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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is a chemically structured delivery system for targeting
drugs, hormones, biologicals or diagnostic materials to the hepatocyte.
2. Description of the Prior Art
Applicant's prior U.S. Pat. No. 4,377,567 is a disclosure of the use of
digalactosyl diglyceride to target vesicles containing drugs, hormones or
other biological and diagnostic materials to the liver. For a full
development of the prior art, the disclosure of that prior patent is
referred to and incorporated by reference.
Hunt U.S. Pat. No. 4,091,088 assesses the hepatobiliary function by a
reagent which is labelled with technetium 0.99 m for use as a
hepatobiliary imaging radiopharmaceutical. This material does not affect
the liver other than to provide scanning capability for diagnoses.
Molter U.S. Pat. Nos. 4,318,898 and 4,350,674 also assess the hepatobiliary
receptors and caused a quick passage to the biliary system for diagnostic,
not treatment, function.
Great Britain Pat. No. 1,545,427, issued in 1979, also uses the
hepatobiliary system to diagnose the biliary system.
Applicant distinguishes over this and all known prior art, including
intensive literature studies, by the discovery that:
(a) the hepatobiliary receptor targeted vesicle will not be merely passed
to the biliary system, as would be expected from prior art teaching.
(b) the hepatocyte directed vesicle system targeted to the hepatobiliary
receptors of the hepatocytes will be utilized to deliver hormones and
drugs to the liver.
SUMMARY OF THE INVENTION
This invention resides primarily in the discovery that a bipolar lipid
vesicle containing a pharmaceutical load can be directed to the
hepatobiliary receptors of a liver. It has been discovered that instead of
passing through to the biliary system as expected, a vesicle directed to
the hepatobiliary receptors will be retained by the hepatocyte with
exceptional efficiency.
With that discovery in place, it is then the function of this invention to
successfully and properly construct a hepatocyte directed vesicle (HDV)
which is directed to the hepatobiliary receptors.
It is, therefore, an object of the invention to enhance the efficiency of
hepatocyte directed vesicles in general, by projection of the target
molecule away from the surface of the vesicle.
Another object of the invention is to enhance the efficiency of liver
medication by accessing the hepatobiliary receptors of the liver.
Yet another object of the invention is to provide a hepatocyte delivery
system comprising a composite of a vesicle and a hepatobiliary targeting
molecular wherein the vesicle's pharmacologic cargo is detached from its
targeting system and retained by the liver cell where it can carry out its
pharmacologic action.
The outstanding object of the invention is implementation of the discovery
that the hepatocyte delivery vesicle can be directed to the hepatobiliary
receptors of the hepatocyte, and not merely pass through to the biliary
system, but the pharmacological cargo is retained by the hepatocytes of
the liver, and enabled to carry out its pharmacologic action.
Another object of the invention is to prevent contact of the pharmacologic
cargo with cells and tissues of the body which are not intended target
tissues, thereby enhancing the specificity and potency of the
pharmacologic cargo, and reducing its nonspecific toxicity.
Another object of the invention is to enable the use of the HDV by
intraduodenal (oral), intravenous, subcutaneous and intramuscular routes
of administration.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph depicting the plasma glucose levels of normal dog models
which were denervated, tested for diabetes, and then normalized by this
invention;
FIG. 2 is a bar graph summarizing the data of FIG. 1;
FIG. 3 is a graph of blood sugar level after a controlled dose of saline on
a diabetic fasting dog, and compared result after administering HDVS;
FIG. 4 is a graph showing the change from glucose output to uptake
following infusion of HDVI;
FIG. 5 is a graph comparison of the effect of correcting the lack of
control serotonine on diabetic animals via an oral dose; and
FIG. 6 is a graph comparison of the effect of correcting the lack of
control serotoine on diabetic animals via intravenous injection.
DETAILED DESCRIPTION
The present invention relates to compositions and processes for delivering
pharmacologically-active agents preferentially to heptatocytes of the
liver. The liver is composed of two types of cells: hepatocytes or
metabolic cells, and reticuloendothelial cells (RE cells) which are
scavenger cells. More specifically, this invention provides Lipid Membrane
Structures (vesicles, liposomes) directed by hepatocyte target molecules
and coupling of molecules to the lipid membrane of the vesicle to carry
drug agents such as insulin preferentially to the hepatobiliary receptors
of the hepatocytes, but not the RE cells.
This invention has generally application for improving the efficiency of
accessing the hepatocyte because of the extension of the target molecule
away from the surface of the vesicle. However, the primary function of the
preferred embodiment, which is detailed herein, is to access the
hepatocyte by utilizing the hepatobiliary receptors of the hepatocyte cell
surface. The hepatobiliary receptors are known to receive substances for
the liver intended to be delivered as bile to the biliary system, whereas
liver (hepatocyte) treatment with liposomes has generally been
accomplished by accessing the Ashwell receptors of hepatocytes by
galactosyl targeting molecules.
The liver is the human body's largest gland and, as such, receives a
massive blood supply through both the portal vein and hepatic artery.
Metabolically, the liver is the most complex organ in the human body and,
among other multiple functions, it metabolizes/distributes drugs which are
introduced into the organism. The liver is also a target organ for
pharmacologically-active agents produced within the body. Accordingly, an
improved means for preferentially delivering drugs to the liver provides a
means for allowing the drug to be handled by the body in a more natural
fashion, thereby improving drug therapy.
The means whereby the liver handles insulin illustrates the activity of
this important target organ.
Insulin is a hormone which affects the metabolism of the animal as a whole.
The most dramatic effect of this hormone is its ability to reduce the
concentration of glucose in plasma. Ingested carbohydrate meals are
normally digested to glucose in the gut and then absorbed in the portal
circulation. The pancreas responds to carbohydrate in the gut with a
release of insulin into the portal circulation. The portal vein carries
the absorbed glucose and the released insulin to the liver. At the liver
the insulin regulates the metabolism of glucose by the heptatocytes. By an
unknown mechanism the liver retains most of the insulin but releases some
to facilitate glucose utilization by muscle and adipose tissue. Reduction
in blood glucose is due to the insulin effect on both liver and peripheral
tissues. Thus, while the pancreas is the source of insulin within the
organism, the liver is the key to its normal utilization.
Insulin therapy for diabetes mellitus began in 1922. In current medical
practice insulin is administered subcutaneously because the oral
administration of the insulin is inefficient, presumably due to
proteolysis. Subcutaneously administered insulin does produce a lowered
level of blood glucose, primarily as a result of its action on muscle and
fat tissue. However, insulin administration by injection can hardly be
classified as a near normal state. Importantly, the anatomic arrangement
of the pancreas in the normal individual is such that high levels of
insulin secreted by the pancreas in response to oral glucose loads pass by
way of the portal circulation to the liver before entering the peripheral
circulation. By comparison, when insulin is administered subcutaneously to
the diabetic patient, the peripheral tissue has first access to the
hormone and may reduce the level of insulin presented to the liver and, in
turn, reduces the effectiveness of the liver as a significant glucose
regulating mechanism. Therefore, insulin administered by injection does
not have the same physiological action as insulin released from the
pancreas.
The present invention provides an improved means whereby insulin or other
pharmacologically-active agents can be delivered preferentially to the
liver in a human or lower animal.
The Lipid Membrane Structures used herein comprise a bipolar lipid and a
target molecule conjugate.
The preferred polar lipid used in the practice of this invention is
distearoyl lecithin. Natural lecithin (phosphatidyl choline; vitellin)
comprises a mixture of the diglycerides of stearic, palmitic and oleic
acids linked to the choline ester of phosphoric acid and is found in all
living plants and animals. Vesicles from such polar lipids are known art.
The term "liposome" is generally referred to by the term "vesicle", but
both are interchangeable for the purposes of this disclosure. The vesicle
may be unilammelar or multilamellar and range in size from 250.degree. A
to 5 microns in diameter.
The HDV is useful in delivering drugs, hormones, biologicals or diagnostic
materials to the liver. The liver is a difficult organ to reach with
exogenously administered drugs, and the HDV presents a unique therapeutic
advance, making it possible to deliver drugs, hormones, biologicals and
diagnostic materials in a more efficient, safer way than those means
currently available. The reason for the "therapeutic unavailability" of
the liver to conventional therapies is that the liver is anatomically
situated in such a way that it is isolated from the rest of the body with
respect to its blood circulation. The majority of the liver's blood comes
to it by way of the portal circulatory system which is a highly localized,
low-pressure, venous system designed to carry absorbed nutrients (products
of digestion) from the intestines to the liver for metabolism. The
arterial blood supply takes care of the rest of the body before a small
portion of it reaches the liver via the portal system.
In view of the fact that the liver is a difficult organ to reach with
exogenously administered drugs, this invention provides a biological
carrier system that utilizes hollow bipolar liposomes in conjunction with
a family of liver molecules to effect delivery of the prescribed drug.
The following diagram schematically sets forth the basic structure of a
functional HDV:
##STR1##
The following structure depicts the basic liposome or bipolar lipid
vesicle:
##STR2##
Where -- is a polar lipid, and the -- represents a lipophilic portion,
and the .circle. represents the hydrophilic portion.
The vesicle is a sphere with an aqueous core, where the sphere is a bipolar
lipid membrane with hydrophilic surfaces and a lipophilic (hydrophobic)
interior of the membrane. The aqueous core can contain water-soluble
substances such as drugs, hormones, minerals, diagnostic agents, and
biologicals. Lipophilic substances are not carried in the core volume but
in the bipolar lipid membranes.
The targeting mechanisms require that the molecular target molecule be
positioned on the surface of the liposome or vesicle in such a manner that
the target molecules are available for interaction with its intended
receptor molecule which is on the surface of the intended cell. The target
molecule is positioned so that it is extended away from the membrane
surface. See the illustration above for a pictorial representation.
The HDV is fashioned in such a way that a connector portion is first
incorporated into the membrane at the time of forming the membrane. The
connector portion must have a lipophilic portion which is firmly embedded
and anchored in the membrane. It must also have a hydrophilic portion
which is chemically available on the aqueous surface of the vesicle. The
hydrophilic portion is selected so that it will be chemically suitable to
form a stable chemical bond with the target molecule which is added later.
Therefore, the connector molecule must have both a lipophilic anchor and a
hydrophilic reactive group suitable for reacting with the target molecule
and holding the target molecule in its correct position, extended out from
the vesicle's surface. In some cases it is possible to attach the target
molecule to the connector molecule directly, but in most instances it is
more suitable to use a third molecule to act as a chemical bridge, thus
linking the connector molecule which is in the membrane with the target
molecule which is extended, three dimensionally, off of the vesicle
surface.
An important aspect of this invention is the fact that the target molecule
can be any molecule that is recognizable by the hepatobiliary receptors of
the hepatocyte. The term "hepatobiliary" is defined in the 1965 edition of
Dorland's Illustrated Medical Dictionary, W. B. Saunders and Company,
Philadelphia, as "pertaining to the liver and the bile or biliary ducts."
The hepatobiliary receptors of hepatocytes are capable of recognizing and
of taking into the hepatocyte a great variety of chemical structure. The
nature of the target molecule is therefore most clearly defined by its
biological specificity for the hepatobiliary receptors of hepatocytes.
DEFINITION
This invention therefore can use as a target molecule, any chemical
substance which can be taken into the hepatocytes and transported into the
biliary system. Because of this diversity it is necessary to have this
biological definition of the target molecule rather than a chemical
definition.
EXAMPLES
A variety of chemical structures are shown below which are chemically
diverse but are generally recognized as hepatobiliary chemicals and act as
effective hepatocyte target molecules. Because of the variety of chemicals
that can be used by this invention as target molecules, it is necessary to
provide an appropriate variety of chemical means to attach these molecules
to the surface of the vesicle. This variety therefore requires a number of
different connectors and chemical bridge systems to permit the use of this
variety of chemical structures commonly referred to as hepatobiliary
materials.
While the majority of hepatobiliary target materials will require the
techniques of attachment generally described above, some will be suitable
for use as a single molecule because they will have a chemical portion
that acts as "connectors" and other portions that act as "target
molecules." Examples of useable systems will therefore include
representative of both the multiple step (connector-bridge-target) system,
and the single step system just described.
The structural formula below is an example of the result of a multiple step
method of constructing a Hepatocyte Directed Vesicle (referred to as HDV).
##STR3##
In the structure above a series of lines and small circles symbolize the
positioning of bipolar lipid membranes which encircle a vesicle core
volume. This structural representation is grossly out of proportion for
convenience of illustration. The bipolar vesicle is actually huge in
comparison to the connector-target molecule.
Under the bracket labelled "connector molecule" is an iminodicarboxylic
acid molecule having a lipophilic and a hydrophilic end. The lipophilic
end is the end containing the benzene ring and is shown embedded into the
bipolar film of the vesicle.
The hydrophilic portion of the connector molecule will extend away from the
fact of the vesicle because it is water loving.
Under the bracket labeled "target molecule" is, in this particular
illustration, an identical iminodicarboxylic acid as that in the
connection molecule. This is one acceptable, and perhaps preferred
embodiment, but by no means is it an exclusive requirement. That is, the
connector and target molecules are illustrated as being identical in this
embodiment, but that is not a requirement. It is a requirement of this
preferred embodiment that the target molecule be a molecule which is
recognized by the hepatobiliary receptors of the liver as opposed to
sugar-type molecules which are normally attracted to the Ashwell
receptors.
Under the bracket labeled "bridging ion" is a chromium bridge which will
connect the hydrophilic charged terminal ends of the two iminodicarboxylic
acid groups. These groups would not normally connect to one another;
therefore, the chromium ion is used for that purpose. There are other
possibilities wherein a hepatobiliary target molecule may be directly
connected to a connector molecule.
The hepatobiliary targeting molecule may be employed to bring a vesicle
into that portion of the liver which normally is concerned only with
creating bile fluids. After this discovery, a wide range of possible
combinations of connectors and target molecules may be visualized. It is
therefore only up to the innovative chemist to select from the great
number of possible combinations to achieve the necessary results.
It is not obvious that the hepatobiliary receptors will accept a system
which includes a vesicle and drop off the vesicle in the hepatocyte where
it releases its pharmacologic cargo and is effective, with the remainder
of the system passing onto the bile duct just as would be expected of
anything taken into the hepatobiliary receptors.
In the study and experiments done to validate this invention, it appears
that much larger and greater number of vesicles may be taken into the
hepatocyte through the hepatobiliary receptors.
I. Chemistry
A. Preferred bulk bipolar lipid constituents (75%-95%)
1. Distearoyl lecithin (DSL)
2. Dipalmatoyl lecithin (DPL)
3. Other lecithins with chain lengths C10-C20
B. Minor constituents (0.1-25%) for stability
1. Cholesterol
2. Dicetyl Phosphate
3. Albumin
C. Target Molecules (0.1%-10%)
Hepatocyte specificity is conveyed by molecules having the following
structure which have both hydrophilic and hydrophobic portions:
##STR4##
Where R.sub.1 has the following structure(s) and n=1-3,
##STR5##
R.sub.2a has the following structure(s):
(1)CH.sub.3, (2)CH.sub.2 CH.sub.3, and (3)CH(CH.sub.3).sub.2
R.sub.2b has the following structure:
CH(C.sub.3).sub.2
Preferred Target Structure #3
N-(3 Bromo-2,4,6-trimethylphenyl carbamoyl methyl)imino diacetic acid
Preferred Target Structure #4
N-(3-Cyano-4,5-dimethyl-2-pyrrl carbamoyl methyl)imino diacetic acid
Preferred Target Structure #5
D. Biliverdin (or Bilirubin) Preferred Bridge Examples
1. Inorganic salts of:
a. Chromium
b. Cobalt
c. Iron
d. Zinc
2. Organic
a. Ethylene Diamine
b. Propylene Diamine
Connectors
With each of the preferred target structures, the preferred connector is
the same as the target. The preference is only for the convenience of the
manufacturer.
It should be noted that in the target structures the diacetic acid portion
provides oxygen bonds for connecting through a bridge to a connector
molecule presenting similar oxygen bonding points. The bridge molecules
provide the necessary ligands to connect to each of the four oxygens and
thereby interconnect the target and connector molecules.
Although the foregoing is preferred, it is not essential that identical
molecules be used for connector and target with a bridge interconnection.
An example of an alternate connector means not using a bridge is to
incorporate albumin into the vesicle membrane then react the proprionyl
group of bilirubin as a target with a lysine amino group to form a Schiff
base. In this example, no bridge molecule is used per se.
Therefore, it is within the penumbra of this invention to form a
single-step target system without separate connector and bridge molecules.
These examples are of materials that are incorporated into the HDV in one
step, not requiring the multiple step addition of bridge and then target
molecule, although portions of the large molecule conjugates can be
designated as "connector", "bridge" and "target".
There are four parts to the completed preferred HDV:
1. Vesicle, which carries the cargo
2. Vesicle connector molecule
3. Bridging molecule
4. Target molecule
In this invention the connector molecules and the target molecules can be
identical. That is to say that two identical molecules which are connected
by a specific molecular bridge, when appropriately attached to the vesicle
surface, form the completed HDV system.
However, it is also possible to connect two dissimilar molecules by a
bridge, thereby enabling selection of various useful combinations of
target and connector molecules which would not otherwise be compatible.
This is within the skill of the biochemist after the concepts of the
invention have been understood.
In the preferred target structures above for the hepatobiliary receptors
where R.sub.1 has the preferred structure of n=1 in order to maximize the
negative charge on the carboxyls, n may be extended with additional
methylenic (CH.sub.2) groups with concomitant reduction of the negative
charge on the carboxyl group, which would result in a progressively weaker
ligand connecting the bridge.
PREPARATION OF THE PREFERRED EMBODIMENT
This development describes the coupling of a liver targeting agent to a
vesicle membrane for the purpose of creating a new targeting system. There
is an orderly sequence of events that is required for the successful
coupling of the targeting agent to the vesicle membrane. The preparation
of the vesicle targeting system initially requires the formation of a
vesicle structure with a connector molecule embedded into the membrane. In
the course of this development there have been two special ligands
utilized to attach the connector molecules to the targeting molecules.
Initially, an inorganic substance, chromium chloride, was used. Concern
about long-term chromium toxicity arising from continual vesicle dosing
has been dispelled by further study, but organic ligands such as H.sub.2
N--CH.sub.2 --(CH.sub.2).sub.n --NH.sub.2 where n=1-4, (i.e. ethylene
diamine) is an alternative for coupling purposes. However, from a
practical point of view, both ligands serve the same function.
Described below is a reaction sequence relating the various steps involved
in the formation of an HDV system. Each step is unique in relation to the
next step and each subsequent step in the reaction sequence. This is the
preferred embodiment.
The first step of the sequence requires that the connector molecule and the
lipid components comprising L-.alpha.-distearoyl lecithin (DSL), dicetyl
phosphate (DCP) and cholesterol (CHOL) in conjunction with human serum
albumin (HSA) be formulated into a vesicle structure with a bipolar
membrane.
As a result, the connector molecule is oriented in three-dimensional space
as noted above. This three-dimensional position of the connector molecule
creates the foundation for the successive reactions resulting in the
structures completing the HDV. At this step the entire vesicle is acting
as a large suspended structure with the vesicle surface interspersed by a
uniform distribution of connector molecules, three-dimensionally poised to
partake in the next reaction sequence. The hydrophilic portion of the
connector molecule projects into the aqueous phase of the media, whereas
the hydrophobic portion of the molecule is buried in the lipophilic
section of the membrane. Since the molecular size of the connector
molecule is small in comparison to that of a vesicle and since only a
portion of the connector molecule is entrapped in the vesicle structure at
the time of preparation, the remaining unentrapped connector molecules can
be removed easily by Sephadex G-100-120 gel filtration chromatography.
In the next step a five-fold molar excess with respect to the initial
concentration of connector molecules, of either CrCl.sub.3 hexahydrate or
ethylene diamine (which are examples of bridging molecules) is reacted
with the vesicle structure containing the embedded connector molecule to
form a vesicle connector molecule-bridging molecule complex. As a result
of this step, the proper three-dimensional orientation of the bridge
molecule is established for the subsequent binding of a targeting molecule
to the vesicle-connector molecule-bridging molecule complex.
In the final step the the target molecules are added to the
vesicle-connector-bridge complex. A five-fold molar excess of target
molecules is reacted with the vesicle structure containing the vesicle
connector molecule-bridging molecule complex to form the vesicle connector
molecule-bridging molecule-target molecule conjugate to be used for
vesicle targeting purposes. The excess unreacted target molecule may then
be removed by Sephadex G-100-120 gel filtration chromatography.
In summary, the unique steric symmetry and structure of the targeting
molecule permits it to be functional and useful in its targeting role.
This unique, three-dimensional symmetry can be achieved through utilizing
the reaction sequences as described in the aforementioned paragraphs.
The new chemistry featured in this targeting system is that a molecule that
is both hydrophilic and hydrophobic can be converted to a hydrophobic
targeting moiety with the concommitant neutralization of the charged
portion of the molecule through ligand formation and, in addition, provide
the correct three-dimensional orientation or projection of its hydrophobic
targeting portion into the hydrophilic aqueous media.
Thus, the sequence in which these molecules are reacted promotes and
establishes the proper orientation of the insoluble hydrophobic group in
spite of the unfriendly environment posed by the aqueous media toward
hydrocarbon structures.
Furthermore, the negative charge contributed by dicetyl phosphate (one of
the membrane constituents) at the surface of the vesicle, creates
charge-charge repulsion between vesicles and facilitates their suspension
and shelf-life stability. The negatively charged phosphate at the
hydrophilic end of the molecule at neutral pH is responsible for the
charge-charge repulsion effect resulting in vesicle stabilization.
This charge-charge repulsion is strong enough (probably by two-orders of
magnitude) to overcome any van der Walls induced dipole interactions
caused by the hydrophobic phenyl ring and the accompanying R-groups.
Method for Preparing HDV
DSL--69.12 mg, --iminodiacetic acid complex 1.07 mg, DCP--14.1 mg,
CHOL--5.0 mg
1.5 ml of CHCl.sub.3.MeOH (2:1 v/v) was added to solubilize the reagents.
The sample was placed on the rotoevaporator and taken to dryness at
60.degree. C..+-.0.5.degree. C. with slow turning under aspirator vacuum.
Then 2.4 ml of freshly prepared 40 mM phosphate buffer pH 7.4 with a
concentration of HSA and serotonin, the active agent at 4.2 mg/ml and 10
mg/ml, respectively, were added to the dried lipid components. The sample
was sonicated using a Heat Systems Ultrasonic Cell Disruptor at setting #4
equipped with a transducer and cup horn at 60.degree. C..+-.0.5.degree. C.
for 15 minutes. Next, the sample was annealed at 60.degree.
C..+-.0.05.degree. C. with slow turning for 15 minutes. Then the sample
was centrifuged in a Triac Clinical Centrifuge at the blood setting for 15
minutes at room temperature.
Then, 1.5 ml of the supernatant was chromatographed on a 1.5.times.25.0 cm
Sephadex G-100-120 column that had been previously equilibrated with 40 mM
phosphate buffer pH 7.4. This first chromatography was performed in order
to remove the unentrapped HSA and serotonin, the active agent. The lipid
vesicles were collected and then, with respect to the initial
concentration of vesicle connector molecules, were reacted with a
five-fold molar excess of CrCl.sub.3. The vesicles were then
rechromatographed using the same buffer to remove unreacted CrCl.sub.3.
The collected vesicles were then reacted with a five-fold molar excess of
connector molecules. Following this step the vesicles were then
rechromatographed using the same buffer system to remove unreacted
connector molecules. Following this final chromatography, the vesicles
were stored under nitrogen in the refrigerator at 5.degree. C.
Phenyl-mercuric nitrate may be used as a preservative (0.001%).
In Vivo Testing of the Preferred Embodiment
To test the invention an in vivo model is used. It must be borne in mind
that the test is for successful delivery of a pharmaceutical dose to the
liver. The delivery of the cargo is established by demonstrating the
desired pharmacological response to the cargo by the liver.
The entire thrust of this invention, hence the disclosure of the many
preferred approach methods, is to establish factually by in vivo testing
that the pharmaceutical load carried within a vesicle can and is delivered
to the liver of a warm-blooded animal and that the pharmacologic cargo is
made available to act at the hepatocyte. In order for the pharmacologic
cargo to act on its receptor, the HDV must be effectively dismantled,
releasing the cargo from the protective coating of the vesicle. The
pharmacologic cargo may then be used by the liver to cause a hormonal
control of the liver as in the natural functioning state of healthy
individuals.
This inventor has also discovered and established beyond doubt that the
liver storage of glucose eaten during a meal requires not only the hormone
insulin, but also that a co-factor is required for proper liver function.
That co-factor is serotonin. Serotonin is a chemical, 5-hydroxytryptamine
(5-HT), present in platelets, gastrointestinal mucosa, mast cells, and in
carcinoid tumors. Serotonin is a potent vasoconstrictor (Taber's
Cyclopedic Medical Dictionary, 14th Edition). This inventor has
established that Serotonin is supplied to the portal vein leading to the
liver while food is being absorbed by the intestines and is controlled by
the central nervous system. Thus, it was discovered that by severing the
vagus nerve the serotonin in the portal vein could be essentially
eliminated. When this is done, it is now established, the liver no longer
will convert the nutrient glucose to glycogen and store the glycogen.
Rather, the liver will allow all of the glucose to proceed into the
peripheral system, thereby producing excess sugar in the blood and
providing the symptoms of diabetes. Although there may be sufficient
insulin available at all times at the liver, a deficiency of serotonin
will result in an excess of glucose in the blood.
By providing a serotonin load in this hepatocyte delivery vesicle and
targeting the vesicle to the hepatobiliary receptors of the liver, and
observing the return to normal liver function, this inventor has clearly
established the ability of the target vesicle system of this invention to
effectively deliver a pharmaceutical load to the liver, whatever the load
may be. If diagnostic material is desired, or insulin, or as in the case
of hypoglycemia, the blocking agents for the serotonin function, the
unique capability of this invention has been established.
Hence, in a dog model having a normal insulin production by the pancreas
glands, it is a superior test of the HDV to denervate the glands producing
serotonin, and after establishing diabetes symptoms of excess blood
glucose, to direct serotonin (5HT) to the liver. Re-establishment of
normal liver glucose control then proves effective HDV delivery to the
hepatocyte.
In a first study testing the HDV system, a chronic dog model was selected
which mimics the early stages of adult onset diabetes mellitus, now
referred to as diabetes mellitus Type II. In this model, the diabetes was
induced by selective denervation. The normal healthy dogs prior to
denervation respond to a standard meal (one-third carbohydrate) by having
slightly lowered levels of peripheral blood glucose. Following
denervation, the dogs maintain normal fasting blood glucose values, but
their peripheral blood glucose levels rise significantly after a
standardized meal, thus responding in a similar manner to adult onset
diabetes.
This particular model was selected for this study because it enabled the
evaluation of the HDV system in alert, unanesthesized animals.
This study was divided into three phases, studying the blood glucose
response of the animals while in the (1) normal state; (2) diabetic-like
state; and (3) successful treatment of the diabetic-like state with
subcutaneously administered HDV containing serotonin.
The experimental plan required four normal, healthy mongrel dogs. This
first phase of the study was to determine the blood glucose response of
these four dogs to a standardized glucose meal, comparable to the oral
glucose tolerance test in people. The graph of FIG. 1 is a comparison
chart for in vivo testing.
The data for this normal phase are shown in the graph as the line indicated
by reference number 10. The data are expressed as a percentage of the
fasting blood glucose value taken prior to feeding. Since four dogs were
used, the average or mean value for the data is plotted. The data are
statistically analyzed, and the variation in the data is shown by the
small vertical bars above and below the data points. These bars are the
standard error of the mean (SEM).
Following the acquisition of the data described above, the dogs were
surgically denervated to induce the diabetic-like state and allowed a week
to recover. At this time the study is again repeated, and the data are
shown as the line indicated by reference number 12, along with the error
bars (SEM). It is clearly seen from the data that the animals' responses
were different following the denervation. The asterisks (*) at the 1, 2, 3
and 4 hour data points along the line 10 indicate that by statistical
analysis of the data by the conventional student's t test, the blood
glucose response of the dogs following surgery is statistically different
from the response of the dogs prior to surgery. The level of significance
is less than 5% (designated, therefore, as p<0.05). This means that there
is less than a 5% chance that the difference observed would have happened
randomly.
The third phase of the study was to repeat the standardized meal in the
diabetic-like dogs, but with the dogs injected subcutaneously with 1.0 ml
of HDV-containing serotonin. The total dose of serotonin in the HDV was
150 ug serotonin, and it was administered one hour prior to eating and
immediately after taking the baseline blood glucose sample. The response
of the HDV-serotonin-treated denervated dogs is shown in line 14. The
abnormal elevation of the blood glucose has been normalized. At one, two
and four hours the data points were p<0.06, p<0.05 and p<0.05 for this
treatment, compared to the post-denervation meal 12.
The data from these studies are summarized in the bar graph of FIG. 2.
The first bar shows that the average value for the blood glucose (hours
1-4) for the dogs prior to surgery decreased about 10% after a meal
compared to their blood glucose value taken prior to eating. In the second
bar, the mean blood glucose response (hours 1-4) was increased after a
meal, and the asterisk indicates that it was statistically significant
(p<0.01, student's t test). The denervation had caused an elevation in
blood glucose following a meal, thus inducing a diabetes-like state. The
third bar shows the effect of the HDV-serotonin treatment. The HDV
serotonin significantly decreased the elevation of the blood glucose
following a meal.
It is most important to know that in studies by the inventor it has been
established that serotonin administered at this dose (150 ug) is not
effective either intravenously or subcutaneously in inducing the effect
seen with the HDV-serotonin in these studies.
The conclusions from these data are:
1. Denervation produced a diabetic-like state.
2. The effects of denervation could be corrected with very low doses of HDV
containing 5-HT.
A second study tested the hypoglycemic (blood glucose-lowering ability)
effectiveness of HDV containing 5-HT in fasting (non-fed) dogs. The graph
of FIG. 3 summarizes the results. Four dogs (denervated as in the first
study) were used in the study on two different days. The first day tested
the effect of saline and the second day, the effect of HDV (with 5-HT) on
fasting plasma glucose levels. The protocol required that a baseline
plasma glucose be obtained and followed by a 1.0 ml subcutaneous injection
of saline (day 1) or HDV/5-HT (day 2). One hour later a second blood
sample was obtained for a post-treatment plasma glucose. The
post-treatment glucose was compared to the baseline plasma glucose values.
The saline treatment (control) experiment resulted in an increased plasma
glucose compared to its baseline values. However, the HDV/5-HT treatment
resulted in a statistically significant (p<0.01) reduction in fasting
plasma glucose.
The conclusions based on this data are:
1. Control saline injections in fasting dogs produced a slight (stastically
insignificant) increase in the fasting plasma glucose.
2. HDV/5-HT injections produced a statistically significant (p<0.01 by
students' t test) reduction of the fasting plasma glucose.
The second conclusion, namely the administration of the HDV-5HT and the
resultant reduction in fasting plasma glucose is quite significant.
Bearing in mind that the purpose for the foregoing test is to establish
conclusively that the HDV delivers pharmacologic agents to the liver.
Therefore, this invention, having first established the function in
serotonin in programming the liver to uptake glucose, and then
administering the serotonin in the HDV with the resultant reduction in
glucose, establishes beyond any reasonable doubt that the HDV has been
taken into the liver and has caused the function expected of Serotonin of
ceasing glucose output and beginning glucose uptake. Following the initial
in vivo testing of four dogs as listed above, addition tests on animals
have been undertaken using four different programs. These four separate
programs are outlined below and then summarized.
Hereafter is a description of four supplemental experiments which document
the versatility of the HDV system. In the original tests above,
experimental data for the hepatocyte delivery of serotonin are included.
This system utilized the connector, bridge and target complex. That
original study utilized the subcutaneous route of administration. The
features of the original experiment and the four supplement experiments
are summarized in Table I. The important variables are:
______________________________________
SUPPLEMENTAL EXPERIMENTS
Experiment de-
scribed in origi-
nal experiments
1 2 3 4
______________________________________
Connector
2,6 diisopropyl
Same Same Same Same
phenyl car-
bamoyl methyl
imino diacetic
acid
Bridge Chromium Chro- Chro- Chro- Chro-
mium mium mium mium
Target 2,6 diisopropyl
Same Same Same Bili-
phenyl car- verdin
bamoyl methyl
imino diacetic
acid
Hormone Serotonin Insulin Growth Sero- Sero-
hormone
tonin tonin
Route of
Subcutaneous
Intra- Subcu- Intra-
Intra-
adminis- venous taneous
duo- venous
tration denal
Fraction of
1/100th 1/200th 1/7th 1/300th
1/100th
dose of to
hormone 1/50th
for effect
______________________________________
1. Target molecule.
The original experiment and the first three supplemental experiments used
the target molecule disclosed hereinabove. Supplemental experiment 4 used
biliverdin. The differences between these two materials are very
significant. The first, N-(2,6-diisopropyl phenyl carbamoyl methyl)imino
diacetic acid is a synthetic material. Biliverdin is a naturally-occurring
metabolite in the body that is used to form bile. Since serotonin HDV
worked with both target molecules, it is established that the
hepatobiliary receptor is an effective target mechanism. There is no known
reference in the medical literature where a naturally-occurring
metabolite, such as biliverdin, has been shown to carry and then render
effective a pharmacologic cargo.
2. Different hormones.
The original experiment used serotonin as its pharmacologic ca | | |
