U.S. patent number RE32,919 [Application Number 07/066,732] was granted by the patent office on 1989-05-09 for method of enhancing the effect of t-pa.
This patent grant is currently assigned to Survival Technology, Inc.. Invention is credited to Stanley J. Sarnoff.
United States Patent |
RE32,919 |
Sarnoff |
May 9, 1989 |
Method of enhancing the effect of t-PA
Abstract
.[.The absorption rate of t-PA in the blood is enhanced by
administering it together.]. .Iadd.A method of enhancing the effect
of t-PA in the blood which comprises the steps of administering
exogenous t-PA and an amount of t-PA inhibitor dissociating agent
effective to increase the dissociation of the inhibitor from the
t-PA, the amount of exogenous t-PA being less than that required to
be administered in the absence of the inhibitor dissociating agent.
The t-PA can be administered outside the blood when administered
.Iaddend.with an absorption enhancing agent, preferably
hydroxylamine or a salt thereof, most preferably hydroxylamine
hydrochloride.Iadd., which constitutes both an enhancing agent and
an inhibitor disassociating agent. .Iaddend.
Inventors: |
Sarnoff; Stanley J. (Bethesda,
MD) |
Assignee: |
Survival Technology, Inc.
(Bethesda, MD)
|
Family
ID: |
24561055 |
Appl.
No.: |
07/066,732 |
Filed: |
June 26, 1987 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
638695 |
Aug 8, 1984 |
4658830 |
|
|
Reissue of: |
708845 |
Mar 6, 1985 |
04661469 |
Apr 28, 1987 |
|
|
Current U.S.
Class: |
514/13.5;
424/719 |
Current CPC
Class: |
A61K
9/0019 (20130101); A61K 31/13 (20130101); A61K
31/415 (20130101); A61K 38/38 (20130101); A61K
38/446 (20130101); A61K 38/49 (20130101); A61K
47/18 (20130101); A61M 5/2066 (20130101); A61K
38/49 (20130101); A61K 31/13 (20130101); A61K
38/49 (20130101); A61K 31/13 (20130101); A61K
33/02 (20130101); A61K 31/17 (20130101); A61K
31/415 (20130101); A61K 31/155 (20130101); A61K
31/15 (20130101); A61K 31/415 (20130101); A61K
38/446 (20130101); A61K 38/49 (20130101); A61M
5/19 (20130101); A61K 2300/00 (20130101); A61K
2300/00 (20130101); A61K 2300/00 (20130101) |
Current International
Class: |
A61K
38/49 (20060101); A61K 38/44 (20060101); A61K
38/43 (20060101); A61K 31/13 (20060101); A61M
5/19 (20060101); A61K 031/00 (); A61K 033/02 () |
Field of
Search: |
;514/2 |
Other References
Iizuka et al-Chem. Abst., vol. 77 (1972) p. 17315u. .
Korninger et al-Chem. Abst., vol. 95 (1981) p. 197233u. .
Belousov et al-Chem. Abst., vol. 92 (1980) p. 39285w. .
Levin, Proc. Natl. Acad. Sci., USA, vol. 80, pp. 6804-6808, Nov.
1983..
|
Primary Examiner: Rosen; Sam
Attorney, Agent or Firm: Cushman, Darby & Cushman
Parent Case Text
This application is a continuation-in-part of application 638,695,
filed Aug. 8, 1984, the entire disclosure of which is hereby
incorporated by reference and relied upon.
Claims
What is claimed is:
1. A method of increasing the .[.absorption.]. .Iadd.effect
.Iaddend.of t-PA in the blood in a mammal in need of t-Pa therapy
comprising administering to the mammal .Iadd.exogenous t-PA and
.Iaddend.an amount of t-PA inhibitor disassociating agent effective
to increase the disassociation of the inhibitor from the t-PA, the
amount of exogenous t-PA being less than that required to be
administered in the absence of the inhibitor disassociating
agent.
2. A method according to claim 1 wherein the inhibitor dissociating
agent is added intramuscularly.
3. A method according to claim 1 wherein the inhibitor
disassociating agent is added intravenously.
4. A method according to claim 1 wherein the inhibitor
disassociating agent is hydroxylamine or a non-toxic salt
thereof.
5. A method according to claim 4 wherein the inhibitor dissociating
agent is added intramuscularly.
6. A method according to claim 4 wherein the inhibitor
disassociating agent is added intravenously.
7. A method according to claim 4 wherein the inhibitor
disassociating agent is hydroxylamine hydrochloride.
Description
This invention relates to the treatment of coronary prone
individuals in the throes of a suspected myocardial infarction in
such a way as to minimize damage to the heart muscle and, more
particularly, to improvements in such treatments enabling the same
to be commenced at the earliest possible time, even before direct
qualified personal care of the individual can be established.
When a clot forms in a blood vessel, the body organ being supplied
with blood by that blood vessel is compromised or totally deprived
of blood supply. Depending on the blood vessel in which this
occurs, the threat to the life of the individual is either small or
very great as in the circumstances to be addressed by the material
below, i.e. certain life threatening circumstances. Clot formation
in a vessel is described as thrombosis. Substances which dissolve
thrombi are called thrombolytic substances. When a coronary artery
clot is dissolved, the resultant establishment of blood flow to the
heart is called reperfusion.
Examples of life threatening or very serious clot formation in
arterial vessels are cerebral thrombosis, renal thrombosis,
opthalmic artery thrombosis, and very importantly, thrombosis of a
coronary artery. In approximately 85% to 90% of cases of acute
myocardial infarction (coronary heart attack), a thrombus is found
in the coronary artery preventing blood from flowing to the heart
muscle (myocardium) and supplying it with essential oxygen and
other nutrients. A consequence of a thrombus or clot forming in a
coronary artery is the danger to the myocardium (heat muscle tissue
that does the pumping of blood). Heart muscle deprived of it's
blood supply does not die immediately but does promptly begin the
process of becoming dead. The extent of the damage which is done to
the heart muscle is, therefore, a function of the time during which
the supply of blood to the infarct zone is restricted by the
occuluding thrombus.
Heretofore, the procedures undertaken to actually establish
reperfusion to the infarct zone have generally been undertaken in a
hospital environment or equivalent. The so-called "prehospital38
treatment was, in general, directed toward keeping the patient
alive and getting the patient into the hospital environment as soon
as possible so that treatment minimizing the heart muscle damage
could be accomplished.
The treatment undertaken in the hospital environment involves
certain procedures for establishing reperfusion in the infarct zone
of the patient's heart. When immediate surgery was not clearly
indicated, the establishment of reperfusion was accomplished by
procedures which had the effect of unblocking the occlusion. The
available procedures included mechanical catheterization and the
administration of thrombolytic agents. Known thrombolytic agents,
such as streptokinase or urokinase required intracoronary infusion
or the slow infeed of the agent within the vessel at the site of
occlusion by means of a catheter. In recent years, intravenous
infusion of streptokinase has been shown to be effective.
More recently a substance called tissue-type plasminogen activator
or t-PA has been utilized experimentally. (The New England Journal
of Medicine, Mar. 8, 1984, Volume 310, No. 10, pages 609-613).
Unlike other plasminogen activators, such as streptokinas or
urokinase, t-PA--which is found in only small amounts in the
body--acts specifically on clots and not on other relevant proteins
in the blood, when maintained at appropriate and effective
levels.
A 1984 Commentary found in Biochemical Pharmacology, Volume 33, No.
12, pages 1831-1838 entitled
"Coronary Thrombolysis: Pharmacological Considerations With
Emphasis On Tissue-Type Plasminogen Activator (t-PA)" contains the
following conclusionary statement:
"Selection of pharmacological agents for induction of coronary
thrombolysis has been determined largely by availability.
Unfortunately, both streptokinase and urokinase induce a systemic
lytic state with depletion of circulating fibrinogen, plasminogen,
and .alpha. 2-antiplasmin, and accumulation of fibrin degradation
products. All of these factors conspire to set the stage for
hemorrhage with a risk of serious bleeding. Intravenous
administration of these agents is limited by a lower success rate,
in part because the upper bound of dose is constrained by the risk
of inducing a severe systemic lytic state.
The probability that progress in recombinant DNA technology will
lead to widespread availability of tissue-type plasminogen
activiator is particularly exciting because of the clot specific
properties of t-PA. For coronary thrombolysis its potential
advantages include: safety and efficacy of intravenous
administration of high doses; effective clot lysis without
induction of a systemic lytic state; prompt implementation without
the need for extensive characterization of the coagulation and
fibrinolytic systems in each patient prior to and during therapy;
avoidance of frank allergic reactions or variations in
dose-response relation due to immune complex formation; ease of
minute-by-minute adjustment of dosage and prompt termination of
fibrinolysis when needed because of the short biological half-life
of t-PA and the lack of induction of a systemic lytic state".
The promise attributable to t-PA administration was discussed at a
news conference at a meeting of the Americal Heart Association and
reported by the New York Times on Nov. 16, 1983, in an article
entitled, "Protein Of Cancer Cells Used To Halt Coronaries." The
article refers to injection of t-PA by stating the following: "The
protein t-PA can simply be injected into the vein in the arm of the
patient seized by a myocardial infarction or heart attack, and it
travels through the blood to dissolve a clot, in much the same way
as Draino clears up stopped plumbing."
The article further indicated under the subheading "Hopes For
Future Application" that many physicians have expressed excitement
about research into the use of t-PA to treat heart attacks because
they hope that some day it may be used in emergency rooms and
ambulances to stop heart attacks at their earliest stages before
they kill or cause permanent damage. Under the "Hopes For Future
Application" subheading there is also included the following
paragraph: "Dr. Burton E. Sobel Of Washington University, one of
the researchers, speculated that patients might some day carry a
vial with them so that the drug could be injected immediately after
they felt chest pain and other early symptoms of a heart
attack."
In medical parlance, a vial is a container for a quantity of liquid
medicine or diluent having a rubber stopper capable of being
pierced by a hypodermic needle of a syringe to enable the operator
of the syringe to withdraw a predetermined dosage of the liquid
from the vial. In the case of t-PA as currently used, the dosage
could then be injected into the mother liquid container of an
infusion assembly. The necessity to administer the drug by
intravenous infusion or by intravenous injection presents a
significant barrier to self-administration from a practical view
point, particularly when considering the disconcerting
circumstances of the individual undergoing the symptoms of a
myocardial infarction.
The development of an effective self-administration procedure for
t-PA sufficient to enable its utilization by a targeted coronary
prone individual immediately following onset of symptoms, would
materially increase the potential efficacy of t-PA as a
thromobolytic agent by insuring its use at the earliest possible
time often before irreversible heart muscle damage has occurred,
and, at the same time, provide a treatment of the pre-hospital or
pre-ambulance type which for the first time is directly effective
to minimize heart muscle damage accompanying myocardial infarction.
It is an object of the present invention to provide such a self
administering treatment.
Another object is to enhance the absorption rate of t-PA in the
blood when introduced intramuscularly.
The invention includes packaging t-PA and an agent enhancing the
absorption of t-PA in the blood. The agent preferably is
hydroxylamine hydrochloride, in a known emergency type automatic
injector and injecting the two medicament agents into the muscle
tissue, e.g. after having received a decision to do so over the
telephone from a qualified source and at a time prior to the
establishment of direct contact qualified personal care.
Even though t-PA may be regarded as a clot selective thrombolytic
agent, when introduced into the blood stream at a predetermined
level, tests thus far performed show that the concentration can be
increased to the point that a systemic lytic state can be induced.
Intramuscular injection involves the introduction of concentrated
dosage of t-PA in an area contiguous to and substantially
surrounding the wound caused by the penetration and withdrawal of
the injection of the hypodermic needle. Consequently, it would be
expected that at least a localized lytic state would be induced
resulting in hemorrhage from the needle wound. Unexpectedly, tests
have shown that no such hemorrhage does in fact occur.
Second, t-PA is a large protein. It would not be expected that it
would be absorbed into the blood stream in discernible quantities.
Extravascular levels of protein are about 1/10 that of
intra-vascular protein. It is thought that this is so because the
capillary pores through which transport of protein can occur are
small relative to the molecular size of protein and limit protein
transport because of electrical charge. It was thus highly
problematical as to whether a large protein such as t-PA, when
given intra-muscularly, i.e. outside the blood vessels, would find
its way rapidly into the blood stream in discernible quantities.
Application tests have indeed shown that by itself t-PA does not
find its way rapidly into the blood stream in therapeutically
significant quantities after intramuscular injection.
The actual treatment of the system must therefore include
intramuscular injection of an absorption enhancing agent
simultaneously or substantially simultaneously with the
intramuscular injection of the t-PA so as to produce effective
thrombolytic blood levels.
Augmentation of absorption of low molecular weight substances
administered topically, subcutaneously, or intramuscularly has been
achieved with vehicles such as dimethylsulfoxide (DMSO) and by
enhancement of skeletal muscle blood and lymph flow.
However, DMSO has proven ineffective as an absorption enhancing
agent for t-PA.
In accordance with the principles of the present invention, the
absorption rate of t-PA in the blood is enhanced by utilizing with
the t-PA dosage, a dosage of an absorption enhancing agent for
t-PA, preferably hydroxylamine hydrochloride. Preferably, the
absorption enhancing agent such as hydroxylamine hydrochloride is
mixed in with the t-PA dosage to form a single mixed dosage which
is then injected intramuscularly (i.m.), e.g. as described in the
parent application. Through the contemplation of the present
invention to inject the absorption enhancing agent as a separate
dosage within the same site as the separate dosage of t-PA, (e.g.
U.S. Pat. No. 4,394,863). An example of an amount of absorption
enhancing agent, such as hydroxylamine hydrochloride, which is
added to the t-PA dosage, as previously described, to form a single
mixed dosage is an amount of from 0.1 to 85 e.g. 0.1 to 40 or 1 to
85 milligrams per kilogram of body weight. As the absorption
enhancing agent hydroxylamine is preferably employed in the form of
a non-toxic water soluble salt. Thus there can be used for example
in place of hydroxylamine salts such as hydroxylamine
hydrochloride, hydroxylamine hyrobromide, hydroxylamine
hydroiodide, hydroxylamine sulfate, hydroxylamine nitrate,
hydroxylamine acetate, and hydroxylamine propionate. Most
preferably there is employed hydroxylamine hydrochloride.
There is also contemplated as absorption enhancing agents for t-PA
in accordance with the invention compounds such as ammonia
(ammonium hydroxide), ammonium carbonate and other ammonium salts,
e.g. ammonium chloride, ammonium acetate, ammonium bromide and
ammonium sulfate, urea, mono and dialkyl ureas, e.g. methyl urea,
ethyl urea, propyl urea, butyl urea, N,N-dimethyl urea, N,N-diethyl
urea, N,N-diisopropyl urea, mono and diaryl ureas, e.g. phenyl
urea, p-tolylurea, N,N-diphenyl urea and, N,N-di-p-tolyl urea,
thiourea, hydantoin, 5-substituted hydantoins, e.g. 5-alkyl,
5-aralkyl, and 5-aryl hydantoins and 5,5-dialkyl and 5,5-diaryl
hydantoins, e.g. 5-methyl hydantoin, 5-ethyl hydantoin,
5,5-dimethyl hydantoin, 1,5-trimethylene hydantoin,
1,5-tetramethylene hydantoin, 5-phenyl hydantoin,
5-p-tolyl-hydantoin, and 5,5-diphenyl hydantoin, guanidine, methyl
guanidine, hydrazine, alkyl and aryl hydrazines, e.g. methyl
hydrazine, ethyl hydrazine, butyl hydrazine, phenyl hydrazine and
diphenyl hydrazine, alkyl and aryl hydroxylamines, e.g. methyl
hydroxylamine, ethyl hydroxylamine and phenyl hydroxylamine. The
substituted ureas, hydrazines and hydroxylamines likewise can be
used in the form of salts, e.g. as hydrochlorides.
Also while the simultaneous administration of t-PA and absorption
enhancing agent is primarily intended for human use, it is within
the scope of the invention that they be administered to other
mammals, e.g. dogs, cats, cattle, and horses.
It is known that hydroxylamine, e.g. as the hydrochloride,
dissociates t-PA from its naturally occurring inhibitor in tissue
culture, Levin. Proc. Natl. Acad. Sci. USA 80, 6804-6808 (1983). It
is also known that hydroxylamine inhibits platelet aggregation, see
Iizuka, Chem. Pharmacol. Bull. 20 614-616 (1972) and elicits smooth
muscle relaxation potentially enhancing vasodilation and hence
absorption at the injection site, see Diamond, J. Pharmacol, Exp.
Therap. 225, 422-426 (1983). These properties may contribute to its
success in the present invention.
While t-PA and the absorption enhancing agent would usually be
administered intramuscularly they can also be administered singly
or in combination intravenously since hydroxylamine has been shown
(see Levin, loc. cit) to disassociate t-PA inhibitor from t-PA,
thereby enhancing the effect of the infused exogenous t-PA or the
hydroxyl amine and thus reducing the amount of t-PA required to
accomplish thrombolysis. As pointed out above the hydroxylamine
will usually be administered in the form of a non-toxic salt,
preferably the hydrochloride. The dosage of absorption enhancing
agent, e.g. hydroxylamine hydrochloride can be in the range
previously mentioned. In place of hydroxylamine in this place of
the invention there can be added other t-PA inhibitor
disassociating agents. In accordance with the teachings of my
copending U.S. application 460,011, filed Jan. 21, 1983 (the
disclosure of which is hereby incorporated by reference into the
present specification), electrical stimulation of the muscle at the
injection site was employed in concert with the inclusion of an
absorption-enhancing agent, specifically hydroxylamine
hydrochloride, in the injectate in a number of the following
examples using intramuscular injection. Electrical stimulation
augments and enhances the absorption of the absorption enhancing
agent of the invention.
Although as pointed out in the parent application an automatic
injector device suitable for intramuscular self-administration of
t-PA can be employed, the examples set forth below were performed
by administering the t-PA and hydroxylamine hydrochloride directly
into the muscle with a conventional needle and syringe.
Administration of the agent with an automatic injector, however, it
is believed will lead to even higher blood levels than those
obtainable by manual injection.
After an approach employing intramuscular injection of t-PA with
hydroxylamine (as the hydrochloride) and electrical stimulation of
skeletal muscle at the injection site in rabbits had been found to
yield peak blood levels of t-PA comparable to or exceeding those
known to elicit coronary thrombolysis after intravenous infusion of
t-PA in dogs and in patients, an analogous approach was evaluated
in dogs subjected to coronary thrombosis. Facilitated absorption of
t-PA after intramuscular injection was found to elicit coronary
thrombolysis as well as therapeutic blood levels of t-PA in these
feasibility experiments.
Large injectate volumes were employed because of the limited
solubility of t-PA in conventional buffers. For consistency the
volumes used in rabbits were selected to be similar to those
planned for use in dogs (1 and 1.5 ml per injection site for
rabbits and dogs respectively) even though they represented large
volumes with respect to rabbit muscle mass. Thus the same
concentration of absorption-enhancing agent per ml of injectate was
used in both species even though they resulted in administration of
markedly greater amounts of hydroxylamine per kg of body weight and
a 10-fold lower concentration of t-PA in the injectates in rabbits
compared with dogs despite administration of comparable proportion
of t-PA administered per kg of body weight in the two species.
Concentrating the t-PA appreciably with solubilizing agents such as
thiocyanate it is believed will permit the volumes to be reduced
substantially.
For studies in rabbits, the t-PA employed was either harvested from
melanoma cell supernatant fractions (mt-Pa) as previously described
(Bergmann, Science 220 1181-1183 (1983) or produced by recombinant
DNA technology, Van der Werf, Circulation 69 605-610 (1984) (rt-PA,
Genentech Corp., lot BH004 DAX). Results with the two preparations
were indistinguishable and therefore the preparations were pooled.
Concentrations of 0.5 mg t-PA per ml buffer (0.3 M NaCl, 0.01%
Tween 80, 0.01M potassium phosphate buffer pH 7.5) were used. For
studies in dogs, rt-PA (Genentech, lot TE031A) was concentrated
20-fold with an Amicon membrane filter system.
DMSO was used in 1% or 3% (v/v) solutions in vitro and in
injectates. Hydroxylamine hydrochloride was used in concentrations
of 43.75 mg per ml of t-PA solution. This concentration was
compatible with a total hydroxylamine hydrochloride dose of
approximately 13 mg/kg shown to be well tolerated
physiologically.
To determine the extent to which the absorption-enhancing agents
evaluated might interact with t-PA, solutions of rt-PA (0.015 to 50
ng/ml) were incubated at 37.degree. C. for 1 hour after addition of
1% DMSO, 3% DMSO, 175 mg/ml hydroxylamine (as the hydrochloride),
or both DMSO and hydroxylamine (as the hydrochloride). No effects
were discernible on t-PA assayed innumoradiometrically or
functionally.
Studies were performed in 56 nonfasted, white male New Zealand
rabbits weighing approximately 2 kg. Endogenous t-PA in these
animals does not react with antibody prepared against human t-PA
and hence does not interfere with the immunoradiometric assay used
to characterize blood levels of exogenously administered t-PA.
Animals were anesthetized with sodium pentobarbital (24 mg/kg) and
ventilated with 95% oxygen administered through a tracheostomy at
2l/min. Skeletal muscle (vastus medialis) at the injection site was
exposed bilaterally and serial blood samples were drawn through an
indwelling femoral venous catheter. To augment skeletal muscle
blood and lymph flow at the injection site, the muscle was
stimulated for 2.0 msec at 14 volts with five pulses per second
with two 27-gauge, 0.5 inch stainless steel needles. A single
negative distal electrode was used as well. A total of 1 mg of
t-PA/kg body weight was injected manually divided in 1 ml aliquots
in each of 4 sites.
Coronary thrombosis was induced in fasted anesthetized dogs
weighing approximately 23 kg, see Bergmann Science 220. 1181-1183
(1983). Occlusive thrombus formed within five to 10 minutes and was
confirmed angiographically. Serial venous blood samples were
obtained through an indwelling inferior vena caval catheter.
Electrical field stimulation at the injection site was implemented
with three 27-gauge stainless steel, one serving as the negative
reference. Parameters were the same as those used in rabbits. t-PA
was injected directly into exposed sartorius muscle in 1.5 ml
aliquots per site such that the total dose was 3 mg/kg body weight
and the total volume of injectate was 6 ml in aggregate for each
dog.
The primary endpoint for experiments in the 56 rabbits studies was
t-PA activity in blood. t-PA antigen levels were assayed serially
as previously described Bergman, loc. cit. and Van der Werf, No.
Engl. 2 Med. 310, 609-613 (1984). Functional t-PA activity was
determined as well Bergman, loc. cit and Tiefenbrunn, Circulation
71, 110-116 (1985). Blood samples were obtained at 0.degree. to
4.degree. C. in sodium citrate vacutainer tubes before
intramuscular injection of t-PA or vehicle alone, immediately after
injection, and at selected intervals from one to 60 minutes
subsequently.
For the feasilibity experiments in dogs, an additional endpoint was
coronary thrombolysis documented angiographically. Blood pressure,
heart rate, the electrocardiogram, arterial blood gases and pH,
hemoglobin and hemoglobin oxygen saturation were monitored.
For experiments in both species, a crude assessment of potential
muscle injury at the site of injections was made by gross
inspection. In addition, serial blood samples were assayed for
plasma creatine kinase (CK) activity spectrophotometrically, Klein,
Cardiovasc. Res. 7, 412-418 (1973) in view of the known prompt and
marked liberation of CK into the circulation when skeletal muscle
is inured.
Serial changes in blood levels of t-PA were evaluated in 56 rabbits
comprising several groups. Blood levels were assessed before and at
selected intervals after intramuscular injection of buffer with or
without absorption-enhancing agent alone; or t-PA in buffer, buffer
with DMSO, buffer with hydroxylamine (as the hydrochloride), or
buffer with DMSO and hydroxylamine (as the hydrochloride).
The same combinations were evaluated with and without concomittant
electrical stimulation of muscle at the injection site throughout
the blood sampling interval. Once it had been determined that
hydroxylamine facilitated absorption of t-PA, experiments were
performed to define the dose-response relations for absorption of
t-PA as a function to the concentrations of t-PA and the
concentration of hydroxylamine in the injectate. Possible systemic
effects of hydroxylamine on absorption of t-PA wer assessed in
rabbits by administering hydroxylamine without t-PA in two
injection sites and t-PA without hydroxylamine in the other two
sites.
The experiments performed in dogs were undertaken after it had been
determined with rabbits therapeutic blood levels could be induced
with amounts of t-PA/kg body weight (1 mg/kg) of the same order of
magnitude as those that had been used previously for intravenous
administration of t-PA in patients (0.5 to 0.75 mg/kg).
Intramuscular t-PA was administered with hydroxylamine (as the
hydrochloride) within five to 45 minutes after angiographic
documentation of formation of an occlusive clot in the left
anterior descending coronary artery, generally occurring within
seven to 10 minutes after introduction of the thrombogenic coil
into the vessel. Serial aniography was performed at approximately
15 minute intervals. Effects of t-PA on coronary thrombi correlated
with plasma t-PA levels. After clot lysis (approximately 15 minutes
after injection of t-PA), heparin (500 U/kg body weight) was given
to prevent reocclusion. In the absence of exogenous activation of
the fibrinolytic system, clots induced by the indwelling
thrombogenic coronary arterial coil invariably persist despite
administration of heparin (n=40 dogs). Statistical comparisons were
performed by analysis of variance with Bonferroni critical limits
or with Students test for paired data. Values are expressed as
means .+-.SE.
Effects of Absorption-Enhancing Media on t-PA Activity in vitro
Neither hydroxylamine (as the hydrochloride) (175 mg/ml), 1% DMSO,
3% DMSO, nor concomitant hydroxylamine (as the hydrochloride) and
DMSO modified immunoradiometrically detectable t-PA or functionally
detectable t-PA activity in samples incubated for 1 hour at
37.degree. C. containing 0.015 to 50 ng rt-PA.
Concentrations of t-PA in Blood
Prior to intramuscular injection of rt-PA, no human t-PA was
detectable by immunoradiometric assay in plasma from any of the
rabbits. No detectable endogenous t-PA activity was evident in
plasma samples assayed with the fibrin plate functional assay
despite the minor surgical procedure performed and the imposed
electrical stimulation of muscle for 60 minutes in any of four
rabbits tested. No human t-PA was detectable after injection of any
of the combinations of vehicles tested when exogenous t-PA was not
included in the injectate. No immunoradiometrically detectable t-PA
was present in plasma samples from sham operated dogs during a 60
minute sampling interval with or without intramuscular injection of
a total of 262 mg/ml of hydroxylamine as the hydrochloride
administered in multiple sites. Fibrin plate assayable functional
activity in sham operated dogs ranged from 10 to 53 IU/ml and did
not increase in any of four animals tested during the 60 minute
sampling interval after electrical stimulation and intramuscular
injection of hydroxylamine hydrochloride in buffer without
t-PA.
In control experiments with hydroxylamine hydrochloride alone (262
mg) injected intramuscularly in dogs, peak methemoglobin levels
ranged from 11 to 13% and occurred within five to 15 minutes after
intramuscular injection (n=3). Arterial oxygen tension decreased to
a minimum of 93 mm Hg. Hemoglobin saturation with oxygen declined
to a minimum of 81%. Except for transitory acceleration of heart
rate, dogs given hydroxylamine hydrochloride with or without t-PA
exhibited no significant hemodynamic or electrocardiographic
abnormalities.
IN THE DRAWINGS
FIG. 1 is a graph of immunoradiometrically detectable and
functionally active plasma t-PA activity in plasma samples from a
rabbit injected with 2 mg t-PA buffer with 43.75 mg/ml
hydroxylamine hydrochloride (total injectate volume=4 ml divided
among 4 sites) followed by electrical stimulation at the injection
sites throughout the sampling interval. Both immuno-reactive and
functionally active t-PA peaked rapidly after intramuscular
injection with facilitated absorption.
FIG. 2 is a graph showing the dependence of the peak concentration
plasma of immunoradiometrically detectable t-PA on the
concentration of hydroxylamine in the injectate. Conditions were
the same as those indicated in the legend to FIG. 1 except that the
amounts of hydroxylamine hydrochloride in the 4 ml aggregate volume
of injectate were varied as indicated in the figure.
FIG. 3 is a chart showing peak plasma t-PA activity as a function
of the amount of t-PA administered intramuscularly in 6 rabbits.
Conditions were the same as those indicated in the legend to FIG. 1
except that the total amount of t-PA administered was varied as
indicated. Panel A depicts immunoradiometrically detectable
activity; panel B depicts amidolytic, functional activity. Dose
related differences throughout the 1 hour interval of measurement
for the entire time-activity aerol (n=30 determinations) were
significant as determined byanalysis of variance (p<0.001).
FIG. 4 is a graph showing early changes in plasma t-PA
concentrations after facilitated absorption of intramuscularly
administered t-PA in each of three rabbits. Conditions were the
same as those indicated in the legend to FIG. 1.
FIG. 5 a graph of serial changes in plasma t-PA assayed
immunoradiometrically in a dog which had been subjected to coronary
thrombosis. Thrombosis was induced with a thrombogenic coil
advanced into the left anterior descending coronary artery at the
tip of a coronary arterial catheter. Coronary thrombolysis was
induced by facilitated absorption of intramuscularly administered
t-PA. (The thrombogenic coil elicited formation of a clot evident
by lack of distal fill with angiographic dye as well as by lack of
opacification of the vessel proximal to the coil that appears as a
bright rectangle). Fifteen minutes after intramuscular
administration of t-PA (3 mg/kg in a total injectate volume of 6 ml
divided among four sites) and electrical stimulation of muscle at
the injection site, lysis of the clot proximal and distal to the
coil was evident with angiographically demonstrable restoration of
patency. As can be seen, plasma t-PA activity peaked soon after
facilitated absorption of intramuscularly administered t-PA.
Elevated levels persisted throughout the sampling interval. A
secondary peak was seen in each of the three dogs studied.
Blood Levels of t-PA After Intramuscular Injection In Rabbits
As shown in Table 1, t-PA injected in buffer alone increased blood
levels only minutely. The addition of DMSO to the injectate did not
increase t-PA levels in plasma. In contrast, hydroxylamine
hydrochloride augmented absorption of t-PA yielding peak blood
levels five minutes after injection approximately 40-fold higher
than those seen in its absence. An example of serial changes of
immunoradiometrically and functional t-PA activity assayed with
fibrin plates after intramuscular absorption of t-PA facilitated by
inclusion of hydroxylamine hydrochloride in the injectate and
electrical stimulation of muscle at the injection site is shown in
FIG. 1.
TABLE 1 ______________________________________
Immunoradiometrically Detectable t-PA In Plasma (ng/ml) After
Intramuscularly Administered t-PA t-PA in t-PA in Buffer + Interval
After t-PA In Buffer + 3% Hydroxylamine Injection Buffer Alone DMSO
Hydrochloride (min) (n = 6) (n = 6) (n = 15)
______________________________________ 0 0 .+-. 0 0 .+-. 0 0 .+-. 0
5 8 .+-. 2 11 .+-. 4 431 .+-. 52* 15 9 .+-. 2 8 .+-. 2 146 .+-. 16*
30 9 .+-. 2 9 .+-. 1 85 .+-. 17* 60 10 .+-. 3 10 .+-. 1 53 .+-. 11*
______________________________________ Values are means .+-. SE.
All injectates contained 2 mg tPA in an aggregate of 4 ml (1 ml per
site). The concentration of hydroxylamine hydrochloride was 43.75
mg/ml. All experiments tabulated were performed with electrical
stimulation of muscle at the infarction site. *P < .01 compared
with tPA in buffer alone or in buffer + DMSO.
To determine whether augmentation of muscle blood flow by
electrical stimulation would enhance absorption of t-PA
administered intramuscularly, experiments were performed with and
without electrical stimulation after injection of t-PA in buffer
alone, t-PA in buffer supplemented with DMSO, and t-PA in buffer
supplemented with hydroxylamine hydrochloride. The very low blood
levels seen when t-PA was administered without hydroxylamine
hydrochloride were not consistently modified by electrical
stimulation (n=11 animals). However, in animals given t-PA with
hydroxylamine hydrochloride (n=15) stimulation augmented peak
levels by an average of 258.+-.32% without altering the time course
of absorption or clearance of t-PA.
As shown in FIG. 2, immunoradiometrically detectable t-PA peak
blood levels were proportional to the amount of hydroxylamine
hydrochloride in the injectate. Addition of 1% or 3% DMSO to
hydroxylamine (as the hydrochloride)-enriched injectates did not
increase peak blood levels of t-PA compared with results with
hydroxylamine hydrochloride alone when the amount of t-PA was held
constant. Both immunoradiometrically detectable and functionally
active t-PA after administration of exogenous t-PA were
proportional to the concentration of t-PA over a four-fold range
when the amount and concentration of hydroxylamine hydrochloride in
the injectate were held constant (FIG. 3). As can be seen in FIG.
4, blood levels rose rapidly and peaked between 4 and 5 minutes
after injection. Appreciable concentrations of t-PA in plasma were
evident as early as one minute after intramuscular injection in
each case.
The augmentation of peak plasma t-PA after facilitated absorption
with hydroxylamine hydrochloride was not caused simply by the
decreased pH of the injectate. In each of two animals, the pH of
the injectate was titrated to 5.9 without hydroxylamine. Plasma
t-PA concentration five minutes after injection was only 6 ng/ml.
No significant increase occurred subsequently. The increment seen
with hydroxylamine hydrochloride was not attributable simply to
systemic effects of hydroxylamine hydrochloride. In two animals in
which hydroxylamine was injected into the right and t-PA in buffer
into the left thigh muscle, peak blood levels did not exceed those
in Table 1 for t-PA injected in buffer alone.
Although the amounts of absorptionenhancing agent per kg body
weight used in rabbits were considerably greater than those used in
dogs or anticipated ultimately for possible clinical studies, the
excessively large quantities were employed to determine whether
high concentrations in the injectate would be deleterious to
skeletal muscle. In rabbits, plasma CK was not significantly
different 30 minutes after the surgical procedure, injection of
t-PA with hydroxylamine hydrochloride and electrical stimulation
compared with values after injection of buffer alone under the same
conditions (690.+-.82 compared with 696.+-.63 IU/l). In dogs given
175 mg hydroxylamine hydrochloride with or without t-PA, plasma CK
increased by less than 18% of baseline at the completion of the
study. No hematoma were evident by gross inspection. Light
microscopy of sections from the injection site obtained two hours
after injection delineated only scanty interstitial hemorrhage and
inflammation.
Effects of Facilitated Absorption of Intramuscularly Administered
t-PA on Coronary Thrombolysis in Dogs.
After demonstrating that facilitated absorption of t-PA could be
achieved in rabbits with hydroxylamine hydrochloride in the
injectate, pilot studies were performed in dogs to determine
whether the approach developed could elicit coronary thrombolysis.
Arterial blood pressure after injection of hydroxylamine
hydrochloride intramuscularly with (n=3) or without (n=3) t-PA
declined only modestly (from an average of 166/121 mm Hg to
144/104) reaching a minimum 2 minutes after injection. Heart rate
increased transiently by an average of 32% peaking also 2 minutes
after injection. Ventricular arrhythmias did not occur with
hydroxylamine hydrochloride alone. Intramuscularly administered
t-PA (3 mg/kg) followed by electrical stimulation initiated
coronary thrombolysis within 15 minutes heralded by reperfusion
arrhythmias. Similar results were obtained in each of the three
animals studied. Plasma t-PA values followed a similar time course
but were lower than those seen in rabbits. The differences may
reflect species differences in the absorption or clearance of human
rt-OA or the larger ratio of injectate volume to muscle mass in
rabbits. In addition, as shown in FIG. 5, a secondary peak of
immunoradiometrically detectable t-PA occurred beginning
approximately 40 minutes after the first peak in each dog
compatible with lat release from the skeletal muscle depot because
of changes in blood flow or slow lymphatic transport of t-PA into
the circulation among other possibilities.
Thus it has been found that therapeutic blood levels of
functionally active t-PA can be achieved and that coronary
thrombolysis can be eliicted by facilitated absorption of
intramuscularly injected material. Plasma activity peaked within
five minutes after injection and subsequently declined rapidly,
consistent with the known half-life of t-PA in the circulation. The
blood levels obtained were sufficient to induce coronary
thrombolysis in dogs within 15 minutes despite the continued
presence of an indwelling, coronary, thrombogenic coil. Absorption
of t-PA was enhanced by inclusion of hydroxylamine in the injectate
and by augmentation of skeletal muscle blood flow by electrical
stimulation. Gross injury to skeletal muscle did not occur.
Because low levels of t-PA in plasma may be adequate to induce clot
lysis of nascent thrombi judging from results of studies in vitro
and because the biological half-life of t-PA bound to fibrin is
substantially longer than the half-life of circulating t-PA, see
Brommer, Thromb. Res. 34, 109-115 (1984), Tran-Thang, Blood 63
1331-1337 (1984), Bergmann, Circulation 70 II:108 (Abstract)
(1984), it is believed that coronary thrombolysis early after the
onset of thrombosis in vivo may be obtained with lower quantities
of t-PA, hydroxylamine hydrochloride, or both than those used in
the examples set forth above. Reduction of the injectate volume
would diminish the dose of hydroxylamine or other absorption
enhancing agent required and minimize potential injury to muscle at
the injection site.
To date, t-PA and other activators of the fibrinolytic system have
been given only by direct injection into the blood stream. This
invention provides an alternative means of administration of t-PA
potentially amendable to prompt implementation by paramedical
personnel or by telephonically supervised patients at high risk
previously instructed in self-medication procedures.
Hydroxylamine was employed after numerous attempts with other
absorption-enhancing media for other compounds failed to yield the
desired results with t-PA. Its major side effect, induction of
methemoglobinemia does not prohibitively limit tissue oxygenation
with the doses used. If the concentration of the hydroxylamine in
the injectate is the critical determinant of absorption of t-PA as
appears likely judging from the present results, the total dose of
hydroxylamine required in human subjects is likely to be so low
that induced methemoglobinemia would be of only trivial extent even
for patients with ischemic heart disease especially if the
injectate volume can be reduced further by increasing the
concentration of t-PA. In those cases where the methemoglobinemia
accompanying use of this absorption-enhancer is deemed to be
unacceptably severe, adjuvant measures such as concomitant
administration of methylene blue or glutathione might be utilized
to minimize or obviate the problem, see Layne, J. Pharmacol. Exp.
Therap. 165, 36-44 (1969).
Blood levels of t-PA comparable to those obtained in the present
investigation induce coronary thrombolysis in experimental animals
and patients without inducing a systemic lytic state predisposing
to bleeding. The time course of elevation of plasma t-PA after
facilitated intramuscular absorption is particularly favorable
because of its sharp peak. With the envisioned application of an
appropriate regimen, subjects would be under direct medical care
soon after self-medication with an automatic injector or treatment
by relatives of paramedical personnel. Thus, as the blood levels
declined promptly after intramuscularly administered t-PA had been
given, intravenous infusions could be initiated along with
anticoagulants or other measures taken to prevent reocclusion while
definitive diagnostic information wa being obtained.
The possibility that myocardial reperfusion induced by facilitated
absorption of intramuscularly administered t-PA might give rise to
reperfusion arrhythmias is easily managed in the setting of the
cardiac catheterization laboratory or coronary care unit but can be
potentially dangerous in the medically unattended patient. Thus,
there is advantage in the concomitant administration of an
antifibrillatory or anti-arrhythmic agent such as lidocaine or an
alpha-adrenergic blocking agent as set forth in the parent
application.
It has also been found that to prevent reocclusions or platelet
aggregation it is desirable to either:
1. inhibit synthesis of thromboxane A *thromboxane A.sub.2) with a
thromboxane synthetase inhibitor, e.g. an imidazole such as
4-(2-[1H-imidazol-1-yl]ethoxy)-benzoic acid hydrochloride
(dazoxiben)
2. introduce an antagonist for the receptor of the thromboxane A
(thromboxane A.sub.2) such as [1.alpha.,2.beta.(5Z), 3.beta.(1E),
4.alpha.]-7-[3-(3-cyclohexyl-3-hydroxy-1-propenyl)-7-oxabicyclo[2.2.1]hept
-2-yl]-5-heptenoic acid) (SQ 27,427).
3. introduce another inhibitor of platelet aggregation, e.g.
aspirin, indomethacin, naproxin, and sulfinpyrazone.
The agent for the prevention of reocclusions or platelet
aggregations could be administered simultaneously or sequentially
in either order with reference to the t-PA and absorption enhancing
agent, e.g. hydroxylamine hydrochloride. The agent for the
prevention of reocclusions or platelet aggregations can be
administered in conventional manner, e.g. intramuscularly,
intravenously, or even orally.
The receptor antagonist or other agent for prevention of platelet
reocclusions can be administered for example in an amount of 0.1-10
mg/kg body weight.
* * * * *