DRY FOAM COMPRISING AGAR-AGAR

Abstract

The present invention relates to a dry foam comprising agar-agar characterized by an elasticity modulus from 0.02 to 0.6 MPa, particularly from 0.15 to 0.6 MPa, more particularly from 0.3 to 0.4 MPa, a manufacturing process thereof and the uses thereof in particular as an embolization agent.

Claims

1. Dry foam comprising at least 95% of agar-agar suitable for embolization characterized by an elasticity modulus from 0.02 to 0.6 MPa.

2. Dry foam comprising agar-agar according to claim 1 further characterized by a maximum compressive strength from 0.008 to 0.11 MPa.

3. Dry foam comprising agar-agar according to claim 1 further characterized by a density from 0.01 to 0.042 g/cm.sup.3.

4. Dry foam comprising agar-agar according to claim 1 having a porosity comprised between 75% and 95%.

5. Dry foam comprising agar-agar according to claim 4 having a porosity comprised between 80% to 85%.

6. Dry foam comprising agar-agar according to claim 4 wherein the foam has an open porosity.

7. Dry foam comprising agar-agar according to claim 1 wherein said dry foam is non-uniform.

8. Dry foam comprising agar-agar according to claim 1 having an average pore size comprised between 250 microns and 700 microns, preferably between 400 and 600 microns, and even more preferably about 540 microns, and having a median pore size comprised between 200 and 400, preferably between 200 and 300 microns and more preferably about 280 microns.

9. Dry foam comprising agar-agar according to claim 1, further comprising a therapeutic drug.

10. Dry foam comprising agar-agar according to claim 9, wherein said therapeutic drug is an anti-inflammatory drug or an anti-cancer drug.

11. Dry foam comprising agar-agar according to claim 1, wherein said dry foam comprises a medical imaging marker.

12. Dry foam comprising agar-agar according to claim 11, wherein said medical imaging marker is a radioactive isotope.

13. (canceled)

14. A method of conducting vascular embolization in a subject in need thereof, comprising introducing the dry foam comprising agar-agar of claim 1 into a blood vessel of the subject so as to occlude the blood vessel and stop blood flow.

15. The method of claim 14, wherein the blood vessel has an abnormal blood flow.

16. The method of claim 14, wherein the blood vessel supplies blood to a cancer.

17. The method of claim 16, wherein the dry agar-agar comprises an anti-cancer drug.

18. Process for manufacturing a dry foam comprising agar-agar according to claim 1, comprising the following steps: a. mixing agar-agar to distilled water at an amount from 1.5 to 7% (w/v); b. heating between 80° C. and 100° C.; c. cooling the mix until a temperature between 45° C. and 80° C.; d. injecting gas to obtain a foam; e. freezing the resulting foam between −120° C. and 40° C.; f. freeze-drying the foam to obtain the dry foam comprising agar-agar.

Description

FIGURES

[0109] FIG. 1: Photos of dry foams comprising agar-agar obtained from a process using an aqueous mix comprising 1.5%, 2.5%, 4%, 6% or 8% (w/v) of agar-agar.

[0110] FIG. 2: Density measurement (g.Math.cm.sup.−3) of the dry agar-agar foam depending on the percentage of agar-agar (w/v) used at the step of mixing with distilled water in the manufacturing process.

[0111] FIG. 3A: Compression curve corresponding to the compression (a) (MPa) against the percentage of deformation (c). Deformation from 0 to 60% is performed on dry foams comprising agar-agar obtained from a process using an aqueous mix comprising 1.5%, 2.5%, 4%, 6% or 8% (w/v) agar-agar.

[0112] FIG. 3B: Maximum compressive strength (MPa) measured at 60% of deformation of a dry agar-agar foam depending on the percentage of agar-agar (w/v) used at the step of mixing with distilled water the manufacturing process of the foam.

[0113] FIG. 4A. Compression curve corresponding to the compression (a) (MPa) against the percentage of deformation (c). Deformation from 0 to 60% is performed on dry foams comprising agar-agar obtained from a process using an aqueous mix comprising 1.5%, 2.5%, 4%, 6% or 8% agar-agar (w/v), after being moistened with a 0.9% saline solution.

[0114] FIG. 4B. Maximum compressive strength (MPa) measured at 60% of deformation of a dry agar-agar foam after being moistened with a 0.9% saline solution, depending on the percentage of agar-agar (w/v) used at the step of mixing with distilled water in the manufacturing process of the foam.

[0115] FIG. 5: Young's modulus (E) (MPa) measurements of a dry foam comprising agar-agar depending on the percentage (%) of agar-agar (w/v) used in the manufacturing process of the foam.

[0116] FIG. 6: Young's modulus (E) (MPa) measurements of a dry foam comprising agar-agar after being moistened with a 0.9% saline solution, depending on the percentage (%) of agar-agar (w/v) used at the step of mixing with distilled water in the manufacturing process of the foam.

[0117] FIG. 7: Fatigue testing (45 cycles) of a dry agar-agar foam obtained from an aqueous mix at 4% of agar-agar (w/v), after being moistened with a 0.9% saline solution. For each cycle, the compression curve corresponding to the compression (σ) against the deformation (ε) is then traced.

[0118] FIG. 8: Pore size distribution in a dry agar-agar foam obtained from an aqueous mix at 4% of agar-agar.

[0119] FIG. 9A: 3D representation of the agar foam network characterized by micro tomography.

[0120] FIG. 9B: Cross-section of the network representing the pore size of the network.

[0121] FIG. 9C: Cross-section of the network representing the wall thickness.

[0122] FIG. 10A: Syringe (10 mL) filled sterilely by saline solution and containing the dry agar-agar foam of the invention (obtained from a water mix at 4% of agar-agar) shaped as a torpedo shaped in the syringe tip.

[0123] FIG. 10B: Syringe (10 mL) filled sterilely by saline solution and containing CuraSpon® shaped as a torpedo shaped in the syringe tip.

[0124] FIG. 11: DSA control before and after arterial embolization of left lower polar artery with a dry agar-agar foam obtained from a water mix at 4% of agar-agar.

[0125] FIG. 12: Computed tomography (CT) scan after arterial embolization of the lower left polar artery by a dry agar-agar foam obtained from a water mix at 4% of agar-agar (w/v) according to the invention, and lower right polar artery by CuraSpon®, and virtual reality reconstruction.

[0126] FIG. 13: macroscopic view (photography) of the left kidney after embolization with the foam according to the invention.

EXAMPLE

Material & Methods

[0127] In the present application, the ambient temperature is considered as 25° C. and the atmospheric pressure as 1013 hPa.

1. Manufacture of a Dry Agar-Agar Foam

[0128] Five type of dry agar-agar foams have been prepared starting from different percentage of agar-agar.

[0129] Agar-agar used comprised 70% of agarose and 30% agaropectin.

[0130] Agar-agar has been added and mixed to distilled water at an amount of 1.5, 2.5, 4, 6 or 8% (w/v). Then, the mix has been heated at 90° C. Once cooled at 55° C., the mix has been placed into a culinary siphon and N.sub.2O gas has been injected at 6 bars to obtain a foam at ambient temperature. The foam has been recovered in a mold which has been immediately placed at −40° C. After 20-30 min, the frozen foam has been placed into a freeze-dryer. The freeze-drying starts with a first cycle at −40° C. with a fast decreasing pressure from atmospheric pressure down to about 0.0035 bar, and then a second cycle at about 0.0035 bar with a slowly and natural increasing temperature from −40° C. up to ambient temperature, and finally break the vacuum. A dry agar-agar foam has been then obtained. The texture differs depending on the agar-agar percentage used in the manufacturing process (FIG. 1). Starting with an agar-agar percentage below 3%, the dry agar-agar foam obtained is very honeycombed and elastic. With an agar-agar percentage between 4 and 6%, the foam texture is fine and elastic. However, with 8% of agar-agar, the dry foam is rigid and breakable.

2. Physical Characterization of the Agar-Agar Foam

[0131] 2.1. Density

[0132] The density (ratio mass of sample/volume of the sample) has been measured for the dry agar-agar foams respectively obtained with 1.5, 2.5, 4, 6 or 8% of agar-agar. Results are reported in the graph at FIG. 2.

[0133] Depending on the initial percentage of agar-agar in the manufacturing process of the foam, the density of the resulting dry agar-agar foam varies greatly, approximately by a factor of 5 when going from 1.5% to 8% agar-agar.

[0134] 2.2. Maximum Compressive Strength

[0135] The maximum compressive strength has been measured for the dry agar-agar foams respectively obtained from water mixes containing 1.5, 2.5, 4, 6 or 8% of agar-agar.

[0136] To measure the maximum compressive strength, a compression is applied with an 8 cm diameter analysis plate, on a 2×2 cm.sup.2 surface of a sample of dry agar-agar foam, having a height of 1 cm, at room temperature and atmospheric pressure. The sample is compressed from 0 to 60% of its initial height. For the experiment, a texturometer Almetek TA1 has been used.

[0137] A compression curve corresponding to the compression (σ) against the deformation (ε) is then traced (FIG. 3A). The maximum compressive strength is then obtained on the curve for a 60% deformation.

[0138] Results of the maximum compressive strength for a 60% deformation depending on the percentage of agar-agar used in the manufacturing process of the foam, are reported in the graph at FIG. 3B.

[0139] Depending on the initial percentage of agar-agar in the manufacturing process, the maximum compressive strength of the resulting dry agar-agar foam varies greatly, approximately by a factor of 10 when going from 1.5% to 8% agar-agar. When the percentage of agar-agar is increased, the resulting foam has an increasingly structured and dense pore network.

[0140] When the dry agar-agar foam is obtained from a percentage of agar-agar below 2.5%, the dry foam has a weak cohesion. When it is obtained from a percentage of agar-agar between 4-6%, the dry foam has a very good cohesion. From 8% of agar-agar, the resulting agar-agar foam becomes rigid and breakable.

[0141] The maximum compressive strength has also been measured for the dry agar-agar foams respectively obtained with 1.5, 2.5, 4, 6 or 8% of agar-agar, after being moistened with a 0.9% saline solution. To moisten the foam, the foam is submerged into a 0.9% saline solution. The foam absorbs water almost instantly, like a sponge. The soaked foam is then placed in the texturometer. The height of the sample is measured, then the compression cycle is carried out (compression of 60% of the initial height) and the compression curve σ(ε) is traced (FIG. 4A). The maximum compressive strength is measured in the same way as for the dry foam, previously described.

[0142] Results of the maximum compressive strength for a 60% deformation measured on a humidified agar-agar foam with a 0.9% saline solution, depending on the percentage of agar-agar used in the manufacturing process of the foam, are reported in the graph at FIG. 4B.

[0143] Depending on the initial percentage of agar-agar in the manufacturing process, the maximum compressive strength of the wet agar-agar foam varies greatly, approximately by a factor of 7 when going from 1.5% to 8% agar-agar.

[0144] Interestingly, the maximum compressive strength varies by a factor 10 between the dry agar-agar foam and said foam after being moistened. This indicates that after humidification the foam is 10 times softer.

[0145] 2.3. Young's Modulus

[0146] The Young's modulus or elastic modulus has been measured for the dry agar-agar foams respectively obtained from water mixes containing 1.5, 2.5, 4, 6 or 8% of agar-agar.

[0147] The Young's modulus is obtained from the compression curve previously obtained for the moistened agar-agar foam (FIG. 3A), by placing in the elastic deformation part, from 0 to 10% of deformation. The directing coefficient of the linear response σ=E.Math.ε, is calculated and corresponds to the Young's Modulus (E).

[0148] Young's modulus measurements depending on the percentage of agar-agar used in the manufacturing process of the foam, are reported in the graph at FIG. 5.

[0149] The Young's modulus has also been measured for the dry agar-agar foams respectively obtained with 1.5, 2.5, 4, 6 or 8% of agar-agar, after being moistened with a 0.9% saline solution. The Young's modulus is obtained from the compression curve previously obtained (FIG. 4A), placing in the elastic deformation part, from 0 to 10% of deformation. The directing coefficient of the linear response σ=Eε is calculated and corresponds to the Young's Modulus (E).

[0150] Young's modulus measurements on a humidified agar-agar foam with a 0.9% saline solution, depending on the percentage of agar-agar used in the manufacturing process of the foam, are reported in the graph at FIG. 6.

[0151] 2.4. Fatigue Testing

[0152] The experiment is the same than the measurement of the maximum compressive strength, except that after compression, the foam is let relax, and a new compression is applied.

[0153] The fatigue testing has been carried out on a dry agar-agar foam obtained with 4% of agar-agar, after being moistened with a 0.9% saline solution, in order to investigate the “sponge” effect.

[0154] A compression is applied with an 8 cm diameter analysis plate, on a 2×2 cm.sup.2 surface of a sample of dry agar-agar foam, having a height of 1 cm, at room temperature and atmospheric pressure. The sample is compressed from 0 to 60% of its initial height and let relax. The cycle of compression/relaxation is repeated 45 times on the same sample. For the experiment, a texturometer Almetek TA1 has been used.

[0155] A compression curve corresponding to the compression (σ) against the deformation (ε) is then traced for each cycle (FIG. 7).

[0156] After 45 cycles of compression/relaxation, the structure is preserved under high mechanical stress. A slight loss of maximum compressive strength is observed after 40 cycles. The reversibility of the foam over 40 compression/relaxation cycles under wet conditions reflects this so-called “sponge” effect.

[0157] 2.5. Foam Microstructure

[0158] X-ray micro-tomography allows to observe the microstructure of the foam, i.e. its porosity and the dispersion of its pores.

[0159] Scans have been performed on a North Star Imaging X50CT, with a XrayWorx XWT-190-TC source and a Dexela 2923 detector. The voltage was 100 kV and the amperage was 50 μA. The images have been taken at a speed of 10 per second for 44 minutes. The data obtained by tomography are processed via the ImageJ software. Different 3D images as well as pore size and network thickness dispersions are obtained.

[0160] Results at FIG. 8 have been obtained from a dry agar-agar foam obtained with 4% of agar-agar, said agar-agar comprising 70% of agarose and 30% of agaropectin, on a cubical foam sample of 2 cm*2 cm*1 cm.

[0161] The network constitutes 17.6% of the foam with an average thickness of 112 microns. The average pore size is 540 microns with a median of 280 microns, indicating the presence of very large bubbles visible in the processed images (FIGS. 9B and 9C).

[0162] Images at FIGS. 9A, 9B and 9C show the details of the network of a dry agar-agar foam obtained from a 4% of agar-agar mix. The pores size is very heterogeneous and the distribution of pores of different sizes is also very heterogeneous. This polydispersity shows that the foam of the invention can be compared to a natural sponge. Indeed, the heterogeneity of the size of the pores allows a fast absorption of water and a swelling, while having a low density and a resistance to strong pressures. As it can be seen, the foam has an open porosity (i.e. the foam is essentially composed of a network of connected pores).

3. Therapeutic Use of the Dry Agar-Agar Foam as Embolization Agent

[0163] 3.1. Animal Settings and Embolization Procedure

[0164] A dry agar-agar foam obtained from the manufacturing process of the invention starting with a water solution at 4% of agar-agar, has been tested in vivo on swine as an embolization agent. CuraSpon®, an embolization agent made of pork gelatin, usually used in clinical practice for transient embolization, has been used as a comparative. The swine has been used as an embolization model because its renal arteries are easy to catheterize. Besides, its anatomy is similar to that of human (with an inferior polar artery and a superior polar artery that can be clearly identified), and the embolization of only one polar artery per kidney remains compatible with life for the 3 months study duration. In addition, double and symmetrical organs such as the renal model also allows having both target and control areas in each kidney while comparing the efficacy test and control agent between the contralateral kidneys.

[0165] Four Petrain swine (30 kg, 6 months old) were used in the study. To anaesthetize the swine during the application of the embolization agent, following intramuscular sedation (20 mg/kg ketamine and 0.03 mg/kg acepromazine), the animals were placed in a dorsal, recumbent position. A venous catheter was inserted into a large ear vein for blood sampling and intravenous access. Induction of anesthesia was obtained by 2 mg/kg propofol. After orotracheal intubation, anesthesia was maintained with gaseous sevoflurane (2%) by mechanical respiration (Dräger Zeus®, Dräger Inc., Telford, PA, US). Aseptic techniques were used throughout the procedure.

[0166] A digital subtraction angiography system (DSA) (Fluorostar, General Electric Medical System, Minneapolis, MN, USA) was used for endovascular procedures. Aseptic percutaneous access was performed by femoral arterial puncture under ultrasound guidance with the placement of a 6 French (F) vascular introducer by the Seldinger method. Catheterization of arterial vascular targets was performed by an experienced interventional radiologist using a Cobra Wirebraid 5F catheter (Cordis, Fremont, California, USA). Embolization was performed once the catheter was placed within each target artery (polar kidney artery).

[0167] The embolization agent, i.e. the agar-agar dry foam according to the invention or CuraSpon®, was shaped in torpedo and placed on tip of syringes containing a saline solution (FIGS. 10A and 10B). The agar-agar dry foam according to the invention was easily introduced into the catheter and then flushed with saline. No adhesion or blockage of the catheter has been noticed.

[0168] Two swine were embolized as acute setting. The dry agar-agar foam according to the invention was tested in torpedo form in different vascular territories (kidney, lumbar and mesenteric arteries). This scheme of embolization was performed to evaluate the ease, reproducibility and feasibility of foam of the invention torpedo manipulation as an embolic agent and releasing through the catheter. The effectiveness (arterial occlusion success) and the safety (risk of off-target embolization) were also examined.

[0169] Two swine were embolized in chronic setting, in which embolization with the dry agar-agar foam according to the invention, in the form of a torpedo, was performed on the left side kidney, whereas the right-side embolization was performed with torpedoes of CuraSpon®, as a comparison. Embolization with torpedoes respectively with the dry agar-agar foam according to the invention and CuraSpon® were realized once the catheter was placed within each target artery. The embolization was performed in the upper or lower polar branch of each kidney depending on the easy-catheterization access.

[0170] The two animals were followed during each endovascular procedure with a bi-daily clinical valuation. Repeated angiographic evaluations (DSA) were also realized to evaluate artery occlusion effectiveness and possible off-target embolization. The efficacy and short- to medium-term (3 months) tolerance of the dry agar-agar foam and CuraSpon® embolizations were assessed for each animal.

[0171] 3.2. Primary Endpoint and Short-Term Evaluation

[0172] The primary endpoint occurred when complete artery occlusion was achieved, or by obtaining significant reflux in the main renal artery. The percentage of embolized extent was estimated by subtracting the contoured renal surface before and after embolization (Horos™ Software, v3.3.6).

[0173] Short-term secondaries endpoints were safety (no off-target embolization), ease and duration of the the dry agar-agar foam embolization procedure compared to that of Curaspon®. This duration was established as the time between the last DSA acquisition before the start of the embolic agent administration, and the final DSA control after having obtained the main endpoint (target artery occlusion). The off-target embolized tissue area, when there was one, was estimated with the same method of the calculation of angiogram defect detailed previously by DSA subtraction method.

[0174] 3.3. Medium-Term Evaluation: Clinical Criteria, CT and DSA Controls, Histology Study

[0175] The 3-month (M3) follow-up was chosen in accordance with reference standard for animal studies ISO10993-6 in order to study the medium-term effects of implanted products.

[0176] After embolization procedure and during the 3 months follow-up, the four swine underwent a bi-daily clinical evaluation, especially to look for signs of infection or inflammation (temperature abnormalities, anorexia, loss of weight and signs of undernutrition, abnormal behavior).

[0177] Computed tomography (CT) explorations were performed one day before embolization (pre-embolization control), at M1 and at M3 after the procedure. Medium-term CT controls evaluated arterial occlusion, possible complications (urinoma, arterial aneurysm, signs of infection: infiltration of peri-renal fat, abscess, local or distant lymphadenopathy), cortical thickness and each kidney's volume (IntelliSpace Portal, Philips, 2015). The volume measure was estimated by repetitive contouring method. The decrease of kidney's volume between before and after embolization was evaluated for each group.

[0178] DSA controls at M3 assessed the persistence of the arterial occlusion, and the residual angiogram defect as detailed for short-term endpoints.

[0179] After this 3-month follow-up period, animals were euthanized in order to perform histological analyzes on explanted kidneys after sacrifice. Kidneys were harvested after careful dissection, and then both medullary and cortical parts were immersed in buffered 4% formalin liquid fixation. Samples were cut (4 μm thick) from paraffin blocks (Microm, France) and examined using hematoxylin-eosin (HE) staining (AutoStainer, DRS 2000 Saqura). The following parameters were evaluated: fibrin, neovascularization, hemorrhage, revascularization, cellular inflammatory parameters (fibroblasts, polynuclear, lymphocytes, macrophages, giant cells) and acute tubular necrosis.

[0180] 3.4. Results

[0181] Each target artery was catheterized according to standard procedure without technical failure. Embolization was feasible in each territory and without any general complication during the different procedures.

[0182] Embolization was performed for each target artery, without any difficulty, demonstrated by non-opacification in downstream arteries. There was none off-target embolization. The bi-daily clinical examination about the 2 swine in chronic setting did not show any sign of significant deterioration of general condition, or infection: the animals showed no significant weight loss, no sign of anorexia, no intrarectal temperature abnormality.

[0183] All vascular targets were effectively occluded, both for the acute and chronic models. DSA controls at D1, D3, D8, M1 and M3 showed persistence occlusion for kidneys arteries embolized with the dry agar-agar foam of the invention. Comparatively, the arteries initially occluded by the Curaspon® were permeable on D8 control and on all the following controls. Thus, 8 days after embolization, Curaspon® is reabsorbed. This confirms that the Curaspon® is not suitable for obliterating an artery over one week and may be only used for transient embolization.

[0184] CT controls at M1 and M3 (FIG. 11) showed cortical atrophy corresponding to the embolized area. For kidneys embolized with the dry agar-agar foam of the invention, a significant decrease of the volume (atrophy) was observed. The average kidney's volume embolized by Curaspon® showed no significant decrease of the volume in comparison of the kidney's volume before embolization. There were no CT signs of complication (abscess, local lymph nodes, peri-renal infiltration, urinoma or renal excretion failure) on each control. This demonstrates an efficient and stable embolization over time with the dry agar-agar foam.

[0185] Similarly, computed tomography (CT) scan after arterial embolization of the lower left polar artery with the foam of the invention, and lower right polar artery by CuraSpon®, associated with virtual reality reconstruction (FIG. 12) shows on a more macroscopic level a significant atrophy of the lower left kidney whereas the right kidney embolized with Curaspon® shows no significant decrease of its volume.

[0186] As can be seen of the photography of the explanted kidney embolized with the foam of the invention (FIG. 13), an atrophy of the inferior part of the kidney is visible by color change. The non-embolized part of the kidney remains unchanged and in good working order.

[0187] The end-of-follow-up histological analysis at M3 did not show any sign of significant tissue inflammation (data not shown). Histological analysis at M3 showed no significant difference of necrosis inflammatory parameters between the two groups, especially no sign of abscess or granuloma. Necrotic rearrangements were found for both groups, associating calcifications, micro-cysts with thin walls, and also non-specific hemorrhagic changes supposed to be of post necrosis origin or related to manipulations during harvesting. The dry agar-agar foam of the invention is then safe and non-toxic for an in vivo use.

[0188] 3.5. Conclusion

[0189] These in vivo results obtained on swine constitute a proof of concept of a new embolization procedure, using biological embolic agent, made from vegetable agar-agar foam. It demonstrates the efficacy and safety of this new agent. This study demonstrates that the dry agar-agar foam of the invention may be used as a permanent embolization agent and can be easily shaped in a torpedo shape like porcine-gelatine Curaspon®.

BIBLIOGRAPHY

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