Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS FOR THE REVERSAL AND DETOXIFICATION
OF ANESTHETICS AND OTHER COMPOUNDS
AND METHODS OF THEIR USE
DESCRIPTION
BACKGROUND OF THE INVENTION
Field of the Invention
This invention focuses on the administration of dispersions of certain
lyotropic liquid
crystal compositions to attenuate the toxic or medically undesirable effect of
one or more
compounds present in the body of a human or other mammal. The particles in
dispersion
comprise reversed cubic phase and/or reversed hexagonal phase liquid
crystalline material in
which a toxin or a drug substance is soluble and partitions substantially. The
particle dispersions
are suitable for administration to a human, and are given preferably by
injection, most preferably
intravenously, in an amount sufficient to be effective in attenuating the
effects of a toxin or
therapeutic drug in the body. The attenuation of the effects of toxins or drug
substances in the
body may result from sequestering the toxin or therapeutic drug from the
plasma, displacing the
toxin from the site of action, inducing redistribution of the toxin, or by
other mechanisms.
Adjusting the composition by various means may increase or decrease the
absorption or
adsorption of a toxin or drug substance to the particles, the rate of uptake
of the particles and
associated toxins or drug substances by the liver, and otherwise impact
attenuation of the effects
of a toxin or drug substance. The invention is especially applicable in
reversing adverse effects
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of local anesthetics delivered systemically, and attenuating the therapeutic
effects of general
anesthetics in the course of treatment.
Background of the Invention
In most clinical applications, local anesthetics are typically injected or
applied at or near
a site intended to render an area or region of the body insensate to painful
stimuli. Once applied
or administered, systemic absorption of the local anesthetic occurs or, in
some instances, a
portion of the injectate may be inadvertently administered directly into the
vascular system. In
any case, local anesthetics may exert varying degrees of systemic toxicity.
Toxicity is usually
directly proportional to the potency of the local anesthetic administered. It
is widely believed
that most local anesthetics exert their effects by binding to the alpha-
subunit and blocking the
voltage-gated sodium channel from an intracellular location, thereby
conformationally
inactivating the sodium channel and disrupting the influx of sodium ions
preventing membrane
depolarization. Local anesthetics are also known to block calcium, potassium
and N-methyl-D-
aspartate (NMDA) receptors to varying degrees. These differences are
associated with the
unique clinical profiles associated with each local anesthetic agent.
The unintentional intravascular administration of local anesthetics,
bupivacaine in
particular, which can occur during procedures designed to effect regional
anesthesia can result in
severe cardiac complications. These reactions include marked hypotension,
atrioventricular
dissociative heart block, idioventricular dysrhythmias as well as ventricular
tachycardia and
ventricular fibrillation. It is widely accepted that the R+ isomer of
bupivacaine has a strong
affinity to binding cardiac sodium channels and that its dissociation from
this site is very slow.
At higher concentrations, calcium and potassium channels can also be blocked,
further
exacerbating the cardiotoxic effects.
Bupivacaine induced cardiac toxicity is a life-threatening emergency in which
aggressive
measures must be undertaken to preserve life. These measures frequently
involve immediate and
repeated administration of vasopressors to maintain normovascular tone as well
as other agents
to control the bradycardia, complete heart block and the various cardiac
dysrhytmias which
ensue. Cardiopulmonary resuscitation may be required to maintain oxygenation
of vital organs
while the myocardium is in arrest. It is not uncommon to attempt to emergently
effect
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extracorporeal oxygenation via cardiopulmonary bypass with membrane
oxygenation (or similar
device) to await the return of a normalized cardiac cycle once the offending
concentration of
bupivacaine has been redistributed, metabolized or otherwise inactivated, and
allow for the
physiologic normalization of the myocardial cycle.
A number of specific therapies for local anesthetic toxicity have been
proposed over the
years including bretylium, glucose/insulin/potassium infusions, emergent
resuscitative efforts
(i.e. cardiopulmonary resuscitation CPR), and cardiopulmonary bypass.
Recently, attempts have
been made to use fatty emulsions, typified by the product marketed under the
name Intralipid ,
to scavenge bupivacaine and thereby reverse toxic effects. [See, e.g.,
Weinburg G, Ripper R,
Feinstein DL, Hoffman W. RA&PM 2003; 28:198; Weinberg et al. (1998)
Anesthesiology
88(4):10711. Similarly Intralipid has been investigated for use in
ropivacaine toxicity. [Litz et
al. (2006) Anaesthesia 61:8001. Tebbutt et al. found increased survival in a
rat model of
verapamil toxicity. [Tebbutt et al., Acac. Emerg. Med. (2006) 13:134]. Bania,
Chu and Stolbach
looked at the use of Intralipid to try to raise the LD50 in mice of an
organophosphate
compound, paraoxon. [Acad Emerg Med (2005) 12(5 Supplement 1):12]. The group
of Bania
and Chu has also looked at the use of Intralipid to treat toxicities from
propanolol, VER, and
amitriptyline. [Reported at the 2006 National SAEM Meeting, San Francisco CA].
Mathy-
Hartert et al. investigated the use of Intralipid against reactive oxygen
species produced by
phorbol myristate acetate, but found only a weak effect. [Mathy-Hartert et
al., Mediators
Inflamm. (1998) 7:3271. Microemulsions made from Pluronic surfactant, ethyl
butyrate, fatty
acid sodium salts and water have been proposed for scavenging bupivacaine by
the group of
Dennis et al. [See Dennis et al., U.S. Patent Application Serial No. 10
/420,608, Varshney et al.
(2004) J. Am. Chem. Soc. 126:5108 and Renehan et al. (2005) Reg. Anes. Pain
Med. 30(4):3801.
Dennis and coworkers have also proposed particles containing detoxifying
enzymes, in U.S.
6,977,171.
Intralipid and related parenteral fatty emulsions have several disadvantages
and
limitations in the present context. Perhaps most importantly, they rely on
triglycerides (soybean
oil in Intralipid , other fats for the other fatty emulsions) for the
solubilization and partitioning
of compounds into the emulsion droplets, and as such they are strongly
limited. Triglycerides
are many-fold higher in molecular weight (on the order of 900Da) than most
compounds that are
known to be effective solubilizers, as the favorable entropies of mixing
associated with low-MW
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compounds is important for the functioning of most solvents. They are
extremely hydrophobic,
and thus very poor solvents for compounds that have one, or particularly more
than one, polar
group. Their propensity to support microbial contamination makes them poorly
suited for field
applications. And the high levels of polyunsaturated fats in lipid emulsions,
combined with low
levels of tocopherol to combat oxidation, lead to growing levels of peroxides
and other free
radical sources that can contribute to toxicities related to reactive oxygen
species.
There is a need for a pharmaceutically acceptable composition and method for
attenuating the effects of common drugs in circumstances in which they are
toxic and in
situations in which for other reasons, such as medical treatment or patient
management, their
attenuation is desirable which can work effectively, against a range of drugs
and other toxins,
and can be adjusted for different characteristics of action and different
toxins.
SUMMARY OF THE INVENTION
In an exemplary embodiment of the invention, a method of attenuating the toxic
effect of
a toxin in a human or other mammal is achieved by administering to the human
or other mammal
in whom a toxin is or is suspected to be present an effective amount of a
composition comprising
particles which are formed from or include reversed cubic or reversed
hexagonal phase material.
The particles are preferably present in a stable dispersion which includes a
liquid comprising a
polar solvent. The particles may be ionically charged (anionic or cationic).
The administration
is preferably by injection (preferably i.v.) and results in the attenuation of
the toxic effects of the
toxin by the particles adsorbing or absorbing or otherwise sequestering the
toxin from the site of
toxic action.
It is another exemplary embodiment of this invention, a method of attenuating
the
therapeutic effect of a drug substance present in a human or other mammal is
achieved by
administering to the human or mammal in whom a drug substance is or is
suspected to be present
an effective amount of a composition comprising particles which are formed
from or include
reversed cubic or reversed hexagonal phase material. The particles are
preferably present in a
stable dispersion which includes a liquid comprising a polar solvent. The
particles may be
ionically charged (anionic or cationic). The administration is preferably by
injection (preferably
i.v.) and results in the attenuation of the therapeutic effects of the drug
substance by the particles
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adsorbing or absorbing or otherwise sequestering the drug substance from the
site of
pharmacologic action.
A further exemplary embodiment of this invention is to provide a method of
attenuating
the effect of a chemical substance in a human comprising administering to the
human a stabilized
composition comprising lipid, tocopherol and a liquid comprising a polar
solvent.
Whatever their specific therapeutic use, drugs are not always present at the
most desired
level in the body, and at times it may be necessary or beneficial to attenuate
their effects. Some
drugs of medical use, most notably the local anesthetics, can become life-
threatening if they are
inadvertently injected into a vein or artery, calling for removal or reduction
to avoid or minimize
neurotoxicity and cardiotoxicity. Other drugs used in medical practice, such
as those associated
with surgical procedures including general anesthetics like propofol and
paralytic agents like
vecuronium, can call for removal or reduction in a number of circumstances:
for example, after
interruption or even completion of a surgical procedure, it may be desirable
to attenuate the
lingering effects of the general anesthetic or paralytic agent whose normal
therapeutic effect has
become, post-operatively, a nuisance, danger or impediment to optimum patient
management. In
other cases, a drug may have side effects on account of generating toxic
metabolites in
circulation, it may be debilitating to the patient, or it may be detrimental
in terms of tolerance or
addiction, and attenuation of the effects of the drug may be an important
therapeutic need.
Certain compounds, such as cocaine or morphine, for example, are of course
used in medical
practice, but also are used as drugs of abuse and as such may require removal
or reduction in the
course of detoxification and rehabilitation. Furthermore, metabolites of
certain drugs, such as
cocaethylene in the case of cocaine and nor-meperedine in the case of
meperedine (Demerol ),
have considerable toxicity and their removal could be of importance in many
settings. Finally, a
toxin can be endogenous, such as an autoantibody or cortisol. In a particular
embodiment of the
invention, the invention permits attenuating the therapeutic effects of drugs
on a selective basis,
as well as in the removal of exogenous drug substances and endogenous toxins
from the site of
action.
Therapeutic agents such as propofol and other general anesthetics are selected
for use and
used, in part, on the basis of the duration and intensity of their therapeutic
effect. In the course
of their use, clinical circumstances may arise making it desirable to
attenuate the therapeutic
effects, for example, by reducing the time to emergence from the effect of a
general anesthetic,
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or the duration of effect of the neuromuscular blocking agent. In an exemplary
embodiment, the
invention provides the ability to attenuate those effects and would provide an
additional tool for
optimum management of, for example, a course of surgery, as well as for the
safety, comfort and
convenience of the patient. In the case of an outpatient surgical procedure
that proceeded
successfully and more promptly than anticipated, for example, there may be
benefits to patient
and provider alike to accelerate the return of the patient to clear
headedness, facilitating the
discharge of the patient. In the case of a surgical procedure which has not
proceeded according
to plan and is being altered or terminated, there may be benefits to
accelerating the patient's
return to a state free from the therapeutic effects of a general anesthetic or
a neuromuscular
blocking agent.
In another setting, the course of delivering an anesthetic or securing an
airway, it is of
critical importance to relax skeletal muscles. This is most commonly
accomplished with the use
of neuromuscular blocking agents of either the depolarizing or non-
depolarizing class.
Neuromuscular blocking agents are used to facilitate endotracheal intubation
in securing and
maintaining a patent airway, effecting relaxation of skeletal muscles to
enable certain operative
procedures and to ensure patient safety in certain clinical settings.
Depolarizing agents, i.e.
succinylcholine, act as acetylcholine (Ach) receptor agonists and quickly
cause short lived
muscle relaxation due to the rapid diffusion away from the neuromuscular
junction and are
hydrolyzed by nonspecific cholinesterases. In contrast, non-depolarizing
agents, i.e.
vecuronium, function as competitive antagonists. The fact that non-
depolarizers are neither
metabolized by acetylcholinesterase nor eluted quickly from the Ach receptor
predictably result
in prolonged depolarization of the neuromuscular endplate, and yield longer
duration of muscle
relaxation. The best clinical practice and safest approach in the care of
those paralyzed with
neuromuscular blocker agents is to reverse, as completely as possible, the
relaxant effects of
these agents, especially non-depolarizing agents once relaxation is no longer
required.
Typically, reversal of the non-depolarizing agents is augmented by the
administration of
cholinesterase inhibitors, also known as anticholinesterases, i.e.,
neostigmine. These agents
reversibly bind to the enzyme that degrades Ach thereby indirectly increasing
the amount of Ach
available to competitively displace non-depolarizing agents from the Ach
receptor by re-
establishing normal neuromuscular function at the neuromuscular endplate.
Complications and
other adverse reactions can occur with the administration cholinesterase
inhibitors. These agents
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may be metabolized or administered in lower doses causing ineffective
displacement of non-
depolarizers from the receptor, yielding latent and unexpected return of
paralysis. Administered
in excessive doses, Achase inhibitors may paradoxically potentiate the effects
of neuromuscular
blockade. Other well known effects of increases in Ach associated with the use
of Achase
inhibitors include vagal mediated bradycardia, bronchospasm initiated by
smooth muscle
contraction, central nervous system effects including diffuse excitation,
intestinal spasms,
increased bladder tone and papillary constriction. Therefore, any strategy to
minimize,
supplement or preferably avoid the use of cholinesterase inhibitors in the
reversal of
neuromuscular blockade offers multiple advantages in the clinical setting. In
an exemplary
embodiment of the invention, this invention provides such a strategy.
Cyclodextrins are known to bind a number of drug molecules, such as rocuronium
and
vecuronium, and as such are being used as reversal agents for compounds like
rocuronium. It
should be noted that bradycardia is sometimes associated with the intravenous
use of
cyclodextrins, which is a distinct problem for the use of such compounds in
connection with
rescue from local anesthetic toxicity, where bradycardia is already a serious
life-threatening
complication. In another exemplary embodiment of the invention, an alternative
to the use of
cyclodextrins is provided.
Another embodiment of the invention relates to the management of the use of
anesthetics
in the clinical setting (e.g., hospital, doctor's office, clinic, nurses
station, etc.). In practice, a
patient will be provided with an anesthetic before, or after, or
simultaneously with a composition
containing particles that include reversed cubic phase or reverse hexagonal
phase material. The
patient will be subject to a medical or surgical procedure or will be simply
observed. The
anesthetizing effect of the anesthetic will be attenuated by the particles
containing the reversed
cubic phase or reverse hexagonal phase material. This allows control of the
anesthetizing effect
and may enhance a patient's recovery time. The type of anesthetics which may
be managed in
this way include both general anesthetics such as propofol, etomidate,
ketamine, thiopental, a
benzodiazepine, a barbiturate, an opioid, haloperidol, droperidol, and
phencyclidine, and local
anesthetics such as bupivacaine, lidocaine, ropivacaine, mepivacaine, and
cocaine. With respect
to general anesthetics, the invention can be used for decreasing a duration of
sedation, reducing a
time to emergence, reducing a duration of apnea, and reducing a time to full
cognition.
In another embodiment of the invention, pharmaceuticals in a wide range of
drug classes
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can have their effects selectively attenuated by providing the patient with a
composition
including particles that have either reversed cubic phase or reversed
hexagonal phase materials.
The methods of the invention can be used in conjunction with, for example, a
patient that has or
is suspected to have taken, or a patient that will be provided with a
benzodiazepine, an opiate, a
central venous system depressant, a respiratory depressant, a cardiovascular
depressant, a
psychomotor stimulant, a psychotropic, a sedative, a hypnotic, a muscle
relaxant, and an
organophosphate.
DETAILED DESCRIPTION OF THE PREFERRED
EMBODIMENTS OF THE INVENTION
A. Definitions.
"Attenuation" of a drug effect or toxic effect of a toxin means that one or
more of the
following occur (as compared to in the absence of treatment with the
invention): substantial
reduction in intensity of the effect, substantial reduction in the duration of
the effect, substantial
reduction in physiological insult and damage, substantially accelerated
clearance of the drug or
toxin from active or otherwise critical sites in the body, substantial
reduction in symptoms of
adverse reaction to the drug or toxin, substantial reduction in the
concentrations of one or more
toxic metabolites of the drug or toxin from critical sites in the body, or
substantial reduction in
pathological binding of exogenous or endogenous substance(s). In this
definition, an effect, such
as a reduction (e.g., in intensity), is considered substantial if it is
clinically or medically useful or
beneficial, or useful or beneficial in the management of a patient, to an
extent that, in the view of
one skilled in the art, it would warrant use of the invention in that
situation. Also in the view of
one skilled in the art, the effect of attenuating the drug or toxin should be
preferential over other
effects or side effects that would otherwise negate or render insignificant
the desirable
attenuating effect.
"Drug" means a compound consisting of or comprising an Active Pharmaceutical
Ingredient (API).
"Toxin", in the context of this disclosure, is much broader than the
connotation usually
implied in day to day speech. In this disclosure "toxin" means any compound,
or closely related
group of compounds (such as different stereoisomers of a drug or different
chain lengths of a
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particular lipid type), that pose a health risk or medical inconvenience to a
mammal or human,
and as such call for removal or reduction in the body, or for redistribution
within the body (e.g.,
from tissues to blood, or from the blood to the liver, etc.). A toxin could be
endogenous, such as
an autoantibody or cortisol for example, though it will more often be an
exogenous compound,
and most typically an exogenous compound recognized to be toxic or antigenic
at least under
some circumstances (for purposes of this application, antigenic materials will
be treated as
toxins). Certain compounds, such as cocaine or morphine, for example, are of
course used in
medical practice, but also are used as drugs of abuse and as such may require
removal or
reduction in the course of detoxification and rehabilitation, and thus can be
toxins in the context
of this invention; furthermore, metabolites of such compounds, such as
cocaethylene in the case
of cocaine, have considerable toxicity and their removal by the present
invention could be of
importance in many settings. Other drugs of medical use, most notably the
local anesthetics, can
become life-threatening if they are inadvertently injected into a vein or
artery, calling for
removal or reduction, and in such cases become toxins in the context of this
disclosure. Other
drugs used in medical practice, such as those associated with anesthesia or
pain control,
including general anesthetics like propofol or paralytic agents like
rocuronium or vecuronium,
can call for removal or reduction in a number of circumstances, including
overdosage, triggering
of dangerous reactions such as malignant hyperthermia (MH) and respiratory
depression. In
addition, there are situations in which the treating physician or clinician
may wish to attenuate
the anesthetic effect for clinical reasons, for example, to allow a patient to
be safely discharged
from a hospital or clinic without having to wait for the entire length of time
required for normal
metabolism/excretion of the drug. Certain long-acting drugs of medical use,
such as reserpine,
or sustained-release formulations such as DepoMorphine, may call for removal
or reduction if
the duration of action needs to be cut, in which case the drug can be deemed a
toxin in the
context of this invention.
"Pharmaceutically-acceptable" generally designates compounds or compositions
in
which each excipient is approved by the Food and Drug Administration, or a
similar body in
another country, for use in a pharmaceutical or vaccine formulation, or
belongs to a succinct
class of compounds for which a Drug Master File is on file with a government
regulatory
agency, usually the FDA, or, less preferably, is known from extensive toxicity
studies to be safe
for the intended route of administration (which in the context of this
invention is typically,
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though not always, intravenous). This also includes compounds that are major
components of
approved excipients. Listings of approved excipients, each with the various
routes of
administration for which they are approved, are published from time to time by
the Division of
Drug Information Resources of the FDA, as in January, 1996 and entitled
"Inactive Ingredient
Guide". The existence of a Drug Master File at the FDA is additional evidence
that a given
excipient is acceptable for pharmaceutical use, at least for certain routes of
administration. For
injectable products, a listing of approved excipients was published in 1997.
See Nema,
Washkuhn and Brendel (1997) PDA J. of Phann. Sci. & Technol. 51(4):166. There
are certain
compounds, such as vitamins and amino acids, which are in injectable products
(typically for
parenteral nutrition) as "actives", and are thus known to be safe upon
injection, and such
compounds are considered herein as pharmaceutically-acceptable as excipients
as well, for
injection. There are also compounds which are not currently listed as an
approved excipient, but
which have been the subject of an extensive toxicity review and have been
shown to be of very
low toxicity, and non-mutagenic, and which, upon application to the proper
authorities for a
specific application, may be approved. A formulation pharmaceutically-
acceptable for injection
must be sterile.
"Site of action of a toxin or drug" is defined herein to be a location, in
molecular terms,
where the toxin or drug manifests a clinically significant toxic or
pharmacologic effect, which in
the context of the invention is a harmful or otherwise undesirable effect
motivating application of
the invention. To be at the site of action in some cases may include being in
the tissue, organ,
blood and/or other body fluids. In some instances, the drug or toxin is in
such proximity, at the
level of molecular binding, to a molecular target as to be capable of exerting
a toxic or
pharmacologic effect at that moment in time, such as acting as agonist or
antagonist at a receptor
protein, or otherwise binding and affecting an enzyme, lipid, saccharide,
lectin, nucleic acid,
vitamin, metal ion, neurotransmitter, hormone, or other target. The site of
action of a toxin or
drug could itself be exogenous, such as in the case of an antibiotic or
antifungal compound
where the site of action is on or in a microbe, existing at the time within
the body of a human
"Recovery" of a toxin or drug by a multitude of particles means that the
particles
substantially (i.e., with clinically desirable effect) displace it from its
site or sites of toxic or
pharmacologic action, or prevent it from interacting with that site before it
ever requires
displacement through interactions between the particles and toxin or drug,
including but not
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limited to adsorption or absorption of the toxin or drug by the particles. An
example of recovery
that is broadly illustrative is where a toxin molecule, existing at or in the
vicinity of a receptor,
enzyme, or other molecular target by virtue of interactions with the molecular
target, is
sequestered by a particle by virtue of interaction(s) with the particle (such
as hydrophobic
interaction), said toxin molecule therefore becoming less prone to interact
with the molecular
target due to its sequestration by the particle and, in some cases, by
movement of the particle
away from the molecular target with subsequent clearance of the toxin and/or
particle-toxin
combination.
"Reversed liquid crystalline phases" in the context of this disclosure
includes reversed
hexagonal phase and reversed cubic phase, which itself includes both reversed
bicontinuous
cubic phase and reversed discrete cubic phase. These phases are known in the
art of surfactant
self-association. All are understood to be as described in detail elsewhere
(e.g. in U.S. Patent
6,638,621
B. Structure of the composition to be administered.
Reversed cubic and reversed hexagonal phase material particles have quite a
distinct
morphology and therefore characteristics in comparison with oil in water
emulsions and
liposomes. The following compares four different 200 nanometer diameter
particles, each made
of one of the four materials above. Particles are compared by morphology,
phase, radius of
monolayer curvature, specific surface area, hydrophobic volume fraction,
farthest distance to
polar or apolar domains, and loading capacity.
Reversed cubic phase material has an intricate long range nanometer-scale
order, and
may exist either in bulk material or particulate form. The fluid lipid bilayer
is arranged with
precise curvature in repeating cubic space groups, creating interlaced polar
and apolar
microdomains. It has a very high interfacial surface area. The radius of
monolayer curvature of
a typical cubic phase is on the order of 3 nanometers. The specific surface
area is approximately
40 m2/mL. The hydrophobic volume fraction approximates 50-75%. The farthest
distance to a
polar or apolar domain from any point in or on the particle is only 3
nanometers. A reversed
cubic phase particle can carry hydrophobic, hydrophilic and amphiphilic
compounds in
hydrophilic domains, in hydrophobic domains, and straddling both types of
domains.
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Similarly, reversed hexagonal phase material also has an intricate long range
nanometer-
scale order, and may be bulk material or particulate form. It also consists of
a fluid lipid layer
arranged with precise curvature creating polar microdomains, but in contrast
to reversed cubic
phase material, the structural units are cylinders arranged in a repeating
pattern conforming to a
hexagonal space group. Reversed hexagonal phase material also has a high
interfacial surface
area, though not as high as the reversed cubic phase. The radius of monolayer
curvature of a
reversed hexagonal phase is on the order of approximately 1.5 nanometers. The
specific surface
area is about 25m2/mL and the hydrophobic volume fraction is roughly 80%. As
in the reversed
cubic phase particle, the farthest distance to a polar or apolar domain is
approximately 3
nanometers from any point in or on the particle. The reversed hexagonal phase
particle, like the
reversed cubic phase particle, can carry hydrophobic, hydrophilic and
amphiphilic compounds in
hydrophilic domains, in hydrophobic domains, and straddling both types of
domains.
By contrast, an oil-in-water emulsion, such as Intralipid , has no long range
nanometer-
scale order. It consists of fat droplets of various sizes and shapes
surrounded by a lipid-rich layer
in an aqueous medium. A 200 nanometer emulsion oil droplet has the lowest
interfacial surface
area of all particles compared here. It is comprised of an oil-rich liquid
phase surrounded by
lipid-rich phase. For such an emulsion particle, the radius of monolayer
curvature is 100
nanometers. The specific surface area is only 6m2/mL. The hydrophobic volume
fraction is
95%, but the farthest distance to polar or apolar domain is 98 nanometers.
Liposomes, too, differ significantly in structure and characteristics from
reversed cubic
and reversed hexagonal phase material particles. As true of emulsions,
liposomes have no long
range nanometer scale order. They occur only in particulate form: the basic
structure is one or
more solid lipid (or less commonly, fluid) bilayers, generally spherical,
encapsulating a large
polar liquid phase (usually aqueous) compartment. Liposomes have a very low
interfacial
surface area. They are derived from lamellar phase material, with a liquid
phase core. The radius
of monolayer curvature of a 200 nanometer liposome is approximately 100
nanometers. The
specific surface area is 12m2/mL. The hydrophobic volume fraction is a very
small, e.g. 5%, and
the farthest distance to polar or apolar domains, like the emulsion droplet,
is very large, 96
nanometers.
Without wishing to be bound by theory, the reversal of toxin and drug effects
by the
invention can be at least partly, if not fully, understood as resulting
directly from the action of
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the particles in absorbing toxin within the particle interior, or adsorbing
toxin at the particle
surface. The former, namely absorption, can occur by virtue of the ability of
the liquid
crystalline material to solubilize the toxin or drug (which is amply
demonstrated by Example 7),
whereas the latter, adsorption, can occur by virtue of particle surface
charge, specific capture
molecules (such as antibodies) at the surface, hydrophobic interaction,
solubilization and
partitioning of the toxin within a particle surface layer such as a PEG-rich
layer, or a number of
other interactions. It should be understood that there is a distinction
between a particle surface
layer, and a distinct surface phase. Once toxin molecules in the immediate
vicinity of the
particles are so taken up by the particles, then due to one or more equilibria
between free toxin
and tissue-bound toxin, tissue-bound toxin molecules may diffuse out of tissue
to fill the void left
by the departed, particle-sequestered toxin molecules, where they can be
ab/adsorbed, and the
cycle continues. A more complete description of the process would take into
account particle
uptake of protein-bound toxin, RES uptake of particles, and other effects, but
in any case the key
step in the process is absorption or adsorption of toxin by the particles. It
is at that point that the
particles provided to a human or other mammal patient according to the
invention step in and
manifest their effect on the pharmacokinetics of the toxin.
The preferred particle architecture is that of a charge-stabilized, uncoated
particle, as
specified in detail in U.S. Patent 7,713,440. The uncoated particle has the
distinct
advantage that no occlusive layer interferes with the direct absorption or
adsorption of toxins
from medium. Thus for example, in the case of a toxin that exhibits slow or
limited diffusion
across bilayers, lamellar coatings are advised against since they would
interfere with the toxin-
absorbing properties of the reversed liquid crystalline phase interior.
Indeed, a bicontinuous
reversed cubic phase in particular, when uncoated, has a microstructure that
allows bilayer-
impermeable toxins to migrate directly into its pore space, where the toxin
can be effectively
maintained there by some combination of permselectivity, hydrophobic
interactions with the
bilayer, electrostatic interactions with the bilayer, van der Waals forces, or
interaction with
interior components of the cubic phase. Other particle architectures are
possible as well
provided that a substantial portion of the particle is a reversed cubic or
reversed hexagonal phase.
Solid-coated particles are antithetical to the purpose of rapid toxin uptake
and are thus
inconsistent with the objects of the invention; this applies to both
crystalline lamellar and solid
(crystalline or amorphous) nonlamellar coatings. Preferably, most or nearly
all of the particle is
13
CA 02672024 2013-12-19
a reversed liquid crystalline phase, with reversed cubic being most preferred
among these.
The reversed cubic or reversed hexagonal phase material in particulate form
should be
readily accessed by the diffusion of toxin molecules from a site in the body
to the material when
the particle is at the site. In particular, the particle should not be
occluded by an impermeable
coating. An impermeable coating would be one which is substantially
crystalline (such as a lipid
in the gel phase), or more generally in which the self-diffusion coefficient
of the toxin within the
coating is strongly reduced relative to the self-diffusion coefficient inside
the liquid crystalline
material; alternatively, the probe of permeability known as pyloPC (chemical
name 1-palmitoy1-
241'-pyrenedecanoyll phosphatidylcholine) can be used, and we take impermeable
to mean that
the self-diffusion coefficient of pyloPC in the coating material as measured
by the fluorescence
photobleaching recovery method as used by Vaz et al. is less than about 1
micron2/sec. [See
Vaz, W. L. C., Z. I. Derzko, and K. A. Jacobson (1982) Cell Surf Rev. 8:83-
136]. In that
publication, Vaz et al. showed that pyloPC exhibits a high D in lipid bilayers
above the gel to
liquid crystalline temperature, and low D below this transition temperature
when the bilayers are
crystalline. The dividing point of 1 micron2/sec for this system is broadly
indicative of coating
fluidity in coated particles generally. The uncoated particles disclosed in
U.S. Patent Application
Ser. No. 10/889,313 are clearly accessible, as are, quite generally, reversed
cubic and reversed
hexagonal phase particles coated with coatings that are in liquid or liquid
crystalline phases at
body temperature (37 C for humans).
The ionically-charged (electrostatically-charged), bilayer-bound components
discussed
above, and described in detail in U.S. Patent 7,713.440 may bind toxin or
pharmacological
substances via electrostatic interactions, free of an impermeable coating, or
coating of any sort.
In many applications, uncoated particles, particularly uncoated ionically
charged
particles, may be preferred to particles having reversed cubic or reversed
hexagonal phase
materials that are coated with solid coatings, such as those described in U.S.
Patent 6,482,517
and U.S. Patent 6,638,621 to Anderson.
Analysis of the toxins of focus in this disclosure reveals that most of them
have at least
one polar group on the molecule, and the vast majority have more than one
polar group per
molecule. In a 200 nrn emulsion droplet within Intralipid , for example, only
a very small
fraction of the droplet volume is within a typical molecular dimension, say 3
nanometers, of a
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strongly polar group of the phospholipid-triglyceride particle composition; by
strongly polar
group we mean a polar group that is, for example, operative as a surfactant
head group as
discussed in, e.g., U.S. Patent 6,638,621. By contrast, within a particle of
the instant invention,
virtually every point in the particle is within 3 nanometers of a polar group
that is operative as a
surfactant head group. Thus, even as hydrophobic groups of the toxin molecule
localize in
hydrophobic bilayer domains, any polar groups on the toxin can simultaneously
localize in close
proximity to such a surfactant polar group, and/or to water molecules (which
hydrate the
surfactant head groups), to experience energetically favorable polar
interactions, without paying
a large entropic price. This in turn leads to higher toxin solubilities, and
higher partition
coefficients, in particles of the instant invention over those of emulsion
droplets, and this can
translate directly into better performance of the instant invention over fat
emulsions in terms of
lower lipid doses (and thus less voluminous injections) required, and more
rapid and complete
removal of toxin from blood and/or tissue, and a wider range or toxins which
can be effectively
attenuated by treatment as described herein.
The surface area of accessible bilayer surface in a 200 nm particle of the
bicontinuous
cubic phase is an order of magnitude higher than that of an Intralipid
emulsion droplet of the
same size. This in itself makes the dispersions of the invention advantageous
over emulsions in
a number of cases: where the toxin adsorbs to the bilayer or binds to a
bilayer component; where
the toxin binds to a component (such as an antibody) which extends from the
bilayer surface,
perhaps with a spacer arm; where uptake of the toxin is limited by diffusion
through a
concentration gradient that extends the full particle radius in the case of an
emulsion droplet
(thus approximately 100 nm), but only half the bilayer thickness in a cubic
phase particle (thus
approximately 2 nm); where the toxin is a macromolecule that binds to the
particle by inserting a
hydrophobic moiety (such as an alpha-helix) into the bilayer; and where the
toxin has a low
partition coefficient and remains in the aqueous domain but interacts
sufficiently with the bilayer
as to remain substantially within any aqueous pores of the particle. As an
example of the first
case listed, Example 12 provides an instance where the toxin, an earth metal
ion, actually binds
to the head groups of the phosphatidylcholine molecule itself.
Particle size for intravenous embodiments of the invention will preferably
have an
average particle size between about 80 and 1,000 nm, most preferably between
about 100 and
700 nm, and wherein 90% of the particle volume is in particles with size less
than about 1,500
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nm and most preferably less than about 1,000 nm. The full particle size
distribution can be
adjusted by manipulating the processing conditions (in particular,
homogenization time and
temperature, and microfluidization if desired), in particular to adjust the
fraction of particles that
are below about 80 nm in size, since evidence indicates that such particles
are cleared more
slowly than particles above 80 nm, and it might be desirable in a particular
application to have a
longer-circulating fraction of particles, so that toxin removal continues for
a longer period than
in the absence of particles smaller than 80 nm.
Dispersions of uncoated particles of the preferred embodiment bind to plasma
protein
much less strongly than do emulsion droplets. This is important because where
a large volume
of emulsion droplets or particles of the invention are given for
detoxification, it is
disadvantageous, if not dangerous, for the particles or droplets to remove
proteins and other
components naturally present in the bloodstream. Moreover, in analogy with
liposome-
disrupting effects of protein opsonization well known in the art, overt uptake
of proteins by lipid-
based particles¨particularly those such as emulsions which are already
nonequilibrium
structures¨can rapidly destabilize particles. Because they are weakly
attracted to plasma
proteins, it is unlikely that a targeting or toxin-capturing compound
associated with the particle
will be interfered with by interaction of the particle and plasma protein.
C. Toxins amenable to attenuation by the administration of the composition of
the invention.
Toxins that are especially amenable to sequestration by particles of the
instant invention
are those that contain at least one hydrophobic group, and are thus considered
either hydrophobic
or amphiphilic. Compounds that are strictly hydrophilic, and contain no
hydrophobic groups
(compounds such as inorganic salts, glycine, and methanol), generally are not
amenable to the
invention since they will not partition preferentially into the particles of
the invention. In the
experience of the inventors, a compound is very likely to partition
significantly into particles of
the invention if one or more of the following criteria is met:
a) it has a contiguous string, or ring, of at least 6 carbon atoms in the
molecule;
b) it has a solubility of at least 1% in alpha-tocopherol;
c) it has an octanol-water partition coefficient equal to or greater than
about 100;
d) it lowers the surface tension of water to less than about 50 dynes/cm at
low
concentrations (e.g., less than 1%); or
16
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e) if a peptide or protein, it contains a contiguous sequence of at least 4
hydrophobic amino
acids.
Bupivacaine and many other local anesthetics clearly fit one or more of these
criteria. For
example, the octanol-water coefficient of bupivacaine at pH 7.4 is greater
than 1,000. It should
be noted that the reference to the traditional octanol-water partition
coefficient (Kow) is made
here because this measure is often tabulated or otherwise available for a wide
range of
compounds. Nevertheless, in the context of this invention, it is the partition
coefficient of the
toxin in particles of the invention, as opposed to octanol, that is the most
relevant measure. This
disclosure will use the nomenclature KQw to indicate the partition coefficient
in a cubic (Q)
phase over water (or aqueous buffer more generally); that is, the ratio of
concentration of the
toxin in the cubic phase to the concentration in water, taken after the toxin
has had time to
equilibrate between contacting water and cubic phase volumes. It should be
noted that this cubic
phase-water partition coefficient, referred to in this paragraph, should be
measured in the
absence of albumin (unless the cubic phase in a particular formulation of the
invention is, in fact,
formulated with albumin as one ingredient) or other proteins. In the Examples
below, a
measurement is performed of partitioning into the cubic phase in the presence
of albumin, in
order to more closely mimic the situation where the invention is applied in
the body, and this
method of measurement is to be distinguished from measurement of the cubic
phase-water
partition coefficient per se. The term "effective partition coefficient" is
used herein when the
aqueous phase contains albumin,and typically this will be an albumin
concentration of about 40
mg/mL..
In addition to these local anesthetics, propofol and other general anesthetics
meet the
above criteria. The invention could be used to attenuate the effects of the
following intravenous
general anesthetics, all of which have octanol-water partition coefficients
greater than one
hundred (100): eltanolone, minaxolone, methohexital, thiamylal, thiopental,
ketamine,
chlormethiazole, alphaxalone, and pentobarbital. As in the case of propofol,
attenuation of these
anesthetics by the invention could be performed either in cases of overdose,
or simply to reverse
anesthesia when it is no longer needed. Vecuronium bromide and other muscle
relaxants also
meet or more or these criteria (for example, by virtue of a steroidal
backbone), and as shown in
Example 15 below, although the octanol-water partition coefficient of
vecuronium bromide is
17
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less than 100, its cubic phase-water partition coefficient is well above 100,
and indeed above
1,000 for at least some cubic phases of use in the invention.
In addition to these drugs, other toxins that can be attenuated by the use of
the instant
invention include: (i) pharmaceutical compounds (some of which are also
substances of abuse)
such as propanolol, amitriptyline, verapamil, digoxin, reserpine, rocuronium,
pancuronium,
propofol, etomidate, halothane, enflurane, and methoxyflurane,
benzodiazepines, N-methy1-3,4-
methylenedioxyamphetamine, paracetamol, vitamin K antagonists (warfarin,
acenocoumarol,
phenindione), opioids (morphine, codeine, methadone, thebaine, heroin,
oxycodone,
hydrocodone, dihydrocodeine, hydromorphone, oxymorphone, nicomorphine),
Clozapine,
Risperidone and Olanzapine (antipsychotics), Paroxetine (antidepressant),
Infliximab and
Etanercept (anti-rheumatics), Paclitaxel (antineoplastic), Interferon beta
(immunomodulator),
phenylheptylamines, piperanilides, phenylpiperidines, diphenylpropylamine
derivatives,
benzomoiphan derivatives, oripavine derivatives, and volatile anesthetics such
as nitrous oxide,
halothane, enflurane, isoflurane, sevoflurane, and desflurane; (ii) vitamins,
particularly the fat-
soluble vitamins; (iii) other drugs of abuse such as cocaine, heroin, LSD,
"ecstasy", mescaline,
amphetamines; (iv) compounds commonly known as poisons such as strychnine,
carbon
monoxide (a rather hydrophobic gas); (v) toxic compounds found in the
environment such as
pesticides, organophosphates, dichlorodiphenyltrichloroethane (DDT), morphinan
derivatives,
carbon tetrachloride, chloroform, trichloroethylene, tetrachloroethylene,
dichloromethane,
chlorofluorocarbons, benzene, toluene, xylene; (vi) toxins used in military
and terrorist weapons
such as VX; (vii) toxins from plant and animal sources such as snake venom,
spider venom,
insect toxins, ricin; allergens such as gluten, peanut oil; and (viii)
substances of microbiological
origin such as endotoxins, cholera toxin, E. coli enterotoxin, or indeed
possibly even portions or
whole bodies of viruses, prions, parasites, fungi, cryptosporidia, or even
bacteria and other
microorganisms. Lipopolysaccharide endotoxins such as Lipid A partition
extremely strongly
into lipid membranes such as those of the instant invention, and could be
especially responsive to
removal by the instant invention. The removal or attenuation of the effect of
endotoxins in this
manner could open up treatment to a variety of conditions, including, for
example, sepsis.
D. Selection and composition of material administered
18
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As demonstrated below in Examples 17 and 18, the particle compositions can be
used in
viva to recover diverse pharmacologic agents from their site of action or the
blood stream to
attenuate the effects of those agents. These results combined with Examples 1-
16 and
discussions herein, and particularly Example 7, suggest a wide variety of
toxins would also be
able to be recovered or captured in vivo using a particle composition which
includes a reversed
cubic phase or reversed hexagonal phase material.
In some specific applications the ability to adsorb, absorb, sequester or
otherwise recover
a pharmaceutical or toxin may be enhanced by choosing particles of specific
constitutions. This
may be accomplished by selection of a reversed liquid crystal composition that
more favorably
solubilizes the toxin or pharmaceutical of interest, and which is
pharmaceutically-acceptable, and
preferably which achieves a high partition coefficient over aqueous buffer at
the relevant pH
(typically about 7.4). Compositions for reversed liquid crystalline phases are
discussed at length
in U.S. Patent No. 6,482,517, filed September 8, 1998, U.S. published
Application No. US
2002/0102280, filed November 28, 2001, and U.S. Patent Publication No. US
2004/0022820, as
well as U.S. Patent 7,713,440.
In summary, the liquid crystal composition should solubilize the toxin (or
toxins), at a
level that provides for clinically significant recovery of the toxin.
Preferably the toxin is soluble
in the reversed cubic or hexagonal phase liquid crystal to a level of at least
0.01%, more
preferably equal to or greater than about 0.1%, and most preferably equal to
or greater than about
1% by weight. The preferred solubility levels are reached by taking advantage
of the inherent
compound-solubilization properties of reversed liquid crystalline materials,
discussed and
demonstrated at length in the disclosures cited in the previous paragraph, and
by further
optimizing the liquid crystal composition through selection of the hydrophobe.
The reversed liquid crystal compositions typically are comprised of a lipid or
surfactant, a
hydrophobe and water. In the context of this invention, the term "hydrophobe"
means the third
major [pseudo-komponent of a surfactant/water/third component liquid crystal
composition
even if this third component is, strictly speaking, amphiphilic by virtue of,
e.g., a hydroxyl
group, as in the case of linalool.
Liquid crystal compositions in which the main structural lipid (which is a
surfactant, in
accordance with the definition of the term "surfactant", see U.S. Patent No.
6,482,517) is
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phosphatidylcholine ("PC"), or a phosphatidylcholine-rich product such as a
purified lecithin, are
strongly preferred in the invention. The current invention makes advantageous
use of naturally-
occurring phospholipids, particularly phosphatidylcholine, and most preferably
phosphatidylcholine that have a transition temperature below body temperature,
or preferably
below ambient temperature¨that is, the compositions employed are above the so-
called Krafft
line, temperatures below which crystalline phases appear and above which
liquid crystalline
and/or liquid phases appear. Phosphatidylcholine is put forth as uniquely well
suited as the
structural basis of injectable particles for toxin removal due to a
constellation of favorable
features, most importantly low toxicity, but including also the fact that it
is endogenous,
biocompatible, biodegradable, non-antigenic, cost-effective, and has a decades-
long history of
safe use in intravenous products at levels of 5 -25 grams per day. While other
surfactants and
lipids, such as poloxamers (e.g., Pluronics), Tweens, cremophors, mono- and di-
glycerides for
example, can be used in the invention, these compounds must be scrutinized for
toxicities that, in
the context of toxin reversal, could lead to dangerous and unpredictable
effects. This is
particularly true since in the practice of this invention, in the face of
toxic challenges to the body,
fairly high volumes of the dispersions may be used, typically on the order of
100-400 mL, which
contain on the order of 5-20 grams of lipid.
Most preferably, the hydrophobe for an injectable product will be one or more
of the
following five hydrophobes: tocopherol, linalool, squalene, benzyl benzoate,
and long-chain
diacetylated monoglycerides.
For an injectable product, in the most preferred method, the solubility of the
toxin is first
determined, using methods well known in the art, in each of the five preferred
hydrophobes
identified above. If desired, pairwise combinations of these five solvents
(numbering ten) can
also be tested, preferably at a 50:50 cosolvent ratio, still keeping the
number of solubility tests to
a manageable number (viz., 15). Generally speaking, for the purposes of this
invention, the
hydrophobe mixture that best solubilizes the toxin will also yield the liquid
crystalline particle
that exhibits the greatest partition coefficient (measured between the liquid
crystal and water).
Partitioning experiments can easily be performed by methods well known in the
art, and as
described in the Examples below. Based on these tests, and on the relative
safety of these five
hydrophobes themselves¨or rather, on their history of safe use in the intended
route of
administration¨a hydrophobe or hydrophobe mixture is selected. Tocopherol has
the most
CA 02672024 2013-12-19
extensive history of safe use particularly in intravenous products, as it has
been used in
parenteral nutrition for many decades, even in neonates, and thus it is the
most preferred of the
five hydrophobes from that perspective. Indeed, tocopherol is widely known to
be of very low
toxicity, with intravenous doses as high as 30 mg/Kg/day being essentially
free from any adverse
effects, and the excipient has a long history of safe use in intravenous
products, both parenteral
nutrition and in liposomal products. Diacetylated monoglycerides, in
particular the brand
Myvacet , which has been used as an excipient in regulatory-approved
intravenous products.
Benzyl benzoate has been used in marketed injectable formulations, though not
yet in
intravenous formulations. Squalene has been used in vaccines as an adjuvant.
Care should be
taken to avoid inclusion of squalene in products intended for use in
indications where adjuvant
stimulation of the immune system may be contraindicated. Linalool has been
found to be safe in
a battery of toxicity studies and nonmutagenic [U.S. National Institute of
Environmental Health
Sciences report, Technical Resources International, Contract No. NO2-CB-50511,
June 19971,
but has not been used in marketed injectable formulations, and could be a
candidate for formal
approval in an appropriate product.
Tocopherol can be included as part of the hydrophobe mixture in most cases,
for a
number of reasons: it helps protect phospholipids from rancidification; it
helps with the
formation and stability of reversed cubic phases; it is a surprisingly
effective solvent for a wide
range of hydrophobic and amphiphilic compounds; and its cleansing effect as a
vitamin is well
known and can serve a helpful role in the detoxification applications of the
invention. It should
be noted that while wax-like components such as ethyl butyrate have been
investigated for use in
injectable products, neither ethyl butyrate nor any of the waxes are on the
FDA Inactive
Ingredient Guide as injectable excipients nor, of course, has any ever been
used as a parenteral
active or component, and the present invention avoids the use of these waxes.
Once the hydrophobe has been selected, the composition of the reversed liquid
crystalline
phase is then found by a simple phase behavior mapping, as described in, e.g.,
U.S. Patent
6,638,821. Quantitative guidelines for determining the composition of a
reversed cubic phase
are given in U.S. Patent Publication No. US 2004/0022820. An examination of
viscosity and
appearance under polarizing optical microscopy are indicative of the phases of
lyotropic liquid
and liquid crystal material.
21
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A dispersant is typically, though not necessarily, incorporated in order to
maintain the
particles dispersed, and stable as such over time, preferably at least two
years without
detrimental increase in effective particle size, and in particular without an
increase in the fraction
of particles larger than about 1.5 micron (and more preferably 1 micron), as
this fraction should
preferably remain less than 10% preferably, and more preferably less than 1%,
by volume. The
term "dispersant" will be used herein even in cases where the compound only
aids in maintaining
stably dispersed particles, and relies on other co-dispersants for achieving
the desired effect; for
example, in a DMPG-containing dispersion of the invention, Myvacet (a long-
chain diacetylated
monoglyceride product from Kerry Bioscience) was necessary as co-dispersant.
(DMPG is the
abbreviation for dimyristoylphosphatidylglycerol). For most applications, the
bile salts
discussed herein are the preferred dispersants, with glycocholate being most
preferred except
possibly in situations where the danger posed by the toxin is low enough that
a product with a
high cost of materials, due to the high cost of glycocholate, is not
justifiable. Bile salts are
preferred for another reason, namely they promote uptake of particles by the
liver, probably
through binding of apolipoprotein E and/or related proteins, resulting in
removal of the toxin
from circulation; alternatively, cholesterol can be incorporated into the
particles, and generally
particles of the invention for intravenous use will contain either a bile salt
or cholesterol, or both.
Other preferred dispersants are sodium oleate (preferably at 0.03% or less of
the dispersion by
weight), and other acidic diacyl phospholipids including
phosphatidylglycerols,
diphosphatidylinositol, and phosphatidic acid. For oral and topical products),
other preferred
dispersants are sodium docusate, and others given in U.S. Patent 7,713.440.
For intraveneous
products with intended use in the United States, salts of fatty acids,
including sodium oleate,
should be avoided since they are not FDA approved as intravenous excipients,
or in the case of
caprylie acid, are approved only at extremely low levels, too low to be
effective.
Preferred tonicity adjusters are neutral amino acids such as glycine or
valine, and other
standard, nonionic tonicity adjusters such as mannitol, glycerol, dextrose and
xylitol.
E. Attenuation of effect from administration of composition.
The administration of an effective amount of the composition intravenously to
a patient
who is suffering neurotoxicity or cardiotoxicity due to the administration of
a local anesthetic, as
for example may occur from the inadvertent introduction directly into the
bloodstream by
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injection of a toxic dose of the local anesthetic, would attenuate the severe
neurological and
cardiological toxicity caused by the local anesthetic. Administration of an
effective amount of
the composition under these circumstances would partially or entirely
attenuate the toxicity, and
mitigate or altogether end the toxic crisis for the patient.
The administration of an effective amount of the composition to a patient
under the
influence of a recently administered therapeutic dose of the general
anesthetic propofol, would
attenuate the effects of that drug, allowing the patient to emerge from
anesthesia and return to
clear headedness more quickly than without the administration of the
composition. The ability to
shorten the duration of effect can be of important medical value and become
part of a standard of
care from a point of view of safety and convenience. Similarly, the
administration of an
effective amount of the composition to a patient who has been treated with and
is experiencing
the therapeutic effects of a skeletal muscle relaxant such as vecuronium can
attenuate the
paralytic effects of that drug more quickly than in the absence of the
administration of the
composition. These and others can serve an important medical need in the
surgical theater, and
therefore facilitate safe use of optimal agents in these practices.
Prophylactic use of the invention is also possible. By administering an
effective amount
of the dispersion prior to or simultaneous with the anticipated ingestion,
contact, or
administration to a human of a drug or toxin, the effect of the toxin or drug
can be attenuated
from the beginning, and either mitigated in severity or duration or indeed
attenuated to such an
extent that clinical indications never appear. This is demonstrated in Example
16 below.
Clearly, in order to successfully attenuate the effect of the toxin (since the
definition of
"attenuate" requires substantial reduction in the effect of the insult), the
dispersion must be
administered fairly close in proximity to the time of the insult, preferably
less than about 15
minutes before the ingestion or administration of the toxin, and more
preferably less than about 5
minutes. However, for purposes of prophylaxis, the invention encompasses
embodiments that
can circulate for considerably longer time periods than minutes. In
particular, by the
incorporation of "stealth" lipids known by those skilled in the art,
circulation times may be
greatly increased. Such lipids, which are readily incorporated into particles
of focus in the
invention as demonstrated in Example 9 below, include PEGylated lipids such as
polyethyleneglycol-2000-phosphatidylethanolamine, Vitamin E TPGS, and other
lipids created
by covalent attachment of hydrophilic polymer chains to hydrophobic lipids.
Settings where
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prophylaxis with the invention could be of medical importance include
administration of local
anesthetics at high-risk sites (guarding the patient against the toxic effect
of a possible
inadvertent injection into the blood stream), chemical or biological warfare
arenas, clean-up
operations at toxic waste sites, as well as other situations where exposure to
toxins is probable or
imminent.
The attenuation of a toxin by adsorbing or absorbing the toxin from the site
of toxic
action stands in contrast with other methods that rely on administering an
antidote, provided that
one is even available. While a competitive agonist or antagonist might
preferentially displace a
toxin from binding to a receptor or enzyme, the toxin can nonetheless remain
at the site and
continue to compete with the antidote for receptor binding, and in the case
where the toxin and
antidote bind to a common enzyme the competition continues as long as toxin
remains in the
organ of toxic insult, or in some cases as long as it remains in circulation.
This is particularly
problematic in cases where the toxin is only slowly detoxified by an organ
such as the liver, and
in such a case, the current invention provides for liver uptake and
detoxification by virtue of liver
uptake of the particles, which can easily be one, or two, or more, orders of
magnitude faster than
liver uptake of individual molecules; lipidic particles in the 80-1000 nm
range can be
substantially taken up by the liver in under 10 minutes. Thus, even in cases
where a toxin can be
displaced from a receptor, it can still be advantageous to administer
dispersions of the current
invention in order to clear the displaced toxin, provided it can be
established that the dispersion
does not clear the antidote preferentially over the toxin; this is unlikely if
the effective partition
coefficient into the particles as measured in the Examples herein is
significantly higher for the
toxin as compared to the antidote, and becomes more and more effective as an
approach the
more strongly the antidote binds to the receptor. In many cases, including the
important case of
bupivacaine, no specific antidote exists, particularly no drug that will
effectively, rapidly and
safely remove the toxin from its molecular target such as receptor or enzyme,
and drugs that
might be administered represent an attempt to counteract the toxic effect,
such as the
administration of vasopressin, epinephrine, norepinephrine, amrinone,
amiodarone, milrinone,
lidocaine, calcium channel blockers, and phenytoin, none of which have been
shown to be
consistently safe and effective. [See Renehen EM et al. (2005) Reg. Anesth.
Pain Med.
30(4):3801. The latter is not surprising because in the application of a non-
displacing antidote
(one that does not exhibit competitive inhibition of the toxin at the receptor
site), the toxic effect
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of the toxin continues in parallel with the counter-effect of the antidote,
and as such the antidote
does not get to the root of the problem, of the toxic insult. Furthermore,
with many toxins the
receptor site may not even be known, and in such cases the current invention
might be found to
be effective in attenuating the toxic effects even in the absence of this
knowledge. The invention
could, in fact, be used to detoxify the body of a mammal thought, but not
known, to be suffering
the effects of a toxin or toxins, such as in cases in man where the patient
might be unconscious,
delusional, or unwilling to admit the ingestion of toxic substances such as
drugs of abuse.
F. Varying the composition
Other methodologies that fit within the spirit of the invention include the
following.
Targeting compounds, such as antibodies, lectins, ligands, bacterial adhesion
receptors,
complementary nucleic acids, bile salts, biotin derivatives, etc., can be
incorporated (as described
in U.S. Patent Application 10/889,313) so as to target particles of the
invention to sites where
toxins may accumulate and/or where they may do the most physiological damage,
or to bind
toxins directly, and/or to direct particles to elimination sites after toxin
recovery. For example, a
cholesterol- and/or bile salt-laden particle of the invention could be
directed to, e.g., the liver by
the ApoE mechanism discussed herein, where it could bring recovered toxin for
detoxification,
or where it could sequester toxin from the liver so as to, e.g., decrease the
local concentration of
free, unbound toxin. And glycolipid bacterial adhesion receptors, such as
Forssman's antigen,
are readily incorporated into the liquid crystalline particles of this
invention, and such tagged
particles could well bind bacteria with considerable specificity, so as to
remove infectious
bacteria, or to remove a bacterial by-product such as an endotoxin. Such
capture could be
coupled with proteases, nucleases, or antibiotics that could render the bound
microbes
noninfectious. Similarly, biospecific capture compounds, again including
antibodies, lectins,
ligands, complementary nucleic acids, bile salts, biotin derivatives, and also
chelating agents,
cyclodextrins, etc., can be incorporated into particles of the invention so as
to amplify their
ability to recover toxin.
Furthermore, the instant invention includes provisions for adjusting the rate
of clearance,
by the liver, of the particles, via a simple adjustment of the composition.
The uptake of these
toxin or drug-laden particles by the liver constitutes a dominant mechanism
for the removal of
toxin or drug away from tissues in the body where it exerts its
pharmacological effect.
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Changing the dispersant from a bile salt, such as deoxycholate, to a charged
phospholipid which
is not recognized by Apolipoprotein E, can have a large effect on the
circulation time of
particles. Thus it is of significant importance that in the instant invention,
the rate of clearance
of the liquid crystalline particles can be increased by raising the
concentration of cholesterol
and/or bile salt in the particles, or decreased by lowering the concentration
of these compounds.
If a bile salt is used for this adjustment, it must satisfy two structural
requirements: first, it must
have a hydroxyl (-OH) group at the 3-position, and second, it must have an
alkyl side chain at the
C17 position of the steroidal ring system. Some of the preferred compounds for
use in the
instant invention, which satisfy these requirements, are the acid and salt
(e.g., sodium salt) forms
of cholic, glycocholic, deoxycholic, and chenodeoxycholic acids most
preferably, and less
preferably glycochenodeoxycholic, glycodeoxycholic, lithocholic, and
ursodeoxycholic acids.
Glycocholate and deoxycholate salts are especially preferred, as they are more
hydrophobic, less
toxic, and more reliably charged than others in the series. Deoxycholate has
the additional
advantage that it is inexpensive relative to glycocholate, although it is less
reliably charged than
glycocholate and the pH must remain above 7.2 to avoid precipitation.
In contrast, lipid emulsions such as Intralipid are poorly suited for
addition of bile salt
or cholesterol without undue experimentation. Addition of bile salt to a fat
emulsion tends to
create mixed micelles, as is well known in the art. And while a small amount
of cholesterol
could, at least in principle, be incorporated in a fat emulsion, this would be
difficult to prepare
due to the extremely low aqueous solubility of cholesterol in water,
precluding the possibility of
mixing the fat emulsion with an aqueous cholesterol solution. More broadly,
the natural
instability of fat emulsion makes them extraordinarily sensitive to changes in
composition.
Also, since reactive oxygen species (ROS) can be detoxified by virtue of the
high
tocopherol concentrations in the particles of the preferred embodiment, the
invention could be
useful in pre- or post-treatment of humans or mammals exposed to hazardous
radiation. It
should be noted that phosphatidylcholine is also reputed to aid in
detoxification of ROS taken
internally, and the preferred embodiment of the invention offers a combination
of tocopherol and
phosphatidylcholine. Furthermore, particles of the invention can reasonably be
expected to
perform better at detoxifying reactive oxygen species than solution
formulations of vitamin E,
for example, because the ability of the particles to sequester ROS compounds
through the
solubilization and partitioning effects discussed herein will translate into a
longer residence time
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of proximity between the toxic compound and tocopherol (and
phosphatidylcholine as well).
Quite broadly, the sequestering effect of particles of the invention can be
used to amplify the
effect of detoxifying agents when those agents are embedded in the particles.
The toxin-attenuating aspect of the invention can be combined with other
measures
known in the medical arts to be of value in the treatment of the particular
toxic insult. For
example, particles of the invention, deliberately selected to attenuate the
effects of a toxin A,
could also be loaded with an Active Pharmaceutical Ingredient B, so as to
deliver the agent B
while at the same time substantially recovering toxin A. For measures that
involve delivery of a
pharmaceutical agent that is hydrophobic or amphiphilic in accordance with the
discussions
above, it must be borne in mind that this pharmaceutical agent may itself be
significantly
sequestered by particles of the invention if co-administered close in time.
Thus it may be
necessary to adjust dosages and dosing schedules of anesthetic agents,
antibiotics, paralytic
agents, and other agents used in the course of treatment when combined with
treatment as per the
instant invention.
G. Other applications.
As discussed above, the effects of anesthetics may be attenuated after
performing a
surgical procedure (e.g., elective or non-elective) or after an observation of
a patient (e.g., simply
observing a patients response to an anesthetic, etc.) by administration of a
particles which
include a reversed cubic phase or reversed cubic phase material. These methods
can be applied
to sequestering a wide variety of other drug substances and toxins. As
discussed in more detail
below, the methods may be employed in a number of other exemplary
applications.
Decompression sickness, one instance of which is commonly called "the bends",
is a
condition in which nitrogen bubbles appear in the bloodstream and occurs in
diving operations,
pressurized mines, aircraft and spacecraft. Administration of dispersions of
the invention,
particularly embodiments in which fluorocarbons are incorporated, could
substantially increase
the solubility of nitrogen in the bloodstream by solubilizing diatomic
nitrogen within the particle
interior (that is, by absorbing nitrogen gas), and attenuate the noxious
effects of nitrogen in
decompression. This is an example of where a normally innocuous substance (N7)
can, under
certain circumstances, become a toxin in the sense of this disclosure. While
the authors are not
aware of any published value for the octanol-water partition coefficient of
diatomic nitrogen, its
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extremely low solubility in water (about 17.3 ppm at 25 C) and its solubility
in organic solvents
indicates that Kow must be extremely high.
Similarly, carbon monoxide is sparingly soluble in water, but appreciably
soluble in some
organic solvents, again suggesting a high Kow, and thus the invention could
potentially be used
to attenuate the toxic effects of carbon monoxide poisoning. The effect of
particles of the
invention in sequestering carbon monoxide, particularly in a mammal already
exposed to the
toxin, might be further enhanced by incorporating hemoglobin or other heme-
containing
compound into the particles, and in contrast with red blood cells carrying
heme-bound carbon
monoxide, particles of the invention carrying CO would be rapidly cleared from
the body.
A number of drugs are associated with QTc prolongation and/or torsade de
pointes (TdP)
including several that have been pulled off the market due to this toxicity
problem, and the
availability of a reversal agent such as provided by the instant invention
could enable the safe use
of these drugs. Some such drugs, potential toxins because of these effects,
include disopyramide,
procainamide, quinidine, amiodarone, bretylium, dofetilide, sotalol,
astemizole, terfenadine,
fluoroquinolone antibiotics such as grepafloxacin, levofloxacin, and
sparfloxacin, macrolide
antibiotics such as clarithromycin and erythromycin, imidazoline antifungals
such as
ketoconazole, antimalarials such as chloroquine, halofantrine and quinine,
other antimicrobials
such as cotrimoxazole, pentamidine and spiramycin, calcium antagonists such as
prenylamine
and terodiline, cisapride, and probucol, tricyclic and related antidepressant
drugs such as
amitriptyline, clomipramine, desipramine, doxepin, imipramine, maprotiline,
nortriptyline, and
antipsychotic drugs such as chlorpromazine, droperidol, thioridazine,
ziprasidone, sertindole and
pimozide.
High atomic weight earth metals, in particular strontium and barium, bind to
phospholipid
head groups, and have been found in the course of this work to bind rapidly
and strongly to
particles of the invention containing phospholipids (See Example 12). Thus the
invention could
be useful in removing such toxins from the body, pointing out yet another
mechanism by which
toxins can be bound by the invention. Similarly, the invention might be useful
in removing
leftover radioactive radionuclides following cancer therapy.
Bile salt accumulation in the blood causes jaundice, and since bile salts
partition strongly
into particles of the invention, another possible use for the invention is in
the treatment of
jaundice.
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A number of compounds used in the operating room and emergency room are known
to
cause, in some people, allergic reactions such as anaphylaxis. Examples of
such compounds,
where the current invention could be used to remove such toxins, are
salicylates, sulfonamides,
cremophor, and penicillins, and to a lesser extent benzyl alcohol.
Due to the inclusion of significant levels of tocopherols in preferred
embodiments of the
invention, it is likely that the damaging effects of reactive oxygen species
from a number of
sources could be ameliorated with the invention. As discussed above,
Intralipid0 has been
tested against reactive oxygen species from produced by phorbol myristate
acetate, but only a
weak effect was found, which is not surprising since Intralipid does not
contain significant
levels of any strongly antioxidant compounds. In contrast, many preferred
embodiments of the
instant invention comprise both high levels of tocopherols and hydrophobic
domains into which
such species may preferentially partition, such domains typically being
tocopherol-rich.
Ischemia and reperfusion are areas where the invention could be useful in a
number of
ways. The ability of tocopherol-containing embodiments of the invention to not
only sequester,
but also detoxify (by quenching radicals) reactive oxygen species is of course
important in this
potential application.
As another example, in organ transplants, the cardioplegia solution used to
bring the heart
to standstill (which typically includes local anesthetic among other toxins)
can be mopped up
with the methods and compositions of the invention administered, e.g., via the
cardiotomy
infusion site to deactivate the cardioplegia solution before attempting to
restart the heart.
Injury also stems from biowaste in the donor organ from its prolonged lack of
normal
blood perfusion. These metabolites are "stunning" to the host upon
reattachment of blood supply
and the start of blood flow, and the current invention could be useful in
removing these
metabolites from the recipient's body.
In some embodiments of the instant invention, the reversed liquid crystalline
phase
material will contain, in its interior, a droplet of a hydrophobe-rich phase
that is distinct from the
reversed liquid crystalline phase; this is not to be confused with hydrophobic
domains that are
structural elements of the reversed liquid crystalline phase itself. This
hydrophobe-rich droplet
will be of a size between about 20 nm and 100 microns, that will contain as a
major component a
hydrophobe, thus a component of low solubility in water (less than about 3%),
and/or of high
octanol-water partition coefficient (Kow greater than or equal to about 10,
more preferably
29
CA 02672024 2013-12-19
greater than about 100), in which are solubilized the toxin and some fraction
(perhaps very
small) of each of the components of the second volume. The solubility of a
given toxin in a
mixture of hydrophobe and lipid is typically a very strongly increasing
function of an increasing
hydrophobe:lipid ratio, because the hydrophobe can generally be chosen
specifically for its
ability to solubilize the particular toxin whereas the choice of lipid has
much more to do with its
ability to form the desired liquid crystal (in the presence of the hydrophobe,
in particular). Such
"oil-core" reversed liquid crystalline particles are described in detail in
U.S. Patent No.
6,991,809, filed June 21, 2002.
Besides intravenous injection, the preferred method for treating systemic
toxicities of
bupivacaine and other local anesthetics, and of general anesthetics quite
broadly, the instant
invention can be administered by a number of routes depending on the source
and nature of the
toxic challenge. Intraarticular injections could be particularly useful for
insults within one or
more joints of a mammal. Intramuscular, subcutaneous, and intraperitoneal
administration may
be used for insults that may be more local in nature. For example, if an
intramuscular or
subcutaneous administration of a drug has undesired consequences, local
injection of dispersions
of the invention could reverse, or at least attenuate, the undesirable effect.
For additional
attenuation, removal of the dispersed particles laden with toxin could
possibly be effected, by,
e.g., removing the particle-containing fluid from the site with a syringe, or
by applying local
pressure or suction, or by using the dispersion of particles more along the
lines of an irrigation
solution in the first place. For toxins taken in by mouth, the preferred route
might be oral, e.g.,
via pills, tablets, lozenges, capsules, troches, syrups and suspensions, but
most preferably as a
simple dispersion in most cases. Intrarectal administration might also be
useful for toxins within
the GI tract. For toxic insults to the eye, particles and particularly
dispersions of this invention
can be applied through a wide range of ophthalmic routes: periocular,
intraocular, conjunctival,
subconjunctival, transconjunctival, peribulbar, retrobulbar, subtenons,
transscleral, topical eye
drop, topical gel, topical dispersion, intraorbital, intrascleral,
intravitreal, subretinal, transretinal,
choroidal, uveal, intracameral, transcomeal, intracorneal, and
intralenticular. And for toxic
insults which may be systemic but also may have a local aspect as well (e.g.,
for a snake bite
where local concentrations at the bite site could be very high and yet
systemic application of the
particles is of course crucial as well), the invention can be applied through
an appropriately
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chosen non-oral route including but not limited to intrathecal, intramuscular,
subcutaneous, intra-
arterial, rectal, intravaginal, intranasal, via inhalation, and topical. For
those applications which
are "local" in nature, i.e., administration of the dispersion is done in such
a way that the
dispersion is primarily confined to a local area where toxin is removed, it
will in some cases be
advantageous to remove, or otherwise displace, the dispersion from the local
site so as to clear
the toxin. This could be accomplished by one of a number of means, such as
removal by a
syringe (pulling the plunger back), flushing with an aqueous buffer or
solution, removal by
suction or blotting, etc.
It should be noted that, in the spirit of this invention, one can in principle
envision the
possibility of emulsion formulations that would utilize the more powerful
solubilizers (i.e., the
preferred hydrophobes) of the instant invention in place of the triglycerides
of emulsions, such as
the soybean oil of Intralipida However, to begin with, such systems would be
inherently
inferior to the particles of the instant invention, due to the very intimate
association of polar
(hydrophilic) and apolar (hydrophobic) groups in the reversed liquid
crystalline phases which, as
discussed above, quite generally results in higher solubilities and partition
coefficients in
reversed liquid crystalline phases, as compared to emulsions. Furthermore, and
perhaps even
more importantly, most, in fact nearly all, of the preferred hydrophobes in
the instant invention
are at least partially miscible with phosphatidylcholine (the overwhelmingly
preferred
surfactant), and thus form water-saturated reversed liquid crystalline phases
when combined with
PC and water¨in sharp contrast with long-chain triglycerides, which due to
near-complete
immiscibility with PC form emulsions, rather than liquid crystals, at high
water content.
EXAMPLES
The following Examples illustrate the present invention but are not to be
construed as
limiting the invention.
Example 1. An 800 mL batch of a dispersion of reversed cubic phase was
prepared
consisting of 862.83 mg/mL sterile water, 6.68 mg/mL sodium deoxycholate,
36.34 mg/mL
alpha-tocopherol, 71.17 mg/mL PC-90G (Phospholipon 90G, from Phospholipid
GmbH), and
22.98 mg/mL glycine. First, a 14% sodium deoxycholate solution was prepared by
sonicating
5.6 g of sodium deoxycholate in 34.4 g of deionized water. An amount 38.62 g
of this solution
was stirred with 64.84 g of the phosphatidylcholine product Phospholipon PC-
90G (from
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Phospholipid GmbH), and 16.55 g of alpha-tocopherol until well-mixed. This was
slowly added
to 480 gm of deionized water, and the mixture homogenized for three hours and
then allowed to
sit overnight without agitation. Later, the sample was re-homogenized for 2
hours and 200 g of a
9.2% glycine solution was subsequently added. The sample was homogenized for
another hour
and 14.55 g of alpha-tocopherol was added over 10 minutes. The dispersion was
homogenized
for another 2 hours followed by addition of 0.6016 g of sodium deoxycholate
and
homogenization for 1 hour. The resulting dispersion consisted of submicron
particles of reversed
cubic phase material, at a volume concentration of approximately 5%, with a
strongly negative
zeta potential to the bilayer surface (both internal and external surfaces
within the particle),
making it well suited for uptake of bupivacaine, or other positively-charged
toxins such as
vecuronium and morphine.
Example 2. Two dispersions per the instant invention were first prepared,
varying in the
composition of the hydrophobe, and then tested in the next Example for their
ability to sequester
bupivacaine, as compared to Intralipid 20%. A 770 mL batch of a dispersion,
hereafter called
"RA5E", of the invention was prepared consisting of 937.38 mg/mL sterile
water, 1.30 mg/mL
sodium deoxycholate, 18.67 mg/mL alpha-tocopherol, 21.85 mg/mL Phospholipon PC-
90G, and
20.80 mg/mL glycine. 20.328 g of alpha-tocopherol and 23.802 g of PC-90G was
stirred into
1.416 g of sodium deoxycholate dissolved in 14.520 g of sterile water. 42.7 g
of this partial
cubic phase was slowly added to 715.4 g of sterile water over 15 minutes. The
mixture was
homogenized at 5400 RPM for 5 hours to create a fine dispersion, followed by a
16.1 g addition
of glycine. The dispersion was sparged, vialed and autoclaved.
In the second dispersion of this Example, hereinafter called "RA5EL", the
hydrophobe in
the reversed cubic phase consisted of a 50:50 mixture of alpha-tocopherol and
linalool. In a
30mL beaker, added 0.3236 grams of sodium deoxycholate, 2.6621 grams of a
50:50 mixture of
alpha-tocopherol and linalool, 2.3052 grams of deionized water, and 2.6682
grams of the
phosphatidylcholine-rich product Phospholipon 90 (from Phospholipid GmbH).
This mixture
was stirred until well-mixed. In a 25mL graduated cylinder, 1.6085 grams of
this mixture and
8.3992 grams of deionized water were combined, and this wash homogenized until
it formed a
fine dispersion, which was vialed and autoclaved at 121 C for 15 minutes.
Example 3. In this Example the dispersions of Example 2 were tested for
bupivacaine
partitioning, as compared to Intralipid 20%. A 50 mL solution of Krebbs-
Ringer buffer was
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prepared with 4% albumin and approximately 0.1 mg/mL bupivacaine. In a 150 mL
beaker,
2.0081 g bovine albumin was stirred until dissolved in 49.998g Krebbs-Ringer
buffer. In a
separate 100 mL beaker, 50.0252g of this Krebbs-Ringer Buffer with albumin was
added to
0.0054 g of bupivacaine. 1 N HC1 solution was added to the beaker until pH
reached 2.0 and
allowed to stir until bupivacaine dissolved. The pH was readjusted to 7.4 with
1 N NaOH
solution.
One mL of formulation (RA5E, RA5EL, as prepared above, or Intralipid 20%) was
volumetrically added to a 15 cm length of 1,000 MW dialysis tubing wherein one
end was tied
shut. The bag was tied on the opposite ends and soaked in deionized water. The
bag was then
placed in a 10 mL graduated cylinders and 10 mL of Krebbs-Ringer buffer with
4% albumin and
approximately 0.1 mg/mL bupivacaine was added volumetrically. The sample was
allowed to sit
without agitation until HPLC analysis was performed on the dialysis bath
water.
In the following Table, the first number gives the percentage of total
bupivacaine that
was sequestered from the reservoir of spiked buffer; the second number gives
the approximate
amount sequestered per gram of lipid; this is a far more important measure;
and the third number
is the approximate effective partition coefficient between the lipid phase and
the buffer:
Dispersion % of bupivacaine Amount sequestered Effective partition
sequestered per gram lipid coefficient
Intralipid control 11% 0.5 mg/gm 6
RASE from Ex. 2 25% 3.7 mg/gm 58
RA5EL from Ex. 2 37% 2.5 mg/gm 46
Thus, the effective partition coefficient of bupivacaine ( in the presence of
albumin, hence
"effective" partition coefficient) into particles of the instant invention was
measured to be an
order of magnitude higher than that into Intralipid emulsion droplets.
Example 4. In this Example of the invention, the hydrophobe in the reversed
cubic phase
consisted of a 50:50 mixture of alpha-tocopherol and a diacetylated
monoglyceride product
marketed under the name Myvacet. A reversed cubic phase was prepared in a 12mL
test tube by
combining 0.944gm of Phospholipon 90 (Phospholipid GmbH, Cologne, Germany)
along with
0.733gm distilled water, and stifling until a homogenous consistency was
reached. Then
0.272gm Myvacet (Kerry Bio-Science, Norwich, NY) and 0.273gm of alpha-
tocopherol (ADM)
were added, and after thorough mixing the material was optically isotropic and
of high viscosity,
indeed a reversed cubic phase. This was then dispersed by homogenizing the
liquid crystal in an
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aqueous solution of sodium deoxycholate, where the deoxycholate constituted 5%
of the weight
of the cubic phase, to create a dispersion of particles of the invention,
which was then
autoclaved. The Myvacet diacetylated monoglyceride behaves qualitatively very
differently
from long-chain triglycerides such as soybean oil when mixed with
phosphatidylcholine and
water: long-chain triglycerides are well known to form emulsions (Intralipid
being an
instance), whereas this Example shows that diacetylated monoglycerides, with
the right
compositions, forms reversed liquid crystalline phases. Furthermore,
diacetylated
monoglycerides are far better solvents in general that long-chain
triglycerides.
Example 5. In this Example, the hydrophobe in the reversed cubic phase
consisted of a
50:50 mixture of alpha-tocopherol and benzyl benzoate. A reversed cubic phase
was prepared in
a 12mL test tube by combining 0.950gm of Phospholipon 90, along with 0.750gm
distilled
water, and stirring until a homogenous consistency was reached. Then 0.276gm
benzyl benzoate
(Sigma Chemical Company) and 0.285gm of alpha-tocopherol were added, and after
thorough
mixing the material was optically isotropic and of high viscosity. This
reversed cubic phase was
then dispersed in a solution of sodium glycocholate, with the glycocholate
being approximately
3.5% of the cubic phase by weight.
Example 6. In this Example, the partition coefficient of the compound
methylene blue,
an antimethemoglobinemic drug which is strongly colored, into particles of the
invention was
estimated using UV-Vis spectrometry. In a 10 mL syringe, 1 mL of an "RA5E"
dispersion of the
invention prepared as in Example 2 was spiked with 5 mL water and 250
microliters of a
methylene blue solution. A small portion of this dispersion was passed through
a 0.1 micron
filter every 30 seconds until 6 minutes, then every minute until 12 minutes,
and then every 2
minutes until 24 minutes. UV/Vis spectrophotometry was used to determine the
concentration of
methylene blue in samples collected at 1 minute and 22 minutes. The one-minute
sample gave
an absorbance of 1.15 and the 22-minute sample gave an absorbance of 1.26,
these being equal
to within the error of the experiment due to idiosyncrasies of the filtration
step. UV/Vis analysis
of the methylene blue stock solution compared with the methylene blue
concentration in test
tubes yielded a log KQw>12 for the dispersion, and no significant difference
of concentration or
KQw at 1 minute and 22 minutes, where KQw stands for the partition coefficient
measured
between the cubic phase and water (or aqueous buffer).
Example 7. In this Example, selected drugs of considerable potency, and thus
potential
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toxins, that are each of very low solubility in water and in soybean oil
triglycerides (and thus in
pharmaceutical emulsions such as Intralipid ) were tested for their
solubilities in the preferred
phosphatidylcholine / tocopherol / water cubic phase of the invention.
For a given compound X, the solubility in soy oil, and in tocopherol, was
first determined
as follows. Volatile organic solvents were first screened for the ability to
dissolve both
compound X and soy oil, or tocopherol. Methanol and dichloromethane were
particularly
effective for this purpose. After solubilizing the compound and soy oil or
tocopherol together
with the common solvent, the common solvent was then evaporated using a
rotovap. At the
resulting concentration of the compound in soy oil or tocopherol, the sample
was monitored over
approximately 4 weeks for any sign of precipitation, using centrifugation and
microscopy; the
microscopy was performed using a combination of Differential Interference
Contrast (DIC),
transmitted dark field, and polarizing modes. For the tocopherol cases, the
solution of compound
X in tocopherol was split into two parts, and phosphatidylcholine plus water
added at the proper
ratios to create a cubic phase (this composition is approximately 34%
phosphatidylcholine, 31%
tocopherol plus compound, and 35% water. This cubic phase was monitored for
signs of
crystallization of the compound. By repeating this process for a range of
concentrations of
compound X, the approximate solubility of the compound was determined to
sufficient accuracy
for the present purpose, in soy oil, tocopherol, and PC-tocopherol-water cubic
phase. In the case
of paclitaxel, the solubility was determined in both the PC-tocopherol-water
cubic phase, and in
another cubic phase with spearmint oil replacing tocopherol. The following
table gives the final
results of the work.
Solubility (mg/mL) in:
cubic phase with
Drug soy oil Water tocopherol tocopherol
Nimodipine -10 0.006 10 20
Paclitaxel <0.1 0.006 11 / 100* 42*
Mycophenolate 3 0.039 200 40
mofetil
Cyclosporin 10 0.023 200 70
Alphaxolone <1 <0.005 120* 42*
Etomidate 80 0.000045 200 105
Hydrocortisone <0.1 0.28 30 3
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Estradiol spar. sol. 0.05 10
Itraconazole <0.5 0.000001 60 20
Gluten <1 25*
*Spearmint oil used in place of tocopherol; tocopherol mixed 50:50 with
linalool.
The results in the table clearly demonstrate the power of the preferred cubic
phase composition
in solubilizing drugs and toxins, and its vast superiority over triglyceride-
based emulsions in this
representative selection of compounds.
Example 8. Reversed cubic phase material was prepared in a 250mL beaker by
combining 2.00gm sodium deoxycholate (Marcor Development), 44.15gm distilled
water,
56.57gm phosphatidylcholine 90G (Phospholipid GmbH), 15.70gm linalool (Aldrich
Chemical)
and 20.39gm vitamin E (Archer Daniels Midland). The mixture was stirred
thoroughly after the
addition of each component and the resulting material was optically isotropic
and of high
viscosity. Of this, 128.5gm of cubic phase was added to a 3L stainless steel
beaker into which
had previously been added 631.0gm of distilled water. The cubic phase/aqueous
solution was
dispersed with a standard homogenizer (SiIverson AX-60) at 5174 rpm for
approximately two
hours, then 40.0gm mannitol (Sigma Chemical) was slowly added to the
dispersion and
homogenization continued for 15 minutes. The pH was measured at 7.6 (Hanna
Instruments). A
portion was filtered through a 5-micron Nylon syringe filter and poured into
10mL sterile
Hollister-Stier glass vials. The vials were capped and autoclaved at 121 C and
15 psi pressure
for 20 minutes. After cooling, the zeta potential was measured at -58mV
(Beckman Coulter
Delsa 440SX) and the particles were substantially submicron in size. The
particle density in this
formulation of the invention is approximately 16 vol%.
Example 9. This Example shows that particles of the instant invention can be
produced
which exhibit dramatically less plasma protein binding than emulsion droplets
of the Intralipid
type. A dispersion of particles loaded with the drug propofol was first
prepared as described in
US 10/889,313. Diprivan emulsion was obtained from the manufacturer. While
these particles
contain on the order of 10% propofol, the effect of the drug on protein
binding is small compared
to the effects of bile salts, surfactants, and cholesterol, as will be seen in
the results below, so that
the experiment is by and large a reasonable model of dispersions of the
invention at least as far
as plasma protein binding. A 10 microliter aliquot of dispersion, or emulsion,
was diluted with
1000 microliters of deionized water, and also 30 microliters of human plasma
was diluted with
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3000 microliters of deionized water. In eight Nanosep Centrifugal devices with
300K MWCO,
200 microliters of dilute human plasma solution was incubated with 200
microliters of either a
dispersion of the instant invention, or a marketed, Intralipid0-based propofol
formulation.
Centrifugal devices were spun down on a microcentrifuge for 20 minutes to
separate unbound
plasma proteins from particle-bound plasma proteins. SDS solution was added to
particle-bound
plasma proteins and heated in a boiling water bath for 1 hour. Then 50
microliters of each
sample was loaded on gel electrophoresis with 950 microliters of Laemmli
Sample Buffer. After
performing SDS-PAGE, gels were fixed, stained, and destained according to
standard
electrophoresis procedure, and photographed, and contained ten lanes. Samples
corresponding
to the ten lanes are as follows, with all samples containing approximately 1%
(10 mg/mL)
propofol overall in the dispersion or emulsion (which would not be expected to
qualitatively
change the relative protein-binding properties of the dispersions or
emulsions):
Lane 1: Long Range Protein Standard (control, i.e., no dispersion or emulsion
involved);
Lane 2: Plasma proteins bound to Diprivan (marketed propofol emulsion, a 1%
propofol
in Intralipid emulsion);
Lane 3: Plasma proteins bound to a dispersion of uncoated, reversed cubic
phase particles
containing propofol and stabilized with a bile salt;
Lane 4: Plasma proteins bound to a dispersion with DMPG
(dimyristoylphosphatidylglycerol) and Myvacet (a long-chain diacetylated
monoglyceride) as dispersants;
Lane 5: Plasma proteins bound to a dispersion as in lane 3 but with 5%
cholesterol
incorporated in the particles;
Lane 6: Plasma proteins bound to a dispersion of phosphatidylcholine /
tocopherol /
propofol / water reversed cubic phase particles with 5% cholesterol in the
cubic phase
and Pluronic F-68 as dispersant;
Lane 7: Plasma proteins bound to a dispersion of phosphatidylcholine /
tocopherol /
propofol / water reversed cubic phase particles with Pluronic F-68 as
dispersant (no
cholesterol);
Lane 8: Plasma proteins bound to a dispersion of phosphatidylcholine /
tocopherol /
propofol / water reversed cubic phase particles with sodium oleate as
dispersant, where
the oleate concentration is quite low (0.03%);
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Lane 9: Plasma proteins bound to Baxter's marketed 1% Propofol emulsion based
on a
fat emulsion;
Lane 10: Plasma proteins (control, i.e., no dispersion or emulsion involved).
Visual analysis of the stained electrophoresis gel showed that plasma protein
binding of
the 8 formulations (lanes 2-9) could be ranked from most to least binding as
follows:
lane 9> lane 2 >> lane 8 >> lane 6 > lane 3 > lane 4 = lane 5 > lane 7, with
the last three
showing almost no binding whatsoever.
It is clear from these data that emulsion droplets bound much more plasma
protein than
any of the dispersions of the instant invention. Furthermore, the most
hydrophobic of the cubic
phase particles, namely that with the low level of sodium oleate as the
dispersant, binds the most
among the cubic phase formulations, whereas the lowest binding is seen with
the "stealth"
particle with a PEGylated surfactant (Pluronic F68) as the dispersant.
The propofol (1%) in each of these dispersions produces a reasonable
representation of
what the particle chemistry might look like after absorbing propofol, a
potential application of
the invention as exemplified in Example 18, and this electrophoresis
measurement demonstrates
that the plasma protein binding of reversed cubic phase particles of the
invention in such an
application can be modulated by manipulating the composition of the particle.
This can be used
to influence the distribution and clearance of particles in the application of
the invention, and
may allow optimization of the particles with respect to opsonization,
targeting, and minimal
disruption of physiological processes.
Example 10. The uptake of 3 toxins¨paraoxon, etomidate, and clomipramine¨into
particles of the invention, in the presence of albumin, was measured in this
Example. Two
dispersions, one at approximately 5.6 vol% particle concentration and the
other approximately
15%, were prepared as follows. An amount 1.4195 gm of sodium deoxycholate was
dissolved in
14.5705 gm of sterile water for injection, to which were added 10.1382 gm of
alpha-tocopherol
and 23.8061 gm of phosphatidylcholine (Phospholipon 90G from Phospholipid
GmbH), and the
mixture was stirred vigorously. From this, 35.66 gm were combined with 707.42
gm of sterile
water for injection, and the mixture homogenized at 5200 RPM with a Silverson
AX60 1/2 HP
homogenizer at ambient temperature, with temperature maintained below 26 C by
ice water
jacketing. After 75 minutes, 7.237 gm of tocopherol were slowly dripped in to
the dispersion.
Homogenization continued until the particle size was such that the dispersion
could be filtered
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through a 2 micron depth filter, follow by a 1.2 micron membrane filter. This
dispersion was
then split in half, and to one half was added 8.07 gm of glycine, and
sufficient NaOH to yield a
pH of 7.6. The other half of the dispersion was placed in a round bottom flask
and on a rotovap,
whereupon the dispersion weight was reduced from 344.3 gm to 119.9 gm; 2.604
gm of glycine
and sufficient NaOH were added to yield a final pH of 7.7. Twenty-milliliter
vials were filled,
sparged and autoclaved at 121 for 20 minutes, yielding two sterile
dispersions, which will be
referred to as "RA5-5E" and "RA5-15E" for the 5.6% and 15% particle
concentrations,
respectively. The zeta potentials were recorded as -46 mV and -32 mV,
respectively.
Three toxin solutions were prepared. These were: 18 mg of etomidate in 9 mL of
Krebbs-Ringer buffer with 40 mg/mL albumin, 20 microliters of paraoxon in 10
mL deionized
water, and 15.1 mg of clomipramine in 30 mL of Krebbs-Ringer buffer with
albumin.
Nanosep Omega centrifuge filters at 300,000 MWCO from Pall Filtron were used
to
separate particles from exterior phase. Thus, into each filter tube were
placed 50 microliters of
either RA5-5E or RA5-15E (also called LT-728(5E) and LT-728(15E)
respectively), 100
microliters of toxin solution, and 150 microliters of distilled water. The
loaded tubes were
incubated at 40 C for 45 minutes, then centrifuged at 10,800 RPM in a Jouan
high-speed
centrifuge for approximately 1 hour.
For HPLC analysis, the bottom phase liquid (filtrate) was injected directly,
and the top
phase (retentate) dissolved in 500 microliters of methanol, then injected. The
mobile phase in
the HPLC was 30% pH 8.1 phosphate buffer, 35% acetonitrile and 35% methanol.
Standards
were run for each of the toxins, and toxin concentrations determined in the
exterior (bottom)
phase and in the top phase retentate.
The following three tables show the data (assay values are in mg/mL) and the
analysis
thereof, for the three toxins. The analysis proceeded as follows. The top
phase dilution factor
was first computed by noting that either 5.6 or 14% liquid crystal was diluted
into 500
microliters of methanol. From this and the top phase assay value, the
concentration of toxin in
the particles was back-calculated. This was then divided by the aqueous phase
(bottom phase)
concentration, since this was undiluted, in order to arrive at an effective
partition coefficient. To
check the validity of the calculation, a mass balance was performed by
properly combining the
two concentrations and respective volumes, and checking how close the result
comes to the
target, or theoretical, value, which is 50.3 in the clomipramine case and 200
in the other two
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cases (reflecting the 4-fold lower toxin concentration in the clomipramine
toxin solution). One
skilled in the art will recognize that this calculation is designed such that
errors due to
incomplete separation of exterior phase from the particles are minimized¨in
particular, by using
the known particle concentration rather than a measured top phase volume, the
need for complete
particle separation is circumvented.
CLOMIPRAMINE
Particle Top ph Eff part Drug
acct
Assay concn dilution coefficient (target=50.3)
Bottom phase
LT-728 (5E) 0.00562 0.056 179.6 3195.2 52.0
LT-728 (15E) 00288 0.15 67.7 2348.6 51.6
Top phase
LT-728 (5E) 0.1
LT-728 (15E) 0.09996
PARAOXON
Particle Top ph Eff part Bup
acct
Assay concn dilution coefficient
(target=200)
Bottom phase
LT-728 (5E) 0.462 0.056 179.6 81.6 242.9
LT-728(15E) 0.207 0.15 67.7 98.4 213.3
Top phase
LT-728 (5E) 0.21
LT-728 (15E) 0.301
ETOMIDA I
Particle Top ph Eff part Etom
acct
Assay concn dilution coefficient (target=200)
Bottom phase
LT-728 (5E) 0.257 0.056 179.6 129.3 169.4
LT-728 (15E) 0.148 0.15 67.7 136.7 195.0
Top phase
LT-728 (5E) 0.185
LT-728 (15E) 0.299
Thus the experiment demonstrates that etomidate and paraoxon, and particularly
clomipramine,
partitioned strongly into the particles of the invention in this experiment.
This is true even
though the experiments were performed in the presence of albumin, which of
course greatly
increases the solubility of the toxins in water, and thus yields a much lower
value for the
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effective partition coefficient than would be found for the true partition
coefficient, that is,
without albumin. Indeed, albumin was included in order to more closely
simulate conditions
under which the invention will be used. When the experiment was repeated with
etomidate in
the absence of albumin, an effective partition coefficient of 309 was recorded
(with the mass
balance within 5% of theoretical).
Example 11. Using the same dispersions and methods as in Example 10, the
effective
partition coefficients of lidocaine, benzocaine, and bupivacaine in the
presence of albumin were
measured. Lidocaine yielded a value of 57 (with the mass balance being within
3% of
theoretical), benzocaine a value of 148 (with the mass balance being 84
whereas 95 was
theoretical), in the case of the RA5-15E dispersion, and the value for
bupivacaine was 62 (mass
balance within 3% of theoretical).
Example 12. In this Example, strontium ions (Sr2+), which are known in the art
to bind to
phosphatidylcholine head groups, were shown to exhibit strong binding to the
bilayer-rich cubic
phase particles of the invention. To demonstrate this,18.9 mM of strontium
chloride was added
to an aliquot of the "RA5-15E" dispersion of the invention prepared in Example
10. A high
degree of binding of strontium ions to the phosphatidylcholine in RA5-15E was
evidenced by the
immediate precipitation of the cubic phase, which centrifuged to the bottom of
the dispersion.
Were the heavy strontium ions not bound, the cubic phase would be less dense
than water and
would have centrifuged to the top of the dispersion, as is the usual case with
other methods of
precipitating the particles. Radiostrontium, and to a lesser extent strontium,
have known toxic
effects, as do related phosphatidylcholine-binding metals. More broadly, this
Example illustrates
how the effect of large bilayer surface areas in cubic phase particles can
result in effective uptake
of toxins that bind to bilayer components.
Example 13. A 15% particle concentration dispersion of the invention was made
in the
Example without requiring rotovapping (as was used in Example 10). An amount
1.9093 gm of
sodium deoxycholate was dissolved in 24.761 gm of sterile water for injection,
to which were
added 15.94 gm of alpha-tocopherol and 24.06 gm of phosphatidylcholine
(Phospholipon 90G
from Phospholipid GmbH), and the mixture was stirred vigorously. From this,
60.01 gm were
combined with 554.99 gm of sterile water for injection plus 2.00 mL of 1N
NaOH, and the
mixture homogenized at 5200 RPM with a SiIverson AX60 1/2 HP homogenizer at
ambient
temperature, with temperature maintained between 19 and 28 C by ice water
jacketing. After 3
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hours, 3.6999 gm of tocopherol were slowly dripped in to the dispersion.
Homogenization
continued for 2 hours and 20 minutes. Glycerol was added to a concentration of
2.3% for
tonicity, the dispersion filtered through a 2 micron depth filter, and
phosphate buffer added to a
concentration of 20 mM, and the pH adjusted. Vials were loaded, sparged with
nitrogen, capped
and autoclaved at 121 C and 15 psi for 15 minutes. The final dispersion had a
mean particle
size, using unimodal analysis, of 193 nm, and a pH of 8Ø
Example 14. In this Example a fluorocarbon was incorporated into the
particles, making
the particles well suited for uptake of diatomic nitrogen, for potential
treatment or prophylaxis of
decompression sickness ("the bends" and related conditions). The specific
fluorocarbon used
was hexafluoropropanol. To start, 0.318 gm of sodium deoxycholate was
dissolved in 4.127 gm
of water. To this were added 4.01 gm of soy phosphatidylcholine (Lipoid E80),
1.96 gm of
alpha-tocopherol, and 0.960 gm of hexafluoropropanol. Mixing this created a
high-viscosity
reversed cubic phase. An amount 10.16 gm of this liquid crystal was combined
with 94.94 gm of
water and 1.837 gm of glycerol (for tonicity), and the mixture homogenized
with an Intron lab-
scale homogenizer. The result was a fine dispersion of the invention, with
approximately 10%
fluorocarbon in the cubic phase, or phrased otherwise, where approximately 17%
of the bilayer
in the cubic phase was fluorocarbon.
Example 15 In this Example, the partitioning of the quaternary ammonium
skeletal
muscle relaxant vecuronium bromide, a water-soluble compound with a low
octanol-water
partition coefficient, between a phosphatidylcholine / tocopherol / water
reversed cubic phase
and water is measured, and found to be very high, sufficiently high as to
enable the use of the
invention in attenuating the effect of the drug in the body of a mammal. An
amount 0.6169 gm
of phosphatidylcholine (Phospholipon 90G, from Phospholipid GmbH) was mixed
with 0.6730
gm of alpha-tocopherol and 0.2194 gm of a 10% vecuronium bromide solution in
water. An
amount 1.1226 gm of this mixture was smeared onto the inner wall of a test
tube, and 6.4495 gm
of water added to the tube so as to contact the mixture. Upon swelling with
water, the mixture
became a reversed cubic phase. After 3 days of contact between the cubic and
aqueous phases to
allow equilibration of the drug between the two phases, HPLC analysis showed
that the
concentration of vecuronium in the cubic phase was 10.0 mg/mL, while that in
the aqueous
phase was only 0.006 mg/mL. The partition coefficient between cubic phase and
water was thus
approximately 1,600 (or log KQw = 3.2). When the content of anionic lipid was
increased
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significantly by incorporating a bile salt, this increased to log KQw
approximately 4, which could
reflect additional binding of drug due to electrostatic interactions with the
bilayer-bound anion.
One with ordinary skill in the art will recognize that the vecuronium mass
balance checks
correctly if one assumes that the water content of the final cubic phase,
after swelling to
equilibrium, is approximately 40%, which is in perfect accord with the phase
diagram derived in
this laboratory for this cubic phase region.
Example 16. The RA5-5E and RA5-15E dispersions prepared in Example 10 were
tested
in vivo as prophylaxis, in a rat model incorporating a normally-lethal
intravenous injection of
bupivacaine. Sixteen adult male rats were randomly arranged into four groups
of four animals
each. Each group of animals were pre-treated with intravenous doses of either
4 ml/Kg normal
physiologic saline (Group A), 4m1/Kg of dispersion RA5-15E of the instant
invention with a 15
percent particle concentration (Group B), 6m1/Kg of dispersion RA5-5E of the
instant invention
with a 5.6 percent particle concentration (Group C) or 4m1/Kg Intralipid , a
commercially
available fat emulsion useful for parenteral nutrition. Animals in all four
groups were then given
intravenously a known lethal dose (12 mg/Kg) of the amide local anesthetic
bupivacaine.
Animals from each group were then monitored for signs and symptoms of
cardiotoxicity
resulting from the administration of bupivacaine. All four animals in Group A
became pulseless
within 10 seconds of the injection of bupivacaine while all of the animals in
the remaining three
groups survived without clinical signs of cardiotoxicity. This example
demonstrates the use of
the invention as prophylaxis to toxic injury.
Example 17. The RA5-5E and RA5-15E dispersions prepared in Example 10 were
subsequently tested in vivo as a rescue agent in a rat model incorporating a
normally-lethal
intravenous injection of bupivacaine. Twelve adult male rats were randomly
arranged into three
groups of four animals each. Animals in each group were given a known lethal
dose (12 mg/Kg)
of the amide local anesthetic bupivacaine intravenously. Each animal was
monitored for signs of
acute cardiotoxicity. All animals became pulseless within 10 seconds of the
injection of
bupivacaine. When each animal became pulseless, it was treated immediately
with mild chest
compression and with one of the following: (i) 4 ml/Kg noimal physiologic
saline (Group A);
(ii) 4m1/Kg of dispersion RA5-15E of the instant invention with a 15 percent
particle
concentration (Group B); or, (iii) 6m1/Kg of dispersion RA5-5E of the instant
invention with a
5.6 percent particle concentration (Group C). All four animals in Group A
succumbed, while all
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four animals in Groups B and C recovered and exhibited normal pulse within
minutes of
administration of the dispersion of the instant invention. The administration
of the dispersion
according to the instant invention rescued the animals from death due to the
systemic toxicity of
an overdose of a common local anesthetic agent.
Example 18. Ten adult rats with previously inserted in-dwelling intra-jugular
(U) venous
cannulae were given 10 mg/kg of the intravenous anesthetic induction agent,
propofol, via the
indwelling U catheter. This constitutes a therapeutic dose. The Time to
Emergence from
anesthesia (defined as the elapsed time from the start of the propofol
injection until the time of
eye-closure and the loss of palpebral or eyelash reflex) was recorded for each
of the animals.
Twenty four hours later, the same group of ten animals was again given the
same dose of
mg/kg of propofol via the indwelling U catheter. Immediately after induction
of anesthesia,
each animal was given an U injection of dispersion RA5-15E from Example 10, at
4 ml/Kg. The
Time to Emergence from anesthesia was recorded as on Day 1 for each animal.
The differences
in Time to Emergence from Day 1 to Day 2 calculated for each animal. The Time
to Emergence
from anesthesia was less for every one of the ten animals on Day 2 than on Day
1. The mean
reduction in Time to Emergence from Day 1 to Day 2 was 23%, with a minimum
reduction of
13% and a maximum reduction of 37%. The administration of the dispersion
decreased the Time
to Emergence, in this way attenuating the therapeutic effect of a therapeutic
dose of the common
general anesthetic propofol.
Example 19. Amphotericin B provides an example of an API with high toxicity
which
has a site of action in the interior of a fungal lipid biomembrane. In this
Example the partition
coefficient of amphotericin B between the preferred cubic phase of the
invention (composition
approximately 30% phosphatidylcholine, 30% alpha-tocopherol and 40% water) and
water (both
phases containing residual DMSO from the experiment) was measured using HPLC,
and this
partition coefficient was found to be approximately 1,000.
An amount 0.0082 g of Amphotericin B and 0.8 mL of DMSO (dimethylsulfoxide)
were
added to a 16 mL test tube. The test tube was heated to 60 C until the
amphotericin B was
completely dissolved. An amount 0.692 g of 2.5% DMPG in the
phosphatidylcholine product
Lipoid E80, 0.8 mL of DMSO, and 0.6mL of ethanol were added to a second 16 mL
test tube.
The mixture was heated to 60 C and vortexed until the Lipoid E80/DMPG was
entirely
dissolved. An amount 0.6873 g of vitamin E and 0.8 mL of DMSO were added to a
third 16 mL
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test tube. The mixture was heated to 60 C and vortexed until the vitamin E was
completely
dissolved. The contents of the second and third test tubes were added to the
first test tube
containing the amphotericin B and DMSO mixture. The test tube containing the
entire mixture
was heated to 60 C for 30 minutes to ensure that all components were
completely dissolved.
An amount of 10 mL of deionized water was added to the test tube. The test
tube was
gently shaken at 40 RPM for 30 minutes on a Lab-Line Junior Orbit Shaker. The
aqueous portion
was decanted and filtered through Aldrich medium speed filter paper into a
clean test tube and
labeled "wash 1". This washing procedure was repeated three additional times
to obtain "wash
2", "wash 3", and "wash 4".
HPLC analysis was then performed to determine the amphotericin B
concentrations in the
4 wash solutions and the final, quadruply-washed cubic phase. The latter
concentration was
found to be 1.76 mg/mL, and it was ascertained that the cubic phase contained
entrapped pockets
of water (as evidenced by an opaque appearance, for example), so that the true
concentration in
the cubic phase itself was estimated to be approximately 3 mg/mL. In contrast,
the amphotericin
B concentrations in the "wash 3" and "wash 4" solutions were both
approximately 0.0035
mg/mL, thus making the partition coefficient on the order of 1,000.
It is known that amphotericin B partitions much more strongly into fungal cell
membranes,
the site of pharmacologic action, than into cell membranes of the human
kidney, the site of toxic
action. It is therefore possible that the application of the instant
invention, using for example a
phospholipid / tocopherol / water composition, could recover amphotericin B
from the site of
toxic action preferentially over the site of desirable pharmacologic action,
thus increasing the
therapeutic index, or relieving a patient of toxic effects while maintaining
antifungal activity.
