WO1992014446A1

WO1992014446A1 - Interaction between bioadhesive liposomes and target sites

Info

Publication number: WO1992014446A1
Application number: PCT/US1991/008112
Authority: WO
Inventors: Rimona Margalit; Theodore Roseman; Ray W. Wood
Original assignee: Baxter International Inc.
Priority date: 1991-02-14
Filing date: 1991-10-30
Publication date: 1992-09-03
Also published as: AU656173B2; EP0525154A1; CA2079450A1; JPH05506040A; AU1259292A

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.

Description

INTERACTION BETWEEN BIOADHESIVE LIPOSOMES AND TARGET SITES

BACKGROUND OF THE INVENTION The present invention relates to a novel drug delivery system, particularly to microscopic drug delivery systems (MDDS) utilizing drug-encapsulating "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 entitles and nondiscriminating 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 proteins or synthetic polymers.

Using these specific systems, drug-loaded MDDS can perform as 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-administered 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

biocompatlble and biodegradable, 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 hydrophilic 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 limited targeting abilities, limited retention and stability in circulation, potential toxicity 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, glycoprotelns 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 entitles can participate in the binding between cells and recognizing substances. These are generally divided into three major types: receptors and non-receptor 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 entitles is usually referred to as "specific binding". Non-receptor cell membrane components also differ in populations and in affinity. Binding to such non-receptor entitles 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 intracellular sites. Currently available liposomes and other MDDS systems do not meet these performance requirements of topical and local drug administration.

SUMMARY OF INVENTION

It has been learned that modifying regular liposomes by covalently anchoring certain recognizing substances to the liposomal surface creates a "bioadhesive" 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 "bioadhesive" recognizing substances can perform either through receptor mechanisms or through associations with components within the extracellular matrix. Regardless of the specific mechanism of adhesion, these substances are referred to as "bioadhesive recognizing substances" based on their common end result.

Through covalent anchoring, the bioadhesive 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 bioadhesive 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 bioavailability 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 DRAWINGS

FIG. 1 shows the binding of bioadhesive liposomes

(EGF-modified; open double triangle) and regular liposomes (asterisk) of the LUVET type to A431 cells in culture (in 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 10⁶ 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 bioadhesive liposomes

(collagen-modified) and regular liposomes of the MLV type to A431 cells in culture (in monolayers). Collagen is

tritium-labeled (³-H) and liposomes are ¹⁴-C labeled. Bound liposomes, denoted as B, are 1n units of ³-H DPM per 10⁵cells (left scale) and in units of ¹⁴-C DPM per 10⁵ cells (right scale). Free ligand concentration, denoted as L, are in units of ³-H or ¹⁴-C DPM per 10⁵ cells. Bloadheslve liposome 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, bioadhesive liposomes have bound to cell cultures having 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 (PE), have been prepared by established procedures.

Recognizing substances, each of which have been accepted for human use, include epidermal growth factor (EGF), hyaluronic acid (HA), 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 bioadhesive liposomes have been disclosed in separate applications concurrently filed with this disclosure and will not be repeated here.

A complete accounting of binding entitles 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:

where n is the number of different cell-associated binding entities that a cellular system has for a specific recognizing substance; [L] is the concentration of free ligand, which can be recognizing substance, free liposomes or bioadhesive liposomes; B is the total quantity of bound recognizing substance per given number of cells, at a given [L]; and, Bmax_i and Kd_i are the total number of sites of a given entity and the corresponding equilibrium dissociation constant. B and B_max 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:

where the last term, K_ns [L], is the contribution of the non-specific binding to B and K_ns 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 (1) and/or (2) above, applying nonlinear regression procedures.

The interaction of the bioadhesive EGF-modified liposomes has been established with cultures of A431 cells, 1n

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 1n their affinities and populations. The first of 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 × 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 line. Two to three days prior to an experiment, the cells were seeded into multiwell culture plates and the experiments were done when the systems were confluent.

For purposes of assaying the modified liposomes, the EGF-recogniz1ng substance was labeled with a generally known radioactive marker. Preparation of EGF-modified LUVET was completed as disclosed in the concurrently filed applications.

Prior to the addition of a reaction mixture of

EGF-modified liposomes, 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 completed 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 liposomes and EGF-modified liposomes to the A431 cells is Illustrated in Figure 1. The EGF-mod1fied 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-modified liposomes to A431 cells were carried out as described in example 1 and the data were processed according to equation (1) above. The experimental conditions were such that the contribution of non-specific binding was negligible. indeed, the data were found to fit unambiguously with a single type of binding site for each liposome system studied. Results for several systems are listed in Table 1.

TABLE 1

BINDING PARAMETERS OF BIOADHESIVE LIPOSOMES TO A431 CELLS IN CULTURE

BIOADHESIVE K_d SITES PER CELL

LIPOSOME SYSTEM (a) (nM) (×10^-5)

EGF-MLV 0.60 ± 0.017 0.17 ± 0.03

EGF-MLV 5.03 ± 1.9 1.07 ± 0.03

EGF-LUVET 2.91 ± 0.003 0.18 ± 0.001

EGF-MEL 0.04 ± 0.007 0.042 ± 0.0042

EGF-MEL 0.40 ± 0.13 3.7 + 0.90

EGF-MEL 0.48 ± 0.05 0.28 ± 0.01

(a) Each bioadhesive liposome system is a different

preparation; recognizing substance in each system is EGF.

An EGF-modified 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-modified liposome systems are expected to be similar to or higher than those of free EGF. For a given class of receptors, the number of receptors per cell that are available for the EGF-modified liposomes is expected to be equal to or lower than the number of available for free EGF. Based on these considerations, the binding data of the present example 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-modified 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 bioadhesive liposomes. Example Three

Binding collagen-modified liposomes 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 binding of collagen-modified liposomes to A431 cells in culture are compared to regular liposome and exemplified in Figure 3. The data were processed according to equation (1) 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 entitles are of the extracellular matrix type of

cell-associated entity. As in the case of EGF-modified

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 available sites in the extracellular matrix available for collagen-modified liposomes is expected to be similar to or lower than free collagen. The example given in Table 2 fits with these considerations. The data for free collagen demonstrate that binding of this bioadhesive recognizing substance to this cellular system does occur and is a measurable phenomena, which can be processed to yield quantitative and meaningful parameters. Moreover, the data in Table 2 show quite clearly that the binding of

collagen-modified liposomes 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 K_d NUMBER OF SITES

LIPOSOME SYSTEM (uM) (×10^-5)

FREE COLLAGEN 8.5 ± 2.3 179 ± 11

COLLAGEN-MLV 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

What we claim is: 1. A method for topical substance administration for a designated cellular target site comprises the topical

application of a encapsulating liposome component and a recognizing substance component bonded to the liposomal surface where the recognizing substance component confers to the liposome component target specificity for and retention at the target site.

2. The method of administration of claim 1 wherein the liposome component is selected from the group consisting of multilamellar vesicles, mlcroemulslfied liposomes and large unllamellar vesicles.

3. The method of administration of claim 1 wherein the liposome component includes phosphatldylethanolamlne.

4. The method of administration of claim 1 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through receptor

mechanisms at the cellular target site.

5. The method of administration of claim 1 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 cellular target site.

6. The method of administration of claim 1 wherein the recognizing substance component is selected from the group consisting of gelatin, collagen, hyaluronlc add and epidermal growth factor.

7. The method of administration of claim 1 where the liposome component and the recognizing substance component are covalently linked.

8. The method of administration of claim 1 where the liposome component and the recognizing substance component are covalently linked by a crossl inking reagent.

9. A method of local substance administration for a designated cellular target site comprises the local application of a liposome component and a recognizing substance component bonded to the liposomal surface where the recognizing substance component confers to the liposome component target specificity for and retention at the target site.

10. The method of administration of claim 9 wherein the liposome component is selected from the group consisting of multilamellar vesicles, mlcroemulslfied liposomes and large unllamellar vesicles.

11. The method of administration of claim 9 wherein the liposome component includes phosphatldylethanolamlne.

12. The method of administration of claim 9 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through receptor

mechanisms at the cellular target site.

13. The method of administration of claim 9 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 cellular target site.

14. The method of administration of claim 9 wherein the recognizing substance component is selected from the group consisting of gelatin, collagen, hyaluronic add and epidermal growth factor.

15. The method of administration of claim 9 where the liposome component and the recognizing substance component are covalently linked by a crosslinking reagent.

16. The method of administration of claim 9 where the liposome component and the recognizing substance component are covalently linked by a crosslinklng reagent.

17. A liposome for substance administration for a designated cellular target site comprising a liposome component and a recognizing substance component bonded to the liposomal surface where the recognizing substance component confers to the liposome component target specificity for and retention at the target site.

18. A liposome of claim 17 wherein 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.

19. A liposome of claim 17 wherein the method of administration of claim 1 wherein the liposome component includes phosphatidylethanolamine.

20. A liposome of claim 17 wherein the method of administration of claim 1 wherein the recognizing substance component confers target specificity and retention by a mechanism of adhesion through receptor mechanisms at the cellular target site.

21. A liposome of claim 17 wherein the method of administration of claim 1 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 cellular target site.

22. A liposome of claim 17 wherein the method of administration of claim 1 wherein the recognizing substance component is selected from the group consisting of gelatin, collagen, hyaluronic add and epidermal growth factor.

23. A liposome of claim 17 wherein the method of administration of claim 1 where the liposome component and the recognizing substance component are covalently linked.

24. A liposome of claim 17 wherein the method of administration of claim 1 where the liposome component and the recognizing substance component are covalently linked by a crosslinklng reagent.