Note: Descriptions are shown in the official language in which they were submitted.
- 1 - Case 118-6968
1323300
IMPR3VEMENTS IN OR RELATING TO oRGANIC COMPCUNDS
The invention relates to the therapeutic lysis of fibrin blood
clots in human patients.
Several naturally-occurring enzymes are known to participate in
the lysis of fibrin clots, and have been used therapeutically to lyse
clots in patients, e.g. coronary patients, in wh~m life-threatening
clots have formed. TwO of these enzymes are pro-urokinase (pro-UX)
and urokinase (UK). UK is believed to be synthesized in vivo in a
single-chain, zymogenic form (pro-UK) which is converted to the
two-chain form (UK) by a proteolytic cleavage. An alternative name
for pro-UK is single chain urinary plasminogPn activator (scu-PA).
Both agents, UK and pro-UR, oan be used for thrcmbolysis in
early myocardial infarction in order to limit or even prevent
definite myocardial necrosis. UK is used in dosages of more than
1 000 000 I.U. for this purpose, preferred intravenous dosages being
2 000 000 to 3 000 000 I.U. per application. The use of pro-UK for
this indication is currently under investigation, and results so far
show that do6ages of above 6 500 000 I.U. are necessary in order to
obtain good results.
me activities of pro-UK and UK were originally expected to be
at best strictly additive. However, it has ncw been faund that UK
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- 2 - Case 118-6968
surprisingly has a synergistic effect on the therapeutic activity of
pro-UR. In particular, when an initial bolus of UK is administered it
is fcund that subsequent administration of pro-UK is much more
effective, resulting in a dramatic reduction of the overall dosage
5 required in order to achieve the beneficial therapeutic effect.
It has also been faund that this synergistic effect is not
confined to the case where initial administration of UK is followed
by subsequent administration of pro-UK, but is also found when pro-UX
is administered first, follawed by UK; or when pro-UK and ~K are
10 administered simultaneously, whether by separate infusions or
injections, by a single injection frcm a double-barelled syringe, or
by a single injection or infusion of a mixture of pro-UX and UK.
Accordingly, the invention now provides a method of lysing
fibrin blood clots in patients in need of such treatment camprising
15 administration of effective amounts of UX and pro-UK, either
simultaneously or sequentially within a time interval close enough to
provide a synergistic effect.
Alternatively, the invention provides the use of UK together
with pra~UX, in free or fixed combination, as a thrambolytic agent.
20 As a further alternative, the invention provides the use of UK for
the preparation of an agent for the potentiation of the thrombolytic
effect of pro-UK.
By ~synergistic effect~ is meant a fibrin clot lysing activity
greater (either in terms of increased rapidity of lysing or a greater
25 amount of lysing, or both) than would be expected fram the additive Y
effect of the two active agents when administered independently.
Urokinase may be used in either the low molecular weight form
(LMWLUK, MW~ 33,000 daltons) or the high molecular weight form
(HMh-UK, MW ~v 55,000 daltans), of which the HMW form is preferred.
3~ m e UK may be obtained fram natural sauroes (e.g. urine) or may be
abtained by recambinant DNA techniques involving cleavage of the
initially formed single-chain form (pro-UK), as reported by W.E.
1323~0(j
- 3 - Case 118-6968
Holmes et al, Bio/Technology 3 923 (1985). HMW~UK is ccmmercially
available as ukidan (R.T.M.) or Actosolv (R.T.M.), LMW-UK as
Abbokinase (R.T.M.).
Similarly pro-UX may be obtained frcm urine as originally
5 described in European Patent 40238, or frcm cultures of natural cell
lines or cell lines transformed by recombinant DNA technology, and
may be obtained in forms differing in degree of glycosylation or in
other respects.
The pro-UK used according to this invention will normally be
human pro-UK having the amino-acid sequence as shown for pro-UK in
Figure A of European Patent Application 92182, ignoring the lead
sequence feom -20 to -1. However, the pro-UK used in the invention
may vary fron this structure by substitution, deletion or addition at
one or more amino acid residues, so long as it retains essentially
the same biological activity. mus for example a non-human pro-UK or
a compaund such as described in PCT Fatent application W~ 86/04351,
having a different amino acid substituted for lys-135 and/or phe-157
or as described in Euro~ean Patent Application 200 451, having
substitutions or deletions at phe-157 or lys-158 is within the
definition of ~pro-UK~ so long as it retains biological activity.
Furthermore, truncated forms of pro-UK for example such as described
by Rijken et al in Thrcmbosis Res2arch 42 749-760 and 761-768, are
included in the definition of ~pro-U~ so long as biological activity
is retained. Such molecules may have their amino-terminal at lys-136,
corresponding to single-chain LMWLUK, or at other convenient sites
for example ala-132, lys-144 and glu-150. Forms of pro-UK having
additional amino acids for example an initial methionine are also
included if active.
Similarly, the UK used in the invention may differ in
corresponding manner from either of the two normal human UK molecules
(~MW-UK and LMW-UK) so long as its biological activity is retained.
Patients receiving particular benefit frcn the invention
include post-Gperative patients, patients who have recently suffered
13233Q~
- 4 - ;e 118-6968
myocardial infarction resulting in clots and patients suffering fram
deep vein thrcmbi. Pro-UK and UK, separately or tagether, are admixed
with a pharmaceutically acceptable carrier substance, e.g. saline,
and administered parenterally, either intravenously or by injection
into affected arteries or the heart. Intravenous administration,
which is preferred, may be by infusion, by a bolus injection, or by a
ccmbination of these.
Preferably, the patient also receives heparin, for example a
bolus injection of 5000 I.U. heparin before administration of any PUK
or UK, optionally followed by infusion of 1000 I.U./hr of heparin.
The heparin may also be given in the form of a mixture with the
pro-UK or the ~K, or as a mixture of all three camponents.
The preferred quantity of UK used in the process according to
the invention is frcm 100,000 I.U. to 300,000 I.U., more preferably
frcn 150,000 I.U. to 250,000 I.U., particularly 200,000 I.U. The
preferred quantity of pro-UK used is less than 6 500,000 I.U., more
preferably frcn 2 000,000 I.U. to 4 000,000 I.U.
The above quantity of UK is most effective when administered as
a bolus injection 15 minutes or less before administration of pro-UK.
However, it may be preferred, for ease of administration, to
administer a mixture of pro-UK and UK in a single injection or
infusion, preferably as a single bolus injection. According to a
further aspect of the invention, therefore, there is provided a
pharmaceutical ccmposition ccmprising a mixture of UR and pro-UK
together with a pharmaceutically acceptable diluent or carrier, or a
- mixture of UK and pro-UK in pure lyophilised form.
The ccmponents of the synergistic mixture of the invention may
also be presented in twin-pack form, that is, as a single package
containing separate unit dosages of UK and pro-UR with instructions
for conccmitant administration.
Preferably the ccmposition is substantially pure, that is, it
contains no significant quantity of any pharmaceutically active
- 5 - case 118-6968
material other than UK and pro-UK. More preferably the camposition is
analytically pure, that is, it contains no protein other than UK and
pro-UK in a quantity detectable by analytical methods available at
the date of this application. When a diluent or carrier is present,
this is preferably sterile water for injection or sterile isotonic
saline.
The weight ratio of UK to pro-UK in the composition according
to the invention is preferably from 1:40 to 1:6.7, more preferably
1:20 to 1:10, particularly 1:15 to 1:10. Preferably the ccmposition
is made up in unit dosage forms e.g. vials for injection or bottles
for infusion, each containing 100,000 - 300,000 I.U. of UK and
2,000,000 - 4,000,000 I.U. of pro-UK, more preferably 200,000 I.U. UK
and 2,000,000 - 3,000,000 I.U. of pro-UK.
Such a unit dosage form contai~s a combined total dosage of UK
and pro-UK of frcm 2,100,000 to 4,300,000 I.U., corresponding to
approximately 21-43 mg of protein. The amounts of each ccnpaund are
thus much lower than the amount of each required for optimal clot
lysis when used in the absence of the other in the same therapeutic
regimen.
According to a further aspect of the invention, the patient is
given a bolus injection of UR and a bolus injection of pro-UK
followed by an infusion of pro-UK. The amount of UR given as a bolus
is as described above, i.e. preferably 100,000 - 300,000 I.U., more
preferably 150,000 - 250,000 I.U., particularly 200,000 I.U. The
25 amount of pro-UK given as a bolus is preferably 300,000 - 1,000,000
I.U., more preferably 400,000 - 60Q,000 I.U., particularly 500,000
I.U. These qyantities may be administered as two separate bolus
injections, delivered in any order, or frcm a twin-barrelled syringe-
or as a single bolus injection of a mixture of UK and PUK, which
represents a further ccmposition according to the invention.
The weight ratio of UK to pro-UK in this ccmposition is
preferably frcm 1:10 to 1:1, more preferably 1:4 to 1:1.6,
particularly 1:2.5. Preferably, however, the UK is administered
~32330n
- 6 - Case 118-6968
before any pro-U~ is given.
It may also be desirable to administer heparin together with
the bolus of UK and/or pro-UK. The amaunt of heparin to be added to
the above quantities of UK, pro-UK or UK/pro-UK mixture is preferably
from 1000 to 10,000 I.U., more preferably about 5000 I.U.
The amount of pro-UK given as a subsequent infusion is
preferably approximately 100,000 I.U./minute, which may be given for
approx. 40 min., i.e. a total of 4,000,000 I.U. In many cases
opening of the occluded artery may occur within 30 minutes or even
less, and infusion may be stopped after this time if arterial opening
is established for example by coronary angiography.
For use in this aspect of the invention, there is provided a
kit comprising a unit dosage of UK for injection, a unit dose of
pro-UK for injection, and a unit dose of pro-UK for infusion. The kit
is preferably presented in a single package, and in association with
instructions for administration. The unit do age forms for injection
may be sterile solutions of UK or pro-UK in pure water or in
physiological saline, or may be in lyophilised or freeze dried solid
form to which sterile water or saline is added before injection. The
unit dosage form of pro-UK for infusion may be a solution in
physiological saline or other sterile aqueous medium for infusion, or
a lyophilised or freeze dried solid or a liquid concentrate frcn
which an infusion solution can be made up. The solution for injection
or infusion may contain other ccmponents, for example buffer salts
such as Na2HP04/NaH2P04 and preservatives e.g. mannitol and human
albumin, and these components may also be present in the lyophilised
or freeze dried solid forms.
The quantity of UK and of pro-UK in the unit dosage forms for
injection is preferably as described above, particularly 200,000 I.U.
of UK and S00,000 I.U. of pro-UK the quantity of pro-UK in the unit
dosage fonm for infusion is preferably 3,000,000 - 5,000,000 I.U.
more preferably 4,000,000 I.U.
132~.u~
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The combination of low-dose UK (200,000 I.U.) and low-dose
pro-UK (3 000 000 I.U.) can achieve effective thrambolysis in the
majority of cases of myocardial infarction. To achieve the same rate
of success with UK alone, dosages of 2,000,000 to 2,500,000 I.U.
5 would be necessary, and at this dosage level side effects such as
systemic fibrinogenolysis and loss of plasminogen are found. To
achieve the same results with PUK alone, dosages well in exoess of
6,500,000 I.U., for example 9,000,000 I.U. may be necessary. These
results indicate a synergistic interaction between UK and pro-UK in
clot lysis.
m e quantity of each compound is expressed herein either by
weight (mg) or in International Units (IU) based on the International
Reference Preparation of UK assayed on a standard fibrin plate
(Brakman, Fibrinolysis, A Standardized Fibrin Plate Method and a
Fibrinolytic Assay of Plasminogen, Scheltema & Holkema NV, Amsterdam,
1967, pp. 1-24). Pro-UK is assayed after activation by plasmin as
described in: Gurewich et al., J. Clin. Invest. (1984) 73, pp.
1731-1739. UK (both HMW and LMW forms) and pro-UK gave similar
specific activities (about 100 000 IU/mg), so that protein
concentrations could be ccmp~red (1 IU = 10 ng protein).
As UK is conveniently available in ampoules containing
250,000 I.U., a dosage of 250,000 I.U. of UK may be substituted for
that of 200,000 I.U., wherever described in this Specification and
Examples. Use of the 250,000 I.U. dose avoids the difficulty of
estimating 4/5 of an ampoule, and gives the same beneficial effects
as for the 200,000 I.U. dose.
~32~ )~;,,
- 8 - - ~ase 118-6968
EXAMPIE 1
A 58-year old waman suffering frcm myocardial infarction
received a bolus injection of 200,000 I.U. of urokinase i.v. followed
by a bolus injection of 1,000,000 I.U. prourokinase and an infusion
of prourokinase at a rate of 5.5 million I.U./hr. Partial perfusion
was attained after 15 minutes, and camplete perfusion after 30
minutes, i.e. after a total of 3,250,000 I.U. of pro-UK had been
administered. There was still camplete perfusion 24 hours after
administration.
EXAMPLE 2
A male patient suffering fram myocardial infarction received a
mixture of 200,000 I.U. urokinase and 2,500,000 I.U. of pro-UK
infused over 15 minutes. Ccnplete perfusion was obtained by the end
of this period.
EXAMPLE 3 Dosage forms
a~ Mixture for injection
200,000 I.U. UK and 2,000,000 I.U. pro-UK are dissolved in 20
ml sterile isotonic saline and packaged in a vial for injection.
b) Lyophili7ed mixture
A solution of 200,000 I.U. UK and 3,000,000 I.U. pro-UK is
lya~hilized and the solid residue sealed in a vial to which saline
for injection can be added.
c) Twin-barrelled syringe
One barrel of the prepackaged syringe contains 200,000 I.U. UX
in 10 ml sterile isotonic saline, the other contains 3,000,000 I.U.
pro-UR in 10 ml sterile isotonic saline.
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- 9 - Case 118-6968
d) Twin-pack
A twin pack comprises one vial containing 200,000 I.U. UK in
lyophilized form and one vial containing 3,000,000 I.U. pro-UK in
lyophilized form, with instructions for conccmitant administration.
EXAMæLE 4
The dosage forms of Example 3 are also made up using
SOO,G00 I.U. pro-UK instead of 2,000,000 or 3,000,000 I.U. pro-UK.
EXAMPLE 5
In a multi-centre, open, prospective clinical trial, the
follawing protocol was used:
Criteria for inclusion
1) pain, characteristic for myocardial inarction, of at
least 30 min. duration
2) onset of symptcms less than five hours before the start of-
lytic therapy
3) ECG findings characteristic of myo~ardial infarction
4) informed consent of ~atient.
Criteria for exclusion
1) contra-indications for coronary angiography
2) contra-indications for lytic therapy
a) commencement of therapy with heparin or coumarin
b) venous or arterial puncture within the last 8 days
c) cerebrovascular accident within the last two months
i ,~330D
- 10 - ~ase 118-6968
d) active bleeding
e) stamach or duodenal ulcer within the last year
f) surgical operation within the last 8 days
g) haemorragic diathesis
3) incamplete blockage of coronary artery (as shown by
coronary angiography)
4) partial or camplete reperfusion after administration of
nitroglycerine
5) cardiac-related cama
6) serious heart valve- or heart disease, hypertrophic
cardicnycpathy
7) serious kidney or liver disease, which could affect the
excretion of pro-urokinase
8) use of oral contraceptives.
Procedure
Patients meeting the above critera for inclusion are given 5000
I.U. heparin, and a coronary angiography is carried out. If a total
arterial blockage is observed, 100-200 ug nitroglycerine is given
intracoronarily, and angiography of the blocked vessel is repeated.
If no opening is observed after administration of nitroglycerine, the
patient is given 200,000 I.U. high ~MW-urokinase in an intravenous
bolus injection, followed by 500,000 I.U. pro-urokinase i.v. in a
bolus injection given over 3 minutes. Immediately thereafter
intravenaus infu~ion of 4,000,000 I.U. pro-UK is carried out over 40
minutes. Coronary angiography is carried out at 15 min. intervals to
check the progress of clot lysis, and this is repeated 60 minutes
after successful lysis, and also 24 hours and 3 wecks after
treabment, to check for re-occlusion.
Results
Of 19 patients treatad according to this protocol, 14 (74 ~)
showed re-opening or patency of the occluded artery after the 40
minutes infusion. Of these, two shcwed re-occlusion after 24 hours.
