HK1152883B - Formulation and method for the prevention and treatment of bone metastases or other bone diseases - Google Patents

Description

Formulations and methods for preventing and treating bone metastases or other bone diseases

Technical Field

The present invention relates to formulations and medicaments for the treatment of bone metastases and other bone diseases. The invention also relates to methods of prevention and treatment.

Background

Bisphosphonates (bisphosphates) are synthetic analogs of pyrophosphates (structure P-O-P) in which the central oxygen atom is replaced by a carbon atom. The chemical structure of which can be represented by the following formula:

bisphosphonates can be divided into two classes.

The first class includes in its side chain R¹And R²A "first generation" compound in which no nitrogen atom is contained. This group includes in particular etidronate (etidronate), clodronate (clodronate) and tiludronate (tiludronate).

The second class includes in its side chain R¹Or R²One of the "second generation" and "third generation" compounds contains one or more nitrogen atoms. Those of the second generation comprising a compound having a nitrogen atom or a terminal NH₂Aliphatic side chains of the group. Mention may be made of pamidronate (pamidronate), alendronate (alendronate), ibandronate (ibandronate) and neridronate (neridronate). Those of the third generation have a heterocyclic nucleus containing a nitrogen atom. Advantageous salts of risedronate and zoledronate (imidazole nucleus) may be mentioned.

Such classifications as first, second and third generation are well known to those skilled in the art. By way of illustration, mention may be made of t.yuasa et al, Current medical chemistry 2007, 14: 2126-2135 and Selvargi et al, Crit. Rev. Oncol. Hematol.2005, 56 (3): 365-; stressing et al, Cancer Letters 2007, 257: 16-35; r.graham g.russell et al, ann.n.y.acad.sci.2007, 1117: 209-257.

In the case of advanced cancer, bone metastasis is common. They are most common in multiple myeloma, breast and prostate cancers, but also in the case of melanoma and bladder, lung and kidney cancers.

Bisphosphonates are necessary in the therapeutic treatment of patients with bone cancer. Thus, clodronate, pamidronate, zoledronate and ibandronate were used.

However, bisphosphonates can be toxic at high doses. They may also undergo considerable renal clearance. For example, in the case of zoledronate, renal clearance produces renal toxicity, necessitating careful supervision of the doses used clinically in humans, which may be found to be below effective doses for treating bone metastases. Thus, the clinical dose of zoledronate is 10 to 40 times less than the effective dose determined in animals (J.Green, Oncoloist 2004, 9: 3-13).

Rossi et al (Journal of Drug Targeting, February 2005, 13 (2): 99-111) describe encapsulation of clodronate in erythrocytes and their use for depleting macrophages. They also describe the use of ZnCl in the presence of ethanolamine and bovine serum albumin₂And BS3 treatment of erythrocytes. The authors confirmed depletion of spleen macrophages in mice. The study neither relates to nor anticipates the use of erythrocytes as bisphosphonate carriers in bone applications.

Disclosure of Invention

It is therefore an object of the present invention to propose a solution for the delivery of bisphosphonates at the bone marrow level, thus limiting renal clearance and avoiding the toxicity problems that currently limit the efficacy of these active ingredients.

It is also an object of the present invention to propose such a solution which increases the bioavailability of bisphosphonates at the level of the bone marrow.

It is also an object of the present invention to propose such a solution which allows to reduce the amount of bisphosphonate administered for a given treatment compared to the free form.

It is also an object of the present invention to propose such a solution which allows an increase in the administrable dose without encountering the limitation of toxicity of the free form.

Thus, one subject of the present invention is a suspension of erythrocytes encapsulating a bisphosphonate, for use as a medicament for targeting the bone marrow and for bringing said bisphosphonate into the bone marrow, limiting or even eliminating any risk of toxicity, in particular nephrotoxicity.

A subject of the present invention is also a suspension of erythrocytes encapsulating a bisphosphonate, for use as a carrier for bringing the bisphosphonate into the bone marrow, in particular for the treatment or prevention of bone diseases such as bone metastases.

By definition, the term "bisphosphonate" encompasses all bisphosphonates, and salts and derivatives thereof, in particular first, second and third generation bisphosphonates, and salts and derivatives thereof. Thus, the term encompasses bisphosphonates, bisphosphonic acids and diphosphonic acids.

Non-limiting examples of bisphosphonates include: etidronate, clodronate, tiludronate, pamidronate (in particular the disodium form of pamidronate), alendronate, incadronate, ibandronate, neridronate, risedronate and zoledronate.

Preferably, the present invention uses bisphosphonates comprising at least one nitrogen atom, such as second and third generation bisphosphonates. In particular, for the second generation, the following compounds were selected: pamidronate, alendronate, ibandronate, and neridronate. Those of the third generation have a heterocyclic nucleus containing a nitrogen atom. Advantageous sertraline phosphonates and zoledronate (imidazole nucleus) should be mentioned.

The invention relates in particular to a suspension of erythrocytes encapsulating a bisphosphonate for use as a medicament for the prevention and treatment of bone and bone marrow pathologies for which bisphosphonates have been indicated. Optimal dosages can be provided for treatment of the targeted lesions, as the treatment regimen is no longer limited by the toxicity associated with the free form bisphosphonate.

Thus, the use may relate to the prevention or treatment of bone metastases, malignant hypercalcemia, paget's disease and osteoporosis.

More particularly, the present invention is a suspension of erythrocytes encapsulating a bisphosphonate, for use as a medicament for the prevention and treatment of bone metastases.

The invention also relates to a medicament comprising a suspension of erythrocytes encapsulating a bisphosphonate, for use as a medicament for the prevention and treatment of these bone or bone marrow diseases, in particular bone metastases.

The invention also relates to the use of a suspension of erythrocytes encapsulating a bisphosphonate for the preparation of a medicament for the prevention and treatment of these bone or bone marrow diseases, in particular bone metastases.

The invention also relates to a suspension of erythrocytes encapsulating a bisphosphonate, for use as a carrier for bringing the bisphosphonate into the bone marrow, for the treatment or prevention of bone metastases.

Preferably, encapsulation of the bisphosphonate is performed by a process known as lysis-resealing (lysine-resealing).

The bisphosphonates are typically prepared in a buffer solution (e.g. PBS) at a pH of 7.2 to 7.6, preferably 7.4.

According to one feature of the method, first, lysis of the erythrocytes is carried out by subjecting the erythrocytes to hypotonic conditions. The red blood cells swell and open pores. Then, bisphosphonate is added, which permeates into the interior of erythrocytes. Preferably, the bisphosphonate solution is gradually added and the mixture is then incubated for, for example, 10 to 60 minutes, typically about 30 minutes. Next, the isotonic conditions are reestablished, thereby resealing or reclosing the pores, allowing the erythrocytes to stably encapsulate the bisphosphonate.

According to an advantageous feature of the invention, the erythrocytes are treated with a chemical agent in a condition that promotes targeting of the bone marrow. The chemical agent facilitates the recognition of phagocytic cells of the bone marrow (macrophages and dendritic cells).

The chemical treatment is performed on erythrocytes encapsulating a bisphosphonate.

One preferred chemical agent suitable for human clinical use is bis (sulfosuccinimidyl) suberate abbreviated as BS3 or BS3³(ii) a CAS 82436-77-9). Solutions of the reagents are advantageously used.

An object of the invention is also a suspension of erythrocytes or a medicament comprising this suspension, comprising erythrocytes encapsulating a bisphosphonate and a membrane which has been treated with a chemical agent, preferably BS3, in order to promote recognition by the phagocytic cells of the bone marrow. BS3 is preferably used alone, without any other chemical or biological agent, such as ZnCl₂. According to a first embodiment, the bisphosphonate is a first generation bisphosphonate. According to a second embodiment, the bisphosphonate is a second generation bisphosphonate. According to a third embodiment, the bisphosphonate is a third generation bisphosphonate. Non-limiting examples of bisphosphonates include: etidronate, clodronate, tiludronate, pamidronate (in particular the disodium form of pamidronate), alendronate, incadronate, ibandronate, neridronate, risedronate and zoledronate. According to one embodiment, the bisphosphonate is selected from: etidronate, tiludronate, pamidronate (in particular pamidronate in disodium form), alendronate, incadronate, ibandronate, neridronate, risedronate and zoledronate.

According to one characteristic of the method of treatment with BS3, the suspension of erythrocytes encapsulating the bisphosphonate is brought into contact with BS3 for a suitable period of time, which may in particular be from about 10 minutes to about 1 hour. This time period is advantageously at a level of about 15 minutes to about 45 minutes, preferably about 20 to about 40 minutes, typically 30 minutes.

According to another characteristic of the method of treatment with BS3, the incubation is preferably carried out at ambient temperature, in particular at a temperature of 18 to 25 ℃.

According to another characteristic of the method of treatment with BS3, the suspension of erythrocytes containing the bisphosphonate is pre-washed with a suitable buffer, for example PBS.

According to another feature, the concentration of the suspension of red blood cells treated with BS3 is brought to about 0.5X 10 before contacting with the BS3 solution⁶To about 5X 10⁶One cell/. mu.l, usually about 1X 10⁶To about 3X 10⁶Cells/. mu.l.

According to another feature, a solution of BS3 is used to obtain a final concentration of BS3 in the suspension of about 0.1 to about 6mM, preferably about 0.5 to about 3mM, usually about 1 mM. In particular, a BS3 solution of about 2mM can be used. Preferably, a BS3 buffer solution preferably comprising glucose and phosphate buffer is used to obtain the desired final concentration of BS3, in particular about 1 mM. According to one feature, the pH of the BS3 buffer solution is advantageously between about 7.2 and about 7.6, preferably about 7.4. According to another feature, the BS3 buffer solution has an osmolality of about 280 to about 320 mOsm.

The incubation can be stopped by using a reagent (e.g., Tris-HCl), then centrifuging the mixture, washing the cells, and resuspending them in a suitable buffer such as SAG-BSA. The mixture is incubated at ambient temperature for a few minutes, in particular 1 to 10 minutes, before centrifugation.

The suspension may be ready-to-use and may have a hematocrit suitable for administration without dilution.

It may also be packaged in such a way that it must be diluted before application.

According to the invention, the hematocrit of the ready-to-use suspension is advantageously from about 40% to about 70%, preferably from about 45% to about 55%, more preferably about 50%.

In its form for dilution, the hematocrit may be high, in particular from about 60% to about 90%.

The suspension is preferably packaged in a volume of about 10 to about 250 ml. The packaging is preferably a blood bag suitable for a transfusion type. The encapsulated amount of bisphosphonate corresponding to the medical prescription is preferably contained entirely in the blood bag.

For example, a suspension corresponding to one dose (e.g. one blood bag) contains 1 to 40mg of bisphosphonate, in particular 2 to 10mg of bisphosphonate.

According to one characteristic of the invention, the erythrocytes to be administered are suspended in a solution of a pharmaceutically acceptable saline, for example a standard medium for erythrocytes, in particular a solution comprising NaCl and one or more components chosen from glucose, dextrose, adenine and mannitol, for example SAG-mannitol or ADsol. The solution is capable of preserving red blood cells and may also contain preservative additives such as L-carnitine.

Accordingly, the object of the present invention is a method for preventing or treating bone metastases or other bone diseases. The method comprises administering to a patient a formulation or medicament according to the invention.

According to the invention, the administration of the formulation or medicament is by intravenous or intra-arterial injection, preferably by instillation from a blood bag or the like. The administration is usually carried out intravenously in the arm or via a central catheter (central catheter).

About 10 to about 250ml of the formulation of the invention (one dose) is particularly administered. Preferably, the amount is 50ml or more by drip.

Treatment includes administration of one dose or several doses according to established protocols. The regimen may provide for several administrations at a frequency of once a month, once every two months, once every three months, once every half year (semestrial), or once a year over the recommended course of treatment.

The techniques for encapsulating active ingredients in erythrocytes are known, and the preferred basic technique of lysis-resealing is described herein in patents EP-A-101341 and EP-A-679101 to which the skilled person is referred. According to this technique, a first compartment of a dialysis element (for example a dialysis bag or a dialysis cartridge) is continuously supplied with a suspension of red blood cells, while a second compartment contains an aqueous solution that is hypotonic with respect to the suspension of red blood cells, in order to lyse the red blood cells; next, in a resealing unit, resealing of the erythrocytes is induced by increasing the osmotic and/or oncotic pressure in the presence of the bisphosphonate, and then a suspension of erythrocytes comprising the bisphosphonate is recovered. According to one feature of the invention, the lysis of the suspension of erythrocytes already containing the bisphosphonate to be encapsulated is preferably carried out.

The suspension of erythrocytes encapsulating the bisphosphonate can be obtained in particular by the following process, which is also a subject of the present invention:

1-suspension of the erythrocyte pellet (pellet) in an isotonic solution with a hematocrit level greater than or equal to 65%, cooling between +1 and +8 ℃,

2-a lysis step constantly maintained at a temperature of between +1 and 8 ℃, comprising bringing a suspension of red blood cells having a hematocrit level of greater than or equal to 65% and a hypotonic lysis solution which has been cooled to between +1 and +8 ℃ into a dialysis bag or cartridge (preferably a dialysis cartridge),

3-an encapsulation step, adding (preferably gradually) a bisphosphonate solution to the lysed suspension, preferably with an incubation period of in particular 10 to 60 minutes, usually about 30 minutes, maintained at a temperature of +1 to +8 ℃, and

4-resealing step at a higher temperature (in particular +30 to +42 ℃) and in the presence of a hypertonic solution.

As A preferred variant, it is possible to inspire from the process described in WO-A-2006016247 that allows for an efficient, reproducible, safe and stable encapsulation of bisphosphonates. Thus, a suspension of erythrocytes encapsulating a bisphosphonate can be obtained by the following process, which is also a subject of the present invention:

1-suspension of the erythrocyte pellet in an isotonic solution with a hematocrit level of greater than or equal to 65%, cooling between +1 and +8 ℃,

2-measuring osmotic fragility using a sample of red blood cells from the same red blood cell pellet, which can be performed in any order (including parallel) for steps 1 and 2,

3-a lysis step, in particular in the same chamber, at a temperature constantly maintained between +1 and +8 ℃, comprising the passage of a suspension of red blood cells having a hematocrit level greater than or equal to 65% and a hypotonic lysis solution which has been cooled to between +1 and +8 ℃ into a dialysis bag or cartridge, preferably a dialysis cartridge; adjusting the lysis parameter as a function of the previously measured osmotic fragility, and

4-an encapsulation step, adding (preferably gradually) a bisphosphonate solution to the lysed suspension, preferably with an incubation period of in particular 10 to 60 minutes, usually about 30 minutes, maintained at a temperature of +1 to +8 ℃, and

5-the resealing step is carried out in the second chamber at a higher temperature (in particular +30 to +42 ℃) and in the presence of a hypertonic solution.

The term "internalization" means that the bisphosphonate permeates into the interior of the red blood cell.

In particular, for dialysis, the red blood cell pellet is suspended in an isotonic solution of high hematocrit level of greater than or equal to 65%, preferably greater than or equal to 70%, and the suspension is cooled to +1 to +8 ℃, preferably +2 to +6 ℃, typically about +4 ℃. According to a particular embodiment, the hematocrit level is between 65% and 80%, preferably between 70% and 80%.

When measuring, the osmotic fragility of the erythrocytes is advantageously measured, in the presence or absence of bisphosphonate in suspension, just before the lysis step. The red blood cells or the suspension containing them are advantageously at a temperature close to or equal to the temperature chosen for lysis. According to an advantageous feature of the invention, the measurement of osmotic fragility is carried out rapidly, i.e. immediately after the sample has been taken. Preferably, the time period between the collection of the sample and the start of lysis is less than or equal to 30 minutes, even more preferably less than or equal to 25 minutes, even less than or equal to 20 minutes.

For the method in which the lysis-resealing step is carried out, for the osmotic fragility measured and considered, the person skilled in the art can refer to WO-A-2006/016247 for further details. This document is incorporated herein by reference.

The invention will now be described in more detail by way of embodiments by way of non-limiting examples.

I-example 1: method for encapsulating zoledronate in mouse and human red blood cells

Ia-materials：

For dialysis: dialysis cartridge (Gambro 280 fiber)

Measurement of: after preparing the samples according to the method described below, the determination of zoledronate in erythrocytes was carried out by High Performance Liquid Chromatography (HPLC). Red Blood Cells (RBC) encapsulating zoledronate were lysed with 2.5 volumes of water, and then zoledronate was extracted by precipitating the proteins and membranes with 12% trichloroacetic acid.

To assess the amount of zoledronate that had been encapsulated, the red blood cells (before encapsulation), the final products RBC-Zol and RBC-LR, treated or not with BS3, and the supernatants thereof at D0 and D1 were determined.

To increase the retention time of zoledronate on the C18 support, the compound tetrabutylammonium hydrogen sulfate was used as ion-pairing agent.

The instrument comprises the following steps: shimadzu UFLC

Column: gemini C185 μ 110A 250 × 4.6mm ID

Temperature: 40 deg.C

Injection volume: 40 μ l

And (4) UV detection: 220nm

Flow rate: 0.7 ml/min

Mobile phase A: 8mM K₂HPO₄-(1.39g/l)，2mM Na₂HPO₄- (0.1g/l), 7mM tetrabutylammonium hydrogen sulfate- (2.7g/l)

Mobile phase B: methanol

Ib-Process：

The red blood cells were centrifuged and then washed three times in PBS. The hematocrit of the suspension was brought to 70% with PBS, after which dialysis was started. Dialyzing RBCs against the low osmolality lysis buffer at a flow rate of 2 ml/min ((counter current 15 ml/min.) the lysed RBCs exiting the column were divided into two equal volumesThe solution (0.8mg/ml zoledronic acid) was gradually added (ten times) to a volume of dialyzed red blood cells to a final concentration of 0.4 mg/ml.

As a control, another volume of dialyzed red blood cells was diluted with a gradually added volume of PBS. Both suspensions were incubated at 4-8 ℃ for 30 minutes.

The erythrocytes were resealed by adding a solution of high osmolality (0.1 volume) and incubated at 37 ℃ for 30 minutes. The resealed cells were washed three times in PBS containing glucose. The hematocrit of the suspension was brought to 50% with SAG-mannitol or PBS containing glucose, supplemented or not with BSA (6%), or the suspension was directly stored at high hematocrit (80%) to constitute the final products RBC-Zol (zoledronate) and RBC-LR (lysis-reseal control without zoledronate).

II-example 2: with bis (sulphosuccinimidyl) suberate (BS) ³ ) P-containing zoledronic acid salt The red blood cells are chemically treated

A suspension of erythrocytes containing zoledronate was obtained as described in example 1. The suspension was washed several times and then diluted to 1.7X 10⁶Cells/. mu.l, then, were mixed with 2mM BS³Solution contact (said BS)³The solution contained 50mM phosphate buffer (pH 7.4) and 0.09% glucose) to obtain 1mM BS³The final concentration. The red blood cells were incubated at ambient temperature for 30 minutes, and then the reaction was stopped by adding a volume of 20mM Tris, 140mM NaCl. After centrifugation for 5 minutes, the erythrocytes were washed once with PBS containing glucose and then once with SAG-mannitol supplemented or not with BSA (6%). The hematocrit of the red blood cells was brought to 50% in SAG-mannitol or glucose-containing PBS, with or without BSA (6%), or stored directly at high hematocrit (80%).

III-results of examples 1 and 2：

a) Encapsulation in human RBCs

The following table gives 4 samples of the human red blood cells (RBC enrichment bags)Evaluation of encapsulation experiments.

The cellular data obtained from the analysis of the final product RBC-Zol at D1 (table 1) confirm that the loaded RBCs retain cellular characteristics close to the pocket RBCs and do not undergo any particular damage.

Table 1:

the haemoglobin concentration of the erythrocytes (MCHC) is still satisfactory, with values greater than 25 g/dl. This demonstrates that there is little loss of content in the erythrocytes during the process. Extracellular haemoglobin is lower than 2g/dl and therefore the final product obtained has injectable properties. The yield of the 1mM BS3 group resulted in a 17% loss of cells. However, the overall cell yield of the final product is compatible with commercial production (> 55%).

And Mean Corpuscular Volume (MCV) of the red blood cells of the bag (97 μm)³) In contrast, the dialysis procedure resulted in a decrease in the mean red blood cell volume of RBC-Zol red blood cells (80-88 μm)³)。

Data on the encapsulation of zoledronate is given in table 2.

TABLE 2

The zoledronate salt has a critical dimension encapsulated in the RBC, the most important criterion to be observed being the amount of compound encapsulated in the RBC at D1 compared to the extracellular ratio. Three experiments were performed without specific treatment of the erythrocyte membranes, and the results showed satisfactory encapsulation of zoledronate at D1 (68% to 83% of zoledronate in the final product was encapsulated in RBCs). The encapsulation efficiency was 71% with BS3 treatment.

Table 3 shows stability data on human erythrocytes comprising zoledronate. On the day of preparation (D0) and the next day (D1), extracellular hemoglobin, hematocrit of suspension, and the amount of intracellular and extracellular zoledronate were measured.

An increase in extracellular zoledronate between D0 and D1 was observed. This may be associated with an increase in extracellular hemoglobin between D0 and D1 and cell loss (hematocrit). Thus, the release of zoledronate into the extracellular medium comes essentially from the rupture of red blood cells after dialysis without proper resealing, rather than from passive or active permeation through the red blood cell membrane. The results show good storage under various conditions.

b) Encapsulation in mouse RBCs

The following table gives the 3 run with mouse red blood cells (whole blood)Evaluation of encapsulation experiments. Like human RBCs, RBC-Zol retains cellular characteristics close to that of whole blood.

TABLE 4

The red blood cell hemoglobin concentration (MCHC) is very satisfactory. Extracellular hemoglobin is higher than in the case of human RBCs because mouse erythrocytes are more fragile. The dialysis procedure yielded a mean red blood cell volume (43-46 μm) for RBC-Zol red blood cells³) Which was consistent from experiment to experiment.

Table 5 shows data regarding mouse RBC encapsulated zoledronate (measured at D1). The final suspension was a concentrated suspension with a hematocrit of 80%.

TABLE 5

The results show that encapsulation at D1 is satisfactory.

V-example 3: by BS ³ Confirmation of treatment targeting bone marrow：

The fluorescent dye FITC-dextran (70kDa) was encapsulated in mouse erythrocytes (OF1 mouse) by the hypotonic dialysis method in a column. Blood was pre-centrifuged and then washed 3 times in PBS. After reaching a hematocrit of 70% in the presence of FITC-dextran added at a final concentration of 8mg/ml, dialysis was started. Dialysis of RBC against low osmolarity lysis buffer at a flow rate of 2 ml/min ((counter current 15 ml/min.) lysed RBC that had left the column by addition of high osmolarity solution resealed and incubated at 37 ℃ for 30 min, RBC diluted to 1.7X 10 after two washes in PBS containing glucose⁶Cells/. mu.l, after which they were contacted with 10mM MBS containing 50mM phosphate buffer (pH 7.4) and 0.09% glucose³And (3) solution. The red blood cells were incubated at ambient temperature for 30 minutes, and then the reaction was stopped by adding a volume of 20mM Tris, 140mM NaCl. After centrifugation for 5 minutes, RBCs were washed once with PBS containing glucose and then once with SAG-mannitol supplemented with BSA (6%). The hematocrit of the red blood cells was brought to 50% to constitute the final product, which was injected into the mice at D1. Mice were sacrificed 1 hour and 30 minutes after injection, and then bone marrow isolated from femurs was placed into Tissue-Tek for freezing in liquid nitrogen. Immunohistochemical analysis was performed by cutting into 10 μm frozen sections. In thatAfter fixation in acetone, double labeling was performed, confirming FITC (DAB, brown) and F4/80 macrophages (New fuchsin, Red).

Microscopic examination of the sections revealed the coexistence of macrophages and dextran (colocalization). Observation of the structure confirmed that macrophages introduced glucan by phagocytosis of erythrocytes.

Flow cytometry analysis (FC500Beckman Coulter) gave the following information.

Table 6: percentage of fluorescent cells in bone marrow 1 hour 30 minutes after intravenous injection of RBCs into mice

Treatment of	Total number of fluorescent cells
		RBC-glucan	5.3％
RBC-dextran-BS 3	7.4％

Flow cytometric analysis showed that about 7% and 5% of bone marrow cells were fluorescent in the case of BS3 treated and untreated, 1 hour and 30 minutes after injection.

Table 7 shows the percentage of phagocytic cells that have phagocytosed treated or untreated red blood cells.

Treatment of	F4/80 macrophage	Dendritic cells
			RBC-glucan	5.2％	7.9％
RBC-dextran-BS 3	9.5％	16.3％

BS3 treatment induced faster and more uptake by phagocytosis of RBCs in the bone marrow than no treatment.

Conclusion

Zoledronate can be stably encapsulated in RBCs, which may or may not have been treated with BS3 membrane.

The encapsulation amount corresponds to a large extent to the amount used clinically. The encapsulation experiment (treated with 1mMBS 3) showed that a final product containing 56.1. mu.g/ml zoledronate was obtained. Therefore, it is necessary to infuse the human with about 71ml of RBC-Zol in order to obtain 4mg equivalents.

Bone marrow cells were targeted by treatment with 1mM BS 3.

All of these demonstrate the use of RBCs as bone marrow targeting vectors for zoledronate and their use in bone metastasis or other bone diseases.

Claims (18)

1. A suspension of erythrocytes encapsulating a bisphosphonate for use as a medicament for the prevention and treatment of bone metastases, wherein the bisphosphonate is a second or third generation bisphosphonate.

2. An erythrocyte suspension according to claim 1, wherein the bisphosphonate-encapsulated erythrocytes have been chemically treated with an agent to facilitate targeting of the bone marrow.

3. The suspension of erythrocytes according to claim 2, wherein the chemical treatment is carried out with a solution of bis (sulfosuccinimidyl) suberate (BS 3).

4. An erythrocyte suspension according to claim 3, wherein the suspension of erythrocytes encapsulating a bisphosphonate is contacted with BS3 for a period of time ranging from 10 minutes to 1 hour.

5. An erythrocyte suspension according to claim 3, wherein the suspension of erythrocytes encapsulating a bisphosphonate is contacted with BS3 for a period of time ranging from 15 minutes to 45 minutes.

6. An erythrocyte suspension according to claim 3, wherein the suspension of erythrocytes encapsulating a bisphosphonate is contacted with BS3 for a period of time ranging from 20 to 40 minutes.

7. An erythrocyte suspension according to claim 3, wherein the erythrocytes have been chemically treated with BS3, wherein the treatment comprises incubating the bisphosphonate-encapsulated erythrocyte suspension with BS3 at ambient temperature.

8. An erythrocyte suspension according to claim 3, wherein the erythrocytes have been chemically treated with BS3, wherein the treatment comprises washing the bisphosphonate-encapsulated erythrocyte suspension with a buffer prior to incubating the erythrocyte suspension with BS 3.

9. A suspension of red blood cells according to claim 8, wherein the buffer is PBS.

10. An erythrocyte suspension according to claim 3, wherein the concentration of the bisphosphonate-encapsulated erythrocyte suspension is brought to 0.5 x 10 prior to contacting the BS3 solution⁶To 5X 10⁶Cells/. mu.l.

11.An erythrocyte suspension according to claim 3, wherein the concentration of the bisphosphonate-encapsulated erythrocyte suspension is 1 x 10 prior to contacting the BS3 solution⁶To 3X 10⁶Cells/. mu.l.

12. Erythrocyte suspension according to claim 3, wherein a solution of BS3 is used to obtain a final concentration of BS3 in the suspension of 0.1 to 6 mM.

13. Erythrocyte suspension according to claim 3, wherein a solution of BS3 is used to obtain a final concentration of BS3 in the suspension of 0.5 to 3 mM.

14. Erythrocyte suspension according to claim 3, wherein a BS3 buffer solution with an osmolality of 280 to 320mOsm and a pH of 7.2 to 7.6 is used.

15. Erythrocyte suspension according to claim 3, wherein a buffer solution of BS3 with an osmolality of 280 to 320mOsm and a pH of 7.4 is used.

16. A suspension of red blood cells according to claim 14 or 15, wherein the BS3 solution comprises glucose and a phosphate buffer.

17. An erythrocyte suspension according to any of claims 1 to 15, wherein the bisphosphonate is pamidronate, alendronate, incadronate, ibandronate, neridronate, risedronate or zoledronate.

18. An erythrocyte suspension according to any of claims 1 to 15, wherein the bisphosphonate is zoledronate.