Patent Application
20220062611
The use of microbubbles has been proposed in a number of applications including in a variety of biomedical applications. Microbubbles generally describe structures comprising a shell surrounding a gas core and having a size (typically expressed as a diameter) from about 0.5 μm to about 25 μm. The shell of microbubbles may be made of different materials, although one contemplated approach includes microbubbles having a lipid shell surrounding a gas core. Regardless of the microbubble type, the use of microbubbles has been explored for use in conjunction with ultrasound devices for a number of purposes. Specifically, application of energy to microbubbles (e.g., after introduction into the body) may result in excitation of the shell and/or gas core of the microbubble to, for instance, resonate the microbubble. This may result in popping or cavitation of the microbubble at a selected location within the body upon selective application of energy thereto.
Accordingly, microbubbles have been proposed for use as contrast agents in medical imaging, drug delivery, extravascular delivery, noninvasive surgery or other approaches. Specifically, ultrasound devices or the like may provide targeted energy delivery to excite bubbles for specific responses in a targeted area in the body. However, continued development of microbubbles and related treatment systems is required to improve clinical outcomes.
The present disclosure generally relates to targeting a drug to a target treatment area. This includes administering to a patient a size-isolated microbubble (SIM) product and administering to a patient a dose of a drug. The targeting also includes introducing ultrasound energy from an ultrasound device to the target treatment area in the presence of the SIMs and the drug. In response, the targeting includes sonoporating the target treatment area in response to the ultrasound energy by cavitating the SIMs of the SIM product to improve delivery of the drug to the target treatment area.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
Other implementations are also described and recited herein.
While the presently disclosed technology is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that it is not intended to limit the presently disclosed technology to the particular form disclosed, but rather, the presently disclosed technology is to cover all modifications, equivalents, and alternatives falling within the scope of the presently disclosed technology as defined by the claims.
As noted above, microbubbles have been proposed for use in a number of applications including as imaging contrast agents and the like. However, the present disclosure specifically contemplates use of microbubbles for sonoporation in connection with drug delivery. As will be shown in greater detail below, it has been found that administration of size isolated microbubbles (SIMs) administered with a therapeutic agent allows for use of an ultrasound device for sonoporation of soft tissue. Specifically, SIMs react to targeted ultrasound energy causing the SIMs to cavitate in a region where the targeted ultrasound energy is applied. The cavitation of the SIMS results in sonoporation of soft tissue, which allows for targeted delivery of a drug to vascularized soft tissue, such as a tumor. As will be described in greater detail below, use of ultrasound driven sonoporation of soft tissue tumors using SIMs in combination with therapeutic agents has been shown in numerous animal model and early-stage human trials to slow or halt tumor growth more effectively than therapeutic agents alone.
Much of the proposed work to date using microbubbles has been performed using ultrasound image enhancement agents, which have not been manufactured or designed for drug delivery applications. These agents include microbubbles having a polydisperse size distribution of microbubbles (referred to herein as polydisperse microbubbles or “PMBs”) in sizes ranging from 0.5-20 μm in diameter. Because the size of a microbubble, in large part, determines the physical response in an ultrasound field of applied ultrasound energy, and therefor mechanical effects on tissue, use of PMBs leads to a broad range of unintended tissue effects. Ultrasound image enhancement agents comprising PMBs also have a very short circulatory half-life. The short circulatory half-life of traditional PMBs can limit the time available to deliver therapeutic ultrasound, although, continuous infusion pumps have been suggested to mitigate this issue.
For example, microbubble contrast agents, such as Definity® (available from Lantheus Medical Imaging of N. Billerica, Massachusetts United States), Optison™ (available from GE Healthcare, Chicago, Ill.) or Vevo MicroMarker (available from FUJIFILM Sonosite, Inc., Toronto, Ontario Canada), are polydisperse microbubbles. It has been found that such PMBs have unpredictable behavior when used for ultrasound driven sonoporation. These PMB contrast agents typically contains a range of microbubbles from less than 1 μm to 20 μm diameter. In addition, the microbubbles often have a size below 2 μm in diameter (e.g., a majority of the volume fraction). High polydispersity may be a consequence of the emulsification method used to generate microbubbles in high quantity. Microbubble production methods based on mechanical agitation (e.g., sonication, shaking, and milling) represent the current standard to create encapsulated microbubbles for biomedical applications. Sonication generates large quantities of microbubbles (1010 per mL) within just a few seconds. However, these methods do not allow homogeneous size distribution of microbubbles, which instead tend to be highly polydisperse due to instability at the water surface causing entrainment of bubbles into the aqueous medium, and subsequent cavitation resulting in bubbles breakup to a critical size (e.g., smaller than 2 μm). Efforts to engineer monodisperse microbubble suspensions have focused on microfluidic technologies including flow focusing, T-junctions, and electro-hydrodynamic atomization. While these techniques may provide low polydispersity, they are rather slow at generating microbubbles and therefore not practical for large-scale manufacturing.
As such, drawbacks in relation to microbubble production and processing have resulted in limitations in the adoption of microbubbles. However, it has been presently recognized that SIMs of a given microbubble target size may be useful in certain contexts. For instance, by controlling the size of microbubbles to a size isolated range, the frequency of the energy applied to SIMS in the body may be adapted for a specific response. Prior proposed methods for microbubble production either result in a polydisperse size distribution or suffer from limitations in microbubble yield such that sufficient quantities may not be feasibly produced for many applications contemplated. In response, certain approaches have been proposed such as those described in U.S. Patent Publication No. 2011/0300078, the entirety of which is incorporated by reference herein.
In addition, PMB agents also exhibit challenges for use in sonoporation of vascularized soft tissues, such as tumors. Sonoporation has shown great potential for drug delivery and gene therapy. Transient plasma membrane perforation achieved by mechanical forces produced from the interaction of focused ultrasound waves with microbubbles increases the permeability of tumor tissues. However, sonoporation of PMBs produces heterogeneous effects leading to complexities and challenges in the realization of controllable and predictable drug delivery. Delivery and monitoring of focused ultrasound energy to tumor tissue has been performed in clinical trials with use of PMB products such as contrast agents that are not optimized for this specific purpose. Effectively monitoring tumor tissue sonoporation in real-time is a critical capability and is important for a successful therapeutic system.
Studies considering the amount of internalized exogenous molecules in sonoporated cells using PMB agents were found to exhibit heterogeneous characteristics. Moreover, sonoporated cells were reported to exhibit heterogeneous and complex concomitant physiological responses, such as calcium-ion transients, calcium oscillations and waves, and non-unitary changes in the levels of plasma membrane potential depolarization. Finally, various cellular developmental effects including proliferation inhibition, cell-cycle arrest, and trends in cell fate (i.e., survival, apoptosis, and necrosis) have been observed in sonoporated cells over several hours following ultrasound exposure. The reason for these heterogeneous effects has yet to be identified. Moreover, for practical applications, the heterogeneity of sonoporation of PMBs poses more challenges in achieving predictable delivery outcome and high delivery efficiency. For instance, some sonicated cells that underwent apoptosis or necrosis are not advantageous to improving the delivery efficiency, because they are essentially the side effects of sonoporation. Also, previous studies have shown that trends in cell fate were correlated with the degree of sonoporation. It is important to address the key acoustic and non-acoustic parameters that are related to the heterogeneity of sonoporation.
The present disclosure recognizes the heterogeneity demonstrated in use of clinically available ultrasound image enhancement PMB products is a major contributing factor to therapeutic effect variability. The need to achieve therapeutic doses of agents within tumors while avoiding the dose dependent side effects requires producing, developing, and deploying a complete system which includes a microbubble agent and hardware. Recognizing this need, the present disclosure includes technology specifically adapted to realize the therapeutic goals of reduced drug dosage requirements in the presence of sonoporation of soft tissue. Specifically, the ability to produce clinically relevant quantities of size iso with requisite qualities of SIMS facilitates the improvements of the sonoporation system presented herein.
Specifically, the sonoporation system contemplated in the present disclosure may provide a SIM solution in connection with a purpose-built ultrasound system to provide for improved sonoporation of vascularized soft tissue tumors. The application of targeted ultrasound energy to vasculature containing the SIM solution causes a sonoporation effect. Specifically, the compressible gas core of the size isolated microbubble product reacts to the oscillating pressure wave by volumetrically expanding and contracting the tissue to temporary (e.g., for a period of 6-24 hours) permeabilize endothelial tissue. In turn, the present system may facilitate increases the permeability of vascularized soft tissue tumors to therapeutic agents. For example, in some examples, permeability of local capillary beds in soft tissue may be limited to molecules larger than around 400 Da. In this regard, the sonoporation contemplated herein may temporarily increase the tissue permeation of circulatory substances to a size than what occurs in the absence of sonoporation. That is, the permeability of local capillary beds in soft tissue in a targeted area may be increased in response to the introduction of ultrasound energy focused on the intended treatment area to cause reversible perforation of capillary walls in specific treatment areas and monitoring said perforation events using diagnostic ultrasound technologies. The increase in permeability in response to the sonoporation may be temporary so as to avoid negative effects such as bleeding or the like in the targeted treatment area. In some examples, sonoporation may temporarily make the local capillary beds in the targeted treatment area to allow molecules larger than the normal limit (e.g., greater than 400 Da) to pass through the capillary interface and encounter the soft tissue cells in the targeted treatment area.
The result of such an increase in the permeability of vascularized soft tissue tumors is to dramatically increase the intratumoral concentration of native therapeutics, thereby, decreasing the required systemic therapeutic dose and associated dose dependent side effects. That is, the sonoporation of local capillary beds in the target treatment area may allow for increased permeability with respect to the therapeutic agent, thus increasing the effective delivery of the therapeutic agent to the targeted treatment area. As will be shown below, use of ultrasound imaging PMB products and generic ultrasound systems have demonstrated variable results and have identified factors driving therapeutic heterogeneity. In turn, the present disclosure directly addresses each major cause of treatment heterogeneity to provide a system purpose-built for tumor sonoporation to facilitate the noted benefits while reducing or eliminating the traditional barriers limiting use of microbubbles for sonoporation. In turn, the sonoporation systems of the present disclosure are specifically designed to increase vascular permeability to circulating drugs in tissue, including tumors.
Although the presence of “leaky” capillary vessels in tumor tissue that are permissive to passive or ultrasound driven transport of therapeutic agents is often discussed, by virtue of clinical outcomes alone, is not always sufficient to permit entry of therapeutic agents into tumor tissue. In turn, the therapeutic advantage provided by the present disclosure is temporarily disrupting epithelial cell tight junctions within vascularized soft tissue tumors to increase the diffusion-limited rate of transfer of circulatory therapeutic agents towards direct contact with target tumor tissue. To this end, the present disclosure presents SIMs with tight size distributions and a longer circulatory half-life along with a purpose-built ultrasound delivery and monitoring of the therapy.
In addition to a specifically formulated SIM product, the present disclosure contemplates a sonoporation system including an ultrasound device to uniquely image, treat, and monitor sonoporation of target tissue regions in targeted clinical settings. The sonoporation system uses diagnostic-level ultrasound pressures focused to a user-selectable target region. The sonoporation system of the present disclosure provide a specifically adapted ultrasound device for sonoporation of tumor tissue and for monitoring this sonoporation in real time.
The presently contemplated sonoporation system will use image processing algorithms to allow the user to confirm that the treatment is being successfully applied to the target tissues. This feature may include full 3D monitoring if utilizing CT or MR registration. In addition, the sonoporation system contemplated herein may function in an outpatient setting, and thus not require the patient to engage multiple systems, specialties, practitioners. The system will image and treat soft tissue tumors. Such tumors may be located throughout the human torso, although other tumor locations may also be treated. The sonoporation system may use diagnostic 3D ultrasound to image the tumor and margins to be treated. In turn, the system may provide focused therapeutic ultrasound at specific tumor locations identified from imaging. The system may also inform clinician as to the defined therapeutic margins and monitor the sonoporation in real time. A user interface may provide data as to the degree of sonoporation delivered throughout the targeted tumor.
Sonoporation systems according to the present disclosure may include SIMs that are produced at commercially viable high yields. As noted above, production of microbubbles in a target size range have not been sufficiently effective to provide sufficient yields. In addition, many microbubble products previously contemplated include PMB distributions. In turn, traditional microbubble production approaches have demonstrated an inability to effectively isolate microbubbles at a target size at sufficiently high yields. Thus, while traditional microbubble production may produce relatively high yields of PMB populations, the process to isolate the target size of microbubbles may significantly reduce the resulting yield of the size isolated microbubbles. Moreover, preservation (e.g., during distribution and storage) may be difficult to achieve, thus resulting in a limited shelf life of PMB microbubbles.
While certain examples presented herein relate to use of liposomal doxorubicin (“L-DOX”) as a chemotherapy agent for targeting vascularized soft tissue tumors, other drug types may also be administered using the techniques described herein. This may extend to other types of chemotherapy drugs (e.g., using metronomic dosing) including doxorubicin, topotecan, cyclophosphamide, vincristine, and/or etoposide. Such chemotherapy agents may be delivered in conjunction with SIMs in the size range of 4-5 μm. This may include SIMs in a size range of about 4μm to about 5μm at a volume percent of 60% or greater, 70% or greater, 80% or greater, 90% or greater, or even 95% or greater. In other examples, any application in which a therapeutic agent provided in a patient's bloodstream is desirably targeted to a specific anatomy may employ sonoporation using SIMS as described herein.
In one example, targeted ultrasound may be provided in a targeted treatment area for the treatment of relapsed and high-risk neuroblastoma. However, other applications in which drugs are desired to be targeted as vascularized soft tissue are contemplated without limitation. The resulting sonoporation increases the permeability of tissue (e.g., tumor tissue) and allows greater infiltration of circulating therapeutic agents (e.g., chemotherapy agents) into the tumor tissue. This enables more effective delivery of therapy at approved doses, leading to increased efficacy. Furthermore, in some examples, use of sonoporation as described herein with SIMs allows a reduction in dose compared to an approved therapy dose of an agent, thus leading to a reduction in systemic side-effects while maintaining current therapy efficacy. In some examples, administration rates of agents with sonoporation in the presence of size isolated microbubbles may allow administration rates at 50% or less, 40% or less, 30% or less, or even 20% or less of approved administration rates in the absence of sonoporation.
Accordingly, the sonoporation system of the present disclosure includes a SIM product produced at high yields.
The method 100 may include establishing 104 conditions for microbubble generation. This may include heating the lipid solution to above the phase transition temperature of the lipid solution to promote mixing of the lipid components in the aqueous medium. By heating the lipid solution to above the phase transition temperature, the lipid solution may be more solvent relative to the aqueous medium to promote mixing. Furthermore, the mixture may also be physically mixed while heated. The lipid solution may be further excited to break up large lipid aggregates into smaller micelles and liposomes. This may include applying energy using an energy applicator such as a sonicator or the like. The energy applicator may be the same as an energy applicator discussed below for excitation to produce microbubbles. However, the energy applicator may be operated at a lower power setting to promote mixing of the lipid component in the aqueous medium prior to generation of microbubbles. In any regard, the suspension may be energized (e.g., sonicated, agitated, or otherwise physically excited) until the solution is translucent and there are no visible aggregates. Once the mixture is translucent and no visible aggregates are present, the lipid solution may be cooled to below the phase transition temperature of the lipid component of the lipid solution. For instance, the temperature of the lipid solution may be set to no less than 5° C. below the phase transition temperature of the main phospholipid in the solution.
The establishing 104 of the conditions for microbubble production may also include introduction of a gas phase into a headspace of a vessel containing the lipid solution. With further reference to
The method 100 may further include applying 106 energy to excite the system and create microbubbles. As described above, this may include physical agitation of the lipid solution 124. With returned reference to
It has been found that the initial microbubble yield may be increased by exciting a relatively warm lipid solution 124. In this regard, the warm lipid solution may be maintained below the main lipid phase transition temperature of the solution but maintained relatively close to the phase transition temperature (e.g., within 5° C. of the main lipid phase transition temperature of the solution). In this regard, the lipid solution 124 may be disposed relative to a thermal regulation device such as a heater and/or cooler to maintain the temperature of the lipid solution 124.
Once initial microbubble production has been completed, the method 100 may include controlling 108 one or more diffusion parameters to maintain preferable diffusion conditions for the microbubble suspension 132. The microbubble suspension 132 may be maintained in the aqueous solution of the lipid solution 124 or may be collected and transferred to a virgin aqueous solution. Further still, the microbubble suspension 132 may be centrifuged to collect the polydisperse microbubble population in a supernatant cake for collection. In any regard, further details regarding the controlling 108 of the diffusion parameters is described in greater detail below. However, the controlling 108 may include establishing conditions in which the diffusion forces acting on the microbubbles in a solution are maintained within a predetermined range to reduce or minimize the diffusion forces in the microbubble suspension 132. Specifically, it is noted that to provide increased yield of lipid stabilized, size isolated microbubbles using differential centrifugation, it is important to optimize initial bubble production while minimizing factors the results in microbubble gas dissolution and breakdown of microbubbles during centrifugation wash cycles of the size isolation process. In this regard, it is presently recognized that it is possible to reduce or impede gas dissolution of the microbubble by minimizing the diffusion force that governs bubble the solution. Specifically, the Stokes-Einstein's Brownian diffusion equation for gas dissolution provides characterization of the gas dissolution of a system comprising microbubbles with a diffusion constant D. The Stokes-Einstein's Brownian diffusion equation can be represented as:
where k is the gas constant, T as the temperature of the ambient fluid, μ* as the effective viscosity of the fluid, and r in the radius of the microbubble. In addition, it is recognized that the effective viscosity of the fluid medium is at least in part based upon the viscosity of the fluid containing the microbubbles and the volume fraction of the microbubbles in the fluid. Specifically, a model to determine the effective fluid viscosity in view of crowding and bubble interaction is provided as:
where μ* is the effective viscosity of the solution, p is the viscosity of the fluid in which the microbubbles are provided, and c as the bubble volume fraction. As will be described in greater detail below, any of the parameters capable of being controlled in relation to the system from Equation 1 may be controlled 108 to reduce or minimize the diffusion forces acting on the microbubbles in the microbubble solution 132.
In any regard, the method 100 may include centrifuging 110 the microbubble solution to isolated target microbubble sizes. This may include a multiple step centrifugal wash cycle in which the solution may be subjected to a number of centrifuging washes to isolate a given target size microbubble. With reference to
The separation column 140 may be subjected to centrifugation to produce a supernatant cake or simply cake 142 and an infranatant 144. In this regard, a number of wash cycles may be applied to selectively isolate various sized microbubbles in the cake 142 or infranatant 144, depending on the wash cycle. For example, the centrifuging 110 may include applying a first centrifugal field having a first field strength to a suspension comprising a polydisperse population of microbubbles for a first duration of time, thereby forming a first infranatant 144 comprising at least a portion of target microbubbles and a first cake 142 comprising microbubbles having a greater size than the target microbubbles. In turn, the first cake 142 may be removed. In turn, the first infranatant 144 may be applied thereto a second centrifugal field having a second field strength to the first infranatant for a second duration of time, the second field strength to form a second supernatant cake 142 comprising at least a portion of the target microbubbles and second infranatant 144 comprising microbubbles having a smaller size than the target microbubbles. Thus, the second supernatant cake 142 that contains the target size microbubbles may be isolated.
In this regard, it may be appreciated that because the diffusion coefficient that describes the diffusion forces acting on a microbubble in the microbubble solution 132 may be at least in part based on the size (e.g., radius) of the microbubble, the controlling 108 of the diffusion factors may be based on the target size of the microbubble targeted in the centrifuging 110. In any regard, once isolated, the target size microbubbles may be concentrated 112.
As will be discussed in greater detail below, by facilitating high-yield size isolated microbubble populations, a relatively large volume of highly concentrated size isolated microbubbles may be realized. In this regard, the resulting size isolated microbubble product may provide very high-volume fractions in a resultant solution. As can be appreciated from the foregoing equations, such a high-volume fraction may result in a high effective viscosity of a final size isolated microbubble product. This may result in a low diffusion coefficient, indicative of low diffusion forces in the concentrated size isolated microbubble product. Accordingly, the resulting high-yield, size isolated microbubble product may demonstrate improved stability as the diffusion forces associated with microbubble gas dissolution and breakdown may be minimized.
As such, the resulting microbubble product may be packaged 114 in an appropriate form. Any suitable vessel may be utilized for containment of the packaged 114 concentrated microbubble product. However, certain product forms are specifically contemplated herein such as a vial or syringe to contain the concentrated microbubble product. Advantageously, when contained in a syringe, the resulting concentrated microbubble product may be injectable without further handling of the microbubbles. By minimizing the handling or transfer of the size isolated microbubbles, the high-yields of the foregoing process may be maintained with minimal degradation of the microbubbles due to transfer between vessels, which may result in breakdown or other degradation of the microbubbles. In an embodiment, the internal volume of the vessel into which the concentrated microbubble product is provided may be the same as the volume of the concentrated microbubble product itself. That is, the container into which the concentrated microbubble product is provided may have substantially no headspace such that the concentrated microbubble product occupies substantially all of the internal volume of the vessel. This may also assist in reduction of the dissolution forces that tend to break down microbubbles during storage.
As such, the improved stability of the concentrated microbubble product may allow for storage 116 of the microbubble product. The storage 116 may be at room temperature or may be at a refrigerated temperature to promote microbubble preservation. For instance, when stored at a refrigerated temperature (e.g., not greater than 4° C.), at least 90% of the initial volume of the concentrated microbubble product disposed in the containment vessel during the packaging 114 may remain after not less than 4 weeks of storage 116. In addition, when stored at room temperature (e.g., not greater than 23° C.), at least 80% of the initial volume of the concentrated microbubble product disposed in the containment vessel during the packaging 114 may remain after not less than 4 weeks storage. As may be appreciated, the increased stability of the microbubble product may allow for enhanced shelf life, thus facilitating maintaining the microbubble product in inventory at a medical facility prior to use. This may allow for increased flexibility in relation to the use of the microbubble product for various medical applications such as use as a contrast agent in medical imaging, ultrasound targeted drug delivery, ultrasound targeted ablation of tissue, ultrasound aided opening of tissue or any other medical application in which size isolated microbubbles may be utilized. Thus, the method 100 may further include administering 118 the microbubble product and a medical application.
With further reference to
In addition, while microbubble fraction is recognized as affecting the effective viscosity of the solution, the viscosity of the fluid in which the microbubbles are contained is also a factor effective viscosity. In this regard, the viscosity of the fluid in which the microbubbles are provided may also be controlled (e.g., to increase the viscosity of the solution and thus reduce the diffusion coefficient of the system). In this regard, a viscosity modifier may be added 156 to the microbubble solution to target effective viscosity. In an embodiment, the viscosity modifier may comprise glycerol.
Furthermore, the temperature of the solution may also affect the diffusion coefficient governing the diffusion forces acting on the microbubbles in the solution. As such, the method 150 may include maintaining 158 a temperature of the solution at a target temperature. As described above, during initial microbubble production, the lipid solution from which the microbubbles are produced may be preferably maintained at below about 5° C. less than the phase transition temperature of the lipid solution. In this regard, the solution during initial microbubble production may be at a relatively elevated temperature. As such, the maintaining 158 of the temperature of the solution may include reducing the temperature of the solution to well below that of the temperature at which the microbubbles are initially produced from the lipid solution. This may include extraction of the microbubbles from the lipid solution remaining after initial bubble production. In this regard, the extracted microbubbles may be refrigerated or may be introduced into a fluid (e.g., during adding 154 of the fluid to the separation column) that is at a refrigerated temperature. For instance, the target temperature of the solution during centrifugation for size isolation may be not greater than about 1° C., not greater than about 4° C., not greater than about 5° C., not greater than about 10° C., or not greater than about 15° C.
The method 150 may also include applying 160 centrifugation to the separation column for size isolation of a target size microbubble. The applying 160 may be performed in view of the prior steps of the method 150 that effectively control the diffusion parameters of the system. As may be appreciated in
The use of SIMS in the presently contemplated sonoporation system is important for a number of reasons. Such a SIM product may carry a pre-formed stabilized gas bubble that can create sustained levels of stable cavitation (bubble expansion and collapse) when exposed to ultrasound energy at the site of a tumor. This purely mechanical effect is used to temporarily disrupt epithelial tight junctions within the capillary bed of soft tissue tumors.
Accordingly, in the sonoporation system contemplated herein, SIMs may be co-administered independently alongside a therapeutic agent (e.g., a native anti-cancer agent). Specifically, SIMs in a size of 4-5 μm may be used as defined above. By virtue of their size, the SIMs circulate in the blood stream and, when exposed to diagnostic ultrasound focused at specific regions of a tumor, temporarily disrupt endothelial tight junctions by popping or cavitating. This may increase permeability to therapeutic agents within solid tumors. Preclinical data supports increased penetration of therapeutic agents into tumor tissue and a concurrent decrease in systemic levels of therapeutic agents needed to produce a therapeutic effect.
SIMs exhibit a number of enabling features that provide specific clinical advantages to the present contemplated sonoporation system. For example, the SIMs provided noted benefits on tissue effects. Specifically, the SIM particles are designed to provide specific, reproducible tissue effects focused on temporary disruption of epithelial tight junctions. SIMs also provide advantages in pharmacokinetics because the in vivo circulation kinetics of SIMS have been designed to be compatible with a broad range, if not all, therapeutic oncology agents. The SIMs also provide sufficient time to administer ultrasound to the anatomic region of interest. In relation to administration, SIMS provide advantages as the SIMs are designed to adopt the same administration route as the therapeutic agent, including intravenous (IV) or intra-tumoral (IT). In addition, SIMs may be independently administered alongside the therapeutic agent such that no drug reformulation is required. In turn, the SIMs fit neatly within current oncology ward out-patient clinical practice. That is, for intravenous administration, the SIMs will be delivered to the patient via an infusion bag and giving set making their administration fit current practice without requiring practitioners to be trained for new administration practices specific to the sonoporation system presented herein.
Furthermore, SIMs may address a number of therapeutic variables that, as noted above, have limited use of prior PMB agents that exhibit a polydisperse population of bubbles. Specifically, PMBs may result in a number of therapeutic heterogeneities that limit the clinical efficacy of sonoporation. The noted therapeutic benefits are reflected in Table 1 below:
Accordingly, the sonoporation system of the present disclosure may advantageously use a minimum amount of force required to temporarily disrupt epithelial cell tight junctions within vascularized soft tissue tumors to increase the diffusion limited rate of transfer of circulatory therapeutic agents towards direct contact with target tumor tissue. To this end, the use of with limited size distributions provide a longer circulatory half-life. As such, SIMs used along with a purpose-built ultrasound delivery and monitoring system facilitates reductions in the heterogeneity demonstrated by prior approaches and improves the clinical efficacy as demonstrated below.
The monodisperse SIMS significantly improved liposomal doxorubicin (“L-DOX”) uptake in the tumor versus L-DOX alone or administration of L-DOX with the use of unsorted (polydisperse) conventional imaging bubbles. In addition, L-DOX delivery covered the tumor in a homogeneous manner in contrast to uptake using conventional imaging PMBs. This treatment demonstrated efficacy after delivery using SIMS and a low dose of a chemotherapy agent. Importantly, the treatment produced preliminary longitudinal efficacy data demonstrating halting or reduction of tumor volume versus controls and versus a regimen in which a drug was administered alone without sonoporation. In studies using mice, the halting of tumor growth was at a very low dose (e.g., 20-fold less than started) of L-DOX without the mice losing weight or exhibiting other symptoms characteristic of L-DOX side effects. The mice appeared healthy after treatment as opposed to the use of higher doses (25 mg/kg) that caused the mice to lose weight or exhibit other symptoms indicative of side effects of the chemotherapy agent.
Accordingly, an example set of operations 900 related to a sonoporation system are shown in
The operations 900 may also include an imaging operation 906 in which an ultrasound device may be used to define a target treatment area. Specifically, definition of a target treatment area may include imaging a soft tissue target to which therapeutic agent is targeted. The imaging operation 906 may include determining margins of a tumor to assist in monitoring the efficacy and/or status of therapy applied to the soft tissue target. In addition, the operations 900 may include a targeting operation 908 in which targeted ultrasonic energy is targeted to the treatment area. The targeting operation 908 may include application of targeted ultrasonic energy using the same ultrasound device used in the imaging operation 906. In this regard, the imaging operation 906 and targeting operation 908 may be conducted simultaneously or at least in an overlapping time period using the same ultrasound device. In addition, the operations 900 may include a monitoring operation 910 in which the ultrasound device may be used to monitor sonoporation resulting from interaction of the targeted ultrasonic energy with the SIM product in the targeted treatment area. The monitoring operation 910 may include quantification of sonoporation, which may assist in determining efficacy of treatment and may provide feedback to help provide guidance for treatment and/or outcome results of the treatment.
Polydisperse microbubbles are prepared by excitation methods such as acoustic emulsification. In the first stage of size-isolation, bubbles below an undesired size are extracted from the polydisperse suspension of high volume fraction (20-30%) using a large capacity column. Since, the bubble population extracted will be at a lower concentration (and lower volume fraction) than the initial population, subsequent separation to remove the undesired smaller sizes is performed by adjusting bubble volume fraction and or fluid viscosity in the next separation column. All centrifugal washing is done with cold aqueous media to control temperature.
Preparation Of The Lipid Suspension
Polydisperse Microbubble Production at High yield
Stage 1: Extracting Target Sizes and Small Sizes from Undesired Large Sizes Using Column at High Bubble Fraction and Cold Media:
Stage 2: Removing Undesired Small Sizes from Target Sizes While Maintaining High Bubble Fraction and Washing with Cold Media:
This method may allow for the production of four different size classes at high yield including 2 micron bubbles at a yield of greater than 2×1010 MB/mL, 3 micron bubbles at a yield of 1×1010 MB/mL, 4 micron bubbles at a yield of 8×109 MB/mL and 6 micron bubbles at a yield of 2×109 MB/mL.
Effect Of Bubble Fraction and Temperature on Yield of Size-Isolated Microbubbles
Example Values for Diffusion Coefficients for High Yield Processing
The following represents example diffusion coefficients for a number of potential conditions for high yield microbubble isolation. Throughout the following, the following, a Boltzmann's constant of k=1.38E−23 J/K is presumed.
In one example, water is used as a fluid medium in which the microbubbles are provided. As such, viscosity values at various temperatures are provided as follows:
As such, diffusion coefficients based on microbubble diameter for a bubble fraction of =5% are provided when the effective viscosity is μ*(5%, 1° C.)=0.00197912 Pa·s, the values being:
With water at an elevated temperature of 25° C., the effective viscosity is μ*(5% , 25° C.)=0.00101816 Pa·s, and the diffusion equation values based on target microbubble size are:
Diffusion coefficients based on microbubble diameter for a bubble fraction of Φ=25% are provided such that the effective viscosity is μ*(25%, 1° C.)=0.003633 Pa·s, the values being:
With water at an elevated temperature of 25° C., the effective viscosity is μ*(25%, 25° C.)=0.001869 Pa·s, and the diffusion constant values based on target microbubble size are:
Diffusion coefficients based on microbubble diameter for a bubble fraction of Φ=50% are provided such that the effective viscosity is μ*(50%, 1° C.)=0.0071795 Pa·s, the values being:
With water at an elevated temperature of 25° C., the effective viscosity is μ*(50%, 25° C.)=0.0036935 Pa·s, and the diffusion constant values based on microbubble size are:
Alternatively, glycerol may be used as a fluid medium in which the microbubbles are provided. As such, viscosity values at various temperatures are provided as follows:
Diffusion coefficients based on microbubble diameter for a bubble fraction of Φ=5% are provided such that the effective viscosity is μ*(5%, 1° C.)=12.232792 Pa·s, the values being:
With glycerol at an elevated temperature of 25° C., the effective viscosity is μ*(5%, 25° C.)=1.03532 Pa·s, and the diffusion constant values based on target microbubble size are:
Diffusion coefficients based on microbubble diameter for a bubble fraction of Φ=25% are provided such that the effective viscosity is μ*(25%, 1° C.)=22.4553 Pa·s, the values being:
With glycerol at an elevated temperature of 25° C., the effective viscosity is μ*(25%, 25° C.)=1.9005 Pa·s, and the diffusion constant values based on target microbubble size are:
Diffusion coefficients based on microbubble diameter for a bubble fraction of Φ=50% are provided such that the effective viscosity is μ*(50%, 1° C.)=44.37595 Pa·s, the values being:
With glycerol at an elevated temperature of 25° C., the effective viscosity is μ*(50%, 25° C.)=3.75575 Pa·s, and the diffusion constant values based on target microbubble size are:
The present inventors have preliminary validated the application of SIMs having a size of 4-5 μm as an enhanced drug delivery system in a preclinical model of high-risk Neuroblastoma (NGP-NB). Data show that in presence of sonoporation, 4-5 μm SIMs allow a more homogeneous delivery of a drug (e.g., liposomal doxorubicin (L-DOX)) at a tumor site compared to administration of the drug alone or in combination with other commercial PMB product. Moreover, administration of a low dose of L-DOX (1 mg/kg vs 25 mg/kg) together with 4-5 μm SIMs and sonoporation in NGP-NB tumor-bearing mice achieved tumor growth control over two weeks without observing any side effects traditionally exhibited in traditional L-DOX doses (e.g., 25 mg/kg). On the contrary, the same quantity of L-DOX administered alone was not able to control tumor growth.
Chart 820 further illustrates the effect on size of microbubble on efficacy of sonoporation using a constant drug dose. As can be seen, polydisperse microbubbles used in sonoporation resulted in a tumor growth of roughly 175% as compared to use of 4-5 μm size isolated microbubbles, which demonstrated tumor growth of less than 50%. Paired T-tests show p-value of 0.3 at day 7 in the results of SIMs alone and SIMS+L-DOX as shown in chart 810 and of 0.045 at day 5 between PMB and SIMS in the chart of 820. As such, the data show that a low dose of the chemotherapy drug L-DOX (1 mg/kg) in combination with sonoporation/SIMS halted tumor growth as compared to L-DOX alone. SIMS allowed a lower and safer dose of L-DOX to be used which resulted in no observable side effects.
These results of tumor growth in relation to treatment using polydisperse sized microbubbles and size isolated microbubbles shows that in the presence of sonoporation, 4-5 μm SIMs allow a more homogeneous delivery of liposomal doxorubicin (L-DOX) at the tumor site compared to administration of the drug alone or in combination with polydisperse microbubbles. Moreover, administration of a low dose of L-DOX (e.g., 1 mg/kg versus a standard dose of 25 mg/kg) together with SIMS having a size of 4-5 μm and sonoporation in NGP-NB tumor-bearing mice achieved tumor growth control over two weeks without observing any side effects. On the contrary, the same quantity of L-DOX administered alone was not able to control tumor growth. To demonstrate SIMs are a more efficient drug delivery system compared to polydisperse microbubbles (PMB), six NGP tumor-bearing mice were divided into two groups treated with low dose L-DOX (1 mg/kg) together with respectively polydisperse microbubbles or 4-5 μm SIMs. On day 0 L-DOX was administered followed by sonoporation with the following parameters 2 MPa peak negative pressure (PNP), 3 W/cm2 power and 10% duty cycle. Tumor volumes were measured by caliper every other day until day five, at day seven tumors were measured by 3D imaging. The data shows that low dose L-DOX administration was more efficient in mice sonoporated with SIMS having a diameter of 4-5 μm compared to those treated with PMB.
Accordingly, one example sonoporation system includes SIMS having a size of 4-5 μm. The SIMs may be provided for intravascular administration at a concentration of (2.5×109 microbubbles/mL) followed by insonification of vascularized tumor tissue using a targeted ultrasound sonoporation device. The ultrasound sonoporation device may include an ultrasound transducer equipped with a focusing lens appropriate for a tumor distance from the ultrasound application site using a frequency of 1 MHz and PNP between 0.4-2 MPa (at tumor depth) where temporary permeation by sonoporation of tumor tissue is desired. SIMs of 4-5 μm offer a location selective technique of temporary tumor sonoporation for increasing permeation of therapeutic agents into sonoporated tumor tissues. Ultrasonic energy delivery and site-specific monitoring of sonoporation may also be performed using the sonoporation system.
One general aspect of the present disclosure includes a method for increasing tissue permeation in a target treatment area. The method includes administering to a patient a size-isolated microbubble (SIM) product and introducing ultrasound energy from an ultrasound device to the target treatment area comprising the SIMs. In turn, the method includes sonoporating the target treatment area in response to the ultrasound energy by cavitating the SIMs of the SIM product to increasing tissue permeation the target treatment area by sonoporation of capillary walls of vasculature of the patient in the target treatment area.
Implementations may include one or more of the following features. For example, the method may also include administering a drug to the patient. The increasing tissue permeation in the target treatment area may result in an increased diffusion of the drug from the vasculature of the patient in the target treatment area. In an example, the dose may be at a lower dose than an approved dose of the drug (e.g., a recommended dose for administration in the absence of the sonoporation of the present method). In an example, the dose may be at least half of the approved dose of the drug. In another example, the dose may be at least one tenth of the approved dose of the drug.
In an example, the drug may be a chemotherapy agent. The target treatment area may be a vascularized soft tissue tumor.
In an example, the SIM product may have at least 80 volume percent microbubbles having a diameter of not less than about 4 μm and not more than about 5 μm.
In an example, the method may also include identifying the target treatment area from ultrasound imaging generated by the ultrasound device. Furthermore, the method may include monitoring the sonoporation of the target treatment area using ultrasound imagining generated by the ultrasound device. The identifying and the monitoring may occur in a common time period.
In an example, the sonoporating may include disrupting epithelial cell tight junctions within vascularized soft tissue.
In an example, the administering the SIM product and the administering the dose of the drug may occur in a common injection to the patient.
Another general aspect of the present disclosure includes a sonoporation system for sonoporation of a target treatment area to tissue permeability in the target treatment area. The system includes a size-isolated microbubble (SIM) product comprising at least 80 volume percent microbubbles having a diameter of not less than about 4 um and not more than about 5 μm at a concentration of not less than about 1.0×109 microbubbles/mL. The system also includes an ultrasound device operative to target the target treatment area with targeted ultrasound energy to cause sonoporation in the target treatment area in response to cavitation of the microbubbles of the SIM product.
Implementations may include one or more of the following features. For example, the system may further include a dose of a therapeutic agent having a dose not greater than about 50% of an approved dose of the therapeutic agent. In an example, the SIM product and the dose of the therapeutic agent may be provided in a common administration vessel for simultaneous administration to a patient. The therapeutic agent may be a chemotherapy agent, and the target treatment area may be a vascularized soft tissue tumor.
In an example, the ultrasound device may be operative to identify the target treatment area from ultrasound imaging generated by the ultrasound device. The ultrasound device may be operative to monitor the sonoporation of the target treatment area using ultrasound imagining generated by the ultrasound device.
In an example, the sonoporation in the target treatment area disrupts epithelial cell tight junctions within vascularized soft tissue.
The description of a feature or features in a particular combination do not exclude the inclusion of an additional feature or features in a variation of the particular combination. Processing steps and sequencing are for illustration only, and such illustrations do not exclude inclusion of other steps or other sequencing of steps to an extent not necessarily incompatible. Additional steps may be included between any illustrated processing steps or before or after any illustrated processing step to an extent not necessarily incompatible.
The terms “comprising”, “containing”, “including” and “having”, and grammatical variations of those terms, are intended to be inclusive and nonlimiting in that the use of such terms indicates the presence of a stated condition or feature, but not to the exclusion of the presence also of any other condition or feature. The use of the terms “comprising”, “containing”, “including” and “having”, and grammatical variations of those terms in referring to the presence of one or more components, subcomponents or materials, also include and is intended to disclose the more specific embodiments in which the term “comprising”, “containing”, “including” or “having” (or the variation of such term) as the case may be, is replaced by any of the narrower terms “consisting essentially of” or “consisting of” or “consisting of only” (or any appropriate grammatical variation of such narrower terms). For example, a statement that something “comprises” a stated element or elements is also intended to include and disclose the more specific narrower embodiments of the thing “consisting essentially of” the stated element or elements, and the thing “consisting of” the stated element or elements. Examples of various features have been provided for purposes of illustration, and the terms “example”, “for example” and the like indicate illustrative examples that are not limiting and are not to be construed or interpreted as limiting a feature or features to any particular example. The term “at least” followed by a number (e.g., “at least one”) means that number or more than that number. As used herein, a range for a feature refers to one or more values for that feature within an upper limit and lower limit, inclusive of the upper and lower limits, and includes situations in which the upper limit and the lower limit are the same, that is when the range includes a single value represented by the equal upper and lower limits.
While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description is to be considered as exemplary and not restrictive in character. For example, certain embodiments described hereinabove may be combinable with other described embodiments and/or arranged in other ways (e.g., process elements may be performed in other sequences). Accordingly, it should be understood that only the preferred embodiment and variants thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/073,249 filed on Sep. 1, 2020 entitled “ULTRASOUND TRIGGERED MONODISPERSE SIZE-ISOLATED MICROBUBBLES (SIMBS) TO IMPROVE DRUG DELIVERY” and U.S. Provisional Patent Application No. 63/147, 967 filed on Feb. 10, 2021 entitled “ULTRASOUND TRIGGERED MONODISPERSE SIZE-ISOLATED MICROBUBBLES (SIMBS) TO IMPROVE DRUG DELIVERY,” the disclosures of which are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 63073249 | Sep 2020 | US | |
| 63147967 | Feb 2021 | US |
