# Patent

## Title
MRI imageable liposomes for the evaluation of treatment efficacy, thermal distribution, and demonstration of dose painting

## Abstract

Methods and compositions useful for detecting an in vivo blood pool, monitoring the distribution of a compound of interest to a desired site in an organism by magnetic resonance imaging, monitoring the accumulation of a compound of interest at a desired site in vivo by magnetic resonance imaging, and monitoring the release of liposomal contents to an external stimulus at a desired site in vivo by magnetic resonance imaging are disclosed. Some compositions comprise envirosensitive or non-sensitive liposomes. Contrast agents, such as manganese-based compounds, are also disclosed.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent application Ser. No. 10/661,977, filed Sep. 11, 2003, which claims the benefit of U.S. Provisional Patent Application Ser. Nos. 60/409,899 filed Sep. 11, 2002 and 60/415,591 filed Oct. 2, 2002; the disclosures of each of which are incorporated herein by reference in their entireties.


GRANT STATEMENT
The presently disclosed subject matter was supported by grants from the Department of Defense Breast Cancer Research Program (DAMD17-03-1-0348) and the National Institutes of Health (CA42745, P4105959, R24 CA092656). Thus, the United States Government has certain rights in the present subject matter.


TECHNICAL FIELD
The methods and compositions disclosed herein generally relate to employing magnetic resonance techniques to target and monitor drug delivery in vivo. More particularly, the disclosure relates to the use of liposomes encapsulating a contrast agent and, in some embodiments, a drug as well, to target and monitor drug delivery to a desired site in vivo wherein the desired site can be characterized by the presence of a non-physiological environmental condition, and monitoring the site utilizing magnetic resonance imaging techniques. The disclosure further relates to blood pool agents, adapted to provide imaging of a site over an extended period of time.
ABBREVIATIONS


    
    
        AP alkaline phosphatase
        CT computerized tomography
        DOX doxorubicin
        ESR electron spin resonance
        HEPES N-2-Hydroxyethylpiperazine-N′-2 ethanesulfonic acid
        HT hyperthermia
        LTSL lysolipid-based temperature sensitive (thermosensitive) liposomes
        MLV multilamellar vesicle
        MR magnetic resonance
        MRI magnetic resonance imaging
        MUGA multiple-gated arteriography
        NMR nuclear magnetic resonance
        NTSL non-temperature sensitive (non-thermally sensitive) liposomes
        PET positron emission tomography
        RES reticuloendothelial system
        RF radio frequency
        SNR signal-to-noise ratio
        TCA trichloroacetic acid
        Te Echo Time
        TI time of inversion
        Tm melting (phase transition) temperature
        Tr repetition time
        T1 spin lattice relaxation time
        T2 spin-spin relaxation time
    
    


BACKGROUND
Medical diagnostic imaging has evolved as an important non-invasive tool for the evaluation of pathological and physiological processes. Presently, nuclear magnetic resonance imaging (“MRI”) and computerized tomography (“CT”) are two of the most widely used imaging modalities. Although both MRI and CT can be performed without the administration of contrast agents, the ability of many contrast enhancement agents to enhance the visualization of internal tissues and organs has resulted in their widespread use.
Proton MRI is based on the principle that the concentration and relaxation characteristics of protons in tissues and organs can influence the intensity of a magnetic resonance image. Contrast enhancement agents that are useful for proton MRI effect a change in the relaxation characteristics of protons, which can result in image enhancement and improved soft-tissue differentiation. Different classes of proton MR imaging agents include paramagnetic metal chelates and nitroxyl spin labeled compounds.
Detection and monitoring of tumor growth and remission is vital for the effective diagnosis and treatment of cancer. Current methods for detecting tumor growth and regression using CT scan, positron emission tomography (“PET”), optical imaging and MRI are limited in their ability to distinguish between normal and tumor tissue. Additionally, the ability to image the vasculature within in a tissue and occlusion of that vasculature finds application in a variety of different scenarios. Vascular occlusion imaging is presently limited by a variety of obstacles, one of which is the inability to selectively image occlusions over other tissues and structures. Further, there is no currently available method of monitoring internal wound healing, nor is there an adequate method of monitoring angiogenic activity in tumor and non-tumor tissue.
The use of envirosensitive liposomes in hyperthermia therapy is a promising approach to targeting a tumor or other tissue. Broadly, envirosensitive liposomes comprise a formulation adapted to lose structural integrity under certain environmental conditions. For example, thermosensitive liposomes lose structural integrity within a given temperature range. Alternatively, the envirosensitive liposome can be a radiation sensitive liposome, which is formulated to lose its structural integrity when contacted with a particular wavelength range of electromagnetic radiation. When an envirosensitive liposome, such as a thermosensitive or a radiation sensitive liposome, loses its structural integrity, the contents of the liposome are released.
One consideration with respect to the use of envirosensitive liposomes as a delivery vehicle (e.g. for a therapeutic compound) is the ability to target the liposomes to a desired location, such as a tumor. Targeting liposomes, both envirosensitive and insensitive species, has been a subject of research for some time. Various approaches have been taken, including the formation of immunoliposomes, which comprise, for example, an antibody or a fragment of an antibody (see, e.g., Sullivan & Huang, (1985) Biochim. Biophys. Acta 812(1): 116-126; Perlaky et al., (1996) Oncol. Res. 8(9): 363-369).
Liposome targeting, however, depends in part on how stably the liposomes administered to a subject circulate through the circulatory system in the normal physiological environment and how effectively the liposomes release their content (e.g. a drug) at a particular desired site (e.g. a tumor). In the case of some thermosensitive liposomes targeting has been problematic. For example, the liposomes described by Yatvin et al. (Yatvin et al., (1978) Science 202: 1290) release only a small amount of the drug at the temperature of hyperthermia. Other liposomes have been observed to release the drug at a temperature lower (e.g., 37-39° C.) than that typically reached in an approach employing hyperthermia (Bassett et al., (1986) J. Urol. 135(3): 612-615; Needham et al., (2000) Cancer Res. 60(5): 1197-1201).
However, an approach for monitoring the release of liposome contents once an envirosensitive liposome has lost structural integrity has not been disclosed in the art. Therefore, absent a method of monitoring liposome opening and content release, a clinician or researcher must assume that the liposome was delivered to the desired site, that it ruptured and released its contents and that the contents were delivered to the desired site.
Thus, what is needed is an envirosensitive liposome, for example a thermosensitive liposome composition that exhibits a desirable phase transition at the typical temperature of hyperthermia (39-45° C.), or a radiation sensitive liposome that exhibits a desirable phase transition when contacted by a particular wavelength range of electromagnetic radiation (e.g., ionizing radiation). Further, what is needed is an envirosensitive liposome that is adapted to entrap a drug at a high concentration for long periods of time when maintained at physiological conditions, for example a temperature lower than that of hyperthermia for thermosensitive liposomes, and that is adapted to reliably release the drug efficiently at a desired site in a very short time after a particular environmental stimulation, for example at the temperature of hyperthermia or higher for thermosensitive liposomes. Such an envirosensitive liposome composition would also be adapted to be tracked to a desired location, wherein content delivery could be monitored in vivo by a non-invasive method.
What is also needed is a non-invasive method of monitoring tumor growth or regression, vascular morphology, vascular occlusion formation and dissolution, angiogenesis and wound healing in a subject that offers superior sensitivity, relative to the currently available methods. These and other problems are addressed by the compositions and methods disclosed herein.
SUMMARY
In one aspect of the presently disclosed methods and compositions, a method of predicting efficacy of a treatment in a subject is disclosed. In one embodiment, the method comprises monitoring accumulation of a compound of interest at a desired site in vivo by magnetic resonance imaging and predicting efficacy of treatment based on accumulation of a compound of interest at the desired site. In some embodiments, the method comprises administering to a subject a liposome composition comprising a contrast agent, a compound of interest, and a non-sensitive liposome or an envirosensitive liposome encapsulating the contrast agent and the compound of interest; and monitoring the accumulation of the compound of interest at the desired site by magnetic resonance imaging. In some embodiments, predicting efficacy comprises predicting efficacy of treatment based on a location of accumulation at the desired site, a rate of accumulation at the desired site, or both location and rate of accumulation at the desired site.
In another aspect of the presently disclosed methods and compositions, a method of enhancing efficacy of a treatment at a desired site in a subject is disclosed. In one embodiment, the method comprises administering to the subject a composition comprising a compound of interest and targeting the composition to a desired location at a desired site in the subject, at a desired rate of accumulation at the desired site, or both a desired location and desired rate of accumulation at the desired site, to thereby enhance efficacy of treatment provided by the compound of interest. In some embodiments, the composition comprises a liposome composition comprising the compound of interest and a non-sensitive liposome or an envirosensitive liposome encapsulating the compound of interest. In some embodiments, the composition further comprises a contrast agent. In some embodiments, a non-physiological environmental condition is present at the desired site, and the composition is targeted to a desired location at the desired site in the subject, at a desired rate of accumulation at the desired site, or both a desired location and desired rate of accumulation at the desired site by the presence of the non-physiological environmental condition. In some embodiments, the desired site is exposed to a non-physiological environmental condition before, after, or both before and after administering the composition. Further, in some embodiments, the method comprises administering the composition in one or more partial doses before, after, or both before and after the desired site is exposed to a non-physiological environmental condition. In some embodiments, the method further comprises predicting efficacy of treatment based on a location of accumulation at the desired site, a rate of accumulation at the desired site, or both the location and the rate of accumulation at the desired site.
In another aspect of the presently disclosed methods and compositions, a method of targeting delivery of a compound of interest at a desired site in vivo is provided. In one embodiment, the method comprises administering to a subject a composition comprising a compound of interest, wherein a non-physiological environmental condition is present at the desired site, and the composition is targeted to a desired location at the desired site in the subject, at a desired rate of accumulation at the desired site, or both a desired location and desired rate of accumulation at the desired site by the presence of the non-physiological environmental condition. In some embodiments, the composition comprises a liposome composition comprising the compound of interest and a non-sensitive liposome or an envirosensitive liposome encapsulating the compound of interest. In some embodiments, the composition further comprises a contrast agent. In some embodiments, the desired site is exposed to a non-physiological environmental condition before, after, or both before and after administering the composition. Further, in some embodiments, the method comprises administering the composition in one or more partial doses before, after, or both before and after the desired site is exposed to a non-physiological environmental condition. In some embodiments, the method further comprises predicting efficacy of treatment based on a location of accumulation at the desired site, a rate of accumulation at the desired site, or both the location and the rate of accumulation at the desired site.
In still another aspect of the presently disclosed methods and compositions, a method of monitoring the accumulation of a compound of interest at a desired site in vivo by magnetic resonance imaging is disclosed. In one embodiment, the method comprises: (a) administering to a subject a non-sensitive liposome composition comprising: (i) a contrast agent; (ii) a compound of interest; and (iii) a non-sensitive liposome encapsulating the contrast agent and the compound of interest; and (b) monitoring the accumulation of the compound of interest at the site of interest by magnetic resonance imaging. In one embodiment, the method comprises increasing blood flow to the desired site. Increasing the blood flow can comprise mechanical stimulation, ultrasound therapy and/or heating the desired site.
In another aspect of the presently disclosed methods and compositions, an in vivo method of monitoring the localization and distribution of a compound of interest to a desired site in an organism by magnetic resonance imaging is disclosed. In one embodiment, the method comprises: (a) administering an envirosensitive liposome composition to the subject, the composition comprising: (i) a contrast agent; (ii) a compound of interest; and (iii) an envirosensitive liposome encapsulating the contrast agent and the compound of interest; and (c) monitoring the distribution of the compound of interest to the desired site by magnetic resonance imaging. In one embodiment, the method comprises increasing blood flow to the desired site.
In yet another aspect, a method of detecting an in vivo blood pool is disclosed. In one embodiment, the method comprises: (a) administering a liposome composition to a subject, the composition comprising: (i) a contrast agent; and (ii) a liposome encapsulating the contrast agent; (b) generating a magnetic resonance image of a site of interest; and (c) detecting the presence of an in vivo blood pool by analyzing the magnetic resonance image.
In a further aspect, a method of monitoring the accumulation of a compound of interest at a desired site in vivo by magnetic resonance imaging is disclosed. In one embodiment, the method comprises: (a) administering to a subject an envirosensitive liposome composition comprising: (i) a contrast agent; (ii) a compound of interest; and (iii) an envirosensitive liposome encapsulating the contrast agent and the compound of interest; and (b) monitoring the accumulation of the compound of interest at a desired site by magnetic resonance imaging.
In another aspect, a method of generating a heating profile of a site of interest is disclosed. In one embodiment, the method comprises: (a) administering to a subject a thermosensitive liposome composition comprising: (i) a contrast agent; and (ii) a thermosensitive liposome encapsulating the contrast agent and the compound of interest and having a melting temperature Tm; (b) heating a site of interest in a subject; (c) monitoring release of the contrast agent from the thermosensitive liposome using magnetic resonance imaging; and (d) generating a heating profile of the site of interest, wherein heating of an area to a temperature of at least Tm is indicated by release of contrast agent, preferably at a periphery of the area.
In the methods disclosed herein, heating can be, for example, provided by a natural process or by a method selected from the group consisting of contacting a heated material with the site of interest, applying electromagnetic energy that heats the desired site, such as, for example microwave or radio frequency energy to the site, applying ultrasonic energy to the site and applying a laser beam to the site. A site of interest can be selected from the group consisting of, for example, a tumor, injury site, and tissue edema.
In the methods disclosed herein, an envirosensitive liposome can comprise, for example, a thermosensitive liposome, a pH-sensitive liposome, a chemosensitive liposome and a radiation-sensitive liposome.
In the methods disclosed herein, a non-sensitive liposome can comprise, for example, DSPC/Cholesterol (55:45, mol:mol). A thermosensitive liposome can comprise, for example, a formulation selected from the group consisting of DPPC-PEG2000, DPPC-DSPE-PEG2000 (95:5, mol:mol); DPPC-MSPC-DSPE-PEG2000 (90:10:4, mol:mol).
In the methods disclosed herein, a contrast agent can be any paramagnetic compound. For example, a contrast agent can comprise a element selected from the group consisting of Gd, Cu, Cr, Fe, Co, Er, Ni, Eu, Dy, Zn, Mg, Mo, Li, Ta, and Mn.
In the methods disclosed herein, various compounds of interest can be employed in the methods of the present invention, and a compound of interest can be, for example, a chemotherapeutic agent.
Accordingly, it is an object to provide MRI imageable liposomes for the evaluation of treatment efficacy, treatment enhancement, thermal distribution, and/or dose painting. This and other objects are achieved in whole or in part by the presently disclosed methods and compositions.
An object of the disclosure having been stated hereinabove, other objects will be evident as the description proceeds, when taken in connection with the accompanying Examples and Drawings as best described hereinbelow.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a plot depicting the dependency of signal on Tr for a non-sensitive liposome-DOX-MnSO4 (temperature=29.93° C.) formulation at various concentrations. In this plot, solid squares represent water alone, circles represent formulations comprising 0.25 mM DOX, solid triangles represent formulations comprising 0.5 mM DOX, solid inverted triangles represent formulations comprising 1.0 mM DOX, solid diamonds represent formulations comprising 2.0 mM DOX and crosses represent formulations comprising 4.0 mM DOX.
FIG. 1B is a plot depicting the relationship between 1/T1 and contrast agent concentration for the same formulations described in FIG. 1A. The slope of the line is a measure of the (T1) relaxivity of the system.
FIG. 1C is a plot depicting the effect of temperature on relaxivity. In this figure inverted solid triangles represent thermosensitive liposomes loaded with contrast agent in the exterior buffer; solid circles represent free contrast agent; stars represent free contrast agent and DOX at a concentration ratio identical to that found in the liposome; and solid triangles represent non-sensitive liposomes loaded with DOX and also represent empty non-sensitive liposomes.
FIG. 1D is a plot depicting the effect of temperature on relaxivity of thermosensitive liposomes as the composition is heated and cooled. In this figure, open squares represent thermosensitive liposomes and closed circles represent thermosensitive liposomes loaded with DOX. Heating and cooling operations are indicated and progress from 30° C. to 45° C. and back to about 32° C. are depicted.
FIG. 2A is a time course of MR images depicting the accumulation of non-sensitive liposomes at a flank tumor in a rat. Panel 0 corresponds to time T=0 minutes (pre-injection); panel 1 corresponds to time T=5 minutes; panel 2 corresponds to time T=30 minutes; and panel 3 corresponds to time T=90 minutes.
FIG. 2B is a time course of MR images depicting the accumulation of thermosensitive liposomes at a flank tumor in a rat. Panel 0 corresponds to time T=0 minutes (pre-injection); panel 1 corresponds to time T=5 minutes; panel 2 corresponds to time T=30 minutes; and panel 3 corresponds to time T=90 minutes.
FIG. 2C is a time course of MR images depicting the accumulation of gadolinium-DTPA at a flank tumor in a rat. Panel 0 corresponds to time T=0 minutes (pre-injection); panel 1 corresponds to time T=1 minutes; panel 2 corresponds to time T=5 minutes; and panel 3 corresponds to time T=15 minutes.
FIG. 2D is a time course of MR images depicting the accumulation of non-sensitive liposomes at a flank tumor in a rat without hyperthermia treatment. Panel 0 corresponds to time T=0 minutes (pre-injection); panel 1 corresponds to time T=5 minutes; panel 2 corresponds to time T=30 minutes; and panel 3 corresponds to time T=90 minutes.
FIG. 2E is a time course of MR images depicting the accumulation of thermosensitive liposomes at a flank tumor in a rat without hyperthermia treatment. Panel 0 corresponds to time T=0 minutes (pre-injection); panel 1 corresponds to time T=5 minutes; panel 2 corresponds to time T=30 minutes; and panel 3 corresponds to time T=90 minutes.
FIG. 3A is a MR image depicting several regions of interest (highlighted by white boxes) in which non-sensitive and thermosensitive liposome signal intensities were measured.
FIG. 3B is a plot depicting signal intensity for non-sensitive liposomes as a function of time for the regions highlighted in FIG. 3A. Solid squares correspond to signal detected in non-heated muscle; solid circles correspond to signal detected in an enhanced tumor region; solid triangles correspond to signal detected in a non-enhanced tumor region; and solid inverted triangles correspond to signal detected in the vessel.
FIG. 3C is a plot depicting signal intensity ratio as a function of time for the non-sensitive liposomes. Solid squares correspond to the signal intensity ratio in an enhanced tumor region; solid circles correspond to the signal intensity ratio in a nonenhanced tumor region; and solid triangles correspond to the signal intensity ratio in the vessel.
FIG. 3D is a plot depicting signal intensity ratio as a function of time as acquired from the femoral vein of the rat. Solid circles correspond to non-sensitive liposomes; solid triangles correspond to thermosensitive liposomes and open squares correspond to gadolinium-DPTA.
FIG. 3E is a plot depicting signal intensity ratio as a function of time in enhanced regions of the tumor. Solid circles correspond to non-sensitive liposomes; solid triangles correspond to thermosensitive liposomes; and open squares correspond to gadolinium-DPTA.
FIG. 3F is a plot depicting signal intensity ratio as a function of time for a nonenhanced tumor region. Solid circles correspond to non-sensitive liposomes; solid triangles correspond to thermosensitive liposomes; and open squares correspond to gadolinium-DPTA.
FIG. 4 is a schematic depicting of an exemplary temperature-sensitive liposome containing doxorubicin (Dox) and contrast agent (manganese sulfate, MnSO4). Doxorubicin is actively loaded and retained in temperature-sensitive liposomes by using a pH gradient-driven loading protocol that includes MnSO4. The phospholipid bilayer undergoes a main melting phase transition at 41° C. Lysolipids can form many stable pores at the transition temperature, enabling release of contents. Polyethylene glycol (PEG) can be grafted onto lipids to help liposomes evade immune recognition. Note that only one pore is shown. DPPC=dipalmitoyl phosphocholine. MSPC=monostearoyl phosphocholine.
FIGS. 5A-5E show tumor drug distribution after administration of doxorubicin- and manganese-containing lysolipid-based temperature-sensitive liposomes (Dox/Mn-LTSLs) and hyperthermia (HT) by three different schedules. FIGS. 5A-5C show axial pelvic magnetic resonance images show rats bearing flank fibrosarcomas (top left). Radial lines in FIG. 5A show the orientations of doxorubicin concentration profiles in FIGS. 5D and 5E. LTSLs administered during steady-state hyperthermia resulted in peripheral enhancement (liposome content release; white) at the edge of the tumor (FIG. 5A); LTSLs administered before hyperthermia resulted in central enhancement (FIG. 5B); and LTSLs administered in two equal doses, half before hyperthermia, and the remainder after steady-state hyperthermia was reached, resulted in uniform enhancement (FIG. 5C). FIG. 5D shows T1-based mean tumor doxorubicin concentration (ng/mg tissue) after treatment with LTSLs during hyperthermia, shown as a function of the normalized tumor radius for each rat. The bold profile is for the tumor shown in FIG. 5A. Mean values are for 80 line profiles from each rat. FIG. 5E shows mean doxorubicin concentration (ng/mg tissue) profiles for each of the three therapeutic groups as a function of the normalized tumor radius (n=6-7 rats per group). Vertical lines in FIGS. 5D and 5E correspond to 95% confidence intervals.
FIG. 6 depicts total amount of doxorubicin delivered to whole tumor as a function of time for three different protocols of lysolipid-based temperature-sensitive liposomes (LTSLs) plus hyperthermia (HT), calculated using magnetic resonance images. Mean amounts of doxorubicin are shown for each group (n=6-7 animals per group); vertical lines represent 95% confidence intervals (staggered for clarity). The zero timepoint marks the image acquired just before both LTSL and HT were present. The line for the LTSL during HT group ends 15 minutes earlier than the lines for the other two groups because hyperthermia was started 15 minutes before time zero (injection). For the other two groups, hyperthermia was initiated at time zero.
FIGS. 7A-7C depict tumor doxorubicin concentration and antitumor effect for various therapeutic protocols. FIG. 7A shows overall tumor doxorubicin concentration (ng/mg) as measured by high performance liquid chromatography after treatment with one of the following (n=3 rats per group): saline only (control); hyperthermia (HT) alone; free doxorubicin alone (free Dox); free doxorubicin during hyperthermia (free Dox+HT); doxorubicin- and manganese sulfate-containing lysolipid-based temperature-sensitive liposomes (Dox/Mn-LTSL) alone; Dox/Mn-LTSL administered before hyperthermia (Dox/Mn-LTSL before HT); Dox/Mn-LTSL administered during hyperthermia (Dox/Mn-LTSL during HT); and Dox/Mn-LTSL administered 15 minutes before initiation of hyperthermia and after thermal steady state was reached (Dox/Mn-LTSL split dose). Saline and doxorubicin were administered intravenously (the therapeutic dose was 5 mg doxorubicin/kg body weight) and local hyperthermia was administered for a total of 1 hour. Tumors were harvested 90 minutes after intravenous injection (comparable to the time that the final magnetic resonance images were taken). Mean values are shown for each group; error bars correspond to 95% confidence intervals. FIG. 7B shows rat fibrosarcoma growth time for each therapeutic group (n=6-7), measured as the median number of days to five times the original tumor volume (error bars correspond to 95% confidence intervals). FIG. 7C is a Kaplan-Meier plot showing cumulative fraction of animals with tumor volume less than five times the treatment volume for each group over time.
FIG. 8 depicts a scatter plot of tumor response as a function of overall tumor doxorubicin concentration ([Dox]) for individual rats treated with lysolipid-based temperature-sensitive liposomes (LTSL) plus hyperthermia (HT). Rats were treated on three different schedules: LTSL before HT (X; n=6), LTSL during HT (Δ; n=7), or LTSL before and during HT (LTSL split dose (□); n=6). Response was defined as the number of days for tumors to reach to five times their original volume and was censored at 60 days. Tumor [Dox] (in ng of doxorubicin/mg of tumor) was calculated from magnetic resonance images acquired before and after therapy.



DETAILED DESCRIPTION
The presently disclosed subject matter pertains in part to selected delivery of therapeutic agents, validated (qualitatively and quantitatively) by MRI, through the selective application of hyperthermia, and/or other non-physiological environmental conditions. Further, evaluation of drug distribution (qualitatively and quantitatively) can be used to predict treatment efficacy. The presently disclosed compositions can also be used to validate temperature distribution in target tissues based on release profiles. In addition, a drug can be targeted to a desired site within a target tissue using selective application of a non-physiological environmental condition.
In some embodiments the presently disclosed subject matter provides methods of quantitatively monitoring the accumulation of a compound in vivo by magnetic resonance imaging. Liposome compositions are provided, which comprise a contrast agent (for example gadolinium- or manganese-based compounds) and a therapeutically active compound of interest. Once this liposome composition is administered to a subject, the release from the liposome and accumulation of the compound can be monitored by magnetic resonance imaging, enabling the real time localization and distribution of the compound to a specific site to be imaged.
Envirosensitive liposomes (e.g., thermally-sensitive, pH-sensitive, chemosensitive, or radiation-sensitive liposomes) can be prepared and employed in selective tissue targeting. Non-thermally sensitive liposomes can be used to act as a blood pool agent, to identify tumors and assess uniformity of tissue perfusion. Imageable liposomes can also be used for temperature measurements during hyperthermia treatment.
The presently disclosed subject matter can provide non-invasive measurement of drug distribution in real time. Qualitative as well as quantitative drug distribution can be assessed. Any desired therapeutic agent can be encapsulated into the presently disclosed liposomes. Selective delivery of a therapeutic drug can be provided, in some embodiments, by sensing, for example, inherent or imposed environmental variation within a tissue of interest. Local hyperthermia is a representative example of an environmental variation.
The presently disclosed subject matter can also provide the ability to monitor and/or predict in vivo concentration distributions, which can further provide for improvement in treatments. The presently disclosed subject matter can impact clinical treatment by providing individualized monitoring of tissue drug concentration distributions, allowing for modification of treatment variables (e.g., selective application of non-physiological environmental conditions) to improve the uniformity or selective targeting of drug delivery. The presently disclosed methods and compositions can provide individualized treatment, which in some applications can increase overall treatment efficacy.
I. Definitions
Following long-standing patent law convention, the terms “a” and “an” mean “one or more” when used in this disclosure, including the claims.
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 ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
As used herein, the term “blood pool” means a localized volume of blood. A blood pool can arise from a normally occurring phenomenon, such as pooling of blood in a subject's heart. A blood pool can also arise from an unnatural vascular condition, such as an aneurysm. The blood in a blood pool can be circulating, prevented from circulating or circulating to some degree. Generally, then, a blood pool is a localized concentration of blood, and a blood pool can comprise any volume of blood.
As used herein, the term “detecting” means confirming the presence of a target entity (which can be a biological structure, such as a vascular blockage, vascular damage or an occlusion) by observing the occurrence of a detectable signal, such as a radiologic or spectroscopic signal or a feature of an image generated by magnetic resonance that will appear exclusively in the presence of the target entity.
As used herein, the term “envirosensitive liposome” means a liposome formulated using physiologically compatible constituents, such as, but not limited to, dipalmitoylphosphatidyl-choline and dipalmitoylphosphatidyl-glycerol phospholipids, which permit preparation of liposomes using art-recognized techniques that are formulated to lose structural integrity and release their contents under specific environmental conditions. The specific environmental conditions under which a particular envirosensitive liposome loses its structural integrity are variable and dependent upon the formulation of the particular liposome. Typically, the environmental conditions differ from normal physiological conditions. For example, thermosensitive liposomes can be formulated to release their contents at temperatures higher than normal mammalian body temperature. Alternatively, radiation-sensitive liposomes can be formulated to release their contents when they interact with electromagnetic radiation within a particular wavelength range, such as x-rays, or other ionizing radiation. While these examples are both categorized as envirosensitive liposomes, as the term is used herein, they are not necessarily structurally vulnerable to the same environmental conditions. For example, a thermosensitive liposome may not lose structural integrity when contacting x-rays, and vice versa for a radiation-sensitive liposome held at a particular temperature. However, envirosensitive liposomes having overlapping environmental sensitivities, for example thermal and pH sensitivities, can also be formulated, and are included within the term “envirosensitive liposome,” as used herein. One of skill in the art will readily recognize and be able to formulate without undue experimentation other types of envirosensitive liposomes, and these formulation are also encompassed by the term as used herein. Non-limiting examples of envirosensitive liposomes include thermosensitive liposomes, radiation-sensitive liposomes, pH-sensitive liposomes, acoustic (e.g. ultrasound)-sensitive liposomes, antigen-sensitive liposomes (e.g. liposomes having recognition molecules, for example, antibodies or antibody fragments (see, e.g., Sullivan & Huang, (1985) Biochim. Biophys. Acta 812(1): 116-126; Perlaky et al., (1996) Oncol. Res. 8(9): 363-369), incorporated into the membrane such that contact of the recognition molecule with its antigen results in loss of structural integrity of the liposome through, for example, a conformational change in the recognition molecule) and chemosensitive liposomes (e.g. liposomes sensitive to particular chemical agents). The present disclosure encompasses the use of envirosensitive liposomes in some embodiments that are less than about 400 nm in diameter, such as, for example liposomes having a diameter of about 200 nm, about 120 nm, 70 nm, 60 nm, or about 50 nm in diameter to facilitate MRI visualization, handling, administration, unhindered progress through mammalian vasculature, and minimize side effects, e.g., interference with the mammalian blood clotting cascade.
As used herein, the term “hyperthermia” means the elevation of the temperature of a subject's body, or a part of a subject's body, compared to the basal temperature of the subject. Such elevation can be the result of a natural process (such as inflammation) or artificially induced for therapeutic or diagnostic purposes. In mammals, a basal body temperature is ordinarily maintained due to the thermoregulatory center in the anterior hypothalamus, which acts to balance heat production by body tissues with heat loss. “Hyperthermia” refers to the elevation of body temperature above the hypothalamic set point due to insufficient heat dissipation. In contrast to hyperthermia, “fever” refers to a systemic elevation of body temperature due to a change in the thermoregulatory center. The overall mean oral temperature for a healthy human aged 18-40 years is 36.8.±0.4° C. (98.2±0.7° F.). See, e.g., Harrison's Principles of Internal Medicine (Fauci et al., eds.) 14th Ed. McGraw-Hill, New York, p. 84 (1998).
As used herein, the term “inner transition elements” means those elements known as lanthanide (or rare earth) and actinide elements. Inner transition elements are also known as f-block transition elements.
As used herein, the term “liposome” means a generally spherical cluster or aggregate of amphipathic compounds, including lipid compounds, typically in the form of one or more concentric layers, for example, bilayers.
As used herein, the term “radiation-sensitive liposome” means a liposome formulated using physiologically compatible constituents, such as, but not limited to, dipalmitoylphosphatidyl-choline and dipalmitoylphosphatidyl-glycerol phospholipids, which permit preparation of liposomes using art-recognized techniques that are formulated to lose structural integrity and release their contents when interacting with electromagnetic radiation having a specific wavelength range. The specific wavelength range under which a particular radiation-sensitive liposome loses its structural integrity is variable and dependent upon the formulation of the particular liposome. For the purposes of example but not limitation, a liposome can be formulated to lose structural integrity and release its contents when interacting with x-rays, that is electromagnetic radiation having a wavelength in the range of about 1×10−11 m to about 1×10−8 m, but not when interacting with radiation having a greater or lesser wavelength. In other embodiments, the wavelength sensitivity may include a different range, or encompass x-rays in a broader range, such as, for example broad sensitivity to all ionizing radiation. The present disclosure encompasses the use of radiation-sensitive liposomes that are less than about 400 nm in diameter, such as, for example liposomes having a diameter of about 200 nm, about 120 nm, 70 nm, 60 nm, or about 50 nm in diameter to facilitate MRI visualization, handling, administration, unhindered progress through mammalian vasculature, and minimize side effects, e.g., interference with the mammalian blood clotting cascade.
As used herein, the term “relaxivity” means the slope of the line drawn between points on a plot of 1/T1 or 1/T2 against contrast agent concentration.
As used herein, the term “subject” means any organism. The term need not refer exclusively to a human being, one example of a subject, but can also refer to animals such as mice, rats, dogs, poultry, and Drosophila and even tissue cultures. The methods disclosed herein are particularly useful in the treatment and diagnosis of warm-blooded vertebrates. Thus, the disclosure concerns mammals and birds. More particularly, provided is the treatment and/or diagnosis of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (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, kept in zoos, as well as fowl, and more particularly domesticated fowl, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans. Thus, provided is the treatment of livestock, including, but not limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.
As used herein, the term “T1” means longitudinal relaxation time, and is also known as the spin lattice relaxation time (1/T1 is a rate constant, R1, the spin-lattice relaxation rate constant).
As used herein, the term “T2” means transverse relaxation time, which arises, in part, from spin-spin relaxation mechanisms. 1/T2 is also a rate constant, R2, the spin-spin relaxation rate constant.
As used herein, the term “non-sensitive liposome” means a liposome formulated using physiologically compatible constituents, such as dipalmitoylphosphatidyl-choline and dipalmitoylphosphatidyl-glycerol phospholipids and cholesterol, that permits preparation of liposomes using art-recognized techniques that do not release their contents as a result of specific environmental stimulation, such as hyperthermic conditions, pH variance, or interaction with electromagnetic radiation. For example, in contrast to thermosensitive liposomes, non-sensitive liposomes (in this case also referred to as non-thermally sensitive liposomes) do not release their contents due to hyperthermic stimulation, such as at temperatures less than about 15 degrees higher than basal mammalian body temperature, i.e., above about 37° C. The present disclosure encompasses the use of non-sensitive liposomes that are less than about 400 nm in diameter, such as, for example liposomes having a diameter of about 200 nm or about 120 nm in diameter to facilitate handling, administration, unhindered progress through mammalian vasculature, ability to target damaged or malformed vasculature, and minimize side effects, e.g., interference with the mammalian blood clotting cascade.
As used herein, the term “thermosensitive liposome” means a liposome formulated using physiologically compatible constituents, such as for example dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol phospholipids, that permits preparation of liposomes using art-recognized techniques that release their contents at temperatures at least about 3 degrees higher than about 37° C. (normal mammalian body temperature). Upon exposure to temperatures at least about 3° C. above basal mammalian body temperature, release of liposome contents occurs by leakage or seepage or by actual lysis (complete or incomplete) of the liposomes. The present disclosure encompasses the use of thermosensitive liposomes that are less than about 400 nm in diameter, such as, for example liposomes having a diameter of about 200 nm, about 120 nm, 70 nm, 60 nm, or about 50 nm in diameter to facilitate MRI visualization, handling, administration, unhindered progress through mammalian vasculature, and minimize side effects, e.g., interference with the mammalian blood clotting cascade.
As used herein, the term “transition elements” means those elements found in columns IIIB, IVB, VB, VIIB, VIIIB IB and IIB of the Periodic Table of Elements. Transition elements are also known as d-block elements.
II. General Considerations
One aspect of the present disclosure involves magnetic resonance-based techniques in general, and magnetic resonance imaging techniques in particular. The magnetic resonance imaging techniques employed herein are known and are described, for example, in Kean & Smith, (1986) Magnetic Resonance Imaging: Principles and Applications, Williams and Wilkins, Baltimore, Md. U.S.A. Representative MR techniques include, but are not limited to, magnetic resonance imaging (“MRI”), nuclear magnetic resonance (“NMR”) and electronic spin resonance (“ESR”).
Standard MR equipment, conditions and techniques can be used to generate images. Appropriate equipment, conditions and techniques can be determined in the course of experimental design. When in vivo MRI experiments are performed in the context disclosed herein, they can be performed on any suitable MRI instrument, such as a 1.5 Tesla or higher whole-body scanner.
According to known principles, nuclei with appropriate spin, when placed in an applied magnetic field (Bo, expressed generally in units of Tesla) align in the direction of the field. In the case of protons, these nuclei precess at a frequency, f, of 42.6 MHz, at a field strength of 1 Tesla (T). At this frequency, an RF pulse of radiation will excite the nuclei and can be considered to tip the net magnetization out of the field direction, the extent of this rotation being determined by the pulse energy (which can be obtained by integrating a time×amplitude curve). After the RF pulse, the nuclei “relax” or return to equilibrium with the magnetic field, emitting radiation at the resonant frequency. The decay of the emitted radiation is characterized by two relaxation times, i.e., T1, the spin-lattice relaxation time or longitudinal relaxation time, that is, the time taken by the nuclei to return to equilibrium along the direction of the externally applied magnetic field, and T2, the spin-spin relaxation time associated with the dephasing of the initially coherent precession of individual proton spins. These relaxation times have been established for various fluids, organs and tissues in different species of mammals.
MRI is a diagnostic and research procedure that uses a large, high-strength magnet and radio frequency signals to produce images. The most abundant molecular species in biological tissues is water. It is the quantum mechanical “spin” of the water proton nuclei that ultimately gives rise to the signal in standard imaging experiments. Other nuclei can be employed in MRI applications, however low signal-to-noise (S/N) ratios are a consideration in these applications. In an MRI experiment, the sample to be imaged is placed in a strong static magnetic field (on the order of 1-12 Tesla) and the spins are excited with a pulse of radio frequency (“RF”) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act on the spins to code spatial information into the recorded signals. The basic MRI experiment can be described, in one frame of reference, as follows. Pre-RF pulse spins can be thought of as collectively aligned along the Z-axis of a Cartesian coordinate system; application of one or a sequence of RF pulses “tip” the spins into the X-Y plane, from which position they will spontaneously relax back to the Z-axis. The relaxation of the spins is recorded as a function of time. Using this basic experiment, MRI is able to generate structural information in three dimensions in a relatively short period of time.
By applying magnetic field gradients so that the magnitude of the magnetic field varies with location inside the subject-receiving space characteristics of the magnetic resonance signals from different locations within the region, such as the frequency and phase of the signals, can be made to vary in a predictable manner depending upon position within the region. Thus, the magnetic resonance signals are “spatially encoded” so that it is possible to distinguish between signals from different parts of the region. After repeating this procedure with various different gradients, it is possible to derive a map showing the intensity or other characteristics of the magnetic resonance signals versus position within the excited region. Because these characteristics vary with concentration of different chemical substances and other characteristics of the tissue within the subject's body, different tissues provide different magnetic resonance signal characteristics. When the map of the magnetic resonance signal characteristics is displayed in a visual format, such as on screen or on a printed image, the map forms a visible picture of structures within the patient's body.
Two characteristic relaxation times are implicated in magnetic relaxation, the basis for MRI. T1 is defined as the longitudinal relaxation time, and is also known as the spin lattice relaxation time (1/T1 is a rate constant, R1, the spin-lattice relaxation rate constant). T2 is known as the transverse relaxation time, or spin-spin relaxation mechanism, which is one of several contributions to T2 (1/T2 is also a rate constant, R2, the spin-spin relaxation rate constant). T1 and T2 have inverse and reciprocal effects on image intensity, with image intensity increasing either by shortening the T1 or lengthening the T2.
In another aspect, the presently disclosed subject matter involves the use of the technique commonly referred to as “hyperthermia”. Hyperthermia, generally, is a technique for locally heating a site of interest to a temperature above normal body temperature. Hyperthermia is an established technique and forms the basis of several therapeutic regimens. For example, typical localized-hyperthermia temperatures required for therapeutic treatment of cancer are in the 42.5-45° C. range, which is maintained for approximately 30 to 60 minutes. Healthy tissue, however, should be kept at temperatures below 42.5° C. during the treatment. For targeted chemotherapy drug delivery, temperatures in the range of about 40 to 45° C. have been demonstrated to be effective on tumors. The presently disclosed subject matter, however, provides methods for using thermosensitive liposomes that can destabilize and release their contents at temperatures above basal temperature and below 42.5° C., thereby avoiding damage to healthy tissue.
In another aspect of the presently disclosed subject matter, a composition (e.g., an envirosensitive or non-sensitive liposome composition) can be introduced into a biological structure disposed in a subject. The mode of administration of a composition to a sample or subject can determine the sites and/or cells in the organism to which an agent will be delivered. The compositions can be administered in admixture with a pharmaceutical diluent (e.g., a buffer) selected with regard to the intended route of administration and standard pharmaceutical practice. The compositions can be injected into a subject parenterally, for example, intra-arterially or intravenously. For parenteral administration, a preparation can be used, e.g., in the form of a sterile, aqueous solution; such a solution can contain other solutes, including, but not limited to, salts or glucose in quantities that will make the solution isotonic. In another aspect, a composition can be injected directly into a tumor. In this aspect, the preparation will be injected in accordance with the above guidelines.
When a composition is administered to humans, the supervising physician or clinician will ultimately determine the appropriate dosage for a given human subject, and this can be expected to vary according to the weight, age and response of the individual as well as the nature of the patient's condition.
III. Contrast Agents
Paramagnetic contrast agents serve to modulate tissue (or intrinsic) T1 and/or T2 values, and are typically designed with regard to a given metal nucleus, which is usually selected based on its effect on relaxation. The capacity to differentiate between regions or tissues that can be magnetically similar but histologically different is a major impetus for the preparation of these agents. Paramagnetic contrast agents provide additional image contrast, and thus enhanced images, of those areas where the contrast agent is localized. For example, contrast agents can be injected into the circulatory system and used to visualize vascular structures and abnormalities (see, e.g., U.S. Pat. No. 5,925,987), or even intracranially to visualize structures of the brain.
The measured relaxivity of the contrast agent is dominated by the selection of the metal atom. Paramagnetic metal ions, as a result of their unpaired electrons, act as potent relaxation enhancement agents. They decrease the T1 relaxation times of nearby spins, exhibiting an r6 dependency, where r is the distance between the two nuclei. Some paramagnetic ions decrease the T1 without causing substantial line broadening, for example copper(II) (“Cu(II)”), zinc(II) (“Zn(II)”), gadolinium(III) (“Gd(III)”) and manganese(II) (“Mn(II), while others induce drastic line broadening, for example, superparamagnetic iron oxide. The mechanism of T1 relaxation is generally a through-space dipole-dipole interaction between the unpaired electrons of a metal atom with an unpaired electron (the paramagnet) and those water molecules not coordinated to the metal atom that are in fast exchange with water molecules in the metal's inner coordination sphere.
When designing or selecting a liposome composition according to the present disclosure, an appropriate paramagnetic ion can be selected as a contrast agent. Any compound that affects the recovery of the magnetic moment of the water protons to the magnetic field, thereby reducing the T1 and T2 relaxation times of an object of interest is suitable for use as a contrast agent with the methods and compounds disclosed herein. Some example metal ions suitable for use include, but are not limited to, the transition, lanthanide and actinide elements. For example, the metal ion is selected from the group consisting of Gd(III), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II), Ni(II), Eu(III), Dy(III), Zn(II), Mg(II), Mo(III), Mo(VI), Li(I), Ta(V), Mn(II), and chelated forms thereof.
IV. Liposome Compositions
Drug delivery systems have been developed in which a drug-entrapping liposome composition is intravenously administered and delivered to a particular target site in the subject's body (see, e.g., Gregoriadis et al.; (1980) Receptor-mediated Targeting of Drugs, Plenum Press, New York, pp. 243-266). A requirement of such systems is that the liposome composition, after being intravenously or intra-arterially administered, should stably circulate along with blood in the subject's body for a longer period of time than provided by conventional systems.
Liposomes are generally not very stable in blood due to interactions between the liposomes' membrane component lipid and blood components such as lipoprotein. Also, intravenously and intra-arterially administered liposomes are sometimes recognized as a foreign substance by the reticuloendothelial system (RES) and are thus likely to be removed from the blood, due to the liposomes' physical morphology and biochemical properties.
Significant efforts have been devoted to solving the problem of stabilizing liposomes in blood to avoid recognition by the RES, and thus, to enhance the liposomes' effective lifetime in the blood. For example, one paper reports a case in which cholesterol was added to liposome membrane composition to increase blood liposome stability (Knight; (1981) Liposomes: From Physical structure to therapeutic applications, Elsevier, North Holland, pp. 310-311). However, the effect thus obtained varies widely depending on the original membrane composition of the liposome (Senior et al., (1985) Biochim. Biophys. Acta 839: 1-8). It has been reported that sialic acid-containing glycolipid, when administered as liposome, is distributed to the liver, a component of the RES (Surolia & Bachhawat, (1977) Biochim. Biophys. Acta 497: 760-765). It has also been reported that a drug was delivered into the brain after increasing the liposome's ability to pass through the blood brain barrier by functionalizing it with sulfatide, a glycolipid and a sulfo group (Naoi & Yagi, (1984) Biochem. Int. 9: 267-272).
Recently, thermosensitive liposomes—liposomes that are stable at mammalian body temperature but become less stable at temperatures higher than mammalian body temperature—have been employed to encapsulate chemotherapy agents and to release these agents into heated tissue (see, e.g., U.S. Pat. No. 6,200,598 to Needham et al., incorporated in its entirety herein by reference). For example, successful targeted chemotherapy delivery to brain tumors in animals using thermosensitive liposomes has been demonstrated (Kakinuma et al., (1996) Int. J. Hyperther 12(1): 157-165). The results of this study indicated that when thermosensitive liposomes are employed as a drug carrier, significant chemotherapy drug levels were measured within brain tumors that were heated to the range of about 41 to 44° C. One formulation for a thermosensitive liposome is described in U.S. Pat. No. 5,094,854, incorporated in its entirety herein by reference.
In one aspect, the presently disclosed methods and compositions comprise envirosensitive or non-sensitive liposomes, e.g. thermosensitive and non-thermally sensitive liposome compositions. These liposomes can comprise virtually any particular combination of lipids, and can further comprise proteins, carbohydrates and other types of compounds as well. A representative non-limiting thermosensitive liposome encapsulating a contrast agent (MnSO4) and an exemplary compound of interest (doxorubicin) is shown in FIG. 4. Other representative, but non-limiting liposome compositions are presented in the following sections and in the Laboratory Examples. Methods of forming the liposomes are also described herein. Generally, the same procedure can be employed for forming both envirosensitive and non-sensitive liposomes (e.g. thermosensitive and non-thermally sensitive liposomes), with one difference being the lipid composition of the liposome.
IV.A. Preparing Non-Sensitive and Envirosensitive Liposomes
Envirosensitive or non-sensitive liposomes can be prepared utilizing techniques such as those employed in the art for conventional liposome preparation. Such conventional techniques are referred to, for example, in Published PCT International Application Serial No. WO 92/21017 and by Papahadjopolous (Papahadiopolous, (1979) Ann. Rep. Med. Chem. 14: 250-260) and include reverse evaporation, freeze-thaw, detergent dialysis, homogenization, sonication, microemulsification and spontaneous formation upon hydration of a dry lipid film. In one embodiment, a film of the lipid is deposited on a glass coverslip and then incubated in a sucrose solution for a predetermined time, such as 12 hours. A thin film of lipid is then deposited on the inside of a round bottom flask and then rehydrated at a temperature above its phase transition temperature (Tm). Then, the hydrated lipids are sonicated in order to form liposomes.
Thermosensitive liposomes can be formed from a combination of lipids. Although almost any combination of lipids can be employed so long as the desired functional characteristic(s) is/are obtained, in one example, a thermosensitive liposome comprises dipalmitoylphosphatidylcholine-polyethylene glycol (DPPC-PEG2000). In another example, a thermosensitive liposome comprises dipalmitoylphosphatidylcholine-distearoylphosphatidylethanolamine-polyethylene glycol (DPPC-DSPE-PEG2000) (95:5, mol:mol), and in yet another example, a thermosensitive liposome comprises polyenylphosphatidylcholine-MSPC-distearoylphosphatidylethanolamine-polyethylene glycol (DPPC-MSPC-DSPE-PEG2000) (90:10:4, mol:mol).
Other embodiments of envirosensitive liposomes include, but are not limited to radiation-sensitive liposomes. The radiation-sensitive liposomes disclosed herein can be formed from a combination of lipids. Although almost any combination of lipids can be employed so long as the desired functional characteristic(s) is/are obtained, in one example, a radiation-sensitive liposome comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or diarachidoylphosphatidylcholine-polyethylene glycol (DAPC) in combination with PEG. In another example, cholesterol is added to one or more of these formulations. The addition of cholesterol reduces membrane fluidity and increases membrane integrity.
The radiation-sensitive formulations can further incorporate a radiation-sensitive lipid, which is selected based on the desired wavelength sensitivity. Thus, in one embodiment, liposomes can be made radiation-sensitive by the incorporation into the liposome wall radiation-sensitive lipids that undergo substantial alterations, such as isomerization, fragmentation or polymerization, upon interation with a particular wavelength range of electromagnetic radiation. For example, incorporation of polymerizable lipids such as 1,2-bis[10-(2′,4′-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC), react directly with ultraviolet radiation in the presence of oxygen to form cross-linked polymer networks that significantly alter bilayer properties in the liposome wall, resulting in destabilization of the liposome and release of its contents (Bondurant et al., (2001) Biochimica et Biophysica Acta 1511: 113-122). Spratt et al. have further shown that using a specific bis-SorbPC (bis-SorbPC19,19) will increase reactivity to ultraviolet radiation by a magnitude of at least two orders (Spratt et al., (2003) Biochimica et Biophysica Acta 1611: 35-43.
Sensitivity to other wavelength ranges of electromagnetic radiation can be achieved by incorporation of any of a variety of other sensitive lipids or even other non-lipid radiation-sensitive molecules. For example, visible light-sensitive liposomes can be formulated by incorporating dyes into the liposome walls. In one embodiment, ballasted cyanine dyes, such as distearoyl indocarbocyanine, in combination with polymerizable lipids, such as bis-SorbPC, are incorporated into liposome walls to produce light-sensitive liposomes that release their contents when contacted with visible light (green wavelength) (Mueller et al., (2000) Macromolecules 33: 4799-4804).
For in vivo biological applications, it can be desirable to use radiation at wavelengths less absorbable by biomolecules than visible or ultraviolet light. Therefore, liposomes incorporating molecules providing radiation-sensitivity at other wavelengths, such as ionizing radiation (e.g. x-rays), or long wavelength radiation (e.g. near-infrared or infrared) can also be formulated similarly to the previously described radiation-sensitive liposomes. One of skill in the art would thus appreciate that radiation-sensitive liposomes providing sensitivity across the electromagnetic spectrum are encompassed by the methods and compositions described herein.
Also included in the methods and compositions described herein are pH-sensitive liposomes. As discussed for thermosensitive liposomes, almost any combination of lipids can be employed so long as the desired characteristics of pH-sensitivity at a particular pH range are obtained. Numerous pH-sensitive liposomes are known and described in the art (see e.g. Litzinger & Huang, (1992) Biochim Biophys Acta 1113: 201-227). In one example, dipalmitoyl phosphatidyl ethanolamine/palmitic acid provides a useful pH-sensitive liposome formulation (Lokling et al., (2001) Magnetic Resonance Imaging 19: 731-738 and Lokling et al. (2003) Magnetic Resonance Imaging 21:531-540). Other formulations having similar properties are also encompassed by the present disclosure. Representative lipid compositions of non-sensitive liposomes include the thermosensitive liposome formulations disclosed herein, with the exception that the non-sensitive formulations often include cholesterol in varying amounts. For example, a non-sensitive liposome can comprise dipalmitoylphosphatidylcholine/cholesterol (DSPC/Cholesterol) (55:45, mol:mol).
Envirosensitive as well as non-sensitive liposomes can be prepared by extrusion methods. Lipids, at certain ratios, such as those described above, can be dissolved in a chloroform-methanol mixture. The solvent can then be removed under a gentle stream of nitrogen gas and the lipid samples subsequently placed under a high vacuum for a time period of at least 4 hours to remove any residual solvent.
The dried samples can then be hydrated such that the final lipid concentration is, for example, about 100 mg/mL. In one embodiment, hydration can be achieved by contacting the dried samples with 300 mM MnSO4 (a contrast agent) adjusted to pH 3.5 by addition of hydrochloric acid. Hydration of the lipid can be performed at a suitable temperature for a desired period of time, for example 55° C. for approximately 30 minutes, and generates multilamellar vesicles (MLVs).
Following hydration, MLVs can be extruded 10-times through stacked polycarbonate filters of 0.1 and 0.08 μm pore size at 55° C. using a water-jacketed extrusion apparatus, such as an EXTRUDERTM apparatus (Northern Lipids Inc., Vancouver, British Columbia, Canada). Extrusion of the MLVs results in liposomes that are ready for loading.
Following preparation, the mean size distribution of a liposome preparation can be determined. For some preparation, a NICOMP Submicron Particle Sizer Model 270 (Pacific Scientific, Santa Barbara, Calif., United States of America) operating at 632.8 nm can be employed, although other methods and apparatuses can also be employed. Phospholipid can also be quantitated, for example by employing a known assay such as the Fiske and Subbarow phosphate assay (Fiske & Subbarow, (1925) J. Biol. Chem. 2: 375-395)
Once an envirosensitive or non-sensitive liposome is prepared, the liposome can be loaded with a contrast agent and/or a compound of interest, such as a drug.
Envirosensitive and non-sensitive liposomes can be loaded with a contrast agent and/or a compound of interest by employing any of a range of techniques known in the art. Various methods include osmotic loading, pH gradient-based loading and ionic gradient-based loading (Kulkarni et al., (1995) J. Microencapsul. 12(3): 229-46).
In one embodiment, for example, liposome loading can be achieved by fractionating a liposome preparation on a SEPHADEX® G-50 (Amersham Biosciences, Piscataway, N.J., United States of America) column. For the fractionation, 1 mL sample volumes can be placed on a column with at least a 20 mL column bed that has been equilibrated with 500 mM sucrose/20 mM HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) buffer at pH 7.5.
Following fractionation, contrast agent and/or a compound of interest can be loaded by generally following the method described by Abraham et al. (Abraham et al., (2002) Biochim. Biophys. Acta 1565: 59-72).
Alternatively, in another embodiment, liposomes can be loaded using a pH gradient-based technique, such as described by Mayer et al. (Mayer et al., (1985) J. Biol. Chem. 260(2): 802-808). Generally, MLVs are produced by extrusion in the presence of K+ and then placed in a Na+ buffer to create a transmembrane Na+/K+ gradient with K+ concentrated within the vesicle and Na+ outside the vesicle. A contrast agent and/or a compound of interest in the buffer solution is accumulated at high concentrations within the liposome as a result of the transmembrane gradient. The rate of uptake of the contrast agent and/or compound of interest can be increased by the presence of an ionophore, such as, for example, valinomycin. Uptake is also sensitive to pH with this system, and can be maximized based on the pK of the contrast agent and/or compound of interest.
By way of example, loaded thermosensitive liposomes can be stored at 4° C. for 3-4 days. Loaded non-sensitive liposomes can be stored for about 1-3 weeks at 4° C. Envirosensitive and non-sensitive liposomes that have not been loaded can be stored for about 3-4 weeks at 4° C. Unloaded envirosensitive and non-sensitive liposomes can also be lyophilized and stored in that form.
This approach can be employed in the preparation of envirosensitive and non-sensitive liposomes. After liposomes are loaded, they can be stored in a suitable buffer solution and will be immediately ready for use without further preparation.
V. Applications
The presently disclosed methods and compositions can be employed in a variety of applications. Several of these applications are described in detail hereinbelow. Additional applications of the presently disclosed methods and compositions will be apparent to those of ordinary skill in the art upon consideration of the present disclosure.
In the following applications, standard magnetic resonance imaging apparatus and methodology can be employed, as would be apparent to those of ordinary skill in the art after a review of the present disclosure.
V.A. Monitoring the Accumulation of a Compound of Interest at a Desired Site In Vivo
In one application, a method of monitoring the accumulation of a compound of interest at a desired site in vivo by magnetic resonance imaging is disclosed. This application can be useful for tracking the delivery of a compound of interest to a site of interest, for example a tumor and for assuring that the compound of interest is delivered to the site in useful quantities.
In one embodiment, the method comprises increasing blood flow to a site of interest. As noted herein, a site of interest can be a tumor. In other examples, a site of interest can comprise a biological organ, such as the brain, liver, kidney or eye. In yet other examples, a site of interest can comprise a specific region or structure associated with the vasculature of a subject, or can even comprise the subject's vascular system in its entirety.
After selecting a site of interest, blood flow is increased to the site of interest. Heating can be used as an effective approach for increasing the blood flow to the site of interest. The heat results in vasodilation at the desired site and a subsequent increase in blood flow to the site. Heating can be achieved by employing any of a variety of techniques. For example, a site can be heated by RF energy, via application of ultrasonic energy or by conduction-based heating methods. When conduction-based heating methods are employed, one convenient method of heating is by contacting the site of interest with a catheter that is heated to a desired temperature, for example, with circulating water. When a site of interest is near an exposed surface of the subject (e.g., skin or eye), a laser can also be employed to heat the site.
A subject is then administered a non-sensitive liposome composition comprising (i) a contrast agent; (ii) a compound of interest; and (iii) a non-sensitive liposome encapsulating the contrast agent and the compound of interest.
Administration can be by an approach adapted to introduce the non-sensitive liposome composition into the bloodstream of the subject. For example, the administration can be by injection into an artery or vein. In one particular example, when a subject is a rat (for example, a Fisher 344 female strain rat), a liposome composition can be injected into the tail vein or femoral vein of the rat. Thus, administration can be, for example, via intravenous, intramuscular, intraperitoneal, intra-tumoral or subcutaneous intra-lesional injection.
A contrast agent can comprise any paramagnetic nucleus containing material, as disclosed herein above. Compounds comprising transition, lanthanide and actinide elements can also be employed. For example, a contrast agent can comprise an atom of manganese or gadolinium. A contrast agent can also comprise a chemical moiety associated with the atom, such as a sulfate moiety, in which case a contrast agent can comprise, for example, MnSO4, which exhibits a linear relationship between concentration and 1/T1 (see FIG. 1B) and 1/T2. A contrast agent can also comprise a chelate of the atom, such as for example gadoteridol, which is a gadolinium (III) chelate of 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (e.g., commercially-available PROHANCE®, Bracco Diagnostics, Inc., Princeton, N.J., U.S.A.).
A compound of interest can comprise any compound. For example, a compound of interest can comprise a pharmaceutically active compound, such as a chemotherapeutic compound (e.g. methotrexate, doxorubicin, cisplatinum, carboplatinum). A compound of interest can also comprise a compound suspected of being pharmaceutically active. In other cases, a compound of interest can generally comprise a compound known or suspected of modulating one or more biological processes. For example, a compound of interest can be a polypeptide or a polynucleotide. A non-sensitive liposome composition also comprises a non-sensitive liposome encapsulating the contrast agent and the compound of interest. Compositions of non-sensitive liposomes are disclosed herein, as are methods for preparing and loading the liposomes.
Continuing with the present embodiment, the accumulation of the compound of interest at the site of interest is monitored by magnetic resonance imaging. As the non-sensitive liposomes that have been administered to the subject circulate in the bloodstream of the subject, they will tend to accumulate at the site of heating. Over a given time interval, the presence of the liposomes at the heated site will increase. Thus, as time progresses, the presence of contrast agent at the heated site will concomitantly increase (see FIG. 2A), since the contrast agent is encapsulated in the non-sensitive liposomes.
Magnetic resonance images of the heated site can be continuously and regularly generated, as depicted in FIG. 2A. Methods of acquiring magnetic resonance images are established and can be employed to generate magnetic resonance images of a heated site. A sequence of images of the heated site shows the accumulation of the contrast agent at the heated site and in the areas surrounding the heated site (see FIG. 2A). Since the contrast agent and the compound of interest are both encapsulated in the non-sensitive liposome, the accumulation of the contrast agent is directly proportional to the accumulation of the compound of interest.
As depicted in FIG. 2A, accumulation of the contrast agent at a heated site is evidenced by an increase in the appearance of white pixels in a timecourse series of images. For example, in FIG. 2A at time point 0, immediately after injection of a non-sensitive liposome composition, in particular, a thermoinsensitve liposome composition, there is no contrast agent found at the site of the tumor, which is heated by hot water flowing through a catheter, shown as a black spot in the image. At time point 1 (corresponding to about 5 minutes), contrast agent, and thus the compound of interest, which is encapsulated with the contrast agent, is accumulating at the periphery of the tumor. This is evidenced by the appearance of white pixelation (i.e., an increase in pixel value/density, or “whiteness” on a gray scale) at the tumor edge. At time point 2 (corresponding to about 30 minutes), the concentration of contrast agent and compound of interest is increased. At time point 3 (corresponding to about 90 minutes), the intensity of pixelation is seen to slightly increase over the concentration at time point 2. By way of comparison, no contrast agent or compound of interest is seen to appear in areas that are not heated.
It is noted that as the non-sensitive liposome compositions accumulate at the heated site, they remain structurally coherent and the contents of the liposomes, namely a contrast agent and a compound of interest, remain inside the liposome. Thus, the accumulation of the compound of interest at a heated site can be monitored.
As FIG. 2A demonstrates, this technique can be performed in vivo, with the liposomes eventually being cleared by the renal and/or hepatic systems of the organism. It is possible to perform the method in vitro, on a tissue culture, for example, but most commonly the method will be performed in vivo on a subject.
V.B. In Vivo Method of Monitoring the Localization and Distribution of a Compound of Interest to a Desired Site in a Subject
Difficulties in delivering drugs to solid tumors in the human body have been documented in the art. For example, abnormal vessels in tumors can restrict local blood flow in tumors and, hence, impede the delivery of drugs to the tumor. Abnormally elevated interstitial pressure within the tumor is also known to retard the passage of drug molecules from the blood stream into the tumor (Baxter & Jain, (1989) Microvasc. Res. 37(1): 77-104; Baxter & Jain, (1990) Microvasc. Res. 40(2): 246-63; Baxter & Jain, (1991) Microvasc. Res. 41(1): 5-23; Baxter & Jain, (1991). Microvasc. Res. 41(2): 252-72)
Effective cancer chemotherapy depends on delivery of therapeutic drugs to cancer cells at cytotoxic concentrations. Due to the inherent perfusion limitations that tumors present, delivery of drugs can be hindered. The ability to monitor and/or predict in vivo concentration distributions could improve treatment. Thus, in one aspect of the present disclosure, envirosensitive liposomes can be employed for in vivo monitoring of drug release and distribution from an envirosensitive liposome using MRI. In vitro results disclosed herein (FIG. 1B) indicate that T1 or T2 shortening correlates with contrast agent (e.g., MnSO4) concentration. Further, contrast agent release from envirosensitive liposomes, for example thermosensitive liposomes, significantly shortens T1 and T2 and thus can be used to monitor content release.
The feasibility of using this drug delivery-imaging agent in vivo was shown in a murine tumor model (FIGS. 2A-2E). These experiments demonstrate that upon tumor heating, thermosensitive and non-sensitive liposomes selectively collect at the site of the tumor, and that thermosensitive liposomes release their contents. In addition, FIG. 2E shows that without heating, a less intense but more uniform signal enhancement occurs, but then fades after five minutes as clearance from the vasculature occurs. However, there is enhancement remaining in the tumor (compared to baseline) indicating that some of the liposomes have collected at the site. The thermosensitive and non-sensitive compositions and methods of the present disclosure, facilitate clinical treatment by providing individualized monitoring of tissue drug concentration distribution. Individualized monitoring provides at least (1) the ability to modify treatment variables to improve the uniformity of drug delivery; and (2) the ability to select patients most likely to benefit from a liposomal treatment as disclosed herein.
Additionally, the methods of loading envirosensitive and non-sensitive liposomes disclosed herein are applicable to a wider spectrum of compounds of interest (e.g., drugs) than was previously possible with pH loading methods, thereby broadening its applicability to other formulations.
In accordance with the present disclosure, an in vivo method of monitoring the distribution of a compound of interest to a desired site in an organism by magnetic resonance imaging is disclosed. In one embodiment, the method comprises increasing blood flow to a site of interest in a subject by, for example, applying heat. The site can be heated by external application of hot water, RF, ultrasound, or IR energy. Alternatively, interstitial application of energy can be obtained using the same physical methods. Further, in some instances, the site of interest can have an increased temperature due to an in vivo process (e.g., inflammation). The heat results in vasodilation at the desired site and an increase in blood flow to the site. Other methods of increasing vasodilatation and blood flow to the site are also acceptable for targeting the liposomes to a desired site. For example, direct mechanical massage or ultrasound treatment to the site can increase blood flow without the use of heat.
As described herein, a subject can be any living organism, for example, a human, mouse, rat, or rabbit, or a subject can be derived from a living organism and can comprise, for example, a tissue culture. A site of interest can comprise any biological structure. For example, a site of interest can comprise a tumor or an organ, such as a brain, liver, kidney, stomach, eye or lung.
Continuing with this embodiment, a thermosensitive liposome composition can be administered to the subject. Again, the administration can be by convenient method, such as injection of the composition into a vein or artery of the subject.
An envirosensitive liposome composition of the present disclosure comprises a contrast agent; a compound of interest; and an envirosensitive liposome encapsulating the contrast agent and the compound of interest. The contrast agent can comprise any paramagnetic material, for example MnSO4 or any compound comprising, for example, a transition element or an inner block element. A contrast agent can comprise a paramagnetic material complexed with an organic material (e.g., a chelator) or an inorganic material (e.g., a sulfate moiety). A contrast agent can also comprise a chelate of the atom, such as for example gadoteridol.
A compound of interest can comprise any compound. Such a compound can comprise a chemotherapeutic agent, pharmaceutically active agent or an agent suspected to be of therapeutic value to the subject. Doxorubicin is employed as a non-limiting embodiment of such a compound in the Laboratory Examples.
Additionally, one embodiment of the method comprises monitoring the localization and distribution of the compound of interest to the desired site by magnetic resonance imaging. The embodiment permits monitoring of both localization to the site of interest and distribution of the compound of interest to the site using magnetic resonance imaging. Distribution of the compound of interest refers to release of the compound from the liposome and dispersion of the compound at the site. The monitoring can be conveniently achieved by acquiring magnetic resonance images at any desired time point. Standard magnetic resonance techniques can be employed to generate such images.
In one embodiment, envirosensitive liposomes in the form of thermosensitive liposomes are used. The thermosensitive liposomes are stable at temperatures near mammalian body temperature, about 37° C. The temperature of the heated site will be several degrees above 37° C. As the thermosensitive liposomes travel to the heated site (e.g., through the circulatory system of the subject), they will accumulate at the heated site due, in part, to their size and release their contents due to their thermoinstability.
After exposure to heat for a period of time, the thermosensitive liposomes will become “leaky”. That is, the thermosensitive liposomes will lose a degree of structural integrity, allowing the contents of the liposomes, namely a contrast agent and a compound of interest, to be released from the thermosensitive liposome. The release of the contents of the thermosensitive liposome can be tracked by monitoring an increase in the presence of contrast agent at a range of points around a given structure. The association of contrast agent with a structure can be denoted by an increase in the pixel density and/or intensity of signal around the structure in a MR-generated image.
Turning now to FIG. 2B, application of one embodiment of the method is depicted in a series of MR images. The images correspond to the effect of injecting loaded thermosensitive liposomes into the tail vein of a rat. The images generally depict association of contrast agent with the periphery of a large flank tumor.
At time point 0, a thermosensitive liposome composition has been injected into the circulatory system of the rat. The flank tumor is heated by inserting a catheter heated with circulating water and appears as a black spot in the upper left of the image.
At time point 1 (corresponding to about 5 minutes), the loaded thermosensitive liposomes have accumulated at the heated site and have released their contents. The association of the contrast agent with the periphery of the tumor is evidenced by the pixelation (which appears white in the figure) surrounding the tumor mass.
At time point 2 (corresponding to about 30 minutes), the pixelation is seen to be more intense, indicating the further release of contrast agent from the thermosensitive liposomes. The periphery of the tumor is even more completely surrounded by contrast agent.
At time point 3 (corresponding to about 90 minutes), the pixelation is less intense, indicating that the contrast agent is being cleared and the concentration in the vicinity of the tumor is decreasing.
In addition, FIG. 2E shows that without heating, a less intense but more uniform signal enhancement occurs, but then fades after five minutes as clearance from the vasculature occurs.
In a similar embodiment, envirosensitive liposomes in the form of radiation-sensitive liposomes are used. Like other envirosensitive liposomes, including the previously described thermosensitive liposomes, radiation-sensitive liposomes are stable under normal physiological conditions.
The radiation-sensitive liposomes also travel to and accumulate at the heated site due, in part, to their size. However, unlike thermosensitive liposomes, the radiation-sensitive liposomes will not be expected to release their contents when heated, unless the heat source is producing a wavelength of electromagnetic radiation within the range of sensitivity of the particular radiation-sensitive liposomes.
Instead, a source of electromagnetic radiation emitting radiation at a wavelength within the range of sensitivity of the particular radiation-sensitive liposomes is directed at the site, which then interacts with susceptible lipids in the liposome wall. The sensitive lipids then either isomerize, fragment or polymerize, which then causes the liposomes to lose structural integrity and increase permeability, in some formulations, sufficiently to release their contents. Namely, the radiation-sensitive liposomes become permeable enough to at least allow exchange of water across the membrane. The membrane in some formulations will become sufficiently permeable to release the contained contrast agent and/or compound of interest. As already described, the release of contents can be tracked by monitoring the presence of contrast agent at a range of points around a given structure.
One of skill in the art will appreciate that other liposome disruption agents can be used, such as pH variance, depending on the disruption characteristics of the particular envirosensitive liposome formulated and the local environment deviation from normal tissue (e.g., from an in vivo process or applied externally).
In another aspect, drug release can also be quantified. One method of quantifying drug release generally involves employing a plot of concentration against 1/T1 or 1/T2 as a calibration curve.
Continuing with this embodiment, before a given experiment is performed, the T1 of a pixel is measured. This T1 measurement can provide additional information, including proton density and base line noise. If all the imaging parameters (e.g., Tr, PD, etc.) are kept constant, which is typical in a dynamic study, a change in signal intensity at a time point later than time t=0 is accompanied by a reduction in T1. Such a change in T1 is indicative of localization and distribution of a drug from an envirosensitive liposome composition. This reduction in T1 can be converted to concentration using the plot of concentration against 1/T1 as a standard curve for the corresponding compound. Thus, observed changes in T1, which are associated with drug release, can be translated into a released drug concentration by indexing the observed T1 with a given concentration on a plot of concentration against 1/T1.
V.C. Method of Detecting an In Vivo Blood Pool
Contrast agents with prolonged presence in the blood (i.e., good resistance to uptake by RES and a relatively low diffusivity into the tissue or extravascular locations) are recognized in the art as useful “blood pool agents” (see, e.g., U.S. Pat. No. 5,464,696, herein incorporated by reference in its entirety). Contrast agents exhibiting long biological half-lives are sometimes desirable for the blood pool agents if a researcher or clinician desires to produce meaningful analytical results and to eliminate repeated injections and the repeated use of a contrast agent.
Several attempts to produce compositions suitable for use as blood pool agents have been made, including some for use with MRI (see, e.g., U.S. Pat. Nos. 5,833,948; 5,464,696; 6,010,681; 5,961,953; and 5,888,476, herein incorporated by reference in its/their entirety). Particularly, there has been an ongoing effort to develop contrast agents with long residence times in the blood circulation, that exhibit high relaxivity and can be completely eliminated from the system of a subject (i.e., agents that can be employed as “blood pool agents”). Some efforts have focused on identifying and preparing paramagnetic substances encapsulated into liposome vesicles, immobilized in the liposome membrane, copolymerized with polyethylene glycol or grafted on a polymeric chain such as albumin, dextran or polylysine. Examples of such compositions include Gd-DTPA-albumin, Gd-DTPA-dextran or Gd-DTPA-polylysine complex molecules (see, e.g., Ogan et al., (1987) Invest. Radiol. 22: 665; Wang et al., (1990) Radiology 175: 483; Schumann-Giampieri et al., (1991) Invest. Radiol. 26: 969; Vexler et al., (1994) Invest. Radiol 29 supl. 2: S62; Dessler et al., (1994) Invest. Radiol. 29 supl. 2: S65; Meyer et al., (1994) Invest. Radiol. 29 supl. 2: S90; Shen et al., (1994) Invest. Radiol. 29 supl. 2: S217).
Notwithstanding, the half-life of contrast agents containing paramagnetic species bonded to macromolecules is often too short to be convenient for blood-pool imaging or have unexpected toxic side effects. In order to solve this difficulty, the use of suspensions of liposomal microvesicles containing encapsulated paramagnetic chelates as carriers of NMR contrast agents has been proposed. Use of liposomes for carriers has been proposed for relative biocompatibility and ease of preparation of liposomes and their suspensions. Encapsulation of known paramagnetic contrast agents into liposomes has been described (see, e.g., Unger et al., (1993) J. Mag. Res. Imag. 3: 195-198).
These known compositions exhibit longer dwelling times in the blood than the water-soluble metal complexes; however, their residence times in the circulation are still not sufficient and some of these compounds have shown unacceptable levels of toxicity for blood-pool imaging. Longer residence times and lower immunogenicity have been reported by Bogdanov et al. (Bogdanov et al., (1993) Radiology 187: 701) for Gd-DTPA-MPEG-polylysine complexes which consist of a methoxy(polyethylene glycol)-shielded macromolecular backbone (polylysine) bearing covalently attached Gd-DTPA. However, these prior art compositions do not offer the advantages of the compositions and methods disclosed herein.
Desirable properties of a blood pool agent include the ability to remain in a subject's bloodstream for protracted periods of time. For contrast agents administered into the systemic vasculature, as a general rule, low molecular weight hydrophilic molecules (e.g. molecular weight beneath about 5000 Da) distribute into the extracellular fluid (ECF) and are relatively rapidly excreted through the kidneys by glomerular filtration. Particulates, liposomes or lipophilic molecules tend to accumulate relatively rapidly in the liver. Thus, an effective blood pool agent would not be recognized by the RES, and would remaining in the bloodstream for an extended period, yet would still provide a magnetic relaxation response. The blood pool agents disclosed herein accomplish this goal.
The use of a blood pool agent can facilitate a wide range of measurements that can be of interest to researchers and clinicians. For example, one role that a blood pool agent can play is as an aid in the measurement of blood volumes and the blood perfusion of various organs, including the brain, using in vivo, non-invasive techniques.
Accordingly, in one aspect of the present disclosure, a method of detecting an in vivo blood pool is disclosed. In one embodiment of the method, a subject is administered a non-sensitive liposome composition. A suitable non-sensitive liposome composition can comprise a contrast agent and a non-sensitive liposome encapsulating the contrast agent. The administering can be carried out by any convenient method, although many times injection of the composition into a subject's vein or artery can be the most convenient approach to administering a composition.
A subject, as described herein, can be any living organism or a subject can even comprise a solution (e.g. blood, plasma, etc.) infused tissue culture. A contrast agent can comprise any paramagnetic material, such as MgSO4. Many such materials are commercially available and can be employed in a non-sensitive liposome composition disclosed herein. Non-sensitive liposomes can be loaded with contrast agent by employing any known technique, such as pH gradient loading, ionic gradient loading and osmotic loading. One method of loading non-sensitive liposomes is disclosed in the Laboratory Examples.
After a non-sensitive liposome composition has been administered to a subject (which can be performed by employing a method disclosed herein or known to those of ordinary skill in the art), a magnetic resonance image of a site of interest can be generated. As noted throughout the present disclosure, a magnetic resonance image can be generated by any known method and can be generated on any available MRI apparatus, such as a 1.5T, 2T, 3T, 4T, or 7T whole-body clinical scanning instrument (e.g., MRI apparatus available from General Electric of Milwaukee, Wis., United States of America or from Siemens, Munich, Germany). One of skill in the art will appreciate, however, that the relaxivity of the liposomes is field strength dependent. Consequently, if a field strength other than 1.5/2T is utilized, it may be desirable to recalibrate T1/T2 relaxivity versus temperature for the exemplary liposome formulations disclosed herein (FIGS. 1b-1d). As the field strength increases the relaxivity at a given temperature decreases. Although this can result in a reduced contrast for a given concentration, there is also an overall increase of the signal to noise from the higher field strength, such that sensitivity to the contrast agent should increase (i.e., signal to noise increases at a faster rate than the rate of relaxivity decrease as field strength is increased).
In one example of the method, a single image can be generated at a time point known or suspected to permit enough time for the envirosensitive liposome composition to circulate through the subject's blood stream to a site of interest. In another example of the method, a time course series of images of a site of interest can be acquired, similar to the time course of images presented in FIGS. 2A and 2B. Such a time course of images can be focused on a particular region of interest, such as the brain, or on a biological structure known or suspected to have a vascular irregularity.
Continuing with the embodiment of the method, the presence of an in vivo blood pool can be detected by analyzing the magnetic resonance image. Such an analysis can comprise an evaluation of one or more MR images to identify the presence or absence of a blood pool at a particular site of interest. The presence of a blood pool is indicated, in a MR image, by the pixelation associated with a contrast agent. When images are black-and-white images, the contrast agent pixelation will show up as white pixelation.
As noted, the presence of a blood pool can be indicative of a vascular irregularity. A vascular irregularity can be, for example, a widening of a vascular structure. In one embodiment, a vascular irregularity can comprise an aneurysm. In another embodiment, a vascular irregularity can comprise an ischemic condition.
Ischemia/reperfusion injury is a significant source of morbidity and mortality in a number of clinical disorders, including myocardial infarction, cerebrovascular disease, and peripheral vascular disease. In addition, ischemia/reperfusion is relevant to the function of transplanted organs and to the recovery expedience following any cardiovascular surgery (see, e.g., Fan et al., (1999) J. Mol. Med. 77:577-596). Often times, ischemic conditions are not identified in a subject until after significant damage or death has resulted. Thus, the presently disclosed subject matter can be employed to monitor the formation, dissolution and properties of a blood pool, which can useful in the diagnosis and prevention of disorders related to vascular diseases and conditions.
V.D. Method of Generating a Heating Profile of a Site of Interest
In another aspect, a method of generating a heating profile of a site of interest is disclosed. The term “heating profile”, as it is used herein, encompasses the heating of a region of tissue surrounding a site of heating. A heating profile reflects the increase and/or decrease in heat as a function of distance from the site of heating or from the source of heat (e.g., a heated catheter or in vivo process, such as inflammation).
In one embodiment, the method comprises administering to a subject a thermosensitive liposome composition comprising: (i) a contrast agent and (ii) a thermosensitive liposome encapsulating the contrast agent and the compound of interest and having a melting temperature, Tm. Thermosensitive liposome compositions can be formed as described herein. The thermosensitive liposomes of such compositions will have a given melting temperature, which can be a function of the composition of the liposome. At temperatures below the Tm, the thermosensitive liposome retains its structural integrity; above the Tm, the thermosensitive liposome loses its structural integrity, allowing release of the liposome's contents. Representative contrast agents are described herein and can comprise, for example, MnSO4.
Continuing with the method, a site of interest in a subject is then heated. Various methods of heating can be employed in the method, such as heating via a catheter warmed by passing hot water through the catheter. Other heating methods are described herein.
The release of the contrast agent from the thermosensitive liposome is then monitored using magnetic resonance imaging. The steps for acquiring such a magnetic resonance image are described herein. Standard MRI methodology can be employed in the acquisition of the image as disclosed herein and also will be known to those of ordinary skill in the art upon consideration of the present disclosure.
A heating profile of the site of interest can then be generated. In such a heating profile, the heating of an area to a temperature of at least Tm can optionally be indicated by release of contrast agent at a periphery of the area. Such a heating profile can reflect the distance from a site of heating (e.g., the radial distance) at which the Tm of the thermosensitive liposome is reached.
By way of example, a heated catheter can be employed to heat a tumor. The tumor tissue will be warmest near the site at which the catheter contacts the tumor, and will be cooler at points further away from the catheter. When the tumor tissue is homogeneous, this decrease in temperature as a function of distance from the catheter can reflect a linear or exponential decrease. At some distance from the catheter, the temperature of the tumor tissue will be equal to the Tm of a given thermosensitive liposome composition. When thermosensitive liposomes reach this distance (as disclosed herein, envirosensitive (e.g. thermosensitive), and non-sensitive, liposomes accumulate at a site of heating) they melt and release their contents (or merely accumulate if non-sensitive), namely a contrast agent. By evaluating MR images acquire as the thermosensitive liposomes approach the site of heating, the distance at which the Tm of the liposomes is reached can be determined. At the distance from the catheter that the tissue is heated to Tm, contrast agent release will be immediate and will resemble a burst release. Thus distances equal to or less than the Tm distance from the catheter can be identified, giving rise to a heating profile.
In another embodiment, two or more thermosensitive liposome compositions can be employed, for example, in succession. In this embodiment, the liposomes can have lipid compositions that impart different melting points. By administering several compositions, each with a different Tm, and compiling the results, a detailed heating profile, similar to a plot of different isotherms can be generated, which reflects the temperature of the tissue at various distances from the catheter or site of heating.
V.E. Methods of Predicting and Enhancing Efficacy of a Treatment
As disclosed herein, novel MRI techniques can be used to observe in vivo content release from liposomes that contain a contrast agent with or without a compound of interest. As disclosed in the Laboratory Examples, local tissue concentrations of the compound of interest can be estimated from the shortening of MR T1 relaxation times. For example, and as set forth in the Laboratory Examples, MRI was used to measure temporal and spatial patterns of drug delivery in a rat fibrosarcoma model during treatment with Dox/Mn-LTSL and hyperthermia administered with different schedules. The data elucidate relationships among temperature profile, tumor drug delivery pattern, and antitumor effect. The findings demonstrate that the pattern of drug delivery with envirosensitive liposomes can be controlled and monitored based on the perfusion pattern at the desired site and the temperature profile at the time of liposome administration. Thus, the use of different protocols of non-physiological condition exposures at a site of interest in conjunction with administration of the envirosensitive liposomes can permit compound of interest distribution to be controlled in real time, which is referred to herein as “drug dose painting.”
In view of the above discussion, in some embodiments of the presently disclosed subject matter a method of predicting efficacy of a treatment in a subject is provided. In some embodiments, the method comprises monitoring accumulation of a compound of interest at a desired site in vivo by magnetic resonance imaging and predicting efficacy of treatment based on accumulation of a compound of interest at the desired site.
In some embodiments, the method comprises administering to a subject a non-sensitive liposome composition and monitoring the accumulation of the compound of interest at the desired site by magnetic resonance imaging. In some embodiments, the non-sensitive liposome composition comprises a contrast agent, a compound of interest, and a non-sensitive liposome encapsulating the contrast agent and the compound of interest.
In other embodiments, the method comprises administering an envirosensitive liposome composition to a subject and monitoring the accumulation of the compound of interest at the desired site by magnetic resonance imaging (e.g., making a pixel density determination). In some embodiments, the envirosensitive liposome composition comprises a contrast agent, a compound of interest, and an envirosensitive liposome encapsulating the contrast agent and the compound of interest. In some embodiments, the method further comprises exposing the envirosensitive liposome at the desired site to a non-physiological environmental condition, which can be selected based on the nature of the envirosensitive liposome utilized. For example, the envirosensitive liposome can be thermosensitive, and the non-physiological environmental condition can be hyperthermia. Additional exemplary envirosensitive liposomes suitable for use include pH-sensitive liposomes, chemosensitive liposomes and radiation-sensitive liposomes.
In some embodiments, predicting efficacy comprises predicting efficacy of treatment based on a location of accumulation at the desired site, a rate of accumulation at the desired site, or both location and rate of accumulation at the desired site. The location of accumulation at the desired site can be a particular targeted region within or proximate to the desired site. For example, in a tumor, it may be desirable to target the periphery or outer regions of the tumor where vasculature feeds the tumor. As disclosed in detail in the Laboratory Examples, the periphery of a tumor can be targeted for delivery of a therapeutic agent by exposing and equalizing the tumor to a non-physiological environmental condition (e.g., hyperthermia when utilizing thermosensitive liposomes) prior to administration of the envirosensitive liposome containing the therapeutic compound such that the liposome is induced to release the therapeutic compound at the periphery of the tumor, when it first encounters the non-physiological environmental condition. In contrast, if distribution to a central region of a tumor is desired, the envirosensitive liposomes can be administered prior to treatment with the non-physiological environmental condition, such that the liposomes accumulate within the central region of the tumor first and are then stimulated to release the therapeutic agent after treatment with the non-physiological environmental condition. In addition, uniform distribution can be accomplished by a combination of the above techniques, that is, a portion of the liposomes are administered prior to non-physiological environmental condition treatment and a portion administered after treatment. Rates of accumulation of the compound of interest at the desired site can also be predicted and manipulated based on the timing and intensity of non-physiological environmental condition treatment. For example, if rapid release of the compound of interest from the envirosensitive liposomes is desired, the treatment can be initiated prior to administration of the liposomes at the site of interest.
Related to methods of predicting efficacy of treatment, in some embodiments of the presently disclosed subject matter, a method of enhancing efficacy of a treatment at a desired site in a subject is provided. In some embodiments, the method comprises administering to the subject a composition comprising a compound of interest and targeting the composition to a desired location at a desired site in the subject, at a desired rate of accumulation at the desired site, or both a desired location and desired rate of accumulation at the desired site, to thereby enhance efficacy of treatment provided by the compound of interest. In some embodiments, composition is targeted to the desired location and/or at the desired rate by exposing the desired site to a non-physiological environmental condition, such as for example hyperthermia, electromagnetic radiation, a chemical agent and non-physiological pH. In some embodiments, the desired site is exposed to a non-physiological environmental condition before, after, or both before and after administering the composition to target the composition. Further, in some embodiments, targeting the composition comprises administering the composition in one or more partial doses before and/or after exposing the desired site to a non-physiological environmental condition.
In some embodiments, the composition comprises a non-sensitive liposome composition comprising the compound of interest and a non-sensitive liposome encapsulating the compound of interest. In other embodiments, the composition comprises an envirosensitive liposome composition comprising the compound of interest and an envirosensitive liposome encapsulating the compound of interest. In some embodiments, the envirosensitive liposome is a liposome selected from the group consisting of a thermosensitive liposome, a pH-sensitive liposome, a chemosensitive liposome and a radiation-sensitive liposome. In some embodiments, the composition further comprises a contrast agent.
In some embodiments, the method further comprises monitoring accumulation of the compound of interest at the desired site in vivo by magnetic resonance imaging. In some embodiments, monitoring the accumulation of the compound of interest at the desired site by magnetic resonance imaging comprises making a pixel density determination.
Additionally, in some embodiments of the presently disclosed subject matter, a method of targeting delivery of a compound of interest at a desired site in vivo is provided. In some embodiments, the method comprises administering to a subject a composition comprising a compound of interest and exposing the desired site to a non-physiological environmental condition to thereby target the composition to a desired location at the desired site in the subject, at a desired rate of accumulation at the desired site, or both the desired location and the desired rate of accumulation at the desired site. In some embodiments, the environmental condition is selected from the group consisting of hyperthermia, electromagnetic and/or ionizing radiation, a chemical agent and non-physiological pH. In some embodiments, the hyperthermia is provided by a method selected from the group consisting of contacting a heated material with the desired site, applying RF energy to the desired site, applying ultrasonic energy to the desired site and applying a laser beam to the desired site. In some embodiments, the desired site is exposed to a non-physiological environmental condition before, after, or both before and after administering the composition to target the composition. Further, in some embodiments, targeting the composition comprises administering the composition in one or more partial doses before and/or after exposing the desired site to a non-physiological environmental condition.
In some embodiments, the composition comprises a non-sensitive liposome composition comprising the compound of interest and a non-sensitive liposome encapsulating the compound of interest. In other embodiments, the composition comprises an envirosensitive liposome composition comprising the compound of interest and an envirosensitive liposome encapsulating the compound of interest. In some embodiments, the envirosensitive liposome is a liposome selected from the group consisting of a thermosensitive liposome, a pH-sensitive liposome, a chemosensitive liposome and a radiation-sensitive liposome. In some embodiments, the composition further comprises a contrast agent.
In some embodiments, the method further comprises monitoring accumulation of the compound of interest at the desired site in vivo by magnetic resonance imaging. In some embodiments, monitoring the accumulation of the compound of interest at the desired site by magnetic resonance imaging comprises making a pixel density determination.
In the above-disclosed methods, a compound of interest can comprise any compound. Such a compound can comprise a chemotherapeutic agent, pharmaceutically active agent or an agent suspected to be of therapeutic value to the subject. Doxorubicin is employed as a non-limiting embodiment of such a compound in the Laboratory Examples. Further, the contrast agent can comprise any paramagnetic material, for example Mn or Gd, or any compound comprising, for example, a transition element or an inner block element. A contrast agent can comprise a paramagnetic material complexed with an organic material (e.g., a chelator, such as for example gadoteridol) or an inorganic material (e.g., a sulfate moiety). Exemplary contrast agents can comprise one or more elements selected from the group consisting of Gd, Cu, Cr, Fe, Co, Er, Ni, Eu, Dy, Zn, Mg, Mo, Li, Ta, and Mn.
In some embodiments of the above-disclosed methods, the non-sensitive liposome can comprise DSPC/Cholesterol (55:45, mol:mol). Further, the thermosensitive liposome can comprise a formulation selected from the group consisting of DPPC-PEG2000, DPPC-DSPE-PEG2000 (95:5, mol:mol) and DPPC-MSPC-DSPE-PEG2000 (90:10:4, mol:mol).
Further, in some embodiments of the above-disclosed methods the desired site is selected from the group consisting of a tumor, an embolism, an injury site, an ischemia, and at a tissue edema.
VI. Advantages of the Present Disclosure
The methods and compositions of the present disclosure offer many advantages over compositions, methods and approaches known and/or employed in state-of-the-art diagnostic and therapeutic procedures. Representative advantages of the present disclosure are described below. Other advantages will be apparent when considered in view of the present disclosure.
VI.A. Selectivity in Targeting
FIGS. 2A and 2B highlight several advantages of the methods and compositions of the presently disclosed subject matter. First, it is seen that in both of these figures, the contrast agent, and thus the compound of interest, is localized to the immediate region surrounding the tumor. There is no contrast agent detected in any region of this or any other slice in the immediate area of the tumor. This advantage of the presently disclosed methods and compositions is highlighted when FIG. 2A and particularly FIG. 2B are compared with FIG. 2C.
FIG. 2C is a MR image depicting the effect of infusing a rat bearing a flank tumor with a prior art contrast agent (MAGNAVIST®, available from Berlex Laboratories, Wayne, N.J., United States of America). When this prior art contrast agent is injected into a rat having a flank tumor not heated by a catheter, the contrast agent is seen to associate not only with the tumor, but with other internal structures as well, including the presumably necrotic tissue in the center of the tumor and non-tumor flank tissue. This figure indicates that prior art contrast agents cannot be employed to specifically target the growing tissue (e.g., the periphery) of a tumor. Consequently, in contrast to the present disclosure, treatments based on this approach will suffer from a decreased concentration of a compound of interest (e.g., a chemotherapeutic agent) at the tumor, where the agent is most needed for therapeutic reasons.
Another related advantage of the methods and compositions disclosed herein is their size. The liposome compositions are typically about 120 nm in diameter. This small size ensures that the liposome compositions can be stably transported through the vasculature of a subject, while being large enough to evade trapping by the renal and/or hepatic systems and passing the normal vasculature wall.
VI.B. Rapid Clearance of Liposomes and Contrast Agent
One problem that can accompany the use of a contrast agent in a living organism is the toxicity of a contrast agent. If the contrast agent is not cleared from the system of a subject in a timely fashion, a toxic reaction can be initiated. This can be complicated by the occasional need to employ relatively high concentrations of contrast agent or repeated injections to generate an interpretable, high resolution image.
The presently disclosed methods and compositions do not suffer from this drawback. The liposome compositions can employ contrast agents that are highly effective at reducing proton relaxation times (i.e. T1's), and can therefore be employed in lower quantities than those that are toxic on a systemic basis. Additionally, in the present methods and compositions, the contrast agent is encapsulated by a liposome, which remains stable until it reaches a target site. Therefore, no contrast agent is released into the bloodstream of the subject as the liposomes travel to their target.
Moreover, as FIG. 3D indicates, the contrast agent is cleared from the circulatory system in about 90 minutes in the case of the exemplary thermosensitive liposomes and about 3-5 hours in the case of the exemplary non-sensitive liposomes. Additionally, the compositions and properties of the presently disclosed liposomes can be modified to vary their circulation time as desired, such as for example by modifying the concentration of polyethylene glycol in the liposome. Increasing polyethylene glycol concentration can increase circulation time, but it can also modify transition temperature, and therefore two properties can be balanced as desired.
VI.C. In Vivo Monitoring
Another advantage of the methods and compositions of the present disclosure is the ability to perform the methods in vivo. Often, diagnostic methods cannot be performed in vivo and are relegated, at least preliminarily, to artificial in vitro studies. In vitro conditions typically do not exactly reproduce in vivo conditions, particularly in the area of drug delivery and drug action/metabolism.
Another advantage is the ability to carry out the methods in a living organism. MRI is particularly suited to this application since it is a non-invasive, yet highly sensitive method. Thus, a study performed to monitor the delivery of a compound to a particular biological structure can be performed in vivo and a researcher can be confident that the observed results will be reproducible in similar living organisms. Further, using MRI, a differential signal can be obtained that provides information as to whether the contrast agent is inside or outside the liposome, thus providing for monitoring of contrast agent/drug release from the liposome.
VI.D. Real Time Monitorinq
Yet another advantage of the present methods and compositions is the ability to perform real time monitoring of events occurring in vivo. A researcher can monitor, for example, the accumulation of liposome compositions at a heated site as the accumulation is occurring. The only delay is that delay associated with obtaining (for example, about 0.5-4 minutes, depending on scan settings) and processing (for example, less than about 3-6 seconds) the nucleus relaxation times to images.
This ability can be of great benefit in determining the time scale upon which events are occurring. This can translate into an estimate of the efficiency with which a given compound of interest is not only delivered to a site of interest, but also how the compound behaves once it has been delivered.
VI.E. A Site can be Heated by Invasive/Noninvasive Techniques
In one aspect of the present disclosure, a site of interest is heated. Heating of the site of interest leads to the accumulation of liposome composition at the heated site over a period of time (FIGS. 2A-2B). Without heating the site of interest, the liposome might at best diffusely accumulate at the site (particularly if it is a tumor) for a brief time (FIG. 2E), or not at all (FIG. 2D). The site can be heated by invasive or non-invasive methods and can depend on the nature of the site being heated.
For example, in one aspect, a site (i.e., a tumor) is heated by inserting a catheter which is heated by circulating water, into the tumor mass. Other similar methods of invasive heating can be employed.
In other instances, it might be desirable to heat a site noninvasively. Such heating can be achieved by employing a method such as directing RF, IR, microwave or ultrasonic energy at the site. In these cases, it can be desirable to monitor the temperature of the heated site for thermal damage to the site.
In yet other instances, a laser can be employed to heat the site. Typically, when the site of interest is near the outer surface of the subject, such as on or near the skin, a laser, for example a laser emitting light at an IR frequency, can be employed to heat a site. In another example, the site is a subject's eye, and a laser can be employed to locally heat an exposed region of the eye.
VI.F. High Resolution Detection
One potential drawback to some MRI techniques is that the images generated are sometimes of low resolution due to a poor signal-to-noise ratio. To overcome this limitation, spatial resolution is sacrificed in order to have enough signal to generate an image. ESR, NMR and resonance frequency shift are several techniques that suffer from the lower S/N ratio or the use of different atom of excitation such as 13C. The presently disclosed methods and compositions, on the other hand, employ the protons of water. The effect of the contrast agent on these protons is minimal. The methods and compositions disclosed herein, on the other hand, routinely provide high resolution images of a site, as depicted in FIGS. 2A and 2B.
VI.G. Low Toxicity
A further advantage is the observation that the liposome compositions are relatively less toxic and/or damaging to a subject than other MR-based contrast agent-containing compositions. Some MR contrast agents, such as gadolinium-based contrast agents, can be toxic in large amounts. Often these amounts are required to generate high-quality images useful for diagnostic purposes.
Yet other imaging techniques require the use of radioactive compositions to monitor in vivo events, such as blood pool detection. Moreover these methods do not yield detailed high resolution images. For example, positron emission tomography (PET) is commonly employed, but cannot be employed to generate high resolution images. In another example, multiple-gated arteriography (MUGA) is typically employed in the study of left-ventricular function and wall motion of the heart. In MUGA, a subject's red cells are infused with 99Tc, a radioactive material. The radiation emitted from the 99Tc is then detected and used to form an image of the blood pool within the subject's heart. Images can be acquired by employing a gamma camera, and data can be acquired at times of interest to a clinician or researcher. While MUGA has been effective, it requires the use of potentially damaging radioactive materials and can lead to toxic anaphylacetic shock.
Yet other methods employ fluorescent dyes, which can themselves be toxic or can lead to undesirable conditions. Additionally, such approaches are limited to sites that are near an exposed surface of a patient, such as the skin or the surface of the eye.
The presently disclosed methods and compositions, on the other hand, employ contrast agents that can be less toxic to a subject. For example, Cu or Zn, which exhibit very low in vivo toxicity and good contrast agent properties, can be used. Even compounds that exhibit some toxicity, such as Mn, are safe to use in vivo with the presently disclosed methods and compounds, as the liposomes shield the contrast agent until the liposome loses structural integrity at the site of interest. Further, since the liposomes are targeted to a specific area of interest, less contrast agent is required to achieve results similar to or superior than those found when the contrast agent is administered at higher (potentially toxic) doses systemically.
LABORATORY EXAMPLES
The following Laboratory Examples have been included to illustrate representative modes. Certain aspects of the following Laboratory Examples are described in terms of techniques and procedures found or contemplated to work well in the practice of the presently disclosed subject matter. These Laboratory Examples are exemplified through the use of standard laboratory practices. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Laboratory Examples are intended to be exemplary only and that numerous changes, modifications and alterations can be employed without departing from the spirit and scope of the present disclosure.
Laboratory Example 1
Liposome Formulation
Liposomes were made from either DMPC/Chol (55:45) mole per mole (mol/mol) or DSPC/Chol (55:45) (mol/mol) and were prepared by extrusion methods. Lipids, at the indicated ratios, were dissolved in chloroform, and then 3H-CHE was added to achieve approximately 10 μCi/100 mg lipid. The chloroform was removed under a gentle stream of nitrogen gas and, subsequently, the lipid samples were placed under a high vacuum for a time period of at least 4 hours to remove any residual solvent. The dried samples were hydrated (such that the final lipid concentration was approximately 100 mg/ml) with 300 mM citrate buffer, 300 mM MnSO4 or 300 mM MnCl2, adjusted to pH 3.5 by addition of hydrochloric acid. Following hydration, the multilamellar vesicles (MLVs) were subjected to five freeze-and-thaw cycles (freezing in liquid nitrogen and thawing at 40° C.). The resulting frozen and thawed MLVs were prepared to optimize the salt distribution across the lipid bilayers (Mayer et al., (1985) Biochim. Biophys. Acta 817: 193-196). The MLVs were extruded 10 times through stacked polycarbonate filters of 0.1 and 0.08 μm pore size at 40° C. using a water-jacketed ExtruderTM (Northern Lipids, Inc., Vancouver, British Columbia, Canada). The mean size distribution of all liposome preparations was determined using a Nicomp Sub-micron Particle Sizer Model 270 (Pacific Scientific, Santa Barbara, Calif.) operating at 632.8 nm. The resulting liposome preparations typically exhibited an average diameter of approximately 100-120 nm. All liposomes, except those used for cTEM analysis and circular dichroism (CD) studies were radiolabeled. For these experiments, the phospholipid was quantitated using the Fiske and Subbarow (Fiske & Subbarow, (1925) J. Biol. Chem. 2: 375-395) phosphate assay. Briefly, 700 μl of 70% perchloric acid was added to lipid samples and heated to approximately 180-200° C. for 2 hours until the samples were colorless. Samples were cooled and 700 μl of Fiske reagent and 7 ml of ammonium molybdate was added and samples were subsequently reheated again to 100° C. for 20 min. Samples were cooled to room temperature and the absorbance was read at 820 nm.
Laboratory Example 2
Doxorubicin Encapsulation
Large unilamellar liposomes in buffer were fractionated on SEPHADEX® G-50 columns (Amersham Biosciences Corp., Uppsala Sweden) (1 ml sample volumes were placed on columns with at least a 20 ml column bed) equilibrated with various buffers at pH 7.5. The buffers used for the external environment included 25 mM HEPES/150 mM NaCl (HBS) for the liposomes with encapsulated 300 mM citrate and 300 mM sucrose/20 mM HEPES/15 mM EDTA (SHE) for the liposomes with encapsulated MnSO4 and MnCl2. The manganese sulfate-based procedure for doxorubicin loading was a modification of the method described by Cheung et al. (Cheung et al., (1998) Biochim Biophys Acta 1414(1-2): 205-16). In particular, it is noted that the solubility of 300 mM MnSO4 is highly variable under the pH conditions described in the original procedure, presumably resulting in the formation of Mn(OH)2(S). In the studies reported here, liposomes were prepared in a 300 mM MnSO4 (and MnCl2) solution at a pH of 3.5; solutions that are stable for months at room temperature.
Following formation of the salt gradients as described above, the liposome lipid concentration was adjusted to 10 mg/ml and, subsequently, doxorubicin was added to achieve a drug-to-lipid ratio (wt/wt) of 0.2:1 at 20, 40 and 60° C. The accumulation of doxorubicin into liposomes was determined at the indicated time points by removing 100 μl aliquots and separating unencapsulated drug from encapsulated drug on 1 ml Sephadex G-50 (medium) spin columns equilibrated with the appropriate buffer. The concentration of doxorubicin in the excluded fraction was determined by measuring absorbance (at 480 nm) of a solution consisting of the sample, adjusted to 100 μl with HBS or SHE, to which 900 μl of 1% Triton X-100 was added. Prior to assessing absorbance at 480 nm, the sample was placed in >90° C. water bath until the cloud point of the detergent was observed. Liposome lipid concentrations were determined by adding a small aliquot of the excluded fraction to 5 ml of scintillation cocktail, where the radioactivity of the sample was subsequently determined by scintillation counting with the Packard 1900TR Liquid Scintillation Analyzer.
Laboratory Example 3
Sample Solution Formation
Liposomes made using the above procedure had their concentration converted to an equivalent MnSO4 molarity using the molecular weight of the lipid and an assumption of a 1.2 μl/μmollipid (Mayer et al., (1985) Biochim. Biophys. Acta 817(1): 193-6) encapsulation volume commonly used to characterize liposome volume from concentration. Consequently, a 1 mg/ml lipid concentration with 300 mM MnSO4 encapsulated concentration resulted in an overall MnSO4 concentration of 0.437 mM for DPPC-MSPC-DSPE-PEG2000 and 0.592 mM for DSPC/Chol for full liposome content release.
Laboratory Example 4
Manganese Sulfate Control Stock Solutions
Three control stock solutions of manganese sulfate (MnSO4.H2O), free DOX and MnSO4.H2O in a molar ratio of 0.902:1 (DOX:Mn), and MnSO4.H2O with thermally sensitive liposomes that were formed without the presence of MnSO4.H2O. These solutions, along with liposome solutions, were then diluted to concentrations from 0.25-8 mM. The solutions were placed in 5 mL NMR tubes cut to a three-inch length.
Laboratory Example 5
In Vitro MRI Scans
All MRI experiments were performed at the Center for In vivo Microscopy at Duke University in a 2-T 30-cm bore diameter magnet (Signa, G.E. Medical Systems, Milwaukee, Wis.). Temperature control within the bore was achieved with an automatic feed back control unit that was initially designed for maintaining temperature of laboratory animals (Qiu, (1997) IEEE Trans. Biomed. Eng. 44(11): 107-1113). Each experiment had six to eight sample tubes, which were contained within an exposed sample holder, and placed inside a 3.81 cm birdcage coil providing RF excitation. A temperature on the heating apparatus was chosen (at random for control experiments) and bore equilibration occurred within ten minutes. A series of T1 images were then taken in the axial plane with a field of view of 5 cm at excitation times (Te) of 8 ms and repetition times (Tr) of 20, 33.3, 66.6, 150, 316, 633, and 1266 msec. Between six and eight temperature relaxivity measurements were obtained. Each experiment was repeated three times with independent formulated reagents. Liposomes were tested sequentially with a minimum of two measurements below-above-and-back below the transition temperature for the LTSL. The NTSLs were tested in the same manner to illuminate any possible hysteresis.
Images that were obtained were analyzed by selecting a region of interest and measuring average intensity for each sample dilution. Intensity data were fit using non-linear least squares data fitting governed by the Gauss-Newton method, MATLABTM (The MathWorks, Inc., Natick, Mass., United States of America), to obtain a T1 for each concentration tested. Reciprocal T1 data were plotted as a function of concentration and fitted to a linear regression yielding relaxivity (mM sec)−1 as the slope. Relaxivity was plotted as a function of temperature.
Laboratory Example 6
In Vivo Experiments
Fibrosarcocomas were transplanted in the flank of fisher 344 rats and allowed to grow to 17-19 mm in diameter. Rats were anesthetized with pentobarbital (source/amount) and a 16 gauge catheter was placed through the center of the tumor. Heated water (56° C., 1.8 ml/s) was passed through the catheter to provided hyperthermia. During MRI scans, the heating water was doped with 0.25 M MnSO4 to prevent flow image artifacts. Gradient recalled echo was used with a Tr of 23 ms, Te of 1.4 ms, and flip angles of 5, 15, 30, 40, 50, 60 degrees for the initial T1 weighted image. Subsequent drug uptake scans were performed at a flip angle of 33°. 3D volume images, 12 slices 1 mm thick, were taken with a field of view of 6 cm with a pixel size of 234×234 μm. Four and signal image averaging was used for liposome and Gd-DTPA experiments yielding scan times of 1 min, 45 sec and 30 sec respectively.
1NEX image averaging was used for the first twenty minutes of liposome or Gd uptake. Subsequent images were taken at 4NEX for the remainder of the experiment (90-120 minutes).
Results and Discussion of Laboratory Examples 1-6
To quantify the concentration of drug in vivo using MRI, in vitro experiments were performed to determine the concentration- and temperature-dependence of T1 shortening. These experiments measured signal intensity resulting from proton spin realignment to the bulk magnetic field after excitation from repetitive radio frequency pulses. The signal received is dependent on the repetition times (Tr) between RF pulses. This dependency of signal on Tr is illustrated in FIG. 1A. MnSO4 liposome-DOX formulation measured over a range of drug concentrations (T=29.9° C.). The curves shown in FIGS. 1A-1D are governed by Equation 1. The data were fit to this equation to obtain relative proton density (PD), relaxation time constant (T1), and baseline noise (C) for each concentration/temperature combination:




  
    
      
        signal
        =
        
          
            PD
            ⁡
            
              (
              
                1
                -
                
                  exp
                  ⁡
                  
                    (
                    
                      
                        -
                        Tr
                      
                      
                        T
                        ⁢
                        
                          
                        
                        ⁢
                        1
                      
                    
                    )
                  
                
              
              )
            
          
          +
          C
        
      
    
    
      
        Equation
        ⁢
        
          
        
        ⁢
        1
      
    
  




The rate of rise of the signal versus Tr (shortening of Tr increases with drug concentration) (FIG. 1). Changes in the curve's asymptotic value, PD, are governed by the number of protons in a sample and by the effects of T2. There is a linear relationship between the reciprocal of T1 (1/T1) and the MnSO4 concentration, and the slope of this line is termed “relaxivity” (mM·sec)−1 (FIG. 1B). It is possible to determine in vivo MnSO4 concentration by measuring T1 shortening and using the in vitro calibration for relaxivity (Lyng, (1998) Mag. Reson. Med. 40(1): 89-98). Since the molar ratio between MnSO4 and DOX is known for the liposome formulations, one can then determine DOX concentration.
Relaxivity was measured for MnSO4±DOX, free MnSO4+empty low temperature sensitive liposomes (LTSL), and MnSO4±DOX loaded into non-thermally-sensitive liposomes (NTSL) over a range of temperatures and drug concentrations. Relaxivity changed as a function of temperature, but this relationship was different for free MnSO4±DOX and the NTSL (FIG. 1C). MnSO4+empty LTSL and free MnSO4±DOX exhibit decreased relaxivity with increased temperature, presumably due to increased thermal motion of H2O and Mn. This increased thermal motion reduces their interaction, thereby limiting the ability of Mn to shorten T1. MnSO4+DOX and MnSO4−DOX have identical relaxivity profiles indicating that DOX, at a concentration expected to be seen in vivo, has no effect on this relationship. In contrast, relaxivity of NTSL±DOX increases as temperature is raised. This inverted temperature dependence might be due to an increase in liposome permeability causing an increase in water exchange across the lipid bilayer (Fossheim, (1999) Magn. Reson. Imaging 17(1): 83-9; Bacic, (1988) Magnet. Reson. Med. 6(4): 445-58).
Linear regressions for relaxivity as a function of temperature were obtained for all in vitro samples (FIG. 1C). MnSO4+LTSL, MnSO4, and NTSL±DOX had slopes±S.E. of −166±0.023, −0.082±0.003, and 0.064±0.005 (mM·sec·° C.)−1 and intercepts±S.E. of 13.67±0.087, 7.28±0.12, −1.41+0.18 (mM·sec)−1, respectively. These results are similar to previous results reported for free MnCl2 and comparable to other liposome formulations using Gd either loaded into the liposomes and/or bound to its surface (Suga, (2001) Invest. Radiol. 36(3): 136-45; Fossheim, (1999) Magn. Reson. Imaging 17(1): 83-9; Unger, (1993) J. Mag. Res. Imag. 3: 195-198; Schwendener, (1990) Invest. Radiol. 25(8): 922-32; Niesman, (1990) Invest Radiol 25(5): 545-51). For comparison, Gd-DTPA (MAGNAVISTTM) has a relaxivity of approximately 44.5 (mM·sec·° C.)−1 (Unger, (1993) J. Mag. Res. Imag. 3: 195-198) at physiological temperatures compared to 4.25 (mM·sec·° C.)−1 for the LTSL.
The relaxivity measurements for the LTSL are measured over a range of temperatures between 30 and 45° C. showing a complex relationship (FIG. 1D). These measurements were taken at several temperature points below and above the transition temperature for this liposome formulation (38-40° C.), which is the temperature at which the liposome goes from solid to liquid phase (Needham, (2000) Cancer Res. 60(5): 1197-201; Magin, (1984) Cancer Drug Delivery 1(2): 109-17). Measurements were started below the transitions temperatures, escalated beyond the transitions, and then were repeated as the solutions were cooled back below the transitions (see arrows, FIG. 1D). During initial heating period, LTSL had relaxivity values similar to those of the NTSL. When the phase transition temperature for LTSL was approached, however, relaxivity dramatically raised to levels nearly equivalent to those for free MnSO4+empty LTSL. This was not unexpected as content release from the LSTL formulation happens within seconds of reaching these temperatures, occurring in a burst like manner (Needharn, (2000) Cancer Res. 60(5): 1197-201). These relaxivity measurements support the previous reports that drug release from this liposome occurs just below the transition temperature (Needham, (2000) Cancer Res. 60(5): 1197-201; Magin & Niesman, (1984) Canc. Drug Del. 1(2): 109-17). Once release of liposomal contents occurred, further heating showed decreased relaxivity, consistent with levels seen for free MnSO4 in solution. Relaxivity increased during subsequent cooling in a manner parallel to MnSO4+empty LTSL; the relaxivity slope of MnSO4 loaded into LTSL, post release, was −0.157±0.0167 (mM·sec·° C.)−1 which is the same as that of MnSO4+empty LTSL at −0.166±0.023 (mM·sec·° C.)−1. However, the intercepts for LTSL and the MnSO4+empty LTSL are somewhat different, suggesting that the liposomes did not release all of their contents. The absence of complete content release from the LTSL formulation has been reported (Needham, (2000) Cancer Res. 60(5): 1197-201).
The liposome formulations were studied in fibrosarcomas implanted in the flank of Fisher 344 rats. The center of the tumor was heated using a catheter through which hot was water circulated. This heating system provided a radial temperature distribution, the profile of which is predicted by the 1D steady state heat equation (insert FIG. 2A, timepoint 1). Steady state temperature distributions were reached after 30 minutes of HT treatment. Liposomes were then administered intravenously, followed by MR imaging.
MR imaging following NTSL administration demonstrated signal enhancement in the tumor when treated with HT (see FIG. 2A), but not with NTSL administration in the absence of HT (see FIG. 2D). Enhanced signal intensity was initially seen in relatively large vessels immediately after injecting liposomes. These vessels remained visible throughout the course of the experiment (90 minutes). This result verifies that these sterically stabilized liposomes have a relatively long circulation time. Signal enhancement progressively increased in the tumor, consistent with time dependent liposome accumulation. There was clear heterogeneity in enhancement, presumably due to heterogeneity in blood perfusion and/or vessel pore size (Kong et al., (2001) Cancer Res. 61(7): 3027-32). There was no enhancement in adjacent normal tissue, suggesting delivery was limited to tissues that were heated.
The results obtained for the LTSL are different from those for the NTSL. As shown in FIG. 2B at identical time intervals, vessel enhancement is also seen with the LTSL as it was with the NTSL, but its intensity begins to decrease after 15-20 minutes. FIG. 2E shows that without heating, a less intense but more uniform signal enhancement occurs, but then rapidly fades after five minutes as clearance from the vasculature occurs. Unlike the NTSL, the LTSL showed strong signal enhancement at the tumor periphery as well as within the tumor parenchyma. Again, although it is not the inventors desire to be bound to any particular theory of operation, it is speculated that the enhancement at the edge of the tumor might be due to the higher vascular density in such regions or intravascular release as liposomes approach the tumor. The temperature at the tumor capsule is between 38.5-41° C., which is high enough to cause MnSO4 and DOX release. There is minimal enhancement of surrounding tissue using the LTSL, further supporting the premise that delivery of the liposome and/or its contents had occurred preferentially in the heated zone. Experiments were also performed using NTSL and LTSL without HT. In this case, signal enhancement was not seen in any tissue.
Serial measurements of signal enhancement following Gd-DTPA injection are shown in FIG. 2C as a typical contrast MR image (without HT) for comparison with liposome results. The panels represent 0, 0.5, 5, and 15 minutes post injection. The rate of signal enhancement occurs over a shorter time scale than with liposomes. Enhancement occurs in all tissues with a high degree of non-uniformity in the tumor. This distribution pattern can be attributed to the high vascular permeability of Gd-DTPA as a result of its low molecular weight (508 g/mol).
In vivo drug relative concentrations were performed by signal intensity image analysis over regions of interest (ROI) within heated tumors. The white boxes present in FIGS. 2A, 2B and 2C indicate these ROIs. ROIs in the tumor represent an enhanced tumor region, +ET, where signal enhancement occurred and a non-enhanced tumor region −ET, where image enhancement did not occur (FIG. 3B). Muscle in the non-tumor bearing leg of the animal was used as a non-heated, normal tissue control. ROI signal intensities for NTSL were normalized with muscle in the non-tumor bearing leg to yield a signal intensity ratio (SIR) (Takehara et al., (2001) Br. J. Cancer 84(12): 1681-5). The procedure was repeated for the LTSL and the Gd-DTPA and the SIR for the vessel, tumor enhancing region, and the non-tumor enhancing region is shown in FIGS. 3D, 3E, and 3F, respectively.
SIR calculations were also performed on the femoral vein in the NTSL, LTSL and Gd-DTPA experiments (see FIG. 3D). Femoral vessel SIR for NTSL increased to a value of 2.25 at 10-15 minutes and remained constant throughout the experiment, consistent with a long circulation time. Femoral vessel SIR for the LTSL shows a rise to 2.5 at 15-20 minutes and subsequent drop off toward base line levels by 60 minutes post injection. This decrease in LTSL SIR might be due to a relatively short circulation time, which has been documented in previous studies. Gd-DTPA also shows a rapid increase to a plateau value of a SIR equal to 2.0 after 10 minutes. This value remains constant through the course of the experiment but would decrease if the experiment had been followed long enough for the contents to wash out of the tissue. It is documented that 83%±14% of the Gd-DTPA is excreted in urine in about 6 hours. The mean elimination half-life of Gd-DTPA is about 0.2±0.13 hours. (Berlex Imaging MAGNAVIST® package insert).
All tumor-enhancing regions tended to reach the same SIR plateau value of 2-2.25, but the rate at which they reached the plateau was different (see FIG. 3E). Gd-DTPA reached a plateau by 10 minutes and remained constant up to 20 minutes. The NTSL had the slowest rise reaching a plateau after 50 minutes, and a maximum of 2.25 after 70 minutes. LTSL had an intermediate response rising to a value of 2 after 20 minutes and a maximum of 2.25 after 30 minutes. The rapid increase of the SIR for Gd-DTPA can be attributed to the small size of the contrast agent. Vascular permeability to molecules than 1000 MW is dominated by the concentration gradient across the vessel wall (Yuan et al., (1995) Cancer Res. 55: 3752-6; Yuan et al., (1994) Cancer Res. 54: 3352-56). At 508 g/mol Gd-DTPA is highly permeable to the vessel walls. The lower rate of increasing SIR for the NRSL is attributed to the relatively low permeability of the liposomes through the vessel. The rapid release kinetics of the LTSL (50% drug release <20 seconds) indicates that at least some of the drug is released intravascularly (Kong et al., (2000) Cancer Res. 60(24): 6950-7). Therefore, the formulation is expected to show SIR kinetics that are intermediate between that of the free contrast agent and the NTSL, which is not thermally sensitive.
The non-tumor enhancing region, FIG. 3F, shows qualitatively similar results when comparing NTSLs with Gd-DTPA experiments. Both experiments have SIRs that rise to 0.4 to 0.6 after 20 minutes. The NTSL SIR remains constant thereafter. The SIR for the LSTL remains constant through the entire experiment. The difference for this response might be due to the rapid release of LTSL contents creating a concentration gradient that drives delivery into regions of the tumor that are poorly perfused.
Laboratory Examples 1-6 demonstrate the ability to image MnSO4 encapsulated within thermally sensitive and non-sensitive liposomes. Additionally these Laboratory Examples show that content release from the liposomes is also imageable. These Laboratory Examples demonstrate the distribution of NTSL and LTSL liposomal drug delivery in animal studies. These results are applicable to other drugs encapsulated in this particular liposome. Because the labeling agent is required for the drug loading, other liposome/drug formulations are possible and can be similarly used for real time monitoring of drug delivery. By employing an in vitro approach, such as that performed in the Laboratory Examples presented herein, quantification of drug concentration distribution can be obtained. This ability allows for monitoring of liposomal drug delivery in individual patients, leading to individualized treatment, which can increase overall efficacy.
Laboratory Examples 7-12
Materials and Methods
Materials
The following phospholipids were purchased from Avanti Polar Lipids Inc. (Alabaster, Ala., U.S.A.): 1,2 dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC); 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (MSPC); and 1,2 distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). Sephadex G-50 medium was purchased from GE Healthcare (Piscataway, N.J.). HEPES sodium salt, manganese sulfate monohydrate, sucrose, ammonium phosphate dibasic, perchloric acid, and ammonium molybdate were purchased from Sigma-Aldrich (St. Louis, Mo., U.S.A.). Doxorubicin was purchased from Pharmacia and Upjohn company (Kalamazoo, Mich., United States of America.
Liposome Preparation
Temperature-sensitive liposomes were prepared from DPPC, MSPC, and DSPE-PEG2000, which were combined in a molar ratio of 90:10:4 as previously described (Viglianti et al. (2004) Magn Reson Med 51:1153-62; Abraham et al. (2002) Biochim Biophys Acta 1565:41-54). The phospholipids were combined in chloroform, and the solvent was removed using nitrogen gas and high vacuum. The dried samples were hydrated with 300 mM manganese sulfate (MnSO4) at pH 3.5 to a lipid concentration of 80 mg/mL. Hydration was performed at 55° C. for 30 minutes. The hydrated lipid vesicles were extruded 10 times through stacked polycarbonate filters of 0.1- and 0.08-μm pore size at 55° C. by using a water-jacketed extruder (Northern Lipids Inc., Vancouver, Canada). The phospholipid concentration was measured using the Fiske and Subbarow phosphate assay, as previously described (Fiske & Subbarow (1925) J Biol Chem 2:375-95; Bartlett (1959) J Biol Chem 234:466-8). Briefly, 70% perchloric acid was added to liposome samples as well as to phosphate dibasic standard solutions (concentration range=0.25-1 mM). The mixtures were heated at 180-200° C. for 2 hours. After cooling, Fiske reagent and ammonium molybdate were added, and the samples were reheated to 100° C. for 20 minutes. Absorbance at 820 nm was linearly related to phosphate concentration and was measured for all samples and standards.
Production of Liposomes Containing Doxorubicin (Dox/Mn-LTSLs)
Liposomes (1-mL aliquots) were fractionated on 20-mL Sephadex G-50 columns that had been pre-equilibrated with 300 mM Sucrose/20 mM HEPES buffer (pH 7.5) to remove unencapsulated MnSO4. Doxorubicin was then added to the fractionated liposome solution at a drug-to-lipid ratio of 0.05:1 (wt:wt), and loading of doxorubicin into liposomes was conducted at 37° C. for 80 minutes (Viglianti et al. (2004) Magn Reson Med 51:1153-62; Viglianti et al. (2006) Magn Reson Med 56:1011-8; Abraham et al. (2002) Biochim Biophys Acta 1565:41-54). Liposomes were fractionated again on Sephadex G-50 columns that had been pre-equilibrated with HEPES buffer to remove unencapsulated doxorubicin, and the concentration of the liposome-encapsulated doxorubicin was estimated using fluorimetry with excitation and emission wavelengths of 480 nm and 550 nm, respectively.
In Vivo Therapeutic Studies in Rats
Tumor model. The tumor model used in this study was a fibrosarcoma (FSA-1, provided by J. Bull, M.D. Anderson Cancer Center, Houston, Tex., U.S.A.) that was originally isolated from the subcutis of rats that were given the carcinogen methylcholanthrene and was maintained by serial transplantation (Viglianti et al. (2004) Magn Reson Med 51:1153-62; Viglianti et al. (2006) Magn Reson Med 56:1011-8; Grant & Wells (1974) J Surg Res 16:533-40). The fibrosarcoma was transplanted subcutaneously in the flanks of 8-week-old female Fischer 344 rats (Charles River Laboratories, Wilmington, Mass., U.S.A.) under anesthesia using intraperitoneal ketamine (9 mg/kg body weight) and xylazine (10 mg/kg body weight). Tumors were allowed to grow for approximately 2 weeks to a diameter of 10-12 mm before treatment.
Treatment protocols. Tumor-bearing rats were anesthetized with an intraperitoneal injection of pentobarbital (45 mg/kg) and randomly assigned to one of the following eight treatment groups (n=9-10 rats per group): saline only (control), hyperthermia alone, free doxorubicin alone, free doxorubicin during hyperthermia, Dox/Mn-LTSL alone, and Dox/Mn-LTSL with hyperthermia given according to one of three protocols. In Protocol 1, hyperthermia was initiated 15 minutes before Dox/Mn-LTSL was administered so that a thermal steady state was reached before the drug was delivered (Dox/Mn-LTSL during hyperthermia). In Protocol 2, Dox/Mn-LTSL was administered 15 minutes before the initiation of hyperthermia (Dox/Mn-LTSL before hyperthermia). In Protocol 3, half of the dose of Dox/Mn-LTSL was administered 15 minutes before initiation of hyperthermia and the other half was administered after thermal steady state was reached (i.e., 15 minutes after the initiation of hyperthermia; Dox/Mn-LTSL split dose).
Each group of rats (except the control group) received an equivalent dose of 5 mg doxorubicin/kg body weight by intravenous injection and/or 1 hour of hyperthermia. Local hyperthermia was delivered by means of an 18-gauge catheter that was placed through the center of the tumor and through which heated water ( ̃50° C., 1.8 mL/s) was passed. This magnetic resonance-compatible heating system provides a reproducible radial temperature distribution that reaches thermal steady state within 15 minutes (Viglianti et al. (2004) Magn Reson Med 51:1153-62). The average temperatures were previously determined to be 45-46° C. adjacent to the catheter and 38.5-39.5° C. at the tumor border (Viglianti et al. (2004) Magn Reson Med 51:1153-62). A rectal thermistor was used to monitor the body temperature of the rats during treatment and during MRI (Qiu et al. (1997) IEEE Trans Biomed Eng 44:1107-13).
For each treatment group, three rats were used to measure doxorubicin concentrations in tumors and hearts by HPLC. The rats were euthanized by carbon dioxide asphyxiation 90 minutes after Dox/Mn-LTSL injection (an interval similar to that between injection and the time the final magnetic resonance images were obtained), and their tumors and hearts were immediately harvested, snap frozen, and stored at −80° C. The remaining 6-7 rats in each treatment group were included in the survival analysis. Magnetic resonance images were acquired before, during, and after therapy for rats in the three Dox/Mn-LTSL plus hyperthermia groups. Final images were taken after tumors cooled to room temperature. Treatment groups that did not receive Dox/Mn-LTSL were not imaged. After therapy, the rats were monitored as they recovered from anesthesia. Tumor volumes were measured three times per week for 60 days or until the tumor volume reached five times the volume on the day of treatment. At these endpoints, the rats were killed by carbon dioxide asphyxiation. The 60-day cutpoint was chosen for comparison to previous studies with Dox-LTSL (2, 3). The five-times-the-initial-volume cutpoint was chosen based on guidelines from the institutional animal care and use committee. No rats were killed before the specified endpoints were reached. This animal protocol was approved by the Duke University Animal Care and Use Committee, Durham, N.C., U.S.A.
MRI protocol. MRI data were acquired before and during therapy with the use of a 2T 30-cm diameter bore magnet (Oxford Instruments, Oxford, UK) and a Signa magnetic resonance console (GE Healthcare, Waukesha, Wis., U.S.A.). A series of axial T1-weighted images were acquired using a three-dimensional spoiled gradient-recalled echo acquisition at flip angles of 2°, 5°, 12°, 17°, 20°, 33°, 45°, and 70° to estimate the initial T1 map (Viglianti et al. (2004) Magn Reson Med 51:1153-62; Viglianti et al. (2006) Magn Reson Med 56:1011-8). These images were acquired by using the following parameters to produce the greatest T1 contrast: repetition time=22 ms; echo time=minimum; matrix=256 256 28; field of view=6 cm; slice thickness=1 mm; bandwidth=15.63 Hz/pixel; and number of excitations=1. Images acquired using these parameters had a voxel size of 234 μm 234 μm 1000 μm. Magnetic resonance scans during and after Dox/Mn-LTSL treatment were performed with a flip angle of 33° and a scan time of 90 seconds. These axial scans were acquired continuously throughout therapy by starting a new scan every 90 seconds (Viglianti et al. (2004) Magn Reson Med 51:1153-62; Viglianti et al. (2006) Magn Reson Med 56:1011-8).
Analysis of Magnetic Resonance Images to Estimate Doxorubicin Concentration
Magnetic resonance images were analyzed to estimate the doxorubicin concentration for each voxel at each time point, as previously described (Viglianti et al. (2004) Magn Reson Med 51:1153-62; Viglianti et al. (2006) Magn Reson Med 56:1011-8). Briefly, the initial T1 and S0 values for each voxel were obtained by fitting the signal intensity at variable flip angles (α) to the following equation:




  
    signal
    =
    
      
        S
        0
      
      ⁢
      
        
          sin
          ⁢
          
            (
            α
            )
          
          ⁢
          
            (
            
              1
              -
              
                exp
                ⁡
                
                  (
                  
                    
                      -
                      
                        T
                        R
                      
                    
                    /
                    
                      T
                      1
                    
                  
                  )
                
              
            
            )
          
        
        
          (
          
            1
            -
            
              
                cos
                ⁡
                
                  (
                  α
                  )
                
              
              ⁢
              
                exp
                ⁡
                
                  (
                  
                    
                      -
                      
                        T
                        R
                      
                    
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                      T
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                  )
                
              
            
          
          )
        
      
    
  
  ,




where S0 accounts for the proton density and T2 effects (Wehrli et al. Biomedical magnetic resonance imaging: principles, methodology, and applications. John Wiley and Sons Ltd. New York; 1988). Each subsequent image was converted to a T1 map with the use of the new signal values and the initial values of S0. Signal enhancement was assumed to be caused by T1 shortening (Wehrli et al. Biomedical magnetic resonance imaging: principles, methodology, and applications. John Wiley and Sons Ltd. New York; 1988). The linear relationship between 1/T1 and the MnSO4 concentration described in the equation:





  
    
      (
      
        
          1
          
            T
            1
            dynamic
          
        
        -
        
          1
          
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            1
            0
          
        
      
      )
    
    =
    
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      ⁢
      
        
      
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            C
            
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was used to convert changes in T1 to manganese concentrations. The relaxivity of manganese





  (
  
    
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is dependent on the temperature and encapsulation state of the manganese ions (Wehrli et al. Biomedical magnetic resonance imaging: principles, methodology, and applications. John Wiley and Sons Ltd. New York; 1988). Therefore, to calculate R1 values for each voxel, we used previously published (Viglianti et al. (2004) Magn Reson Med 51:1153-62) in vivo temperature measurements for this tumor model, and complete release of manganese from the liposomes was presumed. Manganese concentrations (mM) were converted to doxorubicin concentrations (μg/mL) by multiplying CMn2+ by the ratio of encapsulated doxorubicin to encapsulated manganese in the injected liposomes. The final T1-based maps of doxorubicin concentrations were compiled as images similar to the MR images, with the concentration represented by the intensity at each voxel (Viglianti et al. (2004) Magn Reson Med 51:1153-62; Viglianti et al. (2006) Magn Reson Med 56:1011-8). This conversion assumes that doxorubicin and manganese remain colocalized after they are released from the liposome.

Spatial and Temporal Analysis of Drug Delivery
We used the final T1-based maps of doxorubicin concentrations in the tumors to perform a quantitative analysis of the spatial distribution of doxorubicin in the tumor. For each rat, eight radial line profiles of doxorubicin concentrations were acquired from each of the 10 central tumor slices along lines that radiated, at 45° angles, from the central catheter to the edge of the tumor (as illustrated in FIG. 5A). The lengths of these lines were normalized, such that the catheter had a radial value of zero and the edge of the tumor had a radial value of 1.0. The mean doxorubicin concentration at each radial distance was calculated for each rat and then for each treatment group. For each rat, the area under the curve (AUC) in the peripheral portion of the tumor (defined as radial values from 0.5 to 1.0) was compared with the AUC in the central portion of the tumor (defined as radial values from 0 to 0.5) to calculate the doxorubicin distribution (i.e., AUC) ratio. The AUC ratio is thus greater than 1 if the doxorubicin is concentrated at the edge of the tumor and less than 1 if the doxorubicin is concentrated at the center of the tumor.
For each rat, the total amount of doxorubicin that was delivered to the whole tumor at each time point was calculated by multiplying the volume of each voxel (0.055 mm3) by the concentration of doxorubicin and then summing the doxorubicin levels over all voxels. Error bars indicate 95% confidence intervals (CIs) among animals in a given group at a given time point. The initial rate of doxorubicin accumulation in the tumor (μg doxorubicin per minute) was averaged over the first 3 minutes of combined treatment for each rat and each treatment group.
HPLC Analysis of Tumor Doxorubicin Concentration
Frozen tumors and hearts were thawed and homogenized, and doxorubicin concentrations were measured by HPLC as previously described (2, 17). In brief, doxorubicin was extracted from the homogenates using chloroform and silver nitrate. The organic phase was collected, dried, and reconstituted in isopropanol. Doxorubicin concentration was measured by fluorimetric emission at 550 nm following HPLC separation. A standard concentration set was prepared from homogenates of tumors from the control group that were spiked with known amounts of doxorubicin.
Statistical Analysis
Descriptive summary statistics are expressed as mean values with 95% confidence intervals, except for the time-to-five-times-tumor-volume data, which are expressed as median values with 95% confidence intervals. To avoid assuming a normal distribution, nonparametric or semi-parametric statistical methods were used. Group comparisons were conducted with the Kruskal-Wallis test for more than two groups and the Wilcoxon test for between-group differences. For the antitumor efficacy data, the primary endpoint (time to reach five times the original tumor volume) was analyzed by the Kaplan-Meier product-limit method, using the Wilcoxon rank test for between-group differences. Censoring was taken into account for animals that showed complete regression and were therefore killed at 60 days. The Cox proportional hazards regression technique for growth time was also employed to explore whether T1-based doxorubicin concentration was statistically significantly associated with the time to five times the initial tumor volume. This regression model assumed a different baseline hazards function for each treatment group, thereby avoiding the assumption of proportional hazards across different treatment groups. For all tests, P values less than 0.05 were considered statistically significant. Because these analyses were exploratory in nature, no adjustments were made for multiple comparisons. SAS (version 9.1; SAS Institute Inc., Cary, N.C., U.S.A.) was used to implement all of the statistical analyses reported in Laboratory Examples 7-12. All statistical tests were two-sided.
Laboratory Example 7
Magnetic Resonance Images
FIGS. 5A-5E show examples of final axial magnetic resonance images resulting from each Dox/Mn-LTSL plus hyperthermia protocol, with the fibrosarcoma positioned in the upper left corner of FIGS. 5A-5C. The slices were chosen to show each tumor at its greatest diameter; the heating catheter was located near the center of each tumor. The enhanced (i.e., white) areas within the tumors represent free manganese (i.e., manganese released from the liposomes). Full sets of magnetic resonance images over the 75-minute treatment period for these three examples were compiled (not shown). Rats injected with Dox/Mn-LTSLs during steady state hyperthermia displayed a peripheral enhancement pattern near the edge of the tumor (FIG. 5A), similar to what had been observed previously (Viglianti et al. (2004) Magn Reson Med 51:1153-62). Observations over the course of the experiments revealed that the liposomes released their contents quickly into the periphery of the tumor within 10 minutes of injection (FIG. 5A). Rats injected with Dox/Mn-LTSLs before hyperthermia displayed signal enhancement (i.e., content release) that followed the initial wave of heat as it emanated from the catheter, resulting in a central enhancement pattern (FIG. 5B). Among rats treated with the Dox/Mn-LTSL split-dose schedule, the first fraction of liposomes released their contents at the center of the tumor, and the second fraction released at the tumor periphery, yielding a more uniform final enhancement pattern (FIG. 5C).
Laboratory Example 8
Intratumoral Distribution of Doxorubicin
T1-based maps of doxorubicin concentrations were used to examine the intratumoral distribution of doxorubicin in rats that had been treated with each of the three Dox/Mn-LTSL plus hyperthermia protocols. The areas of high doxorubicin concentration were similar to the enhanced areas in the MR images. Each of the six rats that received Dox/Mn-LTSLs during hyperthermia had a higher concentration of doxorubicin at the periphery of the tumor (i.e., near normalized radius 1.0) than near the heating catheter (i.e., near normalized radius 0) (FIGS. 5D and 5E). These six profiles were averaged to produce the “LTSL during HT” profile shown in FIG. 5E. By contrast, rats that received Dox/Mn-LTSL before hyperthermia had higher concentrations of doxorubicin near the center of the tumor (i.e., near the heating catheter) than at the tumor periphery, and rats that received Dox/Mn-LTSL on a split-dose schedule had a relatively uniform concentration of doxorubicin throughout the tumor. The 95% confidence intervals indicated considerable intragroup heterogeneity. The radial drug distribution ratios (AUC ratios) were statistically significantly different in all three pairwise comparisons of the Dox/Mn-LTSL plus hyperthermia groups (LTSL during versus before hyperthermia: P=0.003; LTSL during versus split-dose hyperthermia: P=0.015; LTSL before versus split-dose hyperthermia: P=0.010) (Table 1).







TABLE 1







T1-based tumor drug delivery parameters for three different protocols


of lysolipid-based temperature-sensitive liposomes plus hyperthermia*


















Mean total final

Mean initial rate of

Mean final tumor




Mean radial

doxorubicin

tumor doxorubicin

doxorubicin


Treatment
AUC ratio†

level per tumor,

accumulation,

concentration,


protocol
(95% CI)
P
μg (95% CI)
P
μg/min (95% CI)
P
ng/mg_(95% CI)
P


















LTSL during HT
1.6 (1.3 to 1.9)
<.001‡
39.5 (30.6 to 48.4)
.015‡
9.8 (6.9 to 12.7)
.003‡
15.1 (11.7 to 18.5)
.028‡


LTSL before HT
0.6 (0.4 to 0.8)
.010§
24.5 (19.3 to 29.7)
.292§
1.8 (0.8 to 2.8) 
ND
8.0 (7.2 to 8.8) 
.104§


LTSL split dose
1.0 (0.8 to 1.2)
.016∥
33.5 (20.8 to 46.3)
.557∥
¶
ND
13.5 (8.1 to 18.9) 
.640∥





*Comparisons were conducted using the Kruskal-Wallis test with the Wilcoxon test for between-group differences. All P values are two-sided. LSTL = doxorubicin-containing lysolipid-based temperature-sensitive liposomes; HT = hyperthermia; AUC = area under the curve; CI = confidence interval; ND = not done.


†Peripheral AUC/central AUC.


‡LTSL during HT versus LTSL before HT.


§LTSL before HT versus LTSL split dose.


∥LTSL split dose versus LTSL during HT.


¶Initial rate not determined because of split dosing.






Laboratory Example 9
Total Amount and Concentration of Doxorubicin Delivered to Tumors
MRI data were used to estimate the mean amount of doxorubicin that was delivered to the tumor and the mean initial rate of drug delivery for rats treated with each of the three Dox/Mn-LTSL plus hyperthermia protocols (n=6-7 rats per group). The mean total amount of doxorubicin delivered to tumors of rats treated with Dox/Mn-LTSL during hyperthermia was 39.5 μg (FIG. 6 and Table 1), which was equivalent to 5.5% of the injected dose (mean dose=712 μg). Rats treated with Dox/Mn-LTSL before hyperthermia accumulated statistically significantly less doxorubicin in their tumors than rats treated with Dox/Mn-LTSL during hyperthermia (24.5 μg; difference=15.0 μg, 95% CI=3.9 to 26.1 μg; P=0.015; FIG. 6, Table 1). The split dose of LTSL resulted in an intermediate tumor doxorubicin level (33.5 μg, 95% CI=20.8 to 46.3 μg). Considerable heterogeneity was observed in the final amount of doxorubicin among the tumors in each of the three Dox/Mn-LTSL plus hyperthermia treatment groups.
The initial rate of doxorubicin accumulation over the first 3 minutes was statistically significantly higher for Dox/Mn-LTSL administered during hyperthermia than for Dox/Mn-LTSL administered before hyperthermia (9.8 μg/min versus 1.8 μg/min, difference=8.0 μg/min, 95% CI=6.8 to 12.8 μg/min, P=0.003; FIG. 6, Table 1). (Note that the zero time point marks the magnetic resonance image acquired just before Dox/Mn-LTSL and hyperthermia were administered to the tumor. Rats that received LTSLs before hyperthermia [dashed line] or split dose [gray line] had intact liposomes circulating at this point, so the measured amount of doxorubicin was correspondingly greater than zero.)
The final concentrations of doxorubicin (ng per mg tumor tissue, also by MRI) demonstrated the same trends among the treatment groups as the final amounts of doxorubicin (μg per whole tumor). That is, Dox/Mn-LTSL during hyperthermia yielded a statistically significantly higher average tumor concentration than Dox/Mn-LTSL before hyperthermia (15.1 ng/mg versus 8.0 ng/mg, difference=7.1 ng/mg; 95% CI=3.6 to 10.6 ng/mg, P=0.028; Table 1).
Laboratory Example 10
HPLC Measurements of Doxorubicin Concentration
HPLC was also used to determine the average tumor doxorubicin concentration (in ng of drug per mg of tissue) for three rats from each treatment group. Even though MRI-based doxorubicin concentrations at the end of therapy were lower than HPLC-based doxorubicin concentrations at comparable times (Table 1 versus FIG. 7A), both MRI and HPLC results indicate that Dox/Mn-LTSLs administered during hyperthermia yielded greater doxorubicin concentration in the tumor than Dox/Mn-LTSL administered before hyperthermia (mean tumor doxorubicin concentration by HPLC: 20.0 ng/mg versus 9.6 ng/mg, difference=10.4 ng/mg; 95% CI=1.9 to 18.9 ng/mg, P=0.049; FIG. 7A). The tumor doxorubicin concentration, as measured by either MRI or HPLC, resulting from the Dox/Mn-LTSL split dose was not statistically significantly different from that of Dox/Mn-LTSL administered during hyperthermia. Rats treated with free doxorubicin during hyperthermia had a mean tumor doxorubicin concentration that was higher than rats treated with free doxorubicin alone (mean 6.0 ng/mg versus 2.8 ng/mg, difference=3.2 ng/mg; 95% CI=1.9 to 4.5 ng/mg, P=0.049, FIG. 7A), as has been previously reported (Kawai et al. (1997) Cancer 79:214-9). Rats treated with Dox/Mn-LTSL alone did not show increased tumor uptake of doxorubicin compared with rats treated with free doxorubicin; however, the early timepoint (90 minutes after injection) does not reflect the peak concentration of sterically stabilized liposomes as they extravasate (Drummond et al. (1999) Pharmacol Rev 51:691-743).
Free doxorubicin is known to cause cardiac damage that can lead to congestive heart failure, limiting the cumulative clinical dose to <500 mg/m2 for most patients. Therefore, heart concentrations of doxorubicin were measured and compared among all treatment groups in this study. (Rubin et al. (2003) Cancer Medicine, 6th Ed. BC Decker, Inc). The mean concentration of doxorubicin in rat heart was 9.9 ng/mg (95% CI=8.6 to 11.2 ng/mg) for all treatment groups combined and did not differ statistically significantly between any of the treatment groups.
Laboratory Example 11
Antitumor Effect
The effects of the various treatments on tumor growth were examined by comparing the amount of time required for tumors to reach five times the volume that they were on the day of treatment (n=6-7 rats per group). The rats in the Mn/Dox-LTSL plus hyperthermia groups were the same rats that were used for MRI analyses (FIGS. 5A-5E, FIG. 6, Table 1). The administration of Dox/Mn-LTSL during hyperthermia resulted in the greatest antitumor effect, with a median of 34 days (95% CI=30 days to ∞) to five times the tumor volume on the day of treatment compared with 18.5 days (95% CI=16 to 23 days) for Dox/Mn-LTSL administered before hyperthermia and 22.5 days (95% CI=15 to 25 days) for the Dox/Mn-LTSL split dose (FIGS. 7B and 7C).
The amount of time for the tumors to reach five times the volume at the start of treatment was statistically significantly longer for the group that received Dox/Mn-LTSL during hyperthermia than for all other groups except the Dox/Mn-LTSL split-dose group (P=0.071, Wilcoxon test) (Dox/Mn-LTSL during hyperthermia versus: control or hyperthermia alone, P<0.001; versus free doxorubicin, P=0.004; versus free doxorubicin plus hyperthermia, P=0.032; versus Dox/Mn-LTSL alone, P=0.001; versus Dox/Mn-LTSL before hyperthermia, P=0.007). Free doxorubicin resulted in statistically significantly longer tumor growth times than the saline control, both with (P=0.007) or without hyperthermia (P=0.004), as has been previously observed in this doxorubicin-sensitive tumor model (Newman et al. (1992) Int J Hyperthermia 8:79-85; Baba et al. (1993) Anticancer Res 13:651-4; al-Shabanah et al. (1994) Chemotherapy 40:188-94; Hahn (1975) Proc Natl Acad Sci USA 72:937-40). Hyperthermia alone yielded minimal tumor growth delay, as did Dox/Mn-LTSL alone. Four rats showed complete tumor regression upon censoring at 60 days: two of the seven rats that received Dox/Mn-LTSL during hyperthermia, one of the six rats that received Dox/Mn-LTSL split dose, and one of the six rats that received free doxorubicin plus hyperthermia (FIG. 7C).
Laboratory Example 12
Tumor Drug Delivery and Antitumor Effect
The scatter plot in FIG. 8 shows the relationship between tumor response (as measured by the time to five times the original tumor volume) and overall tumor doxorubicin concentration (as estimated from MRI data) in the three groups of rats that received Dox/Mn-LTSLs and hyperthermia. Rats that had the highest tumor doxorubicin concentrations showed the best tumor responses (i.e., the longest times to five times the initial tumor volume). Conversely, rats that had the lowest tumor doxorubicin concentrations (those receiving Dox/Mn-LTSL before hyperthermia) had the worst tumor responses (i.e., the shortest times to five times the initial tumor volume). A Cox proportional hazards regression analysis of growth time versus tumor doxorubicin concentration among the three treatment groups resulted in an estimated regression parameter (b) of −0.19 (95% CI=−0.35 to −0.03, P=0.023), indicating that MRI-based tumor doxorubicin concentration was statistically significantly associated with the time to five times the original tumor volume. It is notable that the scatter plot shows variable tumor growth times for animals with tumor doxorubicin concentrations that ranged from 13 to 17 ng/mg. At these concentrations, Dox/Mn-LTSL during hyperthermia was more effective than the split-dose approach.
Discussion of Laboratory Examples 7-12
Most systemic drug delivery systems, including commercially available liposomes, such as liposomal doxorubicin HCl and liposomal daunorubicin citrate, target tumors by extravasation from leaky tumor vessels followed by passive drug release into the tumor interstitium over the course of several days (Allen & Cullis (2004) Science; 303:1818-22; Drummond et al. (1999) Pharmacol Rev 51:691-743). Other liposome formulations also use specific antibodies or ligands to promote intracellular uptake of the carriers (Park et al. (2002) Clin Cancer Res 8:1172-81). By contrast, the temperature-sensitive liposomes used in the present Laboratory Examples exhibit rapid intravascular release at the permissive temperature, exposing tumor endothelial and perivascular cells to high concentrations of free drug (Kong et al. (2000); Cancer Res 60:6950-7).
The presently disclosed MRI-based analyses of temporal and spatial drug distribution with Dox/Mn-LTSL plus hyperthermia indicate that tumor drug delivery patterns with this formulation can be governed by perfusion pattern and temperature. In the fibrosarcoma model, the main tumor arteries enter the tumor at its periphery (Viglianti et al. (2004) Magn Reson Med 51:1153-62). As disclosed herein, it was found that intravenous injection of liposomes during hyperthermia resulted in rapid release of contents from the liposomes as they entered the tumor from this peripheral vascular source. Although the entire tumor was heated, the liposomes apparently released almost all contents before they reached the center of the tumor, yielding a peripheral doxorubicin distribution. Conversely, injection of liposomes before hyperthermia allowed the liposomes to perfuse throughout the tumor before releasing. When hyperthermia was initiated, liposome contents were released near the central heat source. In this case, the liposomes were apparently depleted of contents before the periphery was fully heated, yielding a central doxorubicin distribution. By splitting the dose (i.e., by injecting liposomes before and during hyperthermia), a more uniform drug distribution throughout the tumor was achieved. Spatial drug distribution could be controlled in a radial pattern with this tumor model because the perfusion source was peripheral and the heat source was central. Thus, the present Laboratory Examples demonstrate real-time controlled intratumoral drug distribution using targeted local hyperthermia, referred to herein as “drug dose painting”, which can be monitored by MRI.
For all drug delivery systems, the pharmacokinetics and the biodistribution of the drug are identical to that of the carrier until the drug is released (Allen & Cullis (2004) Science 303:1818-22). LTSLs are unique because drug release occurs intravascularly, and accumulation of free doxorubicin in the tumor is dependent on the local rate of drug. In vitro studies have shown that Dox-LTSLs begin to release drug slowly at a temperature of 39° C. and exhibit a sharp increase in the rate of drug release at 41.3° C. (Needham et al. (2000) Cancer Res 60:1197-201; Mills & Needham (2005) Biochim Biophys Acta 1716:77-96). In the current Laboratory Examples, when liposomes were injected during steady-state hyperthermia, most of the tumor had achieved a temperature of 41.3° C. or higher (Viglianti et al. (2004) Magn Reson Med 51:1153-62). Therefore, the local rate of content release in the tumor was very high using this protocol (9.8 μg doxorubicin/min over the first 3 minutes). Conversely, when liposomes were administered before hyperthermia, the tumor did not reach steady-state temperatures until 30 minutes after injection (Viglianti et al. (2004) Magn Reson Med 51:1153-62). As the tumor heated to 41.3° C., the local rate of liposomal drug release was relatively slow (1.8 μg doxorubicin/min over the first 3 minutes); thus, the liposomes may not have released 100% of their contents as they passed through the tumor. The slower local release rate, in addition to any systemic release of contents or liposome clearance, resulted in a lower final doxorubicin concentration in the tumor after administration of Dox/Mn-LTSL before hyperthermia when compared with that after Dox/Mn-LTSL administration during hyperthermia.
It is worth noting that the use of a centrally located heating catheter can potentially make it difficult to achieve identical temperature profiles in tumors of different shapes and sizes. Because the liposome release rate is very sensitive to temperature, the variability in temperature likely contributed to the observed intragroup heterogeneity in drug delivery and tumor response observed in the present Laboratory Examples. For example, the size of the tumor shown in FIG. 5A was smaller than the average size of the tumors in that group of rats, and the heating catheter additionally was displaced toward the periphery. As a result, this tumor attained high peripheral temperatures and drug levels, resulting in complete regression. Thus, in the clinical setting, radiofrequency or microwave devices combined with noninvasive thermometry can be used to provide better control of local hyperthermia and therefore more precise control over delivery of therapeutic agents (Dewhirst et al. Hyperthermia. In: Bast R, Kufe D, Pollock R, Weichselbaum R, Holland J, Frei E, et al., editors. Cancer Medicine. 6th ed. Hamilton, Ontario: B. C. Decker Inc 2003.). In addition, since human tumors can be much larger than rat tumors, these devices are capable of directing heat to specific regions of human tumors.
Injection of liposomes during hyperthermia in the present Laboratory Examples was found to result in the best tumor response in the tested tumor model compared with injection before or before plus during hyperthermia at the same dose. This enhanced response was consistent with a faster accumulation rate and greater final concentration of doxorubicin in the tumor using this protocol. Indeed, tumor drug levels (i.e. final concentrations at the end of therapy) are often used as predictors of chemotherapeutic outcome. In previously published studies in human tumor xenografts (Kong et al. (2000) Cancer Res 60:6950-7), the tumor doxorubicin concentrations resulting from various doxorubicin formulations (including Dox-LTSL) were tightly correlated with tumor growth time (R2=0.98). Likewise, in the present Laboratory Examples, tumor doxorubicin concentration was statistically significantly associated with the time it took for tumors to reach five times the original tumor volume, and tumors with the highest doxorubicin levels displayed complete regression. Although drug concentration in tumors is often measured, the rate of drug accumulation in tumors has rarely been assessed or used to predict outcome. However, it is well known that a gradual (low rate) exposure of tumor cells to chemotherapeutic agents in vitro can select for mechanisms of resistance and survival (Frei & Eder. Principles of dose, schedule, and combination therapy. In: Bast et al., ed. Cancer Medicine. 6th ed. Hamilton, Ontario: B. C. Decker Inc; 2003). Conversely, the rapid rate of doxorubicin exposure achieved with LTSL during hyperthermia may be more likely to overwhelm these mechanisms.
It was also found in the present Laboratory Examples that the pattern of doxorubicin accumulation can influence antitumor efficacy. Specifically, rats treated with Dox/Mn-LTSL during hyperthermia, which resulted in peripheral drug distribution, showed longer tumor growth times than rats treated with Dox/Mn-LTSL split dose, which resulted in uniform drug distribution, even though the tumor doxorubicin concentrations did not differ statistically significantly between these groups. Among the individual rats, this discrepancy in tumor response was most apparent for tumors with intermediate tumor doxorubicin concentrations (13 ng/mg to 17 ng/mg). Chen et al. (Chen et al. (2004) Mol Cancer Ther 3:1311-7) showed that tumors treated with Dox-LTSL during hyperthermia undergo vascular destruction. In the fibrosarcoma model used in the current study, the vascular source is in the periphery of the tumor (Viglianti et al. (2004) Magn Reson Med 51:1153-62). Therefore, and without wishing to be limited by theory, the perfusion-delimited peripheral distribution pattern observed in the present Laboratory Examples likely permitted doxorubicin to target the vessels that feed the tumor, thus achieving a substantial antivascular effect.
The present results indicate that Dox/Mn-LTSL in combination with local hyperthermia causes primarily intravascular drug release as opposed to interstitial drug release. It is well known that 100-nm liposomes (both LTSLs and non-temperature sensitive liposomes [NTSLs]) extravasate in tumors, such that maximum tumor drug concentrations are reached several days after injection. Hyperthermia increases the rate of extravasation, resulting in maximum drug concentrations within hours (Ponce et al. (2006) Future Lipidology 1:25-34; Kong et al. (2001) Cancer Res 61:3027-32; Kong et al. (2000) Cancer Res 60:4440-5). Studies in a human squamous cell carcinoma xenograft model (FaDu) (Kong et al. (2000) Cancer Res 60:6950-7; Needham et al. (2000) Cancer Res 60:1197-201) showed that LTSLs with hyperthermia led to four- to five-fold higher tumor doxorubicin concentrations and better antitumor effects than comparably sized (100 nm) NTSLs with hyperthermia. Because the liposomes themselves (i.e., the drug carriers) should have exhibited similar extravasation and accumulation patterns, it is possible that the higher drug concentration achieved with LTSL plus hyperthermia was due to intravascular release of free doxorubicin. Rapid in vitro release kinetics of this formulation support this interpretation. The present Laboratory Examples further strengthen this interpretation by showing that the rate of drug accumulation in tumors treated with LTSL plus hyperthermia (10 minutes to maximum drug concentration) is much faster than the rate of hyperthermia-mediated extravasation. It was also found in the present Laboratory Examples that this intravascular release resulted in higher tumor drug concentrations and greater anti-tumor effects.
Results from multiple preclinical models suggest that vascular shutdown is part of the mechanism for the enhancement of antitumor effect using LTSL plus hyperthermia compared with free doxorubicin (Kong et al. (2000) Cancer Res 60:6950-7; Needham et al. (2000) Cancer Res 60:1197-201; Chen et al. (2004) Mol Cancer Ther 3:1311-7). The mouse FaDu xenograft studies described above used a water bath to heat tumors, thus achieving more uniform temperatures and a more uniform drug distribution throughout the tumor compared with the hot water catheter heating method used in the presently disclosed rat fibrosarcoma study (Kong et al. (2000) Cancer Res 60:6950-7; Viglianti et al. (2004) Magn Reson Med 51:1153-62; Cope et al. (1990) Cancer Res 50:1803-9). Despite these differences, however, the evidence suggests that intravascular drug release occurred in both models. This intravascular release exposes endothelial cells to the highest drug concentrations, which can lead to endothelial cell damage or death. Using dorsal skin-fold window chamber models to test directly for antivascular effects, it was found that LTSL plus hyperthermia leads to reduced red blood cell velocity and microvessel density in the FaDu tumor model (Chen et al. (2004) Mol Cancer Ther 3:1311-7) and in a 4T1 mouse mammary carcinoma model. These results argue for using a treatment schedule in which LTSLs are administered after thermal steady state has been reached, to expose the vasculature to the highest drug concentration.
The lysolipid-based temperature-sensitive liposomes that were used in the present Laboratory Examples can also be used for local delivery of a variety of hydrophilic or amphiphilic drugs. As emphasized in the present Laboratory Examples, the mechanism of action of a given formulation would depend on the hyperthermia schedule used as well as the activity of the free drug. Although the presently disclosed findings for LTSL during hyperthermia are consistent with intravascular drug release, previous studies have shown that in the absence of heat, LTSLs slowly extravasate in a manner similar to liposomal doxorubicin HCl (Kong et al. (2001) Cancer Res 61:3027-32). Therefore, interstitial drug release can be achieved with LTSLs by allowing the liposomes to extravasate before heating the tumor. It should also be noted that for intravascular targeted release with LTSL, the liposome size need not be limited to 100 nm because transvascular permeability is not required. Therefore, a larger liposome can be used to deliver even greater amounts of drug if desired.
The present Laboratory Examples represent the first longitudinal study of treatment response using a multimodal liposome for both magnetic resonance imaging and therapy. The present Laboratory Examples are also unique in that a T1-based method for calculating tumor doxorubicin concentrations for a quantitative (rather than qualitative) description of tumor drug delivery was used. Several groups have used positron emission tomography to evaluate therapeutic delivery of radiolabeled liposomes (Goins & Phillips (2001) Prog Lipid Res 40:95-123; Gabizon et al. (1990) Cancer Res 50:6371-8; Gabizon et al. (1991) Br J Cancer 64:1125-32; Koukourakis et al. (1999) J Clin Oncol 17:3512-21). However, this imaging method has limited spatial resolution and requires a cyclotron and specialized chemistry. Most magnetic resonance-imageable liposomal formulations have been developed for diagnostic purposes rather than for drug delivery (Glogard et al. (2002) Int J Pharm 233:131-40; Fossheim et al. (1999) Magn Reson Imaging 17:83-9). Recently, Port et al. ((2006) Cancer Chemother Pharmacol 58:607-17) showed that subcutaneous injection of a liposome encapsulating both drug (fludarabine) and magnetic resonance contrast agent resulted in passive release of contents. In addition, co-infusions of imageable liposomes and liposomal doxorubicin have been used to study convection-enhanced drug delivery in rat glioma (Saito et al. (2004) Cancer Res 64:2572-9; Krauze et al. (2005) Exp Neurol 196:104-11; Saito et al. (2005) Exp Neurol 196:381-9; Krauze et al. (2005) Brain Res Brain Res Protoc 16:20-6). A few imageable pH-sensitive and temperature-sensitive liposomes have been developed (McDannold et al. (2004) Radiology 230:743-52; Salomir et al. (2005) J Magn Reson Imaging 22:534-40; Lokling et al. (2001) Magn Reson Imaging 19:731-8; Lokling et al. (2004) J Control Release 98:87-95; Fossheim et al. (2000) Acad Radiol 7:1107-15.), but none have been used to observe triggered release within a tumor.
A technical consideration of the T1-based method for measuring tumor doxorubicin concentration disclosed herein is the requirement that doxorubicin and manganese remain colocalized after liposome release (Viglianti et al. (2004) Magn Reson Med 51:1153-62; Viglianti et al. (2006) Magn Reson Med 56:1011-8). The present analysis assumes complete colocalization, but this assumption does not take into account the tendency of doxorubicin to bind to proteins and DNA (Rubin & Hait. Anthracyclines and DNA intercalators. Cancer Medicine, 6th Ed. BC Decker, Inc., 2003). However, time-course observations show a stable image for at least 30 minutes after therapy, suggesting that manganese is also retained in the specific tumor regions where it is released. Indeed, other studies have shown that free manganese tends to bind to tissues (Ni et al. (1997) Acta Radiol 38(4 Pt 2):700-7; Kusaka et al. (1992) Magn Reson Med 24:137-48; Formasiero et al. (1987) Invest Radiol 22:322-7). Furthermore, a strong linear correlation between T1-based tumor doxorubicin concentration and HPLC-based tumor doxorubicin concentration (slope=0.86±0.07 [standard error], intercept=0.01±1.45 ng/mg, n=48 samples, mixed-effects linear regression model) has been demonstrated, even though tumors were harvested 45 minutes after liposome injection. This published correlation can be utilized to estimate the HPLC-based doxorubicin concentration (i.e., the gold standard for determining tumor drug concentrations) from the MRI-based tumor drug concentration.
Overall, the present Laboratory Examples demonstrate that multimodal liposomes containing both drug and contrast agent such as Dox/Mn-LTSLs permit real-time evaluation of therapeutic protocols in association with outcome on an individual subject basis. Furthermore, the rapid intravascular release that is unique to this formulation facilitates control of drug distribution (i.e., drug dose painting) through perfusion- and/or temperature-limited delivery. Further, the presently disclosed data suggest that a desirable scheduling of Dox-LTSL therapy is to administer liposomes during steady-state local hyperthermia.
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It will be understood that various details disclosed herein may be changed without departing from the scope of the disclosure. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

## Claims

1. A method of predicting efficacy of a treatment in a subject, the method comprising:
administering to a subject a liposome composition comprising:
(i) a contrast agent;
(ii) a compound of interest; and
(iii) a liposome encapsulating the contrast agent and the compound of interest;
monitoring accumulation of the compound of interest at a desired site in vivo by magnetic resonance imaging, wherein the monitoring comprises monitoring release of the contents of the liposome at the desired site by monitoring an increase in the presence of the contrast agent released from the liposome at the desired site as the contents of the liposome are being released at the desired site; and
predicting efficacy of treatment based on accumulation of the compound of interest at the desired site.

2. The method of claim 1, wherein the compound of interest is a chemotherapeutic agent.

3. The method of claim 1, wherein the liposome comprises a non-sensitive liposome.

4. The method of claim 3, wherein the non-sensitive liposome comprises DSPC/Cholesterol (55:45, mol:mol).

5. The method of claim 4, wherein the contrast agent comprises a element selected from the group consisting of Gd, Cu, Cr, Fe, Co, Er, Ni, Eu, Dy, Zn, Mg, Mo, Li, Ta, and Mn.

6. The method of claim 1, wherein the liposome comprises an envirosensitive liposome.

7. The method of claim 6, wherein the envirosensitive liposome is a liposome selected from the group consisting of a thermosensitive liposome, a pH-sensitive liposome, a chemosensitive liposome and a radiation-sensitive liposome.

8. The method of claim 7, wherein the thermosensitive liposome comprises a formulation selected from the group consisting of DPPC-PEG2000, DPPC-DSPE-PEG2000 (95:5, mol:mol), and DPPC-MSPC-DSPE-PEG2000 (90:10:4, mol:mol).

9. The method of claim 6, wherein the contrast agent comprises a element selected from the group consisting of Gd, Cu, Cr, Fe, Co, Er, Ni, Eu, Dy, Zn, Mg, Mo, Li, Ta, and Mn.

10. The method of claim 6, further comprising exposing the envirosensitive liposome at the desired site to a non-physiological environmental condition.

11. The method of claim 10, wherein the environmental condition is selected from the group consisting of hyperthermia, electromagnetic radiation, a chemical agent and non-physiological pH.

12. The method of claim 1, wherein the desired site is selected from the group consisting of a tumor, an embolism, an injury site, an ischemia, and a tissue edema.

13. The method of claim 1, wherein the monitoring the accumulation of the compound of interest at the desired site by magnetic resonance imaging comprises making a pixel density determination.

14. The method of claim 1, wherein the predicting efficacy comprises predicting efficacy of treatment based on a location of accumulation at the desired site, a rate of accumulation at the desired site, or both location and rate of accumulation at the desired site.

15. A method of enhancing efficacy of a treatment at a desired site in a subject, the method comprising:
administering to the subject a composition comprising:
(i) a contrast agent;
(ii) a compound of interest; and
(iii) a liposome encapsulating the contrast agent and the compound of interest; and
controlling the distribution of the compound of interest at a desired site by targeting the composition to a desired location within or proximate to the desired site in the subject, at a desired rate of accumulation at the desired site, or both a desired location and desired rate of accumulation at the desired site, to thereby enhance efficacy of treatment provided by the compound of interest, wherein controlling the distribution comprises monitoring accumulation of the compound of interest at a desired site, wherein monitoring comprises monitoring release of the contents of the liposome at the desired site by monitoring an increase in the presence of the contrast agent released from the liposome at the desired site as the contents of the liposome are being released at the desired site.

16. The method of claim 15, wherein the compound of interest is a chemotherapeutic agent.

17. The method of claim 15, wherein the liposome is a non-sensitive liposome.

18. The method of claim 17, wherein the non-sensitive liposome comprises DSPC/Cholesterol (55:45, mol:mol).

19. The method of claim 15, wherein the contrast agent comprises a element selected from the group consisting of Gd, Cu, Cr, Fe, Co, Er, Ni, Eu, Dy, Zn, Mg, Mo, Li, Ta, and Mn.

20. The method of claim 15, wherein the liposome is an envirosensitive liposome.

21. The method of claim 20, wherein the envirosensitive liposome is a liposome selected from the group consisting of a thermosensitive liposome, a pH-sensitive liposome, a chemosensitive liposome and a radiation-sensitive liposome.

22. The method of claim 21, wherein the thermosensitive liposome comprises a formulation selected from the group consisting of DPPC-PEG2000, DPPC-DSPE-PEG2000 (95:5, mol:mol), and DPPC-MSPC-DSPE-PEG2000 (90:10:4, mol:mol)

23. The method of any one of claims 15, 17 and 20, wherein a non-physiological environmental condition is present at the desired site, and the composition is targeted to a desired location at the desired site in the subject, at a desired rate of accumulation at the desired site, or both a desired location and desired rate of accumulation at the desired site by the presence of the non-physiological environmental condition.

24. The method of claim 23, wherein the non-physiological environmental condition is selected from the group consisting of hyperthermia, electromagnetic radiation, a chemical agent and non-physiological pH.

25. The method of claim 24, wherein the hyperthermia is provided by a natural process or by a method selected from the group consisting of contacting a heated material with the desired site, applying RF energy to the desired site, applying ultrasonic energy to the desired site and applying a laser beam to the desired site.

26. The method of claim 23, wherein the desired site is exposed to a non-physiological environmental condition before, after, or both before and after administering the composition.

27. The method of claim 23, comprising administering the composition in one or more partial doses before, after, or both before and after the desired site is exposed to a non-physiological environmental condition.

28. The method of claim 15, wherein the desired site is selected from the group consisting of a tumor, an embolism, an injury site, an ischemia, and at a tissue edema.

29. The method of claim 15, further comprising monitoring accumulation of the compound of interest at the desired site in vivo by magnetic resonance imaging.

30. The method of claim 29, wherein the monitoring the accumulation of the compound of interest at the desired site by magnetic resonance imaging comprises making a pixel density determination.

31. The method of claim 15, further comprising predicting efficacy of treatment based on a location of accumulation at the desired site, a rate of accumulation at the desired site, or both the location and the rate of accumulation at the desired site.

32. A method of targeting delivery of a compound of interest at a desired site in vivo, the method comprising:
administering to a subject a composition comprising:
(i) a contrast agent;
(ii) a compound of interest; and
(iii) a liposome encapsulating the contrast agent and the compound of interest, wherein a non-physiological environmental condition is present at the desired site,
targeting the delivery of the compound of interest to a desired location within or proximate to the desired site in the subject, at a desired rate of accumulation at the desired site, or both a desired location and desired rate of accumulation at the desired site by the presence of the non-physiological environmental condition, and
monitoring accumulation of the compound of interest at a desired site, wherein monitoring comprises monitoring release of the contents of the liposome at the desired site by monitoring an increase in the presence of the contrast agent released from the liposome at the desired site as the contents of the liposome are being released at the desired site.

33. The method of claim 32, wherein the compound of interest is a chemotherapeutic agent.

34. The method of claim 32, wherein the liposome comprises a non-sensitive liposome.

35. The method of claim 34, wherein the non-sensitive liposome comprises DSPC/Cholesterol (55:45, mol:mol).

36. The method of claim 32, wherein the contrast agent comprises a element selected from the group consisting of Gd, Cu, Cr, Fe, Co, Er, Ni, Eu, Dy, Zn, Mg, Mo, Li, Ta, and Mn.

37. The method of claim 32, wherein the liposome comprises an envirosensitive liposome.

38. The method of claim 37, wherein the envirosensitive liposome is a liposome selected from the group consisting of a thermosensitive liposome, a pH-sensitive liposome, a chemosensitive liposome and a radiation-sensitive liposome.

39. The method of claim 38, wherein the thermosensitive liposome comprises a formulation selected from the group consisting of DPPC-PEG2000, DPPC-DSPE-PEG2000 (95:5, mol:mol), and DPPC-MSPC-DSPE-PEG2000 (90:10:4, mol:mol).

40. The method of claim 32, wherein the non-physiological environmental condition is selected from the group consisting of hyperthermia, electromagnetic radiation, a chemical agent and non-physiological pH.

41. The method of claim 40, wherein the hyperthermia is provided by a natural process or a method selected from the group consisting of contacting a heated material with the desired site, applying RF energy to the desired site, applying ultrasonic energy to the desired site and applying a laser beam to the desired site.

42. The method of one of claims 32 and 40, wherein the desired site is exposed to a non-physiological environmental condition before, after, or both before and after administering the composition.

43. The method of one of claims 32 and 40, comprising administering the composition in one or more partial doses before, after, or both before and after the desired site is exposed to a non-physiological environmental condition.

44. The method of claim 32, wherein the desired site is selected from the group consisting of a tumor, an embolism, an injury site, an ischemia, and at a tissue edema.

45. The method of claim 32, further comprising monitoring accumulation of the compound of interest at the desired site in vivo by magnetic resonance imaging.

46. The method of claim 45, wherein the monitoring the accumulation of the compound of interest at the desired site by magnetic resonance imaging comprises making a pixel density determination.

47. The method of claim 32, further comprising predicting efficacy of treatment based on a location of accumulation at the desired site, a rate of accumulation at the desired site, or both location and rate of accumulation at the desired site.

48. The method of claim 1, further comprising monitoring accumulation of the compound of interest at a desired location at the desired site in vivo.

49. The method of claim 48, wherein the desired site is a tumor and the desired location comprises a vascular source of the tumor.

50. The method of claim 49, wherein the vascular source of the tumor is in a periphery of the tumor.

51. The method of claim 15, wherein the desired site is a tumor and the desired location comprises a vascular source of the tumor.

52. The method of claim 51, wherein the vascular source of the tumor is in a periphery of the tumor.

53. The method of claim 32, wherein the desired site is a tumor and the desired location comprises a vascular source of the tumor.

54. The method of claim 53, wherein the vascular source of the tumor is in a periphery of the tumor.