EP0705096A1 - Surface-modified albumin microspheres and pharmaceutical compositions containing them - Google Patents

Abstract

Internally cross-linked albumin microspheres their surface being modified by attachment of polyoxy(C1-4)alkylene chains having a terminal ether group, especially such containing pharmacologically active agents, usable for pharmaceutical compositions.

Description

SURFACE-MODIFIED ALBUMIN MICROSPHERES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

The invention relates to microspheres, especially to nanospheres and to pharmaceutical compositions containing such particles , loaded with drug compounds. Microspheres have diameters between 10 nanometers and 5000 micrometers ( = 5.10⁶ nanometers). Nanospheres between 10 nanometers and 1 micrometers. If in the following the expression microspheres is used, nanospheres are considered to be included.

An important objective in current drug therapy is the selective delivery of drugs to a specific target site in the body. Adverse reactions and unwanted side effects could be avoided and highly bioactive drugs like analogs of hormones (LH-RH) or cytokines(interleukines, interferons, tumour necrosis factors) entrapped in colloidal carriers could be delivered to their site of action. The mononuclear phagocyte system (MPS) however is one of the major obstacles to prolonged circulation of colloidal carriers in blood. This disadvantage is a common feature of all particulate carriers unless they are modified appropriately to circumvent the host's defence system.

Different approaches have been taken to change the surface properties of carriers to protect them against mononuclear phagocytic clearance or alter the organ distribution. Colloidal carriers from polystyrene polyesters or poiy(methyl-methacrylate) have been modified by adsorption of poloxamers. Biomaterials 8, p. 113-117 (1987). Increasing hydrophilicity of the poloxamers decreased MPS uptake of the particulate carriers. By incorporation of poly(ethylene)glycol (PEG)-lipid derivatives into liposomes, a remarkable enhancement of the circulatory life has recently been reported. Biochim. Biophys.Acta 1066, p. 29-36 (1991).

Proteins covalently modified with PEG derivatives have shown decreased immunogenicity and clearance in vivo. J. Control. Rel. 11 , p. 139-140, (1990). These interesting properties of PEG may be combined with a chemically and physically stable carrier system, albumin microspheres, which are known to be biodegradable. Albumin micropsheres are acceptable for human application. The surface of chemically crosslinked albumin nanospheres provides suitable functionalities for chemical modification, as shown in Fig.1. For a 7 micrometer albumin microparticSe the number of carboxylic acid and amino functionalities were estimated to be in the order of 62 x 10⁵ and 6 x 10^s respectively, [Townes A.B., Martin G.P, James S.L, Mariott C (1989) J. Pharm. Pharmacol. 41.140P]. Amino groups were associated with activated monomethoxypoly(ethylene)glycol (mPEG) to give a carbamyl linkage whereas activated carboxyl residues were amidated by adding methoxypolyoxyethyleneamine (mPEGamine) being modified by attachment of.

The invention provides intemally cross-linked albumin microspheres, especially nanospheres, their surface being modified by attachment of polyoxy(C₁.₄)alkylene chains having a terminal ether group. For example their surface has been modified by covalent attachment of poly(ethylene)glycol or polyoxyethylene amine having one terminal ether group (see Figure 1). The polyoxyalkylene moieties have preferably a molecular weight of from 120 to 40.000 daltons, e.g. 500 to 10.000 daltons, especially 5000 daltons.

The albumin substance is preferably human serum albumin, bovine serum albumin or egg albumin cross-linked by an aliphatic dialdehyde, e.g. glutaraldehyde, glyoxyl, dimethylglyoxal or ketones, e.g. 2,3-butane dione, esters, e.g. ethylene glycol bis- succin-imidyi-succinate, acid chlorides, e.g. terephthalic acid dichloride and diisocyanates, e.g. toluene diisocyanate, or by di-, tri- and tetravalent metallic cations or by heat (90-170"C, 10-60 min). [Tomlinson. E and Burger. J. (1987) in Ilium L. Davis S.S. (ed), Wright, pp 25-48]. These methods may be effected in conventional manner.

The polyoxyalkylene moiety may be reacted with the surface carboxylic acid and amino groups e.g. by condensation with an appropriate functional group, e.g. amino or alcohol group in the presence of a condensation agent e.g. 1 , 1 - c a r b o n y l d i i m i d a z o l a n d N - ( 3 - d i - m e t h y l - aminopropyl)-N'-ethylcarbodiimidehydrochloride (EDC).

The protecting terminal ether group is preferably an (C, alkyl ether, e.g. a methylether group.

The presence of the terminal ether group is a preferred measure for the preparation of the modified microspheres. Without its presence unprotected poly(ethylene)glycol or poiyoxyethyleneamine used for the preparation of the modified microspheres would be bifunctional and thus cross-link the nanospheres externally to a compact water insoluble useless mass.

Phagocytic uptake of colloidal carriers can be quantified by several methods, [Jungi T W, (1988) In: Pal SB (ed.) Macmillan Press, pp. 31-55]. The oxidative burst of polymorphonuclear neutrophils (PMNs) following phagocytosis is a convenient model to study factors influencing this defence mechanism against particulate foreign material. The oxidative burst leads to the production of peroxide radicals which are quantified by chemiiuminescence, [Allen R C, Loose L D (1976) Biochem. Biophys. Res. Com. 69: 245-252].

The surface properties of albumin microspheres were changed by covalently coupling mPEG to their surface in order to reduce phagocytic uptake.

In J. Macromol. Sci. Chem. A 28(8), 743-760 (1991), (CA 115: 189 562) intemally cross-linked albumin microspheres are described, bound via a spacer, e.g. a shorter or longer bifunctional polyoxyethylene chain, to a galactose group, used for liver specific drug targeting. In the intemally cross-linked albumin a pharmacologically active substance, e.g. the antitumor compound 5-fluorouracil is incorporated for the treatment of liver cancer.

The polyoxyethylene spacer is not used for an improved resistance against phagocytes and thus a prolonged residence in the blood circulation, but mainly for providing a better flexibility between the albumin and the galactose group and thus less steric hindrance and thus for improving the binding capacity of the galactose part to the asialoglycoprotein receptors of hepatocytes.

In the intemational PCT-application WO 91/18020 a central nucleus, preferably an intemally cross-linked albumin, is described, bound to antibodies, e.g. antifibrin antibodies, and to drug substances, e.g. thrombolytic agents e.g. streptokinase, each via bifunctional dextran or polyoxyethylene groups e.g. for the treatment of fibrin clots.

The drug substance is thus externally bound to cross-linked albumin.

For analytical reasons, the macromolecular complex is provided with a detectable marker, e.g. a radioactive isotope, preferably introduced into the albumin part.

The complex is contacted and binded via the antibody part with the target and the bound complex is detected, thereby measuring the residence time of the complex at the target site, not primarily the residence time of free, unbounded complex in the blood circulation (see claim 39, page 35, lines 28-34 and page 36, lines 1-5).

A direct measure just as according to the invention, to enlargen the resistance to phagocytes and to prolong the residence time in the blood with a monofunctional polyoxyethylene group on the surface of the albumin part of the macro-molecular complex, is not taken.

EXAMPLE 1 :

Synthesis and characterisation of nanospheres: 5 ml of an aqueous bovine serum albumin solution (4-20, preferably 5%) were injected into 120 ml of methylene chloride circulating through a static mixer and an ultrasonic reaction vessel at a speed of 500 ml/min and the mixture was sonicated for 15 min with an energy flux of 100 Watt cm². (Sonifier B-30, Branson, FRG). The temperature was maintained at 22^*C (+/- 2°C) by refrigerating the reaction vessel. Optionally an active agent e.g. octreotide may be present at a concentration of 1 to 10% w/w. Crosslinking was then inititated by adding 0.2 ml of glutaraldehyde- saturated methylene chloride and accomplished by stirring the emulsion during 60 min. After washing with methanol, acetone and finally with n-hexan, the particles were isolated as free flowing brownish powder wich was dried under high vacuum for 24 h. The diameters of the nanospheres were measured by photon correlation spectrometry (Zetasizer III, Malvem Instr. Ltd.Malvern, FRG).

Example 2:

Albumin microspheres containing octreotide were prepared according to the procedure outlined in Example 1 with the additional measure that 0.05 g octreotide/g albumin was dissoved in the aqueous albumin phase.

Example 3:

Albumin microsphrere≥ containing IL-6 were prepared in the manner according to Example 2 using 0.05 g IL-6 /g albumin.

According to the method described above particles with diameters larger than 200 nm can be produced by reducing the emulsifying intensity e.g. by replacing the high energy ultrasonic processor by a 4-bladed impeller and using higher concentrations of albumin (e.g. 10 - 25 % mV).

To estimate the amount of the charge-shielding effect of the surface modification the electrophoretic mobility of the particles was determined by Laser Doppler Anemometry (Zetasizer III, Malvern Instr. Ltd.Malvern, FRG) and transformed to a zeta potential using the Smoluchowski equation . These measurements were performed (in quadruplicate) in a diluted phosphate buffer saline solution with anionic strength of 0.002 M and a pH of 7.4 at 25^*C.Results are the mean and standard deviation of five measurements. The morphology of the spheres was observed by scanning electron microscopy (Stereoscan 180, Cambridge.UK).

Coupling of mPEG to amino residues, [Beauchamp CO, Gomas SL, Menapace DP, Pizzo SV (1983) Anal. Biochem. 131 : 25-33], m-PEG was activated by 1 ,1'-carbonyldiimidazol in THF under argon at 40^°C for 12 hr. To this solution a suspension of nanoparticles in THF and triethylamine were added. The reaction was carried out at 60°C under argon and while stirring. After 60 hr the PEG-modified nanoparticles were isolated by centrifugation (47800g, 10 min) (Sorvall RC>-5B, Sorvall, Wilmington, U.S.A) and several washing steps in methanol. Finally the nanospheres were collected on a 0.1 m filter and dried under high-vacuum for 24 hours.

Coupling of mPEGamine to carboxylic acid residues. [Townes A B, Martin G P, James S L, Mariott C (1989) J. Pharm. Pharmacol. 41: 140P]: A solution of EDC (1.7 mmol) was added to a suspension of albumin nanospheres in purified water. The reaction mixture was stirred for 20 min at room temperature. 0.17 mmol of mPEGamine was added and the suspension was stirred on a magnetic stirrer at 25^*C for 7 hr. After the neutralization with NaOH (0.1 M) the nanospheres were centrifuged ( 47800 g, 15 min) and washed as described earlier.

Phagocytosis in vitro: Fresh human PMNs from healthy donors were prepared by separation on a percoll gradient [Hansel T T, De Vries I J M, Iff T, Rihs S, Wandzilak M, Betz S, Blaser K, Walker C (1991), J. Immunol. Methods 145: 105-110] and diluted with hanks' buffered salt solution (HBSS). The cell number was adjusted to 7x10⁶ cells /ml. Cell preparations were counted on an automated cell counter (Microcell Counter CC-18,Sysmex,Japan). Cytospins were prepared on a Shandon Cytospin 2 (Shandon.U.K.) and stained with the May-Grϋnwald-Giemsa method prior to light microscopic cell differentiation. The preparation of PMNs was of 96%-98% purity. HBSS and phosphate buffered saline (PBS) was enriched with 2% foetal calf serum (FCS).

The stock solution of Lucigenin was made by dissolving in HBSS to a final concentration of 2.5x10-4 M.

The albumin particles were dispersed in PBS (without FCS) and ultrasonicated (Sonogen, Branson, FRG) for 15 minutes.The particle mass was 0.312 mg/ml (= 2 x 10⁹ 220 nm particles). To measure the chemiluminescence 100 microliter of lucigeninsolution, 100 microliter of particles and 50 microliter of cellsuspension were added to one well of a 96-weil plate (Dynatech, FRG) and the measurement in a MTP-Reader (Hamamatsu Photonics Deutschland GMBH) at 37°C was immediatly started. In general the chemiluminescence (CL) was measured during 40 min with a sampling time of 10 sec. Each experiment was performed quadruplicate (4 wells). The relative standard deviation of the mean was about 5%. The cellresponse was taken as background and subtracted for each experiment from the CL time profile of the particle assays. The intensity/time profiles were analysed using the MTP-Reader software and the AUC (arbitrary units) was calculated as the time integral of CL emission accumulated over 40 min.

Unmodified albumin nanoparticles showed an average diameter of 220 nm, whereas particles with mPEG attached to amino- and carboxylic groups showed an increase of 44nm in mean diameter with no change in polydispersity (Fig. 2). No difference in morphology of the spheres was detected by scanning electron microscopy.

However, no size increase was observed for particles which had attached mPEG only to amino groups. This is probably due to steric requirement, allowing linear PEG to arrange in loops or folds. With a higher surface coverage of PEG by modifying amino- and carboxyl groups, the ethylene oxide chains had to unfold starlikely and lead to an increase of the hydrodynamic radius measured by photon correlation spectroscopy.

By the zeta potential measurement very slight changes in surface properties of colloidal particles can be detected. It was used to quantify the shielding effect of the covalently attached hydrophiiic polymer. Unmodified microspheres with an average diameter of about 220 nm, showed a zeta potential of about - 36 mV, measured in a 2 mMol/l of phosphate buffer. The coupling of activated mPEG with the amino groups reduced the zeta potential by 14 mV (39%) for mPEG-5000, respectively 3.5 mV (10%) for mPEG-2000, and 0 mV (0%) for mPEG-750 (Fig.3). This gradual decrease of zeta potential relates to increased polymer layer thickness based on increasing chain length of PEG. The polymer layer shifts the plane of shear of the diffuse layer to a larger distance from the particle surface which results in a decrease of the measured zeta potential [Napper D H, Netschey A J (1971) Colloid and Interface Science 37: 528-535]. Thus a higher steric barrier activity is observed with increasing chain lenght of PEG.

In a second step mPEGamine was linked with free carboxylic acid residues on the surface of previously with mPEG-5000 modified particles. This procedure resulted in a 94% (35 mV) reduction of the zeta potential (Fig. 3). This additional treatment of the carboxylic acid residues with mPEG led to a nearly complete screening of the surface charge. Direct modification of the carboxylic groups of the particles with EDC without previous attachment of mPEG to the amino groups was followed by interparticulate crosslinking and agglomeration. Hence quenching the amino groups with mPEG causes steric stabilization and prevents interactions between particles during further chemical modifications.

Measuring the phagocytic activity of PMNs by a chemiluminescence assay does not require labelling of the particles with fluorescent dyes or radioactive compounds and therefore possible particle surface alterations can be avoided.

CL was used to monitor time-dependent phagocytic uptake. With regard to reducing the volumes of human blood required for the assay and getting a sufficient sensible response 350O00 cells per well were chosen.

The CL intensity/time profile of differently modified particles is shown in Fig. 4. A very distinct decrease of phagocytic response was seen demonstrating that both, chain length and number of PEG chain influence phagocytic uptake in PMNs.

The CL response expressed as AUC showed for mPEG-750 modified particles a reduction of 13% compared to unmodified particles. Particles grafted with mPEG-2000 showed a 76%, those with mPEG-5000 a 85% decrease respectively. Additional treatment of the mPEG-5000 particles with mPEGamine led to a striking reduction of 92% of the CL response (Fig. 5). Other studies of liposomal formulations with different molecular weights of PEG [Mori A, Klibanov A L, Torchilin V P, Huang L (1991) FEBS Letters 284: 263-266] confirm the observations that the shielding effect of lower molecular weights of PEG was not as effective as higher ones.

Steric barrier activity and hence reduced interaction with human phagocytes are directly correlated to the chain length of PEG.

Blood level curves of albumin nanoparticles marked with ¹²⁵iodine, having a mean diameter of 180 nanometer, modified with PEG-5000 and with mPEG -5000 -amine were during at least 1 hour significantly different from corresponding curves of unmodified nanoparticles (see Fig. 6).

The blood circulation half time values (t 1/2) were numerically fitted to an expression of the form [R(t) = K₀ + K, exp (-tK₂)] and calculated with [t1/2 = In 2/K₂] (Martin A, Swarbrick J., Camarata A, Physikalische Pharmazie, Strieker H, (ed) Wissenschaftliche Verlagsgesellschaft, 1987).

The half-value time of the circulation of the modified particles was increased with a factor 2.6 (unmodified particles 2,13 min, modified particles 5.55 min, calculated over 4 hours), resulting in a 43% increase of the AUC (= Area jJnder Curve).

Albumin nanoparticles generally show a light accumulation in special body organs, liver, spleen, and also the bone marrow which last phenomenon is of interest for cytokines, and other immune modulating substances, if incorporated in the nanoparticles.

Accumulation of m-PEG-modified nanoparticles was also observed in the said organs and was, even to a greater extent, observed in the bone marrow. It is expected, that if higher molecular weight mPEG and mPEGamine is used for modification, more pronounced effects may be shown.

One hour after injection 56% of the injected dose of unmodified particles were found in the liver, 4% in the spleen and 1% in the lungs. The corresponding amounts of the modified particles were 50%, 6.5% and 4.5% respectively. In the kidney with both modifications only small amounts of radioactivity (0.2 and 0.3% respectively) could be observed, presumably caused by free iodine (Fig. 7). After four hours only 21% of the injected dose of unmodified particles were found in the liver, 0.5% in the spleen and 0.5% in the lungs (Fig. 8). The amounts of PEG- modified particles were 26% of the injected dose in the liver, 2% in the spleen and 3% in the lungs (Fig. 8). The biodegradation of the unmodified albumin nanoparticles seems to be much more rapid than the degradation of PEG-modified nanoparticles (see liver and spleen). This can be explained by the fact, that modifying the particle surface with PEG results in a highly flexible polymer-shell around the particle, which sterically hinders the interaction with immune cells. The PEG-modification would thus be favourable to depot formulations.

The modified microspheres according to the invention preferably contain pharmacologically active agents. They are used for sustained release and selective delivery of the agents to a specific target site in the body, e.g. the bone marrow.

The formulations according to the invention may be used to administer a wide variety of classes of active agents, e.g. pharmacologically active agents such as contraceptives, sedatives, steroids, sulphonamides, vaccines, vitamines, anti-migraine drugs, enzymes, bronchodilators, cardiovascular drugs, analgesics, anti-tumor compounds, antibiotics, antigens, anti-convulsive drugs, anti-inflammatory drugs, anti-parkinson drugs, prolaction secretion inhibitors, anti-asthmatic drugs, geriatics and anti-malarial drugs. The active agent may be chosen from a wide variety of chemical compounds, e.g. lipophilic and/or hydrophilic active agents, including peptides, such as octreotide (described in the UK Patent GB 2 234 896A).

The active agent may be incorporated in the albumin particles in conventional manner, e.g. by adding to the pre cross-linking mixture of albumin. Typical final concentrations are from 0.01% to 30% of the resultant microspheres. The invention therefor provides also pharmaceutical compositions containing pharmacologically active agents in intemally cross-linked albumin microspheres, their surface being modified by attachment of polyoxy(C₁.₄)alkylene chains having a terminal ether group.

The formulations may be used for the known indications of the particular drug compound incorporated therein and may have a depot function.

The active proteins or peptides are preferably cytokines, e.g. interleukins, G-CSF, M-CSF or GM-CSF, and cyclosporins or analogs of hormones e.g. octreotide.

The pharmaceutical compositions may be used for

immune stimulation in case of cytokines, e.g. interleukins (IL-3, IL-6), hematopoietic colony stimulating factors (G-CSF, GM-CSF, M-CSF),

accompanying therapy with a cytostatic treatment e.g. cytokines esp. interleukins (IL-3, IL-6) or a lipid derivative e.g. the compound described in EP 0309411 , especially in Example 1 ,

specific immune supression, e.g. with cyclosporins,

cancer treatment e.g. with octreotide, cytokines esp. interleukins, and anti- tumor compounds,

selective targeting e.g. for the treatment of leishmaniasis, of fungus infections, of enzyme storage illnesses (Tay Sachs, Gauckerillness),

aids therapy e.g. targeting in macrophages.

The exact amounts of drug compound and of the depot formulation to be administered depends on a number of factors, e.g. the condition to be treated, the desired duration of treatment, the rate of release of drug compound and the degradability of the albumin matrix.

The desired formulations may be produced in known manner. The amount of the pharmacologically active agent required and the release rate thereof may be determined on the basis of known in vitro or in vivo techniques, e.g. how long a particular active agent concentration in the blood plasma remains at an acceptable level. The degradability of the matrix may also be obtained by in vitro or especially in vivo techniques, for example wherein the amount of matrix materials is determined by measuring the amount of radio-active label in the organs.

The formulations of the invention may be administered, e.g. subcutaneously or intramuscularly, preferably as intravenous suspensions, particularly as a suspension in a suitable liquid carrier.

Repeated admiministration of the formulations of the invention may be effected when the albumin matrix has sufficiently been degraded, e.g. from several hours to several days or weeks.

An advantage of the albumin matrices of the invention is that during and after the release of the drug compound they may be quickly degraded to a molecular size, which may be transported by the body fluids from the site of administration.

Examples of doses for octreotide are e.g. 1 mg per day for human beings of 70 kg of body weight for acromegaly breast cancer or gastroenteropancreatic tumors in a depot formulation having microspheres which contains the peptide in an amount from at least 0.05, preferably 0.05 to 30 percent by weight relative to the albumin matrix, especially 0,1 to 0,5% of weight.

The release time of the peptide from the microspheres may be from several hours to about 2 weeks or longer.

Conveniently the sustained release formulation comprises the octreotide in the albumin carrier which, when administered to a rat subcutaneously at a dosage of 10 mg octreotide per kg of animal body weight, exhibits a concentration of octreotide in the blood plasma of at least 0.3 ng/ml and preferably less than 20 ng/ml during a longer period.

Examples of doses for the preferred compound IL-6 are 0.05 - 0.5 mg per day for human beings for e.g. immune stimulation in chemotherapy and bone marrow transplantation in e.g. a parenteral formulation having microspheres which contain the peptide in an amount of at least 0.05, preferably 0.05 to 30 percent by weight relative to the albumin matrix, especially 0.3 to 5% of weight.

The invention also provides a method of administering a pharmaceutical composition according to the invention to a subject, which comprises a parenteral, oral or ocular administration to a subject in need of such treatment.

Claims

WHAT WE CLAIM IS:

1. Intemally cross-linked albumin microspheres, their surface being modified by attachment of polyoxy(C )alkylene chains having a terminal ether group.

2. Microspheres according to claim 1 containing pharmacologically active agents.

3. Microspheres according to any one of claims 1 or 2 the albumin substance being human serum albumin.

4. Microspheres according to any one of claims 1 to 3, to which polyoxyethylene chains are attached.

5. Microspheres according to any one of claims 1 to 4 to which chains are attached having a terminal (C^)alkyl ether group.

6. Microspheres according to any one of claims 2-5 containing a cytokine.

7. Microspheres according to claim 6 containing an interleukin.

8. Microspheres according to claim 6, containing a hematopoietic colony stimulating factor.

9. Microspheres according to any one of claims 2-5 containing octreotide.

10. Pharmaceutical compositions containing microspheres according to claim 2.

11. A method of administering a composition according to claim 10 to a subject which comprises administering it parenterally to a subject in need of such treatment.