# Patent

## Title
Compositions and methods for reducing or preventing metastasis

## Abstract

Compositions comprising glucopyranosyl lipid adjuvant (GLA) and methods for reducing or preventing the formation of cancer metastasis utilizing same are provided. The compositions may be formulated for local-regional delivery. The compositions may be substantially devoid of a cancer antigen. The treatment with GLA may be combined with treatment with a COX2 inhibitor and a beta-adrenergic blocker.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application claims the priority benefit of U.S. provisional application Ser. No. 61/830,675, filed Jun. 4, 2013, which is incorporated herein by reference in its entirety.


FIELD OF THE INVENTION
The present invention relates to the reduction or even prevention of metastasis development using a glucopyranosyl lipid adjuvant (such as GLA or short, SLA), alone or in combination with a COX2 inhibitor and a beta-adrenergic blocker.
BACKGROUND OF THE INVENTION
Despite improvements in early detection of cancer and in therapeutic interventions, overall cancer survival rates have not been markedly improved along the last decades, and metastasis is still the main cause of mortality in most solid malignancies.
It has recently become recognized that the perioperative period of primary tumor resection, namely the time period immediately before, during and following the surgical operation, enfolds several unattended risk factors for long-term cancer recurrence, and is thus characterized by high risk for the outbreak of pre-existing micrometastases and the initiation of new metastases. Most of these perioperative risks are due to various perturbations induced by the surgical removal of the primary tumor, which are believed to facilitate the progression of pre-existing micrometastases and the initiation of new metastases through several mechanisms, some of which have only recently been identified.
Numerous soluble factors are increased systemically during the perioperative period as a result of patients' neuroendocrine and paracrine responses to (i) the presence of the primary tumor, to (ii) physiological and psychological stress, and (iii) to the surgical procedure itself and its accompanying anesthesia, analgesia, blood transfusion and other intra-operative procedures. These soluble compounds include catecholamines, prostaglandins, glucocorticoids, opioids, and a variety of administered anesthetic and analgesic agents. In recent years, it has become clear that in vitro, many of these factors act directly on malignant cells, activating several molecular processes that are critical for tumor metastatic activity, including tumor cell proliferation, adhesion, locomotion, extracellular matrix invasion capacity, resistance to apoptosis and anoikis, and secretion of pro-angiogenic factors. Additionally, in vitro and human and animal in vivo studies show that many of these soluble factors lead to suppression of anti-metastatic cell mediated immunity (CMI), which is indeed a common perioperative phenomenon.
CMI, particularly cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, is suppressed even before surgery, and significantly more so following it, with the degree of suppression corresponding to the extent of surgical trauma and tissue damage. Notably, the suppression of CMI had been causally shown to mediate promotion of cancer metastasis in animal models, and clinical studies had associated it with increased susceptibility to metastatic development.
Among the various treatments aiming at reducing malignant tissue and/or preventing suppression of CMI, immunotherapy in cancer patients has regained momentum in the past decade. Specifically, type 1 T-helper (Th1) and proinflammatory cytokines (e.g. IL-2, IL-12, IFN-γ) are known to significantly enhance CMI, which plays a crucial role in the in vivo eradication of malignant cells. Immune-stimulatory approaches (ISAs) commonly utilize established biological response modifiers (BRMs), which include natural or synthetic compounds containing pathogen-associated molecular patterns (e.g. LPS, CpG), or the pro-inflammatory/Th1 cytokines that these compounds induce.
However, while animal studies employing anti-tumor ISAs showed promising results, clinical studies in cancer patients were, by-and-large, less successful. Several difficulties in simulating the development of human cancer in animal models were suggested to underlie this discrepancy, most focusing on differences in the biology of the implanted cancerous tissue and host physiology, including immune susceptibility and compatibility to implanted tissue. Stress responses, known to induce an immunossupressing effect, which result from psychological and physiological conditions that characterize cancer patients and absent in animal models, were also suggested to underlie this discrepancy.
Neeman et al. (2012) Clin Cancer Res., 18(18): 4895-902 describe an approach to reducing postsurgical cancer recurrence, by perioperative targeting of catecholamines and prostaglandins using simultaneous beta-adrenergic blockade and COX-2 inhibition.
Avraham et al. (2010) Brain Behav Immun., 24(6):952-8 describe an integration of immunostimulatory therapy with endocrine-blocker pharmacological interventions that prevent postoperative immunosuppression, to reduce post-operative tumor progression.
Despite the acknowledged importance of the perioperative period, and the promising results evident in animal studies employing ISAs, immune-stimulatory therapy has rarely been utilized in patients during the perioperative period, presumably due to the expected pyrogenic and adverse effects of ISAs that cannot be distinguished from signs of life-threatening infections in the context of surgery. The relatively few clinical trials that had attempted this approach utilized a single Th1 (e.g., IL-2, IL-12) or a proinflammatory (e.g., IFN-α) cytokine, and have indeed reported severe adverse reactions to these therapies, including leukopenia, deterioration of performance status, fever, vomiting, and mental depression. Recently FDA-approved synthetic agents, which are based on pathogen-associated molecular patterns (PAMPs), have been shown to cause markedly less adverse reactions while inducing an effective, self-controlled, endogenous, multi-cytokine response. Such ISAs include the TLR-4 agonists, termed glucopyranosyl lipid adjuvants (GLAs as disclosed in U.S. Pat. No. 8,273,361 and WO 2010/141861), which activate T, B, and Dendritic cells.
U.S. Pat. No. 8,273,361 discloses compositions and methods, including vaccines and pharmaceutical compositions for inducing or enhancing an immune response, based on the discovery of useful immunological adjuvant properties of a synthetic, glucopyranosyl lipid adjuvant (GLA) that is provided in a substantially homogeneous chemical form. Also provided are vaccines and pharmaceutical compositions that include GLA and one or more antigens, a Toll-like receptor (TLR) agonist, a co-adjuvant and a carrier such as a pharmaceutical carrier.
WO 2010/141861 discloses compounds, particularly, glucopyranosyl lipid adjuvant (GLA) compounds with an alternate chemical structures and properties. Pharmaceutical compositions, vaccine compositions, and related methods for inducing or enhancing immune responses, are also disclosed.
There still remains a need for more effective treatments against the development of metastases.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for reducing or preventing the formation of cancer metastasis using glucopyranosyl lipid adjuvants (GLA). According to some embodiments, the present invention is directed to the reduction or prevention of metastasis development using local-regional delivery of GLA. According to some embodiments, GLA is used without co-administration of a cancer antigen (whether as a defined peptide, protein, or mixture of peptides, proteins or fragments, or as an inactivated or modified cancer cell preparation). According to some embodiments, GLA is combined with a COX2 inhibitor and a beta-adrenergic blocker.
The present invention discloses for the first time local-regional delivery of GLA to a space or cavity adjacent to a tumor to be treated, or alternatively to a space or cavity adjacent to, or directly into, a draining lymph node of a tumor to be treated. Such local-regional delivery of GLA can also be performed after removal of a primary tumor, to a space or void formed after the resection of a tumor mass. The present invention thus provides a more controlled manner of administration of GLA, to improve treatment outcome. Advantageously, local-regional delivery can provide a more sustained effect of the active ingredient. Additionally, local-regional delivery of GLA can allow for lower total body dosing of GLA and the reduction of deleterious side effects that can be associated with systemic delivery in a cancer setting.
The present invention further discloses the use of GLA against cancer metastasis without co-administration of a cancer antigen, such as an antigen derived from the tumor of the subject being treated, an antigen or antigen composition containing antigens known to be associated with the tumor type of the subject being treated, or a preparation of antigens derived from administration of tumor cells or preparation of tumor cells from the patient or a mixture of patient defined tumors, thus differing from administration of GLA in the context of a composition for a cancer vaccine. Advantageously, in the present invention such use of GLA is not limited to types of cancer for which cancer-specific antigens have been identified.
In some embodiments, GLA is utilized for the prevention of metastasis of a solid tumor, following a tumor excision surgery. It is now disclosed that GLA administered prior to and following surgery, achieves effective inhibition of metastatic development.
The present invention further discloses that GLA treatment can be combined with treatment with a beta-adrenergic blocker and/or a COX2-selective inhibitor, to further optimize the outcome of metastasis inhibition. Without being bound by any particular theory or mechanism of action, it is contemplated that the combination of a beta-adrenergic blocker and a COX2 inhibitor counteracts stress-induced immunosuppression which may occur in cancer patients, thus resulting in improved efficacy of GLA. The combination of active ingredients disclosed herein act at least additively and preferably synergistically.
In one aspect, the invention provides a method of treating a subject having cancer, the method comprising the step of administering to the subject a pharmaceutical composition comprising a glucopyranosyl lipid adjuvant (GLA) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein said administration is effective in treating cancer.
According to one aspect, the present invention provides a method for reducing or preventing metastasis development in a subject, the method comprising the step of administering to the subject a pharmaceutical composition comprising a glucopyranosyl lipid adjuvant (GLA) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein said administration is effective in reducing or preventing metastasis.
As used herein, the terms “metastasis”, “cancer metastasis” or “tumor metastasis” are used interchangeably and refer to the growth of cancerous cells derived from a primary cancerous tumor located in one organ or tissue, in another, non-adjacent organ or tissue. Metastasis also encompasses micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part which is not directly connected to the organ of the original, primary cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from an original tumor site, and migration and/or invasion of cancer cells to other parts of the body.
As used herein, “reduction or prevention of metastasis development” refers to slowing or even completely inhibiting the spread, development and growth of metastasis. The term may also include reducing the number of metastases in an organ or tissue, as well as reducing the size, number or malignancy status of an existing cancer to be treated by the compositions of the invention. As one of ordinary skill in the art would understand a reduction or prevention of metastasis development can be measured by standard methodologies known in the art including a reduction in size or numbers of tumors as measured by a variety of radiographic, imaging, circulating tumor marker, palpitation, direct measurement or observation techniques known in the art. Accordingly a reduction or prevention of metastasis development can also be measured by a reduction of a sign or symptom associated with the disease state of the cancer being treated or a prolongation of survival or reduction in suffering from a disease sign or symptom of the cancer being treated.
In some embodiments, GLA or a pharmaceutically acceptable salt thereof is administered by local-regional delivery.
As used herein “local-regional delivery” indicates delivery to a space or cavity adjacent to a tumor to be treated, or delivery to a space or cavity adjacent to, or directly into, a draining lymph node of a tumor to be treated. If administered after tumor excision surgery, local-regional delivery includes delivery to the space or void formed after the removal of the tumor mass. Local-regional administration can be performed by a single injection to a single or multiple local-regional space, or as a series of injections given concurrently or over a period of at least about a minute, minutes, hours, days or weeks. As used herein, local-regional delivery does not include intra-tumor administration. Additionally, local-regional administration does not include topical administration, where a pharmaceutical composition is applied onto an external body surface of a subject.
In some embodiments, GLA or a pharmaceutically acceptable salt thereof is administered without a cancer-antigen.
In some embodiments, the administered pharmaceutical composition comprising GLA or a pharmaceutically acceptable salt thereof is substantially devoid of a cancer-antigen.
“Substantially devoid” as used herein refers to less than about 1%, preferably less than about 0.1%, less than about 0.01% (w/w), less than about 0.001% (w/w).
In some embodiments, the method is used for reducing or preventing metastasis development in a subject following a tumor excision surgery. In some embodiments, GLA or a pharmaceutically acceptable salt thereof is administered during the perioperative period of said tumor excision surgery.
The “perioperative period” refers to the time period immediately before, during and immediately after surgery. It includes the time preceding an operation, when a patient is being prepared for surgery (“the preoperative period”), followed by the time spent in surgery (“the intraoperative period”) and by the time following an operation when the patient is recovering and usually being monitored for complications (“the postoperative period”). The perioperative period can occur in hospitals, surgical centers and/or health care providers' offices.
As used herein, the perioperative period typically refers to a period beginning 2-10 days prior to a tumor excision surgery and ending 14-21 days following said surgery, for example beginning 4-5 days prior to surgery and ending about 14 days following surgery, or beginning 7 days prior to surgery and ending about 7 days following surgery.
In some embodiments, GLA or a pharmaceutically acceptable salt thereof is administered at least one time (for example two time, three times, four times or more) before the surgery. In some embodiments, GLA or a pharmaceutically acceptable salt thereof is administered at least one time (for example two time, three times, four times or more) after the surgery. In some embodiments, GLA or a pharmaceutically acceptable salt thereof is administered both before and after surgery.
In some embodiments, where GLA or a pharmaceutically acceptable salt thereof is administered a plurality of times, it is administered in constant doses. According to these embodiments, each time GLA or a pharmaceutically acceptable salt thereof is administered, it is administered in the same dose. In other embodiments, where GLA or a pharmaceutically acceptable salt thereof is administered a plurality of times, it is administered in varying doses. According to these embodiments, the administered dose of GLA or a pharmaceutically acceptable salt thereof varies throughout the treatment period.
In some embodiments, GLA has the following structural formula (I):




wherein: R1, R3, R5 and R6 are C11-C20alkyl; and R2 and R4 are C12-C20alkyl.
In some embodiments, R1, R3, R5 and R6 are undecyl and R2 and R4 are tridecyl.
In some embodiments, GLA has the following structural formula (II):




wherein:
L1, L2, L3, L4, L5 and L6 are the same or different and independently —O—, —NH— or —(CH2)—;
L7, L8, L9, and L10 are the same or different and independently absent or —C(═O)—;
Y1 is an acid functional group;
Y2 and Y3 are the same or different and independently —OH, —SH, or an acid functional group;
Y4 is —OH or —SH;
R1, R3, R5 and R6 are the same or different and independently C8-C13 alkyl; and
R2 and R4 are the same or different and independently C6-C11 alkyl.
In certain embodiments, a GLA adjuvant used herein may have the following structural formula (III):




wherein:
R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl.
In a more specific embodiment, the GLA has formula III set forth above wherein R1, R3, R5 and R6 are C11-14 alkyl; and R2 and R4 are C12-15 alkyl.
In a more specific embodiment, the GLA has formula III set forth above wherein R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C13 alkyl.
In a more specific embodiment, the GLA has formula III set forth above wherein R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.
In certain embodiments, the GLA is synthetic and has the following structural formula (IV):




In certain embodiments of the above GLA structure (formula IV), R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl. In certain embodiments, R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.
In certain embodiments, the GLA is synthetic and has the following structural formula (V):




In certain embodiments of the above GLA structure (formula V), R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl. In certain embodiments, R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.
In certain embodiments, the GLA is synthetic and has the following structural formula (VI):




In certain embodiments of the above GLA structure (formula VI), R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl. In certain embodiments, R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.
In certain embodiments, the synthetic GLA has the following structure:




In certain embodiments, the synthetic GLA has the following structure:




In certain embodiments, the synthetic GLA has the following structure:




In some embodiments, the method of the present invention further comprises the step of administering a beta-adrenergic blocker and a COX2 inhibitor.
In some embodiments, administration of the beta-adrenergic blocker and COX2 inhibitor is carried out during the perioperative period of a tumor excision surgery.
In some typical embodiments, the beta-adrenergic blocker and COX2 inhibitor are present within separate pharmaceutical compositions.
In some embodiments, the beta-adrenergic blocker, COX2 inhibitor or both are administered at least one time (for example two time, three times, four times or more) before the surgery. In additional embodiments, the beta-adrenergic blocker, COX2 inhibitor or both are administered at least one time (for example two time, three times, four times or more) after the surgery. In some embodiments, the beta-adrenergic blocker, COX2 inhibitor or both are administered both before and after surgery.
In some embodiments, GLA or a pharmaceutically acceptable salt thereof, beta-adrenergic blocker and COX2 inhibitor are administered on the same days during the perioperative period. In other embodiments they are administered on separate days.
In some embodiments, the beta-adrenergic blocker is selected from the group consisting of acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, nebivolol, oxyprenolol, penbutolol, pindolol, propranolol, sotalol, timolol, or pharmaceutically acceptable salts thereof. Each possibility represents a separate embodiment of the present invention. In particular embodiments, it is propranolol or a pharmaceutically acceptable salt thereof.
In some embodiments, the COX2 inhibitor is selected from the group consisting of celecoxib, cimicoxib, etoricoxib, etodolac, eoricoxib, lumiracoxib, meloxicam, parecoxib, rofecoxib, tiracoxib, valdecoxib, or pharmaceutically acceptable salts thereof. Each possibility represents a separate embodiment of the present invention. In particular embodiments, it is etodolac or a pharmaceutically acceptable salt thereof.
According to another aspect, the present invention provides a pharmaceutical composition comprising GLA or a pharmaceutically acceptable salt thereof as an active ingredient, for use in the reduction or prevention of metastasis development, said composition being substantially devoid of a cancer antigen.
In some embodiments, the pharmaceutical composition consists of GLA or a pharmaceutically acceptable salt thereof as the sole active ingredient.
In some embodiments, the pharmaceutical composition is formulated for local-regional delivery.
In some embodiments, the pharmaceutical composition is for use during the perioperative period of a tumor excision surgery.
In some embodiments, the pharmaceutical composition is for use with a beta-adrenergic blocker and a COX2 inhibitor during the perioperative period of a tumor excision surgery.
These and further aspects and features of the present invention will become apparent from the figures, detailed description, examples and claims which follow.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A and FIG. 1B: Dose curve of the effects of GLA on MADB 106 LTR in male rats with (FIG. 1A) or without (FIG. 1B) epinephrine.
FIG. 2A and FIG. 2B: Time course for initiation and duration of GLA effects in male (FIG. 2A) and female (FIG. 2B) rats.
FIG. 3: GLA effects on the development of MADB 106 metastases in the lungs of rats.
FIG. 4A and FIG. 4B: GLA effects on MADB106 LTR in naive (no-depletion) versus NK-depleted animals (FIG. 4A) and in naive (no-depletion) animals only (FIG. 4B).



DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to the reduction or even prevention of metastasis formation. The approach disclosed herein is based on immune-stimulation with GLA. GLA may optionally be combined with a COX2 inhibitor and a beta-adrenergic blocker.
Pharmaceutical Compositions
In one aspect, the pharmaceutical compositions of the present invention may comprise at least one GLA compound, in combination with pharmaceutically acceptable carriers, excipients or diluents. In another aspect, the pharmaceutical compositions of the present invention generally comprise at least one GLA compound, a beta-adrenergic blocker such as propranolol, or a COX2-selective inhibitor such as etodolac, in combination with pharmaceutically acceptable carriers, excipients or diluents. Pharmaceutically acceptable salts of the active agents described herein are also within the scope of the present invention. “Pharmaceutically acceptable salt” refers to salts of the compounds described herein derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The compositions of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.
Glucopyranosyl Lipid Adjuvant (GLA):
GLA is a synthetic TLR-4 agonist, capable of eliciting an immune response in a host, particularly cell-mediated immune response. GLA differs from other TLR-4 agonists, such as LPS, MPL and 3-DMPL, by being totally synthetic and having a defined number, length, and position of carbon chains. GLA is capable of eliciting an efficient Th1 activation, while causing only minimal adverse Th2 effects. Compositions comprising GLA are described, for example, in U.S. Pat. No. 8,273,361 and WO 2010/141861.
A GLA molecule for use with the compositions of the present invention comprises: (i) a diglucosamine backbone having a reducing terminus glucosamine linked to a non-reducing terminus glucosamine through an ether linkage between hexosamine position 1 of the non-reducing terminus glucosamine and hexosamine position 6 of the reducing terminus glucosamine; (ii) an O-phosphoryl group attached to hexosamine position 4 of the non-reducing terminus glucosamine; and (iii) up to six fatty acyl chains; wherein one of the fatty acyl chains is attached to 3-hydroxy of the reducing terminus glucosamine through an ester linkage, wherein one of the fatty acyl chains is attached to a 2-amino of the non-reducing terminus glucosamine through an amide linkage and comprises a tetradecanoyl chain linked to an alkanoyl chain of greater than 12 carbon atoms through an ester linkage, and wherein one of the fatty acyl chains is attached to 3-hydroxy of the non-reducing terminus glucosamine through an ester linkage and comprises a tetradecanoyl chain linked to an alkanoyl chain of greater than 12 carbon atoms through an ester linkage. GLA is typically not 3′-de-O-acylated.
A GLA as used herein may have the following structural formula (I):




wherein:
R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C12-C20 alkyl.
A particular example is a GLA wherein R1, R3, R5 and R6 are undecyl and R2 and R4 are tridecyl).
In addition, a GLA as used herein may have the following structural formula (II):




wherein:
L1, L2, L3, L4, L5 and L6 are the same or different and independently —O—, —NH— or —(CH2)—;
L7, L8, L9, and L10 are the same or different and independently absent or —C(═O)—;
Y1 is an acid functional group;
Y2 and Y3 are the same or different and independently —OH, —SH, or an acid functional group;
Y4 is —OH or —SH;
R1, R3, R5 and R6 are the same or different and independently C8-C13 alkyl; and
R2 and R4 are the same or different and independently C6-C11 alkyl.
Examples of suitable GLA molecules that can be used are described in WO 2010/141861 noted above.
In certain embodiments, a GLA adjuvant used herein may have the following structural formula (III):




wherein:
R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl.
In a more specific embodiment, the GLA has formula III set forth above wherein R1, R3, R5 and R6 are C11-14 alkyl; and R2 and R4 are C12-15 alkyl.
In a more specific embodiment, the GLA has formula III set forth above wherein R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C13 alkyl.
In a more specific embodiment, the GLA has formula III set forth above wherein R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.
In certain embodiments, the GLA is synthetic and has the following structural formula (IV):




In certain embodiments of the above GLA structure (formula IV), R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl. In certain embodiments, R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.
In certain embodiments, the GLA is synthetic and has the following structural formula (V):




In certain embodiments of the above GLA structure (formula V), R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl. In certain embodiments, R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.
In certain embodiments, the GLA is synthetic and has the following structural formula (VI):




In certain embodiments of the above GLA structure (formula VI), R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C9-C20 alkyl. In certain embodiments, R1, R3, R5 and R6 are C11 alkyl; and R2 and R4 are C9 alkyl.
In certain embodiments, the synthetic GLA has the following structure:




In certain embodiments, the synthetic GLA has the following structure:




In certain embodiments, the synthetic GLA has the following structure:




“Alkyl” means a straight chain or branched, noncyclic or cyclic, unsaturated or saturated aliphatic hydrocarbon containing from 1 to 20 carbon atoms, and in certain preferred embodiments containing from 11 to 20 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, including undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, etc.; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like. Cyclic alkyls are also referred to herein as “homocycles” or “homocyclic rings.” Unsaturated alkyls contain at least one double or triple bond between adjacent carbon atoms (referred to as an “alkenyl” or “alkynyl”, respectively). Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; while representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.
“Acid functional group” means a functional group capable of donating a proton in aqueous media (i.e. a Brønsted-Lowry acid). After donating a proton, the acid functional group becomes a negatively charged species (i.e. the conjugate base of the acid functional group). Examples of acid functional groups include, but are not limited to: —OP(═O)(OH)2 (phosphate), —OS(═O)(OH)2 (sulfate), —OS(OH)2 (sulfite), —C(═O)OH (carboxylate), —OC(═O)CH(NH2)CH2C(═O)OH (aspartate), —OC(═O)CH2CH2C(═O)OH (succinate), and —OC(═O)CH2OP(═O)(OH)2 (carboxymethylphosphate).
GLA be obtained commercially. Methods for the synthesis of GLA are provided, for example, in WO 2010/141861 noted above.
GLA formulations can be prepared in substantially homogeneous form, which refers to a GLA preparation that is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and still more preferably at least 96%, 97%, 98% or 99% pure with respect to the GLA molecule. Determination of the degree of purity of a given GLA preparation can be readily made by those familiar with the appropriate analytical chemistry methodologies, such as by gas chromatography, liquid chromatography, mass spectroscopy and/or nuclear magnetic resonance analysis.
The pharmaceutical compositions comprising GLA of the present invention are preferably substantially devoid of cancer-specific antigen(s), and are administered to a subject in order to stimulate an immune response, e.g., a non-specific immune response.
“Substantially devoid” as used herein refers to less than about 1%, preferably less than about 0.1%, less than about 0.01% (w/w), less than about 0.001% (w/w).
The term “about”, when referring to a measurable value such as an amount, is used herein to encompass variations of +/−10%, more preferably +/−5%, even more preferably +/−1%, and still more preferably +1-0.1% from the specified value, as such variations are appropriate to achieve the intended purpose.
The GLA may be preferably formulated in a stable emulsion. In one particular embodiment, for example, a composition is provided comprising GLA in a stable emulsion substantially devoid of cancer antigens. Emulsion systems include single or multiphase emulsion systems, as known in the art.
In some embodiments, the composition is in the form of an oil-in-water emulsion. In other embodiments, the composition is in the form of a water-in-oil emulsion. In yet other embodiments, the composition is in the form of microparticles.
In a particular embodiment, a composition of the invention comprises an emulsion of oil in water wherein the GLA is incorporated in the oil phase.
In order for any oil in water composition to be suitable for human administration, the oil phase of the emulsion system preferably comprises a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as “being capable of being transformed by metabolism”. The oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts (such as peanut oil), seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE® and others. A non-limiting example of a suitable oil is Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene).
Oil emulsion preparations of the present invention may comprise an antioxidant, for example the oil α-tocopherol (vitamin E, EP 0 382 271 B1).
WO 95/17210 and WO 99/11241 disclose emulsion adjuvant compositions based on squalene, α-tocopherol, and TWEEN® 80. WO 99/12565 discloses an improvement to these squalene emulsions with the addition of a sterol into the oil phase. Additionally, a triglyceride, such as tricaprylin (C27H50O6), may be added to the oil phase in order to stabilize the emulsion (WO 98/56414).
The size of the oil droplets found within the stable oil in water emulsion are preferably less than about 1 micron, may be in the range of about 30-600 nm, preferably about around 30-500 nm in diameter, and most preferably about 150-500 nm in diameter, and in particular about 150 nm in diameter as measured by photon correlation spectroscopy. In this regard, about 80% of the oil droplets by number should preferably be within the preferred ranges, more preferably more than about 90% and most preferably more than about 95% of the oil droplets by number are within the defined size ranges. The amounts of the components present in the oil emulsions of the present invention are conventionally in the range of from about 2 to 10% oil, such as squalene; and when present, from about 2 to 10% alpha tocopherol; and from about 0.3 to 3% surfactant, such as polyoxyethylene sorbitan monooleate. Preferably the ratio of oil:alpha tocopherol is equal or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of about 1%. In some cases it may be advantageous that the GLA compositions of the present invention will further contain a stabilizer.
The method of producing oil in water emulsions is well known to the person skilled in the art. Commonly, the method comprises mixing the oil phase with a surfactant such as a PBS/TWEEN80® solution, followed by homogenization using a homogenizer. For instance, a method that comprises passing the mixture once, twice or more times through a syringe needle would be suitable for homogenizing small volumes of liquid. Equally, the emulsification process in a microfluidiser (M110S microfluidics machine, maximum of 50 passes, for a period of 2 minutes at maximum pressure input of 6 bar (output pressure of about 850 bar)) could be adapted to produce smaller or larger volumes of emulsion. This adaptation could be achieved by routine experimentation comprising the measurement of the resultant emulsion until a preparation was achieved with oil droplets of the required diameter.
Exemplary doses of GLA can range from about 0.01 μg/kg to about 100 mg/kg body weight, such as from about 1 μg/kg to about 1 mg/kg, or about 1 μg/kg to about to about 60 μg/kg, or about 5 μg/kg to about 200 μg/kg.
In some embodiments, the pharmaceutical compositions comprising GLA of the present invention are formulated for local-regional delivery. Examples of suitable routes of administration include intradermal, subcutaneous, intramuscular, intraperitoneal. It will be evident to those skilled in the art that the number and frequency of administration will be dependent upon the response of the host.
Beta-Adrenergic Blocker (Antagonist)—e.g., Propranolol:
Propranolol may be identified by CAS Registry Number 525-66-6. It is commercially available and may also be synthesized by methods known in the art. For pharmaceutical compositions, propranolol hydrochloride is typically used. Suitable formulations include, for example, oral solid dosage forms containing about 10-80 mg, oral liquid dosage forms containing about 20-40 mg/5 ml, extended release oral dosage forms containing about 60-160 mg, and intravenously injectable liquid dosage forms containing about 1 mg/ml of propranolol hydrochloride.
COX2-Selective Inhibitor—e.g., Etodolac:
Etodolac may be identified by CAS Registry Number 41340-25-4. It is commercially available and may also be synthesized by methods known in the art. Suitable formulations include, for example, oral solid dosage forms containing about 200-500 mg, extended release oral dosage forms containing about 400-600 mg.
The amount of compounds in the compositions of the present invention which will be effective in the treatment of a particular condition will depend on the nature of the condition, and can be determined by standard clinical techniques. See, for example, Goodman and Gilman; The Physician's Desk Reference, Medical Economics Company, Inc., Oradell, N.J., 1995; and to Drug Facts and Comparisons, Facts and Comparisons, Inc., St. Louis, Mo., 1993.
The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances.
Methods and Uses
According to an aspect of the present invention, the present invention provides a method for reducing or preventing metastasis development in a subject.
In some embodiments, the method comprises the step of administering a pharmaceutical composition comprising glucopyranosyl lipid adjuvant (GLA) or a pharmaceutically acceptable salt thereof as an active ingredient.
In some embodiments, a method is provided for reducing or preventing metastasis development in a subject by administering to the subject GLA alone or in combination with a beta-adrenergic blocker and a COX2 inhibitor loco-regionally. In some embodiments, a method is provided for reducing or preventing metastasis development in a subject by administering to the subject GLA alone or in combination with a beta-adrenergic blocker and a COX2 inhibitor loco-regionally without tumor excision.
In some embodiments, a method is provided for reducing or preventing metastasis development in a subject following a tumor excision surgery by administering to the subject GLA alone or in combination with a beta-adrenergic blocker and a COX2 inhibitor during the perioperative period of the tumor excision surgery, thereby reducing or preventing metastasis development following the surgery.
In some embodiments, GLA is administered before (pre-operative) and after (post-operative) surgery. In some embodiments, the beta-adrenergic blocker, COX2 inhibitor or both are administered before (pre-operative) and after (post-operative) surgery.
Administration of GLA is typically performed by local-regional delivery. Under certain circumstances, for example during the perioperative period, systemic administration is used. Systemic administration as used herein does not include intravenous administration.
GLA administration can be performed as a single injection or multiple injections.
Typically, the method of the present invention comprises administering GLA without a cancer antigen. The administered pharmaceutical composition of GLA is preferably substantially devoid of a cancer antigen.
In some embodiments, the method of the present invention further comprises co-administration of a beta-adrenergic blocker and a COX2 inhibitor during the perioperative period.
In some embodiments, GLA is administered pre-operatively, while the beta-adrenergic blocker and/or COX2 inhibitor are administered pre- and post-operatively.
In some embodiment, all three active ingredients are administered both before and after surgery.
The particular doses of each of the active ingredients, as well as their specific administration days with respect to the resection surgery should be determined by a practitioner based on the type of the tumor, the severity and the overall patient's circumstances.
The subject treated by the method of the present invention is a mammal, typically a human, inflicted with cancer, including solid and non-solid tumors. Each possibility represents a separate embodiment of the invention. In some embodiments, the subject is a human inflicted with a primary solid tumor that is about to undergo tumor resection. The subject may be inflicted with any type of solid malignancy, for example, carcinomas, such as respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary, and also carcinosarcomas (e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues) and adenocarcinoma (derived from glandular tissue or in which the tumor cells form recognizable glandular structures). Additional examples of malignancies to be treated by the methods disclosed herein include sarcomas, namely malignant tumors of supportive tissues or connective tissue, for example bone or cartilage, and lymphomas, namely tumors of the lymph tissue. In certain embodiments, examples of cancer include but are not limited to, carcinoma, including adenocarcinoma, lymphoma, blastula, melanoma, and sarcoma. More particular examples of such cancers include squamous cell cancer, lung cancer (such as small-cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervical cancer, gloom, ovarian cancer, liver cancer (such as hepatic carcinoma and hematoma), bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (such as renal cell carcinoma and Wilma′ tumors), basal cell carcinoma, melanoma, prostate cancer, thyroid cancer, testicular cancer, esophageal cancer, and various types of head and neck cancer.
The formation of metastasis following tumor resection can be followed up in a subject using methods known in the art, including imaging techniques, biopsies, blood tests and the like.
The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.
EXAMPLES
Materials and Methods
1. Animals
Four to eight months old (age varied between experiments) male and female Fischer 344 (F344) rats were housed 3-4 per cage in our vivarium at Tel Aviv University with ad-libidum access to food and water on a 12:12 light-dark cycle at 22±1° C. Animals were handled a minimum of 4 times prior to experimentation to reduce potential procedural stress. Body weight, sex, and drug administration were counterbalanced across all experimental procedures. Housing conditions were monitored by the Institutional Animal Care and Use Committee of Tel Aviv University, which also approved all studies described herein.
2. Drugs
1. GLA and its Administration
GLA (IDRI, Seattle, Wash.), dissolved in stable emulsion (SE) (2000 μg/ml) was diluted in PBS for a final concentration of 3 μg/ml-200 μg/ml (depending on the relevant experiment). One-hundred μl from the chosen concentration were injected subcutaneously (s.c.) to each animal (0.3 μg-20 μg/animal), immediately, 4 h, 12 h, 24 h, 48 h, or 96 h prior to MADB106 tumor cell inoculation (depending on the relevant experiment).
2. Stable Emulsion (SE)/Adjuvant Emulsion
SE (stable emulsion; IDRI, Seattle, Wash.) was diluted and administered in the exact manner as GLA.
3. Anti-NKR-P1
Anti-NKR-P1 is an IGg monoclonal antibody (mAb) (originally termed mAb 3.2.3) that binds to the NKR-P1 surface antigen expressed on fresh and IL-2-activated natural killer (NK) cells in rats, and, to a much lesser degree, on polymorphonuclear (PMNs) cells. In vivo treatment of rats with anti-NKR-P1 selectively depletes NK cells and eliminates NK- and antibody-dependent non-MHC-restricted cell cytotoxicity. T cell function and the percentages of T cells, peripheral blood mononuclear cells (PBMCs), and PMNs are unaffected. It has been previously shown that this antibody, but not isotype control antibodies, renders NK cells ineffective in vivo immediately upon administration, and selectively depletes NK cells within a day. The antibodies were injected i.v. under light isoflurane anesthesia simultaneously with MADB 106 tumor cell inoculation.
3. Tumor Cell Lines
1. MADB106
MADB 106 is a selected variant cell line obtained from a pulmonary metastasis of a chemically induced mammary adenocarcinoma (MADB 100) in the F344 rat. MADB 106 tumor cells metastasize only to the lungs, and lung tumor retention (LTR), which is highly indicative of the number of metastases that would have developed weeks later, is dependent upon NK cells in this model. Additionally, because the metastatic process of MADB106 is sensitive to NK activity predominantly during the first 24 h following inoculation, LTR is more reflective of in vivo NK activity levels than the number of actual metastases. The MADB 106 cell line was maintained in monolayer cultures in complete media (RPMI-1640 media supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 μg/mL of gentamicin, 2 mM of 1-glutamine, 0.1 mM of non-essential amino-acids, and 1 mM of sodium pyruvate, (Biological Industries, Kibbutz Biet Haemek, Israel) in 100% humidity, 5% CO2 at 37° C. Cells were removed from the culture flask with trypsin solution (0.25% in PBS), and were washed with complete media. This cell line was used for both in vivo assessment of lung tumor retention and in vitro examination of NK cytotoxicity.
2. YAC-1
YAC-1 lymphoma is the standard target cell line used for the assessment of rodent in vitro NK cytotoxicity. The cell line was maintained in suspension cultures in complete media in 100% humidity, 5% CO2 at 37° C.
4. Radiolabeling of MADB 106 Tumor Cells and Assessment of Lung Tumor Retention
Tumor cell DNA radiolabeling for assessment of LTR was accomplished by adding 0.5 μCi/ml of 125Iododeoxyuridine (125IDUR, Danyel Biotech, Rehovot, Israel) to the cell culture for 24 h. For tumor cell injection, rats were lightly anesthetized with isoflurane, and 4×105/kg MADB106 tumor cells in 2 ml/kg PBS containing 0.1% bovine serum albumin (BSA) were injected into their tail vein. Some of the experiments also employed an additional 0.5 ml s.c. injection of epinephrine (1.8 mg/kg in females and 0.6 mg/kg in males) dissolved in a slow-release emulsion (4 parts PBS, 3 parts mineral oil (Sigma, Rehovot, Israel), and 1 part mannide-monooleate (a non-specific surface active emulsifier, Sigma, Rehovot, Israel)), for the amplification of MADB 106 retention in the lungs, allowing better group distinction. For assessment of LTR, animals were sacrificed with CO2, their lungs were removed 24 h after inoculation with 125IDUR-labeled tumor cells, and placed in a γ-counter to assess percent radioactivity retained in this organ. LTR was calculated using the following formula: (radioactivity count of lung−background radioactivity)×100/(radioactivity count of the total injected cell suspension−background radioactivity).
5. EX Vivo Assessment of NK Cytotoxicity
1. Harvesting and Preparing Circulating Leukocytes, Marginating-Pulmonary (MP) Leukocytes, and Marginating-Hepatic (MH) Leukocytes for Assessment of NK Cytotoxicity
Rats were sacrificed with an overdose of isoflurane and the peritoneal and chest cavities opened. Five to 8 ml of blood (females and male respectively) was collected from the right ventricle of the heart into heparinized syringes. One ml of blood was washed once with 3 ml of PBS (400 g for 10 min) and twice with 3 ml of complete medium, and reconstituted to its original volume. MP leukocytes were harvested by perfusing the heart with 30 U/ml of heparinized PBS. PBS was injected into the right ventricle and perfusate was collected from the left ventricle. The first 3 ml of perfusate, which were contaminated with blood, were discarded, and the following 25 ml were collected and concentrated to 1 ml. This was achieved by centrifuging the perfusate (400 g for 10 min), discarding the supernatant, and suspending the pellet in 3 ml of complete medium, centrifuging the perfusate again (400 g for 10 min) and concentrating the perfusate into 1 ml. MH leukocytes were similarly harvested by perfusing the liver with 30 U/ml of heparinized PBS. PBS was injected into the hepatic portal vein and perfusate was collected from the vena cava. The first 5 ml of perfusate, which were contaminated with blood, were discarded, and the following 25 ml were collected and concentrated in the same method as was described for MP leukocytes.
2. Assessment of NK Cytotoxicity
The standard whole blood 51Cr release assay was used. This procedure assesses anti-tumor NK cellular cytotoxicity (NKCC) per ml of effector cells without prior purification of the leukocyte population studied (peripheral blood mononuclear cells, MP or MH leukocytes). Earlier studies have indicated that cytotoxicity measured using this procedure is attributable to NK cells, rather than other cell types or soluble factors, as the selective depletion of NK cells abolishes all target-cell killing. The advantages of this procedure include shorter duration, less interference with the effector cells, and better representation of the original in vivo milieu of cell composition.
Six different effector to target (E:T) ratios were formed by serially diluting 150 μl aliquots of the effector-cell preparation in a microtiter plate. Then, 5000 radiolabeled target cells (MADB 106 or YAC-1) in 100 μl complete medium were added to each well on top of the effector-cell preparation. Radiolabeling of the target cells was conducted by incubating them for 1 h with 100 μCi 51Cr (Rotem Taassiot, Dimona, Israel) in 100 μl saline, 100 μl FCS, and 50 μl complete medium. Following this incubation, target cells were washed 3 times (300 g for 10 min) in complete medium and their concentration adjusted to 5×104/ml. Spontaneous and maximal releases of radioactivity were determined by substituting effector cells with complete medium or Triton-X100 (Sigma, Rehovot, Israel), respectively. Prior to and following a 4 h incubation period (100% humidity, 5% CO2 at 37° C.) plates were centrifuged (400 g for 10 min, at 25 and 4° C., respectively). This creates a “buffy coat” of leukocytes and tumor cells on top of the red blood cells surface, enabling efficient effector-target interaction. Finally, 100 μl samples of supernatant, were recovered for the assessment of radioactivity in a γ-counter. Specific killing was calculated as: 100×[(sample release×HCF−spontaneous release)/(maximal release−spontaneous release)], Hematocrit correction factor (HCF) compensates for changes in the hematocrit-supernatant volume over different E:T ratios. This correction factor is included to consider the changing volume of cell-free medium in which the released radioactive molecules are dispersed.
6. Flow Cytometry
Standard procedures were used to prepare cells for flow cytometric analysis. NK cells in both blood, lung perfusate, and liver perfusate were identified by the APC-conjugated anti-CD161 mAb (Biolegend, San Diego, Calif.) as being CD161bright cells. This criterion has been shown to exclusively identify more than 95% of cells that exhibit NK activity. T cells were identified using a PE-conjugated anti-CD5 mAb (eBioscience, San Diego), and NKT cells were identified as CD161+CD5+ lymphocytes. NK activation markers were identified by the FITC-conjugated NKp46 (BiossUSA, Woburn, Mass.) and the Cy7-conjugate LAMP-1 (BiossUSA, Woburn, Mass.). Granulocytes and lymphocytes were identified based on forward and side scatters. Flow cytometry analysis was conducted using a FACScan (Becton Dickinson). To assess the absolute number of cells per μl of sample (or a specific cell subtype), 300 polystyrene microbeads (20 μm, Duke Scientific, Palo Alto) per μl sample were added to each sample, and the following formula was used: (# of cells in sample/# of microbeads in sample)×300.
7. Statistical Analyses
One- or two-way analysis of variance (ANOVA) with a pre-determined significance level of 0.05 was conducted. Provided significant group differences were found, Fisher's protected least significant differences (Fisher's PLSD) contrasts were performed to compare specific pairs of groups, based on a priori hypotheses.
Example 1
Dose Curve of the Effects of GLA on MADB106 LTR in Male Rats
The experiment was conducted in order to establish a potent dosage for GLA administration for future experiments.
Procedure:
Seventy-five three months old F344 male rats were randomly divided into one of 7 experimental groups administered with PBS, SE, or GLA in a dose/animal of 0.3 μg, 0.7 μg, 2.5 μg, 10 μg, and 20 μg. Each animal was injected s.c. with 100 μl of drug according to its group assignment, and, 24 h later, MADB 106 cells were inoculated (as detailed in section 4 above). Each of these 7 groups was further sub-divided into two separate groups—one that was injected with epinephrine during tumor inoculation, and the second that was injected with vehicle. Twenty-four hours later, animals were sacrificed and lungs were extracted for LTR assessment (as detailed in section 4).
Results:
Significant main effects for treatment (SE, and GLA dose) on LTR were evident both in the epinephrine groups (F(6,31)=14.375, p<0.0001) and in the vehicle groups (F(6,30)=3.354, p<0.05), indicating improved host ability to clear cancer cells from the lung (decreased LTR). See FIGS. 1A and 1B.
Fisher's PLSD post-hoc comparisons in the epinephrine groups indicated a significant improvement for SE over PBS (p<0.001), and for all GLA doses over PBS (p<0.0001). When examined for the additive effects of GLA over SE (due to the fact that GLA was originally dissolved in SE, which is partially responsible for its effects), significant improvements were found for the 0.7 μg, 2.5 μg, 10 μg, and 20 μg doses (p<0.05, p<0.01, p<0.01, and p<0.001, respectively).
Within the vehicle groups, Fisher's PLSD post-hoc comparisons yielded no effect for SE alone, and significant improvement for GLA over PBS in the 0.7 μg, 2.5 μg, 10 μg, and 20 μg doses (p<0.05, p<0.01, p<0.05, and p<0.01, respectively).
These results indicate a partial responsibility for the SE constituency in the beneficial effects of GLA on LTR, and an additive effect for GLA over SE effects for dosages equal or higher than 0.7 μg per animal. Following this experiment, a working dosage of 2 μg GLA per animal was determined, which enables minimal yet effective drug dose that is based on GLA-related effects beyond SE-related ones.
Also, as was already reported previously, the administration of epinephrine along with MADB 106 cell inoculation allowed better distinction between groups, by inducing a more challenging conditions to the host and increasing LTR levels. By analyzing the difference between PBS animals receiving epinephrine and those receiving vehicle, it was possible to calculate the specific epinephrine effect on LTR, and the relevant reduction effects caused by the GLA within the epinephrine groups, which positively correlated with its dosage.
Example 2
Time Course for Initiation and Duration of GLA Effects
The experiment was conducted in order to determine the duration of effect for GLA administration, and for assessing an optimal time point for its use.
Procedure:
In the first experiment, 75 six-month old F344 male rats were randomly divided into one of 5 injection time points preceding tumor inoculation—0 h, 4 h, 12 h, 24 h, and 48 h. Each group was further sub-divided into one of three experimental drug groups—PBS, SE, and 2 μg GLA. Each animal was injected s.c. with 100 μl according to its relevant drug group, in its designated time point. At time 0 h, MADB106 cells were injected (as detailed in section 4), along with epinephrine. Four additional male F344 rats were added to the PBS group and were not injected with epinephrine (received vehicle), to serve as an anchor to establish the effects of epinephrine. Twenty-four hours later, animals were sacrificed and lungs were extracted for LTR assessment (as detailed in section 4)
In the second experiment, a similar experiment was conducted in female rats. Eighty-nine six-month old F344 female rats were randomly divided into one of 6 injection time points—0 h, 4 h, 12 h, 24 h, 48 h, and 96 h. Each group was further sub-divided into one of three experimental drug groups—PBS, SE, and 2 μg GLA. All other procedures were as in males above.
Results:
In both experiments, the different time points within the PBS and SE groups showed no consistent or significant differences, and were thus combined to accumulate sufficient animal in these conditions.
In the first experiment (males), significant main effect for treatment on LTR was evident (F(7,71)=5.990, p<0.0001), improving host resistance. See FIG. 2A. Fisher's PLSD post-hoc comparisons indicated no effect for SE alone, and a significant difference between GLA and PBS in time-points 4 h, 12 h, 24 h, and 48 h (p<0.05 for 4 h, and p<0.0001 for the rest).
In the second experiment (females), significant main effect for treatment on LTR was evident (F(8,84)=3.229, p<0.01). See FIG. 2B. Fisher's PLSD post-hoc comparisons indicated no effect for SE alone, and a significant difference between GLA and PBS in time-points 24 h and 48 h (p<0.05 for both).
These results indicate a quick and a long lasting effect for a single low-dose injection of GLA in both males and females, although suggesting a better response in males to the treatment in this dose.
Example 3
A Three Week Study for the Assessment of GLA Effects on the Actual Development of MADB106 Metastases in the Lungs
This experiment was conducted in order to assess in vivo effects of GLA on the actual development of cancer metastases in the lungs, rather than focusing on the shorter index of LTR.
Procedure:
Eighty-six six-month old F344 rats (44 females) were divided into three experimental groups (2 μg GLA, SE, and PBS). Each animal was injected s.c. with 100 μl according to its group assignment. Twenty-four hrs later, animals were lightly anesthetized with isoflurane and 105 MADB 106 tumor cells (approximately 4×105/kg) were injected into their tail vein in 0.5 ml of PBS (supplemented with 0.1% BSA). Three weeks later, rats were killed, and their lungs removed and placed for 24 h in Bouin's solution (72% saturated picric acid solution, 23% formaldehyde (37% solution) and 5% glacial acetic acid). After being washed in ethanol, visible surface metastases were counted by a researcher uninformed of the origin of each lung.
Results:
Significant main effect for treatment on number of metastases was evident (F(2,83)=5.405, p<0.01). See FIG. 3.
Fisher's PLSD post-hoc comparisons indicated no effect for SE alone, and a significant difference between GLA and PBS (p<0.05) and between GLA and SE (p<0.01)—GLA reducing the number of metastases. No significant sex differences were evident.
Example 4
The Effects of GLA in Naive and in NK-Depleted Animals on MADB106 LTR
This experiment was conducted to assess the role of NK cells in mediating the beneficial impact of GLA administration on LTR.
Procedure:
Fifty-four four-month old F344 male rats were divided into two groups—NK depletion by administration of anti-NKR-P1 mAb, or vehicle administration, and each group was further sub-divided into three (2 μg GLA, SE, and PBS). Each animal was injected s.c. with 100 μl according to its drug condition assignment, and 24 h later MADB 106 cells were administered simultaneously with either the anti-NKR-P1 mAb or vehicle. Twenty-four hours later animals were sacrificed and lungs were removed for LTR assessment (as detailed in section 4).
Results:
A two-by-three ANOVA (depletion×drug injection) revealed a significant effect for depletion (F(1,46)=712.065, p<0.0001), showing an approximately 20-fold higher levels of LTR in NK-depleted animals, and thus the prominent role of NK cells in clearing MADB 106 from the lungs. See FIG. 4A.
When the depletion and non-depletion groups were examined separately, no differences were evident between the depletion groups, indicating no effect for either GLA or SE under this condition, while a significant effect for group was found under the no-depletion condition (F(2,22)=6.447, p<0.01). Fisher's PLSD post-hoc comparisons for the no-depletion condition indicated no effect for SE alone, and a significant difference between GLA and PBS (p<0.01). See FIG. 4B. These findings indicate that the beneficial effects of GLA are mediated prominently by NK cells.
In previous studies employing this experimental approach it also shown that other manipulations increase or decrease LTR in NK-depleted animals, negating a potential methodological obstacle, such as a ceiling or flour effects, in this experimental approach.
Example 5
Testing GLA, a Beta-Blocker and a COX2 Inhibitor in Mouse Models of Spontaneous Postoperative Metastasis
C57BL/6J mice are inoculated intrafootpad with syngeneic B16F10.9-melanoma or Lewis lung carcinoma, and the paw is amputated when a developing tumor exceeds 100 ml. GLA, beta-adrenergic antagonist propranolol, and/or the cyclooxygenase-2 inhibitor etodolac are administered once (or more) before amputation, and recurrence-free survival is monitored. Further experiments are conducted where GLA is administered once before amputation, and propranolol plus etodolac are administered once after amputation.
C57BL/6J male and female mice are purchased at the age of 6 wk and housed 3-4 per cage in a vivarium with ad libitum access to food and water on a 12:12 light/dark cycle at 22 6 1° C. Animals are used at the age of 10-14 wk and age-matched across all groups in each experiment. The order of tumor and drug administration is counterbalanced across all experimental groups, and control animals are injected with vehicle.
Propranolol is injected s.c. (5 mg/kg, 10 ml/kg) in an emulsion of PBS, mineral oil, and Arlacel (8:7:1). Etodolac is dissolved in corn oil and injected s.c. (50 mg/kg, 10 ml/kg). GLA is injected s.c. in a dose/animal ranging from 0.1μ-50 μg.
Each mouse is injected with 5×104 B16F10.9 melanoma cells or d122 Lewis lung carcinoma (in 20 ml PBS containing 0.1% BSA) intrafootpad, and tumors are visually inspected daily. Once a tumor reaches 100-150 ml in volume, the mouse is anesthetized with 2% isoflurane and undergoes a specific drug treatment, and the tumor is excised by paw amputation. Mice in which the designated tumor volume is achieved are assigned to a specific drug treatment group based on a predetermined counterbalanced order, thus ensuring equal distribution of tumor size and tumor age at excision time between the different drug groups. The experimenter conducting the amputation is unaware of the drug treatment group. Mice are subsequently monitored for morbidity signs on a daily basis for an 80 d period (and no less than 2 wk following the last morbidity incidence). Mice that show sickness behavior or manifested cancer recurrence are overdosed with isoflurane and autopsied to determine malignant foci. Sickness behavior is defined by slow body movements, irresponsiveness to environmental stimuli, significant weight loss, or tremor.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

## Claims

1. A method for reducing or preventing metastasis development in a subject following a tumor excision surgery, the method comprising the step of administering to the subject a pharmaceutical composition comprising glucopyranosyl lipid adjuvant (GLA) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein said administration is effective in reducing or preventing metastasis, and wherein said administering is carried out during the perioperative period of said tumor excision surgery.

2. The method of claim 1, wherein said administering is by local-regional delivery.

3. The method of claim 1, wherein said pharmaceutical composition is substantially devoid of a cancer-antigen.

4. The method of claim 1, wherein said administering is carried out at least one time before the surgery (pre-operative).

5. The method of claim 1, wherein said administering is carried out at least one time after the surgery (post-operative).

6. The method of claim 1, wherein said pharmaceutic composition is administered in constant doses.

7. The method of claim 1, wherein said pharmaceutic composition is administered in varying doses.

8. The method of claim 1, wherein GLA has the following structural formula (I):



wherein:
R1, R3, R5 and R6 are C11-C20 alkyl; and R2 and R4 are C12-C20 alkyl.

9. The method of claim 8, wherein R1, R3, R5 and R6 are undecyl and R2 and R4 are tridecyl.

10. The method of claim 1, wherein GLA has the following structural formula (II):



wherein:
L1, L2, L3, L4, L5 and L6 are the same or different and independently —O—, —NH— or —(CH2)—;
L7, L8, L9, and L10 are the same or different and independently absent or —C(═O)—;
Y1 is an acid functional group;
Y2 and Y3 are the same or different and independently —OH, —SH, or an acid functional group;
Y4 is —OH or —SH;
R1, R3, R5 and R6 are the same or different and independently C8-C13 alkyl; and
R2 and R4 are the same or different and independently C6-C11 alkyl.

11. The method of claim 10, wherein GLA has the following structure:

12. The method of claim 1, further comprising the step of administering a beta-adrenergic blocker, a COX2 inhibitor, or both.

13. The method of claim 12, wherein said further administering is carried out during the perioperative period of a tumor excision surgery.

14. The method of claim 12, wherein the beta-adrenergic blocker and COX2 inhibitor are present within separate pharmaceutical compositions.

15. The method of claim 13, wherein the beta-adrenergic blocker, COX2 inhibitor or both are administered at least one time before the surgery (pre-operative).

16. The method of claim 13, wherein beta-adrenergic blocker, COX2 inhibitor or both are administered at least one time after the surgery (post-operative).

17. The method of claim 13, wherein administering said pharmaceutical composition and further administering of a beta-adrenergic blocker, COX2 inhibitor, or both are carried out on the same days during the perioperative period.

18. The method of claim 13, wherein administering said pharmaceutical composition and further administering of a beta-adrenergic blocker, COX2 inhibitor, or both are carried out on separate days during the perioperative period.

19. The method of claim 12, wherein the beta-adrenergic blocker is selected from the group consisting of acebutolol, atenolol, betaxolol, bisoprolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, metoprolol, nadolol, nebivolol, oxyprenolol, penbutolol, pindolol, propranolol, sotalol, timolol, and pharmaceutically acceptable salts thereof.

20. The method of claim 19, wherein the beta-adrenergic blocker is propranolol or a pharmaceutically acceptable salt thereof.

21. The method of claim 12, wherein the COX2 inhibitor is selected from the group consisting of celecoxib, cimicoxib, etoricoxib, etodolac, eoricoxib, lumiracoxib, meloxicam, parecoxib, rofecoxib, tiracoxib, valdecoxib, and pharmaceutically acceptable salts thereof.

22. The method of claim 21, wherein the COX2 inhibitor is etodolac or a pharmaceutically acceptable salt thereof.