AU656173B2 - Interaction between bioadhesive liposomes and target sites - Google Patents

Description

OPT PATE 15/09/92 AOJP DATE 29/10/92 APPLN. ID 12592 92 PCT NUMBER PCT/IIS91/n8112 )N TREATY (PCT)

INTER.

(51) Intt national Patent Classification 5 (11) International Publication Number: WO 92/14446 A61K 9/127 Al A6K 9/127 (43) International Publication Date: 3 September 1992 (03.09.92) (21) International Application Number: PCT/US91/08112 (81) Designated States: AT (European patent), AU, BE (European patent), CA, CH (European patent), DE (Euro- (22) International Filing Date: 30 October 1991 (30.10.91) pean patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, LU (Euro- Priority data: pean patent), NL (European patent), SE (European pa- 655,013 14 February 1991 (14.02.91) US tent).

(71) Applicant: BAXTER INTERNATIONAL INC. [US/US]; Published One Baxter Parkway, Deerfield, IL 60015 With international search report.

Before the expira't."n of the time limit for amending the (72) Inventors: MARGALIT, Rimona 52 Zabotinsky Street, 53 claims and to be republished in the event of the receipt of 318 Givataim ROSEMAN, Theodore 16 Not- amendments.

tingham Drive, Lincolnshire, IL 60069 WOOD, 3 Ray, W. Route 5, Box 258, Elkhorn, WI 53121 (US).

(74) Agents: ROCKWELL, Amy, L. H. et al.; One Baxter Park- 6 way, Deerfield, IL 60015 (US).

(54)Title: INTERACTION BETWEEN BIOADHESIVE LIPOSOMES AND TARGET SITES (57) Abstract Recognizing substances, epidermal growth factor, gelatin, collagen and hyaluronic acid, have been covalently bound to liposomal surfaces and utilized to attach liposomes onto a cellular target site. These "bioadhesive" liposomes offer several advantages in the area of topically and locally administered free drug. These advantages include the mutual protection of both the drug and biological environment; an increase in drug bioavailability and retention at the target site; and improved adherence or adhesion to the designated target site.

WO 92/14446 PCT/US91/08112 1 INTERACTION BETHEEN BIOADHESIVE LIPOSOMES AND TARGET SITES BACKGROUND OF THE INVENTION The present invention relates to a novel drug delivery system, particulasly to microscopic drug delivery systems (MDDS) utilizing drug-enc;psulating "bioadhesive" liposomes for topical and local drug administration.

Currently, the topical and local administration of a drug can be in its free form, dissolved or dispersed in a suitable diluent, or in a vehicle such as a cream, gel or ointment.

Examples of therapeutic or designated targets for topical or local drug administration Include burns; wounds; bone injuries; ocular, skin, intranasal and buccal infections; ocular chronic situations such as glaucoma; and topically and locally accessed tumors. Several difficulties exist with either the topical or local administration of a drug in its free form. For example, short retention of the drug at the designated site of administration reduces the efficacy of the treatment and requires frequent dosing. Exposure of the free form drug to the biological environment in the topical or local region can result In drug degradation, transformation into inactive entities and nondiscrimlnating and uncontrollable distribution of the drug. Such degradation and uncontrollable distribution of.the drug can result In toxicity issues, undesirable side effects and loss of efficacy.

Microscopic drug delivery systems (MDDS) have been developed to overcome some of the difficulties associated with free drug administration. MDDS is divided into two basic classes: particulate systems, such as cells, microspheres, viral envelopes and liposomes; or nonparticulate systems which are macromolecules such as prctels or synthetic polymers.

Using these specific systems, drug-loaded MDDS can perform as WO 92/14446 PCT/US91/08112 sustained or controlled release drug depots. By providing a mutual protection of the drug and the biological environment, MDDS reduces drug degradation or inactivation. As a system for controlled release of the drug, MDDS improves drug efficacy and allows reduction in the frequency of dosing. Since the pharmacokinetics of free drug release from depots of MDDS are different than from directly dminlstered drug, MDDS provides an additional measure to reduce toxicity and undesirable side effects.

Liposomes offer a range of advantages relative to other MDDS systems. Liposomes are lipid vesicles composed of membrane-like lipid layers surrounding aqueous compartments.

Composed of naturally-occurring materials which are biocompatible and bioaegradable, liposomes are used to encapsulate biologically active materials for a variety of purposes. Having a variety of layers, sizes, surface charges and compositions, numerous procedures for liposomal preparation and for drug encapsulation within them have been developed, some of which have been scaled up to industrial levels.

Through appropriate selection of liposome type and size, the encapsulated drug can also range in size. Liposomes can accommodate lipid-soluble drugs, aqueous soluble drugs and drugs with both hydrophlllc and hydrophobic residues.

Liposomes can be designed to act as sustained release drug depots and, In certain applications, aid drug access across cell membranes. Their ability to protect encapsulated drugs and other characteristics make liposomes a popular choice In developing MDDS, with respect to the previous practices of free drug administration.

Despite the advantages offered, utilization of drug-encapsulating liposomes does pose some difficulties. For example, liposomes as MDDS have 1lmited targeting abilities, limited retention and stability in circulation, potential

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r-, -3toxicity upon chronic administration and inability to extravasate. In recent years, attempts have been made to couple different recognizing substances with liposomes to confer target specificity to the liposomes, namely antibodies, glycoproteins and lectins. Although the bonding of these recognizing substances to liposomes occurred, the resulting modified liposomes did not perform as hoped, particularly during in vivo studies. Other difficulties are presented when utilizing these recognizing substances. For example, antibodies can be patient specific and therefore, add cost to the drug therapy.

Several cell-associated entities can participate in the binding between cells and recognizing substances. These are generally divided into three major types: receptors and nonreceptor components of the cellular system and extracellular matrix. Receptors can be present in several species or states, differing in populations per cell and in binding affinity. Binding to such receptor entities is usually referred to as "specific binding". Non-receptor cell membrane components also differ in populations and in affinity. Binding to such non-receptor entities is usually referred to as "non-specific binding".

To perform effectively, the topical or local administration of drug-encapsulating liposomes should have specificity for and the ability to adhere to the designated target area and should facilitate drug access to S intracellular sites. Currently available liposomes and other *9* t rJ 1_ ;L L I 'Y i i -3a- MDDS systems do not meet these performance requirements of topical and local drug administration.

SUMMARY OF THE INVENTION Accordingly, the present invention provides a method of topical substance administration to a designated tissue or tissue-like matrix having a target site comprising the steps of:forming bioadhesive liposomes having the characteristics of specificity for and binding to the target 10 site, the liposomes including a drug-encapsulating liposome component an1 a recognizing substance component selected from the group consisting of epidermal growth factor, hyaluronic acid, gelatin and collagen, in which said recognizing substance component is bonded covalently to the liposomal 15 surface by a cross-linking reagent without disulfide reduction; and, applying the bioadhesive liposomes topically to the target site.

It has been learned that modifying regular liposomes by covalently anchoring certain recognizing substances to the-- WO 92/14446 PCT/US91/0811.2 4 liposomal surface creates a "bloadhesive" liposome with target specificity and retention. The recognizing substances are molecules which can be utilized as an adhesive or glue, attaching a drug-encapsulating liposome onto a therapeutic target site. These "bioadheslve" recognizing substances can perform either through receptor mechanisms or through associations with components within the extracellular matrix.

Regardless of the specific mechanism of adhes'in, these substances are referred to as "bloadhesive recognizing substances" based on their common end result.

Through covalent anchoring, the bloadhesive recognizing substances become an Integral part of the liposome, yet remain accessible to the Interaction counterpart at the target site.

They endow the liposome and encapsulated drug with the ability to adhere to the target site. Hence, "bioadhesive" liposomes have been developed which are target adherent, sustained release drug depots. The identification of recognizing substances and the methodologies of modifying liposomes has been disclosed in concurrently filed applications. These bloadhesive liposomes offer several advantages over previous practices of topically or locally administered free drug and other MDDS, whether with regular liposomes or other MDDS systems. These advantages include the mutual protection of both the drug and biological environment; an increase in drug bioavallability and retention at the target site; and improved adherence or adhesion to the designated target site. These advantages result in the potential reduction of undesirable biological side-effects of the drug being administered.

BRIEF DESCRIPTION OF THE DRAHINGS FIG. 1 shows the binding of bloadhesive liposomes (EGF-modified; open double triangle) and regular liposomes (asterisk) of the LUVET type to A431 cells In culture (in I WO 92/14446 PCT/US91/08112 monolayers), as dependent upon liposome concentration. Bound liposomes, denoted as B, are in units of ng EGF per 106 cells.

Free ligand concentration, denoted as L, are in units of ng EGF per 106 cells for bioadhesive liposome (first row of L values) and in units of umoles lipid per 10 6 cells for the regular liposomes (second row of L values).

FIG. 2 shows a time course of the binding of bioadhesive liposomes (collagen-modified) of the MLV type to A431 cells in culture (in monolayers). Collagen is tritium-labeled. The fraction of liposomes relative to the amount present in the initial reaction mixture at zero-time which is cell-associated is determined over time.

FIG. 3 shows the binding of bloadhesive liposomes (collagen-modified) and regular liposomes of the MLV type to A431 cells in culture (in monolayers). Collagen is tritium-labeled 3 and liposomes are 14 -C labeled. Bound liposomes, denoted as B, are in units of 3 -H DPM per 105cells (left scale) and in units of 14 -C DPM per 10 5 cells (right scale). Free ligand concentration, denoted as L, are in units of 3 -H or 14 -C DPM per 105 cells. Bloadhesive liposomc with collagen labeled is depicted with open double triangles; bioadhesive liposome with the liposome labeled is depicted with crosses; and, regular liposome is depicted with asterisks.

DETAILED DESCRIPTION According to the present invention, bloadhesive liposomes have bound to cell cultures havitg receptors or extracellular matrix which accommodate the recognizing substance bonded to the liposome. Liposomes, in particular, multilamellar vesicles (MLV), microemulsified liposomes (MEL) or large unilamellar vesicles (LUVET), each containing phosphatidylethanolamine have been prepared by established procedures.

Recognizing substances, each of which have been accepted for WO 92/14446 PCT/US91/08112 -6human use, include epidermal 'growth factor (EGF), hyaluronic acid gelatin and collagen. Each of these recognizing substances have a biological origin and are biodegradable and biocompatible. Further, these recognizing substances have functional residues which can be utilized in covalent anchoring to the regular liposomal surfaces.

The methodologies of preparing the specific bloadhesive liposomes have been disclosed In separate applications concurrently filed with this disclosure and will not be repeated here.

A complete accounting of binding entities has been determined by the previously known multi-term Langmuir Isotherm equation, as applied for the quantitative description of the relationship between the free and dependent variables: n Bmax i

[L]

B. I (1) 1.1 Kdi [L1 where n is the number of different cell-associated binding entities that a cellular system has for a specific recognizing substance; is the concentration of free ligand, which can be recognizing substance, free liposomes or bloadhesive liposomes; B is the total quantity of bound recognizing substance per given number of cells, at a given and, Bmaxi and Kdj are the total number of sites of a given entity and the corresponding equilibrium dissociation constant. B and Bmax are normalized for the same number of cells.

For cases In which receptors and non-receptor cell membrane components participate in the recognizing substance binding and in which the dissociation constant of the non-specific binding is sufficiently large with respect to the free ligand concentration, equation 1 can take the form: t i t WO 92/14446 PCTI/US91/08112 n-I Bmax i

[L]

B Kns (2) 1.1 Kd i

[L]

where the last term, Kns is the contribution of the non-specific binding to B and K.s is the ratio of Bmax to Kd corresponding to the non-specific binding.

"Best-fit" values for parameters n, Bmax i and Kd i are obtained by computer-aided data analysis, according to equations and/or above, applying nonlinear regression procedures.

The interaction of the bioadhesive EGF-modlfled liposomes has been established with cultures of A431 cells, in monolayers, as a biological model. This well-established cell line, originating from human epidermoid carcinoma, is enriched with EGF receptors. A431 cells have been repeatedly used for study of the interaction of free EGF and its receptor.

A431 cells have been shown to have three classes of EGF receptors, differing in their affinities and populations. The first o these classes is the ultra-high affinity sites with an equilibrium dissociation constant of 0.07 nM and a population of 150-4000 sites per cell. The next class is the high affinity sites with an equilibrium dissociation constant of 0.7 nM and a population of 1.5 x 105 sites per cell. The final class Is the low affinity sites with an equilibrium dissociation constant of 5.9 nM and a population of 2 x 106 sites per cell.

Example One To compare the binding ability of regular liposomes and bioadhesive liposomes, A431 cell cultures were grown in monolayers, In flasks, applying usual procedures for this cell WO 92/14446 PCT/US91/08112 line. Two to three ,oI- prior to an experiment, the cells were seeded into multlwell culture plates and the experiments were done when the systems were confluent.

For purposes of assaying the modified liposomes, the EGF-recognizing substance was labeled with a generally known radioactive marker. Preparation of EGF-modlfied LUVET was completed as disclosed In the concurrently filed applications.

Prior to the addition of a reaction mixture of EGF-modlfied llposomes, free liposomes or free EGF, media was removed from the A431 cells and the cells were washed with a binding buffer. The reaction mixture and cells were incubated for 1-2 hours, at room temperature. Upon dilution and withdrawal of the reaction mixture at the end of incubation, 2-3 successive washings with a binding buffer,of the wells were completed. Lysis of cells or detachment of cells from the wells was then followed by withdrawal and collection of the well content, denoted as the cell fraction. Assays of the cell fraction were complet by label counting of the fraction as compared with the counting of the immediate products created through the preparation process.

A comparison between the binding of free llposomes and EGF-modified liposomes to the A431 cells is illustrated in Figure 1. The EGF-modifled liposomes adhere to the A431 cells considerably better than free liposomes as no free liposomes were found at cell fraction. It is speculated that If free liposomes do associate with the cells, the dilution brought by the washings is sufficient to cause quantitative dissociation.

Example Two Binding studies of EGF-modifled liposomes to A431 cells were carried out as described In example 1 and the data were processed according to equation above. The experimental conditions were such that the contribution of non-specific 1: r I

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PCT/US91/08112 WO 92/14446 -9binding was negligible.

unambiguously with a sing liposome system studied.

listed In Table 1.

Indeed, the data were found to fit le type of binding site for each Results for several systems are TABLE 1 BINDING PARAMETERS OF BIOADHESIVE LIPOSOMES TO A431 CELLS IN CULTURE

BIOADHESIVE

LIPOSOME SYSTEM (a)

EGF-MLV

EGF-MLV

EGF-LUVET

EGF-MEL

EGF-MEL

EGF-MEL

(nL_ 0.60 0.017 5.03 1.9 2.91 0.003 0.04 0.007 0.40 0.13 0.48 0.05 SITES PE B

CELL

(xlO 0.17 0.03 1.07 0.03 0.18 0.001 0.042 0.0042 3.7 0,90 0.28 0.01 Each bloadhesive liposome system preparation; recognizing substance in Is a different each system is EGF.

AnEGF-modlfled liposome is considerably larger than and different from free EGF, which is expected to affect the binding parameters. For a given class of receptors, the magnitudes of the dissociation constants for EGF-modlfled liposome systems are expected to be similar to or higher than those of free EGF. For a given class of receptors, the n'mber of receptors per cell that are available for the EGF-modifled liposol es is expected to be equal to or lower than the number of available for free EGF. Based on these considerations, the WO 92/14446 PCT/US91/08112 10 binding data of the present ekample fit with the receptor classes of ultra-high and high affinities.

Regardless of the specific cell-associated binding entity involved, the binding data listed in Table 1 show that EGF-modlfied liposomes bind to this cellular system with high affinity and with a sufficient number of sites for these modified liposomes to perform as the desired bloadhesive 11posomes.

Example Three Binding collagen-modified 11posomes to A431 cells was carried out essentially according to the procedures detailed above. The A431 cell line is not known to contain receptors for collagen. The interaction of either free collagen or liposomally bound collagen with the A431 cell line is expected to result from association of collagen with components within the extracellular matrix. Referring to Figure 2, incubation periods up to 4 hours were completed with 3 hours being the optimal period for binding and collagen-liposome concentrations.

Quantitative evaluations of bind,;ig of collagen-modified liposomes to A431 cells In culture are compared to regular liposome and exemplified i,i Figure 3. The data were processed according to equation above. Through double labeling, 3-H-collagen and 14-C-cholesterol, it was possible to monitor the collagen and liposome simultaneously. The binding of the collagen-modified liposomes to the cells is greater than the binding of the corresponding regular liposomes.

For free and collagen-modified liposomes, the binding entities are of the extracellular matrix type of cell-associated entity. As in the case of EGF-modifled liposomes discussed In example 2, the dissociation constant for collagen-modified liposomes is expected to be similar to or higher than those of free collagen. Likewise, the number of I

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LiK WO 92/14446 PCT/US91/08112 11 available sites in the extracellular matrix available for collagen-modified liposomes Is expected to be similar to or lower than free collagen.

with these considerations.

demonstrate that binding of substance to this cellular measurable phenomena, which quantitative and meaningful Table 2 show quite clearly collagen-modified liposomes The example given In Table 2 fits The data for free collagen this bloadhesive recognizing system does occur and Is a can be processed to yield parameters. Moreover, the data in that the binding of to this cellular system Is of sufficiently high affinity and with a large enough number of sites, for the collagen-modified liposomes to perform as the desired bioadhesive liposomes.

TABLE 2 BINDING PARAMETERS OF FREE RECOGNIZING SUBSTANCES AND BIOADHESIVE LIPOSOME TO A431 CELLS IN CULTURE

BIOADHESIVE

LIPOSOME SYSTEM FREE COLLAGEN

COLLAGEN-MLV

K NUMBER O SITES 0) 8.5 2.3 179 11 67.6 31.35 548 160 While the preferred embodiments have been described, various modifications and substitutions may be made without departing from the scope of the invention. For example, the mouse EGF and human urogasterone used In the disclosed examples could be substituted with EGF from other natural or synthetic sources.

Similarly, the collagen, gelatin and HA could come from other natural or synthetic sources. Accordingly, It Is to be understood that the invention has been described by way of illustration and not limitation

Claims (12)

1. 2. The method of administration of claim 1 wherein the liposome component is selected from the group consisting of multilamellar vesicles, microemulsified liposomes and large unilamellar vesicles.
2. 3. The method of administration of claim 1 wherein the liposome component includes phosphatidylethanolamine.
3. 4. The Method of administration of claim 1 wherein the recognizing substance component confers specificity for and binding to the target site by a mechanism of adhesion through receptor mechanisms af the target site. The method of administration or claim i wherein the recognizing substance component confers specificity for and /X binding to the target rite by a mechanism of adhesion through r AN -I 1 Ua-4- -13- 1 'I 4 4 associations with components within an extracellular matrix at the target site.
4. 6. A method of local substance administration to a designated tissue or tissue-like matrix having a target site comprising the steps of:- forming bioadhesive liposomes having the characteristics of specificity for and binding to the target site, the liposomes including a drug-encapsulating liposome component and a recognizing substance component selected from the group consisting of epidermal growth factor, hyaluronic acid, collagen and gelatin, in which said recognizing substance component is bonded covalently to the liposomal surface by a cross-linking reagent without disulfide reduction; and, applying the bioadhesive liposome locally to the target site.
5. 7. The method of administration of claim 6 wherein the liposome component is selected from the group consisting of multilamellar vesicles, microemulsified liposomes and large unilamellar vesicles.
6. 8. The method of administration of claim 6 wherein the liposome component includes phosphatidylethanolamine.
7. 9. The method of administration of claim 6 wherein the recognizing substance component confers specificity for and binding to the target site by a mechanism of adhesion through receptor mechanisms at the target site. The method of administration of claim 6 wherein the recognizing substance component confers specificity for and l~- -14- binding to the target site through associations with the components within the extracellular matrix at the target site.
8. 11. A liposome for topical or local substance administration to a designated tissue or tissue-like matrix having a target site comprising:- a bioadhesive lipcsome having an encapsulated liposome component; i a recognizing substance component covalently bonded to the liposomal surface by a crosslinking reagent without disulfide reduction; and, wherein the recognizing substance component confers to the bioadhesive liposome target specificity for and retention at the target site.
9. 12. The liposome of claim 11 wherein the liposome component is selected from the group consisting of multilamellar i vesicles, microemulsified liposomes and large unilamellar .II. vesicles.
10. 13. The liposome of claim 11 wherein the liposome component includes phosphatidylethanolamine.
11. 14. The liposome of claim 11 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through receptor mechanisms at the target site. The liposome of claim 11 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through associations with components within an extracellular matrix at the target site. III I it
12. 16. The liposome of claim 11 wherein the recognizing substance component is selected from the group consisting of gelatin, collagen, hyaluronic acid and epidermal growth factor. Dated this 30th day of August, 1994. BAXTER INTERNATIONAL INC. Patent Attorneys for the Applicant PETER MAXWELL ASSOCIATES at I i I el I I I A/. i