palpable carcinomas is around 60-90%. A second group of rats receives an estradiol implant (1.2 cm silastic implant containing 9 mg) that is implanted subcutaneously on the back of ACI rats at 8-9 weeks of age. In the group of rats receiving an estradiol implant, the first palpable tumor appears around 3-4 months and nearly 100% of the rats develop palpable mammary tumors by 8 months. In both of these models, one or more phytochemical agents are delivered via a biocompatible polymeric matrix two weeks before the chemical carcinogen treatment. The rats are monitored weekly for palpable tumors. As compared to control groups, postponement of the appearance of the first tumor by approximately 3 to 5 weeks, as well as a reduction in tumor multiplicity and burden by 50-80 percent, is observed in rats receiving a biocompatible polymeric matrix incorporating one or more phytochemical agents, indicating that a composition and method that includes or makes use of a biocompatible polymeric matrix incorporating an effective amount of a phytochemical agent is useful for inhibiting the growth of chemically-induced breast tumors.
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It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

## Claims

1. A device for insertion into a cervical canal, comprising:
a substantially cylindrical shaft having a proximal end and a distal end, and defining a canal that extends from the proximal end to the distal end; and
a cap attached to the proximal end of the cylindrical shaft, said cap including a central opening in registry with the canal defined by the shaft,
wherein the cylindrical shaft is suitably adapted for engaging the walls of the cervical canal and a bottom surface of the cap is suitably adapted for engaging an external orifice of a uterus,
wherein the canal allows for the passage of bodily fluids from the uterus; and
wherein the bottom surface of the cap has a concave shape to suitably engage the external orifice of the uterus and a transformation zone of a cervix.

2. The device of claim 1, wherein the distal end of the shaft is tapered.

3. The device of claim 1, wherein the cylindrical shaft is about 19 to about 25 mm in length and about 9 to about 11 mm in diameter, the cap is about 20 to about 25 mm in diameter, and the canal is about 4 mm to about 5 mm in diameter.

4. A device for insertion into a cervical canal, comprising:
a substantially cylindrical shaft having a proximal end and a distal end, and defining a canal that extends from the proximal end to the distal end; and
a cap attached to the proximal end of the cylindrical shaft, said cap including a central opening in registry with the canal defined by the shaft,
wherein the cylindrical shaft is suitably adapted for engaging the walls of the cervical canal and a bottom surface of the cap is suitably adapted for engaging an external orifice of a uterus,
wherein the canal allows for the passage of bodily fluids from the uterus; and
wherein the device is comprised of a bio compatible polymeric matrix incorporating an effective amount of a phytochemical agent.

5. The device of claim 4, wherein the phytochemical agent is selected from the group consisting of curcumin, green tea polyphenols, punicalagin, diindolylmethane, oltipraz, tocotrienol, tocopherol, plumbagin, cyanidin, delphinidin, lycopene, lupeol, curcurbitacin-B, Withaferin A, indole-3-carbinol, genestein, equol, resveratrol, co-enzyme Q-10, ellagic acid, petunidin, malvidin, peonidin, fennel extract, and combinations thereof.

6. The device of claim 4, wherein the phytochemical agent comprises about 2% to about 50% of the polymer weight.

7. The device of claim 4, wherein the biocompatible polymeric matrix is comprised of polymers selected from the group consisting of polycaprolactone, cyclodextrin, a polyoxyethylene-polyoxypropylene block copolymer, polyethylene glycol, and combinations thereof.

8. The device of claim 4, wherein the bio compatible polymeric matrix comprises polycaprolactone in combination with a second polymer.

9. The device of claim 8, wherein the second polymer is selected from the group consisting of cyclodextrin, a polyoxyethylene-polyoxypropylene block copolymer, and polyethylene glycol.

10. The device of claim 8, wherein the polycaprolactone and the second polymer are combined in a ratio of about 4:1 or about 9:1.

11. The device of claim 4, wherein the biocompatible matrix comprises polycaprolactone and a polyoxyethylene-polyoxypropylene block copolymer in a ratio of about 4:1.

12. The device of claim 11, wherein the phytochemical agent comprises curcumin.

13. The device of claim 4, wherein the bio compatible polymeric matrix is biodegradable.

14. A device for insertion into a cervical canal, comprising:
a substantially cylindrical shaft having a proximal end and a distal end, and defining a canal that extends from the proximal end to the distal end; and
a cap attached to the proximal end of the cylindrical shaft, said cap including a central opening in registry with the canal defined by the shaft,
wherein the cylindrical shaft is suitably adapted for engaging the walls of the cervical canal and a bottom surface of the cap is suitably adapted for engaging an external orifice of a uterus,
wherein the canal allows for the passage of bodily fluids from the uterus; and
wherein the distal end of the shaft is tapered.