CA1293443C - Controlled release of macromolecular polypetides - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

An active agent delivery system for the controlled administration of macromolecular polypeptides which comprises a micro-suspension of water-soluble components in a polylactide matrix.

Description

CONTROLLED RELEASE OF MACROMOLECULAR POLYPEPTIDES

BACKGROUND OF THE INVENTION

Field of the Invention This inven~lon relates to an active agent delivery system for administering macromolecular polypeptide active agents having molecular weights of about lOOO or greater, particularly interferons, at a controlled rate 20 for a prolonged period of time.

Backqround and Related Disclosures The traditional and most widely used method of administration of therapeutic agents is by the oral 25 route. However, in the case of large polypeptides, such delivery is not feasible due to the hydrolysis of the peptides by digestive enzymes. The methods most commonly used for administration of polypeptide therapeutic agents are by repeated injection, intramuscular (IM), 30 subcutaneous (SC) or intravenous (IV) infusion. These methods are acceptable in situations where a very limited number of injections are required, but are undesirable w for chronic administration (for example as ~ith insulin therapy). The nature of many ot the dis~ases, disorders and oonditions susceptible to improvement by polypeptide administration is ohronic rather than acute 9 thus necess~tating frequent injections over a prolonged period of time.
There is, therefore, a need for an ef~icaciDus and economical delivery system tor large poLypeptide agents.
Biodegradable polymer matrices formed ~rom polylactic acid or copolymers of polylactic acid with other comonomers such as polyglycolic acid have been used as sustained release delivery systems for a variety ~
active agents, due to their ability to biodegrade in situ. See, for example, U.S. Patent Nos. 4,29~,539, and 4,419,340. The use o~ these polymers in implants for delivery ot several therapeu~ic agents has been disclosed in scientific publications and in the patent literature.
See, for example, Anderson, L.C. et al, (1976), "An injectablE sustained release fertility control system", 20 Contraception 13: 375-~84; ~eck et al. (1979) "New long-acting injectable microcapsule contraceptive syste~n, Am. J. Obstet. GYnecol. 14~: 799-806; Yolles et al. (1978) "Timed release depot for anti-cancer agents IIn, Acta Pharm. Svec. 15: 382-388, U5 3,773 919, and 25 U.S. Patent ~o 4,675,189.
Sustained delivery of peptides from poly(lactide co-glycolide) systems has been reported by Kent et al. (1982), "In vivo controlled release o~ an LHRH analog from injected polymeric microcapsulesn, 30 ~ E~. Deliv. ~X~ 3: 58; by Sanders et al. ~1984), n Controlled release o~ a luteinizing hormone-releasing hormone analogue from poly (d,l-lactide-co-glyco~ide)-microspheres n ( see also European Patent No. 0052510), J. Pharmaceut. Sci. 73:
~610Y 25570 "` lZ934'~3 , 129~-1297, by T. Chang, "Biodegradeable semipermeable microcapsules containing enzymes, hormones, vaccines and other biologicals", ~. Bioengineerin~, 1, 25-32 (1976), and in EPO Application No. 82300416.3, filed January 27, 1982 (now European Patent No. 0058481). However, the delivery of large polypeptides from polylactide matrices has been very difficult to achieve, for reasons that will be further discussed. Of the publications cited above, only the latter two disclose devices containing 10 polypeptides having molecular weights of 2500 or greater.
Polylactide and poly(lactide-co-glycolide) polymers and copolymers (referred to generically hereinafter as polylactide or PLGA polymers~ are not soluble in water.
In contrast, most polypeptides are soluble in water but 15 not in organic solvents. For this reason, the preparation of polylactide devices in which polypeptide particles are dispersed has, until now, generally followed one of two basic techniques.
One technique involves mixing of the components with 20 the polylactide in the molten state followed by heat extrusion 3 heat pressing, or casting.
The second technique involves the creation of a solution/suspension of the polymer and polypeptide in an organic solvent, which is then pour-cast into a film or 25 slab and the solvent evaporated, The latter method usually requires extensive or rapid stirring of the solution/suspension in order to achieve an acceptable degree of uniformity o~ the polypeptide particles and homogeneity of the polypeptide/polylactide matrix upon 30 solidification. Evaporation of the solvent takes place over several hours to several days unless the film or slab is dried under vacuum, in which case bubbles are invariably created as the solid dries. Additionally, polylactide formulations prepared in this way are not ~610Y 2557n ~ 3~43 , -4- :

su~ficiently uniform for most therapeutic applications;
due ~o ooalescence of the water-soluble particle phase, the polypeptide i 5 unevenly distributed within the polylactide as large aggregat~s of particles. Therefore, formulations prepared in this way must be submitted to further homogenization procedures such as ~rinding the formulat~on to a powder and reforming it under heat, or compressing or extruding the tormulation under heat~ The temperature required ~or for these manipulations is usually at least 70UC.
It is also known to make injectable microcapsul~s of drug in pslylact~de. Surh microcapsules can be prepared by basic techniques such as t~at set ~t in U.S. P~t.~nt No. 3,773,919, and in U.S Patent N~. 4,675,189 and European Patent No. 0052510. The latter method involves dissolving the polymer in a halogenated hydrocarbon solvent, dispersing the aqueous polypeptide containing solution by rapid stirring in the polymer-solvent solution, and adqing a non-solvent 2~ coacervation agent which causes the polymeric excipient to precipitate out of the halogenated hydrocarbon solvent onto the dispersed polypeptide containing water droplets, thereby encapsulating the polypeptide. The resulting microcapsules are then dried by repeated organic solvent Z5 washings~
However, large polypeptides are particularly susoeptible to physical and chemical dena~uration and consequent loss of biological potency from exposure to excessive heat, solvents, and shear forces. For this 30 reason, incorporation of large polypeptides in polylactide polymers has, until now, required either compromise in the degree o~ uniformity of the polypeptide/polymer dispersion, or has resulted in substantial loss of the biological potency of the 361~Y 25570 ,t-.

:~Z~3~`~3 polypeptide 9 nr both. The resultant formulations are generally non-uniform dispersions containing irregularly sized large particles of polypeptide of reduced potency.
The incorporation of large and irregular particles of 5 polypeptide causes an uneven rate of drug delivery, and tends to exacerbate the multiphasic release profiles generally associated with polylactide pharmaceutical preparations.
Preparation of more homogenous monolithic 10 formulations by known techniques, such as mixing of the molten components, grinding, and heat homogenation techniques such as compression and extrusion may result in a substantial, often nearly complete loss of biological activity of the polypeptide. For example, a 15 PLGA/in~erferon formulation formed by heated mixing and extrusion under mild conditions retains less than 1% of the original biological activity of the interferon. (See Example 7, below.) To compensate for the loss in biological activity during manufacturing processes of 2a this type, a large excess of polypeptide must be incorporated in the formulation.
A further disadvantage of formulations which contain denatured polypeptides is the increased immunogenicity which they exhibit. Antibody formation in response to 25 the denatured polypeptide may partially or entirely contravene the desired therapeutic effect.
~ ccordingly, there is a need for a homogeneous polylactide device which provides controlled and regular delivery of macromolecular polypeptides and can be 30 manufactured without significant loss of biological activity.

-`- lZ913~-~3 SUMMARY OF THE IN~ENTION

The present invention provides a novel active agent delivery system for the controlled administration of a water soluble macromolecular polypeptide to a mammal. The system comprises a polymeric matrix comprising not more than about 30 percent by weight of particles of macromolecular polypeptide and other optional water-soluble components dispersed in a polylactide matrix, wherein substantially all of the particles of polypeptide and other water-soluble components have diameters of lO ~ or less and are uniformly and discretely dispersed throughout the matrix, and wherein the polypeptide retains at least about 50 percent of the biological activity which it possessed prior to manufacture of the matrix.
This device provides an economical and relia~le method of delivering controlled and regular quantities of biolo~ically active macromolecular polypeptides to body 20 sites which are capable of making available intracellular and or extracellular ~luids for transfer into the device. The system can be designed to deliver the active agent at an appropriate rate over prolonged periods of time ranging from less than one day to several months.
25 Generally, active agent release periods of about one week to three months are contemplated.
An important advantage of this controlled release device resides in the ~act that it can be manufactured ~ith only minor loss of biological activity of the 30 polypeptide active agent. Maintenance of high biological activity permits the device to be manufactured to contain relatively low initial amounts of the polypeptide.
As a result of maintaining high biological activity of the peptide during manufacture, several further ~39~43 advantages are achieved. First, there is a significant economic advantage to the manufacturer with respect to the cost of active agent incorporated in each dosage form or delivery system. 5econd, due to the relatively low percentage of polypeptide and other water-soluble components in the device, the contribution of these components to the hydration of the system in vivo is minimized, thereby providing a more constant rate of polypeptide release throughout the entire operational 1~ life of the device than can be obtained with previously known biodegradeable systems. Third, the absence of significant quantities of denatured polypeptide reduces the likelihood of undesirable immune responses at the site of polypeptide delivery.
A further advantage o~ this device is that it can be manufactured with little loss of active ingredient. This of course is useful in reducing wastage of active ingredient. Initially the manufacture obtains 80%
incorporation of active ingredient starting material into the device, preferably at least 90%, most preferably substantially 100% incorporation.
Another important advantage of the controlled release device of this invention resides in the novel physical structure of the polymer~polypeptide matrix. The 25 matrix comprises a very fine dispersion, or micro-suspension, of water soluble components in a polylactide polymer, wherein substantially all of the particles of active agent and any other optionally present water soluble components have diameters of lO
30 or less. These particles are uniformly and discretely dispersed throughout the polymer, providing an essentially homogeneous and monoli~hic device. As with previously known systems, the biologically active polypeptide is released through a combination of 39,~3 diffusion and dissolution mechanisms as the device hydrates and subsequently erodes. However, unlike known polymeric matrix systems which deliver macromolecules, the system of this invention does not rely on the formation of aqueous channels, or macropores, in the matrix for release of the macromolecules from the system. The requirement of macropore formation for drug release to occur is known to result in a triphasic release profile characterized by a middle quiescent phase 10 during which little or no drug is released. There may also be a quiescent, or dead period before the initial phase of drug release. In contrast, because the polypeptide and other water soluble components o~ this invention are present as very small and discrete 15 particles within the polylactide matrix, aqueous channels are not formed, if at all, until relatively late in the release period. As a result, a very regular release profile is achieved which can be made to begin with very little initial lag time, and which is continuous 2~ throughout the li~e o~ the system.
Another aspect o~ the invention resides in a method of administering a macromolecular polypeptide active agent, which comprises placing an appropriately sized and shaped device of the above description at a body site 25 which is capable of making available its intracellular and/or extracellular fluids for absorption by the implantO Further aspects of the invention involve novel methods of preparing the devices described and claimed herein.
BRIEF DESCRIPTION OF THE DRAWING
-FIGURE I is a graph of the data obtained from the test described in Example 3, and shows the release 35 profiles of ~-interferon from active agent delivery z~

3~ ~3 systems implanted subcutaneously in mice over a period of 60 to lOO days. The systems were prepared according to the invention as described in Examples l and 2.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

Definitions The term ~biologically active macromolecular polypeptide" refers to any polypeptide having a molecular 10 weight of not less than about lOOO daltons, preferably not less than about 2500 daltons, which possesses useful biological activity when administered to a mammal.
The phrase "wherein the polypeptide retains at least about 50% of its biological activity" means that at least 15 about 50% o~ the biological activity potential of the polypeptide will remain at completion of manufacture, as determined within the accuracy of a biological assay for the particular polypeptide such as the one described in Example 5. Generally, the assay will involve spiking the 20 polypeptide loading stock with a known concentration of radioactively labelled polypeptide, extracting the polypeptide from the manufactured system under mild conditions, and determining both the relative radioactivity (counts per minute per ml), and the 25 relative units/ml biological activity, o~ the loading stock and the extracted polypeptide in a standard biological assay for the polypeptide. The biological assay is per~ormed against a reference standard in serial dilution test wells of the polypeptide samples to be 30 assayed. An arbitrary endpoint is set for scoring the test wells, and the same endpoint is used in scoring the reference samples. The activity of the inter~eron samples is calculated based on the loglO o~ the units/ml biological activity of the loading stock 34 ~3 equivalent for each extracted sample. Systems which fall within the scope of this invention will demonstrate relative 1O91o polypeptide activity values which are one/half, nr greater, than the log10 units/ml of the corresponding polypeptide loading stock. The preferred embodiments of this invention retain at least about 70 of the biological potential of the polypeptide a~ter manufacture, more preferably at least 90%, most preferably substantially all of the biological potential.
The term "substantially all of the particles of polypeptide and other water-soluble components" refers to at a quantity of at least about 75 % of the particles of components so identified, more suitably at least about 90%.
The term "water-soluble" is used herein to refer to macromolecular polypeptides and other optional pharmaceutically acceptable components which are at least "very slightly soluble" by the de~inition given in the United States Pharmacopeia, XX, page 1121, i.e. having 20 water solubilities of at least 0.1-1.0 mg/ml.
The term "micro-suspension" is used herein to describe particles of polypeptide and other water soluble solid components, substantially all of which have diameters o~ 10 microns or less, which are substantially 25 uniformly and discretely dispersed throughout the polymer. The term "uniformly and discretely dispersed"
is used to indicate that the particles are not touching each other, but rather are individually surrounded by polymer, and are approximately equi-distantly spaced.
30 Determination of the size and distribution of the particles of polypeptide and other water-soluble components can be made by a standard microscopic ,. : ,, ~

~; 3L2S~3~3 examination such as that described in Example 4, below.
Preferably, substantially all of the particles will have diameters of 5 ~ or less, and more preferably, l ~ or less.
The term "polylactide" is used herein in a generic sense to describe both homopolymers and copolymers derived from alpha-hydroxycarboxylic acids, particularly -hydroxyacetic (lactic) and ~-hydroxypropionic acid (glycolic) acids. They are usually prepared from the 10 cyclic esters of lactic acids.
The present invention resides in the creation oF a homogeneous matrix of a polylactide in which is incorporated a substantially uniform micro-suspension of a biologically active macromolecular polypeptide. The 15 matrix releases the biologically active polypeptide when placed at a body site which can make available its intracellular and/or extracellular fluid for transfer into the device. As the matrix becomes hydrated, the polypeptide is released by diffusion and erosion 20 mechanisms. Because the polypeptides are water-soluble, the rate of release is governed by the rates o~ hydration and polymer erosion of the device.
The use of polylactide copolymers provides the opportunity to vary the rates of hydration and erosion o~
25 the polymer matrix by appropriate choice of the type and relative amount of comonomer used. Some illustrative examples of suitable comonomers include glycolide, ~-propiolactone, tetramethylglycolide, ~-butyrolactone, 4-butyrolactone, pivalolactone, and intermolecular cyclic 30 esters of a-hydroxy butyric acid, a-hydroxyisobutyric acid, -hydroxyvaleric acid, ~-hydroxyisovaleric acid, ~-hydroxy caproic acid, a-hydroxy-~-ethYl-butyric acid,, ~-hydroxyisopcaproic acid, ., .. . ~ .

`~ ~LZ~39~fl~

-12~

a-hydroxy-3-methylvaleric acid, ~-hydroxy-heptanoic acid, ~-hydroxyoctanoic acid, ~-hydroxydecanoic acid, ~-hydroxymysristic acid, a-hydroxystearic acid, and ~-hydroxylignoceric acid.
Any of these compounds may be used as a comonomer in the preparation of acceptable polymers.
~-butyrolactonelcan be used as the sole monomer or as the principle monomer along with a suitable comonomer.
However it is most preferred to use lactic acid as the 10 sole monomer, or as a copolymer ~ith glycolic acid a~ the comonomer. The term polylactide is used herein to refer to both to those polymers which are prepared soley from the lactic acid monomer and to those which are prepared as copolymers with other comonomers of the type listed 15 above. The terms poly(lactide-co-glycolide) and PLGA are used interchanyeably herein to refer to copolymers which are prepared as copolymers of lactic and glycolic acid.
The alpha hydroxy acid units from which the preferred polymers are prepared may be the optically 20 active (D- and L-) forms or optically inactive (~L-, racemic) forms. For example, lactic acid, whether it is the sole monomer, or a comonomer component, can be present as D-lactic acid, L-lactic acid, DL-lactic acid, or any mixture of D- and L lactic acids.
The combinations of preferred monomer and comonomer which can be prepared are numerous, but the most useful are those polymers prepared from lactic acid alone or lactic acid and glycolic acid wherein the glycolic acid is ,oresent as a comonomer in a mnlar ratio of lactide to 30 glycolide units of 100:0 to 30:70, preferably 100:0 to 40:60, for example 75:25 to 40:60. It is most preferred to use a poly(lactide-co-glycolide) copolymer having a molar ratio of lactide to glycolide of between about 75:25 and 50:50.

~3~ ~3 -Poly(lactide-co-glycolide) polymers pre~erably ~ill range in mole~ular wei~ht from about 20pODO to about 100~000 daltons~ stated as an average. The molecular weight Or a parti~ular copolymer is independent of ~ts monomeric ~akeup. For example, the preferred 50:50 copolymer can have a m~lecular weight which ~alls anywhere within this range.
The invention romprehends the use of polymers which are varied both as to their monomer composi~ion and their 10 molecular weight, including thGse outsi~e the preferred compositions and ranges given above, provided that the polymer is capable of bein~ f ormed as a solid material.
For the purposes ot this invention the molecular weight o~ a particular po}ymer is determined as a 15 tunction o~ ~ts intrinsic viscosity as measured in a capillary viscometer using chloroform or hexafluoroisopropanol at 30C. The intrinsic viscosities o~ polylactides suitable ~or use in this invention range from about 0.2 dl/g to about 1.5 dl~g, and are preferably 20 in the range of about 0.33 to 1.0 dltg. (All viscosities given hereafter were measured in hexafluoroisopropanol.) Methods o~ preparing polylactides are well documented in the scientific and patent literature. The following patents, 25 I provi~e ~etailed descriptions of suitable poly}actides, their physical properties, and methods of preparing them: U.S. 3,77~,919, U.S. 4,293,539, U.$. 3,435,008, U.S. 3,442,871, UOS. 3,468,853, U.S. ~ ~97,450, U.S. 3,781,349.
30 U.S. 3,736,646 and ~ Patent No. 4,675,189 , ;. Also European Patent No. 0052510.

' ~ 3~3 The macromolecular polypeptides which may be incorporated in the device of this invention are biologically active molecules having molecular weights greater than about 1000, suitably greater than about 2500, preferably between about 6,000 and 500,000, and more preferably greater than about 10,000, most preferably greater than about 15,000. The choice of polypeptides which can be delivered in accordance with the practice of this invention is limited only by the re~uirement that they be at least very slightly soluble in an aqùeous physiological media such as plasma, interstitial fluid, and the intra and extracellular ~luids of the subcutaneous space and mucosal tissues.
The term "very slightly soluble" refers to a 15 water-solubility of at least about 0.1-1.0 mg/ml, as defined hereinabove.
~ xemplary classes o~ polypeptides include, among others, proteins, en~ymes, nucleoproteins, glycoproteins, lipoproteins, hormonally active polypeptides, and 20 synthetic analogues including agonists and antagonists of these molecules.
The protein classes which are suitable for use in this invention are numerous, including immune modulators, lymphokines, monokines, cytokines, enzymes, 25 antibodies, growth promotants, growth inhibitory factors, blood proteins, hormones, vaccines (including viral, bacterial, parasitic, and rickettsial antigens), blood coagulation factors and the like, including various precursor protein forms, muteins, and other analogs.
30 Also antibodie5.
Specific examples of polypeptides suitable for incorporation in the delivery system of this invention include the following biologically active macromolecules, and muteins and other analogs thereof: interferons - ~93~ ~

(a , ~-, y- and muteins thereof such ~serl7)~
colony stimulating factors (1, 2, 3, GM, ~ -, y-, and the like), interleukins (IL-l, IL-l~, IL~
IL-2, IL-~, IL-4, IL-5, and the like), macrophage activating factors, macrophage peptides, B cell factors (B cell growth factor and the like), T cell factcrs, protein A, suppressive ~actor of allergy, suppressor factors, cytotoxic glycoprotein, immunocytotoxic agents, immunotoxins, immunotherapeutic polypeptides, lymphotoxins, tumor necrosis ~actors (~ , and the like3, cachectin, oncostatins, tumor inhibitory factors, trans~orming growth ~actors such as TGF-a and TGF-~), albumin, alpha-l-antitrypsin, apolipoprotein-c, erythroid potentiating ~actors, erythropoietin, ~actor VII, factor VIII(c), ~actor IX, fibrinolytic agent, hemopoietin-l, kidney plasminogen activator, tissue plasminogen activator, urokinase, pro-urokinase, streptokinase, lipocortin, lipomodulin, macrocortin, lung surfactant protein, protein C, protein ~, C-reactive protein, renin inhibitors, collagenase inhibitors, superoxide dismutase, epidermal growth factor, growth hormone, platelet derived growth factor, osteogenic growth ~actors, atrial naturetic ~actor, auriculin, atriopeptin, bone morphogenic prote.in, calcitonin, calcitonin precursor, calcitonin gene-related peptide, cartilage inducing factor, connective tissue activator protein, fertility hormones (follicle stimulating hormone~ leutinizing hormone, human chorionic gonadotropin), growth hormone releasing factor, osteogenic protein, insulin, proinsulin, nerve growth factor, parathyroid hormone, parathyroid hormone inhibitors, relaxin, secretin, somatomedin C, insulin-like growth factors, inhibin, adrenocoricotrophic hormone, glucagon, vasoactive intestinal polypeptide, 3~2~a3~3 gastric inhibitory peptide, motilin, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, corticotropin releasing factor, thyroid stimulating hormone, vaccine antigens including antigens of HTLV-I, II, AIDS viruses such as HTLV-III/LAVf~IV and HIV-2, cytomegalovirus, hepatitis A, B, and non-A/non-B, herpes simplex virus-I, herpes simplex virus II, malaria, pseudorabies, retroviruses, feline leukemia virus, bovine leukemia virus, transmissible gastroenteritis virus, infectious bovine rhinotracheitis, parain~luenza, influenza, rotaviruses, respiratory syncytial virus, varicella zoster virus, Epstein-8arr virus, pertussis, and anti-infective antibodies including monoclonal and polyclonal antibodies to gram negative bacteria, pseudomonas, endotoxin, tetanus toxin, and other bacterial or viral or other infectious organisms. Also protease inhibitor.
The lists of macromolecular polypeptides recited above are provided only to illustrate the types of active agents which are suitable for use in practicing the invention, and are not intended to be exclusive.
A particularly preferred class of polypeptides are the naturally occurring and synthetic interferons.
Interferons are polypeptides having monomer molecular 25 weights in the range of about 15,000 to about 28,~00.
They are proteins which are synthesized by mammalian cells in response to viral infection, immune stimulation and other factors. They are presently designated as members of one of three major classes: alpha or 30 leukocyte interferon (IFN-), beta or fibroblast interferon (IFN ~), and gamma or immune interferon (IFN-~) Their biological properties include antiviral, anti-proliferative and immunomodulating activities, which have led to kheir clinical use as 35 therapeutic agents for the treatment of viral infections and malignancies.

~L~934~3 Inter~erons can be obtained from natural sources such as leukocytes, lymphoblastoid cells in continuous suspension or culture, and fibroblast cultures. T
lymphocytes can be stimulated to produce gamma interferon. ~-interferon is derived from mammalian cells such as fibroblast cells. As used herein, "~-interferon"
or "IFN-~" includes ~-interferon derived both from natural sources, including human, bovine, canine, feline, porcine and equine, and by recombinant DNA techniques.
10 It also includes modified forms of ~-interferon; e.g., by glycosylation, methylation, substitution and~or deletion of a limited number of amino acids. As used herein, HuIFN-B refers to human ~-interferon, and rHuIFN-B re~ers to HuIFN-B produced using recombinant techniques.
15 IFN-~ser-17 refers to ~-interferon in which the seventeenth amino acid has been replaced with serine.
Inter~eron concentrations are commonly expressed as standard "units" which are internationally accepted and documented, and relate to the potency of a given quantity 20 of interferon to inhibit virus replication under standard conditions.
IFN-~ser 17~ a known compound, is best produced by modifying DNA sequences which code for IFN-B, and then manipulating microorganisms to express the modified DNA
as protein. When the first base of codon 17 (thymine) of the s0nse strand of the DNA sequence which codes ~or the mature IFN-B is replaced with adenine, the cysteine residue at position 17 in the amino acid sequence of IFN-B is replaced by serine. By changing T to other 30 bases, and by changing other bases in codon 17, cysteine may be replaced with other amino acids. The site-specific mutagenesis is induced using a synthetic 17-nucleotide primer having the sequence ~9~ ~3 GCAATTTTCAGAGTCAG which is identical to a seventeen nucleotide sequence on the sense strand of IFN-~ in the region o~ codon 17 except for a single base mismatch at the ~irst base of codon 17. (As used in this context herein, C is deoxycytidine, T is deoxythymidine, A is deoxyadenosine, and G is deoxyguanosine.) The mismatch is at nucleotide 12 in the primer. The 17-mer is hybridized to single-stranded Ml~ phage DNA which carries the antisense strand of the IFN-~ gene. The 10 oligonucleotide primer is then extended on the DNA using DNA polymerase I Klenow fragment (a fragment o~ DNA
polymerase I lacking the 5'-exonuclease subunit) and the resulting double-strand DNA (dsDNA) is converted to closed circular DNA with T4 ligase. Replication of the 15 resulting mutational heteroduplex yields clones from the DNA strand containing the mismatch. Mutant clones may be identified and screened by the appearance or disappearance of specific restriction sites, antibiotic resistance or sensitivity, or by other methods known in 20 the art. When cysteine is substituted by serine, the substitution of T by A results in the creation o~ a new Hinfl restriction site in the structural gene. ~A
restriction site is a point in a DNA sequence that is recognized and cleaved by a particular restriction 25 enzyme. A HinfI restriction site is a restriction site recognized by HinfI endonuclease.) The mutant clone is identified by using the oligonucleotide primer as a probe in a hybridization screening o~ the mutated phage plaques. The primer will have a single mismatch when 30hybridized to the parent but will have a per~ect match when hybridized to the mutated phage DNA. Hybridization conditions can then be devised where the oligonucleotide primer will preferentially hybridize to the mutated DNA
but not to the parent DNA. The newly generated Hinfl 3ssite also serves as a means o~ con~irming the single base mutation in the IFN-~ gene.

~L2933443 The M13 phage DNA carrying ~he mutated gene is isola~ed and spliced into an appropriate expression vector such as plasmid pTrp3, and a host such as Eo coli strain MM294 is then transformed ~ith the vector.
Suitable growth media ~or culturing the trans~ormants and their progeny are known to th~se skilled in the artO The expressed mutein (protein derived ~rom a mutated gene~ of IFN-B is isolated, puri~ied and characterized.
Further description of this method of synthesizing IFN~B can be ~ound in U.S. Pat. No. 4,51~,814, the teachings of which are incorporated herein by rererence.
U.S. Patent No~4,518,5B4 also discloses muteins ot ~-IFN
and interleukin-2, and teaches methods o~ preparing them.
Recombinant DNA techniques for producing interferons o~ the ~-and ~-classes, as well as muteins o~
interferons are also known. Nagata et al., in Nature 284: 316-320 (1980) teaches a method of preparing bacteria which express ~-interferon. y-lnterferon can be produced by the method disclosed in EP0 2D application 013~087A~

In addition to incorporating one or more biolo~ically ac~ive macromolecular polypeptides, the 25 controlled release device of this invention may contain other water soluble, pharmaceutically acceptable compnnents. ~he ~ptional water-soluble components which may be incorporated in the polylactide matrix are present as particles having diameters Or about 10 microns or 30 less. Ir present, they are intimately mixed with the macromolecular polypeptides, and are uniformly and discretely dispersed throughout the palymer.

~7 ~.~934 ~3 Most macromolecular polypeptides benefit from the presence of small quantities of stabilizers, buffers, salts and the like. Water-soluble components which may be useful in the practice of this invention include, but are not limited to other active agents, proteins or other polypeptides, stabilizers, carbohydrates, buf~ers, salts, surfactants and plasticizers. Examples of suitable stabilizers include human serum albumin (HSA), gelatin, dextrose, other carbohydrates. Examples of other carbohydrates suitable for incorporation in this invention include sucrose, maltose, mannose, glucose, fructose, lactose, sorbitol and glycerol. Suitable sur~actants include Tween (e.g. Tween-20, Tween-80), Pluronic polyols such as Pluronic~ LlOl, Ll21 and 15 Fl27 (see standard works such as Merck Index Tenth Edition for further details of these well known surfactants). Among the suitable plasticizers are the polyethylene glycols, glycerides and ethylcellulose.
The relative proportions of macromolecular 20 polypeptide and other-water soluble components to polylactide and water-insoluble components within the matrix can be varied depending on the polypeptide to be administered and the desired rate and duration of release. The macromolecular active agent and other 25 water-soluble components may comprise up to about 30 weight percent of the system. The precise amount will depend on such factors as the potency of the particular active agent, its physiochemical and pharmacokinetic behaviour, its stability and the desired duration of 30 release.
A preferred composition for the polylactide matrix comprises, by weight:
(a) 80 to 99.9999 % polylactide; and ~ ~Z93~'~3 ~21-(b) 0.0001 to 20 % biologically active macromolecular polypeptide and other optional water-soluble components. For very active polypeptides, the total amount of polypeptide and other ~ater-soluble components may be as low as 10%, 5X, 2% or less of the total weight of the matrix.
The present invention is well-suited to the controlled delivery of interferons. The amount of interferon incorporated in the polylatide matrix will 10 preferably be 20%9 or less, depending on the particular interferon and the other factors listed above. A
presently preferred composition comprises, by weight:
(a) 90 to 99.999 % polylactide; and (b) 0.001 to 2 % HuIFN~~, 15 and may include up to about lQ% of other water-soluble components.
A more preferred composition comprises, by weight:
(a) 95 to 99.9 percent polylactide;
(b) 0.01 to 0.1 percent HuIFN-~, 20 and may include up to about 5 % of other water-soluble components.
A particularly preferred composition comprises, by weight:
(a) 97.47 percent poly(lactide-co-glycolide) having a molar ratio o~ 50:50 and an intrinsic viscosity of 25 about 0.64 dl/g;
(b) 0.03 percent HuIFN-~;
(c) 1.25 percent human serum albumin; and (d) 1.25 percent dextrose.
The preferred interferon for incorporation in the 30 ~oregoing systems is rHuIFN-~serl7.

~g34~3 Methods of Preparation The deliYery systems of this invention may be ~abricated by any method which achieves the desired micro-suspension conformation and substantially maintains the biological activity of the macromolecular polypeptide. A preferred method involves spray-casting of a micro-suspension of the polypeptide in a solution of the polylactide. The skilled chemist will comprehend various methods by which the micro-suspension can be made. Two novel and useful methods are described below.
(The skilled man will appreciate that solvents other than acetone and methylene dichloride may be used, provided the protein is compatible with, and insoluble in, the solvent.) Acetone Method An aqueous, bu~fered solution of the macromolecular polypeptide and other optional water-soluble components buffer is added to a solution of the chosen polylactide in acetone at room temperature. The resulting mixture is 20 vortexed at high speed using a standard laboratory vortex mixer for approximately 5 to 120, preferably about lO, seconds. A precipitate of the polymer, polypeptide, and other components is formed ~hich is then centrifuged for about 0.5 to 30 minutes, preferably about lO minutes, at 500 to lO00, preferably 700 X ~. The resulting 25 supernatant o~ acetone and water is removed, additional acetone added, and the mixture vortexed at high speed until the polymer, for example PLGA, in the pellet is dissolved, leaving a micro-suspension of polypeptide and other water-soluble components in the solution of polymer in acetone.
Methylene Dichloride Method An aqueous, buffered solution of the polypeptide and other optional water-soluble components is added to a solution of the chosen polylactide in methylene dichloride. The resulting mixture is vortexed for approximately 10 to 180 seconds, preferably about 60 seconds, at high speed, until a white emulsion is formed. The emulsion is immediately transferred to an airbrush or other suitable spray device and spray cast as described below.
Formation of the Active A~ent Delivery Systems The active agent delivery systems of this invention are formed so that the final solid polypeptide/polylactide matrix product possesses the required micro-suspension morphology in which substantially all of the particles of polypeptide and other water-soluble components have diameters of 10~ or less and are uniformly and discretely dispersed throughout the matrix. To assure that the liquid micro-suspension of water-soluble components in the polymer solution does not coalesce into a suspension of larger particles upon solidification of the formulation, it is preferable to promptly spray-cast the micro-suspension onto a non-stick surface with an airbrush or other suitable device using appropriate conditions. The airbrush is preferably held about 4 to 6 inches from the surface of the sheet and the film sprayed with a constant motion to achieve an even film. Suitable 25 non-stick surfaces include polypropylene, teflon, nylon, polyethylene or derivatives thereof, and other materials with similar non-stick properties. Polypropylene, teflon and polyethylene are preferred. The spray-cast film can be made as thin as about 5 microns and as thick as 1000 30 microns. For films thicker than about 100 microns it is preferable to allow some time for drying between repeated spray-castings of layers. Thinner films (about 10 to 50 microns) are preferred when it is desirable to minimize the exposure of the polypeptide to the organic solvent.

~Z~3~4~

Generally, the resulting film should be allowed to dry completely be~ore being configured into the final controlled release device or system. Depending on the thickness o~ the film, the drying time to achieve complete dryness will vary from less than one hour to about three days, and can be shortened if desired by drying under vacuum after the matrix has solidified to the point where bubbles will not be caused.
For many polypeptides, parenteral injection is a 10 preferred route of administration. The polypeptide/polylactide matrix formulation of this invention can be prepared in an injectable form by atomizing the liquid micro-suspension and drying the resulting micro-particles in a counter-current or vortex 15 ~ air or inert gas. The resulting particles can be injec~ed directly, or can be incorporated in a compatible and pharmaceutically acceptable inJectable solution or suspension.
The controlled delivery systems of this invention 20 may be structurally reinforced with an inert, pharmaceutically acceptable material such as a fine silk mesh) teflon mesh or other surgically inert material. It is especially advantageous to incorporate a reinforcement material when it is anticipated that the controlled release device will need to be recovered from its active delivery site. Reinforced devices may be made by spraying the polypeptide/polylactide micro-suspension onto the reinforcement material, which is preferably resting on a non-stick surface. The ~ilm is then allowed to dry brie~ly, and can be turned over, and sprayed on 3~ the other side. This procedure is repeated until a film of the desired thickness is achieved. Preferably, the texture of the reinforcement material will be completely covered by a smooth layer of the polymer.

The polymeric film obtained by spray-casting as described above can be configured into any solid article suitable for the intended site Qf use. For example, the film can be cut into pieces of known diMensions and implanted subcutaneously as single segments.
Alternatively, the film can be rolled into a cylindrical device of desired dimensions. Multiple lay@rs of film can be laminated and die-cut to create devices of virtually any size and shape. Integrity of the layers can be assured by light spraying or brushing between lamination of layers with a suitable solvent for the polymer or exposure to solvent vapor.
The controIled release devices of this invention can be designed to deliver the biologically active 15 macromolecular polypeptide, and any accompanying active agents, at a controlled rate over a prolonged period oF
time ranging from less than one day to several months.
Examples of devices which delivered therapeutically useful levels of ~-interferon subcutaneously over a 20 period of 60 to lO0 days are described in Examples l and 2 and shown in Figure I. The actual rate and duration of release can be varied within the practice of this invention by the choice of polylactide polymer (e.g.
choice of monomer or comonomers, molar ratio and 25 intrinsic viscosity) or copolymer, by the shape and configuration of the device (e.g. flat, rolled, single layer or multiple layer), and to a lesser extent, by the amount of active agent which is incorporated.
The amount of active agent incorporated in the 0 device can vary between O.OOOl and 30 percent, by weight, o~ the polymeric system. The optimal amount for any given system will depend on the potency of the agent, the desired physiologic effect, the intended length of treatment, and the rate of active agent release.

3d~3 Pre~erably, the devices of this invention contain about 0.0001 to 20 percent. by weight, of the macromolecular polypeptideO
The size of the device will likewise depend on the amount of active agent which it contains, its release rate9 and the intended duration o~ treatment. For example, i~ it is known that a particular polypeptide/polylactide ~ormulation releases the polypeptide at an average rate of 105 units per day, and the desired duration of treatment is 60 days, the device would require a loading o~ at least 6 x 107 units of polypeptide. ~ased on the weight percent polypeptide in the system, the required size of the device can be calculated.
The following preparations and examples are provided to further illustrate the practice of this invention, and are not intended to in any way limit its scope.

20 Cloning of the IFN-~ gene into M13 Vector:
The use o~ M13 phage vector as a source o~
single-stranded DNA template has been demonstrated by G.
F. Temple et al Nature (1982) 296:537-540. Plasmid p~
trp containing the IFN-~ gene, under control of E. coli 25 trp promoter, is digested with the restriction enzymes HindIII and XhoII. The M13mp8 (J. Messing, "Third Cleveland Symposium on Macromolecules: Recombinant DNA,"
Ed. A. Walton, Elsevier Press, 143 - 153 (1981)) replicative form (RF) DNA is digested with restriction enzymes HindIII and BamHI and mixed with the p~l trp DNA
which have previously been digested with HinDIII and XhoII. The mixture is then ligated with T4 DNA ligase and the ligated DNA transformed into competent cells of Z~39~4~
, E. coli strain JM 103 and plated on Xgal indicator plates ~J. Messing et al, Nucleic Acids Res (1981) 9:309 -321). Plaques containing recombinant phage (white plaques) are picked, inoculated into a fresh culture of JM 103 and minipreps of RF molecules prepared ~rom the infected cells (H. D. 8irnboim and J. Doly, Nucleic Acid Res. (1979) 7:1513 - 1523). The RF molecules are digested with various restriction enzymes to identi~y the clones containing the IFN-~ insert. Single-stranded (ss) 10 phage DNA is prepared from clone M13-~1 to serve as a template for site-speci~ic mutagenesis using a synthetic oligonucleotide.

15 Site specific muta~enesis:
Forty picomoles o~ the synthetic ollgonucleotide GCAATTTTCAGAGTCAG (primer) is treated with T4 kinase in the presence of 0.1 mM adenosine triphosphate (ATP). 50 mM hydroxymethylaminomethane hydrochloride (Tris-HCl) pH
20 8.0, 10 mM MgC12, 5 mM dithiothreitol (DTT) and 9 units of T4 kinase, in 50 ~1 at 37C for 1 hr. The kinased primer (12 pmole) is hybridized to 5 ~9 of ss M13 ~1 DNA in 50~1 of a mixture containing 50 mM NaC1, 10 mM
tris-HCl, pH 8.0, 10 mM MgC12 and 10 mM
25 ~-mercaptoethanol by heating at 67C for 5 min and at 42C for 25 min. The annealed mixture is then chilled on ice and then added to 50 ~1 of a reaction mixture containing 0.5 mM each of deoxynucleotide triphosphate (dNTP), 80 mM Tris-HCl, pH 7.4, 8 mM MgC12, 100 mM
30 NaCl, 9 units of DNA polymerase I Klenow fragment, 0.5 mM
ATP and 2 units of T4 DNA ligase, incubated at 37C for 3 hr and at 25C ~or 2 hr. The reaction is then terminated by phenol extraction and ethanol precipitation. The DNA is dissolved in 10 mM Tris-HCl pH

~ ~33~43 ':
.0, 10 mM ethylenediamine~etraacetic acid (EDTA), 50X
sucrose and 0. 05X bromophenylblue and elertropllorf~sed on 0.8% agarose gel in the presence of 2 ~/ml ot ethidium bromide. The DNA bands corresponding to the RF forms of M13-Bl are eluted from gel slices by the perchlorate method (R. W. Davis, et al, "Advanced Bacterial Genetics," Cold Spring Harbor Laboratory, N.Y., p. 178 -179 (198D)). The eluted DNA is used to trans~orm competent ~M 103 cells, grown overnigh~: and single strand (ss~ DNA isolated trom the culture supernatant. This ssDNA is used as a template in a second cycle o~ primer extension, the gel purified RF ~orms of the DNA are transformed into competent JM 103 cells, plated onto agar plates and incubated overnight to obtain phage plaques.

d identi~ication of mutagenized plaques:
Plates containing mutated M13-~1 plaques as well as two plates containing unmutated M13-Bl phage plaques are 20 chilled to 4C, and plaques from each plate transferred onto two nitrocellulose filter circles by layering a dry filter on the agar plate for S min for the first filter and 15 min for the second ~ilter. The filters are then placed on thick filter papers soaked in 0.2 N NaOH, 1.5 M
25 NaCl and 0.2% Triton~X-100 for 5 min. and neutralized by layering onto filter papers soaked ~i~h 0.5 M Tris-HCl, pH 7.5 and 1.5 M NaCl ~or another 5 ~in. The filters are washed in a similar fashion t~ice on filters soaked in 2xSSC (standard saline citrate), ~ried and then baked in a vacuum oven at ~OCC for 2 hr. The duplicate filters 3~ are prehybridized at 55~ for 4 hr. with 10 ml per filter of DNA hybridization buffer (5xSSC) pH 7.0 4xDenhardt's solution (polyvinyl-pyrrolidine, ficoll an~ bovine serum albumin, 1x=o.o2% o~ each), 0.1% sodium dodecyl sulfate *Trade-~ark t ~~

~ ~ Z~39L~3 (SDS), 50 mM sodium phosphate buffer pH 7.0 and 100 ~g/ml of denatured salmon sperm DNA. A 32P-labeled probe is prepared by kinasing the oligonucleotide primer with 32P-labeled ATP. The filters are hybridized to 3.5x105 cpm/ml of 32P-labeled primer in 5 ml per filter of DNA hybridization buffer at 55C for 24 hr.
The filters are washed at 55C for 30 min. each in washing buffers containing 0.1% SDS and decreasing amounts of SSC. The ~ilters are washed initially with buffer containing 2xSSC and the control filters containing unmutated M13-~1 plaques are checked for the presence of any radioactivity. The concentration of SSC
is lowered stepwise and the filters washed until no detectable radioactivity remains on the control ~ilters with the unmutated M13-~1 plaques. The filters are air dried and autoradiographed at -70C for 2 - 3 days.

Expression of mutated IFN-B in E. coli:
RF DNA from M13-SY2501 is digested with restriction enzymes HindIII and XhoII and the 520 bp insert fragment purified on a 1% agarose gel. The plasmid pTrp3 containing the E. coli trp promoter is digested with the enzymes HindIII and CamHI, mixed with the purified 25 M13-SY2501 DNA fragment and ligated in the presence of T4DNA ligase. The ligated DNA is transformed into E.
coli strain MM294. Ampicillin resistant transformants are screened for sensitivity to the drug tetracycline.
Plasmid DNA from five ampicillin resistant, tetracycline-sensitive clones are digested with Hinfl to screen for the presence of the M13-SY2501 insert.
The plasmid designated as clone pSY2501 is available from the Agricultural Research Culture Collection (NRRL), Fermentation Laboratory, Northern Regional Research 3~3 Center, Sçience and Education Administration, U.S.
Department of Agriculture, 1815 North University Street, Peoria, Illinois 60604 and is assigned accession numbers CMCC No. 1533 and NRRL No. B-15356.
Cultures of pSY2501 and p~ltrp are grown up to an optical density (DD600) of 1Ø Cell free extracts are prepared and the amount of IFN~ antiviral activity assayed on GM2767 (mammalian) cells in a microtiter assay.

Purification of IFN~~rerl7 IFN-~Serl7 is recovered from E. coli which have been transformed to produce IFN-~serl7. The E. coli 15 are grown in the following growth medium to an OD of 10-11 at 680 nm ~dry wt 8.4 9/1).
Ingredient Concentration NH4Cl 20mM
K2S04 16.1 mM
KH2P04 7.8 mM
Na~HPO/l 12.2 mM
M9~04-~H20 3 mM
Na~citrate-2H20 1.5 mM
Mn~04 4H20 30~M
ZnS04 7H2 3~M
Cu504-5H20 3 ~M
L-tryptophan 70 mg/l Feso4-7H2o 72 ~M
thiamine HCl 20 mg/l glucose 40 G/L
pH controlled with NH40H

A 9.9 1 (9.9 kg) harvest of the transformed E. coli is cooled to 20C and concentrated by passing the harvest 30 through a cross-flow filter at an average pressure drop of 110 kPa and steadystate filtrate flow rate of 260 ml/min until the filtrate weight is 8.8 kg. The concentrate (approximately one liter) is drained into a vessel and cooled to 15C. The cells in the concentrate ~3~3 are then disrupted by passing the concentrate through a Mason-Gaulin homogenizer at 5C 69,000 kPa. The homogenizer is washed with one liter phosphate bu~fered saline, pH 7.4 (P~S), and the wash is aclded to the disruptate to give a ~inal volume of two liters. This volurne is continuously centrifuged at 12000xg at a 50 ml~min flow rate. The solid is separated from the supernatant and resuspended in ~our liters of PBS
containing 2% by wt. SDS. This suspension is stirred at room temperature for 15 min a~ter which there should be no visible suspended material. The solution is then extracted with 2-butanol at a 1:1 2-butanol:solution volume ratio. The extraction is carried out in a liquid-liquid phase separator using a flow rate o~ 200 15 ml/min. The organic phase is then separated and evaporated to dryness to yield 21.3 g o~ protein. This may then be resuspended in distilled water at a 1:10 volume ratio.
The E. Coli strains used in these Preparations are 20 known materials commerciably available for example from Culture Collections such as the A.T.C.C., or like serl7 is also a known material that is commercially available. See also U.S. Patent No. 4518584.

EXAMPLE I
Preparation o~ Controlled ~elease Devices Containing Interferon ~Acetone Method) 30 A. Preparation of IFN/PLGA Micro-suspension One gram of D,L-PLGA (molar ratio 50:50, inherent viscosity 0.64 dl/g) was dissolved in 5 ml acetone at room temperature. 0.3 mg o~ recombinant HuIFN-~ in 1 ml ~LZ~3~

of buffer (containing 12.5 mg HSA and 12.5 mg dextrose) was added to the PLGA in acetone and the resulting mixture was vortexed at high speed for approximately 30 seconds. The precipitate of PLGA, HSA, IFN and pcssibly dextrose which formed was then centrifuc~ed for 10 minutes at 700 X 9. The supernatant of acetone and water was removed with a pipet and the residual liquid removed with a cotton swab. Ten ml acetone was added, and the mixture was vortexed at high speed until the PLGA in the pellet was dissolved, leaving a micro-suspension of HuIFN-~, HSA
and dextrose in a solution of PLGA in acetone.
B. Seray-Casting of the IFN/PLGA Micro-suspension The resulting IFN/PLGA micro-suspension, obtained as described in paragraph A, was sprayed with an airbrush, using compressed air at 15 PSI, onto a clean polyethylene sheet. The airbrush was held 4 to 6 inches ~rom the surface of the sheet and the film sprayed with a constant motion to achieve an even ~ilm of the PLGA formulation which was approximately 50 microns thick.
2 C. Reinforcement of Film O
Using IFN/PLGA micro-suspension from paragraph A, a spray-cast film with silk reinforcement was prepared as follows:
Fine woven silk mesh was stretched on a frame and 25 the stretched portion brushed with a solution of 100 mg/m} PLGA (molar ratio 50:50, intrinsic viscosity 0.64) in acetone. The wet mesh was allowed to dry, and then brushed with repeated applications of PLGA solution until the pores in the silk mesh were completely filled. The mesh was then dried, placed on a polyethylene sheet, and spray cast with the IFN/PLGA micro-suspension. After drying for one hour, the coated mesh was turned over, coated side down, and again sprayed with the IFN/PLGA
micro-suspension, applying a layer of polymer about 100 ~34~3 -3~-microns thick. After drying for another hour, the previously coated side was again sprayed, allowed to dry, turned over, and the second side sprayed again. The resulting ~ilm had a thickness of 300 microns.
D. The films obtained in paragraphs B and C were stored at room temperature for 18 hours. They were then removed from the polyethylene sheet and dried at room temperature for three days.
E. Device Configuration Using spray-cast film obtained as described in paragraphs A-D, above, controlled release devices were configured as follows:
a. Flat film segments, 1 x 2 cm, were cut ~rom the non-reinforced film.
b. Flat ~ilm segments, 1 x 2 cm, were cut ~rom the rein~orced film.
c. Flat ~ilm segments, 3 x 5 cm, were cut ~rom non-reinforced ~ilm, were ro1led on an 18 gauge wire and the film secured by a very light application o~ acetone 20 with a cotton swab to the ~inal 5 mm length, or by exposure to acetone vapor. The wire was removed and the rolls sliced into lengths o~ 5 or 10 mm.
d. Flat ~ilm segments, ~ x 5 cm, were cut ~rom reinforced film, were rolled on an 18 gauge wire and the film secured by a very light application of acetone with a cotton swab to the final 5 mm length, or by exposure to acetone vapor. The wire was removed and the rolls sliced into lengths of 5 or 10 mm.
The release pro~iles o~ these devices when implanted subcutaneously in mice over a period of 100 days are 30 shown and identified in Figure I as devices 1, 2, and for configurations a, b9 and c respectively.

,, . , ` " ` ~: .... .

~ ~R~3~3 Preparation of Controlled Release Devices Containing Inter~eron (Methylene Dichloride Method) One gram of D,L-PLGA (molar ratio 50:50, intrinsic viscosity 0.64 dl/g~ was dissolved in 4 ml methylene dichloride. 0.3 mg of recombinant HuIFN-~ in l ml of buffer containing l2.5 mg/ml human serum albumin (HSA) and 12.5 mgtml dextrose was added to the dissolved PLGA
solution. The resulting mixture was vortexed for approximately 60 seconds at high speed until a white emulsion was formed. The emulsion was immediateLy transferred to an airbrush and sprayed onto a 15 polyethylene film, and dried in the same manner as described in Example l, paragraph D.
Controlled release devices were configured by rolling 3 cm x 5 cm film segments on an 18 gauge wire, securing the end o~ the roll by exposure to acetone, 20 removal of the wire, and slicing of each roll into 5 and lO mm lengths.
The release profile of these devices when implanted subcutaneously in mice over a period of 60-lOO days is shown as device 4 in Figure I.

Determination of In Vivo Release Profile When Implanted Subcutaneously In Mice 30 A. Release profile of ~-Interferon Sixty of each of devices 1-4 were prepared as described in Examples l and 2, but the HuIFN-~ was spiked with radioactively-labelled ~-interferon -- , .

.Z~33~ ~3 N ~serl7). The devices were sterilized with 1.25 Mrads o~ gamma-irradiation and implanted subcutaneously in the dorsal region o~ ICR ~emale mice weighing 18-20 gm. One device wes implanted in each mouse. A~ter varying intervals of time (1 to 100 days), the devices were removed ~rom the mice and the radioactivity o~ the remaining 125I-rHuIFN-~se~l7 was determined. Figure I shows release proFiles o~ devices 1-4 over a period of up to 100 days in vivo.

Determination o~ Particle Size and Distribution PLGA/IFN films prepared as described in Examples 1 and ~, above, were analyzed to determine the particle size of the interferon and other macromolecules (human serum albumin and dextrose) in each formulation, according to the following procedures:
A. PLGA/IFN films prepared as described in Lxample 1 One gram of D,L-PLGA (molar ratio 50:50, intrinsic viscosity 0.64 dl/g) was dissolved in 5 ml acetone at room temperature. 0.3 mg of recombinant HuIFN-~ in 1 ml o~ buf~er containing 12.5 mg HSA and 12.5 mg dextrose was 25 added to the PLGA in acetone and the mixture was vortexed at high speed for approximately 10 seconds. The precipitate o~ PLGA, HSA, IFN and possibly dextrose which formed was then centrifuged for 10 minutes at 7no X ~.
The supernatant of acetone and water was removed with a 30 pipet and the residual liquid removed with a cotton swab. Ten ml acetone were added, and the resulting mixture vortexed at high speed until the PLGA in the pellet was dissolved) leaving an IFN, HSA, dextrose precipitate suspended in PLGA dissolved in acetone. A

3~L~3 drop of the suspension was viewed under a polarizing light microscope on a glass slide with cover slip at lOOX
magnification using an ocular reticle with lû micrometer divisions.
The particle sizes of the solid macromolecular components (IFN, HSR, dextrose) suspencled in the PLGA/acetone solution ranged from less than or equal to the limit of detection (approximately lO0 to 500 nanometers) to lO0 microns. Particles having diameters of greater than lO microns were less than lO % of the total numb2r ot particles, and most of the particles had diameters of less than l micron. Less than lO % of the visible particles could be observed to be touching at least one other particle.

B. PLGA/IFN films prepared as described in Exam~le 2 A drop of the PLGA/IfN micro-suspension prepared according the the method described in Example 2 above was viewed under a light microscope on a glass slide with 20 cover slip at lOûX magnification using an ocular reticle with lO micrometer divisions. No particles were observed at lOOX magnification, indicating that all of the IFN and HSA had particle sizes of less than or egual to the limit of detection (lO0 to 500 nanometers).

Assay of Interferon Biological Antiviral Activity The assay for interferon biological antiviral activity measures the effect interferon exerts on cells by monitoring their inhibition o~ the cytopathic effect of vesicular stomatitis virus (VSV) in human wish cells.
Virus caused cell damage can be visualized in the light ~5 ` 3~10Y 25570 .:

3~3 microscope. In cells that are incubated with active interferon, virus growth is reduced. The units of active interferon are determined as reciprocals of endpoint dilutions of an interferon preparation~ and the endpoint is defined as the dilution of interferon which inhibits growth of virus by about fifty percent.
The interferon contained in controlled release systems prepared as described in Examples l and 2 and spiked with a known concentration of [l25I]rHuIFN-~serl7 was extracted from the systems as described in Example 6, below. The extracted interferon samples were assayed to determine the biological activity of the interferon in manufactured systems relative to the biological activity of the loading stock interferon, as described in paragraphs A-D, below.
A. Method 25 ~l of each interferon sample to be assayed and the reference material are pipetted individually into a row of wells of a sterile 96 well microtiter plate 20 containing 50 ~l Eagle's minimum essential media (EMEM) per well. The reference material is the international standard of HuIFN-~ from the National Institutes of Health, Ref. No. G-023-902-527. Each sample is tested in duplicate, and one row column of each plate is reserved 25 for controls to which are added an additional 25 ~l EMEM. The plates are then treated under UV light for 6 minutes to prevent bacterial growth. Serial three-fold dilutions (standard one-half logl0 dilutions) of each sample are then prepared in the remaining wells of the microtiter plate by dilution with EMEM to obtain 50 ~l of diluted sample in each well. 50 ~l of 2 % fetal calf serum (FCS) in EMEM, followed by lO0 ~l of a well-mixed suspension of Human WISH cells in EMEM with 5 ~ FCA, are added to each well, to result in addition of 2.5 X 104 cells~well. The plates are then incubated at 35 37 C in 5% C02 for 24 hours.
~610Y 25570 334~3 Approximately 24 hours a~ter addition of the WISH
cell suspension, 50 ~1 of VSV in EMEM, prepared in a dilution that adds at least one plaque forming unit of VSV per cell, are added to each ~ell, with the exception of four control wells.
The virus-treated plates are incubated at 37 C
under 5 % C02 and are scored approximately 18 hours after addition of the VSV.
B. Scoring The plates are read under a light microscope, and scores recorded when the virus controls reach complete cytopathic effect (CPE) and the endpoint of the references is at the expected titer. Each test well is accorded a score as ~ollows: SP, possible CPE;
1, 25 % o~ cells have CPE; 2, 50% have CPE; 3, 75 % of cells have CPE; 4, 100 % of cells have CPE; C, bacterial contamination; and CT, cell cytotoxicity.
The endpoint of a sample titration is the well which first scores 50 % CPE. The titer in log10 units~ml of IFN corresponds to the dilution of that well, and is corrected according to the reading of the reference standard.
C. Calculation of Interferon Specific Biological Activity The radioactivity of three 1 to 50 ~1 aliquots of each undiluted interferon sample is determined by counting in a Packard gamma counter. From the result in counts per minute (CPM), the counts per unit volume is determined (CPMtml). The IFN activity (IFN units/ml) of each sample, determined according to the method described 30 in paragraphs A and B, above, is divided by the CPM/ml value ~or the sample, giving the activity of the IFN in units/CPM.
The units/CPM value for each sample obtained by extraction from a manufactured polylactide system is 3 divided by the units/CPM value for the corresponding 25'13~

starting stock interferon material (loading stock IFN) used to make the manufactured polylactide/interferon systems, to give the ratio of specific activity o~
extracted interferon to the specific activity of loading stock IFN. The ratio so obtained is multiplied by the IFN units/ml value for the loading stock IFN~ which gives the loading stock IFN units/ml equivalent of the extracted IFN sample.
The logl0 of the loading stock IFN units/ml equivalent for each extracted sample is termed the relative logl0 IFN activity (RLIA). RLIA values for each group of samples tested are averaged and compared to the logl0 IFN units/ml value of the appropriate loading stock. Additional accuracy can be gained by a linear regression analysis o~ RLIA values obtained ~rom a series of systems which have been implanted in a test animal, such as mice, and serially recovered at several intervals over the test period, e~g one month. The Y-intercept of the line determined from a graph of RLIA values (Y axis) 20 versus days of implantation (X axis) indicates the activity of the interferon in the manufactured systems prio~ to implantation.
D. Results The novel controlled release systems claimed herein 25 demonstrate RLIA values following manu~acture, but prior to in vivo application, which are one/half, or more, of the logl0 IFN units/ml of the corresponding polypeptide loading stock.
Interferon/polylactide systems prepared as described 30 in Examples l and 2, when assayed as described in this example, show essentially no loss of biological activity of the incorporated interferon; that is, the average RLIA
values after manufacture but before implantation are essentially indistinguishable from the logl0 IFN
35 units/ml of the IFN loading stock from which they were prepared.
~610Y 25570 ~3~

Extraction of Polypeptide from a Polylactide Matrix A. Extraction of Inter~eron from the Polylactide Matrix of Devices Prepared According to Examples l and 2 Interferon containing systems prepared as described in Examples l and 2 from a loading stock inter~eron spiked with a known concentration o~
[ I]rHuIFN-~serl7 were individually dissolved in acetone (up to 300 mg polylactide to lO ml of acetone), and vortexed at high speed until the polylactide was completely dissolved, and the interferon left as a precipitate suspension. Each suspension was centri~uged at 700 X ~ for lO minutes and the acetone/polylactide supernatant removed. The residual pellet was dried for 24 hours under vacuum at room temperature, and was subsequently extracted ~or l hour with 0.5 ml of 12.5 mg/ml HSA and 12.5 mg/ml dextrose at room temperature 20 with periodic mild agitation. Each tube was centri~uged at 700 X ~ for lO minutes, and the inter~eron-containing supernatant removed and stored at 4C. Ten, 50 and l00 microliter samples of the supernatant were used to determine the radioactivity per unit volume. Following determination of interferon activity, the specific activity (IFN units~radioactivity counts per minute) of the extracted interferon was compared to that of the inter~eron stock starting material. Determination o~ the radioactivity per unit volume and the biological activity of the extracted interferon are described in Example 5.

33~43 Biological Activity of HuIFN-~ in Polylaotide Delivery Devices Prepared by a Known Heat-Formation Me~hod.

A. Preparation o~ Heat-Formed Polylactide Devices Polylactide matrix drug delivery devices containing HuIFN-~ as the active ingredient were prepared according to a known heat-extrusion method outsicle of the scope and practice of this invention, whereby the polypeptide and polylactide are combined and mixed in a heat extrusion apparatus. Ten grams of D,L-PLGA, (molar ratio 50/50, intrinsic viscosity 0.64 dl~gm), was mixed with the contents of 25 vials of lyophilized human recombinant interferon containing 0.3 mg IFN-~ with 4.2 X 107 interferon units, 12.5 mg human serum albumin and 12.5 mg dextrose per vial. The mixture was placed in the loading funnel of a heated extrusion device and extruded at a temperature of approximately 75C through a 3 mm cirucular exit die, and immediately reduced to room temperature by forced air cooling. The resulting rod of interferon/polylactide material was segmented into 7 mm lengths.
B. Extraction of Interferon The interferon/polylactide devices formed by the method described in paragraph A were individually weighed and placed in separate 2 ml glass vials containing 1 ml of buffer solution (74.9 % phosphate buffer pH 7.4, 25 %
ethanol and 0.1 % SDS~. The vials were maintained at 4C
30 with mild circular agitation for 24 hours. Following extraction, the devices ~ere removed from the vial and the extract stored at 4C until they were assayed.
C. Assay for Interferon Ciological Activity The interferon activity, units/ml of extract, was determined by the assay method described in Example 5.
The estimated total interferon units contained in each ~3~43 _42-device was calculated from the product of the dry device weight and the interferon units/gram dry weight of the formulation.
D. Results The interferon extracted from the heat formed devices gave an RLIA value (relative loglQ inter~eron activity) o~ less than 1% of the log10 units/ml of the corresponding interferon loading stock.

Preparation of a Finely Divided Injectable or Implantable Controlled ReLease System A micro-suspension of interferon in a polylactide 15 solution is prepared as described in Example 1 or 2. The solution is then atomized with a spray device, and the resulting particles dried and prilled as they settle in a counter-current or vortex of clean air, nitrogen, or other inert gas. The resulting particles of 20 polypeptide/polylactide matrix are stored under vacuum for 3 days, and then sized for use or storage.
Controlled release systems prepared in this manner may be incorporated in an injectable suspension and administered subcutaneously or intramuscularly.

The following describes a formulation for parenteral injection of finely divided polypeptide/polylactide particles prepared according to the methods disclosed herein.
Finely divided interferon containing polylactide particles prepared as described in Example 8 are suspended in the following solution ~3~ 3 sodium carboxymethylcellulose 0.5~
NaCl 0.6%
Benzyl alcohol 0.9%
Tween 80~ 0.1%
Purified water q.s. 100%

For example, 330 mg of interferon~polylactide particles are suspended in 5.5 ml of the above solution to provide an injectable dose of 9 ~9 of interferon per 0.5 ml of 10 injectable suspension.
The foregoing discussion and specific embodiments are intended to be exemplary of the scope and practice of this invention, and should not be read to limit the practice of the described invention.

~610Y 25570 . .. .

Claims (39)

1. An active agent delivery system for the controlled administration of a macromolecular polypeptide to a mammal, which system comprises a polymeric matrix comprising not more than about 30 percent by weight of particles of macromolecular polypeptide and other optional water-soluble components, dispersed in a polylactide, wherein substantially all of the particles of polypeptide and other water-soluble components have diameters of 10 µ or less and are uniformly and discretely dispersed throughout the matrix, and wherein the polypeptide retains at least about 50 percent of the biological activity which it possessed prior to manufacture of the matrix.

2. The system of claim 1 in which the polypeptide has a molecular weight greater than about 10,000.

3. The system of claim 1 in which the polypeptide is selected from the group consisting of cytokines, lymphokines, monokines and interferons.

4. The system of claim 1 which is formed from one or more layers of a spray-cast film.

5. The system of claim 4 which incorporates a biocompatible inert reinforcement material.

6. The system of claim 1 in which the polylactide is a poly(lactide-co-glycolide) copolymer having a molar ratio of lactide to glycolide units of between about 100:0 and 30:70.

7. The system of claim 2 in which the polylactide is a poly(lactide-co-glycolide) copolymer having a molar ratio of lactide to glycolide units of between about 100:0 and 30:70.

8. The system of claim 3 in which the polylactide is a poly(lactide-co-glycolide) copolymer having a molar ratio of lactide to glycolide units of between about 100:0 and 30:70.

9. The system of claim 4 in which the polylactide is a poly(lactide-co-glycolide) copolymer having a molar ratio of lactide to glycolide units of between about 100:0 and 30:70.

10. The system of claim 5 in which the polylactide is a poly(lactide-co-glycolide) copolymer having a molar ratio of lactide to glycolide units of between about 100:0 and 30:70.

11. The system of anyone of claims 1 to 3 in which the polypeptide is interleukin-1, interleukin-2, or an analog thereof.

12. The system of anyone of claims 4 to 6 in which the polypeptide is interleukin-1, interleukin-2, or an analog thereof.

13. The system of anyone of claims 7 to 9 in which the polypeptide is interleukin-1, interleukin-2, or an analog thereof.

14. The system of claim 10 in which the polypeptide is interleukin-1, interleukin-2, or an analog thereof.

15. The system of anyone of claims 1 or 2, in which the polypeptide is calcitonin or an analog thereof, or parathyroid hormone or an analog thereof.

16. The system of anyone of claims 4 to 6, in which the polypeptide is calcitonin or an analog thereof, or parathyroid hormone or an analog thereof.

17. The system of anyone of claims 7, 9 or 10 in which the polypeptide is calcitonin or an analog thereof, or parathyroid hormone or an analog thereof.

18. The system of anyone of claims 1 or 2, in which the polypeptide is epidermal growth factor, transforming growth factor-a, transforming growth factor-.beta. or an analog thereof.

19. The system of anyone of claims 4 to 6, in which the polypeptide is epidermal growth factor, transforming growth factor-.alpha., transforming growth factor-.beta. or an analog thereof.

20. The system of anyone of claims 7, 9 or 10 in which the polypeptide is epidermal growth factor, transforming growth factor-a, transforming growth factor-.beta. or an analog thereof.

21. The system of anyone of claims 1 to 3 in which the polypeptide is a beta interferon.

22. The system of anyone of claims 4 to 6 in which the polypeptide is a beta interferon.

23. The system of anyone of claims 7 to 9 in which the polypeptide is a beta interferon.

24. The system of claim 10 in which the polypeptide is a beta interferon.

25. The system of anyone of claims 1 to 3, in which the polypeptide is an immune stimulator or an immune depressant.

26. The system of anyone of claims 4 to 6, in which the polypeptide is an immune stimulator or an immune depressant.

27. The system of anyone of claims 7 to 9, in which the polypeptide is an immune stimulator or an immune depressant.

28. The system of claim 10 in which the polypeptide is an immune stimulator or an immune depressant.

29. The system of anyone of claims 1 or 2, in which the polypeptide is superoxide dismutase or a plasminogen activator.

30. The system of anyone of claims 4 to 6, in which the polypeptide is superoxide dismutase or a plasminogen activator.

31. The system of anyone of claims 7, 9 or 10 in which the polypeptide is superoxide dismutase or a plasminogen activator.

32. The system of anyone of claims 1 or 2 in which the polypeptide is a growth hormone or a growth hormone releasing factor.

33. The system of anyone of claims 4 to 6 in which the polypeptide is a growth hormone or a growth hormone releasing factor.

34. The system of anyone of claims 7, 9 or 10 in which the polypeptide is a growth hormone or a growth hormone releasing factor.

35. The system of anyone of claims 1 or 2 in which the polypeptide is bovine growth hormone.

36. The system of anyone of claims 4 to 6 in which the polypeptide is bovine growth hormone.

37. The system of anyone of claims 7, 9 or 10 in which the polypeptide is bovine growth hormone.

38. A process for preparing the system of claim 1, which process includes the step of preparing a microsuspension of the polypeptide, and other optional water-soluble components, in the polylactide solution.

39. The use of the system of claim 1 for the treatment of disorders.