Note: Descriptions are shown in the official language in which they were submitted.
1269931
PROTEIN ABSORPTION ENHANCING AGENTS
This application relates to application WO 86/01118
published 27 February 1986, entitled, "Initiating
Reperfusion Treatment When Heart Attack Symptons
5 are Present". This application also relates to
application WO 86/01114 published 27 February 1986,
entitled, "t-PA Composition and Method of Getting
Into Blood Stream". This application additionally
relates to application WO 86/01109 published
27 February 1986, entitled, "Protein Absorption
Enhancing Agents".
According to the present invention a
package and method have been developed for
enhancement of absorption of proteins with
medicinal properties administered by intramuscular
injection to rapidly achieve high concentrations
of the injected protein in the circulating plasma
permitting rapid implementation of therapeutic
effects without the need for intravenous injection
or infusion. In an exemplary embodiment there is
provided a vehicle in which the protein is
administered in a vehicle comprising an
absorption-enhancement compound, e.g.
hydroxylamine or a salt thereof, such as the
hydrochloride, that markedly increases permeation
of proteins tested into the vascular space leading
to high blood levels within two minutes after
injection in tests as set forth below.
~rA
1269931
The invention is based in part upon the
invention set forth in USP 4,661,46~
which relates to the treatment of coronary prone
individuals in the throes of a suspected
myocardial infarction in sueh a way as to minimize
dama~e 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
.~
^` 126993~
with essential oxygen and other nutrients. A
consequence of a thrombus or clot forming in a
coronary artery is the dan~er to the myocardium
(heart 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
"prehospital" 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' 5 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
1~69931
,
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 En~land Journal
of Medicine, March 8, 1984, Volume 310, No. 10,
pages 609-613). Unlike other plasminogen
activators, such as streptokinase 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
Pharmacoloqv, 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 ~ 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 thece agents
is limited by a lower success
rate, in part because the upper
1269931
-- 5 --
bound of dose is constrained by
the risk of inducing a severe
systemic lytic state.
The probability that proqress
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-like 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 November 16, 1983,
in an article entitiled, "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 art of the patient seized by a myocardial
infarction or heart attack, and it travels through
lZ69931
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. Purton 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 attac~."
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
druq by intravenous infus on 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.
~Z69931
.
-- 7 --
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 myocaridal infarction. It is an object
of the present invention to provide such a self
administering treatment.
It has now been found that the invention
has wider application.
A number of proteins with life-saving,
therapeutic properties presently must be
administered exclusively intravascularly to achieve
therapeutic blood levels. Examples include
activators of the clot-dissolving, fibrinolytic
system including streptokinase and urokinase used
for interrupting heart attacks in progress, and
components of the coagulation system such as anti-
hemophiliac globulin (factor VIII) to arrestbleeding in hemophiliacs. In many circumstances
other proteins such as insulin used to treat
diabetic coma must be administered intravenously to
yield concentrations high enough to manifest the
desired therapeutic effect of promptly enough to do
so within the time-constraints of the medical
emergency. Recently, as pointed out above,
dissolution of clots in the coronary arteries giving
~6~9~33l
-- 8 --
rise to heart attacks has been achieved in
experimental animals and patients by intravenous
administration of tissue-type plasminogen activator
(t-PA), a protein capable of activating the
fibronolytic system at the clot surface without
predisposing the patient to bleeding. Because the
half-life in the circulation of t-PA is so short,
continuous intravenous infusion has been required to
avoid rapid disappearance of the protein from the
circulation with consequently sub-therapeutic
concentrations. In addition, intravenous
administration has been deemed necessary because of
the urgent need for rapid clot lysis to interrupt
- the heart attack in progress before death of a
substantial amount of heart muscle.
In view of the foregoing a method has been
developed with the primary objective of enhancing
absorption in the blood of protein, e.g. t-PA, and
other large proteins injected intramuscularly. The
method utili~es solubilization of the protein in a
vehicle enriched with an agent enhancing absorption
of the protein in the blood that elicits prompt
permeation of the protein into the circulating blood
pool. An additional objective of the method
developed is retention of the pharmacologic and
therapeutic properties of the protein so that the
desired therapeutic effect can be elicited by
intramuscular injection with enhanced absorption. A
further objective is facilitation of absorption over
30 to 60 minutes so that therapeutic blood levels
can be sustained under emergency conditions.
Permeation of the proteins tested into the vascular
spaces led to high blood levels within 2 minutes of
injection. The vehicle developed facilitates
~L269931
absorption of all four proteins in the tests
mentioned below and exerts no deleterious effects on
the functional properties of the two proteins tested
with clot lysing properties. ~he invention is not
limited to use with the proteins employed in the
specific examples but is believed applicable to
other proteins as well, such as those mentioned
above, e.g. insulin.
The invention includes packaging t-PA (and
other proteins with medicinal properties) and an
agent enhancing the absorption of t-PA in the
blood. The agent preferably is hydroxylamine
hydrochloride. There can be used for example a
known emergency type automatic injector and the
lS process comprises injecting the two medicament
agents into the muscle tissue, e.g. af~er 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-
t-PA is a lar~e protein. It would not be
expected that it would be absorbed into the blood
stream in discernible quantities. Extra-vascular
levels of protein are about 1/10 that of intra-
vascular protein. It is thought that this is sobecause 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
~ ~Z69~31
-- 10 --
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 protein, e.g. t-PA.
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 and other
protein molecules.
In accordance with the principles of the
present invention, the absorption rate of t-PA and
other proteins in the blood is enhanced by utilizing
with the t-PA or other protein dosage, a dosage of
an absorption enhancing agent for t-PA or other
protein, e.g. hydroxylamine hydrochloride.
Preferably, the absorption enhancing agent such as
hydroxylamine hydrochloride is mixed in with the t-
PA or other protein dosage to form a single mixed
dosage which is then injected intramuscularly
~i.m.) It is within the conter~lplation cf the
present invention to inject the absorPtion enhancinq
~269931
.
agent as a ~eparate dosage within the same site as
the separate dosage of t-PA or other protein, (e.g.
U.S. patent 4,394,863). An example of an amount of
absorption enhancing agent, such as hydroxylamine
hydrochloride, which is added to the t-PA or other
protein 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 milli~rams 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
hydrobromide, 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 or other proteins 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 dialkyl ureas, e.g. phenyl urea, p-
tolylurea, N,N-diphenyl and urea, 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
Z69931
- 12 -
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.
Likewise there can used as absorption
enhancing agents other amines, e.g. alkyl amines and
dialkyl amines such as lower alkyl amines and
dialkylamines, e.g. methylamine, dimethylamine,
ethylamine, diethylamine, isopropylamine,
sec.butylamine, diisopropylamine, propylamine, n-
butylamine, aralkylamines, e.g. phenylethylamine,
hydroxyaralkylamines, e.g. epinephrine and tyramine,
hydroxyalkylamines, e.g. ethanolamine,
diethanolamine, triethanolamine, propanolamine, and
other amines such as methoxyamine. polyalkylene
amines, e.g. ethylene diamine, diethylene
triamine. These amines also can be used in the form
of salts of non-toxic acids such as salts of the
acids mentioned earlier, e.g. as the hydro-
chlorides. Also there can be used glucoseoxime.
Methylamine and dimethylamine also have the
advantage that they do not induce
methemoglobinemia. This was shown by injecting
rabbits with (a) 0.63 molar methylamine
hydrochloride and ~b) 0.63 molar methylamine
~Z6993
-- 13 --
hydrochloride together with t-PA in an amount of l
mg/kg body weiqht. In each case l ml of injectate
was employed and the solutions had a pH of 6.5.
When using methylamine (as the hydrochloride) t-PA
levels were in the range of 220 to 230 ng/ml. With
dimethylami.,e the levels of t-PA were somewhat lower
but still elevated compared to the case when t-PA
was administered without an absorption enhancing
agent. Methoxyamine (as the hydrochloride) under
the same conditions also enhanced the level of t-PA
but produced methemoglobenemia. The time course of
elevation of t-PA over 30 minutes with methylamine
hydrochloride was virtually the same as with
hyroxylamine hydrochloride. The levels were more
than lO times the plasma t-PA levels in the absence
of the absorption enhancing agent.
Also while the simultaneous administration
of t-PA or other proteins and absorption enhancing
agents 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. In accordance with the earlier
teachings electrical stimulation of the muscle
at the injection site can be 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 auqments and enhances the absorption of
the absorption enhancing agent of the invention.
1269931
- 14 -
~ lthough as pointed out in the earliest
parent application an au~omatic injector device
suitable for intr~muscular self-administration of t-
PA or other protein can be employed, the examples
set forth below were performed by administering the
t-PA or o~her protein 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
~ 2~i993~
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. Concentratinq 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 llBl-1183 (19833 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~,
0.01 M 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/kq chown to be well
tolerated physiologically.
~z6ssal~,
- 16 -
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 37C for 1 hour after addition of
lS DMSO, 34 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 2 l/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 2~ kg, see
Bergmann Science 220. 1181-1183 (1983). Occlusive
thrombus formed within five to 10 minutes and was
` ~269931
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 prima{y 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 to 4C 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 feasability experiments in dogs, an
additional ~ndpoint was coronary thrombolysis
documented angiographically. 3100d 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
-- ~269931
- 18 -
addition, serial blood samples were assayed for
plasma creatine kinase (CK) activity
spectrophotometrically, Klein, Cardiovasc. Res. 7,
412-418 (1973) in view of the ~nown 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 concimitant 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 concentration 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
i9931
,
-- 19 --
order of magnitude as those that had been used
previously for intravenous administration of t-PA in
patients (0.5 to .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 ~ctivation
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 t 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 37C containing 0.015 to 50
ng rt-PA.
-" 1.269931
-- 20 --
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 endoqenous 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
injeotion of any of the combination~ 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 Hq. Hemoglobin saturation with oxygen declined
to a minimum of 81~. Except for transitory
acceleration of heart rate, dogs given hydroxylamine
l~Z6~31
- 21 -
hydrochloride with or without t-PA exhibited no
significant hemodynamic or electrocardiorgraphic
abnormaliites.
In the Drawings:
Figure l is a graph of immunoradio-
metrically 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. 30th immuno-reactive and
functionally active t-PA peaked rapidly after
intramuscular injection with facilitated absorption.
Figure 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 figure 1 except that the amounts of
hydroxylamine hydrochloride in the 4 ml aggregate
volume of injectate were varied as indicated in the
figure.
Figure 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 figure 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 l hour
interval of measurement for the entire time -
activity aerol (n = 30 determinations) were
~Z699~
- 22 -
significant as determined byanalysis of variance (p
<.001) .
Figure 4 is a graph showing early changes
in plasma t-PA concentrations after facilitated
S absorption of intramuscularly administered t-PA in
each of three rabbits. Conditions were the same as
tho~e indicated in the legend to figure 1.
Figure 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 o 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.
Figure 6 is a graph showing the effect of
administering hydroxylamine hydrochloride together
with bovine serum albumin. Administering the bovine
~26993
-- 23 --
serumalbumin in the absence of hydroxylamine
hydrochloride. Thebovie serum albumin was
irradiated with iodine 12S. In a first experiment
it was injected intramuscularly into rabbits.
Samples of plasma were drawnout of the rabiits from
time to time and the counts per minute (cpm) of
radioactive iodine determined. On the graph this is
identified as the curve -HA. In a second experiment
the same amuont of the bovine serum albumin
irradiated with iodine was injected intramuscularly
into rabbits while simultaneously intramuscularly
injecting hydroxylamine hydrocyloride. Samples of
plasma were drawn out from time to time ad the
counts per minute of radioactive iodine
determined. On the graph this is identified as the
curve +HA. It can be seen from the sraph that in
the presence of hydroxylamine hydrochloride the
bovine serum albumin was absorbed more rapidly and
had a sustained peak of absorption after 30 minutes
of over 5 times that of the bovine serum albumin
alone.
Blood Levels of t-PA After Intramuscular
Injection In Rabbits
Judging from results in experimental
animals and patients given t-PA by continuous
intravenous infusion, therapetuic effects are not
elicited unless the concentration in blood exceeds
50 mg/ml of plasma.
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,
1269~31
- 24 -
hydroxylamine hydrochloride augmented absorption of
t-PA yielding peak blood levels five minutes after
injection approximately 40-fold higher or even more
than 50 fold higher than those seen in its
absence. There was noted absorption of
approximately 10~ of aministered protein within 30
minutes after intramuscular injection with enhanced
absorption. Levels within the therapeutic ranqe
persistend throughout the 1 hour observation range
(Table 1). 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 Figure 1.
Table 1
Immunoradiometrically Detectable t-PA In Plasma (ny/ml)
After Intramuscularly Administered t-PA
t-PA in
t-Pa inBuffer +
2G Interval After t-PA In ~uffer + 3~ Hydroxylamine
Injection Buffer Alone DMSO Hydrochloride
(min) (n = 6~ _ ~n = 5)__ _ (n = 15)
O O + O O + O O + O
5 8 + 2 11 + 4 431 + 52*
15 9 + 2 8 + 2 146 + 16*
30 9 + 2 9 + 1 85 + 17*
6010 + 3 10 + 1 53 + 11*
Values are means + SE. A11 injectates contained 2 mg t-PA in
an aggregate of 4 ml (1 ml per site). The concentration of
hydroxylamine hydrochloride was 43.75 mg/ml. All experiments
~X6993~
tabulated were performed with electrical stimulation of muscle
at the infarction site.
* = P < .01 compared with t-PA 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 Figure 2, immunoradiometrically
detectable t-PA peak blood levels were proportional
to the amount of hydroxylamine hydrochloride in the
injectate. Addition of lS 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
~Z~9931
- 26 -
(Figure 3~. As can be seen in Figure 4, blood
levels rose rapidly and peaked between 4 and S
minutes after injection. Appreciable concentrations
of t-PA in plasma were evident as early ac 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 in jectate. 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 absorption-
enhancing 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 va~ues
after injection of buffer alone under the same
conditions (690 + 82 compared with 696 + 63 IU/l).
l~Z69931
-- 27 --
In dogs given 175 mg hydro~ylamine 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
lo Intramuscularly Administered t-PA on
Coronarv 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 whe~her
the approach developed could elicit coronary
thrombolysis. Arterial blood pressure after
injection of hydroxylamine hydrochloride
intramuscularly witn (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 i the
lZ69931
- 28 -
absorption or clearance of human rt-OA or the larger
ratio of injectate volume to muscle mass in
rabbits. In addi~ion, as shown in Figure S, 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 lS 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.
3ecause 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 beeause
the biological half-life of t-PA bound to ~ibrin 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),
~Z6993
-- 29 --
it is believed that coronary thrombolysis early
after the onset of thrombosis in vivo may be
obtained with lower quantites 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 oxyqenation with the doses used. If the
ccncentration 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
"` ~2~9~3~
- 30 -
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).
Methylamine is a non-toxic agent. It was
employed as the hydrochloride to enhance absorption
of t-PA in rabbits. Absorption was measured afte~
intramuscular injections of 2 to 3 mg of
concentrated t-PA (50 mg/ml). Methylamine
hydrochlo~ide (0.63 molar) pl~s electrial field
stimulation elected blood levels of rt-PA within S
minutes after intramuscular injection of rt-PA, with
functional and immunologic activity similar to that
achieved with hydroxylamine (as the hydrochloride),
(specifically 129 vs. 137 mg/ml/mg rt-PA). With the
use of methylamine hydrochloride there was neither
methemoglobinemia nor hemodynamic derangements
occurred, oxygen saturation remained unchanged and
only modest local inflammation and interstitial
hemorrhage were evident microscopically in the
injection site after 48 hours. Vasodilators,
hypertonic media, reduced amounts of hydroxylamine
hydrochloride or rt-PA alone led to much lower blood
levels of rt-PA (14, 65, 46 or 4 ng/me/mg rt-PA
respectively).
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
126993~.
-- 31 --
intramuscular absorption is particuarly 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 treatmentby 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
was being obtained.
The possibility that myocardial reperfusion
induced by facilitated absorption of intramuscularly
administered t-PA might give rise to reperfusion
arrhythmias i~ 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 A2) with a thromboxane synthetase
inhibitor, e.g. an imidazole such as 4-(2-[lH-
imidazol-l-yl]ethoxy)-benzoic acid hydrochloride
(dazoxiben)
2. introduce an antaqonist for the
receptor of the thromboxane A (thromboxane A2) such
~Z69~3~
- 32 -
as [1~, 2~ 15Z), 3B (lE~, 4~]-7-[3-(3-cyclohexyl-3-
hydroxy-l-propenyl)-7~oxabicyclo[2.2.1]hept-2-yl]-5-
heptenoic acid)(SQ 27,427)
3. introduce another inhibitor of
platelet aggresation, 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.
As stated previously the invention is also
useful to enhance the absorption of other proteins
with medicinal properties by administering
intramuscularly. Such proteins include for example
bovine serum albumin, human serum albumin and
insulin. This is shown by the following
experiments:
Two proteins, bovine serum albumin (BSA)
and human serum albumin (HSA), were labeled with
iodine-125 (125I) so that absorption into the
circulation could be characterized by assay of
radioactivity in trichloroacetic acid precipitates
~269931
,,
- 33 -
of plasma.
BSA and HSA proteins were labeled
radioactively with iodine-125 (125I) by the Bolton
and Hunter method to form a radioligand on tyrosine
constituents. In order to verify the independence
of the results on the nature of the radioligand
used, a second procedu~e was employed in which a
125I-glycoconjugate was prepared to form a
radiolabeled protein in which radiolabeled tyramine
is bound to carbohydrate which is bound covalently
to the protein. This type of radioligand is stable
despite exposure to lysosomal enzymes or acidic
conditions. ~urthermore, it does not alter
functional properties of the labeled protein. In
the labeling procedure, dilactitol tyramine (10 nmol
in 24 ul, in 0.5 M potassium phosphate, pH 7.7) was
initially radioiodinated by placing the label in an
iodogen (Pierce Chemical Company, Rockford,
Illinois~ coated tube with Nal25I ~1 mCi in 10 ~1,
Amersham). The reaction mixture was allowed to
stand for 1 hour at room temperature with occasional
vortexing. Contents of the tube were transferred to
another tube containing galactose oxidase (4 units
in 2 ~1, Sigma) and incubated for 45 minutes at
37C. Sodium cyanoborohydride (3 ~1 of a 1 M
solution in potassium phosphate buffer~ and 2 mg of
bovine or human serum albumin (frac~ion V, Sigma) in
200 ~1 of physiological saline were added to the
tube, followed by additional incubation for 45
minutes at 37C. After incubation, 50 ~1 of 1 M
ammonium bicarbonate was added to the tube. 125I-
dilactitol tyramine-albumin was separated from 125I-
dilactitol tyramine by Sephacryl~S300 chromatography
on a 0.9 x 30.0 cm column and dialysis against
~Z69931
,
- 34 -
physioloqical saline. Coupled protein was
concentrated with Aquacide and sterilized by
filtration through a 0.45 um membrane filter. An
aliquot was removed for determination of protein
content, specific radioactivity, and precipitation
by trichloroacetic acid.
Quantification of Absorption of Proteins
after Intramuscular Injection with or without the
Absroption Enhancinq Agent: Radiolabeled BSA and
HSA in plasma were quantified by assay of
radioactivity in a well counter of aliquots of
plasma obtained at selected intervals after
injection and in precipitates formed by
acidification of the plasma with four parts 10%
trichloroacetic acid (TCA). Enhancement of
absorption of BSA and HSA proteins after
intramuscular injection was demonstrated in New
Zealand rabbits. Protein solutions were injected
manually through a 25 guage needle into skeletal
muscle in the thigh, exposed while the animals were
lightly anethetized.
Enhancement of Absorption: Radiolabeled
albumin was administered to rabbits in 1 ml aliquots
with 163.4 to 289.9 million counts per minute
associated with microgram quantities of protein
(specific radioactivity = 1.74 to 3.02 mCi/mg).
Proteins were administered in either 0.9% NaCl
solutions or an absorption enhancing solution of
o.9% NaCl supplemented with hydroxylamine as the
hydrochloride in concentrations of 43.75 to 175
mg/ml.
Enhancement of absorption of protein was
` 126993
-- 3s --
demonstrable with radiolabeled bovine serum albumin
as well (24 determinations). Peak radioactivity in
TCA precipitates from plasma averaged only 3204
counts per minutes per ml when the protein was
injected intramuscularly in rabbits in saline
alone. In contrast, ~CA precipitable radioactivity
averaged 16.580 counts per minute per ml of plasma
with 80% of peak values attained 5 minutes after
intramusuclar injection with absorption enhanced by
hydroxylamine hydrochloride. Similarly, after
injection oE radiolabeled human serum albumin, TCA
precipitable radioactivity averaged only 4176 counts
per ml of plasma during the first 30 minutes after
intramusuclar injection without enhancement of
absorption. In contrast, TCA precipitable
radioactivity avera~ed 32,868 counts per minute per
ml of plasma during the first 30 minutes after
intramuscular injection with absorption enhanced by
inclusion of 43.8 mg of hydroxylamine as the
hydrochloride per ml of injectate (12
determinations). Furthermore, 75% of peak
radioactivity was evident 5 minutes after
injection. The subsequent increment of plasma
radioactivity after the first 5 minutes was
attributable to continued absorption of radiolabeled
albumin at a rate more rapid than its slow rate of
clearance from the circulation.
SafetY of the AbsorPtion Enhancinq Agent:
Administration of hydroxylamine hydrochloride
intramuscularly did not elicit gross or microscopic
evidence of irreversible muscle injury at the
injection site or elevation of plasma creatine
kinase, a marker of muscle injury beyond the
elevation seen when saline alone was used as the
1269931
-- 36 --
injectate in rabbits. In dogs injected intra-
muscularly with 175 mg hydroxylamine hydrochloride
in saline with or without protein, plasma creatine
kinase increased by less than 18~ above baseline and
no hematoma were evident at the injection site by
gross inspection. Light microscopy of sections
obtained from the injection site 2 hours after
injection revealed only modest interstitial
inflammation. Administration of the absorption
enhancing agent to dogs elicited only a modest
decrease in systemic arterial blood pressure (from
166/121 mm Hq to 144/104) with maximal effects
sbserved 2 minutes after injection and~return to
baseline within the next several minutes. Heart
rate increased transiently by an average of 32% with
the maximal effect 2 minutes after injection and a
rapid return to baseline. Hydroxylamine is known to
induce methemoglobinemia. However, in the amounts
utilized for enhancement of absorption of protein it
did not compromise oxygen carriage in the blood.
Methemoglobin peaked at 8~ 2 minutes after injection
in dogs, and arterial oxygen tension did not fall by
more than 7%.
The invention is particularly suitable for
administering proteins with short biological half-
lives. Thus maintenance for 30 to 60 minutes of
therapeutically effective concentrations of proteins
with short biological half-lives administered by
intramuscular injection with enhanced absorption by
utilizing hydroxylamine hydrochloride was shown by
induction of coronary thrombolysis with enhanced
absorption of intramuscularly administered t-PA
which has a half-life in the circulation of 5 to 8
minutes.
