Activatable infusable dispersions containing drops of a superheated liquid for methods of therapy and diagnosis

Dispersions of superheated drops of immiscible liquids in aqueous continuous phase suitable for infusion into a human or other animal, the drops being vaporizable in a selected body location by ionizing radiation or ultrasound. The dispersions can be used to form diagnostic contrast agents, to improve diffusion of drugs, to occlude capillaries and to deliver drugs selectively in a localized body region.

This application claims the benefit of U.S. Provisional application Ser. 
No. 60/009,699, filed Jan. 11, 1996. 
This invention relates to delivery of diagnostic and therapeutic agents in 
humans and animals. 
BACKGROUND 
Selective delivery of drugs to a particular target location in the body of 
a human or other animal has recognized benefits. Cancer chemotherapy is 
the best-known example. Cancer chemotherapy (the treatment of cancer with 
cytotoxic drugs) has produced dramatic improvements in the treatment of 
patients with hematopoietic and lymphoid malignancies; for example, 
childhood leukemia and Hodgkin's disease are now highly curable diseases. 
Antineoplastic drugs are also effective in treating microscopic metatases, 
when given in combination with localized treatment (surgery and/or 
radiotherapy) to control the sites of bulk disease. Cancer chemotherapy 
has proven less effective in the treatment of large solid malignancies. 
Solid tumors (e.g., cancers of the lung, breast, prostate, cervix, brain, 
head and neck) are the most common cancers of adults, and account for the 
vast majority of cancer deaths in the United States today. Almost without 
exception, anticancer drugs are toxic to cells of critical normal tissues, 
as well as to cancer cells. The intensity of treatment with these drugs is 
limited by the ability of normal tissues (and the patient) to tolerate the 
therapy, rather than by the amount of drug needed for optimal treatment of 
the tumor. Perhaps the greatest barrier to the effective treatment of 
solid tumors with antineoplastic drugs is the problem of drug delivery. R. 
K. Jain, "Delivery of novel therapeutic agents in tumors: physiological 
barriers and strategies," Journal of the National Cancer Institute (NIH), 
81, 570-576(1989). 
Selective delivery of anticancer drugs to a needed location, for example, 
the site of a solid tumor, is recognized to have potential value. A number 
of approaches have been tried in laboratory and clinical studies to 
improve the treatment of solid cancers. Direct topical application of 
drugs and intratumoral injection of drugs has had limited success, largely 
because the diffusion of drug from the administration site is inadequate. 
Selective infusion of tumors through a major artery supplying the tumor 
has been effective only in some settings. Solid "slow release polymers" 
containing antineoplastic drugs have been implanted into tumors. These 
attempts to deliver drugs directly to tumors have yielded limited success 
in some contexts, but have not proven to be widely applicable or 
effective. R. Langer, "New methods of drug delivery," Science, 249, 
1527-1533(1990). 
Different approaches have been tried for the purpose of "targeting" 
intravenously injected drugs. Ibid. Drugs have been attached to antibodies 
directed against specific tumor antigens. Drugs have been encapsulated in 
liposomes, starch microspheres, or other encapsulation vehicles in the 
hopes that this would protect the drug from inactivation in the blood and 
that these particles would lodge selectively in the abnormally tortuous 
tumor blood vessels. Attempts have been made to target liposomes, for 
example by developing magnetic liposomes and applying magnets to the 
surface of the tumor or by administering heat-sensitive liposomes and 
delivering heat in order to cause a tumor to become hyperthermic. Limited 
success has been observed. Drug release in non-target tissues remains a 
limitation. 
In diagnostic technology, ultrasound imaging is known. It is known that 
small gas bubbles can be employed as ultrasound contrast agents and that a 
liquid which is immiscible in blood and which boils slightly below body 
temperature (for humans, 37.degree. C.) can create such small bubbles in 
the body. Bubbles may be too large to enter the capillaries of a tumor, 
for example, which is an imaging limitation. 
An aspect of this invention is intravenous dispersions for selective 
intravenous delivery of therapeutic and diagnostic agents to a particular 
location in the body, comprising drops of a liquid that is superheated, 
most preferably highly superheated, at the temperature and pressure of use 
and that can be triggered to vaporize at a particular location by 
radiation or ultrasound. Triggering activates the dispersions for a 
localized purpose. Bubbles formed from vaporized drops may serve as 
therapeutic or diagnostic agents. Alternatively or in addition, the drops 
may carry a drug, which is released at a particular location in the body 
by such vaporization. 
An aspect of this invention is intravenous drug delivery dispersions 
comprising superheated, drug-carrying drops from which a drug can be 
released in a selected "target" location in a living body by application 
of a localized or localizable energy source, namely radiation or 
ultrasound, to activate the dispersions. 
Another aspect of this invention is methods of using intravenous 
dispersions of superheated drops for therapy or diagnosis at a selected 
target location in the body of a human or other animal which includes 
administering the dispersions intravenously and subjecting the selected 
target location with a localized or localizable source of radiation, most 
preferably x-ray or gamma ray, or ultrasound capable of nucleating the 
superheated drops to transform them into the vapor phase. The 
transformation activates the dispersion for a therapeutic or diagnostic 
purpose, such as to serve as a contrast agent or to deliver a drug. 
SUMMARY OF THE INVENTION 
This invention includes dispersions comprising drops of a superheated 
liquid dispersed in an injectable host fluid such as intravenous fluid. 
The dispersions are infusable, that is, suitable for infusion into the 
body of a human or other animal. The drops are "practically immiscible" in 
body fluids (for example, blood or urine) and host fluids with which they 
will be in contact. By this is meant the drops have sufficient 
immiscibility with body fluid of patients, whether human or other animals, 
to retain their integrity as drops after administration to permit 
localized vaporization and sufficient immiscibility with any intravenous 
or other host fluid used for infusion of the drops to retain their 
integrity as drops during preparation, storage, if any, and 
administration. Generally, solubility in aqueous host and body fluids 
should not exceed a few percent during the pertinent time period. 
The drops are a liquid having a boiling temperature below body temperature 
at atmospheric pressure. Preferred components for such drops are organic 
compounds such as fluorocarbons, chlorofluorocarbons and hydrocarbons. In 
some embodiments, inorganic components, such as silicone oils, can be 
used. Mixtures of components or additives such as salts can be used to 
adjust the boiling point of the drop material in known fashion. Additional 
liquid components can be included, for example to dissolve a drug. For 
ease of handling, preferred dispersions are emulsions. Emulsions according 
to this invention also include an emulsifier, a component that protects 
the drops by coating them and preventing their coalescing. 
The drop composition has a degree or amount of superheat rendering the 
drops susceptible to vaporization by a type and amount of radiation or 
ultrasound tolerable by the body. The lower the boiling point, the greater 
the amount of superheat of the drops. The greater the degree of superheat, 
the more susceptible the drops are to nucleation. Preferably, the drop 
material is sufficiently superheated that the drops will readily boil, or 
vaporize, when hit with radiation from a convenient source, or with 
ultrasound, which initiates boiling or "nucleates" the drops. The 
preferred degree of superheat is more than 17 degrees in Celsius units, or 
17 Celsius degrees, for this purpose. Most preferably the degree of 
superheat is very high for nucleation by common medical radiation sources 
such as x-rays or gamma rays, in the range of about 60-80 Celsius degrees, 
near but at least a few degrees below the amount of superheat that causes 
homogeneous nucleation, which occurs typically for pure organic liquids at 
approximately 0.9 Tc, where Tc is the critical temperature of the liquid 
in degrees K. The homogenous nucleation temperature for a given dispersion 
can be determined by gradually warming the dispersion until all the drops 
vaporize, which occurs at the homogenous nucleation temperature. For use 
at human body temperature, 37.degree. C., and pressure (slightly above or 
below one atmosphere), this means the drops preferably have a boiling 
point below 20.degree. C., more preferably below -5.degree. C., and most 
preferably below -15.degree. C. For most liquids a boiling point within 
the range of -15.degree. C. to -30.degree. C. at atmospheric pressure will 
be found to be suitable for nucleation in humans by x-rays. By "radiation" 
I mean ionizing radiation (such as x-rays, alpha, beta, gamma and 
neutron), particles or waves capable of causing an electron to be removed 
from an atom or molecule. "Ultrasound" used herein means acoustic waves 
that can produce physical effects, including nucleation of superheated 
drops into bubbles and the vibration of those bubbles. Generally 
ultrasound is above audible frequencies, that is, above 20 KHz (20,000 
Hertz). Ultrasound for diagnostics is generally 1-20 MHz (megahertz). 
Ultrasound for therapeutic applications is generally 20 KHz to 5 MHz. 
The bubbles formed by vaporization of the superheated drops may serve as 
therapeutic or diagnostic agents. They may occlude blood flow in 
capillaries of a tumor, for example. They may deliver oxygen. Drops may 
vaporize in a selected location to form bubbles that serve as contrast 
agents for diagnostic imaging, including x-ray, ultrasound and MRI. Some 
capillary regions may be entered by drops where larger bubbles, if 
preformed, would not enter. Bubbles may be stimulated with ultrasound to 
impart motion to the bubbles, thereby aiding in dispersal of a drug that 
has been delivered to the selected body location by means of a dispersion 
according to this invention or in some other manner. In certain 
embodiments, a drug is added to the superheated liquid, which may be said 
to be a "carrier" and to be "doped" with the drug. I use the term "drug" 
in the very broadest sense to include a substance intended for use in the 
diagnosis, cure, mitigation, treatment or prevention of disease, or a 
substance other than food intended to affect the structure or function of 
the body. I refer to "drug" in the singular, but it will be understood 
that combinations of drugs are to be included as well. 
Dispersions according to this invention are prepared by dispersing a 
superheatable liquid drop composition in an aqueous liquid under pressure. 
The aqueous liquid may be the infusable host or an aqueous liquid intended 
to be replaced by the ultimate host liquid. Depending on the size of the 
drops formed and their density relative to the aqueous phase, the 
dispersion may or may not be mechanically stable. Drops may tend to settle 
rather rapidly in some embodiments. Coalescing of drops is avoided by 
making the drops sufficiently small (generally less than 1 .mu.m average 
diameter) that Brownian motion maintains the dispersion, by application of 
gentle agitation to maintain the dispersion, by addition of a gelling 
agent or thickening agent to prevent or at least sufficiently retard 
settling, by addition of an emulsifier to coat the drops and prevent their 
coalescing when they settle, or by some combination of those techniques. 
By addition of a gelling or thickening agent, dispersions have been 
maintained without agitation for a period of three months with no 
indication of significant settling. A preferred dispersion technique is to 
mix a drop composition and a host solution or replaceable aqueous phase 
under pressure, and then to shake or sonicate the mixture to disperse the 
drop material. The final dispersion includes an aqueous infusion medium, 
typically an intravenous saline solution. 
Infusion into the body is by conventional means. Infusion can be, for 
example, by means of a catheter inserted into a selected body location, by 
injection directly into a tumor or organ, or by intravenous drip. It is 
noted that injection into the bloodstream closely upstream to the body 
part of interest, sometimes referred to as the "target" or "target 
location," aids in delivering a high concentration of superheated drops as 
compared to more remote injection. 
As the superheated drops are subjected to ionizing radiation or ultrasound, 
they vaporize (boil), thereby forming bubbles and releasing any drug with 
which they are doped into the local environment. I refer to this as 
activation of the dispersion. Vaporization is localized by localizing the 
body portion subjected to radiation or ultrasound. External radiation 
sources and ultrasound are localizable in that they may be directed to 
selected, specific locations in the body. Implanted radiation sources 
("brachytherapy") may also be used for localized effect. As superheated 
drops pass through the target location, a radiotherapy source (either 
external or brachytherapy) or ultrasound is applied to cause the 
superheated drops to boil, thereby forming bubbles and freeing a drug, if 
present, at the selected local site, for example, a tumor. If infusion is 
by intravenous injection, dispersed superheated drops will be recirculated 
naturally through the selected location. At each pass through the site of 
localized energy application (radiation or ultrasound), additional bubbles 
are formed and, if present, additional drug is released. Such localization 
of bubbles and drugs has benefit in cancer chemotherapy and in 
radiation/drug combination or combined modality therapy (especially of 
hard-to-treat solid tumors). Higher doses of a cancer drug can be 
delivered in this way than otherwise can be tolerated by the body, because 
most of the body does not receive the full dose that is delivered locally. 
DETAILED DISCUSSION 
Drop material is a liquid composition that is superheated at body 
temperature and pressure, as has been stated. One composition that has 
been evaluated includes two parts of chloro-pentafluoroethane (C.sub.2 
ClF.sub.5), one part pentane, and one part acetone into which the drug 
mitomycin C had been dissolved. All three ingredients are miscible. 
Pentane modifies both the boiling point and the density of the drop 
material. 
The proportions of materials helps to determine the composition's 
mechanical stability and also the degree of superheat. The greater the 
degree of superheat, the less energy required to initiate the 
transformation of the drops from the metastable liquid state to the vapor 
state. In other words, more greatly superheated drops are more easily 
nucleated. For many superheated liquids, if the degree of superheat is 
sufficiently great (more than about 50 Celsius degrees above its boiling 
temperature at the temperature of use), then the drops of the tested 
composition will be triggered at body temperature by a simple x-ray or 
gamma ray source, commonly available in hospitals. For less superheat, a 
neutron source or perhaps even a proton source is needed to trigger the 
drops at body temperature. The use of x-rays has obvious advantages for 
practical applications. Therefore, the composition selected for testing 
was made to be sensitive to x-rays and gamma rays at body temperature 
(37.degree. C.). 
Drop material is dispersed in an aqueous liquid, which can be the infusable 
host liquid but need not be. Dispersion can be in a first aqueous liquid, 
which is then substantially replaced by another aqueous liquid to form the 
dispersion to be infused. A concentrate can be prepared for dilution to 
the desired concentration in host solution. In preferred embodiments 
dispersion is accomplished in the presence of an emulsifier, so that the 
dispersion is an emulsion. Emulsified drops can be concentrated by 
settling or gentle centrifugation followed by decantation of most of the 
original aqueous phase. This emulsifier coats the drops and prevents them 
from coalescing at this concentrated stage. The final infusable 
composition can then be made by addition of a new aqueous phase, 
preferably one containing a gelling or thickening agent to prevent 
settling, and mixing, for example, stirring. If the dispersion is not an 
emulsion, settling may cause the drops to coalesce. This can be prevented, 
where necessary, by physically maintaining the dispersion, as by gentle 
agitation, until the time of infusion. Dispersion is performed under 
pressure and, preferably, at a temperature below room temperature. After 
the dispersion is prepared, pressure is relieved. At this point the drops 
have a significant degree of superheat but they do not vaporize. The 
amount of superheat of the drop material is below the homogenous 
nucleation temperature, which is approximately 0.9 times the critical 
temperature (in degrees K.) for most organic liquids and approximately 83 
Celsius degrees of superheat. Consequently, the boiling point of the drop 
material is adjusted to be above -30.degree. C. at atmospheric pressure. 
Also nucleation is avoided prior to use. Preparing the dispersion under 
pressure aids in avoiding premature vaporization. 
Some of the techniques useful for preparing and administering the system 
according to this invention are those known for drug carrying liposomes or 
perfluorochemical emulsions. However, drop formation is always done under 
pressure. 
Methods for how to make the superheated drop dispersion include, but are 
not limited to: 
1) A drug, if used, can be either soluble or insoluble in the superheated 
drop liquid composition. In the former case, it is dissolved under 
pressure, in the superheated drop liquid or, if necessary or desired, 
dissolved in one component of the drop material prior to constituting the 
final drop material. In the latter case, it must be in a fine powder form. 
Fineness can be increased as necessary to ensure that the drug is evenly 
dispersed among the superheated drops. Standard grinding techniques and/or 
pulverizing techniques are among several well established procedures for 
making solid materials into powder form. 
2) Liquid drop material is dispersed (oil-in-water dispersion) into an 
aqueous intravenous fluid or other aqueous "host" solution. Dispersion 
occurs at pressures equaling or exceeding the vapor pressure of the drop 
material at the particular temperature of the mixing so that it remains a 
liquid and does not vaporize during this processing. Dispersion can take 
place by many standard techniques, such as rapid stirring of the drop 
material into the host liquid, high intensity ultrasonic waves, and other 
methods generally known. Alternatively, a stream of the drop liquid is 
introduced into a stream of the intravenous fluid so that the stream 
breaks up into drops due to a Rayleigh-Taylor instability as well as the 
shear forces exerted by the outer stream of intravenous fluid. 
The drops have an average diameter in the range of 0.05-20 .mu.m, 
preferably in the range of 0.05-10 .mu.m and more preferably in the range 
of 0.1-5 .mu.m. Drop size varies with the manner of dispersion. Stirring 
or shaking by hand can be used to produce larger drops, for example, 10 
.mu.m and above. Sonication with an ultrasonic cleaner can be used to 
produce smaller drops, in the range of 1-10 .mu.m. For even smaller drops 
an ultrasonic horn emulsifier can be used. In all instances dispersion is 
performed under pressure. As a rough indicator, bubbles can be expected to 
have an average diameter six to nine times larger than the drops. If the 
drops have an average diameter below about 1 .mu.m, they may remain 
suspended owing to Brownian motion. A stable dispersion can be obtained, 
for example, by using an ultrasonic horn emulsifier to produce very small 
dispersed drops having an average diameter of, for example, in the range 
0.05 to 0.5 .mu.m, allowing any large drops to settle and decanting the 
major fraction containing small, non-settling drops. Otherwise, unless the 
drops are coated with an emulsifier to prevent them from coalescing, 
coalescence must be prevented. Addition of a gelling or thickening agent 
may suffice, as may gentle agitation, or a combination of the two 
techniques. 
Superheated drop dispersions according to this invention are physically 
stable from premature vaporization caused by shear stresses in handling 
and infusion. This flow stability was demonstrated by mixing 3 ml of a 
dispersion with 50 ml of water in a 60 ml glass syringe. The mixture 
contained roughly 10.sup.7 drops per ml. The mixture was flowed through a 
1.5 mm glass tube (inside diameter) by a syringe pump. The tube was 20 cm 
in length and was maintained at 37.degree. C. by a thermobath. At flow 
rates of 0.5 ml/min (0.45 cm/sec average), 1 ml/min (0.9 cm/sec average) 
and 2 ml/min (1.8 cm/sec average), no bubble formation was observed using 
a low power microscope. Similar results were obtained using plastic 
tubing. 
The drop material often does not form a stable dispersion in a saline 
solution. The drops either settle or can be made to do so by gentle 
centrifuging. This is not a problem and can be utilized to advantage if 
the dispersion is an emulsion, because concentrated drops remain separate 
due to the encapsulating material. They are nonetheless handled carefully 
to avoid premature vaporization, which at this stage can become a chain 
reaction. 
When an emulsion is used, the drop material can be added to the intravenous 
host fluid in the concentration to be infused. Gentle agitation at the 
time of use can, if needed, redistribute settled drops. If long-term 
stability of the emulsion in the intravenous fluid is not great, addition 
may closely precede administration. Because the drops separate, either 
naturally or with centrifugation, they can be collected by decanting most 
of the original host liquid and then, at a later time, diluted with the 
requisite amount of the particular infusion liquid to be used for a given 
application. 
A more stable emulsion or a stable concentrate can be prepared. Most of the 
original host liquid can be removed, as by simple decantation. It can be 
replaced by host solution containing a gelling or thickening agent, e.g., 
a colloid or polymer which prevents settling. The gelling or thickening 
agent preferably should not be toxic in the amount used, which should not 
be so much as to make administration practically difficult. 
3) Following infusion of the dispersion, the drops are nucleated locally, 
sometimes referred to as "triggering the drops" by radiation or 
ultrasound. The energy source can take several forms, including, but not 
limited to: 
A) An x-ray machine, such as used in diagnostic x-rays, is a directable, 
and hence, localizable source of radiation external to the body; 
B) Radiological sources, for example, cobalt 60, producing gamma x-rays, 
are also directable sources of external radiation; 
C) Any number of imbedded radiation sources (called brachytherapy sources) 
are localized sources of radiation internal to the body; 
D) A medical accelerator source of radiation (either x-rays or high energy 
electrons) is also a directable source of external radiation that will 
penetrate into tissue to trigger drops into bubbles; and 
E) An ultrasound source, such as used for diagnostic ultrasound 
examinations, or more powerful sources of ultrasound used in therapeutic 
applications of ultrasound (for example, to create hyperthermia) can be 
directed to the desired site (either coupled to the skin via a coupling 
gel or by a water standoff), triggering drops to form bubbles. 
An advantage of the dispersion of this invention is that the superheated 
drops are vaporized by a convenient source of radiation or ultrasound 
while circulating through a tumor or other selected location to form 
bubbles there and to release all or nearly all of any drug cargo there. 
Thus, this invention provides a way to increase concentrations of bubbles 
and drugs in a tumor or other selected location. This invention provides a 
way to achieve high concentrations of drugs having very short lifetimes in 
aqueous solutions. Moreover, it has been suggested that reducing tumor 
blood flow completely after agent had been delivered may provide 
therapeutic advantages. R. K Jain, "Delivery of novel therapeutic agents 
in tumors physiological barriers and strategies," Journal of the National 
Cancer Institute (NIH), 81, 570-576(1989), page 575, incorporated by 
reference herein. The transient mechanical disruption of blood flow by 
bubble formation and vessel occlusion offers yet another advantage of this 
invention. 
Ionizing radiation is useful to trigger the drops. The use of ionizing 
radiation as the triggering agent has several advantages. First, there is 
growing evidence than regimens combining concomitant radiation and drugs 
are more effective that regimens using sequential treatments. Preferred 
drugs for combined treatment with radiation are radiation sensitizers or 
bioreductive alkylating agents. Radiosensitizers such as misonidazole or 
etanidazole have proven effective in increasing the radiocurability of 
tumors in experimental animals. They have shown activity in several trials 
with human cancer patients, but the drug doses and number of drug 
treatments have been severely limited by toxicities which reflect the 
cumulative dose of drug delivered to certain normal tissues distant from 
the radiation field. The drug delivery system of this invention minimizes 
drug delivery to normal tissues with such treatment. 
Bioreductive alkylating agents, such as mitomycin C and porfiromycin are 
selectively toxic to radiation-resistant hypoxic tumor cells; in animal 
systems these drugs produce supra-additive effects when given along with 
radiation. Clinical trials at Yale Medical School have shown that 
concomitant treatment with mitomycin C increases the cure of head and neck 
cancer over that seen with radiation alone. Trials with porfiromycin plus 
radiation in head and neck cancer and with mitomycin plus radiation in 
carcinoma of the cervix are ongoing. The drug dose and number of drug 
treatments is limited largely by the toxicity of the drug to marrow and by 
the possibility of toxicity to lung, kidney, and other tissues outside the 
radiation field. Better targeting of drug delivery by the system and 
method of this invention affords a way to reduce the toxic effects and to 
improve these regimens. 
Delivery of drugs to a tumor by vaporizing superheated carrier drops using 
ionizing radiation also offers many technical advantages. Modern 
radiotherapy treatment planning techniques allow the delivery of radiation 
to the tumor volume with excellent precision, either through the use of 
multi-field external beam irradiation or through the use of brachytherapy 
(either low-dose-rate implants or high dose-rate remote afterloading). 
Fluorocarbon carrier materials offer an additional advantage for combined 
chemotherapy and radiotherapy. These materials effectively transport 
oxygen, as well as drugs, to a tumor site which would otherwise be 
hypoxic, thereby increasing the tumor's radiosensitivity. 
Modern ultrasound may also be used as triggering energy for bubble and drug 
delivery to selected regions. Superheated drops can be nucleated with 
sufficient intensities of short-pulse, low duty cycle (less than one 
percent) ultrasound, for example, with diagnostic ultrasound pulses (where 
the peak acoustic pressure exceeds approximately 1-3 MegaPascal, MPa) from 
a commercial scanner, for example, Advanced Technologies Laboratory's High 
Definition Imaging, HDI, system. In a test, superheated 
hexafluoropropylene drops were held in an aqueous host gel with a 
viscosity of a thin syrup and were triggered with ultrasound. Ultrasound 
was directed into the viscous liquid, whereupon bubbles were observed 
visually. In addition, it is known that acoustically-induced mechanical 
agitation of bubbles enhances diffusion, an advantage in distributing a 
delivered drug whether to nearby tumor or other targeted tissue. Moreover, 
the fact that drug-doped drops and triggered bubbles can be imaged by 
ultrasound is of value in non-invasively documenting drug distribution and 
in delineating tissue (e.g. tumor) structure. 
Particular uses and advantages of dispersions and methods according to this 
invention in the treatment of disease and in the diagnostics associated 
with treatment, include: 
a) local drug deposition; 
b) capillary occlusion, (if the bubbles are triggered in small 
capillaries), which in some circumstances will aid in treatment by slowing 
the convection of drug away from the desired region, thereby giving the 
drug more time to diffuse and act in its therapeutic mode; 
c) in-situ creation of contrast agents, because bubbles in vessels and 
capillaries are good contrast agents and, therefore, can be effectively 
imaged (e.g. with x-ray, ultrasound, or MRI); 
d) superadditive effects of drugs and radiation by using therapeutic levels 
of radiation in conjunction with localized drug treatment; 
e) oxygen delivery to hypoxic locations by use of an oxygen-carrying 
perfluorocarbon, thereby increasing the local susceptibility to radiation 
treatment of a tumor; and 
f) increasing drug diffusion by nucleating with ultrasound or radiation at 
a given location, and then agitating bubbles with ultrasound, the presence 
of an acoustic field at sufficient intensities and duty cycles and in an 
appropriate frequency range (e.g. from 20 kHz to 10 MHz) increasing drug 
diffusion into tissue by virtue of mechanical action of the sound field on 
the bubbles, thereby making drug treatment more efficacious.

EXAMPLE 
The following dispersion is presently preferred for drug delivery. It is an 
emulsion, which means that the superheated drops can be concentrated 
without coalescing, as will be related. The described procedure for 
preparing it is a preferred method of preparation of a mechanically stable 
superheated drop drug delivery emulsion. 
Drop material was a mixture. The major component of the drop material was 
chloro-pentafluoroethane (C.sub.2 ClF.sub.5), which is superheatable. 
Another preferred material is hexafluoropropylene (C.sub.3 F.sub.6). The 
drop material also contained acetone, in which the test drug mitomycin C 
was dissolved, and pentane. Both acetone and pentane are soluble in the 
halogenated ethane. Pentane was added to modify the boiling point and the 
density of the drops. Finally, one component of a surfactant combination 
was included, sorbitan monooleate (span 80). 
The major component of the aqueous host solution was water or standard 
intravenous diluent. The host solution contained the coating material, 
such as bovine serum albumin (BSA), or a second component of a surfactant 
combination. With span 80, Tween 80 (poly oxyethylene sorbitan monooleate) 
was used. A minor amount of gelatin or other suspending agent may be added 
at this point, but the preferred procedure is to add it later, as will be 
described. 
The function of each of the materials is summarized in the following table: 
TABLE 1 
______________________________________ 
Material Function 
______________________________________ 
Chlorofluorocarbon 
Volatile liquid component 
which permits drop composition 
to be superheated 
Acetone Dissolves the drug sample 
Pentane Adjusts degree of superheat of 
chlorofluorocarbon and drop 
density 
Surfactant A (optional) 
Aids drop formation when above 
3 ingredients are mixed in 
aqueous phase 
Drug material Kills cancer cells 
Aqueous phase or host 
Diluent for carrying drug- 
carrying drops 
Surfactant B Works by itself or with 
Surfactant A to encapsulate 
drops 
Suspending agent Aids in uniformly suspending 
drops of doped drop material 
in aqueous phase 
______________________________________ 
Assembling these components into the final emulsion requires care. If not 
performed properly, the drops will not be mechanically stable and will 
boil prematurely. Mechanically stable compositions were made under 
pressure by adding the drop phase material to the aqueous phase through a 
two-way pressure-tight valve having fittings for syringe injection. 
Two containers, denoted as A and B, were connected to one another through a 
pressure-tight valve. Acetone into which the test drug had been dissolved, 
pentane and chloropentafluoroethane (C.sub.2 ClF.sub.5) were added to 
container A in nominal proportions 1:1:2. Container A was under the vapor 
pressure of the combined mixture, which I refer to as the "drop phase." 
Container A was cooled to maintain the vapor pressure under about 2-5 
atmospheres (30-75 psi). When the combination of surfactant A and 
surfactant B was used to encapsulate the drops, surfactant A was also 
added to container A. (If BSA, also preferred, is used to encapsulate the 
drops, it is added to container B.) Acetone was chosen, because it 
dissolved the test drug and was soluble in the superheatable drop 
material. Selection of a dissolving agent appropriate for use with a 
particular superheatable material is a routine matter. It is noted that 
acetone is also miscible with the aqueous phase and is believed to bleed 
into that phase, causing at least some of the drug to precipitate out in 
the drops over time. As indicated earlier, the chosen drug may be soluble 
in the volatile component of the drop material, may be solubilized 
therewith, or may be added as a powder. Hence, acetone is optional. 
Pentane was added to adjust the degree of superheat of the 
chlorofluorocarbon: the more pentane added, the less the degree of 
superheat. Pentane also adjusted the density of the drops. It was added to 
minimize the degree of care required in assembling the components. The 
greater the degree of superheat, the greater the chance of premature 
vaporization, particularly when the drops are concentrated, which is a 
chain-reaction environment. Hence, pentane is optional. Pentane was 
soluble in the chlorofluorocarbon. Selection of a vapor pressure-adjusting 
material for a particular superheatable material is a routine matter. 
The aqueous phase, which I refer to as the "host phase" was added to 
container B. In this case it was not the final aqueous phase for infusion. 
In addition to water or intravenous fluid, container B contained the 
encapsulant for the drops (for example, bovine serum albumin, BSA or one 
part of the encapsulant, here surfactant B). The materials in container B 
were maintained at slightly above 0.degree. C. 
The valve between containers A and B was opened. Due to the higher pressure 
of container A, its content flowed to container B, which was then sealed 
by closing the valve. Temperature of container B was maintained at about 
0.degree. C. The pressure in container B was the vapor pressure of the 
contents at that temperature. 
The drop material was dispersed, in this case emulsified, in the host 
material in either of two ways: container B was shaken vigorously by hand, 
or container B was placed in an ultrasonic cleaner bath. Very small drops 
of drop material formed. The drops settled to the bottom, indicating that 
a stable emulsion was not formed. Drops did not coalesce, because they 
were protected by the encapsulant. 
At this point, container B was opened. Despite a degree of superheat of 
about 15-30 Celsius degrees at this cold processing temperature, the drops 
did not vaporize upon decompression. The procedure to this point minimized 
the presence of nucleating gas pockets, so the drops were not triggered. 
It was desired for this example that the emulsion be stable for a 
significant period, to permit storage for 2-3 months without the need to 
re-suspend the drops. Therefore, the drops were settled, and host liquid 
above the drops was carefully decanted. New host material was added, this 
time containing a small amount, less than one percent, of a carbomer to 
raise the viscosity to that of a light syrup, which greatly lowered the 
tendency to settle. Other compatible gelling agents, such as gelatin, can 
be substituted. The sealed container was gently shaken to suspend the 
drops throughout the aqueous phase. 
A further optional stabilizing step may be used to reduce premature 
vaporization. The final emulsion is pressured to about 1000 psi for an 
hour or more to squeeze any undissolved gas in the material or on the 
container walls into solution. This was done by placing the final 
emulsion, held in container B, in a hydraulic pressure chamber, coupled to 
water in the chamber through a flexible plastic membrane (which replaced 
the cap on container B). After the pressurization step, the mixture in 
container B is returned to atmospheric pressure and re-sealed with a cap 
that holds pressure, and is stored in a refrigerator at about 4.degree. C. 
until required for use. 
Emulsification by simple agitation (shaking) will generally result in drops 
in the 10 to 100 .mu.m diameter range. When using an ultrasonic generator 
(.about.21 KHz frequency), the drop size distribution is primarily in the 
1-10 .mu.m range. Using the sonicator technique, a sample was prepared in 
which 94% of the drops had diameters less than 10 .mu.m. This corresponds 
to 70% of the total volume in drops that are smaller than 10 .mu.m. We 
intend to make composition in which 95% of the drops will be below 5.mu.m 
using the sonicator technique. As indicated earlier, bubbles have a 
diameter roughly six times that of the drops. Thus, bubbles formed by the 
method of this invention are not seen to be physically a problem to the 
body but rather to be removable naturally by the lungs, if not absorbed by 
the body. 
Drug concentration in the drops can be measured by spectrophotometric 
techniques. Mitomycin C, the test drug, has a relevant absorbance peak at 
360 nm. A typical test will be described. The test material was 0.4 ml 
superheated liquid (comprising 0.1 ml acetone solution of mitomycin C,0.1 
ml pentane and 0.2 ml C.sub.2 ClF.sub.5) introduced into a water solution 
of Bovine Serum Albumin (BSA), which is one of the test encapsulant 
materials that has been tested. Drops were generated by sonicating this 
mixture. After the drug-bearing drops precipitated to the bottom of the 
container, the residual aqueous solution was removed and 10 ml distilled 
water was added. Then all drops were triggered to boil by heating them to 
over 60.degree. C., which is above the homogeneous nucleation temperature 
of drops of this composition, but which is not high enough to decompose 
the mitomycin. 
Optical absorption of the final water-plus-released drug was performed with 
a Beckman, model DU-2 spectrophotometer. Used as reference liquid was an 
aqueous solution of mitomycin C with a concentration of 1 .mu.M. It was 
determined that the concentration of mitomycin C in the test liquid was 12 
.mu.M. Based on the known ratio of drop material to added water, we thus 
determined that the concentration of drug in the drops themselves was 
approximately 300 .mu.M. These measurements were preliminary and accurate 
to .+-.25%. 
Tests performed with drops not containing any drug were also performed and 
gave optical absorption equivalent to that observed for the control, 
water. 
The bubble production rate for a given dispersion of drops is evaluated 
simply by exposing a known amount of drop material to a source whose 
radiation flux is known at the position of the sample, and just counting 
acoustically the number of bubble "popping" events that occur per unit 
time. A typical test will now be described. 
This test employed a 56.7 millicurie (mCi) Cesium 137 gamma source. The 
contents of container B consist of 0.4 ml of the drops phase and 9.6 ml of 
the aqueous phase. The drops phase contained 0.1 ml acetone, 0.1 ml 
pentane and 0.2 ml C.sub.2 ClF.sub.5. The aqueous phase contained water, 
carbomer gelling agent (Carbopol.RTM. 1342 from B.F. Goodrich) and BSA 
emulsifier. In order to reduce radiation sensitivity, 0.3 ml of the 
emulsion was taken and diluted in 5 ml of additional diluent. It was 
tested by acoustically counting the number of bubble events. The result 
was a sensitivity of 27 (accurate to .+-.15%) counts per millirad per mg 
of drop material. Therefore, for a 100 rad irradiation (1 Gy), which would 
be high for diagnostic uses but low for therapeutic effects, a count of 
roughly 2.7 million bubbles formed per mg of drop material (which does not 
include the aqueous diluent) would be expected. At the drug concentration 
in the test drops of 300 .mu.M, a dose of 1 .mu.g of drug (molecular 
weight 334) would be carried by 10 mg of drop material. As a rough 
calculation, if all drops were 7 .mu.m in diameter (the approximate center 
of the measured distribution), then there would be 5.8 million drops in 
one mg of drop material. Thus, 1 Gy of radiation appears to result in the 
vaporization of approximately half of the drops in the sample. (This 
order-of-magnitude calculation ignores depletion effects.) 
Ultrasound negative pressure (during the rarefaction part of the sound 
wave) is equivalent to the superheat caused by elevated temperature. The 
peak negative pressures of many diagnostic ultrasound machines can exceed 
3 MPa (30 atmospheres). The usefulness of ultrasound as a nucleating agent 
was tested by pouring drop-bearing composition into a water bath at 
37.degree. C., through the focal zone of a 2.5 MHz ultrasound transducer 
from a Hewlett Packard SONOS 100 ultrasound scanner. The drops in the 
composition, which had a density slightly greater than water, fell in the 
bath, whereas the triggered bubbles, which were buoyant, rose, making an 
assessment of ultrasound effectiveness straightforward. If the ultrasound 
is effective, one sees small bubbles rising in the bath. As a control, the 
ultrasound was turned off and the focal zone was observed with an optical 
cathetometer to see if bubbles formed. The SONOS scanner was turned on and 
the results observed. 
At an output intensity of approximately 100 Watts/cm.sup.2 (spatial peak, 
pulse average), or greater, bubbles were nucleated, whereas in the control 
with sound off, no bubbles were generated. One can make an estimate of the 
peak negative pressure, assuming an acoustic plane wave in the focal zone. 
In this way, the peak negative pressure was estimated to be 1.7 MPa (17 
atmospheres). This, of course, is a maximum estimate. An NTR miniature 
immersible hydrophone was also used. With it we measured a nucleation 
threshold of 4-5 atmospheres peak negative pressure. These measurements 
confirmed that diagnostic pulses with duty cycles less than one percent 
can trigger drops to release their drug cargo. 
Before the initiation of tests using biological cell cultures or 
experimental animals, procedures were developed to ensure the sterility of 
the drop composition samples. Since the procedures used in processing the 
composition required certain steps that would prevent standard 
sterilization techniques (gas sterilization or autoclaving) sterilization 
procedures which relied on exposure of pressurized samples (i.e. ones that 
are without superheat and therefore not sensitive to radiation) to gamma 
rays (45 Gy) from a Cs-137 source or exposure of the samples to intense 
ultraviolet irradiation were tested. Irradiation procedures were found, 
both with UV and with gamma irradiation, to result in complete 
sterilization of approximately half of the samples. The sterility of the 
cultures was tested using standard clinical sterility tests and a variety 
of nutrient media and agars, with up to two weeks of incubation. Higher 
irradiations levels are believed to produce 100% bacteria-free samples. 
The effects of the drops on mammalian cells were examined using EMT6 mouse 
breast carcinoma cells, using techniques for studies examining the effects 
of radiation, radiosensitizers, and a wide variety of chemotherapeutic 
drugs. EMT6 cells were grown as monolayer cultures attached to the growth 
surfaces of cell culture dishes and overlaid by a liquid nutrient medium 
(Waymouth's medium supplemented with 15% serum). All studies were 
performed using exponentially growing monolayers. The sterile emulsion 
(.about.20 to 50 .mu.l total amount) was gently pipetted onto the top of 
the medium, and drops were distributed uniformly over the cultures by 
gentle swirling of the dishes. The drops, which were denser than the 
growth medium, fell to rest on the surface of the cells and the dish. 
Preliminary studies included: a) untreated control cultures, b) cultures 
to which drops were added, but no radiation was given to trigger bubble 
formation, c) cultures treated with both the drop composition and 
radiation (1 Gy of x-rays from a 250 kV x-ray source, delivered 5 min 
after the addition of the drops to trigger boiling), and d) cultures 
receiving radiation alone. For the amounts of dispersion tested (20-50 
.mu.l), neither the intact drops nor the drops without drug triggered to 
lysis by x-rays altered in a significant way either the number of intact 
cells in the cultures or the viability of the cells, as tested by 
suspending the cells after treatment and assaying their ability to form 
colonies. These experiments showed that the intact drops were not toxic to 
tumor cells during the 1 hr. incubation. Moreover, neither the process of 
triggering the drops into bubbles near the tumor cells nor the materials 
released by the drops after lysis were toxic to these cells at the 
concentrations used in these experiments. 
In subsequent studies, compositions were prepared analogously, but the 
antineoplastic drug mitomycin C was incorporated into the drops. Mitomycin 
C is a bioreductive alkylating agent which is widely used in the treatment 
of solid tumors. Preliminary studies were performed to examine the effects 
of drops containing mitomycin C. These experiments included a) untreated 
controls, b) unlysed drops, containing no drug, c) radiation-triggered 
drops, containing no drug, and d) radiation alone. In addition, groups 
treated with e) untriggered drug-containing drops, f) radiation-triggered, 
drug-containing drops, and g) free mitomycin C ("MC"), were included: 
TABLE 2 
______________________________________ 
Survival Fraction - Cell Culture Tests 
Treatment Surviving Fraction 
______________________________________ 
a. Untreated controls 
1.00 
b. Unlysed drops, no drug 
1.03 
c. Radiation-triggered drops, no 
0.94 
drug 
d. Radiation alone 0.85 
e. Unlysed drops containing MC 
0.77 
f. Radiation-triggered drops 
0.49 
containing MC 
g. 2 .mu.M MC (drug control) 
0.017 
______________________________________ 
Geometric mean of Surviving Fractions were determined in two independent 
experiments. Because 2 samples were tested in groups e. and f. in each of 
these experiments, these means are for a total of four samples. 
Formulations of the drug-bearing drops were found to contain up to 300 
.mu.M mitomycin. Drops were dispersed in an aqueous diluent at a volume 
fraction of 0.04, so that the effective concentration in the suspension 
was 12 .mu.M. Five drops of suspension pipetted onto the cell culture have 
a volume of approximately 50 .mu.l. Therefore, the maximum total 
concentration of drug deposited on the cell culture, after irradiation, 
would be 0.12 .mu.M if all of the drops were triggered and 0.06 .mu.M if 
50% of the droplets were triggered to bubble formation, as measured in the 
dose-bubble formation studies described above. This amount can be compared 
to the concentration of free mitomycin C used in one of the controls of 
2.0 .mu.M, the range used in reported studies of the biological effects of 
this drug. Based on those studies of the survival of EMT6 cells treated 
with different doses of mitomycin C, it was estimated that a 1 hr. 
treatment with 0.12 .mu.M mitomycin C should result in a surviving 
fraction of approximately 0.8. These predicted survival fractions are in 
fair agreement with that observed in the experiments, shown in Table 2, 
given the uncertainties involved in testing these preliminary 
formulations, and taking into account that the radiation given to the 
cells has a moderate toxicity by itself. 
The tests reported above indicate that superheated drops can be doped with 
drugs and encapsulated in an aqueous diluent, and that these drops can be 
triggered by x-ray irradiation at levels significantly lower than 
therapeutic regimens of radiation (and also by diagnostic levels of 
ultrasound), thereby releasing their cargo of drugs. 
Although the amount of drug released in cell culture tests was only 3% of 
the control with drugs directly used on the cell culture in one test and 
about 6% in a second test, there was a measurable decrease in the growth 
of EMT6 cells. When drops not containing drugs were triggered to boil, no 
similar decrease in cell activity was observed. It was found that much 
greater amounts of drops material tested caused EMT6 cells to react 
negatively, becoming unplanted from the petri dish. Some component, 
perhaps in the particular encapsulant, had a toxic effect on the cells at 
high concentrations. Since a number of different encapsulants can be used 
in this invention or encapsulants can be omitted entirely, this side 
effect is not inevitable. 
Our work indicates the drop material can be triggered in great enough 
proportions with amounts of x-rays significantly lower than therapeutic 
doses. We found that 1 .mu.g of drug carried in approximately 10 mg of 
drop material (carried in 0.25 ml of normal suspension) could be infused 
in 2.5 minutes at a rate of 0.1 ml per minute. If exposed to 1 Gy of 
x-rays at the position of a tumor, over 2 million bubbles, or 
approximately one half of the drops, would be triggered, releasing drug 
into the tumor. These results imply that a bearable radiation exposure to 
the patient will release adequate amounts of chemotherapeutic drug into a 
tumor, releasing significantly less drug outside the tumor region, thereby 
sparing normal tissue and allowing for a spatial partitioning. 
The above examples are given for the purpose of illustration, not as a 
limitation. Other useful embodiments containing no drugs or different 
drugs and other materials, and different proportions of materials, are 
included in this invention and are within the skill of the art.