Disclosed is apparatus for administration of a drug in active form, which includes means defining a prodrug reaction zone including an immobilized enzyme for modifying chemically a prodrug to a physiologically compatible, physiologically active drug form, means in communication with the reaction zone for establishing parenteral access to the body of a patient, and means for transporting a prodrug through the reaction zone at a rate sufficient to convert the prodrug to the active drug form and then into the body of a patient.

The invention relates to an apparatus useful for the parenteral 
administration of a therapeutic agent to a human patient. 
BACKGROUND OF THE INVENTION 
The parenteral administration of a therapeutic agent is used extensively as 
an integral part of the daily treatment of medical and surgical patients. 
Liquids commonly administered parenterally include blood, blood 
substitutes, plasma substitutes, and solutions of dextrose, sodium 
chloride, or other electrolytes. 
U.S. Pat. No. 4,790,820 discloses a parenteral delivery system for 
administering an agent, which includes a drip chamber and a formulation 
chamber. The formulation chamber may include an ion exchange resin to 
which the drug to be administered is releasably bonded. 
U.S. Pat. No. 4,540,403 discloses a parenteral delivery system for 
administering an agent to a recipient, which includes an electrotransport 
apparatus that admits the agent at an electrically controlled rate into 
fluid that flows through the parenteral system over delivery time. 
SUMMARY OF THE INVENTION 
The invention in its broadest aspects implements the concept of modifying a 
prodrug, by chemical reactions, to convert the prodrug to an active, 
physiologically compatible form immediately prior to, e.g., during the 
process of, its parenteral administration to a patient, the prodrug 
typically occurring in some storage stable, soluble, toxic, and/or 
inactive form. Parenteral administration may include infusion via 
intravenous, intraarterial, intraperitoneal, or subcutaneous routes. As 
used herein, "prodrug" refers to any form of a drug or drug precursor, 
either active or inactive, which can be chemically modified to a 
pharmacologically or physiologically desirable form. The conversion of 
prodrug is accomplished by providing at the site of drug treatment, e.g., 
in line between a reservoir of the prodrug and a trocar or other entry 
point into the patient's body, a reactor having a reaction zone comprising 
biological macromolecules, such as an enzyme preparation, or another 
device which chemically modifies the prodrug, e.g., an ion exchange medium 
or ion adsorbing means, both for extracting ions. The modifying agent is 
by nature, construction, or composition designed to be retained 
selectively at or near the site of reaction, and thus prevented from 
accompanying the active drug into the patient' s body. 
The apparatus of the invention can be embodied in many forms, and adapted 
to many specific drug treatment protocols. The invention can be used, for 
example, in an implantable drug pump so as to permit storage in stabilized 
form or at high concentration in solution in a reservoir disposed within 
or adjacent to the pump housing. Passage of the stored prodrug through the 
reaction zone can, for example, enzymatically modify the soluble form to 
active, but less soluble form; or modify the stable but inactive, or low 
activity, or toxic prodrug to active, physiologically compatible form just 
prior to the time the drug is introduced into the body; or modify an 
active drug from a form that is preferred for storage to another active 
form that is physiologically or pharmacologically more desirable. 
Alternatively, the reaction zone can be disposed between a reservoir of 
prodrug and the skin in a transdermal patch. 
Thus, in-one aspect, the invention features an apparatus for administration 
of a drug in active form, the apparatus including means defining a prodrug 
reaction zone comprising an immobilized biological macromolecule, e.g., an 
enzyme, for modifying chemically a prodrug to a physiologically 
compatible, physiologically active drug form, means in communication with 
the reaction zone for establishing parenteral access to the body of a 
patient, and means for transporting a prodrug through the reaction zone at 
a rate sufficient to convert inactive prodrug to active drug and then into 
the body of a patient. 
In preferred embodiments, the reaction zone includes a high surface area 
matrix having immobilized thereon the macromolecule, e.g, an enzyme 
immobilized on the matrix. The matrix may be an insoluble or soluble 
substance capable of binding an enzyme; if insoluble, it may be a porous 
membrane, e.g., nylon, or may comprise insoluble chromatographic particles 
such as those used in column chromatography, e.g., microbeads; if soluble, 
it may include soluble macromolecules capable of being selectively 
retained, e.g., by a filter, within the reaction zone. 
In one preferred species of the invention, a plasminogen activator, e.g., 
urokinase or tissue plasminogen activator immobilized in the reaction zone 
cleaves the prodrug plasminogen, or an analog thereof having the 
properties of plasminogen, to produce the active drug plasmin or an 
enzymatically active analog thereof. Analogs of plasminogen include not 
only recombinant or synthetic versions of the complete molecule that, when 
converted to plasmin, retain the activities of native plasmin, but also 
truncated versions of plasminogen, e.g., mini-, micro-, or 
lys-plasminogen. Alternatively, the enzyme may be a protease, e.g., 
plasmin, and the prodrug a prohormone, e.g., proinsulin, which is 
converted by the protease plasmin into the active hormone insulin. 
In another aspect, the invention features an apparatus for 
fibrinolysis/fibrinogenolysis therapy capable of delivering plasmin to the 
site of an intravascular thrombus in a patient at a rate and concentration 
independent of the patient's vascular plasminogen concentration. The 
apparatus includes a reservoir of plasmin stabilized by the presence of an 
additive, e.g., lauryl sulfate ions, ion removing means for removing 
lauryl sulfate ions from the stabilized plasmin thereby to convert the 
stabilized plasmin to physiologically compatible, 
fibrinolytically/fibrinogenolytically active form, and means defining a 
flow path for establishing flow of stabilized plasmin from the reservoir 
through the ion removing means, and a flow of 
fibrinolytically/fibrinogenolytically active plasmin from the ion removing 
means parenterally into the body of the patient. 
In preferred embodiments, the reservoir includes a reservoir of lauryl 
sulfate-stabilized plasmin; and the ion removing means may include an ion 
exchange resin or an ion adsorbing substance, e.g., a hydrophobic matrix. 
Preferably other hydrophobic anions or cations may also be used in place 
of lauryl sulfate. 
In yet another aspect, the invention features another apparatus for 
fibrinolysis/fibrinogenolysis therapy capable of delivering a 
fibrinolytically/fibrinogenolytically active protein to the site of an 
intravascular thrombus in a patient at a rate and concentration 
independent of the patient's vascular plasminogen concentration. The 
apparatus includes a reservoir of a protein-prodrug selected from the 
group consisting of plasminogen, a serine protease-activatable analog of 
plasminogen, and a mixture thereof, a protein-prodrug reaction zone 
including an immobilized biological macromolecule operative to convert the 
protein-prodrug to a fibrinolytically/fibrinogenolytically active protein, 
and means defining a flowpath for establishing a flow of protein-prodrug 
from the reservoir to the protein-prodrug reaction zone, and a flow of 
fibrinolytically/fibrinogenolytically active protein from the reaction 
zone parenterally into the body of the patient. 
In preferred embodiments, the reaction zone includes a matrix having a 
plasminogen activator bound thereto; preferably, the means defining the 
flowpath includes a conduit communicating between the reaction zone and a 
trocar for accessing the body of the patient. Preferably, the 
protein-prodrug is selected from the group consisting of mini-plasminogen, 
micro-plasminogen, glu-plasminogen, and lys-plasminogen. 
In yet another aspect of the invention, the invention features an apparatus 
for delivering to a site of action an autolytic protease in an active 
form. The apparatus includes a reservoir of an autolytic enzyme 
preparation stabilized by the presence of lauryl sulfate, ion removing 
means for removing lauryl sulfate ions from the preparation thereby to 
convert the stabilized preparation to physiologically compatible, 
enzymatically active form, and means defining a flow path for establishing 
a flow of stabilized preparation from the reservoir and through the 
ion-removing means, and a flow of physiologically compatible, active 
enzyme from the ion-removing means to the site of action. 
In preferred embodiments, the site of action is the human body and the 
apparatus further includes means for parenteral delivery of the active 
enzyme into the body; and the ion removing means may be an ion adsorbing 
or an ion exchange resin. An adsorbing means is capable of removing ionic 
or amphipathic detergents that inhibit the autolysis of plasmin. 
The injection/activation apparatus of the invention is compact, small, and 
economical. Because the prodrug is chemically modified to an active drug 
as it passes through the reaction zone, substantially coincident with 
injection of the drug into the body of the patient, there is little 
elapsed time between modification of the drug and its administration to 
the patient. Consequently, there is little opportunity for loss of the 
enzymatic activity of the active drug prior to its administration to the 
patient. In addition, if an enzyme immobilizer is used that possesses a 
large surface area, the immobilizer will be capable of binding large 
amounts of enzyme, and useful for repeated dosages of drug. Since the 
conversion rate of prodrug to drug by immobilized enzyme using the 
apparatus of the invention is quantifiable using the apparatus of the 
invention, an accurately quantified amount of active drug may be 
administered to the patient, thus providing a precise calculation of the 
level of administered drug and dependable, e.g., thrombolytic, effects.

DESCRIPTION 
The apparatus 1 of the invention, for converting prodrug to drug upon 
delivery of drug to the body, is schematically shown in FIG. 1. Reservoir 
5 contains a prodrug which is delivered via delivery port 6 to reaction 
zone 7. The prodrug is converted to active drug by a biological 
macromolecule within reaction zone 7. The active drug is then delivered 
via delivery port 8 to the patient 2. In addition, container 3 may hold a 
physiologically compatible solution for dissolving or diluting prodrug in 
reservoir 5 which is delivered to reservoir 5 via delivery port 4. 
Alternatively, or in addition, pump 9, e.g., a peristaltic pump, may 
propel prodrug from reservoir 5 through port 6 to reaction zone 7. 
Other embodiments 10 and 10' of the injection/activation apparatus of the 
invention are shown in FIGS. 2A and 2B. The apparatus of FIG. 2A includes 
a reservoir 22 for holding a prodrug, a prodrug reaction zone 12 which 
includes an immobilized biological macromolecule such as an enzyme for 
chemically modifying a prodrug to a physiologically compatible, 
physiologically active drug form; a conduit 14 in communication with the 
reaction zone 12 and capable of establishing parenteral access to the body 
of a patient, e.g., through a trocar (not shown); and a plunger 18 and 
housing 16 for transporting a prodrug through the reaction zone at a rate 
sufficient to convert the prodrug to the active drug form. 
In the embodiment of the invention shown in FIG. 2A, conduit 14 can include 
a trocar, e.g., a beveled needle capable of piercing the patient's skin 
and inserting into a blood vessel. Prodrug reaction zone 12 is contained 
within housing 16 which includes reservoir 22 for containing the prodrug. 
The prodrug is transported by the action of plunger 18 through reaction 
zone 12 within housing 16, into contact with the immobilized enzyme 
immobilized thereon, and then through conduit 14 and into the body 20 of 
the patient. 
The apparatus of the invention may be used to inject a drug in its active 
form into the human body, wherein conversion of the drug from its inactive 
to active form occurs substantially coincident with the introduction of 
the drug into the patient's body, i.e., either during the process or 
immediately prior thereto. The time period between the conversion of 
prodrug to drug and injection of the drug into the body will typically be 
a period less than 10 minutes, preferably less than 5 minutes, and most 
preferably less than 1 minute. "Immediately prior to" means that the 
prodrug to drug conversion and the introduction of drug into the patient 
occur close enough in time such that the active drug maintains at least 
80%, preferably 90-99%, activity during that intervening time period. 
Preferably, plasmin made according to the invention is substantially free 
of elements that interfere with its clot-lysing ability; it may be 98-99% 
pure plasmin, except for the presence of plasminogen in the plasmin 
sample. Thus, in operation, pressure applied to plunger 18 pushes prodrug 
from reservoir 22 through reaction zone 12 and conduit 14 and into the 
body 20 of the patient. As the prodrug is pushed through prodrug reaction 
zone 12, it is converted by the immobilized enzyme into active drug. Thus, 
during the process of injection, the drug is converted from an inactive, 
i.e., a prodrug form, to an active, i.e., a drug form, by exposure to 
immobilized enzyme. The immobilized enzyme, contained within the injection 
apparatus is retained selectively within the reaction zone 12 and normally 
does not accompany the active drug through conduit means 14 to the body; 
i.e., it is only the active drug that is administered to the patient. 
In a similar embodiment, shown in FIG. 2B, wherein corresponding parts are 
identified by primed numbers, prodrug is contained within reservoir 21 and 
solvent is contained within reservoir 19; reservoirs 19 and 21 are 
separated by barrier 23, which may be, e.g., a pressure frangible 
partition. In operation, pressure applied to plunger 18' shatters barrier 
23 by pushing solvent from reservoir 19 into prodrug reservoir 21; solvent 
and prodrug are thus reconstituted before encountering reaction zone 12'. 
Another embodiment of the invention is shown in FIGS. 3A and 3B, which 
schematically illustrate in cross-section (3A) and plan view (3B) a 
transdermal patch 24 in which reservoir 30 contains inactive prodrug. The 
prodrug moves, e.g., by diffusion, from reservoir 30 through a reaction 
zone 32 and into the patient's skin 34. Coincident with movement of the 
prodrug through reaction zone 32, inactive prodrug is converted to active 
drug by a macromolecule that is contained within reaction zone 32. 
Immobilized macromolecules useful in the invention include any biochemical 
entity capable of promoting reactions including, but not limited to, 
electrochemical attractions, chemical reductions, oxidations, 
deacylations, phosphorylations, hydrolytic reactions, and condensations, 
or other enzymatic or catalytic reactions. Such macromolecules may include 
proteins and related cofactors such as coenzymes, polysaccharides, nucleic 
acids, or lipids. 
The immobilized enzyme may be coupled either reversibly or irreversibly to 
a matrix that may include soluble polymers or molecules separable by size 
or by specific ligands (e.g., an affinity ligand or an immunoglobulin 
having specificity for the enzyme) from the drug destined for injection. A 
soluble matrix may be retained in the apparatus upon injection of the drug 
by a filter calibrated to retain the matrix-bound enzyme, but permit 
passage of the drug. Alternatively, the enzyme be attached to an insoluble 
matrix, e.g., made of a synthetic or natural material. An insoluble matrix 
may include a variety of forms, e.g., membranes, porous filters, 
chromatographic support, or magnetic particles, all of which readily and 
selectively are retained by mechanical or magnetic means, but are so 
disposed as to ensure contact with the prodrug. 
The enzyme may be immobilized to a matrix which may be derivatized to bind 
the enzyme according to conventional chemical procedures well-known to 
those skilled in the art. Suitable membranes include those consisting of 
nylon, preferably those having a high density of derivatized chemical 
residues, such as free amino or carboxylate groups. Other examples include 
porous membranes consisting wholly or largely of cellulose-derived 
matrices, also preferably carrying a high density of carboxylate groups. 
Supporting matrices of this type have extensive surface area and may be 
arranged singly, in stacks, or in alternative forms such as sheets or 
discs of desired thickness. These properties enable the immobilization of 
a high density of enzyme and, thus, the achievement of a high prodrug 
conversion rate in a compact volume and at a minimal cost. A porous 
membrane may be prepared for enzyme immobilization as follows. 
Preparation of Immobilization Membrane 
Membrane sheets (Pall Corp., Glen Cove, N.Y., Biodyne C membrane No. BNPCH5 
or BNNCH5) are first cut into the shape of discs of desired diameter, then 
derivatized as follows. A solution of 0.5M spermine tetrahydrocholoride in 
water is brought to pH 7.0-7.1 by the careful addition of NaOH; 
separately, a solution of a water soluble carbodiimide, preferably 
1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDAC), (also 
0.5M in water) is brought to pH 5.0-5.05 by the addition of dilute 
hydrochloric acid; the two solutions are mixed in equal amounts and 
membrane discs are immersed in the mixture and incubated overnight at room 
temperature. The discs are washed copiously first with distilled water and 
then with 1.0M NaHCO.sub.3. The discs are then packed in solid, 
finely-pulverized succinic anhydride (500 mg per cm.sup.2 disc surface 
area), and sufficient dipotassium hydrogen phosphate (e.g., 0.5M) is added 
to thoroughly irrigate the disc and succinic anhydride packing (e.g., 
0.3-0.5 ml/cm.sup.2 disc area). The reaction is allowed to proceed 
overnight at room temperature. Small sample discs are incorporated 
alongside the membranes being treated and are tested for the presence of 
residual free amino groups. This succinylation procedure can be repeated, 
if necessary. The succinylated discs are rinsed free of precipitated 
succinate, washed under suction first with NaHCO.sub.3 (0.5-1.0M) and then 
with water, and dried. The discs may be stored for months at room 
temperature, with no change in properties. 
The dried membrane filter discs are mounted and securely clamped in holders 
that permit them to be perfused, and the entire assembly is incorporated 
into a circuit, driven by a peristaltic pump, in which the discs are 
continuously perfused for at least 1 hour at approximately 
30.degree.-37.degree. C. with a solution of N-hydroxysuccinimide and EDAC, 
both at 50 mM, in pure tert-butanol. At the end of the perfusion, the 
mounted discs are perfused briefly with 1 mM HCl at room temperature, 
blotted, swirled in ice-cold distilled water for a few minutes, then 
placed in a shallow dish, previously rinsed with 0.5% detergent (Triton 
X-100) in water, whose diameter is just sufficient to accommodate the 
membrane discs. At this stage, the discs are capable of immobilizing an 
enzyme. Examples of immobilized enzymes include, but are not limited to, 
those described herein, i.e., immobilized urokinase and immobilized 
plasmin. 
Fibrinolysis/Fibrinogenolysis Treatment 
The injection/activation apparatus of the invention may be used for 
fibrinolysis/fibrinogenolysis, i.e., treatment of a blood vessel 
occlusion, such as occurs in medical conditions such as myocardial 
infarction, thrombophlebitis or other forms of venous thrombosis, or 
pulmonary embolism. A fibrin/fibrinogen dissolving enzyme, e.g., human 
plasmin, may be administered directly using the apparatus of the invention 
to a patient suffering from vessel obstruction by a blood clot or to a 
patient in danger of developing a clot. Prodrug is contained within 
housing 5 of the apparatus (FIG. 1); prodrug reaction zone 7 contains an 
immobilized plasminogen activator. The enzymatically inactive prodrug, 
plasminogen, is converted coincident with its administration to the 
enzymatically active drug, plasmin. Active plasmin then travels through 
the circulatory system and dissolves a blood clot by converting the 
insoluble structural fibrin matrix of the clot to soluble fragments or, 
where a clot has not yet developed, lowers the circulating levels of 
fibrinogen and thus avoids clot formation. The use of the 
injection/activation apparatus of the invention for 
fibrinolysis/fibrinogenolysis is described below. 
Preparation of Immobilized Plasminogen Activators 
The injection/apparatus of the invention may contain a plasminogen 
activator immobilized on a matrix. Plasminogen activators useful in this 
embodiment of the invention include, but are not limited to urokinase (UK) 
and tissue plasminogen activator (tPA). Streptokinase (SK) may also be 
used as a plasminogen activator according to the invention, but would be 
most useful if modified, e.g., using recombinant DNA techniques, so as to 
be insensitive to plasmin degradation. 
Immobilized plasminogen activators may be prepared using a highly porous, 
physically and chemically stable membrane having extensive surface area. 
The membrane may consist of nylon having 1-3 .mu. average pore size, (Pall 
Corporation, Glen Cove, N.Y.). In its preferred version, such a membrane 
bears a high density of unsubstituted carboxylate groups (Pall. No. BNPCH5 
or BNNCH5) which act as starting points for chemical modifications that 
allow anchoring of proteins. 
Highly purified human urinary urokinase is coupled to membrane discs, such 
as those described above, immediately after activation. Lyophilized 
urokinase, e.g., Winkinase (Winthrop Laboratories, Sterling Drug, Inc., 
N.Y., N.Y.), or Ukidan (Serono, Aubonne, Switzerland) is dissolved in 10 
mM HEPES, pH 7.0-7.4 at a concentration of 4-5 mg per ml. Sufficient 
solution is pipetted into small shallow dishes so as thoroughly to 
impregnate the activated membranes, e.g., 15-20 .mu.l/1 cm.sup.2 of 
membrane. A total of approximately 400 .mu.g or 1.2 mg urokinase is used 
to impregnate a membrane of 25 mm or 47 mm diameter, respectively. After 
immersing the membranes in urokinase solution, the dishes are sealed, and 
incubated in a moist chamber at 4.degree. for 14-16 hours, preferably with 
gently rocking agitation. The coupled membrane is rinsed with 1 mM HCl by 
low speed centrifugation in a polypropylene tube, and the rinsing fluid 
collected, pooled with residual incubation medium, and assayed for 
remaining non-membrane-bound enzyme. Membrane bound urokinase may be 
assayed using the urokinase substrate plasminogen, which is converted to 
fibrinolytically/fibrinogenolytically active plasmin upon exposure to the 
plasminogen activator urokinase, or a low molecular weight substrate such 
as N-tosyl-L-arginine methylester. 
Preparation of Substrate Plasminogen 
Human plasminogen is prepared at 4.degree. C. according to a modified 
procedure of Liu et al., Canadian Journal Biochem. 49:1059-1061 (1971). 
Five hundred grams of frozen Cohn fraction III or II & III paste is 
pulverized at 4.degree. C. using a mortar and pestle, then added in 
portions with constant stirring to 5 liters of phosphate buffered saline 
(PBS), containing 1 .mu.M p-nitrophenyl-p-guanidinobenzoate. Stirring is 
continued for 4-5 hours, until the paste is thoroughly and evenly 
suspended. The solution is then centrifuged at 12000.times.g for 20 
minutes at 4.degree., and the gelatinous pellet discarded. The supernatant 
is filtered under gravity through "fast" filter paper, then brought to 10% 
of saturation (e.g. 50 g/l), by the addition of solid ammonium sulfate, 
and centrifuged once again at 12000.times.g for 20 minutes at 4.degree.. 
The resulting pellet is discarded, and the lipid-like material floating on 
the supernatant removed by filtration through a gauze plug. 
The filtered supernatant is then pumped, e.g., at 600-900 ml per hour, into 
e.g., a 230 ml, 4.8.times.15 cm column packed with G-15 Sephadex in PBS, 
and the outflow passed directly into a second column, of, e.g., 750-800 ml 
volume and 10 cm in diameter, packed with lysine-agarose (Pharmacia, 4B or 
6B, Piscataway, N.J.) and pre-equilibrated with PBS. The entire system is 
washed with PBS (e.g., 250 ml), the G-15 column disconnected, and the 
lysine-agarose column is washed with an additional 1.5 column volumes of 
PBS until the A.sub.280 drops below 0.15. The column is then washed with 1 
column volume of a solution consisting of 4 parts of ethylene glycol and 6 
parts of potassium phosphate buffer (0.5M, pH 8.0), followed by one column 
volume of PBS. Plasminogen is then eluted from the column with a linear 
gradient (2.5-3 column volumes) of epsilon aminocaproic acid (0-25 mM) in 
PBS, and collected in fractions. The fractions having the highest 
concentration of protein are pooled and precipitated at 50% saturated 
ammonium sulfate in the presence of benzamidine (50 mM), the pH being kept 
near neutrality by periodic addition of small volumes of 
tris-hydroxymethyl aminomethane (tris) base (1M). 
The precipitated plasminogen can be stored under 50% saturated ammonium 
sulfate containing 50 mM benzamidine for many months with no less of 
activity. After desalting and redissolving in PBS, it can be used directly 
for generating plasmin, as described below. 
Preparation of Substrate Mini-plasminogen 
Truncated forms of plasminogen such as mini- or micro-plasminogens, or 
derivatives thereof, may also be used for the generation of 
correspondingly truncated forms of plasmin. Mini-plasminogen is prepared 
according to a modified procedure of Powell et al., J. Biol. Chem. 
225:5329-5335 (1980). The plasminogen precipitate, e.g., 300 mg, is 
suspended in ammonium sulfate-benzamidine, as described above, centrifuged 
at 10,000.times.g for 30 minutes at 4.degree. C. and the resulting 
supernatant discarded. The pellet is dissolved in a minimum volume e.g., 
15-20 ml, of 100 mM NaCl-50 mM Tris, pH 8.0, at 4.degree. C. and desalted 
by passage through a 230 ml, 4.8.times.15 cm column of G15 Sephadex in the 
cold. Protein-containing fractions eluted from the column are pooled and 
diluted at room temperature with starting NaCl-Tris buffer to 3 mg/ml. 
20,000 kallikrein inhibitor units of aprotinin, and 1.7 mg pancreatic 
elastase are then added to the pooled protein fractions and the solution 
is incubated at room temperature with gentle stirring for 5 hours. The 
reaction is terminated by addition of methoxysuccinyl-(-ala-ala-pro-val) 
chloromethylketone to 10.sup.-4 M, and stirred for a further 30 minutes. 
The solution is dialysed overnight at 4.degree. C. against a large volume 
of 0.1M sodium phosphate buffer, pH 8.0, using tubing with molecular 
weight cutoff at 6500. 300 mg of dialysed plasminogen solution is applied 
to a 4.8.times.15 cm, 230 ml lysine-agarose column equilibrated in 0.3M 
sodium phosphate buffer, pH 8.0, and mini-plasminogen is eluted in 300 ml 
of the same buffer. Protein-containing fractions are pooled, benzamidine 
added to a final concentration of 50 mM, and mini-plasminogen precipitated 
by the addition of solid ammonium sulfate in several portions to a final 
concentration of 80% saturation. 
Assay of Membrane-Bound Enzyme 
Once the immobilized enzyme and the enzyme substrate are prepared, the 
membrane-bound enzyme may be tested for its ability to convert the enzyme 
substrate to product. Membrane-bound enzyme is assayed by pumping 
substrate, either small and synthetic, or macromolecular protein 
substrates (plasminogens), through the membrane; the former measures the 
amount of active enzyme bound, whereas the latter yields an estimate of 
the catalytic capacity of the membrane in plasminogen activation. The 
membrane to be assayed is mounted in a filter holder (for example, 
Millipore Nos. SXOOO2500 and SXOOO4700 for 25 mm and 47 mm diameter, 
respectively), which is connected to a peristaltic pump. Temperature 
control may be achieved by immersing the substrate reservoir and 
connecting tubing in a temperature regulated bath. The substrate solution 
is pumped through the membrane, or several membranes assembled in series, 
e.g., 1 ml aliquots of the effluent are taken. The absorbance change in 
the effluent compared with the substrate solution gives the concentration 
of product which, when multiplied by the flow rate, yields the activity in 
terms of moles per unit time for small substrates; assay of plasmin using 
Kabi S-2251 is used for estimating the rate of plasminogen activation. 
Given the value of apparent K.sub.cat, which is derived from measurements 
of enzyme activity in free solution, the estimate of active bound enzyme 
is easily obtained from the observed rate of product formation. In 
practice, assay of bound enzyme activity should be made under conditions 
of perfusion in which no more than 10% of small substrate are hydrolysed; 
flow rates of 10-15 ml/cm.sup.2 /h give maximum apparent rates of 
substrate hydrolysis. 
The small substrates dissolved in Tris-HCl buffer (0.1M, pH 8.8) are either 
TAME (tosyl-L-arginine methylester, 10 mM) or Kabi S-2160 
(N-benzoyl-phe-val-arg-p-nitroanilide, 0.2 mM). The hydrolysis of TAME is 
measured by change in absorbance at 247 nm, and that of Kabi S-2160 at 405 
nm. The results of such an assay are shown in FIGS. 4A and 4B. FIG. 4A 
shows results of an assay of activity of membrane-immobilized urokinase 
using 10 mm TAME as substrate. Numbers on the horizontal axis denote the 
initiation of perfusion at the indicated flow rate. Estimates of the total 
quality of urokinase are indicated for selected points, after 
equilibration for each flow rate. FIG. 4B shows results of an activity 
assay of membrane-immobilized urokinase using 0.2 mM Kabi S-2160 as 
substrate. Flow rates and estimates of active urokinase are as indicated 
in FIG. 4A. 
The macromolecular substrates, glu- and lys-plasminogens as well as 
truncated forms such as mini- and/or microplasminogen, are dissolved in 90 
mM NaCl, 5 mM NaPO.sub.4, pH 7.3-7.5, 1.8% dextrose, usually at a 
concentration of about 30 .mu.M. Glu-plasminogen is the naturally 
occurring form of plasmin in circulation; the N-terminal amino acid 
residue is glutamic acid. Lys-plasminogen is derived from glu-plasminogen 
by limited proteolysis, usually catalyzed by plasmin, in which a peptide 
fragment of 77 residues is cleaved from the amino terminal domain, leaving 
an N-terminal lysine residue. Mini-plasminogen is derived from either 
glu-or lys-plasminogen by limited proteolysis, catalyzed by pancreatic 
elastase, in which a fragment containing the proenzyme domain of 
plasminogen with a single attached kringle, is generated, the remaining 4 
kringles and intervening peptides having been separated, as described in 
Sottrup-Jensen et al. (Progress in Chem. Fibrinolysis and Thrombosis 
3:191-209, Davison et al., eds., 1978, Raven Press, N.Y.). 
Micro-plasminogen consists of the proenzyme domain of plasminogen with a 
stretch of connecting peptide and a few residues of kringle 5 attached at 
its N-terminus, and is generated by plasmin cleavage of plasminogen (see 
Shi et al., 1980, J. Biol. Chem. 263:17071-17075). 
The rate of plasminogen activation, as well as the fraction that is 
activated to plasmin, are influenced by numerous factors, including 
plasminogen concentration, flow rate, enzyme-binding area within the 
reaction zone, and numbers of membranes in series within the reaction 
zone. These parameters can be adjusted to achieve any desired therapeutic 
goal in terms of plasmin formed per unit time for any fraction of 
plasminogen activated. In a typical run, two 47 mm membranes mounted in 
series will activate approximately 80% of the perfused mini-plasminogen at 
a concentration of 30 .mu.M and a flow rate of 70 ml/hour, yielding about 
1.7 .mu.moles of plasmin/hour. Membranes can function continuously at 
constant rates for at least 3 hours. 
When membrane coupling is performed, 80% of available urokinase is removed 
from solution and bound to the membrane. Of this amount, 20-25% is 
catalytically active in hydrolysis of small substrates, and approximately 
5% is active in plasminogen activation. 
In order to attain therapeutically useful levels of plasmin, the 
plasmin-generating injection/activation apparatus should be easily 
perfusible at low pressures, physically compact, and capable of 
maintaining a relatively high rate of plasminogen activation, i.e., 
conversion of at least 75% of the perfused plasminogen, or at least 2 
.mu.moles of plasmin per hour for several hours. 
Use of Injection/Activation Apparatus 
The injection/activation apparatus may be tested for in vivo use in 
dissolving blood clots by introducing a radioactive clot into, e.g., the 
external jugular vein of a dog, and injecting plasmin using the apparatus 
of the invention into the dog's circulatory system. Dissolution of the 
clot may be followed by monitoring the level of radioactivity; a decrease 
in the level of radioactivity at the site of the clot indicates 
dissolution of the clot. 
Dosage 
The dosage of drug administered to a patient using the apparatus of the 
invention will vary depending upon the type of drug to be administered. 
For example, the duration of fibrinolytic/fibrinogenolytic treatment using 
the apparatus of the invention may vary from a short single dose 
administration of plasmin, e.g., in myocardial infarction, to the longer 
thrombolytic regimens required for thrombophlebitis and pulmonary embolism 
or the prolonged, continuous and/or intermittent treatments which may be 
used to treat coronary occlusion and other conditions, such as 
hyperfibrinogenemia, for which prophylactic therapy may be desirable. A 
longer thrombolytic or fibrinolytic/fibrinogenolytic therapy will require 
adjustment depending upon the size of clot or the ultimate desired level 
of circulating fibrinogen. If a small reduction in fibrinogen 
concentration is required, more frequent administrations of low doses may 
be needed to maintain a given depression of the fibrinogen level. Persons 
at risk for thrombus formation include but are not limited to diabetics 
and pregnant women. Diabetics carry a higher than normal level of 
fibrinogen and, therefore, have a higher risk of developing thrombi. The 
administration of plasmin prophylactically to a diabetic would lower 
fibrinogen levels and thus reduce the risk of clot formation. 
The duration of fibrinolytic and or fibrinogenalytic treatment using the 
injection/activation apparatus of the invention may vary from a short 
single dose administration of plasmin, e.g., 1-30 .mu.moles of plasmin for 
a 150 lb. person within a 6 hour period, depending upon the size and 
location of the clot. For example, if the clot is venous, the duration of 
treatment may be days, whereas if the clot is arterial, only hours of 
treatment may be required. 
Other factors must be taken into account when using the 
injection/activation apparatus of the invention to administer a drug, 
e.g., of primary importance is the nature of the drug itself. The 
following information would be used to assess the administration of the 
drug plasmin to a human patient using the injection/activation apparatus 
of the invention. 
Clot dissolution reflects the fibrinolytic/fibrinogenolytic action of 
plasmin, and the duration and effectiveness of thrombolytic therapy 
following administration of plasmin depend primarily on the balance 
between the rates of plasmin introduction, and plasmin removal and/or 
inhibition by the plasmin inhibitor, .infin.2-antiplasmin, or other 
inhibitors of plasmin. Factors to be taken into account when adjusting 
plasmin dosage for clot dissolution include physical factors such as 
height, weight, and age of the patient; the location of the blood clot, 
and circulating levels of plasmin inhibitors, such as .infin.2-antiplasmin 
and .infin.2-macroglobuiin. .infin.2-antiplasmin, which has a normal range 
of plasma concentration in vivo of approximately 1 .mu.M.+-.20%, is 
ordinarily the dominant factor regulating plasmin action in the 
circulation; plasmin combines irreversibly with .infin.2-antiplasmin to 
form a 1:1 complex and is thereby inhibited before it can attack clots or 
other proteins. The level of circulating .infin.2-antiplasmin is important 
in assessing plasmin dosage, and .infin.2-antiplasmin must be titrated to 
a level at which no more than 15% of the normal circulating concentration 
is present. A high initial level of .infin.2-antiplasmin will require a 
large dose of extracorporeal plasmin to be administered parenterally. 
Hormone Treatment 
Another therapeutic use of the apparatus of the invention involves use of 
an immobilized proteolytic enzyme, e.g., for prohormone conversion, the 
process by which inactive hormone precursors are converted to their active 
forms. Mammalian polypeptide hormone activation typically is mediated by 
limited proteolysis catalyzed by proteolytic enzymes. 
The inactive precursor of the hormone insulin is much more soluble in 
physiological buffer media than the active hormone itself, and therefore 
substantially higher concentrations of proinsulin can be maintained in a 
reservoir of a given size as compared with active form insulin. Proinsulin 
can be perfused through a membrane containing immobilized plasmin, which 
converts proinsulin to insulin by limited proteolysis during the course of 
injection. This procedure allows insulin to be generated during the act of 
infusion and to enter the bloodstream without forming potentially harmful 
polymeric aggregates of low solubility. 
Preparation of Immobilized Plasmin 
Immobilized plasmin is prepared by first immobilizing its inactive 
precursor plasminogen, and then using a plasminogen activator to convert 
the plasminogen to plasmin. Plasminogen, preferably in the form of mini- 
or microplasminogen, is prepared using human or domestic animal blood 
plasma or plasma fractions for starting material. The resulting 
plasminogen is immobilized by coupling either to derivatized nylon 
membrane, e.g., as described above, or to another matrix, e.g., a bead 
matrix, using standardized procedures familiar to those skilled in the 
art. Matrix-bound plasminogen is activated to the corresponding plasmin by 
exposure to a conveniently available plasminogen activator, and washed 
free of the activator. The plasmin membrane may be tested for plasmin 
activity in the manner described for the urokinase membrane, but using the 
plasmin substrate Kabi S-2160 or tosyl-L-arginine methylester, or 
proinsulin (Novo Pharmaceuticals, Copenhagen, Denmark; Eli Lilly & 
Company, Indianapolis, Ind.; or produced by conventional recombinant DNA 
techniques (Nature 282:525-527 (1979)). For prohormone therapy, the 
plasmin membrane mounted in a filter holder, or a plasmin-containing bead 
matrix in a cylindrical cartridge, is attached to a reservoir containing a 
concentrated solution of proinsulin and a pump capable of perfusing the 
immobilized plasmin. The flow rate is determined by varying the diameter 
of the plasmin membrane or the length of the column of plasmin beads in 
the cartridge, the rate of pumping, and the concentration of the 
proinsulin substrate solution. The flow rate may be adjusted empirically 
to achieve the desired rate of conversion of single chain proinsulin to 
two-chain insulin coincident with the injection of insulin into the 
patient's body. 
Drug Activation by Sequestration of Inhibitor on an Insoluble Matrix: 
Lauryl Sulfate Ion-stabilized Plasmin 
Plasmin exhibits, like any proteases, a strong tendency to self-digestion, 
especially under the conditions of high concentration that are encountered 
during its preparation, storage and formulation for delivery. It is 
desirable to prevent autolysis in order to preserve catalytic activity. A 
preferred way of accomplishing such suppression is by the addition of 
suitable concentrations of lauryl sulfate ions, in the form of sodium 
lauryl sulfate, which inhibits plasmin-catalyzed autolysis. Lauryl sulfate 
can be toxic, and therefore should be substantially removed from the 
plasmin preparation prior to injection. This is conveniently accomplished 
by passing the solution of plasmin/lauryl sulfate over a matrix or 
membrane capable of removing lauryl sulfate, either by hydrophobic 
adsorption and/or ion exchange. Thus, the plasmin is activated coincident 
with its injection into the patient's body. Other hydrophobic anions or 
cations which may have the same effect and are removable are sarcosyl, 
deoxycholate, and cetyltrimethyl ammonium bromide. 
All operations are performed under sterile conditions using sterile, 
pyrogen-free reagents. Plasmin, or one of its truncated forms (mini- or 
micro-plasmin) is produced by perfusing the corresponding plasminogen 
through one or more urokinase membranes, prepared as described above. The 
flow rate, plasminogen concentration, temperature and number of membranes 
are selected to activate 80-95% of the perfused plasminogen to plasmin; 
for example, two 47 mm membranes perfused with approximately 30 .mu.M 
mini-plasminogen at about 50 ml per hour and at 25.degree. C. The effluent 
may be collected directly into a chilled vessel containing a solution of 
sodium lauryl sulfate sufficient to yield a final lauryl sulfate 
concentration of 0.05-1.0%, e.g., 0.5-10% lauryl sulfate in a volume of 
H.sub.2 O one tenth that of the anticipated final volume of effluent to be 
produced. 
Alternatively, to achieve a greater proportion of plasmin in the final 
product, the effluent may be led through a refrigeration bath and directly 
onto a column bed of immobilized plasmin inhibitor (e.g. aprotinin), where 
it is bound in an inhibited state; the material emerging from this bed 
contains the remaining, unactivated plasminogen which may be recycled onto 
the urokinase membrane to achieve substantially complete conversion to 
plasmin. At the termination of plasminogen activation any residual 
unactivated plasminogen is removed by washing the column bed with PBS 
buffer, and the plasmin product recovered by elution with 90 mM NaCl-1 mM 
HCl, collected into a buffered solution of lauryl sulfate, as indicated 
above, calculated to neutralize the HCl. The inactive plasmin is 
concentrated by addition of solid ammonium sulfate to 80% of saturation, 
the precipitated plasmin collected by centrifugation, and the ammonium 
sulfate removed by dialysis against large volumes of water containing 
sodium lauryl sulfate at approximately 0.1% and 90 mM NaCl, and the 
dialyzed plasmin solution lyophilized. For therapeutic administration, the 
inactive plasmin is reconstituted by addition of sterile, pyrogen-free 
water containing 1.8% dextrose, 90 mM NaCl and 0.1% lauryl sulfate, final 
concentrations. The inactive plasmin solution is then perfused through a 
matrix capable of retaining lauryl sulfate ions and thus removing them 
from the plasmin preparation. For example, the inactive plasmin solution 
may be pumped through a column of Extractigel (Pierce Chemical Co., 
Rockford, Ill.), an adsorbing matrix, at a rate not exceeding 1 ml per 
cm.sup.2 per minute. The lauryl sulfate ions are thus retained by the 
matrix and reactivated plasmin is produced. 
Alternatively, the lauryl sulfate-stabilized plasmin may be activated by 
contacting it with a plurality of ion exchange resin particles. The 
particles may vary in size, e.g., from 10-350 mesh, and the resin may be 
any conventional material capable of attracting lauryl sulfate ions, e.g., 
AG11 A8 (Bio-Rad Laboratories, Richmond, Calif.). 
Other Embodiments 
The invention may be embodied in other specific forms without departing 
from the spirit or essential characteristics thereof. The present 
embodiments are therefore to be considered in all respects as illustrative 
and not restrictive, the scope of the invention being indicated by the 
appended claims rather than by the foregoing description, and all changes 
which come within the meaning and range of equivalency of the claims are 
therefore intended to be embraced therein. 
Other embodiments of the invention are within the following claims.