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.

Description of the Invention

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 (B.sub.0, 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.times.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., T.sub.1, 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 T.sub.2, 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. T.sub.1 is defined as the longitudinal relaxation time, and is also known as the spin lattice relaxation time (1/T.sub.1 is a rate constant, R.sub.1, the spin-lattice relaxation rate constant). T.sub.2 is known as the transverse relaxation time, or spin-spin relaxation mechanism, which is one of several contributions to T.sub.2 (1/T.sub.2 is also a rate constant, R.sub.2, the spin-spin relaxation rate constant). T.sub.1 and T.sub.2 have inverse and reciprocal effects on image intensity, with image intensity increasing either by shortening the T.sub.1 or lengthening the T.sub.2.

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.degree. C. range, which is maintained for approximately 30 to 60 minutes. Healthy tissue, generally, should be kept at temperatures below 42.5.degree. C. during the treatment. For targeted chemotherapy drug delivery, temperatures in the range of about 40 to 45.degree. C. have been demonstrated to be effective on tumors.

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) T.sub.1 and/or T.sub.2 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 T.sub.1 relaxation times of nearby spins, exhibiting an r.sup.6 dependency, where r is the distance between the two nuclei. Some paramagnetic ions decrease the T.sub.1 without causing substantial linebroadening, for example copper(II) ("Cu(II)"), zinc(II) ("Zn(II)"), gadolinium(III) ("Gd(III)") and manganese(II) ("Mn(II), while others induce drastic linebroadening, for example, superparamagnetic iron oxide. The mechanism of T.sub.1 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 T.sub.1 and T.sub.2 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), and Mn(II).

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.degree. 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. Some 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 (T.sub.m). 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-PEG.sub.2000). In another example, a thermosensitive liposome comprises dipalmitoylphosphatidylcholine-distearoylphosphatidylethanolamine-polyeth- ylene glycol (DPPC-DSPE-PEG.sub.2000) (95:5, mol:mol), and in yet another example, a thermosensitive liposome comprises polyenylphosphatidylcholine-MSPC-distearoylphosphatidylethanolamine-polye- thylene glycol (DPPC-MSPC-DSPE-PEG.sub.2000) (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-SorbPC.sub.19,19) 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 MnSO.sub.4 (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.degree. 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 .mu.m pore size at 55.degree. C. using a water-jacketed extrusion apparatus, such as an EXTRUDER.TM. 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.RTM. 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.sup.+ and then placed in a Na.sup.+ buffer to create a transmembrane Na.sup.+/K.sup.+ gradient with K.sup.+ concentrated within the vesicle and Na.sup.+ 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.degree. C. for 3-4 days. Loaded non-sensitive liposomes can be stored for about 1-3 weeks at 4.degree. C. Envirosensitive and non-sensitive liposomes that have not been loaded can be stored for about 3-4 weeks at 4.degree. 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 an inner block element, e.g. a manganese atom. 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 MnSO.sub.4, which exhibits a linear relationship between concentration and 1/T.sub.1 (see FIG. 1B (see Original Patent)) and 1/T.sub.2.

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 (see Original Patent)), 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 (see Original Patent). 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, 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 at the tumor edge. At time point 2, the concentration of contrast agent and compound of interest is increased. At time point 3, 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 (see Original Patent)) indicate that T.sub.1 or T.sub.2 shortening correlates with contrast agent (e.g., MnSO.sub.4) concentration. Further, contrast agent release from envirosensitive liposomes, for example thermosensitive liposomes, significantly shortens T.sub.1 and T.sub.2 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 (see Original Patent)). 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. 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 MnSO.sub.4 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 compound of interest can comprise any compound. Such a compound can comprise a chemotherapeutic agent, pharmaceutically active agent or an agent suspect 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.degree. C. The temperature of the heated site will be several degrees above 37.degree. 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 is denoted by an increase in the pixelation 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 15 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.

In another aspect, drug release can also be quantified. One method of quantifying drug release generally involves employing a plot of concentration against 1/T.sub.1 or 1/T.sub.2 as a calibration curve.

Continuing with this embodiment, before a given experiment is performed, the T.sub.1 of a pixel is measured. This T.sub.1 measurement can provide additional information, including proton density and base line noise. If all the imaging parameters (e.g., T.sub.r, 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 T.sub.1. Such a change in T.sub.1 is indicative of localization and distribution of a drug from an envirosensitive liposome composition. This reduction in T.sub.1 can be converted to concentration using the plot of concentration against 1/T.sub.1 as a standard curve for the corresponding compound. Thus, observed changes in T.sub.1, which are associated with drug release, can be translated into a released drug concentration by indexing the observed T.sub.1 with a given concentration on a plot of concentration against 1/T.sub.1.

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 MgSO.sub.4. 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 2T whole-body clinical scanning instrument (e.g., a Signa Medical Systems scanner, available from General Electric of Milwaukee, Wis., United States of America).

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).

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, T.sub.m. 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 T.sub.m, the thermosensitive liposome retains its structural integrity; above the T.sub.m, 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, MnSO.sub.4.

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 T.sub.m 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 T.sub.m 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 T.sub.m 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, namely a contrast agent. By evaluating MR images acquired as the thermosensitive liposomes approach the site of heating, the distance at which the T.sub.m of the liposomes is reached can be determined. At the distance from the catheter that the tissue is heated to T.sub.m, contrast agent release will be immediate and will resemble a burst release. Thus distances equal to or less than the T.sub.m 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 T.sub.m, 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.

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.RTM., 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 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 (see Original Patent) indicates, the contrast agent is cleared from the circulatory system in about 90 minutes in the case of the thermosensitive liposomes and about 3-5 hours in the case of the non-sensitive liposomes. Additionally, the compositions and properties of these liposomes can be modified to vary their circulation time 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.

VI.D. Real Time Monitoring

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 .sup.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 .sup.99Tc, a radioactive material. The radiation emitted from the .sup.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 anaphylactic 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, 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.
 

Claim 1 of 26 Claims

1. 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, the method comprising: (a) administering an envirosensitive liposome composition to a 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 (b) monitoring the localization and distribution of the compound of interest to a desired site 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.

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